Synthetic mRNA for treating EBV-related diseases

Abstract

In particular, synthetic mRNA is provided that can specifically induce transcription of one or more Epstein-Barr virus (EBV) lysis genes in an EBV infected cell, thereby destroying the cell. Also described herein are methods of synthesizing the mRNA and methods of using the mRNA for the treatment of EBV-related diseases, such as EBV-positive cancer.

Description

[0001] Related applications

[0002] This application claims priority to U.S. Provisional Patent Application No. 63 / 462,197, filed April 26, 2023, the contents of which are incorporated herein by reference in their entirety for all purposes. Background of the Invention

[0004] A wide range of lymphocytic malignancies and two distinct types of human epithelial carcinoma, nasopharyngeal carcinoma (NPC) and EBV-associated gastric cancer (EBVaGC), are driven by persistent latent infection with Epstein-Barr virus (EBV). In total, these EBV-associated tumors accounted for an estimated 265,186 new cases of disease burden globally in 2017 alone. Of these EBV-associated cancers, over 40% are nasopharyngeal carcinoma (NPC), prevalent in southern China and Southeast Asia. A well-documented literature on virus-cell interactions in tumorigenesis suggests that targeting EBV is an effective approach to eradicating these cancers. Notably, the unique episodic nature of the EBV genome in tumor cells implies that inducing cell death through reactivation of the viral lysis cycle is an attractive approach for treating EBV-associated cancers. When latent EBV is induced into the lysis cycle, the immediate early (IE) proteins BZLF1 and BRLF1 are expressed, and transcription of both early and late proteins is further activated to propel the virus forward. Reactivation of the virus from its latent period depends on the expression of viral BZLF1 and BRLF1 proteins. Previous studies have demonstrated the usefulness of strategies in cancer therapy that kill cancer cells carrying viral genomic sequences by activating the viral cleavage cycle, see, for example, WO2021 / 173977A9. This disclosure relates to the application of synthetic mRNAs to efficiently activate EBV cleavage genes in latently infected cells. These synthetic nucleoside-modified mRNAs can be encapsulated in lipid nanoparticles (LNPs) or other non-viral delivery systems and then delivered to tumor cells to translate artificial proteins containing linked DNA-binding and transcriptional activation domains that specifically activate EBV-encoded cleavage genes (e.g., EBV-encoded cleavage genes). BZLF1 , BRLF1 and BGLF4The present invention utilizes mRNA nanomedicine technology to overcome the highly complex regulatory mechanisms of EBV cleavage gene expression. By leveraging the high copy number of EBV episomes in EBV-infected cancer cells, synthetically designed mRNAs efficiently express BZLF1 and BRLF1 to reactivate the EBV cleavage cycle. This artificial activation of EBV cleavage gene transcription (including transcription of the EBV-encoded protein kinase BGLF4) enhances the efficient conversion of the non-toxic antiviral prodrug form of ganciclovir to its cytotoxic DNA replication inhibitor form for the rapid killing of cancer cells and bystander cells. This disclosure provides a method for highly specific induction of EBV immediate early genes and early cleavage cycle genes using synthetically modified mRNAs. These synthetic mRNAs are first-in-class therapeutic agents that slow or stop cancer cell growth in vitro and in vivo, representing an innovative therapeutic strategy for the efficient activation of viral cleavage genes for cleavage induction therapy in EBV-related diseases. Summary of the Invention

[0005] This invention describes a first-of-its-kind synthetic mRNA drug for use in cleavage-inducing therapy that specifically kills EBV-positive cells (particularly EBV-positive cancer cells) associated with symptoms or diseases. The synthetic nucleoside-modified mRNA is designed to efficiently activate EBV cleavage genes in all EBV-related cells, while inducing cleavage cycles with existing chemical inducers (e.g., HDAC inhibitors, gemcitabine) is cell environment-specific and exhibits varying levels of efficiency across different lesions (e.g., different tumors). Currently, the clinical application of EBV cleavage-inducing therapy in cancer patients is limited by its broad-spectrum cytotoxicity and low cleavage reactivation efficiency. The synthetic nucleoside-modified mRNA efficiently activates the EBV cleavage promoter, bypassing the highly complex regulatory mechanisms in all EBV-related cancer cells. Furthermore, preferential transcriptional activation and cytotoxicity against EBV-related malignant cells (but not normal cells) have been demonstrated in vitro and in vivo. Instead of DNA-based transcriptional activation constructs, the synthetic modified mRNA shows advantages in small size, efficient encapsulation and delivery, and low-risk induction of genomic recombination and aberrant immune responses.

[0006] In a first aspect, the present invention provides a composition for treating EBV-related lesions (e.g., EBV-related cancers) in human subjects. The composition comprises (1) a nucleic acid containing a polynucleotide sequence encoding a fusion protein, the fusion protein comprising (i) at least one nuclear localization signal (NLS); and (ii) targeting Epstein-Barr virus (EBV). BZLF1, BRLF1 or BGLF4 (iii) Transcription activator-like effector (TALE) of promoter sequence; and (iii) capable of initiating BZLF1, BRLF1 or BGLF4(1) a transactivation domain for gene transcription, and (2) one or more physiologically acceptable excipients. In some embodiments, the nucleic acid of the present invention, as described above and herein, is DNA. In some embodiments, the nucleic acid of the present invention is RNA. In some embodiments, the uracil (U) residues of the RNA are at least partially substituted with pseudouridine (e.g., N1-methylpseudouridine), and in some cases completely substituted with said pseudouridine. In some embodiments, the nucleic acid is DNA and comprises an expression cassette containing a polynucleotide sequence encoding such a fusion protein operatively linked to a promoter sequence. In some embodiments, the TALE is encoded by a nucleotide sequence having at least 90%, 95%, or up to 100% sequence identity with any segment of the TALE specified in Table 7 as SEQ ID NO:31-60, such segments being, for example, segments 649 to 2178 of SEQ ID NO:31; segments 649 to 2382 of SEQ ID NO:32; segments 649 to 2280 of SEQ ID NO:33; or segments 649 to 2281 of SEQ ID NO:34. In some embodiments, the trans-activating domain is encoded by a nucleotide sequence having at least 90%, 95%, or up to 100% sequence identity with segments 2722 to 3564 of SEQ ID NO:33. In some embodiments, the NLS is encoded by a nucleotide sequence having at least 90%, 95%, or up to 100% sequence identity with segments 91 to 114 of SEQ ID NO:33. In some embodiments, multiple NLSs (e.g., 3xNLSs) are used in the nucleotide sequence, such as segments 91 to 162 of SEQ ID NO:33. Further examples of coding sequences for TALE, transactivation domain, and NLS can be found in SEQ ID NO:31-60 as labeled in Tables 4 to 6. In some embodiments, the fusion protein includes an epitope tag, such as a FLAG tag. For example, an exemplary fusion protein includes, from its N-terminus to its C-terminus, a FLAG optionally encoded by segments 1 to 69 of SEQ ID NO:33, at least one NLS (e.g., 3xNLS) encoded by segments 91 to 162 of SEQ ID NO:33, a TALE encoded by segments 649 to 2280 of SEQ ID NO:33, and a transactivation domain encoded by segments 2722 to 3564 of SEQ ID NO:33. Exemplary polynucleotide sequences encoding the fusion proteins of the present invention are presented in SEQ ID NO:31-60, and their functional regions are labeled in Tables 4 to 6.Furthermore, the NLS, TALE, and trans-activation domain coding sequences labeled and shown in Tables 4 to 6 of each of SEQ ID NO:31-60 can be individually selected to form one or more additional NLS-TALE-trans-activation domain combinations to generate novel polynucleotide sequences encoding fusion proteins that can be used to efficiently activate EBV lysis genes in all EBV-related cells and induce cell lysis. In particular, by selecting and combining the coding sequences of the TALE and trans-activation domains, additional fusion proteins for this purpose based on additional TALE-trans-activation domain pairings derived from the group of sequences shown in SEQ ID NO:31-40, or similarly from the group of sequences shown in SEQ ID NO:41-50 or the group of sequences shown in SEQ ID NO:51-60. For example, any one of the TALE coding sequences shown in segments 649 to 2178 of SEQ ID NO:31, segments 649 to 2382 of SEQ ID NO:32, segments 649 to 2280 of SEQ ID NO:33, and segments 649 to 2281 of SEQ ID NO:34 can be used interchangeably in combination with other NLS and / or trans-activation domain coding sequences (particularly those from SEQ ID NO:31-40) to produce other fusion proteins for activating EBV cleavage genes. In some embodiments, the compositions of the present invention are specifically formulated for treating EBV-related diseases in subjects of need, said compositions being, for example, nucleic acids, which may be RNA or DNA as described above, and the nucleic acids described herein are present in lipid nanoparticles. In some embodiments, the lipid nanoparticles comprise [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2000), and cholesterol. For nucleic acids in RNA form, for example, for the purpose of improving the stability and / or bioavailability of the RNA molecule, at least some or possibly all of the U residues of the molecule are replaced with pseudouridine, thereby ensuring adequate expression of the fusion protein encoded by the RNA. In some embodiments, the composition is formulated for injection, for example, in the form of a liquid, solution, suspension, or emulsion. In some implementations, EBV-related cancers are Burkitt lymphoma, Hodgkin's lymphoma, natural killer cell lymphoma, T-cell lymphoma, post-transplant lymphoma, nasopharyngeal carcinoma, or gastric cancer.

[0007] In a second aspect, the present invention provides a method for treating an EBV-related lesion, such as EBV-related cancer, in a subject by administering to a subject an effective amount of the composition described above and herein, i.e., a composition containing an effective amount of (1) the nucleic acid described above or herein; and (2) one or more physiologically acceptable excipients. In some embodiments, the nucleic acid is RNA, which optionally has at least some, and possibly all, of its U residues replaced by pseudouridine (e.g., N1-methylpseudouridine) to improve stability and / or bioavailability. Whether the nucleic acid is a DNA or RNA molecule, in some embodiments, it is present in the composition within lipid nanoparticles. According to the invention, the treatment methods in some embodiments are carried out by administering the composition of the invention to the subject via systemic administration or by oral ingestion or by nasal inhalation, said systemic administration being, for example, by intravenous, intratumoral, intramuscular, or subcutaneous injection. In some embodiments, the EBV-related cancer is Burkitt lymphoma, Hodgkin lymphoma, natural killer cell lymphoma, T-cell lymphoma, post-transplant lymphoma, nasopharyngeal carcinoma, or gastric cancer.

[0008] In a related aspect, the present invention provides novel uses of the composition for treating EBV-related lesions (e.g., EBV-related cancers). The composition contains an effective amount of (1) the nucleic acid described above or herein; and (2) one or more physiologically acceptable excipients. In some embodiments, the nucleic acid is RNA, which optionally has at least some, and possibly all, of its U residues replaced by pseudouridine (e.g., N1-methylpseudouridine). Whether the nucleic acid is DNA or RNA, in some cases it is present in the composition within lipid nanoparticles. Depending on the manner of administration, in some embodiments, the composition of the present invention is formulated for systemic administration, for example, in liquid or semi-liquid form for intravenous, intratumoral, intramuscular, or subcutaneous injection, or in powder or aerosol form for nasal inhalation, or in liquid / semi-liquid or solid / semi-solid form, such as solution, emulsion, paste, cream, powder, tablet, or capsule, for oral intake. In some embodiments, the EBV-related cancer is Burkitt lymphoma, Hodgkin lymphoma, natural killer cell lymphoma, T-cell lymphoma, post-transplant lymphoma, nasopharyngeal carcinoma, or gastric cancer.

[0009] In a third aspect, the present invention provides a kit for treating EBV-related lesions (e.g., EBV-related cancers) in patients in need. Typically, the kit comprises multiple containers, a first container containing a first composition having an effective amount of the nucleic acid described above and herein, and a second container containing a second composition having an effective amount of at least one therapeutic agent known to be effective in treating EBV-related lesions, such as an anticancer therapeutic agent. In some embodiments, the nucleic acid is RNA, optionally at least some, and possibly all, of its U residues are replaced with pseudouridine (e.g., N1-methylpseudouridine). In some embodiments, the polynucleotide sequence is as shown in any one of SEQ ID NO:31-60. In some embodiments, the nucleic acid is RNA and the polynucleotide sequence is as shown in any one of SEQ ID NO:51-60, and other anticancer agents include ganciclovir (GCV). In some embodiments, nucleic acids are present in lipid nanoparticles, which, for example, comprise [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000), and cholesterol. In some embodiments, EBV-related cancers are Burkitt lymphoma, Hodgkin lymphoma, natural killer cell lymphoma, T-cell lymphoma, post-transplant lymphoma, nasopharyngeal carcinoma, or gastric cancer. Attached Figure Description

[0010] Figure 1. Reactivation of EBV cleavage genes in EBV-related cancers via CRISPR-based transcriptional activators. Figure 1a Quantitative RT-PCR demonstrated the endogenous expression of Zta / BZLF1, Rta / BRLF1 and BGLF4 in SNU719 and C666-1 cells 24 h after transient transfection with HA-dCas9-2A-EGFP, 3xFLAG-PUFa-p65HSF1 and sgRNA. Figure 1b Western blotting demonstrated endogenous expression of Zta / BZLF1, Rta / BRLF1, and BGLF4 in SNU719 and C666-1 cells 24 h (n = 3 / group) following transient transfection with HA-dCas9-2A-EGFP, 3xFLAG-PUFa-p65HSF1, and sgRNA. Data are presented as mean ± SD. P < 0.001. Figure 1cIn SNU719, C666-1, and C17 cells stably transfected with sgRNA3, HA-dCas9-2A-EGFP, and inducible 3xFLAG-PUFa-p65HSF1 transactivator, the expression of Zta and other EBV cleavage proteins (Rta, BGLF4, and VCAp18) was detected by Western blotting after 48 h of doxycycline (Dox) treatment (n = 3 / group). Figure 1d Immunofluorescence staining showed the expression of Zta, EA-D, and gp350 lysate proteins in Dox-treated, stably transfected SNU719, C666-1, and C17 cells (scale bar = 10 mm) (n = 3 / group). Figure 1e The percentage of Zta-expressing cells in stably transfected SNU719, C666-1, and C17 cells was determined using flow cytometry 48 h after Dox treatment. Representative flow cytometry plots are shown (n = 3 / group). Figure 1f The inhibition of cell viability was detected in stable transfected SNU719, C666-1 and C17 cells treated with Dox using the CCK8 assay (n = 3 / group). Figure 1g , BZLF1 Induction significantly inhibited the colony-forming ability of stably transfected EBV-positive cancer cells treated with Dox (n = 3 / group). Data are presented as mean ± SD. P < 0.01; P < 0.001.

[0011] Figure 2. Synthetic BZLF1 Specific TALE transcription activator. Figure 2a Western blotting was used to detect the endogenous expression of immediate early (Zta, Rta), early (BGLF4), and late (VCAp18) cleavage proteins in SNU719 and C666-1 cells, which were used with designed targeted proteins. BZLF1 Transient transfection with TALE plasmid, TZ3, and other TALE plasmids (TZ1, TZ2, and TZ4) was performed. P3HR1 cells treated with NaB and TPA were included as controls for lysate protein expression (n = 3 / group). Figure 2b Zta expression was detected in SNU719 and C666-1 cells transiently transfected with TZ3 using immunofluorescence (IF) staining (n = 3 / group). Representative IF images are shown. Figure 2c This shows wild-type EBV BZLF1Promoter sequences of (B95-8, Zp-P) and reported variants (Zp-V3, Zp-V4, and Zp-V1). Cis-regulatory element sequences and sequence variations are shown in uppercase letters and red font, respectively. The Z3 binding sequence is highlighted in yellow and is conserved in all EBV strains. Figure 2d The interaction between TZ3 transcription activator and C666-1 cells was detected by EMSA. BZLF1 Specific binding affinity of the target sequence in the promoter (arrow). Besides... BZLF1 In addition to the wild-type (WT) target sequence in the promoter, which contains specific probes, three mutant probes are also included. Their sequences are shown in the boxes. Cells transfected with the vector alone served as controls (n = 3 / group). Figure 2e RNA sequencing was used to determine differentially expressed genes between TZ3-transfected HK1 cells and control HK1 cells (n = 3 / group). In TZ3-transfected HK1 cells, a few genes showed significant expression changes. Figure 2f No significant decrease in cell viability was detected in HK1 cells transfected with TZ3 (n = 3 / group). Data are expressed as mean ± SD. ns: not significant, P > 0.05. Figure 2g Ectopic transfection with TZ3 did not significantly alter the cell cycle of HK1 cells (n = 3 / group). Data are presented as mean ± SD. ns: not significant.

[0012] Figure 3. Reactivation of the EBV cleavage gene by nucleoside-modified mRNA (mTZ3-LNP) encapsulated by LNP. Figure 3a The particle size, polydispersity index, and zeta potential of mTZ3-LNP were analyzed using dynamic light scattering. The data are representative of four independent experiments. Figure 3b The uptake of LNP-encapsulated Cy5-labeled mTZ3 mRNA by SNU719 cells at 1 h, 3 h, and 6 h was observed using an LSM 880 confocal laser scanning microscope. Fluorescence signals were measured in three channels: Cy5, excitation / emission wavelengths (ex / em) of 633 / 697 nm; Dnd-26, ex / em of 488 / 524 nm; and Hoechst, ex / em of 405 / 460 nm. Figure 3c Western blotting was used to detect Zta expression in SNU719 and C666-1 cells treated with control mRNA (control-LNP) encapsulated with mTZ3-LNP and LNP for 24 h. The EBV-negative NPC cell line HK1 was used as a negative control. Figure 3dWestern blotting was used to detect the expression of Zta, Rta, downstream early (BGLF4) and late (VCA) cleavage proteins and caspase 3 in SNU719 and C666-1 cells treated with mTZ3-LNP for 3 h to 96 h. Figure 3e Immunofluorescence staining and ( Figure 3f Flow cytometry analysis revealed the expression of Zta, EA-D, and gp350 induced in mTZ3-LNP treated SNU719 and C666-1 cells.

