RU2730663C2 - Gene-therapeutic dna-vector based on gene-therapeutic dna-vector vtvaf17, carrying target gene cas9 for heterologous expression of this target gene in human and animal cells in implementing various methods of genomic editing, method of producing and using gene-therapeutic dna vector, escherichia coli scs110-af/vtvaf17-cas9 strain, carrying gene-therapeutic dna vector, method for preparing thereof, method for production on industrial scale of gene-therapeutic dna vector - Google Patents

Abstract

FIELD: biotechnology; medicine; agriculture.

SUBSTANCE: invention refers to genetic engineering and can be used in biotechnology, medicine and agriculture to develop gene therapy preparations. Presented is a gene-therapeutic DNA vector based on the gene-therapeutic DNA vector VTvaf17, carrying the target Cas9 gene for heterologous expression of this target gene in human and animal cells when implementing various methods of genomic editing. Gene-therapeutic DNA vector VTvaf17-Cas9 has the nucleotide sequence SEQ ID No. 1. Invention also discloses a method of producing said vector, use of a vector, an Escherichia coli strain carrying said vector, as well as a method of producing said vector on an industrial scale.

EFFECT: invention can be used for safe application for human and animal genetic therapy.

8 cl, 12 dwg, 14 ex

Description

Technology area

The invention relates to genetic engineering and can be used in biotechnology, medicine and agriculture to create drugs for gene therapy.

State of the art

Gene therapy is a modern medical approach aimed at treating hereditary and acquired diseases by introducing new genetic material into the patient's cells in order to compensate or suppress the function of the mutant gene and / or correct the genetic defect. The end product of gene expression can be an RNA or protein molecule. However, the implementation of most of the physiological processes in the body is associated with the functional activity of protein molecules, while RNA molecules are either an intermediate product in protein synthesis or carry out regulatory functions. Thus, the goal of gene therapy is, in most cases, the introduction into the body of genes that provide transcription and subsequent translation of the protein molecules encoded by these genes. In the framework of the description of the present invention, the expression of a gene means the production of a protein molecule, the amino acid sequence of which is encoded by this gene. Mutations in genes can lead to complete or partial loss of protein expression, or the expression of variants of protein molecules that have undesirable functional activity. Introduction into the body of gene therapy vectors encoding a particular gene can restore the expression of target proteins. However, this approach is compensatory and is not aimed at correcting genetic defects. With the discovery of systems for directed (targeted) editing of nucleotide sequences, it became possible, by introducing various nucleases with specific properties (for example, Cas9) into gene therapy vectors, to implement the approach of therapeutic genomic editing, which is aimed at correcting mutations in the DNA sequence, which is also targeted gene therapy. ... In this case, function is restored by correcting genetic defects (Memi F, Ntokou A, Papangeli I, 2018; Hussain W et al., 2019).

The Cas9 gene encodes a nuclease protein. The CRISPR / Cas9 system was initially discovered as a component of the bacterial immune system, which allows bacterial cells to get rid of the nucleotide sequences of bacteriophages in a targeted manner (Sapranauskas R, 2011; Mougiakos I, 2017). Since this system has a certain degree of versatility of the principle of operation, it has found wide application in biomedical and biotechnological research. At present, the CRISPR / Cas9 system is widely used in scientific research for editing genomes in mammalian and laboratory animal cell cultures and has the potential to develop tools and methods for gene therapy. The principle of operation of this system is that the Cas9 endonuclease, using gRNA complementary to a specific sequence in the genome, introduces breaks in DNA strands, cutting out a section of the targeted DNA. The integrity of DNA at the site of breaks is then restored using cellular repair systems, which can use a homologous DNA strand containing the correct nucleotide sequence as a template for repair, or restore breaks by directly connecting adjacent nucleotides without repairing the cut DNA region (Salsman J, 2017). Designing gRNA in such a way that this molecule is complementary to the DNA region containing this or that mutation allows using Cas9 to specifically cut out this particular region, which determines the potential of this mechanism of action in correcting genetic material, that is, editing genomes (Wilson LOW , 2018).

Nevertheless, one of the main problems of using the CRISPR / Cas9 system for editing genomes remains the problem of delivery of the endonuclease and gRNA complex into the cell nucleus and, in general, the problems of pharmacokinetics that limit the penetrating ability of molecules into various organs and tissues or require the introduction of too high concentrations and the use of special compositions that provide cell penetration (Dowdy SF, 2017). These limitations can be overcome using genetic vectors for heterologous expression of the Cas9 gene. The most studied vectors in this area are lentiviral, adenoviral, adeno-associated and other vectors of viral origin.

Another problem of using the CRISPR / Cas9 system is the risk of nonspecific endonuclease action. In this vein, the use of vectors that do not integrate into the genome and provide only transient gene expression is potentially safer than, for example, the use of lentiviral and adeno-associated vectors (Li L, Hu S, Chen X, 2017). However, the use of any viral vectors for the delivery of certain sequences into the body is limited by the tropism of pseudoviral particles to various tissues, which does not always allow for effective penetration into target cells and organs (Maginnis MS, 2017). Also, the potential for using any viral vectors is limited, including their relatively high immunogenicity, preexisting immunity, and the risks associated with gene therapy vectors of viral origin in general (Lukashev AN, Zamyatnin AA, 2016).

Thus, the prior art indicates that there is a need to develop effective and safe gene therapy approaches for the delivery of Cas9 to target organs and tissues.

Gene therapy vectors are known to be divided into viral, cellular and DNA vectors (Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal Products EMA / CAT / 80183/2014). Recently, in gene therapy, more and more attention is paid to the development of non-viral systems for the delivery of genetic material, among which plasmid vectors are in the lead. Plasmid vectors are devoid of the disadvantages inherent in cellular and viral vectors. In the target cell, they exist in episomal form, do not integrate into the genome, their production is quite cheap, the absence of an immune response and side reactions to the introduction of a plasmid vector make them a convenient tool for gene therapy and genetic prevention (DNA vaccine) (Li L, Petrovsky N . // Expert Rev Vaccines. 2016; 15 (3): 313-29).

Nevertheless, the limitations for the use of plasmid vectors for gene therapy are: 1) the presence of antibiotic resistance genes for production in bacterial strains, 2) the presence of various regulatory elements represented by the sequences of viral genomes, 3) the size of the therapeutic plasmid vector, which determines the efficiency of vector penetration into target cell.

It is known that the European Medicines Agency considers it necessary to avoid the introduction of antibiotic resistance markers into developing plasmid vectors for gene therapy (Reflection paper on design modifications of gene therapy medicinal products during development / 14 December 2011 EMA / CAT / GTWP / 44236/2009 Committee for advanced therapies). This recommendation is associated, first of all, with the potential danger of penetration of the DNA vector or horizontal transfer of antibiotic resistance genes into the cells of bacteria present in the body as part of normal or opportunistic microflora. In addition, the presence of antibiotic resistance genes significantly increases the size of the DNA vector, which leads to a decrease in the efficiency of its penetration into eukaryotic cells.

It should be noted that antibiotic resistance genes also make a fundamental contribution to the method of obtaining DNA vectors. In the case of the presence of antibiotic resistance genes, strains for the production of DNA vectors are usually cultivated in a medium containing a selective antibiotic, which creates the risk of traces of antibiotic in insufficiently purified DNA vector preparations. Thus, obtaining DNA vectors for gene therapy, which lack genes for antibiotic resistance, is associated with obtaining strains that have such a distinctive feature as the ability to stable amplification of target DNA vectors in a medium without antibiotics.

In addition, the European Medical Agency recommends avoiding the presence of regulatory elements in the composition of therapeutic plasmid vectors to increase the expression of target genes (promoters, enhancers, post-translational regulatory elements), which are the nucleotide sequences of the genomes of various viruses (Draft Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products, http: //www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2015/05/WC500187020.pdf). Although these sequences can increase the level of expression of the target transgene, however, they pose a risk of recombination with the genetic material of wild-type viruses and integration into the genome of a eukaryotic cell. Moreover, the feasibility of overexpression of one or another gene for therapy purposes remains an unresolved issue.

Also, the size of the therapeutic vector is essential. It is known that modern plasmid vectors are often overloaded with non-functional regions, which significantly increase the size of the vector (Mairhofer J, Grabherr R. // Mol Biotechnol. 2008.39 (2): 97-104). For example, the ampicillin resistance gene in vectors of the pBR322 series, as a rule, consists of at least 1000 bp, which is more than 20% of the size of the vector itself. At the same time, an inverse relationship is observed between the size of the vector and its ability to penetrate into eukaryotic cells - DNA vectors with a small size penetrate more effectively into human and animal cells. For example, in a series of experiments on the transfection of HeLa cells with DNA vectors ranging in size from 383 to 4548 bp. it was shown that the difference in penetration efficiency can reach two orders of magnitude (differ by a factor of 100) (Hornstein BD et al. // PLoS ONE. 2016; 11 (12): e0167537.).

Thus, when choosing a DNA vector, for the sake of safety and maximum efficiency, preference should be given to those constructs that do not contain antibiotic resistance genes, sequences of viral origin and the size of which allows efficient penetration into eukaryotic cells. The strain for obtaining such a DNA vector in quantities sufficient for gene therapy purposes must provide the possibility of stable amplification of the DNA vector using culture media that do not contain antibiotics.

An example of the use of recombinant DNA vectors for gene therapy is the method for producing a recombinant vector for genetic immunization according to US patent 9550998 B2. The plasmid vector is a super-bored plasmid DNA vector and is intended for the expression of cloned genes in animal and human cells. The vector consists of the origin of replication, regulatory elements including the promoter and enhancer of human cytomegalovirus, regulatory elements from the human T-lymphotropic virus.

The accumulation of the vector is carried out in a special strain of E. coli without the use of antibiotics due to antisense complementation of the sacB gene introduced into the strain by means of a bacteriophage. The disadvantage of this invention is the presence of regulatory elements in the DNA vector, which are sequences of viral genomes.

The prototypes of the present invention in terms of the use of gene therapy approaches for the expression of Cas9 in eukaryotic cells are the following patents.

US8795965B2 discloses a DNA molecule that encodes an expression cassette containing the sequence encoding the Cas9 protein. The disadvantage of this invention is the uncertainty of the requirements for the presence of regulatory sequences of viral origin in the DNA molecule, as well as the uncertainty of methods for obtaining these molecules and their industrial applicability.

CN103981216B discloses a plasmid vector expressing the Cas9 gene. The disadvantage of this invention is the use of regulatory elements in the vector that ensure the expression of the Cas9 gene in plant cells, but not mammals, as well as the presence of antibiotic resistance genes in the vector.

US9914939B2 describes a plasmid vector expressing the Cas9 gene. The disadvantage of this invention is the uncertainty of the safety requirements and manufacturability with respect to the plasmid vector used, in particular the presence / absence of viral sequences and antibiotic resistance genes in the vector.

Disclosure of invention

The objective of the invention is to construct a gene therapeutic DNA vector for heterologous expression of the Cas9 gene in human and animal cells, combining the following properties:

I) Efficiency of gene therapy DNA vector for heterologous expression of the target gene in eukaryotic cells.

II) The possibility of safe use for the implementation of various methods of genomic editing of human and animal genomes, including in genetic therapy of humans and animals due to the absence of regulatory elements in the composition of the gene therapy DNA vector, which are the nucleotide sequences of viral genomes.

III) Possibility of safe use for the implementation of various methods of genomic editing of human and animal genomes, including in genetic therapy of humans and animals due to the absence of antibiotic resistance genes in the composition of the gene therapy DNA vector.

IV) Manufacturability of obtaining and the possibility of developing a gene therapy DNA vector on an industrial scale.

Items II and III are provided for in this technical solution in accordance with the recommendations of state regulators for drugs for gene therapy, in particular, the European Medicines Agency regarding the refusal to introduce antibiotic resistance markers into plasmid vectors for gene therapy being developed (Reflection paper on design modifications of gene therapy medicinal products during development / 14 December 2011, EMA / CAT / GTWP / 44236/2009 Committee for advanced therapies) and regarding the refusal to introduce elements of viral genomes into plasmid vectors for gene therapy under development (Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products / 23 March 2015, EMA / CAT / 80183/2014, Committee for Advanced Therapies).

The object of the invention is also the design of strains carrying this gene therapy DNA vector for the development and production on an industrial scale of a gene therapy DNA vector.

