KR102726438B1 - Genetically engineered bacterial expressing multiple miRNA targeting cancer cells - Google Patents

Abstract

The present invention relates to bacterial strains genetically engineered to express multiple miRNAs targeting cancer cells and to uses thereof.

Description

{Genetically engineered bacterial strain expressing multiple miRNA targeting cancer cells}

The present invention relates to bacterial strains genetically engineered to express multiple miRNAs targeting cancer cells and to uses thereof.

Among various diseases, various treatments have been developed to treat cancer, which has the highest mortality rate both domestically and internationally. However, treatment using traditional small-molecule anticancer drugs has non-specific effects that do not distinguish between cancer cells and normal cells, and causes the emergence of cancer cells with increased resistance to anticancer treatment, or serious side effects due to excessive cytotoxicity.

However, due to the advancement of anticancer drug development technology, the specificity for tumors has been greatly increased compared to the past, and the therapeutic effect has been improved. Nevertheless, improved anticancer drugs still have problems. For example, Bevacizumab, a single antibody that binds to VEGF-A, and Cetuzimab, a single antibody that binds to the EGF receptor, have been used to treat colon cancer, lung cancer, and brain tumors, but their specificity is reduced due to frequent mutations in the target protein. In addition, immunotherapy using immune checkpoint inhibitors such as Pembrolizumab, Ipilimumab, and Nivolumab, which have recently attracted attention, has very limited effects on cold tumor patients with extremely low T cell activity. Therefore, the need for a new concept of treatment that can solve the problems of these treatments is being demanded in the medical field. Recently, targeted therapy at the gene level is being developed to overcome the problems related to target protein mutations found in tumors, and in particular, the correlation between miRNA and disease is receiving attention.

MicroRNAs (miRNAs) are short RNA fragments of approximately 19-25 nucleotides in length that do not contain genetic information on their own, but are known to regulate the expression of various genes. miRNAs regulate the final function of specific genes by participating in various biological processes such as the cell cycle, differentiation, proliferation, death, stress resistance, metabolism, and immune response. Gene expression induced by miRNA is regulated by the final gene expression by the miRNA-induced silencing complex (miRISC) binding to the 3'-untranslated regions (UTR) of the target mRNA to inhibit mRNA cleavage or translation. In this way, miRNAs have been found to regulate the expression of approximately 30% of human genes, about half of which are involved in inducing tumors, and it is thought that reduced expression of miRNAs in tumor cells contributes to the development of tumors.

On the other hand, some pathogenic microorganisms, including Salmonella and Listeria, cause diseases when infected, but many studies have shown that these attenuated microorganisms, when administered to laboratory mice, attack tumors, inhibit their growth, and extend the survival period of laboratory mice with tumors. In particular, since the possibility of using Salmonella for tumor treatment was suggested through preclinical experiments, research on tumor treatment using Salmonella has been actively conducted. Salmonella is known to form colonies in tumor tissues and directly kill tumor cells, or change the immune microenvironment around the tumor, causing the patient's immune cells to recognize the tumor and further induce the death of tumor cells. Nevertheless, there was controversy over the anticancer effect of Salmonella in clinical trials, and for this reason, the approach has developed from treating tumors with Salmonella alone to delivering therapeutic drugs.

One object of the present invention is to provide a recombinant nucleic acid construct comprising a polynucleotide encoding a plurality of miRNAs targeting cancer cells.

Another object of the present invention is to provide a system for expressing the recombinant nucleic acid construct provided in the present invention.

Another object of the present invention relates to a bacterial strain genetically engineered to express said plurality of miRNAs by being transfected with the expression system provided by the present invention.

Another object of the present invention relates to a composition for preventing, improving or treating cancer comprising the genetically engineered bacterial strain provided in the present invention.

However, the technical problems to be achieved by the present invention are not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

According to one embodiment of the present invention, the present invention relates to a recombinant nucleic acid construct comprising a polynucleotide encoding each of miR-138 and miR-153.

In the present invention, "miR" or "microRNA (miRNA)" is a small non-expressed RNA molecule consisting of about 21 to 23 nucleotides found in plants, animals, viruses, etc., and is a non-coding RNA that negatively regulates gene expression at the post-transcriptional level by triggering a process called RNA interference (RNAi, see below). MicroRNAs are involved in almost all biological processes, including development, differentiation, proliferation, and apoptosis (Xiao and Rajewsky, 2009). In addition, they are known to play an important role in diseases such as cancer, heart failure, and metabolic disorders (Xiao et al., 2009; Divaka et al., 2008; Krutzfeldt et al., 2006). MicroRNA genes are scattered throughout all human chromosomes except the Y chromosome. They can be located in non-coding regions of the genome or within introns of protein-coding genes. Approximately 50% of miRNAs occur in clusters, which are transcribed as polycistronic primary transcripts. Similar to protein-coding genes, miRNAs are typically transcribed from polymerase II promoters, producing so-called primary miRNA transcripts (pri-miRNAs). Pri-miRNAs are then processed through a series of endonucleolytic cleavage steps, which are performed by two enzymes belonging to the RNAse type III family, Drosha and Dicer. From pri-miRNA, a stem loop of about 60 nucleotides in length, called pre-miRNA, is cleaved by a specific nuclear complex consisting of Drosha and DiGeorge syndrome critical region gene (DGCR8), which cleaves two strands near the base of the primary stem loop, leaving a 5' phosphate and a 2 bp long 3' overhang. The pre-miRNA is then actively transported from the nucleus to the cytoplasm by RAN-GTP and Exportin. Dicer then performs a double-strand cleavage at the end of the stem loop that is not defined by the Drosha cut, generating a 19-24 bp duplex, which consists of the mature miRNA and the opposite strand of the duplex, called miRNA* (Bartel DP., 2004). According to the thermodynamic asymmetry rule, only one strand of the duplex is selectively loaded into the RNA-induced silencing complex (RISC) and accumulated as a mature microRNA.

In the present invention, the "miR-138" is one of a family of miRNA precursors found in animals including humans, and miRNAs are generally transcribed into a 70 nucleotide precursor and then processed by the dicer enzyme to form a 22 nucleotide product. At this time, the excised region or mature product of the miR-138 precursor corresponds to miR-138. For example, two basic transcripts, pri-miR-138-1 and pri-miR-138-2, are encoded in chromosomes 3 (3p21) and 16 (16q13), and generate mature miR-138. In the present invention, the miR-138 has the function of suppressing the expression of a gene encoding human PD-L1 (Programmed Cell Death-Ligand-1), which is one of the immune checkpoint proteins.

In the present invention, it is known that changes in the expression of the "miR-153" lead to invasion, metastasis, angiogenesis, and progression of various types of tumors. In the present invention, the miR-153 has the function of suppressing the expression of a gene encoding human IDO (indoleamine dioxygenase), which is one of the immune checkpoint proteins.

