WO2020139156A1

WO2020139156A1 - Gene therapy dna vector and its application

Info

Publication number: WO2020139156A1
Application number: PCT/RU2019/000996
Authority: WO
Inventors: Natalia SAVELIEVA
Original assignee: Cell End Gene Therapy Ltd
Current assignee: Cell End Gene Therapy Ltd
Priority date: 2018-12-27
Filing date: 2019-12-23
Publication date: 2020-07-02
Anticipated expiration: 2021-06-27
Also published as: CN113490743A; RU2018147083A3; WO2020139156A8; RU2730663C2; RU2018147083A

Abstract

The invention is related to genetic engineering and can be used in biotechnology, medical science and agriculture to create gene therapy medicinal products. Gene therapy DNA vector based on the VTvaflV gene therapy DNA vector is proposed, the vector carrying the target Cas9 gene for heterologous expression of this target gene in human and animal cells upon implementation of various genome editing methods. Gene therapy DNA vector VTvafl7-Cas9 having a nucleotide sequence SEQ ID No. 1. Gene therapy DNA vector based on gene therapy DNA vector VTvaflV carrying the Cas9 target gene, said DNA vector is unique due to the fact that the constructed gene therapy DNA vector VTvafl7-Cas9, due to the limited size of VTvafl7 vector part not exceeding 3200 bp, has the ability to efficiently penetrate into human and animal cells and express the Cas9 target gene cloned to it. A method of production of the specified vector, the method of usage of the vector, Escherichia coli strain carrying the specified vector, as well as a method of production of the specified vector on an industrial scale are also provided.

Description

Gene therapy DNA vector and its application

Field of the invention

The invention refers to genetic engineering and can be used in biotechnology, medicine, and agriculture for the manufacture of gene therapy products.

Background of the Invention

Gene therapy is an innovative approach in medicine aimed at treating inherited and acquired diseases by means of delivery of new genetic material into a patient’s cells to compensate for or suppress the function of a mutant gene and/or treat a genetic disorder. The final product of gene expression may be an RNA molecule or a protein molecule. However, most physiological processes in the body are associated with the functional activity of protein molecules, while RNA molecules are either an intermediate product in the synthesis of proteins or perform regulatory functions. Thus, the objective of gene therapy in most cases is to inject the organism with genes that provide transcription and further translation of protein molecules encoded by these genes. Within the description of the invention, gene expression refers to the production of a protein molecule with amino acid sequence encoded by this gene. Mutations in genes can result in complete or partial loss of protein expression, or expression of variants of protein molecules that have adverse functional activity. Injection of gene therapy vectors into the body that encode a particular gene can restore the expression of therapeutic proteins. However, this approach is compensatory and not aimed at correcting genetic defects. Following the discovery of directed (targeted) editing of nucleotide sequences the implementation of therapeutic genome editing approach that aims to correct mutations in the DNA sequence that also constitutes targeted gene therapy was made possible by injecting various nucleases with specific properties (for example, Cas9) into gene therapy vectors. In this case, the function is restored due to the correction of 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 originally discovered as a component of the bacterial immune system, which enables bacterial cells to targetedly remove the nucleotide sequences of bacteriophage (Sapranauskas R, 2011; Mougiakos I, 2017). Since this system has a certain genericity of the action principle, it is widely used in biomedical and biotechnological researches. Currently, the CRISPR/Cas9 system is widely used within scientific studies for genome editing in mammalian and laboratory animal cell cultures and has the capacity to design drugs and methods for gene therapy. The operating principle of this system is that the Cas9 endonuclease with the help of gRNA complementary to a specific sequence in the genome cleaves the DNA chains, cutting out a region of the targeted DNA. The DNA integrity in the breakpoints is then restored using cellular repair systems that can use a homologous DNA strand containing the correct nucleotide sequence as a matrix for recovery or repair the breaks through the direct connection of adjacent nucleotides without repairing the excised DNA region (Salsman J, 2017). Construction of gRNA in such a way that this molecule is complementary to the DNA region that contains one or another mutation allows targetedly cutting out that particular region using Cas9, which determines the capacity of this mechanism of action in the correction of genetic material, i.e. genome editing (Wilson LOW, 2018).

Nevertheless, one of the main problems of using the CRISPR/Cas9 system for genome editing is the problem of delivering the endonuclease and gRNA complex to the cell nucleus and, in general, pharmacokinetic problems that limit the penetrating capability of molecules into various organs and tissues or require extremely high concentrations and use of special compositions that allow for cell penetration (Dowdy SF, 2017). The use of gene vectors for the heterologous expression of Cas9 gene helps to overcome these limitations. The most studied vectors in this field are lentiviral, adenoviral, adeno-associated, and other virus- related vectors.

The risk of non-specific endonuclease action is another problem of the usage of CRISPR/Cas9 system. In this context, 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 particular sequences to the organism is limited by the tropism of pseudoviral particles to various tissues, which does not always allow for efficient 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 risks associated with gene therapy virus-related vectors in general (Lukashev AN, Zamyatnin AA, 2016).

Thus, the background of the invention indicates that there is a need to develop effective and safe gene therapy approaches for delivering Cas9 to target cells and tissues.

It is known that gene therapy vectors are divided into viral, cell, and DNA vectors (Guideline on the quality, non-clinical, and clinical aspects of gene therapy medicinal Products EMA/CAT/80183/2014). Recently, gene therapy has paid increasingly more attention to the development of non-viral gene delivery systems with plasmid vectors topping the list. Plasmid vectors are free of limitations inherent in cell and viral vectors. In the target cell, they exist as an episome without being integrated into the genome, while producing them is quite cheap, and there is no immune response or side effects caused by the administration of plasmid vectors, which makes them a convenient tool for gene therapy and prevention of the genetic diseases (DNA vaccination) (Li L, Petrovsky N. // Expert Rev Vaccines. 2016;15(3):313-29).

However, limitations of plasmid vectors use in gene therapy are: 1) presence of antibiotic resistance genes for the production of constructs in bacterial strains; 2) the presence of various regulatory elements represented by sequences of viral genomes; 3) length of therapeutic plasmid vector that determines the efficiency of vector delivery to the target cell.

It is known that the European Medicines Agency deems it necessary to refrain from adding antibiotic resistance marker genes to newly engineered 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 primarily related to the potential danger of the DNA vector penetration or horizontal antibiotic resistance gene transfer into the cells of bacteria found in the body as part of normal or opportunistic microflora. Furthermore, the presence of antibiotic resistance genes significantly increases the length of DNA vector, which reduces the efficiency of its penetration into eukaryotic cells.

It is important to note that antibiotic resistance genes also make a fundamental contribution to the method of production of DNA vectors. If antibiotic resistance genes are present, strains for the production of DNA vectors are usually cultured in medium containing a selective antibiotic, which poses risk of antibiotic traces in insufficiently purified DNA vector preparations. Thus, production of DNA vectors for gene therapy without antibiotic resistance genes is associated with the production of strains with such distinctive feature as the ability for stable amplification of therapeutic DNA vectors in the antibiotic-free medium.

In addition, the European Medicines Agency recommends avoiding the presence of regulatory elements in therapeutic plasmid vectors to increase the expression of therapeutic genes (promoters, enhancers, post-translational regulatory elements) that constitute nucleotide sequences of 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_gui deline/2015/05/WC500187020.pdf). Although these sequences can increase the expression level of the therapeutic transgene, however, they pose risk of recombination with the genetic material of wild-type viruses and integration into the eukaryotic genome. Moreover, the relevance of overexpression of the particular gene for therapy remains an unresolved issue.

The size of the therapy vector is also essential. It is known that modem plasmid vectors often have unnecessary, non-functional sites that increase their length substantially (Mairhofer J, Grabherr R. // Mol Biotechnol. 2008.39(2):97- 104). For example, ampicillin resistance gene in pBR322 series vectors, as a rule, consists of at least 1000 bp, which is more than 20% of the length of the vector itself. A reverse relationship between the vector length and its ability to penetrate into eukaryotic cells is observed; DNA vectors with a small length effectively penetrate into human and animal cells. For example, in a series of experiments on transfection of HeLa cells with 383—4548 bp DNA vectors it was shown that the difference in penetration efficiency can be up to two orders of magnitude (100 times different) (Homstein BD et al. // PLoS ONE. 2016;11(12): e0167537.).

Thus, when selecting a DNA vector, for reasons of safety and maximum effectiveness, preference should be given to those constructs that do not contain antibiotic resistance genes, the sequences of viral origin and length of which allows for the effective penetration into eukaryotic cells. A strain for production of such DNA vector in quantities sufficient for the purposes of gene therapy should ensure the possibility of stable DNA vector amplification using antibiotic- free nutrient media.

Example of usage of the recombinant DNA vectors for gene therapy is the method of producing a recombinant vector for genetic immunisation (Patent No. US 9550998 B2). The plasmid vector is a supercoiled plasmid DNA vector that is used for the expression of cloned genes in human and animal cells. The vector contains an origin of replication, regulatory elements comprising human cytomegalovirus promoter and enhancer, and regulatory sequences from the human T-cell lymphotropic virus.

The vector is accumulated in a dedicated E. coli strain free of antibiotics through antisense complementation of sacB gene inserted into the strain by means of bacteriophage. The disadvantage of this invention is the presence of regulatory elements in the composition of DNA vector that constitute sequences of viral genomes.

The following patents are prototypes of this invention with regard to the use of gene therapy approaches for the Cas9 expression in eukaryotic cells.

Patent No. US8795965B2 describes a DNA molecule that encodes an expression cassette containing the sequence encoding the Cas9 protein. The disadvantages of this invention are vague requirements for the presence of regulatory virus-related sequences in the DNA molecule composition, as well as the uncertainty of methods of these molecules production and their industrial applicability.

