Multilamellar RNA nanoparticles

Abstract

To provide novel RNA nanoparticles.SOLUTION: The present disclosure provides a nanoparticle comprising a positively charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between cationic lipid bilayers.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Grant Disclosure This invention was made with U.S. government support under Grant No. K08 CA199224 awarded by the National Institutes of Health and Grant No. W81XWH-17-1-0510 awarded by the U.S. Army Medical Command. The government has certain rights in this invention.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 876440, filed July 19, 2019, U.S. Provisional Patent Application No. 62 / 877598, filed July 23, 2019, U.S. Provisional Patent Application No. 62 / 884983, filed August 9, 2019, and U.S. Provisional Patent Application No. 62 / 933326, filed November 8, 2019, the disclosures of which are incorporated herein by reference in their entireties. [Background technology]

[0003] Due to the severe and nonspecific adverse effects of radiation therapy and chemotherapy, targeted therapies that can selectively kill tumor cells in patients with glioblastoma (GBM) are essential (Stupp et al., The New England Journal of Medicine. 2005;352(10):987-96; Stupp et al., The Lancet Oncology. 2009;10(5):459-66; Sampson et al., Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology. 2010;28(31):4722-9). Tumor-specific immunotherapy can be utilized to eradicate malignant brain tumors with exquisite precision and without collateral damage to normal tissue (Schuster et al., Journal of Clinical Oncology: Official Journal of the American Society of Clinical Oncology. 2011;29(20):2787-94; Ribas et al., Anti-CTLA4 Antibody Clinical Trials in Melanoma. Update Cancer Ther. 2007;2(3):133-9; Paller et al., Hum Vaccin Immunother. 2012;8(4):509-19). Immunotherapy relies on the cytotoxic potential of activated T cells, which scavenge for tumor-associated or specific antigens (TAAs or TSAs) to recognize and reject. Unlike most drugs, activated T cells can cross the blood-brain barrier (BBB) ​​via ICAM / VCAM integrin (i.e., LFA-1, VLA-4) binding (Sampson et al., Neuro Oncol. 2011;13(3):324-33; Ransohoff et al., Nature Reviews Immunology. 2003;3(7):569-81; Miao et al., PloS one. 2014;9(4):e94281).T cells can be activated ex vivo by co-culture with dendritic cells (DCs) presenting TAA / TSA (Mitchell et al., Nature. 2015;519(7543):366-9) or by transduction with chimeric antigen receptors (CARs) (Grupp et al., The New England Journal of Medicine. 2013;368(16):1509-18). Alternatively, T cells can be activated endogenously using cancer vaccines. However, in a randomized phase III trial in patients with primary GBM, a peptide vaccine targeting the tumor-specific EGF-RVIII surface antigen did not mediate improved survival over a control vaccine (Weller et al., The L. Int Oncology. 2017;18(10):1373-85). The failure of the EGF-RVIII vaccine to mediate antitumor effects highlights the challenges of therapeutic cancer vaccines. While prophylactic cancer vaccines function to prevent malignancies (i.e., HPV vaccines to prevent cervical cancer), the vaccines require multiple booster immunizations over months to years to confer protection in immune-rich patients. Furthermore, therapeutic cancer vaccines must induce immune responses much more rapidly against rapidly developing malignancies (i.e., GBM) (Sayour et al., Int J Mol Sci. 2018;19(10)). Furthermore, GBM is a highly aggressive and heterogeneous tumor associated with severe systemic or intratumoral suppression, which may hinder early immunotherapy responses (Chongsathidkiet et al., Nature Medicine. 2018;24(9):1459-68; Learn et al., Clinical Cancer Research: an official journal of the American Association for Cancer Research. 2006;12(24):7306-15).

[0004] RNA vaccines offer several advantages over conventional modalities. RNA exerts a potent influence on both the innate and adaptive immune systems. RNA functions as a toll-like receptor (TLR) agonist for receptors 3, 7, and 8, inducing potent TLR-dependent innate immunity (24). RNA also stimulates intracellular pathogen-recognition receptors (i.e., melanoma differentiation antigen 5 (MDA-5) and retinoic acid-inducible gene I (RIG-I)), maximally activating both helper CD4 and cytotoxic CD8 T cell responses (Strobel et al., Gene therapy. 2000;7(23):2028-35; Mitchell et al., The Journal of Clinical Investigation. 2000;106(9):1065-9; Kim et al., Oncogene. 1998;17(24):3125-35). Unlike DNA vaccines, which must cross both the cell and nuclear membranes, RNA cannot integrate into the host genome and therefore only requires access to the cytoplasm, offering significant safety advantages (Sayour et al., Immunotherapy for Pediatric Brain Tumors. Brain Sci. 2017;7(10). Epub 2017 / 10 / 27). Unlike many peptide vaccines developed exclusively for specific HLA haplotypes (i.e., HLA-A2), RNA bypasses MHC class restriction and can be utilized in the general population (Sayour et al., Immunotherapy for Pediatric Brain Tumors. Brain Sci. 2017;7(10). Epub 2017 / 10 / 27). One drawback of RNA is its lack of stability, making it difficult to administer "naked" RNA directly to patients. Because cancer vaccines require localization to antigen-presenting cells (APCs) where the RNA must be translated, processed, and presented to MHC class I and II molecules, degradation remains a strong barrier to the development of new mRNA technologies.To overcome these limitations, researchers within the University of Florida Brain Tumor Immunotherapy Program (UFBTIP) have developed an RNA-loaded dendritic cell (DC) vaccine for the treatment of brain tumors (NCT03334305, PI: Sayour) (Sampson et al., Journal of clinical oncology: official journal of the American Society of Clinical Oncology. 2010;28(31):4722-9; Fecci et al., an official journal of the American Association for Cancer Research. 2007;13(7):2158-67; Mitchell et al., Blood. 2011;118(11):3003-12. Epub 2011 / 07 / 20; Nair et al., Clinical Cancer Research: an official journal o). f the American Association for Cancer Research.2014. doi:10.1158 / 1078-0432.CCR-13-3268. PubMed PMID:24658154;Sanchez-Perez et al.,PloS one.2013;8(3):e59082;Fecci (2006;12(14 Pt 1):4294-305). Total tumor-derived mRNA (autologously prepared to represent a personalized tumor-specific transcriptome) has been shown to be amplified to clinical scale from a small number of cells (approximately 500 tumor cells), providing a renewable, antigen-specific resource for DC vaccine production. While ex vivo production of RNA-loaded DCs shows considerable promise, advances in cell therapy have come with developmental challenges, making it difficult to generate vaccines for the entire population.

[0005] To circumvent the challenges of cell therapy, nanocarriers have been developed as RNA delivery vehicles. However, the unknown biological reactivity of novel nanoparticle (NP) designs has delayed the translation of NPs into human clinical trials. Alternatively, simple biodegradable lipid-NPs have been developed as cationic and anionic cancer vaccine formulations. Cationic formulations have been created to shield mRNA within the lipid core, while anionic formulations have been created to tether mRNA to the particle surface. However, cationic formulations suffer from low immunogenicity, and anionic formulations remain hindered by severe intratumoral and systemic immunosuppression, which can hinder activated T cell responses.

[0006] Therefore, there is a need in the art for new RNA-NPs that overcome the aforementioned limitations. Summary of the Invention