In the present invention, the polynucleotide encoding the miR-138 or miR-153 may be a polynucleotide encoding the mature form of these miRNAs, as well as a polynucleotide encoding the miRNA mimic. Here, the miRNA mimic may include a miRNA precursor, an early transcript miRNA (pri-miRNA), or a miRNA precursor in the form of a plasmid.

In the present invention, the mature sequence of the above miR-138 may be hsa-miR-138 derived from human (homo sapiens) and may be hsa-miR-138-5p consisting of a base sequence represented by sequence number 1, but is not limited thereto.

In the present invention, the sequence encoding the mature sequence of miR-138 may include a base sequence represented by SEQ ID NO: 2, preferably, may be composed of a base sequence represented by SEQ ID NO: 2, but is not limited thereto.

In the present invention, the sequence encoding the precursor (pre-miRNA) of miR-138 may include a base sequence represented by SEQ ID NO: 3, preferably, may be composed of a base sequence represented by SEQ ID NO: 3, but is not limited thereto.

In the present invention, the sequence encoding the initial transcript (pri-miRNA) of miR-138 may include a base sequence represented by SEQ ID NO: 4, preferably, may be composed of a base sequence represented by SEQ ID NO: 4, but is not limited thereto.

In the present invention, the mature sequence of the above miR-153 may be hsa-miR-153 derived from human (homo sapiens) and may be hsa-miR-153-5p consisting of a base sequence represented by sequence number 5, but is not limited thereto.

In the present invention, the sequence encoding the mature sequence of miR-153 may include a base sequence represented by SEQ ID NO: 6, preferably, may be composed of a base sequence represented by SEQ ID NO: 6, but is not limited thereto.

In the present invention, the sequence encoding the precursor (pre-miRNA) of miR-153 may include the base sequence represented by SEQ ID NO: 7, preferably, may be composed of the base sequence represented by SEQ ID NO: 7, but is not limited thereto.

In the present invention, the sequence encoding the initial transcript (pri-miRNA) of miR-153 may include the base sequence represented by SEQ ID NO: 8, preferably, may be composed of the base sequence represented by SEQ ID NO: 8, but is not limited thereto.

The recombinant nucleic acid construct of the present invention may simultaneously comprise a sequence encoding miR-138 or a mimic thereof and a sequence encoding miR-153 or a mimic thereof in a single construct, but it is also within the scope of the present invention to separately comprise a polynucleotide comprising a sequence encoding miR-138 or a mimic thereof and a polynucleotide comprising a sequence encoding miR-153 or a mimic thereof.

In the present invention, when a sequence encoding miR-138 or a mimic thereof and a sequence encoding miR-153 or a mimic thereof are simultaneously included in one construct, the connection order of the two genes is not particularly limited, and may be connected in the order of miR-138 (or mimic thereof) coding sequence-miR-153 (or mimic thereof) coding sequence from 5' to 3', or may be connected in the order of miR-153 (or mimic thereof) coding sequence-miR-138 (or mimic thereof) coding sequence.

According to another embodiment of the present invention, the present invention relates to an expression vector comprising a recombinant nucleic acid construct provided by the present invention.

In order to introduce the recombinant nucleic acid construct of the present invention into an appropriate host cell, the construct may be inserted so that it can be cloned into a vector.

In the present invention, the "vector" refers to a gene expression system that is a recombinant vector that can be introduced into a suitable host cell to express a target protein and includes essential regulatory elements operably linked to allow the gene insert to be expressed.

In the present invention, a number of expression systems such as chromosomes, episomes, and induced viruses can be used as vectors for inserting the recombinant nucleic acid construct. More specifically, recombinant expression vectors that can be used include, but are not limited to, those derived from bacterial plasmids, transposons, yeast episomes, insertion elements, yeast chromosomal elements, viruses such as baculovirus, papillomaviruses such as SV40, vaccinia virus, adenovirus, adeno-associated virus, retroviruses, fowlpox virus, and pseudorabies virus. These recombinant expression vectors may also be cosmid or phagemid derivatives.

In exemplary aspects, one type of vector is a “plasmid,” which refers to a circular double-stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a phage vector. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication within a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are capable of integrating into the genome of a host cell upon introduction into the host cell, thereby replicating along with the host genome. Additionally, certain vectors are capable of directing the expression of a gene to which the vector is operably linked. Such vectors are referred to herein as “recombinant expression vectors” (or simply, “recombinant vectors”).

In the present invention, the term “operatively linked” means a functional linkage between an expression control site (e.g., promoter, signal sequence, ribosome binding site, transcription termination sequence, transcription regulatory factor binding site, etc.) of a target nucleic acid molecule and another nucleic acid sequence, whereby the control sequence controls transcription and/or translation of the other nucleic acid sequence.

The vector system of the present invention can be constructed by various methods known in the art, and specific methods thereof are disclosed in Sambrook et al., Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory Press (2001), which is incorporated herein by reference.

As an example of the present invention, a vector that can be used in the present invention can be produced by manipulating a plasmid (e.g., pSC101, ColE1, pBR322, pUC8/9, pHC79, pUC19, pET, etc.), a phage (e.g., λgt4λB, λ-Charon, λΔz1, λGEM.TM.-11, and M13, etc.) or a virus (e.g., SV40, etc.) that is often used in the art.

As an example of the present invention, the recombinant nucleic acid construct can be inserted into a host cell using a viral expression system (vaccinia or other poxvirus, retrovirus or adenovirus). For example, viral vectors can include, but are not limited to, retroviral vectors derived from HIV, SIV, murine retroviruses, gibbon ape leukemia virus, adeno-associate viruses (AAVs) and adenoviruses (Miller et al., 1990, Mol. Cell Biol. 10:4239; J. Kolberg 1992, NIH Res. 4:43; Cornetta et al., 1991, Hum. Gene Ther. 2:215). Retroviral vectors derived from murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), ecotropic retroviruses, simian immunodeficiency virus (SIV), and human immunodeficiency virus (HIV) are widely used.

As an example of the present invention, when the recombinant nucleic acid construct is DNA, the so-called ‘naked DNA’ method can be utilized, or a plasmid vector or a viral vector can be utilized, and the nucleic acid can be included in a form in which it can be expressed as a vector targeting prokaryotic or eukaryotic cells.

The expression vector of the present invention may include, in addition to the nucleic acid sequence of the recombinant nucleic acid construct as the expression target sequence, a functional sequence for expressing it. In the present invention, the functional sequence may include one or more promoters capable of conducting transcription, one or more introns, one or more transcription termination regions, one or more initiation codons, and one or more termination or stop codons. When a prokaryotic cell is used as the host cell in the present invention, the vector may include a ribosome binding site (RBC) and/or a transcription/translation termination sequence to initiate translation into a peptide. In addition, the expression vector of the present invention may include a nucleic acid sequence encoding a protease or peptidase cleavage site, and may also include one or more leader sequences. Here, each leader sequence encodes a signal peptide. In addition, the expression vector of the present invention may include one or more linker or tag sequences. Here, the tag sequence may encode a hemagglutinin (HA) tag.