The problem is solved due to the fact that a gene therapy DNA vector based on the gene therapy DNA vector VTvaf17 has been created, which carries the target Cas9 gene for heterologous expression of this target gene in human and animal cells when implementing various methods of genomic editing, while the gene therapy DNA vector VTvaf17 -Cas9 has the nucleotide sequence of SEQ ID No. 1. At the same time, the created gene therapy DNA vector based on the gene therapy DNA vector VTvaf17 carrying the target Cas9 gene, characterized in that the created gene therapy DNA vector VTvaf17-Cas9, due to the limited size of the vector part VTvaf17, not exceeding 3200 bp, has the ability efficiently penetrate human and animal cells and express the target Cas9 gene cloned into it.

A gene therapy DNA vector based on the gene therapy DNA vector VTvaf17 carrying the target Cas9 gene, characterized by the fact that the gene therapy DNA vector does not contain nucleotide sequences of viral origin and there are no antibiotic resistance genes, providing the possibility of its safe use for the implementation of various methods of genomic editing of human genomes , animal, including in genetic therapy of humans and animals.

A method for producing a gene therapy DNA vector based on a gene therapy DNA vector VTvaf17 carrying the target Cas9 gene has also been created, which consists in that the gene therapy DNA vector VTvaf17-Cas9 is obtained as follows: the coding part of the target Cas9 gene is cloned into the DNA vector VTvaf17 and receive gene therapy DNA vector VTvaf17-Cas9, SEQ ID No. 1.

A method of using the created gene therapy DNA vector based on the gene therapy DNA vector VTvaf17 carrying the target Cas9 gene for heterologous expression of this target gene in human and animal cells, which consists in introducing the gene therapy DNA vector into the cells, organs and tissues of a human or animal together with molecules gRNA or genetic constructs that provide gRNA expression, and / or in the introduction into organs and tissues of a human or animal autologous human or animal cells transfected with a gene therapeutic DNA vector together with gRNA molecules or genetic constructs that provide gRNA expression or in a combination of the indicated methods.

The method of obtaining the Escherichia coli SCS110-AF / VTvaf17-Cas9 strain consists in electroporation of the competent cells of the Escherichia coli SCS110-AF strain with the created gene therapy DNA vector and subsequent selection of stable strain clones using a selective medium.

The claimed strain of Escherichia coli SCS110-AF / VTvaf17-Cas9, which carries a gene therapeutic DNA vector for its production with the possibility of cultivating the strain without using antibiotics.

The method of industrial-scale production of a gene therapy DNA vector consists in scaling the bacterial culture of the strain to the amounts required for increasing the bacterial biomass in an industrial fermenter, after which the biomass is used to isolate the fraction containing the target DNA product - the gene therapeutic DNA vector VTvaf17-Cas9, in multistage filtered and purified by chromatographic methods.

The invention is illustrated by drawings, where:

Figure 1

shows a diagram of a gene therapy DNA vector VTvaf17 carrying the target Cas9 gene, which is a circular double-stranded DNA molecule capable of autonomous replication in the cells of Escherichia coli bacteria.

Figure 1 shows a diagram of a gene therapy DNA vector VTvaf17-Cas9.

The following structural elements of the vector are marked on the diagram:

EF1a is the promoter region of the human elongation factor gene EF1A with its own enhancer contained in the first intron of the gene. Serves to ensure a high level of transcription of the recombinant gene in most human tissues;

Reading frame of the target gene corresponding to the coding part of the Cas9 gene;

hGH-TA - transcription terminator and polyadenylation site of the human growth factor gene;

ori - the origin of replication, which serves for autonomous replication with a single nucleotide substitution to increase the copy number of the plasmid in the cells of most Escherichia coli strains;

RNA-out is a regulatory element of the RNA-out transposon Tn 10, which provides the possibility of positive selection without the use of antibiotics when using the Eshcerichia coli SCS 110 strain.

Unique restriction sites are marked.

Figure 2

shows the graphs of the accumulation of cDNA amplicons of the target gene, namely, the Cas9 gene, in the primary culture of human skin fibroblasts HDFa (ATCC PCS-201-01) before their transfection and 48 hours after transfection of these cells with the gene therapy DNA vector VTvaf17-Cas9 in order to assess the ability to penetrate into eukaryotic cells and functional activity, that is, the expression of the target gene at the mRNA level.

Figure 2 shows the accumulation curves of amplicons during the reaction, corresponding to:

1 - cDNA of the Cas9 gene in HDFa cell culture before transfection with the VTvaf17-Cas9 DNA vector;

2 - cDNA of the Cas9 gene in HDFa cell culture after transfection with the VTvaf17-Cas9 DNA vector;

3 - cDNA of the B2M gene in HDFa cell culture before transfection with the VTvaf17-Cas9 DNA vector;

4 - cDNA of the B2M gene in HDFa cell culture after transfection with the VTvaf17-Cas9 DNA vector.

The B2M gene (Beta-2 microglobulin) listed in the GenBank database under the number NM 004048.2 was used as a reference gene.

Figure 3

shows the graphs of accumulation of cDNA amplicons of the target gene, namely the Cas9 gene, in the primary culture of keratinocytes of the human epidermis HEKa (ATCC PCS-200-011) before their transfection and 48 hours after transfection of these cells with the DNA vector VTvaf17-Cas9 in order to assess the ability to penetrate in eukaryotic cells and functional activity, that is, the expression of the target gene at the mRNA level.

Figure 3 shows the accumulation curves of amplicons during the reaction, corresponding to:

1 - cDNA of the Cas9 gene in the primary culture of keratinocytes of the human epidermis HEKa before transfection with the VTvaf17-Cas9 DNA vector;

2 - cDNA of the Cas9 gene in the primary culture of keratinocytes of the human epidermis HEKa after transfection with the DNA vector VTvaf17-Cas9;

3 - cDNA of the B2M gene in the primary culture of keratinocytes of the human epidermis HEKa before transfection with the DNA vector VTvaf17-Cas9;

4 - cDNA of the B2M gene from the primary culture of keratinocytes of the human epidermis HEKa after transfection with the DNA vector VTvaf17-Cas9.

The B2M gene (Beta-2-microglobulin) listed in the GenBank database under the number NM 004048.2 was used as a reference gene.

Figure 4

shows the graphs of accumulation of cDNA amplicons of the target gene, namely the Cas9 gene, in the primary culture of human epidermal melanocytes cells of the Primary Epidermal Melanocytes line; Normal, Human, Adult (HEMa) (ATCC ^® PCS-200-013 ™) before their transfection and 48 hours after transfection of these cells with the DNA vector VTvaf17-Cas9 in order to assess the ability to penetrate into eukaryotic cells and functional activity, that is, expression the target gene at the mRNA level.

Figure 4 shows the accumulation curves of amplicons during the reaction, corresponding to:

1 - cDNA of the Cas9 gene in the primary culture of HEMa human epidermal melanocytes cells before transfection with the VTvaf17-Cas9 DNA vector;

2 - cDNA of the Cas9 gene in the primary culture of HEMa human epidermal melanocytes cells after transfection with the VTvaf17-Cas9 DNA vector;

3 - cDNA of the B2M gene in the primary culture of HEMa human epidermal melanocytes cells before transfection with the VTvaf17-Cas9 DNA vector;

4 - cDNA of the B2M gene in the primary culture of human epidermal melanocytes HEMa after transfection with the VTvaf17-Cas9 DNA vector.

The B2M gene (Beta-2-microglobulin) listed in the GenBank database under the number NM 004048.2 was used as a reference gene.

Figure 5

shows a diagram of the Cas9 protein concentration in the cell lysate of the primary culture of human skin fibroblasts HDFa (ATCC PCS-201-01) after transfection of these cells with the VTvaf17-Cas9 DNA vector in order to assess the functional activity, that is, expression at the protein level, by changing the amount of Cas9 protein in the cell lysate.

Figure 5 shows the following elements:

culture A - culture of HDFa human skin fibroblasts cells transfected with an aqueous solution of dendrimers without plasmid DNA (control);

culture B - culture of HDFa human skin fibroblasts cells transfected with the VTvaf17 DNA vector;

culture C - culture of HDFa human skin fibroblasts cells transfected with the VTvaf17-Cas9 DNA vector.

FIG. 6

shows a diagram of the Cas9 protein concentration in the lysate of cells of the primary culture of human epidermal keratinocytes HEKa (ATCC PCS-200-01), after transfection of these cells with the DNA vector VTvaf17-Cas9 in order to assess the functional activity, that is, the expression of the target gene at the protein level, and the possibility increasing the level of protein expression using a gene therapy DNA vector based on a gene therapy DNA vector VTvaf17 carrying the target Cas9 gene.

Figure 6 shows the following elements:

culture A - primary culture of HEKa human epidermal keratinocytes cells transfected with an aqueous solution of dendrimers without plasmid DNA (control);

culture B - primary culture of HEKa human epidermal keratinocytes cells transfected with the VTvaf17 DNA vector;

culture C - primary culture of HEKa human epidermal keratinocytes cells transfected with the VTvaf17-Cas9 DNA vector.

FIG. 7

shows a diagram of the Cas9 protein concentration in the lysate of cells of the primary culture of melanocytes of the human epidermis of the Primary Epidermal Melanocytes line; Normal, Human, Adult (HEMa) (ATCC ^® PCS-200-013 ™) after transfection of these cells with the VTvaf17-Cas9 DNA vector in order to assess the functional activity, that is, the expression of the target gene at the protein level, and the possibility of increasing the level of protein expression with using a gene therapy DNA vector based on a gene therapy DNA vector VTvaf17 carrying the target Cas9 gene.

7, the following elements are marked:

culture A - culture of HEMa human epidermal melanocytes cells transfected with an aqueous solution of dendrimers without plasmid DNA (control);

culture B - a culture of HEMa human epidermal melanocytes cells transfected with a VTvaf17 DNA vector;

culture C is a culture of HEMa human epidermal melanocytes cells transfected with the VTvaf17-Cas9 DNA vector.

FIG. 8

shows a diagram of the Cas9 protein concentration in the lysate of Syrian hamster ovary cells CHO-K1 (ATCC ^® CCL-61 ™) after transfection of these cells with the gene therapeutic DNA vector VTvaf17-Cas9 in order to assess the functional activity, that is, the expression of the target gene at the protein level, and the possibility increasing the level of protein expression using a gene therapy DNA vector based on a gene therapy DNA vector VTvaf17 carrying the target Cas9 gene.

Figure 8 shows the following elements:

culture A - culture of cells of the ovary of the Syrian hamster CHO-K1, transfected with an aqueous solution of dendrimers without plasmid DNA (control);

culture B - culture of cells of the ovary of the Syrian hamster CHO-K1, transfected with the DNA vector VTvaf17;

culture C - culture of cells of the ovary of the Syrian hamster CHO-K1, transfected with the DNA vector VTvaf17-Cas9.

FIG. nine

shows a diagram of the Cas9 protein concentration in the culture lysate of human peripheral blood mononuclear cells (PBMC) after transfection of these cells with the VTvaf17-Cas9 gene therapeutic DNA vector in order to assess the functional activity, that is, the expression of the target gene at the protein level, and the possibility of increasing the level of protein expression using gene therapy DNA vector based on gene therapy DNA vector VTvaf17 carrying the target Cas9 gene.

In Fig. 9, the following elements are marked:

culture A - culture of human PBMC cells transfected with an aqueous solution of dendrimers without plasmid DNA (control);

culture B — culture of human PBMC cells transfected with the VTvaf17 DNA vector;

culture C - culture of human PBMC cells transfected with the VTvaf17-Cas9 DNA vector.

Figure 10

shows a diagram of the Cas9 protein concentrations in skin biopsies of three rats in a pre-epilated area of Wistar rats after introducing a culture of autologous fibroblasts transfected with the VTvaf17-Cas9 gene therapeutic DNA vector into the skin to demonstrate the method of application by introducing autologous cells transfected with the VTvaf17 gene therapeutic DNA vector -Cas9.

In Fig. 10, the following elements are marked:

K1I - biopsy specimen of rat skin K1 in the zone of administration of the culture of autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17-Cas9;

K1II - biopsy specimen of rat skin in the area of injected autologous fibroblasts transfected with gene therapy DNA vector VTvaf17, not carrying the Cas9 gene;

K1III - rat skin biopsy from an intact site;

K2I - biopsy of the skin of a K2 rat in the zone of administration of a culture of autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17-Cas9;

K2II - biopsy of the skin of a K2 rat in the injection zone of autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17, not carrying the Cas9 gene;

K2III - biopsy of the skin of a K2 rat from an intact site;

K3I - biopsy specimen of the skin of rat K3 in the injection zone of the culture of autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17-Cas9;

K3II - biopsy of the skin of rat K3 in the area of injection of autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17, which does not carry the Cas9 gene;

K3III - biopsy of the K3 rat skin from an intact site.

