Modified mrna for multicellular transformation

Abstract

Synthetic bacterial messenger RNA can be used to make autologous, allogeneic or direct nucleic acid cancer vaccines. Cancer cells are transfected in vitro or in vivo with mRNA obtained from DNA encoding immunogenic bacterial proteins. The immune response to cancer is generated by direct administration of the mRNA in vivo or administration of a vaccine made from cancer cells in vitro. Codon modification of the mRNA can optimize expression of the immunogenic polypeptide in cancer cells.

Description

[0001] Cross-references to related applications

[0002] This application is a continuation to U.S. Application Serial No. 16 / 869,642, filed May 8, 2020; U.S. Application Serial No. 15 / 583,599, filed May 1, 2017, now U.S. Patent No. 10,682,401; U.S. Application Serial No. 15 / 114,943, filed July 28, 2016, now U.S. Patent No. 9,636,388; U.S. National Phase Application for International Patent Application No. PCT / US2016 / 033235, filed May 19, 2016; and U.S. Provisional Application Serial No. 62 / 163,446, filed May 19, 2015. The disclosures of all the above applications are incorporated herein by reference in their entirety, including all figures, tables, and amino acid and nucleic acid sequences.

[0003] The sequence list in this application is labeled "Seq-List.txt", created on April 12, 2020, and is 31KB in size. The entire contents of the sequence list are incorporated herein by reference in their entirety. Technical Field

[0004] This invention relates generally to vaccines, and more particularly to cancer vaccines prepared by transfection of cancer cells or by direct intratumoral administration of synthetic bacterial messenger RNA (mRNA).

[0005] The present invention describes more specifically the synthetic RNA for the efficient expression of selected polypeptides in mammalian cells and the use of the RNA for cell transformation in vivo or in vitro.

[0006] This invention provides the development and use of an effective mRNA vaccine for cancer treatment. While deoxyribonucleic acid (DNA) vaccines have several drawbacks (including low transfection efficiency and time-consuming delivery methods), the mRNA vaccine of this invention is administered directly to tumor cells and is immediately translated into immunogenic proteins that elicit a multi-tumor antigen response. Compared to plasmid DNA, mRNA has a shorter in vivo half-life, making it less likely to integrate into the host genome and thus considered safer. Unlike DNA, mRNA vaccines do not need to cross the nuclear membrane, thus typically resulting in faster and higher levels of protein expression. Furthermore, the expression of transfected mRNA is independent of the cell cycle, and because mRNA levels are not promoter-driven, protein expression and vaccine dosage can be modulated by altering the levels of transfected mRNA. Compared to peptides, mRNA vaccines lack major histocompatibility complex (MHC) haplotype restriction and can be designed for self-regulation through the addition of MHC I transport signals or combination with protamine. While the efficacy of mRNA vaccines may benefit from complexes that protect RNA from degradation and enhance cellular uptake, in vivo cells can be transfected with mRNA in the absence of other reagents or physical transduction methods. Messenger RNA used as a vaccine can be produced from plasmid DNA through in vitro transcription. Background Technology

[0007] Cancer treatment is based on the specific type diagnosed. Some common cancers include bladder cancer, breast cancer, colon cancer, lymphoma, melanoma, and prostate cancer. Treatment plans are developed by doctors based on an assessment of many factors, including but not limited to the stage of the disease, its cause, and the patient's age and overall health. For many cancers, treatment options may include one or a combination of surgery, chemotherapy, radiation therapy, bone marrow / stem cell transplantation, anticancer drugs, or immunotherapy. The most common treatments include surgery, chemotherapy, radiation therapy, and oral medications. While these treatments can be effective, they often have many side effects. Chemotherapy specifically targets all newly dividing cells in the body, not just cancer cells.

[0008] Some immunotherapies have the advantage of targeting diseased cells while leaving healthy cells intact. Cancer cells originate from the breakdown of normal growth regulation mechanisms; therefore, the body still recognizes many of these cancer cells as its own. Cancer immunotherapy overcomes the body's tolerance to these diseased, self-contained cells and enables the body to distinguish them as foreign. Cancer can also evade immune detection by directly suppressing the body's immune system by reducing the expression of immune activation markers on cells, such as MHC molecules. MHC is one of the components that helps the body distinguish which cells are self-contained and which are foreign or diseased.

[0009] Treatment for solid tumors typically involves chemotherapy and / or surgery. Recently, there has been interest in developing vaccines to stimulate the body's own immune defenses. U.S. Patent No. 7,795,020 details a lymphoma vaccine for treating advanced lymphoma, which transforms autologous or non-autologous cells isolated from a subject diagnosed with lymphoma. The isolated cells are transfected with a plasmid vector carrying the *Streptococcus pyogenes* emm55 gene. Bacterial proteins are expressed on the cell surface, and when the transfected cells are introduced into a subject with cancer, an immune response against lymphoma cells is generated.

[0010] To date, the FDA has only approved the cell-based cancer immunotherapy vaccine Provenge for the treatment of prostate cancer; however, several vaccines are currently being tested in clinical trials. BiovaxId, an autologous tumor-derived immunoglobulin idiotype vaccine, is undergoing Phase III clinical trials for the treatment of indolent follicular non-Hodgkin lymphoma.

[0011] In principle, both exogenous DNA and RNA can express proteins in mammals. Whether proteins expressed from DNA and mRNA can produce similar immune activity remains uncertain. Traditionally, DNA is considered more suitable for vaccine production and gene therapy due to its stability and ease of use. An example of a plasmid DNA vaccine is Merial's Oncept, developed for the treatment of oral canine melanoma.

[0012] Work on mRNA vaccines has been reported. In one case, an effective mRNA vaccine was delivered using liposomes. This specific vaccine induced cytotoxic T lymphocytes in mice after administration of mRNA encoding influenza virus proteins. Other studies at CureVac GMH have shown that mRNA vaccines elicit humoral and cellular immune responses when delivered intradermally. This vaccine is administered naked and conjugated with protamine, a protein that enhances mRNA stability and improves protein expression. This vaccine is currently undergoing clinical trials for castration-resistant prostate cancer.

[0013] Human trials using mRNA on both liquid and solid tumors have been conducted. These cancers include acute myeloid lymphoma, metastatic melanoma, prostate cancer, renal cell carcinoma / ovarian cancer, neuroblastoma, brain cancer, lung cancer, colon cancer, and renal cell carcinoma. Most ongoing clinical trials involve transfecting mRNA into autologous dendritic cells, rather than into cancer cells. Furthermore, there have been no clinical trials attempting intratumoral administration of mRNA. Figure 3 This is a table of published clinical trials using mRNA vaccines.

[0014] Delivery vectors such as liposomes and cationic polymers appear to hold promise for enhancing transfection. Once the liposome or polymer complex enters the cytoplasm, the mRNA must be able to dissociate from the delivery vector for antigen translation to occur; unfortunately, these vectors may fail to properly compound with the mRNA and therefore fail to properly translate the encoded protein. Antigen may be produced, but in insufficient quantities to produce the desired effect.

[0015] Many immunotherapies are disease-specific, conceptually complex, and even more complex and expensive to produce. Their commercial viability remains to be seen. Directly administering mRNA to a patient's tumor has profound implications, as the mRNA is immediately translated into an immunogenic protein within the tumor, triggering a multi-tumor antigen response. For example, a single synthetic mRNA can be used to treat multiple types of cancer in multiple species. mRNA is easy to deliver, cost-effective, easy to transport and store, and easy to administer. Along with its excellent safety profile, these properties of mRNA make it possible to treat cancer patients worldwide, even in developing countries.

[0016] Guiding the immune system to kill cancer cells is the foundation of all cancer immunotherapies. For any type of immunotherapy to be successful, an immune response to tumor-associated antigens must be triggered and amplified. Immune responses can involve any number of immune cells, including antigen-presenting cells, neutrophils, natural killer cells, T helper cells, T cytotoxic cells, and B cells. However, in human cancer vaccine trials, the triggering and activation of an immune response to a single tumor antigen has not been sufficient to translate into beneficial clinical efficacy, likely due to immune escape variants; nor has the use of intact tumor cells or tumor cell lysates with exogenous adjuvants as suppliers of multiple associated tumor antigens been avoided. This is why it is essential to be able to provide triggers in the context of tumor antigens, as they are expressed on the patient's tumor cells. The only way to achieve this is to provide tumor cells with encoding nucleic acids so that the cellular structure can express trigger antigens alongside the tumor antigens, thereby exposing all of these antigens to the cells of the immune system. This exposure leads to interantigen epitope diffusion, thereby culturing and activating an adaptive immune response against all tumor cells carrying these antigens, even in the absence of trigger antigens.

[0017] Using nucleic acids as vaccines offers several other advantages. Nucleic acid vaccines can induce humoral and cellular immune responses; require low effective doses; are simple to administer; can be tested rapidly; are cost-effective and reproducible in large-scale production and isolation; can be produced at high frequencies and are easy to isolate; are more temperature-stable than traditional vaccines; have a long shelf life; are easy to store and transport; and are unlikely to require a cold chain.

[0018] DNA has been successfully used in vaccines. DNA is a double-stranded molecule that serves as the blueprint for organisms, containing their genetic instructions. DNA is suitable for use as a vaccine because it is relatively stable and non-reactive, and can be stored for long periods. However, DNA is self-replicating and is easily damaged by ultraviolet radiation.

[0019] On the other hand, RNA is single-stranded and its function is to execute the instructions of DNA, that is, RNA transfers genetic code to produce proteins. RNA is more reactive and less stable than DNA, but it is resistant to ultraviolet radiation. This latter property has proven to make RNA more suitable for use as a vaccine. Generally, the chance of mRNA integrating into the host chromosome is zero. mRNA delivery leads to faster expression of the antigen of interest and requires fewer copies for expression. The transient nature of mRNA expression, which might seem like a disadvantage, actually increases its safety. For protein production in postmitotic and non-dividing cells, mRNA is more efficient than DNA because DNA requires translocation through nuclear members and plasmid membranes, while mRNA only requires translocation through the plasmid membrane. mRNA is not only a template for translation but also a ligand for toll-like receptors and is sensitive to nucleases; therefore, it presents fewer concerns about horizontal transmission. Summary of the Invention

[0020] This invention is based on the use of ribonucleic acid information (mRNA) encoding immunogenic bacterial proteins (SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:16). The information can be delivered to the cytoplasm using any of a variety of known techniques. Once the mRNA reaches the cytoplasm, it is translated into the encoded protein using existing cellular mechanisms. The bacterial protein, such as an M-like protein having the amino acid sequence SEQ ID NO:2 or SEQ ID NO:14, is then expressed in the cell, thereby conferring immunogenicity to the cancer cell. For example, the M-like protein can be of bacterial origin, such as group A and group G streptococci (GAS and GGS), and is therefore considered foreign by the mammalian body. Immune surveillance cells, such as antigen-presenting cells (APCs), are attracted to the foreign protein. The APCs engulf the entire cancer cell and then present all foreign / mutant proteins (including M-like proteins) to other immune cells.

[0021] The production of bacterial proteins in cells is achieved by inserting corresponding genetic code. The gene for an M-like protein is called emmL. Once the emmL information is delivered to a cancer cell containing an abnormal protein resulting from a mutation in the cell's DNA, the M-like protein will be expressed in the cell, attracting immune cells to engulf it and causing the previously masked mutated protein to be presented to the immune system. Abnormal proteins may have been present for a long time, but because they originate from "self" proteins, the body may not necessarily recognize them as foreign or a threat. Bacterial protein antigens act as primers or triggers for the immune system to process cells that it might not have previously recognized as damaged and harmful.

[0022] mRNA is generated as described in the examples. Once obtained, the mRNA containing immunogenicity information can be delivered to autologous or allogeneic cells that require the initiation action described in this invention. The mRNA can also be delivered directly within the tumor, or, in the case of certain cancers (e.g., lymphoma), within the nodule.

[0023] An M-like protein encoded by the emmL gene has previously been delivered into cells via DNA and shown to be expressed in cells to produce an immunological effect. Due to concerns about DNA delivery, including the possibility of gene integration into chromosomes, delivery via RNA is a safer option because it cannot integrate into host DNA. This ability of DNA to integrate into host DNA becomes particularly important in medical applications where exogenous DNA integration can have adverse effects. In contrast to DNA expression, mRNA expression persists within cells for at most a few hours to a few days. mRNA that is not delivered into cells is rapidly degraded by RNases present in the environment and therefore does not pose a risk of horizontal spread. When emmL mRNA is successfully transfected into cancer cells, it can express immunogenic bacterial proteins both within and on the surface of cancer cells, thus inducing an immunogenic response. Attached Figure Description

[0024] Figure 1 This describes the plasmid backbone designed for the production of M-like protein mRNA.

[0025] Figure 2 This describes what is used to connect to Figure 1 The plasmid DNA from which the emmL gene is derived in the linearized vector.

[0026] Figure 3 This is a summary of mRNA trials performed in solid tumors.

[0027] Figure 4 This is a diagram illustrating the differences in cellular production pathways between mRNA and DNA.

[0028] Figure 5 The diagram shows the generation of a recombinant DNA vector to produce mRNA encoding bacterial antigens.

[0029] Figure 6 The image shows a Western blot of isolated Emm55 against an anti-M-like antibody.

[0030] Figure 7 The results show a comparison of protein expression from DNA and RNA transfections.

[0031] Figure 8 This is an agarose gel photograph of emm55 mRNA synthesized using the Flashgel system.

[0032] Figure 9 This is a graph showing the antibodies that react with EmML protein in mice inoculated with emmL mRNA or water (control) (C2) after weeks 1, 2, 3, and 4.

[0033] Figure 10 It is a protein blot showing the presence of antibodies that react with EmML protein in the blood of mice before and after inoculation with emmL mRNA.