In the present invention, the promoter as a structural promoter or an inducible promoter can be used in the present invention according to the needs of a particular situation that can be identified by a person skilled in the art. A number of promoters recognized by various possible host cells are well known. The selected promoter can be operably linked to a cistronic DNA encoding a peptide described herein by removing the promoter from the source DNA via a restriction enzyme recognition sequence and inserting the isolated promoter sequence into a selection vector. Both native promoter sequences and a number of heterologous promoters can be used to direct amplification and/or expression of the target gene. However, heterologous promoters can generally achieve transcription and expression efficiency of the expressed target gene compared to the native target polypeptide promoter.

As an example of the present invention, when a prokaryotic cell is used as a host cell, it is common to include a strong promoter capable of driving transcription (e.g., tac promoter, lac promoter, lacUV5 promoter, lpp promoter, pLλ promoter, pRλ promoter, rac5 promoter, amp promoter, recA promoter, SP6 promoter, trp promoter, T7 promoter, etc.), a ribosome binding site for initiation of translation, and a transcription/translation termination sequence. When E. coli is used as a host cell, the promoter and operator region of the E. coli tryptophan biosynthetic pathway (Yanofsky, C., J. Bacteriol., 158:1018-1024(1984)) and the left-hand promoter of phage λ (pLλ promoter, Herskowitz, I. and Hagen, D., Ann. Rev. Genet., 14:399-445(1980)) can be used as regulatory regions. As such plasmid vectors, plasmids commonly used in the art, such as pSC101, pBR322, pUC19, and pET-22, can be used.

As an example of the present invention, when a eukaryotic cell is used as a host cell, a promoter derived from a mammalian cell (metallotyanin promoter) or a promoter derived from a mammalian virus can be used. In other words, when a virus is to be used as a vector of the present invention, not only an attenuated virus such as vaccinia or flowpox, but also an adenovirus vector, an adeno-associated virus vector, a retrovirus vector, a herpes simplex virus, a non-toxic anthrax toxin vector, etc. can be used to express a recombinant nucleic acid construct.

As an example of the present invention, in addition to the types described above, the promoter may include, for example, a promoter derived from a mammalian virus such as a cytomegalovirus (CMV) promoter, an adenovirus late promoter, a vaccinia virus 7.5K promoter, an SV40 promoter, a tk promoter of HSV, an RSV promoter, an EF1 alpha promoter, a metallothionine promoter, a beta-actin promoter, a promoter of a human IL-2 gene, a promoter of a human IFN gene, a promoter of a human IL-4 gene, a promoter of a human lymphotoxin gene, a promoter of a human GM-CSF gene, a cancer cell-specific promoter (e.g., a TERT promoter, a PSA promoter, a PSMA promoter, a CEA promoter, an E2F promoter, and an AFP promoter), a tissue-specific promoter (e.g., an albumin promoter), and/or a bacteriophage T7. Promoters may be used, but are not limited to these.

The expression vector of the present invention may further include an enhancer sequence to increase transcriptional activity. The enhancer sequence is a nucleic acid base sequence located at various sites in the promoter and increases transcriptional activity compared to the transcriptional activity by the promoter when the enhancer sequence is absent. At this time, the enhancer may be, but is not limited to, an enhancer of human actin, human myosin, human hemoglobin, human muscle creatine, or a viral enhancer derived from CMV, SV40, RSV, and EBV.

In the present invention, the expression vector may comprise a sequence encoding one or more signal peptides/secretion signal peptides to promote and/or increase the expression or post-translational secretion of the recombinant nucleic acid construct. In the present invention, the signal sequence or peptide is a secretion signal, also known as a leader sequence or peptide, or a localization signal or sequence, which is a short peptide at the N-terminus of a newly synthesized protein to be secreted. Signal peptides generally promote the cell to translocate the protein to the cell membrane. The efficiency of protein secretion is strongly determined by the signal peptide. Therefore, the immunostimulatory bacteria of the present invention contain a plasmid which may comprise a signal peptide/secretion signal peptide to promote and/or increase the expression or secretion of the encoded therapeutic agent(s). In the present invention, the signal sequence may include an Hly signal sequence, an ActA signal sequence, a PhoA signal sequence, an OmpA signal sequence, an α-amylase signal sequence, an LLO signal sequence, a subtilisin signal sequence, an MF-α signal sequence, a SUC2 signal sequence, an insulin signal sequence, an α-interferon signal sequence, an antibody molecule signal sequence, and the like, but may also be any other known signal sequence.

The expression vector of the present invention may further include a transcription termination sequence to increase the stability of the transcript or facilitate cytoplasmic transport. At this time, the transcription termination sequence may include, but is not limited to, a polyadenylation sequence (e.g., bovine growth hormone terminator and SV40-derived polyadenylation sequence).

Additionally, if the expression vector of the present invention is a replicable expression vector, it may include a replication origin, which is a specific nucleic acid sequence at which replication is initiated.

The expression vector of the present invention can include a DNA nuclear targeting sequence (DTS). A DNA nuclear targeting sequence (DTS), such as the SV40 DTS, mediates translocation of a DNA sequence through the nuclear pore complex. The mechanism of this transport is reported to depend on the binding of a DNA binding protein comprising a nuclear localization sequence. Inclusion of expression vectors to increase nuclear transport and expression has been demonstrated (see, e.g., Dean, D.A. et al. (1999) Exp. Cell Res. 253(2):713-722) and has been used to increase gene expression from plasmids delivered by Salmonella typhimurium (see, e.g., Kong et al. (2012) Proc. Natl. Acad. Sci. U.S.A. 109(47):19414-19419).

As an example of the present invention, the DNA nuclear targeting sequence may include, but is not limited to, a base sequence represented by SEQ ID NO: 9.

In addition, the expression vector of the present invention may further include a selection marker to facilitate purification of the expressed peptide. The selection marker is for selecting cells transformed with the vector, and a marker that confers a selectable phenotype such as drug resistance, nutrient requirement, resistance to cytotoxic agents, or expression of surface proteins may be used. Examples of such markers include, but are not limited to, glutathione S-transferase (Pharmacia, USA), maltose binding protein (NEB, USA), FLAG (IBI, USA), and 6 x His (hexahistidine; Quiagen, USA).

Several in vitro amplification techniques are known for amplifying sequences cloned into the expression vector of the present invention. These techniques include polymerase chain reaction (PCR), ligase chain reaction (LCR), Qβ-replicase amplification, and other techniques using RNA polymerases.

