Nucleic acids having promoter activity and their use

Abstract

This application relates to the fields of genetics, gene therapy, and molecular biology. More specifically, the present invention relates to nucleic acids (variants) having promoter activity, expression cassettes and vectors based thereon, target products or host cells for producing expression vectors.

Description

Technical Field

[0001] This application relates to the fields of genetics, gene therapy, and molecular biology. More specifically, the present invention relates to nucleic acids (variants) having promoter activity, expression cassettes and vectors based thereon, and host cells for producing target products or expression vectors.

Background Art

[0002] A promoter is a DNA element that promotes the transcription of a polynucleotide to which the promoter is operably linked. A promoter can also be part of a promoter / enhancer element. The physical boundary between a promoter element and an enhancer element is not always clear, but a promoter typically refers to a site on a nucleic acid molecule to which RNA polymerase and / or any associated factors bind and where transcription is initiated. An enhancer enhances promoter activity both temporally and spatially. A number of promoters are known to be transcriptionally active in a wide range of cell types. Promoters can be divided into two classes: those that function constitutively and those that are regulated by induction or derepression. Both classes are suitable for protein expression. Promoters used for high-level production of polypeptides in eukaryotic cells, particularly mammalian cells, should be strong and preferably active in a wide range of cell types. Strong constitutive promoters capable of driving expression in a number of cell types are well known in the art and thus need not be described in detail herein.

[0003] Examples of promoters and / or enhancers include promoters and / or enhancers derived from retroviral LTRs, cytomegalovirus (CMV) (e.g., CMV promoter / enhancer), simian virus 40 (SV40) (e.g., SV40 promoter / enhancer), adenovirus (e.g., adenovirus major late promoter (AdMLP)), the CAG promoter, and strong mammalian promoters such as the TTR promoter or EF1-α promoter.

[0004] The CMV promoter has a length of 589 - 1650 bp depending on the subtype (Changyu Zheng et al., All Human EF1α Promoters Are Not Equal: Markedly Affect Gene Expression in Constructs from Different Sources, Int J Med Sci. 2014;11(5):404 - 408, doi:10.7150 / ijms.8033). The CAG promoter (CMV early enhancer / chicken β-actin) has a length of 868 bp (Nieuwenhuis, B., Haenzi, B., Hilton, S. et al., Optimization of adeno-associated viral vector-mediated transduction of the corticospinal tract: comparison of four promoters. Gene Ther. 2021;28:56 - 74, https: / / doi.org / 10.1038 / s41434-020-0169-1).

[0005] All of the above promoters have a length exceeding 588 bp. A promoter with a length exceeding 588 bp in an expression cassette is not an optimal choice for the expression of large transgenes using multiple expression vectors.

[0006] That is, it is necessary to create a short promoter. A number of promoters, including the CMV promoter, are not tissue-specific, i.e., they do not provide selective expression of a therapeutic transgene in cells of a particular organ, and this fact is a clear drawback of these promoters when used to produce gene therapy products.

[0007] That is, it is necessary to create a tissue-specific promoter that provides selective expression of a therapeutic transgene in cells of a particular organ.

Summary of the Invention

Means for Solving the Problems

[0008] The inventors of the present invention surprisingly found that a nucleic acid having a nucleotide sequence selected from the group consisting of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 has promoter activity. The promoter having the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 has a length in the range of 123 to 252 bp. Furthermore, the inventors of the present invention surprisingly found that a promoter having the nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 has tissue-specific promoter activity in cells of hepatocyte origin.

[0009] Definitions and General Methods Unless otherwise defined herein, all technical and scientific terms used in connection with the present invention have the same meaning as commonly understood by one of ordinary skill in the art.

[0010] Furthermore, unless the context requires otherwise, singular terms shall include plural terms and plural terms shall include singular terms. Typically, current classifications and methods in cell culture, molecular biology, immunology, microbiology, genetics, analytical chemistry, organic synthetic chemistry, medicinal chemistry and pharmaceutical chemistry, as well as the hybridization and chemistry of proteins and nucleic acids described herein, are well known to those skilled in the art and widely used in the art. Enzyme reactions and purification methods are carried out according to the manufacturer's guidelines, as is common in the art or as described herein.

[0011] As used in this specification and the following claims, unless the context indicates otherwise, the words "include" (and its variations "includes", "including", "comprise" and "comprising") shall be understood to imply the inclusion of the stated integer or group of integers but not the exclusion of any other integer or group of integers.

[0012] Nucleic acid molecule The terms "nucleic acid", "nuclear sequence", "nucleic acid sequence", "polynucleotide", "oligonucleotide", "polynucleotide sequence" and "nucleotide sequence", which are used interchangeably herein, mean the precise sequence of nucleotides that determines a fragment or region of a nucleic acid, which may or may not contain unnatural nucleotides and which is either a modified form or not, and which is any of double-stranded DNA or RNA, single-stranded DNA or RNA, or the transcription product of said DNA.