[0034] Figure 11 This is a map of a novel double-stranded DNA molecule for the production of synthetic mRNA, which can be efficiently translated in mammalian cells. This DNA molecule contains the sequence of T7 RNA polymerase (SEQ ID NO:8), a portion of the 5' untranslated region of the Xenopus β-globin gene (SEQ ID NO:9), a multi-connector with restriction endonuclease recognition sites of SacI, NotI, BglII, EcoRV, and SpeI for insertion into the emmL coding region (SEQ ID NO:7), a portion of the 3' untranslated region of the Xenopus β-globin gene (SEQ ID NO:10), and then a multi-connector with restriction endonuclease recognition sites of BamHI, EcoRI, and XbaI for linearizing the plasmid before in vitro transcription (SEQ ID NO:11). Corresponding to... Figure 11 The DNA sequence is SEQ ID NO:12.

[0035] Figure 12A-12B A relative fitness plot of emm55 is shown. The Y-axis is the fitness index of emm55 for each codon, where 1.0 is highly suitable for human cell expression.

[0036] Figure 12A It is the original emm55 sequence.

[0037] Figure 12B The image shows the emm55 optimized using the JCat algorithm.

[0038] Figures 13A-13C It is derived from Emm55 cells expressed after transient transfection of HEK293T and B16-F10 cells with mRNA containing wild-type uridine (WT) and N1-methylpseudouridine. TM Simulated Western blot analysis by capillary electrophoresis. Lysates were 0.5 μg / μL. ERK1 was used as a normalized control. - Cells transfected with EGFP mRNA. * HEK293T cell lysates were cultured with rabbit anti-ERK1 only 6 hours after Emm55FreqDist transfection.

[0039] Figure 13A It is emm55Jcat.

[0040] Figure 13B It is emm55MostFreq.

[0041] Figure 13C It is emm55FreqDist. * Six hours after transfection with Emm55FreqDist, HEK293T cell lysates were cultured only with rabbit anti-ERK1.

[0042] Figures 14A-14B The normalized representation of Emm55 from the image shown in Figure 13 is displayed. The codon fitness index is used to determine codon optimization in the JCat algorithm, where 1.0 is perfectly optimized.

[0043] Figure 14A Expression in HEK293T cells was shown.

[0044] Figure 14B Expression in B16-F10 cells was shown.

[0045] Figures 15A-15B The time course of Emm55 expression in HEK293T cells was shown.

[0046] Figure 15A In Wes TM Initial analysis performed at maximum signal strength.

[0047] Figure 15B These are repeated experiments under reduced protein loading. Significance was determined by one-way ANOVA and Bonferroni post-hoc test. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 (n = 3). Significance between emm55JCat-N1 and pAc / emm55 for hollow star-shaped experiments. Significance between emm55JCat-WT and pAc / emm55 for solid star-shaped experiments. Detailed Implementation

[0048] This invention provides a cancer vaccine that is more efficient and less expensive to prepare than previously used vaccines. The cancer vaccine is prepared by directly introducing plasmid DNA into the cell nucleus. Using emmL-encoding mRNAs (SEQ ID NO:1, SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:16) inserted into the cytoplasm is more effective in transfecting tumor cells.

[0049] This invention further provides several novel emmL-encoding RNAs designed to optimize increased expression in transformed cells. More robust expression of the Emm55 protein was achieved using several codon optimization algorithms designed to adapt the emm55 nucleotide sequence for mammalian expression.

[0050] mRNA can deliver antigenic protein information to cancer cells. Not only is mRNA a safer alternative because it cannot integrate into the host DNA, but its expression is also limited to a few hours to a few days at most. mRNA delivery further embeds antigenic information into the cellular protein production process, thus providing faster expression within the cell. mRNA only needs to be delivered to the cytoplasm, while DNA must ultimately enter the nucleus to function. Using mRNA is advantageous for producing protein antigens because mRNA can induce protein production in both post-mitotic and non-dividing cells.

[0051] While mRNA delivery of antigen proteins is a safer alternative to DNA delivery, the stability and immunogenicity of the mRNA must be addressed. Many elements that contribute to increasing stability and immunogenicity are engineered into recombinant vector templates. If a suitable element is not present in the vector, it can be added; for example, if the template vector does not contain a poly(A) tail coding sequence, the tail is added during transcription.

[0052] To increase stability in the cytoplasm, mRNA must contain both a 5'-methylguanosine cap and a 3'-poly(A) tail (SEQ ID NO:3). These elements are responsible for attracting and attaching components of the cellular apparatus responsible for translating mRNA into proteins. The absence of these components reduces the time mRNA has available for protein translation before degradation. Therefore, as illustrated in the examples, these elements have been incorporated into the mRNA.

[0053] High immunogenicity can be enhanced by utilizing techniques such as viral vectors, nanoparticles, cationic polymers, lipids, and electroporation for improved delivery. Viral vectors are widely used for plasmid DNA (pDNA) delivery, but they carry several risks and increase costs. Electroporation using mRNA is less cytotoxic to cells due to its less stringent electrical setup and is a preferred method for mRNA delivery. DNA requires a higher charge to pass through both the outer and nuclear membranes, while mRNA only needs to pass through the outer cell membrane.

[0054] Compared to pDNA, the production of mRNA for vaccines offers economic and production advantages. mRNA is synthesized in vitro from linearized pDNA templates and requires only a small amount of DNA. On the other hand, the production of large quantities of pDNA is labor-intensive and requires equipment such as large fermenters to cultivate enough bacteria to produce the large amounts of pDNA needed for vaccine production. Although pDNA isolated from large cultures is pure, the final product appears in three structural forms due to the circular nature of plasmids: relaxed, linear, and supercoiled. While each form is capable of producing antigenic proteins upon entering the cell, each DNA form has a different ability to cross the plasma membrane and enter the cell. mRNA production yields only one structural form. Furthermore, batch-to-batch reproducibility is high due to the synthetic methods used in its production.

[0055] From a production perspective, mRNA is synthesized from DNA and is highly reproducible. This is important for its use as a vaccine because it does not require large-scale growth, meaning it needs less time and materials, and the risk of contamination is lower. These factors contribute to reduced costs. Furthermore, mRNA synthesis leads to higher yields, as it requires only one linearized plasmid DNA to produce one hundred mRNA molecules. Since mRNA is produced in vitro, there is no E. coli contamination (genomic DNA or endotoxins) after isolation. This results in fewer purification steps and quality control tests. The synthetic nature of in vitro transcription also ensures better batch-to-batch reproducibility and a purer product, as the vector sequence, including selection markers, is not part of the final product. Also unlike DNA, mRNA has a single molecular conformation, while plasmid DNA has three. mRNA is also easier to transfect than plasmid DNA and experiences less cell death during electroporation due to the lower voltage required. Like DNA, mRNA can also be lyophilized. From a regulatory perspective, mRNA is safer because it is non-replicable and transient. mRNA also does not pose environmental problems because it is easily degraded and does not induce antibiotic resistance.

[0056] The following comparison illustrates the advantages of using mRNA instead of DNA to deliver antigenic EMmL information into cancer cells. The comparison is divided into three parts: upstream production, downstream production, and cell delivery. Most of the benefits, including reduced production costs, shorter manufacturing times, superior information delivery, and improved safety, are visible in upstream production and cell delivery. Each part shows the significant differences between DNA and mRNA processes, as well as the similar steps in each process.

[0057] Upstream production:

[0058] The upstream production of the two nucleic acid products is almost identical before bacterial culture amplification. Only a small amount of DNA is needed to produce approximately 100 times the amount of mRNA. For example, in vitro transcription experiments produced 25 μg of mRNA from just 0.2 μg of DNA. This is 25 times more mRNA than the same amount of DNA produced. Culture amplification can be very expensive and time-consuming, and can lead to an increased risk of DNA contamination or mutation.

[0059] The benefits of culturing only small amounts of bacterial cultures are significant. The small amount of DNA in such cultures requires minimal isolation. This scale-up saves time and resources and reduces the risk of contamination. The production of the final mRNA product requires an additional step of transcription from a DNA template. This is a synthetic step performed in vitro. Due to the synthetic nature of transcription, batch-to-batch reproducibility is excellent, and the entire process takes only a few hours. Culturing bacteria containing DNA can take up to several days.

[0060] A significant drawback of using pDNA instead of mRNA is that the final product may be contaminated with genomic DNA (gDNA). Furthermore, isolated pDNA forms three conformations: linear, supercoiled, and round, which do not transfect cells with equal efficiency. The final mRNA product is pure, has a single conformation, and is free from contamination by either gDNA or pDNA.

[0061] Table 1 compares the steps used for DNA and mRNA production.

[0062] Table 1

[0063]

[0064]

[0065] Downstream production (autologous preparation):

[0066] The downstream production of DNA and mRNA is largely the same. One difference lies in the electroporation step. mRNA requires a lower voltage because it only needs to pass through the plasma membrane and not the nuclear membrane, unlike DNA, which must pass through both the plasma and nuclear membranes. The lower voltage is advantageous because it results in less cell death during electroporation. The increased viability of mRNA-transfected cells translates into a sufficient proportion of vaccine cells that readily express M-like proteins.

[0067] Table 2 compares the processing of DNA and mRNA in tumor tissues, from preparation to inoculation in transfected cells.

[0068] Table 2

[0069]

[0070]

[0071] Cell delivery:

[0072] The cell delivery flowchart below illustrates the significant advantages of using mRNA delivery. As shown in the table, the mRNA delivered into the cell jumps to immediate translation into an antigenic M-like protein. The transfected DNA must not only cross the additional cell membrane but also be transcribed into mRNA for delivery back to the cytoplasm, which is the starting point for protein synthesis initiated by the mRNA vaccine.

[0073] Depending on the desired effect, mRNA vaccines can be combined with compatible immune adjuvants or inhibitors. Adjuvants such as TriMix (a mixture of immunostimulatory molecules) can be added to mRNA-based vaccines to trigger an enhanced immune response against the encoded immunogen. Immunorepressors can be used to counteract other elements of immunosuppressive enzymes that may hinder the body's ability to produce a sufficient immune response. These immunosuppressive elements can be silenced by using silencing RNA (siRNA), which can be co-delivered during the immune process. Another type of immunorepressor that can be co-administered with mRNA-based cancer vaccines is a checkpoint inhibitor. These are typically composed of antibodies, such as anti-PD1 and anti-CTLA4, which bind to receptors present on tumor cells or immune-activated cells and, if not blocked, induce immunosuppression. This process is called "applying the brakes," and as it implies, the release of these "brakes" allows immunotherapies, such as mRNA cancer vaccines, to hone the immune system's efforts to attack cancer cells.

[0074] The aforementioned vaccines can be used not only in combination with checkpoint inhibitor therapy, but also before or simultaneously with chemotherapy, radiotherapy, whole-cell vaccines, other nucleic acid therapies, natural killer cell therapy, or chimeric antigen receptor therapy.

[0075] In other cases, cancer patients are treated with a regimen that alters the tumor microenvironment before or during vaccination. This regimen includes, but is not limited to, cytokines, anti-fugetaxis agents, chemokines, and rhythmic doses of chemicals.

[0076] Table 3 compares the processing of DNA and mRNA that enter the cell for translation.

[0077] Table 3

[0078]

[0079] In Examples 23-25, an in vitro transcription reaction can be used to generate mRNA encoding emmL. In this reaction, the resulting mRNA can be modified to improve the stability and translation efficiency of the mRNA and EmmL protein, and to reduce the immunogenicity of the mRNA. For example, modified nucleic acids can be ligated to the 5' end of the emmL mRNA, such as, but not limited to, anti-reverse cap analogs [ARCA, P1-(5'-(3'-O-methyl)-7-methyl-guanosine)P3-(5'-(guanosine))triphosphate], N1-methylguanosine, 2'-fluoroguanosine, 7-denitroguanosine, inosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine, and 2-azidoguanosine.

[0080] In another example, a poly(A) tail of approximately 50-200 monophosphate adenosine monophosphates in length can be attached to the 3' end of emmL mRNA, or a 5' modified nucleotide cap and a poly(A) tail can be added to emmL mRNA.

[0081] emmL mRNA can be synthesized using ribonucleotide analogs. Chemical modifications can be performed at this stage to further improve translation efficiency and stability. Examples include 5-methylcytidine-5'-triphosphate, pseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, and N1-methylpseudouridine-5'-triphosphate.

[0082] There are several methods to deliver mRNA into cells, thereby producing EmmL protein at high levels. For example, emmL mRNA produced after in vitro transcription can be directly injected into tissues or tumors.

[0083] Complexes such as lipids or polymers can be used to protect RNA from degradation, enhance cellular uptake, and improve the delivery of translational structures to the cytoplasm. In one embodiment, emmL mRNA is complexed with liposomes prepared from lipophilic substances such as cholesterol and synthetic phospholipids.

[0084] In one embodiment, emmL mRNA is complexed with liposomes prepared from lipophilic substances such as cholesterol and natural phospholipids.

[0085] In one embodiment, emmL mRNA is complexed with cationic lipids, such as, but not limited to, N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylmethylammonium sulfate (DOTAP).

[0086] In one embodiment, emmL mRNA is complexed with zwitterionic lipids, such as, but not limited to, 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS).

[0087] In one embodiment, emmL mRNA is complexed with polyethylene glycol-modified lipids, such as, but not limited to, N-(carbonyl-methoxy polyethylene glycol_2000)-1,2-distearate-sn-glycerol-3-phosphate ethanolamine (DSPE-PEG).

[0088] In one embodiment, emmL mRNA is complexed with a mixture of cationic, zwitterionic and polyethylene glycol-modified lipids.

[0089] In one embodiment, emmL mRNA is complexed with protamine.

[0090] In one embodiment, emmL mRNA is compounded with liposomes prepared from a specific substance (e.g., Japanese hemagglutinin virus), which has been used to prepare mRNA vaccines for the treatment of melanoma

[26] .