In the present invention, bacteria can be used as a host cell, and a bacterial strain transfected with the expression vector of the present invention can be used for the prevention, improvement, or treatment of cancer.

Accordingly, the expression vector of the present invention may additionally include a polynucleotide encoding a toxin protein having a function of directly or indirectly inducing the death of cancer cells. In the present invention, the toxin protein may be at least one selected from the group consisting of Ricin, Saporin, Gelonin, Momordin, Debuganin, Diphtheria toxin, Pseudomonas toxin, Hemolysin (HlyA), FAS ligand (FASL), Tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), streptolysin O (SLO), pneumolysin (PLO), listeriolysin O (LLO), and Cytolysin A (ClyA), but is not limited thereto.

As an example of the present invention, the expression vector may further include a polynucleotide encoding a toxin protein, listeriolysin O (LLO). In the present invention, the “listeriolysin O (LLO)” is a hemolysin produced by a bacterium, Listeria monocytogenes, which is a pathogen that causes listeriosis. Listeriolysin O (LLO) lyses and destroys phagosomes, and then the bacteria escape into the cytoplasm, where they can grow within the cell. After escaping from the phagosome, the toxin has little activity in the cytoplasm. The listeriolysin O (LLO) is encoded by the Hly gene, which is part of a pathogenicity island called LIPI-1.

As an example of the present invention, the listerilysine O (LLO) may include an amino acid sequence represented by SEQ ID NO: 10, and the Hly gene may include a base sequence represented by SEQ ID NO: 11, but is not limited thereto.

When the expression vector of the present invention further comprises a polynucleotide encoding a toxin protein, the present invention includes a case in which both the recombinant nucleic acid construct and the polynucleotide encoding the toxin protein are inserted into a single vector, as well as a case in which two types of vectors are included, namely a vector comprising the recombinant nucleic acid construct and a vector comprising the polynucleotide encoding the toxin protein.

In the present invention, when the recombinant nucleic acid construct and the polynucleotide encoding the toxin protein are simultaneously included in one vector, they may be operably linked by one promoter, or they may be operably linked by two or more promoters.

FIG. 1 is a schematic diagram of a plasmid system comprising a recombinant nucleic acid construct comprising polynucleotides (SEQ ID NOS: 4 and 8) encoding a miR-138 early transcript and a miR-153 early transcript in one embodiment of the present invention.

FIG. 2 is a schematic diagram of a plasmid system comprising the recombinant nucleic acid construct and the Hly gene sequence (SEQ ID NO: 11) encoding listerilysin O (LLO) in one embodiment of the present invention.

As an example of the present invention, DTS (SEQ ID NO: 9) may be additionally cloned into the vector shown in FIG. 1 or 2, but is not limited thereto.

According to another embodiment of the present invention, the present invention relates to a bacterial strain genetically engineered to express miR-138 and miR-153.

In the present invention, the mature sequence of miR-138 may be composed of a base sequence represented by sequence number 1, but is not limited thereto.

In the present invention, the mature sequence of miR-153 may be composed of a base sequence represented by SEQ ID NO: 5, but is not limited thereto.

In the present invention, the bacterial strain may be genetically engineered to additionally express listerilysin O (LLO).

In the present invention, the listerilysine O (LLO) may include an amino acid sequence represented by sequence number 10, but is not limited thereto.

In the present invention, the genetically engineered bacterial strain may be transformed by the expression vector provided in the present invention.

In the present invention, the bacterial strain may be an immunostimulatory bacterial strain. Herein, the term "immunostimulatory bacteria" refers to therapeutic bacteria that, when introduced into a subject, accumulate in immunoprivileged tissues and cells, such as tumors, tumor microenvironment, and tumor-resident immune cells, and replicate and/or express products that are immunostimulatory or elicit immune stimulation.

In the present invention, the immunostimulatory bacteria can be any suitable species. For example, Salmonella, Listeria, Rickettsia, Klebsiella, Bordetella, Neisseria, Aeromonas, Francisella, Corynebacterium, Citrobacter, Chlamydia, Haemophilus, Brucella, Mycobacterium, Mycoplasma, Legionella, Rhodococcus, Pseudomonas, Helicobacter, Vibrio, Bacillus or Erysipelothrix. May include, but is not limited to, strains. For example, Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella infantis, Salmonella paratyphi, Salmonella typhi, Listeria monocytogenes, Rickettsia rickettsiae, Rickettsia prowazekii, Rickettsia tsutsugamuchi, Rickettsia mooseri, Rickettsia sibirica, Bordetella bronchiseptica, Neisseria Neisseria meningitidis, Neisseria gonorrhoeae, Aeromonas eucrenophila, Aeromonas salmonicida, Francisella tularensis, Corynebacterium pseudotuberculosis, Citrobacter freundii, Chlamydia pneumoniae, Haemophilus somnus, Brucella abortus, Mycobacterium intracellulare, Legionella pneumophila, Rhodococcus equi, These may include, but are not limited to, Pseudomonas aeruginosa, Helicobacter mustelae, Vibrio cholerae, Bacillus subtilis, Erysipelothrix rhusiopathiae, Yersinia enterocolitica, Rochalimaea quintana, or Agrobacterium tumerfacium.

As a preferred example of the present invention, the bacterial strain may be preferably a Salmonella strain, and more preferably, may be derived from at least one selected from the group consisting of Salmonella typhimurium, Salmonella choleraesuis, Salmonella enteritidis, Salmonella infantis, Salmonella paratyphi, and Salmonella typhi. The listed Salmonella strains are gram-negative facultative anaerobic bacilli. They are distinguished from Escherichia coli in that they do not have lactose decomposition ability, do not form indole, and do not produce hydrogen sulfide. The Salmonella strains may have filaments and thus be motile.

In the present invention, the “Salmonella Typhimurium” is a causative agent of typhoid fever in the genus Salmonella. The Salmonella Typhimurium is a rod-shaped bacterium with flagella and is gram-negative. The Salmonella Typhimurium is sensitive to heat and dies in 20 minutes at 60℃, and can cause salmonellosis, a type of food poisoning, by being primarily contaminated by livestock, wild animals, carriers, etc., or by milk, eggs, etc. and easily receiving secondary infection from contaminated raw meat, etc. Exemplary Salmonella Typhimurium strains in the present invention include, but are not limited to, attenuated and wild-type strains, for example, Salmonella Typhimurium strains derived from strains designated AST-100, VNP20009, YS1646 (ATCC #202165), RE88, SL7207, χ8429, χ8431, χ8468, or the wild-type strain having ATCC Accession No. 14028. Here, the VNP20009 strain is a genetically modified strain of an attenuated Salmonella typhimurium having deletions at msbB and purI sites, which is described in detail in Luo et al., Oncol Res. 12(11-12):501-8, 2001.

