RU2533817C1 - Improving genetic constructs for providing high efficacy of anti-hiv therapy - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medical and molecular genetics. There are described genetic constructs expressing RNA sequences and genes coding proteins possessing the antiviral activity on human immunodeficiency virus. The genetic constructs contain a sequence coding the human TRIM5a modified protein. The invention can be used in scientific investigations.

EFFECT: presented constructs enable providing the higher expression of the modified gene of the human TRIM5a.

40 cl, 4 dwg, 3 tbl, 13 ex

Description

Technical field

The invention relates to medical and molecular genetics, in particular to genetic constructs expressing RNA sequences and / or genes encoding proteins having antiviral activity against human immunodeficiency virus, and can be used in scientific research.

State of the art

HIV infection remains a huge problem for health and society as a whole. The number of HIV-infected continues to grow steadily. Modern highly active antiretroviral therapy (HAART) can effectively inhibit the progression of the disease and significantly increase the standard of living of an HIV-infected patient. However, HAART is an expensive lifelong treatment and leads to an accumulation of side effects associated with the toxicity of the chemicals used. In addition, the problem of HAART is the emergence of drug-resistant mutant strains, which leads to the need for constant development and use of an increasing number of new chemicals.

Gene therapy for HIV involves the introduction into the cells of genes that suppress the development of infection. At the moment, many genetic constructs have been tested on cell cultures that, to one degree or another, enhance the resistance of cells to HIV. Improving the clinical methods of cell isolation and transplantation allows us to consider gene therapy as a promising area of anti-HIV therapy.

Patent RU 2426788 ("Genetic constructs for anti-HIV therapy", 08/20/2011; IPC C12N 15/86) discloses genetic constructs based on a modified lentiviral vector containing a sequence encoding a modified human TRIM5α protein (tripartite motif 5 a), where the modification includes a change the amino acid sequence of TRIM5α in the SPRY domain, which allows the TRIM5α protein to recognize the HIV viral capsid. TRIM5α is a protein factor of innate immunity that causes resistance to retroviruses. The difference between primates in the amino acid composition of the SPRY domain of the TRIM5 protein determines the difference in the set of retroviruses to which this species is resistant. The TRIM5α homologue in rhesus monkeys ensures the resistance of the cells of these animals to HIV infection. Human TRIM5α is not able to recognize HIV, however, a small change in the amino acid sequence allows the protein to be modified so that it acquires antiviral anti-HIV activity.

The objective of the invention according to the patent RU 2426788 is the expansion of the arsenal of new generation drugs intended for the treatment of HIV infection. It is aimed at creating more effective anti-HIV drugs based on RNA and proteins produced in cells using the introduced genetic constructs of the invention. RU 2426788 also demonstrated that the combination of two or more antiviral agents listed below in one genetic construct provides a synergistic effect: each agent individually suppresses the reproduction of HIV to varying degrees (from 50% to 90%); the simultaneous use of several antiviral agents slows down the formation of mutant strains, and a combination of antiviral agents with agents that confer resistance to cells on HIV makes it possible to obtain antiviral activity close to 100% and prevent the emergence of resistant mutants. As one of the agents that confer HIV resistance to the cell, the authors selected short interfering RNAs directed against the CCR5 chemokine receptor gene, which is involved in the process of HIV penetration into the cell. The technical result is the creation of the proposed structures and their implementation of the specified purpose. Particular embodiments of the invention include genetic constructs for anti-HIV therapy based on a lentiviral vector, including a self-inactivating lentiviral vector, where the lentiviral vector is based on one or more of lentiviruses: HIV-1, HIV-2, VIO (monkey immunodeficiency virus, SIV ), cattle immunodeficiency virus (BIV), goat arthritis encephalitis lentivirus (CAEV), equine infectious anemia virus (EIAV), cat immunodeficiency virus (FIV), etc.

US patent application US2012076763 ("Combination anti-HIV vectors, targeting vectors, and methods of use"; 03/29/2012; IPC A61K 48/00, C12N 5/071, C12N 15/49, A61P 31/18, C12N 5 / 10, C12Q 1/70, C12N 15/63, C12N 7/01) discloses recombinant lentiviral vectors comprising the following elements: a lentiviral base containing sequences for integration into the genome of the target cell; nucleic acids encoding an interfering RNA directed against the human CCR5 receptor gene; and also a control element for the expression of nucleic acid encoding RNAi CCR5. The vectors can also contain polynucleotides encoding the sequences of TRIM5α and TAR traps of HIV together with elements of regulation of gene expression, and can be combined with packaging plasmids and conjugated specific antibodies against target cells. The description of this application describes a polynucleotide encoding the sequence of the chimeric TRIM5α, where the 13 amino acid sequence of Rhesus monkeys is inserted into the sequence of the human TRIM5α protein (SEQ ID NO 17, 994-1032). The resulting chimeric TRIM5α effectively inhibits the HIV-1 virus at the stage after the virus enters the cell and before its integration. The application also provides diagnostic and therapeutic methods for using these structures.

In US patent application US2012270773 ("TRIM5alpha mutants and uses thereof; 10.25.2012; IPC C07H 21/04, A61K 31/7088, C12N 5/02, C12Q 1/70, C07K 16/18, C07K 14/435, A61K 38/00, C12N 15/63, C12N 5/10) describes a mutant TRIM5α polypeptide that confers greater resistance to lentiviruses compared to the wild type TRIM5α human. This mutant TRIM5α polypeptide contains a mutation in an amino acid (substitution or deletion) corresponding to amino acid 324, 328, 330, 333, 335, 336, 336 or 337 wild-type human TRIM5α. This application also describes the selection of nucleic acids containing sequences encoding the above TRIM5α polypeptides, or homologues thereof. The application discloses compositions containing mutant TRIM5α polypeptides encoding their nucleic acids, gene transfer vectors, as well as methods for determining (in vivo or in vitro) resistance of cells to lentiviral infection, including determining the presence or the absence of mutant TRIM5α and / or nucleic acids encoding them. Cells containing mutant TRIM5α polypeptides have been shown to have increased resistance to lentiviral infection.

Anderson (JS Anderson, J. Javien, JA Nolta, and G. Bauer "Preintegration HIV-1 Inhibition by a Combination Lentiviral Vector Containing a Chimeric TRIM5α Protein, and CCR5 shRNA, and a TAR Decoy", Molecular Therapy, 2009, vol. 17 (12), pp. 2103-2114) describes a combined lentiviral vector encoding three highly effective anti-HIV genes and functioning at individual stages of the virus life cycle:

- CCR5 short hairpin RNA (shRNA) (before virus penetration),

- chimeric TRIM5α human / Rhesus monkeys (at the stage after the penetration of the HIV virus into the cell and before its integration),

- TAR traps (after integration).

The main efforts of scientists in the design of this anti-HIV vector were focused on blocking the productive infection of HIV-1 and inhibiting any formation of provirus that will support the viral reservoir.

E. Bativelli and colleagues (E. Battivelli, J. Migraine, D. Lecossier et al. "Modulation of TRIM5α Activity in Human Cells by Alternatively Spliced TRIM5 Isoforms", J. Virol., 2011, vol. 85 (15), pp. .7828-7835) found that the activity of TRIM5α in a particular cell type may depend on the ratio of expressed TRIM5 isoforms. In addition to TRIM5α, TRIM5ι, TRIM5γ, TRIM5δ and TRIM5κ transcripts may be present. They do not inhibit HIV-1 replication, but have dominant negative activity against TRIM5α. The authors showed that the expression of TRIM5i reduces the antiviral activity of TRIM5α in U373-X4 human cells, which indicates that physiological levels of expression of truncated TRIM5 isoforms in human cells can reduce TRIM5α activity.

An object of the present invention is to improve genetic constructs containing a sequence encoding a modified human TRIM5α protein in order to increase the level of gene expression and, consequently, the amount of protein, which will increase the antiviral efficacy of these constructs with respect to human immunodeficiency virus. The technical result is the creation of improved genetic constructs and their implementation of the specified purpose. The genetic constructs encoding the modified TRIM5α protein proposed in the research object differ from the known genetic constructs in that the sequence of the modified TRIM5α gene is 1488 bp in size. (496 codons), synonymous nucleotide substitutions were made along the entire length so that the percentage of the sum of all guanines (G) and cytosines (C) in relation to the total number of nucleotides (GC-composition) of the sequence increased.

According to the results of the study, anti-HIV activity of a construct comprising a sequence (SEQ ID N1) encoding a modified human TRIM5α protein is almost 40 times higher than the activity of a similar construct comprising a sequence (SEQ ID N1) encoding a modified human TRIM5α protein described in patent RU 2426788. In addition, increased cell viability in the presence of HIV.

Disclosure of invention

In the present invention, “nucleic acid molecule” and “polynucleotide” mean the polymeric form of nucleotides of any length, or deoxyribonucleotides, or ribonucleotides, or their analogues. Polynucleotides can have a three-dimensional structure and can perform any function. Non-limiting examples of polynucleotides are a gene, gene fragment, exons, introns, open reading frame, messenger RNA (mRNA), transport RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, cosmids, vectors, isolated DNA with isolated RNA with any sequence, nucleic acid probes and primers.

A polynucleotide usually consists of a specific sequence of four nucleotide bases: adenine (A), cytosine (C), guanine (G) and thymine (T) (and if the indicated polynucleotide is RNA, then uracil (U) is present instead of thymine) . Thus, the term "polynucleotide sequence" means a literal representation of a polynucleotide molecule. This alphabetic representation can be entered into the database of a computer having a central processor and is used in bioinformatics, for example, in functional genomics and in the search for homology.

The term “construct” means any molecule capable of transferring nucleic acid sequences (for example, non-viral vectors, carrier particles, liposomes and viral vectors) to target cells. The term "plasmid construct" means an extrachromosomal genetic element capable of self-replication in a host cell. Typically, the terms “vector”, “construct”, “expression vector” and “vector for gene transfer” mean any nucleic acid construct capable of regulating the expression of a desired gene and transferring gene sequences to target cells. Thus, this term includes cloning and expression vehicles, as well as viral vectors.