FIG. eleven

shows a diagram of the decrease in the number of fluorescent cells 293 / GFP Cell Line (Cell Biolabs, Cat. AKR-200) after combined transfection of these cells with the VTvaf17-Cas9 DNA vector carrying the Cas9 gene and the GFP-targeting guide RNA for CRISPR (Genaxxon bioscience, Cat. P2008.0010) in order to assess the functional activity, that is, the expression of the target gene at the protein level and the ability of this protein to carry out targeted endonuclease activity with the participation of a specific gRNA, which leads to editing the DNA sequence.

In Fig. 11, the following elements are marked:

culture A - 293 / GFP cells transfected with water, followed by transfection with GFP-targeting guide RNA for CRISPR;

culture B - 293 / GFP cells transfected with the VTvaf17 DNA vector, which does not contain the Cas9 gene cDNA, followed by transfection with the GFP targeting guide RNA for CRISPR;

culture C - 293 / GFP cells transfected with the VTvaf17-Cas9 DNA vector carrying the Cas9 gene, followed by transfection with the GFP targeting guide RNA for CRISPR;

culture D - 293 / GFP cells transfected with the VTvaf17-Cas9 DNA vector carrying the Cas9 gene, followed by transfection with water.

FIG. 12

Shown are the results of PCR sequencing of fragments of several clones of the MCF-7 lung adenocarcinoma cell line (ATCC® HTB-22 ™), in which the genome was edited after transfection with VTvaf17-Cas9 carrying the Cas9 gene, with the gRNA_Cloning Vector carrying the gRNA sequences of the TLR9 gene.

The figure shows the DNA region corresponding to the region of the double-stranded break introduced by sgRNA TLR_1 and TLR_2, where Tlr12 is the consensus sequence; WT - the sequence of the TLR9 gene fragment not subjected to genomic editing or with the recovered sequence; al1, al2, G2 are different alleles in heterozygous clones (both alleles are shown in the diagram).

Implementation of the invention

Based on the DNA vector VTvaf17 with a size of 3165 bp. a gene therapy DNA vector carrying the target Cas9 gene, designed for heterologous expression of this target gene in human and animal cells, has been created. The method of obtaining a gene therapy DNA vector carrying the target gene consists in the fact that the protein-coding sequence of the target gene Cas9 (encodes the endonuclease Cas9) is cloned into the polylinker of the gene therapy DNA vector VTvaf17. It is known that the ability of DNA vectors to penetrate into eukaryotic cells is mainly determined by the size of the vector. In this case, DNA vectors with the smallest size have a higher penetrating ability. Thus, it is preferable that the vector contains no elements that do not carry a functional load, but at the same time increase the size of the DNA vector. These features of DNA vectors were taken into account when obtaining a gene therapy DNA vector based on a gene therapy DNA vector VTvaf17 carrying the target Cas9 gene, due to the absence of large non-functional sequences and antibiotic resistance genes in the vector, which made it possible, in addition to technological advantages and advantages in terms of safety of use. , significantly reduce the size of the resulting gene therapy DNA vector VTvaf17 carrying the target Cas9 gene. Thus, the ability to penetrate into eukaryotic cells of the obtained gene therapy DNA vector is due to its small size.

The gene therapy DNA vector VTvaf17-Cas9 was obtained as follows: the coding part of the target Cas9 gene was cloned into the gene therapy DNA vector VTvaf17 and the gene therapy DNA vector VTvaf17-Cas9, SEQ ID NO. The coding part of the Cas9 gene is 4221 bp. obtained by enzymatic synthesis from chemically synthesized oligonucleotides. Cleavage of the synthesis product with specific restriction endonucleases was carried out taking into account the optimal procedure for further cloning, and cloning into the gene therapeutic DNA vector VTvaf17 was carried out at the restriction sites BamHI, EcoRI located in the polylinker of the VTvaf17 vector. The choice of restriction sites was carried out so that the cloned fragment fell into the reading frame of the expression cassette of the VTvaf17 vector, while the protein-coding sequence did not contain restriction sites for the selected endonucleases. At the same time, specialists in this field of technology understand that the methodical implementation of obtaining a gene therapeutic DNA vector VTvaf17-Cas9 can vary within the selection of known methods of molecular cloning of genes, and these methods fall within the scope of the present invention. For example, different sequences of oligonucleotides can be used to amplify the Cas9 gene, different restriction endonucleases, or laboratory techniques such as ligase-free gene cloning.

Gene therapy DNA vector VTvaf17-Cas9 has the nucleotide sequence SEQ ID No. 1. At the same time, those skilled in the art are aware of the degeneracy property of the genetic code, from which it follows that the scope of the present invention also includes variants of nucleotide sequences differing by insertion, deletion or substitution of nucleotides that do not lead to a change in the polypeptide sequence encoded by the target gene, and / or do not lead to a loss of functional activity of the regulatory elements of the VTvaf17 vector. At the same time, specialists in this field of technology know the phenomenon of genetic polymorphism, from which it follows that the scope of the present invention also includes variants of the nucleotide sequences of the Cas9 gene, which at the same time encode various variants of the amino acid sequences of the Cas9 protein, which do not differ from those given in their functional activity in physiological conditions.

The ability to penetrate into eukaryotic cells and functional activity, that is, the ability to express the target gene, of the obtained gene therapeutic DNA vector VTvaf17-Cas9 is confirmed by introducing the obtained vector into eukaryotic cells and subsequent analysis of the expression of specific mRNA and / or protein product of the target gene. The presence of specific mRNA in cells into which the gene therapy DNA vector VTvaf17-Cas9 was introduced indicates both the ability of the resulting vector to penetrate into eukaryotic cells and its ability to express mRNA of the target Cas9 gene. Moreover, as is known to those skilled in the art, the presence of the mRNA of the gene is a prerequisite, but not proof of translation of the protein encoded by the target gene. Therefore, to confirm the property of the gene therapy DNA vector VTvaf17-Cas9 to express the target gene at the protein level in eukaryotic cells into which the gene therapy DNA vector was introduced, the concentration of the protein encoded by the target gene is analyzed using immunological methods. The presence of the Cas9 protein confirms the efficiency of expression of the target gene in eukaryotic cells using a gene therapy DNA vector based on the gene therapy DNA vector VTvaf17 carrying the target Cas9 gene. Thus, to confirm the efficiency of expression of the created gene therapy DNA vector VTvaf17-Cas9 carrying the target gene, namely the Cas9 gene, the following methods were used:

A) Real-time PCR - changing the accumulation of cDNA amplicons of the target gene in the lysate of human and animal cells, after transfection of various human and animal cell lines with a gene therapeutic DNA vector;

B) Enzyme-linked immunosorbent assay - a change in the quantitative level of the target protein in the lysate of human and animal cells after transfection of various human and animal cell lines with a gene therapy DNA vector;

C) Enzyme-linked immunosorbent assay - a change in the quantitative level of the target protein in the supernatant of biopsies of animal tissues, after the introduction into these tissues of autologous cells of this animal, transfected with a gene therapeutic DNA vector;

D) Measurement by flow fluorometry of the expression of the GFP gene in cells subjected to genomic editing after combined transfection of these cells with the gene therapeutic DNA vector VTvaf17-Cas9 and gRNA, which led to inactivation of the GFP gene and the absence of expression of fluorescent protein in cells or its significant decrease;

F) Sequencing of a DNA segment of human or animal cells that underwent genomic editing after combined transfection of these cells with a VTvaf17-Cas9 gene therapy DNA vector and a gRNA expression vector directed to the TLR9 gene sequence.

To confirm the feasibility of the method of using the created gene therapy DNA vector VTvaf17-Cas9 carrying the target gene, namely, the Cas9 gene, we performed:

A) transfection with a gene therapy DNA vector of various human and animal cell lines;

B) combined transfection with a gene therapeutic DNA vector and gRNA of various mammalian cell lines;

C) combined transfection with a gene therapy DNA vector and a vector encoding gRNA of various mammalian cell lines;

D) introducing autologous cells transfected with a gene therapy DNA vector into animal tissues;

E) demonstration of changes in the expression of the edited gene in a cell line subjected to the genomic editing procedure;

F) Demonstration of changing the sequence of a region of the edited gene in a cell line subjected to genomic editing.

These methods of application are characterized by the absence of potential risks for genetic therapy of humans and animals due to the absence of regulatory elements in the composition of the gene therapeutic DNA vector, which are the nucleotide sequences of viral genomes, and due to the absence of genes for antibiotic resistance in the composition of the gene therapeutic DNA vector, which is confirmed by the absence of regions homologous to viral genomes and antibiotic resistance genes in the nucleotide sequence of the gene therapy DNA vector VTvaf17-Cas9 (SEQ ID No. 1).

As is known to those skilled in the art, antibiotic resistance genes in gene therapy DNA vectors are used to obtain these vectors in preparative quantities by increasing bacterial biomass in a nutrient medium containing a selective antibiotic. Within the framework of the present invention, in order to safely use the VTvaf17 gene therapeutic DNA vector carrying the target Cas9 gene, it is not possible to use selective culture media containing the antibiotic. As a technological solution for obtaining a gene therapy DNA vector VTvaf17 carrying the target Cas9 gene, for the possibility of scaling up to an industrial scale for obtaining a gene therapy vector, a method is proposed for obtaining a strain for the production of these gene therapy vectors based on the bacterium Escherichia coli SCS110-AF. The method of obtaining the Escherichia coli strain SCS110-AF / VTvaf17-Cas9 consists in obtaining competent cells of the Escherichia coli strain SCS110-AF with the introduction into these cells of the gene therapeutic DNA vector VTvaf17-Cas9 using transformation methods (electroporation) well known to those skilled in the art. The resulting Escherichia coli SCS110-AF / VTvaf17-Cas9 strain is used to develop a gene therapy DNA vector VTvaf17-Cas9 with the possibility of using media without antibiotics.

To confirm the receipt of the Escherichia coli strain SCS110-AF / VTvaf17-Cas9, transformation, selection and subsequent growth with the isolation of plasmid DNA were carried out.

To confirm the manufacturability of obtaining and the possibility of scaling up to industrial production of the gene therapy DNA vector VTvaf17-Cas9 carrying the target gene, namely the Cas9 gene, the Escherichia coli strain SCS110-AF / VTvaf17-Cas9, which contains the gene therapy DNA vector, was fermented on an industrial scale. VTvaf17 carrying the target Cas9 gene.

The method of scaling up the production of bacterial mass to an industrial scale for the isolation of the gene therapy DNA vector VTvaf17 carrying the target gene, Cas9, is that the seed culture of the Escherichia coli SCS110-AF / VTvaf17-Cas9 strain is incubated in a volume of culture medium without antibiotic content, which provides suitable dynamics accumulation of biomass, upon reaching a sufficient amount of biomass in the logarithmic phase of growth, the bacterial culture is transferred to an industrial fermenter, after which it is grown until the stationary growth phase is reached, then a fraction containing the target DNA product is isolated - the gene therapy DNA vector VTvaf17-Cas9 is filtered and purified in many stages chromatographic methods. At the same time, it is clear to those skilled in the art that the cultivation conditions of the strains, the composition of the nutrient media (excluding the content of antibiotics), the equipment used, the DNA purification methods may vary within the framework of standard operating procedures, depending on the individual production line, but known approaches to scaling , industrial production and purification of DNA vectors using Escherichia coli strain SCS110-AF / VTvaf17-Cas9 fall within the scope of the present invention.

The described disclosure of the invention is confirmed by examples of implementation of the present invention.

The invention is illustrated by the following examples.

Example 1 .

Obtaining a gene therapy DNA vector VTvaf17-Cas9 carrying the target gene, namely the Cas9 gene.

The gene therapy DNA vector VTvaf17-Cas9 was constructed by cloning the 4221 bp coding part of the Cas9 gene. into the DNA vector VTvaf17 with a size of 3165 bp. at restriction sites BamHI and EcoRI. The coding part of the Cas9 gene is 4221 bp. obtained by enzymatic synthesis from chemically synthesized oligonucleotides.