Patent No. CN103981216B describes a plasmid vector expressing the Cas9 gene. The disadvantage of this invention is the use of regulatory elements in the vector composition, ensuring the Cas9 gene expression in plant cells, but not in mammalian cells, as well as the presence of antibiotic resistance genes in the vector.

Patent No. US9914939B2 describes a plasmid vector expressing the Cas9 gene. The disadvantages of this invention include vague safety, producibility, and constructability requirements applied to the used plasmid vector, in particular the presence/absence of virus-related sequences and antibiotic resistance genes in the vector composition.

Disclosure of the Invention

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

I) Efficiency of gene therapy DNA vector for the heterologous expression of therapeutic genes in eukaryotic cells. II) The possibility of safe use for the implementation of various methods of genome editing of human and animal genomes, including human and animal gene therapy due to the lack of regulatory elements in the gene therapy DNA vector that constitute the nucleotide sequences of viral genomes.

III) The possibility of safe use for the implementation of various methods of genome editing of human, animal genomes, including human and animal gene therapy due to the lack of antibiotic resistance genes in the gene therapy DNA vector.

IV) Producibility and constructability of gene therapy DNA vector on an industrial scale.

Item II and III are provided for herein in line with the recommendations of the state regulators for gene therapy medicines and, specifically, the requirement of the European Medicines Agency to refrain from adding antibiotic resistance marker genes to newly engineered 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) and refrain from adding viral genomes to newly engineered plasmid vectors for gene therapy (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 purpose of the invention also includes the construction of strains carrying this gene therapy DNA vector for the development and production of this gene therapy DNA vector on an industrial scale.

The specified purpose is achieved by using the produced gene therapy DNA vector based on the gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene for the heterologous expression of this therapeutic gene in human and animal cells in the implementation of various genome editing methods, while gene therapy DNA vector VTvafl7-Cas9 has the nucleotide sequence of SEQ ID No. 1. The constructed gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene that is unique due to the fact that the constructed gene therapy DNA vector VTvafl7-Cas9 due to the limited size of VTvafl7 vector part not exceeding 3200 bp has the ability to efficiently penetrate into human and animal cells and express the Cas9 therapeutic gene cloned to it.

Gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene that is unique due to the fact that the gene therapy DNA vector contains no nucleotide sequences of viral origin and no antibiotic resistance genes, which ensures its safe use for the implementation of various methods of genome editing of humans, animals, including the gene therapy of humans and animals.

The method of production of the gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene has been developed that involves obtaining gene therapy DNA vector VTvafl7-Cas9 as follows: the coding region of the Cas9 therapeutic gene is cloned to DNA vector VTvafl7, and gene therapy DNA vector VTvafl 7-Cas9, SEQ ID No. 1 is obtained.

The method of usage of the constructed gene therapy DNA vector based on gene therapy DNA vector VTvafl 7 carrying the Cas9 therapeutic gene for heterologous expression of this gene in human and animal cells that involves introduction of a gene therapy DNA vector into human or animal cells, organs, and tissues in combination with gRNA molecules or genetic constructs that provide gRNA expression and/or introduction of autologous human or animal cells into human or animal organs and tissues transfected with gene therapy DNA vector in combination with gRNA molecules or genetic constructs that enable the gRNA expression or a combination of the indicated methods.

The method of production of Escherichia coli strain SCS110-AF/VTvafl7- Cas9 involves electroporation of competent cells of Escherichia coli strain SCS110-AF by the constructed gene therapy DNA vector and subsequent selection of stable clones of the strain using selective medium.

Escherichia coli strain SCSI 10-AF/VTvafl7-Cas9 carrying the gene therapy DNA vector for its production allowing for antibiotic-free selection is claimed.

The method of gene therapy DNA vector production on an industrial scale involves scaling-up the bacterial culture of the strain to the quantities necessary for increasing the bacterial biomass in an industrial fermenter, after which the biomass is used to extract a fraction containing the therapeutic DNA product, i.e. the gene therapy DNA vector VTvafl7-Cas9, and then multi-stage filtered, and purified by chromatographic methods.

The essence of the invention is explained in the drawings, where:

Figure 1

shows the structure of gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene that constitutes a circular double-stranded DNA molecule capable of autonomous replication in Escherichia coli cells.

Figure 1 shows the structure of gene therapy DNA vector VTvafl7-Cas9.

The following structural elements of the vector are indicated in the structures:

EFla - the promoter region of human elongation factor EF1A with an intrinsic enhancer contained in the first intron of the gene. It ensures efficient transcription of the recombinant gene in most human tissues,

The reading frame of the therapeutic gene corresponding to the coding region of Cas9 gene,

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

ori - the origin of replication for autonomous replication with a single nucleotide substitution to increase plasmid production in the cells of most Escherichia coli strains,

RNAout - the regulatory element RNA-OUT of transposon Tn 10 allowing for antibiotic-free positive selection in case of the use of Escherichia coli strain SCS 110-AF.

Unique restriction sites are marked.

Figure 2

shows diagrams of cDNA amplicon accumulation of the therapeutic gene, namely the Cas9 gene, in HDFa primary human dermal fibroblast cell culture (ATCC PCS-201-01) before its transfection and 48 hours after transfection of these cells with gene therapy DNA vector VTvafl7-Cas9 in order to assess the ability to penetrate into eukaryotic cells and functional activity, i.e. expression of the therapeutic gene at the mRNA level.

Curves of accumulation of amplicons during the reaction are shown in Fig.

2 corresponding to:

1 - cDNA of Cas9 gene in HDFa cell culture before transfection with DNA vector VTvafl7-Cas9,

2 - cDNA of Cas9 gene in HDFa cell culture after transfection with DNA vector VTvafl7-Cas9,

3 - cDNA of B2M gene in HDFa cell culture before transfection with DNA vector VTvafl7-Cas9,

4 - cDNA of B2M gene in HDFa cell culture after transfection with DNA vector VTvafl7-Cas9.

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

Figure 3

shows diagrams of cDNA amplicon accumulation of the therapeutic gene, namely the Cas9 gene, in HEKa primary human epidermal keratinocyte cell culture (ATCC PCS-200-011) before its transfection and 48 hours after transfection of these cells with gene therapy DNA vector VTvafl7-Cas9 in order to assess the ability to penetrate into eukaryotic cells and functional activity, i.e. expression of the therapeutic gene at the mRNA level.

Curves of accumulation of amplicons during the reaction are shown in Fig.

3 corresponding to:

1 - cDNA of Cas9 gene in HEKa primary human epidermal keratinocyte cell culture before transfection with DNA vector VTvafl7-Cas9,

2 - cDNA of Cas9 gene in HEKa primary human epidermal keratinocyte cell culture after transfection with DNA vector VTvafl7-Cas9,

3 - cDNA of B2M gene in HEKa primary human epidermal keratinocyte cell culture before transfection with DNA vector VTvafl7-Cas9,

4 - cDNA of B2M gene in HEKa primary human epidermal keratinocyte cell culture after transfection with DNA vector VTvafl 7-Cas9. B2M (beta-2-microglobuline) gene listed in the GenBank database under number NM 004048.2 was used as a reference gene.

Figure 4

shows diagrams of cDNA amplicon accumulation of the therapeutic gene, namely the Cas9 gene, in Primary Epidermal Melanocytes; Normal, Human, Adult (HEMa) (ATCC® PCS-200-013™) before their transfection and 48 hours after transfection of these cells with the DNA vector VTvafl7-Cas9 in order to assess the ability to penetrate into eukaryotic cells and functional activity, i.e. expression of the therapeutic gene at the mRNA level.

Curves of accumulation of amplicons during the reaction are shown in Fig. 4 corresponding to:

1 - cDNA of Cas9 gene in HEMa primary human epidermal melanocyte cell culture before transfection with DNA vector VTvafl7-Cas9,

2 - cDNA of Cas9 gene in HEMa primary human epidermal melanocyte cell culture after transfection with DNA vector VTvafl7-Cas9,

3 - cDNA of B2M gene in HEMa primary human epidermal melanocyte cell culture before transfection with DNA vector VTvafl7-Cas9,

4 - cDNA of B2M gene in HEMa primary human epidermal melanocyte cell culture after transfection with DNA vector VTvafl7-Cas9.

Figure 5

shows the plot of Cas9 protein concentration in the cell lysate of HDFa primary human dermal fibroblasts (ATCC PCS-201-01) after transfection of these cells with DNA vector VTvafl7-Cas9 in order to assess the functional activity, i.e. expression at the protein level based on the Cas9 protein concentration change in the cell lysate.

The following elements are indicated in Figure 5:

culture A - HDFa human dermal fibroblast cell culture transfected with aqueous dendrimer solution without plasmid DNA (reference), culture B - HDFa human dermal fibroblast cell culture transfected with DNA vector VTvafl7,

culture C - HDFa human dermal fibroblast cell culture transfected with DNA vector VTvafl7-Cas9.

Figure 6

shows the plot of Cas9 protein concentration in the lysate of HEKa primary human epidermal keratinocyte cells (ATCC PCS-200-01) after transfection of these cells with gene therapy DNA vector VTvafl7-Cas9 in order to assess the functional activity, i.e. the therapeutic gene expression at the protein level, and the possibility of increasing the level of protein expression by gene therapy DNA vector based on gene therapy vector VTvafl7 carrying the Cas9 therapeutic gene.

The following elements are indicated in Figure 6:

culture A - HEKa primary human epidermal keratinocyte cell culture transfected with aqueous dendrimer solution without plasmid DNA (reference), culture B - HEKa primary human epidermal keratinocyte cell culture transfected with DNA vector VTvafl7,

culture C - HEKa primary human epidermal keratinocyte cell culture transfected with DNA vector VTvafl7-Cas9.