[0091] In one embodiment, emmL mRNA can be complexed with a polymer that is rationally designed to contain a variety of virus-mimicking components for efficient transfection of specific cells. These will include, but are not limited to, membrane-disrupting peptides, nucleic acid-binding components, protective coatings, and external targeting ligands.

[0092] In one embodiment, composite components can be combined and formulated into individual nanoparticles.

[0093] In one embodiment, the emmL mRNA or emmL mRNA complex may be combined with interfering RNA or an interfering RNA complex targeting an immune checkpoint molecule, such as programmed cell death protein 1 (PD-1), programmed death ligand 1 (PD-L1), cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), or a T-cell immune receptor (TIGIT) having Ig and ITIM domains. A non-limiting example is an emmL mRNA complex and a short interfering RNA (siRNA) complex targeting PD-1.

[0094] In one embodiment, the emmL coding region can bind to a viral RNA replication gene to form a self-replicating linear RNA molecule. This linear RNA molecule can then be formulated with lipophilic compounds and lipids to form liposomes capable of transfecting cells with self-replicating mRNA.

[0095] In one embodiment, emmL mRNA, or emmL mRNA formulated with lipids, protamine, or in liposomes, is complexed with a biodegradable polymer. A non-limiting example is polycaprolactone, which has been approved by the U.S. Food and Drug Administration for use as a drug delivery device. The advantage of biodegradable polymer complexes is that the mRNA or mRNA complex can be delivered over a long period as the polymer degrades. This sustained delivery can enhance vaccine efficacy.

[0096] In one embodiment, emmL mRNA or emmL mRNA complexes can be formulated in a biodegradable polymer containing tissue- or tumor-specific factors that enhance the efficacy of emmL mRNA vaccines. One example could be an emmL mRNA vaccine formulated with a biopolymer containing factors that inhibit angiogenesis or vasculogenesis, thereby providing a synergistic antitumor effect.

[0097] In one embodiment, emmL mRNA or emmL mRNA complexes may be formulated in a biodegradable polymer containing a factor that reduces the expression of immune checkpoint molecules, such as PD-1, PD-L1, CTLA-4, or TIGIT. As a non-limiting example, this may include existing or novel pharmaceutical agents, such as small molecules or antibodies that reduce PD-1 expression, thereby providing a synergistic antitumor effect.

[0098] In one embodiment, emmL mRNA or emmL mRNA complexes can be formulated in a biodegradable polymer containing interfering RNA or interfering RNA complexes, which are factors that reduce the expression of immune checkpoint molecules such as PD-1, PD-L1, CTLA-4, or TIGIT. A non-limiting example is an emmL mRNA complex and a short interfering RNA (siRNA) complex.

[0099] In one embodiment, emmL mRNA or emmL mRNA complex can be combined with an adjuvant, such as synthetic double-stranded RNA polyribosinosine polyribonucleotide [poly(I:C)].

[0100] Analysis of pSFCMVT / emmL Design Components

[0101] Several design elements can improve Emml protein production levels.

[0102] The mRNA vaccine (SEQ ID NO: 1-3) detailed in Example 1 was cloned into the Oxford Genetics pSF-CMV_T7 plasmid DNA vector to generate pSFCMVT7 / emmL. Although mRNA expression was detected after transfection into mammalian cells, only low levels of Emm55 protein were detected on Western blotting. Retrospective analysis of pSFCMVT7 / emmL identified design elements that could improve translation efficiency in mammalian cells.

[0103] At least two different types of design elements can improve EmmL protein production levels. Proximal elements are located close to the emmL coding region and are added during pAc / emm55 emmL polymerase chain reaction (PCR) amplification. Distal elements are located away from the emmL coding region and are versatile in enhancing protein expression derived from mRNA.

[0104] The proximal element includes introducing a translation initiation sequence optimized for mammalian cell expression into the N-terminus of the emmL coding region. This sequence has been identified as RYMRMVATGGC, where R is A or G, Y is C or T, M is A or C, and V is A, C, or G (SEQ ID NO:4). In one embodiment, the optimal translation initiation sequence ATAGCCATGGC (SEQ ID NO:5) replaces the natural start codon of emmL.

[0105] In one embodiment of this proximal design element, oligonucleotide PCR primers are synthesized such that each nucleotide at degenerate positions (R, Y, M, and V in SEQ ID NO:4) has an equimolar concentration, and the final oligonucleotide synthesis product contains all possible primer sequences. In the current embodiment, after the emmL gene is subcloned into a mammalian expression plasmid DNA vector, the optimal translation initiation sequence is empirically determined by comparing EmmL protein expression levels in analyses such as Western blotting.

[0106] Another proximal design element that can be used to modify the C-terminus of the emmL coding region is to add a stop codon after the natural stop codon. Many eukaryotic expression plasmid vectors contain DNA sequences of all three stop codon variants, TAG, TAA, and TGA, immediately following the last codon of the expressed gene. Since the DNA sequence of the natural emmL stop codon is TAG, SEQ ID NO:6 can be included as an added proximal design element.

[0107] Another proximal design element is the addition of a restriction endonuclease recognition site, which can be added to the N and C ends of the emmL coding region to facilitate its insertion into the plasmid cloning vector. The selection of the restriction endonuclease recognition site is based on the target plasmid. Some examples of restriction endonucleases that can be used are SacI, NotI, BglII, EcoRV, and SpeI. In a preferred embodiment, the SacI and SpeI sites are added to the N and C ends of the emmL coding region because they are not cleaved within the emmL coding region and share reaction conditions; therefore, they can be used simultaneously to prepare emmL coding region PCR amplicones for insertion into the target plasmid.

[0108] Design elements distal to the emmL coding region are added to plasmid cloning vectors so that any variations in the emmL coding region, or other genes adapted to the proximal design elements, can be inserted. These distal elements can be engineered to produce mRNA expression vectors capable of driving high-level mRNA and protein expression of emmL or any other mammalian mRNA.

[0109] One distal design element is the DNA sequence of the promoter region of a phage RNA polymerase. Examples of phage RNA polymerases are T7, T3, and SP6. In one embodiment, the promoter of the T7 RNA polymerase (SEQ ID NO:8) is the first of several distal elements added in such a way that it is ultimately located upstream of the emmL coding region relative to the action of the T7 RNA polymerase.

[0110] The RNA sequences immediately adjacent to the 5' and 3' of the coding region of a transcribed but untranslated eukaryotic gene are called untranslated regions (UTRs) and can have a strong positive or negative impact on protein translation of in vitro transcribed mRNA. Generally, UTRs supporting high levels of protein expression are unstructured, lack negative regulatory sequences, and may contain microRNA binding sites for further refining gene expression patterns. UTRs of genes typically highly expressed in mammalian cells, or those from genes with expression patterns matching those of tissues requiring mRNA vaccine expression, can be used to optimize the delivery of gene products (e.g., Emml proteins). The UTR from the Xenopus β-globin gene has been widely used to mediate high levels of translation in various eukaryotic cells, including for designing RNA for mammalian immunotherapy. An example of a tissue-specific UTR is the tryptophan hydroxylase (TPH) isotype, which drives differential expression in the pineal gland and brainstem.

[0111] In one embodiment, the 5' UTR of the Xenopus β-globin gene may be located upstream of the emmL coding region in terms of RNA polymerase activity. SEQ ID NO:9 is a non-limiting example of the 5' untranslated region of the Xenopus β-globin gene.

[0112] In one embodiment, the 3'UTR of the Xenopus β-globin gene may be located downstream of the emmL coding region in relation to RNA polymerase activity. SEQ ID NO:10 is a non-limiting example of the 3' untranslated region of the Xenopus β-globin gene.

[0113] In one embodiment, both the 5' and 3' UTRs of the Xenopus β-globin gene can be located on the flanks of the emmL coding region.

[0114] In one embodiment, the 5' and 3' UTRs are selected based on the type of tumor cells targeted by the vaccine. In a set of non-limiting examples, when used to treat melanoma, the 5' and 3' UTRs of genes highly expressed in melanoma cells can be used to provide high levels of cell-specific expression for emmL-based vaccines, such genes include tyrosinase (TYR), melanin production-associated transcription factor (MITF), melanocortin receptor 1 (MC1R), telomerase (TERT), cyclooxygenase 2 (COX2), CXC motif chemokine receptor 4 (CXCR4), and a baculovirus IAP repeat sequence (BIRC5) containing five genes.

[0115] In one embodiment, additional genetic elements not located in the primary gene transcript may be included before, after, or within the 5' and 3' UTRs to impart the desired expression level and specificity, in order to further improve vaccine expression.

[0116] Another distal design element is a synthetic DNA sequence that provides a restriction endonuclease recognition site, which can be used to insert the emmL coding region into other flanking distal design elements. In one embodiment, restriction endonuclease recognition sites of SacI, NotI, BglII, EcoRV, and SpeI (SEQ ID NO:7) are used.

[0117] Another distal design element is a synthetic DNA sequence that provides restriction endonuclease recognition sites to cleave the target plasmid immediately after the emmL coding region to produce linear plasmid DNA molecules. Linearization provides efficient termination for RNA polymerase activity, thereby increasing the production of consistent-length mRNA transcripts. An example of a sequence is SEQ ID NO:11, which contains restriction endonuclease recognition sites for BamHI, EcoRI, and XbaI. In a preferred embodiment, BamHI can be used to linearize plasmids containing the emmL coding region because it does not cleave within the emmL coding region and leaves a 5' nucleotide overhang, which can allow for higher transcription efficiency than a 3' nucleotide overhang.

[0118] In one embodiment, a double-stranded DNA molecule containing the aforementioned distal design element is synthesized by using overlapping complementary synthetic single-stranded oligonucleotide molecules. These molecules are annealed, and the remaining gaps are filled with a DNA polymerase, such as Taq polymerase.

[0119] In one embodiment, two complementary synthetic single-stranded oligonucleotide molecules can be synthesized to contain all the desired distal design elements. These oligonucleotide molecules are then annealed. In either of the above embodiments, the resulting blunt-ended synthetic DNA molecule can be inserted into a PCR cloning vector, such as Invitrogen, by adding template-nonspecific adenosine. The DNA was synthesized in the II-TOPO plasmid. This was achieved by incubating blunt-ended synthetic DNA molecules with Taq polymerase and deoxyadenosine triphosphate at a final concentration of approximately 200 μM at 720 °C for 10 minutes.

[0120] In one embodiment, the plasmid containing the aforementioned distal design element is named pT7XLUTR. Inserted into The map of synthetic DNA molecules in II-TOPO is as follows: Figure 11 As shown, and the nucleotide sequence is shown as SEQ ID NO:12. This sequence contains the aforementioned distal design elements, but does not represent the full or complete range of elements that can be added to support the desired expression level or specificity.

[0121] pT7XLUTR can be used to transform *E. coli*. Bacteria transformed with pT7XLUTR can be selected on agar plates containing an antibiotic compatible with the PCR cloning vector, such as agar plates containing Luria Bertani medium supplemented with 50-100 μg / mL kanamycin or carbenicillin. Bacterial cultures can be amplified by growth in a liquid antibiotic-selective medium, such as Luria Bertani medium supplemented with 50-100 μg / mL kanamycin or carbenicillin, and plasmid DNA prepared using methods known to those skilled in the art.

[0122] RNA containing several of the aforementioned design elements was found to increase Emm55 expression. The sequences are shown in SEQ ID NO:13, SEQ ID NO:15, and SEQ ID NO:16.

[0123] Example

[0124] The following examples are provided as illustrations of the invention and are in no way intended to be limiting.

[0125] Example 1. Autologous mRNA vaccine for canine lymphoma

[0126] A 75-pound male, neutered Rhodesian Ridgeback was taken to the veterinarian with swollen lymph nodes in its jaw and groin. Fine-needle aspiration was performed on one of the swollen lymph nodes. Upon examination by a pathologist, it was diagnosed with low-grade diffuse lymphoma.

[0127] Because immunotherapy reported the fewest side effects, the patient's owner opted for immunotherapy instead of chemotherapy and steroids. The veterinarian removed the right submandibular lymph node while the patient was under general anesthesia. Tissue samples were transported overnight for laboratory processing.

[0128] Upon receiving the tissue sample in the laboratory, the following steps are performed: 1) check the transfer medium for any bacterial contamination; 2) measure the tissue size; 3) repeatedly aspirate the intact lymph node using several bolus washes to release tumor cells; and 4) collect and count the aspirated cells.

[0129] An appropriate number of cells were used for electroporation with emmL-encoding mRNA. Using the BioRad Gene Pulse machine, 120 × 10⁶ cells were transfected with 80 μg mRNA. 6 A small fraction of the transfected cells were cryopreserved, while the remainder were placed in culture for approximately 24 hours. After 24 hours, the cells were irradiated and divided into 10 × 10⁻⁶ cells. 6 Each cell-based vaccine dose is frozen and stored until needed.

[0130] The patient received a total of eight doses of vaccine. Each dose was transported overnight from the laboratory to the veterinary clinic, arriving on the scheduled administration date. The veterinarian administered each dose intradermally using a syringe with a needle. The eight doses were administered every seven days (plus / minus one day) for four weeks, followed by monthly administrations for four months. Blood samples were collected before the first dose. Subsequent blood samples were collected before the fifth and eighth vaccinations, and eight weeks after the final vaccination. The blood samples were processed into peripheral blood and plasma and stored in the laboratory. They were later used to assess the antitumor immune response.

[0131] Throughout the treatment process, the patient's lymph node size and overall quality of life will be monitored. Overall disease status will be assessed through tumor burden reduction and antitumor immune response. Tumor burden will be assessed by measuring each lymph node throughout the treatment process. Antitumor immune response will be measured using standard enzyme-linked immunosorbent assay (ELISA) to assess antibody levels and flow cytometry to assess cytotoxic T-cell (CTL) responses.

