Cancer oncogene mRNA vaccine

Abstract

An mRNA vaccine composition is described. The vaccine composition includes an exosome comprising an expression cassette comprising mRNA encoding chimeric / fusion / hybrid oncogene (CFHON). Methods of treating or preventing cancer in a subject by administering a therapeutically effective amount of the mRNA vaccine composition are also described.

Description

Tarolli Ref. No. KJK-034481 WO ORDCANCER ONCOGENE mRNA VACCINESTATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0001] This invention was made with government support under Grant Numbers R01CA248019 and R01CA266256, awarded by the National Institutes of Health. The Government has certain rights in this invention.CROSS REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application No. 63 / 645,199, filed on May 10, 2024, which is incorporated herein by reference.SEQUENCE LISTING

[0003] The instant application contains Sequence Listing XML (Name: KJK-034481 WO ORD.xml; size: 14,097 bytes; and Date of Creation: May 12, 2025) which has been submitted in XML format via EFS-Web and is hereby incorporated by reference in its entirety.BACKGROUND

[0004] The non-random chromosomal translocation t(8;21)(q22;q22), involving ETO (known as MTG8) on chromosome 8q22 and AML1 (known as RUNX1) on chromosome 21q22, generates a chimeric protein AML1 / ETO (also termed RUNX1 / RUNX1T1; A / E) in acute myeloid leukemia (AML). Erickson el al., Blood 80, 1825-1831 (1992). A / E is generally thought to be a transcriptional repressor by recruiting corepressors, like NCOR, HDACs and DNMTs, to AML1 targets or by interacting with other transcriptional factors, like the ETS family proteins, C / EBP, GATA and E proteins. A / E, a non-druggable fusion protein, is a leukemia initiation factor, which influences differentiation, proliferation and apoptosis in both in vitro and in vivo models. Mulloy et al., Blood 99, 15-23 (2002). The incidence of the A / E abnormality is approximately 5%- 10% of all AML cases and 10%-22% of AML-M2. Valk et al., N Engl J Med 350, 1617-1628 (2004). The A / E9a, a spliced isoform of AE, is highly leukemogenic when expressed in vivo, but the full-length AE requires a “second-hit” to trigger leukemia. Yuan et al., Proc Natl Acad Sci USA 98, 10398-10403 (2001). Over 95% A / E patients carry A / E9a. Co-expression of A / E and A / E9a induces a more immature leukemicTarolli Ref. No. KJK-034481 WO ORD phenotype with a rapid onset of AML. Yan et al., Nat Med 12, 945-949 (2006) Although A / E patients have a higher complete remission (CR) rate with a relatively good prognosis and appear to benefit from high dose cytarabine consolidation therapy in younger patients, up to 70% relapse rates have been observed, particularly in elderly patients who are not candidates for standard chemotherapy, and over 40% patients die from their disease. Notably, AML patients with p53 mutations (although only 5% to 10% patients) have even worse outcomes, including treatment resistance, poorer prognosis and lower overall survival.

[0005] Emerging evidence reveals the profound changes in the bone marrow immune environment of AML patients. Such changes enhance the severity of the disease but also offer opportunities to prompt or rewire a proficient anti-tumor immune surveillance. In line, allogeneic hematopoietic stem cell transplantation (alloHSCT) frequently benefits AML patients, but this therapy is associated with graft-versus-host disease. Further, alloHSCT has the limited effects on preventing relapses in most patients who undergo transplantation with active disease. The chimeric antigen receptor (CAR) T-cell therapy has been applied to leukemia patients, which induce high CR rates in patients with poor prognosis and few therapeutic options. However, CAR-T therapy faces these pitfalls, the time-consuming process of CAR-T engineering, limited number of targets (only CD- 19 with a test of CG22 and CD20), cytokine release syndrome (CRS) and ineffectual in pediatric and adult patients with solid tumors etc. Particularly, CRS is a potentially life-threatening toxicity affecting majority of patients receiving CAR-T therapy, which, together with neurotoxicity, remain one of the major challenges in the clinics. While CAR-T therapy has yielded remarkable clinical success in certain types of B-cell malignancies, applications to myeloid malignancies including A / E+ AML are very challenging, due to the absence of an indispensable antigen, in which myeloid antigens are often shared by normal hematopoietic stem / progenitor cells (HSPCs) or even non- hematopoietic tissues. L. Vago, I. Gojo, J Clin Invest 130, 1552-1564 (2020). Previous studies suggest that translocations (AML1-ETO, DEC-CAN, PML-RARa, BCR-ABL) and gain-of- function mutations (FLT3-ITD, NPM1, IDH1R132H) may be AML-specific immunogenic proteins, representing ideal antigen targets. For example, NPM1 mutation has been shown to induce CD4+ and CD8+ T cell responses (van der Lee etal., J Clin Invest 129, 774-785 (2019)), and the IDH1R132H mutation generates an HLA-DR-restricted neoantigen recognized by CD4+ T cells (Schumacher el al., Nature 512, 324-327 (2014). However, the success of immunotherapeutic strategies targeting these proteins in AML has not been reported.Tarolli Ref. No. KJK-034481 WO ORD

[0006] The success of mRNA vaccination during the COVID- 19 pandemic raises new hopes for a mRNA vaccine in cancers like myeloid malignancies. Stamatatos et al., Science, 372(6549): 1413-8 (2021). mRNA-based vaccines are well tolerated, safe administration, high potential for rapid development, low-cost manufacture, and do not integrate into the host genome. Thess et al., Mol Ther 23, 1456-1464 (2015). mRNAs provide platforms for rapidly expressing proteins (not only peptides of a given gene) of multiple antigens in nondividing and hard-to-transfect cells including dendritic cells (DCs). Several clinical trials (e.g., NCT04534205, NCT03313778, NCT04503278) are enrolling patients for various mRNA- based cancer vaccine therapy studies. These trials have reported durable objective responses in cancer patients. Lorentzen etal., Lancet Oncol 23, e450-e458 (2022). Mechanistically, the anti- cancer effects of mRNA vaccines occur through a reduction of effector T cells and the development of regulatory T cell (Treg cell) populations, induction of strong CD4+ and CD8+ T cell immunity against the vaccine antigens, or Toll-like receptor 4 signaling etc.

[0007] The fusion / chimeric oncogenic (CFON) proteins are frequently identified in solid and blood cancers serving as cancer drivers, yet these proteins are largely undruggable. It is well known that mRNA vaccines tremendously benefit human patients with COVID-19, yet vaccines targeting CFON proteins have not been developed.SUMMARY OF THE INVENTION

[0008] The inventors generated an AML1 / ETO (A / E) mRNA vaccine to specifically target acute myeloid leukemia (AML) cells expressing A / E fusion gene. The A / E mRNA vaccine can be coupled with exosome carriers (with / without tumor cell receptor targeting) in the future for cancer specific delivery.

[0009] The AML1 / ETO (AE) fusion gene was cloned into an expression cassette containing T7 promoter; A / E mRNA was synthesized in vitro, uploaded into exosomes isolated from red blood cells and characterized for protein expression by Western blotting. The exosomes-loaded A / E mRNA was intramuscularly injected into healthy or leukemic (carrying A / E+ cells) C57BL / 6 mice to determine safety, immune response, or therapeutic benefits. To characterize the activated immune response, CD4+ and CD8+ cells were isolated by magnet-sorting with anti-CD4 or anti-CD8 antibodies and subjected to RNA sequencing followed by PCR validation. Serum levels of cytokines were determined by cytokine microarrays.Tarolli Ref. No. KJK-034481 WO ORD

[0010] A / E mRNA synthesized in vitro was efficiently loaded into exosomes and efficiently translated into A / E proteins in 293T cells. Post intramuscular injection, one dose and shortterm treatment activates immune cells via cytokine expression; multiple-doses and long-term treatment is safe, because it did not induce obvious damage to mouse organs, including lung, liver, spleen and muscle. Importantly, leukemic mice treated with multiple-doses for 5 weeks have the reduced leukemic disease burden with reduction of white blood cells, appearance of dendritic cells and longer survival in A / E mRNA vaccine-treated group. Mechanistically, A / E mRNA vaccine-primed mice have more activated immune systems with higher number of CD4+ and CD8+ T cells and upregulation of anticancer cytokines, including interferon gamma (INF-y). RNA-seq identified over 6,000 differentiated genes in CD4+ and CD8+ T cells from A / E mRNA vaccine-treated vs vehicle mice. KEGG analysis identified JAK-STAT and TNF signaling pathways that mediate CD4 and CD8 T cell-initiated leukemia killing. This was substantiated by 13 up- and 3 down-regulated serum cytokines (144 examined), where TNF signaling is centered on the top. Finally, T cell-initiated leukemia cell killing was demonstrated by the less detection of A / E+ cells in liver, bone marrow, and spleen from A / E mRNA vaccine- primed mice.

[0011] The results obtained by the inventors demonstrate that this A / E mRNA vaccine platform provides potent anti-tumor effectiveness through eliciting robust anti-tumor immune responses in leukemia, possible other CFON-associated cancers. The studies establish a customizable and scalable approach to produce mRNA vaccine-based therapeutics effectively targeting CFON protein in solid and blood cancers.

[0012] Compared with traditional plasmid and viral-based approaches, this approach allows design of patient-personalized mRNAs that also benefit from eliminating needing to pass through the nuclear membrane (unlike DNA) and thus carries little to no risk of genomic integration. Furthermore, mRNA vaccines are safe, simple, and inexpensive and possess maximum flexibility. Particularly compared with peptide vaccines, they have self-adjuvanting properties, lack of MHC haplotype restriction, and do not need to enter the nucleus (Schlake et al., RNA Biol. 2012; 9(11): 1319-1330]. mRNA does not integrate into the genome and therefore it avoids oncogenesis and mutagenesis (McNamara et al., J Immunol Res. 2015; 2015:794528]. These vaccines are temporary information carriers due to early metabolic degradation within a few days.Tarolli Ref. No. KJK-034481 WO ORDBRIEF DESCRIPTION OF THE FIGURES

[0013] The present invention may be more readily understood by reference to the following figures, wherein:

[0014] Figs. 1A-1G provide images showing In vitro and in vivo translation assays of EVs- encapsulated mRNA. (A and B) Charge (A) or size (B) distribution of EVs determined by a Nanosight nanoparticle analyzer. (C) Morphological characterization of exosomes by transmission electron microscopy (TEM). Representative TEM image of EVs (l l,000x, 13,000x, 49,000x). Scale bar: 200 nm. (D) Representative dSTORM images of pan-tetraspanin (CD9 / 63 / 81). The digits 23, 12 and 29 on right panel indicate the molecule numbers of CD81, CD63 and CD9 markers on each exosome. Scale bar: 200 nm. (E and F) 293T cells were transfected with AE expression DNA plasmids (delivered by LPN) or mRNA (delivered by EVs or LPN). The AE protein was detected by Western blot. The data are representative of three independent experiments. (G) Health C57BL / 6J mice (n = 3 mice / group) were primed by intramuscular injection (IM) with EV, LPN-GFP or EV-GFP mRNA. The muscles were harvest 48 hours after injection and subjected to Western blot using GFP antibody. indicates GFP protein bands. EV, exosomes; LPN, lipofectamine nanoparticles.

[0015] Figs. 2A-2G provide graphs and images showing EVs-encapsulated mRNA vaccination significantly reduces leukemic burden with no obvious cytotoxicity. (A) The schedule of vaccination and tissue collection. (B) Representative images of H&E section staining of spleen, lung, liver, muscle, heart, and kidney from healthy mice (n = 3 mice / group) vaccinated with EV or EV-mRNA. Health: same age of mice with PBS injection. (C) Bar-charts showing the total white blood cell (WBC) counts in leukemic mice measured at week 4 following the administration of EV, LPN or delivered mRNA. Healthy mice were used as controls. (D) The weight of body and organs in mice after mRNA vaccination. Healthy mice were used as control. (E) Representative images of organs like spleen, lung, liver, muscle, heart, and kidney from health and treated leukemic mice (n = 3 mice / group). (F) Representative H&E staining sections of spleen, lung, liver, muscle, heart, and kidney showing tissue damage after vaccination (n = 3 mice / group). (G) Log rank test for comparison of Kaplan-Meier survival curves indicated a significant increase in the survival of the mice treated with EVs-delivered mRNA. *P <0.05; ns, no statistically significant; WBC, white blood cell counts; EV, exosomes; LPN,Tarolli Ref. No. KJK-034481 WO ORD lipof ectamine nanoparticles; AE, AE mRNA in PBS; EV+AE, AE mRNA delivered by EV; LPN-AE, AE mRNA delivered by LPN.