[0013] Figure 4. Efficient EBV lysis reactivation in a cohort of EBV-positive cancer cells treated with mTZ3-LNP. Representative flow cytometry plots show the high efficiency of mTZ3-LNP treatment (48 h) in inducing Zta expression in a cohort of EBV-positive tumor cell lines, including ( Figure 4a NPCs (C666-1, NPC76c, C17, NPC43 and NPC-M81), ( Figure 4b ) EBVaGC (SNU719, YCCLE1 and AGS-EBV) and ( Figure 4c Burkitt lymphoma (P3HR1 and Akata-EBV). SNU719 and C666-1 cells treated with NaB are included as a reference for chemically induced lysis and reactivation. Figure 4d The percentage of Zta-positive cells in NPC, EBVaGC, and BL cell lines treated with mTZ3-LNP for 48 h is shown. Data are expressed as mean ± SD. Figure 4e The expression of Zta, its downstream cleavage proteins (Rta, BGLF4, and EA-D), and cleavage caspase 3 in YCCEL1, NPC31M81, and C17 cells treated with mTZ3-LNP was detected by Western blotting.

[0014] Figure 5. Effect of mTZ3-LNP treatment on EBV-positive epithelial cancer cells. Figure 5a Using RNA sequencing, a few significantly differentially expressed genes were detected in EBV-negative HK1 cells treated with mTZ3-LNP. Figure 5b Differentially expressed genes identified in SNU719 and C666-1 cells treated with mTZ3-LNP for 48 h were compared with those identified in cells treated with control mRNA-LNP for 48 h. Transcripts encoded by BZLF1 and EBV are shown as blue and red dots, respectively. Figure 5cThe EBV transcriptome profile revealed multiple EBV cleavage genes induced in SNU719 and C666-1 cells after 48 hours of mTZ3-LNP treatment. Figure 5d The genome specificity of TZ3 TALE transcription activator was assessed in mTZ3-LNP-treated C666-1 cells using ChIP sequencing and anti-FLAG antibody. EBV genomes from C666-1 cells treated with mTZ3-LNP for 6 hours were analyzed. BZLF1 A single peak was observed in the promoter region. C666-1 cells treated with control LNP were included as a control. The experiment was repeated twice. Figure 5e The viability of SNU719, C666-1, and HK1 cells treated alone with mTZ3-LNP and in combination with GCV was measured at 24 h, 48 h, 72 h, and 96 h. Significant growth inhibition was observed in EBV-positive SNU719 and C666-1 cells treated alone with mTZ3-LNP and in combination with GCV. Data are expressed as mean ± SD. ns: not significant; p < 0.0001. One-way ANOVA.

[0015] Figure 6. mTZ3-LNP reactivates EBV cleavage gene expression in an in vivo EBV-positive tumor model. Figure 6a Immunohistochemical staining was used to detect the expression of Zta, EA-D, and gp350 in representative tissue sections of SNU719 tumors from the NOD-SCID mouse model at 12 h, 24 h, and 48 h following intravenous administration of mTZ3-LNP. Tumor cells expressing gp350 are shown with red arrows. Scale bar = 50 μm. Figure 6b The percentage of tumor cells expressing Zta, EA-D, and gp350 in mouse tumors was determined at 12 h, 24 h, and 48 h following intravenous administration of mTZ3-LNP. At least four distinct representative fields of view (x200 magnification) were counted from slices obtained from each of the three replicate experiments. Data are presented as mean ± SD. Figure 6c At 12 h (hours) and 48 h (hours) after treatment with mTZ3-LNP, RNAscope RNA in situ hybridization was used to determine the immediate early (EBV) levels in representative FFPE sections of SNU719 tumors from the NOD-SCID mouse model. BZLF1 ), early stage ( BMRF1 and BGLF4 ) and late ( BLLF1Expression of lysed gene transcripts. Representative tumor cells expressing high and low copy numbers of EBV lysed gene transcripts are indicated by blue and red arrows, respectively. Scale bar = 50 μm. Figure 6d Expression of mTZ3-LNP in mouse tumors was measured at 12 h, 24 h, and 48 h after treatment. BZLF1, BGLF4, BMRF1 and BLLF1 The percentage of tumor cells with mRNA. Counts were performed on at least four distinct representative fields of view (x200 magnification) obtained from slices from each of the three replicate experiments. Data are expressed as mean ± SD.

[0016] Figure 7. In vivo inhibition of EBV-positive epithelial carcinoma by mTZ3-LNP-based lysis-induced treatment. Figure 7a This demonstrates a protocol for in vivo treatment of EBV-positive EBVaGC (SNU719) and NPC (C666-1, C17, Xeno-76) preclinical xenograft NOD-SCID mouse models with mTZ3-LNP and GCV. Figure 7b Tumor volume was measured throughout the treatment period (n = 6 to 7). Data are presented as mean ± SEM. ns: not significant. Figure 7c ,use EBER In situ hybridization was used to detect EBV-positive tumor cells in representative FFPE sections of residual tumors collected after treatment. Scale bar = 250 μm. Representative images from n = 6 to 7 mice / groups are shown. Figure 7d Tumor index was measured for each collected EBV-positive tumor after treatment with mTZ3-LNP alone, combined treatment with mTZ3-LNP and GCV, and treatment with GCV and a mediator control. Tumor index = tumor volume × EBER Percentage of positive area. Significant tumor growth inhibition was observed in SNU719, C666-1, C17, and Xeno-76 xenografts treated alone or in combination with mTZ3-LNP and GCV (n = 6 to 7 / group). Data are presented as mean ± SD. ns: not significant; p < 0.001; p < 0.0001. One-way ANOVA.

[0017] Figure 8 Targeting EBV-positive epithelial carcinoma with mRNA-LNP-based cleavage induction therapy. A schematic diagram illustrating the synthesis of mTZ3-LNP and its application in cleavage induction therapy against EBV-positive epithelial carcinoma. Modified mRNA encapsulated with LNP encapsulates mTZ3-LNP. BZLF1Specific TALE transcription activator TZ3 induces transcription of EBV cleavage genes in EBV-positive tumor cells. Treatment induces cell cycle cleavage to directly kill EBV-infected cells and activates GCV cytotoxicity and bystander killing effects in tumors. mTZ3-LNP treatment can also activate the host's innate and adaptive immune responses to EBV-positive cancers.

[0018] definition

[0019] The term “Epstein-Barr virus” or “EBV” refers to a member of the herpesvirus family, also known as human herpesvirus 4. EBV is commonly associated with three malignancies: Burkitt lymphoma, B-cell lymphoproliferative syndrome, and nasopharyngeal carcinoma. In later studies, EBV has been identified as associated with Hodgkin's disease, T-cell lymphoma, and gastric cancer, and is a causative agent of EBV infectious mononucleosis. As used herein, the term “EBV-associated cancer” includes any pathological symptom or disease, including malignancies, in which clonal EBV episomes are detected in cells of infected or diseased (e.g., malignant) tissue. Cells obtained from relevant tissues of EBV-associated diseases / lesions (e.g., EBV-associated cancer) are validated by polymerase chain reaction (PCR)-based assays, with each cell containing at least one copy of the EBV genome, and possibly multiple copies.

[0020] The term "nucleic acid" or "polynucleotide" refers to deoxyribonucleic acid or ribonucleic acid in single-stranded or double-stranded form and polymers thereof. Unless specifically defined, the term includes nucleic acids containing known analogs of natural nucleotides that possess desired properties, such as binding characteristics similar to a reference nucleic acid, being metabolized in a manner similar to naturally occurring nucleotides, and / or encoding a specific amino acid sequence. Unless otherwise specified, a specific nucleic acid sequence also implicitly includes variants of its conserved modifications (e.g., degenerate codon substitutions), alleles, homologous sequences, SNPs, and complementary sequences, as well as explicitly stated sequences. Specifically, degenerate codon substitutions can be achieved by generating a sequence in which the third position of one or more selected (or all) codons is replaced with a mixture of bases and / or deoxyinosine residues (Batzer). et al ., Nucleic Acid Res. 19:5081 (1991); Ohtsuka et al ., J. Biol. Chem. 260:2605-2608 (1985); andRossolini et al ., Mol. Cell. Probes 8:91-98 (1994)). The terms nucleic acid and gene, cDNA and mRNA encoded by a gene are used interchangeably.

[0021] The term "gene" refers to a segment of DNA involved in producing a polypeptide chain. It can include regions before and after coding regions (leader and tail regions) as well as insertion sequences (introns) between the individual coding regions (exons).

[0022] As used herein, in the context of describing two or more polynucleotide or amino acid sequences, the term "identity" or "percentage of identity" means that two or more identical sequences or subsequences, or those with a specified percentage of identical amino acid residues or nucleotides (e.g., the transactivation domain amino acid sequence having at least 80% identity with a reference sequence (e.g., the reference sequence encoded by segments 2722 to 3564 of the polynucleotide sequence shown in SEQ ID NO: 33), preferably 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity), measured using one of the sequence comparison algorithms described below or by manual alignment and visual inspection. Such sequences are then referred to as "substantially identical." Regarding polynucleotide sequences, this definition also refers to the complementary sequence of the test sequence. Preferably, the identity exists in a region of at least about 50 amino acids or nucleotides, in a region of 75 to 100 amino acids or nucleotides, in a region of 200 to 500 amino acids or nucleotides, or in a region of 500 to 1000 amino acids or nucleotides.

[0023] For sequence comparisons, a reference sequence is typically used, and the test sequence is compared to this reference sequence. When using a sequence comparison algorithm, the test and reference sequences are input into the computer, and if necessary, subsequence coordinates and sequence algorithm program parameters are specified. Default program parameters can be used, or optional parameters can be specified. The sequence comparison algorithm then calculates the percentage of sequence identity between the test sequence and the reference sequence based on the program parameters. For nucleic acid and protein sequence comparisons, the BLAST and BLAST 2.0 algorithms and the default parameters discussed below are used.

[0024] As used herein, a “comparison window” includes a segment selected from any number of consecutive positions, such as about 20 to 2000, about 20 to 1000, about 20 to 750, about 20 to 600, or about 20 to 500, typically about 50 to 200, and more typically about 100 to 150, which, after optimal alignment of two sequences, is compared with a reference sequence of the same number of consecutive positions. Alignment methods for the sequences used for comparison are well known in the art. Optimal alignment of the sequences used for comparison can be performed, for example, by Smith & Waterman, Adv. Appl. Math2:482 (1981) Local homology algorithm; by Needleman & Wunsch, J. Mol. Biol. The homology alignment algorithm of 48:443 (1970); through Pearson & Lipman, Proc. Nat'l. Acad. Sci. USA Similarity search methods as described in 85:2444 (1988); by computer implementation of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Computer Group (575 Science Dr., Madison, WI); or by manual comparison or visual inspection (see, for example...). Current Protocols in Molecular Biology (Ausubel et al (., eds. 1995 supplement).

[0025] Examples of algorithms suitable for determining sequence identity percentages and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul respectively. et al (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res25: 3389-3402. The software used for BLAST analysis is publicly available at the National Center for Biotechnology Information (NCBI) website, ncbi.nlm.nih.gov. The algorithm involves first identifying high-scoring sequence pairs (HSPs) by recognizing short words of length W in the query sequence. When said short word aligns with words of the same length in the database sequence, it either matches perfectly or satisfies some positive threshold score T. T is called the neighborhood word score threshold (Altschul et al., ibid.). These initial neighborhood word matches serve as seeds for starting the search to find longer HSPs containing them. The word matches are then extended in both directions along each sequence until the cumulative alignment score reaches a level that can be increased. For nucleotide sequences, parameters M (reward score for matching residue pairs; always > 0) and N (penalty score for non-matching residues; always < 0) are used. For amino acid sequences, a scoring matrix is ​​calculated to compute the cumulative score. The extension of word matching in each direction stops when: the cumulative alignment score decreases by X from its maximum value; the cumulative score becomes zero or lower due to the accumulation of one or more negatively scored residues; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses a word length (W) of 28, an expected value (E) of 10, M = 1, N = -2, and two-strand comparisons as default values. For amino acid sequences, the BLASTP program uses a word length (W) of 3, an expected value (E) of 10, and a BLOSUM62 scoring matrix as default values ​​(see Henikoff & Henikoff). Proc. Natl. Acad. Sci. USA 89:10915 (1989)).

[0026] The BLAST algorithm also performs statistical analysis of the similarity between two sequences (see, for example, Karlin & Altschul). Proc. Natl. Acad. Sci. USA 90:5873-5787 (1993)). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P(N)), which provides an indication of the probability that a match will occur by chance between two nucleotide or amino acid sequences. For example, if the minimum sum probability in a comparison of the test nucleic acid with a reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001, then the nucleic acid is considered similar to the reference sequence.

[0027] An indication that two nucleic acid sequences or peptides are substantially identical is that the peptide encoded by the first nucleic acid has an immunological cross-reactivity with an antibody generated against a peptide encoded by the second nucleic acid, as described below. Therefore, the peptide is usually substantially identical to the second peptide, for example, where the two peptides differ only in conserved substitutions. Another indication that two nucleic acid sequences are substantially identical is that the two molecules or their complementary sequences hybridize under stringent conditions, as described below. Yet another indication that two nucleic acid sequences are substantially identical is that the same primers are available for amplifying the sequence.

[0028] The term "nuclear localization signal" or "NLS" refers to an amino acid sequence, typically a short peptide sequence responsible for directly introducing proteins, particularly newly synthesized proteins, into the cell nucleus. These sequences typically contain a high proportion of the basic amino acids lysine and arginine. Amino acids that disrupt the helical domain, such as proline, are also often present. Numerous NLS sequences have been identified in the analysis of various viral proteins, in which the nuclear localization peptide is covalently linked to the 5' end of the DNA-coding sequence. Well-known viral NLS peptides include those from SV40, HIV, influenza virus, and adenovirus, such as the NLS present in the 92-kDa SV40 large T antigen. Further exemplary NLS sequences are provided in this disclosure, such as the 3x NLS encoded by segments 91 to 162 of any one of SEQ ID NO: 31-60.

[0029] The term "recombinant" when used to refer to, for example, cells, nucleic acids, proteins, or vectors, indicates that the cells, nucleic acids, proteins, or vectors have been modified by introducing heterologous or exogenous nucleic acids or proteins or by altering native nucleic acids or proteins, or that the cells are derived from such modified cells. Thus, for example, recombinant cells express genes not found in the natural (non-recombinant) form of the cell, or express native genes that are normally abnormally expressed, poorly expressed, or not expressed at all.

[0030] A “promoter” is defined as an array of polynucleotide control sequences that direct the transcription of another polynucleotide sequence. As used herein, a promoter includes an essential polynucleotide sequence near the transcription start site, for example, a TATA element in the case of a polymerase II promoter. A promoter may also optionally include a distal enhancer or repressor element, which can be located up to several thousand base pairs away from the transcription start site. A “constitutive” promoter is a promoter that is active under most environmental and developmental conditions. Conversely, an “inducible” promoter is a promoter that is regulated by environmental or developmental factors and exhibits activity under certain specified environmental or developmental conditions. The term “operably linked” refers to a functional link between a polynucleotide expression control sequence (e.g., a promoter, or an array of transcription factor binding sites) and another polynucleotide sequence (e.g., a protein-coding sequence), wherein the expression control sequence directs the transcription of the second polynucleotide sequence.

[0031] An "expression cassette" is a recombinant or synthetically produced nucleic acid construct that has a series of designated polynucleotide elements that allow a specific polynucleotide sequence to be transcribed in a host cell or in an in vitro transcription system (partial recombinant cell lysate). An expression cassette can be whole or part of a plasmid, viral genome, or other reproducible nucleic acid construct (e.g., an episome). Typically, an expression cassette comprises the polynucleotide to be transcribed, operatively linked to a promoter.

[0032] The term “cancer” refers to any of the various malignant tumors characterized by the proliferation of anaplastic cells that tend to invade surrounding tissues and metastasize to new anatomical sites within the patient’s body. Non-limiting examples of different types of cancer suitable for treatment using the compositions and methods of the present invention include colorectal cancer, colon cancer, anal cancer, liver cancer, ovarian cancer, breast cancer, lung cancer, bladder cancer, thyroid cancer, pleural cancer, pancreatic cancer, cervical cancer, prostate cancer, testicular cancer, bile duct cancer, gastrointestinal carcinoid tumors, esophageal cancer, gallbladder cancer, rectal cancer, appendix cancer, small bowel cancer, gastric cancer, kidney cancer (e.g., renal cell carcinoma), central nervous system cancers, skin cancer, oral squamous cell carcinoma, choriocarcinoma, head and neck cancer, bone cancer, osteosarcoma, fibrosarcoma, neuroblastoma, glioma, melanoma, leukemia (e.g., acute lymphoblastic leukemia, chronic lymphoblastic leukemia, acute myeloid leukemia, chronic myeloid leukemia, or hairy cell leukemia), lymphoma (e.g., non-Hodgkin lymphoma, Hodgkin lymphoma, B-cell lymphoma, or Burkitt lymphoma), and multiple myeloma.

