CN113490743A

CN113490743A - Gene therapy DNA vector and application thereof

Info

Publication number: CN113490743A
Application number: CN201980093145.1A
Authority: CN
Inventors: N.萨韦利瓦
Original assignee: Prolivne Innovation Technology Co ltd; Cell Gene Therapy Co ltd
Current assignee: Prolivne Innovation Technology Co ltd; Cell Gene Therapy Co ltd; Cell and Gene Therapy Ltd
Priority date: 2018-12-27
Filing date: 2019-12-23
Publication date: 2021-10-08
Also published as: RU2018147083A; RU2018147083A3; WO2020139156A8; RU2730663C2; WO2020139156A1

Abstract

The present invention relates to genetic engineering and can be used in biotechnology, medicine and agriculture for the manufacture of gene therapy medicinal products. The implementation of various genome editing methods proposed a gene therapy vector based on the VTvaf1V gene therapy DNA vector, i.e. a vector carrying the target Cas9 gene, for heterologous expression of the target gene in human and animal cells. The gene therapy DNA vector VTvaf17-Cas9 has a nucleotide sequence of SEQ ID No. 1. A gene therapy DNA vector based on the gene therapy DNA vector VTvaf1V carrying a Cas9 target gene, which is unique due to the following facts: due to the limited size of the VTvaf17 vector portion not exceeding 3200bp, the constructed gene therapy DNA vector VTvaf17-Cas9 has the ability to efficiently penetrate human and animal cells and express the Cas9 target gene cloned thereto. Also proposed are methods for producing specific vectors, methods for using the vectors, E.coli strains harboring the specific vectors, and methods for producing the specific vectors on an industrial scale.

Description

Gene therapy DNA vector and application thereof

Technical Field

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

Background

Gene therapy is an innovative approach in medicine aimed at treating genetic and acquired diseases by delivering new genetic material into the cells of patients to compensate or suppress the function of mutated genes and/or to treat genetic disorders. The end product of gene expression may be an RNA molecule or a protein molecule. However, most physiological processes in vivo are associated with the functional activity of protein molecules, while RNA molecules are either intermediates in protein synthesis or perform regulatory functions. Thus, in most cases, the goal of gene therapy is to inject into an organism genes that provide for the transcription and further translation of the protein molecules encoded by these genes. In the description of the present invention, gene expression refers to the production of a protein molecule having an amino acid sequence encoded by the gene. Mutations in a gene can result in the complete or partial loss of protein expression, or in the expression of variants of protein molecules with unfavorable functional activity. Injection of a gene therapy vector encoding a particular gene into the body can restore expression of the therapeutic protein. However, this is compensatory and is not intended to correct genetic defects. With the discovery of targeted (targeted) editing of nucleotide sequences, the implementation of therapeutic genome editing methods aimed at correcting mutations in DNA sequences that also constitute targeted gene therapy is made possible by the injection of various nucleases with specific properties (such as Cas9) into gene therapy vectors. In this case, the function is restored by correction of the genetic defect (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 that enables bacterial cells to target the removal of the nucleotide sequence of a phage (Sapranauskasas R, 2011; Mougiakos I, 2017). The action principle of the system has certain universality, so the system is widely applied to biomedical and biotechnological research. At present, the CRISPR/Cas9 system is widely used in genome editing scientific research in mammalian and experimental animal cell cultures and has the ability to design drugs and methods for gene therapy. The working principle of this system is that Cas9 endonuclease cleaves DNA strands with the help of grnas complementary to specific sequences in the genome, cutting out the region of the targeted DNA. The integrity of the DNA in the breakpoint is then restored using a cellular repair system that can restore or repair the break by direct ligation of adjacent nucleotides using homologous DNA strands containing the correct nucleotide sequence as substrates, without repairing the excised DNA region (Salsman J, 2017). Constructing the gRNA in such a way that the molecule is complementary to a DNA region containing one or another mutation allows targeted excision of that specific region using Cas9, which determines the ability of this mechanism of action to correct genetic material, i.e., genome editing (Wilson LOW, 2018).

However, one of the major problems with genome editing using the CRISPR/Cas9 system is the problem of delivering endonuclease and gRNA complexes to the nucleus, and the pharmacokinetic problems that often limit the ability of molecules to penetrate into various organs and tissues or require extremely high concentrations and use of special compositions that allow cellular penetration (Dowdy SF, 2017). The use of gene vectors for heterologous expression of Cas9 gene helps to overcome these limitations. The most studied vectors in the art are lentiviral vectors, adenoviral vectors, adeno-associated vectors and other virus-associated vectors.

The risk of non-specific endonuclease action is another problem with the use of CRISPR/Cas9 systems. In this context, the use of vectors that do not integrate into the genome and only provide 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 vector for delivering specific sequences to an organism is limited by the tropism of pseudovirions to various tissues, which does not always allow efficient penetration into target cells and organs (Maginnis MS, 2017). Furthermore, the potential for using any viral vector is limited, including their relatively high immunogenicity, pre-existing immunity, and risks associated with gene therapy virus-associated vectors (lukashiev AN, Zamyatnin AA, 2016).

Thus, the background of the invention suggests that there is a need to develop effective and safe gene therapy methods to deliver Cas9 to target cells and tissues.

It is known to classify gene therapy vectors into viral, cellular, and DNA vectors (EMA/CAT/80183/2014 guidelines on the quality, non-clinical, and clinical aspects of gene therapy drug products). Recently, gene therapy has focused on the development of non-viral gene delivery systems, in which the plasmid vector is the leader. Plasmid vectors have no limitations inherent to cellular and viral vectors. In target cells, they are present as episomes, do not integrate into the genome, but they are rather inexpensive to produce, and do not have the immune response or side effects caused by the administration of plasmid vectors, which makes them a convenient tool for gene therapy and prevention (DNA vaccination) of genetic diseases (Li L, Petrovsky N.// Expert Rev vaccines.2016; 15(3): 313-29).

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

It is well known that the european medicines agency considers that it is necessary to avoid adding antibiotic resistance marker genes to newly engineered plasmid vectors for gene therapy (post-read report on design modification of gene therapy drug products during development)/2011 12/14/day EMA/CAT/GTWP/44236/2009 advanced therapy Committee (Committee for advanced therapies)). This suggestion is primarily related to the potential risk of DNA vector penetration or horizontal transfer of antibiotic resistance genes into bacterial cells present in the body, which are part of a normal or opportunistic community of microorganisms. Furthermore, the presence of the antibiotic resistance gene significantly increases the length of the DNA vector, which reduces its efficacy in penetrating into eukaryotic cells.

It is to be noted that antibiotic resistance genes also make a fundamental contribution to the method of producing DNA vectors. If an antibiotic resistance gene is present, the strain used to produce the DNA vector is usually cultured in a medium containing a selective antibiotic, which poses the risk of antibiotic traces in DNA vector preparations that are not sufficiently purified. Thus, the production of DNA vectors for gene therapy without antibiotic resistance genes is linked to the production of strains with unique characteristics, such as the ability of stable amplification of the therapeutic DNA vector in antibiotic-free medium.

Furthermore, the European drug administration suggests avoiding the presence of regulatory elements (promoters, enhancers, post-translational regulatory elements) in the therapeutic plasmid vector that constitute the genomic nucleotide sequences of various viruses that increase the expression of therapeutic genes (Draft guide on the quality, non-clinical and clinical aspects of gene therapy drug products, http:// www.ema.europa.eu/docs/en _ GB/document _ library/Scientific _ guidine/2015/05/WC500187020. pdf) for the quality, non-clinical and clinical aspects of gene therapy drug products. Although these sequences may increase the expression level of the therapeutic transgene, they pose a risk of recombination with the genetic material of the wild-type virus and integration into the eukaryotic genome. Furthermore, the relevance of overexpression of specific genes for therapy remains an open question.

The size of the therapeutic vehicle is also necessary. It is well known that modern plasmid vectors often have unnecessary non-functional sites that significantly increase their length (Mairhofer J, Grabherr R.// Mol Biotechnol.2008.39(2): 97-104). For example, ampicillin resistance genes in pBR322 series vectors usually consist of at least 1000bp, accounting for more than 20% of the length of the vector itself. An inverse relationship between vector length and its ability to penetrate eukaryotic cells was observed; DNA vectors having a small length efficiently penetrate into human and animal cells. For example, in a series of experiments on transfection of HeLa cells with 383-19-4548 bp DNA vector, it was shown that the difference in infiltration efficacy can be up to two orders of magnitude (100-fold difference) (Hornstein BD et al// PLoS ONE.2016; 11(12): e 0167537.).

Therefore, in selecting DNA vectors, those constructs which do not contain antibiotic resistance genes, viral-derived sequences, and which are of a length that allows efficient infiltration into eukaryotic cells should be considered preferentially for reasons of safety and maximum efficiency. Strains producing such DNA vectors in sufficient amounts for gene therapy purposes should ensure the possibility of stable DNA vector amplification using antibiotic-free nutrient media.

An example of the use of a recombinant DNA vector for gene therapy is a method of producing a recombinant vector for genetic immunization (patent No. US 9550998B 2). Plasmid vectors are supercoiled plasmid DNA vectors that are used for expression of cloned genes in human and animal cells. The vector contains an origin of replication, regulatory elements including a human cytomegalovirus promoter and enhancer, and regulatory sequences from a human T-cell lymphotropic virus.

The vector was accumulated in antibiotic-free dedicated E.coli strains by antisense complementation of the sacB gene inserted in the strain using phage. The disadvantage of this invention is the presence of regulatory elements in the DNA vector composition that constitute the sequences of the viral genome.