[0013] As used herein, polynucleotides include, by way of non-limiting example, all nucleic acid sequences obtained by recombinant means, i.e., any means available in the art including the cloning of nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, etc., and by synthetic means.

[0014] It should also be included here that the present invention is not related to its natural chromosomal environment, i.e., the nucleotide sequence in its natural state. The sequences of the present invention are isolated and / or purified, i.e., sampled directly or indirectly, for example by copying, and their environment is at least partially modified. That is, for example, isolated nucleic acids obtained by recombinant genetics using host cells or obtained by chemical synthesis should also be mentioned here.

[0015] Unless otherwise indicated, the term nucleotide sequence includes its complement. That is, a nucleic acid having a specific sequence should be understood to include its complementary strand having the complementary sequence.

[0016] Vector As used herein, the term "vector" means a nucleic acid molecule capable of transporting another nucleic acid linked thereto. Further, the term "vector" herein means a recombinant viral particle capable of transporting a nucleic acid.

[0017] As used in this description, the term "expression" is defined as the transcription and / or translation of a specific nucleotide sequence driven by its promoter. Detailed Description of the Invention Nucleic Acid In one aspect, the present invention relates to a nucleic acid having promoter activity and comprising a nucleotide sequence selected from the group consisting of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15.

[0018] In some embodiments of the present invention, the nucleic acid is an isolated nucleic acid. An "isolated" nucleic acid molecule is one that is identified and separated from at least one nucleic acid molecule impurity. An isolated nucleic acid molecule is different from the form or set found under natural conditions. That is, an isolated nucleic acid molecule is different from the nucleic acid molecule present in a cell under natural conditions.

[0019] All nucleic acids having a nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 have promoter activity. All promoters having a nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 have tissue-specific promoter activity in cells of hepatocyte origin.

[0020] All promoters having a nucleotide sequence of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15 are strong promoters, and their use as part of an expression vector results in an increase in the level of production and activity of the target protein.

[0021] Expression cassette. Expression vector. In one aspect, the present invention relates to an expression cassette comprising any one of the above nucleic acids having promoter activity.

[0022] As used herein, the term "cassette that expresses ~" or "expression cassette" particularly means, in a suitable setting, a DNA fragment capable of causing the expression of a polynucleotide encoding a polypeptide of interest whose sequence is included in the expression cassette. When introduced into a host cell, the expression cassette can, among other things, cause a polynucleotide encoding a polypeptide of interest to be transcribed into RNA by the cellular machinery, and the RNA is typically subsequently further processed and ultimately translated into the polypeptide of interest. The expression cassette can be included in an expression vector.

[0023] In some embodiments, the expression cassette comprises the following elements in the 5' to 3' direction: Left (first) ITR (inverted terminal repeat); Any one of the above nucleic acids having promoter activity; Transgene; Polyadenylation signal; Right (second) ITR.

[0024] The above structural elements of the expression cassette are operably linked to each other. As used herein, the term "operably linked" means the linkage of polynucleotide (or polypeptide) elements that are in a functional relationship. A nucleic acid is "operably linked" when it is present under conditions that allow for a functional relationship with another nucleic acid sequence. For example, a transcriptional regulatory sequence is operably linked to a coding sequence if it affects the transcription of the coding sequence. The term "operably linked" means that the linked DNA sequences are typically contiguous and, where necessary to join two protein-coding regions, contiguous and in the reading frame.

[0025] In some embodiments, the expression cassette comprises a left (first) ITR having the nucleotide sequence of SEQ ID NO: 16. In some embodiments, the expression cassette comprises a polyadenylation signal having the nucleotide sequence of SEQ ID NO: 17.

[0026] In some embodiments, the expression cassette comprises a right (second) ITR having the nucleotide sequence of SEQ ID NO: 18. In some embodiments, the expression cassette comprises the following elements in the 5' to 3' direction: A left (first) ITR (inverted terminal repeat) having the nucleotide sequence of SEQ ID NO: 16; A promoter having a nucleotide sequence selected from the group of SEQ ID NO: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15; A transgene; A polyadenylation signal having the nucleotide sequence of SEQ ID NO: 17; A right (second) ITR having the nucleotide sequence of SEQ ID NO: 18.

[0027] In some embodiments, the expression cassette comprises a transgene encoding a protein or small molecule inhibitory nucleic acid. In one aspect, the expression cassette comprises a transgene encoding a protein selected from the group consisting of Factor VIII or a functional variant thereof, Factor IX or a functional variant thereof, SMN1 protein (survival motor neuron protein), the RBD-S polypeptide of SARS-cov2, or a therapeutic antibody.