[0007] The present disclosure provides nanoparticles comprising a positively charged surface and (i) a core and (ii) an interior comprising at least two nucleic acid layers, each nucleic acid layer being interposed between cationic lipid bilayers. In exemplary embodiments, the nanoparticles of the present disclosure comprise an interior comprising alternating nucleic acid layers and cationic lipid bilayers. In exemplary embodiments, the nanoparticles comprise at least three nucleic acid layers, each of which is interposed between cationic lipid bilayers. In exemplary aspects, the nanoparticles comprise at least four or five or more nucleic acid layers, each of which is interposed between cationic lipid bilayers. In various aspects, the outermost layer of the nanoparticle comprises a cationic lipid bilayer. In various examples, the surface comprises multiple hydrophilic portions of the cationic lipids of the cationic lipid bilayer. In exemplary aspects, the core comprises a cationic lipid bilayer. In various aspects, the outermost region of the core comprises a cationic lipid bilayer. In some examples, the outermost region of the core comprises a cationic lipid bilayer comprising DOTAP. Optionally, the core comprises less than about 0.5% by weight of nucleic acid. In exemplary embodiments, the core comprises (i) a therapeutic agent or (ii) a diagnostic agent (e.g., an imaging agent), or (iii) a combination thereof. Suitable therapeutic and diagnostic agents are described herein. In exemplary embodiments, the therapeutic agent comprises or is a nucleic acid. Optionally, the therapeutic agent is an antisense oligonucleotide (ASO) or siRNA. In various examples, the ASO or siRNA is not the same nucleic acid present in the alternating nucleic acid layers (cationic lipid bilayer). In exemplary examples, the ASO or siRNA is the same nucleic acid present in the alternating nucleic acid layers (cationic lipid bilayer). In various embodiments, the core comprises iron oxide nanoparticles (IONPs), useful for imaging tissues or cells, e.g., via magnetic resonance imaging (MRI). Optionally, the IONPs are coated with a fatty acid, e.g., a C8-C30 fatty acid. In various embodiments, the fatty acid is oleic acid. In various embodiments, the core comprises a plurality of IONPs (optionally coated with oleic acid), where the plurality is held together by a lipid, e.g., a cationic lipid. Optionally, multiple IONPs (optionally coated with oleic acid) are held together by DOTAP. The diameter of the nanoparticles is, in various embodiments, about 50 nm to about 250 nm in diameter, optionally about 70 nm to about 200 nm in diameter. In illustrative examples, the nanoparticles are characterized by a zeta potential of about +40 mV to about +60 mV, optionally about +45 mV to about +55 mV. In various cases, the zeta potential of the nanoparticles is about 50 mV. In some embodiments, the nucleic acid molecules are present at a nucleic acid molecule:cationic lipid ratio of about 1 to about 5 to about 1 to about 20, optionally about 1 to about 15, about 1 to about 10, or about 1 to about 7.5. In various embodiments, the nucleic acid molecules are RNA molecules, optionally messenger RNA (mRNA). In various embodiments, the mRNA is in vitro transcribed mRNA, and the in vitro transcription template is cDNA made from RNA extracted from tumor cells. In various embodiments, the nanoparticles comprise a mixture of RNA molecules, and the mixture of RNA is RNA isolated from human tumors, optionally the human tumor is malignant brain tumor, optionally glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma, or peripheral tumor with metastatic invasion into the central nervous system.

[0008] The present disclosure also provides a method for making nanoparticles comprising a positively charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, each nucleic acid layer disposed between cationic lipid bilayers, the method comprising: (A) mixing nucleic acid molecules and liposomes at an RNA:liposome ratio of about 1 to about 5 to about 1 to about 20, optionally about 1 to about 15, about 1 to about 10, or about 1 to about 7.5, to obtain RNA-coated liposomes, wherein the liposomes are made by a process for making liposomes comprising drying a lipid mixture comprising cationic lipids and an organic solvent by evaporating the organic solvent under vacuum; and (B) mixing the RNA-coated liposomes with an excess amount of liposomes. In exemplary embodiments, the lipid mixture comprises cationic lipid and organic solvent in a ratio of about 40 mg cationic lipid per mL of organic solvent to about 60 mg cationic lipid per mL of organic solvent, optionally about 50 mg cationic lipid per mL of organic solvent. In various examples, the process for producing liposomes further comprises rehydrating the lipid mixture with a rehydration solution to form a rehydrated lipid mixture, then stirring, resting, and sizing the rehydrated lipid mixture. Optionally, sizing the rehydrated lipid mixture comprises sonicating, extruding, and / or filtering the rehydrated lipid mixture.

[0009] Further provided herein are nanoparticles produced by the method of producing nanoparticles disclosed herein. Further provided herein are cells comprising the nanoparticles of the present disclosure. Optionally, the cells are antigen-presenting cells (APCs), such as dendritic cells (DCs). The present disclosure also provides a population of cells, wherein at least 50% of the population are cells according to the present disclosure.

[0010] The present disclosure provides a pharmaceutical composition comprising a plurality of nanoparticles according to the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient. In various embodiments, the composition comprises about 10 nanoparticles per mL. 10 Nanoparticles ~ approx. 10 per mL 15 Nanoparticles, optionally about 10 per mL 12Contains nanoparticles ±10%.

[0011] The present disclosure provides a method for increasing an immune response, such as an immune response against a tumor, in a subject. In an exemplary embodiment, the method includes administering a pharmaceutical composition of the present disclosure to the subject. In an exemplary aspect, the nucleic acid molecule is mRNA. Optionally, the composition is administered systemically to the subject. For example, the composition is administered intravenously. In various aspects, the pharmaceutical composition is administered in an amount effective to activate dendritic cells (DCs) in the subject. In various examples, the immune response is a T cell-mediated immune response. Optionally, the T cell-mediated immune response includes activity by tumor-infiltrating lymphocytes (TILs).

[0012] The present disclosure also provides a method for delivering an RNA molecule to a tumor microenvironment, a lymph node, and / or a reticuloendothelial organ. In an exemplary embodiment, the method comprises administering a pharmaceutical composition disclosed herein to a subject. Optionally, the reticuloendothelial organ is the spleen or liver.

[0013] Further provided herein is a method for treating a subject with a disease. In an exemplary embodiment, the method comprises delivering an RNA molecule to cells of a subject by a method of the present disclosure, which delivers the RNA molecule to the tumor microenvironment, lymph nodes, and / or reticuloendothelial organs. In various aspects, the RNA molecule is delivered to cells ex vivo, and the cells are administered to the subject. In an exemplary embodiment, the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the subject's disease. In various examples, the subject has cancer or a tumor, optionally a malignant brain tumor, optionally a glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma, or a peripheral tumor with metastatic infiltration into the central nervous system.

[0014] Additional embodiments and aspects of the presently disclosed pharmaceutical compositions and methods are provided below. [Brief explanation of the drawings]

[0015] [Figure 1A]A series of illustrations of the general schemes leading to lipid bilayers, liposomes, and multilamellar (ML) RNA NPs (boxed). [Figure 1B] A pair of CEM images of uncomplexed NPs (left) and ML RNA NPs (right). [Figure 2A] 1 is an illustration of the general scheme leading to cationic RNA lipoplexes. [Figure 2B] 1 is an illustration of the general scheme leading to cationic RNA lipoplexes. [Figure 2C-2D] Figure 2C is a CEM image of uncomplexed NPs, Figure 2D is a CEM image of RNA LPX, and Figure 2E is a CEM image of ML RNA NPs. [Figure 2F] 1 is a graph of %CD86+ of CD11c+MHC class II+ splenocytes present in the spleens of mice treated with ML RNA NP (ML RNA-NP), RNA LPX, anionic LPX, or untreated. [Figure 2G] 1 is a graph of %CD44+CD62L+ of CD8+ splenocytes present in the spleens of mice treated with ML RNA NP (ML RNA-NP), RNA LPX, anionic LPX, or untreated. [Figure 2H] 1 is a graph of %CD44+CD62L of CD4+ splenocytes present in the spleens of mice treated with ML RNA NP (ML RNA-NP), RNA LPX, anionic LPX, or untreated. [Figure 2I] 1 is a graph of the survival rate of mice treated with ML RNA NP (ML RNA-NP), RNA LPX, anionic LPX, or untreated. [Figure 2J] 1 is a graph of the amount of IFN-α produced in mice treated with ML RNA NP (ML RNA-NP), RNA LPX, anionic LPX, or untreated mice. [Figure 3A] 10 is a pair of photographs of lungs from mice treated with ML RNA NPs or untreated. [Figure 3B]Graph of % central memory T cells (CD62L+CD44+ of CD3+ cells) present in mice treated with ML RNA NPs loaded with tumor-specific RNA or ML RNA NPs containing non-specific RNA (GFP RNA), or untreated. [Figure 3C] 1 is a graph of the survival rate of mice treated with ML RNA NPs loaded with tumor-specific RNA or ML RNA NPs containing non-specific RNA (GFP RNA), or untreated mice. [Figure 3D] This is a graph of the survival rate of mice treated with ML RNA NPs loaded with tumor-specific RNA or ML RNA NPs containing nonspecific RNA (GFP RNA), or untreated mice. This model is different from the model used to obtain the data in Figure 3C. [Figures 4A-4D] Figure 4A is a graph of the percentage expression of CD8 or CD44 and CD8 on CD3+ cells plotted as a function of time after administration of ML RNA NPs. Figure 4B is a graph of the percentage expression of PDL1, MHC II, CD86, or CD80 on CD11c+ cells plotted as a function of time after administration of ML RNA NPs. Figure 4C is a graph of the percentage expression of CD44 and CD8 on CD3+ cells plotted as a function of time after administration of ML RNA NPs. Figure 4D is a graph of the survival rate of dogs treated with ML RNA NPs compared to median survival (dotted line). [Figure 5] CEM images of ML RNA NPs, showing an example with several layers. [Figure 6]This cartoon depicts the generation of NPs loaded with personalized tumor mRNA. From as few as 100–500 biopsied brain tumor cells, total RNA is extracted and a cDNA library is generated. From this library, large amounts of mRNA (representing the personalized tumor-specific transcriptome) can be amplified. The negatively charged tumor mRNA is then encapsulated into positively charged lipid NPs. The NPs encapsulate the RNA via electrostatic interactions and are administered intravenously (i.v.) for uptake by dendritic cells (DCs) in the reticuloendothelial organs (i.e., liver, spleen, and lymph nodes). The RNA is then translated and processed by the intracellular machinery of the DCs to present peptides to MHC class I and II molecules, which activate CD4 and CD8+ T cells. [Figure 7A]

