RU2771605C2 - Peptides for intracellular delivery of nucleic acids - Google Patents

Abstract

FIELD: genetic engineering and biochemistry.SUBSTANCE: invention relates to the field of genetic engineering and biochemistry, as well as to medicine and pharmaceuticals, and is aimed at creating low-toxic cationic dendrimeric peptides as intracellular transporters of nucleic acids (NA) of various structures. A means for intracellular delivery of nucleic acids into mammalian cells is proposed, which is a dendrimeric cationic peptide.EFFECT: resulting agent provides delivery of both plasmid DNA and small interfering RNA (siRNA), which makes it possible to use it as a promising agent for gene therapy, including therapy based on RNA interference.1 cl, 15 dwg, 2 tbl, 3 ex

Description

The invention relates to genetic engineering, biochemistry, medicine and pharmaceutical technologies and is associated with the creation of low-toxicity cationic dendrimeric peptides as intracellular transporters of nucleic acids (NA) of various structures. Transfection of NK cells is an effective way to introduce a new gene or, conversely, to purposefully suppress the expression of a native or foreign gene. The last method, the so-called RNA interference, implemented by intracellular introduction of small interfering RNA (siRNA), can cover a very wide range of diseases, including congenital, infectious, oncological and immune diseases. The discovery of the phenomenon of RNA interference as a way to regulate gene expression prompted researchers and pharmaceutical companies to develop various variants of gene therapy drugs containing miRNA molecules. To manifest specific activity, NA must penetrate into the cytoplasm of the target cell, but the fulfillment of this condition has a number of difficulties. First of all, free NA in the biological environment is very quickly destroyed by the action of endogenous nucleases, and due to the high hydrophilicity and significant negative charge, NA molecules are practically unable to pass through the cell membrane barrier. Therefore, the main emphasis in the development of such drugs is associated with the creation of effective NA delivery systems.

Viruses, recombinant virions, and various classes of substances, including inorganic substances, organic amphiphilic polymers, biopolymers, lipids, liposomes, polysaccharides, nanoparticles, and peptides, have been studied as possible NA transporters. Although viruses are effective penetrating agents, however, due to the potential biological hazard (possible pathogenic mutations, unwanted immune response), the use of replicating agents is generally avoided in practice. Synthetic polymers and inorganic nanoparticles are often highly toxic and tend to accumulate in the body. One of the attractive solutions in terms of safety is the use of non-viral systems based on cationic biodegradable vectors of peptide and/or lipid nature due to their low immunogenicity and biodegradability. Such agents form complexes with negatively charged NAs and condense them into compact nanostructures, which, on the one hand, protects NAs from the action of nucleases, and, on the other hand, promotes their translocation through cell membranes due to endocytosis. Usually, the cationic carrier is in excess in the composition of the complex, providing a total positive charge of the entire complex, which increases the efficiency of its penetration into cells, because the cell surface usually carries a negative charge.

Cationic peptides (CPs) should be considered as promising NA transporters of this group due to their distinctive advantages. These include: high transmembrane activity, the presence of a well-defined composition/structure, storage stability, which allows for the analysis of purity and structure by modern methods (mass spectrometry, high-performance chromatography, NMR), providing the necessary standardization of the drug. At the same time, the possibilities of modern peptide chemistry provide great opportunities in the design of peptide structures based on the variation of amino acid sequences, which makes it possible to regulate their topology, change the hydrophilic-lipophilic balance, the distribution of negative and positive charges, and change the enantiomeric composition. In addition, the comparative availability of methods for their synthesis and scalability are attractive; production can be carried out automatically. Thus, in terms of these parameters, peptides as a delivery vehicle have a clear competitive advantage over liposomes. Considering the classification of PCs capable of penetrating cells, the following classes should be distinguished: protein fragments that specifically bind DNA/RNA molecules, protein transduction domains and signal structures, natural antimicrobial peptides (more often their fragments), sequences from viral and bacterial adhesion proteins, and peptides of the original design, including peptides from phage libraries [1].