The following patents are prototypes of the present invention, and relate to gene therapy methods using Cas9 expression in eukaryotic cells.

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

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

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

Disclosure of Invention

The object of the present invention is to construct gene therapy DNA vectors heterologously expressing the Cas9 gene in human and animal cells, which combine the following properties:

I) efficiency of gene therapy DNA vectors for heterologous expression of therapeutic genes in eukaryotic cells.

II) the possibility of safe use for implementing various genome editing methods of human and animal genomes, including human and animal gene therapy, due to the absence of regulatory elements constituting viral genome nucleotide sequences in gene therapy DNA vectors.

III) the possibility of being safe for implementing various genome editing methods for human and animal genomes, including human and animal gene therapy, due to the absence of antibiotic resistance genes in gene therapy DNA vectors.

VI) Productibility and constructability of gene therapy DNA vectors on an industrial scale.

According to the recommendations of the national regulatory authorities for gene therapy drugs, and in particular the requirements of the european drug administration, i.e. avoiding the addition of antibiotic resistance marker genes to newly engineered plasmid vectors for gene therapy (post-read report on design modification of gene therapy drug products during development/EMA/CAT/GTWP/44236/2009 advanced therapy Committee for advanced therapies 12/14/2011), and avoiding the addition of viral genomes to newly engineered plasmid vectors for gene therapy (guidelines on quality, non-clinical and clinical aspects of gene therapy drug products/23/2015 3/80183/2014, advanced therapy Committee), items II and III are provided herein.

The object of the present invention also includes the construction of strains carrying the gene therapy DNA vectors for the development and production of these gene therapy DNA vectors on an industrial scale.

Specific objects are achieved by using a gene therapy DNA vector produced based on the gene therapy DNA vector VTvaf17 carrying a Cas9 therapeutic gene for heterologous expression of this therapeutic gene in human and animal cells in enabling various genome editing methods, wherein the gene therapy DNA vector VTvaf17-Cas9 has the nucleotide sequence SEQ ID No. 1. Gene therapy DNA vectors constructed based on gene therapy DNA vector VTvaf17 carrying Cas9 therapeutic gene are unique due to the following facts: the constructed gene therapy DNA vector VTvaf17-Cas9 has the ability to efficiently penetrate human and animal cells and express Cas9 therapeutic genes cloned thereto due to the limited size of the VTvaf17 vector portion not exceeding 3200 bp.

Gene therapy DNA vectors based on gene therapy DNA vector VTvaf17 carrying Cas9 therapeutic gene are unique due to the fact that they do not contain virus-derived nucleotide sequences nor antibiotic resistance genes, which ensures their safe possibility for implementing various genome editing methods for humans, animals, including human and animal gene therapy.

A method for producing a gene therapy DNA vector based on gene therapy DNA vector VTvaf17 carrying a Cas9 therapeutic gene was developed, which method involves obtaining gene therapy DNA vector VTvaf17-Cas9 as follows: the coding region of the Cas9 therapeutic gene was cloned onto DNA vector VTvaf17 to obtain gene therapy DNA vector VTvaf17-Cas9, SEQ ID No. 1.

The present invention discloses a method for the heterologous expression of Cas9 therapeutic genes in human and animal cells using gene therapy DNA vectors constructed based on gene therapy DNA vector VTvaf17, which carry the gene, involving the introduction of the gene therapy DNA vector into human or animal cells, organs and tissues in combination with a gRNA molecule or genetic construct providing expression of the gRNA, and/or the introduction of autologous human or animal cells into human or animal organs and tissues transfected with the gene therapy DNA vector in combination with a gRNA molecule or genetic construct, or combinations of the indicated methods, which achieve expression of the gRNA.

The production method of the E.coli SCS110-AF/VTvaf17-Cas9 strain involved electroporating competent cells of the E.coli SCS110-AF strain with the constructed gene therapy DNA vector and then selecting stable clones of the strain using a selective medium.

The E.coli strain SCS110-AF/VTvaf17-Cas9 is claimed, which carries gene therapy DNA vectors for its production, allowing antibiotic-free selection.

A method of producing gene therapy DNA vectors on an industrial scale involves scaling up the bacterial culture of the strain in an industrial fermentor to the amount necessary to increase bacterial biomass, then using the biomass to extract the fraction containing the therapeutic DNA product, i.e. gene therapy DNA vector VTvaf17-Cas9, performing a multi-stage filtration and purification by chromatographic methods.

Drawings

The essence of the invention is explained in the following figures, wherein:

FIG. 1 shows a schematic view of a

Shows the structure of a gene therapy DNA vector VTvaf17 carrying a Cas9 therapeutic gene, which constitutes a circular double stranded DNA molecule capable of autonomous replication in e.

FIG. 1 shows the structure of gene therapy DNA vector VTvaf17-Cas 9.

The following structural elements of the carrier are indicated in the following structure:

the promoter region of EF1a, human elongation factor EF1A, contains the internal enhancer contained in the first intron of the gene. It ensures efficient transcription of recombinant genes in most human tissues,

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

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

ori-origin of replication for autonomous replication, with single nucleotide substitutions to increase plasmid production in most E.coli strain cells,

RNAout-in the case of the use of the E.coli strain SCS110-AF, the regulatory element RNA-OUT of transposon Tn10, which allows antibiotic-free positive selection.

Unique restriction sites are labeled.

FIG. 2

Graph showing the accumulation of cDNA amplicons of the therapeutic gene (i.e., Cas9 gene) in HDFa primary human dermal fibroblast cultures (ATCC PCS-201-01) before and 48 hours after transfection of these cells with the gene therapy DNA vector VTvaf17-Cas9 in order to assess the ability to infiltrate eukaryotic cells and functional activity, i.e., expression of the therapeutic gene at the mRNA level.

The following curves of amplicon accumulation during the reaction are shown in fig. 2, corresponding to:

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

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

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

4-cDNA of the B2M gene in HDFa cell culture after transfection with the DNA vector VTvaf17-Cas 9.

The B2M (β -2-microglobulin) gene listed under accession No. NM 004048.2 in GenBank database was used as reference gene.

FIG. 3

Graph showing the accumulation of the cDNA amplicon of the therapeutic gene (i.e., Cas9 gene) in a culture of HEKa primary human epidermal keratinocytes (ATCC PCS-200-011) before 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 the functional activity, i.e., the expression of the therapeutic gene at the mRNA level.

The following curves of amplicon accumulation during the reaction are shown in fig. 3, corresponding to:

1-cDNA of Cas9 gene in HEKa primary human epidermal keratinocyte culture before transfection with DNA vector VTvaf17-Cas9,

2-cDNA of Cas9 gene in HEKa primary human epidermal keratinocyte culture after transfection with DNA vector VTvaf17-Cas9,

3-cDNA of the B2M gene in HEKa primary human epidermal keratinocyte culture before transfection with the DNA vector VTvaf17-Cas9,

4-cDNA of the B2M gene in HEKa primary human epidermal keratinocyte cell culture after transfection with the DNA vector VTvaf17-Cas 9.

FIG. 4

Shows in normal adult primary epidermal melanocytes (HEMA) ((

PCS-200-013^TM) Graph of the accumulation of cDNA amplicons of the therapeutic gene (i.e. the Cas9 gene) before and 48 hours after transfection of these cells with the gene therapy DNA vector VTvaf17-Cas9, in order to assess the ability to infiltrate eukaryotic cells and the functional activity, i.e. the expression of the therapeutic gene at the mRNA level.

The following curves of amplicon accumulation during the reaction are shown in fig. 4, corresponding to:

1-cDNA of Cas9 gene in HEMA primary human epidermal melanocyte culture before transfection with DNA vector VTvaf17-Cas9,

2-cDNA of Cas9 gene in HEMA primary human epidermal melanocyte culture after transfection with DNA vector VTvaf17-Cas9,

3-cDNA of the B2M gene in HEMA primary human epidermal melanocyte culture before transfection with the DNA vector VTvaf17-Cas9,

4-cDNA of the B2M gene in HEMa primary human epidermal melanocyte cultures after transfection with the DNA vector VTvaf17-Cas 9.

FIG. 5

Graph showing the concentration of Cas9 protein in HDFa primary human dermal fibroblast (ATCCPCS-201-01) cell lysates after transfection of these cells with the DNA vector VTvaf17-Cas9 in order to assess functional activity, i.e. expression at the protein level based on changes in Cas9 protein concentration in the cell lysates.

The following elements are indicated in fig. 5:

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

culture B-HDFa human dermal fibroblast culture transfected with DNA vector VTvaf17,

culture C-HDFa human dermal fibroblast culture transfected with DNA vector VTvaf17-Cas 9.

FIG. 6

Graph showing the concentration of Cas9 protein in HEKa primary human epidermal keratinocytes (ATCC PCS-200-01) cell lysate after transfection of these cells with gene therapy DNA vector VTvaf17-Cas9 in order to evaluate the functional activity (i.e. therapeutic gene expression at the protein level) and the possibility of increasing the protein expression level by gene therapy-based DNA vector VTvaf17 vector carrying Cas9 therapeutic gene.

The following elements are indicated in fig. 6:

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

culture B-HEKa primary human epidermal keratinocyte culture transfected with DNA vector VTvaf17,

culture C-HEKa primary human epidermal keratinocyte culture transfected with DNA vector VTvaf17-Cas 9.

FIG. 7

In normal adult primary epidermal melanocytes (HEMA) ((

PCS-200-013^TM) In cell lysatesGraph of Cas9 protein concentration after transfection of these cells with gene therapy DNA vector VTvaf17-Cas9 to facilitate evaluation of functional activity, i.e., expression of therapeutic gene at the protein level and the possibility of increasing the protein expression level by gene therapy-based DNA vector VTvaf17-Cas9 carrying Cas9 therapeutic gene.