In the present invention, the “Salmonella choleraesuis” is a bacterium well known as a swine cholera bacterium of the genus Salmonella, which infects both humans and animals. The Salmonella choleraesuis is the main causative agent of acute sepsis caused by Salmonella. This bacterium is a gram-negative, facultative anaerobic rod with motility due to its filamentous hairs. It is distinguished from Escherichia coli in that it has no lactose decomposition ability, does not form indole, and does not produce hydrogen sulfide. The optimal growth temperature is 35 to 37°C, the possible growth temperature range is 10 to 43°C, and it is killed by heating at 60°C for 20 minutes. The optimal pH is 7.2 to 7.4, and the size is 0.5 to 0.8Х3 to 4μm.

In the present invention, the “Salmonella enteritidis” is a bacterial food poisoning causative agent of the genus Salmonella, also called enterobacteriaceae. The Salmonella enteritidis is a representative bacterium of Salmonella, can cause infection in all animals, and has very high host adaptability. It is a gram-negative facultative anaerobic rod with motility due to its filamentous hairs. It is distinguished from Escherichia coli in that it does not have the ability to decompose lactose, does not form indole, and does not produce hydrogen sulfide. The optimal growth temperature is 35 to 37°C, the temperature range in which it can proliferate is 10 to 43°C, and it is killed by heating at 60°C for 20 minutes. The optimal pH is 7.2 to 7.4, and the size is 0.5 to 0.8Х3 to 4μm.

In the present invention, the “Salmonella infantis” is a strain that infects eggs or poultry meat, and Salmonella paratyphi and Salmonella typhi are causative strains of typhoid fever.

According to another embodiment of the present invention, the present invention relates to a pharmaceutical composition for preventing, improving or treating cancer, comprising the genetically engineered bacterial strain provided by the present invention as an active ingredient.

In the present invention, the "cancer" is a disease characterized by rapid and uncontrolled growth of mutant cells, and may be at least one selected from the group consisting of melanoma, fallopian tube cancer, brain cancer, small intestine cancer, esophageal cancer, lymphoma, gallbladder cancer, blood cancer, thyroid cancer, endocrine cancer, oral cancer, liver cancer, biliary tract cancer, colon cancer, rectal cancer, cervical cancer, ovarian cancer, kidney cancer, stomach cancer, duodenal cancer, prostate cancer, breast cancer, brain tumor, lung cancer, undifferentiated thyroid cancer, uterine cancer, colon cancer, bladder cancer, ureteral cancer, pancreatic cancer, bone/soft tissue sarcoma, skin cancer, non-Hodgkin's lymphoma, Hodgkin's lymphoma, multiple myeloma, leukemia, myelodysplastic syndrome, acute lymphoblastic leukemia, acute myelogenous leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, and solitary myeloma, but is not limited thereto.

In the present invention, “prevention” means any act of suppressing or delaying the progression of cancer by administering the composition of the present invention.

In the present invention, “treatment” and “improvement” mean all acts in which the symptoms of cancer are improved or beneficially changed by administration of the composition of the present invention.

The pharmaceutical composition of the present invention can be formulated as a pharmaceutical composition by additionally including one or more pharmaceutically acceptable carriers in addition to the effective ingredients described above for administration. Pharmaceutically acceptable carriers include saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposomes, and mixtures of one or more of these components, and other conventional additives such as antioxidants, buffers, and bacteriostatic agents can be added as needed. In addition, diluents, dispersants, surfactants, binders, and lubricants can be additionally added to formulate the composition as an injectable formulation such as an aqueous solution, suspension, or emulsion, pills, capsules, granules, or tablets, and a target organ-specific antibody or other ligand can be combined with the carrier to act specifically on the target organ. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the relevant technical field or a method disclosed in Remington's literature (Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA).

The pharmaceutical composition of the present invention may be in the form of a solution, suspension, dispersion, emulsion, gel, injectable solution, and sustained-release preparation of an active compound, and is preferably an injection.

When the pharmaceutical composition of the present invention is formulated as an injection, the pH can be adjusted using a buffer solution such as an acid solution or phosphate solution that can be used as an injection to ensure product stability according to distribution of the injection prescription, thereby producing an injection that is very stable both physically and chemically.

More specifically, the injection can be prepared by dissolving the solution in water for injection together with a stabilizer or a solubilizer, and then sterilizing it by a sterilization process, particularly a high-temperature and reduced pressure sterilization process or an aseptic filtration process. The water for injection can be distilled water for injection or an injection buffer solution, for example, a phosphate buffer solution having a pH range of 3.5 to 7.5 or a sodium dihydrogen phosphate (NaH2PO4)-citric acid buffer solution. The phosphate used may be in the form of a sodium salt or a potassium salt, or in the form of an anhydrous or hydrated form, and may also be in the form of citric acid or an anhydrous or hydrated form.

In addition, the stabilizer used in the present invention includes sodium pyrosulfite, sodium bisulfite (NaHSO ₃ ), sodium metabisulfite (Na ₂ S ₂ O ₃ ), or ethylenediaminetetraacetic acid, and the dissolution aid includes a base such as sodium hydroxide (NaOH), sodium bicarbonate (NaHCO ₃ ), sodium carbonate (NaCO ₃ ), or potassium hydroxide (KOH), or an acid such as hydrochloric acid (HCl) or acetic acid (CH ₃ COOH).

The injectable composition according to the present invention can be formulated as bioabsorbable, biodegradable, and biocompatible. Bioabsorbable means that the injectable composition can disappear from the body upon initial application without decomposition or degradation of the dispersed injectable composition. Biodegradability means that the injectable composition can be broken down or decomposed in the body by hydrolysis or enzymatic degradation. Biocompatibility means that all of the components are non-toxic in the body.

The injection according to the present invention can be manufactured using a diluent such as a conventional filler, weighting agent, binder, wetting agent, surfactant, or excipient.

The composition or effective ingredient of the present invention may be administered in a conventional manner, depending on the purpose, via intravenous, intraarterial, intraperitoneal, intramuscular, intrasternal, transdermal, intranasal, subcutaneous, intrauterine epidural, inhalation, topical, rectal, oral, intraocular, or intradermal routes, and is preferably administered intravenously. The composition or effective ingredient of the present invention may be administered by injection or catheter.

