Composition and method for organ-protective expression and regulation of coding ribonucleic acid

Abstract

An isolated mRNA sequence for the expression of one or more polypeptides in one or more target organs, comprising at least one coding sequence encoding at least one polypeptide, at least one first untranslated region (UTR) sequence and a plurality of microRNA (miRNA) binding site sequences. Each miRNA binding site sequence is located immediately adjacent to the 5' or 3' of the first UTR sequence; the miRNA binding site sequence enables differential expression of the coding sequence in at least first and second cell types within the target organ or organs. A method of using the above composition is provided, in particular, for the treatment of diseases such as liver cancer, brain cancer, lung cancer, breast cancer, pancreatic cancer, colon cancer, and kidney cancer.

Description

Technology Field

[0001] The present invention relates to messenger ribonucleic acid (mRNA) delivery technology and methods for using such mRNA delivery technology in various therapeutic, diagnostic, and prophylactic measures. Such delivery systems may be used as standalone interventions or in combination with other therapeutic components. Background Technology

[0002] Gene therapy is a method of introducing coding polynucleotides into a patient's cells to treat a disease. For example, it can replace mutated and / or non-functional genes in target cells with intact copies. While gene therapy often relies on viral vectors to introduce coding polynucleotides into target cells, other techniques exist to deliver polynucleotides to cells without the use of viruses. The advantages of viruses include a relatively high possible transfection rate, as well as the ability to target the virus to specific cell types by controlling binding proteins that allow the virus to enter the target cells. In contrast, non-viral methods of introducing coding polynucleotides into cells may present problems such as low transfection rates and limited choices for targeting expression to specific organs and cell types. However, the nature of viral intervention entails risks of toxicity and inflammation, and control over the duration and extent of expression of the introduced factor is limited.

[0003] Tumor therapies based on biological approaches offer advantages over traditional chemotherapy because they can utilize a wide variety of mechanisms—specifically direct cell lysis, cytotoxic immune effector mechanisms, and angiogenesis—to more accurately target and destroy cancer. Consequently, the number of clinical studies investigating the potential of these approaches has increased significantly. However, preclinical and clinical research is complex because the diverse range of therapeutic activity means that multiple parameters can influence its therapeutic potential; thus, it can be difficult to define the reasons for treatment failure or methodologies that could enhance therapeutic activity. Maintaining targeted activity and tumor specificity, as well as reducing side effects, are also major challenges for these experimental and potent therapies.

[0004] Even in non-clinical contexts, the ability to induce the expression of specific gene products, such as polypeptides, in specific target tissues or organs is frequently required. In many cases, the target tissue or organ will contain more than one cell type; in such cases, it is also frequently required to express gene products to different degrees in different cell types—that is, to provide differential expression in different cell types. While methods for introducing polynucleotides exist in vitro and in vivo, they have the same limitations as discussed above.

[0005] WO 2017 / 132552 A1 describes a recombinant tumor cell disruption virus having an engineered genome containing a microRNA binding site.

[0006] US 2013 / 156849 A1 relates to a method for expressing a polypeptide of interest in a mammalian cell or tissue, comprising the step of contacting said mammalian cell or tissue with a formulation comprising a modified mRNA encoding the polypeptide of interest. WO 2016 / 011306 A2 describes the design, formulation, manufacture, and / or formulation of a nucleic acid comprising at least one terminal modification that may include a microRNA binding site. The aforementioned prior art does not address the issue of ensuring effective protection of a single or multiple organ types within the body of a subject being treated with a co-administered therapeutic agent or therapeutic factor.

[0007] Therefore, there is a need to further develop methods and compositions for delivering polynucleotide sequences, such as mRNA, to specific organs and / or tissues, and methods for regulating the expression of the delivered polynucleotide sequences in specific cells. means of solving the problem

[0008] In a first embodiment, an isolated mRNA sequence for the expression of one or more polypeptides within one or more target organs is provided. The sequence comprises at least one coding sequence encoding at least one polypeptide, at least one first non-translational region (UTR) sequence, and a plurality of microRNA (miRNA) binding site sequences. Each miRNA binding site sequence is located immediately adjacent to the 5' or 3' of the first UTR sequence. The miRNA binding site sequence enables differential expression of the coding sequence in at least first and second cell types within the target organ or organs.

[0009] The mRNA sequence may include more than two, suitably more than three, typically more than four binding site sequences. The plurality of miRNA binding site sequences may include at least two substantially similar sequences and / or the plurality of miRNA binding site sequences may include at least two substantially different sequences.

[0010] In some embodiments, a plurality of miRNA binding site sequences are substantially complementary to a miRNA sequence selected from at least one or more of the group consisting of miRNA-122, miRNA-125a, miRNA-125b, miRNA-199, miRNA-124a, Let-7, miRNA-148a, miRNA-148b, miRNA-375, miRNA-143, miRNA-145, miRNA192, miRNA194, miRNA-204, miRNA215, miRNA-30b, and miRNA-30c. At least one of the plurality of miRNA binding site sequences may comprise one or more of SEQ ID NOs 1 to 7, suitably SEQ ID NO. 1.

[0011] In some embodiments, the binding site sequences each include SEQ ID NOs 1, 2, 3, 4 and 5; or each include SEQ ID NOs 1, 2, 5, 6 and 7.

[0012] In some embodiments, the first and second cell types are different selections from the group consisting of non-neoplastic cells, transformed cell phenotypes, precancerous phenotypes, and neoplastic phenotypes. Target organs or organs may be selected from the group consisting of liver, brain, lung, breast, pancreas, colon, and kidney.

[0013] In some embodiments, at least one of the one or more polypeptides comprises a therapeutic enhancer. The therapeutic enhancer may be selected from the group consisting of the following:

[0014] (i) a cytokine (or its ligand) involved in immune response and inflammation, selected from one or more of TNFα, TNFβ, IFNα, IFNβ, IFN-gamma, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10;

[0015] (ii) a dendritic cell activator selected from one or more of GM-CSF, TLR7 and TLR9;

[0016] (iii) a molecule targeting a cell receptor and its ligand, selected from one or more of CD40, CD40L, CD160, 2B4, Tim-3, GP-2, B7H3 and B7H4;

[0017] (iv) TGF β inhibitor;

[0018] (v) T-cell membrane protein 3 inhibitor;

[0019] (vi) inhibitors of programmed death 1 (PD1), programmed death-ligand 1 (PDL1), programmed death-ligand 2 (PDL2), cytotoxic T-lymphocyte antigen 4 (CTLA4) and lymphocyte-activating gene 3 (LAG3); and

[0020] (vii) NF-κB inhibitors.

[0021] mRNA may contain more than one open reading frame (ORF).

[0022] In some embodiments, the mRNA comprises a sequence selected from one of the group consisting of sequence numbers 18 to 29.

[0023] In another embodiment, a pharmaceutical composition is provided comprising an isolated mRNA sequence as described herein, a delivery particle (said that the sequence is contained within the delivery particle), and a pharmaceutically acceptable carrier. The delivery particle may be selected from at least one of the group consisting of aminoalcohol lipidoid particles, liposomes, exosomes, cell-derived vesicles, and polymeric particles.

[0024] In some embodiments, the delivery particle is targeted to one or more target organs or organs. In such cases, the delivery particle may include a targeting agent selected from proteins, peptides, carbohydrates, glycoproteins, lipids, small molecules, and nucleic acids, wherein the targeting agent preferentially associates with cells of the target organs or organs.

[0025] In another additional aspect, a polynucleotide expression vector structure encoding an mRNA sequence as described herein is provided.

[0026] In another aspect, a viral vector comprising an mRNA sequence or a polynucleotide expression vector structure as described herein is provided.

[0027] Another aspect provides a method for treating cancer, comprising the step of administering to a subject in need a composition comprising an isolated mRNA sequence, a composition, a vector structure, or a viral vector as described herein.

[0028] The above method may further include the step of administering a therapeutic regimen or therapeutic agent to the subject. The therapeutic regimen or therapeutic agent may be selected from chemotherapy, radiotherapy, biological agents, tumor cell destructive viruses, small molecule drugs, CAR-T or adoptive cell therapy, and combinations thereof. In an embodiment of the above method, the subject is a human or non-human animal.

[0029] In a specific embodiment, the cancer is selected from at least one of the group consisting of liver cancer, brain cancer, lung cancer, breast cancer, pancreatic cancer, colorectal cancer, and renal cancer, suitably liver cancer. The liver cancer may include primary liver cancer or secondary liver cancer. Primary liver cancer may be selected from the group consisting of hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma, and angiosarcoma. Secondary liver cancer may be metastatic liver cancer derived from a known or unknown primary solid tumor.

[0030] The above method may further include the step of administering a tumor cell disintegrating virus to the subject. In such an embodiment, the isolated mRNA sequence may encode a therapeutic agent that increases the efficacy of the tumor cell disintegrating virus. The tumor cell disintegrating virus may be attenuated by mutations in one or more virulence genes, and in such an embodiment, the mRNA sequence may encode one or more virulence genes, or equivalents or homologs thereof. In some embodiments, the tumor cell disintegrating virus is selected from any one of groups 1 to 7 of the Baltimore virus classification. The tumor cell disintegrating virus may be selected from the group comprising one or more of bullous stomatitis virus, marabar virus, polio virus, reovirus, measles virus, Newcastle disease virus, Coxsackievirus A21, parvovirus, herpes simplex virus type 1, vaccinia virus, and adenovirus. Typically, the tumor cell disintegrating virus is a herpes simplex virus.

[0031] The method described above further comprises, in some embodiments, the step of administering CAR-T or adoptive cell therapy to the subject. In these embodiments, the isolated mRNA sequence, composition, vector structure, or viral vector may encode one or more immunomodulatory molecules selected from the group consisting of:

[0032] (i) a cytokine (or its ligand) involved in immune response and inflammation, selected from one or more of TNFα, TNFβ, IFNα, IFNβ, IFN-gamma, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10;

[0033] (ii) a dendritic cell activator selected from one or more of GM-CSF, TLR7 and TLR9;

[0034] (iii) a molecule targeting a cell receptor and its ligand, selected from one or more of CD40, CD40L, CD160, 2B4, Tim-3, GP-2, B7H3 and B7H4;

[0035] (iv) TGF β inhibitor;

[0036] (v) T-cell membrane protein 3 inhibitor;

[0037] (vi) inhibitors of programmed death 1 (PD1), programmed death-ligand 1 (PDL1), programmed death-ligand 2 (PDL2), cytotoxic T-lymphocyte antigen 4 (CTLA4) and lymphocyte-activating gene 3 (LAG3); and

[0038] (vii) NF-κB inhibitors.

[0039] In another embodiment, the above method may further include the step of administering a cell checkpoint inhibitor to the target. Again, in such an embodiment, the isolated mRNA sequence, composition, vector structure, or viral vector may encode one or more immunomodulatory molecules selected from the group consisting of:

[0040] (i) a cytokine (or its ligand) involved in immune response and inflammation, selected from one or more of TNFα, TNFβ, IFNα, IFNβ, IFN-gamma, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10;

[0041] (ii) a dendritic cell activator selected from one or more of GM-CSF, TLR7 and TLR9;

[0042] (iii) a molecule targeting a cell receptor and its ligand, selected from one or more of CD40, CD40L, CD160, 2B4, Tim-3, GP-2, B7H3 and B7H4;

[0043] (iv) TGF β inhibitor;

[0044] (v) T-cell membrane protein 3 inhibitor;

[0045] (vi) inhibitors of programmed death 1 (PD1), programmed death-ligand 1 (PDL1), programmed death-ligand 2 (PDL2), cytotoxic T-lymphocyte antigen 4 (CTLA4) and lymphocyte-activating gene 3 (LAG3); and

[0046] (vii) NF-κB inhibitors.

[0047] In a further embodiment, a composition comprising an isolated mRNA sequence, a vector structure, or a virus as described herein for use in medicine, or a composition as described herein is provided.

[0048] In another embodiment, a composition comprising an isolated mRNA sequence, a vector structure, or a virus as described herein for use in the treatment of cancer, or a composition as described herein, wherein the cancer is suitably selected from the group consisting of liver cancer, brain cancer, lung cancer, breast cancer, pancreatic cancer, colorectal cancer, and kidney cancer. Brief explanation of the drawing

[0049] The present invention is further described with reference to the accompanying drawings: FIG. 1 shows a schematic diagram of a method for administering a lipidoid-encapsulated mRNA composition according to one embodiment of the present invention. FIG. 2 shows an example of a cloning method for preparing a DNA synthesis vector, which is a vector used to prepare an mRNA structure according to an embodiment of the present invention. FIG. 3 shows three variations of the mRNA structure shown in FIG. 4 used in an embodiment of the present invention, and possible selections for the insertion site of a pair of miRNA binding sequences (sequences binding to miR-122 in the present invention) located within or adjacent to the UTR sequence at the 3' of the coding sequence. Figure 4 shows an example of a synthetic vector for manufacturing a DNA plasmid, a template plasmid, as well as an mRNA structure shown in Figure 3. Figures 5, 6, and 7 show examples of methods that can be used to manufacture a synthetic vector for manufacturing a modified mRNA structure as illustrated in Figure 3. FIG. 8 shows the chemical formula of an example of a constituent compound that can be used to manufacture a transfer particle according to one embodiment of the present invention. FIG. 9a shows a method for preparing a nanoformulation of a delivery particle containing mRNA according to one embodiment of the present invention. FIG. 9b shows a cross-sectional structure of a delivery particle comprising mRNA according to one embodiment of the present invention and further comprising the encapsulating constituent compound shown in FIG. 8. FIG. 10a is a fluorescence microscope image showing the experimental results of transfecting cells from a healthy human hepatocyte culture (Human Plateable Hepatocyte, HMCPP5), human hepatocellular carcinoma (Hep3B), and human hepatoblastoma (HepG2) cells in vitro with a composition according to an embodiment of the present invention. Two delivery particles were administered: one was a delivery particle containing mRNA encoding the fluorescent protein mCherry (mRNA-mCh-DMP). CTx ) and the other is a delivery particle (mRNA-mCh-122-DMP) containing mRNA encoding the fluorescent protein mCherry, but whose differential expression in target cells is controlled by the miRNA-122 content. CTx )lim. Figure 10b shows the quantification of fluorescence intensity after 48 hours of transfected cells according to the experiment of Figure 10a. Results are presented as mean ± SD (standard deviation). Statistical significance is t It was determined using a assay. An asterisk indicates a statistically significant difference between mRNA-mCherry and mRNA-mCherry-122 expression in transfected cells (****p < 0.0001, ***p < 0.001). Figure 11 shows a graph of the experimental results in which human hepatocytes (HMCPP5) were transfected multiple times (MPT) or once (ST) with the delivery particles used in Figure 10a. mCherry expression is determined by fluorescence intensity levels measured at 24, 28, 72, 96, and 144 hours after transfection. Results are presented as mean ± SD. Statistical significance t It was determined using a assay. An asterisk indicates a statistically significant difference between mRNA-mCherry and mRNA-mCherry-122 expression in transfected cells (*p < 0.01, **p < 0.05). Figure 12a shows a fluorescence microscope image representing the experimental results of transfecting healthy mouse hepatocytes (AML12 cell line) in vitro with the delivery particles used in Figure 10a, showing the relative expression levels of mCherry after 24 hours of transfection. Figure 12b shows a graph providing the quantification of fluorescence intensity as a pixel% counted for the results at a time point 72 hours after transfection, in addition to the results of Figure 12a. FIG. 13 shows Western blot results from two experiments (indicated as Run 1 and Run 2) in which human hepatocytes (HMCPP5), human hepatoblastoma (HepG2), and human hepatocarcinoma (Hep3B) cells were transfected with a composition according to one embodiment of the present invention comprising mRNA encoding an exemplary human polypeptide with a molecular mass of 25 kDa under miRNA differential expression control. Figure 14 shows the effect of herpes simplex virus variant R7041 on the viability of human cells from hepatocellular carcinoma (Hep3B) and hepatoblastoma (HepG2) models. The effect of virus application on relative cell viability is presented. FIG. 15 shows a timetable for an in vitro experiment in which human cells from a liver carcinoma model were treated with a composition and method according to one embodiment of the present invention and then tested by MTS colorimetric assay. FIGS. 16a and 16b show human cells from hepatoblastoma (Fig. 16a) and hepatocarcinoma (Fig. 16b) models treated with the virus alone or in combination with a composition according to one embodiment of the present invention after the timetable of FIG. 15. In vitro The experimental results are shown. The above composition comprises a delivery particle (US3 mRNA DMP) containing mRNA encoding US3. CTx ) is. The above treatment effect on cell viability is presented. Figures 17a and 17b show the results of in vivo experiments using a mouse model of human hepatocarcinoma. Figure 17a shows tumor growth (Hep3B cells were labeled with luciferase). Figure 17b shows a fluorescence microscopy image of a healthy mouse liver using mRNA encoding mCherry (mCherry-DMP). CTx - No fluorescence was detected when the miRNA122 composition was used). Figure 18 shows the results of immunohistochemical microscopy of an in vivo experiment using the same mouse model as presented in Figure 17a. A delivery particle containing mRNA encoding US3 (US3 mRNA DMP) CTx miRNA-122) is administered via the tail vein and provides differential expression between healthy hepatocytes and tumor liver tissue, as evidenced by the darker staining of US3 protein in tumor tissue. The boundary between tumor tissue and healthy tissue is indicated by a dotted line. FIG. 19a shows an example of a miRNA binding site sequence that can be used in an mRNA structure according to the present invention. FIG. 19b shows a general schematic diagram of an example of an mRNA structure according to some embodiment of the present invention, which includes five or fewer binding sites. FIGS. 19c and 19d show examples of combinations of binding site sequences that can be used in mRNA structures according to the present invention. FIG. 19e shows some specific combinations of coding sequences, including coding sequences and binding sites for a plurality of polypeptides according to some embodiments. Specific details for implementing the invention

[0050] Unless otherwise indicated, the practice of the present invention utilizes the ordinary techniques of chemistry, molecular biology, microbiology, recombinant DNA technology, and chemical methods that are within the capacity of those skilled in the art. Such techniques are also described, for example, in the following literature: [MR Green, J. Sambrook, 2012, Molecular Cloning: A Laboratory Manual, Fourth Edition, Books 1-3, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY]; [Ausubel, FM et al. (1995 and periodic supplements; Current Protocols in Molecular Biology, ch. 9, 13, and 16, John Wiley & Sons, New York, NY)]; [B. Roe, J. Crabtree, and A. Kahn, 1996, DNA Isolation and Sequencing: Essential Techniques, John Wiley & Sons]; [JM Polak and James O'D. [McGee, 1990, In Situ Hybridisation: Principles and Practice, Oxford University Press]; [MJ Gait (Editor), 1984, Oligonucleotide Synthesis: A Practical Approach, IRL Press]; and [DMJ Lilley and JE Dahlberg, 1992, Methods of Enzymology: DNA Structure Part A: Synthesis and Physical Analysis of DNA Methods in Enzymology, Academic Press]. These general documents are each incorporated herein by reference.