The following elements are indicated in fig. 7:

culture a-HEMa human primary epidermal melanocyte culture transfected (reference) with plasmid DNA-free aqueous dendrimer solution,

culture B-HEMA human epidermal melanocyte culture transfected with DNA vector VTvaf17,

culture C-HEMA human epidermal melanocyte culture transfected with DNA vector VTvaf17-Cas 9.

FIG. 8

(iii) shown in CHO-K1 Syrian hamster ovary cells

CCL-61^TM) Graph of Cas9 protein concentration in cell lysates after transfection of these cells with gene vector VTvaf17-Cas9 to facilitate evaluation of functional activity (i.e., therapeutic gene expression at the protein level) and the possibility of increasing protein expression levels by gene therapy-based DNA vector VTvaf17 carrying Cas9 therapeutic gene.

The following elements are indicated in fig. 8:

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

culture B-CHO-K1 Syrian hamster ovary cell cultures transfected with the DNA vector VTvaf17,

culture C-CHO-K1 Syrian hamster ovary cell cultures transfected with the DNA vector VTvaf17-Cas 9.

FIG. 9

A graph showing the protein concentration of Cas9 in human peripheral blood mononuclear cell culture (PBMC) lysates after transfection of these cells with DNA vector VTvaf17-Cas9, in order to assess the functional activity (i.e. therapeutic gene expression at the protein level) and the possibility of increasing the protein expression level by gene therapy-based DNA vector VTvaf17 carrying Cas9 therapeutic gene.

The following elements are indicated in fig. 9:

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

culture B-PBMC human cell culture transfected with DNA vector VTvaf17, culture C-PBMC human cell culture transfected with DNA vector VTvaf17-Cas 9.

FIG. 10 shows a schematic view of a

A graph showing the concentration of Cas9 protein in skin biopsy samples of primary depilated areas of three Wistar rats injected with autologous fibroblast cell culture transfected with gene therapy DNA vector VTvaf17-Cas9 to illustrate the method of use of autologous cells transfected with gene therapy DNA vector VTvaf17-Cas9 by injection.

The following elements are indicated in fig. 10:

K1I-rat K1 skin biopsy in the injection area of autologous fibroblast cultures transfected with gene therapy DNA vector VTvaf17-Cas9,

k1 II-rat skin biopsy in the injection area of autologous fibroblast cultures transfected with the gene therapy DNA vector VTvaf17 that does not carry the Cas9 gene,

k1 III-rat skin biopsy from intact sites,

K2I-rat K2 skin biopsy in the injection area of autologous fibroblast cultures transfected with gene therapy DNA vector VTvaf17-Cas9,

k2 II-rat K2 skin biopsy in the injection area of autologous fibroblast cultures transfected with gene therapy DNA vector VTvaf17 that does not carry the Cas9 gene,

k2 III-rat K2 skin biopsy from an intact site,

K3I-rat K3 skin biopsy in the injection area of autologous fibroblast cultures transfected with gene therapy DNA vector VTvaf17-Cas9,

k3 II-rat K3 skin biopsy in the injection area of autologous fibroblast cultures transfected with gene therapy DNA vector VTvaf17 that does not carry the Cas9 gene,

k3 III-rat K3 skin biopsy from intact sites.

FIG. 11

A plot of the fluorescent cytoreductive capacity of the 293/GFP Cell line (Cell Biolabs, cat. akr-200) after co-transfection of these cells with the DNA vector VTvaf17-Cas9 carrying the Cas9 gene and the GFP targeting guide RNA for CRISPR (Genaxxon bioscience, cat. p2008.0010) is shown in order to assess the functional activity (i.e. expression of the therapeutic gene at the protein level) and the ability of the protein to perform a target-specific endonuclease activity involving a particular gRNA, which leads to DNA sequence editing.

The following elements are indicated in fig. 11:

culture A-293/GFP cells transfected with water, then transfected with GFP targeting guide RNA for CRISPR,

culture B-293/GFP cells transfected with a DNA vector VTvaf17 without Cas9 gene cDNA, then transfected with GFP targeting guide RNA for CRISPR,

culture C-293/GFP cells transfected with the DNA vector VTvaf17-Cas9 carrying the Cas9 gene, then transfected with GFP-targeted guide RNA for CRISPR,

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

FIG. 12

Shows a cancer cell line for MCF-7 lung adenocarcinoma ((

HTB-22^TM) The result of PCR sequencing of several cell clone fragments of (a), wherein the genome was edited after transfection of DNA vector VTvaf17-Cas9 carrying Cas9 gene and gRNA _ cloning vector carrying TLR9 gene gRNA sequence.

DNA regions corresponding to double stranded breaks introduced by sgrnas TLR _1 and TLR _2 are shown, where TLR12 is a consensus sequence; WT is a TLR9 gene fragment sequence that has not been genome edited or has a restored sequence; al1, al2, G2 are different alleles of a heterozygous clone (two alleles are shown in the figure).

Detailed Description

Based on the 3165bp DNA vector VTvaf17, a gene therapy DNA vector carrying a Cas9 therapeutic gene was generated, intended for heterologous expression of this therapeutic gene in human and animal cells. A method of generating a gene therapy DNA vector carrying a therapeutic gene involves cloning the protein-encoding sequence of the Cas9 therapeutic gene (encoding Cas9 endonuclease) into the polylinker of the gene therapy DNA vector VTvaf 17. It is well known that the ability of a DNA vector to penetrate eukaryotic cells depends largely on the size of the vector. The smallest size DNA carrier has a higher permeability. Thus, it is preferred that no elements which do not bear a functional load, but which at the same time increase the size of the vector DNA, are present in the vector. These features of the DNA vector are taken into account in the production of gene therapy DNA vectors based on the gene therapy DNA vector VTvaf17 carrying the Cas9 therapeutic gene, where there are no large non-functional sequences and antibiotic resistance genes in the vector, allowing a significant reduction in the size of the resulting gene therapy DNA vector VTvaf17 carrying the Cas9 therapeutic gene, in addition to technical advantages and safe use. 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 VTvaf17-Cas9 was produced as follows: the coding region of the Cas9 therapeutic gene was cloned into gene therapy DNA vector VTvaf17 and gene therapy DNA vector VTvaf17-Cas9, SEQ ID No.1 was obtained. The coding region of the 4221bp Cas9 gene was generated by enzymatic synthesis from chemically synthesized oligonucleotides. The amplified product was cleaved by specific restriction endonucleases taking into account the optimal procedures for further cloning and cloned into the gene therapy DNA vector VTvaf17 via BamHI and EcoRI restriction sites located in the VTvaf17 vector polylinker. The restriction sites are selected in such a way that the cloned fragment enters the reading frame of the expression cassette of the vector VTvaf17, whereas the protein coding sequence does not contain the restriction sites for the chosen endonuclease. The expert in the field recognizes that the methodological implementation of the generation of the gene therapy DNA vector VTvaf17-Cas9 can vary within the selection framework of known molecular gene cloning and that these methods are included within the scope of the present invention. For example, different oligonucleotide sequences can be used to amplify Cas9 gene, different restriction endonucleases or laboratory techniques (e.g., independent of ligated gene cloning).

The gene therapy DNA vector VTvaf17-Cas9 has a nucleotide sequence of SEQ ID No. 1. At the same time, the degeneracy of the genetic code is known to the expert in the field and this means that variants of the nucleotide sequences are also included within the scope of the invention, differing by the insertion, deletion or substitution of nucleotides which do not lead to a change in the sequence of the polypeptide encoded by the therapeutic gene and/or do not lead to a loss of functional activity of the regulatory elements of the VTvaf17 vector. Meanwhile, genetic polymorphisms are known to experts in the field and it is meant that the scope of the present invention also includes variants of the nucleotide sequence of the Cas9 gene, which also encode different variants of the amino acid sequence of the Cas9 protein, which do not differ in their functional activity under physiological conditions from those listed.

The ability to penetrate eukaryotic cells and express functional activity, i.e. the ability to express the therapeutic gene of the obtained gene therapy DNA vector VTvaf17-Cas9, was demonstrated by introducing the obtained vector into eukaryotic cells and subsequently analyzing the expression of specific mRNA and/or the protein product of the therapeutic gene. The presence of specific mRNA in cells introduced with gene therapy DNA vector VTvaf17-Cas9 demonstrates the ability of the obtained vector to penetrate eukaryotic cells and express mRNA of Cas9 therapeutic gene. Furthermore, experts in the field know that the presence of mRNA genes is a mandatory condition, but not evidence for translation of the protein encoded by the therapeutic gene. Therefore, to confirm the property of the gene therapy DNA vector VTvaf17-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 the protein encoded by the therapeutic gene was performed using immunological methods. The presence of Cas9 protein demonstrates the efficacy of therapeutic gene expression in eukaryotic cells using a gene therapy DNA vector based on the gene therapy DNA vector VTvaf17 carrying the Cas9 therapeutic gene. Thus, to demonstrate the efficacy of the resulting gene therapy DNA vector carrying the therapeutic gene (i.e., Cas9 gene), VTvaf17-Cas9, the following method was used:

A) real-time PCR, i.e., the change in cDNA accumulation of therapeutic genes in human and animal cell lysates following transfection of different human and animal cell lines with gene therapy DNA vectors;

B) enzyme-linked immunosorbent assay, i.e. the change in the quantitative level of therapeutic proteins in human, animal cell lysates after transfection of different human and animal cell lines with gene therapy DNA vectors.