It should be noted that the resistance of linear PCs to proteolysis in a biological environment is severely limited, since the α-amide bonds of the amino acid chain are easily cleaved by extra- and intracellular proteases [2]. Moreover, linear PCs with a strong positive charge can be quite toxic and can destroy cell membranes, up to complete cell lysis. These features are of decisive importance in the case of the use of PC as a component of therapeutic agents. Various approaches have been taken to increase proteolytic stability, including the replacement of L-amino acids with D-forms, the introduction of non-natural fragments, and chemical modification of the peptide skeleton [3, 4]. In the free state, due to the electrostatic repulsion of charged atoms, especially in the case of lysine-containing peptides, CPs tend to acquire an elongated conformation, while the introduction of a fatty acid into them promotes the formation of micelles or liposomes, which greatly increases their affinity for lipid membranes [5, 6]. However, the introduction of highly hydrophobic residues significantly reduces the solubility of the peptides in an aqueous medium. This issue is usually resolved by converting such lipopeptides into liposomes, but again the problem of stability and storage of liposomes arises, since they exist only in an aqueous environment. It should be noted that in some works and patents, not electrostatic complexes are used for transfection, but covalent conjugates of NA with the carrier peptide, and covalent binding is sometimes carried out through the cysteine mercapto group, which is introduced into the peptide and which is able to interact with the activated NA molecule. This approach is due to the fact that when the conjugate enters the cell, cytoplasmic reductases can cleave this bond, thereby releasing NA. However, such a nucleic acid delivery system significantly complicates the practical implementation of the transfection method, since an additional complex chemical modification of the target NA is required to introduce a special SH-reactive group, which can disrupt the functional specificity of the NA.

A promising alternative to linear peptides as delivery vehicles are cationic dendrimeric peptides (DCPs). DCTs are branched three-dimensional structures with a dense core and an outer layer consisting of positively charged groups. The presence of a large number of terminal charged groups allows DCT to effectively bind and condense NA. The use of peptide dendrimers for transfection largely eliminates the problem of proteolytic stability, since they contain unnatural ε-amide bonds in their structure [2, 7, 10]. All functional features of such peptides are mainly concentrated in the terminal regions of the LTR, where the main charge is concentrated (Fig. 1).

It should be noted that these peptides are much less toxic than linear peptides with the same amino acid composition, although they are also able to penetrate cell membranes and can interact with both surface and intracellular cellular targets [8, 9]. Most importantly, they are much more resistant to enzymatic biodegradation due to their branched structure and the presence of unnatural amide bonds. The high solubility of CDP in an aqueous medium, even in the presence of hydrophobic amino acids, is of decisive importance for the systemic administration of such a drug [10]. By changing the structure of the terminal groups, it is possible to control its properties: charge, hydrophobicity, pharmacokinetics. The introduction of cysteine into the LCT structure allows one to selectively attach a specific label to study the behavior of such a molecule in the body.

Our previous experiments have shown that arginine-containing dendrimeric peptides, such as LTP (Fig. 1) with 8-terminal arginine residues, have a markedly higher transfection activity compared to linear analogs. Initially, it was thought that import into the cell occurs by direct penetration through the plasma membrane, but then it was found that endocytosis plays a major role in it, which includes macropinocytosis, clathrin-mediated and caveolin/raft-mediated endocytosis. But, probably, transport can go in parallel along these pathways, and further study showed that import into the cell can also go along an endocytosis-independent pathway due to interaction with heparin sulfate, which plays the role of a low-affinity receptor for cationic peptides. And in order to ensure cross-linking of molecules of such a receptor for the internalization of the peptide, obviously, a certain specific length of the peptide is required. Experimentally, we have previously established that the 17-membered dendrimeric peptide with terminal arginines (R) was much more active than the peptide where the terminal arginine was replaced by lysine (K). The structural feature of the arginine guanidine group can serve as a key to understanding such an increased activity of R peptides [17]. In an aqueous medium, the guandine cation containing three charged amino groups forms a planar pseudoaromatic structure with three conjugated bonds with a very high pK (13.6). Such an ion forms strong hydrogen bonds with water molecules located in the same plane, thus forming an amphiphilic cation with a hydrophobic front. The hydrophobic plane/front face of the cation is able to interact with hydrophobic aromatic amino acids, tryptophan, tyrosine, and at the same time, such a cation can also actively bind to anionic amino acids due to the coordination of oxygen atoms, and all this contributes to the disorganization/denaturation of the protein structure. Surprisingly, in contrast to K-peptides, where the cation has an ammonium structure, R-peptide molecules in an aqueous medium do not exhibit mutual electrostatic repulsion. Moreover, it was found that in an aqueous environment, an energetically favorable pairing of guanidine groups in planar orientation (stacking interaction) is observed. Therefore, R-peptides are capable of forming associates in water, while K-peptides, where repulsive forces dominate, are not. The binding of the R-peptide to the membrane is two orders of magnitude stronger than that of the K-peptide. Thus, branched dendrimer constructs containing only two arginine residues are able to bind and penetrate into cells more efficiently than constructs with two lysines. The degree of binding and uptake is enhanced if additional arginine residues are included in the peptide construct.