Figure 7

shows the plot of Cas9 protein concentration in the cell lysate of Primary Epidermal Melanocytes; Normal, Human, Adult (HEMa) (ATCC® PCS-200- 013™) after transfection of these cells with gene therapy DNA vector VTvafl7- Cas9 in order to assess the functional activity, i.e. the therapeutic gene expression at the protein level, and the possibility of increasing the level of protein expression by gene therapy DNA vector based on gene therapy vector VTvafl7 carrying the Cas9 therapeutic gene.

The following elements are indicated in Figure 7:

culture A - HEMa human primary epidermal melanocyte cell culture transfected with aqueous dendrimer solution without plasmid DNA (reference), culture B - HEMa human epidermal melanocyte cell culture transfected with DNA vector VTvafl 7, culture C - HEMa human epidermal melanocyte cell culture transfected with DNA vector VTvafl7-Cas9.

Figure 8

shows the plot of Cas9 protein concentration in the lysate of CHO-K1 Syrian hamster ovary cells (ATCC® CCL-61™) after transfection of these cells with DNA vector VTvafl7-Cas9 in order to assess the functional activity, i.e. the therapeutic gene expression at the protein level, and the possibility of increasing the level of protein expression by gene therapy DNA vector based on gene therapy vector VTvafl7 carrying the Cas9 therapeutic gene.

The following elements are indicated in Figure 8:

culture A - CHO-K1 Syrian hamster ovary cell culture transfected with aqueous dendrimer solution without plasmid DNA (reference),

culture B - CHO-K1 Syrian hamster ovary cell culture transfected with DNA vector VTvafl7,

culture C - CHO-K1 Syrian hamster ovary cell culture transfected with DNA vector VTvafl7-Cas9.

Figure 9

shows the plot of Cas9 protein concentration in the lysate of human peripheral blood mononuclear cell culture (PBMC) after transfection of these cells with DNA vector VTvafl7-Cas9 in order to assess the functional activity, i.e. the therapeutic gene expression at the protein level, and the possibility of increasing the level of protein expression by gene therapy DNA vector based on gene therapy vector VTvafl7 carrying the Cas9 therapeutic gene.

The following elements are indicated in Figure 9:

culture A - PBMC human cell culture transfected with aqueous dendrimer solution without plasmid DNA (reference),

culture B - PBMC human cell culture transfected with DNA vector VTvafl 7,

culture C - PBMC human cell culture transfected with DNA vector VTvafl 7-Cas9. Figure 10

shows the plot of Cas9 protein concentration in skin biopsy samples of three Wistar rats in the preliminary epilated area after subcutaneous injection of autologous fibroblast cell culture transfected with the gene therapy DNA vector VTvafl7-Cas9 in order to demonstrate the method of use by injecting autologous cells transfected with the gene therapy DNA vector VTvafl7-Cas9.

The following elements are indicated in Figure 10:

K1I - rat K1 skin biopsy in the region of injection of autologous fibroblast culture transfected with gene therapy DNA vector VTvafl7-Cas9,

Kill - rat skin biopsy in the region of injection of autologous fibroblasts transfected with gene therapy DNA vector VTvafl 7 not carrying the Cas9 gene,

K1 III - rat skin biopsy from intact site,

K2I - rat K2 skin biopsy in the region of injection of autologous fibroblast culture transfected with gene therapy DNA vector VTvafl 7-Cas9,

K2II - rat K2 skin biopsy in the region of injection of autologous fibroblasts transfected with gene therapy DNA vector VTvafl 7 not carrying the Cas9 gene,

K2III - rat K2 skin biopsy from intact site,

K3I - rat K3 skin biopsy in the region of injection of autologous fibroblast culture transfected with gene therapy DNA vector VTvafl 7-Cas9,

K3II - rat K3 skin biopsy in the region of injection of autologous fibroblasts transfected with gene therapy DNA vector VTvafl 7 not carrying the Cas9 gene,

K3III - rat K3 skin biopsy from intact site.

Figure 11

shows the fluorescent cell reduction diagram 293/GFP Cell Line (Cell Biolabs, Cat. AKR-200) after combined transfection of these cells with DNA vector VTvafl 7-Cas9 carrying the Cas9 gene and GFP-targeting guide RNA for CRISPR (Genaxxon bioscience, Cat. P2008.0010) in order to assess the functional activity, i.e. the expression of therapeutic gene at the protein level and ability of this protein to carry out target-specific endonuclease activity involving specific gRNA, which leads to the DNA sequence editing.

The following elements are indicated in Figure 11 : 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 DNA vector VTvafl7 devoid of cDNA of Cas9 gene, followed by transfection with the GFP-targeting guide RNA for CRISPR,

culture C - 293/GFP cells transfected with DNA vector VTvafl7-Cas9 carrying the Cas9 gene, followed by transfection with the GFP-targeting guide RNA for CRISPR,

culture D - 293/GFP cells transfected with DNA vector VTvafl7-Cas9 carrying the Cas9 gene, followed by transfection with water.

Figure 12

shows the results of PCR sequencing of fragments of several cell clones of MCF-7 lung adenocarcinoma cell line (ATCC® HTB-22 ™), in which the genome was edited after transfection of DNA vector VTvafl7-Cas9 carrying the Cas9 gene with gRNA_Cloning Vector carrying the gRNA sequence of TLR9 gene.

The figure shows the DNA region corresponding to the double-stranded break area introduced by sgRNA TLR l and TLR_2, where Tlrl2 is a consensus sequence; WT is a TLR9 gene fragment sequence that has not undergone genomic editing or with the restored sequence; all, al2, G2 are different alleles of heterozygous clones (the diagram shows both alleles).

Embodiment of the Invention

Gene therapy DNA vector carrying the Cas9 therapeutic gene intended for heterologous expression of this therapeutic gene in human and animal cells was produced based on 3165 bp DNA vector VTvafl7. The method of production of gene therapy DNA vector carrying the therapeutic gene involves cloning of the protein coding sequence of the Cas9 therapeutic gene (encodes the Cas9 endonuclease) to the polylinker of the gene therapy DNA vector VTvafl7. It is known that the ability of DNA vectors to penetrate into eukaryotic cells is due mainly to the vector size. DNA vectors with the smallest size have higher penetration capability. Thus, the absence of elements in the vector that bear no functional load, but at the same time increase the vector DNA size is preferred. These features of DNA vectors were taken into account during the production of gene therapy DNA vectors based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene with no large non-functional sequences and antibiotic resistance genes in the vector, which, in addition to technological advantages and safe use, allowed for the significant reduction of size of the produced gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene. Thus, the ability of the obtained gene therapy DNA vector to penetrate into eukaryotic cells is due to its small length.

Gene therapy DNA vector VTvafl7-Cas9 was produced as follows: the coding region of the Cas9 therapeutic gene was cloned to gene therapy DNA vector VTvafl7 and gene therapy DNA vector VTvafl7-Cas9, SEQ ID No. 1, was obtained. The coding region of 4221 bp Cas9 gene was produced by enzymic synthesis from chemically synthesised oligonucleotides. The synthesis product was cleaved by specific restriction endonucleases taking into account the optimal procedure for further cloning, and cloning to the gene therapy DNA vector VTvafl7 was performed by BamHI and EcoRI restriction sites located in the VTvafl7 vector polylinker. The selection of restriction sites was carried out in such a way that the cloned fragment entered the reading frame of expression cassette of the vector VTvafl7, while the protein coding sequence did not contain restriction sites for the selected endonucleases. Experts in this field realise that the methodological implementation of gene therapy DNA vector VTvafl7-Cas9 production can vary within the framework of the selection of known methods of molecular gene cloning and these methods are included in the scope of this invention. For example, different oligonucleotide sequences can be used to amplify Cas9 gene, different restriction endonucleases or laboratory techniques, such as ligation independent cloning of genes.

Gene therapy DNA vector VTvafl7-Cas9 has the nucleotide sequence SEQ ID No. 1. At the same time, degeneracy of genetic code is known to the experts in this field and means that the scope of this invention also includes variants of nucleotide sequences differing by insertion, deletion, or replacement of nucleotides that do not result in a change in the polypeptide sequence encoded by the therapeutic gene, and/or do not result in a loss of functional activity of the regulatory elements of VTvafl7 vector. At the same time, genetic polymorphism is known to the experts in this field and means that the scope of this invention also includes variants of nucleotide sequences of Cas9 gene that also encode different variants of the amino acid sequences of Cas9 protein that do not differ from those listed in their functional activity under physiological conditions.