C) Enzyme-linked immunosorbent assay, i.e.the change in the quantitative level of therapeutic proteins in the supernatant of animal tissue biopsy after injection of autologous cells in these tissues transfected with gene therapy DNA vectors,

D) flow cytofluorometric measurement of GFP gene expression in cells undergoing genome editing after combined transfection of these cells with gene therapy DNA vectors VTvaf17-Cas9 and gRNAs, which transfection results in inactivation of the GFP gene and lack or significant reduction in fluorescent protein expression in the cells,

E) DNA regions were sequenced after transfection of gene therapy DNA vector VTvaf17-Cas9 in combination with a vector expressing a gRNA targeting the TLR9 gene sequence in human and animal cells that had undergone genome editing.

To confirm the feasibility of the constructed gene therapy DNA vector VTvaf17-Cas9 carrying the therapeutic gene, Cas9 gene, the following operations were performed:

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

B) transfecting different mammal cell lines by combining a gene therapy DNA vector and gRNA,

C) transfection of different mammalian cell lines with gene therapy DNA vectors in combination with vectors encoding gRNAs,

D) autologous cells transfected with a gene therapy DNA vector are injected into animal tissues,

E) shows changes in the expression of the editing gene in a cell line in which genome editing is performed,

F) changes in the sequence of the edited gene region are shown in the cell line undergoing genome editing.

As demonstrated by the lack of regions of homology to the viral genome and antibiotic resistance genes in the nucleotide sequence of the gene therapy DNA vector VTvaf17-Cas9(SEQ ID No.1), these methods of use lack the potential risk of gene therapy for humans and animals due to the lack of regulatory elements that make up the nucleotide sequence of the viral genome in the gene therapy DNA vector and the lack of antibiotic resistance genes in the gene therapy DNA vector.

It is known to experts in the field that the use of antibiotic resistance genes in gene therapy DNA vectors allows to obtain a preparative scale of these vectors by increasing the bacterial biomass in a nutrient medium containing selective antibiotics. Within the framework of the present invention, to ensure safe use of the gene therapy DNA vector VTvaf17 carrying the Cas9 therapeutic gene, it was not possible to use selective nutrient media containing antibiotics. A method to obtain strains for producing these gene therapy vectors based on the escherichia coli strain SCS110-AF is proposed as a technical solution for obtaining the gene therapy DNA vector VTvaf17 carrying the Cas9 therapeutic gene in order to scale up the production of gene therapy vectors on an industrial scale. The method of production of the e.coli strain SCS110-AF/VTvaf17-Cas9 involves the production of competent cells of e.coli strain SCS110-AF, into which the gene therapy DNA vector VTvaf17-Cas9 is injected using a transformation (electroporation) method well known to the expert in the art. The obtained E.coli strain SCS110-AF/VTvaf17-Cas9 was used for the production of gene therapy DNA vector VTvaf17-Cas9, allowing the use of antibiotic-free medium.

To confirm the production of the E.coli SCS110-AF/VTvaf17-Cas9 strain, transformation, selection, subsequent biomass growth, and extraction of plasmid DNA were performed.

To demonstrate the producibility and constructability of the gene therapy DNA vector VTvaf17-Cas9 carrying the therapeutic gene (i.e., Cas9 gene) on an industrial scale, large-scale fermentation was performed on the e.coli strain e.coli SCS110-AF/VTvaf17-Cas9 containing the gene therapy DNA vector VTvaf17 carrying the Cas9 therapeutic gene.

A method of expanding the production of bacterial consortia to an industrial scale for the isolation of gene therapy DNA vector VTvaf17 carrying Cas9 therapeutic gene, which method involves incubating a seed culture of escherichia coli strain SCS110-AF/VTvaf17-Cas9 in antibiotic-free nutrient medium that provides suitable biomass accumulation dynamics. After a sufficient amount of biomass was reached in the logarithmic growth phase, the bacterial culture was transferred to an industrial fermentor and then cultured to stationary phase before fractions containing the therapeutic DNA product (i.e. gene therapy DNA vector VTvaf17-Cas9) were extracted, multi-stage filtered and purified by chromatographic methods. It is known to experts in the field that the culture conditions of the strain, the composition of the nutrient medium (except for the absence of antibiotics), the equipment used, and the DNA purification method can vary within the framework of standard operating procedures with the specific production line, but known methods of expansion, industrial production, and purification of DNA vectors using the escherichia coli strain SCS110-AF/VTvaf17-Cas9 fall within the scope of the present invention.

The disclosure as claimed herein is illustrated by way of example of embodiments of the invention.

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

Example 1.

Generation of gene therapy DNA vector VTvaf17-Cas9 carrying the therapeutic gene, Cas9 gene.

The coding region (4221bp) of the Cas9 gene was cloned into the 3165bp DNA vector VTvaf17 by BamHI and EcoRI restriction sites to construct the gene therapy DNA vector VTvaf17-Cas 9. The 4221bp Cas9 gene coding region was generated by enzymatic synthesis from chemically synthesized oligonucleotides.

The gene therapy DNA vector VTvaf17 was constructed by integrating six fragments of DNA derived from different sources:

(a) the origin of replication was generated by PCR amplification of the pBR322 region of a commercially available plasmid with point mutations;

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

(c) hGH-TA transcriptional terminator was generated by PCR amplification of the human genomic DNA locus;

(d) the RNAout regulatory site of transposon Tn10 was synthesized from oligonucleotides;

(e) the kanamycin resistance gene was generated by PCR amplification of a site of the commercially available human plasmid pET-28;

(f) polylinkers are generated by annealing two synthetic oligonucleotides.

According to the manufacturer's instructions, use the commercially available kit

PCR amplification was performed with high fidelity DNA polymerase (New England Biolabs, USA). The fragments have overlapping regions allowing them to be combined with subsequent PCR amplification. The oligonucleotides Ori-F and EF1-R integration fragments (a) and (b) were used, and the oligonucleotides hGH-F and Kan-R integration fragments (c), (d), and (e) were used. The resulting fragments were then integrated by restriction with sites BamHI and NcoI, followed by ligation. This results in a plasmid still lacking polylinkers. To add it, the plasmid was cut through BamHI and EcoRI sites and then ligated with fragment (f). Thus, a 4182bp vector carrying the kanamycin resistance gene flanked by SpeI restriction sites was constructed. The gene was then cleaved by the SpeI restriction site, and the remaining fragment was ligated to itself. This resulted in the recombinant 3165bp gene therapy DNA vector VTvaf17 and allowed antibiotic-free selection.

The amplified product of the coding region of Cas9 gene and DNA vector VTvaf17 were cleaved with BamHI and EcoRI restriction enzymes (New England Biolabs, USA).

This resulted in a 7351bp DNA vector VTvaf17-Cas9 having the nucleotide sequence SEQ ID No.1 and the general structure is shown in FIG. 1.

Example 2.

Evidence of the ability of the gene therapy DNA vector VTvaf17-Cas9 carrying the therapeutic gene, Cas9 gene, to penetrate eukaryotic cells and its functional activity at the therapeutic gene mRNA expression level. This example also demonstrates the feasibility of using gene therapy DNA vectors carrying therapeutic genes.

Changes in mRNA accumulation of Cas9 therapeutic gene were evaluated 48 hours after transfection with gene therapy DNA vector VTvaf17-Cas9 carrying the human Cas9 gene in HDFa primary human dermal fibroblast cultures (ATCC PCS-201-01). The amount of mRNA was determined dynamically by accumulation of cDNA amplicons in real-time PCR.

HDFa primary human dermal fibroblast cultures were used to evaluate changes in the accumulation of therapeutic Cas9 mRNA. Serum-free fibroblast growth kit was used under standard conditions (37 ℃, 5% CO2) (iii)

PCS-201-040). The culture medium was changed every 48 hours during the culture.

To achieve 90% confluence, at 5 × 10 per well⁴Amount of individual cells were seeded into 24-well plates 24 hours prior to the transfection procedure. Transfection of gene therapy DNA vector expressing human Cas9 gene VTvaf17-Cas9 was performed using Lipofectamine3000 (ThermoFisher Scientific, USA) according to manufacturer's recommendations. In

test tube

1, 1. mu.L of the DNA vector VTvaf17-Cas9 solution (concentration 500 ng/. mu.L) and 1. mu.L of reagent P3000 were added to 25. mu.L of medium Opti-MEM (Gibco, USA). The preparations were mixed by gentle shaking. In

tube

2, 1. mu.L of Lipofectamine3000 solution was added to 25. mu.L of culture medium Opti-MEM (Gibco, USA). The preparations were mixed by gentle shaking. The contents from tube 1 were added to the contents of tube 2 and the mixture was incubated for 5 minutes at room temperature. The resulting solution was added dropwise to the cells in a volume of 40. mu.l.

HDFa cells transfected with gene therapy DNA vector VTvaf17 lacking the inserted therapeutic gene (cDNA of Cas9 gene is not shown in the figure before and after transfection with gene therapy DNA vector VTvaf17 lacking the inserted therapeutic gene) were used as reference. The reference vector VTvaf17 was produced for transfection as described above.

Total RNA was extracted from HDFa cells using Trizol reagent (Invitrogen, USA) according to the manufacturer's recommendations. 1ml of Trizol reagent was added to the wells containing the cells, homogenized, and heated at 65 ℃ for 5 minutes of addition. The sample was centrifuged at 14,000g for 10 minutes and heated again at 65 ℃ for 10 minutes. Then 200. mu.L of chloroform was added, the mixture was gently stirred, and centrifuged at 14,000g for 10 minutes. The aqueous phase was then separated and mixed with 1/10 volumes of 3M sodium acetate, ph5.2 and an equal volume of isopropanol. The samples were incubated at-20 ℃ for 10 minutes and then centrifuged at 14000g for 10 minutes. The precipitated RNA was washed in 1ml of 70% ethanol, air-dried, and dissolved in 10. mu.L of RNase-free water. Expression levels of Cas9 mRNA after transfection were determined by real-time PCR to assess the cumulative dynamics of cDNA amplicons. For the generation and amplification of cDNA specific for the human Cas9 gene, Cas9_ SF and Cas9_ SR oligonucleotides (sequence listing (1), (2)) were used.