[0033] Figure 1. Timeline of long-term survivor treatment. First and second tumor inoculations are shown. [Figure 7B] Graph of animal survival rates after the second tumor inoculation for each of three groups of mice: two groups treated with ML RNA NPs containing nonspecific RNA (RNA not specific to the tumor of interest, green fluorescent protein (GFP) or pp65) before the second tumor inoculation, and one group treated with ML RNA NPs containing tumor-specific RNA before the second tumor inoculation, or untreated animals before the second tumor inoculation. The survival rate of the control group is shown as "untreated." [Figure 8] 1 is a series of images illustrating the localization of anionic LPX in mice upon administration. [Figure 9] 1 is an image of iron oxide nanoparticles bound by a lipid coating of DOTAP. [Figure 10] Multilayered RNA NPs form complex structures that wrap mRNA around multilayered vesicles to facilitate payload delivery. The bar graphs show gene expression (luminescence) for anionic RNA-LPS (first bar on the left), RNA-lipoplexes (second bar), RNA-NPs (low) (third bar), and RNA-NPs (high) (fourth bar). [Figure 11]Multilayered RNA NPs mediate DC activation and increased IFN-α release. RNA / anionic lipoplexes (LPX) or RNA-NPs were administered intravenously to C57Bl / 6 mice once a week (×3). Spleens were harvested one week later for evaluation of activated DCs (left). Serum was collected 6 hours after the first treatment for evaluation of IFN-α by ELISA (right). [Figure 12] Multilayered RNA-NPs are superior to LPX and peptide-based vaccines in eliciting antigen-specific T cells. RNA / anionic lipoplexes (LPX) (left) or peptide-based vaccines (right) formulated in complete Freund's adjuvant (CFA) were compared with OVA-specific RNA-NPs. Animals (N = 5-8 / group) received 107 OT-Is prior to evaluation of tetramer-positive (OVA-specific) T cells 1 week after the last vaccination. [Figure 13] We show that RNA-NPs induce a memory restimulation response to the CMV matrix protein pp65. Weekly pp65 RNA-NPs (x3) were administered to naive C57 / Bl / 6 mice, and splenocytes were harvested 1 week later for culture with overlapping pp65 peptide pools and IFN-γ assessment (*p<0.05, **p<0.01, Mann Whitney). [Figure 14] This shows that multilayered tumor-specific mRNA-NPs mediate superior efficacy. Different lipoplexes (LPX) or RNA-NPs were loaded with tumor-specific mRNA and compared in a therapeutic lung cancer model (K7M2) (n=8 / group). Each vaccine was administered intravenously weekly (x3). **p<0.01, Gehan-Wilcoxon test. [Figures 15A-15C]We demonstrate that charge-modified RNA-NPs can be targeted to, for example, the lungs or spleen. RNA-NPs were intravenously injected into C57Bl / 6 mice (n = 3-4 per group). Reticuloendothelial organs (lymph nodes, spleen, and liver) were harvested within 24 hours, and CD11c cells expressing the activation marker CD86 (*p < 0.05, **p < 0.01, Mann-Whitney test) were assessed from lymph nodes (Figure 15A), splenocytes (Figure 15B), or hepatocytes (Figure 15C). The data confirm that the constructs of this disclosure can be delivered to the reticuloendothelial organ with only a single administration. [Figure 16] This shows that full-length LAMP-conjugated pp65 appears to induce a higher percentage of antigen-specific T cells. Full-length LAMP-conjugated pp65 RNA was administered intravenously to naive mice (n=5 / group) once a week (x3), and spleens were harvested and restimulated with overlapping pp65 peptide pools (*p<0.05, Mann-Whitney test). The graph compares IFN production in subjects receiving NP alone, RNA-NP, or LAMP RNA-NP. [Figures 17A-17B] Graphs illustrating the percentage of OVA-specific tetramer+ CD8 cells in subjects receiving NP alone and RNA-NP in MDAS knockout subjects. Figure 17A - T cells alone. Figure 17B - After restimulation assay with B16F10-OVA. [Figure 18] RNA-NP mediates efficacy independent of TLR7. Wild-type TLR7+ / + C57Bl / 6 mice (n=8 / group) were implanted subcutaneously with B16F10-derived tumors, and tumor volume was compared with TLR7- / - knockout (KO) mice (n=5-8 / group) on a C57Bl / 6 background. Both groups of animals were treated intravenously with RNA-NP weekly (x3), whereas KO mice received NP alone (***p<0.001, two-way ANOVA). [Figures 19A-19B]RNA-NP mediates IFNAR1-dependent responses independently of TLR7. (Figure 19A) K7M2 (1.25x106 cells) were inoculated into the lungs of Balb / c mice (n=7 / group) via tail vein injection and treated intravenously with RNA-NP weekly (x3) with or without IFN-α-blocking antibodies (IFNAR1 mAbs) every other week. ***pWild TLR7+ / + C57Bl / 6 mice (n=8) were implanted with B16F0 melanoma, and survival outcomes were compared with TLR7- / - knockout (KO) mice (n=5 / group) on a C57Bl / 6 background. Both groups of animals were treated intravenously with RNA-NP weekly (x3), whereas KO mice received NP only (*p). [Figures 20A-20B] RNA-NP mediates memory recall responses. (Figure 20A) Balb / c mice (5-8 / group) inoculated with K7M2 lung tumors were subsequently intravenously vaccinated with RNA-NP for 3 weeks, and spleens were harvested 1 week after the third vaccination for analysis of ex vivo memory recall responses to tumor antibodies. IFN-γ was administered to the antigen (K7M2) versus the control tumor (B16F0) (*p<0.05, Mann-Whitney test). (Figure 20B) Long-term survivors (n=7) previously treated with RNA-NP were rechallenged with intravenous administration of K7M2 tumor cells (1.25x106 cells) and compared with a new cohort of untreated mice (n=8) inoculated with K7M2 tumors (****p<0.001, Gehan-Breslow-Wilcoxon test). DETAILED DESCRIPTION OF THE INVENTION

[0016] The present disclosure relates to nanoparticles comprising cationic lipids and nucleic acids. As used herein, the term "nanoparticle" refers to a particle having a diameter of less than about 1000 nm. Because the nanoparticles of the present disclosure comprise cationic lipids treated to induce liposome formation, in various embodiments, the nanoparticles disclosed herein comprise liposomes. Liposomes are artificially prepared vesicles, which in exemplary embodiments are composed primarily of lipid bilayers. In various examples, liposomes are used as delivery vehicles for the administration of nutrients and pharmaceuticals. In various embodiments, the liposomes of the present disclosure are of different sizes, and the composition may include one or more of: (a) multilamellar vesicles (MLVs), which may be several hundred nanometers in diameter and comprise a series of concentric bilayers separated by narrow aqueous compartments; (b) small unicellular vesicles (SUVs), which may be less than 50 nm in diameter; and (c) large unicellular vesicles (LUVs), which may be 50-500 nm in diameter. In various examples, liposomes are designed to contain opsonins or ligands to improve liposome attachment to unhealthy tissue or to activate events such as, but not limited to, endocytosis. In exemplary embodiments, liposomes contain a low or high pH to improve delivery of the formulation. In various examples, liposomes are formulated according to, but not limited to, the encapsulated formulation and liposome components, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the encapsulated substance and its potential toxicity, the use of the vesicles and / or additional processes involved during delivery, optimizing the size, polydispersity, and shelf life of the vesicles for the intended use, and the batch-to-batch reproducibility and feasibility of large-scale production of safe and efficient liposome products.

[0017] In exemplary embodiments, nanoparticles comprise a surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, optionally three or more nucleic acid layers. In exemplary examples, each nucleic acid layer is disposed between lipid layers, e.g., cationic lipid layers. In exemplary aspects, nanoparticles are multilayered, comprising alternating layers of nucleic acid and lipid. In exemplary embodiments, nanoparticles of the present disclosure comprise an interior comprising alternating nucleic acid layers and cationic lipid bilayers. In exemplary embodiments, nanoparticles comprise at least three nucleic acid layers, each of which is disposed between cationic lipid bilayers. In exemplary aspects, nanoparticles comprise at least four or five nucleic acid layers, each of which is disposed between cationic lipid bilayers. In exemplary aspects, nanoparticles comprise at least five or more (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleic acid layers, each of which is disposed between cationic lipid bilayers. As used herein, the term "cationic lipid bilayer" refers to a lipid bilayer that comprises, essentially consists of, or consists of cationic lipids or mixtures thereof.Suitable cationic lipids are described herein.As used herein, the term "nucleic acid layer" refers to a layer of the nanoparticles disclosed herein that comprises, essentially consists of, or consists of nucleic acid, such as RNA.