Gene therapy DNA vector VTvaf17 was constructed by combining six DNA fragments obtained from different sources:

(a) the origin of replication was obtained by PCR amplification of a portion of the commercial plasmid pBR322 with the introduction of a point mutation;

(b) the EF1a promoter region was obtained by PCR amplification of a region of human genomic DNA;

(c) the hGH-TA transcriptional terminator was obtained by PCR amplification of a region of human genomic DNA;

(d) the regulatory region of the Tn10 transposon RNA-out was obtained by synthesis from oligonucleotides;

(e) a kanamycin resistance gene was obtained by PCR amplification of a portion of the commercial human plasmid pET-28;

(f) a polylinker was obtained by annealing two synthetic oligonucleotides.

PCR amplification was performed using a commercial Phusion® High-Fidelity DNA Polymerase kit (New England Biolabs, USA) according to the manufacturer's instructions. Fragments have overlapping regions for the possibility of combining them with subsequent PCR amplification. Fragments (a) and (b) were combined using oligonucleotides Ori-F and EF1-R, as well as fragments (c), (d), and (e) using oligonucleotides hGH-F and Kan-R. Then the resulting regions were combined by restriction, followed by ligation at the BamHI and NcoI sites. The result was a plasmid that does not yet contain a polylinker. For its introduction, the plasmid was cleaved at the BamHI and EcoRI sites, and ligated to fragment (e). Thus, a vector of 4182 bp was obtained, carrying the kanamycin resistance gene, which is flanked by SpeI restriction sites. Further, this region was cut at the SpeI restriction sites, after which the remaining fragment was ligated onto itself. Thus, a gene therapy DNA vector VTvaf17 with a size of 3165 bp was obtained, which is recombinant, with the possibility of selection without antibiotics.

Cleavage of the amplification product of the coding region of the Cas9 gene and the VTvaf17 DNA vector was performed with restriction endonucleases BamHI and EcoRI (New England Biolabs, USA).

As a result, a 7351 bp DNA vector VTvaf17-Cas9 was obtained. with the nucleotide sequence SEQ ID No. 1 and the general structure depicted in figure 1.

Example 2.

Confirmation of the ability of the gene therapy DNA vector VTvaf17-Cas9 carrying the target gene, namely, the Cas9 gene, to penetrate into eukaryotic cells and confirmation of its functional activity at the level of mRNA expression of the target gene. This example also demonstrates the feasibility of a method for using a gene therapy DNA vector carrying the target gene.

Changes in the accumulation of mRNA of the target Cas9 gene were evaluated in the primary culture of human skin fibroblasts HDFa (ATCC PCS-201-01) 48 hours after their transfection with the VTvaf17-Cas9 gene therapeutic DNA vector carrying the human Cas9 gene. The amount of mRNA was determined by the dynamics of accumulation of cDNA amplicons in the real-time PCR reaction.

To assess changes in the accumulation of mRNA of the target Cas9 gene, we used a primary culture of human skin fibroblasts HDFa. HDFa cell culture were grown under standard conditions (37 ° С, 5% СО2) using a Fibroblast Growth Kit – Serum-Free culture medium (ATCC® PCS-201-040). During cultivation, the growth medium was changed every 48 h.

To obtain 90% confluence, 24 hours before transfection, the cells were seeded in a 24-well plate at the rate of 5 × 10⁴cells / well. Transfection with the VTvaf17-Cas9 gene therapy DNA vector expressing the human Cas9 gene was performed using Lipofectamine 3000 (ThermoFisher Scientific, USA) according to the manufacturer's recommendations. In test tube No. 1, 1 μL of a solution of the VTvaf17-Cas9 DNA vector (concentration 500 ng / μL) and 1 μL of P3000 reagent were added to 25 μl of Opti-MEM medium (Gibco, USA). Mix gently with gentle shaking. In test tube No. 2, 1 μl of Lipofectamin 3000 solution was added to 25 μl of Opti-MEM medium (Gibco, USA). Mix gently with gentle shaking. The contents of tube No. 1 were added to the contents of tube No. 2, incubated for 5 min at room temperature. The resulting solution was added dropwise to the cells in a volume of 40 μl.

As a control, HDFa cells transfected with the gene therapy DNA vector VTvaf17, which did not contain the insert of the target gene, were used (cDNA of the Cas9 gene before and after transfection with the gene therapy DNA vector VTvaf17, which did not contain the insert of the target gene, not shown in the figures). The preparation of the control vector VTvaf17 for transfection was performed as described above.

Total RNA from HDFa cells was isolated using Trizol Reagent (Invitrogen, USA) according to the manufacturer's recommendations. 1 ml of Trizol Reagent was added to a well with cells and homogenized, followed by heating for 5 min at 65 ° C. Then the sample was centrifuged at 14000g for 10 min and heated again for 10 min at 650C. Then, 200 μL of chloroform was added, mixed smoothly, and centrifuged at 14000g for 10 min. Then the aqueous phase was taken, 1/10 volume of 3M sodium acetate, pH 5.2 and an equal volume of isopropyl alcohol were added to it. The sample was incubated at -20 ° C for 10 min, followed by centrifugation at 14000g for 10 min. The precipitate was washed with 1 ml of 70% ethanol, dried in air, and dissolved in 10 μl of RNase-free water. The expression level of Cas9 gene mRNA after transfection was determined by assessing the dynamics of accumulation of cDNA amplicons using real-time PCR. To obtain and amplify cDNA specific for the human Cas9 gene, we used the Cas9_SF and Cas9_SR oligonucleotides (sequence listing (1), (2)).

Amplification product length - 275 bp.

The reverse transcription reaction and PCR amplification were performed using the SYBR GreenQuantitect RT – PCR Kit (Qiagen, USA) for real-time PCR. The reaction was carried out in a volume of 20 μl containing: 25 μl of QuantiTect SYBR Green RT-PCR MasterMix, 2.5 mM magnesium chloride, 0.5 μM of each primer, 5 μl of RNA. The reaction was carried out on a CFX96 amplifier (Bio-Rad, USA) under the following conditions: 1 cycle of reverse transcription at 42 ° C for 30 minutes, denaturation at 98 ° C for 15 min, then 40 cycles, including denaturation at 94 ° C for 15 sec, annealing primers 60 ° C - 30 sec and elongation 72 ° C - 30 sec. The B2M gene (Beta-2-microglobulin) listed in the GenBank database under the number NM 004048.2 was used as a reference gene. As a positive control, we used amplicons obtained by PCR on templates, which are plasmids in known concentrations containing the cDNA sequences of the Cas9 and B2M genes. Deionized water was used as a negative control. The dynamics of accumulation of cDNA amplicons of the Cas9 and B2M genes was assessed in real time using the Bio-RadCFXManager 2.1 thermocycler software (Bio-Rad, USA). The analysis plots are shown in FIG. 2.

From figure 2 it follows that as a result of transfection of the primary culture of human fibroblast cells HDFa with the gene therapy DNA vector VTvaf17-Cas9, the presence of mRNA of the Cas9 gene was established, which confirms the ability of the vector to penetrate into eukaryotic cells and express the Cas9 gene at the mRNA level. The presented results also confirm the feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 for heterologous expression of the Cas9 gene in eukaryotic cells.

Example 3.

Confirmation of the ability of the gene therapy DNA vector VTvaf17-Cas9 carrying the target gene, namely, the Cas9 gene, to penetrate into eukaryotic cells and confirmation of its functional activity at the level of mRNA expression of the target gene. This example also demonstrates the feasibility of a method for using a gene therapy DNA vector carrying the target gene.

Changes in the accumulation of mRNA of the target Cas9 gene in the primary culture of keratinocytes of the human epidermis HEKa (ATCC PCS-200-011) were evaluated 48 hours after their transfection with the gene therapy DNA vector VTvaf17-Cas9 carrying the human Cas9 gene. The amount of mRNA was determined by the dynamics of accumulation of cDNA amplicons in the real-time PCR reaction.

A primary culture of HEKa human epidermal keratinocytes was grown in Keratinocyte Growth Kit (ATCC® PCS-200-040 ™) medium under standard conditions (37 ° C, 5% CO2). To obtain 90% confluence, 24 hours prior to transfection, cells were seeded in a 24-well plate at a rate of 5 × 10 ⁴ cells / well. Lipofectamine 3000 reagent (ThermoFisher Scientific, USA) was used for transfection. Transfection with the VTvaf17-Cas9 gene therapy DNA vector expressing the human Cas9 gene was performed as described in example 2. The B2M gene (Beta-2-microglobulin) listed in the GenBank database under the number NM 004048.2 was used as a reference gene. As a control, we used a culture of HEKa cells transfected with the VTvaf17 gene therapy DNA vector, which does not carry the target gene (cDNA of the Cas9 gene before and after transfection with the VTvaf17 gene therapy DNA vector, which does not contain the target gene insert is not shown in the figures). RNA isolation, reverse transcription reaction and real-time PCR were performed as described in example 2.

As a positive control, we used amplicons obtained by PCR on templates, which are plasmids in known concentrations containing the cDNA sequences of the Cas9 and B2M genes. Deionized water was used as a negative control. Number of PCR products - cDNA of Cas9 genes and B2M obtained as a result of amplification were evaluated in real time using the Bio-RadCFXManager 2.1 thermocycler software (Bio-Rad, USA). The analysis plots are shown in FIG. 3.

From figure 3 it follows that as a result of transfection of HEKa cell culture with the gene therapy DNA vector VTvaf17-Cas9, the presence of mRNA of the Cas9 gene was established, which confirms the ability of the vector to penetrate into eukaryotic cells and express the Cas9 gene at the mRNA level. The presented results also confirm the feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 for heterologous expression of the Cas9 gene in eukaryotic cells.

Example 4.

Confirmation of the ability of the gene therapy DNA vector VTvaf17-Cas9 carrying the target gene, namely, the Cas9 gene, to penetrate into eukaryotic cells and confirmation of its functional activity at the level of mRNA expression of the target gene. This example also demonstrates the feasibility of a method for using a gene therapy DNA vector carrying the target gene.

Changes in the accumulation of mRNA of the target Cas9 gene in the cell culture of human epidermal melanocytes of the Primary Epidermal Melanocytes line were evaluated; Normal, Human, Adult (HEMa) (ATCC® PCS-200-013 ™) 48 hours after their transfection with a gene therapy DNA vector VTvaf17-Cas9 carrying the human Cas9 gene. The amount of mRNA was determined by the dynamics of accumulation of cDNA amplicons in the real-time PCR reaction.

A culture of human epidermal melanocytes of the Primary Epidermal Melanocytes line was grown in a medium prepared using the Adult Melanocyte Growth Kit (ATCC® PCS-200-042 ™) under standard conditions (37 ° C, 5% CO2). To obtain 90% confluence, 24 hours before transfection, cells were seeded in a 24-well plate at a rate of 5 × 10 ⁴ cells / well. Lipofectamine 3000 reagent (ThermoFisher Scientific, USA) was used for transfection. Transfection with the VTvaf17-Cas9 gene therapeutic DNA vector expressing the human Cas9 gene was performed as described in example 2. The B2M gene (Beta-2 microglobulin) listed in the GenBank database under the number NM 004048.2 was used as a reference gene. A culture of HEMa human epidermal melanocytes cells transfected with the VTvaf17 gene therapeutic DNA vector, which does not carry the target gene (cDNA of the Cas9 gene before and after transfection with the VTvaf17 gene therapeutic DNA vector, which does not contain the insert of the target gene, not shown in the figures), was used as a control. RNA isolation, reverse transcription reaction and real-time PCR were performed as described in example 2.

As a positive control, we used amplicons obtained by PCR on templates, which are plasmids in known concentrations containing the cDNA sequences of the Cas9 and B2M genes. Deionized water was used as a negative control. Number of PCR products - cDNA of Cas9 genes and B2M obtained as a result of amplification were evaluated in real time using the Bio-RadCFXManager 2.1 thermocycler software (Bio-Rad, USA). The analysis plots are shown in FIG. 4.

From figure 4 it follows that as a result of transfection of the culture of cells of melanocytes of the human epidermis of the HEMa line with the gene therapy DNA vector VTvaf17-Cas9, the presence of mRNA of the Cas9 gene was established, which confirms the ability of the vector to penetrate into eukaryotic cells and express the Cas9 gene at the mRNA level. The presented results also confirm the feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 for heterologous expression of the Cas9 gene in eukaryotic cells.

Example 5.

Confirmation of the efficiency and feasibility of the method of using the VTvaf17-Cas9 gene therapy DNA vector carrying the Cas9 gene for heterologous expression of the Cas9 protein in mammalian cells.