The dosage of the bacteria to a subject upon administration of the composition of the present invention can depend on many factors, including the species, size, body surface area, age, sex, immunocompetence, and general health of the subject, the specific bacteria to be administered, the duration and route of administration, the type and stage of the disease, e.g., tumor size, and other compounds, such as concomitant drugs, as is known in the medical art. In addition to the above factors, such levels can be influenced by the infectivity of the bacteria and the properties of the bacteria, as can be determined by one of skill in the art. In the present methods, an appropriate minimum dosage level of the bacteria can be a level sufficient for the bacteria to survive, grow, and replicate in the tumor or metastasis. Exemplary minimum levels for administering the bacteria to a 65 kg human can include at least about 5×10 ⁶ colony forming units (CFU), at least about 1×10 ⁷ CFU, at least about 5×10 ⁷ CFU, at least about 1×10 ⁸ CFU, or at least about 1×10 ⁹ CFU. In the present methods, an appropriate maximum dosage level of the bacteria can be a level that is nontoxic to the host, does not cause splenomegaly greater than 3x, and does not produce colonies or plaques in normal tissues or organs after about 1 day, or after about 3 days, or after about 7 days. Exemplary maximum levels for administering the bacteria to a 65 kg human can include no more than about 5×10 ¹¹ CFU, no more than about 1×10 ¹¹ CFU, no more than about 5×10 ¹⁰ CFU, no more than about 1×10 ¹⁰ CFU, or no more than about 1×10 ⁹ CFU.

In the pharmaceutical composition of the present invention, the active ingredient may be contained in an amount of 0.001 to 50 wt% based on the total weight of the composition. However, the content is not limited thereto.

In addition, the pharmaceutical composition of the present invention may further include one or more anticancer agents.

In the present invention, the anticancer agent is nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nilotinib, semasanib, bosutinib, axitinib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, carboplatin, bevacizumab, cisplatin, cetuximab, viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab ozogamicin, ibritumab tuscetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxifluridine, Pemetrexed, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, fluorouracil, fludagabine, enocitabine, flutamide, decitabine, mercaptopurine, thioguanine, cladribine, carpur, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide, doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pirarubicin, aclarubicin, pepromycin, temsirolimus, tezolomide, busulfan, ifosfamide, One or more selected from the group consisting of cyclophosphamide, melphalan, altretamine, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglutethimide, anagrelide, navelbine, padrazol, tasifen, toremifene, testolactone, anastrozole, letrozole, vorozole, bicalutamide, lomustine, and carmustine may be used, but is not limited thereto.

In addition to administering the composition of the present invention for the prevention, improvement or treatment of the cancer in the present invention, combination therapy with other anti-tumor agents and/or treatments may be performed. For example, cell therapy, such as administration of modified immune cells; CAR-T therapy; CRISPR therapy; immunotherapy, such as immune checkpoint inhibitors, such as antibodies and antibody fragments; chemotherapy and chemotherapeutic compounds, such as nucleoside analogs; surgery; oncolytic virus therapy; and radiotherapy, but are not limited thereto.

The genetically mutated bacterial strain provided in the present invention, particularly the mutant strain of the genus Salmonella, has the advantage of excellent anticancer activity due to the synergistic action of miR-138 and miR-153, which target cancer cells expressed in the strain, in addition to the anticancer activity of the strain itself.

Figure 1 is a cleavage map of the pcDNA3-miRNA-Pdl1-IDO vector produced in Example 1 of the present invention.
Figure 2 is a cleavage map of the pcDNA3-miRNA-Pdl1-IDO-Hly vector produced in Example 2 of the present invention.
Figure 3 shows the results of electrophoresis of a fragment obtained after treating the vector produced in Example 1 of the present invention with a restriction enzyme.
Figure 4 shows the results of electrophoresis of a fragment obtained after treating the pcDNA3-miRNA-Pdl1-IDO-Hly vector produced in Example 2 of the present invention with a restriction enzyme.
Figure 5 shows the results of Western blot analysis of the LLO expression level in cell lysates of Salmonella strains transformed with the pcDNA3-miRNA-Pdl1-IDO-Hly vector produced in Example 4 of the present invention.
Figure 6 shows the results of Western blotting to determine the expression levels of PD-L1 and IDO in cell lysates infected with a Salmonella strain transformed with a vector in Example 6 of the present invention.
Figure 7 shows the results of Western blot analysis of the expression levels of PD-L1 and IDO in cell lysates infected with a vector-transformed Salmonella strain of colon cancer HT29 cells in Example 6 of the present invention.
Figure 8 shows the results of Western blotting to determine the expression levels of PD-L1 and IDO in cell lysates after infecting liver cancer Huh7 cells with a vector-transformed Salmonella strain in Example 6 of the present invention.
Figure 9 shows the results of Western blotting to determine the expression levels of PD-L1 and IDO in cell lysates infected with a vector-transformed Salmonella strain of breast cancer MDA-MB-231 cells in Example 6 of the present invention.
Figure 10 shows the results of Western blot analysis of the expression levels of PD-L1 and IDO in cell lysates infected with a vector-transformed Salmonella strain of pancreatic cancer Pan1 cells in Example 6 of the present invention.
Figure 11 is a graph showing the ratio of changes in the expression levels of PD-L1 and IDO in liver cancer cells treated with the Salmonella strain compared to untreated liver cancer cells, after confirming the expression levels of PD-L1 and IDO in the cell lysate after infecting liver cancer cells with a vector-transformed Salmonella strain in Example 7 by Western blotting.
Figure 12 is a graph showing the ratio of changes in the expression levels of PD-L1 and IDO in breast cancer cells treated with the Salmonella strain compared to untreated breast cancer cells, after confirming the expression levels of PD-L1 and IDO in cell lysates infected with the vector-transformed Salmonella strain in Example 7 by Western blotting.
Figure 13 is a graph showing the ratio of changes in the expression levels of PD-L1 and IDO in pancreatic cancer cells treated with the Salmonella strain compared to untreated pancreatic cancer cells, after confirming the expression levels of PD-L1 and IDO in the cell lysate after infecting pancreatic cancer cells with the vector-transformed Salmonella strain in Example 7 by Western blotting.
In each drawing, “Neg” or “Neg. no Hly” is the result of treating a Salmonella strain transformed with a pcDNA3 mock vector in which the Hly gene is not cloned, “Neg. Hly” is the result of treating a Salmonella strain transformed with a pcDNA3 vector in which only the Hly gene is cloned, “P/I” or “PI” is the result of treating a Salmonella strain transformed with the pcDNA3-miRNA-Pdl1-IDO-Hly vector of Example 2, and “DP/I” or “DPI” is the result of treating a Salmonella strain transformed with the pcDNA3-DTS-miRNA-Pdl1-IDO-Hly vector of Example 3, which is a vector in which DTS is additionally cloned into the vector of Example 2.

Hereinafter, the present invention will be described in more detail through examples. These examples are only intended to explain the present invention more specifically, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention.