[0028] In one aspect, the present invention relates to an expression vector comprising any of the above nucleic acids having promoter activity or any of the above expression cassettes. In some embodiments of the invention, the vector is capable of autonomous replication in the host cell into which it is introduced (e.g., bacterial vectors and episomal vectors having a bacterial origin of replication site). In a further embodiment of the invention, the vector (e.g., a non-episomal vector) can be integrated into the genome of the host cell upon introduction into the host cell, thereby replicating with the host genome. Further, some vectors can be directed to the expression of a gene to which the vector is operably linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors").

[0029] Examples of expression vectors include plasmids, retroviruses, adenoviruses, adeno-associated viruses (AAV), plant viruses such as cauliflower mosaic virus, tobacco mosaic virus, cosmids, YACs, EBV, etc. The DNA molecule can be inserted into the vector such that the transcriptional and translational control sequences within the vector perform their intended function of regulating the transcription and translation of the DNA. The expression vector and the expression control sequences can be selected to be compatible with the expression host cell used. The DNA molecule can be introduced into the expression vector by standard methods (e.g., ligation of complementary restriction sites, or blunt-end ligation if no restriction sites are present).

[0030] In some embodiments of the invention, the vector is a plasmid, AAV, adenovirus or lentivirus. In some embodiments of the present invention, the vector is a plasmid, i.e., a circular double-stranded DNA into which additional DNA segments can be inserted.

[0031] In some embodiments of the present invention, the vector is a viral (expression) vector, wherein additional DNA segments can be inserted into the viral genome. In some embodiments, the expression vector is a recombinant adeno-associated virus (AAV).

[0032] In some embodiments, the AAV is selected from the group consisting of the following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, rAAV.rh8, rAAV.rhlO, rAAV.rh20, rAAV.rh39, rAAV.Rh74, rAAV.RHM4-1, AAV.hu37, rAAV.Anc80, rAAV.Anc80L65, rAAV.7m8, rAAV.PHP.B, rAAV2.5, rAAV2.6, rAAV2.8, rAAV2.9, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15 or AAV.HSC16.

[0033] In some aspects of the invention, the vector or cassette can include other expression control sequences, in addition to the promoter. As used herein, the term "other expression control sequences" refers to polynucleotide sequences necessary to achieve the expression and processing of the inserted coding sequence. Those skilled in the art will understand that the design of the expression vector or cassette, including the selection of the expression control sequences, can depend on factors such as the type of host cell to be transformed and the required level of protein expression. Expression control sequences, in addition to the promoter, can include corresponding transcription initiation sequences, termination sequences, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequences); sequences that enhance protein stability; and, if desired, sequences that enhance protein secretion. The nature of such control sequences varies depending on the host organism; in prokaryotes, such control sequences generally include a promoter, a ribosome binding site, and a transcription termination sequence; in eukaryotes, such control sequences typically include a promoter and a transcription termination sequence. Expression control sequences encompass at least all components whose presence is important for expression and processing.

[0034] In addition to the genes and expression control sequences described above, the recombinant expression vectors according to the invention can carry additional sequences such as sequences that control the replication of the vector in the host cell (e.g., origin of replication) and selectable marker genes. The selectable marker gene facilitates the selection of host cells into which the vector or cassette has been introduced.

[0035] In one aspect of the invention, the expression vector relates to a vector that includes one or more polynucleotide sequences of interest, a gene of interest, or a transgene flanked by parvovirus sequences or inverted terminal repeat (ITR) sequences.

[0036] Neither the cassette nor the vector of the present invention contains the nucleotide sequences of the genes encoding the non-structural protein (Rep) and the structural protein (Cap) of adeno-associated virus.

[0037] Host cell In one aspect, the present invention relates to a host cell for producing a target product or for producing any one of the above expression vectors, which contains any one of the above nucleic acids having promoter activity.

[0038] As used herein, the term "host cell" means a cell into which a recombinant expression vector or cassette according to the present invention has been introduced. It should be understood that the term "host cell" means not only a specific target cell but also the progeny of such a cell. Such progeny may not actually be identical to the parental cell because modifications due to either mutation or environmental influences may occur in subsequent generations; however, such cells are still included within the scope of the term "host cell" as used herein.

[0039] The expression vector or cassette according to the present invention can be used for transfection of mammalian cells, plant cells, bacteria or yeast host cells. Transfection can be carried out by any known method for introducing a polynucleotide into a host cell. Methods for introducing a heterologous polynucleotide into mammalian cells are well known in the art and include dextran-mediated transfection, cationic polymer-nucleic acid complex transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, encapsulation of the polynucleotide in liposomes, and direct microinjection of DNA into the nucleus. In addition, nucleic acid molecules can be introduced into mammalian cells by viral (expression) vectors.