The ability to penetrate into eukaryotic cells and express functional activity, i.e. the ability to express the therapeutic gene of the obtained gene therapy DNA vector VTvafl7-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 therapeutic gene. The presence of specific mRNA in cells into which the gene therapy DNA vector VTvafl7-Cas9 was introduced shows the ability of the obtained vector to both penetrate into eukaryotic cells and express mRNA of the Cas9 therapeutic gene. Furthermore, it is known to the experts in this field that the presence of mRNA gene is a mandatory condition, but not an evidence of the translation of protein encoded by the therapeutic gene. Therefore, in order to confirm properties of the gene therapy DNA vector VTvafl7-Cas9 to express the therapeutic gene at the protein level in eukaryotic cells into which the gene therapy DNA vector was introduced, analysis of the concentration of protein encoded by the therapeutic gene was carried out using immunological methods. The presence of Cas9 protein confirms the efficiency of expression of therapeutic genes in eukaryotic cells using the gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene. Thus in order to confirm the efficiency of the produced gene therapy DNA vector VTvafl7-Cas9 carrying the therapeutic gene, namely the Cas9 gene, the following methods were used:

A) real-time PCR, i.e. change in cDNA accumulation of therapeutic gene in human and animal cell lysate after transfection of different human and animal cell lines with gene therapy DNA vector,

B) Enzyme-linked immunosorbent assay, i.e. change in the quantitative level of therapeutic protein in the human, animal cell lysate after transfection of different human and animal cell lines with gene therapy DNA vector, C) Enzyme-linked immunosorbent assay, i.e. change in the quantitative level of therapeutic protein in the supernatant of animal tissue biopsies after the injection of autologous cells in these tissues transfected with gene therapy DNA vector,

D) Flow cytofluorimetry measurement of GFP gene expression in cells subjected to genome editing after combined transfection of these cells with the gene therapy DNA vector VTvafl7-Cas9 and gRNA resulting in inactivation of GFP gene and lack of fluorescent protein expression in cells or its considerable reduction,

E) Sequencing of DNA region of human and animal cells that has undergone a genome editing after combined transfection of these cells with gene therapy DNA vector VTvafl7-Cas9 and vector expressing gRNA targeted at the TLR9 gene sequence.

In order to confirm the practicability of use of the constructed gene therapy DNA vector VTvafl7-Cas9 carrying the therapeutic gene, namely the Cas9 gene, the following was performed:

A) transfection of different human and animal cell lines with gene therapy DNA vector,

B) combined transfection with gene therapy DNA vector and gRNA of different mammalian cell lines,

C) combined transfection with gene therapy DNA vector and vector encoding gRNA of different mammalian cell lines,

D) injection of autologous cells transfected with gene therapy DNA vector into animal tissues,

E) demonstration of changes in the expression of edited gene in the cell line subjected to the genome editing,

F) demonstration of changes in the sequence of the edited gene region in the cell line subjected to genome editing.

These methods of use lack potential risks for gene therapy of humans and animals due to the absence of regulatory elements in the gene therapy DNA vector that constitute the nucleotide sequences of viral genomes and absence of antibiotic resistance genes in the gene therapy DNA vector as confirmed by the lack of regions homologous to the viral genomes and antibiotic resistance genes in the nucleotide sequences of gene therapy DNA vector VTvafl7-Cas9 (SEQ ID No. 1).

It is known to the experts in this field that antibiotic resistance genes in the 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 this invention, in order to ensure the safe use of gene therapy DNA vector VTvafl7 carrying Cas9 therapeutic gene, the use of selective nutrient media containing an antibiotic is not possible. A method for obtaining strain for production of these gene therapy vectors based on Escherichia coli strain SCS110-AF is proposed as a technological solution for obtaining the gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene in order to scale up the production of gene therapy vector to an industrial scale. The method of production of Escherichia coli strain SCS 110-AF/VTvafl 7- Cas9 involves production of competent cells of Escherichia coli strain SCSI 10- AF with the injection of gene therapy DNA vector VTvafl7-Cas9 into these cells using transformation (electroporation) methods widely known to the experts in this field. The obtained Escherichia coli strain SCS110-AF/VTvafl7-Cas9 is used to produce the gene therapy DNA vector VTvafl7-Cas9 allowing for the use of antibiotic-free media.

In order to confirm the production of Escherichia coli strain SCSI 10- AF/VTvafl7-Cas9 transformation, selection, and subsequent biomass growth with extraction of plasmid DNA were performed.

To confirm the producibility and constructability of gene therapy DNA vector VTvafl7-Cas9 carrying the therapeutic gene, namely the Cas9 gene, on an industrial scale, large-scale fermentation of Escherichia coli strain Escherichia coli SCS110-AF/VTvafl7-Cas9 containing gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene was performed.

The method of scaling the production of bacterial mass to an industrial scale for the isolation of gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene involves incubation of the seed culture of Escherichia coli strain SCS110-AF/VTvafl7-Cas9 in the antibiotic-free nutrient medium that provides suitable biomass accumulation dynamics. Upon reaching a sufficient amount of biomass in the logarithmic phase, the bacterial culture is transferred to an industrial fermenter and then grown to a stationary phase, then the fraction containing the therapeutic DNA product, i.e. the gene therapy DNA vector VTvafl7-Cas9 is extracted, multi-stage filtered, and purified by chromatographic methods. It is known to the experts in this field that culture conditions of strains, composition of nutrient media (except for antibiotic-free), equipment used, and DNA purification methods may vary within the framework of standard operating procedures depending on the particular production line, but known approaches to scaling, industrial production, and purification of DNA vectors using Escherichia coli strain SCSI 10-AF/VTvafl7-Cas9 fall within the scope of this invention.

The described disclosure of the invention is illustrated by examples of the embodiment of this invention.

The essence of the invention is explained in the following examples.

Example 1.

Production of gene therapy DNA vector VTvafl7-Cas9 carrying the therapeutic gene, namely the Cas9 gene.

Gene therapy DNA vector VTvafl7-Cas9 was constructed by cloning the coding region of Cas9 gene (4221 bp) to a 3165 bp DNA vector VTvafl7 by BamHI and EcoRI restriction sites. The coding region of 4221 bp Cas9 gene was produced by enzymic synthesis from chemically synthesised oligonucleotides.

Gene therapy DNA vector VTvafl7 was constructed by consolidating six fragments of DNA derived from different sources:

(a) the origin of replication was produced by PCR amplification of a region of commercially available plasmid pBR322 with a point mutation,

(b) EFla promoter region was produced by PCR amplification of a site of human genomic DNA,

(c) hGH TA transcription terminator was produced by PCR amplification of a site of human genomic DNA,

(d) the RNAout regulatory site of transposon TnlO was synthesised from oligonucleotides, (e) kanamycin resistance gene was produced by PCR amplification of a site of commercially available human plasmid pET-28,

(f) the polylinker was produced by annealing two synthetic oligonucleotides.

PCR amplification was performed using the commercially available kit Phusion® High-Fidelity DNA Polymerase (New England Biolabs, USA) as per the manufacturer’s instructions. The fragments have overlapping regions allowing for their consolidation with subsequent PCR amplification. Fragments (a) and (b) were consolidated using oligonucleotides Ori-F and EF1-R, and fragments (c), (d), and (e) were consolidated using oligonucleotides hGH-F and Kan-R. Afterwards, the produced fragments were consolidated by restriction with subsequent ligation by sites BamHI and Ncol. This resulted in a plasmid still devoid of the polylinker. To add it, the plasmid was cleaved by BamHI and EcoRI sites followed by ligation with fragment (f). Therefore, a 4182 bp vector was constructed carrying the kanamycin resistance gene flanked by Spel restriction sites. Then this gene was cleaved by Spel restriction sites and the remaining fragment was ligated to itself. This resulted in a 3165 bp gene therapy DNA vector VTvafl7 that is recombinant and allows for antibiotic-free selection.

The amplification product of the coding region of Cas9 gene and DNA vector VTvafl7 was cleaved by BamHI and EcoRI restriction endonucleases (New England Biolabs, USA).

This resulted in a 7351 bp DNA vector VTvafl7-Cas9 with the nucleotide sequence SEQ ID No. 1 and general structure shown in Fig. 1.

Example 2.

Proof of the ability of gene therapy DNA vector VTvafl7-Cas9 carrying the therapeutic gene, namely Cas9 gene, to penetrate eukaryotic cells and its functional activity at the level of therapeutic gene mRNA expression. This example also demonstrates practicability of use of gene therapy DNA vector carrying the therapeutic gene.

Changes in the mRNA accumulation of the Cas9 therapeutic gene were assessed in HDFa primary human dermal fibroblast cell culture (ATCC PCS-201- 01) 48 hours after its transfection with gene therapy DNA vector VTvafl7-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.

HDFa primary human dermal fibroblast cell culture was used for the assessment of changes in the therapeutic Cas9 mRNA accumulation. HDFa cell culture was grown under standard conditions (37°C, 5% C02) using the Fibroblast Growth Kit-Serum-Free (ATCC® PCS-201-040). The growth medium was replaced every 48 hours during the cultivation process.

To achieve 90% confluence, 24 hours before the transfection procedure, the cells were seeded into a 24-well plate in the quantity of 5x 10⁴ cells per well. Transfection with gene therapy DNA vector VTvafl7-Cas9 expressing the human Cas9 gene was performed using Lipofectamine 3000 (ThermoFisher Scientific, USA) according to the manufacturer’s recommendations. In test tube 1, Imΐ of DNA vector VTvafl7-Cas9 solution (concentration 500ng/pl) and Imΐ of reagent P3000 was added to 25m1 of medium Opti-MEM (Gibco, USA). The preparation was mixed by gentle shaking. In test tube 2, Imΐ of Lipofectamine 3000 solution was added to 25m1 of medium Opti-MEM (Gibco, USA). The preparation was mixed by gentle shaking. The contents from test tube 1 were added to the contents of test tube 2, and the mixture was incubated at room temperature for 5 minutes. The resulting solution was added dropwise to the cells in the volume of 40 mΐ.

HDFa cells transfected with the gene therapy DNA vector VTvafl7 devoid of the inserted therapeutic gene (cDNA of Cas9 gene before and after transfection with gene therapy DNA vector VTvafl7 devoid of the inserted therapeutic gene is not shown in the figures) were used as a reference. Reference vector VTvafl7 for transfection was prepared as described above.