The formation of a complex between the dendrimer and NA prevents the interaction of the latter with serum albumin, which is known to be the main obstacle to the delivery of free NA to tissues during gene therapy. Dendrimers can also be used for gene transfer for the treatment of malignant neoplasms. Thus, in the gene therapy of breast cancer, dendrimers were used to deliver the carcinostatic angiostatin and the tissue inhibitor gene for metalloproteinase (TIMP-2). As a result, a significant reduction in the proliferation of endothelial cancer cells was shown. The efficiency of delivery of genetic constructs to tumor tissue was tested in mice, which showed a significant reduction in malignant growth. Dendrimers have also been shown to be effective in suppressing gene expression based on the principle of RNA interference [11].

Synthesis of DCT can be carried out by conventional standard methods of solid phase synthesis. In practical terms, such peptides are synthesized faster than linear ones, since each addition step leads to an exponential increase in the number of amino acids. For example, a 17-member DCT requires only 6 condensation steps (FIG. 1). The only problem is that in solid-phase synthesis at high degrees of branching (generation), for example, more than 3-4 generations, steric hindrances and slowing down of the reaction rates can occur. However, this problem can be overcome by using low cross-linking, high swelling solid state synthesis resins having a gel structure (PEG chains), another option is whole solution synthesis.

Currently known inventions for intracellular delivery of NA for transfection, including RNA interference, based on dendrimeric molecules. It should be noted that many methods for obtaining complexes of dendrimers with miRNAs and plasmids and protocols for their delivery into cells are published in the public scientific (medical and biochemical) literature. True, most of the claimed methods, due to the ease of their synthesis, are associated not with the use of DCT, but dendrimer polymers, synthetic polyamidoamines (PAMAM), with various terminal structures. For example, in the patent PCT/GB 02/04706 dated 24.04.2003 "Dendrimers for use in targeted delivery" describes the use of cationic dendrimers for the delivery of NA, peptides and pharmaceutical agents. Polymers derived from diamino-ethane/propane/butane, which can be modified with hydrophobic, hydrophilic and amphiphilic groups, are used as the dendrimer base. However, such compounds have a carbochain structure without a definite clear structure and are incapable of biodegradation.

The invention described in US patent 6376248 dated April 23, 2002 "Peptide-enhanced transfections" is known, in which the authors propose numerous compositions for transfection of eukaryotic cells, including NA complexes with peptides, where the nucleic acid is covalently linked to a linear cationic peptide with the amino acid sequence TAT- peptide (from HIV), dendrimer, or lipopeptide The dendrimers herein are derived from a commercial product, the high molecular weight PAMAM dendrimer (from Dendritech Inc.), terminated with lysine (LysDmer) or arginine (ArgDmer) residues. However, the known solution has increased toxicity, difficulty in obtaining, has a limited shelf life.

A well-known publication [2] describes the use of DKP, a variant of a polylysine dendrimer, where phenyl-imidazole residues are introduced into the terminal branches of the dendrimer to obtain an amphipathic structure. The highest efficiency in terms of protecting NA (by the example of plasmid DNA) from degradation by nucleases was obtained using a polylysine dendrimer with a branching degree of 5 (generation G5), the same preparation, the only one, showed acceptable transfection efficiency. In another work by the same authors [12], G5 generating dendrimers with terminal arginine residues showed high efficiency in transfection of cells with plasmid DNA and subsequent expression of the GFP (green fluorescent protein) or pGL3 (luciferase) gene. The G6 generating dendrimer, although also effective, was much more toxic, probably due to the high positive charge density. It should be noted that the yield during the synthesis of such dendrimers is quite low, and the drugs were not used to suppress gene expression (miRNA interference).

The document CN 102911252 B dated February 6, 2013 "Cationic lipid containing peptide dendrimer, transgenic carrier and preparation method and application of transgenic carrier" describes compounds as carriers for intracellular delivery of plasmids containing GFP or pGL3 genes. The structure of the described media will contain a short arginine-containing dendrimer with a lipid fragment containing cholesterol. However, transfection in the format of RNA interference (miRNA) is not given in the patent.

Patent RU 2127125 dated March 10, 1999 (“Biologically active and/or target dendrimer conjugates”) describes dense star polymer conjugates associated with a biological modulator, and in one of the embodiments, a dense star polymer is a dendrimer, but obtained not on the basis of a branched peptide, but based on PAMAM polymers, polyethyleneimine and others.