A vector may contain either DNA or RNA. For example, either a DNA or an RNA virus can be used to produce a vector. Based on the genomic RNA virus, a copy of cDNA can be obtained. Conversely, a cDNA fragment (or viral genomic DNA) can be transcribed in vitro to form RNA. These techniques are well known in the art.

The term "coding sequence" or a sequence that "encodes" a selected polypeptide means a nucleic acid molecule that is transcribed (in the case of DNA) and translated (in the case of mRNA) into the in vivo polypeptide when it is controlled by the corresponding regulatory sequences (or "regulatory elements "). The boundaries of the coding sequence are determined by the start codon at the 5 '(amino) end and the translation termination codon at the 3' (carboxy) end. The coding sequence may include, but is not limited to, cDNA derived from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences derived from viral or prokaryotic DNA, and even synthesized DNA sequences. The transcription termination sequence can be localized at the 3'-end with respect to the coding sequence. Transcription and translation of coding sequences are usually regulated by "regulatory elements", including, but not limited to, transcription promoters, transcription enhancer elements, Shine-Dalgarno sequences, transcription termination signals, polyadenylation sequences (localized from the 3'-end to the termination codon translation), sequences for optimizing translation initiation (localized from the 5'-end with respect to the coding sequence) and the sequence translation termination. The term "gene expression regulation element" means a nucleotide sequence containing at least a promoter. Such an element may also contain other sequences necessary for the expression of coding sequences that are functionally attached to the promoter, or affecting such expression. The components of these elements do not have to be contiguous, that is, they can be separated by intermediate sequences. The components of these elements can affect the expression of coding sequences at the level of transcription, RNA stability, processing and / or translation of RNA. Typically, such an element does not contain coding sequences with which they are functionally associated. In some cases, the element of regulation of gene expression may contain a natural gene or, mainly, consist of such a gene, in addition to the coding sequence of this gene.

HIV Gene Therapy Vectors

The main requirement of gene therapy is the stable expression of therapeutic genes, which does not cause negative clinical effects. It is highly desirable to have a high titer vector (a sequence enabling the insertion of DNA sequences into the genome of a human cell) that can stably integrate into target cells (including dividing and non-dividing cells) and which is not pathogenic and does not cause an immune response.

Retroviral Vector Systems

One of the first systems for gene therapy was a retroviral vector constructed from the Moloney mouse leukemia virus (MoMLV). This vector has the ability to infect dividing cells. MoMLV DNA provirus is able to integrate into the genome of dividing cells and be inherited along with the cellular genome. In general, clinical trials using vector systems based on the MoMLV virus account for more than 23% of the clinical trials of all gene therapy systems for various human diseases.

Spumovirus-based vectors (foamy virus)

Currently, vectors based on spumavirus have been developed for gene therapy. Spumaviruses are not pathogenic, integrating retroviruses with properties that distinguish them from lentiviruses and gamma retroviruses. Such vectors allow the packaging and transfer of large DNA fragments and effectively transduce hematopoietic cells. In addition, the advantage of this vector for gene therapy of HIV infection is the low homology of the nucleotide sequence with the human immunodeficiency virus and, due to which, the risk of vector mobilization is sharply reduced, ensuring the safety of therapy. The ability to efficiently transfer anti-HIV transgenes that cannot be transferred using HIV-1 vectors is a key advantage of this vector. It has been shown that the titer of a vector based on spumavirus is not affected by transgenic cassettes, which significantly reduce the titer of the lentiviral vector. The integration sites of this vector are significantly different from the integration sites of lentiviruses and gamma retroviruses, which can reduce the risk of leukemia when used in gene therapy.

Non-viral gene delivery systems - transposop based vector

Non-viral gene delivery systems (using DNA plasmids) are the simplest and safest, but the effectiveness of such methods has long remained very low. There are currently several transposon-based expression vectors, some of which have DNA transfer efficiencies comparable to retroviral systems. An example of such a vector is the transposon system of Sleeping Beauty. This system consists of an artificial transposon and the corresponding transposase, an enzyme that has the function of cutting and embedding DNA fragments. The gene transfer process consists of two steps: on the first transposase, it recognizes short inverted or direct repeats in the transposon, and on the second, it cuts and embeds the transposon into the genomic DNA, in the region of TA repeats. Two independent plasmids encoding transposon and transposase can be used in the Sleeping Beauty transposon system, or both components can be combined on one plasmid. Improvement of the transposase enzyme has led to an increase in the efficiency of gene delivery using this system. The possibility of gene transfer to various mammalian cells, including mouse embryonic cells, has been shown. The use of transposon / transposase vectors is an alternative transgene delivery method for HIV gene therapy.

Lentiviral vectors

To date, HIV-1-based lentiviral vectors are most often used in gene therapy and molecular biological research, since the genome structure of this virus has been studied in detail, which greatly facilitates genetic engineering manipulations. However, vectors based on other lentiviruses have also been constructed: HIV-2, VIO, BIV, CAEV, EIAV, FIV, Jambran disease virus (JDV). Such vectors can potentially have certain advantages, such as increased safety, since they cannot infect humans, which means that the likelihood of replication-competent viral particles is reduced. In addition, many anti-HIV agents acting against the virus at the packaging stage significantly reduce the titer of viral particles when using vectors based on HIV-1, but do not affect the titer of viral particles based on other lentiviruses.

The viruses of the lentivirus subfamily have the unique ability to stably transfer genetic material to non-dividing cells. Unlike MuLV and other γ-retroviruses, lentiviral vectors are usually inserted after the transcription start point, and thus cannot activate oncogenes. Large-scale screening of lentivirus integration sites has shown the absence of malignant cell degeneration. The development of new vectors based on lentiviruses (in particular HIV) opens up wide possibilities in the gene therapy of HIV. Lentivirus subfamily RNA viruses include human immunodeficiency viruses types 1 and 2 (i.e., HIV-1 or HIV-2, while HIV-1 was previously called lymphadenopathy virus 3 (HTLV-III) and viruses associated with acquired Immunodeficiency (AIDS) (ARV)), or other viruses close to HIV-1 or HIV-2 that have been identified and associated with AIDS or an AIDS-like disease. In addition, Lentivirus subfamily RNA viruses include the Visna / Maedi virus (e.g., an infected sheep), feline immunodeficiency virus (FIV), bovine lentivirus, monkey immunodeficiency virus (SIV), equine infectious anemia virus (EIAV), and arthritis-encephalitis virus goats (CAEV).

Requirements for the viral vector include the presence of promoter elements, the size of the genome that allows packaging of foreign genetic material, and the absence of virulent determinants. To reduce pathogenicity in the production of recombinant viral vectors, the supply of structural genes (encoding proteins that form the envelope of the virus, nucleocapsids, etc.) is provided in trans by integration into the genome of packaging cells, or on a plasmid introduced into cells together with the vector.

Replication, integration, and packaging of viral particles requires the presence of RNA (DNA) regions that do not encode proteins. Most of these cis-elements must be included in the lentiviral vector (Table 1).

Table 1. Cis Elements Vector Item name Function in vector LTR (long terminal repeat) Contains sequences required for reverse transcription and integration PBS (primer binding site) Needed to initiate synthesis minus DNA strands Ψ Required for packaging vector RNA RRE (rev responsive element) Interacts with Rev protein, is required for processing and transport of the RNA vector

PPT (polypurine tract) Needed for seed synthesis plus chains cPPT (central polypurine tract - flap) Provides effective reverse transcription and nuclear transport of vector DNA

Structural genes that are supplied in trans include three groups of proteins gag, pol, and env (Table 2). These genes are part of several individual packaging plasmids. Removing such genes from the composition of a vector does not allow it to replicate and produce viral particles on its own, thereby increasing the safety of using such vectors.

Table 2. Trans Elements Vector Item name Function gag / pol Encodes the structural proteins of the capsid and nucleocapsid and the enzymes necessary for reverse transcription and integration. env Determines the binding and entry of a viral particle into the cell. rev Essential for the expression of unspliced and partially spliced HIV mRNA in the cell. Increases the titer of viral particles containing the vector when packaged in an in vitro cell line. It is advantageous when there is an RRE element in a vector.

The vectors of the present invention can also be self-inactivating lentiviral vectors, which give the constructs the property of self-inactivation and provide additional safety during therapy. When constructing self-inactivating vectors in a 3'-LTR vector DNA (provirus), a deletion is introduced that affects the enhancer-promoter region (it is located in the U3 region of the LTR), i.e. construct a 3'LTR with a deletion in the U3 region. After infection of the target cell, a provirus is formed due to the peculiarities of reverse transcription, in which both LTRs lack the promoter-enhancer region. Thus, after integration into the genome of the target cell, the self-inactivating vector is not capable of replication, therefore, the likelihood of a replication-competent virus (RCV) in the culture of transduced cells is negligible. In addition, the possibility of insertional mutagenesis is excluded, since the absence of strong enhancer-promoter regions at the 3 'end of the vector (which are located in the delegated LTR region) prevents the activation of the cell’s own genes, including oncogenes.

Instead of the viral promoter-enhancer in the U5-pauone LTR, the RSV / 5'-LTR hybrid promoter is used to obtain vector transcripts in packaging cells. This eliminates the need to introduce an additional plasmid encoding the tat viral protein, and makes the system even safer.

According to the present invention, the proposed genetic constructs can use any known vectors with a sequence that allows the integration of DNA sequences into the human cell genome, with a high titer, which can stably integrate into target cells (including dividing and non-dividing cells) and which are not pathogenic and Do not cause an immune reaction.

Gene delivery at predetermined genome loci using endonucleases

Currently, artificial endonucleases have been created that are able to specifically recognize and bind to extended sections of DNA. Such endonucleases can introduce a gap in a unique part of the genome, which leads to activation of chromosome repair processes, which can either follow the path of the non-homologous connection of DNA ends or the path of homologous recombination. A non-homologous connection of the ends is in most cases accompanied by a deletion of nucleotides or the insertion of additional nucleotides, which leads to a shift in the reading frame and impaired function of the corresponding gene. Homologous recombination can take place if donor double-stranded DNA is present in the nucleus, which has homology with sequences located in the immediate vicinity of the gap site. As a result, the donor sequence is inserted into the gap region. Thus, endonucleases that introduce a break in a unique part of the genome can be used to introduce mutations in certain genes, correct mutations in genes by homologous recombination, or delivery of target genes to specific regions of the genome. The advantage of this delivery of target genes is the known localization of the inserted sequence, which is very important for understanding the further regulation of the target gene and avoids the genotoxicity, the probability of which exists when the DNA sequence is accidentally inserted into the genome.