[0051] Before describing the present invention, a number of definitions are provided to aid in understanding the invention. All references cited herein are incorporated herein by reference in their entirety. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains.

[0052] As used herein, the term “comprising” means that any mentioned element is necessarily included, and other elements may be optionally included. “essentially consisting of” means that any mentioned element is necessarily included, elements that may substantially affect the basic and novel features of the enumerated elements are excluded, and other elements may be optionally included. “consisting of” means that all elements other than those enumerated are excluded. Embodiments defined by each of these terms fall within the scope of the present invention.

[0053] When applied to polynucleotide sequences, the term 'isolated' indicates that the sequence has been removed from an organism of natural origin and is free of irrelevant or unwanted coding or regulatory sequences. Isolated sequences are suitable for use in recombinant DNA processes and genetically engineered protein synthesis systems. Such isolated sequences include cDNA, mRNA, and genomic clones. Isolated sequences may be limited solely to protein-coding sequences, or may also include 5' and 3' regulatory sequences, such as promoters and transcription terminators. Before further describing the invention, a number of definitions are provided to aid in understanding the invention.

[0054] A “polynucleotide” is a single or double-stranded covalently bonded sequence of nucleotides in which the 3’ and 5’ ends of each nucleotide are connected by phosphodiester bonds. Polynucleotides may consist of deoxyribonucleotide bases or ribonucleotide bases. Polynucleotides include DNA and RNA and may be synthesized in vitro or isolated from natural sources. The size of a polynucleotide is typically expressed as the number of base pairs (bp) for double-stranded polynucleotides or the number of nucleotides (nt) for single-stranded polynucleotides. 1,000 bp or nt is equal to a kilobase (kb). Polynucleotides with a length of about 40 nucleotides or less are typically referred to as “oligonucleotides.” As used herein, the term “nucleic acid sequence” is a single or double-stranded covalently bonded sequence of nucleotides in which the 3’ and 5’ ends of each nucleotide are connected by phosphodiester bonds. Polynucleotides may consist of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acid sequences may include DNA and RNA, and may be synthesized in vitro or isolated from natural sources. The size of a nucleic acid sequence, also referred to herein as a 'polynucleotide,' is typically expressed as the number of base pairs (bp) for double-stranded polynucleotides or the number of nucleotides (nt) for single-stranded polynucleotides. 1,000 bp or nt is equal to a kilobase (kb). Polynucleotides with a length of about 40 nucleotides or less are typically referred to as 'oligonucleotides' and may contain primers for use in DNA manipulation, such as through a polymerase chain reaction (PCR).

[0055] As used herein, the term “nucleic acid” is a single or double-stranded covalently bonded sequence of nucleotides in which the 3’ and 5’ ends of each nucleotide are connected by phosphodiester bonds. Polynucleotides may consist of deoxyribonucleotide bases or ribonucleotide bases. Nucleic acids may include DNA and RNA, which may be synthesized in vitro or isolated from natural sources. Nucleic acids may further include modified DNA or RNA, e.g., methylated DNA or RNA, or RNA subjected to post-translational modification, such as 5’-capping to 7-methylguanosine, cleavage, and 3’-treatment, such as polyadenylation, and splicing. Nucleic acids may also include synthetic nucleic acids (XNA), e.g., hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), threose nucleic acid (TNA), glycerol nucleic acid (GNA), lock nucleic acid (LNA), and peptide nucleic acid (PNA). The size of a nucleic acid, also referred to herein as a “polynucleotide,” is typically expressed as the number of base pairs (bp) for double-stranded polynucleotides or the number of nucleotides (nt) for single-stranded polynucleotides. 1,000 bp or nt is equal to a kilobase (kb). Polynucleotides with a length of about 100 nucleotides or less are typically referred to as “oligonucleotides” and may contain primers for use in DNA manipulation, such as through a polymerase chain reaction (PCR). In a specific embodiment of the present invention, the nucleic acid sequence comprises messenger RNA (mRNA).

[0056] According to the present invention, homology for the nucleic acid sequences described herein is not limited to simply 100% sequence identity. Many nucleic acid sequences may demonstrate biochemical or functional equivalence to one another despite having clearly low sequence identity. In the present invention, homologous nucleic acid sequences are considered to be sequences that hybridize to each other under low-strictness conditions (see Sambrook J. et al. above). In this regard, the term "substantially similar" relating to two sequences means that the sequences have at least 70%, 80%, 90%, 95%, or 100% similarity. Likewise, the term "substantially complementary" relating to two sequences means that the sequences are completely complementary to the bases, or that at least 70%, 80%, 90%, 95%, or 99% of the bases are complementary. That is, a mismatch may occur between the bases of the sequence to be hybridized, and this may occur between at least 1%, 5%, 10%, 20%, or up to 30% of the bases.

[0057] For example, when applied to nucleic acid sequences in expression constructs, the term 'operationally linked' indicates that the sequences are arranged to function cooperatively to achieve an intended purpose. For example, in a DNA vector, a promoter sequence enables the initiation of transcription that proceeds through the linked coding sequence to the termination sequence. In the case of RNA sequences, one or more untranslated regions (UTRs) may be arranged in relation to a linked protein-coding sequence referred to as an open reading frame. A given mRNA may contain more than one ORF, so-called polycistronic RNA. UTRs may be located at the 5' or 3' position in relation to the operationally linked coding sequence ORF. UTRs may include sequences typically found in naturally occurring mRNA sequences, such as the Kozak common sequence, initiation codon, cis-action regulatory element, polyA tail, internal ribosome entry site (IRES), structures regulating mRNA lifetime, sequences directing mRNA localization, etc. mRNA may contain multiple identical or different UTRs.

[0058] In the context of the present invention, the term 'expression of polypeptide' refers to the production of a polypeptide encoding the polynucleotide sequence described herein. Typically, this includes the translation of a supplied mRNA sequence by the ribosomal machinery of the cell to which the sequence is delivered.

[0059] As used herein, the term “delivery particle” refers to a particle capable of delivering a therapeutic component, such as a coding nucleic acid sequence, to a target cell, which may contain a therapeutic component by encapsulation, retention within a matrix, formation of a complex, or other means. The delivery particle may be microscale, but in certain embodiments, it may be nanoscale, i.e., nanoparticle. The size of the nanoparticle is typically at least 50 nm (nanometers), suitably at least approximately 100 nm, typically up to 150 nm, 200 nm, optionally 300 nm or less in diameter. In one embodiment of the present invention, the nanoparticle has an average diameter of at least approximately 60 nm. The advantage of this size is that it means the particle is below the threshold for reticuloendothelial system (monuclear phagocyte system) clearance, that is, the particle is small enough not to be destroyed by phagocytes as part of the body’s defense mechanism. This enables the use of an intravenous delivery route for the composition of the present invention.

[0060] Possible alternatives for the composition of nanoparticles include lipid- or phospholipid-based particles such as polylactic acid (PLA), poly(lactic-co-glycolic acid) (PLGA), and liposomes; particles based on proteins and / or glycoproteins such as collagen, albumin, gelatin, elastin, gliadin, keratin, legumin, zein, soy protein, and milk protein such as casein ([Lohcharoenkal et al. BioMed Research International; Volume 2014 (2014)]); and particles based on metals or metallic compounds such as gold, silver, aluminum, copper oxide, etc.

[0061] In particular, polymers containing polyethyleneimine (PEI) have been investigated for the delivery of nucleic acids. Nanoparticle vectors composed of poly(β-amino ester) (PBAE) have also been found to be suitable for nucleic acid delivery, particularly in formulations with polyethylene glycol (PEG) ([Kaczmarek JC et al Angew Chem Int Ed Engl. 2016; 55(44): 13808-13812]). These formulations have been used to deliver mRNA to the lungs.

[0062] Particles based on polysaccharides and their derivatives, such as cellulose, chitin, and chitosan, are also considered. Chitosan is a cationic linear polysaccharide obtained by the partial deacetylation of chitin, having nanoparticles containing these materials that possess promising properties for drug delivery, such as biocompatibility, low toxicity, and small size ([Felt et al., Drug Development and Industrial Pharmacy, Volume 24, 1998 - Issue 11]). Combinations of the above components are considered to be usable.

[0063] US2010 / 0331234, US2011 / 0293703, and US2015 / 0203439 (these documents are incorporated herein by reference) describe the preparation of aminoalcohol lipidoids by the reaction of amines with epoxide-terminated compounds. Complexes, micelles, liposomes, and particles (including nanoparticles) can be prepared from these lipidoids, and their chemical structures make them particularly suitable for delivering 'cargo', such as nucleic acids like coding mRNA, to target cell types within the body of humans or animals. Because delivery platforms comprising aminoalcohol lipidoid compounds are particularly suitable for use in the delivery of a given purely negatively charged carrier molecule, the tertiary amine available for protonation forms a cationic moiety. For example, aminoalcohol lipidoid compounds may be used in the preparation of particulate compositions for delivering DNA, RNA, or other polynucleotide carriers to a target, or target cell or tissue. Suitable particles may be in the form of microparticles, nanoparticles, liposomes, exosomes, or micelles.

[0064] Aminoal lipidoid-based delivery particles have a tertiary amine available for interacting with a polynucleotide carrier, such as coding mRNA. A polynucleotide or a derivative thereof is contacted with an aminoal lipidoid compound under conditions suitable for forming a polynucleotide / lipidoid complex. The lipidoid is preferably at least partially protonated to form a complex with a negatively charged polynucleotide. In this way, the polynucleotide / lipidoid complex forms a particle useful for delivering the carrier polynucleotide to cells and tissues. In certain embodiments, a plurality of aminoal lipidoid molecules may be associated with a polynucleotide molecule. The complex may comprise at least 1, at least 5, at least 10, at least 20, at least 50, or suitably at least 100 aminoal lipidoid molecules. The above complex may contain up to 10,000, up to 5,000, up to 2,000, up to 1,000, up to 500, or typically up to 100 aminoalcohol lipoid molecules.

[0065] Those skilled in the art will understand that a population of particles follows the principles of particle size distribution. Methods widely used in the art to describe particle size distribution include, for example, average diameters and D values ​​such as the D50 value, which is commonly used to indicate the average diameter of a particle size range of a given sample. In certain embodiments, the diameter of the nanoparticles is in the range of 10 to 500 nm, and more suitably, the diameter of the nanoparticles is in the range of 10 to 1200 nm, particularly 50 to 150 nm. In some embodiments, the nanoparticles have an average diameter of at least about 10 nm, suitably at least about 30 nm. In some embodiments, the nanoparticles have an average diameter of less than about 150 nm and greater than 50 nm.

[0066] The above particles may be further associated with a targeting agent that facilitates the binding of the delivery particles to the target cell type. As used herein, the term 'targeted' refers to an object or composition, such as one comprising a delivery particle intended to associate with cells within a specific organ, tissue, or cell type in the body and to promote its transfection. In certain embodiments, the delivery particle, e.g., a delivery nanoparticle, may be targeted to deliver the target only to a specific organ, tissue, or cell type. Targeting may be geographical, for example, by delivering the targeted object directly to a specific tissue, or may be chemically mediated through a targeting agent or binding moiety that preferentially associates with the target cell or tissue.

[0067] Various targeting agents that induce pharmaceutical compositions to specific cells are known in the art (see, e.g., [Cotten et al. Methods Enzym. 217:618, 1993]; [Wagner et al. Advanced Drug Delivery Reviews, Volume 14, Issue 1, April-May 1994, 113-135]; [Fiume et al. Advanced Drug Delivery Reviews, Volume 14, Issue 1, April-May 1994, 51-65]). Targeting agents may be included throughout the entire particle or may be limited only to the surface. Targeting agents may be proteins, peptides, carbohydrates, glycoproteins, lipids, small molecules, nucleic acids, etc. Targeting agents may be used to target specific cells or tissues, or to promote endocytosis or phagocytosis of the particle. Examples of targeting agents include, but are not limited to, antibodies, antibody fragments, low-density lipoprotein (LDL), transferrin, sialoglyph, gp120 envelope protein of human immunodeficiency virus (HIV), carbohydrates, receptor ligands, sialic acid, aptamers, etc. If the targeting agent is distributed throughout the particle, the targeting agent may be included in the mixture or composite material used to form the particle. If the targeting agent is confined only to the surface, the targeting agent may be associated with the particle formed using standard chemical techniques, for example, by covalent bonding, hydrophobicity, hydrogen bonding, van der Waals, biotin-avidin linkage, or other interactions.

[0068] The microparticle composition of a specific embodiment of the present invention can suitably deliver an encapsulated mRNA carrier over time, which can be controlled by a specific selection or formulation of a biodegradable non-toxic polymer or biocompatible material for encapsulation. For example, the microparticle composition can release the encapsulated mRNA carrier over a period of at least 30 minutes, at least 1 hour, at least 2 hours, at least 6 hours, at least 12 hours, or at least 1 day. The microparticle composition can release the encapsulated mRNA carrier over a period of up to 2 days, up to 3 days, or up to 7 days.

[0069] As used herein, such as in ‘diseased cells’ and / or ‘diseased tissues,’ the term ‘diseased’ refers to tissues and organs (or parts thereof) and cells that are abnormal, unhealthy, or exhibit disease pathology. For example, diseased cells may be infected by viruses, bacteria, prions, or eukaryotic parasites; may contain harmful mutations and / or may be cancerous, precancerous, neoplastic, or neoplastic. Diseased cells may contain an altered intracellular miRNA environment compared to other normal or so-called healthy cells. In certain examples, diseased cells may contain an altered intracellular miRNA environment that is pathologically normal but indicates a prodromal state of disease. Diseased tissue may include healthy tissue infiltrated by diseased cells from another organ or organ system. For example, many inflammatory diseases involve pathology in which otherwise healthy organs are infiltrated by immune cells such as T cells and neutrophils. As an additional example, organs and tissues affected by stenotic or sclerotic lesions may contain both healthy and diseased cells in close proximity.

[0070] As used herein, the term “cancer” refers to a neoplasm within a tissue, including a malignant tumor, which may be a primary cancer originating in a specific tissue or a secondary cancer spread by metastasis from elsewhere. The terms cancer, neoplasm, and malignant tumor are used interchangeably herein. Cancer may refer to tissues or cells located within a neoplasm or possessing characteristics associated with the neoplasm. Neoplasms typically possess features that distinguish them from normal tissues and normal cells. These features include, but are not limited to, a degree of anaplasticity, morphological changes, irregular shape, reduced cell adhesion, metastatic ability, and increased cell proliferation. Terms associated with and often synonymous with “cancer” include sarcoma, carcinoma, malignant tumor, epithelioma, leukemia, lymphoma, transformation, neoplasm, etc. As used herein, the term “cancer” includes not only malignant cancer but also premature and / or precancerous tumors.

[0071] As used herein, such as in ‘healthy cells’ and / or ‘healthy tissues,’ the term ‘healthy’ refers to tissues and organs (or parts thereof) and cells that are not diseased and typically resemble a normal functional phenotype. In the context of the present invention, it may be understood that the term ‘healthy’ is relative, as non-neoplastic cells within tissues affected, for example, by a tumor, may not be completely healthy in an absolute sense. Thus, ‘unhealthy cells’ are used to refer to cells that are not neoplastic, cancerous, or precancerous in themselves, but may be, for example, sclerotic, inflamed, or infected, or otherwise diseased. Similarly, ‘healthy or unhealthy tissues’ are used to refer to tissues or parts thereof that are free from tumors, neoplastic, cancerous, or precancerous cells; or other diseases mentioned above, regardless of overall health. For example, in the context of an organ containing cancerous and fibrous tissues, cells contained within the fibrous tissue may be considered relatively ‘healthy’ compared to the cancerous tissue.

[0072] In an alternative embodiment, the health status of a cell, cell type, tissue, and / or organ is determined by the quantification of miRNA expression. In certain disease types, such as cancer, the expression of specific miRNA species is affected and may be upregulated or downregulated compared to unaffected cells. These differences in miRNA transcripts can be used to identify relative health status and / or to track progression to a disease state in healthy cells, cell types, tissues, and / or organs. A disease state may include various stages of transformation into neoplastic cells. In an embodiment of the present invention, differential variations in the miRNA transcripts of cell types contained within a given organ or organ system are utilized to control protein expression in different cell types.