[0132] During treatment, the size of the patient's lymph nodes increased and then decreased as treatment progressed. This observation may be due to immune cell infiltration into the tumor site, in this case, the lymph nodes. ELISA and flow cytometry results showed increased antibody production and CTLs after the fourth vaccination, which persisted after the completion of the series of vaccines.

[0133] Example 2. Direct mRNA vaccine for equine melanoma

[0134] A 15-year-old Andalusian horse was taken to the vet with black lesions on its neck, mane, and perianal area. After examining fine-needle aspiration, a pathologist diagnosed melanoma. Due to the complexity of removing the perianal lesions, the owner opted for immunotherapy.

[0135] Three vaccine doses were prepared, each containing 100 μg mRNA in 100 μL of sterile, nuclease-free H₂O. The three doses and three needle-free injection devices (J-Tip) were delivered to a veterinarian. Three lesions on the patient were selected for the treatment course, with a total of 300 μg mRNA at each time point. As before, three additional doses were delivered to the veterinary clinic every two weeks, and each dose was administered to the same three lesions using the J-Tip device. The patient received a total of six doses of vaccine per lesion.

[0136] Blood samples were collected before the start of the vaccine series, before the fifth vaccination, and two weeks after the series was completed. The blood samples were processed into peripheral blood and plasma and stored. They were then used to assess the antitumor immune response.

[0137] Overall disease status was assessed through tumor burden reduction and antitumor immune response. Tumor burden was assessed by measurements of the lesions prior to each of the six doses of vaccine. Antitumor immune response was assessed using standard ELISA measurements to evaluate antibody levels and flow cytometry measurements to evaluate CTL response.

[0138] As seen in other patients receiving immunotherapy, melanoma lesions initially increase in size, then decrease as the vaccine series progresses. ELISA and FACS results showed increased antibody production and CTLs after the second vaccination, which persisted after the completion of the vaccine series.

[0139] Example 3. Overview of emmL mRNA generation methods

[0140] Method Overview:

[0141] Restriction enzyme digestion of vectors and inserts

[0142] To generate suitable recombinant plasmids for optimal mRNA production, a plasmid backbone including dual prokaryotic and eukaryotic promoters, untranslated 3' and 5' regions, and selection markers was used. Vectors of this type, such as pSFCMVT7, possess several features that facilitate the production and stabilization of mRNA encoding antigenic M-like proteins (e.g., Emm55). The vector pSFCMVT7 and the insert containing plasmid pAc / emmL were digested using the restriction enzymes SacI and EcoRV. See the plasmid map below. Figure 1 and 2 .

[0143] DNA fragments were separated using gel electrophoresis.

[0144] After restriction digestion with a suitable enzyme, DNA fragments are separated by gel electrophoresis. A reference DNA gradient solution is run with both digestion reactants to assess DNA band lengths, thus aiding in the identification of bands of interest. DNA-containing bands are then extracted from the gel.

[0145] Gel extraction / DNA separation

[0146] Dissolve the gel slice containing the target DNA and extract the DNA so that the vector and insert can be ligated together to produce the recombinant plasmid pSFCMVT7 / emmL.

[0147] Carrier and insert connection

[0148] During the restriction digestion of the vector and the plasmid-containing insert, "sticky ends" are generated, which are then spliced ​​together by a ligation reaction. "Sticky ends" are unpaired nucleotides that can form hydrogen bonds with complementary nucleotides. Since the vector pSFCMVT7 and the insert emmL are cut with the same restriction endonuclease, they contain complementary ends that are ligated upon exposure to T4 DNA ligase.

[0149] Transfection into bacteria

[0150] After producing the mRNA-producing plasmid pSFCMVT7 / emmL, it is transformed—that is, transfected into competent bacteria capable of producing sufficient DNA. This DNA is then isolated and used for in vitro mRNA synthesis. Invitrogen's Stbl3 *E. coli* is one example of a bacterial type that can be used for transfection. Transformation is induced by heat-shocked bacteria creating pores in the cell membrane, allowing the plasmid to enter the cell and eventually the nucleus.

[0151] Growth and amplification of bacterial cultures

[0152] Bacteria transfected with plasmids are placed on an appropriate growth medium containing a selective antibiotic. In the case of pSFCMVT7, this is kanamycin. If the bacteria are correctly transformed with the plasmid, they will produce a protein that inhibits the antimicrobial properties of kanamycin and allows kanamycin-resistant bacteria to selectively grow on the medium.

[0153] Plasmid isolation and purification

[0154] After a sufficient number of bacteria containing pDNA have grown, the cells are lysed, releasing the plasmids from the cell interior. The pDNA is then separated from gDNA, proteins, and other cellular debris by filtration and anion exchange column separation.

[0155] Template DNA preparation: Plasmid DNA linearization

[0156] The isolated DNA contains template DNA for mRNA production. For transcription to occur, the plasmid must be linearized. Importantly, linearization occurs downstream of the open reading frame gene of interest.

[0157] mRNA transcription response

[0158] Once the template is prepared, information is generated via an in vitro transcription reaction. This reaction mimics the transcription of mRNA in cells, including adding a 5' cap and a poly(A) tail to increase stability.

[0159] mRNA purification

[0160] Once the information is transcribed into mRNA, the remaining DNA template is degraded, allowing the pure mRNA product to be used for transfection into autologous cells, allogeneic cells, or intratumoral transfection. Once inside the cell, the mRNA will produce and display M-like proteins on the cell surface for immune activation.

[0161] Transfecting cancer cells with mRNA

[0162] One way to deliver mRNA into cancer cells is through electroporation. This method uses a weak electric current to create small pores in the cell membrane, allowing the mRNA to pass through the membrane and enter the cytoplasm.

[0163] Example 4. Restriction enzyme digestion

[0164] Table 1 shows the rapid digestion procedure for pDNA.

[0165] Table 1

[0166]

[0167] Example 5. Separating DNA fragments using gel electrophoresis.

[0168] Table 2 shows the procedure for DNA fragment isolation.

[0169] Table 2

[0170]

[0171] Example 6. Gel extraction / DNA separation

[0172] Table 3 shows the procedures for extraction and DNA separation.

[0173] Table 3

[0174]

[0175]

[0176] Example 7. Connection between carrier and insert.

[0177] Table 4 shows the procedure for inserting and connecting the carrier.

[0178] Table 4

[0179]

[0180]

[0181] Example 8. Transforming DNA into E. coli.

[0182] Table 5 shows the transformation procedure for Escherichia coli.

[0183] Table 5

[0184]

[0185]

[0186] Example 9. Growth and amplification of bacterial cultures

[0187] Table 6 shows the growth and amplification procedures for bacterial cultures.

[0188] Table 6

[0189]

[0190] Example 10. Plasmid isolation and purification

[0191] Table 7 shows the procedures for plasmid isolation and purification.

[0192] Table 7

[0193]

[0194]

[0195] Example 11. Preparation of template DNA.

[0196] Table 8 shows the procedures for template DNA preparation and plasmid linearization.

[0197] Table 8

[0198]

[0199]

[0200] Example 12. mRNA transcription

[0201] Table 9 shows the procedure for transcribing mRNA.

[0202] Table 9

[0203]

[0204] Example 13. mRNA purification

[0205] Table 10 shows the procedure for purifying mRNA.

[0206] Table 10

[0207]

[0208]

[0209] Example 14. Transfecting cancer cells with mRNA

[0210] Table 11 below shows the procedure for transfecting mammalian cancer cells with emmL mRNA.

[0211] Table 11

[0212]

[0213] Example 15. Cloning steps for DNA pSFCMVT7 / emmL.

[0214] Figure 5The procedure for generating a recombinant DNA vector to produce mRNA encoding bacterial antigens is shown.

[0215] Example 16. Direct binding of antibody to M-like protein.

[0216] Figure 6 The Western blot shown demonstrates the specificity of the anti-M-like protein antibody for isolated M-like proteins, particularly Emm55.

[0217] Proteins were separated by SDS-PAGE (10%) using a loading buffer containing 130 mM β-ME. Samples were boiled at 100 °C for 3 minutes and then rotated at 13,000 x g for 2 minutes at room temperature. The blot (far left) was probed with primary antibody (α-M-like protein) in 5% milk at room temperature for 1.5 hours. The primary antibody dilution was 1:500. The secondary antibody (goat α-mouse conjugated HRP) was diluted 1:5000. The empty blot (second from left) shows nonspecific binding of the secondary antibody.

[0218] Proteins on nitrocellulose blots were visualized using chemiluminescence (exposure: 10 minutes).

[0219] Example 17. Fluorescence microscopy images and graphs show increased mRNA expression compared to DNA. Results from experiments in which RNA and DNA were transfected into mammalian cells and protein expression was analyzed were compared. The results showed that an equal amount of transfected RNA produced a five-fold increase in expression. (See...) Figure 7 ).

[0220] Example 18. Synthesized emmL mRNA, tailless and tailed.

[0221] Used for execution Figure 8 The denaturing agarose gel procedure shown demonstrates the visualization of synthesized emmL mRNA, particularly emm55 mRNA, using the Lonza FlashGel system.

[0222] 20 ng samples and 100 ng gradient solutions were prepared by diluting the total volume to 2.5 μL using DEPC-treated water. An equal volume of formaldehyde sample buffer was added to each sample. The samples were mixed and then incubated at 65 °C for 15 min, followed by incubation on ice for 1 min. The samples were loaded into 1.2% RNA gel cartridges and then run at 225 V for 8 min. The gels were incubated at room temperature for 10 min and then visualized using a FlashGel camera. mRNA size was determined using RNA MillenniumMarker.

[0223] Example 19

[0224] Table 4 shows the results of an experiment in which RNA (emmL mRNA) and DNA (pSFCMVT17 / emmL) were transfected into mammalian cells, stained with α-M-like proteins, and analyzed using flow cytometry. The results showed that RNA-transfected cells produced a signal equivalent to that produced by DNA-transfected cells, i.e., 9%.

[0225] Table 4

[0226]

[0227] Example 20

[0228] The presence of antibodies that react with the emmL protein was tested in blood samples from mice inoculated with emmL mRNA (treatment) or sterile water (control). Figure 9 As shown, blood samples from control mice (C2) did not contain α-M-like protein antibodies, while samples from treated mice (T2) showed a slight increase.

[0229] Example 21

[0230] Table 5 shows the results of an experiment in which mice were transplanted with melanoma tumor cells and subsequently injected with either emmL mRNA (treatment) or sterile water (control). The injection regimen began 10 days after tumor implantation. The regimen consisted of three treatment or control injections every seven days. All five mice survived injection #2. At this point, two of the three treated mice had tumors smaller than those in the control mice. Three of the five mice survived injection #3, at which point the remaining two treated mice still had tumors smaller than those in the remaining control mice.

[0231] Table 5

[0232]

[0233] Example 22

[0234] Figure 10 The results from the experiment are shown, in which the presence of antibodies reacting with emmL proteins was tested in blood samples from mice before and after inoculation with emmL mRNA. Western blot images indicate that antibody binding was increased in blood samples collected after inoculation compared to samples collected before inoculation.

[0235] Example 23

[0236] PCR amplification of the emmL coding region from the pAc / emm55 plasmid was performed using high-fidelity Taq DNA polymerase. In one embodiment, this amplicon was designed to have a proximal element including a restriction endonuclease site compatible with the pSF-CMV_T7 vector to minimize potential repressive elements within the 5' and 3' UTRs of pSFCMVT7 / emmL.

[0237] The proximal design elements include NcoI and XhoI restriction endonuclease recognition sites, an optimal translation initiation sequence (SEQ ID NO: 4 or 5), and two additional stop codons downstream of the emmL coding region (SEQ ID NO: 6), relative to the activity of T7 RNA polymerase. The resulting amplicon can be inserted into a PCR cloning vector, for example... II-TOPO. The resulting plasmid can be used to transform *E. coli*, with positive transformation performed on agar plates containing a selective antibiotic that matches the PCR cloning vector, for example... In the case of II-TOPO, agar plates containing Luria Bertani culture medium supplemented with 50-100 μg / mL kanamycin or carbenicillin.

[0238] Bacterial cultures containing the obtained plasmids can be amplified by growth in liquid antibiotic-selective media, such as... In the case of II-TOPO, Luria Bertani culture medium supplemented with 50-100 μg / mL kanamycin or carbenicillin is used. Plasmid DNA can be prepared from these bacterial cultures using methods known to those skilled in the art. Then, restriction endonucleases NcoI and XhoI can be used to extract the plasmid DNA. The emmL coding region flanked by proximal design elements was excised from the II-TOPO plasmid DNA. The emmL coding region of the DNA fragment flanked by proximal design elements could then be inserted into the pSF-CMV_T7 vector, which had been digested with NcoI and XhoI. Following ligation, bacterial transformation, selection of positive transformations on agar plates containing 50–100 μg / mL kanamycin, amplification in Luria Bertani medium supplemented with 50–100 μg / mL kanamycin, and plasmid DNA preparation using methods known to those skilled in the art, the resulting plasmid pSF / emmL could be used as a template for in vitro mRNA synthesis after linearization with the restriction endonuclease XhoI. The resulting mRNA and predicted amino acid sequences are shown in SEQ ID NO: 13 and 14, respectively.

[0239] Example 24

[0240] High-fidelity Taq DNA polymerase is used to amplify the emmL coding region from a pAc / emm55 plasmid with a proximal design element comprising a restriction endonuclease site compatible with the multi-connector region of pT7XLUTR. In one embodiment, this may further include an optimal translation initiation sequence (SEQ ID NO: 4 or 5) and two additional stop codons (SEQ ID NO: 6). The complete sequence of this embodiment is shown in SEQ ID 15. The resulting amplicons can be inserted into a PCR cloning vector, such as... In II-TOPO. Following bacterial transformation, selection, amplification, and plasmid DNA preparation as described in Example 23, the resulting plasmids can be digested with restriction endonucleases SacI and SpeI to extract... The emmL coding region containing proximal design elements was excised from II-TOPO. This emmL coding region could then be ligated into a pT7XLUTR plasmid vector digested with SacI and SpeI to generate pT7XLUTR / emmL. The resulting pT7XLUTR / emmL plasmid could then be used to transform *E. coli*.