Until recently, two classes of nucleases were used - meganucleases and nucleases with zinc fingers. A significant limitation for the use of this method of delivery was the complexity of creating these nucleases, introducing a gap in the selected region of the genome. The discovery of transcriptional activator-like (TALE) proteins that were isolated from the Xanthomonas bacteria formed the basis for the creation of a new class of TALE nucleases or TALEN nucleases. Such nucleases have a modular structure. They consist of repeating domains of 33-35 amino acids, and the two amino acids in the middle of the repeat are variable and determine the nucleotide with which the domain binds. A serial connection of domains uniquely determines the DNA sequence with which nuclease binds. Thus, the use of the described nucleases allows the delivery of genetic constructs to a given part of the genome.

Promoters

Sequences encoding genetic antiviral agents can be incorporated into a vector particle under the control of appropriate promoters known to those skilled in the art.

The term "promoter" means a nucleotide sequence that controls the initiation and transcription rate of a polynucleotide. The promoter contains elements with which regulatory proteins and molecules, such as RNA polymerase and other transcription factors, can bind to initiate specific transcription of the nucleic acid sequence. The expressions “functionally located”, “functionally linked”, “under control” and “under transcriptional control” mean that the promoter is in the correct functional arrangement and / or orientation with respect to the nucleic acid sequence to control the initiation of transcription and / or expression of such a sequence .

Human RNA Polymerase II Promoter

Transcription of all coding sequences of eukaryotes is under the control of the RNA polymerase II promoter and leads to the formation of a fully mature mRNA having a cap at the 5 'end and polyA at the 3' end of the mRNA sequence. The promoter, in General, contains a sequence that determines the position of the beginning of the synthesis of RNA. The best known example of such a sequence is the TATA box, but in some promoters lacking a TATA box, such as, for example, the mammalian terminal deoxynucleotidyl transferase gene promoter and the SV40 late gene promoter, another element adjacent to the start site facilitates precise determination of the initiation site. Additional promoter elements regulate the frequency of transcription initiation. Usually they are localized in the region at 30-110 bp above the start site, although some promoters have been shown to also contain functional elements below the start site. To place the coding sequence “under the control of the promoter”, the 5 ′ end of the transcription initiation site is located in the transcriptional reading frame “below” (ie, in the 3 ′ direction) of the selected promoter.

The action of the promoter can be enhanced by an “enhancer”. By enhancer is meant a specific cis-acting nucleotide sequence that greatly enhances gene transcription by RNA polymerase II; for example, the SV40 virus enhancer (size - 72 pairs of nucleotides) can enhance the transcription of the beta-globin gene 200 times, even at a considerable distance from it and in any orientation with respect to the promoter. The ability of a number of enhancers to interact with specific proteins in differentiated cells provides tissue-specific expression of the corresponding genes.

Naturally, it is important to use a promoter and / or enhancer that efficiently directs the expression of the DNA segment in the organelle, cell type, tissue, organ or organism selected for expression. Specialists in the field of molecular biology are generally aware of the use of promoters, enhancers and combinations of cell types for protein expression (see, for example, Sambrook et al., 1989, incorporated herein by reference). The promoters used may be constitutive, tissue-specific, inducible and / or may be used under conditions suitable for directing high-level expression of the introduced DNA segment. The promoter may be heterologous or endogenous.

Constitutive promoters

To constitutive promoters, i.e. the promoters working in all types of cells, regardless of the conditions, include promoters of the housekeeping genes. The genes of the “household” include genes that ensure the vital activity of the eukaryotic cell and function everywhere, at all stages of the life cycle of the body. They provide the process of glycolysis, biosynthesis of amino acids and nucleotides, protein catabolism, etc. Non-limiting examples of such promoters are the promoter of the gene for the elongation factor factor EF1α, the gene for phosphoglycerate typease (PGK), the β-actin gene, the ubiquitin promoter UbC, the histone promoter H2 B (TH2 B), etc.

Many viral promoters perform a constitutive function in eukaryotic cells. These promoters include:

- early and late SV40 promoters (see Bemoist and Chambon, Nature, 290: 304 (1981));

- Long terminal repeats (LTR) of the Molonei leukemia virus and other retroviruses (see R. Weisset et al., eds., Molecular Biology of Tumor Viruses, 2 ^nd ed., RNA Tumor Viruses. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1985);

the herpes simplex virus thymidine kinase (HSV) promoter (see Wagner et al., Proc. Natl. Acad. Sci. USA, 1981, vol. 78, p. 1441);

- the earliest promoter of cytomegalovirus (IE1) (see Karasuyama et al., J. Exp. Med., 1989, vol. 169 p. 13;

the promoter of Routh sarcoma virus (RSV) (see Yamamoto et al., Cell, 1980, vol. 22, P.787);

- the main late promoter of adenovirus (see Yamada et al., Proc. Natl. Acad. Sci. USA, 1985, vol. 82, p. 3567) and many others.

Therefore, any of the above constitutive promoters can be used to control transcription of the gene insert.

Tissue-specific promoters

Tissue-specific are promoters that initiate gene transcription only in cells of a particular tissue. The tissue-specific nature of gene expression is provided by various mechanisms of interaction of protein transcription factors with regulatory nucleic acid sequences. The distinctive features of tissue-specific promoters or elements, as well as analyzes to characterize their activity, are well known to those skilled in the art. Non-limiting examples of genes controlled by such promoters include the human LIMK2 gene (Nomoto et al., 1999), the somatostatin receptor 2 gene (Kraus et al., 1998), the retinoic acid binding protein of the mouse testicular appendages (Lareyre et al., 1999), human CD4 (Zhao-Emonet et al., 1998), mouse alpha-2- (XI) -collagen (Tsumaki, et al., 1998), dopamine receptor gene D1A (Lee, et al., 1997), insulin-like growth factor II (Wu et al., 1997) and the platelet-1 adhesion molecule of human endothelial cells (Almendro et al., 1996).

The following are non-limiting examples of tissue-specific RNA polymerase II elements / promoters that can be used in the context of the present invention to regulate RNA expression: promoter of the human immunoglobulin heavy chain gene, T cell receptor gene, interferon β gene, IL2 gene. IL2 receptor gene, MHC class II genes, a-fetoprotein gene, γ-globin gene, β-globin gene, genes encoding the human hemoglobin α and β subunits, albumin gene, collagenase gene, metallothionein gene (MTII), elastase I gene , c-HA-ras gene, insulin gene, etc.

Adjustable promoters

Regulated or inducible promoters allow you to enable and / or disable gene transcription. The activity of such promoters depends on the introduction of additional factors normally absent in expressing cells. There are several reversible expression control systems that enable you to turn the gene on and off. The most used and studied is the tetracycline-dependent switching system, which consists of three main parts: the tetracycline molecule Tet or its analogue, the tetracycline repressor protein molecule TetR or the tTA activator, and the promoter site (the so-called tetOcycline operator tetO) binding the repressor or activator. There are two options: a) the Tet-On system uses a TetR repressor that communicates with the operator and blocks transcription; the addition of tetracycline leads to the removal of the repressor and the inclusion of gene expression b) in the Tet-OFF system, a tTA activator is used, which promotes expression in the absence of tetracycline and turns off the gene when tetracycline is added. The tetracycline analog doxycycline penetrates better through the cell membrane and has a great affinity for the repressor and activator, therefore it is often used as a switch. Other examples of regulated promoters include the LacZ regulated system or the ecdysone regulated system. The only example of irreversible induction (or repression) is the Cre-Lox-based recombination of bacteriophage P1. Using Cre-Lox recombination allows you to irreversibly turn on or off the expression of the sequence of interest in the gene construct after introducing it into the cell.

Human polymerase III promoters

For the expression of short RNA sequences (in our case, ribozyme, TAR, shRNA), it is convenient to use promoters for RNA polymerase III. The promoters for RNA polymerase III are divided into three types based on the composition of the promoter elements and their position relative to the start of transcription (PAULE MR, WHITE RJ. Survey and summary: transcription by RNA polymerases I and III. Nucleic Acids Res 2000; 28 (6) : 1283-98). Type 3 promoters for RNA polymerase III are most suitable for transcription of short RNA sequences, since in nature they are used by a cell to produce small nuclear RNA molecules. Unlike type 1 and type 2 promoters, type 3 promoters are completely located above the transcriptional start and contain 3 specific promoter elements necessary for their functioning: 1. TATA sequence at -20 bp, 2. PSE the sequence of the proximal element at position -50 bp, 3. DSE sequence of the distal element at position -240 bp (where +1 is the first transcribed nucleotide). Both DSE and PSE elements contain an enhancer and a transcription factor binding sequence that are absolutely necessary and a deletion of which completely eliminates transcription. The distance between these elements is strictly defined and critical to achieve maximum transcription rate. A characteristic feature of these promoters is a clearly defined transcription initiation site and a simple termination signal consisting of 4 or 5 consecutive thymidines (SCHRAMM L et al. Recruitment of RNA polymerase III to its target promoters. Genes Dev 2002; 16 (20): 2593-620). The relative simplicity and high level of transcription (4 * 10 ⁵ transcripts per cell) makes them as convenient as possible for use in genetic constructs.

Type 3 promoters for RNA polymerase III include promoters of the HI, U6, and 7SK genes, which are commonly used in experiments for shRNA expression. The HI gene encodes RNA, which is a component of nuclear RNase P and is involved in tRNA maturation. The human HI promoter is extremely compact - all the elements necessary for transcription are within 100 bp. above start. RNA encoded by the U6 gene plays a central role in RNA processing and is an integral part of the spliceosome (BERGET SM, ROBBERSON BL. U1, U2, and U4 / U6 small nuclear ribonucleoproteins are required for in vitro splicing but not polyadenylation. Cell 1986; 46 (5 ): 691-6). In vivo and in vitro experiments show that the U6 promoter is much more active than the HI. However, by analogy with U1 RNA, U6 RNAs suggest a regulatory mechanism that prevents the excessive accumulation of U6 RNA in Noonberg SB, Scott GK, Benz CC cells. Evidence of posttranscriptional regulation ofU6 small nuclear RNA. J Biol Chem 1996; 271 (18): 10477-81). Therefore, overexpression of shRNA controlled by the U6 promoter can lead to a decrease in the amount of endogenous U6 RNA and a change in the phenotype of the cell. The 7SK promoter is becoming more and more popular, as it is stronger compared to U6, but does not have a self-regulation mechanism. 7SK RNA is evolutionarily conserved and is abundant in the cell. There are artificially created regulated promoters of RNA polymerase III based on the tetracycline and Cre-Lox systems described above.