[0073] As used herein, the term "organ" is synonymous with "organ system" and refers to a combination of tissue and / or cell types that may be compartmentalized within the body of a subject to provide biological functions, such as physiological, anatomical, homeostatic, or endocrine functions. Suitably, an organ or organ system may refer to an internal organ with blood vessels, such as the liver or pancreas. Typically, an organ comprises at least two tissue types and / or multiple cell types that exhibit the phenotypic characteristics of the organ.

[0074] As used herein, the term "therapeutic virus" refers to a virus capable of infecting and killing cancer cells, sometimes through direct viral lysis (tumor cell disruption) as well as indirect cell death caused by stimulation of a host anti-tumor response. Tumor-disrupting viruses are often characterized by having increased activity in diseased cells, including cancer cells, compared to healthy cells.

[0075] Examples of tumor cell disrupting viruses include those provided in Table 1 and their subtypes.

[0076]

[0077] In an embodiment of the present invention, the virus may be selected from any one of groups 1 to 7 of the Baltimore virus classification ([Baltimore D (1971) "Expression of animal virus genomes". Bacteriol Rev.35 (3): 235-41]). In a specific embodiment of the present invention, a suitable virus may be selected from Baltimore group 1, characterized by having a double-stranded DNA virus genome; group 4, having a single-stranded positive RNA genome; and group 5, having a single-stranded negative RNA genome.

[0078] As used herein, the terms 'virulence gene' or 'virulence factor' refer to genes or gene products that aid in the replication of therapeutic viruses, such as tumor cell-disintegrating viruses, within infected cells, or in the lysis of infected cells. The term 'replication factor' is used herein by analogy. Virulence factors may typically be viral genes encoded by the viral genome. Virulence factors may be involved in functions such as intracellular immune system suppression and evasion, viral genome replication, virion dispersal or transmission, generation or assembly of structural coat proteins, viral activation in a latent state, prevention of viral latency, and acceptance of host cell processing. Some virulence factors possess cells or other equivalents capable of compensating for the function of these genes in the absence of the viral genome. Some viruses may be modified with exogenous virulence genes that increase their replication, cell lysis, and dispersal capabilities.

[0079] In a specific embodiment of the present invention, the mRNA sequence enhances or maintains the tumor cell disruption efficacy of the co-administered virus in a tumor located within an organ through differential expression of one or more proteins or polypeptides that preferentially enhance virion replication in the tumor. In this way, the mRNA may encode one or more factors that enhance the biological activity of the tumor cell disruption virus by increased replication and / or increased direct tumor cell disruption efficacy and / or increased viral progeny and / or acquired anti-tumor immune response. In a further embodiment of the present invention, the composition may encode a gene product that controls the interaction between the tumor cell disruption virus and the host immune cells within the tumor. In another further embodiment, the composition of the present invention may be used to produce a gene product that regulates the differential pattern of tumor cell disruption virus activity as well as the expression of an immunoco-stimulating molecule administered exogenously or through the delivery particle of the present invention via the virion.

[0080] As used herein, the term "polypeptide" is a polymer of amino acid residues linked by peptide bonds, which is produced in vitro, either naturally or by synthetic means. Polypeptides with a length of about 12 amino acid residues or less are typically referred to as "peptides," and polypeptides with a length of about 12 to about 30 amino acid residues may be referred to as "oligopeptides." As used herein, the term "polypeptide" refers to a naturally occurring polypeptide, a precursor form, or a product of a precursor protein. Polypeptides may also undergo maturation, or, to a non-limiting degree, post-translational modification processes that may include glycosylation, proteolytic cleavage, lipidation, signal peptide cleavage, propeptide cleavage, phosphorylation, etc. The term "protein" is used herein to refer to a macromolecule comprising one or more polypeptide chains.

[0081] As used herein, the term “gene product” refers to a product of at least one coding sequence or ORF contained within an mRNA construct of the present invention as described herein. The gene product(s) may comprise polypeptides or proteins. Polycistronic mRNA constructs that induce the production of multiple gene products may be used. It will be understood that multiple ORFs enable the in situ production of various products that can functionally cooperate or form complexes and / or multimeric proteins having various biological and potential therapeutic effects.

[0082] Direct delivery of mRNA into cells enables direct and controllable translation of desired gene products, such as intracellular polypeptides and / or proteins. The delivery of mRNA specifically enables the use of cell expression regulatory mechanisms (described in detail in the specific embodiments below), such as miRNA-mediated control, as well as represents a finite and exhaustible supply of the product rather than potentially permanent changes to the transcript of the target cell to which the DNA vector inserted into the episome or genome may be delivered.

[0083] In an embodiment of the present invention, an mRNA sequence is provided comprising a sequence encoding at least one polypeptide in operative combination with one or more untranslated regions (UTRs) capable of imparting tissue specificity and stability to the nucleic acid sequence as a whole. 'Tissue specificity' means that the translation of the protein product encoded by the mRNA is regulated according to the presence of the UTRs. Regulation may include allowing, reducing, or even blocking detectable translation of the mRNA into the protein product. The UTRs may be cis-translated, that is, directly linked to the mRNA on the same polynucleotide strand. In an alternative embodiment, a first sequence encoding a gene product is provided, and an additional second sequence is provided that hybridizes with a portion of the first sequence, comprising one or more UTRs that impart tissue specificity to the nucleic acid sequence as a whole. In such an embodiment, the UTRs are operatively linked to a sequence encoding the gene product trans-translated.

[0084] According to a specific embodiment of the present invention, an mRNA comprising an associated nucleic acid sequence operatively linked thereto is provided as required to prevent or reduce the expression of a gene product in undiseased liver tissue, e.g., healthy hepatocytes. Thus, an mRNA construct or transcript is provided comprising a 5' cap and a UTR (typically, but not exclusively located at the 3' of the ORF) required for ribosome recruitment and tissue and / or organ-specific expression, as well as start and stop codons defining one or more ORFs. When said construct is introduced into an undiseased liver, lung, pancreas, breast, brain, kidney and / or colon gastrointestinal tract, the expression of the gene product is prevented or reduced. In contrast, neoplastic or otherwise diseased cells contained within the aforementioned organs do not typically follow the normal, undiseased cell expression pattern and possess significantly different miRNA transcripts. The gene product(s) contained within the mRNA are specifically translated in these abnormal cells rather than in neighboring healthy cells. Delivery of mRNA constructs to the aforementioned organs can be achieved via a microparticle delivery platform in any suitable manner known in the art, as described herein. Cell type-specific expression can be mediated through microRNA regulatory mechanisms as described in more detail below.

[0085] As defined herein, a “therapeutic component” or “therapeutic agent” refers to a molecule, substance, cell, or organism that contributes to a therapeutic effect on an individual human or other animal when administered to such individual human or other animal as part of a therapeutic intervention. The therapeutic effect may be caused by the therapeutic component itself or by another component of the therapeutic intervention. The therapeutic component may be a coding nucleic acid component, in particular mRNA. The coding nucleic acid component(s) may code for a therapeutic enhancer as defined below. The therapeutic component may also include chemotherapy drugs, such as drugs, optionally small molecules or monoclonal antibodies (or fragments thereof). In some embodiments, the therapeutic component may include recombinantly modified immune effector cells, such as CAR-T cells. In other embodiments of the invention, the therapeutic agent includes a therapeutic virus, such as a tumor cell disrupting virus or a viral vector.

[0086] The term "therapeutic effect" refers to a local or systemic effect in animal subjects, typically in humans, caused by a pharmacological or therapeutic agent comprising a substance, molecule, composition, cell, or organism administered to a subject, and the term "therapeutic intervention" refers to the administration of such substance, molecule, composition, cell, or organism. Accordingly, these terms refer to any agent intended for use in the diagnosis, cure, alleviation, treatment, or prevention of a disease, or for the enhancement of desirable physical or mental development and condition in animal or human subjects. The phrase "therapeutic effective dose" refers to an amount of such agent that produces the intended local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. In certain embodiments, the therapeutic effective dose of the agent will vary depending on the therapeutic index, solubility, etc. For example, a specific therapeutic agent of the present invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment. In specific contexts of cancer treatment, 'therapeutic effect' may be manifested by various means, including, but not limited to, a reduction in solid tumor volume, a reduction in the number of cancer cells, a reduction in the number of observed metastases, an increase in lifespan, a reduction in cancer cell proliferation, a reduction in cancer cell survival, a reduction in tumor cell marker expression, and / or the alleviation of various physiological symptoms associated with cancerous conditions.

[0087] In one embodiment, the subject to whom the therapeutic regimen is administered is a mammal (e.g., mouse, rat, primate, non-human mammal, domestic animal or livestock, e.g., dog, cat, rabbit, cattle, horse, sheep, goat, etc.), and suitably a human. In a further embodiment, the subject is an animal model of cancer. For example, the animal model may be an orthotopic xenograft animal model of human-derived cancer, suitably liver cancer, lung cancer, pancreatic cancer, breast cancer, brain cancer, kidney cancer and / or colorectal cancer.

[0088] In a specific embodiment of the method of the present invention, the subject has not yet received therapeutic treatment such as therapeutic viral therapy, chemotherapy, radiation therapy, targeted therapy, and / or anti-immunotherapy checkpoint therapy. In another embodiment, the subject has received therapeutic treatment such as the aforementioned therapies.

[0089] In an additional embodiment, the subject underwent surgery to remove cancerous or precancerous tissue. In another embodiment, the cancerous tissue was not removed, and, for example, the cancerous tissue may be located in an inoperable area of ​​the body, such as a tissue or organ where surgical intervention could endanger the subject's life, or in an area where a surgical procedure could cause a significant risk of permanent injury or even death.

[0090] In some embodiments, the provided mRNA may encode a 'therapeutic enhancer'. According to the present invention, a therapeutic enhancer is a gene product or polypeptide capable of enhancing or promoting the ability of another co-administered therapeutic agent to exert a therapeutic effect on a given cell, suitably a target cell. When introduced into or near a target cell, the expression of the therapeutic enhancer may cooperate with the co-administered therapeutic agent to enable or enhance the therapeutic activity of the agent. In some embodiments, the therapeutic enhancer may enhance the ability of a co-administered tumor cell-disrupting virus to lyse cancer cells. In other embodiments of the present invention, the therapeutic enhancer may influence changes in the tumor microenvironment to assist or recruit the target's own immune response. In such embodiments, changes in the tumor microenvironment may assist the co-administration of a tumor cell-disrupting virus or a CAR-T or other adoptive cell-based therapy. In some embodiments, the therapeutic enhancer may convert a prodrug into an active form.

[0091] Multiple therapeutic enhancers may be combined in a composition according to a specific embodiment of the present invention. In such embodiments, the coding sequence for each therapeutic enhancer may exist in separate mRNA molecules. In some embodiments, sequences for more than one therapeutic enhancer may exist in the same mRNA molecule. In such cases, the polycistronic mRNA molecule additionally includes a sequence necessary for the expression of the entirely coded sequence, such as an internal ribosome entry site (IRES).

[0092] In an embodiment in which a plurality of different mRNA molecules are contained in one or more delivery systems, each delivery system (e.g., particle, liposome, viral vector system) may contain one or more types of mRNA molecules as a 'payload'; that is, it is considered that in a particular embodiment, all delivery payloads may not necessarily contain all mRNA molecules provided in the embodiment. In this way, it is considered that different delivery systems and their associated sequences can be directed to different target cells using the targeting agent described herein.

[0093] mRNA structures of specific embodiments of the present invention may be synthesized from polynucleotide expression structures, which may be, for example, DNA plasmids. Such expression structures may include any promoter sequence and a corresponding termination sequence necessary for the initiation of transcription so that transcription of the mRNA structures may occur. Such polynucleotide expression structures are naturally considered to include embodiments of the present invention.

[0094] The gene product encoded by the mRNA is typically a peptide, polypeptide, or protein. Where a particular protein consists of more than one subunit, the mRNA may encode one or more subunits within one or more ORFs. In an alternative embodiment, the first mRNA may encode the first subunit, but the second co-administered mRNA may encode the second subunit, which, when translated in situ, leads to the assembly of a multi-subunit protein gene product.

[0095] The gene product encoded by mRNA can be any type suitable for producing a therapeutic effect. In the context of cancer treatment, the gene product encoded by mRNA may suitably include genes that cause or assist in the destruction of cancer cells when expressed by cancer cells.

[0096] Tumor suppressor genes such as p53 may be provided by the structures of the present invention. p53 plays a role in cellular processes including apoptosis and genomic stability. It is involved in the activation of DNA repair processes in response to genomic damage and can halt cell growth and regeneration.

[0097] Genes that promote cell death by apoptosis, so-called suicide genes, which cause cells to activate the process of apoptosis when expressed, may also be provided by the composition or structure of the present invention. Cancer cells often possess mutated and / or non-functional versions of these apoptosis-related genes, thereby being unable to undergo apoptosis in response to external signals. Suicide gene therapy may also involve the introduction of genes that enable the conversion of non-toxic compounds or prodrugs into lethal drugs ([Duarte et al. Cancer Letters, 2012]). According to embodiments of the present invention, such gene products may be selectively introduced into diseased cells, such as neoplastic cells, to destroy them through induced apoptosis or by the delivery of other non-toxic compounds or prodrugs.

[0098] In a specific embodiment of the present invention, mRNA may encode inhibitors of programmed cell death pathways, such as inhibitors of the PD-1 receptor (CD279) or its ligands PD-L1 (B7-H1; CD274) and PD-L2 (B7-DC; CD273). Thus, mRNA may encode proteins or polypeptides that bind to the PD-1 / PDL-1 or PD-1 / PDL-2 axis within diseased or neoplastic cells in target organs, or otherwise interfere with its function. Suitable proteins or polypeptides may include antibodies, which may be monoclonal or polyclonal, or their antigen-binding fragments, or other antigen-binding microproteins that bind to the PD-1 receptor, PDL-1, PDL-2, or the complex of the ligand and the receptor. These effects may also be observed by the use of protein or polypeptide inhibitors of the cytotoxic T lymphocyte antigen 4 (CTLA4) pathway, which is another so-called immune checkpoint. It is known that inhibition of either or both of the two pathways induces changes in immune responses within the tumor microenvironment that may be positively beneficial to the patient's health. Additionally, by modulating the immune response in the subject, the compositions of the present invention may exhibit specific utility in combination therapy with other anticancer treatment approaches, such as radiotherapy or chemotherapy. FDA-approved anti-PD1 pathway inhibitors include pembrolizumab and nivolumab. Known anti-PDL-1 inhibitors include MPDL-3280A, BMS-936559, and atezolizumab. Anti-CTLA4 therapeutic inhibitors include ipilimumab and tremelimumab. The composition of the present invention can be used to selectively deliver these inhibitors of these programmed cell death pathways or functional mimics thereof to diseased cells within the target organ by utilizing the differential miRNA environment of these cells.

[0099] Chimeric antigen receptor T-cells (CAR-T cells) are immune cells, specifically T-lymphocytes modified to express receptors that target cancer cells.

[0100] Adoptive immunotherapy involving the migration of in vitro autoantigen-specific T cells is a promising strategy for treating viral infections and cancer. T cells used in adoptive immunotherapy can be generated through genetic engineering by expanding antigen-specific T cells or reorienting T cells (see, e.g., [Park, TS, SA Rosenberg, et al. (2011). "Treating cancer with genetically engineered T cells." Trends Biotechnol 29(11): 550-7]).

[0101] Novel specificity of T cells, also known as immune effector cells, has also been successfully generated through the gene transfer of a genetically modified T cell receptor or a chimeric antigen receptor (CAR) (see, e.g., Jena, B., G. Dotti, et al. (2010). "Redirecting T-cell specificity by introducing a tumor-specific chimeric antigen receptor." Blood 116(7): 1035-44). A CAR is a synthetic receptor composed of at least three parts: an extracellular antigen recognition domain (also known as an ectodomain), a transmembrane domain, and an intracellular T-cell activation domain (also known as an endodomain). In some embodiments, the engineered T cells include a specific class of T cells, such as gamma delta T cells, which are a subtype of T cells that selectively target tumor cells without affecting healthy cells. CARs have successfully reoriented T cells toward antigens expressed on the surface of tumor cells from various malignancies, including lymphomas and solid tumors (see Jena, Dotti et al. above). In some embodiments, the engineered T cells comprise a population of autologous T cells engineered to eliminate the expression of an endogenous αβ T-cell receptor (TCR) so that the CAR-T cells prevent graft-versus-host response without impairing CAR-dependent effector function. In some embodiments, the engineered T cells comprise at least a population of allogeneic T cells. In some embodiments, the engineered T cells comprise at least a population of autologous T cells and a population of allogeneic T cells.