[0033] As used herein, “inhibiting” or “inhibition” refers to any detectable negative impact on a target biological process, such as RNA / protein expression of a target gene, biological activity of a target protein, cell signal transduction, cell proliferation, presence / level of an organism (particularly microorganisms), any measurable biomarker, biological parameter, or symptom in the subject. Typically, inhibition is reflected as a reduction of at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater in the target process or signal (e.g., the subject's weight, or blood glucose / cholesterol levels, or any detectable symptom or biomarker in the subject, such as the rate of infection caused by a pathogenic infectious agent, or the rate of cancer cell proliferation / metastasis) or any of the downstream parameters mentioned above, compared to a control. “Inhibition” also includes a 100% reduction, i.e., the complete elimination, prevention, or abolition of the target biological process or signal. Other relative terms, such as “suppressing,” “suppression,” “reducing,” and “reduction,” are used in a similar manner in this disclosure to refer to a reduction of a target biological process or signal to different levels (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or greater, compared to a control level) up to complete elimination. On the other hand, terms such as “activate,” “activating,” “activation,” “increase,” “increasing,” “promote,” “promoting,” “enhance,” “enhancing,” or “enhancement” are used in this disclosure to include positive changes (e.g., at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 200% or more, such as 3-fold, 5-fold, 8-fold, 10-fold, 20-fold increases) in a target process, signal, or parameter at different levels compared to a control level.

[0034] As used herein, the terms "treatment" or "treating" include therapeutic and preventative measures taken in response to the presence of a disease or condition or the risk of its subsequent development. This includes measures to alleviate persistent symptoms, suppress or slow disease progression, delay the onset of symptoms, or eliminate or reduce downstream effects and / or side effects caused by such disease or condition. In this context, preventative measures and their variations do not require the complete elimination of the event; rather, they refer to suppressing or reducing the likelihood or severity of such occurrence or postponing its occurrence.

[0035] The term "severity" of a disease refers to the level and extent to which the progression of the disease has an adverse impact on the well-being and health of the patient suffering from it, such as short-term and long-term physical, mental, and psychological impairments, up to and including the patient's death. Disease severity can be reflected in the nature and quantity of necessary treatment and care measures, the duration required for the patient's recovery, the possible degree of recovery, the percentage of patients who achieve full recovery, the percentage of patients requiring long-term care, and the mortality rate.

[0036] The “patient” or “object” receiving the compositions or treatments of the present invention is a human being, including adults and minors of any age, sex, and ethnic background, who may have been diagnosed with any specific disease or condition or may not have been diagnosed with any specific disease or condition (e.g., EBV-related cancer), but is at high risk of developing such a disease and therefore requires preventive or therapeutic medical intervention (e.g., to reduce or eliminate the development of EBV). + (Risk of cancer). Generally, patients or subjects receiving treatment according to the method of the invention to treat or prevent cancer do not otherwise require treatment with the same therapeutic agent. For example, if the subject is receiving a nucleic acid composition according to the claimed method, the subject does not have any disease known to be treated with the same therapeutic agent. Although the patient can be of any age, in some cases the patient is at least 40, 45, 50, 55, 60, 65, 70, 75, 80, or 85 years old; in some cases the patient can be 40 to 45 years old, or 50 to 65 years old, or 65 to 85 years old. A “child” subject is a subject under 18 years of age, for example, about 5 to 17 years old, 9 to 17 years old, or 10 to 17 years old, or 12 to 17 years old, including “infants” whose age is less than about 12 months, for example, less than about 10, 8, 6, 4, or 2 months old, while an “adult” subject is a subject over 18 years of age.

[0037] As used herein, the term "effective amount" refers to the amount that produces the intended (e.g., therapeutic or preventative) effect of the applied composition. This effect includes prevention, correction, or inhibition of the progression of symptoms and associated complications of a particular disease / symptom to any detectable extent, such as the incidence of EBV-related diseases, severity levels including mortality, and one or more symptoms of such diseases (e.g., the incidence, metastasis rate, and 5- or 10-year survival rate of EBV-related cancers). The exact amount of an "effective amount" of a specific substance will depend on the purpose of treatment and can be determined by someone skilled in the art using known techniques (see, e.g., Lieberman). Pharmaceutical Dosage Forms (vols. 1-3, 1992); Lloyd, The Art, Science and Technology of Pharmaceutical Compounding (1999); and Pickar, Dosage Calculations (1999)).

[0038] The term “about” when used to refer to a given value indicates a range that includes ±10% of that value. For example, “about 10” means a range of 10 + / - 1, that is, 9 to 11.

[0039] "Pharmaceuticalally acceptable" or "pharmacologically acceptable" excipients are non-biologically harmful or non-adverse substances, meaning that the excipient can be administered to an individual together with a bioactive agent without causing any adverse biological effects. The excipient also will not interact harmfully with any component of the composition containing the excipient.

[0040] The term "excipient" refers to any substantially auxiliary substance that may be present in the final dosage form of the compositions of the present invention. For example, the term "excipient" includes media, binders, disintegrants, fillers (diluents), lubricants, flow aids (flow enhancers), compression aids, colorants, sweeteners, preservatives, suspending / dispersing agents, film-forming / coating agents, flavoring agents, and printing inks.

[0041] When used in the context of describing a composition containing one or more active ingredients, the term "consistently composed of..." means that the composition does not contain other ingredients that have any similar or related biological activity of the active ingredient or that can enhance or inhibit its activity, while one or more inactive ingredients, such as physiologically or pharmaceutically acceptable excipients, may be present in the composition. For example, a composition consisting essentially of an active agent for an effective treatment of EBV-related disease (e.g., the mRNA LNP formulation of the present invention) is a composition that does not contain any other reagents that may have any detectable positive or negative effect on the same target process (e.g., inhibit EBV-related disease) or may increase or decrease the severity of the disease in the recipient to any measurable extent.

[0042] As used herein, the term "particle" refers to a structured entity formed of molecules or molecular complexes, which can be a micrometer or nanometer-sized structure, such as a dense micrometer or nanometer-sized structure dispersed in a medium. For example, a "particle" can be a nucleic acid-encapsulated particle, such as a particle containing DNA, RNA, or a mixture thereof. Electrostatic interactions between positively charged molecules (e.g., polymers and lipids) and negatively charged nucleic acids are involved in particle formation. This leads to the complexation and spontaneous formation of nucleic acid particles. Depending on size, such nucleic acid-containing particles can be nanoparticles. As used herein, a "nanoparticle" is a particle having an average diameter suitable for parenteral administration, such as particles in the nanometer range. "Nucleic acid particles" can be used to deliver nucleic acids to a target site (e.g., cells, tissues, organs, etc.). Nucleic acid particles can be formed from at least one cationic or cationic ionizable lipid or lipid-like material, at least one cationic polymer (e.g., protamine), or a mixture thereof, and nucleic acids. The nucleic acid particles of the present invention include formulations based on lipid nanoparticles (LNPs) and lipid complexes (LPXs).

[0043] Detailed description

[0044] I. Introduction

[0045] This invention describes a novel method for treating cancers associated with latent virus infection, such as EBV-related cancers, in which cancer cells carry EBV episomes by inducing latent viruses into their lysis cycle and thus destroying cancer cells. For example, to induce latent EBV into the lysis cycle, one or more immediate-early (IE) proteins, such as BZLF1 and BRLF1, must be expressed, which sequentially activate the transcription of early and late proteins to further drive the lysis cycle. This invention relates to the use of synthetic mRNA encoding fusion proteins containing a DNA-binding domain linked to a transcriptional activation domain, and thus capable of specifically activating EBV-encoded lysis genes (e.g., EBV-encoded lysis genes). BZLF1, BRLF1 and BGLF4 The transcription of EBV lysis genes is effectively triggered to induce expression of these genes in latently infected cancer cells, leading to cell lysis. Synthetic mRNA molecules, preferably modified mRNA molecules, can be encapsulated in lipid nanoparticles (LNPs) or other suitable formulations for delivery to EBV-associated cancer cells. See [link to relevant documentation]. Figure 8 .

[0046] II. General Recombination Techniques

[0047] Foundational textbooks that published general methods and techniques in the field of recombinant genetics include those by Sambrook and Russell. Molecular Cloning, A Laboratory Manual (3rd ed. 2001); Kriegler, Gene Transfer and Expression: A Laboratory Manual(1990); and Ausubel et al. ,eds., Current Protocols in Molecular Biology (1994).

[0048] For nucleic acids, sizes are given in kilobases (kb), base pairs (bp), or nucleotides (nt). These estimates are derived from agarose or acrylamide gel electrophoresis, from sequenced nucleic acids, or from publicly available DNA sequences. For proteins, sizes are given in kilodaltons (kDa) or the number of amino acid residues. Protein sizes are estimated from gel electrophoresis, from sequenced proteins, from derived amino acid sequences, or from publicly available protein sequences.

[0049] Oligonucleotides that are not commercially available can be chemically synthesized, for example, according to Beaucage & Caruthers. Tetrahedron Lett. 22: 1859-1862 (1981) first described the solid-phase phosphoramidite method, using an automated synthesizer, such as Van Devanter. et. al. , Nucleic Acids Res. As described in 12: 6159-6168 (1984). Purification of oligonucleotides was performed using any strategy known in the art, such as natural acrylamide gel electrophoresis or anion exchange HPLC, as in Pearson & Reanier. J. Chrom. As described in 255: 137-149 (1983).

[0050] Wallace can be used, for example et al. , Gene Methods for chain termination of double-stranded templates for sequencing, as described in 16: 21-26 (1981), after cloning or subcloning, to verify the sequence of the target gene, the polynucleotide sequence encoding the target recombinant polypeptide, and the synthesized oligonucleotide or polynucleotide sequence.

[0051] III. RNA Synthesis and Modification

[0052] The RNA molecule of this invention is designed to encode a fusion protein that is recombinantly expressed by a host cell upon receipt of an mRNA transcript, the fusion protein specifically targeting or binding to EBV IE and E proteins (e.g. BZLF1, BRLF1 and BGLF4 The promoter sequence of at least one of the following is used to activate the transcription of IE and E proteins, thereby enabling the body to respond to EBV infection and maintain EBV. +The EBV lysis cycle is induced in cells (e.g., by retaining at least a portion of the EBV genome sequence in exosomes). Therefore, the mRNA of the present invention comprises the following segments: a first segment is a coding sequence for a nuclear localization signal that directs the newly synthesized fusion protein into the host cell nucleus to activate the transcription of the EBV IE or E protein. Exemplary 3x NLS can be found in segments 91 to 162 of any of SEQ ID NO:31-60 (indicated by dotted lines in Tables 2 to 4). The second segment is a coding sequence for a transcription activator-like effector (TALE) domain that ensures the fusion protein specifically recognizes and binds to a preselected portion of the promoter sequence of the BZLF1, BRLF1, or BGLF4 protein. TALE is originally derived from a naturally occurring protein secreted by the plant pathogenic bacterium Xanthomonas. Recent descriptions of the TAL-DNA code now allow for the design of TALE sequences as DNA-binding domains for artificial transcriptional activators, with the aim of enabling said proteins to target and bind to any known promoter sequence of a specific gene (e.g., any preselected portion of the promoter directing transcription of EBV IE proteins BZLF1 or BRLF1, or E protein BGLF4), thereby activating transcription of that specific gene. The bold portion of any one of SEQ ID NO:31-60 provides the targeting... BZLF1, BRLF1 or BGLF4 An exemplary TALE coding sequence for a gene promoter. The third segment is a coding sequence for a transactivation domain capable of initiating gene transcription and is typically derived from known naturally occurring transcription factors such as p53 and p65. The underlined portion of any one of SEQ ID NO:31-60 provides an exemplary coding sequence for the transactivation domain. Furthermore, the fusion protein encoded by the mRNA of the present invention may optionally further include one or more epitope tags, typically located at the N-terminus or C-terminus of the fusion protein, or both. An example of such epitope tags is a FLAG tag, such as a FLAG tag encoded by segments 1 to 69 of any one of SEQ ID NO:31-60. In addition to those shown in SEQ ID NO:31-60 (especially the combinations of each TALE transactivation domain of SEQ ID NO:31-40; SEQ ID NO:41-50; or SEQ ID NO:51-60), these coding sequence elements can be selected to form combinations to produce other fusion proteins that have the function of activating the EBV lysis cycle, thereby leading to the lysis of EBV-positive cells (e.g., EBV-positive cancer cells).

[0053] Polynucleotide sequences, including RNA or any derivatives or modified forms thereof, can be chemically synthesized according to methods known in the relevant art. RNA molecules can be modified by substitution with one or more nucleotide analogs, and / or by substitution of the base moiety, sugar moiety, or phosphate backbone, for example, to improve molecular stability, hybridization or binding ability, or bioavailability, etc. Polynucleotides may include other additional groups, such as peptides (e.g., for targeting host cell receptors), or agents that promote transmembrane transport (see, e.g., Letsinger). et al. , 1989, Proc. Natl. Acad. Sci. USA 86:6553-6556; Lemaitre et al. , 1987, Proc. Natl. Acad. Sci. 84:648-652; WO 88 / 09810) or blood-brain barrier (see, e.g., WO89 / 10134), hybridization-triggered cleavage agents (see, e.g., Krol) et al. , 1988, BioTechniques 6:958-976) or an insert (see, for example, Zon, 1988, Pharm. Res. (5:539-549). Therefore, RNA molecules can be conjugated to another molecule for purposes such as tissue / cell targeting, stability, and bioavailability.

[0054] The RNA molecules of this invention can be synthesized using standard methods known in the art, such as by using an automated polynucleotide synthesizer (e.g., commercially available from Biosearch, Applied Biosystems, etc.). As an example, they can be synthesized using Stein... et al. (1988, Nucl. Acids Res. Phosphothiophosphate polynucleotides can be synthesized using a method described in 16:3209. Methylphosphonate polynucleotides can be prepared using a glass polymer support with controllable pore size (Sarin). et al. , 1988, Proc. Natl. Acad. Sci. USA 85:7448-7451), etc.

[0055] A. In vitro RNA synthesis

[0056] In addition to chemical synthesis methods, the RNA molecules of the present invention can be generated using recombinant nucleic acid technology. To obtain high levels of RNA transcripts encoding the desired polypeptide, a polynucleotide encoding the polypeptide is typically subcloned into an expression vector containing a strong promoter for directing transcription, a transcription / translation terminator, and a ribosome-binding site for translation initiation. Suitable bacterial promoters are well known in the art and described, for example, by Sambrook and Russell (ibid.) and Ausubel. et al. (Same as above)

[0057] For example, the RNA molecule of the present invention is in vitro transcribed RNA (IVT-RNA) and can be obtained by in vitro transcription using a suitable DNA template. The promoter used to control transcription can be any promoter of any RNA polymerase. The DNA template for in vitro transcription can be obtained by cloning a polynucleotide sequence encoding a target recombinant protein and introducing it into a suitable vector for in vitro transcription.

[0058] In some embodiments, the RNA according to this disclosure comprises a 5'-UTR and / or a 3'-UTR. The terms "untranslated region" or "UTR" refer to a region in a DNA molecule that is transcribed but not translated into an amino acid sequence, or to a corresponding region in an RNA molecule (e.g., an mRNA molecule). An untranslated region (UTR) may be present at the 5' (upstream) end of an open reading frame (5'-UTR) and / or the 3' (downstream) end of an open reading frame (3'-UTR). If present, the 5'-UTR is located at the 5' end upstream of the start codon in a protein-coding region. The 5'-UTR is downstream of the 5'-cap (if present), for example, directly adjacent to the 5'-cap. If present, the 3'-UTR is located at the 3' end downstream of the stop codon in a protein-coding region, but the term "3'-UTR" preferably does not include the poly(A) sequence. Thus, the 3'-UTR is upstream of the poly(A) sequence (if present), for example, directly adjacent to the poly(A) sequence.

[0059] In some embodiments, the RNA of the present invention comprises a 3'-poly(A) sequence. As used herein, the terms "poly(A) sequence" or "poly(A) tail" refer to a continuous or discontinuous string of adenosine residues located at the 3' end of an RNA molecule. The RNA molecule of the present invention may have a poly(A) sequence that is attached to the free 3' end of the RNA post-transcriptionally by template-independent RNA polymerase or a poly(A) sequence encoded by DNA and transcribed by template-dependent RNA polymerase. A poly(A) sequence of approximately 120 A nucleotides has been shown to have a beneficial effect on RNA levels in transfected eukaryotic cells, as well as on protein levels translated from an open reading frame upstream (5') of the poly(A) sequence (Holtkamp). et al. , 2006, Blood ( , vol.108, pp. 4009-4017). For the purposes of this invention, the poly(A) sequence can be of any length. In some embodiments, the poly(A) sequence comprises, is substantially composed of, or is composed of at least 20, at least 30, at least 40, at least 80, or at least 100, and up to 500, up to 400, up to 300, up to 200, or up to 150 A nucleotides, and specifically about 120 A nucleotides. In this document, “substantially composed of” means that the majority of the nucleotides in the poly(A) sequence, typically at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% of the nucleotides in the poly(A) sequence are A nucleotides, but the remaining nucleotides are permitted to be nucleotides other than A nucleotides, such as U nucleotides (uridine monophosphate), G nucleotides (guanylic acid), or C nucleotides (cytidine monophosphate). In this document, "composed of" means all nucleotides in the poly(A) sequence, i.e., 100% of the nucleotides in the poly(A) sequence are A nucleotides. In some embodiments, during RNA transcription, such as during the preparation of RNA transcribed in vitro, the poly(A) sequence is linked to a DNA template containing repeating dT nucleotides (deoxythymidines) in a strand complementary to the coding strand. The DNA sequence (coding strand) encoding the poly(A) sequence is referred to as the poly(A) box.