In general, promoters and enhancers that control transcription in eukaryotic cells are composed of multiple genetic elements. The cellular apparatus is able to combine and integrate the regulatory information that each element carries, which allows the formation of complex transcriptional regulation profiles. Activation or repression of promoter and enhancer elements can be accomplished by contacting these elements with suitable protein activators or transcriptional repressors.

In the context of the present invention, any combination of promoter / enhancer elements (according to the EPDB eukaryotic promoter database) can be used to efficiently initiate and maintain transcription of the sequence in hematopoietic cells. For the expression of transgenes, regulated promoters can be used to enable / disable transcription during the production of viral particles and / or during the treatment of HIV infection. Based on this description and the prior art, one skilled in the art is able to select an appropriate promoter for each of the antiviral agents listed below or a combination thereof.

Genetic Antivirus Agents

Genetic antiviral constructs that are used in gene therapy can be divided into 2 types: agents that interact with viral elements after penetration into the cell and start replication, and agents that prevent the virus from entering the cell and / or integration into the genome, and thus imparting to the cell - the host resistance (resistance) to infection.

The present invention provides genetic constructs comprising one or more of the antiviral agents described below, one of which must be a nucleotide sequence encoding a modified human TRIM5α protein (SEQ ID N3) and different from the original nucleotide sequence of SEQ ID N2 by multiple synonymous substitutions providing an increase in percentage GC pairs in the DNA sequence.

Modified Human TRIM5α Gene Sequence

TRIM5α (tripartite motif 5α) is a protein factor that determines the resistance of rhesus monkeys to HIV, containing 3 different domains (RING, B-box and RBCC domains). In addition to these domains, TRIM5α includes the C-terminal SPRY domain, which binds to the HIV capsid and is responsible for the cell's resistance to HIV infection. The difference in the amino acid composition of this domain between primates determines the difference in the set of retroviruses to which this species is resistant. Protein trimers form multiple contacts with the surface of the viral capsid and recognize, apparently, the spatial structure. Thus, TRIM5α can be called a structure recognizing receptor (PRR) of the innate immune system, aimed at recognizing the multimeric protein surface of the virus capsid. The mechanisms of action of the protein have not been sufficiently studied, it is known that it prevents the integration of viral DNA into the chromosome, i.e. blocks HIV at an early stage. It is assumed that the blocking of the virus occurs at two stages of the viral cycle: blocking the formation of cDNA (before or after reverse transcription) and blocking entry into the nucleus of the preintegration complex.

The human TRIM5α protein homologue is ineffective against HIV, probably due to changes in the viral capsid during evolution (Luban J., "Cyclophilin A, TRIM5, and resistance to human immunodeficiency virus type 1 infection", J. Virol., 2007, vol .81 (3), pp. 1054-1061). Changes in just one amino acid in the sequence of the human TRIM5α homologue makes cells resistant to HIV infection. Thus, the modified TRIM5α is promising for use in gene therapy - the necessary minor change makes it non-immunogenic and at the same time, the introduction of such a modification in target cells gives them resistance to HIV infection. Based on the TRIM5α human isoform, a chimeric protein is created in which 11 a.a. replaced by 13 from TRIM5α rhesus macaque (Sawyer SL et al., "Positive selection of primate TRIM5α identifies a critical species-specific retroviral restriction domain", Proc. Natl. Acad. Sci. USA, 2005, vol. 102 (8), pp. 2832-2837).

In application W02005081911 ("Methods and compositions for the treatment and prevention ofHIV infection using TRIM5α"; 09.09.2005; IPC C07H 21/02) TRIM5α and their genetic constructs are disclosed.

Patent RU2426788 describes genetic constructs based on a modified lentiviral vector containing a sequence encoding a modified human TRIM5α protein, where the modification involves changing the amino acid sequence of TRIM5α in the SPRY domain (see SEQ ID N3), which allows TRIM5α protein to recognize the HIV viral capsid.

An object of the present invention is to improve genetic constructs containing a sequence encoding a modified human TRIM5α protein in order to increase the level of gene expression and, consequently, the amount of protein, which will increase the antiviral efficacy of these constructs with respect to HIV.

The amount of protein produced as a result of TRIM5α gene expression directly affects the efficiency of suppressing HIV replication. Therefore, it is very important to achieve the highest level of gene expression. In (Kudia G., Lipinski L., Caffin F., Helwak A., Zylicz M. "High Guanine and Cytosine Content Increases mRNA Levels in Mammalian Cells", PLoS Biol., 2006, vol. 4 (6), 180 ; Bauer AP, Leikam D., Krinner S., et al. "The impact of intragenic CpG content on gene expression", Nucleic Acids Res., 2010, vol. 38 (12), pp. 3891-3908; Al-Saif M., Khabar KSA, "UU / UA Dinucleotide Frequency Reduction in Coding Regions Results in Increased mRNA Stability and Protein Expression", Mol. Ther., 2012, vol. 20 (5), pp. 954-959), it was shown that the level of mRNA expression depends on the GC composition of the nucleotide sequence of the gene: an increase in the number of GC due to synonymous substitutions led to an increase in gene expression by orders of magnitude.

Thus, the main objective of the present invention is the creation of genetic constructs containing a GC rich nucleotide sequence encoding a modified human TRIM5α protein.

The modified human TRIM5α gene can be controlled by the RNA polymerase II promoter, which transcribes this transgene into a human cell. In a preferred embodiment, the promoter is active in particular cell types or descending germs of hematopoietic cell precursors. The action of the promoter may be activated or suppressed by transcription control factors, activators or repressors.

As a promoter, the promoter of the gene for elongation factor EF1α, which is a constitutive promoter, can be used. The activity of this promoter in human lymphocytes is significantly higher than the activity of some other constitutive promoters, in particular the promoters of the UbC and Pgk genes. This property of the EF1α promoter allows it to be effectively used to direct the expression of anti-HIV genes, since it is the lymphocytes that are cells sensitive to the human immunodeficiency virus and a high level of expression of antiviral genes must be achieved in these cells. However, the present invention is not limited to this promoter, and other promoters that can be used without limitation are known to a person skilled in the art, for example, PGK, EF1α, CD4, CD2, IL-2 gene promoters, beta-globin gene promoter, MHC gene promoters. Hybrid promoters can be used, in addition to the natural sequence, enhancer or silencer sequences are introduced, as well as sequences that allow the promoter activity to be regulated depending on binding to cytokines / chemokines or other regulatory molecules.

Another objective of the present invention is the creation of genetic constructs producing an antiviral agent based on a modified TMM5a gene with a GC rich nucleotide composition with increased expression, in combination with one or more of the following antiviral agents.

The combination of two or more antiviral genes in one genetic construct is an advantage over the use of a single agent, in particular, a synergistic effect is manifested. Different antiviral agents prevent infection of cells at different stages by blocking the penetration of the virus (CCR5 siRNA), preventing the integration of the virus into the genome (TRIM5α), slowing down the replication of the virus (TAR) and leading to the formation of non-infectious viral offspring (env antisense). Each agent individually suppresses the reproduction of HIV to varying degrees (from 50% to 90%). The simultaneous use of several antiviral agents slows down the formation of mutant strains, and the combination of antiviral agents with agents that confer resistance to cells on HIV makes it possible to obtain antiviral activity close to 100% and prevent the emergence of resistant mutants.

The present invention provides genetic constructs that, in addition to the above-described agent based on the TRIM5a gene, include at least one of the antiviral agents listed below.

Short interfering RNA directed against the human receptor gene CCR5 (CCR5si)

As one of the agents that confer HIV resistance to the cell, the authors selected short interfering RNAs directed against the CCR5 chemokine receptor gene, which is involved in the process of HIV penetration into the cell.

RNA interference is a regulatory mechanism present in most eukaryotic cells that uses double-stranded RNA molecules as a signal to regulate the activity of genes homologous to these RNAs. In mammalian cells, the endogenous mechanism of RNA interference is triggered by the so-called miRNA molecules. The miRNA gene sequences form a hairpin structure (primary miRNA), which is recognized and processed by the Drosha cell enzyme in the nucleus, after which the formed short RNA hairpins (miRNA precursors) are exported to the cytoplasm. In the cytoplasm, a special protein complex (RISC) removes the hairpin loop, forming short RNA duplexes. These duplexes known as short interfering RNAs - siRNAs (short interfering RNAs, siRNAs) are double-stranded RNAs with a size of 21-22 pairs of nucleotides; at the 3 'ends of both chains of which there are two unpaired nucleotides. siRNAs are widely used to inhibit gene expression when working with cell cultures in vitro. Further processing of the RNA duplex leads to the selection of an effector short (~ 21 bp) RNA chain, which is directed to homologous mRNA and hybridizes with a complementary region of the target mRNA. In the case of 100% homology, binding leads to cleavage of mRNA and subsequent complete degradation of mRNA.

The intracellular mechanism of RNA interference, aimed at suppressing viral or cellular genes, can be triggered by introducing transgenes expressing short hairpin RNAs (short hairpin RNA, shRNA) into the cell, which form dense hairpins in the secondary structure and are analogues of microRNA precursors. As well as miRNA precursors, they are exported to the cytoplasm and further processed using the RNA interference mechanism. If such a hairpin RNA contains a sequence complementary to the CCR5 gene sequence, this will lead to mRNA degradation and the absence of receptor expression. Short hairpin-forming RNAs are introduced into cells using a vector using the RNA polymerase III promoter to provide constitutive or inducible expression.