[0241] Following the selection, amplification, and plasmid DNA preparation described in Example 23, the resulting pT7XLUTR / emmL plasmid was used as a template for in vitro mRNA synthesis after linearization with the restriction endonuclease BamHI. The resulting mRNA and the predicted amino acid sequences are shown in SEQ ID NO:15 and 14, respectively.

[0242] Example 25

[0243] The emmL coding region was amplified from the pAc / emm55 plasmid with minimum 5' and 3' UTRs using high-fidelity Taq DNA polymerase. The T7 RNA polymerase promoter sequence (SEQ ID NO:8) was added as a proximal design element along with the optimal translation initiation sequence (SEQ ID NO:4 or 5), two additional stop codons (SEQ ID NO:6), and an XhoI restriction endonuclease site.

[0244] The resulting PCR product can be inserted into a PCR cloning vector, for example... In II-TOPO, following bacterial transformation, selection, amplification, and plasmid DNA preparation as described in Example 23, the resulting plasmid pT7 / emmL was used as a template for in vitro mRNA synthesis after linearization with the restriction endonuclease XhoI. The resulting mRNA and the predicted amino acid sequences are shown as SEQ ID NO:16 and 14, respectively.

[0245] In Examples 23-25, an in vitro transcription reaction can be used to generate mRNA encoding emmL. In this reaction, the resulting mRNA can be modified to improve the stability and translation efficiency of the mRNA and EmmL protein, and to reduce the immunogenicity of the mRNA. For example, modified nucleic acids can be ligated to the 5' end of the emmL mRNA, such as, but not limited to, anti-reverse cap analogs [ARCA, P1-(5'-(3'-O-methyl)-7-methyl-guanosine)P3-(5'-(guanosine))triphosphate], N1-methylguanosine, 2'-fluoroguanosine, 7-denitroguanosine, inosine, 8-oxoguanosine, 2-aminoguanosine, LNA-guanosine, and 2-azidoguanosine.

[0246] In another example, a poly(A) tail of approximately 50-200 monophosphate adenosine monophosphates in length can be attached to the 3' end of emmL mRNA, or a 5' modified nucleotide cap and a poly(A) tail can be added to emmL mRNA.

[0247] emmL mRNA can be synthesized using ribonucleotide analogs. Chemical modifications can be performed at this stage to further improve translation efficiency and stability. Examples include 5-methylcytidine-5'-triphosphate, pseudouridine-5'-triphosphate, 2-thiouridine-5'-triphosphate, and N1-methylpseudouridine-5'-triphosphate.

[0248] SEQ ID NO:4. Degenerate DNA sequence of the optimal translation initiation sequence. R is A or G, Y is C or T, M is A or C, and V is A, C, or G.

[0249] RYMRMVATGGC

[0250] SEQ ID NO:5. DNA sequence of the optimal translation initiation sequence.

[0251] ATAGCCATGGC

[0252] SEQ ID NO:6. A DNA sequence containing two stop codons.

[0253] TAATGA

[0254] SEQ ID NO:7. DNA sequences of restriction endonuclease recognition sites of SacI, NotI, BglII, EcoRV and SpeI.

[0255] GAGCTCGCGGCCGCAGATCTGATATCACTAGT

[0256] DNA sequence of RNA polymerase promoter SEQ ID NO:8.T7.

[0257] TAATACGACTCACTATAG

[0258] SEQ ID NO:9. 50 ribonucleotides in the 5' untranslated region of the Xenopus laevis β-globin gene.

[0259] aagcuucuuguucuuuuugcagaagcucagaauaaacgcucaacuuuggc

[0260] SEQ ID NO:10. 74 ribonucleotides in the 3' untranslated region of the Xenopus laevis β-globin gene.

[0261] cuuuuugaugccauugccgacgcccuuggcaaggguuaccacuaaaccagccucaagaacacccgaauggaguc

[0262] SEQ ID NO:11. DNA sequence of restriction endonuclease recognition sites of BamHI, EcoRI and XbaI.

[0263] GGATCCGAATTCTCTAGA

[0264] SEQ ID NO:12. DNA sequence for preparing a synthetic double-stranded DNA molecule for pT7XLUTR. Restriction endonuclease recognition sites for T7 RNA polymerase, SacI, NotI, BglII, EcoRV, SpeI, and BamHI, EcoRI, and XbaI are underlined.

[0265] TAATACGACTCACTATAG AAGCTTCTTGTTCTTTTTGCAGAAGCTCAGAATAAACGCTCAACTTTGGC G AGCTCGCGGCCGCAGATCTGATATCACTAGT CTTTTTGATGCCATTGCCGACGCCCTTGGCAAGGGTTACCACTAAACCAGCCTCAAGAACACCCGAATGGAGTC GGATCCGAATTCTCTAGA

[0266] SEQ ID NO:13 (RNA sequence of Example 23). The synthetic mRNA sequence after in vitro mRNA synthesis from the plasmid template, written relative to the mRNA molecule from 5' to 3'. During in vitro transcription, an anti-reverse cap analog (ARCA) was added to the 5' end of the transcript, and approximately 50–200 adenosine monophosphate (ATP) molecules were added. 50-200The 5' lowercase letter is added to the 3' end of the transcript. The 5' lowercase letter represents the 5' untranslated region (UTR) from the pSF-T7_CMV plasmid vector. The guanine (g) immediately following the ARCA cap is a +1 ribonucleotide produced by T7 RNA polymerase. Bold text indicates the added ribonucleotide to form the 5' NcoI restriction endonuclease site (underlined) and the optimal translation initiation sequence. Uppercase letters indicate the coding sequence of emmL. At the 3' end of the mRNA sequence, bold text indicates two additional stop codons. The 3' XhoI restriction endonuclease site is underlined.

[0267]

[0268] AGACGCAAGCCGUAAAGGUCUUCGUCGUGACUUGGACGCAUCACGUGAAGCUAAGAAACAAGUUGAAAAAGCUUUAGAAGAAGCAAACAGCAAAUUAGCGGCUCUUGAAAAACUUAACAAAGAGCUUGAAGAAAG CAAGAAAUUAACAGAAAAAGAAAAAAGCUGAGCUACAAGCGAAACUUGAAGCAGAAGCAAAAGCACUCAAAGAACAAUUAGCGAAACAAGCUGAAGAACUUGCAAAACUAAGAGCUGGAAAAGCAUCAGACUCACA AACCCCUGAUGCAAAACCAGGAAACAAAGUUGUUCCAGGUACAGGUCAAGCACCACAAGCAGGCACAAAACCUAACCAAAACAAAGCACCAAUGAAGGAAACUAAGAGACAGUUACCAUCAACAGGUGAAGCAGC UAAUCCAUUCUUUACAGCGGCAGCCCUUACUGUUAUGGCAACAGCUGGAGUAGCAGCAGUUGUAAAACGCAAAGAAGAAAACGAAGCUGAAUUCUGCAGAUAUCCAUCACACUGGCGGCCGCGACUCUAGuaauga cucgag (a 50-200 )

[0269] SEQ ID NO:14 (Predicted protein sequences for Examples 23-25). One letter predicts the amino acid sequence of the emmL transcripts generated in Examples 2-4. * indicates a stop codon.

[0270] *

[0271] SEQ ID NO:15 (RNA sequence of Example 24). Synthetic mRNA sequence derived from an emmL in vitro mRNA synthesis from a pT7XLURT plasmid template linearized with the restriction endonuclease BamHI, written from 5' to 3' relative to the mRNA molecule. During in vitro transcription, an anti-reverse cap analog (ARCA) was added to the 5' end of the transcript, and approximately 50–200 adenosine monophosphate (ATP) molecules were added. 50-200 The guanine (g) is added to the 3' end of the transcript. Immediately following the ARCA cap is a +1 ribonucleotide produced by T7 RNA polymerase. The lowercase 5' comes from the 5' untranslated region of the Xenopus β-globin gene. Bold text indicates ribonucleotides added to form the optimal translation initiation sequence. The SacI restriction endonuclease site at the 5' of the emmL coding region is underlined. Uppercase letters indicate the coding sequence of emmL. The lowercase 3' of the coding region is the 3' untranslated region of the Xenopus β-globin gene. At the 3' end of the mRNA sequence, bold text indicates two additional stop codons. The SpeI and BamHI restriction endonuclease sites are underlined.

[0272] gaagcuucuuuguucuuugcagaagcucagaauaaacgcucaacuuuggc gagcuc augccAUGGCUAAAAAUACCACGAAUAGACACUAUUCGCUUAGAAAAUAAAAACAGGAACGGCUUCAGUAGCAGUAGCUUUGACUUUUAGGGACAGGUCUGGUAGCAGGCAGACAGUAAAAAGCAAGCCAAACAGACCAUCUCAGACCAAUAACAGAUAUAUAUCAAGAAAGACAACGUUUACAGGAUUUAAAGUAAGUAGUUUUCAAGACCUGGAUAAAUCGUUCAGGAAUACAUUCAGCAAUACUACGACGAAAGAAAAAGACGUGGAAGUACUCUACUGGUACGCAACCUACUUAAGAAUUAUGACGAAUUUGACAAAAUUAUGACGAAUUGUAAAAAUUAGCUGGUAAAAAAUUAGCUGUAAAAAAUUAGCUGUAAAAUUAGCUUAGA

[0273] AAACCAGGAAACAAAGUUGUUCCAGGUACAGGUCAAGCACCACAAGCAGGCACAAAACCUAACCAAAAAGCACCAAUGAAGGAAACUAAGAGACAGUUACCAUCAACAGGUGAAGCCAGCCUUACUUACUUAGGCAACAGCUGGAGUAGCAGCAGUUGUAAAAGCCAAAGAAAAACGAAGCUGAAUUCUGCAGAUAUCCAUCACACUGGCGCCGCGACUUACUAGuaauga acuagu cuuuuuugaugccauugccgacgcccuuggcaaggguuaccacuaaaccagccucaagaacacccgaauggaguc ggaucc (a 50-200 )

[0274] SEQ ID NO:16 (RNA sequence of Example 24). The synthetic mRNA sequence after in vitro mRNA synthesis from the plasmid template, written relative to the mRNA molecule from 5' to 3'. During in vitro transcription, an anti-reverse cap analog (ARCA) was added to the 5' end of the transcript, and approximately 50–200 adenosine monophosphate (ATP) molecules were added. 50-200 A guanine (g) is added to the 3' end of the transcript. Immediately following the ARCA cap is a +1 ribonucleotide produced by T7 RNA polymerase. Bold text indicates ribonucleotides added to form the optimal translation initiation sequence. Capital letters indicate the coding sequence for emmL. At the 3' end of the mRNA sequence, bold text indicates two additional stop codons. The 3' XhoI site is underlined.

[0275] gauagccAUGGCUAAAAAUACCACGAAUAGACACUAUUCGCUUAGAAAAUUAAAAACAGGAACGGCUUCAGUAGCAGUAGCUUUGACUGUUUUAGGGACAGGACUGGUAGCAGGGCAGACAGUAAAAGCAAGCCAAACAGAACCAUCUCAGACCAAUAACAGAUUAUAUCAAGAAAGACAACGUUUACAGGAUUUAAAAAGUAAGUUC AAGACCUGAAAAAAUCGUUCAGAGGGAUACAUUCAGCAAUACUACGACGAAGAAAAGAACAGUGGAAGUAACUCUAACUGGUACGCAACCUACUUAAAAGAAUUAAAUGACGAAUUUGAACAAGCUUAUAAUGAACUUAGUGGUGAUGGUGUAAAAAAAUUAGCUGCAAGUUUGAUGGAAGAAAGAGUCGCUUUAAGAGACGAAAUCGAUC

[0276]

[0277]

[0278] Example 26

[0279] Codon optimization algorithms were also used to synthesize emm55 mRNA. Emm55 expression was measured and compared to determine the relative increase in protein synthesis and stability in mammalian cells. Each mRNA was synthesized in vitro using wild-type uridine (WT), and N1-methylpseudouridine (N1) was shown to improve protein expression.

[0280] Three different algorithms were used to optimize the nucleotide sequence of emm55, from expression in *Streptococcus pyogenes* to expression in humans. Codon optimization was performed using the JCat algorithm (Grote 2005) and two proprietary algorithms (FreqDist and MostFreq) run by the Karolinska Institute. mRNAs designed using these three algorithms were... Biotechnologies used wild-type (WT) uridine and chemically modified N1-methylpseudouridine (N1) for in vitro synthesis, which improved mRNA stability and protein expression levels in vitro (Svitkin 2017) and in vivo (Pardi 2015). The template DNA sequences for each mRNA are shown as SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19. As part of the design, the BbsI and BspQI restriction endonuclease sites were removed by base substitution from emm55JCat and emm55MostFreq to facilitate cloning for in vitro mRNA synthesis. In plasmid vectors.

[0281] mRNA and Biotechnologies receives each tube of mRNA together and adds it as a stock item so that each tube can be tracked independently. The mRNA is stored at -80°C. Quality control testing is performed using RD 3-75.1 to verify purity and integrity, the difference being that the mRNA is produced by… Biotechnologies were divided equally, instead of following the SOP (Standard Operating Procedure).