[0060] In some embodiments, the RNA of the present invention comprises one or more nucleosides as described herein and modified by methods in the art. For example, the RNA may comprise modified nucleosides in place of at least one (e.g., each) uridine.

[0061] Once RNA transcripts are generated in a satisfactory quantity in an in vitro system, they can be purified using standard nucleic acid purification procedures, including size difference filtering and column chromatography. The identity of the RNA molecules can be further verified using methods such as nucleic acid sequence analysis and mass spectrometry.

[0062] B. Modified nucleotides, nucleosides and polynucleotides

[0063] When referring to nucleotides, nucleosides, or polynucleotides (such as the nucleic acids of this invention, e.g., mRNA molecules), the terms "modified" and "modified" describe modifications of A, G, U, and C ribonucleotides. Generally, these terms are not intended to refer to ribonucleotide modifications in the naturally occurring 5' end mRNA cap portion.

[0064] These modifications can be of various kinds. In some implementations, when the nucleic acid is mRNA, the coding region, flanking region, and / or terminal region may contain one, two, or more (optionally different) nucleoside or nucleotide modifications.

[0065] Polynucleotides may include any useful modifications, such as modifications to sugars, nucleobases, or nucleoside linkages (e.g., linkages between phosphate / phosphodiester bonds / phosphodiester backbones). For example, the major groove of a polynucleotide, or the major groove face of a nucleobase, may include one or more modifications. One or more atoms of a pyrimidine nucleobase (e.g., on the major groove face) may be replaced or substituted with an optionally substituted amino group, an optionally substituted thiol, an optionally substituted alkyl group (e.g., methyl or ethyl), or a halogen (e.g., chlorine or fluorine). In some embodiments, the modification (e.g., one or more modifications) is present in each of the sugar and nucleoside linkages. Modifications according to the invention may be modifications of ribonucleic acid (RNA) to deoxyribonucleic acid (DNA), such as replacing the 2'OH of the furanyl ribose ring with 2'H, threonium nucleic acid (TNA), ethylene glycol nucleic acid (GNA), peptide nucleic acid (PNA), locked nucleic acid (LNA), or hybrids thereof.

[0066] The polynucleotides of the present invention substantially do not induce an innate immune response in cells that have introduced polynucleotides (e.g., mRNA). An induced innate immune response is characterized by 1) increased expression of pro-inflammatory cytokines; 2) activation of intracellular PRRs (RIG-I, MDA5, etc.); and / or 3) termination or reduction of protein translation.

[0067] In some embodiments, it may be necessary to degrade the modified nucleic acid molecules introduced into the cells within the cells. For example, degradation of the modified nucleic acid molecules may be preferred if precise protein production timing is required. Therefore, in some embodiments, the present invention provides modified nucleic acid molecules containing degradation domains that are capable of functioning in a directed manner within the cells. In other embodiments, the modified polynucleotides introduced into the cells exhibit reduced degradation in the cells compared to unmodified polynucleotides. In another aspect, this disclosure provides polynucleotides comprising nucleosides or nucleotides capable of disrupting the binding of the major groove interaction (e.g., binding) partner to the polynucleotide (e.g., wherein the modified nucleotide has a reduced binding affinity to the major groove interaction partner compared to the unmodified nucleotide).

[0068] Nucleosides and nucleotides (e.g., building blocks) that can be incorporated into polynucleotides (e.g., RNA or mRNA, as described herein) can be modified on the sugars of ribonucleic acid. For example, the 2' hydroxyl group (OH) can be modified or substituted with a number of different substituents. Exemplary substitutions at the 2'-position include, but are not limited to, H, halogen, optionally substituted C1-6 alkyl; optionally substituted C1-6 alkoxy; optionally substituted C6-10 aryloxy; optionally substituted C3-8 cycloalkyl; optionally substituted C3-8 cycloalkoxy; optionally substituted C6-10 aryloxy; optionally substituted C6-10 aryl-C1-6 alkoxy; optionally substituted C1-12 (heterocyclic)oxy; sugars (e.g., ribose, pentose, or any sugar described herein); "locked" nucleic acids (LNAs) wherein the 2'-hydroxyl group is connected to the 4'-carbon of a sugar of the same ribose via a C1-6 alkylene or C1-6 heteroalkylene bridge, wherein exemplary bridges include methylene, propylene, ether, or amino bridges; aminoalkyl as defined herein; aminoalkoxy as defined herein; amino as defined herein; and amino acids as defined herein.

[0069] Typically, RNA comprises a glycosylribose, which is a 5-membered ring with oxygen. Exemplary non-restrictive modifications to nucleotides include the substitution of oxygen in the ribose (e.g., with S, Se, or alkylene, such as methylene or ethylene); addition to the double bond (e.g., replacing the ribose with cyclopentenyl or cyclohexenyl); ring contraction of the ribose (e.g., to form a 4-membered ring of cyclobutane or oxetane); ring expansion of the ribose (e.g., to form a 6- or 7-membered ring with additional carbon or heteroatoms, such as anhydride hexitol, atroitol, mannitol, cyclohexyl, cyclohexenyl, and morpholino with a phosphoramide backbone); polycyclic forms (e.g., tricyclic; and "unlocked" forms, such as glycol nucleic acids (GNAs)). (For example, R-GNA or S-GNA, where the ribose is replaced by an ethylene glycol unit linked to a phosphodiester bond), threonine nucleic acid, and peptide nucleic acid (PNA, where the 2-aminoethylglycine linker replaces the ribose and phosphodiester backbone). The glycosyl group may also contain one or more carbons with a stereochemical configuration opposite to that of the corresponding carbon in ribose. Therefore, polynucleotide molecules can include nucleotides containing, for example, arabinose as a sugar.

[0070] This disclosure provides modified nucleosides and nucleotides. As described herein, a "nucleoside" is defined as a compound containing a sugar molecule (e.g., a pentose or ribose) or a derivative thereof combined with an organic base (e.g., a purine or pyrimidine) or a derivative thereof (also referred to herein as a "nucleobase"). As described herein, a "nucleotide" is defined as a nucleoside comprising a phosphate group. In some embodiments, the nucleosides and nucleotides described herein are typically chemically modified on their great groove surfaces. Exemplary non-limiting modifications include amino groups, thiol groups, alkyl groups, halogen groups, or any modifications described herein. Modified nucleotides can be synthesized by any useful method described herein (e.g., chemical, enzymatic, or recombinant synthesis to include one or more modified or non-natural nucleosides).

[0071] Modified nucleotide base pairings include not only standard adenosine-thymine, adenosine-uracil, or guanosine-cytosine base pairs, but also base pairs formed between nucleotides and / or modified nucleotides containing non-standard or modified bases, where the arrangement of hydrogen bond donors and acceptors allows hydrogen bonding between non-standard and standard bases or between two complementary non-standard base structures. An example of such non-standard base pairings is the base pairing between the modified nucleotide inosine and adenine, cytosine, or uracil.

[0072] Modified nucleosides and nucleotides may include modified nucleobases. Examples of nucleobases found in RNA include, but are not limited to, adenine, guanine, cytosine, and uracil. Examples of nucleobases found in DNA include, but are not limited to, adenine, guanine, cytosine, and thymine. These nucleobases may be modified or completely substituted to provide polynucleotide molecules with enhanced properties (e.g., resistance to nucleases, stability), and these properties may be manifested by disrupting the binding of the major groove binding partner. For example, the nucleosides and nucleotides may be chemically modified on the major groove surface. In some embodiments, major groove chemical modifications may include amino groups, thiol groups, alkyl groups, or halogen groups.

[0073] In some embodiments, the modified nucleobase is a modified uracil. Exemplary nucleobases and nucleosides having modified uracil include pseudouridine, pyridin-4-ketoribonucleoside, 5-azauridine, 6-azauridine, 2-thio-5-azauridine, 2-thiouridine, 4-thiouridine, 4-thiopseudouridine, 2-thiopseudouridine, 5-hydroxyuridine, 5-aminoallyluridine, 5-halouridine (e.g., 5-iodouridine or 5-bromouridine), 3-methyluridine, 5-methoxyuridine, uridine-5-oxoacetic acid, uridine-5-oxoacetic acid methyl ester, 5-carboxymethyluridine, 1-carboxymethylpseudouridine, and 5-carboxyhydroxyuridine. Methyluridine, 5-carboxyhydroxymethyluridine methyl ester, 5-methoxycarbonylmethyluridine, 5-methoxycarbonylmethyl-2-thiouridine, 5-aminomethyl-2-thiouridine, 5-methylaminomethyluridine, 5-methylaminomethyl-2-thiouridine, 5-methylaminomethyl-2-selenouridine, 5-carbamoylmethyluridine, 5-carboxymethylaminomethyluridine, 5-carboxymethylaminomethyl-2-thiouridine, 5-propynyluridine, 1-propynyl pseudouridine, 5-taurylmethyluridine, 1-taurylmethyl pseudouridine, 5-taurylmethyl-2-thiouridine, 1- Taurylmethyl-4-thiopseudouridine, 5-methyluridine, 1-methylpseudouridine, 5-methyl-2-thiouridine, 1-methyl-4-thiopseudouridine, 4-thio-1-methylpseudouridine, 3-methylpseudouridine, 2-thio-1-methylpseudouridine, 1-methyl-1-deazopseudouridine, 2-thio-1-methyl-1-deazopseudouridine, dihydrouridine, dihydropseudouridine, 5,6-dihydrouridine, 5-methyldihydrouridine, 2-thiodihydrouridine, 2-thiodihydrouridine, 2-methoxyuridine, 2-methoxy-4-thiouridine, 4- Methoxypseudouridine, 4-methoxy-2-thiopseudouridine, N1-methylpseudouridine, 3-(3-amino-3-carboxypropyl)uridine, 1-methyl-3-(3-amino-3-carboxypropyl)pseudouridine, 5-(isopentenaminomethyl)uridine, 5-(isopentenaminomethyl)-2-thiouridine, α-thiouridine, 2'-O-methyluridine, 5,2'-O-dimethyluridine, 2'-O-methyl-pseudouridine, 2-thio-2'-O-methyluridine, 5-methoxycarbonylmethyl-2'-O-methyluridine, 5-carbamoylmethyl-2' -O-methyluridine, 5-carboxymethylaminomethyl-2'-O-methyluridine, 3,2'-O-dimethyluridine, and 5-(isopentenaminomethyl)-2'-O-methyluridine, 1-thiouridine, deoxythymidine, 2'-F-uridine, 2'-F-uridine, 2'-OH-uridine, 5-(2-methoxycarbonylvinyl)uridine and 5-[3-(1-E-propenylamino)]uridine.

[0074] In some embodiments, the modified nucleobase is a modified cytosine. Exemplary nucleobases and nucleosides having modified cytosine include 5-azacytidine, 6-azacytidine, pseudocytidine, 3-methylcytidine, N4-acetylcytidine, 5-formylcytidine, N4-methylcytidine, 5-methylcytidine, 5-halocytidine (e.g., 5-iodocytidine), 5-hydroxymethylcytidine, 1-methylpseudocytidine, pyrrolocytidine, pyrrolopseudocytidine, and 2-thiocytidine. 2-Thio-5-methylcytidine, 4-thiopseudocytidine, 4-thio-1-methylpseudocytidine, 4-thio-1-methyl-1-deazopseudocytidine, 1-methyl-1-deazopseudocytidine, zebularine, 5-azazebularine, 5-methylzebularine, 5-aza-2-thiozebularine, 2-thiozebularine, 2-methoxycytidine, 2-methoxy-5-methylcytidine, 4-methoxypseudocytidine, 4-methoxy-1-methylpseudocytidine, α-thiocytidine, 2'-O-methylcytidine, 5,2'-O-dimethylcytidine, N4-acetyl-2'-O-methylcytidine, N4,2'-O-dimethylcytidine, 5-formyl-2'-O-methylcytidine, N4, N4,2'-O-trimethylcytidine, 1-thiocytidine, 2'-F-cytarabine, 2'-F-cytidine and 2'-OH-cytarabine.

[0075] In some embodiments, the modified nucleobase is a modified adenine. Exemplary nucleobases and nucleosides having modified adenine include 2-aminopurine, 2,6-diaminopurine, 2-amino-6-halopurine (e.g., 2-amino-6-chloropurine), 6-halopurine (e.g., 6-chloropurine), 2-amino-6-methylpurine, 8-azidoadenosine, 7-deadenine, 7-deaden-8-azaadenine, 7-deaden-2-aminopurine, 7-deaden-8-aza-2-aminopurine, 7-deaden-2,6-diaminopurine, 7-deaden-8-aza-2,6-diaminopurine. Aminopurine, 1-methyladenosine (m'A), 2-methyladenosine, N6-methyladenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis-hydroxyisopentenyl)adenosine, N6-glycylcarbamoyladenosine, N6-threonylcarbamoyladenosine, N6-methyl-N6-threonylcarbamoyladenosine, 2-methylthio-N6-threonylcarbamoyladenosine, N6, N6-Dimethyladenosine, N6-hydroxyn-valinecarbamoyladenosine, 2-methylthio-N6-hydroxyn-valinecarbamoyladenosine, N6-acetyladenosine, 7-methyladenosine, 2-methylthioadenosine, 2-methoxyadenosine, α-thioadenosine, 2'-O-methyladenosine, N6,2'-O-dimethyladenosine, N6,N6,2'-O-trimethyladenosine, 1,2'-O-dimethyladenosine, 2'-O-ribosyladenosine, 2-amino-N6-methylpurine, 1-thioadenosine, 8-azidoadenosine, 2'-F-arabinogalactadenosine, 2'-F adenosine, 2'-OH-arabinogalactadenosine, and N6-(19-aminopentaenoyl)adenosine.

[0076] In some embodiments, the modified nucleobase is a modified guanine. Exemplary nucleobases and nucleosides having modified guanine include inosine, 1-methylinosine, wyosine, methylwyosine, 4-demethylwyosine, isowyosine, wybutosine, peroxywybutosine, hydroxywybutosine, incompletely modified hydroxybutosine, 7-deazoguanosine, queuosine, epoxyqueuosine, galactosylqueuosine, mannosylqueuosine, 7-cyano-7-deazoguanosine, 7-aminomethyl-7-deazoguanosine, archopurinol, 7-deazo-8-azaguanosine, 6-thioguanosine, 6-thio-7-deazoguanosine, 6-thio-7-deazo-8-azaguanosine, 7-methylguanosine, 6-thio-7-methylguanosine. 7-Methylinosine, 6-methoxyguanosine, 1-methylguanosine, N2-methylguanosine, N2,N2-dimethylguanosine, N2,7-dimethylguanosine, N2,N2,7-trimethylguanosine, 8-oxoguanosine, 7-methyl-8-oxoguanosine, 1-methyl-6-thioguanosine, N2-methyl-6-thioguanosine, N2,N2-dimethyl-6-thioguanosine, α-thioguanosine, 2'- O-methylguanosine, N2-methyl-2'-O-methylguanosine, N2,N2-dimethyl-2'-O-methylguanosine, 1-methyl-2'-O-methylguanosine, N2,7-dimethyl-2'-O-methylguanosine, 2'-O-methylinosine, 1,2'-O-dimethylinosine, 2'-O-ribosylguanosine, 1-thioguanosine, O6-methylguanosine, 2'-F-arasylguanosine, and 2'-F-guanosine.

[0077] In some implementations, the nucleotide can be modified on the major groove face. For example, such modifications include replacing the hydrogen at C-5 of uracil or cytosine with an alkyl group (e.g., methyl) or a halogen.

[0078] The nucleobases of a nucleotide can be independently selected from purines, pyrimidines, purine analogs, or pyrimidine analogs. For example, each nucleobase can be independently selected from adenine, cytosine, guanine, uracil, or hypoxanthine. In another embodiment, the nucleobase may also include, for example, naturally occurring and synthetic derivatives of the base, including pyrazolo[3,4-d]pyrimidine, 5-methylcytosine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-propynyluracil and cytosine, 6-azauracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halogenated (e.g., 8-bromo), 8-amino, 8-thiol, 8-thio Alkyl, 8-hydroxy and other 8-substituted adenine and guanine, 5-halogenated (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracil and cytosine, 7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine, deadenine, 7-deadenine, 3-deadenine, deadenine, 7-deadenine, 3-deadenine, pyrazolo[3,4-d]pyrimidine, imidazo[1,5-a]1,3,5-triazinone, 9-deadenine, imidazo[4,5-d]pyrazine, thiazo[4,5-d]pyrimidine, pyrazin-2-one, 1,2,4-triazine, pyridazine; and 1,3,5-triazine. When abbreviations A, G, C, T, or U are used to describe nucleotides, each letter refers to a representative base and / or its derivatives. For example, A includes adenine or adenine analogs (e.g., 7-deadenine).

[0079] After the RNA molecule has been synthesized and isolated / purified, it can be tested to confirm that it activates EBV. + EBV lysis cycle in cells leads to EBV + Cell death or inhibition of EBV + The ability of cells to proliferate. For example, by contacting EBV-positive cells with a sufficient amount of test RNA molecules in vitro, a cell-based assay is performed to observe any inhibitory effect of the test RNA on the proliferation of EBV-positive cells. When an inhibitory effect is detected (e.g., at least 25%, 50%, 80%, 90% or more inhibition in the proliferation or viability of treated EBV-positive cells compared to untreated EBV-positive cells of the same type), the RNA molecule is considered to be used in the method of the present invention to treat EBV-related lesions (e.g., EBV-related lesions). + It is effective against cancer. To further validate its activity, the RNA molecule can optionally be tested in animal models, for example, by administering (e.g., by injection) a sufficient amount of the RNA molecule to individuals carrying xenograft EBV.+ The activity was confirmed in tumor-immune-deficient animals to observe the inhibitory effect of the RNA molecule on tumor growth and / or metastasis (e.g., at least 25%, 50%, 80%, 90% or more inhibition) compared with untreated control animals.