A sequence complementary to the sequence of the CCR5 gene can also be inserted into the body of endogenous miRNA; such artificially modified primary miRNAs will be processed and will lead to mRNA cleavage of the corresponding gene. The expression of miRNAs can be regulated by both the promoter of RNA polymerase II and the promoter of RNA polymerase III.

An integrated vector containing shRNA or miRNA is transmitted to daughter cells and provides heritable gene shutdown (Paddison P. et al., "Short hairpin RNAs (shRNAs) induce sequence-specific silencing in mammalian cells", Genes Dev., 2002, vol.16 (8), pp. 948-958).

RNA interference is a very effective mechanism of suppressing gene expression, with its help it is possible to achieve 99% inhibition, that is, practically turn off the gene.

The main problem that arises when using RNA-interfering molecules directed against HIV genes lies in the very nature of the virus, which leads to the emergence of resistant mutant strains. Successful use of RNA interference requires strict complementarity of the siRNA and the target mRNA. The occurrence of a single nucleotide replacement in HIV RNA, corresponding to 9-11 nucleotides of siRNA, leads to complete failure of the RNA interference mechanism and the absence of replication suppression. In cell culture, the emergence of resistant viral mutants carrying a single nucleotide replacement usually occurs on day 25. siRNAs directed against highly conserved sites only partially improve the situation, requiring a longer time for the appearance of mutants. The most promising approach is to use siRNAs directed against cellular genes whose protein products interact with HIV and which are highly conservative.

In applications WO 2004065549 ("Small interference RNA gene therapy"; 01/15/2004; IPC C12N 15/11, A61K 48/00) and WO 2003022052 ("Method for expression of small RNA molecules within a cell"; 09/13/2002; IPC C12N 15/11, C12N 15/867, A61K 48/00) disclose lentiviral siRNA delivery systems to cells for treating cancer.

In accordance with the present invention, there are provided constructs comprising a siRNA coding sequence directed against a cell gene, the CCR5 chemokine receptor gene. CCR5-coreceptor necessary for the entry of tropic viruses into the R5 cell. Natural coreceptor mutations are widely found in the population of both HIV-infected and uninfected people. In some people, such mutations lead to a complete absence of the receptor molecule, which makes these people resistant to HIV infection. It is noteworthy that the absence of CCR5 receptor in such people does not lead to phenotype disturbances.

The sequence encoding this siRNA can be cloned into the lentiviral vector as an artificial miRNA (miCCR5) based on human miRNA, the transcription of which is initiated by the Pol II promoter.

The advantage of siRNA data (in the form of miRNAs) is the high efficiency of suppressing CCR5 gene expression and, at the same time, the absence of cellular toxicity compared to shRNA. Since the presence of even several molecules on the cell surface makes it susceptible to infection by the virus, only the complete suppression of receptor synthesis, due to the high efficiency of siRNA, protects the cell from virus penetration. Structures proposed in accordance with the present invention, including, in particular, miCCR5, provide excellent antiviral activity in combination with a complete absence of toxicity.

RNA hairpin similar to the TAR sequence

Another group of antiviral agents are RNA aptamers. Aptamers, polynucleotide molecules that bind protein ligands of the virus with high efficiency. Aptamers synthesized in vitro exhibit a high inhibitory activity, however, when expressed in a cell, their efficiency decreases markedly. The problem is the formation of the correct spatial structure in the cellular context. An exception is the use of two sequences present in HIV RNA - the TAR (trans-activating response region) and RRE (Rev response element) regions, the so-called TAR and RRE traps, which are natural aptamers for tat and rev viral proteins.

Tat, one of the earliest viral proteins produced during HIV infection, plays a key role in the regulation of HIV replication. By binding to the TAR sequence, Tat activates the expression of viral genes more than 100 times. The TAR element is located at the 5th end of all HIV transcripts; its structure is a hairpin with a length of 59 nucleotides. Studies have shown that the protrusion of three nucleotides is responsible for the binding of the tat protein, and the loop at the end of the hairpin is necessary for cooperative interaction with the cellular protein cyclin T1 (cyclinT1). The long double-stranded region - the hairpin trunk - plays only a structural role.

Among several gene therapy strategies aimed at blocking tat gene activity, one of the most successful is the use of short RNA molecules that act as traps. Such RNAs mimic the tat binding RNA sequence of a virus, resulting in a decrease in free tat protein (Bohjanen PR et al., "A small circular TAR RNA decoy specifically inhibits Tat-activated HIV-1 transcription", Nucleic Acids Res., 1996, vol. 24 (19), pp. 3733-3738).

Since the design based on the sequence (SEQ ID N4) encoding an RNA hairpin similar to the TAR sequence slows down HIV replication, its use in combination with other anti-HIV agents will enhance the antiviral effect and avoid the appearance of resistant mutants.

Antisense sequence to the HIV env gene region

Antisense (antisense) RNA - short or long RNA sequences, complementary HIV RNA sequences, form non-functional duplexes with viral RNA and are capable of blocking virus replication in cells. Although the exact mechanism of inhibition is not clear, it may include triggering the deamination of HIV cytosine: antisense of duplex RNA, leading to a delay in transcripts in the nucleus and their subsequent degradation or to the formation of numerous non-viable viral mutants. Other possible mechanisms (which depend on the structure of antisense RNA) are inhibition of expression at the splicing level, inhibition of translation, packaging of a chimeric duplex with the formation of defective viral particles.

Many in vitro antisense constructs have been shown to inhibit virus reproduction, including antisense RNAs that block regulatory cis elements such as packaging signal (Ψ), tat protein binding sequence (TAR), and primer binding site (PBS) (Cohli H., Fan B., Joshi RL, et al. "Inhibition of HIV-1 multiplication in a human CD4 + lymphoid cell line expressing antisense and sense RNA molecules containing HIV-1 packaging signal and RREs", Antisense Res. Dev., 1994 , vol. 4 (1), pp. 19-26; Ding SF, Noronha J., and Joshi S., "Co-packaging of sense and antisense RNAs: a novel strategy for blocking HIV-1 replication", Nucleic Acids Res ., 1998, vol. 26, pp. 3270-3278; Chadwick DR, and Lever AM, "Antisense RNA sequences targe ting the 5 leader packaging signal region of human immunodeficiency virus type-1 inhibits viral replication at post-transcriptional stages of the life cycle ". Gene Ther., 2000, vol. 7, pp. 1362-1368) or RNA antisense corresponding to coding areas of viral genes such as rev, env, vif, etc. It is known that the antiviral effect is heavily dependent on the promoter and the vector used for delivery.

Levine's article (Levine BL et al., "Gene transfer in humans using a conditionally replicating lentiviral vector", Proc. Natl. Acad. Sci. USA, 2006, vol. 103 (46) pp.17372-17377) discloses constructions on based on lentiviral vectors expressing antisense genes against HIV envelope.

In the constructions proposed in accordance with the present invention, the sequence (SEQ ID N5) obtained from the cDNA isolate of human immunodeficiency virus subtype A is used as an antisense sequence for the HIV env gene. This isolate was obtained from clinical material from a Russian HIV-infected patient. Since in Russia the vast majority of HIV-positive patients are infected with HIV subtype A (more than 90%), it can be said that an antiviral agent derived from this isolate will be more preferable in the treatment of Russian patients.

Based on the information disclosed herein, one skilled in the art can readily obtain genetic constructs including any combination of the above genetic antiviral agents.

Vector constructs in accordance with the present invention can be created, for example, based on the pLVX-Puro vector (Clontech) (Fig. 1) containing lentiviral LTR and a packaging signal (t). The vector also contains a 124 bp CPPT sequence, which increases the efficiency of the transport of the viral genome into the nucleus. This leads to more efficient integration of the genome, and, consequently, to more efficient transduction. The presence of RRE (Rev-responsive element) in the vector contributes to an increase in titer due to increased export of unspliced viral RNA from the nucleus in the presence of rev. The presence of the North American forest marmot hepatitis virus in the vector WPRE (post-transcriptional regulatory element) allows to increase the efficiency of events related to RNA processing and export from the nucleus. WPRE provides a double effect. Firstly, being a part of the viral transcript, this element increases the efficiency of vector packaging, which helps to increase the titer of the virus produced by 293T packaging cells. Secondly, it enhances gene expression (gene of interest) by facilitating the formation of mature matrix mRNA from a transcript initiated from an internal promoter.

According to another embodiment of the invention, the genetic constructs can be made on the basis of a self-inactivating lentiviral vector, for example, on the basis of the pLV-neo vector (FIG. 2). The indicated vector contains the same basic elements as the pLVX-Puro vector, with the exception of LTR sequences. Instead of the 5'LTR sequence, the RSV / 5'-LTR hybrid promoter is used, which eliminates the need for additional plasmid encoding the tat viral protein. 3'LTR contains a deletion in the U3 region, affecting the enhancer-promoter region. Since, due to the features of reverse transcription, the provirus integrating into the genome has U3 LTR regions identical to the long terminal repeat located at the 3 'end, in this case it will be deprived of the promoter-enhancer region.

Unexpectedly, it was additionally discovered that the modification of the lentiviral vector, which consists in cloning the sequence of SEQ ID N10 directly in front of the cPPT region, makes it possible to further stabilize gene expression, increases the infectivity of viral particles, and improves the efficiency of introducing genetic material into target cells.

If necessary, a different sequence can be introduced into the vector instead of the gene for puromycin selection as a molecular marker for the selection and / or monitoring of modified cells. A marker gene is a sequence that is attached to the elements of a heterologous promoter or enhancer, encoding a product that can be easily and quantitatively analyzed when this construct is introduced into tissue or cells. Various sequences for monitoring and / or selection are known to those skilled in the art, for example, the green fluorescent protein (EGFP) gene. The marker gene / sequence can be deleted by using the vector in HIV therapy.