Total RNA from HDFa cells was extracted using Trizol Reagent (Invitrogen, USA) according to the manufacturer’s recommendations. 1ml of Trizol Reagent was added to the well with cells and homogenised and heated for 5 minutes at 65°C. Then the sample was centrifuged at 14,000g for 10 minutes and heated again for 10 minutes at 65°C. Then 200m1 of chloroform was added, and the mixture was gently stirred and centrifuged at 14,000g for 10 minutes. Then the water phase was isolated and mixed with 1/10 of the volume of 3M sodium acetate, pH 5.2, and an equal volume of isopropyl alcohol. The sample was incubated at -20°C for 10 minutes and then centrifuged at 14,000g for 10 minutes. The precipitated RNA were rinsed in 1ml of 70% ethyl alcohol, air-dried and dissolved in 10m1 of RNase-free water. The level of Cas9 mRNA expression after transfection was determined by assessing the dynamics of the accumulation of cDNA amplicons by real-time PCR. For the production and amplification of cDNA specific for the human Cas9 gene, the Cas9_SF and Cas9_SR oligonucleotides were used (list of sequences (1), (2)).

The length of amplification product is 275 bp.

Reverse transcription reaction and PCR amplification was performed using SYBR GreenQuantitect RT-PCR Kit (Qiagen, USA) for real-time PCR. The reaction was carried out in a volume of 20m1, containing: 25m1 of QuantiTect SYBR Green RT-PCR Master Mix, 2.5mM of magnesium chloride, 0.5 mM of each primer, and 5m1 of RNA. For the reaction, CFX96 amplifier (Bio-Rad, USA) was used under the following conditions: 1 cycle of reverse transcription at 42°C for 30 minutes, denaturation at 98°C for 15 minutes, followed by 40 cycles comprising denaturation at 94°C for 15s, annealing of primers at 60°C for 30s and elongation at 72°C for 30s. B2M (beta-2-microglobuline) gene listed in the GenBank database under number NM 004048.2 was used as a reference gene. Positive control included amplicons from PCR on matrices represented by plasmids in known concentrations containing cDNA sequences of Cas9 and B2M genes. Negative control included deionised water. Real-time quantification of the dynamics of accumulation of cDNA amplicons of Cas9 and B2M genes was conducted using the Bio-Rad CFX Manager 2.1 software (Bio-Rad, USA). Diagrams resulting from the assay are shown in Figure 2.

Figure 2 shows that the Cas9 gene was found after transfection of HDFa primary human fibroblast cell culture with gene therapy DNA vector VTvafl7- Cas9, which confirms the ability of the vector to penetrate eukaryotic cells and express the Cas9 gene at the mRNA level. The presented results also confirm the practicability of use of gene therapy DNA vector VTvafl7-Cas9 for heterologous expression of Cas9 gene in eukaryotic cells.

Example 3.

Changes in the mRNA accumulation of the Cas9 therapeutic gene were assessed in HEKa primary human epidermal keratinocyte cell culture (ATCC PCS-200-011) 48 hours after its transfection with gene therapy DNA vector VTvafl7-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.

HEKa primary human epidermal keratinocyte cell culture was grown in Keratinocyte Growth Kit (ATCC® PCS-200-040™) under standard conditions (37°C, 5% C02). To achieve 90% confluence, 24 hours before the transfection procedure, the cells were seeded into a 24-well plate in the quantity of 5xl0⁴ cells per well. Lipofectamine 3000 (ThermoFisher Scientific, USA) was used as a transfection reagent. The transfection with gene therapy DNA vector VTvafl7- Cas9 expressing the human Cas9 gene was performed according to the procedure described in Example 2. B2M (beta-2 -microglobuline) gene listed in the GenBank database under number NM 004048.2 was used as a reference gene. HEKa cell culture transfected with the gene therapy DNA vector VTvafl7 devoid of the therapeutic gene (cDNA of Cas9 gene before and after transfection with gene therapy DNA vector VTvafl7 devoid of the inserted therapeutic gene is not shown in the figures) was used as a reference. RNA isolation, reverse transcription reaction, and real-time PCR were performed as described in Example 2.

Positive control included amplicons from PCR on matrices represented by plasmids in known concentrations containing cDNA sequences of Cas9 and B2M genes. Negative control included deionised water. Real-time quantification of the PCR products, i.e. Cas9 and B2M gene cDNAs obtained by amplification, was conducted using the Bio-Rad CFX Manager 2.1 software (Bio-Rad, USA). Diagrams resulting from the assay are shown in Figure 3.

Figure 3 shows that the Cas9 gene was found after transfection of HEKa cell culture with gene therapy DNA vector VTvafl7-Cas9, which confirms the ability of the vector to penetrate eukaryotic cells and express the Cas9 gene at the mRNA level. The presented results also confirm the practicability of use of gene therapy DNA vector VTvafl7-Cas9 for heterologous expression of Cas9 gene in eukaryotic cells.

Example 4.

Changes in the mRNA accumulation of the Cas9 therapeutic gene were assessed in Primary Epidermal Melanocytes; Normal, Human, Adult (HEMa) (ATCC® PCS-200-013™) 48 hours after their transfection with gene therapy DNA- vector VTvafl7-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.

Primary Epidermal Melanocyte human cell culture was grown in medium prepared using Adult Melanocyte Growth Kit (ATCC® PCS-200-042™) under standard conditions (37°C, 5% C02). To achieve 90% confluence, 24 hours before the transfection procedure, the cells were seeded into a 24-well plate in the quantity of 5x10⁴ cells per well. Lipofectamine 3000 (ThermoFisher Scientific, USA) was used as a transfection reagent. The transfection with gene therapy DNA vector VTvafl7-Cas9 expressing the human Cas9 gene was performed according to the procedure described in Example 2. B2M (beta-2-microglobuline) gene listed in the GenBank database under number NM 004048.2 was used as a reference gene. HEMa human epidermal melanocyte cell culture transfected with the gene therapy DNA vector VTvafl7 devoid of the therapeutic gene (cDNA of Cas9 gene before and after transfection with gene therapy DNA vector VTvafl7 devoid of the inserted therapeutic gene is not shown in the figures) was used as a reference. RNA isolation, reverse transcription reaction, and real-time PCR were performed as described in Example 2.

Positive control included amplicons from PCR on matrices represented by plasmids in known concentrations containing cDNA sequences of Cas9 and B2M genes. Negative control included deionised water. Real-time quantification of the PCR products, i.e. Cas9 and B2M gene cDNAs obtained by amplification, was conducted using the Bio-Rad CFX Manager 2.1 software (Bio-Rad, USA). Diagrams resulting from the assay are shown in Figure 4.

Figure 4 shows that the Cas9 gene was found after transfection of HEMa human primary epidermal melanocyte cell culture with gene therapy DNA vector VTvafl7-Cas9, which confirms the ability of the vector to penetrate eukaryotic cells and express the Cas9 gene at the mRNA level. The presented results also confirm the practicability of use of gene therapy DNA vector VTvafl7-Cas9 for heterologous expression of Cas9 gene in eukaryotic cells.

Example 5.

Proof of the efficiency and practicability of use of gene therapy DNA vector VTvafl7-Cas9 carrying the Cas9 gene for heterologous expression of Cas9 protein in mammalian cells.

The change in the Cas9 protein concentration in the lysate of HDFa primary human dermal fibroblast cell culture (ATCC PCS-201-01) was assessed after transfection of these cells with DNA vector VTvafl7-Cas9 carrying the human Cas9 gene. Cells were grown as described in Example 2.

The 6th generation SuperFect Transfection Reagent (Qiagen, Germany) was used for transfection. The aqueous dendrimer solution without DNA vector (A) and DNA vector VTvafl7 devoid of cDNA of Cas9 gene (B) were used as a reference, and DNA vector VTvafl7-Cas9 carrying the human Cas9 gene (C) was used as the transfected agent. Transfection of HDFa cells was performed according to the procedure described in Example 2.

After transfection, 0.1ml of IN HC1 were added to 0.5ml of the culture broth, mixed thoroughly, and incubated for 10 minutes at room temperature. Then, the mixture was neutralised by adding 0.1ml of 1.2M NaOH/0.5M HEPES (pH 7-7.6) and stirred thoroughly. Supernatant was collected and used to assay the therapeutic protein. The Cas9 protein was assayed by 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 method with optical density detection using ChemWell Automated EIA and Chemistry Analyser (Awareness Technology Inc., USA).

To measure the numerical value of concentration, the calibration curve constructed using the reference samples from the kit with known concentrations of Cas9 protein was used. The sensitivity was at least 1.5pg/ml, measurement range - from 1.56pg/ml to lOOpg/ml. R-3.0.2 was used for the statistical treatment of the results and data visualization (https://www.r-project.org/). Diagrams resulting from the assay are shown in Figure 5.

Figure 5 shows that the Cas9 gene was found compared to its lack in reference samples after transfection of HDFa primary human cell culture with gene therapy DNA vector VTvafl7-Cas9, which confirms the ability of the vector to penetrate eukaryotic cells and express the Cas9 gene at the protein level. The presented results also confirm the practicability of use of gene therapy DNA vector VTvafl7-Cas9 for heterologous expression of Cas9 gene in eukaryotic cells.

Example 6.

The change in the Cas9 protein concentration in the cell lysate of HEKa primary human epidermal keratinocyte cell culture (ATCC PCS-200-011) was assessed after transfection of these cells with the DNA vector VTvafl7-Cas9 carrying the human Cas9 gene. Cells were grown as described in Example 3.

The 6th generation SuperFect Transfection Reagent (Qiagen, Germany) was used for transfection. The aqueous dendrimer solution without DNA vector (A) and DNA vector VTvafl7 devoid of cDNA of Cas9 gene (B) were used as a reference, and DNA vector VTvafl7-Cas9 carrying the human Cas9 gene (C) was used as the transfected agent. The DNA-dendrimer complex was prepared and cells were transfected according to the manufacturer’s procedure.