The change in the amount of Cas9 protein in the lysate of cells of the primary culture of human skin fibroblasts HDFa (ATCC PCS-201-01) after transfection of these cells with the VTvaf17-Cas9 DNA vector carrying the human Cas9 gene was evaluated. The cells were grown as described in example 2.

The 6th generation SuperFect Transfection Reagent (Qiagen, Germany) was used for transfection. An aqueous solution of dendrimers without a DNA vector (A), a DNA vector VTvaf17 that does not contain the cDNA of the Cas9 gene (B) were used as controls, and a DNA vector VTvaf17-Cas9 carrying the human Cas9 gene (C) was used as transfected agents. Transfection of HDFa cells was performed as described in example 2.

After transfection, 0.1 ml of 1N HCl was added to 0.5 ml of the culture fluid, mixed thoroughly and incubated for 10 minutes at room temperature. Then the mixture was neutralized by adding 0.1 ml of 1.2M NaOH / 0.5M HEPES (pH 7-7.6) and mixed thoroughly. The supernatant was taken and used to quantify the target protein. Quantitative determination of the Cas9 protein was carried out by the method of enzyme-linked immunosorbent assay (ELISA) using the Cas9 (CRISPR Associated Protein 9) ELISA Kit (Cell Biolabs Inc, Cat. PRB-5079) according to the manufacturer's procedure with optical density detection using an automatic biochemical and enzyme-linked immunosorbent assay ChemWell (Awareness Technology Inc., USA).

The numerical value of the concentration was determined using a calibration curve constructed using standard samples with a known concentration of Cas9 protein included in the kit. The sensitivity of the method was no less than 1.5 ng / ml, the measurement range was from 1.56 ng / ml to 100 ng / ml. Statistical processing of the results was carried out using software for statistical processing and data visualization R, version 3.0.2 (https://www.r-project.org/). The analysis plots are shown in FIG. five.

From figure 5 it follows that as a result of transfection of the primary culture of human HDFa cells with the gene therapy DNA vector VTvaf17-Cas9, the presence of the Cas9 protein was found in comparison with the absence of such in control samples, which confirms the ability of the vector to penetrate into eukaryotic cells and express the Cas9 gene at the protein level. The presented results also confirm the feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 for heterologous expression of Cas9 in eukaryotic cells.

Example 6.

Confirmation of the efficiency and feasibility of the method of using the VTvaf17-Cas9 gene therapy DNA vector carrying the Cas9 gene for heterologous expression of the Cas9 protein in mammalian cells.

The change in the amount of Cas9 protein in the lysate of cells from the primary culture of human epidermal keratinocytes HEKa (ATCC PCS-200-011) after transfection of these cells with the VTvaf17-Cas9 DNA vector carrying the human Cas9 gene was evaluated. The cells were grown as described in example 3.

The 6th generation SuperFect Transfection Reagent (Qiagen, Germany) was used for transfection. An aqueous solution of dendrimers without a DNA vector (A), a DNA vector VTvaf17 that does not contain the cDNA of the Cas9 gene (B) were used as controls, and a DNA vector VTvaf17-Cas9 carrying the human Cas9 gene (C) was used as transfected agents. The DNA-dendrimer complex was prepared and the cells were transfected according to the manufacturer's procedure.

After transfection, 0.1 ml of 1N HCl was added to 0.5 ml of the culture fluid, mixed thoroughly and incubated for 10 minutes at room temperature. Then the mixture was neutralized by adding 0.1 ml of 1.2M NaOH / 0.5M HEPES (pH 7-7.6) and mixed thoroughly. The supernatant was taken and used to quantify the target protein. Quantitative determination of the Cas9 protein was carried out by ELISA using the Cas9 (CRISPR Associated Protein 9) ELISA Kit (Cell Biolabs Inc, Cat.PRB-5079) according to the manufacturer's method with optical density detection using an automatic biochemical and enzyme-linked immunosorbent assay ChemWell (Awareness Technology Inc., USA).

The numerical value of the concentration was determined using a calibration curve constructed using standard samples with a known concentration of Cas9 protein included in the kit. The sensitivity of the method was no less than 1.5 ng / ml, the measurement range was from 1.56 ng / ml to 100 ng / ml. Statistical processing of the results was carried out using software for statistical processing and data visualization R, version 3.0.2 (https://www.r-project.org/). The analysis plots are shown in FIG. 6.

From figure 6 it follows that as a result of transfection of the primary culture of keratinocytes of the human epidermis HEKa with the gene therapy DNA vector VTvaf17-Cas9, the presence of the Cas9 protein was established in comparison with the absence thereof in the control samples, which confirms the ability of the vector to penetrate into eukaryotic cells and express the Cas9 gene on protein level. The presented results also confirm the feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 for heterologous expression of Cas9 in eukaryotic cells.

Example 7.

Confirmation of the efficiency and feasibility of the method of using the VTvaf17-Cas9 gene therapy DNA vector carrying the Cas9 gene for heterologous expression of the Cas9 protein in mammalian cells.

The change in the amount of Cas9 protein in the lysate of cells of the primary culture of melanocytes of the human epidermis, Primary Epidermal Melanocytes, was assessed; Normal, Human, Adult (HEMa) (ATCC® PCS-200-013 ™) after transfection of these cells with a VTvaf17-Cas9 DNA vector carrying the human Cas9 gene. The cells were grown as described in example 4.

The 6th generation SuperFect Transfection Reagent (Qiagen, Germany) was used for transfection. An aqueous solution of dendrimers without a DNA vector (A), a DNA vector VTvaf17 that does not contain the cDNA of the Cas9 gene (B) were used as controls, and a DNA vector VTvaf17-Cas9 carrying the human Cas9 gene (C) was used as transfected agents. Preparation of the DNA-dendrimer complex and transfection of HEMa cells were performed according to the manufacturer's procedure.

After transfection, 0.1 ml of 1N HCl was added to 0.5 ml of the culture fluid, mixed thoroughly and incubated for 10 minutes at room temperature. Then the mixture was neutralized by adding 0.1 ml of 1.2M NaOH / 0.5M HEPES (pH 7-7.6) and mixed thoroughly. The supernatant was taken and used to quantify the target protein. Quantitative determination of the Cas9 protein was carried out by the method of enzyme-linked immunosorbent assay (ELISA) using the Cas9 (CRISPR Associated Protein 9) ELISA Kit (Cell Biolabs Inc, Cat. PRB-5079) according to the manufacturer's procedure with optical density detection using an automatic biochemical and enzyme-linked immunosorbent assay ChemWell (Awareness Technology Inc., USA).

The numerical value of the concentration was determined using a calibration curve constructed using standard samples with a known concentration of Cas9 protein included in the kit. The sensitivity of the method was no less than 1.5 ng / ml, the measurement range was from 1.56 ng / ml to 100 ng / ml. Statistical processing of the results was carried out using software for statistical processing and data visualization R, version 3.0.2 (https://www.r-project.org/). The analysis plots are shown in FIG. 7.

From figure 7 it follows that as a result of transfection of the primary culture of human HEMa cells with the gene therapy DNA vector VTvaf17-Cas9, the presence of the Cas9 protein was found in comparison with the absence of such in the control samples, which confirms the ability of the vector to penetrate into eukaryotic cells and express the Cas9 gene at the protein level. The presented results also confirm the feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 for heterologous expression of Cas9 in eukaryotic cells.

Example 8.

Confirmation of the effectiveness and feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene for heterologous expression of the Cas9 protein in animal cells.

The change in the amount of Cas9 protein in the lysate of Syrian hamster ovary cells CHO-K1 (ATCC® CCL-61 ™) after transfection of these cells with the VTvaf17-Cas9 DNA vector carrying the human Cas9 gene was evaluated. Cells were grown in F-12K Medium (Kaighn's Modification of Ham's F-12 Medium) (ATCC® 30-2004 ™) supplemented with 10% Fetal Bovine Serum (FBS) (ATCC® 30-2020 ™) under standard conditions.

The 6th generation SuperFect Transfection Reagent (Qiagen, Germany) was used for transfection. An aqueous solution of dendrimers without a DNA vector (A), a DNA vector VTvaf17 that does not contain the cDNA of the Cas9 gene (B) were used as controls, and a DNA vector VTvaf17-Cas9 carrying the human Cas9 gene (C) was used as transfected agents. The DNA-dendrimer complex was prepared and the cells were transfected according to the manufacturer's procedure.

After transfection, 0.1 ml of 1N HCl was added to 0.5 ml of the cell suspension, mixed thoroughly and incubated for 10 minutes at room temperature. Then the mixture was neutralized by adding 0.1 ml of 1.2M NaOH / 0.5M HEPES (pH 7-7.6) and mixed thoroughly. The supernatant was taken and used to quantify the target protein. Quantitative determination of the Cas9 protein was carried out by the method of enzyme-linked immunosorbent assay (ELISA) using the Cas9 (CRISPR Associated Protein 9) ELISA Kit (Cell Biolabs Inc, Cat. PRB-5079) according to the manufacturer's procedure with optical density detection using an automatic biochemical and enzyme-linked immunosorbent assay ChemWell (Awareness Technology Inc., USA).

The numerical value of the concentration was determined using a calibration curve constructed using standard samples with a known concentration of Cas9 protein included in the kit. The sensitivity of the method was no less than 1.5 ng / ml, the measurement range was from 1.56 ng / ml to 100 ng / ml. Statistical processing of the results was carried out using software for statistical processing and data visualization R, version 3.0.2 (https://www.r-project.org/). The analysis plots are shown in FIG. 8.

From figure 8 it follows that as a result of transfection of the CHO-K1 cell culture with the gene therapy DNA vector VTvaf17-Cas9, the presence of Cas9 protein was found in comparison with the absence of it in the control samples, which confirms the ability of the vector to penetrate into eukaryotic animal cells and express the Cas9 gene at the protein level ... The presented results also confirm the feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 for heterologous expression of Cas9 in eukaryotic animal cells.

Example 9.

Confirmation of the effectiveness and feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene for heterologous expression of the Cas9 protein in human cells.

The change in the amount of Cas9 protein in the lysate of the culture of primary human peripheral blood mononuclear cells (PBMC) after transfection of these cells with the VTvaf17-Cas9 DNA vector carrying the human Cas9 gene was evaluated. PBMC cells were isolated from 10 ml of human venous blood by fractionation in a Ficoll gradient 1.119 (PanEko, P051-1) The cells were grown in RPMI-1640 medium (PanEko, C310n) under standard conditions (37C, 5% CO2).

The 6th generation SuperFect Transfection Reagent (Qiagen, Germany) was used for transfection. An aqueous solution of dendrimers without a DNA vector (A), a DNA vector VTvaf17 that does not contain the cDNA of the Cas9 gene (B) were used as controls, and a DNA vector VTvaf17-Cas9 carrying the human Cas9 gene (C) was used as transfected agents. The DNA-dendrimer complex was prepared and the cells were transfected according to the manufacturer's procedure.

24 hours after transfection, 1 ml of cell culture was precipitated by centrifugation, 0.1 ml of 1N HCl was added to the cell pellet, mixed thoroughly and incubated for 10 minutes at room temperature. Then the mixture was neutralized by adding 0.1 ml of 1.2M NaOH / 0.5M HEPES (pH 7-7.6) and mixed thoroughly. The supernatant was taken and used to quantify the target protein. Quantitative determination of the Cas9 protein was carried out by the method of enzyme-linked immunosorbent assay (ELISA) using the Cas9 (CRISPR Associated Protein 9) ELISA Kit (Cell Biolabs Inc, Cat. PRB-5079) according to the manufacturer's procedure with optical density detection using an automatic biochemical and enzyme-linked immunosorbent assay ChemWell (Awareness Technology Inc., USA).

The numerical value of the concentration was determined using a calibration curve constructed using standard samples with a known concentration of Cas9 protein included in the kit. The sensitivity of the method was no less than 1.5 ng / ml, the measurement range was from 1.56 ng / ml to 100 ng / ml. Statistical processing of the results was carried out using software for statistical processing and data visualization R, version 3.0.2 (https://www.r-project.org/). The analysis plots are shown in FIG. nine.

From figure 9 it follows that as a result of transfection of PBMC cell culture with the gene therapy DNA vector VTvaf17-Cas9, the presence of Cas9 protein was established in comparison with the absence of such protein in control samples, which confirms the ability of the vector to penetrate into human cells and express the Cas9 gene at the protein level. The presented results also confirm the feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 for the heterologous expression of Cas9 in eukaryotic mammalian cells.