Brief Description of the Drawings

Figure 1 presents a map of the plasmid pLVX-puro, characterized by:

5'LTR: 1-635

PBS: 636-653

Ψ: 685-822

RRE: 1303-1536

cPPT: 2028-2151

P _{CMV IE} : 2185-2788

MCS: 2816-2880

P _PGK : 2882-3390

Puro ^r : 3411-4010

WPRE: 4024-4615

3'LTR: 4819-5455

pUC: 5925-6595

Amp ^r : 6740-7736

Figure 2 presents a map of the plasmid pLV-neo, characterized by:

RSV / 5'-LTR Hybrid Promoter: 1-416

PBS: 418-435

Ψ: 467-604

RRE: 1081-1314

cPPT: 1810-1928

P _{CMV IE} : 1940-2525

Histone promoter: 2576-2933

Neomycin resistance gene: 2942-3736

WPRE: 3750-4291

3'LTR, with a deletion in the field of U3: 4428-4662

SV40 polyA: 4734-4864

Amp ^r : 5828-6688

Figure 3 shows the sequences encoding the modified human TRIM5α protein with a GC rich nucleotide composition (trim enh, SEQ ID N1) and the original sequence described in patent RU 2426788 (trim, SEQ ID N2).

Figure 4 presents a comparison of anti-HIV activities of a construct comprising a sequence encoding a modified human TRIM5α protein with a GC rich nucleotide composition (trim enhanced) and a construct including the original sequence (trim).

The following are examples of carrying out the invention with reference to the accompanying figures.

Examples of the invention and the implementation of the purpose

Example 1. Obtaining vectors for genetic constructs

1a. Lentiviral vector

The pLVX-puro vector is available from Clontech, a length of 8102 nucleotide pairs (FIG. 1) was modified using the sequence SEQ ID N10. Using primers cPPT 1g Hpa for (TTTGGTTAACCCAGTAAAAACAGTACATACAG) and cPPT 1g Cla Barn rev (TTTTGGATCCATCGATAAATTTTGAATTTTTGTAATTTG), a plasmid pLP1 (Invitrogen) was used as a template for the production of a vector that was used for the preparation of vector . After transformation and subsequent selection, this fragment was cloned into the plasmid pLV-neo, previously hydrolyzed at the HpaI and BamHI sites. The obtained plasmid was produced in preparative quantities and cut using endonucleases MfeI and ClaI, and the resulting fragment was 1532 bp in length. ligated into the pLVX-Puro vector previously hydrolyzed at the same sites. The pLVX-Puro cPPT 1g vector obtained after ligation was prepared in preparative amounts and isolated from E. coli using the Axygen kit. For the convenience of cloning, a linker was introduced into the pLVX-Puro cPPT 1g vector, instead of the CMV promoter, at the ClaI and EcoRI sites

Link For (TTGATATCGATCTGAGTGAGACTCTGACGAATTCTTCT)

Link Rev (AGAAGAATTCGTCAGAGTCTCACTCAGATCGATATCAA).

The resulting vector pLVX Puro cPPT lg ACMV was used for subsequent cloning procedures of the listed sequences.

1b. Self-inactivating antiviral vector

Modification of the pLV-neo vector with a length of 7907 nucleotide pairs (FIG. 2) using the sequence SEQ ID N10 was carried out similarly to the previous one. Using primers cPPT 1g Hpa for (TTTGGTTAACCCAGTAAAAACAGTACATACAG) and cPPT 1g Cla Barn rev (TTTTGGATCCATCGATAAATTTTGAATTnTGTAATTTG), using the pLP1 plasmid (Invitrogen), the vector was used to test the vector for the P template. Promega). After transformation and subsequent selection, this fragment was cloned into the plasmid pLV-neo, previously hydrolyzed at the HpaI and BamHI sites. The resulting plasmid pLV-neo cPPT 1g contains the sequence of SEQ ID N10 in front of the cPPT region. Instead of the CMV promoter, a linker was introduced into plasmid pLV-neo cPPT 1g as in Example 1a, and pLV-neo cPPT 1g ACMV was obtained.

Example 2. Modification of the lentiviral vector - obtaining constructs without the PURO gene with the EGFP marker gene

The PCR fragment containing the EGFP gene and the internal IRES ribosome binding site was obtained by amplification of the plasmid pIRES-EGFP (Clontech # 6064-1) with primers RI-ires for GACGGAATTCAGTGGATCCACTAGTAACGGC Sal-egfp rev TTTGTCGACCTTTTTTTTGT. This fragment is embedded in the pLVneo vector at EcoRI-SalI sites. The cassette containing the IRES site and the EGFP gene was cloned into the pLVX-Puro cPPT 1g vector instead of the puromycin resistance gene at the EcoRI-KpnI restriction sites. This vector pLVX Egfp cPPT1g instead of the selective puromycin resistance gene contains a green fluorescent protein gene as a marker that allows cells to be sorted by the presence of fluorescence.

Example 3. Obtaining the design of TMM5a with GC rich nucleotide composition.

In the sequence of the modified Trim5a gene (SEQ ID N2) of 1488 bp (496 codons), 200 synonymous nucleotide substitutions were designed along the entire length so that the GC composition of the sequence increased from 48% to 61%. A comparison of the original and GC-rich sequences (SEQ ID N1) of the TRIM5α gene is shown in FIG. 3. To construct a GC-rich sequence, the de novo gene was assembled using a ligase chain reaction followed by PCR in accordance with the standard protocol (Au LC, Yang FY, Yang WJ, Lo SH, Kao CF, "Gene synthesis by a LCR-based approach : high-level production of leptin-L54 using synthetic gene in Escherichia coli ", Biochem. Biophys. Res. Sottip., 1998, vol. 248 (1), pp.200-203). Fermentas Tth ligase was used for assembly. The 5'-phosphorylated oligonucleotides were designed using the Gene201igo program (Rouillard JM, Lee W., Truan G., Gao X., Zhou X., Gulari E., “Gene201igo: oligonucleotide design for in vitro gene synthesis.” Nucleic Acids Res., 2004, vol. 32 (Web Server issue), W176-80). The resulting amplicon containing the new modified Trim5a sequence was cloned into the pGEM vector (Promega). The correct assembly of the gene in individual clones was verified by sequencing. The created plasmid was designated pGEM-TRIM enh.

Example 4. Cloning of the GC rich sequence of the modified TRIM5α gene into a lentiviral vector.

The PCR product of amplification of the plasmid pGEM-TRIM enh using the primers ACTTCTAGATTACGAGCTCGGCGAGCACAG and TTTTCTCGAGACTAGTGATGCCGCCACCATG was cut at the XhoI and XbaI restriction enzyme sites and ligated into the pLVX-Puro cPPT1 vector. The resulting plasmid is designated pLVX-Puro cPPT 1g CMV-Trim enh.

Example 5. Cloning of the GC rich sequence of the modified TRIM5α gene into a self-inactivating lentiviral vector.

The PCR product of amplification of the plasmid pGEM-TRIM enh using the primers ACTTCTAGATTACGAGCTCGGCGAGCACAG and TTTTCTCGAGACTAGTGATGCCGCCACCATG was cut at the XhoI and XbaI restriction enzyme sites and ligated into the pLV-neo vector cPPTg 1. The resulting plasmid is designated SIV CMV-Trim enh.

Example 6. Cloning of a sequence encoding miRNA directed against the CCR5 gene into a vector.

The nucleotide sequence of the miR155 mouse miRNA SEQ ID N6 was used to create artificial miRNAs: SEQ ID N7, SEQ ID N8 or SEQ ID N9 directed against the CCR5 receptor gene (miCCR5).

Primers O1-1 TGCTGTAAGAGGTAGTTTTCTGAACGTTTTG and O3-1 GTGGCCAAAACGTTCAGAAACTACCTCTTAC were used to obtain SEQ ID N7 miRNAs, which were annealed on each other to obtain a short double-stranded olig1-3 DNA fragment with sticky ends. Primers O2-1 GCCACTGACTGACGTTCAGAATACCTCTTA and O4-1 CCTGTAAGAGGTATTCTGAACGTCAGTCA were annealed, to give the olig2-4 double chain olig. A mixture consisting of pcDNA6.2-GW / miR plasmid (Block-iT PolII miR RNA expression Vector Kit, Invitrogen) and two olig1-3 and olig2-4 oligates were ligated for 1 hour at room temperature according to the instructions from recruitment. The ligase mixture was transformed into E. Coli cells, the grown clones were selected by sequence. Transformants containing the desired plasmid were expanded and the plasmid miR-CCR5-1 DNA was isolated. A fragment containing miRNAs (SEQ ID N7) was excised from the plasmid at SalI-BgIII restriction sites. The pLVX Puro vector cPPTIg was digested with XhoI-BamHI enzymes and ligated with a fragment containing miRNAs. The resulting Puro / miR-CCR5-1 construct was checked by restriction analysis and sequenced. For transfection, preparative amounts were obtained in E. coli and isolated using the QIAGEN plasmid maxi kit.

Similarly, constructs containing miRNA SEQ ID N8 or SEQ ID N9 were obtained. The following primers were used to obtain the microRNAs of SEQ ID N8:

TgCTgCATAgATTggACTTgACACgTTTTg

CCTgCATAgATTgCTTgACACgTCAgTCA

gCCACTgACTgACgTgTCAAgCAATCTATg

gTggCCAAAACgTgTCAAgTCCAATCTATgC

The following primers were used to obtain microRNA SEQ ID N9:

TgCTgAATTgATgTCATAgATTggACTTgACACgTTTTg

CCTgAATTgATgTCATAgATTgCTTgACACgTCAgTCA

gCCACTgACTgACgTgTCAAgCAATCTATgACATCAATT

gTggCCAAAACgTgTCAAgTCCAATCTATgACATCAATTC

Example 7. Obtaining constructs containing both the GC-rich modified gene TRIM5α and miRNA against CCR5.

The pLVX Egfp vector cPPT1g was restricted at the XbaI and ClaI sites. The microRNA containing fragment was excised from the Puro / miR-CCR5-l plasmid at the ClaI and XbaI sites, and the fragment containing the GC-rich modified TRIM 5a gene was excised from the pLVX-Puro cPPT 1g CMV-Trim enh plasmid at the NheI and XbaI sites . To obtain a combined construct, the vector was ligated with two fragments and transformed into E. Coli cells. Clones containing both fragments were selected and the isolated plasmids were analyzed by sequencing. The resulting construct was designated pLVX-Puro miR-CCR5-1-Trim enh.

Similarly, constructs containing Trim enh and miRNA SEQ ID 8 or SEQ ID 9 were obtained.

Example 8. Transfection of packaging cells and obtaining lentiviral particles.