[0040] Mammalian cell lines used as hosts for transfection are well-known in the art and include a plurality of immortalized cell lines that are available. These include, for example, Chinese hamster ovary (CHO) cells, NS0 cells, SP2 cells, HEK-293T cells, FreeStyle293 cells (Invitrogen), NIH-3T3 cells, HeLa cells, baby hamster kidney (BHK) cells, African green monkey kidney cells (COS), human hepatocellular carcinoma cells (e.g., HepG2), A549 cells, SK-HEP1, HUH7, Hep-RG, and many other cell lines. The cell line is selected by determining that the cell line has a high expression level and provides the necessary characteristics of the protein to be produced. Other cell lines that can be used are insect cell lines such as Sf9 or Sf21 cells. When a recombinant expression vector according to the present invention is introduced into a mammalian host cell, the target protein is produced by culturing the host cell for a period sufficient for the target protein to be expressed in the host cell or, more preferably, secreted into the culture medium in which the host cell is cultured. The target protein can be isolated from the culture medium using standard protein purification techniques. Examples of plant host cells include those of the genus Nicotiana, Arabidopsis, duckweed, corn, wheat, and potato. Examples of bacterial host cells include bacterial species of the genus Escherichia and Streptomyces. Examples of yeast host cells include Schizosaccharomyces pombe, Saccharomyces cerevisiae, and Pichia pastoris.

[0041] The above host cells are not related to host cells generated using human embryos. The above host cells are not related to host cells generated by modifying the genetic integrity of human germline cells. BRIEF DESCRIPTION OF THE DRAWINGS

[0042]

Figure 1

Figure 2

[0043] Percentage of EGFP-expressing cells after transfection: 1 - using an expression cassette containing the EGFP transgene and not containing the promoter region (control); 2 - using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 4; 3 - using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 5; 4 - using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 6; 5 - using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 7; 6 - using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 13; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 14; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 12; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 3.

Figure 3

[0044] Percentage of EGFP-expressing cells after transfection: Using an expression cassette containing the EGFP transgene and not containing the promoter region (control); Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 8; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 10; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 9; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 15.

Figure 4

[0045] Fluorescence intensity of cells expressing EGFP after transfection: Using an expression cassette containing the EGFP transgene and not containing the promoter region (control); Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 4; Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 5; Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 6; Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 7; Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 13; Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 14; Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11; Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 12; 10-Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 3.

Figure 5

[0046] Fluorescence intensity of cells expressing EGFP after transfection: 1-Using an expression cassette containing an EGFP transgene and not containing a promoter region (control); 2-Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 8; 3-Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 10; Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 9; Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 15.

Figure 6

[0047] Level of production of clotting proteins during transfection: 1 - Using an expression cassette containing a coagulation factor transgene and not containing a promoter region (control); 2 - Using an expression cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 3; 3 - Using an expression cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11; 4 - Using an expression cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 14.

Figure 7

[0048] Level of activity of clotting proteins during transfection: 1 - Using an expression cassette containing a coagulation factor transgene and not containing a promoter region (control); 2 - Using an expression cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 3; 3 - Using an expression cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11; 4 - Using an expression cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 14.

Figure 8

[0049] Percentage of EGFP-expressing cells after transfection: 1-Using an expression cassette containing the EGFP transgene and not containing the promoter region (control); 2-Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 4; 3-Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 5; 4-Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 6; 5-Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 7; 6-Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 13; 7-Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 14; 8-Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11; 9-Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 12; 10-Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 3.

Figure 9

[0050] Percentage of EGFP-expressing cells after transfection: 1 - Using an expression cassette containing the EGFP transgene and not containing a promoter region (control); 2 - Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 7; 3 - Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11.

Figure 10

[0051] Fluorescence intensity of cells expressing EGFP after transfection: 1 - Using an expression cassette containing the EGFP transgene and not containing a promoter region (control); 2 - Using an expression cassette containing the EGFP transgene and supplemented with the cytomegalovirus (CMV) virus promoter region (control); 3 - Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 2; 4 - Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 1.

Figure 11

[0052] Fluorescence intensity of cells expressing EGFP after transfection: 1 - Using an expression cassette containing the EGFP transgene and not containing a promoter region (control); Using an expression cassette containing the 2-EGFP transgene and supplemented with the cytomegalovirus (CMV) virus promoter region (control); Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 2; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 1.

Figure 12

[0053] Fluorescence intensity of cells expressing EGFP after transfection: Using an expression cassette containing the 1-EGFP transgene and no promoter region (control); Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 4; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 5; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 6; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 7; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 13; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 14; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11; Using an expression cassette containing the EGFP transgene under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 12; Using an expression cassette containing an EGFP transgene under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 3.