[0016] Figs. 3A-3D provide graphs and images showing Treatment with EVs-delivered AE mRNA ameliorates immune cells in mice. (A) Blood smears from vaccinated leukemic mice visualized with Wright Giemsa staining. The enlarged image at right upper comer indicates dendritic cell-like cells, which appear more frequent in EV-AE than other groups. The images represent multiple mice in each group: 3 in healthy group, 5 in EV group, 8 in EV-AE group, 5 in AE mRNA only group, and 7 in LPN-AE group. (B) Bar-chart showing the frequency of dendritic cell-like cells derived from blood smears in mice treated with various formulated mRNA vaccination. (C and D) Bar graphs depict % of CD4+, CD8+, CD80, CDl lc, MHC-II cells in splenic cells across different conditions. T cells were enriched from frozen mouse splenic cells using column- free magnetic separation method. The cells were stained with antibodies for mouse CD4+, CD8+, CD80, CDl lc, MHC-II surface markers. *P < 0.05; **P <0.01; EVs, exosomes; LPN, lipofectamine nanoparticles; AE, AE mRNA only. EV, exosomes; AE, AE mRNA in PBS; EV+AE, AE mRNA delivered by EV; PBS, leukemic mice injected with PBS as control.

[0017] Figs. 4A-4E provide graphs showing Cytokine production in various T cells in mice treated with AE mRNA vaccines. The splenic T cells expressing CD4, CD8, CD80, CD11c, or MCH-II were enriched by magnet sorting, and the levels of indicated cytokines were determined by qPCR. Fold changes in cytokine RNA expression levels normalized by [Lactin were calculated using the 2-AACt method. (A, B and C) qPCR measuring cytokine productions in CD8+, CD4+ or CD80+ T cells. (D) Nest-qPCR to measure AE expression in splenic, and BM cells (n = 3 mice / group). (E) Representative Western blot to detect AE protein expression in splenic cells (n = 3 mice / group). ****p <0.0001; ***P <0.001, **P <0.01, *P <0.05; ns, not statistically significant; EV, exosomes; AE, AE mRNA in PBS; EV+AE, AE mRNA delivered by EV; PBS, leukemic mice injected with PBS as control.

[0018] Figs. 5A-5I provide graphs and images showing Bulk RNA-Seq of splenic CD8+ and CD4+ T cells from C57BL / 6 mice treated with EV-AE, EV or PBS. The leukemic C57BL / 6 mice were treated with EV-AE, EV or PBS (5 does in total) for 3 weeks. The spleens were collected 2 weeks post vaccination, and single splenic cells were made. T cells with the immunophenotype CD4+ or CD8+ were purified by magnet sorting and subjected to scRNA-Tarolli Ref. No. KJK-034481 WO ORD seq (n = 3 mice / group). (A and B) Volcano map highlighting significant differences of gene expression (log2 (fold change); >1.5 fold) in EV-AE vs EV or PBS-treated cells. Red indicates expression upregulation, blue indicates expression down-regulation, in EV-AE with respect to EV or PBS. (C and D) Heatmap of 1,000 up- or down-regulated genes (log2 (fold change); >1.5 fold). (E and F) The bubble plot showing the top 15 most enriched KEGG terms based on all differentially expressed genes (log2 (fold change); >1.5 fold). The different colors of each circle are the adjusted FDR values, the size of each circle means the amounts of genes under a specific term, and RichFactor means the number of genes in the key modules that belong to this pathway. (G) Venn diagram analysis illustrating overlap and differences of cytokine expression between our differentially expressed gene signature (log2 (fold change); >1.5 fold) and the published cytokine profile. (H and I) KEGG circle network plot (left) showing a chord dendrogram of the clustering of the expression spectrum of significantly changed cytokines derived from “G”. The names of the top KEGG signaling pathways are marked on the right of the chord diagram, the adjusted P values are marked with different colors. KEGG, Kyoto Encyclopedia of Genes and Genomes; EV, exosomes; EV-AE, EV-delivered AE mRNA.

[0019] Figs. 6A-6F provide graphs and images showing Comparison of various plasma cytokines levels between EVs-AE and EV-treated leukemic mice. (A) Scanned images from cytokine arrays. One spot is a serum pool of 4 mice, and each group has 4 spots (4 technical repeats). Red rectangles are downregulated cytokines, green rectangles are up-regulated cytokines. (B) Heatmaps show the changes in 144 cytokines (n = 4 mice / lane). (C and D) Bars show the mean ±SD, and indicate the significantly up- or down-regulated cytokines (P <0.05). The serum from 4 mice in each group was pooled and measured in quadruplicate. (E) The KEGG pathway -based network analysis of significantly changed cytokines in EV-AE vs EV comparison (P <0.05). Purple denotes gene nodes that were identified by cytokine array, and light blue denotes gene nodes that were defined by KEGG in EV-AE compared to EV samples. The red circle (e.g., TNF) denotes the most shared node in comparisons. Larger circles represent larger enrichment scores. (F) Selected enriched Gene Ontology cellular / disease- enriched functions associated with up-regulated differentially expressed cytokines (DEGs). The top 16 by DEG counts are presented (FDR <0.05). The color of the dot depends on the value of FDR, and its size is determined by the number of DEGs related to the respective pathway in the analyzed set of DEGs (map color keys along with dot size ones are shown on the right). *P <0.05, **P <0.01, ***P <0.001; EV, exosomes; EV-AE, EV-delivered AE mRNA.Tarolli Ref. No. KJK-034481 WO ORDDETAILED DESCRIPTION OF THE INVENTION

[0020] The present invention provides an mRNA vaccine composition. The vaccine composition includes an exosome comprising an expression cassette comprising mRNA encoding chimeric / fusion / hybrid oncogene (CFHON). Methods of treating or preventing cancer in a subject by administering a therapeutically effective amount of the mRNA vaccine composition are also provided.

[0021] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which these exemplary embodiments belong. The terminology used in the description herein is for describing particular exemplary embodiments only and is not intended to be limiting of the exemplary embodiments. As used in the specification and the appended claims, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.

[0022] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0023] A “subject,” as used herein, can be any animal, and may also be referred to as the patient. Preferably the subject is a mammal, such as a research animal (e.g., a monkey, rabbit, mouse or rat) or a domesticated farm animal (e.g., cow, goat, horse, pig) or pet (e.g., dog, cat). In some embodiments, the subject is a human.

[0024] “Treat", "treating", and "treatment", etc., as used herein, refer to any action providing a benefit to a subject at risk for or afflicted with a condition or disease such as cancer, including improvement in the condition through lessening or suppression of at least one symptom, delay in progression of the disease, prevention or delay in the onset of the disease, etc.Tarolli Ref. No. KJK-034481 WO ORD

[0025] “Preventing,” as used herein, refers to any action that decreases the risk that a subject will develop cancer, inhibits the growth of cancer, or decreases the incidence of cancer recurrence. Cancer prevention can be done in subjects who have an increased risk of developing cancer. Also intended to be encompassed by this definition is the prevention of metastasis of malignant cells or to arrest or reverse the progression of malignant cells. Subjects can have an increased risk of developing cancer as a result of, for example, a genetic predisposition or exposure to carcinogens.

[0026] The terms “therapeutically effective” and “pharmacologically effective” are intended to qualify the amount of each agent which will achieve the goal of decreasing disease severity while avoiding adverse side effects such as those typically associated with alternative therapies. The therapeutically effective amount may be administered in one or more doses.

[0027] An "effective amount" of the mRNA vaccine composition can be determined, based at least in part, on the target tissue, target cell type, means of administration, physical characteristics of the polynucleotide (e.g., size, and extent of modified nucleosides) and other components of the mRNA vaccine, and other determinants. In general, an effective amount of the mRNA vaccine composition provides an induced or boosted immune response as a function of antigen production in the cell, preferably more efficient than a composition containing a corresponding unmodified polynucleotide encoding the same antigen. Increased antigen production may be demonstrated by increased cell transfection, increased protein translation from the polynucleotide, decreased nucleic acid degradation (as demonstrated, e.g., by increased duration of protein translation from a modified polynucleotide), or altered innate immune response of the host cell.

[0028] As used herein, a "vaccine" refers to a composition, for example, a substance or preparation that stimulates, induces, changes, causes or improves immunity in an organism, e.g., an animal organism, for example, a mammalian organism (e.g., a human.) Preferably, a vaccine provides immunity against one or more diseases or disorders in the organism, including prophylactic and / or therapeutic immunity.

[0029] The term “polynucleotide” as used herein is defined as a chain of nucleotides. Furthermore, nucleic acids are polymers of nucleotides. Thus, nucleic acids and polynucleotides as used herein are interchangeable. One skilled in the art is well aware thatTarolli Ref. No. KJK-034481 WO ORD nucleic acids are polynucleotides, which can be hydrolyzed into the monomeric “nucleotides.” The monomeric nucleotides can be hydrolyzed into nucleosides. As used herein polynucleotides include, but are not limited to, all nucleic acid sequences which are obtained by any means available in the art, including, without limitation, recombinant means, i.e., the cloning of nucleic acid sequences from a recombinant library or a cell genome, using ordinary cloning technology and PCR, and the like, and by synthetic means.

[0030] The term “a regulatory sequence”, “an expression control element” or “promoter” as used herein, intends a polynucleotide that is operatively linked to a target polynucleotide to be transcribed or replicated, and facilitates the expression or replication of the target polynucleotide.

[0031] All scientific and technical terms used in the present application have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present application.RNA Vaccine Compositions

[0032] In one aspect, the present invention provides an mRNA vaccine composition, comprising an exosome comprising an expression cassette comprising mRNA encoding chimeric / fusion / hybrid oncogene (CFHON). In some embodiments, the CFHON is AML1 / ETO (A / E). In further embodiments, mRNA encoding a plurality of CFHON are included in the mRNA vaccine composition.

[0033] The mRNA vaccine composition comprises an open reading frame (ORF) encoding one or more CFHON. An oncogene is a gene that has the potential to cause and / or promote cancer. Chimeric / fusion / hybrid oncogenes (CFHON) are oncogenes that are formed by combining partial or complete coding sequences from two or more previously independent genes, including those located in different chromosomes. For example, AML1 / ETO is a CFHON that results from combining the AMLl(RUNXl) gene on chromosome 21 and the ETO gene on chromosome 8.

[0034] In some embodiments, the mRNA vaccine composition encodes the oncogene AML1 / ETO. Acute myeloid leukemia 1 protein (AML1), also known as RUNX1, is aTarolli Ref. No. KJK-034481 WO ORD transcription factor encoded by the AMLl(RUNXl) gene. The full-length AML1 is comprised of a N-terminal Runt-homology domain responsible for DNA-binding, C-terminal transactivation domain (TAD) that consists of a number of activating and inhibitory domains, e.g. Etsl interacting domain (EID). ETO, also known as RUNX1T1, is a nuclear Zinc-binding protein encoded by the ETO gene, and functions as a transcriptional co-repressor. The ETO protein domain structure consists of four highly conserved functional domains termed Nervy Homology domains 1-4 (NHR 1-4): (1) TATA-binding protein- associated factor homology domain (eTAFH = NHRI); (2) the hydrophobic heptad repeat domain (HHR = NHR2); (3) an a-helical domain (NHR3), and (4) the myeloid-Nervy-DEAFl domain (MYND = NHR4). CFHON AML1 / ET0 protein structure consists of the Runt DNA-binding domain of AML1 and the four functional domains (NHR 1-4) of ETO. See Rejeski, K., et al., Oncogene 40, 5665-5676 (2021).

[0035] “Messenger RNA” (mRNA) refers to any polynucleotide that encodes a polypeptide and can be translated to produce the encoded polypeptide / proteins in vitro, in vivo, in situ or ex vivo. One of ordinary skill in the art will appreciate that, except where otherwise noted, polynucleotide sequences set forth in the instant application will recite “T”s in a representative DNA sequence but where the sequence represents RNA (e.g., mRNA), the “T”s would be substituted for “U”s. Thus, any of the RNA polynucleotides encoded by a DNA identified by a particular sequence identification number may also comprise the corresponding RNA (e.g., mRNA) sequence encoded by the DNA, where each “T” of the DNA sequence is substituted with “U.” The basic components of an mRNA molecule typically include at least one coding region, a 5 '-untranslated region (UTR), a 3'-UTR, a 5 '-cap and a poly- A tail. Polynucleotides of the present disclosure may function as mRNA but can be distinguished from wild-type mRNA in their functional and / or structural design features, which serve to overcome existing problems of effective polypeptide expression using nucleic-acid based therapeutics. In some embodiments, the mRNA is single strand mRNA (ssmRNA), while in other embodiments the mRNA is double stranded mRNA (dsmRNA).