In patent RU 2575603 dated February 20, 2016 “Production of complexes of nucleic acids and cationic components cross-linked with disulfide bonds intended for transfection and stimulation”, the authors propose to use a composition containing a polymeric carrier and a carrier molecule as an immunostimulatory agent or adjuvant. Including oligopeptides and proteins with the general formula (Arg) ₁ ;(Lys) _m ;(His) _n ;(Orn) _o ;(Xaa) _x , however, the authors do not indicate whether these are linear peptides or dendrimers. As NK, single-stranded RNA is used to induce innate or adaptive immunity.

The article [13] describes LCT with G3 generation for cell transfection with miRNA molecules. Lysine-based dendrimeric peptides contain a hydrophobic region represented by either hydrophobic amino acids or fatty acids in order to more efficiently exit the endosome into the cytoplasm after internalization of the NA/peptide complex by the cell. Among them, the most active LTR in miRNA transfection were: (KL) ₈ (KKL) ₄ (KLL) ₂ KK(C ₁₆ )K(C ₁₆ ) and (KL) ₈ (KKL) ₄ (KLL) ₂ KLLLL) However, it must be emphasized that the synthesis and purification of such peptides (length 37 and 39 amino acids + fatty acids) is a rather complicated and extremely expensive procedure (done by hand), resulting in very low yields (less than 10 percent).

The article [14] describes the use of polylysine dendrimers containing more than 40 lysine residues for improved delivery of DNA (plasmids) into cells as agents for antitumor therapy, indicates the high speed and efficiency of transfection, as well as relatively low toxicity to healthy tissue. Their use for RNAi has not been described. Obviously, due to the high molecular weight, the yields of peptides during solid-phase synthesis were very low.

The closest analogue of the claimed invention is the LTP dendrimeric peptide described in patent RU 2572575 C1 dated January 20, 2016, which has transfection activity when complexed with NA, low toxicity to mammalian cells. However, compared with the liposomal delivery vehicle Lipofectamine ^® 2000, used as the gold standard, LTP has a transfection activity 2-3 orders of magnitude lower.

The objective of this invention is to create low-toxic dendrimeric peptides capable of providing efficient transport of oligonucleic acids into mammalian cells and stimulating DNA and RNA transfection in order to both selectively suppress the expression of the target gene by RNA interference and stimulate the expression of the target gene.

The problem is solved due to the fact that the specified tool is a tool for intracellular delivery of nucleic acids into mammalian cells, containing an effective amount of dendrimeric cationic peptides, which are synthetic branched structures with lysines at the branching sites, characterized by the structural formula:

R _4n K _2n X _2n K _n X _n K(ZZ)C-Lip _m , where

n=1 or 2;

R - arginine;

K - lysine;

X=0-5 amino acids in any order, where the amino acids are: K (lysine), glycine (G), tryptophan (W), leucine (L), isoleucine (I), threonine (T), proline (P), glutamic acid (E), valine (V), alanine (A) or histidine (H);

(ZZ)C - C-terminal linear amino acid sequence, any 1-5 amino acids with cysteine (C) at the end;

Lip _m is a lipophilic hydrocarbon chain, where the length of the hydrocarbon chain is from C6 to C18 and is linked through a maleimide group to a cysteine residue (C), where m=1 or 0 is the absence of Lip.

The technical result of the present invention is to create a modular construction of dendrimeric peptides, consisting of an N-terminal cationic module, a central part and a reactive module at the C-terminus. The cationic module is a charged cluster of N-terminal arginines and provides interaction with nucleic acids and a negatively charged cell surface and translocation of the peptide-nucleic acid complex into the cell. The central part is a hydrophobic core of lysine residues, which provides additional affinity for the cell membrane and the necessary three-dimensional structure. It is adjacent to a short hydrophilic region containing a cysteine residue, which, due to the free thiol group, acts as a reactive module, providing the possibility of further modification of the peptide by various molecules. In particular, in the present invention, a modification was carried out with hydrophobic molecules with a hydrocarbon chain length from C6 to C18 according to the thiol-maleimide click reaction, which contributes to an increase in the transfection/transport activity of the dendrimer. The mass ratio of the positively charged peptide to the negatively charged NA in the range from 10 to 50 ensures the formation of complexes of the claimed DCTs with NA, which are capable of effectively penetrating various types of cells. Thus, the resulting agent provides the delivery of both plasmid DNA and small interfering RNA (siRNA), which makes it possible to use them as a promising agent for gene therapy, including therapy based on RNA interference.