The length of the amplification product is 275 bp.

Reverse transcription reactions and PCR amplifications were performed using SYBR GreenQuantitect RT-PCR kit for real-time PCR (Qiagen, USA). The reaction was carried out in a volume of 20 μ L, comprising: 25 μ L QuantiTect SYBR Green RT-PCR Master Mix, 2.5mM magnesium chloride, 0.5 μm each primer and 5 μ L RNA. For the reaction, 1 cycle of reverse transcription at 42 ℃ for 30min and denaturation at 98 ℃ for 15min was followed by 40 cycles comprising denaturation at 94 ℃ for 15s, primer annealing at 60 ℃ for 30s and extension at 72 ℃ for 30s using a CFX96 amplification apparatus (Bio-Rad, USA) under the following conditions. The B2M (microglobulin of β 2M) gene listed under accession No. NM 004048.2 in GenBank database was used as reference gene. The positive control included amplicons from PCR on a matrix represented by plasmids at known concentrations containing cDNA sequences of Cas9 and B2M genes. The negative control included deionized water. Real-time quantification of the cDNA amplification accumulation dynamics of Cas9 and B2M genes was performed using Bio-Rad CFX Manager 2.1 software (Bio-Rad, USA). The analysis results are shown in FIG. 2.

Figure 2 shows that Cas9 gene was found after transfection of HDFa primary human fibroblasts with gene therapy DNA vector VTvaf17-Cas9, confirming the ability of the vector to penetrate eukaryotic cells and express Cas9 gene at mRNA level. The presented results also demonstrate the feasibility of using the gene therapy DNA vector VTvaf17-Cas9 to heterologously express the Cas9 gene in eukaryotic cells.

Example 3.

Changes in the accumulation of Cas9 therapeutic gene mRNA were evaluated in HEKa primary human epidermal keratinocyte culture (ATCC PCS-200-. The amount of mRNA was determined dynamically by accumulation of cDNA amplicons in real-time PCR.

In the use of a keratinocyte growth kit (

PCS-200-040^TM) HEKa primary human epidermal keratinocyte cultures were cultured under standard conditions (37 ℃, 5% CO 2). To achieve 90% confluence, at 5 × 10 per well 24 hours prior to transfection procedure⁴Amount of individual cells were seeded into 24-well plates. Lipofectamine3000 (ThermoFisher Scientific, USA) is the transfection reagent. Transfection was performed with the gene therapy DNA vector VTvaf17-Cas9 expressing the human Cas9 gene according to the procedure described in example 2. The B2M (microglobulin of β 2M) gene listed under accession No. NM 004048.2 in GenBank database was used as reference gene. HEKa cell cultures transfected with the gene therapy DNA vector VTvaf17 lacking the therapeutic gene (cDNA for Cas9 gene is not shown in the figure before and after transfection with the gene therapy DNA vector VTvaf17 lacking the inserted therapeutic gene) were used as reference. RNA isolation, reverse transcription reaction and real-time PCR were performed as described in example 2.

The positive control included amplicons from PCR on a matrix represented by plasmids at known concentrations containing cDNA sequences of Cas9 and B2M genes. The negative control included deionized water. Real-time quantification of PCR products (i.e., Cas9 and B2M gene cDNAs obtained by amplification) was performed using Bio-Rad CFX Manager 2.1 software (Bio-Rad, USA). The analysis results are shown in FIG. 3.

Figure 3 shows that Cas9 gene was found after transfection of HEKa cell culture with gene therapy DNA vector VTvaf17-Cas9, confirming the ability of the vector to penetrate eukaryotic cells and express Cas9 gene at the mRNA level. The presented results also demonstrate the feasibility of using the gene therapy DNA vector VTvaf17-Cas9 to heterologously express the Cas9 gene in eukaryotic cells.

Example 4.

(HEMA) in normal adult primary epidermal melanocytes 48 hours after transfection with Gene therapy DNA vector carrying the human Cas9 Gene VTvaf17-Cas9

PCS-200-013^TM) In (e), changes in the accumulation of Cas9 therapeutic gene mRNA were evaluated. The amount of mRNA was determined dynamically by accumulation of cDNA amplicons in real-time PCR.

Under standard conditions (37 ℃, 5% CO2), using an adult melanocyte growth kit (

PCS-200-042^TM) The prepared culture medium is used for culturing the primary epidermal melanocyte human cells. To achieve 90% confluence, at 5 × 10 per well 24 hours prior to transfection procedure⁴Amount of individual cells were seeded into 24-well plates. Lipofectamine3000 (ThermoFisher Scientific, USA) is the transfection reagent. Transfection was performed with the gene therapy DNA vector VTvaf17-Cas9 expressing the human Cas9 gene according to the method described in example 2. The B2M (microglobulin of β 2M) gene listed under accession No. NM 004048.2 in GenBank database was used as reference gene. HEMa cell cultures transfected with the gene therapy DNA vector VTvaf17 lacking the therapeutic gene (cDNA of Cas9 gene is not shown in the figure before and after transfection with the gene therapy DNA vector VTvaf17 lacking the inserted therapeutic gene) were used as reference. RNA isolation, reverse transcription reaction and real-time PCR were performed as described in example 2.

The positive control included amplicons from PCR on a matrix represented by plasmids at known concentrations containing cDNA sequences of the cDNA genes of Cas9 and B2M genes. The negative control included deionized water. Real-time quantification of PCR products (i.e., Cas9 and B2M gene cDNAs obtained by amplification) was performed using Bio-Rad CFX Manager 2.1 software (Bio-Rad, USA). The graph derived from the assay is shown in figure 4.

Figure 4 shows the discovery of the Cas9 gene after transfection of HEMa-representative melanophore cultures with gene therapy DNA vector VTvaf17-Cas9, demonstrating the ability of the vector to penetrate eukaryotic cells and express the Cas9 gene at the mRNA level. The presented results also demonstrate the feasibility of using the gene therapy DNA vector VTvaf17-Cas9 to heterologously express the Cas9 gene in eukaryotic cells.

Example 5.

Evidence of the efficacy and feasibility of using a gene therapy DNA vector carrying the Cas9 gene, VTvaf17-Cas9, for heterologous expression of Cas9 protein in mammalian cells.

After transfection of HDFa primary human dermal fibroblast cultures (ATCC PCS-201-01) with DNA vector VTvaf17-Cas9 carrying the human Cas9 gene, lysates of these cells were evaluated for changes in Cas9 protein concentration. Cells were cultured as described in example 2.

Transfection was performed using the 6 th generation SuperFect transfection reagent (Qiagen, Germany). The aqueous dendrimer solution without DNA carrier (a) and the DNA carrier VTvaf17(B) lacking Cas9 gene cDNA were used as reference, and the DNA carrier VTvaf17-Cas9(C) carrying the human Cas9 gene was used as transfection agent. HDFa cells were transfected according to the method described in example 2.

After transfection, 0.1ml1N HCl was added to 0.5ml of the culture broth, mixed well, and incubated at room temperature for 10 minutes. The mixture was then neutralized by adding 0.1ml of 1.2M NaOH/0.5M HEPES (pH7-7.6) and stirred well. The supernatant was collected and used to assay for therapeutic proteins. Cas9 protein was detected by enzyme-linked immunosorbent assay (ELISA) using Cas9 (CRISPR-associated protein 9) ELISA kit (Cell Biolabs Inc, cat. prb-5079) and densitometry detection using ChemWell Automated EIA and Chemistry analyzer (aware Technology Inc., USA) according to the manufacturer's method.

To measure the value of concentration, a calibration curve was used, constructed using a reference sample from the kit with a known concentration of Cas9 protein. The sensitivity is at least 1.5pg/ml, measured in the range of from 1.56pg/ml to 100 pg/ml. R-3.0.2 was used to statistically process the results and to visualize the data (https:// www.r-project. org /). The graph derived from the assay is shown in figure 5.

Figure 5 shows that the Cas9 gene was found after transfection of HDFa primary human cell cultures with gene therapy DNA vector VTvaf17-Cas9, whereas Cas9 gene was absent in the reference sample, confirming the ability of the vector to penetrate eukaryotic cells and express Cas9 gene at the protein level. The presented results also demonstrate the feasibility of using the gene therapy DNA vector VTvaf17-Cas9 to heterologously express the Cas9 gene in eukaryotic cells.

Example 6.

Evidence of the efficacy and feasibility of heterologous expression of Cas9 protein in mammalian cells using a gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene.

After transfection of HEKa primary human epidermal keratinocyte culture (ATCC PCS-200-011) with DNA vector VTvaf17-Cas9 carrying human Cas9 gene, the cell lysates of these cells were evaluated for changes in the concentration of Cas9 protein. Cells were cultured as described in example 3.

Transfection was performed using the 6 th generation SuperFect transfection reagent (Qiagen, Germany). The aqueous dendrimer solution without DNA carrier (a) and the DNA carrier VTvaf17(B) lacking cDNA of Cas9 gene were used as reference, and the DNA carrier VTvaf17-Cas9(C) carrying human Cas9 gene was used as transfection agent. DNA dendrimers were prepared and cells were transfected according to the manufacturer's method.