[0102] Generally, the extracellular antigen recognition domain is a targeting moiety associated with one or more signaling domains in a single fusion molecule from an antibody, receptor, or ligand domain that binds to a specific target, typically a tumor-associated target. In some embodiments, the extracellular antigen recognition domain is a short-chain fragment variant (scFv) of the antigen-binding domain of a short-chain antibody (scFv), comprising light and heavy chain variable fragments of a monoclonal antibody linked by a flexible linker, or is derived therefrom. In some embodiments, the extracellular antigen recognition domain is linked to a transmembrane domain by a linker, such as a flexible linker like an IgG1 hinge linker. In some embodiments, the transmembrane is a CD28 transmembrane domain or is derived therefrom. In some embodiments, the endodomain comprises a co-stimulation domain designed to enhance the immune response by, for example, enhancing the survival of CAR-modified T cells and increasing their proliferation, and an endogenous T-cell activation domain designed to activate T cells when bound to a target. In some embodiments, the co-stimulation domain is a CD28 co-stimulation domain, an OX-40 (CD134) co-stimulation domain, an ICOS co-stimulation domain, a 4-1BB (CD137) co-stimulation domain, or any combination thereof, or is derived therefrom. In some embodiments, the intracellular T-cell activation domain comprises a CD3 zeta (CD3ζ) domain or a biologically active portion thereof. In some embodiments, T-cell activation induces immune cell activation in which inflammatory cytokines are released by T cells to promote inflammation and / or immune responses. In some embodiments, T-cell activation induces cytotoxic activity in which cytotoxins are released by T cells to promote cancer cell apoptosis. In some embodiments, T-cell activation induces proliferation in which interleukins are released by T cells to promote cell development and division.In some embodiments, T cell activation induces a combination of at least two of immune cell activation, cytotoxic activity, and / or proliferation.

[0103] In some embodiments, the extracellular antigen recognition domain specifically binds to CD19. Since most B-acute lymphoblastic leukemias (B-ALL) uniformly express CD19 but expression is absent in non-hematopoietic cells, as well as in bone marrow, erythrocytes, T cells, and bone marrow stem cells, CD19 is an attractive target for immunotherapy. Clinical trials targeting CD19 in B-cell malignancies are progressing with encouraging anti-tumor responses. Many of the current CAR-T therapies being evaluated in clinical trials use T cells genetically engineered to express a chimeric antigen receptor (CAR) with specificity derived from the scFv region of the CD19-specific mouse monoclonal antibody FMC63 (e.g., [Nicholson, Lenton et al. (1997); "Construction and characterisation of a functional CD19 specific single chain Fv fragment for immunotherapy of B-lineage leukemia and lymphoma." Mol Immunol. 1997 Nov-Dec;34(16-17):1157-65]; [Cooper, Topp et al. (2003). "T-cell clones can be rendered specific for CD19: toward the selective augmentation of the graft-versus-B-lineage leukemia effect." Blood. 2003 Feb 15;101(4):1637-44]; Cooper, Jena et al. (2012) (International Application: (Refer to WO2013 / 126712).

[0104] In some embodiments, the extracellular antigen recognition domain specifically binds to CD22. CD22 is a transmembrane phosphoglycoprotein belonging to the Siglec family of lectins that specifically binds sialic acid to seven extracellular immunoglobulin domains at its N-terminus. It acts primarily as an inhibitory receptor for B cell activation and signaling, and regulates the interactions between B cells, T cells, and antigen-presenting cells (APCs). Similar to CD19, CD22 is a B cell lineage-restriction marker that is clearly expressed by B lymphoid cells from the progenitor-B to the mature B cell stage. However, it is lost during differentiation into plasma cells. CD22 is universally expressed in most B cell malignancies, including acute lymphoblastic leukemia (ALL), chronic lymphoblastic leukemia (CLL), and various subtypes of non-Hodgkin lymphoma (NHL), such as diffuse large B-cell lymphoma. Targeting CD22 as an attractive therapeutic target for B-cell malignancies has been demonstrated by positive results in clinical trials of anti-CD22 monoclonal antibodies (e.g., epratuzumab) and immunotoxins (e.g., BL22, HA22). CD22 was shown to be expressed in all cells that lost CD19 expression after treatment with anti-CD19 CAR-T cells, which makes anti-CD22 CAR-T cells suitable for combination with and / or subsequent therapy with anti-CD19 CAR-T cells.

[0105] However, while the adoptive potential of CAR-T cells for cancer treatment has been demonstrated in numerous clinical studies, risks associated with cytokine-releasing syndrome (CRS) and "on-target off-tumor" effects have also been raised.

[0106] The mRNA delivery compositions provided in some embodiments of the present invention are useful for improving the safety and efficacy of CAR-T cells. For example, the mRNA nanoparticle delivery system of the embodiments described herein can be used to recruit specific immune cells, or modified subsets of immune cells such as CAR-T cells, into the tumor microenvironment. Additionally, the mRNA nanoparticle delivery system can be used to inhibit the expression of endogenous T cell receptors (TCRs) to evade graft-versus-host disease and / or selectively delete immune checkpoint genes in these cells to enhance their anticancer activity in a suppressive tumor environment (see, e.g., [Moffett, Coon, et al. (2017) "Hit-and-run programming of therapeutic cytoreagents using mRNA nanocarriers." Nature Communications. 8:389]).

[0107] In some embodiments, coding mRNA is used to attract CAR-T cells from a target to a specific site. In some embodiments, coding mRNA is used to overcome insufficient migration of immune cells into the tumor microenvironment. In response to specific chemokines, different subsets of immune cells migrate into the tumor microenvironment and modulate the tumor immune response in a spatiotemporal manner. In some embodiments, coding mRNA is used to enhance CAR-T cell activation. Additionally, chemokines can directly target non-immune cells, including tumor cells and vascular endothelial cells, in the tumor microenvironment, which has been shown to modulate tumor cell proliferation, cancer stem-like cell characteristics, cancer invasiveness, and metastasis. In some embodiments, immune cells are antigen-presenting cells (APCs), such as T cells, natural killer (NK) cells, B cells, macrophages, or dendritic cells, or any combination thereof.

[0108] In some embodiments, coding mRNA may be used to overcome insufficient migration of CAR-T cells into the tumor microenvironment and to prevent off-target CAR-T activity. In some embodiments, the mRNA is delivered to the tumor microenvironment, and the coding mRNA encodes a gene product that attracts or otherwise recruits CAR-T cells into the tumor microenvironment. In some embodiments, the coding mRNA expresses a chemokine. As a non-limiting example, one or more coding mRNAs may encode one or more chemokines that attract T-cells, such as CCL2, CCL3, CCL4, CCL5, CCL20, CCL22, CCL28, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, XCL1, and any combination thereof. When intended for the opposite effect, as in autoimmune diseases, the coding mRNA may express a blocker, antagonist, and / or inhibitor of the aforementioned factors.

[0109] In some embodiments, the coding mRNA is transiently expressed in the tumor microenvironment. In some embodiments, the coding mRNA encodes cytokines or other gene products involved in regulating the survival, proliferation, and / or differentiation of immune cells in a tumor response, such as, for example, activated T cells and NK cells. As a non-limiting example, the coding mRNA may encode cytokines such as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IL-33, IL-35, TGF-beta, and any combination thereof. Again, when intended for the opposite effect, as in autoimmune diseases, the coding mRNA may express blockers, antagonists, and / or inhibitors of the aforementioned factors.

[0110] In some embodiments of the present invention, the coding mRNA is delivered together with a CAR-T or other adoptive cell therapy to provide transient expression of the coding mRNA.

[0111] In some embodiments, an mRNA delivery system as described herein delivers mRNA encoding a gene-editing agent to a target cell population. In some embodiments, the mRNA codes for a sequence-specific nuclease that targets a gene locus and interferes with the expression of one or more endogenous gene products in the target cell population. In some embodiments, the mRNA interferes with the expression of one or more domains in the TCR by encoding a sequence-specific nuclease that targets a T cell receptor (TCR)-associated gene locus.

[0112] In some embodiments, the described mRNA delivery system may be used to deliver mRNA encoding one or more agents that program engineered T cells into a desired phenotype. In some embodiments, the mRNA may be used to induce transcription patterns and markers characteristic of the desired T cell phenotype. In some embodiments, the mRNA may be used to promote the development of CD26L+ central memory T cells (Tcm), which have been shown to improve CAR-T therapy (e.g., see Moffett, Coon above).

[0113] MicroRNAs (miRNAs) are a class of non-coding RNAs each containing about 20 to 25 nucleotides, some of which are believed to be involved in the post-transcriptional regulation of gene expression by binding to sequences complementary to the 3' untranslating region (3' UTR) of target mRNAs, thereby inducing their silencing. Such sequences are also referred to herein as miRNA binding sites or miRNA binding site sequences. Specific miRNAs are highly tissue-specific in their expression; for example, miR-122 and its variants are abundant in the liver and rarely expressed in other tissues ([Lagos-Quintana (2002), Current Biology, Vol.12, Apr]).

[0114] Therefore, miRNA systems provide a powerful platform for nucleic acids introduced into cells to be silenced in selected cell types of target tissues and expressed in other cells. When a binding site for a specific given miRNA sequence is incorporated into an mRNA construct, specifically within or right next to the 5' or 3' of a UTR, and introduced into target cells, the expression of the specific introduced gene may be reduced or substantially eliminated in some cell types, but maintained in others ([Brown and Naldini, Nature Reviews Genetics volume 10, pages 578-585 (2009)]). The use of the term 'right next to' is understood as synonymous with terms such as 'very close' or 'very near'. When referring to the 5' or 3' positioning of a UTR sequence, this typically includes variants in which about 20 or fewer, suitably 50 or fewer, intervening nucleotides may be located between the miRNA binding sequence and the adjacent UTR. It is considered that one or more such miRNA binding site sequences may be included in an mRNA construct. Where multiple miRNA binding site sequences are present, such plurality may include, for example, more than two, more than three, and typically more than four miRNA binding site sequences. These miRNA binding site sequences may be arranged sequentially, in tandem, or at a pre-set position within the 3' or 5' of a specific UTR within the mRNA construct. The multiple binding site sequences may be separated by a linker sequence that may be variable, or by a specific sequence, for example, " uuuaaa It may include. In some embodiments, a linker sequence may not exist between the binding site sequences. Other parts of the mRNA sequence, such as between the stop codon of the ORF and the UTR or binding site, may include a linker sequence.

[0115] miRNA-122, despite being abundant in healthy, non-diseased liver tissue, is reduced in diseased cells as well as in most liver cancers ([Braconi et al. [2011, Semin Oncol; 38(6): 752-763], [Brown and Naldini Nature 2009;10 578]). By the method mentioned above, it was confirmed that when the target tissue is the liver, the translation of the introduced mRNA sequence can be promoted in cancerous liver cells and reduced or substantially eliminated in transfected healthy cells by including the miRNA-122 binding site (e.g., SEQ ID NO. 1) within or adjacent to the 3' UTR.

[0116] In a similar manner, by using different miRNA binding site sequences, differential translation of these mRNAs between cancer cells and healthy cells of different organs is also possible. Suitable candidates include (without limitation) binding sites for miRNA-125, miRNA-124a, miRNA-Let7, miRNA-375, miRNA-143, miRNA-145, miRNA-192, miRNA-194, miRNA-204, miRNA-215, and miRNA-30b,c. Table 2 shows additional (without limitation) examples of miRNA sequences for which differential expression has been demonstrated.

[0117] miRNA-125 is used in hepatocellular carcinoma ([Coppola et al. Oncotarget 2017;8]); breast cancer ([Mattie et al. Mol Cancer 2006;5]), lung cancer ([Wang et al. FEBS J 2009]), ovarian cancer ([Lee et al. Oncotarget 2016;7]), gastric cancer ([Xu et al. Mol Med Rep 2014;10]), colon cancer ([Tong et al. Biomed Pharmacother 2015;75]) and cervical cancer ([Fan et al Oncotarget 2015;6]); It is downregulated in some solid tumors such as neuroblastoma, medulloblastoma ([Ferretti et al. Int J Cancer 2009;124]), glioblastoma ([Cortez et al. Genes Chromosomes Cancer 2010;49]), and retinoblastoma ([Zhang et al; Cell signal 2016;28]).

[0118] Several miRNA species are also differentially expressed in glioblastoma pleomorphic cells compared to healthy brain cells (e.g., neurons) ([Zhangh et al. J Miol Med 2009;87]; [Shi et al. Brain Res 2008;1236], where miRNA-124a is one of the most unregulated ([Karsy et al. Gene Cancer 2012;3]; [Riddick et al. Nat Rev Neurol 2011;7]; [Gaur et al. Cancer Res 2007;67]; [Silber et al. BMC Med 2008;6]).

[0119] In lung cancer, a recent meta-analysis confirmed the downregulation of Let-7 (also miRNA-148a and miRNA-148b) in non-small cell lung cancer ([Lamichhane et al. Disease Markers 2018]).

[0120] Similarly, miRNA-375 expression was found to be downregulated in pancreatic cancer cells compared to healthy pancreatic cells ([Shiduo et al. Biomedical Reports 2013;1]).

[0121] In another embodiment, IL-12-secreting cells can be used to enhance the activity of CAR-T cells. Without being constrained by theory, it is believed that IL-12-induced IFNγ accumulation in a tumor can promote the infiltration of CAR-T or other host immune cells (e.g., NK cells) into the tumor, thereby enhancing the therapeutic effect ([Chinnasamy D. et al. Clin Cancer Res 2012:18]; [Chmielewski M. et al. Cancer Res 2011;71]; [Kerkar SP. Et al. J Clin Invest 2011;121]; [Jackson HJ. Et al. Nat Rev Clin Oncol 2016;13]). Accordingly, according to one embodiment of the present invention, mRNA can be used to promote the exogenous expression of IL-12 in the tumor microenvironment by encoding IL-12 or its functional equivalent or analog.

[0122] In the context of the disease-specific expression of introduced polynucleotides, binding sequences for any miRNA sequence that are disrupted in a specific disease—that is, upregulated or downregulated in diseased cells (e.g., tumor cells) compared to healthy cells—are considered suitable for use in the present invention. Table 2 further discusses non-limiting examples of such tumor-associated miRNA binding sequences that may be used in embodiments of the present invention. However, it will be understood that the present invention is not limited only to cases where a given miRNA or class of miRNAs is downregulated in a first cell type compared to a second cell type within a given organ or organ system. On the contrary, it is required that there exist a differential expression pattern of the regulatory miRNA between the first cell type and the second cell type contained within the organ or organ system. Differential expression of miRNA using the compositions and methods described herein may be utilized to enable corresponding differential translation of protein products in these cells.

[0123] Examples of cancers for which evidence of similar differential miRNA expression between healthy and cancer cells has been identified include breast cancer ([Nygaard et al, BMC Med Genomics, 2009 Jun 9;2:35]), ovarian cancer ([Wyman et al, PloS One, 2009;4(4):e5311]), prostate cancer ([Watahiki et al, PloS One, 2011; 6(9):e24950]), and cervical cancer ([Lui et al. Cancer Research, 2007 Jul 1;67(13):6031-43]). WO 2017 / 132552 A1 describes a wide range of miRNAs with different expression levels in various cancer cells.

[0124]

[0125] In the pancreas, miRNA-375 expression was found to be high in normal pancreatic cells but significantly low in diseased and / or cancerous tissues ([Song, Zhou et al. 2013]). This expression was shown to be associated with the stage of cancer progression, with expression further decreasing in more advanced cancers. This suggests that miRNA-375 is involved in regulating glucose-induced biological responses in pancreatic β-cells by targeting 3-phosphoinositide-dependent protein kinase-1 (PDK1) mRNA and influencing the PI 3-kinase / PKB cascade ([El Ouaamari et al. Diabetes [57:2708-2717, 2008]). The antiproliferative effect of miRNA-375 is implied by this mode of behavioral estimation, which may explain its downregulation in cancer cells.

[0126] Variants and polymorphisms of miRNA sequences may be found, and miRNA families are known to have similar characteristics. In the present invention, it is considered that, where appropriate, all suitable variants and family members of a specific miRNA sequence, and their associated binding sites, may be used. Meanwhile, since clearly closely related miRNA sequences may have different expression profiles ([Sun et al, World J Gastroenterol. 2017 Nov 28]), in some cases, it may be necessary to refer to the literature to determine whether a specific substitution is appropriate. For example, Let-7 is part of a broad family with numerous related variants, which may be denoted as Let-7a through Let-7k, etc.

[0127] Treating patients with immunotherapies, such as tumor cell-disrupting viruses or CAR-T therapy, may present safety concerns due to the potential for off-target effects. The expression of specific polypeptides resulting from the provision of coding mRNA sequences can also have adverse effects on specific organs. Therefore, protecting healthy tissues, such as the liver, brain, breast, lung, pancreas, colon / gastrointestinal tract, and kidneys, is of paramount importance for successful clinical application. Accordingly, the present composition may represent an enabling technology platform capable of enhancing and facilitating the successful adoption of conventional 'experimental' cell or viral therapies.

[0128] The presence of multiple miRNA binding site sequences within an mRNA construct can improve the efficacy of differential expression of the supplied polypeptide or polypeptides. Regardless of theory, it is believed that as the number of binding sites increases, the potential for miRNA binding and subsequent degradation increases. Multiple miRNA binding sites can introduce redundancy by including multiple copies of substantially the same binding site sequence. Alternatively or additionally, multiple binding site sequences may include substantially different sequences, allowing the mRNA construct to be targeted by more than one miRNA species. In this way, differential expression of the supplied mRNA construct can be achieved for more than one cell type and / or more than one organ, as is evident in the discussion of the organ and its associated miRNA sequences. Both approaches are considered possible within the same sequence or multiple sequences. An intermediate approach is also considered in which multiple binding sequences are included that differ to be targeted by the same miRNA sequence but bind to different variants of the same miRNA sequence.

[0129] Some advantages associated with the use of multiple binding sites include increased efficiency of differential expression of polypeptides supplied by the mRNA sequence of the present invention within a single organ. The use of different binding site sequences, or sequences applicable to more than one tissue or organ type, enables differential expression to be achieved in different cell types within more than one organ or tissue. This may be desirable when systemic administration of the composition according to the present invention is used, and there is a need to avoid off-target effects in more than one organ.