[0018] The unique structure of the nanoparticles of the present disclosure provides a mechanistic difference in how multilayered nanoparticles exert their biological effects. Previously described RNA-based nanoparticles exert their effects, at least in part, through the Toll-like receptor 7 (TLR7) pathway. Surprisingly, the multilayered nanoparticles of the present disclosure mediate efficacy independent of TLR7. See, e.g., Figures 18 and 19A-19B. Without wishing to be bound by any particular theory, intracellular pathogen recognition receptors (PRRs), such as MDA-5, appear to be more relevant to the biological activity of multilayered nanoparticles than TLRs. See, e.g., Figure 17. This suggests that ML RNA-NPs stimulate multiple intracellular PRRs (i.e., RIG-I, MDA-5) as opposed to a single TLR (i.e., TLR7 in endosomes), resulting in a greater expression of type I interferons. This may result in the release of mRNA and the induction of stronger innate immunity (Figure 11). This allows mRNA from unrelated species to induce antitumor activity (Figures 3B, 3C, and 7B), and tumor-specific RNA-NPs to show superior efficacy and long-term survivorship benefits (Figure 14).

[0019] In various embodiments, the nanoparticles disclosed herein comprise a positively charged surface. In some examples, the positively charged surface comprises a lipid layer, e.g., a cationic lipid layer. In various embodiments, the outermost layer of the nanoparticle comprises a cationic lipid bilayer. Optionally, the cationic lipid bilayer comprises DOTAP. In various examples, the surface comprises a plurality of hydrophilic portions of the cationic lipids of the cationic lipid bilayer. In some embodiments, the core comprises a cationic lipid bilayer. In various embodiments, the outermost region of the core comprises a cationic lipid bilayer. In some examples, the outermost region of the core comprises a cationic lipid bilayer comprising DOTAP. In various examples, the core lacks nucleic acid. Optionally, the core comprises less than about 0.5% by weight of nucleic acid. In exemplary embodiments, the core comprises (i) a therapeutic agent or (ii) a diagnostic agent (e.g., an imaging agent), or (iii) a combination thereof. Suitable therapeutic and diagnostic agents are described herein. In exemplary embodiments, the therapeutic agent comprises or is a nucleic acid. Optionally, the therapeutic agent is an antisense oligonucleotide (ASO) or siRNA. In various examples, the ASO or siRNA is not the same nucleic acid as that present in the alternating nucleic acid layers (cationic lipid bilayers). In illustrative examples, the ASO or siRNA is the same nucleic acid as that present in the alternating nucleic acid layers (cationic lipid bilayers). In various embodiments, the core comprises iron oxide nanoparticles (IONPs), which are useful for imaging tissues or cells, for example, via magnetic resonance imaging (MRI). Optionally, the IONPs are coated with a fatty acid, for example, a C8-C30 fatty acid. In various embodiments, the fatty acid is oleic acid. In various embodiments, the core comprises a plurality of IONPs (optionally coated with oleic acid), where the plurality is held together by a lipid, for example, a cationic lipid. Optionally, the plurality of IONPs (optionally coated with oleic acid) are held together by DOTAP. Further description of cores comprising therapeutic and diagnostic agents is provided below.

[0020] In exemplary embodiments, the nanoparticles have diameters in the nanometer range, and therefore, in certain instances, are referred to herein as "nanoliposomes" or "liposomes." In exemplary embodiments, the nanoparticles have diameters of about 50 nm to about 500 nm, e.g., about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 50 nm to about 150 nm, about 50 nm to about 100 nm, about 100 nm to about 500 nm, about 150 nm to about 500 nm, about 200 nm to about 500 nm, about 250 nm to about 500 nm, about 300 nm to about 500 nm, about 350 nm to about 500 nm, or about 400 nm to about 500 nm. In exemplary embodiments, the liposomes have a diameter of about 50 nm to about 300 nm, e.g., about 100 nm to about 250 nm, about 110 nm ± 5 nm, about 115 nm ± 5 nm, about 120 nm ± 5 nm, about 125 nm ± 5 nm, about 130 nm ± 5 nm, about 135 nm ± 5 nm, about 140 nm ± 5 nm, about 145 nm ± 5 nm, about 150 nm ± 5 nm, about 155 nm ± 5 nm, about 160 nm ± 5 nm, about 165 nm ± The nanoparticles have a diameter of about 5 nm, about 170 nm ± 5 nm, about 175 nm ± 5 nm, about 180 nm ± 5 nm, about 190 nm ± 5 nm, about 200 nm ± 5 nm, about 210 nm ± 5 nm, about 220 nm ± 5 nm, about 230 nm ± 5 nm, about 240 nm ± 5 nm, about 250 nm ± 5 nm, about 260 nm ± 5 nm, about 270 nm ± 5 nm, about 280 nm ± 5 nm, about 290 nm ± 5 nm, or about 300 nm ± 5 nm. In exemplary embodiments, the nanoparticles have a diameter of about 50 nm to about 250 nm. In some embodiments, the nanoparticles have a diameter of about 70 nm to about 200 nm.

[0021] In exemplary embodiments, the nanoparticles have a diameter, for example, from about 50 nm to about 500 nm or The pharmaceutical composition comprises a heterogeneous mixture of nanoparticles ranging from about 50 nm to about 250 nm in diameter. Optionally, the pharmaceutical composition comprises a heterogeneous mixture of nanoparticles ranging from about 70 nm to about 200 nm in diameter.

[0022] In illustrative examples, the nanoparticles are characterized by a zeta potential of about +40 mV to about +60 mV, e.g., about +40 mV to about +55 mV, about +40 mV to about +50 mV, about +40 mV to about +50 mV, about +50 mV, about +40 mV to about +45 mV, about +45 mV to about +60 mV, about +50 mV to about +60 mV, or about +55 mV to about +60 mV. In exemplary embodiments, the nanoparticles have a zeta potential of about +45 mV to about +55 mV. In various cases, the zeta potential of the nanoparticles is about +50 mV. In various embodiments, the zeta potential is greater than +30 mV or +35 mV. Zeta potential can be used to characterize the nanoparticles of the present disclosure and Sayour This is one parameter that distinguishes them from the nanoparticles described in [Illegible Text] et al., Oncoimmunology 6(1):e1256527 (2016).

[0023] In exemplary embodiments, the nanoparticles comprise cationic lipids. In some embodiments, the cationic lipids are low-molecular-weight cationic lipids, such as those described in U.S. Patent Application No. 2013 / 0090372 (the contents of which are incorporated herein by reference in their entirety). In exemplary embodiments, the cationic lipids are cationic fatty acids, cationic glycerophospholipids, cationic glycerophospholipids, cationic sphingolipids, cationic sterol lipids, cationic prenol lipids, cationic glycolipids, or cationic polyketides. In exemplary embodiments, the cationic lipids comprise two fatty acyl chains, each of which is independently saturated or unsaturated. In some examples, the cationic lipids are diglycerides. For example, in some examples, the cationic lipids can be cationic lipids of Formula I or Formula II.

[0024] [ka]

[0025] [ka]

[0026] wherein each of a, b, n, and m is independently an integer between 2 and 12 (e.g., between 3 and 10). In some embodiments, the cationic lipid is a cationic lipid of Formula I, wherein each of a, b, n, and m is independently an integer selected from 3, 4, 5, 6, 7, 8, 9, and 10. In an illustrative example, the cationic lipid is a DO In an exemplary embodiment, the cationic lipid is TAP (1,2-dioleoyl-3-trimethylammonium-propane), or a derivative thereof. In an exemplary embodiment, the cationic lipid is DOTMA (1,2-di-O-octadecenyl-3-trimethylammonium propane), or a derivative thereof.

[0027] In some embodiments, the nanoparticles comprise liposomes formed from 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US 2010 / 0324120, incorporated herein by reference in its entirety). In some embodiments, the nanoparticles comprise liposomes formed from the synthesis of stabilized plasmid lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have previously been described and shown to be suitable for in vitro and in vivo oligonucleotide delivery. In some embodiments, the nanoparticles are composed of three to four lipid components in addition to the nucleic acid molecule. In an exemplary embodiment, the liposomes comprise 55% cholesterol, 20% distearoylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al. In an exemplary example, the liposomes comprise 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic lipid can be 1,2-distearoyl-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA) as described by Heyes et al.