To measure the value of concentration, a calibration curve was used, constructed using a reference sample from the kit with a known concentration of Cas9 protein. The sensitivity is at least 1.5pg/ml, measured in the range of from 1.56pg/ml to 100 pg/ml. R-3.0.2 was used to statistically process the results and to visualize the data (https:// www.r-project. org /). The graph derived from the assay is shown in fig. 6.

Figure 6 shows that Cas9 gene was found after transfection of HEKa primary human epidermal keratinocyte cultures with gene therapy DNA vector VTvaf17-Cas9, whereas Cas9 gene was absent in the reference sample, confirming the ability of the vector to penetrate eukaryotic cells and express Cas9 gene at the protein level. The presented results also demonstrate the feasibility of using the gene therapy DNA vector VTvaf17-Cas9 to heterologously express the Cas9 gene in eukaryotic cells.

Example 7.

In transfecting normal adult (HEMA) with gene therapy DNA vector VTvaf17-Cas9 carrying human Cas9 gene

PCS-200-013^TM) Following primary epidermal melanocyte cultures, cell lysates of these cells were evaluated for changes in Cas9 protein concentration. Cells were cultured as described in example 4.

Transfection was performed using the 6 th generation SuperFect transfection reagent (Qiagen, Germany). The aqueous dendrimer solution without DNA carrier (a) and the DNA carrier VTvaf17(B) lacking Cas9 gene cDNA were used as reference, and the DNA carrier VTvaf17-Cas9(C) carrying the human Cas9 gene was used as transfection agent. Production of DNA dendrimers and transfection of HEMa cells were performed according to the manufacturer's method.

After transfection, 0.1ml of 1N HCl was added to 0.5ml of the culture broth, mixed well and incubated at room temperature for 10 minutes. The mixture was then neutralized by adding 0.1ml of 1.2M NaOH/0.5M HEPES (pH7-7.6) and stirred well. The supernatant was collected and used to assay for therapeutic proteins. Cas9 protein was detected by enzyme-linked immunosorbent assay (ELISA) using Cas9 (CRISPR-associated protein 9) ELISA kit (Cell Biolabs Inc, cat. prb-5079) and densitometry detection using ChemWell Automated EIA and Chemistry analyzer (aware Technology Inc., USA) according to the manufacturer's method.

To measure the value of concentration, a calibration curve was used, constructed using a reference sample from the kit with a known concentration of Cas9 protein. The sensitivity is at least 1.5pg/ml, measured in the range of from 1.56pg/ml to 100 pg/ml. R-3.0.2 was used to statistically process the results and to visualize the data (https:// www.r-project. org /). The graph derived from the assay is shown in fig. 7.

Figure 7 shows that the Cas9 gene was found after transfection of HEMa primary human cell cultures with gene therapy DNA vector VTvaf17-Cas9, whereas Cas9 gene was absent in the reference sample, confirming the ability of the vector to penetrate eukaryotic cells and express Cas9 gene at the protein level. The presented results also demonstrate the feasibility of using the gene therapy DNA vector VTvaf17-Cas9 to heterologously express the Cas9 gene in eukaryotic cells.

Example 8.

Evidence of the efficacy and feasibility of heterologous expression of Cas9 protein in human cells using gene therapy DNA vector VTvaf17-Cas9 carrying Cas9 gene.

CHO-K1 Syrian hamster ovary cells transfected with DNA vector VTvaf17-Cas9 carrying human Cas9 gene (

CCL-61^TM) Thereafter, changes in Cas9 protein concentration in cell lysates of these cells were evaluated. In the presence of 10% Fetal Bovine Serum (FBS) (FBS) under standard conditions

30-2020^TM) F-12K Medium (Kaighn modification of Ham F-12 Medium) ((II))

30-2004^TM) Culturing the cells.

Transfection was performed using the 6 th generation SuperFect transfection reagent (Qiagen, Germany). The aqueous dendrimer solution without DNA carrier (a) and the DNA carrier VTvaf17(B) lacking Cas9 gene cDNA were used as reference, and the DNA carrier VTvaf17-Cas9(C) carrying the human Cas9 gene was used as transfection agent. DNA dendrimers were prepared according to the manufacturer's method and cells were transfected.

To measure the value of concentration, a calibration curve was used, constructed using a reference sample from the kit with a known concentration of Cas9 protein. The sensitivity is at least 1.5pg/ml, measured in the range of from 1.56pg/ml to 100 pg/ml. R-3.0.2 was used to statistically process the results and to visualize the data (https:// www.r-project. org /). The graph derived from the assay is shown in fig. 8.

Figure 8 shows that Cas9 protein was found after transfection of CHO-K1 cell cultures with gene therapy DNA vector VTvaf17-Cas9, whereas Cas9 protein was absent in the reference sample, demonstrating the ability of the vector to penetrate eukaryotic animal cells and express Cas9 gene at the protein level. The presented results also demonstrate the feasibility of using the gene therapy DNA vector VTvaf17-Cas9 to heterologously express the Cas9 gene in eukaryotic animal cells.

Example 9.

After transfection of primary human Peripheral Blood Mononuclear Cells (PBMCs) with the DNA vector VTvaf17-Cas9 carrying the human Cas9 gene, changes in Cas9 protein concentration in cell lysates of these cells were evaluated. PBMC cells were isolated from 10ml human venous blood by fractionating in a Ficoll gradient 1.119(PanEco, P051-1). Cells were cultured in RPMI-1640 medium (PanEco, C310p) under standard conditions (37 ℃, 5% CO 2).

Transfection was performed using the 6 th generation SuperFect transfection reagent (Qiagen, Germany). Aqueous dendrimer solution of DNA vector (a) and DNA vector VTvaf17(B) lacking Cas9 gene cDNA were used as reference, and DNA vector VTvaf17-Cas9(C) carrying human Cas9 gene was used as transfection agent. DNA dendrimers were prepared according to the manufacturer's method and cells were transfected.

After 24 hours of transfection, 1ml of cell culture was pelleted, 0.1ml of 1N HCl was added to the pelleted cells, mixed well, and incubated at room temperature for 10 minutes. The mixture was then neutralized by adding 0.1ml of 1.2M NaOH/0.5M HEPES (pH7-7.6) and stirred well. The supernatant was collected and used to assay for therapeutic proteins. Cas9 protein was detected by enzyme-linked immunosorbent assay (ELISA) using Cas9 (CRISPR-associated protein 9) ELISA kit (Cell Biolabs Inc, cat. prb-5079) and densitometry detection using ChemWell Automated EIA and Chemistry analyzer (aware Technology Inc., USA) according to the manufacturer's method.

To measure the value of concentration, a calibration curve was used, constructed using a reference sample from the kit with a known concentration of Cas9 protein. The sensitivity is at least 1.5pg/ml, measured in the range of from 1.56pg/ml to 100 pg/ml. R-3.0.2 was used to statistically process the results and to visualize the data (https:// www.r-project. org /). The graph derived from the assay is shown in fig. 9.

Figure 9 shows that Cas9 protein was found after transfection of PBMC cell cultures with gene therapy DNA vector VTvaf17-Cas9, whereas Cas9 protein was absent in the reference sample, confirming the ability of the vector to penetrate human cells and express the Cas9 gene at the protein level. The presented results also demonstrate the feasibility of using the gene therapy DNA vector VTvaf17-Cas9 to heterologously express the Cas9 gene in eukaryotic mammalian cells.

Example 10.

Efficacy of gene therapy DNA vector VTvaf17-Cas9 carrying Cas9 gene and feasibility of increasing expression levels of Cas9 protein in animal tissues by injection of autologous fibroblasts transfected with gene therapy DNA vector VTvaf17-Cas 9.

To demonstrate the efficacy of the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene and the feasibility of its use, changes in rat skin Cas9 protein concentration were evaluated after injection of autologous fibroblast cultures of the same animals transfected with the gene therapy DNA vector VTvaf17-Cas 9.

Suitable autologous fibroblast cultures transfected with the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene were injected into the skin of 3 Wistar rats, along with placebo, in the form of animal autologous fibroblast cultures transfected with the gene therapy DNA vector VTvaf17 not carrying the Cas9 gene.

Rat primary fibroblasts were isolated from animal skin biopsy samples. Skin biopsy samples weighing approximately 11mg were collected using skin biopsy Epithesasy 3.5 (Memax SRL, Italy). Primary cell cultures were cultured at 37 ℃ in the presence of 5% CO2 in DMEM medium containing 10% fetal bovine serum and 100U/ml ampicillin. Medium passage and replacement were performed every 2 days. The total duration of culture growth does not exceed 25-30 days. Then 5x10 was removed from the cell culture broth⁴Aliquots of individual cells. Rat fibroblast cultures were transfected with the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene or placebo, i.e., VTvaf17 vector which does not carry the Cas9 therapeutic gene.

Transfection is carried out using cationic polymers such as polyethyleneimine JETPEI (Polyplus transfection, France) according to the manufacturer's instructions. Cells were cultured for 72 hours and then injected into animals. Autologous fibroblast cultures were injected into animals transfected with gene therapy DNA vector VTvaf17-Cas9, and non-transfected rat autologous fibroblast cultures of gene therapy DNA vector VTvaf17 were injected at the primary dehaired area to a depth of about 1mm using 27G needle tunnel method as placebo. The concentration of the modified autologous fibroblasts in the injection suspension is about 5mln cells per 1ml of suspension, and the dose of the injected cells does not exceed 10 mln. The injection points of the autologous fibroblast cultures are 3-5cm apart.