[0282] mRNA was transiently transfected into attachment-enhanced HEK sublines HEK293T-AE and B16-F10 cells using RD 3-79.1 (2.5 μg mRNA / well). Cells were transfected with pAc / emm55 using RD 3-34.2 (4 μg pDNA / well). Cells were lysed in RIPA buffer 2–48 h post-transfection using RD 3-83.1, and protein concentrations of the lysates were determined by BCA analysis using RD 3-14.2.

[0283] EGFP mRNA and pDNA were used as a positive control for transfection and a negative control for Emm55 expression. Initially, lysates were analyzed using denaturing SDS-PAGE, Western blotted, stained with chicken anti-Emm55, and detected using chemiluminescence on RD 3-74.1. The bands corresponding to the Emm55 size consistently overwhelmed the peroxidase detection reaction, resulting in unquantifiable bands. Suitable dilutions for chicken anti-Emm55 or goat anti-chicken horseradish peroxidase could not be identified to increase the high end of the detection range. Therefore, ProteinSimple Wes using chicken anti-Emm55 was used. TM Capillary electrophoresis system was used for Emm55 protein detection. This system resolves proteins and protein / antibody complexes in a reducing SDS-PAGE capillary to provide antibody binding quantification, such as Western blot data. These analyses were performed using the manufacturer's provided protocols.

[0284] Wes TM The analysis measured the level of Emm55 in cell lysates, which was normalized to the level of ERK1, a serine / threonine kinase regulated by phosphorylation rather than protein levels, thus normalizing the data within the appropriate protein for the cell or tissue type. This method has advantages over internal total protein normalization because it can be reused with Emm55 detection, provides sample loading control, and reduces the number of sample assays that need to be performed by half. To establish the linearity of this analysis, a standard curve was constructed using 10 ng to 30 pg rEmm55 from 1.5 μg of untransfected HEK293T-AE lysate. Wes TM The antibody dilutions analyzed were 1:100 for chicken anti-Emm55 and 1:100 for goat anti-chicken-HRP.

[0285] Wes TM The analysis assembly instructions refer to inputting protein concentrations, not masses. Cell lysates of 0.1, 0.25, and 0.5 μg / μL were used for Wes... TM The experiment. Although 0.5 μg / μL of B16-F10 cell lysate in Wes TM Results were obtained within the dynamic range, but the expression levels produced by 0.25 and 0.5 μg / μL HEK293T lysates exceeded the detection capability at peak expression time points. A concentration of 0.1 μg / μL lysate was found to be optimal throughout the time course, although lower concentrations could be used to provide additional dynamic range when high expression levels were expected. However, such lower input concentrations might miss low but biologically significant expression levels, particularly at early and late time points.

[0286] By using the Wes of Emm55 TMData standardization was performed by dividing the numerical output value by the ERK1 output value. In the data replication experiment, individual samples were standardized, and then the mean ± standard deviation (n = 3) was calculated. At one time point (12 hours after 0.1 μg / μL emm55 JCat-WT transfection), abnormally low ERK1 levels resulted in a single drop in standardized data point in the Grubbs-based extreme studentity bias test. In this case, the mean was calculated based on the remaining two replicate data points. Significant differences between the means were determined using one-way ANOVA and Bonferroni post-hoc tests performed with GraphPad Prism software. The important findings were confirmed using Tukey post-hoc tests.

[0287] The JCat algorithm was used to calculate the codon fitness index (CAI) for each codon in emm55. This algorithm returns a score of 1.0 if the mRNA sequence is perfectly optimized for translation in human cells. Figure 12 shows the relative fitness of emm55 before and after optimization using JCat. The relative fitness graph of emm55 is shown below. Figure 12A and 12B As shown.