Brief description of graphic materials, figures:

Figure 1. Structural diagram of the LTP dendrimeric peptide (terminal amino acids are arginine (R) bearing a positive charge).

Figure 2. Structure and mass spectrum of the synthesized TA-22 peptide (R ₈ K ₄ K ₂ KAC-mal-N-5-carboxypentyl)* xTFA (trifluoroacetate), M=2547 g/mol, Charge +15, Cationic amphiphilic peptide dendrimer (salt form, high charge density).

Figure 3. Structure and mass spectrum of the synthesized peptide KK-50 (R ₄ K ₂ G ₂ KAWWC) xTFA, M=1687 g/mol, Charge +8, Cationic amphiphilic peptide dendrimer (salt form, average charge density).

Figure 4. Structure and mass spectrum of the synthesized peptide KK-61 (R ₄ K ₂ G ₂ KAWWC-mal-C ₂ H ₄ -CO-NH-C ₁₆ H ₃₃ )* xTFA, M=2078 g/mol, Charge + 8, Cationic amphiphilic lipopeptide dendrimer (salt form, medium charge density).

Figure 5. Structure and mass spectrum of the synthesized peptide KK-46 (R ₈ K ₄ W ₄ T ₄ P ₄ E ₄ V ₄ K ₂ H ₂ KFC)* xTFA, M=5140 (+6H) g/mol, Charge + 16, Cationic amphiphilic peptide dendrimer (salt form, high charge density).

Figure 6. Structure and mass spectrum of the synthesized peptide SA-32 (R ₈ K ₄ K ₂ Y ₂ KPWC)* xTFA, M=2875 g/mol, Charge +16, Cationic amphiphilic peptide dendrimer (salt form, high charge density) .

Figure 7. Structure and mass spectrum of synthesized peptide TA-11 (R ₈ K ₄ K ₂ KARGDP)* xTFA, M=2660 g/mol, Charge +16, Cationic amphiphilic peptide dendrimer (salt form, high charge density).

Figure 8. Transfection activity of LCT on the HeLa cell line (human cervical carcinoma), where the (y) axis represents the relative light units per 100 thousand cells, the (x) axis shows the mass ratio of the peptide to NA; KK61, KK46, TA11, KK50, SA32, TA22 - synthesized DCTs; bLTP - control peptide, P1 - negative control, pGL3 plasmid without transfection agent, Lf - positive control, Lipofectamine ^® 2000 in the ratio recommended by the manufacturer to the plasmid.

Figure 9. Transfection activity of LCT on the Vero C1008 (E6) cell line (African green monkey kidney), where the (y) axis represents the relative light units per 100 thousand cells, the (x) axis shows the mass ratios of the peptide to NA; KK61, KK46, TA11, KK50, SA32, TA22 - synthesized DCTs; bLTP - control peptide, P1 - negative control, pGL3 plasmid without transfection agent, Lf - positive control, Lipofectamine ^® 2000 in the ratio recommended by the manufacturer to the plasmid.

Figure 10. Transfection activity of LCT on the cell line FECh (human embryonic fibroblasts), where the (y) axis represents the relative light units per 100 thousand cells, the (x) axis shows the mass ratio of the peptide to NA; KK61, KK46, TA11, KK50, SA32, TA22 - synthesized DCTs; bLTP - control peptide, P1 - negative control, pGL3 plasmid without transfection agent, Lf - positive control, Lipofectamine ^® 2000 in the ratio recommended by the manufacturer to the plasmid.

Figure 11. Transfection activity of LTP on the HEK293 cell line (human embryonic kidney), where the (y) axis represents the relative light units per 100 thousand cells, the (x) axis shows the mass ratio of the peptide to NA; KK61, KK46, TA11, KK50, SA32, TA22 - synthesized DCTs; bLTP - control peptide, P1 - negative control, pGL3 plasmid without transfection agent, Lf - positive control, Lipofectamine ^® 2000 in the ratio recommended by the manufacturer to the plasmid.

Figure 12. Transfection activity of LCT on the HepG2 cell line (human hepatocellular carcinoma), where the (y) axis represents the relative light units per 100 thousand cells, the (x) axis shows the mass ratio of the peptide to NA; KK61, KK46, TA11, KK50, SA32, TA22 - synthesized DCTs; bLTP - control peptide, P1 - negative control, pGL3 plasmid without transfection agent, Lf - positive control, Lipofectamine ^® 2000 in the ratio recommended by the manufacturer to the plasmid.