Biopsy samples were taken on day 4 after injection of autologous fibroblast cell culture transfected with gene therapy DNA vector VTvaf17-Cas9 carrying the therapeutic gene (i.e., Cas9 gene) and placebo. Biopsy samples were taken from animal skin at sites injected with autologous fibroblast cell culture (I) transfected with gene therapy DNA vector VTvaf17-Cas9 carrying the therapeutic gene (i.e., Cas9 gene), autologous fibroblast cell culture (placebo) (II) transfected with gene therapy DNA vector VTvaf17 not carrying the Cas9 therapeutic gene, and intact skin (III) using skin biopsy device Epitheasy 3.5(Medax SRL, Italy). The biopsy sample size was about 10mm3 and weighed about 11 mg. The sample was placed in a buffer solution containing 50mM Tris-HCl, pH7.6, 100mM NaCl, 1mM EDTA and 1mM phenylmethylsulfonyl fluoride, and homogenized to obtain a homogenized suspension. The suspension was then centrifuged at 14,000g for 10 minutes. The supernatant was collected and used to assay for therapeutic proteins as described in examples 5-9.

The analysis results are shown in FIG. 10.

Figure 10 shows that in the injected region of autologous fibroblast cultures transfected with the gene therapy DNA vector VTvaf17-Cas9 carrying the Cas9 gene in rat skin, Cas9 protein was found compared to its absence in the reference sample: in the injection region injected with autologous fibroblasts transfected with the gene therapy DNA vector VTvaf17 not carrying the Cas9 gene (placebo), and in samples from intact sites, this demonstrates the efficacy of the gene therapy DNA vector DNA VTvaf17-Cas9 and demonstrates its feasibility of use in order to increase Cas9 expression levels in mammalian tissues (particularly after injection of autologous fibroblasts transfected with the gene therapy vector DNA VTvaf17-Cas 9).

Example 11.

The gene therapy DNA vector carrying the Cas9 gene, VTvaf17-Cas9, was used to facilitate efficacy and feasibility of genome editing in mammalian cells.

After combined transfection of 293/GFP cell line cells (cell Biolabs, cat. akr-200) with the DNA vector VTvaf17-Cas9 carrying the human Cas9 gene and the GFP targeting guide RNA for CRISPR, the decrease in cellular fluorescence caused by the interruption of GFP gene expression in these cells was evaluated (genaxon bioscience, cat. p2008.0010). Cells were cultured in DMEM medium under standard conditions.

For transfection of cells with the DNA vector carrying the Cas9 gene VTvaf17-Cas9, Lipofectamine3000 (Thermfisher scientific, USA) was used according to the manufacturer's recommendations, as described in example 2. Cells were transfected 24 hours after transfection with the DNA vector carrying the Cas9 gene, VTvaf17-Cas9, using GFP-targeting guide RNA for CRISPR. For transfection of GFP-targeted guide RNAs for CRISPR, the CRISPRfect E transfection reagent (Genaxxon bioscience, cat. p2002.0035) was used according to the manufacturer's instructions.

As an experimental sample, water transfection was used, followed by transfection of GFP targeting guide rna for CRISPR (a), transfection of DNA vector VTvaf17 without Cas9 gene cDNA, followed by transfection of GFP targeting guide rna for CRISPR (b), transfection of DNA vector VTvaf17-Cas9 carrying Cas9 gene, followed by transfection of GFP targeting guide rna for CRISPR (c), transfection of DNA vector VTvaf17-Cas9 carrying Cas9 gene, followed by water transfection (D).

48 hours after the second transfection, the medium was removed, the cells were resuspended in physiological saline, and the number of cells expressing GFP fluorescent protein was assessed by flow fluorimetry using Beckman Coulter's Cytomics FC 500(Beckman Coulter's, USA). The analysis results are shown in FIG. 11.

Figure 11 shows that the result of transfecting 293/GFP primary cell culture with gene therapy DNA vector VTvaf17-Cas9 followed by transfection with GFP targeting guide RNA for CRISPR is a reduction in the number of GFP-expressing cells (about 80%) compared to no reduction in the number of GFP-expressing cells in the reference sample, confirming the ability of the vector to penetrate eukaryotic cells and express an active Cas9 endonuclease to edit the GFP gene through the GFP targeting guide RNA, which leads to GFP fluorescent protein silencing. The presented results also demonstrate the feasibility of the gene therapy DNA vector VTvaf17-Cas9 for eukaryotic targeted genome editing.

Example 12.

Use of gene therapy DNA vector VTvaf17-Cas9 carrying Cas9 gene to facilitate evidence of efficacy and feasibility of mammalian cell genome editing.

Transfection of MCF7 cell line clones with a mixture of DNA vector VTvaf17-Cas9 carrying Cas9 gene and gRNA _ cloning vector carrying oligonucleotides directed against the exon regions of TLR9 gene(s) (

HTB-22^TM) These cells were then screened to identify clones with genome editing.

Specific oligonucleotides corresponding to 20 nucleotide loci of the TLR9 gene exon (Toll-like receptor 9) were selected as sgrnas. sgRNA selection was performed using CRISPR design service (http:// CRISPR. mit. edu /). Two synthetic oligonucleotides, tlrg4f and tlrg4r (sequence listings (3) and (4)) were mixed in equimolar amounts in T4 DNA ligase buffer (Thermo scientific, USA), and the samples were heated at 94 ℃ for 2 minutes and then slowly cooled to room temperature to form duplexes. The oligonucleotide duplex was then cloned into a gRNA _ cloning vector (AddGene, # 41824). Plasmids containing the cloned oligonucleotides were amplified and purified in preparative quantities using a plasmid DNA purification kit (Qiagen, USA).

In EMEM Eagle Minimum Essential Medium (EMEM) (EMEM)

30-2003^TM) MCF-7 cells were cultured under standard conditions (37 ℃, 5% CO 2). Transfection was performed using the Lipofectamine3000 kit (Thermoscientific, USA) according to the manufacturer's instructions. Transfection was performed using an equimolar mixture of DNA vector VTvaf17-Cas9 carrying Cas9 gene and gRNA _ cloning vector carrying oligonucleotides directed against the exon regions of TLR9 gene. Water or a gRNA _ cloning vector carrying an oligonucleotide against the exon region of the TLR9 gene, or a DNA vector VTvaf17-Cas9 carrying the Cas9 gene were used as references.

The cells were then seeded into 96-well plates using a FACS sorter. Cells grown in 96-well plates were washed with PBS, lysed with 50 μ Ι _ of DNA expression reagent (Lytech, Russia) to analyze clones grown after sorting, and then sample preparation was performed according to the manufacturer's recommendations. The obtained sample was used as a substrate to amplify a locus region containing a sgRNA recognition site in the TLR9 gene by real-time PCR. The obtained PCR fragment was subjected to sequencing analysis using ABI Prism 3730xl genetic Analyzer (Application Biosystems, USA). As a result, 4 clones were identified that contained changes in the nucleotide sequence of the TLR9 gene and were derived from targeted genome editing. Clones containing any changes in the TLR9 gene sequence were not identified in the reference sample.

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

Figure 12 shows that as a result of transfecting MCF7 cell line with DNA vector VTvaf17-Cas9 carrying Cas9 gene and a gRNA _ cloning vector mixture carrying oligonucleotides directed to TLR9 gene exon regions, several clones with targeted genome editing were identified, confirming the ability of DNA vector VTvaf17-Cas9 to penetrate eukaryotic cells and express an active Cas9 endonuclease, thereby enabling introduction of mutations into therapeutic genes, such as TLR9 gene, through the gRNA, as shown by targeted sequence sequencing. This result also demonstrates the feasibility of using the gene therapy DNA vector VTvaf17-Cas9 to facilitate targeted genome editing in eukaryotic cells.

Example 13.

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

Construction of a strain of gene therapy DNA vector based on gene therapy DNA vector VTvaf17 for the production of Cas9 therapeutic gene on an industrial scale, i.e. e.coli strain SCS110-AF/VTvaf17-Cas9 carrying gene therapy DNA vector VTvaf17-Cas9, allowing antibiotic-free selection, involves the preparation of electrocompetent cells of e.coli strain SCS110-AF and electroporation of these cells with gene therapy DNA vector VTvaf17-Cas 9. Thereafter, the cells were poured into agar plates (petri dishes) with a selective medium containing yeast extract, peptone, 6% sucrose, and 10 μ g/ml chloramphenicol. Wherein the production of the escherichia coli strain SCS110-AF to produce the gene therapy DNA vector VTvaf17 or a gene therapy DNA vector based on the gene therapy DNA vector VTvaf17, allows for antibiotic-free positive selection, which involves the construction of a 64bp linear DNA fragment containing the transposon Tn10 regulatory element RNA-IN that allows for antibiotic-free positive selection; the 1422bp fructan sucrase gene sacB (the product of which ensures selection in a medium containing sucrose), the 763bp chloramphenicol resistance gene catR required for cloning of the strain undergoing homologous recombination and the two homologous sequences 329bp and 233bp (ensuring homologous recombination in the region of the gene recA concurrent with the inactivation of the gene) were selected, then the e.coli cells were transformed by electroporation and clones surviving in a medium containing 10 μ g/ml chloramphenicol were selected.

The obtained strains used for production were included in the collections of National center for Biological resources (National Biological Resource Centre), Russian National collections of Industrial Microorganisms (NBRC RNCIM), RF patent deposit services in the UK under the following accession numbers:

coli strain SCS110-AF/VTvaf17-Cas 9-registered at Russian national collections of Industrial microorganisms, accession No. B-, deposit date: .

Example 14.

Method for expanding gene therapy DNA vectors based on gene therapy DNA vector VTvaf17 carrying Cas9 therapeutic gene to industrial scale.