[0288] The results showed that codon-modified emm55 mRNA drove robust expression of the Emm55 peptide after transient transfection of human HE293T cells and mouse B16-F10 melanoma cells in vitro. The comparison of protein levels with pAc / emm55 (DNA) transfection levels was not quantitative because a single copy of pDNA produces multiple copies of mRNA, but it did demonstrate the presence of WT and N1-modified emm55JCat in HEK293 cells between 4 and 12 hours post-transfection. The peak Emm55 expression of emm55JCat mRNA occurred at approximately 12 hours post-transfection and was detectable at 48 hours. sequence list <110> TuHURA Biosciences, Inc. <120> Modified mRNA for multi-cell transformation <130> MOR.205XC3 <150> US 15 / 114,943 <151> 2016-07-28 <150> US 15 / 583,599 <151> 2017-05-01 <150> US 62 / 163,446 <151> 2015-05-19 <160> 19 <170> PatentIn version 3.5 <210> 1 <211> 1854 <212> RNA <213> Artificial Sequence <220> <223> Theoretical emm55 mRNA sequence <220> <221> misc_feature <222> (1) (2) <223> 5' ARCA methylguanosine cap <220> <221> misc_feature <222> (3).(132) <223> 5' untranslated region <220> <221> misc_feature <222> (133).(135) <223> Start codon <220> <221> misc_feature <222> (133).(1804) <223> emm55 gene (emm55 gene) <220> <221> misc_feature <222> (1806).(1854) <223> Polyadenylate tail, with a minimum of 50 adenosine molecules. <400> 1 gggagaagau cuuugucgau ccuaccaucc acucgacaca cccgccagcg gccgcugcca 60 agcuuccgag cucaagcuuc gaauucugca gucgacggua ccgcggggccc gggauccaua 120 Aggagcauaa aaoggcuaaaaaccacg aaagacacu auucgcuuag aaaaaaaaa 180 ashaggaacgg cucaguagc aguagcuuug acuguuuuag ggagacu ggquagggg 240 cagacaguaa aagcaagcca aacagaacca ucucagacca auaacagauu auaucaagaa 300 agacaacgu uacaggauu aaaaaguag uacagacc ugaaaaucg ucagaggga 360 uacauucagc auaacuacga cgaagaaag aacaguggaa guaacuuaa cugguacgca 420 accuacuuaa aaaaaaaa ugacgaauu u gacaagcuu auaaugaacu uauggogau 480 gguuaaaaaaauaagcugc aaoooogag gagaagag ucgcuuuaag agacgaaac 540 gaucagaua agaaaauauc agagaaua aaaaaaaagc ugagagcaaa agagaagaa 600 uaaaaaaaaaaaaaaaaaaaaaagg acgugagcuu gagcaugcug ccuaugcagc agaugcaag 660 aaacaugaag auauguca auccaugucu cucguacuaa uggauaaga agaggagcgu 720 cauaaacuag agcaucauu agacacggcu aaagcugagc uuguuaaaaa agagcagag 780 uacaguuag ucaaggcaa ucuagauaaaaaaag aaaaaaaaa ugagagcua 840 gcgaaagaaa gugcuauuag ugauuugacu gagcagauua cugcuaagaa ggcugaagua 900 gaaaaaaaa cucaagauuu agcugcuaag ucugcugaa ucaggaaaa agagcugaa 960 aaagaucgcc aacagcauau guacgaagcg uuauugagcc aguacaaga aaagougag 1020 aaaacaagagc aagagcuugc uaagcuaaaa caucauaa caucauaa 1080 gguaaugcua aggaauugau agcuaaguug ucugcugaa augaaaugcua agcaagcgac 1140 aaagcaaac ugagagaca aaacaagau ucagaagcga gccguaaagg ucuucgucgu 1200 gacuuggacg caucacguga agcuaagaa caaguagaa aagauuuagc aaacuugacu 1260 gcugaacuug auaagguuaa agagauaaa caauuucag acgcaagccg uaaaggucuu 1320 cgucgugacu uggacgcauc acgugaagcu aaaaaaaag uuuuagaagaa 1380 gcaacagca auuuagcggc ucuuaaaa cuaaaaag agcuagaag aagcaaaa 1440 uaaaaaaaaaaaaaagc ugagcuaaaa gcgaaacuug aagcagaagc aaaagcacuc 1500 aaagaacaau uagcgaaaca agcugaagaa cuugcaaaac uaagagcugg aaaagcauca 1560 gacucacaaa ccccugaugc aaaaccagga aacaaaguug uuccagguac aggucaagca 1620 ccacaagcag gcacaaaacc uaaccaaaac aaagcaccaa ugaaggaaac uaagagacag 1680 uuaccaucaa caggugaagc agcuaaucca uucuuuacag cggcagcccu uacuguuaug 1740 gcaacagcug gaguagcagc aguuguaaaa cgcaaagaag aaaacgaagc ugaauucugc 1800 agauaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaa 1854 <210> 2 <211> 557 <212> PRT <213> Artificial Sequence <220> <223> Emm55 Protein Sequence <400> 2 Met Ala Lys Asn Thr Thr Asn Arg His Tyr Ser Leu Arg Lys Leu Lys 1 5 10 15 Thr Gly Thr Ala Ser Val Ala Val Ala Leu Thr Val Leu Gly Thr Gly 20 25 30 Leu Val Ala Gly Gln Thr Val Lys Ala Ser Gln Thr Glu Pro Ser Gln 35 40 45 Thr Asn Asn Arg Leu Tyr Gln Glu Arg Gln Arg Leu Gln Asp Leu Lys 50 55 60 Ser Lys Phe Gln Asp Leu Lys Asn Arg Ser Glu Gly Tyr Ile Gln Gln 65 70 75 80 Tyr Tyr Asp Glu Glu Lys Asn Ser Gly Ser Asn Ser Asn Trp Tyr Ala 85 90 95 Thr Tyr Leu Lys Glu Leu Asn Asp Glu Phe Glu Gln Ala Tyr Asn Glu 100 105 110 Leu Ser Gly Asp Gly Val Lys Lys Leu Ala Ala Ser Leu Met Glu Glu 115 120 125 Arg Val Ala Leu Arg Asp Glu Ile Asp Gln Ile Lys Lys Ile Ser Glu 130 135 140 Glu Leu Lys Asn Lys Leu Arg Ala Lys Glu Glu Glu Leu Lys Asn Lys 145 150 155 160 Lys Glu Glu Arg Glu Leu Glu His Ala Ala Tyr Ala Ala Asp Ala Lys 165 170 175 Lys His Glu Glu Tyr Val Lys Ser Met Ser Leu Val Leu Met Asp Lys 180 185 190 Glu Glu Glu Arg His Lys Leu Glu Gln Ser Leu Asp Thr Ala Lys Ala 195 200 205 Glu Leu Val Lys Lys Glu Gln Glu Leu Gln Leu Val Lys Gly Asn Leu 210 215 220 Asp Gln Lys Glu Lys Glu Leu Glu Asn Glu Glu Leu Ala Lys Glu Ser 225 230 235 240 Ala Ile Ser Asp Leu Thr Glu Gln Ile Thr Ala Lys Lys Ala Glu Val 245 250 255 Glu Lys Leu Thr Gln Asp Leu Ala Ala Lys Ser Ala Glu Ile Gln Glu 260 265 270 Lys Glu Ala Glu Lys Asp Arg Gln Gln His Met Tyr Glu Ala Phe Met 275 280 285 Ser Gln Tyr Lys Glu Lys Val Glu Lys Gln Glu Gln Glu Leu Ala Lys 290 295 300 Leu Lys Gln Leu Glu Thr Ile Asn Asn Asn Leu Leu Gly Asn Ala Lys 305 310 315 320 Asp Met Ile Ala Lys Leu Ser Ala Glu Asn Glu Gln Leu Ala Ser Asp 325 330 335 Lys Ala Lys Leu Glu Glu Gln Asn Lys Ile Ser Glu Ala Ser Arg Lys 340 345 350 Gly Leu Arg Arg Asp Leu Asp Ala Ser Arg Glu Ala Lys Lys Gln Val 355 360 365 Glu Lys Asp Leu Ala Asn Leu Thr Ala Glu Leu Asp Lys Val Lys Glu 370 375 380 Asp Lys Gln Ile Ser Asp Ala Ser Arg Lys Gly Leu Arg Arg Asp Leu 385 390 395 400 Asp Ala Ser Arg Glu Ala Lys Lys Gln Val Glu Lys Ala Leu Glu Glu 405 410 415 Ala Asn Ser Lys Leu Ala Ala Leu Glu Lys Leu Asn Lys Glu Leu Glu 420 425 430 Glu Ser Lys Lys Leu Thr Glu Lys Glu Lys Ala Glu Leu Gln Ala Lys 435 440 445 Leu Glu Ala Glu Ala Lys Ala Leu Lys Glu Gln Leu Ala Lys Gln Ala 450 455 460 Glu Glu Leu Ala Lys Leu Arg Ala Gly Lys Ala Ser Asp Ser Gln Thr 465 470 475 480 Pro Asp Ala Lys Pro Gly Asn Lys Val Val Pro Gly Thr Gly Gln Ala 485 490 495 Pro Gln Ala Gly Thr Lys Pro Asn Gln Asn Lys Ala Pro Met Lys Glu 500 505 510 Thr Lys Arg Gln Leu Pro Ser Thr Gly Glu Ala Ala Asn Pro Phe Phe 515 520 525 Thr Ala Ala Ala Leu Thr Val Met Ala Thr Ala Gly Val Ala Ala Val 530 535 540 Val Lys Arg Lys Glu Glu Asn Glu Ala Glu Phe Cys Arg 545 550 555 <210> 3 <211> 179 <212> RNA <213> Artificial Sequence <220> <223> Theoretically untranslated mRNA sequence of SEQ ID NO: 1 <220> <221> misc_feature <222> (1) (2) <223> 5' ARCA methylguanosine cap <220> <221> misc_feature <222> (3).(132) <223> 5' untranslated region <220> <221> misc_feature <222> (133).(135) <223> Start codon <220> <221> misc_feature <222> (136).(179) <223> Polyadenylate tail, with a minimum of 50 adenosine molecules. <400> 3 gggagaagau cuuugucgau ccuaccaucc acucgacaca cccgccagcg gccgcugcca 60 120 aggagcauaa aaaugaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 179 <210> 4 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> Degenerate DNA sequence with optimal translation start sequence (Degenerate DNA sequence of an optimal translation initiation sequence) <220> <221> misc_feature <222> (1).(1) <223> R is either A or G <220> <221> misc_feature <222> (2).(2) <223> Y is C or T <220> <221> misc_feature <222> (3).(3) <223> M is either A or C <220> <221> misc_feature <222> (4).(4) <223> R is either A or G <220> <221> misc_feature <222> (5).(5) <223> M is either A or C <220> <221> misc_feature <222> (6).(6) <223> V is A, C, or G <400> 4 rymrmvatgg c 11 <210> 5 <211> 11 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of an optimal translation initiation sequence. <400> 5 atagccatgg c 11 <210> 6 <211> 6 <212> DNA <213> Artificial Sequence <220> <223> A DNA sequence containing two stop codons. <400> 6 taatga 6 <210> 7 <211> 32 <212> DNA <213> Artificial Sequence <220> <223> DNA sequences for restriction endonuclease recognition sites of SacI, NotI, BglII, EcoRV, and SpeI. (DNA sequence of the restriction endonuclease recognition sites for SacI,NotI, BglII, EcoRV and SpeI) <400> 7 gagctcgcgg ccgcagatct gatatcacta gt 32 <210> 8 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence of the T7 RNA polymerase promoter <400> 8 taatacgact cactatag 18 <210> 9 <211> 50 <212> RNA <213> African clawed frog (Xenopus laevis) <220> <221> misc_feature <222> (1).(50) <223> 5' untranslated region <400> 9 aagcuucuug uucuuuuugc agaagcucag aauaaacgcu caacuuuggc 50 <210> 10 <211> 74 <212> RNA <213> African clawed frog (Xenopus laevis) <220> <221> misc_feature <222> (1).(74) <223> 3' untranslated region <400> 10 cuuuuugaug ccauugccga cgcccuuggc aaggguuacc acuaaaccag ccucaagaac 60 acccgaaugg aguc 74 <210> 11 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> DNA sequences for restriction endonuclease recognition sites of BamHI, EcoRI, and XbaI. (DNA sequence of the restriction endonuclease recognition sites for BamHI,EcoRI and XbaI) <400> 11 ggatccgaat tctctaga 18 <210> 12 <211> 192 <212> DNA <213> Artificial Sequence <220> <223> DNA sequence for preparing the synthetic double-stranded DNA molecule pT7XLUTR (DNA sequence of the synthetic double stranded DNA molecule used to preparepT7XLUTR) <400> 12 taatacgact cactatagaa gcttcttgtt ctttttgcag aagctcagaa taaacgctca 60 actttggcga gctcgcggcc gcagatctga tatcactagt ctttttgatg ccattgccga 120 cgcccttggc aagggttacc actaaaccag cctcaagaac acccgaatgg agtcggatcc 180 gaattctcta ga 192 <210> 13 <211> 1851 <212> RNA <213> Artificial Sequence <220> <223> synthesis <400> 13 gggagagau cuuugucgau ccuaccaucc acucgacaca cccgccagcg gccgcugcca 60 agcuuccgag cuaagcuuc gaauucgca gucgacggua ccgcgggcccc gggauccaua 120 aggagcauaa aaauagccau ggcuaaaaau accacgaaua gacacuauuc gcuuagaaaa 180 uuaaaaacag gaacggcuuc aguagcagua gcuuugacug uuuaagggac aggacuggua 240 gcagggcaga caguaaaagc aagccaaaca gaaccaucuc agaccaauaa cagauuauau 300 caagaaagac aacguuuaca gguauuaaaa aguaauguuuc aagaccugaa aaaucguuca 360 gaggggauaca uucagcaaua cuacgacgaa gaaaagaaca guggaaguaa cucuaacugg 420 uacgcaaccu acuaaaaga auuaaugac gauuugaac aagcuuauaa ugaacuagu 480 ggugauggug uaaaaaaaauu agcugcaagu uugauggaag aagagucgc uuuaagagac 540 gaaucgauc agauuaagaa aauacagaa gaauuaaaaa auaagcugag agcaaaagaa 600 gaagaauuaa aaaauaaaaa agagaacgu gagcuugagc augcugccua ugcagcagau 660 gcaaagaaac augaagaaua ugucaaaucc augucucug uacuaaugga uaaagaagag 720 gagcgucaua aacuagagca aucauuagac acggcuaaag cugagcuugu uaaaaaagag 780 caagaguuac aguuagucaa aggcaaucua gaucaaaaag aaaaagaacu agaaaaugaa 840 gagcuagcga aagaaagugc uauuagugau uugacugagc agauuacugc uaagaaggcu 900 gaaguagaaa aauuaacuca aguauuagcu gcuaagucug cugaaauuca ggaaaaagaa 960 gcugaaaaag aucgccaaca gcauauguac gaagcguuuua ugagccagua caaagaaaaa 1020 guugagaaac aagagcaaga gcuugcuaag cuaaaacaac uugaaaccau caacaacaau 1080 cuauuaggua augcuaagga uaugauagcu aaguugucug cugaaaauga acaauuagca 1140 agcgacaaag caaaacuuga agaaccaac agauuuucag aagcgagccg uaaaggucuu 1200 cgucgugacu uggacgcauc acgugaagcu aagaaacaag uugaaaaaga uuuagcaac 1260 uugacugcug aacuugauaa gguuaaaga gauaaacaaa uuucagacgc aagccguaaa 1320 ggucuucguc gugacuugga cgcaucacgu gaagcuaaga aacaaguuga aaaagcuua 1380 gaagaagcaa acagcaaauu agcggcucuu gaaaaacuua acaaagagcu ugaagaaagc 1440 aagaaauuaa cagaaaaaga aaaagcugag cuacaagcga aacuugaagc agaagcaaaa 1500 gcacucaaag aacaauuagc gaaacaagcu gaagaacuug caaaacuaag agcuggaaaa 1560 gcaucagacu cacaaacccc ugaugcaaaa ccaggaaaca aaguuguucc agguacaggu 1620 caagcaccac aagcaggcac aaaaccuaac caaaacaaag caccaaugaa ggaaacuaag 1680 agacaguuac caucaacagg ugaagcagcu aauccauucu uuacagcggc agcccuuacu 1740 guuauggcaa cagcuggagu agcagcaguu guaaaacgca aagaagaaaa cgaagcugaa 1800 uucugcagau auccaucaca cuggcggccg cgacucuagu aaugacucga g 1851 <210> 14 <211> 566 <212> PRT <213> Artificial Sequence <220> <223> Synthetic <400> 14 Met Ala Lys Asn Thr Thr Asn Arg His Tyr Ser Leu Arg Lys Leu Lys 1 5 10 15 Thr Gly Thr Ala Ser Val Ala Val Ala Leu Thr Val Leu Gly Thr Gly 20 25 30 Leu Val Ala Gly Gln Thr Val Lys Ala Ser Gln Thr Glu Pro Ser Gln 35 40 45 Thr Asn Asn Arg Leu Tyr Gln Glu Arg Gln Arg Leu Gln Asp Leu Lys 50 55 60 Ser Lys Phe Gln Asp Leu Lys Asn Arg Ser Glu Gly Tyr Ile Gln Gln 65 70 75 80 Tyr Tyr Asp Glu Glu Lys Asn Ser Gly Ser Asn Ser Asn Trp Tyr Ala 85 90 95 Thr Tyr Leu Lys Glu Leu Asn Asp Glu Phe Glu Gln Ala Tyr Asn Glu 100 105 110 Leu Ser Gly Asp Gly Val Lys Lys Leu Ala Ala Ser Leu Met Glu Glu 115 120 125 Arg Val Ala Leu Arg Asp Glu Ile Asp Gln Ile Lys Lys Ile Ser Glu 130 135 140 Glu Leu Lys Asn Lys Leu Arg Ala Lys Glu Glu Glu Leu Lys Asn Lys 145 150 155 160 Lys Glu Glu Arg Glu Leu Glu His Ala Ala Tyr Ala Ala Asp Ala Lys 165 170 175 Lys His Glu Glu Tyr Val Lys Ser Met Ser Leu Val Leu Met Asp Lys 180 185 190 Glu Glu Glu Arg His Lys Leu Glu Gln Ser Leu Asp Thr Ala Lys Ala 195 200 205 Glu Leu Val Lys Lys Glu Gln Glu Leu Gln Leu Val Lys Gly Asn