After transfection, 0.1ml of IN HC1 were added to 0.5ml of the culture broth, mixed thoroughly, and incubated for 10 minutes at room temperature. Then, the mixture was neutralised by adding 0.1ml of 1.2M NaOH/0.5M HEPES (pH 7-7.6) and stirred thoroughly. Supernatant was collected and used to assay the therapeutic protein. The Cas9 protein was assayed by enzyme-linked immunosorbent assay 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 ChemWell Automated EIA and Chemistry Analyser (Awareness Technology Inc., USA).

To measure the numerical value of concentration, the calibration curve constructed using the reference samples from the kit with known concentrations of Cas9 protein was used. The sensitivity was at least 1.5pg/ml, measurement range - from 1.56pg/ml to lOOpg/ml. R-3.0.2 was used for the statistical treatment of the results and data visualization (https://www.r-project.org/). Diagrams resulting from the assay are shown in Figure 6.

Figure 6 shows that the Cas9 gene was found compared to its lack in reference samples after transfection of HEKa primary human epidermal keratinocyte cell culture with gene therapy DNA vector VTvafl7-Cas9, which confirms the ability of the vector to penetrate eukaryotic cells and express the Cas9 gene at the protein level. The presented results also confirm the practicability of use of gene therapy DNA vector VTvafl7-Cas9 for heterologous expression of Cas9 gene in eukaryotic cells.

Example 7.

The change in the Cas9 protein concentration in the cell lysate of Primary Epidermal Melanocyte cell culture; Normal, Human, Adult (HEMa) (ATCC® PCS-200-013™) was assessed after transfection of these cells with gene therapy DNA- vector VTvafl7-Cas9 carrying the human Cas9 gene. Cells were grown as described in Example 4.

The 6th generation SuperFect Transfection Reagent (Qiagen, Germany) was used for transfection. The aqueous dendrimer solution without DNA vector (A) and DNA vector VTvafl7 devoid of cDNA of Cas9 gene (B) were used as a reference, and DNA vector VTvafl7-Cas9 carrying the human Cas9 gene (C) was used as the transfected agent. The DNA-dendrimer complex was prepared and HEMa cells were transfected according to the manufacturer’s procedure.

To measure the numerical value of concentration, the calibration curve constructed using the reference samples from the kit with known concentrations of Cas9 protein was used. The sensitivity was at least 1.5pg/ml, measurement range - from 1.56pg/ml to 100pg/ml. R-3.0.2 was used for the statistical treatment of the results and data visualization (https://www.r-project.org/). Diagrams resulting from the assay are shown in Figure 7.

Figure 7 shows that the Cas9 gene was found compared to its lack in reference samples after transfection of HEMa primary human cell culture with gene therapy DNA vector VTvafl7-Cas9, which confirms the ability of the vector to penetrate eukaryotic cells and express the Cas9 gene at the protein level. The presented results also confirm the practicability of use of gene therapy DNA vector VTvafl7-Cas9 for heterologous expression of Cas9 gene in eukaryotic cells.

Example 8.

Proof of the efficiency and practicability of use of gene therapy DNA vector VTvafl7-Cas9 carrying the Cas9 gene for heterologous expression of Cas9 protein in human cells.

The change in the Cas9 protein concentration in the lysate of CHO-K1 Syrian hamster ovary cells (ATCC® CCL-61™) was assessed after transfection of these cells with DNA vector VTvafl7-Cas9 carrying the human Cas9 gene. Cells were grown in F-12K Medium (Kaighn’s Modification of Ham’s F-12 Medium) (ATCC® 30-2004™) with the addition of 10% Fetal Bovine Serum (FBS) (ATCC® 30-2020™) under standard conditions.

After transfection, 0.1ml of IN HC1 were added to 0.5ml of the cell-rich fluid, mixed thoroughly, and incubated for 10 minutes at room temperature. Then, the mixture was neutralised by adding 0.1ml of 1.2M NaOH/0.5M HEPES (pH 7- 7.6) and stirred thoroughly. Supernatant was collected and used to assay the therapeutic protein. The Cas9 protein was assayed by 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 method with optical density detection using ChemWell Automated EIA and Chemistry Analyser (Awareness Technology Inc., USA).

To measure the numerical value of concentration, the calibration curve constructed using the reference samples from the kit with known concentrations of Cas9 protein was used. The sensitivity was at least 1.5pg/ml, measurement range - from 1.56pg/ml to lOOpg/ml. R-3.0.2 was used for the statistical treatment of the results and data visualization (https://www.r-project.org/). Diagrams resulting from the assay are shown in Figure 8.

Figure 8 shows that the Cas9 protein was found compared to its lack in reference samples after transfection of CHO-K1 cell culture with gene therapy DNA vector VTvafl7-Cas9, which confirms the ability of the vector to penetrate eukaryotic animal cells and express the Cas9 gene at the protein level. The presented results also confirm the practicability of use of gene therapy DNA vector VTvafl7-Cas9 for heterologous expression of Cas9 gene in eukaryotic animal cells.

Example 9. Proof of the efficiency and practicability of use of gene therapy DNA vector VTvafl7-Cas9 carrying the Cas9 gene for heterologous expression of Cas9 protein in human cells.

The change in the Cas9 protein concentration in the lysate of (PBMC) primary human peripheral blood mononuclear cell culture was assessed after transfection of these cells with DNA vector VTvafl7-Cas9 carrying the human Cas9 gene. PBMC cells were isolated from 10ml of human venous blood by separating fractions in Ficoll gradient 1.119 (PanEco, P051-1). The cells were grown in RPMI-1640 medium (PanEco, C310p) under standard conditions (37°C, 5% C02).

24 hours after transfection, 1ml of cell culture was pelleted, 0.1ml of IN HC1 was added to the precipitated cells, mixed thoroughly and incubated for 10 minutes at room temperature. Then, the mixture was neutralised by adding 0.1ml of 1.2M NaOH/0.5M HEPES (pH 7-7.6) and stirred thoroughly. Supernatant was collected and used to assay the therapeutic protein. The Cas9 protein was assayed by 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 method with optical density detection using ChemWell Automated EIA and Chemistry Analyser (Awareness Technology Inc., USA).

To measure the numerical value of concentration, the calibration curve constructed using the reference samples from the kit with known concentrations of Cas9 protein was used. The sensitivity was at least 1.5pg/ml, measurement range - from 1.56pg/ml to lOOpg/ml. R-3.0.2 was used for the statistical treatment of the results and data visualization (https://www.r-project.org/). Diagrams resulting from the assay are shown in Figure 9.

Figure 9 shows that the Cas9 protein was found compared to its lack in reference samples after transfection of PBMC cell culture with gene therapy DNA vector VTvafl7-Cas9, which confirms the ability of the vector to penetrate human cells and express the Cas9 gene at the protein level. The presented results also confirm the practicability of use of gene therapy DNA vector VTvafl7-Cas9 for heterologous expression of Cas9 gene in eukaryotic mammalian cells.

Example 10.

Proof of the efficiency of gene therapy DNA vector VTvafl7-Cas9 carrying the Cas9 gene and practicability of its use in order to increase the expression level of the Cas9 protein in animal tissues by injecting autologous fibroblasts transfected with gene therapy DNA vector VTvafl7-Cas9.

To confirm the efficiency of gene therapy DNA vector VTvafl7-Cas9 carrying the Cas9 gene and practicability of its use, changes in the Cas9 protein concentration in rat skin upon injection of autologous fibroblast culture of the same animal transfected with gene therapy DNA vector VTvafl7-Cas9 were assessed.

The appropriate autologous fibroblast culture transfected with the gene therapy DNA vector VTvafl7-Cas9 carrying the Cas9 gene was injected into the skin of three Wistar rats with concurrent injection of a placebo in the form of animal autologous fibroblast culture transfected with gene therapy DNA vector VTvafl 7 not carrying the Cas9 gene.

The primary rat fibroblast culture was isolated from the animal skin biopsy samples. The skin biopsy sample weighing about 11 mg was taken using the skin biopsy device Epitheasy 3.5 (Medax SRL, Italy). The primary cell culture was cultivated at 37°C in the presence of 5% C02, in the DMEM medium with 10% fetal bovine serum and lOOU/ml of ampicillin. The passage and change of culture medium were performed every 2 days. Total duration of culture growth did not exceed 25-30 days. Then an aliquot of 5><10⁴ cells was taken from the cell culture. The rat fibroblast culture was transfected with the gene therapy DNA vector VTvafl 7-Cas9 carrying the Cas9 gene or placebo, i.e. VTvafl7 vector not carrying the Cas9 therapeutic gene.

The transfection was carried out using a cationic polymer such as polyethyleneimine JETPEI (Polyplus transfection, France), according to the manufacturer’s instructions. The cells were cultured for 72 hours and then injected into the animals. Injection of autologous fibroblast culture into the animal transfected with gene therapy DNA vector VTvafl7-Cas9, and autologous rat fibroblast culture non-transfected with gene therapy DNA vector VTvafl7 as a placebo was performed in the preliminary epilated area using the tunnel method with a 27G needle to the depth of approximately 1mm. The concentration of the modified autologous fibroblasts in the injected suspension was approximately 5 min cells per 1ml of the suspension, the dose of the injected cells did not exceed 10 min. The points of injection of the autologous fibroblast culture were located at 3 to 5cm intervals.