Example 10.

Confirmation of the efficacy of the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene and the feasibility of its application to increase the expression level of the Cas9 protein in animal tissues by introducing autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17-Cas9.

To confirm the efficacy of the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene and the feasibility of the method of its application, the changes in the amount of Cas9 protein in the skin of rats were assessed when a culture of autologous fibroblasts of this animal transfected with the gene therapeutic DNA vector VTvaf17-Cas9 was injected into the skin of rats.

Three Wistar rats were injected into the skin with an appropriate culture of autologous fibroblasts transfected with the VTvaf17-Cas9 gene therapeutic DNA vector carrying the Cas9 gene, and in parallel were injected with placebo, which was a culture of autologous animal fibroblasts transfected with the VTvaf17 gene therapeutic DNA vector not carrying the Cas9 gene.

The primary culture of rat fibroblasts was isolated from biopsies of animal skin. Using an Epitheasy 3.5 skin biopsy device (Medax SRL, Italy), a skin biopsy sample weighing about 11 mg was taken. Cultivation of the primary cell culture was carried out at 37 ° C in an atmosphere containing 5% CO2 in DMEM medium with 10% fetal calf serum and ampicillin 100 U / ml. Subculture of culture and change of culture medium was carried out every 2 days. The total duration of culture growth did not exceed 25-30 days. An aliquot containing 5 × 10 ⁴ cells was taken from the cell culture. The culture of rat fibroblasts was transfected with the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene or placebo - the vector VTvaf17 not carrying the target Cas9 gene.

Transfection was carried out using a cationic polyethyleneimine polymer JETPEI (Polyplus Transfection, France) according to the manufacturer's instructions. The cells were cultured for 72 hours and then injected into the animals. The introduction to the animal of a culture of autogenous fibroblasts transfected with the gene therapy DNA vector VTvaf17-Cas9 and placebo, which is a culture of autologous rat fibroblasts transfected with the gene therapy DNA vector VTvaf17, was carried out by the tunnel method into the area of pre-epilated skin with a 27G needle to a depth of about 1 mm. The concentration of modified autologous fibroblasts in the injected suspension was approximately 5 million cells per 1 ml of suspension, the number of injected cells did not exceed 10 million. The foci of injected culture of autogenous fibroblasts were located at a distance of 3 - 5 cm from each other.

Biopsy samples were taken on the 4th day after the introduction of the culture of autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17-Cas9 carrying the target gene, namely the Cas9 gene and placebo. Biopsies were taken from the skin areas of animals in the zone of administration of the culture of autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17-Cas9 carrying the target gene, namely the Cas9 (I) gene, cultures of autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17, which did not carry the target Cas9 gene (placebo) (II), as well as from areas of intact skin (III), using an Epitheasy 3.5 biopsy device (Medax SRL, Italy). The biopsy sample size was about 10 cc. mm, weight - about 11 mg. The sample was placed in a buffer solution containing 50 mM Tris-HCl pH 7.6, 100 mM NaCl, 1 mM EDTA, and 1 mM phenylmethylsulfonyl fluoride, and homogenized until a homogeneous suspension was obtained. The resulting suspension was centrifuged for 10 min at 14000g. The supernatant was collected and used to quantify the target protein as described in Examples 5-9.

The analysis diagrams are shown in FIG. ten.

From figure 10 it follows that in the skin of rats in the area of introduction of the culture of autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene, the presence of Cas9 protein was established in comparison with the absence of it in the control samples: in the area of introduction of the culture of autologous fibroblasts transfected gene therapy DNA vector VTvaf17, which does not carry the Cas9 gene (placebo) and in the sample from the intact zone, which indicates the effectiveness of the gene therapy DNA vector VTvaf17-Cas9 and confirms the feasibility of the method of its use to increase the expression level of Cas9 in mammalian tissues, in particular, when administered autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17-Cas9.

Example 11.

Confirmation of the efficiency and feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene for genome editing in mammalian cells.

The decrease in cell fluorescence due to the disruption of the expression of the GFP gene in 293 / GFP Cell Line cells (Cell Biolabs, Cat. AKR-200) after combined transfection of these cells with the VTvaf17-Cas9 DNA vector carrying the human Cas9 gene and GFP-targeting guide RNA for CRISPR (Genaxxon bioscience Cat. P2008.0010). The cells were grown in DMEM medium under standard conditions.

For transfection of cells with a DNA vector VTvaf17-Cas9 carrying the Cas9 gene, Lipofectamine 3000 (ThermoFisher Scientific, USA) was used according to the manufacturer's recommendations as described in Example 2. 24 hours after transfection with a DNA vector VTvaf17-Cas9 carrying the Cas9 gene, cells transfected with the GFP targeting guide RNA for CRISPR. The CRISPRfect E transfection reagent (Genaxxon bioscience, Cat. P2002.0035) was used for transfection of the GFP-targeting guide RNA for CRISPR according to the manufacturer's instructions.

The experimental samples were water transfection followed by transfection with GFP-targeting guide RNA for CRISPR (A), transfection with a VTvaf17 DNA vector that does not contain the cDNA of the Cas9 gene, followed by transfection with GFP-targeting guide RNA for CRISPR (B), transfection with DNA- vector VTvaf17-Cas9 carrying the Cas9 gene, followed by transfection with the GFP targeting guide RNA for CRISPR (C), transfection with the DNA vector VTvaf17-Cas9 carrying the Cas9 gene, followed by transfection with water (D).

48 hours after the second transfection, the culture medium was removed, the cells were resuspended in saline and used to assess the number of cells expressing the fluorescent protein GFP by flow fluorometry using a Beckman Coulter's Cytomics FC 500 instrument (Beckman Coulter's, USA). The resulting analyzes are shown in FIG. eleven.

From figure 11 it follows that as a result of transfection of the primary culture of 293 / GFP cells with the gene therapy DNA vector VTvaf17-Cas9, followed by transfection with the GFP-targeting guide RNA for CRISPR, a decrease (about 80%) in the number of cells expressing GFP compared with the absence of a decrease in control samples, which confirms the ability of the vector to penetrate into eukaryotic cells and express the functionally active endonuclease Cas9, which, using the GFP-targeting guide RNA, performs directed editing of the GFP gene, which leads to suppression of the expression of the fluorescent protein GFP. The presented results also confirm the feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 to perform targeted editing of the genome of eukaryotic cells.

Example 12.

Confirmation of the efficiency and feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene for genome editing in mammalian cells.

Screening of clones of MCF7 cells (ATCC® HTB-22 ™) was performed in order to identify clones in which directed genome editing occurred after transfection of these cells with a mixture of VTvaf17-Cas9 DNA vector carrying the Cas9 gene with gRNA_Cloning Vector carrying oligonucleotides to the regions exon of the TLR9 gene.

Specific oligonucleotides corresponding to 20 nucleotide regions of the TLR9 gene exon (Toll-like receptor 9) were selected as sgRNAs. The sgRNA selection was performed using the CRISPR DESIGN service (http://crispr.mit.edu/). Two synthesized oligonucleotides tlrg4f and tlrg4r (sequence list (3) and (4)) were mixed in equimolar amounts in buffer T4 DNA ligase (Thermo Scientific, USA), the samples were heated at 94 ° C for 2 min, after which they were slowly cooled to room temperature. temperatures to form duplexes. Next, the oligonucleotide duplexes were cloned into the gRNA_Cloning Vector (AddGene, # 41824). Plasmids with cloned oligonucleotides were grown and purified in preparative quantities using the Plasmid DNA Purification Kit (Qiagen, USA).

MCF-7 cells were cultured using Eagle's Minimum Essential Medium (EMEM) (ATCC® 30-2003 ™) standard conditions (37C, 5% CO2). Transfection was performed using the Lipofectamine 3000 reagent kit (Thermoscientific, USA) according to the manufacturer's instructions. For transfection, an equimolar mixture of the VTvaf17-Cas9 DNA vector carrying the Cas9 gene was used with the gRNA_Cloning Vector carrying oligonucleotides to the regions of the TLR9 gene exon. Water or gRNA_Cloning Vector carrying oligonucleotides to regions of the TLR9 gene exon, or a VTvaf17-Cas9 DNA vector carrying the Cas9 gene were used as controls.

The cells were then plated using a FACS sorter into the wells of a 96-well plate. For the analysis of clones grown after sorting, cells growing in 96-well plates were washed with PBS, lysed in 50 μl of DNA-express reagent (Litekh, Russia), after which sample preparation was carried out according to the manufacturer's recommendations. The obtained samples were used as a template for PCR amplification of the locus region containing the sgRNAs recognition regions in the TLR9 gene. The obtained PCR fragments were sequenced for analysis on an ABI Prism 3730xl Genetic Analyzer (Applied Biosystems, USA). As a result, four clones were identified containing changes in the nucleotide sequences of the TLR9 gene, which were the result of directed genomic editing. In the control samples, no clones were identified containing any changes in the TLR9 gene sequences.

The data obtained as a result of the analysis are presented in FIG. 12.

It follows from figure 12 that as a result of transfection of the MCF7 cell line with a mixture of the VTvaf17-Cas9 DNA vector carrying the Cas9 gene, with gRNA_Cloning Vector carrying oligonucleotides to the regions of the TLR9 gene exon, and subsequent screening of cell clones, several clones were identified in which the directed genome editing, which confirms the ability of the VTvaf17-Cas9 vector to penetrate into eukaryotic cells and express the functionally active endonuclease Cas9, which, using gRNA, is capable of introducing a mutation into the target gene, for example, into the TLR9 gene, as shown by sequencing the target sequence. The presented results also confirm the feasibility of the method of using the gene therapy DNA vector VTvaf17-Cas9 to perform targeted editing of the genome of eukaryotic cells.

Example 13.

Escherichia coli strain SCS110-AF / VTvaf17-Cas9 carrying a gene therapy DNA vector and a method for its production.

Construction of a strain for industrial-scale production of a gene therapy DNA vector based on a gene therapy DNA vector VTvaf17 carrying the target gene, Cas9, namely the Escherichia coli SCS110-AF / VTvaf17-Cas9 strain carrying a gene therapy DNA vector VTvaf17-Cas9 for its production with the possibility of selection without the use of antibiotics consists in obtaining electrocompetent cells of the Escherichia coli strain SCS110-AF with electroporation of these cells with the gene therapeutic DNA vector VTvaf17-Cas9, after which the cells are seeded on Petri dishes with agar selective medium containing yeast extract, peptone, 6% sucrose as well as 10 μg / ml chloramphenicol. In this case, the Escherichia coli strain SCS110-AF for the development of a gene therapy DNA vector VTvaf17 or gene therapy DNA vectors based on it with the possibility of positive selection without the use of antibiotics was obtained by constructing a linear DNA fragment containing the regulatory element RNA-in of the transposon Tn10 for selection without the use of antibiotics size 64 bp, the levansurase sacB gene, the product of which provides selection on a sucrose-containing medium with a size of 1422 bp, the chloramphenicol resistance gene catR, which is necessary for the selection of clones of the strain in which homologous recombination of 763 bp has taken place. and two homologous sequences providing the process of homologous recombination in the region of the recA gene with its simultaneous inactivation of 329 bp in size. and 233 bp, after which the transformation of Escherichia coli cells was carried out by electroporation and clones that survived on a medium containing 10 μg / ml of chloramphenicol were selected.

The resulting strain for development was included in the collection of the National Bioresource Center - All-Russian Collection of Industrial Microorganisms (NBC VKPM), RF with the assignment of registration numbers:

Escherichia coli strain SCS110-AF / VTvaf17-Cas9 - registration number VKPM: B-, date of deposit:

Example 14.

A method for scaling the production of a gene therapy DNA vector based on a gene therapy DNA vector VTvaf17 carrying the target Cas9 gene to an industrial scale.

To confirm the manufacturability and the possibility of industrial-scale production of the gene therapy DNA vector VTvaf17-Cas9 (SEQ ID No. 1), a large-scale fermentation of Escherichia coli strain SCS110-AF / VTvaf17-Cas9, which contains the gene therapy DNA vector VTvaf17 carrying the target gene, and namely Cas9. The Escherichia coli SCS110-AF / VTvaf17-Cas9 strain was obtained on the basis of the Escherichia coli SCS110-AF strain (Cell and Gene Therapy LLC, UK) according to example 13 by electroporation of the competent cells of this strain with the VTvaf17-Cas9 gene therapeutic DNA vector carrying the target gene, namely, Cas9, followed by plating the transformed cells onto Petri dishes with agar selective medium containing yeast extract, peptone, 6% sucrose, and selecting individual clones.