Figure 13

Figure 14

[0054] Level of production of coagulation proteins during transduction: 1 - using an expression vector containing a cassette containing the coagulation factor transgene and not containing the promoter region (control); 2 - using an expression vector containing a cassette in which the coagulation factor transgene is under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 8; 3 - using an expression vector containing a cassette in which the coagulation factor transgene is under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11; 4 - using an expression vector containing a cassette in which the coagulation factor transgene is under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 14; 5 - using an expression vector containing a cassette in which the coagulation factor transgene is under the control of the promoter corresponding to the nucleic acid sequence of SEQ ID NO: 3;

Figure 15

[0055] Level of coagulation protein activity during transduction: 1 - Using an expression vector containing a cassette that includes a coagulation factor transgene and does not include a promoter region (control); 2 - Using an expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 8; 3 - Using an expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11; 4 - Using an expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 14; 5 - Using an expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 3;

Figure 16

[0056] Level of coagulation protein production during transduction: 1 - Using an expression vector containing a cassette that includes a coagulation factor transgene and does not include a promoter region (control); 2 - Using an expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11; 3 - Using an expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 8.

Figure 17

[0057] Level of coagulation protein activity during transduction: 1 - Using an expression vector containing a cassette that includes a coagulation factor transgene and does not include a promoter region (control); 2 - Using an expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11; 3 - Using an expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 8.

Figure 18

[0058] Level of coagulation factor protein in animal plasma after injection: 1 - AAV - free control solution (negative control). 2 - Using an AAV - based expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 8.

[0059] 3 - Using an AAV - based expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11.

Figure 19

[0060] Levels of coagulation factor proteins in animal plasma after injection: 1 - A control solution without AAV (negative control). 2 - Using an AAV - based expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 8.

[0061] 3 - Using an AAV - based expression vector containing a cassette in which the coagulation factor transgene is under the control of a promoter corresponding to the nucleic acid sequence of SEQ ID NO: 11.

Mode for Carrying Out the Invention

[0062]

Examples

[0063] The following examples are provided for a better understanding of the present invention. These examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention in any way.

[0064] All publications, patents, and patent applications cited herein are incorporated herein by reference. Although the above - described invention has been described in some detail by way of illustration and example for the purpose of clarity of understanding, it will be readily apparent to those skilled in the art that certain changes and modifications can be made thereto without departing from the spirit or scope of the appended claims in light of the teachings of the present invention.

[0065] Materials and General Methods Recombinant DNA technology Standard methods were used to manipulate DNA as described in Sambrook, J. et al., Molecular cloning: A laboratory manual; Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 2012. Reagents for molecular biology were used according to the manufacturer's protocols. Briefly, plasmid DNA was generated for further manipulation in Escherichia coli (E. coli) cells grown under selective antibiotic pressure so that the plasmid was not lost in the cell population. The inventors isolated plasmid DNA from cells using a commercially available kit, measured the concentration, and used this for cloning by restriction endonuclease treatment or PCR amplification. DNA fragments were ligated to each other using ligase and transformed into bacterial cells for clone selection and further generation. All obtained gene constructs were confirmed by restriction pattern and full Sanger sequencing.

[0066] DNA Sequencing The DNA sequence was determined by Sanger sequencing. DNA and protein sequences were analyzed, and the sequence data were processed in SnapGene Viewer 4.2 or higher for sequence generation, mapping, analysis, annotation, and illustration.

[0067] Culture of Cell Cultures The experiments were conducted using the following cell lines: HEK293 (human embryonic kidney clone 293 cell line), HUH7 (human hepatocellular carcinoma cell line), U87-MG (Uppsala 87 malignant glioma cell line), CHO-K1-S (Chinese hamster ovary cell line), and HepG2 (human hepatocellular carcinoma cell line). The suspension HEK293 cells used to generate AAV were cultured under standard conditions at 37 °C and 5% CO2 in a complete culture medium without FBS and antibiotics. The adherent HEK293, HUH7, and HepG2 cells used to test the effectiveness of EGFP and AAV product expression were cultured under standard conditions at 37 °C and 5% CO2 in complete DMEM medium supplemented with 10% FBS and antibiotics / antifungal agents. The adherent U87-MG and CHO-K1-S cells used for tissue specificity tests were cultured under standard conditions at 37 °C and 5% CO2 in complete EMEM nutrient medium supplemented with 10% FBS and antibiotics / antifungal agents, and DMEM / F12 medium supplemented with 5% FBS and antibiotics / antifungal agents, respectively.

[0068] The cells were passaged when they reached 80 - 90% confluence. TrypLE Select enzyme (10×) was used to disperse the cell monolayer. Cell viability was evaluated using trypan blue staining and a disposable cell counting chamber with an automatic Countess II counter.