Figure 13. Transfection activity of LTP on cell line A549 (human lung carcinoma), where the (y) axis represents relative light units per 100 thousand cells, the (x) axis shows the mass ratio of the peptide to NA; KK61, KK46, TA11, KK50, SA32, TA22 - synthesized DCTs; bLTP - control peptide, P1 - negative control, pGL3 plasmid without transfection agent, Lf - positive control, Lipofectamine ^® 2000 in the ratio recommended by the manufacturer to the plasmid.

Figure 14. Transfection activity of LCT on the CHO-Luc cell line (Chinese hamster ovary cell line transfected with a plasmid encoding the luciferase gene), where the (y) axis represents relative light units per 100 thousand cells, the (x) axis shows mass ratio of peptide to NA; KK61, KK46, TA11, KK50, SA32, TA22 - synthesized DCTs; bLTP - control peptide, siLuc - negative control, siLuc siRNA without transfection agent, Lf - positive control, Lipofectamine ^® 2000 in the manufacturer's recommended ratio to siRNA.

Figure 15. Modular design of dendrimeric peptides (example of TA-22 peptide). The cationic cluster represented by L-arginine residues is highlighted in red, hydrophobic regions are marked in yellow, and the hydrophilic fragment is marked in blue.

The invention is illustrated by the examples below.

Example 1. Solid phase synthesis of peptides R _4n K _2n X _2n K _n X _n K(ZZ)C-Lip _m

The reagents and solvents used were obtained from Sigma-Aldrich/Merck, Anaspec, ApexBio, Gyros Proteins, Alfa Aesar, Merck, Fluka AG, Reachem, Novabiotech, Protein Technologies Inc, Carl Roth GmbH, Scharlau, Fisher Chemical in either peptide quality synthesis, or HPLC, or GLC/MS, or for analysis. The peptides were synthesized using a PS3 automated peptide synthesizer (Protein Technologies Inc., USA).

High performance liquid chromatography (HPLC) was used to purify the peptides. One of the systems used consisted of a 321-H binary pump (Gilson), a Vydac 218TP1022 column (Grace), a Spectroflow 757 photometric detector (Kratos Analytical), a 2210 recorder (LKB), and a Multirac 2111 (LKB) fraction collector, another system was the LC Series -20 Prominence (Shimadzu, Japan). The elution used a linear gradient of 5-70% eluent B (25 minutes) at 10 ml/min with detection at 226 nm. Eluent A contained 0.1% TFA/H ₂ O; eluent B contained pure acetonitrile (Sigma-Aldrich). For preparative HPLC, an LC-20 Prominence Shimadzu system (Japan) was used.

All peptides were synthesized using solid phase synthesis methods with Fmoc-protected α-amino groups on Rink Amide MBHA resin using an automated synthesizer or manually using Fmoc chemistry protocols and N-hydroxybenzotriazole/diisopropylcarbodiimide (HBT/DIC) or tetramethyluronium benzotriazole hexafluorophosphate (HBTU) amino acid activation methods. The functional groups of the Fmoc-amino acid side chains (AnaSpec) were protected with oxy-tert-butyl (Ot-Bu) for carboxyl groups, tert-butyl (t-Bu) for hydroxyl groups, tert-butyloxycarbonyl (Boc) or 9-Fluorenylmethoxycarbonyl (Fmoc) for ε-amino groups of lysine, a trityl group (Trt) for the SH group of cysteine and the imidazole group of histidine, and 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (Pbf) for the guanidine group of arginine. Synthesis was performed either manually or automatically using Rink-amide-MBHA (NH ₂ -P) resin (NovaBiochem) and 2-fold excess of activated Fmoc-amino acids and appropriate reagents.

The scheme of solid-phase synthesis is based on a repeating cycle of standard operations (RU 2572575 C1 dated January 20, 2016), which includes:

1) At the start, a portion of the original Rink Amide resin is added to the reactor, dimethylformamide (DMF) is added, the suspension is vigorously stirred until the resin is completely swollen, the operation is repeated 3 times for 3 minutes, removing the solvent by pressure filtration.

2) Removal of the Fmoc protection with a 20% solution of 4-methylpiperidine (MPP) in DMF for 10 min.

3) Washing the resin with DMF.

4) Pre-activation of a 2-fold molar excess of Fmoc-amino acid with DIC/HBT or HBTU in DMF or N-methylpyrrolidone (MP) medium.

5) Amino acid condensation by adding activated Fmoc-amino acid to the reactor in DMF or N-methylpyrrolidone (MP) medium and incubating for 1 hour.

6) Washing the resin with DMF peptide.

7) Operations on points 2-6 are repeated.