[0080] IV. Pharmaceutical Composition and Administration

[0081] This invention provides a pharmaceutical composition comprising an effective amount of RNA encoding a recombinant protein capable of activating the expression of EBV immediate early (IE) proteins (such as BZLF1 and BRLF1) and thereby inducing the EBV cleavage cycle, for the purpose of treating EBV-related diseases, particularly EBV-related diseases. + This type of disease in people with cancer. The pharmaceutical compositions of the present invention are suitable for a variety of drug delivery systems. Suitable formulations for use in the present invention can be found, for example, Remington's Pharmaceutical Sciences , Mack Publishing Company, Philadelphia, PA, 17th ed. (1985). For a brief overview of drug delivery methods, see Langer, Science 249: 1527-1533 (1990).

[0082] The pharmaceutical compositions of the present invention can be administered via various routes, such as systemic administration via oral ingestion or injection (e.g., intravenous, intramuscular, or subcutaneous injection) and local delivery, such as intratumoral, intracranial, or intraperitoneal injection, or by direct (e.g., local) administration or by using a suitable suppository. A preferred route of administration of the pharmaceutical compositions is intravenous administration of the RNA of the present invention at a daily dose of about 1 μg to about 1000 μg, about 5 μg to about 500 μg, about 10 μg to about 250 μg, about 20 μg to about 100 μg, or about 25 μg to about 50 μg. Additionally, the compositions can be formulated for administration to a subject in daily, weekly, or monthly doses. Suitable doses can be administered as a single, once-daily dose or in separate doses provided at suitable intervals, such as every two, three, four, five, six, or more months, or, for example, every 12 months.

[0083] To prepare pharmaceutical compositions containing the RNA molecules of the present invention, one or more inert and pharmaceutically acceptable carriers are used. Depending on the method of administration, the pharmaceutical carrier can be solid or liquid. Solid formulations include, for example, powders, creams / pastes, tablets, dispersible granules, capsules, suppositories, and sachets. The solid carrier can be one or more substances that can also act as diluents, flavoring agents, solubilizers, lubricants, suspending agents, binders, or tablet disintegrants; it can also be an encapsulating material.

[0084] Powders and other forms of solid compositions contain a sufficient amount of the active ingredient (e.g., the mRNA of the present invention, optionally together with another anticancer therapeutic agent) and one or more carriers. Suitable carriers include, for example, magnesium carbonate, magnesium stearate, talc, lactose, sugar, pectin, dextrin, starch, astragalus gum, methylcellulose, sodium carboxymethyl cellulose, low-melting-point wax, cocoa butter, etc.

[0085] Liquid pharmaceutical compositions include, for example, solutions, suspensions, and emulsions suitable for oral administration or local delivery. Examples of liquid or semi-liquid compositions suitable for oral administration or local delivery (e.g., the mRNA of the present invention, optionally together with another anticancer agent) or sterile solutions of the active ingredient in a solvent are sterile aqueous solutions of the active ingredient in a solvent, including water, buffered water, saline, PBS, ethanol, or propylene glycol. The compositions may contain pharmaceutically acceptable excipients close to those required for physiological conditions, such as pH adjusters and buffers, tonic modifiers, wetting agents, detergents, etc.

[0086] Sterile solutions can be prepared by dissolving the active component (e.g., the RNA of the present invention, optionally further combined with one or more anticancer therapeutic agents) in a desired solvent system, followed by sterilizing the resulting solution by passing it through a membrane filter, or alternatively, by dissolving the sterile active component in a pre-sterilized solvent under sterile conditions. The resulting aqueous solution can be packaged for use as is or lyophilized, with the lyophilized formulation combined with a sterile aqueous carrier prior to administration. The pH of the formulation is typically from about 3 to about 11, for example from about 5 to about 9, or from about 7 to about 8.

[0087] The dosage level and pattern of the composition may be selected by the treating physician for single or multiple administrations. In any case, the pharmaceutical formulation should provide an amount of active agent sufficient to trigger the EBV cleavage cycle (e.g., an mRNA molecule encoding a recombinant protein capable of activating EBV IE protein expression as described herein).

[0088] In some embodiments, the composition containing the RNA of the present invention is formulated as a composition of nucleic acid particles, particularly in the form of lipid nanoparticles (LNPs) containing RNA. One or more types of lipids and other ingredients may be used in the formulation. For example, the LNP may contain cationic lipids, neutral lipids, steroids, polymer-conjugated lipids, and RNA. In some cases, the LNP may further contain at least one lipid or lipid-like material other than cationic or cationic ionizable lipids or lipid-like materials, at least one polymer other than cationic polymers, or mixtures thereof. In some embodiments, the ratio of mRNA to total lipids (N / P) is 5 to 10, for example, about 6 or about 7. The nucleic acid particles of the present invention may have an average diameter ranging from about 30 nm to about 1000 nm, about 50 nm to about 800 nm, about 70 nm to about 600 nm, about 90 nm to about 400 nm, or about 100 nm to about 300 nm. The nucleic acid particles may exhibit a polydispersity index of less than about 0.5, less than about 0.4, less than about 0.3, or about 0.2 or less. As an example, nucleic acid particles may exhibit a polydispersity index in the range of about 0.1 to about 0.3 or about 0.2 to about 0.3.

[0089] The preferred administration method for this RNA-encapsulated LNP composition is intratumoral or intravenous administration, more preferably, the composition is administered intratumorally or intravenously in an aqueous cryoprotectant buffer. The composition is typically a preservative-free, sterile RNA dispersion formulated in lipid nanoparticles (LNPs) for intratumoral or intravenous administration in an aqueous cryoprotectant buffer.

[0090] V. Other treatments

[0091] In carrying out this invention, other known anticancer therapeutic agents can be combined with the mRNA described herein to treat EBV-related cancers by inducing EBV lysis. In such applications, one or more of these previously known effective anticancer therapeutic agents can be administered to a patient as a single composition with an effective amount of the RNA formulation, or separately as two or more different compositions. They can be used in combination with the active agents of this invention (e.g., mRNA lipid nanoparticles) to inhibit cancer growth, inhibit cancer metastasis, and promote disease remission.

[0092] For example, various chemotherapeutic agents are known to be effective in treating a variety of cancers. As used herein, “chemotherapeutic agent” includes any compound that shows inhibitory effects on cancer cells and is therefore suitable for treating cancer. Categories of chemotherapeutic agents include, but are not limited to: alkylating agents, antimetabolites, kinase inhibitors, spindle alkaloids, cytotoxic / antitumor antibiotics, topoisomerase inhibitors, photosensitizers, anti-estrogens and selective estrogen receptor modulators (SERMs), antiprogestins, estrogen receptor downregulators (ERDs), estrogen receptor antagonists, luteinizing hormone-releasing hormone agonists, antiandrogens, aromatase inhibitors, EGFR inhibitors, VEGF inhibitors, and antisense oligonucleotides that inhibit the expression of genes involved in abnormal cell proliferation or tumor growth. Chemootherapeutic agents used in the treatments disclosed herein also include cell growth inhibitors and / or cytotoxic agents.

[0093] Exemplary anticancer therapeutic agents include alkylating agents such as altretamine, bendamustine, busulfan, carboquone, carmustine, chlorambucil, chlormethine, chlorozotocin, cyclophosphamide, dacarbazine, fotemustine, and ifosfamide. Phosphamide, lomustine, melphalan, melphalanflufenamide, mitobronitol, nimustine, nitrosoureas, pipobroman, ranimustine, semustine, streptozotocin, temozolomide Drugs containing thiotepa, treosulfan, triaziquone, triethylenemelamine, trofosfamide, and uramustine; anthracyclines such as aclarubicin, daunorubicin, doxorubicin, epirubicin, idarubicin, and mitoxantrone. Oxantrone, pirarubicin, valrubicin, and zorubicin; cytoskeleton disruptors (taxanes), such as albumin-bound paclitaxel, cabazitaxel, docetaxel, larotaxel, paclitaxel, taxotere, and tesetaxel; epothilone, such as ixabepilone;Histone deacetylase inhibitors, such as vorinostat and romidepsin; and topoisomerase I inhibitors, such as belotecan, camptothecin, exatecan, gimatecan, irinotecan, and topotecan; and topoisomerase II inhibitors, such as etoposide, teniposide, and taflupository. (de); kinase inhibitors, such as bortezomib, erlotinib, gefitinib, imatinib, vemurafenib, and vismodegib; nucleotide analogs and pro-analytes, such as azacitidine, azathioprine, capecitabine, cytarabine, deoxyfluorouridine, fluorouracil, gemcitabine, hydroxyurea, mercaptopurine, methotrexate, and tioguanine. (Formerly thioguanine); peptide antibiotics, such as actinomycin and bleomycin; platinum-based drugs, such as carboplatin, cisplatin, bicycloplatin, oxaliplatin, nedaplatin, and satraplatin; retinoids, such as retinoic acid, bexarotene, and retinoic acid; and vinblastine alkaloids and their derivatives, such as vincristine, vinblastine, vinorelbine, and vinorelbine.

[0094] In one specific embodiment of the invention, the anticancer drug ganciclovir (GCV) is used in combination with one or more of the RNA compositions described herein, particularly those containing RNA encoding a recombinant protein (e.g., any one of SEQ ID NO: 21-30), which targets and activates the EBV BGLF4 protein to enhance and promote its anticancer effects in inhibiting and eliminating EBV-positive cancer cells.

[0095] VI. Reagent Kit

[0096] The present invention also provides a kit for treating EBV-related diseases, particularly EBV-related cancers, according to the methods disclosed herein. The kit typically comprises multiple containers, each containing a pharmaceutical composition containing one or more RNA preparations (e.g., the RNA LNP of the present invention). Optionally, the kit may include additional containers, each containing one or more known cancer drugs to be administered concurrently with the RNA composition disclosed herein. The kit may also include informational material providing instructions on how to dispense the pharmaceutical composition, including a description of the type of patient who can be treated (e.g., a human patient who has already received a diagnosis of any of these diseases, or who has been identified as having an elevated risk of developing any of these diseases at a later time, and therefore seeks treatment for such diseases or to reduce the risk of future development of such diseases), and a description of the dosing and administration regimen for dispensing the pharmaceutical composition to the patient.

[0097] Example

[0098] The following embodiments are provided by way of illustration only and not by way of limitation. Those skilled in the art will readily recognize that various non-critical parameters can be changed or modified to produce substantially the same or similar results.

[0099] The unique virus-cell interactions in Epstein-Barr virus (EBV)-associated malignancies suggest that targeting the viral latency-lysis switch is a promising therapeutic strategy. However, the lack of specific and effective therapeutic agents to induce the lysis cycle in these cancers is a major challenge for clinical implementation. We have developed a synthetic transcriptional activator that specifically activates endogenous [viruses / cells]. BZLF1 It effectively induced lysis and reactivation in EBV-positive cancer cells. An encapsulation-encoding... BZLF1 Lipid nanoparticles of nucleoside-modified mRNA containing specific transcription activators (mTZ3-LNP) were used for EBV-targeted therapy. Compared with conventional chemical inducers, mTZ3-LNP more effectively activated EBV cleavage gene expression in EBV-associated epithelial carcinoma. We demonstrated the efficacy and safety of mTZ3-LNP treatment to inhibit tumor growth in an EBV-positive tumor xenograft model. The combination of mTZ3-LNP and ganciclovir produced a highly selective cytotoxic effect against EBV-positive tumor cells using mRNA-based cleavage induction therapy, demonstrating the potential of mRNA nanomedicines in the treatment of EBV-associated epithelial carcinoma.

[0100] introduction

[0101] Epstein-Barr virus (EBV) was the first cancer-associated virus identified in humans, and it affects more than 90% of the global population. Although most EBV carriers remain asymptomatic throughout their lives, latent EBV infection contributes to the transformation and progression of various human malignancies, including endemic Burkitt lymphoma (BL), Hodgkin's lymphoma, natural killer (NK)- / T-cell lymphoma, post-transplant lymphoma, nasopharyngeal carcinoma (NPC), and a subset of gastric cancer (GC). These cancers have a global incidence of more than 265,000 people per year and a mortality of more than 164,000 people per year (1,2). All EBV-associated tumors share unique characteristics, including the persistent presence of multiple viral genomes (up to 100 genomes / cell) and a restricted latency program. In these malignancies, EBV maintains episome genomes and expresses multiple latent genes to modulate cancer traits. The presence of viral episome genomes in all EBV-associated cancer cells serves as a tumor-specific target for developing effective therapeutic strategies against these cancers (2,3).

[0102] Transducing EBV-infected cells from the latent cycle to the lytic cycle can induce growth arrest, promote apoptosis, and cause cell lysis, thus presenting an attractive approach for treating EBV-related malignancies (4-9). When latent EBV is induced into the lytic cycle, it is respectively... BZLF 1 and BRLF1 The immediately early (IE) proteins Zta and Rta are necessarily expressed. These proteins then activate the transcription of a group of early proteins (e.g., EA-D, BGLF4) and late proteins (e.g., VCA, gp350) to promote the lysis and replication of the EBV genome and the production of infectious virions (7-9). Reactivation of EBV from the latent period depends on the expression of the viral Zta protein. Separate ectopic... BZLF1Expression can trigger the transition from the latency phase to the lysis phase and drive the completion of the EBV lysis cycle in EBV-infected cells. Therefore, cytolytic viral activation (CLVA) has been developed as a therapeutic strategy specifically targeting EBV-related cancers, in which chemical lysis inducers (e.g., gemcitabine, valproic acid, sodium butyrate, and other histone deacetylase inhibitors) are used to trigger EBV into the lysis phase. In this type of lysis-inducing therapy, antiviral ganciclovir (GCV) is co-administered with the chemical inducer to the patient to mediate specific cell killing and prevent viral production in EBV-infected cells. GCV is non-cytotoxic to EBV-positive tumors confined to viral latency. However, BGLF4, an EBV-encoded serine / threonine kinase, is expressed during lysis reactivation and can convert non-cytotoxic GCV into an active cytotoxic form via phosphorylation. This BGLF4-converted cytotoxic GCV, or phosphorylated GCV, rapidly kills cancer cells and has been evaluated in clinical trials as an oncolytic cell therapy for EBV-related cancers (3, 7-9). In addition to mediating the direct killing of EBV-positive tumor cells after lysis and reactivation, phosphorylated GCV can translocate to neighboring cells, leading to a “bystander kill” effect. Importantly, phosphorylated GCV can inhibit EBV-encoded DNA polymerase, interrupting the production of infectious virions and preventing viral spread during lysis-induction therapy (7-11).

[0103] CLVA therapy has been evaluated in phase I / II clinical trials involving patients with recurrent NPC and has elicited clinical responses in some patients (10,11). However, the efficacy of chemoactivators used to induce lysis reactivation in EBV-associated tumors is often low and inconsistent. These activators also exhibit low specificity and broad-spectrum cytotoxicity against EBV activation, meaning that treated cells may die from the toxic effects of the chemicals before viral lysis reactivation is induced. Recent studies have revealed that chemoactivator-induced EBV lysis reactivation is cellular environment-specific. The efficiency of chemoactivator-based lysis induction therapy depends on a variety of acquired epigenetic changes and cellular transcription factors in tumor cells (7-9). In native EBV-associated gastric cancer (EBVaGC) and NPC cell lines (e.g., SNU719, C666-1, NPC43, and C17), weak or no lysis gene expression was detected after treatment with various chemoactivators. Even in tumors that respond to chemoactivator therapy, only a small proportion of tumor cells express lysis genes. None of the reported chemical inducers could universally reactivate the lysis cycle in all natural EBV-positive epithelial cancer cell lines.

[0104] In this study, we explored the synthesis of BZLF1Can specific transcriptional activators bypass the limitations of various cytokines on EBV reactivation and improve the specificity of cleavage-induced therapy? Using the CRISPR-Casilio activator system, we demonstrated the feasibility of using artificial activators to reactivate EBV cleavage genes and their cytotoxic effects in NPC and EBVaGC cells (12). A set of designed single-stranded guide RNAs (sgRNAs) were also used in this system to identify… BZLF1 Potential artificial transcription activator binding sequences in the promoter. The CRISPR-Casilio system is a hybrid system combining CRISPR / dCas9 and Pumilio RNA-binding domain fusion effectors, consisting of the dCas9 protein, the Pumilio / fem-3 mRNA-binding factor (PUF)-p65HSF1 activator module, and an sgRNA with five PUF binding site type a (PBSa) encoded by three lentiviral constructs (12). The complexity of the system potentially poses delivery challenges, which reduce the likelihood of delivery. BZLF1 The in vivo activation efficiency in EBV-positive epithelial carcinoma limits its clinical application. In contrast to the CRISPR-Casilio system, transcription activator-like effector (TALE)-based activator systems encode only synthetic proteins with a designed DNA-binding domain fused to the p65HSF1 transactivator. The simplicity of TALE-based transcription activator systems implies their potential effectiveness in developing EBV cleavage induction therapies for clinical application (13). To achieve... BZLF1 The successful in vivo therapeutic delivery of the specific artificial transcription activation system is based on our... BZLF1 The sgRNA binding sequence identified in the promoter was used to synthesize a TALE-based transcriptional activator, which was used to efficiently induce EBV cleavage and reactivation. Then, a sequence encoding this was synthesized. BZLF1 Nucleoside-modified mRNA of a specific TALE-transcriptional activator was encapsulated in formulated lipid nanoparticles (LNPs) for efficient delivery to EBV-associated epithelial carcinoma and to induce immediate early cleavage of EBV genes. BZLF1 Transcription, followed by cleavage cycle transition in tumor cells. In in vitro and in vivo EBV-positive tumor models, the encoding... BZLF1 The LNP-encapsulated mRNA of the specific TALE transcription activator efficiently induces EBV cell cycle lysis and is universally applicable to all EBV-positive epithelial carcinomas. The potent and specific cytotoxicity of this EBV-targeting mRNA drug in in vitro and in vivo tumor models suggests its potential as a promising nanomedicine therapy for EBV-related malignancies.