To obtain lentiviruses, the HEK 293T cell line and the Lenti-X HT Packaging Mix (Clontech) packaging plasmid set were used. HEK 293T growth medium is: Dulbecco's Modified Eagle's Medium (DMEM) containing 4 mmol L-glutamine, 4.5 g / L glucose, 1 mmol sodium pyruvate, 1.5 g / L sodium bicarbonate and 10% fetal bovine serum . This level of sodium bicarbonate (NaHCO ₃ ; 1.5 g / l) is used in incubators containing 5% CO ₂ .

The day before transfection, 4-5 * 10 cells were added per 100 mm plate in 10 ml of complete medium containing tetracycline-free serum (Tet System Approved FBS Clontech Cat # 631106). On the day of transfection, 15 μl of Lenti-X HT Packaging Mix and 3 μg vector DNA were mixed in a 15 ml tube. Then, 1 ml of OptiMEM medium (Invitrogen) and 20 μl of Turbofect transfection reagent (Fermentas) were added to the tube. The mixture was incubated for 20 minutes at room temperature. During incubation, the cell medium was replaced with 10 ml of OptiMEM medium containing 10% tetracycline-free serum. After incubation, a mixture of plasmids, OptiMEM and Turbofect was added dropwise to the cell medium. Cells were incubated overnight at 37 ° C in a CO ₂ incubator. The next day, the medium containing the Turbofect DNA complex was removed, and replaced with 10 ml of culture medium containing tetracycline-free serum. Cells were incubated at 37 ° C in a CO ₂ incubator for 48-72 hours. The supernatant containing the lentiviral particles was selected. The supernatant was centrifuged at 3000 rpm for 15 minutes at + 4 ° C. The supernatant was filtered through 0.45 μm. The finished product was stored at -80 ° C.

To obtain self-inactivating lentiviral particles, constructs based on a self-inactivating vector and a set of packaging plasmids ViraPower Packaging Mix (Invitrogen) were used. Cell transfection was carried out similarly, tetracycline-free serum was replaced with Invitrogen serum.

Example 9. Determination of virus titer

The day before transduction (1 day), HT1080 line fibroblasts (approximately 2 × 10 ⁵ cells per well) were plated and incubated overnight at 37 ° C. in a CO ₂ incubator. The next day (day 2), the lentiviral flow was thawed and sequential 10-fold dilutions from 10 ^-2 to 10 ^{-6 were} performed. For each dilution, the volume was adjusted to 1 ml with culture medium. The medium was removed from the cells and each dilution of the virus was added to its well (in a volume of 1 ml). Incubated overnight at 37 ° C in a CO ₂ incubator.

The next day (day 3), the virus containing medium was removed and replaced with 2 ml of culture medium. Incubated overnight at 37 ° C in a CO ₂ incubator.

Day 4: cells were treated with puromycin antibiotic. The medium was removed, replaced with a medium containing 2 μg / ml puromycin. Replaced the medium containing the antibiotic every 3-4 days.

After 7-10 days of selection, the medium was removed and PBS cells were washed 2 times. A crystal violet solution was added and incubated for 10 minutes at room temperature. Washed PBS cells 2 times. Blue colonies were counted and the titer of viral runoff was determined.

Example 10. Transduction of MT-4 cells

Cells were grown in RPMI + 10% FBS culture medium to a concentration of 0.5 × 10 ⁶ cells per ml. On the first day, the lentiviral stock was thawed and diluted in fresh culture medium to the required MOI. To a 3 ml suspension of cells with a concentration of 0.5 × 10 ⁶ cells / ml was added 1.5 × 10 ⁶ viral particles in 2 ml of culture medium + 5 μl polybrene 4 mg / ml.

Incubated overnight at 37 ° C in a CO ₂ incubator.

The next day (day 2), the cells were centrifuged, the medium containing the virus was removed, and it was replaced with fresh culture medium. Incubated overnight at 37 ° C in a CO ₂ incubator.

On day 3, an antibiotic was added to the medium to select transduced cells. We changed the medium to fresh with an antibiotic every 3-4 days.

Puromycin-resistant cells were expanded to a titer of 0.9 × 10 ⁶ . The cells were then used to test for resistance to HIV infection.

Example 11. Evaluation of the effectiveness of the design based on the modified TRIM5a gene on MT-4 cells.

Methodology

The suspension of the modified MT-4 cells was divided into two culture vessels and diluted to a concentration of 5 * 10 ⁵ cells per milliliter. One part of the cells was infected with human immunodeficiency virus (pNL4-3) to determine antiviral activity, the other served as a control of the cytotoxicity of the drug. Cells were cultured in parallel.

Cells were infected with a virus with a multiplicity of infection of 0.01 infectious units per cell. Cell cultures were incubated at 37 ° C for 1 hour and washed twice. After that, RPMI-1640 nutrient medium (final cell concentration of 400,000 cells / ml) was added to the cell culture. Further, the medium was changed on the 2nd, 4th, 8th, 12th, 15th, and 19th day of cultivation. When changing the medium, the old medium was completely removed, the cells were washed with fresh RPMI-1640 medium without serum, after which fresh nutrient medium was added (final cell concentration 400,000 cells / ml). The results of antiviral activity were taken into account by cell viability by staining the cells with trypan blue dye. Using enzyme-linked immunosorbent assay, using a commercial vector enzyme immunoassay kit from Vector-Best (Russia) and diluting a standard p24 antigen preparation (Aalto, Ireland), the amount of p24 antigen (ng / ml) in the culture fluid was determined according to the manufacturer's instructions. The results were taken into account using a Multiscan photometer at a wavelength of 630 nm.

Results.

Figure 4 shows the anti-HIV activity of a construct comprising a sequence encoding a modified human TRIM5α protein with a GC rich nucleotide composition (trim enh), compared with a construct comprising a sequence encoding a modified human TRIM5α protein described in patent RU 2426788.

The activity of the construct, including the sequence encoding the modified human TRIM5α protein with a GC rich nucleotide composition, exceeds almost 40 times (on the 7th day of cultivation) the activity of the construct with the original nucleotide composition. In addition, cell viability in the presence of HIV has increased.

Example 12. Transduction of cells U373-MAGI-CCR5

On the first day, U373-MAGI-CCR5 cells scattered 1 * 10 ⁵ cells per well of a 48-well plate in 0.5 ml of DMEM medium with 10% fetal calf serum and 8 μg / ml polybrene. On the second day, the medium was completely removed and lentiviral particles containing the antiviral gene / genes or control insert were added in an amount of 1 infectious particle per cell in 100 μl of complete DMEM with polybrene. After 2 hours, another 300 μl of DMEM was added and left overnight. On the third day, a change of medium was carried out. On the fifth day, the number of transduced cells was estimated by flow cytometry, measuring the percentage of cells containing green fluorescent protein. If the percentage of transduced cells was not less than 50, the obtained lines were used for subsequent evaluation of antiviral activity.

Example 13. Evaluation of the effectiveness of combined designs on cells U373-MAGI-CCR5.

Methodology

1 * 10 ⁶ cells of each line were seeded in 2 vials (for infection and control) with an area of 25 cm ² in 10 ml of complete DMEM growth medium and 4 μg / ml polybrene. The next day, cells were infected with the HIV pNL (AD) 8 virus, which is tropic for the CCR5 receptor. To do this, the growth medium was removed from the vials and 1 ml of the viral suspension was added, containing 1 * 10 ⁴ infectious units in 1 of the vials of each line. 1 ml of growth medium was added to control vials. Incubated for 2 hours at 37 ° C, swaying every 30 minutes. After 2 hours, the cells were washed once with PBS and a second time with maintenance medium. Then, 7 ml of complete growth medium was added to each vial. Starting from the next day, daily sampling was carried out for 9 days. For this, 4 samples of 500 μl were taken from each vial and 2 ml of fresh growth medium was added in exchange. The resulting samples were used to measure the viral load of HIV and the level of p24 viral antigen.

Results.

Table 3 presents the results of measuring the p24 level of HIV antigen within 9 days after infection of U373-MAGI-CCR5 cells transduced using combined constructs. In all cases, a slowdown in the development of infection was observed compared with the control (vector without insertion). The use of a triple combination had a stronger antiviral effect than a double.

Table 3. Accumulation of p24 antigen in MAGI cells (pg / ml). Day after infection Vector without insert Trim-miR ccr Trim-miR ccR-TAR Trim-miR ccR-env one 1.29 1.19 1.19 1,11 2 4.15 4.32 2.29 2.15 3 22.6 11.2 8.8 7.5 four 115 16 15.7 13.7 5 810 67.6 62,4 58 6 2020 378 286 216 7 4630 833 634 375 8 17600 1160 885 550 9 24500 3350 2020 1200

Thus, the present experimental data have demonstrated that the anti-HIV activity of a construct comprising a sequence encoding a modified human TRIM5α protein with a GC rich nucleotide composition is almost 40 times higher than the activity of a similar construct including a sequence encoding a modified human TRIM5α protein described in patent RU 2426788 (without synonymous replacements). Adding one or two anti-HIV agents to this construct increased the antiviral effect.