[0028] In some embodiments, the liposomes contain about 25.0% to about 40.0% cholesterol, about 30.0% to about 45.0% cholesterol, about 35.0% to about 50.0% cholesterol, and / or about 48.5% to about 60% cholesterol. In some embodiments, the liposomes may contain a percentage of cholesterol selected from the group consisting of 28.5%, 31.5%, 33.5%, 36.5%, 37.0%, 38.5%, 39.0%, and 43.5%. In some embodiments, the liposomes may contain about 5.0% to about 10.0% DSPC and / or about 7.0% to about 15.0% DSPC.

[0029] In some embodiments, the liposomes are DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine)-based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713), incorporated herein by reference in its entirety), and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

[0030] In various examples, the cationic lipid includes 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), or di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), further including a neutral lipid, a sterol, and a molecule capable of reducing particle aggregation, such as PEG or a PEG-modified lipid.

[0031] In various embodiments, the liposomes include DLin-DMA, DLin-K-DMA, 98N Liposomes may include, but are not limited to, cationic lipids such as DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and amino alcohol lipids. In some embodiments, the liposomes include cationic lipids such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and amino alcohol lipids. In some embodiments, the amino alcohol cationic lipids include those described and / or prepared by the methods described in U.S. Patent Application Publication No. 2013 / 0150625 (incorporated herein by reference in its entirety). As a non-limiting example, the cationic lipid in certain embodiments is 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)- octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 1 of US2013 / 0150625), 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (compound 2 of US2013 / 0150625), 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]- 2-[(octyloxy)methyl]propan-1-ol (compound 3 of US2013 / 0150625), and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (compound 4 of US2013 / 0150625), or a pharmaceutically acceptable salt or stereoisomer thereof.

[0032] In various embodiments, the liposomes comprise (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE, and SM; (iii) a sterol, such as cholesterol; and (iv) a PEG-lipid, such as PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid:5-25% neutral lipid:25-55% sterol. 0.5-15% PEG-lipid.

[0033] In some embodiments, the liposomes comprise about 25% to about 75% by molar amount of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl)9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., about 35% to about 65%, about 45% to about 65%, about 60%, about 57.5%, about 50%, or about 40% by molar amount.

[0034] In some embodiments, the liposomes contain about 0.5% to about 15% neutral lipid on a molar basis, e.g., about 3% to about 12%, about 5% to about 10%, or about 15%, about 10%, or about 7.5% on a molar basis. Examples of neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE, and SM. In some embodiments, the formulation contains about 5% to about 50% sterol on a molar basis (e.g., about 15% to about 45%, about 20% to about 40%, about 40%, about 38.5%, about 35%, or about 31%). An exemplary sterol is cholesterol. In some embodiments, the formulation comprises about 0.5% to about 20% PEG or PEG-modified lipid on a molar basis (e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis). In some embodiments, the PEG or PEG-modified lipid is and PEG molecules having an average molecular weight of 2,000 Da. In other embodiments, the PEG or PEG-modified lipid comprises PEG molecules having an average molecular weight of less than 2,000, for example, about 1,500 Da, about 1,000 Da, or about 500 Da. Examples of PEG-modified lipids include, but are not limited to, PEG-distearoylglycerol (PEG-DMG) (also referred to herein as PEG-C14 or C14-PEG), PEG-cDMA (Reyes et al. J. Controlled Release, 107, 276-287 (2005) (the contents of which are incorporated herein by reference in their entirety).

[0035] In exemplary embodiments, the cationic lipid is (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z)-N,N-dimethylpentacosa-16,19-dien-8-amine, (13Z,16Z)-N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z)-N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-diamine (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z)-N,N-dimethylheptacosa-19,22-dien-9-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-dien-8 -amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z)-N,N-dimethylhentriaconta-22,25-dien-10-amine, (21Z,24Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z)-N,N-dimethylheptacosa-18-en-10-amine, (17Z)-N,N-dimethylhexacosa-17-en-9-amine, (19Z,22Z) -N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosa-10-amine, (20Z,23Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z)-N,N-dimethylheptacosa-20-en-10-amine, (15Z)-N,N-dimethyleptacosa-15-en-10-amine, (14Z)-N,N-dimethylnonacosa-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltritriacont-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylpentriacont-22-en-10-amine, (16Z)-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl- 1-[(1S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21-[(1S,2R)-2-octylcyclopropyl]henicosan-10-amine, N,N-dimethyl-1-[(1S,2S)-2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1-[(1S,2R) -2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1, -[(1S,2R)-2-Decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, SN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)- Octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine; (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine; N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine; N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-1- [(6Z,9Z,12Z)-Octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docosa- 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R)-N,N-dimethyl-H(1-methyloxy) (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S)-2-{[(1R,2R)-2- The compound may be selected from N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, N,N-dimethyl-1-{[8-(2-octylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine, and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

[0036] In some embodiments, the nanoparticles comprise lipid-polycation complexes. Formation of lipid-polycation complexes can be achieved by methods known in the art and / or by methods described in U.S. Patent Application Publication No. 2012 / 0178702, which is incorporated herein by reference in its entirety. By way of non-limiting example, polycations include, but are not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the composition may comprise a lipid-polycation complex, which may further comprise a non-cationic lipid, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0037] In some embodiments, the nucleic acid molecules are present at a nucleic acid molecule:cationic lipid ratio of about 1:5 to about 1:20, optionally about 1:15, about 1:10, or about 1:7.5. As used herein, the term "nucleic acid molecule:cationic lipid ratio" refers to a mass ratio, where the mass of the nucleic acid molecules is relative to the mass of the cationic lipid. Also, in exemplary embodiments, the term "nucleic acid molecule:cationic lipid ratio" refers to the mass ratio of nucleic acid molecules, e.g., RNA, added to liposomes containing cationic lipids during the process of making the ML RNA NPs of the present disclosure. In exemplary embodiments, the nanoparticles contain less than or about 10 μg of RNA molecules per 150 μg of lipid mixture. In exemplary embodiments, the nanoparticles are made by incubating about 10 μg of RNA with about 150 μg of liposomes. In other embodiments, the nanoparticles contain more RNA molecules per mass of lipid mixture. For example, the nanoparticles may contain more than 10 μg of RNA molecules per 150 μg of liposomes. In some instances, the nanoparticles contain more than 15 μg of RNA molecules per 150 μg of liposomes or lipid mixture.

[0038] In various embodiments, the nucleic acid molecule is an RNA molecule, such as transfer RNA (tRNA), ribosomal RNA (rRNA), or messenger RNA (mRNA). In various embodiments, the RNA molecule comprises tRNA, rRNA, mRNA, or a combination thereof. In various embodiments, the RNA is the total RNA isolated from cells. In exemplary embodiments, the RNA is the total RNA isolated from diseased cells, such as tumor cells or cancer cells. Methods for obtaining total tumor RNA are known in the art and are described in Example 1 herein.

[0039] In an exemplary embodiment, the RNA molecule is mRNA. In various embodiments, the mRNA is in vitro transcribed mRNA. In various examples, the mRNA molecule is generated by in vitro transcription (IVT). Suitable techniques for performing IVT are known in the art. In an exemplary embodiment, an IVT kit is used. In an exemplary embodiment, the kit includes one or more IVT reaction reagents. As used herein, the term "in vitro transcription (IVT) reaction reagent" refers to any molecule, compound, factor, or salt that functions in an IVT reaction. For example, the kit may include a prokaryotic phage RNA polymerase and a promoter (T7, T3, or SP6) with a eukaryotic or prokaryotic extract for synthesizing a protein from an exogenous DNA template. In an exemplary embodiment, the RNA is in vitro transcribed mRNA, and the in vitro transcription template is cDNA made from RNA extracted from tumor cells. In various embodiments, the nanoparticles comprise a mixture of RNA, which is RNA isolated from human tumors, optionally malignant brain tumors, optionally glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma, or peripheral tumors with metastatic infiltration into the central nervous system. In various embodiments, the RNA comprises a sequence encoding a poly(A) tail, such that the in vitro transcribed RNA molecules comprise a poly(A) tail at the 3' end. In various embodiments, the method for producing nanoparticles comprises an additional processing step, such as capping the in vitro transcribed RNA molecules.