Example

[Example 1] Production of pcDNA3-miRNA-Pdl1-IDO vector

In order to clone hsa-mir-138, which suppresses the expression of PD-L1, and hsa-mir-153, which regulates the expression of IDO, into the plasmid of pcDNA3, oligonucleotides were synthesized so that hsa-mir-138 (SEQ ID NO: 4) and hsa-mir-153 (SEQ ID NO: 8) were positioned sequentially in that order. The oligonucleotides were inserted between the EcoRI and XhoI restriction enzyme sites of the pcDNA3 so that the hsa-mir-138 (SEQ ID NO: 4) and hsa-mir-153 (SEQ ID NO: 8) genes were cloned downstream of the T7 promoter present in the pcDNA3 (see the cleavage map in FIG. 2). The plasmid includes the base sequence of SEQ ID NO: 4 encoding the miR-138 early transcript (pri-miRNA) and the base sequence of SEQ ID NO: 8 encoding the miR-153 early transcript (pri-miRNA).

Thereafter, the obtained plasmid was cleaved with HindIII/XhoI restriction enzymes, and the obtained fragment was subjected to electrophoresis on a 0.9% agarose gel. As a result, a band corresponding to 585 bp was confirmed, as shown in Fig. 3. This indicated that hsa-mir-138 and hsa-mir-153 were successfully cloned into the vector, resulting in the acquisition of a recombinant plasmid.

[Example 2] Production of pcDNA3-miRNA-Pdl1-IDO-Hly vector

In order to additionally insert the Hly gene (SEQ ID NO: 11) into the plasmid constructed in Example 1, the sense primer 5’-CCCGGGCCCTCCTTTGATTAG-3’ having a restriction enzyme recognition site at the 5’ terminus and the antisense primer 5’-TTCGAAGCTTATATTATATGGATAAACAGTC-3’ having a restriction enzyme recognition site at the 3’ terminus were purchased from IDT. The bacterial plasmid pT7RNAi-Hly-Inv(TRIP) was used as a gene template for amplification of the Hly gene (Xiang, S., Fruehauf, J., & Li, C. J. (2006). Short hairpin RNA-expressing bacteria elicit RNA interference in mammals. Nature Biotechnology, 24(6), 697-702. doi:10.1038/nbt1211). For Hly gene amplification, 0.5 ng of TRIP and 2 pmoles of sense and antisense primers and Phusion DNA polymerase (Invitrogen, USA) were mixed, and PCR was performed according to the manufacturer's method. The PCR reaction conditions were 98°C for 30 sec, followed by 35 cycles of 98°C for 5 sec, 55°C for 10 sec, and 72°C for 30 sec, and then an extension reaction was performed at 72°C for 5 min. After PCR, the amplified Hly gene was extracted using Qiagen's Extraction kit after the exact size was confirmed by DNA electrophoresis.

In order to insert the Hly gene into the plasmid produced in Example 1, the plasmid and the amplified Hly gene were treated with restriction enzymes XmaI and BstBI. Each gene segment was ligated to produce the pcDNA3-miR-PDL1-IDO-Hly plasmid (see the cleavage map in Fig. 2).

The plasmid thus obtained was cleaved with XmaI/BstBI restriction enzymes, and the obtained fragment was subjected to electrophoresis on a 0.9% agarose gel. As a result, a band corresponding to 2016 bp, the size of the Hly gene, was confirmed, as shown in Fig. 4. This indicated that the Hly gene was successfully inserted and cloned into the vector.

[Example 3] Production of pcDNA3-DTS-miRNA-Pdl1-IDO-Hly vector

In order to clone the DNA nuclear targeting sequence (DTS) into the vector constructed in Example 2, a DTS template (SEQ ID NO: 9) was purchased from IDT and used as a PCR template. The sense primer used for PCR was 5'-AGGCGTTTTGCGCTGCTTCG-3', and the antisense primer was 5'-TATATCTGGCCCGTACATCGGGAAAGTC-3', and PCR was performed using Phusion DNA polymerase (Invitrogen, USA) according to the manufacturer's method. The PCR reaction conditions were 98°C for 30 seconds, 35 cycles at 98°C for 5 seconds, 68°C for 10 seconds, and 72°C for 2 seconds, and then an extension reaction was performed at 72°C for 5 minutes.

To produce an amplified DTS gene assembly, the vector produced in Example 2 was treated with NruI restriction enzyme at 37°C for 30 minutes to obtain a linear vector. Subsequently, the amplified DTS gene fragment and the linear vector were produced using the NEBuilder HiFi DNA Assembly kit provided by New England BioLabs according to the manufacturer's recommended method. The two gene fragments were reacted at 50°C for 30 minutes.

[Example 4] Production of Salmonella strain transformed with vector

In order to transform Salmonella typhimurium VNP20009 strain with the vector constructed in Examples 1 to 3, VNP20009 strain cultured on agar medium was washed with 1 ml of cold 10 mM HEPES and centrifuged at 3000 rpm. After removing the supernatant, the centrifuged bacterial cells were washed again with 1 ml of 10 mM HEPES and placed on ice for 10 minutes. The washing process was performed a total of 3 times, and the remaining centrifuged bacterial cells were mixed with 100 ng of the vector to be transformed (the vector of Examples 1 to 3) in 40 ㎕ of cold 10% glycerol and placed on ice for 15 minutes. After that, the vector was transferred to the strain using an Eppendorf Eporator at 1700 V. Afterwards, 250 ㎕ of SOC culture medium was collected at 37℃ in a 200 rpm shaking incubator for 1 hour, and cultured on agar medium containing ampicillin at 37℃ for more than 24 hours. Recombinant Salmonella colonies cultured on the agar medium were selected and transferred to 10 ml of BHI medium containing ampicillin, and then cultured again at 37℃. Recombinant Salmonella cultured on the BHI medium were centrifuged at 3000 rpm for 10 minutes, and the resulting bacterial bodies were washed with 5 ml of serum-free Dulbeccos Modified Eagles Media (DMEM) three times in total.

[Example 5] Verification of LLO expression in Salmonella strains transformed with vectors

The Salmonella strain transformed with the pcDNA3-miRNA-Pdl1-IDO-Hly vector of Example 2 obtained in the above Example 4 was cultured in LB medium containing 50 ug/ml of ampicillin for 24 hours, and then the whole cell lysate was obtained. The same volume of 2X SDS-PAGE sample solution was added to the whole cell lysate, boiled for 5 minutes, and 10 ul was subjected to electrophoresis on a 4-20% gradient SDS-PAGE gel together with a protein size marker at 120 V for about 1 hour and transferred to a nitrocellulose membrane at 100 V for about 50 minutes. Thereafter, the expression of LLO was confirmed at the protein level by performing a primary reaction with anti-Hly rabbit monoclonal antibody (Abcam) and using HRP-conjugated anti-rabbit mouse polyclonal antibody (Sigma). The results are shown in Fig. 5. For the same samples, loading control was performed with anti-DnaK mouse monoclonal antibody (Abcam) and HRP-conjugated anti-rabbit mouse polyclonal antibody (Sigma).