[0069] Transfection of cell cultures To evaluate the functional activity of the novel promoter variants during transfection, the inventors used plasmids containing expression cassettes for the expression of various variants of EGFP and coagulation factor transgenes. The model cell lines were seeded at 10000 cells / cm 2They were pre-seeded into the wells of a 12-well plate at a density of . One day later, the inventors added plasmid DNA of test and control samples with equal copy numbers as part of the complex with Lipofectamine 3000. Regarding the mutants of the transgene based on the gene encoding green fluorescent protein (EGFP), on the third day after transfection, the inventors determined the proportion of EGFP-expressing cells and the intensity of cell fluorescence by flow cytometry. Regarding the mutants of the transgene based on the coagulation factor, on the seventh day after transfection, the inventors determined the content and activity of the coagulation protein in the culture medium by ELISA and colorimetric assay. An intact model cell line was used as a negative control.

[0070] Production and purification of viral particles of AAV recombinant vectors To generate recombinant AAV viral particles containing a transgene based on a coagulation factor, the inventors used HEK293 producer cells transfected with three plasmids: · A plasmid containing an AAV expression cassette for the expression of the coagulation factor; · A plasmid containing the Cap gene of serotype AAV6 / AAV5 and the Rep gene of serotype AAV2. Using alternative reading frames, each gene encodes several protein products; · A plasmid containing the Ad2 adenovirus gene required for the assembly and packaging of the AAV capsid.

[0071] 72 hours after transfection, the cells were lysed, followed by purification of the viral particles, which were concentrated using filtration, chromatography, and ultracentrifugation. The titer of the viral particles was determined by quantitative PCR using primers and samples specific for the region of the recombinant viral genome and expressed as the number of copies of the viral genome per milliliter.

[0072] Transduction of cell cultures The Huh7 cell line was seeded at 10,000 cells / cm 2They were pre-seeded into the wells of a 12-well plate at a density of . After the cells adhered to the adhesion substrate, the AAV preparation was introduced at an MOI of 500,000 vg / cell. On the 7th day after transduction, the levels and activities of coagulation proteins in the culture medium were determined by ELISA and a colorimetric assay. Studies involving the evaluation of the levels and activities of coagulation proteins in the culture medium were conducted in 6 independent experiments. Untreated cells were used as negative controls.

[0073] Determination of the percentage of cells expressing the EGFP reporter protein and the intensity of cellular fluorescence using flow cytometry To evaluate the expression of the EGFP reporter protein in a flow cytometer fluorometer, on the 3rd day after transfection, a pre-prepared mixture of buffer and propidium iodide (PI) was added to the cell pellet at a ratio of 1 μL of PI at a concentration of 10 μg / mL per 1 mL of buffer. Cells that were not stained with PI and not transfected with plasmid DNA (isotype) were transferred to the wells of a 96-well microplate to assess compensation, and the buffer / PI mixture was added to the remaining wells of the microplate to measure the controls and samples containing PI. After transferring the cell samples to the microplate, they were incubated for 5 minutes in the absence of light. After incubation, the microplate containing the test cell samples was loaded into a flow cytometer for analysis. Using flow cytometry, the percentage of cells containing EGFP, as well as the average intensity of the fluorescence signal, was determined. Each sample was measured in 3 technical replicates (the number of events analyzed was 10,000 or more).

[0074] Determination of the amount of coagulation factor protein by ELISA The content of coagulation factor proteins in the culture medium after HUH7 cell transfection and transduction using the target candidate was evaluated by the sandwich method of non-competitive solid-phase enzyme immunoassay (ELISA). Briefly, culture fluid samples diluted in a commercially available buffer were transferred to 96-well microplates sensitized with a coagulation factor-specific primary antibody. Standards and controls for plotting the calibration curve were loaded onto the same microplate. The plate was incubated at a temperature of 37 °C for 1 hour. Prior to the introduction of the biotinylated antibody, horseradish peroxidase-conjugated streptavidin solution, and tetramethylbenzidine (TMB) substrate, the microplate wells were washed with buffer. Next, a solution containing the biotinylated detection antibody specific for the coagulation factor was introduced, and the microplate was incubated at a temperature of 37 °C for 30 minutes. The streptavidin horseradish peroxidase-conjugated solution was added to the resulting complex, and the plate was incubated at a temperature of 37 °C for 30 minutes. To visualize the enzyme reaction, the TMB solution was introduced. Once the required staining intensity was achieved, the stop solution was added to all wells to stop the reaction. After stopping the reaction, the optical density was measured. The concentration of the coagulation factor in the test sample was measured by normalization of the colorimetric staining according to the standard sample calibration curve, taking into account the pre-dilution factor.