[0036] In some embodiments, the CFHON is derived from chromosomal translocations, inversion, or other genetic rearrangement. Mechanisms causing DNA rearrangements include (a) reciprocal translocation, i.e., the interchromosomal exchange of DNA between regions, which can be equal (balanced) or unequal (unbalanced), (e.g., SLC34A2-ROS1); (b) insertions, i.e., inter- or intrachromosomal movement of a DNA fragment from one region to another; (c)Tarolli Ref. No. KJK-034481 WO ORD deletions (e.g., ATG7-RAF1); (d) tandem duplication (in which a duplicated genomic region fuses with a gene in its original region) (e.g., FGFR3-TACC3); (e) inversion (in which segments of a chromosome flip relative (pericentric) or not relative (paracentric) to the centromere (e.g., KIF5B-RET ); (f) chromothripsis (i.e., the fragmentation and inaccurate reassembly of one chromosome or chromosomal region). Liu, S. V. et al., Sig Transduct Target Ther 10, 111 (2025). CFHON can also be derived without DNA rearrangements. For example, CFHON can be formed by read-through transcription, in which the transcription process does not properly terminate at the end of the gene and continues into the next gene (e.g., SCNN1A- TNFRSF1A). As another example. CFHON can be formed cis or trans splicing of mRNA, with exons from one or two different primary RNA transcripts, respectively, being joined.

[0037] In some embodiments, the CFHON A / E is variant 9a (A / E9a), derived from chromosomal translocation (t(8;21)). CFHON AML1 / ETO can be derived from chromosomal translocation t(8;21) by combining AMLl(RUNXl) gene on chromosome 21 and the ETO gene on chromosome 8. Less common than (t(8 ;21)), CFHON AML1 / ETO can be derived from other events, e.g. inversions (i.e., inv(8)(q22q24)), or insertions (i.e., ins(21;8) and ins(8;21)) involving the derivative chromosome 8. Rejeski, K. et al., Oncogene 40, 5665-5676 (2021).

[0038] While the t(8;21) translocation generates a canonical genomic breakpoint that lies between AML1 exon 5 and ETO exon 2, CFHON AML1 / ETO transcript variants exist. For example, CFHON AML1 / ETO 9a (A / E9a) is an alternative spliced isoform, harboring an additional ETO exon, termed exon 9a, while lacking exons 9, 10, and 11 at the C-terminal. The sequence information of AML1 / ETO 9a (AE9a) mRNA can be accessed through NCBI’s database (NCBI Reference Sequence: NM_001198628.2) (SEQ ID NO:1). Protein encoded by A / E9a variant contains only the NHRI and NHR2 functional domains of ETO. The sequence information of AML1 / ETO 9a (AE9a) protein can be found in SEQ ID NO:2. Other CFHON AML1 / ETO transcript variants include an ETO variant containing an additional exon I la that produces a protein with an additional 27 amino acids in-frame instead of the MYND domain at the C-terminal; or an AMLl(exon 6) / ETO variant harboring AML1 exons 1 to 6 and ETO exons 2 to 11 that encodes a protein containing additional amino acids downstream of the Runt domain of AMLL

[0039] In addition to the t(8;21) translocation, other chromosomal translocations can occur to AML1, e.g. t (12;21) translocation, generating the TEL / AML1 (ETV6 / RUNX1) fusion geneTarolli Ref. No. KJK-034481 WO ORD product; t(3;21) translocation, generating AML1 / MDS / EVI1 (RUNX1 / MEC0M) fusion gene product.

[0040] In some embodiments, the mRNA vaccine composition further comprises a CFHON other than A / E. CFHON can drive cancers by joining a strong promoter that drives overexpression and a second proto-oncogene, e.g. TRABD-DDR2, PML-RARa, ETS gene fusions, TMPRSS2-ERG fusions, PAX3-FOXO1. CFHON can drive cancers also by activating receptor tyrosine kinase (RTK)s, e.g. NRG1 ligand gene fusions, EGFR fusions. CFHON can drive cancers further by driving aberrant signaling in neighboring cells beyond the fusionpositive cancer cells themselves, e.g. PAX3-FOXO1, Rab22a-NeoFl, BRD4-NUT. (Liu, S.V.et al., Sig Transduct Target Ther 10, 111 (2025)). Examples of CFHON include, but not limited to DEC-CAN, BCR-ABL, EWS-FLI, TLS-FUS, PAX3-FKHR, BCL-2, AML1-MTG8, CTNNB1-PLAG1, CBFP-MYH11, EWSR-FLI1, KIF5B-RET, RET fusion, CCDC6-RET, ROS 1 fusion, NTRK fusion, ETV6-NTRK3, PDGFR fusions, TMPRSS2-ETS, BRAF fusion, KIAA1549-BRAF, EML4-ALK, VTI1A-TCF7L2, NTRK1 fusions, FGFR fusions, FGFR3- TACC3, FGFR2-BICC1, NRG1 fusions, CD74-NRG1, FGFR2 fusion, RET and ROS1 fusions, HER2 fusion, or EGFR fusions etc.

[0041] In some embodiments, the mRNA of the mRNA vaccine is modified mRNA. Modified mRNA comprises at least one modification to a base of the polynucleotide. For example, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, about 90%, or at least 100% of the bases of the polynucleotide may be modified. In polynucleotide modifications of this disclosure, a specific base may comprise at least one modification. For example, the base adenine can be modified in polynucleotides of this disclosure, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the adenine bases can be modified. For example, the base guanine can be modified in polynucleotides of this disclosure, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the guanine bases can be modified. For example, the base cytosine can be modified in polynucleotides of this disclosure, e.g., at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the cytosine bases can be modified. For example, the base uracil can be modified in polynucleotides of this disclosure, e.g., at least 20%, at least 30%, at least 40%, at least 50%,Tarolli Ref. No. KJK-034481 WO ORD at least 60%, at least 70%, at least 80%, at least 90%, or at least 100% of the uracil bases can be modified.

[0042] In an aspect, the at least one modification is pyridin-4-one ribonucleoside, 5 -azauridine, 2-thio-5-aza-uridine, 2-thiouridine, 4-thio-pseudouridine, 2-thio-pseudouridine, 5- hydroxyuridine, 3 -methyluridine, 5-carboxymethyl-uridine, 1-carboxymethyl-pseudouridine, 5-propynyl-uridine, 1-propynyl-pseudouridine, 5-taurinomethyluridine, 1-taurinomethyl- pseudouridine, 5-taurinomethyl-2-thio-uridine, l-taurinomethyl-4-thio-uridine, 5-methyl- uridine, 1-methyl-pseudouridine, 4-thio-l-methyl-pseudouridine, 2-thio- 1 -methyl - pseudouridine, 1 -methyl- 1 -deaza-pseudouridine, 2- thio- 1 -methyl- 1 -deaza-pseudouridine, dihydrouridine, dihydropseudouridine, 2-thio-dihydrouridine, 2-thio-dihydropseudouridine, 2- methoxyuridine, 2-methoxy-4-thio-uridine, 4-methoxy-pseudouridine, and 4-methoxy-2-thio- pseudouridine, 5-aza-cytidine, pseudoisocytidine, 3-methyl-cytidine, N4-acetylcytidine, 5- formylcytidine, N4-methylcytidine, 5-hydroxymethylcytidine, 1-methyl-pseudoisocytidine, pyrrolo-cytidine, pyrrolo-pseudoisocytidine, 2-thio-cytidine, 2-thio-5-methyl-cytidine, 4-thio- pseudoisocytidine, 4-thio- 1 -methyl-pseudoisocy tidine, 4-thio- 1 -methyl- 1 -deaza- pseudoisocytidine, 1-methyl-l-deaza-pseudoisocytidine, zebularine, 5-aza-zebularine, 5- methyl-zebularine, 5-aza-2-thio-zebularine, 2-thio-zebularine, 2-methoxy-cytidine, 2- methoxy-5-methyl-cytidine, 4-methoxy-pseudoisocytidine, and 4-methoxy- 1-methyl- pseudoisocytidine, 2-aminopurine, 2, 6-diaminopurine, 7-deaza-adenine, 7-deaza-8-aza- adenine, 7-deaza-2-aminopurine, 7-deaza-8-aza-2-aminopurine, 7-deaza-2, 6-diaminopurine, 7-deaza-8-aza-2, 6-diaminopurine, 1-methyladenosine, N6-methyladenosine, N6- isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio-N6-(cis- hydroxyisopentenyl) adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyladenosine, 2-methylthio-N6-threonyl carbamoyladenosine, N6,N6- dimethyladenosine, 7-methyladenine, 2-methylthio-adenine, and 2-methoxy-adenine, inosine, 1-methyl-inosine, wyosine, wybutosine, 7-deaza-guanosine, 7-deaza-8-aza-guanosine, 6-thio- guanosine, 6-thio-7-deaza-guanosine, 6-thio-7-deaza-8-aza-guanosine, 7-methyl-guanosine, 6- thio-7-methyl-guanosine, 7-methylinosine, 6-methoxy-guanosine, 1 -methylguanosine, N2- methylguanosine, N2,N2-dimethylguanosine, 8-oxo-guanosine, 7-methyl-8-oxo-guanosine, 1- methyl-6-thio-guanosine, N2-methyl-6-thio-guanosine, or N2,N2-dimethyl-6-thio-guanosine. In some embodiments, the uridine-5’ -triphosphate nucleotides are replaced by pseudouridine l-methylpseudouridine-5’-triphosphate (m 1 TP) in the modified mRNA.Tarolli Ref. No. KJK-034481 WO ORD

[0043] The nucleotide sequences encoding CFHON mRNA, as described herein, can alternatively comprise sequence variations with respect to the original nucleotide sequences, for example, substitutions, insertions and / or deletions of one or more nucleotides, with the condition that the resulting polynucleotide encodes a polypeptide according to the invention. Therefore, the scope of the present invention includes nucleotide sequences that are substantially homologous to the nucleotide sequences recited herein and encode CFHON mRNA.

[0044] A nucleotide sequence that is substantially homologous to a nucleotide sequence encoding CFHON mRNA can typically be isolated from a producer organism based on the information contained in the nucleotide sequence by means of introducing conservative or nonconservative substitutions, for example. Other examples of possible modifications include the insertion of one or more nucleotides in the sequence, the addition of one or more nucleotides in any of the ends of the sequence, or the deletion of one or more nucleotides in any end or inside the sequence. The degree of identity between two polynucleotides is determined using computer algorithms and methods that are widely known for the persons skilled in the art.

[0045] In one aspect, the mRNA molecule further comprises a 3'-UTR, a 5'-UTR and optionally further comprises additional elements that (a) stabilize the molecule and (b) enhance expression of the polypeptide encoded by the ORF. In a further aspect, the 5'-UTR comprises a cap such as an m7G cap structure and / or a start codon. In a further aspect, the 3'-UTR comprises a stop codon and / or a polyA tail. In a further aspect, the m7G cap comprises an m7GpppG structure or an m7GpppGm structure. In another aspect, the 3'-UTR comprises a stop codon and a polyA tail. The length of the tail can range from 10 to 150 A.

[0046] The invention relates to a construct comprising an mRNA encoding a CFHON. In another particular embodiment, the construct is operatively bound to a translational control element. The construct can incorporate an operatively bound regulatory sequence for the expression of the nucleotide sequence of the invention, thus forming an expression cassette.

[0047] A promoter is an example of an expression control element or a regulatory sequence. Promoters can be located 5' or upstream of a gene or other polynucleotide, that provides a control point for regulated gene transcription. In some embodiments, a promoter as used herein corresponds to the RNA polymerase. In further embodiments, a promoter as sued hereinTarolli Ref. No. KJK-034481 WO ORD comprises, or consists essentially of, or yet further consists of a T7 promoter, or a SP6 promoter, or a T3 promoter. Non-limiting examples of suitable promoters are provided in W02001009377A1.

[0048] In some embodiments, the mRNA vaccine nucleotide sequence is codon-optimized. Codon optimization methods are known in the art and may be used as provided herein. Codon optimization, in some embodiments, may be used to match codon frequencies in target and host organisms to ensure proper folding; bias GC content to increase mRNA stability or reduce secondary structures; minimize tandem repeat codons or base runs that may impair gene construction or expression; customize transcriptional and translational control regions; insert or remove protein trafficking sequences; remove / add post translation modification sites in encoded protein (e.g. glycosylation sites); add, remove or shuffle protein domains; insert or delete restriction sites; modify ribosome binding sites and mRNA degradation sites; adjust translational rates to allow the various domains of the protein to fold properly; or to reduce or eliminate problem secondary structures within the polynucleotide. Codon optimization tools, algorithms and services are known in the art. Exemplary services include, but are not limited to, services from GeneArt (Life Technologies), DNA2.0 (Menlo Park CA) and / or proprietary methods. In some embodiments, the open reading frame (ORF) sequence is optimized using optimization algorithms.Delivery Vehicles and Formulations

[0049] The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single- or multi-dose unit.

[0050] The mRNA vaccine can be administered together with a delivery vehicle. In certain embodiments, delivery of mRNA of the invention to a subject comprises mixing the mRNA with a delivery vehicle prior to the step of contacting the subject with the pharmaceutical composition. Examples of deliver)' vehicles include lipid-based delivery vehicles, proteinbased delivery vehicles, carbohydrate-based delivery vehicles, cationic lipid-based deliveryTarolli Ref. No. KJK-034481 WO ORD vehicles, cationic polymer-based delivery vehicles, membrane-derived extracellular vesicles, red blood cell-based delivery vehicles, and nanoparticle delivery vehicles.