To demonstrate the producibility and constructability of the gene therapy DNA vector VTvaf17-Cas9(SEQ ID No.1) on an industrial scale, large-scale fermentation of the e.coli strain SCS110-AF/VTvaf17-Cas9 comprising the gene therapy DNA vector VTvaf17 carrying the therapeutic gene, i.e. the Cas9 gene, was performed. Coli strain SCS110-AF/VTvaf17-Cas9 was produced based on E.coli strain SCS110-AF (Cell and Gene Therapy LLC, United Kingdom) as described in example 13 by: competent cells of this strain were electroporated with the gene therapy DNA vector VTvaf17-Cas9 carrying the therapeutic gene (i.e., Cas9 gene), where the transformed cells were further inoculated in agar plates (petri dishes) with selective medium containing yeast extract, peptone, and 6% sucrose, and selection of individual clones was performed.

Fermentation of E.coli SCS110-AF/VTvaf17-Cas9 carrying gene therapy DNA vector VTvaf17-Cas9 was performed in a 10l fermentor, followed by extraction of gene therapy DNA vector VTvaf17-NOS 2.

For the fermentation of the E.coli strain SCS110-AF/VTvaf17-Cas9, a medium containing the following components per 10l volume was prepared: 100g tryptone and 50g yeast extract (Becton Dickinson, USA); the medium was then diluted to 8800ml with water and autoclaved at 121 ℃ for 20 minutes and then 1200ml of 50% (w/v) sucrose was added. Thereafter, seed cultures of the E.coli strain SCS110-AF/VTvaf17-Cas9 were inoculated in culture flasks in a volume of 100 ml. The cultures were incubated at 30 ℃ for 16 hours in a shaker incubator. The seed culture was transferred to a Techfors S bioreactor (Infors HT, Switzerland) and cultured to stationary phase. The process was controlled by measuring the optical density of the culture at 600 nm. Cells were pelleted at 5,000-10,000g for 30 min. The supernatant was removed and the cell pellet was resuspended in 10% (by volume) phosphate buffered saline. The cells were centrifuged again at 5,000-10,000g for 30 min. The supernatant was removed, a solution containing 20mM TrisCl, 1mM EDTA, 200g/l sucrose (pH 8.0) was added to the cell pellet in a volume of 1000ml, and the mixture was stirred well to a homogenized suspension. The egg lysozyme solution was then added to a final concentration of 100. mu.g/ml. The mixture was incubated on ice for 20 minutes with gentle stirring. Then 2500ml of 0.2M NaOH, 10g/l Sodium Dodecyl Sulfate (SDS) was added, the mixture was incubated on ice for 10 minutes while stirring gently, then 3500ml of 3M sodium acetate, 2M acetic acid (pH 5-5.5) were added, and the mixture was incubated on ice for 10 minutes while stirring gently. The resulting sample was centrifuged at 15,000g or more for 20-30 minutes. The solution was carefully decanted and the residual precipitate was removed by a strainer (filter paper). RNase A (Sigma, USA) was then added to a final concentration of 20. mu.g/ml and the solution was incubated overnight at room temperature for 16 hours. The solution was then centrifuged at 15,000g for 20-30 minutes and passed through a 0.45 μm membrane filter (Millipore, USA). Then, ultrafiltration was performed with a 100kDa membrane (Millipore, USA), and the mixture was diluted to the initial volume with a buffer solution of 25mM TrisCl (pH 7.0). This operation was performed three to four times. The solution was applied to a column containing 250ml DEAE Sepharose HP (GE, USA) and equilibrated with 25mM TrisCl (pH 7.0). After loading, the column was washed with three volumes of the same solution, and then the gene therapy DNA vector VTvaf17-Cas9 was eluted using a linear gradient of 25mM Tris-HCl (pH 7.0) to obtain a solution of 25mM TrisCl (pH 7.0), 1M NaCl, five times the column volume. The elution process was controlled by measuring the optical density of the effluent solution at 260 nm. The chromatogrAN _ SNhic fractions containing the gene therapy DNA vector VTvaf17-Cas9 were pooled 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 the optical density of the effluent solution at 260nm and the fractions were analyzed by agarose gel electrophoresis. Fractions containing gene therapy DNA vector VTvaf17-Cas9 were pooled and stored at-20 ℃. To evaluate the reproducibility of the process, the specified treatment operations were repeated five times.

The process reproducibility and quantitative properties of the final product yield confirm the productivity and constructability of the gene therapy DNA vector VTvaf17-Cas9 on an industrial scale.

Thus, the constructed gene therapy DNA vectors containing therapeutic genes can be used to inject human, animal and mammalian cells, providing heterologous expression of Cas9 endonuclease, which can be used for human and animal genomic sequence editing in the presence of specific grnas.

The aim set by the invention is to construct gene therapy DNA vectors for heterologous expression of Cas9 protein in human and animal cells, combining the following properties:

I) efficacy of gene therapy DNA vectors for heterologous expression of therapeutic genes in eukaryotic cells;

II) the possibility of safe use in various methods of genome editing of human and animal genomes due to the absence of regulatory elements as nucleotide sequences of viral genomes in gene therapy DNA vectors;

III) the possibility of safe use in various methods of genome editing of the human and animal genome due to the absence of antibiotic resistance genes in gene therapy DNA vectors;

IV) the producibility and constructability of gene therapy DNA vectors on an industrial scale,

and to achieve the construction objectives of strains carrying these gene therapy DNA vectors for the production of these gene therapy DNA vectors, which are supported by the following examples:

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

for II and III-examples 1, 11, 12;

for IV-examples 1, 13, 14.

INDUSTRIAL APPLICABILITY

All the examples listed above demonstrate the industrial applicability of the proposed gene therapy DNA vector based on the gene therapy DNA vector VTvaf17 carrying the Cas9 therapeutic gene, the escherichia coli strain SCS110-AF/VTvaf17-Cas9 carrying the gene therapy DNA vector and the production method thereof for the heterologous expression of the Cas9 gene in human and animal cells, as well as the production method of the gene therapy DNA vector on an industrial scale.

And (3) a abbreviation list:

VTvaf 17: gene therapy vectors lacking viral genome and antibiotic resistance marker (virus-free: antibiotic therapeutic vector) sequences

gRNA: guide RNA

PMBC: peripheral blood mononuclear cells

DNA: deoxyribonucleic acid

cDNA: complementary deoxyribonucleic acid

RNA: ribonucleic acid

Mrna: messenger ribonucleic acid

bp: base pairing

And (3) PCR: polymerase chain reaction

ml: ml, μ l: microlitre

mm 3: cubic millimeter

l: lifting of wine

μ g: microgram of

mg: milligrams of

g: keke (Chinese character of 'Keke')

μ M: micromolar

And (mM): millimole

min: minute (min)

s: second of

rpm: revolutions per minute

nm: nano meter

cm: centimeter

mW: milliwatt meter

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_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.

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,Vol.45,No.1,p.44–55。

Claims

1. a gene therapy DNA vector based on gene therapy DNA vector VTvaf17 carrying a Cas9 therapeutic gene for heterologous expression of this therapeutic gene in human and animal cells in carrying out various genome editing methods, wherein the gene therapy DNA vector VTvaf17-Cas9 has the nucleotide sequence of SEQ ID No. 1.

2. A gene therapy DNA vector based on gene therapy DNA vector VTvaf17 carrying a Cas9 therapeutic gene according to claim 1, which is unique due to the fact that the gene therapy DNA vector VTvaf17-Cas9 constructed according to claim 1 has the ability to efficiently penetrate human and animal cells and express Cas9 therapeutic gene cloned thereto due to the limited size of the VTvaf17 vector portion not exceeding 3200 bp.

3. A gene therapy DNA vector carrying a Cas9 therapeutic gene based on gene therapy DNA vector VTvaf17 according to claim 1, which is unique due to the fact that it does not contain a nucleotide sequence of viral origin nor an antibiotic resistance gene, thus ensuring its safety for enabling various methods of genome editing in humans and animals, including gene therapy in humans and animals.

4. A method of producing a gene therapy DNA vector based on gene therapy DNA vector VTvaf17 carrying a Cas9 therapeutic gene according to claim 1, the method involving obtaining the gene therapy DNA vector VTvaf17-Cas9 according to claim 1 as follows: the coding region of the Cas9 therapeutic gene was cloned into DNA vector VTvaf17 and gene therapy DNA vector VTvaf17-Cas9 was obtained.

5. Method for the heterologous expression of a Cas9 therapeutic gene in human and animal cells using a gene therapy DNA vector based on gene therapy DNA vector VTvaf17 according to claim 1, which involves introducing the gene therapy DNA vector according to claim 1 in combination with a gRNA into human or animal cells, organs and tissues, and/or introducing autologous human or animal cells into human or animal organs and tissues transfected with the gene therapy DNA vector according to claim 1 together with the gRNA, or a combination of said methods.

6. A method for the production of the e coli strain SCS110-AF/VTvaf17-Cas9, said method involving electroporation of competent cells of e coli strain SCS110-AF by means of the gene therapy DNA vector according to claim 1, followed by selection of stable clones of said strain using a selective medium.

7. The E.coli strain SCS110-AF/VTvaf17-Cas9 carrying a gene therapy DNA vector produced according to claim 6, carrying the gene therapy DNA vector according to claim 1 for its production, allowing antibiotic-free selection.

8. A method for producing a gene therapy DNA vector according to claim 1 on an industrial scale, the method involving: the fraction containing the therapeutic DNA product, i.e. the gene therapy DNA vector VTvaf17-Cas9 according to claim 1, was extracted by expanding the bacterial culture of the strain according to claim 7 to the amount necessary for increasing the bacterial biomass in industrial fermentors, then using the biomass, then multi-stage filtration and purification by chromatographic methods.