Fermentation of the obtained Escherichia coli strain SCS110-AF / VTvaf17-Cas9, carrying the gene therapy DNA vector VTvaf17-Cas9, was carried out in a 10 L fermenter, followed by isolation of the gene therapy DNA vector VTvaf17-Cas9.

For fermentation of Escherichia coli strain SCS110-AF / VTvaf17-Cas9, a medium was prepared containing 10 L: 100 g tryptone, 50 g yeast extract (Becton Dickinson, USA), brought up to 8800 ml with water and autoclaved at 121 ° C for 20 min, then added 1200 ml 50% (w / v) sucrose. Then, a seed culture of Escherichia coli SCS110-AF / VTvaf17-Cas9 in a volume of 100 ml was inoculated into a flask. Incubated in an incubator shaker for 16 h at 30 ° C. The seed culture was transferred to a Techfors S fermenter (Infors HT, Switzerland), grown until the stationary phase was reached. The control was carried out by measuring the optical density of the culture at a wavelength of 600 nm. The cells were pelleted by centrifugation for 30 min at 5000-10000 g. The supernatant was removed, the cell mass was resuspended in 10% by volume phosphate-buffered saline. They were re-centrifuged for 30 min at 5000-10000g. The supernatant was removed, a solution of 20 mM TrisCl, 1 mM EDTA, 200 g / L sucrose, pH 8.0 in a volume of 1000 ml was added to the cell mass, and thoroughly mixed until a homogeneous suspension was formed. Egg lysozyme solution was added to a final concentration of 100 μg / ml. Incubated for 20 min on ice with gentle stirring. Next, 2500 ml of a solution of 0.2 M NaOH, 10 g / L sodium dodecyl sulfate were added, incubated for 10 min on ice with gentle stirring, then 3500 ml of a solution of 3M sodium acetate, 2M acetic acid, pH 5-5.5 were added, incubated 10 min on ice with gentle stirring. The obtained sample was centrifuged for 20-30 min at 15000 g or more. The solution was carefully decanted, and the residual sediment was removed by filtration through a coarse filter (filter paper). RNase A (Sigma, USA) was added to a final concentration of 20 ng / ml, and the mixture was incubated overnight for 16 h at room temperature. The solution was centrifuged for 20-30 min at 15000 g, then filtered through a membrane filter with pores of 0.45 μm (Millipore, USA). Then ultrafiltration was carried out through a membrane with a cutoff size of 100 kDa (Millipore, USA) and diluted to the initial volume with 25 mM TrisCl buffer, pH 7.0. The operation was repeated three to four times. The solution was applied to a column with 250 ml of DEAE sepharose HP sorbent (GE, USA) equilibrated with a solution of 25 mM TrisCl, pH 7.0. After the sample was applied, the column was washed with three volumes of the same solution, and then the gene therapy DNA vector VTvaf17-Cas9 was eluted with a linear gradient from a 25 mM TrisCl solution, pH 7.0 to a 25 mM TrisCl solution, pH 7.0, 1 M NaCl in a volume of five column volumes. Elution was monitored by the optical density of the descending solution at 260 nm. Chromatographic fractions containing the gene therapy DNA vector VTvaf17-Cas9 were pooled and gel filtration was performed on a Superdex 200 sorbent (GE, USA). The column was equilibrated with phosphate buffered saline. Elution was monitored by the optical density of the descending solution at 260 nm, fractions were analyzed by electrophoresis in agarose gel. The fractions containing the gene therapy DNA vector VTvaf17-Cas9 were pooled and stored at -20 ° C. To assess the reproducibility of the technical process, the indicated technological operations were repeated five times.

The reproducibility of the technical process and the quantitative characteristics of the final product yield confirm the manufacturability of the preparation and the possibility of industrial production of the gene therapy DNA vector VTvaf17-Cas9.

Thus, the created gene therapy DNA vector with the target gene can be used for introduction into human, animal, and mammalian cells, providing heterologous expression of Cas9 endonuclease, which can be used to edit the sequence of the human and animal genome in the presence of a gRNA of a given specificity.

The problem posed in this invention, namely: construction of a gene therapeutic DNA vector for heterologous expression of the Cas9 gene in human and animal cells, combining the following properties:

I) The effectiveness of the gene therapy DNA vector for heterologous expression of the target gene in eukaryotic cells;

II) The possibility of safe use for the implementation of various methods of genomic editing of human and animal genomes, including in genetic therapy of humans and animals due to the absence of regulatory elements in the composition of the gene therapy DNA vector, which are the nucleotide sequences of viral genomes;

III) Possibility of safe use for the implementation of various methods of genomic editing of human and animal genomes, including in genetic therapy of humans and animals due to the absence of antibiotic resistance genes in the gene therapy DNA vector;

IV) Manufacturability of obtaining and the possibility of developing a gene therapy DNA vector on an industrial scale,

as well as the problem of constructing a strain carrying this gene therapy DNA vector for the production of this gene therapy DNA vector has been solved, which is confirmed by examples:

for item I - example 1, 2, 3, 4, 5; 6; 7; 8; nine; ten; eleven;

for item II and item III - example 1, 11, 12;

for item IV - example 1, 13, 14.

Industrial applicability:

All the above examples confirm the industrial applicability of the proposed gene therapy DNA vector based on the gene therapy DNA vector VTvaf17 carrying the target Cas9 gene for heterologous expression of the Cas9 gene in human and animal cells, the Escherichia coli SCS110-AF / VTvaf17-Cas9 strain carrying the gene therapeutic DNA vector, a method for producing a gene therapy DNA vector, a method for industrial-scale production of a gene therapy DNA vector.

List of abbreviations

VTvaf17 is a vector therapeutic virus-antibiotic-free vector that does not contain sequences of viral genomes and markers of antibiotic resistance

gRNA - guided RNA

PBMC - Peripheral Blood Mononuclear Cells

DNA - deoxyribonucleic acid

cDNA - complementary deoxyribonucleic acid

RNA - ribonucleic acid

mRNA - matrix ribonucleic acid

p.n. - nucleotide pairs

PCR - polymerase chain reaction

ml - milliliter, μl - microliter

cub. mm - cubic millimeter

l - liter

mcg - microgram

mg - milligram

g - gram

μM - micromole

mM - millimole

min - minute

sec - second

rpm - revolutions per minute

nm - nanometer

cm - centimeter

mW - milliwatts

o.e. f-relative unit of fluorescence

PBC - phosphate buffered saline

List of references

1. Dowdy SF. Overcoming cellular barriers for RNA therapeutics. Nat Biotechnol. 2017 Mar; 35 (3): 222-229.

2. Draft Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal products, http://www.ema.europa.eu/docs/en_GB/document_library/Scientific_guideline/2015/05/WC500187020.pdf

3. Guideline on the quality, non-clinical and clinical aspects of gene therapy medicinal Products EMA / CAT / 80183/2014

4. Hornstein BD, Roman D, Arévalo-Soliz LM, Engevik MA, Zechiedrich L. Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells. Ceña V, ed. PLoS ONE. 2016; 11 (12): e0167537.

5. Hussain W, Mahmood T, Hussain J, Ali N, Shah T, Qayyum S, Khan I. CRISPR / Cas system: A game changing genome editing technology, to treat human genetic diseases. Gene. 2019 Feb 15; 685: 70-75. doi: 10.1016 / j.gene.2018.10.072. Epub 2018 Oct 26. Review. PubMed PMID: 30393194.

6. Li L, Hu S, Chen X. Non-viral delivery systems for CRISPR / Cas9-based genome editing: Challenges and opportunities. Biomaterials. 2018 Jul; 171: 207-218.

7. Li L, Petrovsky N. Molecular mechanisms for enhanced DNA vaccine immunogenicity. Expert Rev Vaccines. 2016; 15 (3): 313-29

8. Lukashev AN, Zamyatnin AA Jr. Viral Vectors for Gene Therapy: Current State and Clinical Perspectives. Biochemistry (Mosc). 2016 Jul; 81 (7): 700-8.

9. Maginnis MS. Virus-Receptor Interactions: The Key to Cellular Invasion. J Mol Biol. 2018 Aug 17; 430 (17): 2590-2611.

10. Mairhofer J, Grabherr R. Rational vector design for efficient non-viral gene delivery: challenges facing the use of plasmid DNA. Mol Biotechnol. 2008.39 (2): 97-104

11. Memi F, Ntokou A, Papangeli I. CRISPR / Cas9 gene-editing: Research technologies, clinical applications and ethical considerations. Semin Perinatol. 2018 Dec; 42 (8): 487-500. doi: 10.1053 / j.semperi.2018.09.003. Epub 2018 Oct 2. Review. PubMed PMID: 30482590.

12. Mougiakos I, Mohanraju P, Bosma EF, Vrouwe V, Finger Bou M, Naduthodi MIS, Gussak A, Brinkman RBL, van Kranenburg R, van der Oost J. Characterizing a thermostable Cas9 for bacterial genome editing and silencing. Nat Commun. 2017 Nov 21; 8 (1): 1647.

13. Reflection paper on design modifications of gene therapy medicinal products during development / 14 December 2011 EMA / CAT / GTWP / 44236/2009 Committee for advanced therapies

14. Salsman J, Dellaire G. Precision genome editing in the CRISPR era. Biochem Cell Biol. 2017 Apr; 95 (2): 187-201.

15. Sapranauskas R, Gasiunas G, Fremaux C, Barrangou R, Horvath P, Siksnys V. The Streptococcus thermophilus CRISPR / Cas system provides immunity in Escherichia coli. Nucleic Acids Res. 2011 Nov; 39 (21): 9275-82.

16. Wilson LOW, O'Brien AR, Bauer DC. The Current State and Future of CRISPR-Cas9 gRNA Design Tools. Front Pharmacol. 2018 Jul 12; 9: 749.

17. Molecular Biology, 2011, Volume 45, No. 1, p. 44-55

List of abbreviations

VTvaf17 is a vector therapeutic virus-antibiotic-free vector that does not contain sequences of viral genomes and markers of antibiotic resistance

gRNA - guided RNA

PBMC - Peripheral Blood Mononuclear Cells

DNA - deoxyribonucleic acid

cDNA - complementary deoxyribonucleic acid

RNA - ribonucleic acid

mRNA - matrix ribonucleic acid

p.n. - nucleotide pairs

PCR - polymerase chain reaction

ml - milliliter, μl - microliter

cub. mm - cubic millimeter

l - liter

mcg - microgram

mg - milligram

g - gram

μM - micromole

mM - millimole

min - minute

sec - second

rpm - revolutions per minute

nm - nanometer

cm - centimeter

mW - milliwatts

o.e. f-relative unit of fluorescence

PBC - phosphate-buffered saline.

--->

LIST OF SEQUENCES

<110> CELL and GENE THERAPY Ltd, Disruptive Innovative Technologies Limited Liability Company,

<120> Gene therapy DNA vector based on gene therapy DNA vector VTvaf17, carrying the target Cas9 gene for heterologous expression of this target gene in human and animal cells when implementing various methods of genomic editing, a method for producing and using a gene therapeutic DNA vector, Escherichia coli SCS110 strain -AF / VTvaf17-Cas9, carrying a gene therapy DNA vector, a method for its production, a method for industrial-scale production of a gene therapy DNA vector.