[0051] The mRNA vaccine is preferably delivered in an extracellular vesicle. Extracellular vesicles (EVs) are lipid bound cell membrane-derived vesicles secreted by cells into the extracellular space. The three main subtypes of EVs are microvesicles (MVs), exosomes, and apoptotic bodies, which are differentiated based upon their biogenesis, release pathways, size, content, and function. Exosomes (30-400 nm) are produced by the endosomal pathway, whereas microvesicles (100 nm-1 pm) and apoptotic bodies (1-4 pm) are produced by direct shedding from the plasma membrane. The content of EVs consists of lipids, nucleic acids, and proteins, and in particular proteins associated with the plasma membrane, cytosol, and those involved in lipid metabolism. Zhang et al., Cell Biosci., 9: 19 (2019). Likewise, exosomes are small, single-membrane, secreted organelles of ~30 to ~200 nm in diameter that have the same topology as the cell and are enriched in selected proteins, lipids, nucleic acids, and glycoconjugates. Pegtel D. and Gould, S., Annu Rev Biochem, 88:487-514 (2019). Types of cells from which vesicles can be obtained include mammalian cells, plant cells, bacterial cells, and yeast cells. In some embodiments, the mRNA is delivered using an exosome, while in further embodiments the exosomes are human red blood cell exosomes. The use of exosomes can overcome the pitfalls of lipid nanoparticles and other artificial nanoparticles that have been used for mRNA delivery.

[0052] In some embodiments, a dosage of between 10 ug / kg and 400 ug / kg of the vaccine is administered to the subject. In some embodiments, a dosage of 25 micrograms of the mRNA is included in the vaccine administered to the subject. In some embodiments, a dosage of 100 micrograms of the mRNA is included in the vaccine administered to the subject. In some embodiments, a dosage of 400 micrograms of the mRNA is included in the vaccine administered to the subject. In some embodiments, the mRNA accumulates at a 100 fold higher level in the local lymph node in comparison with the distal lymph node.

[0053] Route of administration can also be determined and method of determining the most effective route of administration are known to those of skill in the art and will vary with the composition used for treatment, the purpose of the treatment, the health condition or disease stage of the subject being treated, and target cell or tissue. Non-limiting examples of route of administration include oral administration, intraperitoneal, infusion, nasal administration,Tarolli Ref. No. KJK-034481 WO ORD inhalation, injection, and topical application. In some embodiments, the administration is an infusion (for example to peripheral blood of a subject) over a certain period of time, such as about 30 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, about 12 hours, about 24 hours or longer.

[0054] The term administration shall include without limitation, administration by oral, parenteral (e.g., intramuscular, intraperitoneal, intravenous, intracerebroventricular (ICV), intrathecal, intracisternal injection or infusion, subcutaneous injection, or implant), by inhalation spray nasal, vaginal, rectal, sublingual, urethral (e.g., urethral suppository) or topical routes of administration (e.g., gel, ointment, cream, aerosol, etc.) and can be formulated, alone or together, in suitable dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, excipients, and vehicles appropriate for each route of administration. In some embodiments, the mRNA vaccine is administered by intramuscular injection. The disclosure is not limited by the route of administration, the formulation or dosing schedule.

[0055] Although the description of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as non-human primates, cattle, pigs, horses, sheep, cats, and dogs.Cancer Prevention using an mRNA Vaccine

[0056] Another aspect of the present invention provides a method of preventing cancer in a subject, comprising administering an effective amount of an mRNA vaccine composition to the subject having an increased risk of developing CFHON driven cancer, wherein theTarolli Ref. No. KJK-034481 WO ORD composition comprises an exosome comprising an expression cassette comprising mRNA encoding a CFHON. In some embodiments, the CFHON of the mRNA vaccine is AML1 / ET0 (A / E), while in further embodiments the CFHON A / E is variant 9a (A / E9a).

[0057] Vaccination is the most successful medical approach to disease prevention and control. The successful development and use of vaccines has saved thousands of lives and large amounts of money. A key advantage of RNA vaccines is that RNA can be produced in the laboratory from a DNA template using readily available materials, less expensively and faster than conventional vaccine production, which can require the use of chicken eggs or other mammalian cells. In addition, mRNA vaccines have the potential to streamline vaccine discovery and development, and facilitate a rapid response to emerging infectious diseases, see, for example, Maruggi et al., Mol Ther. 2019; 27 (4):757 -772.

[0058] Aspects of the invention provide methods of creating, maintaining or restoring antigenic memory to cancer in an individual or population of individuals comprising administering to said individual or population an antigenic memory booster nucleic acid vaccine.

[0059] The present invention produces mRNA for use as a vaccine. Construction of mRNA vaccines typically involves the insertion of the encoded oligonucleotide in a DNA template from where the mRNA is transcribed in vitro, isolated and used as an active vaccine component. The mRNA when administered to a subject, is incorporated into cells of the subject and produces the desired protein to stimulate the subject's immune response. Unlike DNA, mRNA only needs to reach the cytosol, where it can be transcribed into the antigen in vivo, using cell machinery. In general, any cells can be used to produce mRNA. Production of mRNA for vaccine use is well known to one skilled in the art, for example, production of SARS-Cov- mRNA vaccines are well known, and a similar process can be used to produce mRNA vaccine of the present invention.

[0060] The mRNA vaccines of the present invention can be used as therapeutic or prophylactic agents. They are provided for use in medicine and / or for the priming of immune effector cells, e.g., stimulate / transfect PBMCs ex vivo and re-infuse the activated cells. For example, an mRNA vaccine described herein can be administered to a subject, wherein the polynucleotides is translated in vivo to produce a chimeric / fusion / hybrid oncogene. The active therapeuticTarolli Ref. No. KJK-034481 WO ORD agents of the invention include mRNA vaccine, cells containing an mRNA vaccine, or polypeptides translated from the polynucleotides contained in said mRNA vaccines.

[0061] Provided herein are methods of inducing translation of an oncoprotein in a cell, tissue or organism using the polynucleotides of the mRNA vaccines described herein. Such translation can be in vivo, ex vivo, in culture, or in vitro. The cell, tissue or organism is contacted with an effective amount of a composition containing an mRNA vaccine which contains a polynucleotide that has at least one a translatable region encoding the oncoprotein.

[0062] Cancer cells, as defined herein, are cells that contain genetic damage that has resulted in the relatively unrestrained growth of the cells. The genetic damage present in a cancer cell is maintained as a heritable trait in subsequent generations of the cancer cell line. The cancer treated by the method of the invention may be any of the forms of cancer known to those skilled in the art or described herein. Cancer that manifests as both solid tumors and cancer that instead forms non-solid tumors as typically seen in leukemia / blood cancer can be treated. Examples of different types of cancers include carcinomas, sarcomas, lung cancer, and lymphomas.

[0063] The fusion / chimeric / hybrid oncogenic proteins are frequently identified as cancer drives in both solid and blood cancers. They are present in approximately 16.5% of all types of cancer, >55% in leukemia, about 40% in sarcoma, about 18.8% in childhood solid tumors, about 90% of Ewing’s sarcoma, about 50-70% of prostate cancer, and about 13% of thyroid cancers. Overall, CFHON genes are responsible for about 20% of global cancer morbidity.

[0064] In some embodiments, Adjuvants or immune potentiators may also be administered with the mRNA vaccine. Advantages of adjuvants include the enhancement of the immunogenicity of antigens, modification of the nature of the immune response, the reduction of the antigen amount needed for a successful immunization, the reduction of the frequency of booster immunizations needed and an improved immune response in elderly and immunocompromised vaccinees. These may be co-administered by any route, e.g., intramusculary, subcutaneous, IV or intradermal injections.

[0065] Adjuvants useful in the present invention may include, but are not limited to, natural or synthetic. They may be organic or inorganic.Tarolli Ref. No. KJK-034481 WO ORD

[0066] Adjuvants may be selected from any of the classes (1) mineral salts, e.g., aluminum hydroxide and aluminum or calcium phosphate gels; (2) emulsions including: oil emulsions and surfactant based formulations, e.g., microfluidized detergent stabilized oil-in-water emulsion, purified saponin, oil-in- water emulsion, stabilized water-in-oil emulsion; (3) particulate adjuvants, e.g., virosomes (unilamellar liposomal vehicles incorporating influenza haemagglutinin), structured complex of saponins and lipids, polylactide co-glycolide (PLG); (4) microbial derivatives; (5) endogenous human immunomodulators; and / or (6) inert vehicles, such as gold particles; (7) microorganism derived adjuvants; (8) tensoactive compounds; (9) carbohydrates; or combinations thereof. Adjuvants for DNA nucleic acid vaccines (DNA) have been disclosed in, for example, Kobiyama et al, Vaccines, 2013, 1(3), 278-292, the contents of which are incorporated herein by reference in their entirety. Any of the adjuvants disclosed by Kobiyama may be used with the mRNA vaccines of the present invention.Cancer Treatment using an mRNA Vaccine

[0067] Another aspect of the present invention provides a method of treating cancer in a subject, comprising administering a therapeutically effective amount of an mRNA vaccine composition to a subject having a CFHON driven cancer, wherein an exosome comprising an expression cassette comprising mRNA encoding a CFHON. In some embodiments, the CFHON of the mRNA vaccine is AML1 / ETO (A / E), while in further embodiments the CFHON A / E is variant 9a (A / E9a). Cancer treatment using an mRNA vaccine is a type of immunotherapy designed to train the body’s immune system to recognize and attack cancer cells.

[0068] In some embodiments, the cancer being treated is selected from CFHON-driven blood or solid cancers such as brain, colon, lung, pancreatic, ovarian, skin, breast, head, and neck cancers. In some embodiments, the cancer being treated is leukemia.

[0069] In some embodiments, the method further comprises treating the subject with surgical resection, chemotherapy, cryotherapy, radiation therapy, immunotherapy. The use of an additional method of cancer treatment can be used before, during, or after treatment with the mRNA vaccine.

[0070] Chemotherapy involves treatment of cancer with an anticancer agent. Examples of chemotherapeutic agents include alkylating agents (e.g., doxorubicin), antimetabolites (e.g.,Tarolli Ref. No. KJK-034481 WO ORD methotrexate), topoisomerase inhibitors (e.g., etoposide), mitotic inhibitors (e.g., vincristine), platinum-based compounds (e.g., carboplatin), and anthracycline antibiotics (e.g., epirubicin).

[0071] Surgical resection refers to the surgical removal of all or part of the cancer. Cryotherapy includes, but is not limited to, therapies involving decreasing the temperature, for example, hypothermic therapy.

[0072] Radiation therapy includes, but is not limited to, exposure to radiation, e.g., ionizing radiation, UV radiation, as known in the art. Exemplary dosages include, but are not limited to, a dose of ionizing radiation at a range from at least about 2 Gy to not more than about 10 Gy or a dose of ultraviolet radiation at a range from at least about 5 J / m2to not more than about 50 J / m2, usually about 10 J / m2.

[0073] Immunotherapy either modulates the immune system or in some embodiments regulates immune checkpoints. In further embodiments, the immunotherapy comprises, or consists essentially of, or yet further consists of an immune checkpoint inhibitor, such as a Cytotoxic T-Lymphocyte Associated Protein 4 (CTLA4) inhibitor, or a Programmed Cell Death 1 (PD-1) inhibitor, or a Programmed Death Ligand 1 (PD-L1) inhibitor. In yet further embodiments, the immune checkpoint inhibitor comprises, or consists essentially of, or yet further consists of an antibody or an equivalent thereof recognizing and binding to an immune checkpoint protein, such as an antibody or an equivalent thereof recognizing and binding to CTLA4 (for example, Yervoy (ipilimumab), CP-675,206 (tremelimumab), AK104 (cadonilimab), or AGEN1884 (zalifrelimab)), or an antibody or an equivalent thereof recognizing and binding to PD-1.

[0074] The effectiveness of cancer treatment may be measured by evaluating a reduction in tumor load or decrease in tumor growth in a subject in response to the administration of the mRNA vaccine composition. The reduction in tumor load may represent a direct decrease in mass, or it may be measured in terms of tumor growth delay, which is calculated by subtracting the average time for control tumors to grow over to a certain volume from the time required for treated tumors to grow to the same volume.

[0075] An Example has been included to more clearly describe a particular embodiment of the invention and its associated cost and operational advantages. However, there are a wide varietyTarolli Ref. No. KJK-034481 WO ORD of other embodiments within the scope of the present invention, which should not be limited to the particular example provided herein.EXAMPLE

[0076] The inventors hypothesized that the use of nucleoside-modified, purified mRNA (mlY mRNA) for in vivo delivery of oncogenic fusion proteins would enable systemic tolerogenic antigen presentation in immune cells resulting in cancer cell killing. To test it, they engineered nanoparticle-like mRNA-exosomes consisting of nonimmunogenic (mlY) A / E mRNA (herein referred to as A / E-EVs).