[0130] Even when using local or targeted administration, there is a possibility that the supplied mRNA construct may occur or accumulate in unintended organs, tissues, and / or cells. In particular, liver and kidney tissues may accumulate the administered composition due to the physiological functions of these organs. In such cases, to avoid off-target effects, it may be advantageous for the supplied construct to contain a miRNA binding site sequence that can reduce expression in these tissues. Conversely, it may be desirable to recommend expression only in certain organs, tissues, and / or cell types, which can be achieved by selecting the miRNA binding site sequence.

[0131] In some embodiments, more than one different mRNA sequence may be provided as a single composition. These different sequences may encode different polypeptides and / or different miRNA binding sequences. In this way, a single composition may express multiple different polypeptides. By using different combinations of miRNA binding sequences in distinct mRNA sequences, different cell types or target organs may express specific polypeptides or be protected from their expression according to the intended purpose. For example, if healthy cells in the liver and brain need to be protected from the expression of polypeptide 'A', but it is desirable for the healthy brain, not the liver, to express polypeptide 'B', the first mRNA sequence may include the sequence of 'A' having binding sites for miRNA-122, miRNA-125a, and miRNA-124a, while the second mRNA sequence may include the sequence of 'B' having binding sites for miRNA-122 and miRNA-125a.

[0132] It is understood that to achieve any combination of expression in a given series of organs and cell types, a person skilled in the art may devise combinations of binding sites, polypeptide sequences, and multiple mRNA sequences. Some examples are presented in FIGS. 19a, 19b, 19c, 19d, and 19e, and SEQ ID NOs. 1 to 29. FIG. 19a shows possible miRNA binding site sequences for miRNA-122, miRNA-125a, miRNA-124a, Let-7, miRNA-375, miRNA-192, and miRNA-143 (each associated with SEQ ID NOs. 1 to 7). The relevant organs and tissues associated with these sequences are discussed above and in Table 2. FIG. 19b shows a schematic diagram of an mRNA construct according to some embodiments of the present invention. The ORF begins with a start codon and ends with a stop codon, followed by a series of up to five binding site sequences. As shown in these drawings, the binding site sequence may be separated by a linker or may be without a linker. The ORF may code for, for example, US3, or be any ORF. Variability of the stop codon is expected in any embodiment, and in all embodiments, there may be no stop codon between the ORF and the binding site sequence.

[0133] Specific combinations of miRNA binding site sequences are presented in FIGS. 19c and 19d, along with organs with improved protection as a result of these selections. These can be seen as exemplified by SEQ ID NOs. 8 to 16. Protection is relative rather than absolute, and levels of microRNA expression are understood to vary between different tissues within an organ and between organs. Variability may also exist within a population based on race, age, sex, health status, and pathology, as well as genetic variation and inter-individual polymorphisms.

[0134] In one embodiment, the mRNA sequence may include SEQ ID NO. 11. This sequence includes binding sequences for the microRNA sequences miRNA-122, miRNA-125, miRNA-124a, Let-7, and miRNA-375. These sequences provide protection against unwanted expression of the provided polypeptide in healthy tissues of organs such as the liver, brain, lungs, pancreas, and breast (without limitation). Similarly, a sequence including binding sequences for the miRNA sequences miRNA-122, miRNA-124a, Let-7, miRNA-375, and miRNA-192 will provide enhanced protection for the liver, brain, lungs, pancreas, breasts, and kidneys. For the treatment of gastrointestinal tumors such as colorectal cancer, protecting healthy tissues in the liver, lungs, breasts, pancreas, kidneys, and colon will be important for medical use.

[0135] FIG. 19e illustrates specific combinations of polypeptide coding sequences and binding sites. That is, the US3 coding sequence is represented in combination with any binding site sequence presented in FIG. 19b and 19c (e.g., FIG. 8 to 16), as exemplified by FIG. SEQ ID NOs. 18 to 26, and the RR1 coding sequence is represented in combination with the binding site sequences of FIG. 11 and 16, for example, as exemplified by FIG. SEQ ID NOs. 27 and 28. The IL-12 coding sequence is represented in combination with the binding site sequence included in FIG. 11, for example, as exemplified by FIG. 29. An ORF comprising coding sequences for US3 and RR1, US3 and anti-PDL1, US3 and anti-PDL1, and RR1 and IL12 is represented in combination with the binding site sequence of FIG. 11, for example. However, all combinations of coding sequences and multiple binding site sequences are considered herein. In particular, the coding sequences for US3, RR1, anti-PDL1, and IL12 can be combined with any binding site sequence.

[0136] In some embodiments, it may be desirable for the binding site sequence to have a mismatch with the miRNA sequence targeting it. For example, the mismatch may occur in relation to up to 5%, up to 10%, up to 20%, or up to 30% of the bases of the binding site sequence relative to the total target miRNA sequence. Regardless of theory, excessive uptake of intracellular miRNA sequences is believed to induce dysregulation of the endogenous miRNA system in some cell types. This dysregulation of miRNA profiles is associated with different liver diseases ([Szabo G et al. Gastroenterol Hepatol 2013;10]; [Schueller F. et al. Int J Mol Sci 2018;19]). Reduced complementarity between the provided binding site sequence and the miRNA sequence may mitigate potential side effects.

[0137] The UTR of the mRNA sequence supplied by the present invention may be selected to have similarity, for example, more than 90% similarity, to part or all of the UTR sequence expressed in one of the cell types within the target organ. The specific cell type may have genes whose expression is upregulated or downregulated, and the UTR sequence may mediate such regulation, for example, by promoting the stability or degradation of the related mRNA sequence.

[0138] For example, a UTR associated with a gene known to be upregulated in cancer cells may possess one or more features, such as a miRNA binding site sequence that promotes its stability and translation in these cancer cells. Including a similar sequence in a supplied mRNA sequence may improve stability and translation in cancerous cells, as opposed to non-cancerous or healthy cells.

[0139] Furthermore, the cancer intended to be treated by the present invention is considered to be a secondary cancer in the target tissue, that is, a metastasis from cancer located elsewhere other than the target tissue. For example, liver metastasis may originate from esophageal cancer, gastric cancer, colon cancer, rectal cancer, breast cancer, kidney cancer, skin cancer, pancreatic cancer, or lung cancer, and may be an adenocarcinoma or another type of cancer. In such cases, it may be necessary to select alternative miRNA sequences to provide differential expression in healthy, non-cancerous, and / or cancerous cells. In fact, in such cases, the selection of candidate miRNA sequences may be increased due to the different tissue origins of the metastatic cells.

[0140] In certain cases, it is possible that there may be more than one candidate for miRNA sequences exhibiting differential expression in different cell types of the target tissue. In such cases, it may be advantageous to include multiple miRNA binding site sequences in the mRNA construct, and for these sequences to be substantially different. However, it is also expected that the multiple miRNA binding site sequences may each be substantially identical.

[0141] Combination therapy

[0142] Tumor cell disruption virus

[0143] As mentioned above, oncolytic virus therapy is a method of using viruses to infect and kill cancer cells, sometimes by direct viral lysis and sometimes by indirect killing through the stimulation of a host anti-tumor response. Oncolytic viruses are often characterized by increased activity in cancer cells compared to healthy cells, but off-target effects caused by damage to healthy cells have been reported ([Russell et al. Nature Biotechnology, 2012]).

[0144] To increase safety and reduce off-target effects, oncolytic viruses can be modified or selected to reduce their virulence by deleting virulence factors or genes involved in functions such as intracellular immune system suppression and evasion, viral genome replication, and takeover of host cell processes. Historical production of safe forms of live viruses used for vaccination is another source of attenuated viruses. In other cases, specific mutations or even additional genes have been found to enhance oncolytic activity in specific oncolytic viruses. Non-limiting examples of virulence genes typically added, mutated, or deleted in oncolytic viruses can be found in Table 3.

[0145]

[0146] Attenuation or modification of oncolytic viruses in this manner can play a role in the selectivity of oncolytic viruses toward cancer cells: since the carcinogenesis process often involves the inactivation of genes that serve a protective role against both cancer (e.g., by regulating cell division or apoptosis) and viral infection, as described, attenuated oncolytic viruses can maintain their virulence in cancer cells due to the absence of normal antiviral genes within these cells. Thus, in healthy cells, the attenuated virus is eliminated because it cannot defend against the normal antiviral response, but in cancer cells, there is no such response, and the virus can lyse the cancer cells. However, this approach is rarely effective because, firstly, partial inactivation of the antiviral response in cancer cells is more common than a complete lack of antiviral activity ([Haralambieva et al, Mol. Ther., 2007]) (meaning that virulence can still be reduced in these cells), and secondly, infection of healthy cells can still occur.

[0147] Similarly, viruses typically utilize the cellular machinery of host cells to replicate their genomes; however, since this machinery is typically downregulated in healthy, quiescent, non-replicating cells that do not replicate their own genomes, many viruses possess genes to reactivate or compensate for host machinery. For example, ribonucleotide reductase enzymes are required for the production of deoxyribonucleotides from ribonucleotides; these enzymes are typically downregulated in quiescent host cells, and some viruses possess genes for this type of enzyme itself to provide a source of deoxyribonucleotides. Since replicating in cancer cells can reactivate these enzymes, attenuated oncolytic viruses lacking their own ribonucleotide reductase enzyme genes can still replicate in cancer cells. However, for reasons similar to those mentioned above, this approach may not be entirely effective in protecting healthy cells from infection or restoring virulence in cancer cells. For example, not all cells in a tumor replicate at any given time, and therefore sufficient deoxyribonucleotides may not be available for viral replication in most cancer cells.

[0148] In addition to the foregoing, when a composition or method according to the present invention is used in conjunction with a tumor cell disruption virus therapy, the therapeutic enhancer provided by the structure of the present invention may be a factor that enhances the ability of the virus to increase the efficacy of the tumor cell disruption virus in cancer cells, for example, the replication of the virus, or the ability of the virus to lyse cells in which the virus is present. In particular, if the tumor cell disruption virus is modified to attenuate its function, for example, by deletion of one or more genes for a virulence factor, the therapeutic enhancer may replace the deleted gene with a gene product that is a copy of the viral gene product, or with mRNA for a gene product that is substantially homologous to the deleted gene or otherwise compensates for the deletion of the gene. In such an embodiment, by means of differential expression in healthy and cancerous cells made possible by the present invention, the replacement gene product may be expressed only in cancer cells to enhance viral activity and lysis in cancer cells rather than healthy cells, wherein the expression of the provided mRNA is inhibited by the presence of a miRNA binding site.

[0149] By similar means, mRNA encoding a factor that increases cell resistance to tumor cell destructive viruses is preferentially expressed in healthy cells, which can then promote viral activity in cancerous cells compared to healthy cells.

[0150] The advantage of this approach is that, unlike previous therapies using oncolytic viruses, it does not rely on which cellular antiviral genes and processes may be inactivated by carcinogenesis, nor on cell replication processes that may be activated in some cancer cells but not in others. As a result, a greater range is allowed for the deletion of virulence genes from oncolytic viruses. Thus, oncolytic viruses can be modified to completely lack replication activity in both healthy and cancer cells, and if the function of the deleted virulence genes is replaced by the means of the present invention, the virus can be restored to full efficacy. Consequently, side effects can be reduced and efficacy increased. Similarly, since the differential expression of the provided mRNA depends on the difference in miRNA expression between cancer cells and healthy cells, virulence can be restored not only in all transfected cancer cells but also, for example, in cells that replicate upon administration.

[0151] In a specific embodiment, the tumor cell disrupting virus is HSV-1, which is part of the herpesvirus family. An attenuated version of HSV can be engineered or selected to be deficient in ICP6, which encodes viral ribonucleotide reductase ([Aghi et al, Oncogene. 2008]) and / or US3, which encodes serine / threonine-protein kinase, and plays several roles in the viral lifecycle, including blocking host cell apoptosis ([Kasuya et al, Cancer Gene Therapy, 2007]).

[0152] In another embodiment, the tumor cell disruption virus is part of the poxvirus family, specifically, it may be a vaccinia virus. An attenuated version of vaccinia may be engineered or selected to be deficient in one or more subunits of ribonucleotide reductase (RR1 and RR2) and / or thymidine kinase (TK) ([Buller et al. Nature (London) 1985;317]; [Puhlamm M et al. Cancer Gene Ther 2000;7]; [Slabaugh MB et al. J Virol 1984;52]).

[0153] Examples of sequences including a specific coding sequence for a specific polypeptide associated with a tumor cell disruption virus, as well as multiple miRNA binding site sequences, are presented, for example, in SEQ ID NOs 18 to 28 and FIGS. 19b to 19d.

[0154] cytokines

[0155] Compositions and methods as described herein are considered to be capable of inducing an immune response to a disease. In particular, the immune response may be induced against cancer cells. The carcinogenic process often involves ways in which cancer cells attempt to evade the immune system, including changes to antigens produced and presented by cancer cells.

[0156] In some embodiments, the mRNA provided by the present invention comprises at least one polynucleotide encoding a bispecific T-cell agent (BiTE), an anti-immunosuppressive protein, or an immunogenic antigen. As used herein, the term "anti-immunosuppressive protein" is a protein that inhibits an immunosuppressive pathway.

[0157] The present invention comprises a composition for supplying mRNA encoding an anti-immunosuppressive protein, which is an anti-regulatory T-cell (Treg) protein or an anti-myeloid-derived suppressor cell (MDSC) protein. In some embodiments, the anti-immunosuppressive protein is a VHH-derived blocker or a VHH-derived BiTE.

[0158] As used herein, the term “immunogenic antigen” refers to a protein that increases an inflammatory or immunogenic immune response. In certain embodiments, the anti-immunosuppressive and immunogenic antigens induce an anti-tumor immune response. Examples of such proteins include antibodies or antigen-binding fragments thereof that bind to and inhibit immune checkpoint receptors (e.g., CTLA4, LAG3, PD1, PDL1, etc.), pro-inflammatory cytokines (e.g., IFNγ, IFNα, IPNβ, TNFα, IL-12, IL-2, IL-6, IL-8, GM-CSF, etc.), or proteins that bind to and activate activating receptors (e.g., FcγRI, FcγIIa, FcγIIIa, co-stimulatory receptors, etc.). In a specific embodiment, the protein is selected from EpCAM, IFNβ, anti-CTLA-4, anti-PD1, anti-PDL1, A2A, anti-FGF2, anti-FGFR / FGFR2b, anti-SEMA4D, CCL5, CD137, CD200, CD38, CD44, CSF-1R, CXCL10, CXCL13, endothelin B receptor, IL-12, IL-15, IL-2, IL-21, IL-35, ISRE7, LFA-1, NG2 (also known as SPEG4), SMAD, STING, TGFβ, and VCAM1.

[0159] The present invention comprises a composition for supplying mRNA encoding a functional macromolecule to a targeted cell population used in cell-based therapy. In some embodiments, the targeted cell population is a genetically engineered T cell population. In some embodiments, the targeted cell population is a population of chimeric antigen receptor T cells (CAR-T cells).

[0160] Coding mRNA can be used to attract a population of immune cells or a combination of immune cell populations to a specific site of a target. In some embodiments, coding mRNA and delivery particles are used to attract immune cells into the tumor microenvironment. In some embodiments, coding mRNA and delivery particles are used to overcome insufficient migration of immune cells into the tumor microenvironment. In some embodiments, immune cells are antigen-presenting cells (APCs), such as T cells, natural killer (NK) cells, B cells, macrophages, or dendritic cells, or any combination thereof. In some embodiments, coding mRNA and delivery particles are used to attract CAR-T cells into the tumor microenvironment.

[0161] Coding mRNA can be used to overcome insufficient migration of CAR-T cells into the tumor microenvironment. In some embodiments, the delivery particle specifically targets the tumor microenvironment, and the coding mRNA encodes a gene product that attracts or otherwise recruits CAR-T cells into the tumor microenvironment. In some embodiments, the coding mRNA expresses a chemokine. As a non-limiting example, the coding mRNA may encode T-cell attracting chemokines such as CCL2, CCL3, CCL4, CCL5, CCL20, CCL22, CCL28, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, XCL1, and any combination thereof. When the opposite effect is intended, as in autoimmune diseases, the coding mRNA may express blockers, antagonists, and / or inhibitors of the aforementioned factors.

[0162] Coding mRNA is delivered to the tumor microenvironment and may be transiently expressed within the tumor microenvironment. In some embodiments, the coding mRNA encodes cytokines or other gene products involved in regulating the survival, proliferation, and / or differentiation of immune cells in a tumor response, such as activated T cells and NK cells, for example. As a non-limiting example, the coding mRNA may encode cytokines such as IL-1, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-17, IL-33, IL-35, TGF-beta, and any combination thereof. Again, when intended for the opposite effect, as in autoimmune diseases, the coding mRNA may express blockers, antagonists, and / or inhibitors of the aforementioned factors.

[0163] Compositions that deliver mRNA can target specific cell subtypes and, upon binding to them, stimulate receptor-mediated endocytosis, thereby introducing the synthetic mRNA they carry into the cell and enabling the expression of the synthetic mRNA. Since nuclear transport and transcription of transgenes are not required, this process is rapid and efficient.

[0164] In some embodiments, an mRNA delivery system delivers mRNA encoding a gene-editing agent to a target cell population. In some embodiments, the mRNA codes for a sequence-specific nuclease that targets a gene locus and interferes with the expression of one or more endogenous gene products in the target cell population. In some embodiments, the mRNA interferes with the expression of one or more domains in the TCR by encoding a sequence-specific nuclease that targets a T cell receptor (TCR)-associated gene locus.