[0105] result

[0106] CRISPR-Casilio activator system induces EBV cleavage and reactivation

[0107] To reactivate the EBV cleavage cycle in EBVaGC and NPC cells, we first utilized an efficient CRISPR-based Casilio activator system to induce the EBV-encoded immediate early cleavage gene. BZLF1 The Casilio activator system consists of dCas9 protein, a PUFa-p65HSF1 activator module, and sgRNA with 5 copies of PBSa attached (12). The Casilio activator system is specifically designed to recruit multiple PUF-p65HSF1 activators to achieve potent transcriptional activation of target genes. Considering the high feasibility of sgRNA synthesis, the system can be used to screen multiple target sequences for effective transcriptional activation. Here, the EBVaGC cell line SNU719 and the NPC cell line C666-1 were used with dCas9 protein, 3XFLAG-PUFa-p65HSF1 activator module, and a group of sgRNAs that can bind to PBSa. BZLF1 Co-transduction of promoter sgRNA-5xPBSa (sgRNA1, sgRNA2, sgRNA3, sgRNA4) with lentiviral vectors (Tables 8-9). Among the sgRNAs used for co-transfection in SNU719 and C666-1 cells, sgRNA3 induced... BZLF1 The highest expression was observed in Zta, as well as in another immediate early cleavage protein, Rta, and the early cleavage protein BGLF4. Figures 1a to 1b The high efficiency of sgRNA3 in inducing EBV cleavage reactivation was further demonstrated in SNU719, C666-1, and C17 cells engineered using the inducible Casilio (iCasilio) activator system. The iCasilo system was delivered via transduction using a lentiviral vector constitutively expressing sgRNA3, HA-dCas9-2A-EGFP, and the piggyBac transposon containing the Tet-On 3xFLAG-PUFa-p65HSF transactivator (Table 2). Treatment of stably transfected SNU719 and C17 cells with doxycycline (Dox) resulted in transactivator induction and expression of Zta, Rta, and downstream cleavage proteins (e.g., BGLF4, EA-D, VCA, and gp350) in these EBV-positive epithelial carcinoma cells. Figures 1c to 1e The supernatant of the processed cells was collected and analyzed by Akata cells successfully infected with EBV. EBV-阴性 Cellular evidence demonstrates the presence of infectious EBV virions. Transcripts of the EBV-encoding gene were detected in reinfected Akata cells. This finding confirms... BZLF1Endogenous activation of [the substance] induced complete lysis cycle and the production of infectious EBV virions in SNU719 and C17 cells. As previously described, we observed the induction of an abortive early lysis cycle pattern in C666-1 cells (6,14). In stably transfected C666-1 cells treated with Dox, no late lysis proteins (VCA and gp350) were detected, while immediate early (Zta, Rta) and early (BGLF4) lysis proteins were induced. Figures 1c to 1e ). BZLF1 Endogenous activation of Casilio showed significant in vitro cell lysis in EBVaGC and NPC cells. BZLF1 Artificial activation of expression significantly inhibited cell viability and colony formation ability of EBV-positive cancer cells. Figures 1f to 1g Surprisingly, BZLF1 The potent cytotoxicity of artificially activated GCV was also demonstrated in vivo in a nude mouse model implanted with iCasilio-engineered EBV-positive epithelial cancer cells. In the mouse model of EBV-positive epithelial cancer, daily Dox treatment, alone or in combination with GCV, significantly inhibited tumor formation. These findings demonstrate that endogenous GCV... BZLF1 Artificial activation of expression can effectively reactivate the lysis cycle and induce cell lysis in EBV-positive epithelial carcinoma.

[0108] Development of TALE activator for artificial activation of BZLF1

[0109] Using the established Casilio activation system with designed sgRNAs, we identified a method for artificial activation. BZLF1 The target sequence in the promoter was selected to support attempts to develop a synthetic transcriptional activator for effective EBV cleavage-induced therapy. However, efficient delivery of a complex Casilio activator system containing three complex, large constructs to target EBV-positive tumors in patients will be challenging. We assembled a system encoding a precise and efficient TALE transcriptional activator, a nuclear localization signal (NLS), and an artificial activation... BZLF1 A single construct of the transcriptional activation domain of transcription, the TALE transcription activator contains only the central repeat domain for DNA recognition. We designed and constructed the TALE activator plasmid TZ3, which specifically targets… BZLF1Predicted binding sequences in the promoter were used to activate endogenous Zta expression in EBV-positive tumor cells (Tables 8-10). The constructed TALE transcription activator had an open reading frame of approximately 3.6 kb, encoding a FLAG tag and a designed TALE DNA-binding domain and NLS domain fused with the p65HSF1 transactivator. In addition to TZ3, we also constructed TALE transcription activators targeting regions overlapping with the binding sequences of sgRNA1, sgRNA2, and sgRNA4 (TZ1, TZ2, and TZ4, respectively) to determine the consistency of the Casilio and TALE-based artificial transcription activation systems. Figures 2a to 2b As shown, in the use of targeted BZLF Transient transfection of the TALE plasmid into SNU719 and C666-1 cells induced Zta expression, but not into cells transfected with a control vector lacking a DNA-binding domain. In addition to Zta, downstream immediate early (Rta), early (BGLF4), and late (VCA) cleavage proteins were detected in EBV-positive SNU719 cells transfected with TZ3. Furthermore, TZ3 induced high levels of expression of immediate early (Zta), Rta, and early (BGLF4) cleavage proteins in C666-1 cells. These findings confirm that ectopic expression of the synthetic transcription activator TZ3 successfully reactivated the viral cleavage cycle in EBV-positive tumor cells. TZ3 induced the highest level of Zta expression compared to other TALEs (TZ1, TZ2, and TZ4), consistent with findings obtained using the Casilio activator system with sgRNA3.

[0110] like Figure 2c As shown, BZLF1 The TZ3 targeting sequence in the promoter is conserved in all reported EBV variants (15). Using electrophoretic mobility shift assay (EMSA) analysis, we showed that the TZ3 activating protein is conserved in all EBV genome variants. BZLF1 It binds to the promoter sequence, but not to the mutant probe. Figure 2d These findings demonstrate that the synthesized TZ3 protein specifically binds to the EBV genome. BZLF1 The promoter activates transcription of this immediately early cleavage gene. Furthermore, transient transfection of HK1 cells (EBV-negative NPC cell line) with TZ3 did not induce significant changes in transcriptome or cell viability and cell cycle regulation. Figures 2e to 2g These findings demonstrate the high specificity of the synthetic TALE transcription activator in targeting EBV-positive tumor cells. Next, TZ3 was used to further develop TALE-based lysis-induction therapy for EBV-associated epithelial carcinoma.

[0111] Synthesized nucleoside-modified mRNA encoding TZ3 cleavage activator

[0112] In order to effectively activate endogenous... BZLF1 For clinical applications, we synthesized a nucleoside-modified mRNA encoding a TALE-based transcriptional activator for in vivo delivery using LNPs. The T7 promoter in the TZ3 construct is used to initiate [the process / delivery]. TZ3 In vitro transcription (IVT) of mRNA. For in vitro and in vivo studies, a nucleoside-modified mRNA with a 5' cap and a 3'-poly(A) tail was synthesized. TZ3 mRNA, and the nucleoside-modified mRNA was encapsulated in an LNP formulation containing ALC-0315, 1,2-distearyl-sn-glycerol-3-phosphocholine (DSPC), cholesterol, and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2000). TZ3 mRNA. These components have previously been used in LNP, which is formulated for in vivo delivery of modified mRNA in BNT162b2, a SARS-CoV-2 vaccine approved by the U.S. Food and Drug Administration (FDA) (16). Dynamic light scattering analysis (DLS) was used to determine the formulated encapsulation. TZ3 The size of the LNP (mTZ3-LNP) of the mRNA is approximately 124 nm, and its polydispersity index is 0.124 ± 0.032. Figure 3a The synthesized mTZ3-LNP achieved an encapsulation efficiency of up to 90%. Figure 3b In this study, we used confocal laser scanning microscopy to demonstrate the uptake of mTZ3-LNP by EBV-positive SNU719 cells and TZ3 Intracellular release of mRNA.

[0113] After incubation with mTZ3-LNP for 24 hours, effectively induced Zta expression was detected in EBV-positive SNU719 and C666-1 cells by Western blotting. Figure 3c Induced Zta expression was observed in SNU719 and C666-1 cells at 12 h and 18 h following mTZ3-LNP treatment, respectively. Figure 3d Using quantitative reverse transcription polymerase chain reaction (qRT-PCR), we revealed that as early as 4 h (hours) and 8 h (hours) after mTZ3-LNP treatment, respectively, in SNU719 and C666-1 cells... BZLF1 Transcript upregulation. At a later time point, induction of Zta-downstream EBV immediate early (Rta), early (BGLF4, EA-D), and late (VCA) cleavage proteins was observed in these cells. Figure 3dEarly cleavage proteins BGLF4 and EA-D were expressed in C666-1 cells 48 to 72 hours after mTZ3-LNP treatment. Early (BGLF4, EA-D) and late (VCA) cleavage proteins were induced in SN719 cells 18 hours after mTZ3-LNP treatment. Figure 3d Immunofluorescence staining confirmed the prevalence of Zta and downstream early cleavage protein EA-D expression in SNU719 and C666-1 cells. Expression of the late cleavage protein gp350 was observed in SNU719 cells 96 h (hours) after mTZ3-LNP treatment. Figure 3e Flow cytometry analysis detected up to 71.9% and 83.7% Zta-positive tumor cells in mTZ3-LNP-treated SNU719 and C666-1 cells, respectively. Figure 3f Compared to Zta-positive cells, a smaller proportion of EA-D-positive and gp350-positive cells were detected in mTZ3-LNP-treated SNU719 and C666-1 cells at 72 h and 96 h post-treatment, indicating a heterogeneous state of the lytic cycle in these lysed and reactivated EBV-positive cancer cells. Notably, caspase-3 cleavage was induced in mTZ3-LNP-treated EBV-positive cancer cells, including C666-1 cells that underwent only abortive lytic cycle reactivation, from 72 h to 96 h post-treatment. These findings demonstrate that the production of synthetic mTZ3-LNP can induce lysis in EBV-positive cancer cells. BZLF1 The expressed functional TZ3 transcriptional activator.

[0114] In addition to SNU719 and C666-1, the ability of mTZ3-LNP to reactivate Zta expression was also tested in a group of EBV-positive cancer models, including four NPC (C17, NPC43, NPC43-M81, and NPC76c), two EBVaGC (AGS-EBV and YCCLE1), and two BL (Akata-EBV and P3HR1) cell lines (Figure 4). Surprisingly, mTZ3-LNP effectively induced Zta expression in all EBV-positive cancer cell lines. Flow cytometry analysis after 48 hours of treatment with mTZ3-LNP revealed that the proportion of cells expressing Zta in this group of EBV-positive cancer models ranged from approximately 26.4% to 92.2%. Figures 4a to 4d Western blotting also demonstrated the induced expression of Zta and its downstream EBV cleavage protein in mTZ3-LNP-treated YCCLE1 and NPC43-M81 cells. Figure 4eCompared with chemoactivators, mTZ3-LNP more effectively reactivated the EBV lysis cycle in EBV-positive epithelial carcinoma. At 48 h post-MTZ3-LNP treatment, Zta expression was detected in up to 86.2% of SNU719 cells and 78.9% of C666-1 cells, respectively, while Zta expression was detected in only 19.5% of SNU719 cells and 3% of C666-1 cells, respectively, after sodium butyrate (NaB) treatment. Figures 4a to 4b Therefore, our study demonstrates that mTZ3-LNP is an effective lysis activator for different types of EBV-positive malignant tumor cells.

[0115] Using RNA sequencing (RNA-seq), we revealed that almost no cellular genes were transcribed and activated in EBV-negative HK1 cells treated with mTZ3-LNP, indicating the high specificity of the TZ3 transcription activator. Figures 5a to 5b We observed similar results in HK1 cells transiently transfected with the TZ3 construct. Figure 2e Notably, significant upregulation of EBV and other cellular transcripts was observed in mTZ3-LNP-treated SNU719 and C666-1 cells. Figure 5b ).Apart from BZLF1 In addition, EBV transcriptome profiling demonstrated the presence of multiple cleaved gene transcripts (e.g., in EBV-positive tumor cells treated with mTZ3-LNP) in these cells. BRLF1, BGLF4, BXLF1, LF3, BALF2, BHLF1, BMRF1 The extensive expression of ) Figure 5c Furthermore, mTZ3-LNP mediated... BZLF1 Expression and cleavage reactivation induced the expression of multiple cellular genes in EBV-positive SNU719 and C666-1 cells. Although differentially expressed genes are involved in multiple cellular mechanisms, few of these genes were detected in mTZ3-LNP-treated SNU719 and C666-1 cells. These distinct transcriptional patterns can be attributed to unique genomic variations in each cell line and the abortive cleavage cycle in C666-1 cells.

[0116] In addition to RNA-seq studies, we also confirmed the absence of off-target effects in mTZ3-LNP treatment through chromatin immunoprecipitation (ChIP) sequencing analysis using anti-FLAG antibodies. Figure 5d As shown, from the EBV genome BZLF The predicted TZ-binding sequence in the promoter was enriched in EBV-positive SNU719 cells treated with mTZ3-LNP. ChIP sequencing analysis did not identify any potential TZ3-binding sequences in the exons, introns, and regulatory regions of the human gene. Furthermore, the synthetic mTZ3-LNP exhibited specific cytotoxicity against EBV-positive cancer cells. Figure 5d The results showed that, specifically, a significant decrease in cell viability was observed in EBV-positive SNU719 and C666-1 cells treated with mTZ3-LNP. Notably, mTZ3-LNP did not significantly affect the viability of the EBV-negative NPC cell line HK1 alone, nor did it significantly affect the viability of the EBV-negative NPC cell line HK1 in combination with GCV.

[0117] Reactivation of EBV cleavage gene via in vivo delivery of mTZ3-LNP

[0118] This study investigated the in vivo delivery of luciferase mRNA to EBV-positive tumors via a formulated ALC-0315-LNP in a non-obese diabetic severe combined immunodeficiency disease (NOD-SCID) mouse model. For this purpose, luciferase mRNA-encapsulated LNPs were intravenously injected into mice. Notably, luciferase protein signaling was detected in the tumors 24 hours (hours) post-injection. No luciferase protein signaling was found in other normal organs of the mice, except for the liver. The delivery of luciferase mRNA to EBV-positive tumors via a formulated ALC-0315-LNP was investigated. TZ3 After intravenous injection of mRNA into NOD-SCID mice, we determined that the circulating half-life of the formulated mTZ3-LNP was 8.34 h. Three h after injection of Dil C18-labeled LNP-encapsulated TZ3 mRNA, Dil C18 fluorescence was also detected in tumor tissue from the mice.

[0119] We demonstrated the efficient induction of endogenous Zta expression in EBVaGC and NPC tumors in vivo by intravenous injection of mTZ3-LNP into NOD-SCID mice implanted with EBV-positive cancer cells. Immunohistochemical staining revealed significant induction of Zta, EA-D / BMRF1, and gp350 expression in SNU719 xenograft tumor cells at 12 h, 24 h, and 48 h post-mTZ3-LNP injection; however, we did not observe similar findings in the control group. Figure 6a At 48 h post-treatment, expression of Zta, EA-D, and gp350 cleavage proteins was detected in 10%, 12%, and 9% of tumor cells, respectively. These findings indicate that mTZ3-LNP treatment successfully induced the expression of early and late cleavage proteins in EBV-positive tumor xenografts in a NOD-SCID mouse model. In addition... BZLF1 In addition, RNA in situ hybridization assays using RNAscope detected [various components] in mTZ3-LNP treated tumors. BGLF4, BMRF1 and BLLF1 Large amounts of transcription of other downstream cleavage genes ( Figure 6cVarious amounts of immediate early or early cleavage gene transcripts were observed in tumor cells, indicating heterogeneity in terms of cleavage cycle phase. Our study demonstrated that 48 h (hours) after intravenous injection of mTZ3-LNP, these transcripts were present in 15.1%, 12.7%, 18.3%, and 8.1% of tumor cells, respectively. BZLF1, BGLF4, BMRF1 and BLLF1 Transcriptional activation of genes ( Figure 6d It is worth noting that, BGLF4 It is not only an important marker of the lysis cycle process, but also encodes the serine / threonine kinase that converts non-cytotoxic GCV into its cytotoxic form.