[0077] The inventors describe herein nanoparticulate (EVs) delivery of nucleoside-modified A / E encoding mRNA (A / E-EVs) into muscles APCs as a therapeutic approach for killing A / E+ AML cells. The results of an exploratory interim analysis focused on the immune responses induced by A / E-EVs are summarized. They show that this mRNA vaccine is efficient in suppressing A / E+ cell proliferation in vivo leading to a reduction of leukemia burden, and a longer survival time. Mechanistically, such anti-leukemia activity occurs through activation of immune responses, including the robust enrichment of MHCII+, CD80+, CD4+ and CD8+ cells, which is attributable to the upregulation of cytokines or toll-like receptors(TLRs), such as IL-6, INF-y, TLR-3 or TLR7, in these immune cells.Materials and MethodsCell lines and cell culture

[0078] HEK293 and C1498 cells were newly purchased from American Type Culture Collection with no further authentication or testing for mycoplasma. Cell lines were grown in DMEM (GE Healthcare #SH30027.01) supplemented with 10% fetal bovine serum (FBS, Gibco by Life Technologies™ #16140-071) and Antibiotic-Antimycotic (Gibco by Life Technologies™ #15240062) at 37 °C under 5% CO2.Retrovirus vector, virus production, virus infection and GFP sorting

[0079] For virus production, HEK-293 (3.8 x 106) cells were planted in a 10 cm cell culture dish for 24 hours, and transfected with 6 pg of targeted or scrambled plasmids (with GFP)Tarolli Ref. No. KJK-034481 WO ORD using calcium phosphate transfection reagent (CalPhos™ Mammalian Transfection Kit), following the manufacture’s instruction. The retroviruses were harvested at 48 and 72 hours after transfection and concentrated using the protocol of the Lenti-X™ Concentrator (Clotech #631232). For virus infection, HEK-293 or C1498 cells (1 x 106) were infected by the retroviruses using Polybrene (final concentration 4 pg / ml) in 1 ml medium. The GFP positive cells were sorted at 72 hours post-infection, expanded and sorted again for further investigations.EV purification from RBCs

[0080] Exosomes (EVs) were purified from red blood cells (RBCs) as previously reported. Usman et al., Nat Commun 9, 2359 (2018) Briefly, RBCs were treated with 10 pM calcium ionophore (Sigma Aldrich) overnight. To purify EVs, RBCs and cell debris were removed by centrifugation at 600 x g for 20 min, 1600 x g for 15 min, 3260 x g for 15 min, and 10,000 x g for 30 min at 4 °C. The supernatants were passed through 0.45 iim-syringe filters. EVs were concentrated by ultracentrifugation with a TY50.2Ti rotor (Beckman Coulter, USA) at 56,000 x g for 24 hours at 4 °C, and resuspended in cold PBS. All ultracentrifugation experiments were performed with a Beckman XE-90 ultracentrifuge (Beckman Coulter). Purified EVs were stored at -80 °C.Staining and visualization of exosomes by Nanoimager

[0081] The EVs samples were incubated with fluorescently labeled primary antibodies against tetraspanin(s) (CD63, CD81 or CD9) following the manufacturers’ instructions. Images were acquired using the super-resolution microscope Nanoimager (ONI) with the NimOS software.Negative staining EM

[0082] Negative staining EM was performed to assess the homogeneity and size of the human exosome. Four microliters of the sample solution (~0.3 pM) were applied to freshly glow- discharged, 200-mesh carbon-coated copper grids (EM Sciences) and incubated for 1 min, followed by blotting to remove excess liquid. The grids were washed three times with water and stained with 0.75 % uranyl formate solution for 30 seconds. Excess liquid was gently blotted from the side of the grids using filter paper, and the grids were air-dried before imaging. Imaging was conducted using a Biotwin Tecnai Spirit 120 kV electron microscopeTarolli Ref. No. KJK-034481 WO ORD(ThermoFisher Scientific) equipped with a Gatan 4K x 4K CCD camera, at a defocus of -2 pm and nominal magnifications ranging from 18,500 x to 98,000 x.RNA constructs, in vitro transcription, and purifications

[0083] Full length A / E gene was amplified from MigRl-A / E by PCR using High-Fidelity 2x Master Mix (NEB, #M0492).

[0084] pGEM4z-Frag01_fwd (SEQ ID NO:3) aggatgaAGCGGCCGCGGATCCCCG.

[0085] pGEM4z-Frag01 rev (SEQ ID NO:4) tacgggaCACCATGGTGGCGACCGGT.

[0086] AE9a-FragO2_fwd (SEQ ID NO:5) ccatggtgTCCCGTATCCCCGTAGA.

[0087] AE9a-FragO2_rev (SEQ ID NO : 6) cggccgctTC ATCCTAGTGCAACTG.

[0088] The fragments were cloned into the multiple-cloning site (MCS) of T7 promoter plasmids pGEM4z-GFP-63A using Gibson Assembly Cloning Kit. The generated pGEM4z- A / E-63A vector was confirmed by sequencing. The 3' end poly (A) tail and 5' end was modified according to previous reports. The modification of 3' end includes a 63-bp poly(A) tail and two serial fragments (UTR) in front of the poly(A) tail.

[0089] For in vitro mRNA transcription from the DNA template, the plasmid pGEM4z-A / E- 63A was linearized by Spel-HF (NEB # R3133S), the linearized pGEM4z-A / E-63A plasmid was used as a template. The in vitro mRNA transcription was performed using E2040S HiScribe™ T7 High Yield RNA Synthesis Kit. The 5 ’CAP is S 141 IL 3'-0-Me- m7G(5')ppp(5’)G RNA Cap Structure Analog. The uridine-5’ -triphosphate (UTP) was 100% replaced by l-methylpseudouridine-5’ -triphosphate (m l TTP) (TriLink). The reactions were incubated at 37 °C for 2 hours. Then the M0303L DNase I (RNase-free) was added and incubated at 37 °C for 15 min to remove template DNA. mRNA quality control was performed by spectrophotometry on a 2100 Bioanalyzer (Agilent technologies).

[0090] The dsRNA in 100 to 500 mg IVT mRNA was removed as previously described (Baiersdorfer et al., Mol Ther Nucleic Acids 15, 26-35 (2019)) by using microcentrifuge spin columns (NucleoSpin Filters, Macherey-Nagel, Duren, Germany), cellulose fibers (C6288, SigmaAldrich), and a chromatography buffer containing 10 mM HEPES (pH 7.2), 0.1 mMTarolli Ref. No. KJK-034481 WO ORDEDTA, 125 mM NaCl, and 16% (v / v) ethanol. Finally, a T2050S Monarch® RNA Cleanup Kit (500 pg) was used to clean the mRNA, electrophoresis to determine RNA quality and stored mRNA in pure water at -80°C.Preparation and characterization of mRNA-encapsulated EVs

[0091] The A / E-EVs were prepared using Exo-Fecf™ Exosome Transfection Reagent (EXFT20A-1) with the manufacturer’s protocol. In brief, 10 pl Exo-Fect solution, 5 pl mRNA (10 pg), 133 ul sterile lx PBS and 2 pl purified exosomes (2x 108) were added into 1.5 ml tube (150 pl total transfection reaction). The components were mixed well by flicking / inversion three times, but not vortex. The mixture was incubated at 37°C in a shaker for 10 minutes and then immediately placed on ice. The reaction was stopped by adding 30 pl of the ExoQuick- TC reagent provided in the Kit and mixed by inverting 6 times. The transfected exosomes were placed on ice (or at 4°C) for 30 minutes, and centrifuged for 5 minutes at 13,000-14,000 rpm in a microcentrifuge (top speed). The supernatant was removed, and the transfected exosome pellets were resuspended in 150 pl lx PBS.RNA loading efficiency and stability in EVs

[0092] To quantify the amount of unbound A / E mRNA, 2 pg mRNA were uploaded into EVs (l x 107) via the Kit. After centrifuge, the mRNA was precipitated from the supernatant and concentrations were determined by Nano-Drop. Same amount of mRNA was separated in 10% Tris-acetate-EDTA (TBE) native gel at 150 V for 30 min, and visualized using SYBR-Gold staining for 30 min at room temperature (ThermoFisher Scientific). The SYBR Gold bands of the ASOs were quantified using imageJ (NIH, USA) and normalized to the background. The quantity of mRNA was also determined by PCR, and normalized by respective controls.In vitro transfection of HEK293 cells by A / E-mRNA

[0093] HEK293 cells were seeded in 35-mm dishes at a density of 5 x 104cells / well and cultured overnight. Cells were transfected with A / E-mRNA by different delivery vehicles. Approximately 150 pl of transfected exosomes was added to 105cells per well in a 6-well culture plate grown in opti-MEM media. Scale this ratio up or down depending upon experimental requirements. The experiment groups included EVs only, A / E-mRNA only, Lipfectamine 2000- A / E-mRNA, and the EV-A / E-mRNA group (A / E-EVs). Then the cellsTarolli Ref. No. KJK-034481 WO ORD were incubated for 2-24 h and analyzed by fluorescent microscopy for GFP expression or harvested for Western blot to validate target protein expression.Western blotting

[0094] The whole cellular lysates were prepared by harvesting the cells in 1 x cell lysis buffer (20 mM HEPES (pH 7.0), 150 mM NaCl and 0.1% NP40) supplemented with 1 mM phenylmethane sulfonyl fluoride (PMSF, Sigma #10837091001), lx Phosphatase Inhibitor Cocktail 2 and 3 (Sigma #P5726, P0044), and lx protease inhibitors (protease inhibitor cocktail set III, Calbiochem-Novabiochem #539134). The proteins were resolved by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, transferred onto PVDF membranes (GE Healthcare #10600023), blocked by 5% non-fat milk followed by probing with anti-GFP (Novus; NB600-308), anti-AMLl-ETO (GeneTex; GTX60339) or anti- -Actin (Cell Signaling; 8H10D10; #3700). The secondary antibodies are: horse anti-mouse IgG, HRP- linked antibody (Cell Signaling #7076), goat anti-rabbit IgG, HRP-linked antibody (Cell Signaling #7074), rabbit anti-goat IgG HRP-linked antibody (Invitrogen #31402).RNA isolation and quantitative PCR (qPCR)

[0095] Total RNA was isolated from various types of cells using RNeasy Mini Kit (Qiagen, Valencia, CA) according to the manufacturer’s recommendation. The first strand cDNA synthesis was conducted using the SuperScript® III First-Strand Synthesis System (Invitrogen). Expression of A / E9a was detected using the TaqMan® Gene Expression Assay (Applied Biosystems, Foster City, CA). The expression of interferon gamma, IL-6, IL-4, IL- 10, TLR3, and other genes were measured using Power SYBR® Green PCR Master Mix (Applied Biosystems). Expression of all genes was determined by the comparative Ct method using ABL1 or GAPDH levels for normalization.Local administration of mRNA-exo / lip Complex

[0096] C57BL / 6I mice (4-6 weeks; male; female) were purchased from Jackson Laboratory. All mice were maintained under specific pathogen-free conditions. All animal experiments were approved by the Institutional Animal Care and Use Committees of the University of Minnesota and were in accordance with the US National Institutes of Health Guide for Care and Use of Laboratory Animals. Mice were euthanized and sacrificed using CO2 as per theTarolli Ref. No. KJK-034481 WO ORD institutional guidelines in all experiments, when they showed any signs of distress (i.e., breathing disorders, weight loss, immobility or oversized tumors), for tissue collections and survival duration.