Biopsy samples were taken on the 4th day after the injection of autologous fibroblast culture transfected with the gene therapy DNA vector VTvafl7-Cas9 carrying the therapeutic gene, namely Cas9 gene, and placebo. Biopsy was taken from the animal skin in the site of injection of autologous fibroblast culture transfected with gene therapy DNA vector VTvafl7-Cas9 carrying the therapeutic gene, namely Cas9 gene (I), autologous fibroblast culture transfected with gene therapy DNA vector VTvafl7 not carrying the Cas9 therapeutic gene (placebo) (II), as well as from intact skin site (III) using the skin biopsy device Epitheasy 3.5 (Medax SRL, Italy). The biopsy sample size was ca. 10mm3, and the weight was approximately 11 mg. The sample was placed in a buffer solution containing 50mM of Tris-HCl, pH 7.6, lOOmM of NaCl, ImM of EDTA, and ImM of phenylmethylsulfonyl fluoride, and homogenised to obtain a homogenised suspension. The suspension was then centrifuged for 10 minutes at 14,000g. Supernatant was collected and used to assay the therapeutic protein as described in Examples 5-9.

Diagrams resulting from the assay are shown in Figure 10.

Figure 10 shows that Cas9 protein was found compared to its lack in reference samples in the skin of rats in the injection area of autologous fibroblast culture transfected with gene therapy DNA vector VTvafl7-Cas9 carrying the Cas9 gene: in the region of injection of injection of autologous fibroblasts transfected gene therapy DNA vector VTvafl7 not carrying the Cas9 gene (placebo) and in the sample from the intact site, which indicates the efficiency of gene therapy DNA vector DNA VTvafl7-Cas9 and confirms the practicability of its use in order to increase the Cas9 expression level in mammalian tissues, in particular upon injection of autologous fibroblasts transfected with the gene therapy vector DNA VTvafl 7-Cas9.

Example 11.

Proof of the efficiency and practicability of use of gene therapy DNA vector VTvafl 7-Cas9 carrying the Cas9 gene for genome editing in mammalian cells.

The cell fluorescence reduction due to the disruption of GFP gene expression in 293/GFP Cell Line cells (Cell Biolabs, Cat. AKR-200) was assessed after combined transfection of these cells with DNA vector VTvafl 7-Cas9 carrying the human Cas9 gene and GFP-targeting guide RNA for CRISPR (Genaxxon bioscience, Cat. P2008.0010). Cells were grown in DMEM medium under standard conditions.

For transfection of cells with DNA vector VTvafl 7-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 DNA vector VTvafl 7-Cas9 carrying the Cas9 gene, cells were transfected with the GFP-targeting guide RNA for CRISPR. For the transfection of GFP-targeting guide RNA for CRISPR, CRISPRfect E transfection reagent (Genaxxon bioscience, Cat. P2002.0035) was used according to the manufacturer's instructions.

Transfection with water was used as experimental samples, followed by transfection with the GFP-targeting guide RNA for CRISPR (A), transfection with DNA vector VTvafl7 devoid of cDNA of Cas9 gene, followed by transfection with GFP-targeting guide RNA for CRISPR (B), transfection with DNA vector VTvafl 7-Cas9 carrying the Cas9 gene, followed by transfection with GFP- targeting guide RNA for CRISPR (C), transfection with DNA vector VTvafl 7- Cas9 vector carrying the Cas9 gene, followed by transfection with water (D).

48 hours after the second transfection, the culture medium was removed, cells were resuspended in physiological saline and used for the assessment of the number of cells expressing GFP fluorescent protein by flow fluorometry method using a Beckman Coulter’s Cytomics FC 500 (Beckman Coulter’s, USA). Diagrams resulting from the assay are shown in Figure 11. Figure 11 shows that the number of cells expressing GFP was reduced (approximately 80%) compared to no reduction in reference samples as a result of transfection of the 293/GFP primary cell culture with gene therapy DNA vector VTvafl7-Cas9 followed by the transfection of GFP-targeting guide RNA for CRISPR, which confirms the ability of the vector to penetrate into eukaryotic cells and express the active Cas9 endonuclease editing the GFP gene by GFP- targeting guide RNA, which leads to the GFP fluorescent protein silencing. The presented results also confirm the practicability of use of gene therapy DNA vector VTvafl7-Cas9 for targeted genome editing in eukaryotic cells.

Example 12.

Proof of the efficiency and practicability of use of gene therapy DNA vector VTvafl7-Cas9 carrying the Cas9 gene for genome editing in mammalian cells.

MCF7 cell line clones (ATCC® HTB-22™) were screened in order to identify clones with genome editing after transfection of these cells with a mixture of DNA vector VTvafl7-Cas9 carrying the Cas9 gene with gRNA Cloning Vector carrying oligonucleotides to the exon regions of TLR9 gene.

Specific oligonucleotides corresponding to 20 nucleotide sites of the TLR9 gene exon (Toll-like receptor 9) were selected as sgRNA. SgRNA selection was performed using the CRISPR DESIGN service (http://crispr.mit.edu/). Two synthesised oligonucleotides tlrg4f and tlrg4r (list of sequences (3) and (4)) were mixed in equimolar amounts in T4 DNA ligase buffer (Thermo Scientific, USA), samples were heated at 94°C for 2 min, then slowly cooled down to room temperature for the formation of duplexes. Then oligonucleotide duplexes were cloned into gRNA_Cloning Vector (AddGene, #41824). Plasmids with cloned oligonucleotides were expanded and purified in preparative amounts using the Plasmid DNA Purification Kit (Qiagen, USA).

MCF-7 cells were cultured using EMEM Eagle’s Minimum Essential Medium (EMEM) (ATCC® 30-2003™) under standard conditions (37°C, 5% C02). Transfection was performed using the Lipofectamine 3000 kit (Thermoscientific, USA) according to the manufacturer's instructions. An equimolar mixture of DNA vector VTvafl7-Cas9 carrying the Cas9 gene with gRNA Cloning Vector carrying the oligonucleotides to exon regions of the TLR9 gene was used for transfection. Water or gRNA Cloning Vector carrying oligonucleotides to exon regions of the TLR9 gene, or DNA vector VTvafl7-Cas9 carrying the Cas9 gene were used as a reference.

Then the cells were seeded into a 96-well plate by FACS sorter. The cells growing in 96-well plates were rinsed with PBS, lysed in 50m1 of DNA express reagent (Lytech, Russia) in order to analyse the clones grown after sorting, and then sample preparation was performed according to the manufacturer's recommendations. The obtained samples were used as a matrix for amplification of locus region containing the sgRNAs recognition sites in the TLR9 gene by realtime PCR. The obtained PCR fragments were sequenced for analysis using the ABI Prism 3730x1 Genetic Analyser (Applied Biosystems, USA). As a result, four clones containing changes in the nucleotide sequences of TLR9 gene and resulting from targeted genome editing were identified. No clones containing any changes in the TLR9 gene sequences were identified in reference samples.

The data obtained from the analysis are shown in Figure 12.

Figure 12 shows several clones with targeted genome editing identified due to the transfection of MCF7 cell line with a mixture of DNA vector VTvafl7- Cas9 carrying the Cas9 gene and gRNA Cloning Vector carrying oligonucleotides to the exon regions of TLR9 gene, which confirms the ability of DNA vector VTvafl7-Cas9 to penetrate eukaryotic cells and express active Cas9 endonuclease, which is able to introduce a mutation into the therapeutic gene by gRNA, for example, into TLR9 gene, as shown by sequencing of targeted sequence. The presented results also confirm the practicability of use of gene therapy DNA vector VTvafl7-Cas9 for targeted genome editing in eukaryotic cells.

Example 13.

Escherichia coli strain SCSI 10-AF/VTvafl7-Cas9 carrying the gene therapy DNA vector, and the method of its production.

The strain construction for the production of gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying Cas9 therapeutic gene on an industrial scale, namely, Escherichia coli strain SCS 110-AF/VTvafl 7-Cas9 carrying the gene therapy DNA vector VTvafl7-Cas9 for its production allowing for antibiotic-free selection involves making electrocompetent cells of Escherichia coli strain SCS110-AF and subjecting these cells to electroporation with gene therapy DNA vector VTvafl7-Cas9. After that, the cells were poured into agar plates (Petri dishes) with a selective medium containing yeastrel, peptone, 6% sucrose, and 10pg/ml of chloramphenicol. At the same time, production of Escherichia coli strain SCS110-AF for the production of gene therapy DNA vector VTvafl7 or gene therapy DNA vectors based on it allowing for antibiotic- free positive selection involves constructing a 64 bp linear DNA fragment that contains regulatory element RNA-IN of transposon TnlO allowing for antibiotic- free positive selection, a 1422 bp levansucrase gene sacB, the product of which ensures selection within a sucrose-containing medium, a 763 bp chloramphenicol resistance gene catR required for the selection of strain clones in which homologous recombination occurs, and two homologous sequences, 329 bp and 233 bp, ensuring homologous recombination in the region of gene recA concurrent with gene inactivation, and then the Escherichia coli cells are transformed by electroporation, and clones surviving in a medium containing 10pg/ml of chloramphenicol are selected.

The obtained strain for production was included in the collection of the National Biological Resource Centre - Russian National Collection of Industrial Microorganisms (NBRC RNCIM), RF under the following registration numbers:

Escherichia coli strain SCS 110-AF/VTvafl 7-Cas9 — registered at the Russian National Collection of Industrial Microorganisms under number: B-, date of deposit:

Example 14.

The method for scaling up of the gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene to an industrial scale.