Claims (40)

1. A genetic construct for anti-HIV therapy that includes a nucleotide sequence (SEQ ID N1) encoding a modified human TRIM 5α protein (SEQ ID N3) and different from the original SEQ ID N2 sequence by multiple synonymous substitutions providing an increase in the percentage of GC pairs in the DNA sequence . 2. The genetic construct according to claim 1, where the percentage of GC pairs in the sequence encoding the modified human TRIM 5α protein (SEQ ID N3) is from 49 to 64%. 3. The genetic construct according to claim 1, where as the sequence encoding the modified human TRIM 5α protein (SEQ ID N3), the sequence SEQ ID N1 is used. 4. The construct according to claims 1, 2 or 3, further comprising a molecular marker, which is a puromycin resistance gene, a green fluorescent protein gene, or another sequence for selection and / or monitoring of modified cells. 5. The genetic construct according to claims 1, 2 or 3, where the sequence encoding the modified human TRIM5a protein is under the control of a constitutive, tissue-specific or inducible promoter. 6. The genetic construct according to claims 1, 2 or 3, where one of the following promoters is used as a promoter: promoter of the PGK gene, EF1α gene, UbC gene, MHC class II gene promoter, T-cell receptor gene promoter, β- gene promoter interferon, IL2 gene promoter, IL2 receptor gene promoter, or tetracycline dependent promoter. 7. Genetic design for anti-HIV therapy, which includes:
a vector sequence for embedding DNA sequences in the genome of a human cell, and
a sequence (SEQ ID N1) encoding a modified human TRIM 5α protein (SEQ ID N3) and different from the original sequence SEQ ID N2 by multiple synonymous substitutions providing an increase in the percentage of GC pairs in the DNA sequence. 8. The genetic construct according to claim 7, where the percentage of GC pairs in the sequence encoding the modified human TRIM 5α protein (SEQ ID N3) is from 49 to 64%. 9. The genetic construct according to claim 7, where as the sequence encoding the modified human TRIM 5α protein (SEQ ID N3), the sequence SEQ ID N1 is used. 10. The genetic construct according to claims 7, 8 or 9, wherein the sequence encoding the modified human TRIM5a protein is under the control of a constitutive, tissue-specific or inducible promoter. 11. The genetic construct according to claims 7, 8 or 9, where one of the following promoters is used as a promoter: promoter of the PGK gene, EF1α gene, gene, UbC, MHC class II gene promoter, T-cell receptor gene promoter, β- gene promoter interferon, IL2 gene promoter, IL2 receptor gene promoter, or tetracycline dependent promoter. 12. The genetic construct according to claims 7, 8 or 9, where the sequence encoding the modified human TRIM5a protein can be in the forward or reverse direction with respect to the vector. 13. The genetic construct according to claims 7, 8 or 9, further comprising a molecular marker, which is a puromycin resistance gene, a green fluorescent protein gene, or another sequence for selection and / or monitoring of modified cells. 14. The genetic construct according to claims 7, 8 or 9, wherein the lentiviral vector based on HIV-1 is used as a vector, which includes at least the following areas:
5′-LTR and 3′LTR,
specific tRNA binding site (PBS),
packing signal (ψ),
rev-response element (RRE),
North American woodchuck hepatitis virus post-transcriptional regulatory element (WPRE),
CPPT area. 15. The genetic construct according to claims 7, 8 or 9, wherein the lentiviral vector based on HIV-1 is used as a vector, which includes at least the following areas:
5′-LTR and 3′LTR,
specific tRNA binding site (PBS),
packing signal (ψ),
rev-response element (RRE),
North American woodchuck hepatitis virus post-transcriptional regulatory element (WPRE),
CPPT region,
and where the vector sequence is modified using the sequence of SEQ ID N10 in front of the CPPT region. 16. The genetic construct according to claims 7, 8 or 9, where a self-inactivating lentiviral vector is used as a vector, which includes at least the following areas:
RSV / 5′-LTR hybrid promoter,
3′LTR, with a deletion in the U3 region,
specific tRNA binding site (PBS),
packing signal (ψ),
rev-response element (RRE),
North American woodchuck hepatitis virus post-transcriptional regulatory element (WPRE),
CPPT area. 17. The genetic construct according to claims 7, 8 or 9, where a self-inactivating lentiviral vector is used as a vector, which includes at least the following areas:
RSV / 5′-LTR hybrid promoter,
3′LTR, with a deletion in the U3 region,
specific tRNA binding site (PBS),
packing signal (ψ),
rev-response element (RRE),
North American woodchuck hepatitis virus post-transcriptional regulatory element (WPRE),
CPPT area
and where the vector sequence is modified using the sequence of SEQ ID N10 in front of the CPPT region. 18. The genetic construct according to claims 7, 8 or 9, where a lentiviral vector based on one or more of the lentiviruses is used as a vector: HIV-1, HIV-2, VIO, cattle immunodeficiency virus, goat arthritis-encephalitis lentivirus, horse infectious anemia virus, feline immunodeficiency virus, and / or membrane disease virus, or a vector based on spumavirus. 19. The genetic construct according to claims 7, 8 or 9, wherein a non-viral vector based on the transposon / transposase vector system is used as a vector. 20. The genetic construct according to claims 7, 8 or 9, where the insertion of the DNA sequence into a specific location of a human chromosome is achieved by introducing a gap into a given region of the chromosome using a specific endonuclease, and subsequent homologous recombination between the vector sequences included in the constructs of claims. 7, 8 or 9 and adjacent to the gap sections of the chromosome. 21. A genetic construct for anti-HIV therapy, which includes:
a sequence (SEQ ID N1) encoding a modified human TRIM 5α protein (SEQ ID N3) and different from the original sequence SEQ ID N2 by multiple synonymous substitutions providing an increase in the percentage of GC pairs in the DNA sequence,
and one or more sequences selected from SEQ ID N 7, 8, 9 encoding an artificial miRNA that inhibits expression of the human CCR5 receptor gene (miRNA CCR5). 22. The genetic construct of claim 21, wherein the percentage of GC pairs in the sequence encoding the modified human TRIM 5α protein (SEQ ID N3) is from 49 to 64%. 23. The genetic construct according to item 21, where as the sequence encoding the modified human TRIM 5α protein (SEQ ID N3), the sequence SEQ ID N1 is used. 24. The genetic construct according to claims 21-23, further comprising a sequence of SEQ ID N4 encoding an RNA hairpin similar to a TAR sequence (TAR) and / or an antisense sequence to a region of the HIV env gene SEQ ID N5 (a / s env). 25. The genetic construct according to claims 21-23, wherein at least one of the sequences: CCR5 miRNA and / or a sequence encoding a modified human TRIM 5α protein is under the control of a constitutive, tissue-specific or inducible promoter. 26. The genetic construct according to claims 21, 22 or 23, where one of the following promoters is used as a promoter: promoter of the PGK gene, EF1α gene, UbC gene, promoter of the MHC class II genes, promoter of the T-cell receptor gene, promoter of the β-interferon gene , IL2 gene promoter, IL2 receptor gene promoter, tetracycline dependent promoter or RNA polymerase III promoter. 27. The construction according to claims 21-23, further comprising a molecular marker, which is a puromycin resistance gene, a green fluorescent protein gene, or another sequence for selection and / or monitoring of modified cells. 28. Genetic design for anti-HIV therapy, which includes:
a vector sequence that allows the insertion of DNA sequences into the genome of a human cell,
a sequence (SEQ ID N1) encoding a modified human TRIM 5α protein (SEQ ID N3) and different from the original sequence SEQ ID N2 by multiple synonymous substitutions providing an increase in the percentage of GC pairs in the DNA sequence,
and one or more sequences selected from SEQ ID N 7, 8, 9 encoding an artificial miRNA that inhibits expression of the human CCR5 receptor gene (miRNA CCR5). 29. The genetic construct of claim 28, wherein the percentage of GC pairs in the sequence encoding the modified human TRIM 5α protein (SEQ ID N3) is from 49 to 64%. 30. The genetic construct of claim 28, wherein the sequence encoding the modified human TRIM 5α protein (SEQ ID N3) is the sequence SEQ ID N1. 31. The genetic construct of claims 28-30, further comprising a sequence of SEQ ID N4 encoding an RNA hairpin similar to a TAR sequence (TAR) and / or an antisense sequence to a region of the HIV env gene SEQ ID N5 (a / s env). 32. The genetic construct according to claims 28-30, wherein at least one of the sequences: CCR5 miRNA and / or a sequence encoding a modified human TRIM 5α protein is under the control of a constitutive, tissue-specific or inducible promoter. 33. The genetic construct according to claims 28-30, wherein one of the following promoters is used as a promoter: promoter of the PGK gene, EF1α gene, UbC gene, MHC class II gene promoter, T cell receptor gene promoter, β-interferon gene promoter, IL2 gene promoter, IL2 receptor gene promoter, tetracycline dependent promoter, or RNA polymerase III promoter. 34. The genetic construct according to claims 28-30, wherein the lentiviral vector based on HIV-1 is used as a vector, which includes at least the following areas:
5′-LTR and 3′LTR,
specific tRNA binding site (PBS),
packing signal (ψ),
rev-response element (RRE),
North American woodchuck hepatitis virus post-transcriptional regulatory element (WPRE),
CPPT area. 35. The genetic construct according to claims 28-30, wherein the lentiviral vector based on HIV-1 is used as a vector, which includes at least the following areas:
5′-LTR and 3′LTR,
specific tRNA binding site (PBS),
packing signal (ψ),
rev-response element (RRE),
North American woodchuck hepatitis virus post-transcriptional regulatory element (WPRE),
CPPT area
and where the sequence of the vector is modified using the sequence of SEQ ID N1 in front of the CPPT region. 36. The genetic construct according to claims 28-30, wherein a self-inactivating lentiviral vector is used as a vector, which includes at least the following areas:
RSV / 5′-LTR hybrid promoter,
3′LTR, with a deletion in the U3 region,
specific tRNA binding site (PBS),
packing signal (ψ),
rev-response element (RRE),
North American woodchuck hepatitis virus post-transcriptional regulatory element (WPRE),
CPPT area. 37. The genetic construct according to claims 28-30, wherein a self-inactivating lentiviral vector is used as a vector, which includes at least the following areas:
RSV / 5′-LTR hybrid promoter,
3′LTR, with a deletion in the U3 region,
specific tRNA binding site (PBS),
packing signal (ψ),
rev-response element (RRE),
North American woodchuck hepatitis virus post-transcriptional regulatory element (WPRE),
CPPT area
and where the sequence of the vector is modified using the sequence of SEQ ID N1 in front of the CPPT region. 38. The genetic construct according to claims 28-30, wherein a lentiviral vector based on one or more of the lentiviruses is used as a vector: HIV-1, HIV-2, VIO, cattle immunodeficiency virus, goat arthritis-encephalitis lentivirus, infectious virus equine anemia, feline immunodeficiency virus, and / or membrane disease virus, or a vector based on spumavirus. 39. The genetic construct according to claims 28-30, wherein a non-viral vector based on the transposon / transposase vector system is used as a vector. 40. The genetic construct according to claims 28-30, where the insertion of the DNA sequence into a specific location of a human chromosome is achieved by introducing a gap into a given region of the chromosome using a specific endonuclease, and subsequent homologous recombination between the vector sequences included in the constructs according to claims 28, 29 or 30 and adjacent to the gap parts of the chromosome.

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