[0097] About 5 x 104A / E9a liver cells were injected into C57BL / 6 mouse via the tail veil. After 5 days, the hind leg of the mice was shaved, 75 pl of A / E9a, A / E9a-exosome, A / E9a- exosome-mRNA solution, and PBS control was administrated by intramuscular injection into the musculus gastrocnemius, every four days with 5 injections in total. Mice were sacrificed in 3 weeks after the final mRNA injection. Cytospin preparations of bone marrow cells were processed for Giemsa staining. The plasma was prepared and stored at -80°C. The lungs, spleens, and livers were harvested and immediately fixed in 10% neutral-buffered formalin and stained with H&E.Hematoxylin and eosin (H&E) and Immunohistochemistry (IHC) staining

[0098] Tissues collected from the animal studies were immediately fixed in 10% neutral buffered formalin. The paraffin-embedded samples were subjected to H&E and IHC staining at the MetroHealth pathological center.Cvtospin / Wright-Giemsa staining

[0099] About 0. 1 x 106BM cells were harvested from vaccinated mice and placed in Shandon EZ Single Cytofunnel (Thermo Electron Corporation). Samples were centrifuged at 1 ,000 rpm for 8 min. The slides were air-dried and stained using Hema-3 Kit (Fisher Scientific). Stained slides were viewed and photographed using a Leica microscope mounted with a high-resolution spot camera with Image-Pro Plus software. Morphologic differentiation was determined by calculating the percentage of post-mitotic cells containing metamyelocytes, bands and segmented neutrophils within six visual fields per slide.Characterization of immune cells by magnetic or flow cell sorting

[0100] After vaccination, spleens were isolated and prepared into single cell suspension. Spleens were mashed through a 70-pm cell strainer (BD-Falcon) using the plunger of a 5-ml syringe (BD Biosciences) while rinsing with PBS. Erythrocytes were removed by hypotonic lysis. Fluorescence- activated cell sorting (FACS) surface and intracellular antibodies wereTarolli Ref. No. KJK-034481 WO ORD purchased from eBioscience, Bioledgend or BD Pharmingen and used in accordance with the manufacturer’s protocol. Single cell suspensions were stained for 30 min at 4 °C for extracellular markers. All cell-based analyses were performed on single cell suspensions. Immune cells from spleen or liver were subjected to surface and intracellular staining. Flow cytometric data were collected with a BD FACS Calibur™. Single immune cell population from spleens or liver was separated with a BD FACS Aria II Cell Sorter. Flow cytometric data were analyzed with Flowjo software.

[0101] Regarding the magnet sorting, the single cells were incubated with microbeads coated by antibodies against CD11c, CD4, CD8, CD80 or MHCII. The respective positive cells were isolated using L3T4 microbeads and MACS LS columns (Miltenyi Biotec) as manufacturer’s recommendations.Measurement of serum cytokines

[0102] Blood samples were collected from leukemic mice treated with Exo or Exo+AE mRNA in serum tubes, and allowed to clot at room temperature for ~30 min. Serum samples were prepared by centrifuging at 3,000g at 4 °C for 5 minutes, and the supernatants were transferred to a polypropylene tube individually and stored at -80 °C for downstream applications. For simultaneous quantitation of cytokines, four individual mouse samples were combined / pooled and subjected to RayBio® Mouse Cytokine Antibody Array G-Series 2000 (RayBiotech Life; #AAM-CYT-G2000-4; 144 cytokines detected) analysis in accordance with the manufacturers’ instructions. All samples were assayed in quadruplicate and averaged to calculate concentrations in the serum. The captured immunoassay was then read and quantified via the Array Scanning and Analysis Services provided by RayBiotech Life. Statistical differences in measured values were analyzed using unpaired t test with welch correction. P values were calculated based on 4 signal outputs. P values less than 0.05 were considered statistically significant, and no fold change cut-off was used. All data are presented. A complete list of 144 cytokines and chemokines is referred to the reagent kits (#AAM-CYT-G2000-4).Immune monitoring with bulk RNA sequencing

[0103] The effects of mRNA-Exo versus Ctrl on immune cells were investigated by RNA sequencing for the expression of immune-relevant factors (e.g., cytokines). The splenic cells were incubated with antibodies against CD4+ or CD8+, and sorted by magnet isolation. TotalTarolli Ref. No. KJK-034481 WO ORDRNA was extracted from these CD4+ or CD8+ T cells using RNeasy Mini Kit (Qiagen #74104). The strand specific transcriptome library construction was completed by enriching mRNA from total RNA, and sequenced by DNBSEQ high-throughput platform at BGI (https: / / www.bgi.com). Briefly, mRNA molecules were purified from total RNA using oligo(dT)-attached magnetic beads, and fragmented into small pieces using fragmentation reagent after reaction a certain period in proper temperature. First strand cDNA synthesis was done by adding an appropriate amount of primers to the interrupted sample, mixing well, and reacting at a suitable temperature on a thermal cycler for a certain period of time to open the secondary structure and combine with the primers. Then second strand cDNA synthesis was done by preparing a second-strand synthesis reaction system (using dUTP instead of dTTP). The second-strand cDNA was purified by magnetic beads. The end repair & add ‘A’ was done by repairing the sticky ends of the cDNA double-stranded by reverse transcription, and add A base to the 3 'end. Adaptor ligation was done by connecting the linker to the A base, and the reaction product was purified by magnetic beads. PCR amplification was done by digesting the U-labeled second-strand template with UDG enzyme. PCR products were purified with XP Beads, and dissolved in EB solution. Library was validated on the Agilent Technologies 2100 bioanalyzer. The double stranded PCR products were heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) were formatted as the final library. The library was amplified with phi29 to make DNA nanoball (DNB) which had more than 300 copies of one molecular. The DNBs were load into the patterned nanoarray and single end 50 (pair end 100 / 150) bases reads were generated in the way of combinatorial Probe- Anchor Synthesis (cP AS). Triplicate samples were sequenced.

[0104] Data were then processed, and basic quality metrics were checked using Illumina Genome Studio. For a gene to be considered significantly up- or down-regulated, the fold difference is >1.5 (log2 (fold change)). This “cutoff” was used for further analysis, including the signaling pathway by GO or DAVID bioinformatics resources (Version 6.7; https: / / david.ncifcrf.gov / tools.jsp). All images were made online using Sangerbox 3.0. The FDR or P values of <0.05 were used to select top signaling pathways and genes involved in for further verification.Statistical analysisTarolli Ref. No. KJK-034481 WO ORD

[0105] All graphs were generated using the Student’s t test, and the Kaplan-Meier survival curves were created by the log-rank test. Correlation data were acquired using the Pearson correlation coefficients. The sample sizes for each study were chosen to be sufficient to allow statistical analysis of the outcomes of the experimental vs control of the studies based on literature documentation of similar well-characterized experiments. In vitro experiments, such as qPCR, cell proliferation assays, and clonogenic assays, were routinely repeated three times unless indicated otherwise in figure legends or main text. The statistical analysis was conducted using the Student’s t test. All analyses were conducted using the GraphPad Prism 5 Software. All P values were two-tailed. No samples or animals were excluded from the analysis. All criteria were pre-established. No randomization was used in our studies. No blinding for all experiments. Variations were compatible between groups. The statistical tests were justified as appropriate for every figure. Significance was determined at P <0.05 is *, P <0.01 is **, and P <0.001 is ***.ResultsIn vitro mRNA synthesis and characterization of mRNA encapsulated EVs

[0106] To synthesize mRNA in vitro, we cloned full length of A / E9a, or GFP gene into T7 promoter plasmids pGEM4z-GFP-64A, which was linearized by enzyme Spel-HF digestion. The mRNA was transcribed using linearized plasmids as a template that was removed by DNAse T digestion after mRNA synthesis. Because 'P replacement during in vitro transcription was shown to lower the immunogenicity of synthetic mRNAs with improving mRNA stability and translation efficiency, (46) the uridine residues were replaced 100% with the pseudouridine ( ). The CAP2 (3'-O-Me-m7G(5’)ppp(5')G RNA Cap Structure Analog; S 141 IL) was added to the 5’ of mRNA to enhance the translatability. (47) The 3’ and 5’ UTRs, which are known to be important for translation, was also added to the expression cassettes. Further, removal of double-stranded mRNA (dsRNA) contaminants is known to reduce inflammatory properties of single-stranded mRNA (Baiersdorfer et al., Mol Ther Nucleic Acids 15, 26-35 (2019)), the dsRNA was removed. About 50% loss was seen after removing dsRNA, but with no negative effects on the quality and integrity of mRNAs.

[0107] Lipid nanoparticles (LNPs) are, clinically, one of the most advanced mRNA carriers delivering mRNA to cells but avoiding mRNA degradation (Eygeris et al., Nano Lett 20, 4543-Tarolli Ref. No. KJK-034481 WO ORD4549 (2020)), which have been actively used in COVID-19 mRNA delivery and approved for human use. However, the expression efficacy of mRNA after intramuscular delivery in human is very low (1-3% of delivered mRNA) with pronounced cellular toxicity. Thus, we hypothesized that mRNA delivered by exosomes (EVs) could overcome some pitfalls of LPN delivery, because EVs have high stability, high biocompatibility, and low immunogenicity. Lu et al., Pharmaceutics 15(2):598 (2023). To this end, EVs were isolated from red blood cells (RBCs) through sequential centrifugation steps. A homogenous population of EVs was produced with an average diameter of -80-200 nm (with a peak at 200 nm) determined by a Nanosight particle analyzer (Fig. 1A) and negatively charged with a Zeta potential of 20-30 mV on average (Fig. IB). The transmission electron microscopy (TEM) revealed that the EV morphology appears to be heterogeneous with a mixture of both small exosome-like and large microvesicle-like particles within 50-200 nm in diameter (Fig. 1C), together supporting the identity and purity of RBCEVs. Further, findings from Nanoimager demonstrated that these exosomes are strongly stained by antibodies again exosome markers like CD9, CD63 or CD81 (Fig. ID).

[0108] The mRNAs of A / E9a or GFP were uploaded into EVs using Exo-Fect™ Exosome Transfection Reagent (EXFT20A-1). To examine the loading efficacy, we run an arose gel of supernatants, and found that all mRNA disappeared in samples of Kit buffer, even without EVs, suggesting that those mRNAs are precipitated by the Kit buffer, which was verified by the removal of flocculent materials using RNase treatment. The successful loading of mRNAs into EVs was further verified by the release of mRNAs from the broken EVs. These findings support the efficient and successful loading of mRNA into EVs.Translational capacity of mRNA-containing EVs in vitro

[0109] To determine the translational capacity of mRNA-containing EVs, the inventors first used EVs to deliver TX-Red (positive control) into 293T cells. We found that 2 pl of EVs (2x 1010EVs in total) has the highest delivery efficacy, but too much EVs led to less protein expression. When GFP mRNA was introduced into 293T cells, GFP proteins were efficiently translated at 8 hours with a stronger expression at 24 hours post transfection. The dsRNA removal and pseudo-U replacement did not affect the production of proteins. Notably, GFP protein expression showed that 2 pl of EVs achieves the highest protein translation, consistent with the positive controls. Translation of GFP was profoundly higher in LPN compared to EVsTarolli Ref. No. KJK-034481 WO ORD in vitro. A / E protein translation was further confirmed by Western blot (Fig. IE and IF), in agreement with GFP protein expression pattern. Finally, C57BL / 6J mice were intramuscularly injected with EVs or liposome-delivered GFP mRNAs (50-75 pl). In 48 hours, the muscular protein lysates were subjected to Western blot. It was found that GFP proteins are successfully translated in vivo, in which EVs delivery achieves higher efficacy of protein translation than LPNs (Fig. 1G). Together, these results support the efficient delivery of mRNA by EVs and the success of mRNA translation into protein in vitro and in vivo.A / E mRNA vaccination leads to significant therapeutic benefits with no adverse reactions

[0110] To evaluate the safety profile of the A / E9a mRNA vaccines in vivo, the healthy C57BL / 6 mice were treated with PBS, EVs and EVs-delivered mRNA via intramuscular injection every 4 days for 5 injections in total. On day 21 after immunization, the main organs were collected for further investigation (Fig. 2A). Histopathological examinations revealed no discernable effects / damages on heart, liver, spleen, lung, kidney and skeletal muscle tissues from mice with the A / E9a mRNA vaccine (Fig. 2B). Also we did not see any obvious changes in body weight, behaviors, capability of moving and getting food and water. These results suggest that the A / E9a-mRNA vaccine does not cause notable toxicity in vivo.

[0111] To explore the therapeutic potential of A / E9a-mRNA vaccines in vivo, we injected A / E9a+ liver cells (0.5 x 105) through the tail vein into C57BL / 6 mice (male and female). Five days post injection, these mice were randomly grouped, and treated with EVs only, mRNA only, mRNA delivered by liposome or EVs, intramuscularly every four days for a total of 5 doses (see Fig. 2a). The PBS-given healthy mice served as controls. First, bone marrow (BM) cells were immobilized onto glass microscope slides by the cytospin and subjected to Giemsa staining. The BM histopathology from mice treated with A / E9a-mRNA-delivered by EVs identified the lowest number of white blood cells (WBC) compared to other groups (Fig. 2C). Notably, like EVs and mRNA only controls, liposome-delivered A / E mRNA did not change WBC counts, indicating the limited therapeutic benefits. To further dissect the therapeutic effects of mRNA vaccines, the mouse organs, including lung, heart, liver, spleen, kidney, and skeletal muscle, from treated leukemic mice were harvested and subjected to histopathological examinations. While the weight of body and organs did not show obvious difference across different groups (Fig. 2D), H&E staining disclosed that, compared to the mice treated with A / E-EVs, the control mice displayed an increased infiltration of A / E leukemia cells into theTarolli Ref. No. KJK-034481 WO ORD spleen, lung and liver of recipients, leading to more considerable damages to these organs. The differences in other organs including kidney and muscles were not obviously seen (Fig. 2E and 2F). Accordingly, the survival of mice treated with A / E-EVs was significantly longer than that of the control group (Fig. 2G). Thus, targeting A / E+ leukemia cells by A / E-EVs might represent a promising approach in AML management.Exosome-based A / E mRNA vaccination expands antigen-specific immune cells

[0112] Evaluating immunogenicity is critical to define the safety profile of mRNA therapeutics and their use in humans. DCs are efficient antigen-presenting cells (APCs) and potent activators of naive T cells. They play a central role in immune-mediated cancer elimination via antigen presentation and T-cell priming. They act as a connective ring between innate and adaptive immunity, and are targets for infection by DNA and RNA viruses even in the application of cancer mRNA vaccines. In line with this, we observed that BM cells from A / E- EVs-treated mice have the highest number of DCs as supported by cell counts in BM cells, when compared to groups of healthy, EVs only, mRNA only and lipo+mRNA (P <0.01; Fig. 3A and 3B). Notably, compared to healthy mice, EVs only, mRNA only and lipo+mRNA did not significantly change DC number. This implies that DCs detect danger signals associated with “infection” (A / E9a mRNA delivered by EVs), which may effectively capture, process and present antigens to initiate adaptive T cell immune response or adjust activation or maturation status in response to the signal associated with these antigens.