Leu 210 215 220 Asp Gln Lys Glu Lys Glu Leu Glu Asn Glu Glu Leu Ala Lys Glu Ser 225 230 235 240 Ala Ile Ser Asp Leu Thr Glu Gln Ile Thr Ala Lys Lys Ala Glu Val 245 250 255 Glu Lys Leu Thr Gln Asp Leu Ala Ala Lys Ser Ala Glu Ile Gln Glu 260 265 270 Lys Glu Ala Glu Lys Asp Arg Gln Gln His Met Tyr Glu Ala Phe Met 275 280 285 Ser Gln Tyr Lys Glu Lys Val Glu Lys Gln Glu Gln Glu Leu Ala Lys 290 295 300 Leu Lys Gln Leu Glu Thr Ile Asn Asn Asn Leu Leu Gly Asn Ala Lys 305 310 315 320 Asp Met Ile Ala Lys Leu Ser Ala Glu Asn Glu Gln Leu Ala Ser Asp 325 330 335 Lys Ala Lys Leu Glu Glu Gln Asn Lys Ile Ser Glu Ala Ser Arg Lys 340 345 350 Gly Leu Arg Arg Asp Leu Asp Ala Ser Arg Glu Ala Lys Lys Gln Val 355 360 365 Glu Lys Asp Leu Ala Asn Leu Thr Ala Glu Leu Asp Lys Val Lys Glu 370 375 380 Asp Lys Gln Ile Ser Asp Ala Ser Arg Lys Gly Leu Arg Arg Asp Leu 385 390 395 400 Asp Ala Ser Arg Glu Ala Lys Lys Gln Val Glu Lys Ala Leu Glu Glu 405 410 415 Ala Asn Ser Lys Leu Ala Ala Leu Glu Lys Leu Asn Lys Glu Leu Glu 420 425 430 Glu Ser Lys Lys Leu Thr Glu Lys Glu Lys Ala Glu Leu Gln Ala Lys 435 440 445 Leu Glu Ala Glu Ala Lys Ala Leu Lys Glu Gln Leu Ala Lys Gln Ala 450 455 460 Glu Glu Leu Ala Lys Leu Arg Ala Gly Lys Ala Ser Asp Ser Gln Thr 465 470 475 480 Pro Asp Ala Lys Pro Gly Asn Lys Val Val Pro Gly Thr Gly Gln Ala 485 490 495 Pro Gln Ala Gly Thr Lys Pro Asn Gln Asn Lys Ala Pro Met Lys Glu 500 505 510 Thr Lys Arg Gln Leu Pro Ser Thr Gly Glu Ala Ala Asn Pro Phe Phe 515 520 525 Thr Ala Ala Ala Leu Thr Val Met Ala Thr Ala Gly Val Ala Ala Val 530 535 540 Val Lys Arg Lys Glu Glu Asn Glu Ala Glu Phe Cys Arg Tyr Pro Ser 545 550 555 560 His Trp Arg Pro Arg Leu 565 <210> 15 <211> 1856 <212> RNA <213> Artificial Sequence <220> <223> Synthetic <400> 15 gaagcuucuu guucuuuuug cagaagcuca gaauaaacgc ucaacuuugg cgagcucaua 60 gccauggcua aaaauaccac gaauagacac uauucgcuua gaaaauuaaa aacaggaacg 120 gcuucaguag caguagcuuu gacuguuuua gggacaggac ugguagcagg gcagacagua 180 aaagcaagcc aaacagaacc aucucagacc aauaacagau uauaucaaga aagacaacgu 240 uuacaggauu uaaaaaguaa guuucaagac cugaaaaauc guucagaggg auacauuucag 300 360 aaagaauuaa augacgaauu ugaacaagcu uauaugaac uaagugguga ugguguaaaa 420 aaauuagcug caauguugau ggagaaaga gucgcuuuua gagacgaaau cgaucagauu 480 Keep going, keep going, keep going, keep going, keep going, keep going, keep going, keep going, keep going aaaaaagagg aacgugagcu ugagcaugcu gccuaugcag cagaugcaa gaagaugaa 600 gaauaguaca aauccauguc ucucguacua auggauaaag aagaggagcg ucauaaacua 660 gagcaaucau uagacacggc uaaagcugag cuuguuaaaa aagagcaaga guuacaguua 720 gucaaggca aucuaguauca aaaagaaaaa gaacuaagaaa augaagagcu agcgaaaagaa 780 agugcuauua gugauuugac ugagcagauu aucucuaaga aggcugaagu agaaaaaauua 840 900 caacagcaua ugaacgaagc guuuaugagc caguacaaag aaaaaguuga gaacaagag 960 cagagcuug cuagcuaaaacuaaaacuaaaacuaaaacuauu agguaugcu 1020 aaggaauga uagcuaagugu gucugcuga auagaaaaaaaaaaagaaagcga aaagcaaa 1080 cugagaach aaaaaaaaacagau ucagaagcg agccguaag gucuucgucg ogacuggc 1140 gcaucacgug aagcuagaa acagougaa aaagauuuag aaacuugac ugcugacuu 1200 gauaagguua aagauaa aaauuuca gacgcaagcc guaaaggucu ucgucgugac 1260 uuggacgcau cacgugaagc uaagaaaaaaaaag cacgugaagc 1320 aaauuagcgg cucuuaaaaaaaaaaaaaaaaaaaaaaaaaaaaaacaaaaaaacagaa 1380 aaaaaaaaaagg agcgaaaaaaaaaaaaaaaaaaaaaggchaagg 1440 uuagcgaaac aagcugaga acuugcaaaquaagcug gaaagcuac agacucacaa 1500 accccugaug aaaaccagg aaaaaaagu guuccaggua caggucaagc accacagca 1560 ggcacaaaac cuaaccaaaaaagcacca augaaggaaa cavagaca guuaccauca 1620 acaggugaag cagcuaaucc auucuuuaca gcggcagccc uuacuguuau ggcaacagcu 1680 ggaguagcag caguuguaaa acgcaaagaa gaaaacgaag cugaauucug cagauaucca 1740 ucacacuggc ggccgcgacu cuaguaauga acuagucuuu uugaugccau ugccgacgcc 1800 cuuggcaagg guuaccacua aaccagccuc aagaacaccc gaauggaguc ggaucc 1856 <210> 16 <211> 1720 <212> RNA <213> Artificial Sequence <220> <223> Synthetic <400> 16 gauagccaug gcuaaaaaua ccacgaauag acacuauucg cuuagaaaau uaaaaacagg 60 aacggcuuca guagcaguag cuuugacugu uuuagggaca ggacugguag cagggcagac 120 aguaaaagca agccaaacag aaccaucuca gaccaauaac agauuauauc aagaaagaca 180 acguuuacag gauuuaaaaa guaaguuuca agaccugaaa aaucguucag agggauacau 240 ucagcaauac uacgacgaag aaaagaacag uggaaguaac ucuaacuggu acgcaaccua 300 cuuaaaagaa uuaaaugacg aauuugaaca agcuuauaau gaacuuagug gugauggugu 360 aaaaaaauua gcugcaaguu ugauggaaga aagagucgcu uuaagagacg aaaucgauca 420 gauaaagaaaauaaaaaaaaaaaaaaaaaagcugaga gcaaaagaag aagauaaaa 480 aaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaaa haveaaaaaaaaaaaaaaaaaaaaaa out from outside 540 ugaagaauau cuaaucca ugucucgu acuauggau aaagaagagg agcgucauaa 600 acuagagcaa ucauuagaca cggcuaaagc ugagcuuguu aaaaagagc aagaguuaca 660 guuagucaaa ggcaaucuag aucaaaaga aaaagacua gaaaagaag agcuagcgaa 720 agaaagcu auuagugauu ugacugagca gauaacugcu aagaggcug aaguaaaaaaaaaaaaaaaaaaaaaaaa haveing ​​ha being being 780 auuaacuca gauuuagcug quaagucugc ugaaauucag gaaaaagag cugaaaaaga 840 ucgccacag cauaguacg agcguuuau gagccaguac aaagaaaag ugagaaaca 900 agagchaagg cugcuaagc uaaaaaaacu uaaaaaaacua 960 ugcuaggau augauagcua aguugucugc ugaaaugaa cauuagcaa gcgacaaagc 1020 aaaacuugaa gacaaca agauuucaga agcgagccgu aaggcuuc gucgugacuu 1080 ggacgcauca cgugaagcua agaacaagu ugaaaagau uuagcaacu agacugcua 1140 acuugauaag guuaaagaag auaaacaaau uucagacgca agccguaaag gucuucgucg 1200 ugacuuggac gcaucacgug aagcuaagaa acaaguugaa aaagcuuuag aagaagcaaa 1260 cagcaaauua gcggcucuug aaaaacuuaa caaagagcuu gaagaaagca agaaauuaac 1320 agaaaaagaa aaagcugagc uacaagcgaa acuugaagca gaagcaaaag cacucaaaga 1380 acaauuagcg aaacaagcug aagaacuugc aaaacuaaga gcuggaaaag caucagacuc 1440 acaaaccccu gaugcaaaac caggaaacaa aguuguucca gguacagguc aagcaccaca 1500 agcaggcaca aaaccuaacc aaaacaaagc accaaugaag gaaacuaaga gacaguuacc 1560 aucaacaggu gaagcagcua auccauucuu uacagcggca gcccuuacug uuauggcaac 1620 agcuggagua gcagcaguug uaaaacgcaa agaagaaaac gaagcugaau ucugcagaua 1680 uccaucacac uggcggccgc gacucuagua augacucgag 1720 <210> 17 <211> 1707 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 17 gccaccatgg ccagaacacc caccaaccgc cactacagcc tgcgcaagct ccagaccggc 60 accgccagcg tggccgtggc cctgaccgtg ctggcaccg gcctgtggc cggccagacc 120 gtgaaggcca gccagaccga gcccagccag accacaacc gcctgtacca ggagcgccag 180 cgcctgcagg acctgaaag caagttccag gacctgaaga accgcagcga gggctacatc 240 cagcagtact axacgagga gagaacagc ggcagcaca gcaactggta cgccacctac 300 ctgaaggagc tgaacgacga gttcgagcag gcctacaacg agctgagcgg cgacggcgtg 360 aagaagctgg ccgccagcct gatgaggag cgcgtggccc tgcgcgacga gatcgaccag 420 atcaagaga tcagcgagga gctgagaac aagctgcgcg ccaaggagga ggagctgaag 480 aacaagaagg aggagcgcga gctggagcac gccgcctacg ccgccgacgc siagaagcac 540 gaggagtacg tgaaaagcat gagcctggtg ctgatggaca aggagga gcgccacaag 600 ctggagcaga gcctggacac cgccaaggcc gagctggtga agaggagca ggagctgcag 660 ctggtgaagg gcaacctgga ccagaggag aaggagctgg agaacgagga gctggccaag 720 gagagcgcca tcagcgacct gaccgagcag atcaccgcca agaaggccga ggtggagaag 780 ctgacccagg acctggccgc cagagcgcc gagatccagg agaggaggc cgagaggaggc 840 cgccagcagc acatgtacga ggccttcatg agccagtaca aggaaggt ggagaagcag 900 gagcaggagc tggccaagct gaagcagctg gagaccatca acaacct gctgggcaac 960 gccaaggaca tgatcgccaa gctgagcgcc gagaacgagc agctggccag cgacaaggcc 1020 aagctggagg agcagaaca gatcagcgag gccagccgca agggctgcg ccgcgacctg 1080 gacgccagcc gcgaggccaa gagcaggtg gagagaggc tggccacct gaccgccgag 1140 ctggacaagg tgaggagga caagcagatc agcgacgcca gccgcaaggg cctgcgccgc 1200 gacctggacg ccagccgcga ggcagaag caggtggaga aggccctgga ggaggccac 1260 agcaagctgg ccgccctgga gaagctgaac aaggagctgg aggagca gaagctgacc 1320 gagaaggagaagccgagct gcaggccaag ctggaggccg agccaggc cctgaggagg 1380 cagctggcca agcaggccga ggagctggcc aagctgcgcg ccggcaggc cagcgacagc 1440 cagacccccg acgccaagcc cggcaacaag gtggtgcccg gcaccggcca ggccccccag 1500 gccggcacca agcccaacca gaacaaggcc cccatgaagg agaccaagcg ccagctgccc 1560 agcaccggcg aggccgccaa ccccttcttc accgccgccg ccctgaccgt gatggccacc 1620 gccggcgtgg ccgccgtggt gaagcgcaag gaggagaacg aggccgagtt ctgccgctac 1680 cccagccact ggcgcccccg cctgtaa 1707 <210> 18 <211> 1707 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 18 gccaccatgg ccaagaacac caccaacaga cactacagcc tgagaaagct caagaccggc 60 accgccagcg tggccgtggc cctgaccgtg ctgggcaccg gcctggtggc cggccagacc 120 gtgaaggcca gccagaccga gcccagccag accaacaaca gactgtacca ggagagacag 180 agactgcagg acctgaaaag caagttccag gacctgaaga acagaagcga gggctacatc 240 cagcagtact acgacgagga gaagaacagc ggcagcaaca gcaactggta cgccacctac 300 ctgaaggagc tgaacgacga gttcgagcag gcctacaacg agctgagcgg cgacggcgtg 360 aagaagctgg ccgccagcct gatgaggag agagtggccc tgagacga gatcgaccag 420 attack tcagcgag gctgagaac aagctgagag ccaaggag ggagctgag 480 aacaagaagg aggagaga gctggagcac gccgcctacg ccgccgacgc siagaagcac 540 gaggagtacg tgaaaagcat gagcctggtg ctgatggaca agggagga gagacacaag 600 ctggagcaga gcctggacac cgccaaggcc gagctggtga agaggagca ggagctgcag 660 ctggtgaagg gcaacctgga ccagaggag aaggagctgg agaacgagga gctggccaag 720 gagagcgcca tcagcgacct gaccgagcag atcaccgcca agaaggccga ggtggagaag 780 ctgacccagg acctggccgc cagagcgcc gagatccagg agaggaggc cgagaggaggc 840 agcagcagc acatgtacga ggccttcatg agccagtaca aggagaggt ggagaagcag 900 gagcaggagc tggccaagct gaagcagctg gagaccatca acaacct gctgggcaac 960 gccaaggaca tgatcgccaa gctgagcgcc gagaacgagc agctggccag cgacaaggcc 1020 aagctggagg agagaaca gatcagcgag gccagcagaa agggcctgag agagacctg 1080 gacgccagca gagaggccaa gaagcaggtg gagagaggc tggccacct gaccgccgag 1140 ctggacaagg tgaggagga caagcagatc agcgacgcca gcagaaaggg cctgagaga 1200 gacctggacg ccagcagaga ggccagaag caggtggaga aggccctgga ggaggccaac 1260 agcaagctgg ccgccctgga gaagctgaac aaggagctgg aggagca gaagctgacc 1320 gagaaggagaagccgagct gcaggccaag ctggaggccg agccaggc cctgaggagg 1380 cagctggcca agcaggccga ggagctggcc aagctgagag ccggcaggc cagcgacagc 1440 cagacccccg acgccaagcc cggcacaag gtggtgcccg gcaccggcca ggcccccg 1500 gccggcacca agcccaacca gacaaggcc cccatgaagg aggaccagag acagctgccc 1560 agcaccggcg agccgcca cccttctc accgccgccg cccgaccgt gatggccacc 1620 gccggcgtgg ccgccgtggt gagagaag gaggagaacg aggccgagtt ctgcagatac 1680 cccagccact gaggacccag actgtaa 1707 <210> 19 <211> 1707 <212> DNA <213> Artificial Sequence <220> <223> Synthetic <400> 19 gccaccatgg caaagaatac cacgaataga cattattcat taagaaagct caagaccggc 60 acggcatccg tggcagtggc tttgacagtg ctgggcacag gcctcgtggc cggccagaca 120 gttaaggcca gccagacaga gccatcacaa acgaataacc ggctgtacca ggagcggcaa 180 agactgcagg acttaaagtc taaatttcag gatcttaaaa accggagtga aggttatatc 240 cagcagtatt atgacgagga gaagaactcc gggagcaata gcaactggta cgcaacctat 300 ctgaaggaac ttaatgacga gtttgaacag gcttataatg aactgtccgg tgatggagta 360 aagaaattgg ccgctagtct catggaagag cgagtggctt tgcgggatga gattgaccag 420 attaagaaaa tctccgaaga gctgaaaaat aaacttaggg ctaaggagga ggagctcaaa 480 aataaaaagg aagaaagaga actggagcac gccgcgtacg ccgctgatgc taaaaaacat 540 gaggagtatg tcaagagcat gtccctggtc ctgatggaca aggaggaaga gcggcacaag 600 660 ctagtgaaag gcaattaga ccagaaggag aaagcaattag aaaatgaaga gctggcgaaa 720 gagtctgcca tatcagactt gacagagcag atcactgcta agaaggccga agtggaaaag 780 ttgacccaag acttagcagc taaatcggcc gagattcaag agaaggaggc tgagaaggac 840 cgccagcagc acatgtacga agcttttatg tcacagtaca aagagaaagt cgagaaacag 900 gagcaggaac tggccaaact gaagcagctg gagacaataa ataaataatct actgggcaac 960 gtaaggata tgatcgctaa actgagcgct gaaacgagc agttagcctc cgataaagcg 1020 aaattggaag agcaaaacaa gatctccgag gccagcagga aaggccttag aagggaccta 1080 gacgcctga gagaggccaa aaagcaggtc gagaaggacc tagcaaacct taccgccgag 1140 1200 1260 tccaaattag cggcattgga gaagctgaac aaggagctgg aggagtcgaa gaagttaacg 1320 gagaaggaa aagcagagtt gcaagcaaag ttggaggccg aggcaaaggc attaaaaga cagctcgcta aacaggcgga ggaactggca aaactgagag caggcaaggc ctccgacagt cagacgcctg atgcaaagcc tgggaataag gtggtgcctg gcacggggca ggccccccag gctggcacga agccaaatca aaacaaggct ccaatgaagg agactaagcg ccagctgcct agtaccggag aggccgccaa tccttttttc accgccgccg cccttactgt tatggccact 1620 gctggagttg cggctgtggt gaacggaa gaggaaacg aagccgaatt ttgccggtac cctagtcatt ggagaccag attgtaa

Claims

1. A nucleic acid selected from one or more of SEQ ID NO:17, SEQ ID NO:18 and SEQ ID NO:19, wherein the nucleic acid expresses a polypeptide in transfected mammalian cells.

2. The nucleic acid according to claim 1, wherein the nucleic acid is as shown in SEQ ID NO:

17.

3. The nucleic acid according to claim 1, wherein the mammalian cell is a cancer cell.

4. The nucleic acid according to claim 1, wherein the polypeptide expressed in cells is as shown in SEQ ID NO:

14.

5. The nucleic acid according to claim 1, wherein the expression of Emm55 peptide is increased in cells transformed with said nucleic acid compared to using pAC / emm55.

6. The nucleic acid according to claim 1, wherein the nucleic acid is as shown in SEQ ID NO:

19.

7. The nucleic acid according to claim 1, wherein the nucleic acid is as shown in SEQ ID NO:18.

Images