The cleavage of peptides from the resin was carried out in a 95% TFA solution with the addition of thioanisole, 1,2-ethanedithiol, and deionized water, which resulted in the complete removal of the remaining protective groups from the side chains (all the protective groups of the side chains, except for Fmoc, were labile for TFA). The crude peptide products were precipitated with diethyl ether, extracted with dilute acetic acid and then freeze-dried before purification. The crude peptide was purified by gradient HPLC using 0.1% aqueous trifluoroacetic acid and acetonitrile as the mobile phase. Declare DKP - stable substances when stored in a dry form. Their synthesis can be carried out using an automatic peptide synthesizer or in manual mode.

As a result, 6 dendrimeric cationic peptides were synthesized and purified: KK-50 (R ₄ K ₂ G ₂ KAWWC), SA-32 (R ₈ K ₄ K ₂ Y ₂ KPWC), TA-11 (R ₈ K ₄ K ₂ KARGDP ), KK-46 (R ₈ K ₄ W ₄ T ₄ P ₄ E ₄ V ₄ K ₂ H ₂ KFC), TA-22 (R ₈ K ₄ K ₂ KAC(C ₄ H ₂ NO ₂ )C ₅ H ₁₀ COOH), KK-61 (R ₄ K ₂ G ₂ KAWWC-(C ₄ H ₂ NO ₂ )-C ₂ H ₄ -CO-NH-C ₁₆ H ₃₃ ) (Table 1, Figs. 2-7).

where, arginine (R); lysine (K); glycine (G), tryptophan (W), threonine (T), proline (P), glutamic acid (E), valine (V), alanine (A) or histidine (H);

In particular, the sequence of operations in the synthesis of the TA22 peptide is as follows:

Fmoc-C(Trt)-OH+NH ₂ -P-(Rink-Amid) → Fmoc-C(Trt)-P → H-Cys(Trt)-P → Fmoc-AC(Trt)-P → H-AC (Trt)-P → Fmoc-K(Fmoc)-AC(Trt)-P → H-KAC(Trt)-P → (Fmoc-K(Fmoc)) ₂ -KAC(Trt)-P → (HK) ₂ KAC(Trt)-P → (Fmoc-K(Fmoc)) ₄ K ₂ KAC(Trt)-P → K ₄ K ₂ KAC(Trt)-P → (FmocR(Pbf)) ₈ K ₄ -K ₂ KAC( Trt)-P → R ₈ K ₄ K ₂ KAC-NH ₂ + N-maleimidehexanoic acid → R ₈ K ₄ K ₂ KAC-(C ₄ H ₂ NO ₂ ) -C ₅ H ₁₀ COOH.

amino acids used: C - Cysteine, A - Alania, K - Lysine, R - Arginine.

Thus, a modular construction of dendrimeric peptides was created, consisting of an N-terminal cationic module, a central part, and a reactive module at the C-terminus. The cationic module is a charged cluster of N-terminal arginines and provides interaction with nucleic acids and a negatively charged cell surface and translocation of the peptide-nucleic acid complex into the cell. The central part is a hydrophobic core of lysine residues, which provides additional affinity for the cell membrane and the necessary three-dimensional structure. It is adjacent to a short hydrophilic region containing a cysteine residue, which, due to the free thiol group, acts as a reactive module, providing the possibility of further modification of the peptide by various molecules. In particular, in the present invention, a modification was carried out with hydrophobic molecules with a hydrocarbon chain length from C6 to C18 according to the thiol-maleimide click reaction, which contributes to an increase in the transfection/transport activity of the dendrimer.

Example 2. Analysis of the transfection activity of the peptide when delivered to cells with plasmid DNA or miRNA.