[0113] CD4+ and CD8+ T cells are the main types of lymphocytes in cell-mediated immunity and essential for the induction of efficient immune responses against cancer cells. Given their roles in directly eliminating tumor cells, the immunotherapies and vaccine studies mainly focused on exploiting CD8+ T cells. However, CD4+ T cells could also play a critical role in the antitumor immune response due to diverse cytokine production and immune activation functions (e.g. IFN-y for CD8+T cell activation). In line, all treatments, including EVs only, mRNA only and LPN-AE, restored immune functions by increasing the number of CD4+, CD8+, CDl lc+, CD80+ and MHC-II+ cells when compared to leukemic / sick mice treated with PBS in spleens (Fig. 3C and 3D). Among them, administration of A / E-EVs led to significantly strong activation of CD4+ T cells with increased cell counts (Fig. 3D, left). By contrast, the inventors did not observe significant activation of CDl lc+, CD8+, MHCII+, and CD80+ in A / E-EVs-treated vs. EVs mice as supported by no apparent changes of cell countsTarolli Ref. No. KJK-034481 WO ORD in spleens (Fig. 3C and 3D). The changes in T cell counts in A / E-EVs vs PBS groups inform an activated immunity within the tumor microenvironment.The activation of immune cells by A / E mRNA vaccination is accompanied by upregulation of pro-inflammatory cytokines in immune cells

[0114] Previous studies show that the toll-like receptors (TLRs) 3 and 7 stimulate innate immune responses upon recognizing pathogen-derived nucleic acids. TLR3, the only RNA sensor, mediates the transcriptional induction of type I interferons (IFNs), and proinflammatory cytokines, thereby collectively establishing an antiviral host response. TLR3 works in antigen- presenting DCs to induce lymphocyte-mediated antigen-specific immune responses. TLR7 is mainly expressed in antigen-presenting cells (APCs), such as plasmacytoid dendritic cells (pDCs) and B-cells. To determine if A / E mRNA vaccine affects immune response, leukemia C57B / L6 mice were primed with multiple doses of PBS, EVs, EV-A / E mRNA and mRNA only. The spleens were collected, and single cell suspension was made. CDl lc+ and MHC- 11+ dendritic cells (DCs) were isolated by magnet-sorting and the extracted RNA was subjected to qPCR. In CDl lc+ cells, the expression of IL-6, IFN-y, IL-10, TLR-3 and TLR7, but not TLR-8, was significantly higher in all treated than healthy and sick mice. When compared to EVs, mice treated with EV-A / E mRNA have significant upregulation of IFN-y, TLR-3 and TLR7, downregulation of IL-6 with no obvious changes in the expression of IL- 10 and TLR-8, supporting the specific effects of A / E mRNA vaccination. Moreover, in MHC-II+ cells, marked increase of TLR3, TLR7 and TLR8 as well as significant decrease of IL-6, while no significant difference in the expression of IL- 10 and IFN- y, was observed in EV-A / E mRNA-treated mice when compared to EVs only or sick mice. However, both TLR3 and TLR7 were significantly decreased in treatment vs PBS. TLR7 expression was much lower with no changes in TLR3 in exosomes-A / E mRNA when compared to exosomes only. Expression of TRL-10 and IL-6 is not detectable in all groups.

[0115] Although an increase of T cells is a notable indicator for active immune response, T cell abundance is not an adequate marker of whether tumors will regress. To understand the nature of tumor progression, it is desirable to example T cell function over T cell abundance. Notably, T cells are the primary sources of cytokines. Thus, a combination of cytokine markers can predict the capacity of T cells to act within the tumor. Raphael et al., Cytokine 74, 5-17 (2015). To probe further the effects of A / E mRNA vaccination on immune functions, CD4+Tarolli Ref. No. KJK-034481 WO ORD and CD8+ T cells were isolated by magnet sorting from spleen of leukemic mice treated with 5 doses of PBS, EVs or EVs-A / E mRNA. To investigate how the A / E-mRNA vaccines influence the activation status of T cells, we assessed the expression of several cytokines, including IFN-y, IL-6, IL- 10, TLR-3, TLR-7, and TLR-8, which are known as indicator of active T cell functions, in sorted CD4+, CD8+ or CD80+ splenic T cells. Compared with those receiving PBS and exosome only, upregulation by 2-3 folds of IFN-y, IL-10 and TLR-3 / 7 / 8, which are indicators of effective therapy, but downregulation by 5-6 folds of IL-6 that is cancer- derived and immune-suppressive cytokine, was seen in both CD4+ and CD8+ T cells from mice immunized with A / E-mRNA (Fig. 4A, 4B and 4C). The recruitment and activation of DCs, CD4+ or CD8+ T cells in blood and organs like spleen, liver and BM could initiate tumor cell killing, because the A / E+ blasts cells detected in these tissues are much less when compared to EVs only and leukemic / sick mice, supported by the nest qPCR (Fig. 4D) in mouse BM, spleen and liver cells showing that A / E-mRNA-treated mice have significant decrease of A / E+ cells with a trend of reduction in liver compared to PBS and EVs only. These results were further confirmed in splenic cells by Western blot showing that A / E protein is reduced in AE vaccines vs exosome treated mic (Fig. 4E). These findings imply that A / E-mRNA vaccine can initiate a specific killing of AE+ AML cells leading to the less infiltration of A / E+ AML cells into liver, spleen and BM.Immune pathway clusters mediate the therapeutic effects of A / E mRNA vaccination

[0116] To further dissect the molecular mechanisms by which A / E mRNA vaccination activates T cells, the inventors isolated CD4+ or CD8+ T cells from spleen of leukemic mice treated with PBS, EVs and EVs-delivered AE9a mRNA (EvoRNA). The extracted bulk RNA was subjected to RNA sequencing. When comparing EV-mRNA to PBS (fold change; >1.5 or <-1.5), over 18,242 or 16,777 transcripts were identified in CD4+ and CD8+ T cells. In contrast, when comparing Exo-mRNA to EVs (fold change; >1.5 or <-1.5), over 17,336 or 15,353 transcripts were identified in CD4+ and CD8+ T cells, respectively, which is illustrated in the volcano plot (Fig. 5A and 5B). Among the changed transcripts, which 7,658 or 7,140 genes were significantly down- or upregulated comparing EvoRNA to PBS, and 7,183 or 6,525 genes were altered comparing EvoRNA to EVs only. The fold change of expression ranged from ~1.5 to 25 -fold or -22.5 to -1.5-fold. Based on log2 (fold change; >1.5 or <-1.5), top 1,000 downregulated and upregulated genes were illustrated in heatmap (Fig. 5C and 5D).Tarolli Ref. No. KJK-034481 WO ORD

[0117] To identify the key biological processes and pathways that are affected by mRNA vaccination, we first performed GO term enrichment among the differentially upregulated and downregulated genes based on log2 (fold change; >1.5 or <-1.5). In both CD4+ and CD8+ T cells, comparing EvoRNA to PBS or EVs, GO analysis revealed that the majority of differentially expressed genes are enriched in top pathways like positive / negative regulation of transcription from polymerase II promoter, immune system process, and protein phosphorylation (FDR <0.0000005), which is known to be highly active in functional T cells, because T cell expansion and proliferation require expression of many genes. Consistently, KEGG pathway analysis revealed that immune-relevant pathways are centered on the top, including MAPK signaling pathway, pathways in cancer, cytokine-cytokine receptor interaction, and Herpes simplex virus 1 infection (FDR <0.0001), which were remarkably regulated by vaccines in both CD4+ and CD8+ T cells (Fig. 5E and 5F). Notably, about 320 differentially expressed genes are enriched in MAPK signaling pathway and pathways in cancer.

[0118] Given that treatment with EVs only influences immune responses, to precisely understand the molecular rules behind the activated T cells, we focused on the comparison between EVs only and EvoRNA to reduce the background interference to a maximum level. Further, we mainly targeted cytokine signaling to dissect the altered T cell behaviors, because cytokine expression in T cells predicts the active immunogenicity, and T cell derived cytokines critically regulate T cell differentiation, maturation, migration and T cell-mediated tumor cell killing. To this end, we overlapped our seq data (7,178 genes in CD4 and 6,533 genes in CD8+ cells) with previously reported cytokine profiles (over 130 mouse cytokines included) (Carrasco Pro et al., Nucleic Acids Res 46, 9321-9337 (2018)) and identified 21 in CD8+ and 26 in CD4+ T cells (Fig. 5G). The majority of altered cytokines were shared by both CD8+ and CD4+ T cells. The inventors first performed GO pathway enrichment in these cytokines and identified many immune-relevant pathways shared by CD4+ and CD8+ T cells (P <0.05), including immune responses, positive regulation of tyrosine phosphorylation of STAT protein, inflammatory response, positive regulation of cell proliferation, immunoglobulin production, and macrophage differentiation. In line with these, KEGG pathway analysis identified the top signaling pathways including cytokine-cytokine receptor interaction, viral protein interaction with cytokine and cytokine receptor, JAK-STAT signaling pathway, TNF signaling pathway, PI3K-AKT signaling, Toll-like receptor signaling pathways and T cell receptor signaling pathways in mRNA vaccine-treated CD4+ and CD8+ T cells (Fig. 5H and 51).Tarolli Ref. No. KJK-034481 WO ORDJAK-STAT and TNF signaling pathways facilitate T cell-mediated cancer cell killing

[0119] While GO and KEGG revealed numerous pathways involved in T cell activation, JAK- STAT and TNF signaling pathway are particularly important for antigen recognition and direct tumor cell killing. JAK-STAT signaling mediates almost all immune regulatory processes, including those involved in tumor cell recognition and tumor-driven immune escape. (55) CD4+ T cell-derived TNF mediates tumor killing via multiple pathways, including recruitment of CD8+ T cells, activation of macrophages and B cells. Richardson el al., Cancers (Basel) 13, (2021). In contrast, TNF produced from activated CD8+ T cells can directly kill tumor cells through TNFR1 -dependent cell death.

[0120] To further dissect the molecular rules behind CD4+ or CD8+ T cell-mediated tumor cell killing, using DAVID 6.8 for functional annotation, KEGG data for differentially expressed cytokines in mRNA vaccine-treated T cells identified numerous downstream effector pathways. In JAK-STAT signaling, cytokines like IL-2, IL- 10, IL-3, IL-6 and IL- 15 seem to be upstream activators. The activation of both CD4+ and CD8+ T cells was supported by upregulation of STAT1, an essential regulator for Thl cell differentiation, in mRNA vaccine-treated T cells. In TNF signaling pathway, TNF seems to be an upstream regulator. TNF binding to TNFR1 led to changes at the levels of cytokines including IL-6, IL-15 and CSF. The apoptosis induced by TNF-TNFR1 interaction partially occurs through RIP pathway. In agreement with this concept, the inventors found that TNFR1 is highly expressed in AE+ AML cells when compared to health donors or other types of chromosome translocations. These findings imply that the activated CD4+ or CD8+ T cells with TNF overexpression recognize AE+ AML cells with TNFR1 upregulation, and initiate AML cell killing likely through TNFR1 -dependent cell death.Diverse cytokine profile in EVs vs EV-AE treated leukemic mice

[0121] In cancer vaccine therapy, serum cytokines can modulate immune cell activity and promote a more effective immune response against cancer cells. To identify serum factors supporting an activation of CD4+ and CD8+ T cells, we performed a cytokine screen of 144 factors, using serum from EVs vs EV-AE treated leukemic mice and the RayBio® Mouse Cytokine Antibody Array G-Series 2000 (Fig. 6A). The heatmap reflected the changes in all 144 factors without using fold cut-off (Fig. 6B). Among them, 80 factors were upregulated, butTarolli Ref. No. KJK-034481 WO ORD64 factors were downregulated in EVs vs EV-AE groups. Using P <0.05 as cut-off, twelve were significantly upregulated, including Granzyme B (Gzmb), Galectin-1 (Lgalsl), P-selectin (Selp), VEGF-R1 (Fltl), Leptin (Lep), Troy (Tnfrsfl9), ALK-1 (Acvrll), LTAC (Cxclll), Amphiregulin (Areg), CD36, SCF (Kitl) and TNFa (Tnf), but three are downregulated, including BLC (Cxcll3), Axl and Epigen (Epgn), when compared to EVs only (Fig. 6C and 6D).