[0040] The mRNA in exemplary embodiments encodes a protein. Optionally, the protein is selected from the group consisting of a tumor antigen, a cytokine, or a costimulatory molecule. In some embodiments, the RNA molecule encodes a protein. The protein, in some embodiments, is selected from the group consisting of a tumor antigen, a costimulatory molecule, a cytokine, a growth factor, a lymphokine, (e.g., cytokines and growth factors effective to inhibit tumor metastasis, anti-proliferative agents for at least one cell population, The cytokine, lymphokine, growth factor, or other hematopoietic factor is selected from the group consisting of M-CSF, GM-CSF, TNF, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IFN, TNFα, TNF1, TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin.Additional growth factors for use herein include angiogenin, bone morphogenetic protein-1, bone morphogenetic protein-2, bone morphogenetic protein-3, bone morphogenetic protein-4, bone morphogenetic protein-5, bone morphogenetic protein-6, bone morphogenetic protein-7, bone morphogenetic protein-8, bone morphogenetic protein-9, bone morphogenetic protein-10, bone morphogenetic protein-11, bone morphogenetic protein-12, bone morphogenetic protein-13, bone morphogenetic protein-14, bone morphogenetic protein-15, bone morphogenetic protein-16, bone morphogenetic protein-17, bone morphogenetic protein-18, bone morphogenetic protein-19, bone morphogenetic protein-20, bone morphogenetic protein-21, bone morphogenetic protein-22, bone morphogenetic protein-23, bone morphogenetic protein-24, bone morphogenetic protein-25, bone morphogenetic protein-26, bone morphogenetic protein-27, bone morphogenetic protein-28, bone morphogenetic protein-29, bone morphogenetic protein-30, bone morphogenetic protein-31, bone morphogenetic protein-32, bone morphogenetic protein-33, bone morphogenetic protein-34, bone morphogenetic protein-35, bone morphogenetic protein-36, bone morphogenetic protein-37, bone morphogenetic protein-38, bone morphogenetic protein-39, bone morphogenetic protein-40, bone morphogenetic protein-41, bone morphogenetic protein-42, bone morphogenetic protein-43, bone morphogenetic protein-44, bone morphogenetic protein-45, bone morphogenetic protein-46, bone morphogenetic protein-47, bone morphogenetic protein-48, bone morphogenetic protein-49, bone morphogenetic protein-50, bone morphogenetic protein-5 Protein receptor IA, bone morphogenetic protein receptor IB, brain-derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor α, cytokine-induced neutrophil chemoattractant 1, cytokine-induced neutrophil, chemoattractant 2α, cytokine-induced neutrophil chemoattractant 2β, beta-endothelial growth factor, endothelin 1, epithelial-derived neutrophil attractant, glial cell line-derived neurotrophic factor receptor α1, glial cell line-derived neurotrophic factor receptor α2, growth-associated protein, growth-associated protein α, growth-associated protein β, growth-associated protein γ, heparin-binding epithelial growth factor growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor alpha, nerve growth factor, nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor alpha, transforming growth factor beta, transforming growth factor beta 1, transforming growth factor beta 1.2, transforming growth factor beta 2, transforming growth factor beta 3, transforming growth factor beta 5, latent transforming growth factor beta 1, transforming growth factor beta binding protein I, transforming growth factor beta binding protein II, transforming growth factor beta binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor II, urokinase-type plasminogen activator receptor, and chimeric proteins and biologically or immunologically active fragments thereof.In exemplary embodiments, the tumor antigen is an antigen derived from a viral protein, an antigen derived from a point mutation, or an antigen encoded by a cancer-germline gene. In exemplary embodiments, the tumor antigen is pp65, p53, KRAS, NRAS, MAGEA, MAGEB, MAGEC, BAGE, GAGE, LAGE / NY-ESO1, SSX, tyrosinase, gp100 / pmel17, Melan-A / MART-1, gp75 / TRP1, TRP2, CEA, RAGE-1, HER2 / NEU, or WT1. In exemplary embodiments, the costimulatory molecule is selected from the group consisting of CD80 and CD86. In some embodiments, the protein is not expressed by tumor cells or humans. In exemplary examples, the protein is not related to tumor or cancer antigens. In some embodiments, the protein is non-specific for tumors or cancer. For example, the non-specific protein can be green fluorescent protein (GFP) or ovalbumin (OVA).

[0041] In various cases, RNA molecule is antisense molecule, optionally siRNA, shRNA, miRNA, or any combination thereof.Antisense molecule can be mediating RNA interference (RNAi).As known to those skilled in the art, RNAi is a ubiquitous mechanism of gene regulation in plants and animals, in which target mRNA is degraded in a sequence-specific manner (Sharp, Genes Dev., 15, 485-490 (2001); Hutvagner et al., Curr. Opin. Genet. Dev. 12, 225-232 (2002); Fire et al., Nature, 391, 806-811 (1998), Zamore et al., Cell, 101, 25-33 (2000)). The natural RNA degradation process is initiated by the dsRNA-specific endonuclease Dicer, which promotes the cleavage of long dsRNA precursors into 21-25 nucleotide double-stranded fragments called small interfering RNAs (also known as siRNAs) (Zamore et al., Cell, 101, 25-33 (2000); Elbashir et al., Genes Dev., 15, 188-200 (2001); Hammond et al., Nature, 404, 293-296 (2000); Bernstein et al., Nature, 409, 363-366 (2001)). siRNAs are incorporated into large protein complexes that recognize and cleave target mRNAs (Nykanen et al., Cell, 107, 309-321 (2001)). It has been reported that introduction of dsRNA into mammalian cells does not lead to efficient Dicer-mediated siRNA generation and therefore does not induce RNAi (Caplen et al., Gene 252, 95-105 (2000), Ui-Tei et al., FEBS Lett, 479, 79-82 (2000)). The requirement for Dicer for cellular siRNA maturation can be circumvented by introducing synthetic 21-nucleotide siRNA duplexes that inhibit the expression of transfected and endogenous genes in various mammalian cells (Elbashir et al., Cell, 107, 309-321 (2001)). et al., Nature, 411:494-498(2001)).

[0042] In this regard, the RNA molecule in some embodiments is an siRNA molecule that mediates RNAi, and in some embodiments, is specific for inhibiting protein expression. As used herein, the term "siRNA" refers to an RNA (or RNA analog) comprising about 10 to about 50 nucleotides (or nucleotide analogs) that can induce or mediate RNAi. In exemplary embodiments, the siRNA molecule comprises about 15 to about 30 nucleotides (or nucleotide analogs) or about 20 to about 25 nucleotides (or nucleotide analogs), e.g., 21 to 23 nucleotides (or nucleotide analogs). The siRNA can be double-stranded or single-stranded, preferably double-stranded.

[0043] In an alternative embodiment, the RNA molecule is a short hairpin RNA (shRNA) molecule specifically designed to inhibit protein expression. As used herein, the term "shRNA" refers to a single-stranded RNA molecule of about 20 or more base pairs, containing a partially palindromic base sequence and forming a double-stranded structure (i.e., a hairpin structure). The shRNA may be an siRNA (or siRNA analog) folded into a hairpin structure. The shRNA typically contains about 45 to about 60 nucleotides, including about 21-nucleotide antisense and sense portions of the hairpin, an optional overhang on the non-loop side of about 2 to about 6 nucleotides in length, and a loop portion that may be, for example, about 3 to 10 nucleotides in length. The shRNA may be chemically synthesized. Alternatively, the shRNA may be produced by ligating the sense and antisense strands of a DNA sequence in reverse orientation and synthesizing the RNA in vitro using T7 RNA polymerase with the DNA as a template.

[0044] Without wishing to be bound by any theory or mechanism, it is believed that after shRNA is introduced into cells, it is degraded to approximately 20 or more bases (e.g., typically 21, 22, or 23 bases), triggering RNAi and resulting in an inhibitory effect. Therefore, shRNA can be used as an effective component of the present disclosure to induce RNAi. The shRNA may preferably have a 3'-overhanging end. The length of the double-stranded portion is not particularly limited, but is preferably 10 or more nucleotides, more preferably 20 or more nucleotides. Here, the 3'-overhanging end is preferably DNA, more preferably DNA at least 2 nucleotides in length, and even more preferably DNA 2 to 4 nucleotides in length.

[0045] In an exemplary embodiment, the antisense molecule is a microRNA (miRNA). As used herein, the term "microRNA" refers to a small (e.g., 15-22 nucleotide) non-coding RNA molecule that base pairs with mRNA molecules to silence gene expression through translational repression or targeted degradation. MicroRNAs and their therapeutic potential have been reported in the art. See, for example, Mulligan, MicroRNA: Expression, Detection, and Therapeutic Strategies, Nova Science Publishers, Inc., Hauppauge, NY, 2011; Bader and Lammers, "The Therapeutic Potential of microRNAs," Innovations in Pharmaceutical Technology, pages 52-55 (March 2011).