For analysis, Vero C1008 (E6), HEK293, A549, HepG2, HEF, HeLa, CHO-Luc cells were seeded in a 48-well plate at 4×10 ⁴ cells per well in 300 µl of the appropriate complete nutrient medium (DMEM, or F12 , or Needle-Mem, or RPMI containing 10% fetal bovine serum, 2% HEPES buffer, 0.1% antibiotic gentamicin, 0.6% L-glutamine) the day before transfection and incubated overnight at 37°C in a CO ₂ incubator until reaching 75% confluence. Transfection agent dispersion in 3 dilutions (with mass ratios of peptide to NA - 50:1, 25:1 and 12.5:1) with a total volume of 80 µl, consisting of 0.3 µg of pGL3 plasmid or siLuc siRNA (duplex GGAAAGACGAUGACGGAAAtt/UUUCCGUCAUCGUCUUUCCtt) and a solution of the peptide in the appropriate concentration according to the mass ratios to NA were prepared in OPTIMEM serum-free medium. The commercial transfection agent Lipofectamine2000 (Lf) was used as a positive control. The unsupported pGL3 plasmid (P1) was used as a negative control. The closest synthetic analogue, the LTP peptide (bLTP - control peptide) (RU 2572575 C1), was used as a reference agent. The prepared mixtures were kept for 15 min at room temperature and added to the wells with a monolayer of cells. Before the introduction of the transfection mixture in the wells with cells, the complete nutrient medium was replaced with a serum-free one. Cells were incubated at 37°C in a CO ₂ incubator for 4 hours. Then 30 μl of 10% fetal bovine serum was added to the cells. The cells were incubated at 37° C. in a CO ₂ incubator for 24 hours and then the luciferase activity was determined by the luciferase test method. The test was performed using a commercial kit "Luciferase Assay System" (Promega) according to the manufacturer's recommendations. To do this, the growth medium was removed from the wells, then 100 µl of lysis buffer (Glolysis buffer, 1x, USA) was added. Cells were kept for 15 minutes at 37° C. in a CO ₂ incubator to achieve complete lysis. They were then scraped off from the bottom of the wells and the cell suspension was transferred into 1.5 ml eppendorf tubes. To precipitate cell debris, the resulting lysate was centrifuged for 2 minutes at 10,000 rpm. 50 µl of the supernatant were collected into separate 1.5 ml eppendorf tubes and the luciferase substrate was added to them in a ratio of 1:1. Transfection efficiency was assessed by the level of luminescence on a Glomax 20/20 luminometer. For all quantitative data, the arithmetic mean (M) and standard error of the mean (m) were calculated. Intergroup differences were determined and the significance of differences between groups was assessed using Student's t-test using the Statistica 8.0 program. The transfection activity of LTP on various cell lines is shown in FIG. 8-14).

From the presented data, it can be seen that the mass ratio of the positively charged peptide to the negatively charged NA in the range from 10 to 50 ensures the formation of complexes of the claimed DCT with NA, which can effectively penetrate into various cell types. Thus, the resulting agent provides the delivery of both plasmid DNA and small interfering RNA (siRNA), which makes it possible to use them as a promising agent for gene therapy, including therapy based on RNA interference.

Example 3 Peptide cytotoxicity assay.

Vero C1008 (E6), HEK293, A549, HepG2, HEF, HeLa, CHO-Luc cells were seeded in a 96-well plate at 3×10 ⁴ cells per well in 100 µl of the appropriate complete nutrient medium (DMEM, or F12, or Igla -Mem, or RPMI containing 10% fetal bovine serum, 2% HEPES buffer, 0.1% antibiotic gentamicin, 0.6% L-glutamine) and incubated overnight at 37°C in a CO ₂ incubator to 75% confluence. Peptide concentrations in wells were varied (5500, 2750, 1375, 688, 344, 172, 86, 43, 21, 11, 5 μg/mL) by serial dilution. The plate was then incubated at 37° C. in a CO ₂ incubator. A day later, 25 μl of tetrazolium dye MTT in phosphate-buffered saline at a concentration of 4 mg/ml was added to each well, and the plate was placed in a CO ₂ incubator. After 4 hours, 50 μl sodium dodecyl sulfate (20% SDS in water with 0.02N H ₂ SO ₄ ) was added to the wells and incubated overnight. Then the difference in the optical density of the solution at 570 and 650 nm was determined on a spectrophotometer (Thermo Scientific™ Multiskan™ GO Microplate Spectrophotometer). The number of living cells was calculated as the ratio of the optical density (A) in the well with the peptide to the optical density in the positive control (100% live cells) according to the formula, where both values were normalized to the negative control (100% dead cells):

number of living cells, % = (A _peptide -A _-control )/(A _+control -A -control) _⋅100 %

Each value was obtained by averaging several parallel measurements in duplicate. We built a graph of the obtained values from the concentration, determined the IC ₅₀ in μg / ml, the values \u200b\u200bare presented in table 2.

Analysis of the cytotoxicity of the claimed DCTs (LD50>0.2 mg/ml) indicates their low toxicity.

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Claims (5)

An agent for intracellular delivery of nucleic acids into mammalian cells, which is a dendrimeric cationic peptide selected from:

where amino acids are: arginine - R, lysine - K, glycine - G, tryptophan - W, threonine - T, proline - P, glutamic acid - E, valine - V, alanine - A, histidine - H, phenylalanine - F, tyrosine - Y.

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