[0165] In some embodiments, the mRNA delivery system may be used to deliver mRNA encoding one or more agents that program engineered T cells into a desired phenotype. In some embodiments, the mRNA nanoparticle delivery composition may be used to induce transcription patterns and markers characteristic of the desired T cell phenotype. In some embodiments, the mRNA nanoparticle delivery composition may be used to promote the development of CD26L+ central memory T cells (Tcm), which have been shown to improve CAR-T therapy (e.g., see Moffett, Coon above). In some embodiments, the composition delivers mRNA encoding one or more transcription factors that control cell differentiation in a target cell population. In some embodiments, the transcription factor is Foxo1, which controls developmental effector versus memory transfer in CD8 T-cells.

[0166] In some embodiments, the mRNA delivery composition comprises a surface-immobilized targeting domain specific to T cell markers, such as surface antigens found on T cells. In some embodiments, the surface-immobilized targeting domain is specific to an antigen that selectively binds the nanoparticle to a T-cell and initiates receptor-induced endocytosis to internalize the mRNA nanoparticle delivery composition. In some embodiments, the surface-immobilized targeting domain selectively binds CD3, CD8, or a combination thereof. In some embodiments, the surface-immobilized targeting domain is an antibody that selectively binds CD3, CD8, or a combination thereof, or is derived therefrom.

[0167] By means of the present invention, differential expression of the aforementioned gene product can be achieved in different cell types, e.g., healthy, disease-free, diseased, and cancer cells. By this method, a targeted immune response against diseased cells can be triggered while preserving disease-free or healthy cells.

[0168] The introduction of coding nucleotide sequences into target cells often requires the use of a delivery agent to transport the target substance from the extracellular space to the intracellular environment. Typically, these delivery agents are in the form of delivery particles capable of undergoing phagocytosis or fusing with the target cell. Delivery particles can contain the target substance by encapsulating or including the substance within a matrix or structure.

[0169] Delivery particles can target cells in target tissues. This targeting may be mediated by a targeting agent on the surface of the delivery particle, which may be a protein, peptide, carbohydrate, glycoprotein, lipid, small molecule, nucleic acid, etc. The targeting agent may be used to target specific cells or tissues, or to facilitate the endocytosis or phagocytosis of the particle. Examples of "targeting agents" include, but are not limited to, antibodies, antibody fragments, low-density lipoprotein (LDL), transferrin, asialycorpotein, gp120 envelope protein of human immunodeficiency virus (HIV), carbohydrates, receptor ligands, sialic acid, aptamers, etc.

[0170] The delivery particles may include amino alcohol lipoids. These compounds may be used to form particles, including nanoparticles, liposomes, and micelles, which are particularly suitable for the delivery of nucleic acids. Exemplary examples of the preparation of nanoformulations containing particles according to some embodiments of the present invention can be found in the examples.

[0171] When administered to a subject, the therapeutic component is administered suitably as part of a composition comprising a pharmaceutically acceptable vehicle. The acceptable pharmaceutical vehicle may be water and liquids such as oils of petroleum, animal, plant, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil, etc. The pharmaceutical vehicle may be saline solution, acacia gum, gelatin, starch paste, talc, keratin, colloidal silica, urea, etc. Additionally, adjuvants, stabilizers, thickeners, lubricants, and coloring agents may be used. When administered to a subject, it is preferable that the pharmaceutically acceptable vehicle be sterilized. Water is a suitable vehicle when the compound of the present invention is administered intravenously. Saline solutions, and aqueous solutions of dextrose and glycerol may also be used as liquid vehicles, particularly for injectable solutions. Suitable pharmaceutical vehicles also include excipients such as starch, glucose, lactose, sucrose, gelatin, malt, rice, wheat flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene glycol, water, ethanol, etc. If desired, the pharmaceutical composition may also contain trace amounts of a wetting agent or emulsifier or buffer.

[0172] The pharmaceuticals and pharmaceutical compositions of the present invention may take the form of liquids, solutions, suspensions, gels, modified release formulations (e.g., sustained-release or sustained-release), emulsions, capsules (e.g., capsules containing liquid or gel), liposomes, microparticles, nanoparticles, or any other suitable formulation known in the art. Other examples of suitable pharmaceutical vehicles are described in [Remington's Pharmaceutical Sciences, Alfonso R. Gennaro ed., Mack Publishing Co. Easton, Pa., 19th ed., 1995 (see, for example, pages 1447 to 1676)].

[0173] For any compound or composition described herein, the therapeutically effective dose is initially in vitro It can be determined from a cell culture assay. The target concentration will be the concentration of the active ingredient(s) that can achieve the method described herein when measured using a method described herein or known in the art.

[0174] As is widely known in the art, the therapeutically effective dose for use in humans can also be determined from animal models. For example, the dose for humans may be formulated to achieve a concentration found to be effective in animals. The dosage in humans may be adjusted by monitoring the effect of the compound as described above and by up- or down-adjusting the dosage. Adjusting the dosage to achieve maximum efficacy in humans based on the methods described above and other methods is within the capabilities of those skilled in the art.

[0175] Embodiments of the present invention are considered to include compositions formulated for use in medicine. Thus, compositions of the present invention may be suspended in a biocompatible solution to form compositions that can be targeted to locations within tissues or on cells within the body of a patient or animal (i.e., said compositions may be used in vitro, in vitro, or in vivo). Suitably, the biocompatible solution may be phosphate-buffered saline or any other pharmaceutically acceptable carrier solution. One or more additional pharmaceutically acceptable carriers (e.g., diluents, adjuvants, excipients, or vehicles) may be combined with the compositions of the present invention in the pharmaceutical composition. Suitable pharmaceutical carriers are described in [EW Martin, 'Remington's Pharmaceutical Sciences']. Pharmaceutical formulations and compositions of the present invention may be formulated to comply with control standards and may be administered orally, intravenously, topically, into a tumor, subcutaneously, or via other standard routes. Administration may be systemic, local, intranasal, or intraspinal.

[0176] Furthermore, the compositions of some embodiments of the present invention are intended to be administered separately or in combination with alternative antitumor or other anticancer therapeutic components. Such components may include tumor cell-disrupting viruses, small molecule drugs, chemotherapy agents, radiotherapy agents, or biological agents. Such components may be administered simultaneously with the composition of the present invention, may be contained within delivery particles, or may be administered separately before or after the administration of the composition of the present invention by any suitable means.

[0177] Additionally, compositions of some embodiments of the present invention are considered to be usable in in vitro and / or in vitro methods, for example in a laboratory setting. An example of an in vivo method is a method of administering a composition comprising a delivery system comprising an mRNA sequence as described herein to target in vitro cells, such that the miRNA binding site sequence included in the mRNA sequence enables differential expression of the coding sequence of the mRNA in different cell types within the target in vitro cells. Similarly, a method is considered of administering a composition comprising a delivery system and an mRNA sequence as described herein to target in vitro samples taken from animals, such that the miRNA binding site sequence included in the mRNA sequence enables differential expression of the coding sequence of the mRNA in different cell types within the target samples.

[0178] The device of the present invention is illustrated by the following examples, but is not limited thereto.

[0179] Examples

[0180] General protocol

[0181] cell line

[0182] Human Hepatocarcinoma (HCC) HepG2 (ATCC) ® HB-8065 ™ ) and Hep3B(ATCC ® HB-8064 ™ Cells were purchased from ATCC. Cells were fed Eagle's Minimum Essential Medium (EMEM) (Cellgro, USA), 10% FBS (HyClone, USA), streptomycin (100 μg / mL), and penicillin (100 U / mL). -1 HepG2 cells were cultured as a monolayer in Cellgro at 37°C under a 5% CO2 atmosphere. 5 μg / cm² 2 It was grown on collagen-coated plates (Gibco, USA) at a collagen concentration.

[0183] HMCPP5 (pooled plating-ready human hepatocytes; a mixture of plating-ready primary hepatocytes generated by combining cells from five individual donors) was purchased from ThermoFisher Scientific (USA). Cells were plated in Williams E Medium (WEM) supplemented with 5% FBS, 1 μM dexamethasone, and Cocktail A (penicillin / streptomycin, human recombinant insulin, GlutaMax, and HEPES, pH 7.4). After 24 hours of plating, the WEM / Cocktail A medium was changed to maintenance / incubation medium WEM supplemented with 0.1 μM dexamethasone and Cocktail B (penicillin / streptomycin, ITS (human recombinant insulin, human transferrin, selenic acid, BSA, linoleic acid), GlutaMax, and HEPES, pH 7.4), and cultured as a monolayer at 37°C under a 5% CO2 atmosphere. Throughout all experiments, except during transfection with nanoformulated mRNA, cells were cultured in WEM / Cocktail B medium. The WEM / Cocktail B medium was replaced with fresh medium every 3 days. HMCPP5 cells were cultured at 5 μg / cm² 2 It was grown on collagen (Gibco) coated plates at a protein concentration.

[0184] Aml12 (healthy mouse hepatocytes) were purchased from ATCC (USA). Cells were 1 x 10⁶ 5 Seeded into a 12-well plate at a density of / well.

[0185] Vector structure

[0186] Construction of pMRNA-CTx-mRNA template

[0187] Plasmid pMRNA-CTx-mRNA template formation matrices for the in vitro synthesis of all mRNAs used in the experiment were constructed according to the commercially available mRNAExpress™ mRNA synthesis kit (SBI, USA). All plasmids were amplified in E. coli (Invitrogen, USA) and purified using the Qiagen Mini or Maxi Kit (Qiagen, USA). Restriction maps for all plasmids were generated using pDRAW32 software (www.acaclone.com).

[0188] Cloning Method 1 - Restriction Endonuclease

[0189] As shown in FIG. 2, sequences of one or more genes flanked by a Kojak sequence for optimal translation immediately prior to the 5'-terminal ATG codon or the 3'-terminal stop codon (TAA, TAG, TGA) of the gene were synthesized by GeneArt (without codon optimization) and delivered as plasmid DNA (referred to as DNA plasmid or vector), as shown in FIG. 2 as pMA-T-CTx-Gene. Exclusive 5' and 3' UTR regions flanked by the coding sequence were included in all synthesized sequences (not presented in the attached sequence). The 5' UTR is synthetic and contains a Kojak sequence, and the 3' UTR is based on a mouse alpha-globin UTR and also contains a 120-base polyA tail. To generate the synthetic vector shown in Fig. 2 as pMRNA-CTx-mRNA, a gene or a nucleotide fragment containing genes was cut from a DNA plasmid using restriction endonucleases (here, EcoRI and NheI) and subcloned into the EcoRI / NheI restriction site of a pMRNA template plasmid, wherein the template plasmid contained a T7 RNA polymerase, 5'- and 3'-UTRs, and a T7 promoter recognized by a polyA sequence.

[0190] Cloning Method 2 - Low-temperature Fusion

[0191] As in Cloning Method 1, the sequences of one or more genes flanked by Kojak sequences and stop codons (TAA, TAG, TGA) were synthesized by GeneArt (without codon optimization) and delivered as plasmid DNA pMAT-CTx-Gene having the backbone of these plasmids as described above. To construct the pMRNA-CTx-mRNA template vector, a cryo-fusion cloning kit (SBI, USA) was used. Briefly, the gene sequences from the DNA plasmid were amplified by PCR using specific primers, wherein the primers added a 14-base homology extension to each end of the gene sequence. These 14 bases were designed to be homologous to the end of the linearized vector produced by the digestion of the template plasmid using restriction endonuclease cleavage at the multiple cloning site located between the 5' and 3' UTRs. To generate the synthetic vector, the predicted PCR product was purified using a PCR purification kit (Qiagen, USA) and incorporated into a pMRNA template plasmid after a low-temperature fusion reaction (homologous recombination) according to the manufacturer's protocol.

[0192] Construction of a template containing miRNA binding site sequences

[0193] An exemplary method for generating three variants for generating mRNA sequences containing miRNA binding site sequences (an example using miR-122 is shown in FIG. 3) is discussed below. In variant 1, two copies of the miRNA binding site sequence are contained between the stop codon and the +1 position of the 3' UTR. In variant 2, two copies of the miRNA binding site sequence are contained at the beginning or the 5' end of the 3' UTR, and in variant 3, two copies of the miRNA binding site sequence are contained at the end or the 3' end of the 3' UTR.

[0194] Figure 4 shows an example of a synthetic vector containing these three variants, using a gene of approximately 1,400 base pairs as example protein B.

[0195] Variant 1

[0196] As illustrated in FIG. 5, sequences of one or more genes having a Kojak sequence and stop codons (TAA, TAG, TGA) as in the method above, and additionally containing two copies of a miRNA binding site sequence after the stop codon, were synthesized by GeneArt (without codon optimization), and pMAT-CTx-Gene having the backbone of such plasmid as described above was delivered as plasmid DNA (here again shown as protein A). Subsequently, these sequences were cloned into a template plasmid to generate a synthetic vector by any of the methods described above.

[0197] Variants 2 and 3

[0198] As in the method above, sequences of one or more genes containing 3' UTRs with two copies of a miRNA binding site sequence at the start / 5' end (variant 2, as shown in FIG. 6) or the end / 3' end (variant 3, as shown in FIG. 7) of these regions were synthesized by GeneArt (without codon optimization), and pMAT-CTx-Gene having the backbone of these plasmids as described above was delivered as plasmid DNA. Subsequently, these sequences were cloned into a template plasmid to generate a synthetic vector by any of the methods described above, wherein restriction enzymes (here, EcoRI and NotI) were selected and modified to remove the 3' UTR from the template vector so that the 3' UTR from the supplied DNA sequence could be present in the final synthetic vector (since it contains the miRNA binding site sequence).

[0199] In vitro transcription of mRNA using in vitro mRNA synthesis (IVT)

[0200] To perform IVT of mRNA with or without the miRNA-modified 3' UTR, the commercially available mRNAExpress™ mRNA synthesis kit was used. A DNA template for the IVT vector was constructed as described in the protocol presented above. The in vitro mRNA synthesis procedure was performed according to the manufacturer's protocol. Briefly, a polyA tail was added to the DNA sequence using a PCR reaction with specific 5' and 3' primers (supplied with the kit). During in vitro transcription, the mRNA synthesized on the DNA template was capped with an anti-reverse cap analog (ARCA)-modified nucleotide (5-methylcytidine-5'-triphosphate). The incorporation of the cap analog, pseudouridine-5'-triphosphate, and the polyA tail into the in vitro transcribed mRNA enhanced stability and reduced the host cell immune response.

[0201] DMP CTx and synthesis of mRNA formulations

[0202] Delivery and Regulation Platform for Combined Therapeutics (DMP) CTx The formulation is a multicomponent nanoparticle of a mixture of ionizable lipid analog C12-200, phospholipid DOPE, cholesterol, and lipid-immobilized polyethylene glycol C14-PEG-DSPE2000. This DMP CTx The specific composition of, and the specific weight ratio of C12-200:mRNA (10:1), and the molar [%] composition of lipid-like substances, phospholipids, cholesterol, and PEG are ( Table 4It was optimized, and high efficiency of the formulation in vivo was confirmed ([Kauffman KJ, Nano Letter. 2015, 15, 7300-7306]). The chemical structure of this exemplary component is shown in Fig. 8.

[0203] DMP CTx To synthesize, an ethanolic solution (Mix A) of C12-200 (WuXi, China) as shown in Fig. 9a, and phospholipid DOPE (1,2-dioleyl- sn A buffered aqueous solution of mRNA (Mix B) (in 10 mM citrate, pH 4.5) and C14-PEG-DSPE2000 (Avanti Polar Lipids, Alabaster, AL, USA), cholesterol (Sigma, USA), and C14-PEG-DSPE2000 (Avanti Polar Lipids, Alabaster, AL, USA) was prepared. Ethanolic Mix A and aqueous Mix B were mixed / combined in a 3:1 ratio using a syringe pump and a microfluidic chip device ([Chen D, at al J. Am. Chem. Soc. 2012, 134 (16), 6948-6951]). The alcoholic solution of the nano-formulated mRNA from the microfluidic chip was collected in a 1.5 mL tube.

[0204]

[0205] To remove alcohol after formulation, DMP CTx The mRNA mixture was transferred to a Slide-A-Lyzer® dialysis cassette G2 and dialyzed in PBS on a magnetic stirrer at room temperature for 4 hours. Subsequently, the formulation mRNA was transferred to a new 1.5 mL tube using an 18-gauge syringe with a 1-inch inclined needle and prepared for characterization.

[0206] To calculate the efficacy of mRNA encapsulation, the RiboGreen RNA assay (Invitrogen) was used according to the manufacturer's protocol. The polydispersity density (PDI) and size of lipid nanoparticles were measured using dynamic light scattering (ZetaPALS, Brookhaven, Instruments). DMP CTx The surface charge (zeta potential) was measured using the same instrument. Solutions of mRNA sequences linked by a linker (SEQ No. 30) and containing or not containing two copies of the miR-122 sequence inserted after the stop codon of the coding mRNA sequence were prepared from 1.05 and 1 mg / ml stocks, respectively. An example of parameters after encapsulation of an mRNA sequence containing the mCherry (mCh) sequence, which is the sequence of Protein A, a human protein of approximately 25 kDa, is the Delivery and Regulation Platform of Combined Therapeutics (DMP). CTx The size, encapsulation efficiency, and polydispersity of DMP are presented in Table 5. CTx An exemplary diagram of the transport particle according to this can be seen in Fig. 9b.

[0207]

[0208] In vitro differential expression of the delivered mRNA construct

[0209] To investigate the potential of the present invention to successfully transfect target cells with construct mRNA and subsequently induce differential expression in different cell types, a DMP modified with a miRNA-122 binding site CTx The mRNA platform was used in a model of liver carcinoma.