[0046] In particular examples, the RNA molecule is an antisense molecule, optionally an siRNA, shRNA, or miRNA, that targets and reduces the expression of a protein in an immune checkpoint pathway. In various embodiments, the protein in the immune checkpoint pathway is CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, TIGIT, LAG3, CD112, TIM3, BTLA, or costimulatory receptors: ICOS, OX40, 41BB, or GITR. In particular examples, the protein in the immune checkpoint pathway is CTLA4, PD-1, PD-L1, B7-H3, B7H4, or TIM3. Immune checkpoint signaling pathways are reviewed in Pardoll, Nature Rev Cancer 12(4):252-264 (2012).

[0047] In exemplary embodiments, the NPs of the present disclosure comprise a mixture of RNA molecules. In exemplary aspects, the mixture of RNA molecules is RNA isolated from cells from a human, optionally the human having a tumor. In some aspects, the mixture of RNA is RNA isolated from a human tumor. In exemplary aspects, the human has cancer, optionally any of the cancers described herein. Optionally, the tumor from which the RNA is isolated is selected from the group consisting of glioma (including but not limited to glioblastoma), medulloblastoma, diffuse intrinsic pontine glioma, or peripheral tumors with metastatic infiltration into the central nervous system (e.g., melanoma or breast cancer). In exemplary aspects, the tumor from which the RNA is isolated is a cancer tumor, for example, any of these cancers described herein.

[0048] In various embodiments, the nucleic acid molecule (e.g., RNA molecule) further comprises a nucleotide sequence encoding a chimeric protein comprising a LAMP protein. In certain embodiments, the LAMP protein is a LAMP1, LAMP2, LAMP3, LAMP4, or LAMP5 protein.

[0049] core In exemplary embodiments, nanoparticles of the present disclosure function as delivery vehicles for therapeutic agents or diagnostic agents, or a combination thereof. In various aspects, nanoparticles of the present disclosure function as delivery vehicles for therapeutic agents that function as both therapeutic and diagnostic agents. In exemplary embodiments, nanoparticles of the present disclosure comprise a core comprising a therapeutic agent or diagnostic agent, or a combination thereof. In exemplary examples, the therapeutic agent is a chemotherapeutic agent or an immunotherapeutic agent. Optionally, the immunotherapeutic agent is a PD-L1 or PD-1 inhibitor. In various aspects, the PD-L1 or PD-1 inhibitor is an antisense oligonucleotide or siRNA. In various aspects, the diagnostic agent is an imaging agent, such as any one of those described herein. Optionally, the imaging agent comprises iron oxide nanoparticles.

[0050] chemotherapy drugs Chemotherapeutic agents suitable for inclusion in the multilayered RNA NPs disclosed herein are well known in the art. These include, but are not limited to, platinum coordination compounds, topoisomerase inhibitors, antibiotics, antimitotic alkaloids, and difluoronucleosides, as are known and described in U.S. Pat. No. 6,630,124, incorporated herein by reference.

[0051] In some embodiments, the chemotherapeutic agent is a platinum coordination compound. The term "platinum coordination compound" refers to any tumor cell growth inhibitory compound that provides platinum in ionic form. In some embodiments, the platinum coordination compound is selected from the group consisting of cis-diamminediaquaplatinum(II)-ion, chloro(diethylenetriamine)-chloroplatinum(II), dichloro(ethylenediamine)-platinum(II), diammine(1,1-cyclobutanedicarboxylato)platinum(II) (carboplatin), spiroplatin, iproplatin, diammine(2-ethylmalonato)-platinum(II), ethylenediaminemalonatoplatinum(II), aqua(1,2-di (1,2-diaminocyclohexane)-sulfatoplatinum(II), (1,2-diaminocyclohexane)malonatoplatinum(II), (4-carboxyphthalato)(1,2-diaminocyclohexane)platinum(II), (1,2-diaminocyclohexane)-(isocitrate)platinum(II), (1,2-diaminocyclohexane)cis(pyruvato)platinum(II), (1,2-diaminocyclohexane)oxalatoplatinum(II), ormaplatin, or tetraplatin.

[0052] In some embodiments, cisplatin is the platinum coordination compound used in the compositions and methods of the present disclosure. Cisplatin is commercially available from Bristol Myers-Squibb Corporation under the name PLATINOL™ and is available as a powder for constitution with water, sterile saline, or other suitable vehicle. Other platinum coordination compounds suitable for use in the context of the present disclosure are known, commercially available, and / or can be prepared by known techniques. Cisplatin, i.e., cis-dichlorodiammineplatinum II, has been used successfully for many years as a chemotherapeutic agent in the treatment of various human solid malignancies. More recently, other diaminoplatinum complexes have also demonstrated efficacy as chemotherapeutic agents in the treatment of various human solid malignancies. Such diaminoplatinum complexes include, but are not limited to, spiroplatinum and carboplatin. Cisplatin and other diaminoplatinum complexes have been widely used as chemotherapeutic agents in humans, but have required delivery at high dose levels that can cause toxicity issues, such as kidney damage.

[0053] In some embodiments, the chemotherapeutic agent is a topoisomerase inhibitor. Topoisomerases are enzymes that can alter DNA topology in eukaryotic cells. Topoisomerases are important for cell function and proliferation. Generally, eukaryotic cells contain two classes of topoisomerases: type I and type II. Topoisomerase I is a monomeric enzyme with a molecular weight of approximately 100,000. This enzyme binds to DNA, introduces a transient single-strand break, unwinds (or allows unwinding) the double helix, and then reseals the break before dissociating from the DNA strand. Various topoisomerase inhibitors have shown clinical efficacy in treating humans with ovarian cancer, breast cancer, esophageal cancer, or non-small cell lung cancer.

[0054] In some embodiments, the topoisomerase inhibitor is camptothecin or a camptothecin analogue. Camptothecin is a water-insoluble cytotoxic alkaloid produced by the Camptotheca accuminata tree, which is endemic to China, and the Nothapodytes foetida tree, which is endemic to India. Camptothecin suppresses the growth of many tumor cells. Compounds of the camptothecin analogue class are typically specific inhibitors of DNA topoisomerase I. Compounds of the camptothecin analogue class include, but are not limited to, topotecan, irinotecan and 9-amino-camptothecin.

[0055] In additional embodiments, the chemotherapeutic agent is any tumor cell growth inhibiting camptothecin analogue claimed or described in: U.S. Pat. No. 5,004,758 and European Patent Application No. 88311366.4, published as EP 0 321 122; U.S. Pat. No. 4,604,463 and European Patent Application Publication No. EP 0 137 145; U.S. Pat. No. 4,473,692 and European Patent Application Publication No. EP 0 074 256; U.S. Pat. No. 4,545,880 and European Patent Application Publication No. EP 0 074 256, European Patent Application Publication No. EP 0 088 642; Wani et al., J. Med. Chem., 29, 2358-2363 (1986); Nitta et al., Proc. 14th International Congress of Chemotherapy, Kyoto, 1985, Tokyo Press, Anticancer Section. 1, pp. 28-30, specifically a compound called CPT-11. CPT-11 is a camptothecin analogue in which a 4-(piperidino)-piperidine side chain is attached to C-10 of 10-hydroxy-7-ethylcamptothecin via a carbamate bond. CPT-11 is currently undergoing human clinical trials and is also known as irinotecan. Wani et al. al, J. Med. Chem., 23, 554 (1980); Wani et al., J. Med. Chem., 30, 1774 (1987), U.S. Patent No. 4,342,776, U.S. Patent Application No. 581,916 filed September 13, 1990, and European Patent Application Publication No. EP 418 099, U.S. Patent Application No. 4,513,138, and European Patent Application Publication No. EP 0 074 770, U.S. Patent No. 4,399,276, and European Patent Application Publication No. 0 056 692 (the entire disclosures of each are incorporated herein by reference). All of the above-described compounds in the camptothecin analog class are commercially available and / or can be prepared by known techniques, including those described in the above-mentioned references. The topoisomerase inhibitor may be selected from the group consisting of topotecan, irinotecan, and 9-aminocamptothecin.

[0056] The preparation of numerous compounds of the camptothecin analog class, including pharmaceutically acceptable salts, hydrates, and solvates thereof, and the preparation of oral and parenteral pharmaceutical compositions comprising such compounds of the camptothecin analog class and an inert, pharmaceutically acceptable carrier or diluent, has been broadly described in U.S. Pat. No. 5,004,758, published as European Patent Application Publication No. EP 0 321 122, and European Patent Application No. 88311366.4, the teachings of which are incorporated herein by reference.

[0057] In yet other embodiments, the chemotherapeutic agent is an antibiotic compound. Suitable antibiotics include, but are not limited to, doxorubicin, mitomycin, bleomycin, daunorubicin, and streptozocin.