To confirm the producibility and constructability of gene therapy DNA vector VTvafl7-Cas9 (SEQ ID No. 1) on an industrial scale, a large-scale fermentation of Escherichia coli strain SCSI 10-AF/VTvafl7-Cas9 containing gene therapy DNA vector VTvafl7 carrying the therapeutic gene, namely Cas9 gene, was performed. Escherichia coli strain SCS110-AF/VTvafl7-Cas9 was produced based on Escherichia coli strain SCS110-AF (Cell and Gene Therapy LLC, United Kingdom) as per Example 13 by electroporation of competent cells of this strain by gene therapy DNA vector VTvafl 7-Cas9 carrying the therapeutic gene, namely Cas9 gene, with further inoculation of transformed cells into agar plates (Petri dishes) with a selective medium containing yeastrel, peptone, and 6% sucrose, and selection of individual clones.

Fermentation of Escherichia coli strain SCS110-AF/VTvafl7-Cas9 carrying gene therapy DNA vector VTvafl 7-Cas9 was performed in a 101 fermenter with subsequent extraction of gene therapy DNA vector VTvafl 7-Cas9.

For the fermentation of Escherichia coli strain SCS110-AF/VTvafl7-Cas9, medium containing the following ingredients per 101 of volume was prepared: lOOg of tryptone and 50g of yeastrel (Becton Dickinson, USA); then the medium was diluted with water to 8800ml and autoclaved at 121°C for 20 minutes, and then 1200ml of 50% (w/v) sucrose was added. After that, the seed culture of Escherichia coli strain SCSI 10-AF/VTvafl7-Cas9 was inoculated into a culture flask in the volume of 100ml. The culture was incubated in an incubator shaker for 16 hours at 30°C. The seed culture was transferred to the Techfors S bioreactor (Infors HT, Switzerland) and grown to a stationary phase. The process was controlled by measuring optical density of the culture at 600nm. The cells were pelleted for 30 minutes at 5,000-10,000g. Supernatant was removed, and the cell pellet was re-suspended in 10% (by volume) phosphate buffered saline. The cells were centrifuged again for 30 minutes at 5,000-10,000g. Supernatant was removed, a solution of 20mM TrisCl, ImM EDTA, 200g/l sucrose, pH 8.0 was added to the cell pellet in the volume of 1000ml, and the mixture was stirred thoroughly to a homogenised suspension. Then egg lysozyme solution was added to the final concentration of 100pg/ml. The mixture was incubated for 20 minutes on ice while stirring gently. Then 2500ml of 0.2M NaOH, lOg/1 sodium dodecyl sulphate (SDS) was added, the mixture was incubated for 10 minutes on ice while stirring gently, then 3500ml of 3M sodium acetate, 2M acetic acid, pH 5-5.5 was added, and the mixture was incubated for 10 minutes on ice while stirring gently. The resulting sample was centrifuged for 20-30 minutes at 15,000g or a greater value. The solution was decanted delicately, and residual precipitate was removed by passing through a coarse filter (filter paper). Then, RNase A (Sigma, USA) was added to the final concentration of 20pg/ml, and the solution was incubated overnight for 16 hours at room temperature. The solution was then centrifuged for 20-30 minutes at 15,000g and passed through a 0.45pm membrane filter (Millipore, USA). Then, ultrafiltration was performed with a lOOkDa membrane (Millipore, USA) and the mixture was diluted to the initial volume with a buffer solution of 25mM TrisCl, pH 7.0. This manipulation was performed three to four times. The solution was applied to the column with 250ml of DEAE Sepharose HP (GE, USA), equilibrated with 25mM TrisCl, pH 7.0. After the application of the sample, the column was washed with three volumes of the same solution and then gene therapy DNA vector VTvafl7-Cas9 was eluted using a linear gradient of 25mM TrisCl, pH 7.0, to obtain a solution of 25mM TrisCl, pH 7.0, 1M NaCl, five times the volume of the column. The elution process was controlled by measuring optical density of the run-off solution at 260nm. Chromatographic fractions containing gene therapy DNA vector VTvafl7-Cas9 were joined together and subjected to gel filtration using Superdex 200 (GE, USA). The column was equilibrated with phosphate buffered saline. The elution process was controlled by measuring optical density of the run-off solution at 260nm, and the fractions were analysed by agarose gel electrophoresis. The fractions containing gene therapy DNA vector VTvafl7-Cas9 were joined together and stored at - 20°C. To assess the process reproducibility, the indicated processing operations were repeated five times.

The process reproducibility and quantitative characteristics of final product yield confirm the producibility and constructability of gene therapy DNA vector VTvafl7-Cas9 on an industrial scale.

Thus, the constructed gene therapy DNA vector with therapeutic gene can be used for the injection into human, animal and mammalian cells, providing heterologous expression of Cas9 endonuclease, which can be used for the human and animal genome sequence editing in the presence of specific gRNA.

The purpose set in this invention, namely, the construction of gene therapy DNA vector for the heterologous expression of Cas9 gene in human and animal cells, combining the following properties:

I) Efficiency of gene therapy DNA vector for the heterologous expression of therapeutic genes in eukaryotic cells, II) The possibility of safe use for the implementation of various methods of genome editing of human and animal genomes, including human and animal gene therapy due to the lack of regulatory elements in the gene therapy DNA vector, which are the nucleotide sequences of viral genomes,

III) The possibility of safe use for the implementation of various methods of genomic editing of the genomes of humans and animals, including the genetic therapy of humans and animals due to the lack of antibiotic resistance genes in the gene therapy DNA vector,

IV) Producibility and constructability of gene therapy DNA vector on an industrial scale,

as well as the purpose of the construction of strain carrying these gene therapy DNA vector for the production of this gene therapy DNA vector is achieved, which is supported by the following examples:

for Item I - Example 1, 2, 3, 4, 5; 6; 7; 8; 9; 10; 11;

for Item II and Item III - Example 1 , 11, 12;

for Item IV - Example 1, 13, 14.

Industrial Applicability

All the examples listed above confirm industrial applicability of the proposed gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene for the heterologous expression of Cas9 gene in human and animal cells, Escherichia coli strain SCS 110-AF/VTvaf 17-Cas9 carrying gene therapy DNA vector, method of its production, method of gene therapy DNA vector production on an industrial scale.

List of Abbreviations

VTvafl 7 - Gene therapy vector devoid of sequences of viral genomes and antibiotic resistance markers (vector therapeutic virus-antibiotic-free)

gRNA - guided RNA

PBMC - peripheral blood mononuclear cells

DNA - Deoxyribonucleic acid

cDNA - Complementary deoxyribonucleic acid

RNA - Ribonucleic acid

mRNA - Messenger ribonucleic acid

bp - base pair

PCR - Polymerase chain reaction

ml - millilitre, mΐ - microlitre

mm3 - cubic millimetre

1 - litre

pg - microgram

mg - milligram

g - gram

mM - micromol

mM - millimol

min - minute

s - second

rpm - rotations per minute

nm - nanometre

cm - centimetre

mW - milliwatt

RFU - Relative fluorescence unit

PBS - 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_guideli ne/2015/05/WC500187020.pdf

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

4. Homstein BD, Roman D, Arevalo-Soliz LM, Engevik MA, Zechiedrich L.

Effects of Circular DNA Length on Transfection Efficiency by Electroporation into HeLa Cells. Cena V, ed. PLoS ONE. 2016;1 l(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.

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

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

. 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

1. 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, Vol. 45, No. 1, p. 44-55

Claims

1. Gene therapy DNA vector based on the gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene for heterologous expression of this therapeutic gene in human and animal cells in the implementation of various genome editing methods, while gene therapy DNA vector VTvafl7-Cas9 has the nucleotide sequence SEQ ID No. 1.

2. Gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene as per claim 1. Said DNA vector is unique due to the fact that the constructed gene therapy DNA vector VTvafl7-Cas9 as per claim 1 due to the limited size of VTvafl7 vector part not exceeding 3200 bp and has the ability to efficiently penetrate into human and animal cells and express the Cas9 therapeutic gene cloned to it.

3. Gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene as per claim 1. Said DNA vector is unique due to the fact that the gene therapy DNA vector contains no nucleotide sequences of viral origin and no antibiotic resistance genes, which ensures its safe use for the implementation of various methods of genome editing of humans and animals, including the gene therapy of humans and animals.

4. The method of production of gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene as per claim 1 that involves obtaining gene therapy DNA vector VTvafl7-Cas9 as per claim 1 as follows: the coding region of Cas9 therapeutic gene is cloned to DNA vector VTvafl7, and gene therapy DNA vector VTvafl7-Cas9 is obtained.

5. The method of usage of the gene therapy DNA vector based on gene therapy DNA vector VTvafl7 carrying the Cas9 therapeutic gene as per claim 1 for heterologous expression of this therapeutic gene in human and animal cells that involves introduction of a gene therapy DNA vector as per claim 1 into human or animal cells, organs and tissues in combination with gRNA, and/or introduction of autologous human or animal cells into human or animal organs and tissues transfected with gene therapy DNA vector as per claim 1 together with gRNA, or a combination of the indicated methods.

6. The method of production of Escherichia coli strain SCSI 10- AF/VTvafl 7-Cas9 involves electroporation of competent cells of Escherichia coli strain SCSI 10- AF by the gene therapy DNA vector as per claim 1 and subsequent selection of stable clones of the strain using selective medium.

7. Escherichia coli strain SCSI 10-AF/VTvafl7-Cas9 carrying the gene therapy DNA vector produced as per claim 6 and carrying the gene therapy DNA vector as per claim 1 for its production allowing for antibiotic-free selection.

8. The method of gene therapy DNA vector production on an industrial scale as per claim 1 that involves scaling-up the bacterial culture of the strain as per claim 7 to the quantities necessary for increasing the bacterial biomass in an industrial fermenter, after which the biomass is used to extract a fraction containing the therapeutic DNA product, i.e. the gene therapy DNA vector VTvafl7-Cas9 as per claim 1, and then multi-stage filtered, and purified by chromatographic methods.