[0210] Cell line transfection

[0211] Fluorescence imaging and quantification

[0212] Single transfection of human hepatocarcinoma cell lines HepG2 and Hep3B was performed as follows: One day prior to transfection, HepG2 and Hep3B cells were, respectively, 2.7 x 10⁶ 5 / well and 2x10 5 Cells were seeded separately into 12-well plates at a density of / well (EMEM / 10%FCS). The following day, cells were treated with a vehicle control of PBS alone and 0.5 μg / well mRNA-mCherry-DMP. CTx or 0.5 μg / well of mRNA-mCherry-122-DMP CTx Transfection was performed with (sequence containing SEQ ID NO. 31). Transfection was performed by adding mRNA-DMP to the culture medium of the wells, along with a moderate mixture of cultured cells if necessary. CTx It was performed by directly adding.

[0213] Single transfection of HMCPP5 (pooled plating-ready human hepatocytes) was performed as follows: 1 day prior to transfection, 2.5 x 10⁶ HMCPP5 cells 5 Cells were seeded into 12-well plates at a density of / well (WEM / Cocktail B). The next day, cells were DMP CTx Vehicle control of (PBS), 0.5 μg / well mRNA-mCherry-DMP CTx or 0.5 μg / well of mRNA-mCherry-122-DMP CTx Transfection was performed using mRNA-DMP in the culture medium of the wells, along with a mild mixture of cultured cells if necessary. CTx It was performed by directly adding [the substance]. During the transfection of HMCPP5, 5% FBS was supplemented to the WEM / Cocktail B medium. Transfection was performed on the liver cancer cells in the manner described above. After 24 hours of transfection, the medium was changed back to WEM / Cocktail B.

[0214] To evaluate the constitutive activity and expression of miRNA-122 in healthy human hepatocytes, multiple transfection of HMCPP5 cells was performed as follows: HMCPP5 cells were seeded and cultured as above, and mRNA-mCherry-DMP CTx or mRNA-mCherry-122-DMP CTx Thus, a total of three transfections (MPT) were performed with a 48-hour interval between each transfection. The transfections were performed in the same manner as described above for a single transfection of HMCPP5.

[0215] Single transfection of healthy mouse hepatocytes (Aml12, ATCC, USA) was performed as follows: One day prior to transfection, 1 x 10⁶ Aml12 cells were formed. 5 The cells were seeded into 12-well plates at a density of / well. The next day, the cells were DMP CTx Vehicle control of (PBS), 0.5 μg / well mRNA-mCherry-DMP CTx or 0.5 μg / well of mRNA-mCherry-122-DMP CTx Transfection was performed using mRNA-DMP in the culture medium of the wells, along with a mild mixture of cultured cells if necessary. CTx It was performed by directly adding.

[0216] After transfection, mCherry expression in the cell line was detected using a fluorescence imaging system (EVOS® FL Imaging Systems). Photographs showing mCherry fluorescence were taken 16, 24, 48, 72, 96, and 144 hours after transfection.

[0217] Quantification of mCherry fluorescence signals was performed using ImageJ software (NIH, USA) from three randomized fields on culture plates (mRNA-mCherry, mRNA-mCherry-122). The results of this quantification are shown in Figures 10b, 11, and 12b. The pixel count of mCherry transfected wells was set to 100% (mCherry fluorescence). Statistical significance was Student t -Test(Student t It was determined using the p-test. Results are presented as mean ± SD. Significant differences were defined as p-values ​​less than 0.05. Asterisks indicate statistically significant differences in mCherry fluorescence in cells transfected with mRNA-mCherry compared to cells transfected with mRNA-mCherry-122 (****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05).

[0218] Example 1: Tumor-specific gene expression by miRNA-122 regulation

[0219] miRNA-122 is an abundant liver-specific miRNA, and its expression is significantly reduced in human primary hepatocellular carcinoma (HCC) and HCC-derived cell lines such as Hep3B and HepG2. The purpose of this study was to demonstrate that modification of the 3'-untranslated region (UTR) of an mRNA sequence by insertion of a miRNA-122 targeting sequence (e.g., as shown in SEQ ID NO. 30, upper part of Fig. 3, variant 1) can induce translation repression and / or deadenylation, followed by decapping of exogenous mRNA in normal hepatocytes other than the tested HCC cell lines.

[0220] To investigate endogenous miRNA-122 activity in healthy hepatocytes, HMCPP5 cells (pooled plating-ready human hepatocytes, a mixture of plating-ready primary hepatocytes generated by combining cells from five individual donors) were transfected with mRNA-mCherry or mRNA-mCherry-122 prepared according to the general protocol using mCherry (red fluorescent protein) as the gene of interest, and then mCherry (red fluorescent protein) was expressed over time. As shown in Fig. 10a, mCherry (mCh) expression was analyzed by fluorescence microscopy 48 hours after transfection. Throughout the entire time after transfection, mCherry expression was observed in HMCPP5 cells transfected with mRNA-mCherry (i.e., without 3'UTR modifications to introduce the miR-122 sequence), which indicates successful transfection and translation. In contrast, in healthy hepatocytes known to be miRNA-122 positive, the expression of mRNA-mCherry-122 was downregulated to a practically undetectable level even 3 days after transfection, compared to what is seen in untransfected control cells. This suggests that the presence of the miRNA-122 targeting sequence in mRNA-mCherry-122 (variant 1) inserted into the 3' UTR blocks the translation of the mRNA, which appears to be due to the repression of translation in the receptor cells.

[0221] Quantification of the fluorescence signal produced by these cells confirmed the above. As shown in Fig. 10b, the fluorescence intensity was dramatically reduced in healthy cells transfected with mRNA-mCherry-122 compared to cells transfected with mRNA-mCherry.

[0222] The results obtained from the above experiment demonstrated that the intrinsic expression of miRNA-122 and co-localization with the miRNA-122-targeted sequence (Variant 1) effectively regulate protein expression in healthy hepatocytes, thereby significantly increasing tumor-specific gene expression. In the following experiment, the constitutive expression and activity of miRNA-122 in HMCPP5 cells were evaluated. HMCPP5 cells were transfected a total of three times with mRNA-mCherry or mRNA-mCherry-122 at 48-hour intervals. Six days after the first transfection (i.e., 48 hours after the last transfection), mCherry expression was determined by fluorescence microscopy. As described above, cells transfected with the mRNA-mCherry construct exhibited clear red fluorescence, whereas cells transfected with the mRNA-mCherry-122 construct did not. In Figure 11, a comparison between cells transfected with mRNA-mCherry-122 and cells transfected with mRNA-mCherry is presented for both single (ST) and multiple-transfected (MPT) cells over a period of 5 days after final transfection. Multiple-transfected cells can be seen to exhibit the same rapid decline in fluorescence intensity when transfected with mRNA-mCherry-122 as single-transfected cells, but with the effect lasting longer after multiple transfection. As expected, this indicates that the differential expression effect induced by the miRNA control mechanism is robust for repeated transfection events, and that the amount of miRNA-122 available within the cell to induce this mechanism is not exhausted within this timeframe.

[0223] To investigate the activating effects of endogenous miRNA-122 using human hepatocellular carcinoma Hep3B and hepatoblastoma HepG2 cell lines, experiments similar to the above were performed. Cells were transfected with the mRNA sequences mRNA-mCherry, mRNA-mCherry-122 (variant 1), or the control group. As described above, after 48 hours, Fig. 10a As illustrated in Fig. 10, mCherry expression in transfected Hep3B and HepG2 cells was determined using fluorescence microscopy. In Hep3B cells (Fig. 10a, middle column), mCherry fluorescence was evident in both mRNA-mCherry and mRNA-mCherry-122 transfected cell lines, indicating that miRNA-122-mediated translation repression is inactive in these cells. In HepG2 cells, mCherry fluorescence was evident in mRNA-mCherry transfected cell lines, but only partial fluorescence was evident in mRNA-mCherry-122 transfected cells, which was only partially reduced and appeared significantly greater than that seen in normal hepatocytes. Further evidence suggests that the quantification of mCherry fluorescence in mRNA-mCherry-122 transfected cell lines indicates no reduction in fluorescence in Hep3B cells, but a reduction of approximately 50% in HepG2 cells. Fig. 10b It is depicted in.

[0224] The partial downregulation observed in HepG2 cells further implies a miRNA-122-mediated effect on translation, as cells from these cell lines were shown to retain residual miRNA-122 activity ([Demonstration of the Presence of the "Deleted" MIR122 Gene in HepG2 Cells, PLoS One. 2015; 10(3)]).

[0225] miRNA-122 is strongly conserved among vertebrate species, and as in humans, reduced levels of miRNA-122 are associated with hepatocellular carcinoma in mice (Kutay et al, 2006). Therefore, the endogenous effects of miRNA-122 activity were investigated using the healthy mouse hepatocellular cell line Aml12.

[0226] Healthy mouse liver cells also use the mRNA-Cherry sequence described above, namely DMP CTx Transfected with mRNA-mCherry or mRNA-mCherry-122 encapsulated in the molecule. A similar effect of miRNA-122 binding site sequence insertion on mCherry fluorescence was observed 24 and 72 hours after transfection.

[0227] As shown in Fig. 12a, fluorescence was observed after transfection to mRNA-mCherry. Although a significant decrease in fluorescence was observed after transfection to mRNA-mCherry-122, some signal was still visible.

[0228] Quantification of mCherry fluorescence 24 and 72 hours after transfection was performed on three randomized fields on culture plates from each treatment group (Fig. 12b), which showed that more than 70% translation inhibition was observed when transfected with mRNA-mCherry-122.

[0229] As a preliminary conclusion, the above examples demonstrate that the dual-targeting features of the nanoparticle delivery system and the inclusion of the miRNA-122 target sequence within the mRNA construct are sufficient to obtain significantly significant differential expression of protein products in hepatocellular carcinoma and hepatoblastoma cells compared to healthy hepatocytes. The observation of differential expression was evident in both human and mouse cell lines.

[0230] Example 2: Protein expression levels after tumor-specific gene expression

[0231] In another experiment, Western blotting was used to determine the ultimate protein expression levels after transfection as follows.

[0232] Cell line transfection and immunoblot - Protein A

[0233] To evaluate the tumor-specific expression levels of an exemplary 25 kDa human protein (denoted as 'Protein A'), both liver cancer cells (HepG2 and Hep3B) and healthy liver cells (HMCPP5) were seeded in 12-well plates, and 0.5 μg / well of nanoformulated mRNA expressing human Protein A, 25 kDa (mRNA-A-DMP), as described above in Example 1 for mCherry transfection, was added. CTx ), or variant 1 (mRNA-A-miRNA122-DMP), which is an mRNA expressing human protein A (a human protein of approximately 25 kDa) containing two miRNA122 binding sequences at the 3' UTR (SEQ ID NO. 30). CTx Transfected with ). 24 hours after transfection, total protein was extracted and an immunoblot was performed.

[0234] For immunoblotting, the culture medium was removed, cells were washed with cooled PBS (Cellgro), and the cell pellet was dissolved in RIPA (radioimmunoprecipitation assay) buffer (Boston Bioproducts) containing a cocktail of protease inhibitors (Sigma). Protein concentration was determined by the colorimetric Bradford assay. A total of 10 mg of protein was separated onto a Novex™ 4-12% mini-gel (ThermoFisher Scientific) and transferred to a PVDF (polyvinylidene difluoride) membrane by electroblotting (iBlot® 2 Gel Transfer Device, Invitrogen). After blocking the membrane with 5% non-fat milk powder in TBS-Tween 20 (Boston Bioproducts), the membrane was incubated overnight at 4°C with anti-protein A antibody (1:2000, Abcam) or β-actin (cell signaling), followed by incubation for 1 hour at room temperature with an appropriate HRP (horseradish peroxidase)-conjugated goat anti-rabbit secondary antibody (1:10000; Abcam). The protein-antibody complexes were visualized and imaged using Clarity™ Western ECL substrate (Bio Rad) and the LI-COR® system (LI-COR), respectively.

[0235] The above results can be confirmed in Fig. 13, and DMP after transfection CTx A structure encapsulated in is presented:

[0236] Lanes 1, 4, and 7 of Fig. 13: Vehicle (mock group processing, PBS alone),

[0237] Lanes 2, 5, and 8 of Fig. 13: mRNA-A (mRNA containing a sequence for protein A), and

[0238] Lanes 3, 6, and 9 of Fig. 13: mRNA-A-122 construct (containing sequences for protein A and miRNA122 inserted at variant 1 position as shown in Fig. 3).

[0239] As described above, in cells from healthy hepatocytes (HMCPP5) in lanes 1 to 3, the hepatocellular carcinoma model Hep3B in lanes 7 to 9, and the hepatoblastoma model HepG2 in lanes 4 to 6, 0.5 μg mRNA-DMP per well CTx Transfection was performed using [the appropriate method]. Proteins were extracted from each cell line 24 hours after transfection. 10 μg of protein was loaded into each lane, and data were taken from two independent experiments. Protein A was detected in all tested cell lines when transfected with mRNA-A, indicating that successful transfection was achieved. When transfected with mRNA-A-122, translational repression was observed only in healthy liver cells (lanes 3, 6, and 9), excluding Hep3B and HepG2 cells, indicating that miRNA-122 does not perform its function in the tested liver cancer cells. However, in HepG2 cells transfected with mRNA-A-122, the expression of Protein A was slightly downregulated compared to cells transfected with mRNA-A, similar to the pattern previously identified for mCherry expression in Example 1. This is clearly visible in the enhanced exposure photograph (bottom) showing incomplete downregulation in HepG2 cells. The partial downregulation observed in HepG2 cells further implies a miRNA-122-mediated effect on translation, as cells from these cell lines were shown to retain residual miRNA-122 activity ([Demonstration of the Presence of the "Deleted" MIR122 Gene in HepG2 Cells, PLoS One. 2015; 10(3)]).

[0240] In summary, modification of 3'UTR mRNA by insertion of liver-specific miRNA-122 target sequences can significantly restrict mRNA translation for hepatocellular carcinoma Hep3B and hepatoblastoma HepG2, rather than normal human hepatocytes.

[0241] Example 3: In vitro combination therapy with tumor cell disintegrating viruses

[0242] It is described herein that differential expression of the provided mRNA construct, permitted by the method of the present invention and presented in the above examples, can be used in combination with oncolytic virus therapy. In particular, when an oncolytic virus is modified to attenuate its replication ability in healthy cells by removing a virulence gene, the present invention can be used to restore the function of a gene or its equivalent in diseased cells, such as cancer cells. To investigate this possibility, a US3-deficient oncolytic virus HSV-1 (R7041) (see [Leopardi et al, 1997, PNAS 94; 7891-7896]) and a DMP providing an mRNA construct encoded by US3 and modified with a miRNA-122 binding site. CTx A combination of platforms was used in a model of liver carcinoma (sequence number 32).

[0243] General Protocol:

[0244] cell culture

[0245] Human hepatocarcinoma (HCC) HepG2 and Hep3B cells were cultured as a monolayer at 37°C under a 5% CO2 atmosphere in Eagle Minimal Essential Medium (EMEM, Cellgro, USA), 10% FBS, streptomycin (100 μg / mL), and penicillin (100 U / mL-1). HepG2 cells were cultured at 5 μg / cm² 2 It was grown on a collagen-coated plate at a collagen concentration.

[0246] Virus manufacturing

[0247] The frozen R7041 virus was thawed in a water bath at 37°C, sonicated using an ultrasonic bath (Q500 ultrasonic disperser, Qsonica, USA) for 30 seconds, and then transferred to ice for use.

[0248] Toxicity of R7041 alone to human HCC

[0249] The US3 mutant R7041 virus is considered to be virtually apathogenic to healthy cells (Leopardi et al. 1997) and has demonstrated good safety even in immunodeficient athymic mice ([Liu et al. 2007, Clin Cancer Res 2007;13(19)]). To establish a baseline for the efficacy of the R7041 virus against hepatocellular carcinoma cells, model cell lines were treated with the oncolytic virus alone. Cells from Hep3B and HepG2 cell lines were seeded in triples in 96-well plates at 15,000 and 17,000 cells per well, respectively.

[0250] After 24 hours, cells were infected with 3-fold serial dilutions of the virus with an MOI of 0.37 to 0.0001694. 96 hours after infection, the viability of the tested cell lines was measured by the MTS assay according to the supplier's instructions (CellTiter 96® AQueous One Solution Cell Proliferation Assay, Promega, USA). Absorbance was measured at 490 nm using a 96-well plate reader (BioTek, Cytation 3, USA). Dose-response curves and 50% effective dose values ​​(ED) were obtained using a GraphPad Prism 7.03. 50 ) obtained.

[0251] As shown in Fig. 14, both Hep3B and HepG2 cell lines, respectively, ED 50With MOIs of 0.01 and 0.02, similar sensitivity to R7041 was observed. However, the Hep3B cell line was found to be slightly more sensitive to R7041 than HepG2 cells.

[0252] R7041 and mRNA-DMP on human HCC viability CTx Combination effect of

[0253] R7041 virus and mRNA-US3-DMP against human hepatocarcinoma cells CTx Before evaluating the combined effect, the inventors [discussed] mRNA-US3-DMP at 0.04 μg / mL mRNA-US3 in Hep3B and HepG2 cells when measured by the MTS assay. CTx It was confirmed that transfection of the rheumatology did not have a significant effect on cell viability.