As shown in Figure 5, it was confirmed that LLO was expressed in the Salmonella strain transformed with the pcDNA3-miR-PDL1-IDO-Hly vector.

[Example 6] Verification of inhibition of PD-L1 and IDO protein expression in cancer cells treated with vector-transformed Salmonella strain (1)

In order to confirm the inhibitory effect of the expression of PD-L1 and IDO proteins by the vectors constructed in the above examples in tumor cells, SW480 colon cancer cells, HT29 colon cancer cells, Huh7 liver cancer cells, MBA-MB-231 breast cancer cells, and Panc1 pancreatic cancer cells were infected with the recombinant Salmonella strain of Example 4 transformed with each vector. Specifically, tumor cells cultured in a 12-well culture plate were infected with 1 ml of DMEM in which the cell:recombinant Salmonella ratio was adjusted to the desired ratio for 2 hours, and then the Salmonella strains that did not penetrate into the tumor cells were removed for 1 hour with DMEM containing 200 ㎕/ml of gentamicin. The tumor cells infected with the recombinant Salmonella were cultured in fresh DMEM medium for 48 hours, lysed on ice for 30 minutes with RIPA hemolysis buffer, centrifuged, and only the supernatant was collected and mixed with an equal volume of 2X SDS sample. 20 μg of protein was quantified and separated according to molecular weight using the SDS-PAGE method described above on an SDS-PAGE gel at 100 V for 1 hour, and then transferred to a nitrocellulose membrane at 100 V for 50 minutes. The primary antibody for confirming the expression of PD-L1 protein was a rabbit monoclonal antibody against PD-L1 (R&D systems), the primary antibody for confirming the expression of IDO protein was a rabbit monoclonal antibody against IDO (Cell Signaling), and as a control, a mouse monoclonal antibody against GAPDH (Abcam) was used. HRP-conjugated mouse anti-rabbit polyclonal antibody and rabbit anti-mouse polyclonal antibody were used as secondary antibodies.

As a result, as shown in FIGS. 6 to 10, it was confirmed that the expression levels of PD-L1 and IDO were decreased in each tumor cell infected with the Salmonella strain transformed with the pcDNA3-miR-PDL1-IDO-Hly vector (Example 2) and pcDNA3-DTS-miR-PDL1-IDO-Hly vector (Example 3) of the present invention compared to the control group.

[Example 7] Verification of inhibition of PD-L1 and IDO protein expression in cancer cells treated with transformed Salmonella strain (2)

In order to quantitatively confirm the effect of inhibiting the expression of PD-L1 and IDO proteins by the vectors produced in the above examples in tumor cells, liver cancer cells, breast cancer cells, and pancreatic cancer cells were infected with the recombinant Salmonella strains of Example 4 transformed with the respective vectors in the same manner as in Example 5. Thereafter, the expression levels of PD-L1 (target A) and IDO (target B) expressed in the tumor cells were confirmed by Western blot, and the ratio of the change in the expression level according to the treatment of each strain compared to the expression level in the untreated negative control group was measured, and the results are shown in Figs. 11 to 13.

As shown in FIGS. 11 to 13, when the Salmonella strains transformed with the pcDNA3-miR-PDL1-IDO-Hly vector (Example 2) and pcDNA3-DTS-miR-PDL1-IDO-Hly vector (Example 3) of the present invention were treated in all of the liver cancer cells, breast cancer cells, and pancreatic cancer cells, it was confirmed that the expression levels of PD-L1 and IDO were significantly reduced compared to the untreated negative control group or the case where the wild-type Salmonella strain was treated.

The above description of the present invention is for illustrative purposes only, and those skilled in the art will understand that the present invention can be easily modified into other specific forms without changing the technical idea or essential characteristics of the present invention. Therefore, it should be understood that the embodiments described above are exemplary in all respects and not restrictive.

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Claims (22)

An attenuated Salmonella strain genetically engineered to express miR-138 and miR-153. In the first paragraph,
An attenuated Salmonella strain, wherein the Salmonella strain is transfected with an expression vector comprising a recombinant nucleic acid construct comprising a polynucleotide encoding miR-138 and a polynucleotide encoding miR-153. In the second paragraph,
An attenuated Salmonella strain, wherein the polynucleotide encoding the above miR-138 encodes a mature form of miR-138, a miRNA precursor, an early transcript miRNA (pri-miRNA), or a miRNA precursor in the form of a plasmid. In the second paragraph,
An attenuated Salmonella strain, wherein the polynucleotide encoding the above miR-153 encodes a mature form of miR-153, a miRNA precursor, an early transcript miRNA (pri-miRNA), or a miRNA precursor in the form of a plasmid. In the first paragraph,
An attenuated Salmonella strain, wherein the above miR-138 is composed of a base sequence represented by sequence number 1. In the first paragraph,
An attenuated Salmonella strain, wherein the above miR-153 is composed of a base sequence represented by sequence number 5. In the second paragraph,
An attenuated Salmonella strain, wherein the polynucleotide encoding the above miR-138 comprises a base sequence represented by SEQ ID NO: 2. In the second paragraph,
An attenuated Salmonella strain, wherein the polynucleotide encoding the above miR-153 comprises a base sequence represented by SEQ ID NO: 6. In the second paragraph,
An attenuated Salmonella strain, wherein the expression vector comprises at least one promoter, at least one intron, at least one transcription termination region, at least one initiation codon, and at least one termination or stop codon. In the first paragraph,
The above Salmonella strain is an attenuated Salmonella strain that has been genetically engineered to additionally express a toxin protein. In Article 10,
The above toxin protein is at least one attenuated toxic protein selected from the group consisting of Ricin, Saporin, Gelonin, Momordin, Debuganin, Diphtheria toxin, Pseudomonas toxin, Hemolysin (HlyA), FAS ligand (FASL), Tumor necrosis factor-alpha (TNF-alpha), TNF-related apoptosis-inducing ligand (TRAIL), streptolysin O (SLO), pneumolysin (PLO), listeriolysin O (LLO) and cytolysin A (ClyA). Salmonella strains. In the first paragraph,
The above Salmonella strain is an attenuated Salmonella strain genetically engineered to express listerilysin O (LLO). In Article 12,
An attenuated Salmonella strain, wherein the above Listeriol O (LLO) comprises an amino acid sequence represented by sequence number 10. In the second paragraph,
An attenuated Salmonella strain, wherein the expression vector further comprises a DNA nuclear targeting sequence (DTS). In Article 14,
An attenuated Salmonella strain, wherein the DNA nuclear targeting sequence (DTS) comprises a base sequence represented by SEQ ID NO: 9. A pharmaceutical composition for preventing or treating cancer, comprising an attenuated Salmonella strain according to any one of claims 1 to 15 as an active ingredient. delete delete delete delete delete delete