[0075] Determination of the activity level of coagulation factor proteins by ELISA The activity of coagulation factor proteins in the culture medium after transfection and transduction of Huh7 cells using target candidates and control samples was evaluated using a chromogenic assay. The assay is based on the fact that in the presence of calcium ions, phospholipids, and factor IXa, factor X is converted to activated Xa, factor VIII functions as a cofactor in the reaction, and the rate of factor X activation is linearly related to the level of factor VIII. Briefly, culture medium samples diluted in a commercially available buffer, standards, and controls for plotting a calibration curve were transferred to a 96-well microplate. The samples were then subjected to incubation at a temperature of 37 °C for 3 minutes, after which a factor reagent solution containing factor IXa, factor X, thrombin, CaCl2, and phospholipids was introduced into the microplate wells. The microplate was then incubated at a temperature of 37 °C for 4 minutes, after which a chromogenic substrate solution S-2765+I-2581 was introduced into the wells. Subsequently, the plate was incubated at a temperature of 37 °C for 7 minutes. When the required degree of staining intensity was achieved, a 20% solution of acetic acid was added to all the wells to stop the reaction. Subsequently, the optical density of the solution in the microplate wells was measured. The activity of the coagulation factors in the test samples was determined by normalization of chromogenic staining with a standard sample calibration curve, taking into account preliminary dilution.

[0076] In vivo studies on experimental animals B6.129S-F8tm1Smoc mice (male, 6 - 8 weeks old) with a defect in coagulation factor were used for the experiment. The product was administered to the animals using a single intravenous injection into the tail vein. A buffer solution without AAV was administered to the negative control group of animals. Plasma samples were collected on the day of injection before administration of the product and subsequently on the 70th day after introduction of the expression vector. All animal tests were conducted in full compliance with the ARRIVE guidelines.

[0077] Statistical data analysis The results are presented as mean ± standard deviation (SD), and the experimental results were compared using one-way analysis of variance (ANOVA) followed by Dunnett's multiple pairwise comparison, and it was determined that they were statistically significant.

[0078] Example 1. In order to increase the expression level of the target gene, while designing the gene construct, the inventors used a panel of promoters (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) developed based on transcription factor binding sites. These nucleotide sequences as part of an expression cassette consisting of a left (first) ITR (inverted terminal repeat) corresponding to the sequence of SEQ ID NO: 16, a promoter selected from the group comprising SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, the target gene, a polyadenylation signal (SEQ ID NO: 17), and a right (second) ITR (SEQ ID NO: 18), wherein the target gene can be a green fluorescent protein (GFP) sequence or a coagulation cascade protein sequence, were tested in vitro by transfecting model cell lines (HEK293, HUH7, U87-MG, CHO-K1-S and HepG2). An expression cassette containing a left (first) ITR (SEQ ID NO: 16), the target gene, a polyadenylation signal (SEQ ID NO: 17), and a right (second) ITR (SEQ ID NO: 18) was used as a control. Figures 1 and 2 also show additional controls containing the same elements of the expression cassette supplemented with the constitutive cytomegalovirus (CMV) viral promoter.

[0079] All nucleic acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, which are promoter regions, provide an increase in the percentage of cells expressing the EGFP reporter protein (Figs. 1 - 3) and an increase in the intensity of EGFP fluorescence (Figs. 4 - 5) when using the green fluorescent protein sequence as the gene of interest. When using the coagulation factor sequence as the gene of interest, the inventors also observed an increase in the levels of production (Fig. 6) and activity (Fig. 7) of the coagulation protein when compared to the use of the nucleic acid of the gene of interest without the promoter region. That is, the nucleic acids (SEQ ID NOs: 1 - 15) exhibit promoter activity as part of an expression cassette containing various variants of the transgene during transfection of model cell lines (Huh7, CHO - K1 - S and HepG2).

[0080] Example 2. To measure the tissue specificity of the developed nucleic acid sequences (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) having promoter activity, a nucleotide sequence was tested during transfection of in vitro model cell lines (HEK293, HUH7, U87 - MG, CHO - K1 - S and HepG2) as part of an expression cassette consisting of the left (first) ITR (inverted terminal repeat) corresponding to the sequence of SEQ ID NO: 16, a promoter (SEQ ID NOs: 1 - 15), the gene of interest, a polyadenylation signal (SEQ ID NO: 17), and the right (second) ITR (SEQ ID NO: 18). The gene of interest can be represented by the EGFP protein sequence or a nucleic acid encoding a coagulation cascade protein. An expression cassette containing the left (first) ITR (SEQ ID NO: 16), the gene of interest, the polyadenylation signal (SEQ ID NO: 17), and the right (second) ITR (SEQ ID NO: 18) was used as a control. Also, in Figs. 8, 10, 11 and 12, a control containing the same elements of the expression cassette supplemented with the cytomegalovirus (CMV) virus promoter region was also used.