<160> 1

<170> BiSSAP 1.3.6

<210> 1

<211> 7351

<212> DNA

<213> Homo Sapiens

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aaagtgatgt cgtgtactgg ctccgccttt ttcccgaggg tgggggagaa ccgtatataa 180

gtgcagtagt cgccgtgaac gttctttttc gcaacgggtt tgccgccaga acacaggtaa 240

gtgccgtgtg tggttcccgc gggcctggcc tctttacggg ttatggccct tgcgtgcctt 300

gaattacttc cacgcccctg gctgcagtac gtgattcttg atcccgagct tcgggttgga 360

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tttttctggc aagatagtct tgtaaatgcg ggccaagatc tgcacactgg tatttcggtt 600

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ctggtgcctg gcctcgcgcc gccgtgtatc gccccgccct gggcggcaag gctggcccgg 780

tcggcaccag ttgcgtgagc ggaaagatgg ccgcttcccg gccctgctgc agggagctca 840

aaatggagga cgcggcgctc gggagagcgg gcgggtgagt cacccacaca aaggaaaagg 900

gcctttccgt cctcagccgt cgcttcatgt gactccacgg agtaccgggc gccgtccagg 960

cacctcgatt agttctcgag cttttggagt acgtcgtctt taggttgggg ggaggggttt 1020

tatgcgatgg agtttcccca cactgagtgg gtggagactg aagttaggcc agcttggcac 1080

ttgatgtaat tctccttgga atttgccctt tttgagtttg gatcttggtt cattctcaag 1140

cctcagacag tggttcaaag tttttttctt ccatttcagg tgtcgtgaaa actaccccta 1200

aaagccaagg atccgccacc atggccccaa agaagaagcg gaaggtcggt atccacggag 1260

tcccagcagc cgacaagaag tacagcatcg gcctggacat cggcaccaac tctgtgggct 1320

gggccgtgat caccgacgag tacaaggtgc ccagcaagaa attcaaggtg ctgggcaaca 1380

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tcttcggcaa catcgtggac gaggtggcct accacgagaa gtaccccacc atctaccacc 1680

tgagaaagaa actggtggac agcaccgaca aggccgacct gcggctgatc tatctggccc 1740

tggcccacat gatcaagttc cggggccact tcctgatcga gggcgacctg aaccccgaca 1800

acagcgacgt ggacaagctg ttcatccagc tggtgcagac ctacaaccag ctgttcgagg 1860

aaaaccccat caacgccagc ggcgtggacg ccaaggccat cctgtctgcc agactgagca 1920

agagcagacg gctggaaaat ctgatcgccc agctgcccgg cgagaagaag aatggcctgt 1980

tcggaaacct gattgccctg agcctgggcc tgacccccaa cttcaagagc aacttcgacc 2040

tggccgagga tgccaaactg cagctgagca aggacaccta cgacgacgac ctggacaacc 2100

tgctggccca gatcggcgac cagtacgccg acctgtttct ggccgccaag aacctgtccg 2160

acgccatcct gctgagcgac atcctgagag tgaacaccga gatcaccaag gcccccctga 2220

gcgcctctat gatcaagaga tacgacgagc accaccagga cctgaccctg ctgaaagctc 2280

tcgtgcggca gcagctgcct gagaagtaca aagagatttt cttcgaccag agcaagaacg 2340

gctacgccgg ctacattgac ggcggagcca gccaggaaga gttctacaag ttcatcaagc 2400

ccatcctgga aaagatggac ggcaccgagg aactgctcgt gaagctgaac agagaggacc 2460

tgctgcggaa gcagcggacc ttcgacaacg gcagcatccc ccaccagatc cacctgggag 2520

agctgcacgc cattctgcgg cggcaggaag atttttaccc attcctgaag gacaaccggg 2580

aaaagatcga gaagatcctg accttccgca tcccctacta cgtgggccct ctggccaggg 2640

gaaacagcag attcgcctgg atgaccagaa agagcgagga aaccatcacc ccctggaact 2700

tcgaggaagt ggtggacaag ggcgcttccg cccagagctt catcgagcgg atgaccaact 2760

tcgataagaa cctgcccaac gagaaggtgc tgcccaagca cagcctgctg tacgagtact 2820

tcaccgtgta taacgagctg accaaagtga aatacgtgac cgagggaatg agaaagcccg 2880

ccttcctgag cggcgagcag aaaaaggcca tcgtggacct gctgttcaag accaaccgga 2940

aagtgaccgt gaagcagctg aaagaggact acttcaagaa aatcgagtgc ttcgactccg 3000

tggaaatctc cggcgtggaa gatcggttca acgcctccct gggcacatac cacgatctgc 3060

tgaaaattat caaggacaag gacttcctgg acaatgagga aaacgaggac attctggaag 3120

atatcgtgct gaccctgaca ctgtttgagg acagagagat gatcgaggaa cggctgaaaa 3180

cctatgccca cctgttcgac gacaaagtga tgaagcagct gaagcggcgg agatacaccg 3240

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caatcctgga tttcctgaag tccgacggct tcgccaacag aaacttcatg cagctgatcc 3360

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acatcgtgat cgaaatggcc agagagaacc agaccaccca gaagggacag aagaacagcc 3600

gcgagagaat gaagcggatc gaagagggca tcaaagagct gggcagccag atcctgaaag 3660

aacaccccgt ggaaaacacc cagctgcaga acgagaagct gtacctgtac tacctgcaga 3720

atgggcggga tatgtacgtg gaccaggaac tggacatcaa ccggctgtcc gactacgatg 3780

tggaccatat cgtgcctcag agctttctga aggacgactc catcgacaac aaggtgctga 3840

ccagaagcga caagaaccgg ggcaagagcg acaacgtgcc ctccgaagag gtcgtgaaga 3900

agatgaagaa ctactggcgg cagctgctga acgccaagct gattacccag agaaagttcg 3960

acaatctgac caaggccgag agaggcggcc tgagcgaact ggataaggcc ggcttcatca 4020

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tctacagcaa catcatgaac tttttcaaga ccgagattac cctggccaac ggcgagatcc 4440

ggaagcggcc tctgatcgag acaaacggcg aaaccgggga gatcgtgtgg gataagggcc 4500

gggattttgc caccgtgcgg aaagtgctga gcatgcccca agtgaatatc gtgaaaaaga 4560

ccgaggtgca gacaggcggc ttcagcaaag agtctatcct gcccaagagg aacagcgata 4620

agctgatcgc cagaaagaag gactgggacc ctaagaagta cggcggcttc gacagcccca 4680

ccgtggccta ttctgtgctg gtggtggcca aagtggaaaa gggcaagtcc aagaaactga 4740

agagtgtgaa agagctgctg gggatcacca tcatggaaag aagcagcttc gagaagaatc 4800

ccatcgactt tctggaagcc aagggctaca aagaagtgaa aaaggacctg atcatcaagc 4860

tgcctaagta ctccctgttc gagctggaaa acggccggaa gagaatgctg gcctctgccg 4920

gcgaactgca gaagggaaac gaactggccc tgccctccaa atatgtgaac ttcctgtacc 4980

tggccagcca ctatgagaag ctgaagggct cccccgagga taatgagcag aaacagctgt 5040

ttgtggaaca gcacaagcac tacctggacg agatcatcga gcagatcagc gagttctcca 5100

agagagtgat cctggccgac gctaatctgg acaaagtgct gtccgcctac aacaagcacc 5160

gggataagcc catcagagag caggccgaga atatcatcca cctgtttacc ctgaccaatc 5220

tgggagcccc tgccgccttc aagtactttg acaccaccat cgaccggaag aggtacacca 5280

gcaccaaaga ggtgctggac gccaccctga tccaccagag catcaccggc ctgtacgaga 5340

cacggatcga cctgtctcag ctgggaggcg acaaaaggcc ggcggccacg aaaaaggccg 5400

gccaggcaaa aaagaaaaag taagaattcc ctgtgacccc tccccagtgc ctctcctggc 5460

cctggaagtt gccactccag tgcccaccag ccttgtccta ataaaattaa gttgcatcat 5520

tttgtctgac taggtgtcct tctataatat tatggggtgg aggggggtgg tatggagcaa 5580

ggggcaagtt gggaagacaa cctgtagggc ctgcggggtc tattgggaac caagctggag 5640

tgcagtggca caatcttggc tcactgcaat ctccgcctcc tgggttcaag cgattctcct 5700

gcctcagcct cccgagttgt tgggattcca ggcatgcatg accaggctca gctaattttt 5760

gtttttttgg tagagacggg gtttcaccat attggccagg ctggtctcca actcctaatc 5820

tcaggtgatc tacccacctt ggcctcccaa attgctggga ttacaggcgt gaaccactgc 5880

tcccttccct gtccttacgc gtagaattgg taaagagagt cgtgtaaaat atcgagttcg 5940

cacatcttgt tgtctgatta ttgatttttg gcgaaaccat ttgatcatat gacaagatgt 6000

gtatctacct taacttaatg attttgataa aaatcattaa ctagtccatg gctgcctcgc 6060

gcgtttcggt gatgacggtg aaaacctctg acacatgcag ctcccggaga cggtcacagc 6120

ttgtctgtaa gcggatgccg ggagcagaca agcccgtcag ggcgcgtcag cgggtgttgg 6180

cgggtgtcgg ggcgcagcca tgacccagtc acgtagcgat agcggagtgt atactggctt 6240

aactatgcgg catcagagca gattgtactg agagtgcacc atatgcggtg tgaaataccg 6300

cacagatgcg taaggagaaa ataccgcatc aggcgctctt ccgcttcctc gctcactgac 6360

tcgctgcgct cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaata 6420

cggttatcca cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaa 6480

aaggccagga accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccct 6540

gacgagcatc acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataa 6600

agataccagg cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccg 6660

cttaccggat acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctca 6720

cgctgtaggt atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaa 6780

ccccccgttc agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccg 6840

gtaagacacg acttatcgcc actggcagca gccactggta acaggattag cagagcgagg 6900

tatgtaggcg gtgctacaga gttcttgaag tggtggccta actacggcta cactagaaga 6960

acagtatttg gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagc 7020

tcttgatccg gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcag 7080

attacgcgca gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgac 7140

gctcagtgga acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatc 7200

ttcacctaga tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgag 7260

taaacttggt ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgt 7320

ctatttcgtt catccatagt tgcctgactc c 7351

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LIST OF SEQUENCES OF OLIGONUCLEOTIDES

(1) Cas9_SF CATCGAGCAGATCAGCGAGT

(2) Cas9_FR CGATCCGTGTCTCGTACAGG

(3) tlrg4f CACCGACTGGGTGTACAACGAGCTT

(4) tlrg4r AAACAAGCTCGTTGTACACCCAGTC

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Claims (8)

1. A gene therapy DNA vector based on the gene therapy DNA vector VTvaf17 carrying the target Cas9 gene for heterologous expression of this target gene in human and animal cells when implementing genomic editing methods, while the gene therapy DNA vector VTvaf17-Cas9 has the nucleotide sequence SEQ ID # 1. 2. Gene therapy DNA vector based on gene therapy DNA vector VTvaf17, carrying the target Cas9 gene according to claim 1, characterized in that the created gene therapy DNA vector VTvaf17-Cas9 according to claim 1 due to the limited size of the vector part of VTvaf17, not exceeding 3200 bp, has the ability to effectively penetrate human and animal cells and express the target Cas9 gene cloned into it. 3. Gene therapy DNA vector based on the gene therapy DNA vector VTvaf17 carrying the target Cas9 gene according to claim 1, characterized in that the gene therapy DNA vector contains no nucleotide sequences of viral origin and no antibiotic resistance genes, providing the possibility of its safe use for implementation various methods of genomic editing of human and animal genomes, including those for genetic therapy of humans and animals. 4. A method of obtaining a gene therapy DNA vector based on a gene therapy DNA vector VTvaf17 carrying the target Cas9 gene according to claim 1, comprising that the gene therapy DNA vector VTvaf17-Cas9 according to claim 1 is obtained as follows: the coding part of the target Cas9 gene cloned into DNA vector VTvaf17 and receive gene therapy DNA vector VTvaf17-Cas9. 5. A method of using a gene therapy DNA vector based on a gene therapy DNA vector VTvaf17 carrying the target Cas9 gene according to claim 1 for heterologous expression of this target gene in human and animal cells, which consists in introducing the gene therapy DNA vector according to claim 1 into cells , human or animal organs and tissues together with gRNA and / or in the introduction into human or animal organs and tissues of autologous human or animal cells transfected with a gene therapeutic DNA vector according to claim 1 together with gRNA or in a combination of the indicated methods. 6. The method of obtaining the Escherichia coli strain SCS110-AF / VTvaf17-Cas9, which consists in electroporation of competent cells of the Escherichia coli strain SCS110-AF with a gene therapy DNA vector according to claim 1 and subsequent selection of stable clones of the strain using a selective medium. 7. The strain obtained according to claim 6, Escherichia coli SCS110-AF / VTvaf17-Cas9, carrying the gene therapy DNA vector according to claim 1 for heterologous expression of the target gene in human and animal cells. 8. A method of industrial-scale production of a gene therapeutic DNA vector according to claim 1, which consists in scaling the bacterial culture of the strain according to claim 7 to the amounts required for the growth of bacterial biomass in an industrial fermenter, after which the biomass is used to isolate a fraction containing the target DNA product - gene therapy DNA vector VTvaf17-Cas9 according to claim 1, is filtered and purified by chromatographic methods in many stages.

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