[0122] GO and KEGG enrichment analysis was conducted for the 16 cytokines (P <0.05) selected from 144 changed serum cytokines) to investigate the involved pathways and biological functions. The KEGG pathway network composed of the significantly enriched pathways is presented in Fig. 6E. The direct pathway that is associated with 16 cytokines is TNF signaling, and the indirect ones are MAPK and PI3K-AKT pathways. GO analysis revealed that these 16 cytokines affect 96 cellular functions, and 16 out of 96 cellular functions, including MAPK and PI3K signaling, are significant (FDR <0.05) and displayed (Fig. 6F). Given the crucial roles of MAPK, PI3K and TNF signaling in regulating immune responses, these findings, collectively, imply that AE mRNA vaccination may alter cytokines from both serum (systematic) and infiltrating immune cells to evoke a coordination between tumor and immune cell communications.Discussion

[0123] Fusion / chimeric genes, resulting from chromosomal rearrangements, are frequently identified in both blood and solid cancers (PMCID: PMC3675181 PMID: 23376639). Their contributions to cancers are complicated, because these genes usually interact with many cofactors to initiate cancer transformation and promoter drug resistance. Gao et al., PLoS One 10, e0124241 (2015). Typically, most of chimeric genes themselves, including A / E, are not druggable, and pharmacological targeting of their co-factors haven’t consistently demonstrated improved outcomes. J. S. Ungerstedt, Int J Mol Sci 19, (2018). Given the success of the CO VID- 19 mRNA vaccines, these non-druggable chimeric genes might become druggable. Here in A / E+ AML, the inventors show that A / E mRNA-exosome vaccine, which targets A / E fusion protein itself and is administered intramuscularly in mice bearing A / E+ AML cells with a competent immune system, may offer a composite solution to this challenge.Tarolli Ref. No. KJK-034481 WO ORD

[0124] The bone marrow (BM) is both a hematopoietic and immune organ. Changes in BM immune environment significantly enhance the severity of the leukemic disease offering a theoretical base for implementing immunotherapy, including alloHSCT (Stern et al., Leukemia 28, 2235-2240 (2014)) and CAR-T (Sheykhhasan et al., Cancer Gene Ther 29, 1080-1096 (2022)). However, many drawbacks exist in these therapies, including GVHD, lethal CRS, ineffectual in solid tumors and challenging in myeloid malignancies due to the absence of an indispensable antigen. Because of many advantages including the broad availability of antigens (mutated and overexpressed genes), well tolerance and rapid development with low cost, mRNA-based vaccines could be promising in treating leukemia. Indeed, the inventors used exosomes as delivery vehicles in the therapeutic A / E mRNA vaccine against AML leukemia mouse model. They present strong evidence, for the first time, showing that A / E mRNA vaccines delivered by exosomes exhibit excellent therapeutic effects on AML leukemia. This is supported by less damaged organs, fewer A / E+ leukemia cells in BM, spleen and liver and longer survival time in mice treated with A / E mRNA vaccines. Mechanistically, priming leukemic mice with A / E mRNA vaccines elicits robust CD4+ and CD8+ T cell responses, which was mediated by upregulation of IFN-y, IL- 10, IL-21, and CSFs in CD4+ and CD8+ T cells, the major components within the activated JAK-STAT or TNF signaling pathway in immune cells. Although we demonstrate that A / E mRNA-exosome can potently evoke CD4+ T cells in general, it remains to be seen whether A / E mRNA vaccines can trigger lasting immunity in a timely manner via sequentially potentiating dendritic cells, CD4+ T cells and CD8+ T cells throughout the treatment procedures. It also warrants further investigations about which subpopulations of CD4+ regulatory T cells actually initiate A / E+ cell killing. Notably, the significance of our findings is limited by the small sample size and non-longitude tracking of changes in A / E+ cells and immune cells. While the results indicate the potential therapeutic application prospects of the A / E mRNA vaccine (vaccination post leukemia cell injection), it remains unknown whether A / E mRNA vaccines have preventive effects, which the vaccination should be initiated in mice before AE+ cell inoculation. Finally, the inventors cannot rule out that vaccines also activate CD80+ T cells, CD11C+ dendritic cells, and MHC-II+ macrophages to kill A / E+ cells by themselves or coordinating with CD4+ and CD8+ T cells, given the upregulation of cytokines like TLR-8, TLR-3 or TLR-7 in these cells.

[0125] A / E results from one of the most frequent chromosome translocation (t(8;21) in AML. It is leukemia-initiating transcription factor (Burel et al., Mol Cell Biol 21, 5577-5590 (2001)), serves as a scaffold protein cooperating with many transcription repressors (i.e., HDACs,Tarolli Ref. No. KJK-034481 WO ORDDNMTs) (Wang et al., Science 333, 765-769 (2011)) and activators (i.e., p300, HIFla, PRMT), and / or driver mutations / “second hits” (FLT3-ITD, KIT, or JAK2 mutation), in order to promote leukemic transformation and growth. While such complexity makes A / E be hard- or nondruggable, it offers opportunities to develop unique treatments, like mRNA vaccines, for specifically recognizing and eradicating A / E+ leukemia cells. In line, previous studies support the AML-specific immunogenicity of translocations like A / E, together, representing ideal antigen targets. L. Vago, I. Gojo, J Clin Invest 130, 1552-1564 (2020) As expected, mice treated with exosome-delivered A / E mRNA have much lower levels of A / E expression in BM, spleen and liver, arguing for the less infiltration of A / E+ cells into these organs or higher killing of AE+ cells, given that CD4+ and CD8+ T cells are more active in these organs. Given that fusion proteins also drive solid cancers, and as exosomes efficiently deliver RNAs, our findings expand the theoretical vaccine-eligible pool to nearly all cancers driven by fusion / chimeric genes. However, the present studies did not demonstrate whether A / E mRNA induces an A / E specific or a broad-spectrum immunity to kill A / E+ ells. Given the relatively small magnitude of A / E mRNA vaccine, it remains to be exploited whether the combination between AE mRNA vaccines with other treatment options, particularly inhibitors for A / E partner proteins (DNMTs, HDACs, HIFla, P300, PRMT) (Liu et al., J Pharmacol Exp Ther 321, 953-960 (2007)), synergistically improve AML outcomes.

[0126] To date, LNPs are frequently used to deliver mRNA in vitro and in vivo, and also approved for human use. Yet the expression efficacy of mRNA is relatively low with pronounced cellular toxicity. As EVs have high stability, high biocompatibility, and low immunogenicity (Lu et al., Pharmaceutics 15, 15(2):598 (2023)), present studies employed EV delivery. While AE delivered by LPNs has higher expression in vitro, intramuscular delivery by EVs in mice achieved more pronounced expression. Importantly, A / E mRNA delivered by LPNs in our mouse models did not achieve apparent therapeutic outcomes compared to EVs, including the lower counts of dendritic cells, less activation of CD4+ and CD8+ cells and less expression of “good” cytokines, demonstrating that EVs could be a more promising delivery tool for mRNA vaccine in human, although it has not been approved by FDA. Further, we used class II cap to increase protein translation, and replaced uridinel00% with the pseudouridine (VP) to enhance RNA stability and reduces non-specific immunogenicity. Kim et al., Mol Cell Toxicol 18, 1-8 (2022) This could be further improved by modifying RNA with (N6- methyladenosine (m6A) and 5-methylcytosine (m5C)) in different ratios or / and using different types of cap.Tarolli Ref. No. KJK-034481 WO ORD

[0127] The inventors find that priming leukemic mice with A / E mRNA vaccines increase levels of cytokines (e.g., IFN-y, IL-10, TLR-3, TLR-7, TLR-8, IL-21, and CSFs with antitumor properties) in serum and infiltrating immune cells (CD4+, CD8+, CD80+ or CD1 lc+) in spleen or livers. Upregulation in these cytokines indicates the active status of T cell functions potentiating the effects of A / E mRNA immunotherapy. However, it warrants future investigation whether serum and cellular cytokines are positively correlated and coordinately empower T cell-initiated A / E+ cell killing. Of note, the measurement of all these cytokines was done at the later stage of disease progression and treatment, a key limitation of current study, because cytokine production and changes in immune cell functions are dynamic with large variations throughout cancer treatment. The findings from one-point measurement may not accurately reflect the contribution of tested cytokines to the activation of CD4+ and CD8+ T cells thereby the treatment efficacy. This issue will be addressed in future study by examining longitude alterations of cytokine productions and immune cell activation, recruitment phenotype and function at multiple stages in the treatment procedures. Such studies will also address whether and how CD4+ / CD8+ T cells, dendritic cells and macrophages sequentially and coordinately kill A / E+ cells post vaccination. Despite these limitations, our findings further support the hypothesis that A / E mRNA vaccines kill A / E+ cells via cytokine (e.g. TNF)- initiated communication between immune cells (CD4+ / CD8+ T cells) and A / E+ cells.

[0128] In summary, these findings highlight the critical role of indirectly targeting A / E protein in AML cells to achieve potent anti-tumor effects. This study not only introduces a novel strategy for repairing chromosomal abnormalities in blood and solid cancers but also underscores the importance of enhancing tumor cell immunogenicity in anti-tumor therapy. Overall, this work offers a promising approach to accelerate the clinical translation of A / E mRNA vaccines without the need for personalized vaccines.

[0129] The complete disclosure of all patents, patent applications, and publications, and electronically available material cited herein are incorporated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood there from. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

Tarolli Ref. No. KJK-034481 WO ORDCLAIMSWhat is claimed is:

1. An mRNA vaccine composition, comprising an exosome comprising an expression cassette comprising an mRNA encoding a chimeric / fusion / hybrid oncogene (CFHON).

2. The mRNA vaccine composition of claim 1 , wherein the CFHON is derived from chromosomal translocations, inversion, or other genetic arrangement.

3. The mRNA vaccine composition of claim 1, wherein the CFHON is AML1 / ET0 (A / E)4. The mRNA vaccine composition of claim 3, wherein the CFHON A / E is variant 9a (A / E9a), derived from chromosomal translocation (t(8;21 )).

5. The mRNA vaccine composition of claim 1, wherein the composition comprises a CFHON other than A / E.

6. The mRNA vaccine composition of claim 1 , wherein the composition comprises a plurality of mRNA encoding different CFHON.

7. The mRNA vaccine composition of claim 1, wherein the mRNA is modified mRNA.

8. The mRNA vaccine composition of claim 1, wherein the uridine-5’ -triphosphate nucleotides are replaced by pseudouridine 1 -methylpseudouridine-5’ -triphosphate (ml TP) in the modified mRNA.

9. The mRNA vaccine composition of claim 1 , wherein the mRNA is single strand mRNA (ssRNA).

10. The mRNA vaccine composition of claim 1, wherein the exosomes are human red blood cell exosomes.

12. The mRNA vaccine composition of claim 1, wherein the expression cassette further comprises a T7 promoter.Tarolli Ref. No. KJK-034481 WO ORD13. A method of treating cancer in a subject, comprising administering a therapeutically effective amount of an mRNA vaccine composition to a subject having a CFHON driven cancer, wherein the composition comprises an exosome comprising an expression cassette comprising mRNA encoding a CFHON.

14. The method of claim 13, wherein the cancer is leukemia.

15. The method of claim 13, wherein the cancer is a CFHON-driven blood or solid cancer.

16. The method of claim 13, wherein the CFHON is AML1 / ET0 (A / E).

17. The method of claim 16, wherein the CFHON A / E is variant 9a (A / E9a).

18. The method of claim 13, wherein the subject is a human subject.

19. The method of claim 13, wherein the mRNA vaccine is administered by intramuscular injection.

20. The method of claim 13, wherein the method further comprises treating the subject with surgical resection, chemotherapy, cryotherapy, radiation therapy, and / or immunotherapy .

21. A method of preventing cancer in a subject, comprising administering an effective amount of an mRNA vaccine composition to the subject having an increased risk of developing CFHON driven cancer, wherein the composition comprises an exosome comprising an expression cassette comprising mRNA encoding a CFHON.

22. The method of claim 21 , wherein the cancer is leukemia.

23. The method of claim 21, wherein the cancer is a CFHON-driven blood or solid cancer.

24. The method of claim 21, wherein the CFHON is AML1 / ETO (A / E).

25. The method of claim 24, wherein the CFHON A / E is variant 9a (A / E9a).

26. The method of claim 21, wherein the subject is a human subject.

Images