[0120] In vivo therapeutic efficacy of mRNA-based cleavage induction therapy

[0121] The therapeutic efficacy of combined mTZ3-LNP and GCV treatment against EBV-associated epithelial carcinoma was evaluated in NOD-SCID mouse models implanted with SNU719, C666-1, and C17 cells and NPC patient-derived xenografts (PDX) (Xeno-76). When the tumor size reached 80 mm... 3 Up to 100 mm 3 At that time, mRNA-based cleavage induction treatment was initiated. mTZ3-LNP was injected via tail vein every 2 to 3 days, while GCV was injected intraperitoneally daily. Figure 7a ).like Figures 7b to 7d As shown, potent growth inhibition of SNU719, C666-1, C17, and xeno-76 tumors was observed in mice treated with mTZ3-LNP alone or in combination with GCV compared to the control (P < 0.005). Similar tumor growth inhibition was observed in all EBV-positive xenograft models with mTZ3-LNP alone and with combination with GCV. No significant changes in mouse body weight were observed during treatment. Notably, residual tumors harvested from mice treated with mTZ3-LNP alone or with combination with GCV contained significantly fewer tumor cell components, but increased proportions of necrotic lesions, lymphocytes, and fibroblasts. Figure 7bAlthough NOD-SCID mice lack B and T lymphocytes, the increased inflammation can be induced by EBV lysis reactivation and persistent cell death processes. This observation reinforces our observation of the potent in vivo antitumor effect of mTZ3-LNP against EBV-positive cancers. Apart from no change in body weight, neither organ tissue damage nor significant changes in serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), and creatinine concentrations were observed in mice treated with mTZ3-LNP and GCV, highlighting the safety of mTZ3-LNP-mediated lysis-inducing therapy in a preclinical model of EBV-related cancers.

[0122] discuss

[0123] Persistent latency in EBV-positive epithelial cancer cells is closely controlled by acquired genetic alterations and epigenetic modifications in both the virus and the host genome. A variety of viruses and cytokines have been shown to regulate the latency-lysis switch to prevent cell death through lysis induction during transformation and clonal expansion (7-9). Over the past few decades, a variety of drugs or agents with lysis-inducing capabilities in EBV-positive tumor cells have been reported and described. These chemical lysis inducers include histone deacetylase inhibitors (e.g., sodium butyrate, valproic acid, vorinostat (suberanilohydroxamic acid), and romidepsin), DNA methyltransferase inhibitors (e.g., 5-aza-2'-deoxycytidine and 5-azacytidine), protein kinase C activators (e.g., TPA), chemotherapeutic agents (e.g., gemcitabine), antimicrobial antibiotics (e.g., clofoxetine), and several novel compounds (e.g., C7 and E11). These compounds reactivate EBV lysis genes through different mechanisms, targeting epigenetic regulation or cellular signaling pathways (8). Although phase I / II clinical trials of CLVA therapy using a combination of gemcitabine, valproic acid, and valganciclovir have demonstrated its safety and clinical response in patients with recurrent NPC, response rates have been reported to be less than 30% (10,11). Previous in vitro studies have shown that chemical lysis inducers elicit cellular environment- and cell type-specific lysis-inducing responses in EBV-positive epithelial cancer cells. Most chemical lysis inducers are far less efficient at reactivating EBV lysis genes in patient-derived EBV-positive epithelial cancer cell lines (e.g., SNU719, C666-1, NPC43, and C17) compared to EBV-reinfected cancer cells (e.g., HK1-EBV, AGS-BX1, and HONE-1-EBV). It is believed that lysis-inducing responses in EBV-infected tumor cells are influenced by epigenetic modifications and aberrant oncogenic signaling pathways acquired during clonal expansion. In addition to their low efficiency in lysis reactivation and intertumor heterogeneity, the clinical implementation of chemical lysis inducers for EBV lysis induction therapy is limited by the broad-spectrum cytotoxicity of these compounds. At low doses, these drugs cannot effectively induce EBV lysis reactivation. However, at high doses, both EBV-infected and uninfected normal cells can be killed indiscriminately by the drugs.

[0124] To overcome the high level of complexity in the regulatory mechanism of EBV latency-cleavage switch, we developed a TALE-based transcriptional activator to artificially activate the immediate early EBV gene in EBV-positive tumor cells. BZLF1 The expression ( Figure 8By utilizing mRNAs encapsulated with highly specific LNPs encoding the TALE transcription activator, we demonstrated the artificial activation of endogenous [activation] in multiple models of EBV-positive epithelial carcinoma. BZLF1 The expression demonstrated effective in vivo antitumor activity. We further demonstrated the highly specific cytotoxicity of this method against EBV-positive cells. The synthesized transcription activator specifically targeted EBV-positive cancer cells. BZLF1 The promoter is absent in EBV-negative cells and does not induce transcriptional activity. In addition to reported growth arrest and cytotoxic effects, Zta can upregulate the transcription of various cellular genes that contribute to multiple cancer features. Our approach avoids the safety concerns arising from the cytotoxicity and potential oncogenicity of ectopic Zta expression in uninfected cells (18).

[0125] Unlike Casilio CRISPR-dCas9-based platforms with three essential components, it is feasible to deliver TALE transcription activator factors in vivo to patients' primary and metastatic tumors using LNP-encapsulated mRNA technology. We utilized an LNP formulation used in FDA-approved mRNA vaccines to develop a novel mRNA drug, mTZ3-LNP, for highly efficient cleavage-inducing therapy against EBV-associated epithelial carcinoma. In our study, the safety and specificity of mTZ3-LNP were demonstrated in in vitro and in vivo preclinical models of EBVaGC and NPC. In addition to its ability to specifically bind to target sequences, the strong transcriptional activity of TALE transcription activator TZ3 is likely due to the high copy number and low methylation of EBV epithelial cells in EBV-positive tumor cells. BZLF1 Promoter-induced (7-9). Importantly, when Zta expression is induced by mTZ3-LNP, Zta can cis- or trans-activate the BZLF1 promoter, which encodes multiple EBV episomes in tumor cells. Zta also drives the expression of the trans-activator Rta. BRLF1 The expression of Rta; Rta then induces BZLF1 Transcription, forming a positive feedback loop to activate BZLF1 Promoter. These two mechanisms are maintained after transient expression of the synthesized TZ3 transcription activator in EBV-positive tumor cells. BZLF1 Activation and accumulation of Zta (9). In C17, the NPC cell line contains only 2 to 3 copies of the EBV genome per cell, and treatment with artificial activators also induces Zta expression. As demonstrated in our study, this was observed in C17 cells after Dox treatment. BZLF1This finding indicates that tumor cells with low EBV genome copy numbers require a longer Zta accumulation period to activate the expression of downstream early and late cleavage proteins. Notably, significant growth inhibition was observed in an in vivo C17 CDX model following multiple doses of mRNA-TZ3. Furthermore, the high efficiency of mTZ3-LNP in inducing cleavage reactivation in different types of EBV-related cancers is attributed to its mechanism of action, which is independent of host epigenetic status or aberrant signaling pathways.

[0126] Using a suite of tumor xenograft models, our in vivo studies highlight the therapeutic efficacy of this newly developed mRNA-based lysis-induction therapy against EBV-positive epithelial carcinoma. The safety of long-term intravenous administration of mTZ3-LNP was demonstrated in a NOD-SCID mouse xenograft model. For clinical implementation of lysis-induction therapy, co-administration with GCV is essential for the rapid and specific killing of EBV-positive tumor cells and the inhibition of infectious virion production. In vivo studies showed no significant difference in growth inhibition between tumors treated alone with mTZ3-LNP or in combination with GCV. The lack of a significant bystander effect with the combination treatment is likely due to the high efficiency of lysis reactivation and the potent cytotoxicity of mTZ3-LNP against EBV-positive tumors. Despite the limited bystander killing effect observed, GCV administration is a necessary procedure for the rapid and specific killing of EBV-positive cells and the inhibition of infectious virion production during EBV lysis induction. In addition to direct cytotoxic effects, potent innate and adaptive immune responses induced by large amounts of immunogenic lysed proteins can also help to effectively eradicate EBV-positive cancers during lysis-induction therapy (19,20).

[0127] Note that the NOD-SCID mouse model used in this study lacked B lymphocytes and T lymphocytes, which are key immune cells that trigger the host's immune response to highly expressed EBV cleavage antigens. However, we observed NK cells accumulating in adjacent necrotic areas and infiltrating into residual tumors in the mTZ3-LNP-treated mouse model. In future studies, we will establish EBV-positive tumor xenografts in humanized mouse models, allowing us to precisely elucidate the innate and adaptive immune responses induced by mTZ3-LNP treatment and the potential therapeutic effects of combined treatment with mTZ3-LNP and immune checkpoint blockade (21). The combination of mTZ3-LNP with immunotherapeutic strategies (e.g., immune checkpoint blockade) and NK cell therapy could further enhance the treatment response in patients with EBV-related cancers.

[0128] With the widespread use of mRNA COVID-19 vaccines during the COVID-19 pandemic, the development of other mRNA therapies targeting various human diseases, including cancer, is on the rise. In this proof-of-concept study, we successfully developed a first-in-class mRNA drug for lysis-inducing therapy against EBV-associated epithelial carcinoma. By developing nucleoside-modified mRNA technology, a non-viral delivery strategy, and the TALE artificial activator system, we produced mTZ3-LNP, a highly effective inducer of lysis reactivation and selective killing of EBV-positive tumor cells. This novel mRNA nanomedicine offers promising clinical opportunities for lysis-inducing therapy against EBV-associated epithelial carcinoma.

[0129] method

[0130] Cell lines and patient-derived xenografts

[0131] The EBVaGC cell line SNU719 was obtained from the Korean Cell Line Bank, Seoul, Republic of Korea. The EBVaGC cell lines YCCEL1 and AGS-EBV were provided by Professor Qian Tao and Professor Jun Yu, respectively, from the University of Hong Kong (22,23). EBV-positive NPC cell lines C666-1, C17, NPC43, NPC43-M81, and NPC76c were established in our laboratory (24-26). These cell lines were used in various in vitro experiments. EBV-positive NPC PDX and Xeno-76 were used in our in vivo studies (26). The EBV-negative NPC cell line HK1 was included as a control (27). Two EBV-positive BL cell lines, P3HR1 and Akata-EBV, preserved in our laboratory, were also used in this experiment. Except for C17, NPC43, NPC43M81, and NPC76c, all cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium (Sigma, St. Louis, MO, USA) supplemented with 10% fetal bovine serum (Gibco, Waltham, MA, USA). To maintain the growth of the C17 and NPC76c cell lines, 0.5 μM of Y-27632 (a ROCK inhibitor) (Enzo Life Sciences Inc., Farmingdale, NY, USA) was added to the RPMI-1640 medium. All cell cultures and all biological experiments were performed at 37°C and 5% CO2. Short tandem repeat (STR) mapping analysis and... EBERIn situ hybridization was used to identify all cell lines used in this study. Mycoplasma contamination in all cells was tested by PCR using primer sets 5′-YGCCTGVGTAGTAYRYWCGC-3′ (MYCO5) and 5′-GCGGTGTGTACAARMCCCGA-3′ (MYCO3). No cell lines listed in the International Committee for Cell Line Identification (ICLAC) that are prone to misclassification were used in this study.

[0132] Constructing TALE plasmids

[0133] The Golden Gate assembly protocol (Golden Gate TAL Effector Kit 2.0, #1000000024; Addgene, Watertown, MA, USA) was used to design and construct the TAL Effector Kit in vitro. BZLF1 Promoter-targeted TALE, as previously described (28). Based on BZLF1 The target sequences identified on the promoter were used to design the amino acid sequences of the binding domains of TZ3 and other TALEs (TZ1, TZ2, and TZ4), as shown in Tables 9 and 10. The constructed TALEs were then fused with the p65 activation domain to ensure transcription. In short, the DNA sequence designed for expression of FLAG-NLS-lacZ-p65HSF1 was first cloned into the pcDNA3.1 vector. Customizable polymorphic amino acid repeat sequences from TAL effectors targeting the EBV BZLF1 promoter sequence were used to replace lacZ using multi-round Golden Gate cloning. The TZ3 plasmid or other TALE plasmids (TZ1, TZ2, and TZ4) were transiently transfected into cells for expression or the plasmids were linearized for in vitro transcription.

[0134] Preparation of nucleoside-modified mRNA

[0135] pass XmaI Digestion linearized and purified the TZ3 plasmid. The linearized TZ3 plasmid was then used as a template under T7 promoter regulation. Nucleoside-modified mRNA was synthesized as described in

[29] . Briefly, in vitro transcription was performed using the T7 High Yield RNA Synthesis Kit (New England BioLabs, Ipswich, MA, USA), in which UTP was 100% replaced with N1meΨTP and 1 μg template. The reaction mixture was incubated at 37°C for 4 h (hours) followed by treatment with RNase-free DNase I. Then, mRNA capping was performed using the vaccinia virus capping system (New England BioLabs), followed by Escherichia coli (E. coli) E. coliPoly(A) polymerase (New England BioLabs) was added to the 3' poly(A)-tail. mRNA purification was performed using the Monarch RNA Cleansing Kit (New England BioLabs).

[0136] Preparation and characterization of LNP-encapsulated mRNA

[0137] mTZ3-LNP was prepared by mixing an ethanol phase containing lipids with purified mTZ3 mRNA in an aqueous phase in a microfluidic device. Briefly, the ethanol phase was prepared by dissolving an ionizable lipid ALC-0315 (Cayman Chemical, Ann Arbor, MI, USA), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC; Avanti, Alabaster, AL, USA), cholesterol (Sigma), and 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG2000; Avanti) in ethanol at a molecular ratio of 50:10:38.5:1.5. The aqueous phase was prepared by diluting mTZ3 mRNA or luciferase mRNA (Trilink, San Diego, CA, USA) 8-fold in 200 mM acetate buffer (pH 5.0). The aqueous and ethanol phases were mixed using a syringe pump at a 3:1 ratio. At 4°C, the obtained LNPs were dialyzed for 2 h in 1x phosphate-buffered saline (PBS) using a 20,000 MWCO box (Invitrogen, Carlsbad, CA, USA). The encapsulation efficiency of mRNA-LNPs was calculated according to previous reports (29). Briefly, samples were treated alone with PBS buffer (as unencapsulated mRNA) or with 2% Triton X-100 (as total mRNA). RNA concentration was measured using the Qubit RNA HS assay kit (Invitrogen) and a Qubit 4 fluorometer (Invitrogen). Encapsulation efficiency (EE) was calculated using the following formula: EE = [1 - (unencapsulated mRNA / total mRNA) × 100%] (30). The mean size, polydispersity index, and zeta potential of the prepared mTZ3-LNPs were determined using dynamic light scattering and a Zetasizer Nano ZS90 system (Malvern PANalytical, Worcestershire, UK). Samples were diluted with PBS before measurement. To investigate the cellular uptake of the formulated mTZ3-LNP, Cy5-labeled mTZ3 mRNA encapsulated with LNP was incubated with SNU719 cells for 1 h, 3 h, and 6 h. Fluorescence signals emitted by endosomes stained with Cy5-mTZ3-LNP and LysoTracker Green (Invitrogen) were measured using an LSM 880 confocal laser scanning microscope (Zeiss, Jena, Germany) equipped with an AxioObserver system.Fluorescence signals were measured in three channels: Cy5, excitation / emission wavelengths (ex / em) 633 / 697 nm; Dnd-26, ex / em, 488 / 524 nm; and Hoechst, ex / em 405 / 460 nm.

[0138] Quantitative real-time PCR

[0139] Total RNA was extracted from cells using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The extracted RNA was then reverse transcribed into complementary DNA using an RT kit with a gDNA Eraser (TaKaRa, Kyoto, Japan), and quantitative real-time PCR was performed using SYBR Green master mix (Thermo Fisher Scientific). The mRNA expression level of the EBV cleavage gene was normalized relative to the mRNA expression level of GAPDH. Primer sequences are listed in Table 11.

[0140] Western blot assay

[0141] Protein extracts were prepared using RIPA lysis buffer supplemented with protease inhibitors (Roche, Basel, Switzerland). Protein concentrations were determined using a protein assay kit based on a bovine serum albumin (BSA) standard curve (Bio-Rad, Hercules, CA, USA). Equal amounts of protein from each extract were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a 0.45 μm nitrocellulose membrane. The blocked membrane was incubated with an appropriate primary antibody. The primary antibodies used in this study included anti-BZLF1 / Zta (BZ1, Santa Cruz Biotechnology, Dallas, TX, USA; 1:1000), anti-EA-D (1108-1, Santa Cruz Biotechnology; 1:1000); anti-BGLF4 (1:1000), VCAp18 (# PA1-73003, Invitrogen; 1:500); anti-caspase 3 (Asp175, Cell Signaling; 1:1000); and anti-actin (13E5, Cell Signaling; 1:4000). After washing, the antibodies were incubated with secondary antibodies. Signal in the blots was detected using a ChemiDoc imaging system (Bio-Rad).

[0142] Fluorescence-activated cell sorting (FACS) analysis

[0143] Cells treated with chemical inducers, mTZ3-LNP, or control-LNP were trypsinized, collected, and washed with cold PBS. Cells were then fixed in freshly prepared 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 PBS. The cell pellet was stained with Alexa-647-conjugated mouse anti-BZLF1 / Zta antibody (BZ1, Santa Cruz Biotechnology; 1:100), Alexa-594-conjugated anti-EA-D antibody (1108-1, Santa Cruz Biotechnology; 1:100), or Alexa-488-conjugated anti-gp350 antibody (Santa Cruz Biotechnology; 1:100), and analyzed using a BD LSRFortessa cell analyzer (Becton Dickinson, Franklin Lakes, NJ, USA). Data were analyzed using FlowJo software (version 10) (FlowJo, LLC, Ashland, OR, USA).