[0254] Hep3B and HepG2 cells were seeded in triples in 96-well plates at 15,000 and 17,000 cells per well, respectively. After 24 hours, cells were infected with 3-fold serial dilutions of the virus with an MOI of 0.37 to 0.0001694. All tested cell lines were administered a fixed dose of 0.04 μg / mL mRNA-US3-DMP 24 and 48 hours after infection with R7041, according to the experimental timeline shown in Fig. 15. CTx Transfection was performed twice. Three days after transfection, the viability of the tested cell lines was measured by the MTS assay as described above. In both human HCC tested, two different compounds were used: the tumor cell-disrupting R7041 and a non-toxic dose of mRNA-US3-DMP. CTx The combination of (0.04 μg / mL) significantly enhanced tumor destruction at low viral titers, as shown in Figure 16. In this figure, the effect on viability is mRNA-US3-DMP at 0.04 μg / mL CTx It is illustrated for R7041 alone (y-axis intersection), alone (gray triangle / diamond), and in combination (black circle) at various dilutions.

[0255] The above examples indicate that a combination of an attenuated tumor cell disintegrating virus with a deleted virulence gene and a substitute that differentially expresses the deleted gene can significantly increase the efficacy of in vitro tumor cell disintegrating virus therapy. In particular, a greater effect was observed at low viral titers when combined with the composition of the present invention.

[0256] Example 4: In vivo expression of the delivered fluorescent protein mCherry mRNA construct

[0257] To determine the applicability of the present invention to an in vivo approach, a mouse model of human hepatocellular carcinoma was used. Differential expression induced by the miRNA-122 binding site is applicable to healthy mouse Aml12 liver cells in vitro, as presented above (see Example 1).

[0258] orthotopic human hepatocellular carcinoma (HCC) model

[0259] animal

[0260] Female (CB17 / Ics-PrkdcSCID / IcrIcoCrl) Fox Chase SCID mice aged 6 to 8 weeks were purchased from Charles River (UK). All in vivo procedures were approved by the Subcommittee on Research Animal Care of CrownBio, UK.

[0261] cell

[0262] To generate an HCC model of the same, a bioluminescent variant (Hep3B-cLuX) of the human Hep3B cell line expressing firefly luciferase was used. Cells were cultured in EMEM medium (Sigma, UK) supplemented with 10% heat-inactivated FBS, 2 mM L-glutamine, and 1% NEAA; cells were treated weekly with 2 μg / mL puromycin (Sigma).

[0263] Intrahepatic injection and tumor growth monitoring

[0264] Under anesthesia, 20 μL of 1:1 PBS:Matrigel TM Human Hep3B-cLuX cells (2x10⁻¹⁰) suspended in 6 A 29G needle was used to inject the liver into the upper left lobe. The injection site was covered with an absorbable gelatin sponge (AGS), the liver was repositioned into the abdominal cavity without damaging the AGS, and the skin was sutured. Tumor growth was monitored twice a week using bioluminescence imaging (BLI).

[0265] Briefly, mice were anesthetized, and 150 mg / kg D-luciferin was injected subcutaneously 15 minutes prior to imaging. BLI images were captured and processed using Living Image 4.3.1 software (Caliper LS, US). Mice were weighed three times a week or once a week prior to drug administration. On the indicated days, mice were sacrificed, and livers were fixed in 2 or 4% paraformaldehyde solution (PFA) and frozen in OCT (Optimal Cutting Temperature Compound - Embedded Medium) for further histopathological analysis.

[0266] Evaluation of mRNA formation and tumor targeting efficiency

[0267] An mRNA sequence containing an mCherry sequence, and an mCherry sequence containing miRNA-122 (Sequence No. 31), wherein the 'DMP CTx It was formulated as described in the 'Synthesis and mRNA Formulation' section and Table 4. To evaluate selective tumor targeting and preservation of healthy liver cells, the formulated mRNA was injected into the tail vein of mice with orthotopic liver cancer. Briefly, 20 μL of 1:1 PBS:Matrigel TM 2 x 10 suspended in 6Canine human Hep3B-cLuX cells were injected into the upper left lobe of the liver as described above. Subsequently, tumor growth was monitored by BLI imaging as described above. After 8 days, when the tumor was established (BLI ≥ 6 x 10⁶ 6 ), 20 μg of formulated mRNA-mCherry-DMP per mouse CTx , mRNA-mCherry-122-DMP CTx or mRNA-A-122-DMP CTx The drug was injected through the tail vein, and the delivery particles were delivered to the liver by restoring blood flow. After 24 hours, the final BLI was performed, the mice were euthanized, the livers were resected, and localized liver lesions were imaged by in vitro BLI.

[0268] histology

[0269] Briefly, in vitro After imaging, the left lobe of the tumor-containing liver was removed, fixed with 2% PFA, immersed in a 30% sucrose solution (in PBS; pH 7.4) at 4°C, embedded in an OCT, frozen in isopentane pre-cooled using a dry ice bath, and stored at -80°C. 5 μm frozen sections (Leica CM300, USA) were nuclear counterstained with DAPI (VECTASHIELD, Vector Laboratories, USA) or H&E (hematoxylin and eosin). Tumor targeting was evaluated by determining mCherry versus mCherry-122 expression levels in tumors and healthy livers using a fluorescence microscope and / or software. Tumor-like and healthy tissues were identified by H&E staining.

[0270] Simulated group and mRNA-mCherry-DMP CTx and mRNA-mCherry-122-DMP CTxExample of tumor growth monitored by BLI imaging in treated mice. D-luciferin was subcutaneously injected into the animals, and imaging was performed after 15 minutes. The signal was present only in the midsection of the animals, as shown in Fig. 17a (top panel), and the presented animals were prior to treatment with the composition. All animals with similar intensity in the midsection were dissected, and the liver was imaged in vitro (Fig. 17a, bottom panel). The left lobe of the liver containing the tumor was dissected and counterstained with DAPI. Using a fluorescence microscope, the expression of mCherry in healthy liver cells and liver tumor cells was determined 24 hours after injection of the formulated mRNA, as shown in Fig. 17b. Mice mRNA-mCherry-DMP CTx mCherry fluorescence was detected in healthy hepatocytes when treated with mRNA-mCherry-122-DMP (Fig. 17b, middle panel). CTx Translational inhibition was observed upon treatment with (Variant 1) (Fig. 17b, left panel). Fig. 17b shows, (from left to right) mock group treatment, mRNA-mCherry-DMP CTx and mRNA-mCherry-122-DMP CTx This shows healthy liver cells from a mouse.

[0271] In conclusion, the composition of the present invention can be administered in vivo and successfully transfect targeted liver cells. When modified with a miRNA binding site, differential expression can be achieved in healthy cells and tumor cells.

[0272] Example 5: In vivo differential expression of transferred US3 mRNA constructs between healthy and diseased tissues in the liver

[0273] US3 mRNA DMP in vivo mouse model described in Example 4 CTxThe delivery particle composition containing the miRNA-122 construct was applied. Differential expression of US3 in the livers of mice containing Hep3B human cancer was analyzed using immunohistochemistry with an anti-US3 polyclonal antibody. The results are shown in Fig. 18, where visible differences in US3 protein levels can be observed between tumor cells (darker staining) and healthy cells (lighter staining). Differential expression tracks the boundaries of the tumor, as independently verified by a pathologist. Therefore, it can be concluded that the composition of the present invention can successfully induce differential expression of potential therapeutic enhancers in vivo in mammals.

[0274] Immunohistochemistry

[0275] Fresh frozen sections were cut into 5 μm (microns) and air-dried for approximately 1 hour, then fixed with 4% paraformaldehyde at room temperature (RT) for 15 minutes. The sections were washed with running tap water and transferred to PBS-0.1% Tween. The sections were incubated with 2.5% normal horse serum (ready-to-use, ImmPRESS HRP Anti-Rabbit IgG Peroxidase Polymer Detector Kit, Vector MP-7401) for 20 minutes. The slides were drained and incubated with the primary antibody against US3 (Acris AP55266SU-N) diluted 1:400. The antibody was diluted with PBS-0.1% Tween, and a negative control was included, in which the primary antibody was omitted and the slides were incubated with the antibody diluent PBS-0.1% Tween at room temperature for 1 hour. Slides were washed with PBS-0.1% Tween, and endogenous peroxidase was blocked for 10 minutes with 0.3% hydrogen peroxide diluted with Elga water. Slides were washed with PBS-0.1% Tween and incubated at room temperature for 30 minutes with ImmPress anti-rabbit IgG reagent (ready-to-use, ImmPRESS HRP anti-rabbit IgG peroxidase polymer detection kit, vector MP-7401). Slides were washed with PBS-0.1% Tween and incubated for 5 minutes with the chromogen ImmPACT DAB (ImmPACT DAB peroxidase (HRP) substrate, vector SK-4105), then washed with Elga water and appropriately counterstained with Mayer's hematoxylin. Additionally, they were briefly washed with Elga water and washed under running tap water for 5 minutes. After dehydrating, cleaning, and fixing the slides (95% IMS, 99% IMS x2 and xylene x2), they were covered with coverslips.

[0276] Although specific embodiments of the present invention are disclosed herein in detail, they are disclosed by way of example and merely for illustrative purposes. The embodiments mentioned above are not intended to limit the scope of the claims appended below. The inventors take into account that various substitutions, changes, and modifications may be made without departing from the spirit and scope of the invention as defined by the claims. Any non-human nucleic acid and / or polypeptide sequences included in the structures and vectors according to embodiments of the present invention were obtained from sources within the United Kingdom, the United States, and the European Union. To the best of the inventors' knowledge, access to and benefit-sharing agreements for genetic resources, or related prior art, were not utilized in the creation of the present invention.

Claims

Claim 1 A composition comprising an isolated mRNA sequence for the expression of one or more cytokines in one or more target organs, wherein the mRNA sequence comprises: at least one coding sequence encoding one or more proinflammatory cytokines; at least one first non-translational region (UTR) sequence; and at least three different binding site sequences for microRNAs (miRNAs), wherein each of the miRNA binding site sequences is located immediately adjacent to the 5' or 3' of the first UTR sequence, and the miRNA binding site sequences enable differential expression of the coding sequence in at least first and second cell types in the target organ or organs. Claim 2 A composition according to claim 1, wherein the mRNA sequence comprises at least four miRNA binding site sequences. Claim 3 A composition according to claim 1, wherein one or more of the above-mentioned pro-inflammatory cytokines are involved in immune responses and inflammation, and are selected from one or more of TNFα, TNFβ, IFNα, IFNβ, IFN Gamma, GM-CSF, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10 or their ligands. Claim 4 delete Claim 5 A composition according to claim 1, wherein the sequence of at least three miRNA binding sites is substantially complementary to a miRNA sequence selected from at least one or more of the group consisting of miRNA-122, miRNA-125a, miRNA-125b, miRNA-199, miRNA-124a, Let-7, miRNA-148a, miRNA-148b, miRNA-375, miRNA-143, miRNA-145, miRNA192, miRNA194, miRNA-204, miRNA215, and miRNA-30. Claim 6 A composition according to claim 1, wherein at least one of the at least three miRNA binding site sequences comprises one or more of SEQ ID NOs 1 to 7. Claim 7 A composition according to claim 6, wherein at least one of the at least three miRNA binding site sequences comprises SEQ ID NO.

1. Claim 8 A composition according to claim 1, wherein the miRNA binding site sequences each comprise SEQ ID NOs 1, 2, 3, 4, and 5; or wherein the binding site sequences each comprise SEQ ID NOs 1, 2, 5, 6, and 7. Claim 9 A composition according to claim 1, wherein the first and second cell types are different selections from the group consisting of non-neoplastic cells, transformed cell phenotypes, precancerous phenotypes, and neoplastic phenotypes. Claim 10 A composition according to claim 1, wherein the target organ or organs are selected from the group consisting of the liver, brain, lung, breast, pancreas, colon, and kidney. Claim 11 A composition according to claim 1 or 3, wherein the mRNA comprises one or more open reading frames (ORFs). Claim 12 In claim 11, the ORF or OFRs are a composition encoding one or more cytokines or their ligands involved in immune responses and inflammation, selected from one or more of TNFα, TNFβ, IFNα, IFNβ, IFN gamma, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10; or one or more dendritic cell activators selected from one or more of GM-CSF, TLR7, and TLR9. Claim 13 A composition according to claim 1, wherein the mRNA comprises SEQ ID NO.

29. Claim 14 The composition according to claim 1 or 3, wherein the composition comprises an additional mRNA sequence, wherein the additional mRNA sequence comprises at least one coding sequence encoding one or more cytokines or their ligands involved in immune responses and inflammation, selected from one or more of TNFα, TNFβ, IFNα, IFNβ, IFN gamma, IL1, IL2, IL3, IL4, IL5, IL6, IL7, IL8, IL9, IL10, IL11, IL12, CCL2, CCL3, CCL4, CCL5, CXCL9, and CXCL10; at least a first non-translational region (UTR) sequence; and at least three different microRNA (miRNA) binding site sequences, wherein each miRNA binding site sequence is located immediately adjacent to the 5' or 3' within the first UTR sequence. Claim 15 The composition according to claim 1 or 3, wherein the composition comprises an additional mRNA sequence, said additional mRNA sequence comprising: at least one coding sequence encoding one or more dendritic cell activators selected from one or more of GM-CSF, TLR7 and TLR9; at least one first non-translational region (UTR) sequence; and at least three different microRNA (miRNA) binding site sequences, wherein each miRNA binding site sequence is located immediately next to the 5' or immediately next to the 3' within the first UTR sequence. Claim 16 The composition of claim 1, wherein the composition further comprises a delivery particle and a pharmaceutically acceptable carrier, and the mRNA sequence is contained within the delivery particle. Claim 17 A composition according to claim 16, wherein the delivery particle is selected from at least one of the group consisting of aminoalcohol lipidoid particles, liposomes, exosomes, cell-derived vesicles, and polymeric particles. Claim 18 A composition according to claim 16, wherein the above-mentioned delivery particle is targeted toward one or more target organs or organs. Claim 19 A composition according to claim 18, wherein the delivery particle comprises a targeting agent selected from proteins, peptides, carbohydrates, glycoproteins, lipids, small molecules, and nucleic acids, and the targeting agent preferentially associates with target organs or cells of organs. Claim 20 A DNA expression vector construct encoding an mRNA sequence, wherein the mRNA sequence comprises: at least one coding sequence encoding one or more pro-inflammatory cytokines; at least one first non-translational region (UTR) sequence; and at least three different binding site sequences for microRNAs (miRNAs), wherein each of the miRNA binding site sequences is located immediately adjacent to the 5' or 3' of the first UTR sequence, and the miRNA binding site sequences enable differential expression of the coding sequence in at least first and second cell types within a target organ or organs. Claim 21 A viral vector comprising an mRNA sequence or a DNA expression vector structure encoding said mRNA sequence, wherein the mRNA sequence comprises: at least one coding sequence encoding one or more pro-inflammatory cytokines; at least one first non-translational region (UTR) sequence; and at least three different binding site sequences for microRNAs (miRNAs), each said miRNA binding site sequence located immediately adjacent to the 5' or 3' of the first UTR sequence, and said miRNA binding site sequence enables differential expression of the coding sequence in at least first and second cell types within a target organ or organs. Claim 22 In claim 1, the composition is intended for use in cancer treatment, comprising administering the composition to a subject in need. Claim 23 A composition according to claim 22, wherein the above treatment further comprises a therapeutic regimen or administration of a therapeutic agent to a subject. Claim 24 A composition according to claim 23, wherein the therapeutic method or therapeutic agent is selected from chemotherapy, radiotherapy, biological agents, therapeutic viruses, small molecule drugs, CAR-T or adoptive cell therapy, and combinations thereof. Claim 25 A composition according to claim 22, wherein the cancer is selected from at least one of the group consisting of liver cancer, brain cancer, lung cancer, breast cancer, pancreatic cancer, colorectal cancer and kidney cancer. Claim 26 A composition in which the cancer in item 25 is liver cancer. Claim 27 A composition according to claim 26, wherein the liver cancer is primary liver cancer or secondary liver cancer. Claim 28 A composition according to claim 27, wherein the primary liver cancer is selected from the group consisting of hepatocellular carcinoma, hepatoblastoma, cholangiocarcinoma, and angiosarcoma. Claim 29 A composition according to claim 28, wherein the secondary liver cancer is metastatic liver cancer from a known or unknown primary solid tumor. Claim 30 A composition according to claim 23, wherein the treatment further comprises administering a tumor cell-disrupting virus to the subject. Claim 31 A composition according to claim 30, wherein the mRNA sequence codes for a therapeutic agent that increases the efficacy of the tumor cell-disrupting virus. Claim 32 A composition according to claim 30, wherein the tumor cell destructive virus is attenuated by a mutation of one or more virulence genes. Claim 33 A composition according to claim 32, wherein the mRNA sequence codes for one or more virulence genes, or equivalents or homologs thereof. Claim 34 A composition according to claim 30, wherein the tumor cell disrupting virus is selected from any one of groups 1 to 7 of the Baltimore virus classification. Claim 35 A composition according to claim 30, wherein the tumor cell destructive virus is selected from the group comprising one or more of bullous stomatitis virus, marabar virus, polio virus, reovirus, measles virus, Newcastle disease virus, Coxsackievirus A21, parvovirus, herpes simplex virus type 1, vaccinia virus, and adenovirus. Claim 36 A composition according to claim 30, wherein the tumor cell destructive virus is a herpes simplex virus. Claim 37 A composition in which, in paragraph 30, the tumor cell destructive virus is a vaccinia virus. Claim 38 In paragraph 22, the above treatment comprises administering the above composition orally, intravenously, topically, into a tumor, or subcutaneously. Claim 39 delete Claim 40 delete Claim 41 delete Claim 42 delete Claim 43 delete Claim 44 delete Claim 45 delete

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