[0081] All nucleic acids of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15, which are promoter regions, provide an increase in the percentage of cells expressing the EGFP reporter protein (Figs. 8 and 9) and an increase in the intensity of EGFP fluorescence (Figs. 10 - 13) in cell lines of hepatocyte origin, when compared to the use of nucleic acids that do not contain a promoter region. That is, the nucleic acids (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) have tissue - specific promoter activity in cells of hepatocyte origin.

[0082] Example 3. The inventors generated an AAV - based expression vector containing an expression cassette encoding a transgene (coagulation factor) under the control of the nucleic acids of SEQ ID NOs: 8, 11 and 14, which have promoter activity. The expression vector was confirmed by transducing Huh7 cells in vitro. An AAV - based expression vector that does not contain a promoter region was used as a control.

[0083] The use of expression vectors containing the nucleic acids of SEQ ID NOs: 8, 11 and 14 results in an increase in the levels of production (Fig. 14) and activity (Fig. 15) of the coagulation protein, when compared to the use of expression vectors that do not contain a promoter region. The use of expression vectors based on various serotypes of AAV containing the nucleic acids of SEQ ID NOs: 8 and 11 showed an increase in the levels of production (Fig. 16) and activity (Fig. 17) of the coagulation protein, when compared to the control ones. The results correspond to the data obtained during the transfection of Huh7 cells (Figs. 6 and 7). That is, the nucleic acids (SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or 15) exhibit promoter activity while being delivered as expression vectors containing various variants of the transgene during the transduction of the model cell line.

[0084] Example 4. To measure the promoter activity of the nucleic acids of SEQ ID NO: 8 and 11 as part of an in vivo expression vector, the inventors generated an AAV-based product in which the transgene is a coagulation factor under the control of the nucleic acids of SEQ ID NO: 8 or 11. Next, the test product was administered to B6.129S-F8tm1Smoc experimental mice. A buffer for diluting the virus product without AAV particles was used as a negative control. The AAV product was administered to the animals once using intravenous hydrodynamic injection into the tail vein. Plasma samples were collected on the day of injection (day 0) before product administration and subsequently on day 70 after administration.

[0085] In vivo studies showed that when using a test drug containing a coagulation factor gene sequence under the control of the nucleic acids of SEQ ID NO: 8 and 11, a significant increase in the amount and activity of the coagulation factor in the plasma of the animals was observed on day 70 after product administration (Figs. 18-19). That is, the nucleic acids of SEQ ID NO: 8 and 11 exhibit promoter activity when delivered as an AAV-based expression vector in vivo.

Claims

1. A nucleic acid having promoter activity, comprising a nucleotide sequence selected from the group including SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

2. An expression cassette comprising nucleic acid molecules and operably linked transgenes as described in claim 1.

3. The expression cassette according to claim 2, wherein the introduced gene encodes a protein or small molecule inhibitory nucleic acid.

4. The expression cassette according to claim 3, wherein the transgene encodes a protein selected from the group including factor VIII or a functional variant thereof, factor IX or a functional variant thereof, SMN1 protein (survival motor neuron protein), SARS-cov2 RBD-S polypeptide, or a therapeutic antibody.

5. An expression vector comprising the nucleic acid described in claim 1 or the expression cassette described in any one of claims 2 to 4.

6. The expression vector according to claim 5, which is a plasmid, AAV, lentivirus, or adenovirus.

7. The following AAV serotypes: AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, rAAV. rh8, rAAV. rhlO, rAAV. rh20, rAAV. rh39, rAAV. Rh74, rAAV. RHM4-1, AAV. hu37, rAAV. Anc80, rAAV. Anc80L65, rAAV. 7m8, rAAV. PHP. The expression vector according to claim 6, which is an AAV selected from the group including B, rAAV2.5, rAAV2.6, rAAV2.8, rAAV2.9, rAAV2tYF, rAAV3B, rAAV.LK03, AAV.HSC1, AAV.HSC2, AAV.HSC3, AAV.HSC4, AAV.HSC5, AAV.HSC6, AAV.HSC7, AAV.HSC8, AAV.HSC9, AAV.HSC10, AAV.HSC11, AAV.HSC12, AAV.HSC13, AAV.HSC14, AAV.HSC15, or AAV.HSC16.

8. A host cell for producing a target product comprising the nucleic acid described in Claim 1, wherein the target product encodes a protein or small molecule inhibitory nucleic acid.

9. A host cell for generating an expression vector comprising the nucleic acid described in Claim 1 or the expression cassette described in any one of Claims 2 to 4, wherein the host cell comprises the nucleic acid described in Claim 1 or the expression cassette described in any one of Claims 2 to 4.

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