[0144] Immunofluorescence staining

[0145] For immunofluorescence staining, cells were seeded onto coverslips in 6-well plates the day before treatment. Cells were then washed with PBS, fixed in 4% paraformaldehyde, and permeabilized in PBS with 0.1% Triton X-100 for 30 min. Cells were then incubated in the dark at room temperature for 2 h (hours) with Alexa-647-conjugated anti-BZLF1 antibody (BZ1, Santa Cruz Biotechnology; 1:100), Alexa-594-conjugated anti-EA-D (1108-1, Santa Cruz Biotechnology; 1:100), or Alexa-488-conjugated anti-gp350 (0221, Santa Cruz Biotechnology; 1:100). Finally, the stained cells were counterstained with DAPI and fixed onto slides using Dako fluorescent mounting medium (Agilent, Santa Clara, CA, USA). Images were processed using an LSM 880 confocal laser scanning microscope (Zeiss, Oberkochen, Germany) with Zen software.

[0146] Immunohistochemical staining

[0147] Immunohistochemical staining was used to detect the expression of Zta, EA-D, and gp350 proteins in EBV-positive xenograft tumor sections. Briefly, 4 μm sections were obtained from tumors grown in mice treated with PBS, GCV, mTZ3-LNP, or a combination of mTZ3-LNP and GCV. Paraffin-embedded sections were dewaxed, rehydrated, and washed with water. After antigen retrieval, samples were incubated with primary antibodies against BZLF1 / Zta (BZ1, Santa Cruz Biotechnology; 1:100), anti-EA-D (1108-1, Santa Cruz Biotechnology; 1:100), or anti-gp350 (0221, Santa Cruz Biotechnology; 1:100). Sections were then incubated with horseradish peroxidase-labeled secondary antibody, developed with 3,3'-diaminobenzidine, and counterstained with hematoxylin (Sigma). The percentage of cells expressing EBV cleavage proteins was assessed from tumor sections from mice treated with mTZ3-LNP and controls. Representative images were obtained using a Nikon ECLIPSE Ni-E microscope (Nikon, Tokyo, Japan) equipped with a Ds-Ri2 microscope camera and NIS-Elements software. At least four different images (x200 magnification) were obtained in three replicates for each group and analyzed using ImageJ software to determine the percentage of tumor cells expressing EBV cleavage proteins.

[0148] EBER in situ hybridization

[0149] EBV-positive cancer cells were detected in tumor samples using EBER in situ hybridization assay. This was performed according to the manufacturer's instructions. EBER The probe ISH kit (Leica, Newcastle, UK) is used to confirm the presence of EBV in formalin-fixed paraffin-embedded (FFPE) tumor sections.

[0150] RNAscope RNA in situ hybridization

[0151] Detection was performed using RNAscope 2.0 RISH assay and a suite of EBV cleavage gene-specific probes (Advanced Cell Diagnostics, USA). BZLF1 and a group of lysed gene transcripts ( BMRF1, BGLF4 and BLLF1 The expression of EBV cleavage gene transcripts was as described previously (26). The percentage of cells expressing EBV cleavage gene transcripts in tumor sections of mice treated with mTZ3-LNP and controls was evaluated as described in the immunohistochemical staining section.

[0152] Detection of infectious EBV particles

[0153] Supernatant from cultures of EBV-positive tumor cells induced for artificial EBV lysis and reactivation was collected and centrifuged at 800 rpm for 5 min, then filtered through a 0.45 μm cellulose acetate filter to remove cell debris. The centrifuged and filtered supernatant containing EBV particles was further ultracentrifuged at 20,000 rpm for 4 h at 4 °C to precipitate the EBV particles. The supernatant was discarded, and the precipitate was resuspended in RPMI-1640 medium supplemented with 10% fetal bovine serum at 1 / 30 of the original supernatant volume. These procedures increased the EBV concentration in the culture supernatant by 30-fold. EBV-negative Akata cells were then incubated with the concentrated EBV supernatant for 3 days. Infected Akata cells were then collected, and DNA and RNA were extracted separately to detect the presence of EBV genome and EBV gene expression (25).

[0154] RNA sequencing

[0155] To assess the RNA profile of cancer cells after TZ3 transfection or mTZ3-LNP treatment, total RNA was extracted from cells using TRIzol reagent (Invitrogen). RNA sequencing libraries were prepared using the Swift RNA Library Kit (Swift Biosciences, AnnArbor, MI, USA) after DNase I treatment and rRNA and globin depletion. Next-generation sequencing (150 bp, paired ends) was performed using an Illumina HiSeq1500 sequencing system (Illumina, San Diego, CA, USA). Emporiomic and low-quality sequences in the total sequencing reads were filtered before downstream analysis. Briefly, the reads were mapped, aligned, and annotated to the human reference genome (GRCh38) and the EBV genome (chrEBV_Akata_inverted) using Hisat2 (2.1.0) with the "-rna-strandness RF" parameter and StringTie (1.3.6) (32). Downstream analysis was performed on the R software platform (v4.1.0). Differentially expressed genes between control and TZ3-treated samples were identified using DEseq2 (1.32.0), with a false discovery rate of less than 0.05 as the criterion (33). Expression levels of protein-coding genes were further determined by gene set enrichment analysis (GSEA) using Hallmark and GO:BP gene sets obtained from molecular feature databases and the clusterProfiler package (4.0.5) (34–35). Volcano plots were generated using the ggplot2 package (3.4.1). Raw data from the RNA sequencing fastq files were archived in the Sequence Reading Archive (RA) of the National Center for Biotechnology Information (NCBI) with accession number PRJNA1007461.

[0156] Chromatin immunoprecipitation sequencing

[0157] As previously described, the genome specificity of TALE transcription activator TZ3 was assessed in mTZ3-LNP-treated C666-1 cells using ChIP sequencing (36). mTZ3-LNP-treated and control cells were fixed in 1% formaldehyde and quenched with glycine. Chromatin was prepared using the truCHIP Chromatin Disruption Kit (Covaris, Woburn, USA) and fragmented into 100 bp to 500 bp fragments using a Covaris S220 focused sonicator (Covaris). Protein-DNA complexes were immunoprecipitated overnight at 4°C on a spinner with 5 μg anti-FLAG antibody (F1804, Sigma), followed by purification using magnetic beads (26162, Pierce; ThermoFisher). After washing, crosslinking was reversed and DNA was purified. 8 ng of immunoprecipitated DNA and input DNA were used for each Illumina sequencing library construction according to the manufacturer's protocol (Kapa Hyper Prep Kit, KK8504, Roche). Each library was sequenced on the Nextseq 500 platform (Illumina) to obtain 150-base paired-end reads. Sequencing tags were mapped to the Akata reference genome (accession number KC207813) using Bowtie 2. Broad peak identification was performed using MACS2 analysis with unique FLAG tag mapping identifiers. The raw data from the ChIP-sequencing fastq files have been archived in the Sequence Reads Archive (RA) of the National Center for Biotechnology Information (NCBI) with accession number PRJNA1007461.

[0158] EMSA

[0159] As previously described (37), EMSA was performed to determine the TZ3 trans-activator and its activity in the NPC cell line C666-1. BZLF1 Binding to the promoter's target sequence. In addition to the wild-type sequence, three mutant sequences were included to demonstrate binding specificity.

[0160] Cell viability assay

[0161] Approximately 10 4100 μL of cells / well was seeded in 96-well plates and transfected with TZ3 plasmid or treated with mTZ3-LNP and GCV one day later. For mTZ3-LN treatment, 100 ng / well of mRNA was added to 96-well plates with or without 10 μg / mLGCV the next day, for a total volume of 100 μL. At the end of treatment, the medium was replaced and 10 μl of CCK-8 reagent (Dojindo Molecular Technologies, Rockville, MD, USA) was added to each well to determine cell viability. After incubation at 37°C for 3-4 hours, absorbance was measured at 450 nm and 650 nm using a 96-well SpectraMax plate reader (Molecular Devices, San Jose, CA, USA). Cell growth inhibition in each well was calculated as follows: (viability) 对照 -vitality 药物 ) / vitality 对照 ×100%. Each sample was analyzed three times.

[0162] Cell cycle analysis

[0163] Cells were detached from the culture plate using trypsin, washed with cold PBS, and fixed overnight in 70% ethanol at 4°C. The cells were then washed with PBS and incubated for 30 minutes with propidium iodide (1 μg / mL; Invitrogen, P3566) and RNase (10 μg / mL; Roche). After washing, the cells were analyzed using a FACSCalibur flow cytometer (BDBiosciences). 4 DNA content was measured in individual cells / samples. Data analysis was performed using FlowJo software.

[0164] In vivo mouse experiments

[0165] Disassemble Xeno-76 PDX tumor tissue or 5×10 6 SNU719 or C666-1 cells were subcutaneously injected into the flank of 3- to 4-week-old NOD-SCID mice with an initial body weight of approximately 18 to 22 g; tumor growth was allowed to reach approximately 100 mm. 3Mice were housed under the following conditions: temperature 20°C to 23°C, relative humidity 40% to 60%, and a 12-hour light / dark cycle (7:00 am to 7:00 pm). Mice were randomly assigned to different experimental groups and received intravenous injections of either a mediator (PBS) or mTZ3-LNP every 2 days for 2 weeks. GCV was administered intraperitoneally only to mice in the GCV group and the group receiving a combination of mTZ3-LNP and GCV. Mice were weighed, and their tumors were measured using calipers every 3 days. Tumors exceeding 1000 mm in size were considered tumors. 3 At that time, the mice were euthanized, and tumor and blood samples were collected for analysis. Using the formula... 0.5×l ×w 2 Calculate tumor volume, where l and w These represent tumor length and width, respectively. At the end of the experiment, serum samples and organs, including the heart, lungs, liver, spleen, and kidneys, were collected to assess the in vivo cytotoxicity of mTZ3-LNP treatment. Serum aspartate aminotransferase (ALT), alkaline phosphatase (AST), and creatinine concentrations were measured. Formalin-fixed paraffin-embedded sections of organs were stained with hematoxylin and eosin (H&E). Histological characteristics were assessed by a pathologist (KF To).

[0166] To assess the circulating lifetime of LNP-encapsulated mTZ3 mRNA in a NOD-SCID mouse model, LNPs were fluorescently labeled with DilC18 (Invitrogen). Four mice per group were intravenously injected with either DilC18-labeled LNP-encapsulated mTZ3 mRNA at a dose of 0.5 mg RNA / kg or PBS as a control. 50 μL of blood was collected from the facial vein in EDTA-treated tubes at 0 h, 8 h, 24 h, and 48 h post-injection. Circulating LNPs were measured by detecting the DilC18 fluorescence signal in the blood samples, and plasma was extracted. Circulating LNPs were measured by detecting the DilC18 signal in the plasma using a SpectraMax Molecular Devices reader (38). All animal care and experimental procedures were approved by the University Animal Ethics Committee (AEEC) of the Chinese University of Hong Kong.

[0167] Statistical analysis

[0168] All graphs were generated using GraphPad 8 software (GraphPad Inc, San Diego, CA, USA), and all statistical analyses were performed using one-way ANOVA or two-tailed Student's t-tests. All in vitro experiments were repeated three times. Error bars indicate standard deviation (SD), and unless otherwise specified, standard error (SEM) of the mean is indicated. A p-value < 0.05 was considered statistically significant.

[0169] All patents, patent applications and other publications cited in this application, including GenBank accession numbers and equivalents, are incorporated herein by reference in their entirety for all purposes.

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[0209] Table 1. Genomic sequence of human herpesvirus type 1 (NC_007605): TALE target region of BZLF1 promoter

[0210] Table 2. Genomic sequence of human herpesvirus type 1 (NC_007605): BRLF1 promoter TALE target region

[0211] Table 3. Genomic sequence of human herpesvirus type 1 (NC_007605): TALE target region of BGLF4 promoter.

[0212] Table 4. Sequences of mRNAs that activate BZLF1 synthesis

[0213] Table 5. Sequences of mRNAs that activate BRLF1 synthesis

[0214] Table 6. Sequences of mRNAs that activate BGLF4 synthesis

[0215] Table 7 Elements of mRNA Sequence

[0216] Nucleotides 1 to 69: FLAG sequence (double underscore)

[0217] Nucleotides 91 to 162: 3x NLS sequence (dotted underline)

[0218] Table 8. Plasmids used in this study

[0219] Table 9. sgRNA binding sequence and TALE binding sequence in the BZLF1 promoter.

[0220] Table 10. Amino acid sequence of TALE and RNA sequence of TZ3 mRNA

[0221] Table 11 Primer sequences used for quantitative real-time PCR

Claims

1. A nucleic acid comprising a multinucleotide sequence encoding a fusion protein, said fusion protein comprising (i) at least one nuclear localization signal (NLS); (ii) a transcription activator-like effector (TALE) targeting an Epstein-Barr virus (EBV) BZLF1, BRLF1, or BGLF4 promoter sequence; and (iii) a transactivation domain.

2. The nucleic acid according to claim 1, wherein it is RNA.

3. The nucleic acid according to claim 2, wherein the U residue of the RNA is replaced by pseudouridine.

4. The nucleic acid of claim 1, which is DNA and comprises an expression cassette, the expression cassette comprising the polynucleotide sequence operatively linked to a promoter sequence.

5. The nucleic acid according to any one of claims 1 to 4, wherein the TALE is encoded by a nucleotide sequence having at least 90% sequence identity with segments 649 to 2280 of SEQ ID NO:

33.

6. The nucleic acid according to any one of claims 1 to 5, wherein the transactivation domain is encoded by a nucleotide sequence having at least 90% sequence identity with segments 2722 to 3564 of SEQ ID NO:

33.

7. The nucleic acid according to any one of claims 1 to 6, wherein the at least one NLS is encoded by a nucleotide sequence having at least 90% sequence identity with segments 91 to 114 of SEQ ID NO:

33.

8. The nucleic acid according to any one of claims 1 to 7, wherein the fusion protein further comprises an epitope tag.

9. The nucleic acid according to any one of claims 1 to 8, wherein the fusion protein comprises, from its N-terminus to its C-terminus, a FLAG encoded by segments 1 to 69 of SEQ ID NO: 33, at least one NLS encoded by segments 91 to 162 of SEQ ID NO: 33, a TALE encoded by segments 649 to 2280 of SEQ ID NO: 33, and a transactivation domain encoded by segments 2722 to 3564 of SEQ ID NO:

33.

10. The nucleic acid according to any one of claims 1 to 8, wherein the polynucleotide sequence is shown in any one of SEQ ID NO:31-60.

11. A composition for treating EBV-related diseases in a subject, comprising an effective amount of (1) the nucleic acid of any one of claims 1 to 10; and (2) a physiologically acceptable excipient.

12. The composition according to claim 11, wherein the nucleic acid is RNA.

13. The composition of claim 12, wherein the U residue of the RNA is replaced by pseudouridine.

14. The composition according to any one of claims 11 to 13, wherein the nucleic acid is present within the lipid nanoparticles.

15. The composition according to any one of claims 11 to 14, wherein the lipid nanoparticles comprise [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycerol-3-methoxy polyethylene glycol-2000 (DMG-PEG2000) and cholesterol.

16. The composition according to any one of claims 11 to 15, wherein it is formulated for injection into said object.

17. A method for treating EBV-related disease in a subject, the method comprising administering to the subject an effective amount of the composition of any one of claims 11 to 16.

18. The method of claim 17, wherein the EBV-related disease is EBV-related cancer.

19. The method of claim 18, wherein the EBV-related cancer is Burkitt lymphoma, Hodgkin lymphoma, natural killer cell lymphoma, T-cell lymphoma, post-transplant lymphoma, nasopharyngeal carcinoma, or gastric cancer.

20. The method according to any one of claims 17 to 19, wherein the nucleic acid is RNA.

21. The method of claim 20, wherein the U residue of the RNA is replaced by pseudouridine.

22. The method according to any one of claims 17 to 21, wherein the nucleic acid is present in lipid nanoparticles.

23. The method of claim 22, wherein the lipid nanoparticles comprise [(4-hydroxybutyl)azadiyl]bis(hexane-6,1-diyl)bis(2-hexyldecanoate) (ALC-0315), 1,2-distearate-sn-glycerol-3-phosphocholine (DSPC), 1,2-dimyristoyl-rac-glycerol-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000) and cholesterol.

24. The method according to any one of claims 17 to 23, wherein the application comprises injecting the composition into the object.

25. A kit for treating EBV-related diseases in a subject, comprising a first container containing a first composition and a second container containing a second composition, the first composition comprising an effective amount of at least one nucleic acid according to any one of claims 1 to 10, and the second composition comprising an effective amount of at least one other anticancer therapeutic agent for said EBV-related diseases.

26. The kit according to claim 25, wherein the nucleic acid is RNA, and the polynucleotide sequence is shown in any one of SEQ ID NO:31-60.

27. The kit according to claim 25 or 26, wherein the nucleic acid is RNA, and the polynucleotide sequence is as shown in any one of SEQ ID NO:51-60, and wherein the other anticancer agent comprises ganciclovir (GCV).

28. The kit according to any one of claims 25 to 27, wherein the EBV-related disease is EBV-related cancer, and wherein the second composition comprises an effective amount of at least one other anticancer therapeutic agent.

29. The kit according to claim 28, wherein the EBV-related cancer is Burkitt lymphoma, Hodgkin lymphoma, natural killer lymphoma, T-cell lymphoma, post-transplant lymphoma, nasopharyngeal carcinoma, or gastric cancer.

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