[0058] In some embodiments, the chemotherapeutic agent is a mitotic inhibitor alkaloid. In general, antimitotic alkaloids can be extracted from Cantharanthus roseus and have been shown to be effective as anticancer chemotherapeutic agents. Numerous semisynthetic derivatives have been studied both chemically and pharmacologically (see O. Van Tellingen et al., Anticancer Research, 12, 1699-1716 (1992)). Mitotic inhibitor alkaloids of the present invention include, but are not limited to, vinblastine, vincristine, vindesine, paclitaxel (PTX; Taxol®), and vinorelbine. The latter two mitotic inhibitor alkaloids are commercially available from Eli Lilly and Company and Pierre Fabre Laboratories, respectively (see U.S. Pat. No. 5,620,985). In an exemplary embodiment of the present invention, the mitotic inhibitor alkaloid is vinorelbine.

[0059] In another embodiment of the invention, the chemotherapeutic agent is a difluoronucleoside. 2'-deoxy-2',2'-difluoronucleosides are known in the art to have antiviral activity. Such compounds are disclosed and taught in U.S. Patent Nos. 4,526,988 and 4,808,614. European Patent Application Publication No. 184,365 discloses that these same difluoronucleosides have oncolytic activity. In certain embodiments, the 2'-deoxy-2',2'-difluoronucleoside used in the compositions and methods of the present invention is 2'-deoxy-2',2'-difluorocytidine hydrochloride, also known as gemcitabine hydrochloride. Gemcitabine is commercially available or can be synthesized by a multi-step process as disclosed and taught in U.S. Patent Nos. 4,526,988, 4,808,614 and 5,223,608, the teachings of which are incorporated herein by reference.

[0060] In an exemplary embodiment, the chemotherapeutic agent is a hormone therapy agent. In an exemplary example, the hormone therapy agent is, for example, letrozole, tamoxifen, bazedoxifene, exemestane, leuprolide, goserelin, fulvestrant, anastrozole, or toremifene. In an exemplary embodiment, the hormone therapy agent is a luteinizing hormone (LH) blocker, for example, gosarelin, or an LH-releasing hormone (RH) agonist. In exemplary embodiments, the hormone therapy agent is an ER-targeted agent (e.g., fulvestrant or tamoxifen), rapamycin, a rapamycin analog (e.g., everolimus, temsirolimus, ridaforolimus, zotarolimus, and 32-deoxorapamycin), an anti-HER2 agent (e.g., trastuzumab, pertuzumab, lapatinib, T-DM1, or neratinib), or a PI3K inhibitor (e.g., taselisib, alpelisib, or buparlisib).

[0061] immunotherapy agents As used herein, the term "immunotherapeutic agent" refers to any therapeutic agent that enhances the body's natural defenses to fight disease, e.g., cancer. In various embodiments, the immunotherapeutic agent is a cell or molecule, e.g., a nucleic acid molecule, a protein, or a peptide. Optionally, the cell is an engineered cell that is made to express a nucleic acid molecule, a protein, or a peptide. The immunotherapeutic agent can be, for example, a monoclonal antibody, an oncolytic virus therapy, a T-cell therapy, or a cancer vaccine. The monoclonal antibody can be, for example, ipilimumab, nivolumab, pembrolizumab, atexolizumab, avelumab, or durvalumab. In various examples, the immunotherapeutic agent is a CAR T-cell therapy, e.g., tisagenlecleucel, axicabutagene, or ciloleucel. In various embodiments, the immunotherapeutic agent is a tumor-agnostic agent, e.g., raclotrectinib. In various embodiments, the immunotherapeutic agent is a cytokine, optionally an interferon or an interleukin. In various aspects, the cytokine is IFN-α (IFN-α (IFN-α[2a], Intron A[2b], Alferon[2a]) or IL-2 (Aldesleukin)).

[0062] In exemplary embodiments, the therapeutic agent comprises or is a nucleic acid. Optionally, the therapeutic agent is an antisense oligonucleotide (ASO) or siRNA. In various examples, the ASO or siRNA is not the same nucleic acid present in the alternating nucleic acid layer (cationic lipid bilayer). In exemplary examples, the ASO or siRNA is the same nucleic acid present in the alternating nucleic acid layer (cationic lipid bilayer). In exemplary examples, the ASO or siRNA targets a protein that functions in an immune checkpoint pathway. In exemplary examples, the ASO or siRNA reduces the expression of a protein that functions in an immune checkpoint pathway. In various embodiments, the protein that functions in an immune checkpoint pathway is any of PD-1, PD-L1, CTLA-4, CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, and B7-H3. one of H4, CEACAM-1, TIGIT, LAG3, CD112, CD112R, CD96, TIM3, BTLA, ICOS, OX40, 41BB, CD27, or GITR.

[0063] contrast agents We designed multifunctional RNA-loaded magnetic liposomes that initiate potent antitumor immunity and serve as early MRI-based imaging biomarkers of therapeutic response. These liposomes were shown to activate dendritic cells (DCs) more effectively than electroporation, resulting in superior inhibition of tumor growth in a therapeutic model. The inclusion of iron oxide enhanced DC transfection and enabled tracking of DC migration by MRI. T2 lymph node tumors were also observed. * Weighted MRI hypointensity has been shown to be strongly correlated with DC trafficking, and T2 *It has been suggested that weighted MRI hypointensity may be an early predictor of anti-tumor response. In preclinical tumor models, MRI-predicted "responders" identified 2 days after vaccination had significantly smaller tumors 2 to 5 weeks after treatment and lived 100% longer than MRI-predicted "non-responders." Therefore, these studies provide a simple and scalable nanoparticle formulation for generating potent anti-tumor immune responses and predicting individual treatment outcomes by MRI. Without being bound by any particular theory, the disclosed multilayered RNA NPs containing iron oxide nanoparticles may be used to activate DCs, inhibit tumor growth, enhance DC transfection, and enable tracking of DC migration by MRI. Accordingly, the present disclosure further provides nanoparticles comprising a positively charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, each nucleic acid layer disposed between cationic lipid bilayers, and the core comprising a diagnostic agent, such as an imaging agent (e.g., contrast agent), optionally gadolinium, perfluorocarbon microbubbles, iron oxide nanoparticles, gold colloids, or gold nanoparticles (see, e.g., Mahan and Doiron, J. Nanomaterials, volume 2018, article ID 5837276, provides nanoparticles. In various embodiments, the core comprises a radiopharmaceutical (e.g., carbon-11, fluorine-18, gallium-67 or -68, indium-111, iodine-123, -125, -131, krypton-81m, lutetium-177, nitrogen-13, oxygen-15, phosphorus-32, selenium-75, technetium-99m, thallium-201, xenon-133, yttrium-90). In various embodiments, the core comprises iron oxide nanoparticles (IONPs), useful for imaging tissues or cells, e.g., via magnetic resonance imaging (MRI). In various aspects, the IONPs are Combidex®, Resovist®, Endorem®, or Sinerem®. Optionally, the IONPs are coated with a fatty acid, for example, a C8-C30 fatty acid. In various embodiments, the fatty acid is stearic acid, palmitic acid, myristic acid, lauric acid, capric acid, caprylic acid, palmitoleic acid, cis-citric acid, or oleic acid. In various embodiments, the core comprises a plurality of IONPs (optionally, each IONP is coated with oleic acid), where the plurality is held together by a lipid, for example, a cationic lipid. Optionally, the plurality of IONPs (optionally, coated with oleic acid) are held together by DOTAP. Methods for making such IONPs held together by DOTAP coating are described herein.

[0064] Production method The present disclosure also provides a method for making nanoparticles comprising a positively charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is disposed between cationic lipid bilayers, the method comprising: (A) mixing nucleic acid molecules and liposomes at an RNA:liposome ratio of about 1:5 to about 1:25, about 1:5 to about 1:20, optionally about 1:15, to obtain RNA-coated liposomes, wherein the liposomes are made by a process for making liposomes comprising drying a lipid mixture comprising cationic lipids and an organic solvent by evaporating the organic solvent under vacuum; and (B) mixing the RNA-coated liposomes with an excess amount of liposomes.

[0065] In exemplary embodiments, nanoparticles produced by the methods disclosed herein conform to the description of the nanoparticles disclosed herein. For example, nanoparticles produced by the methods disclosed herein have a zeta potential of about +40 mV to about +60 mV, optionally about +45 mV to about +55 mV. Optionally, the zeta potential of nanoparticles produced by the methods disclosed herein is about +50 mV. In various embodiments, the core of a nanoparticle produced by the methods disclosed herein comprises less than about 0.5% by weight of nucleic acid, and / or the core comprises a cationic lipid bilayer, and / or the outermost layer of the nanoparticle comprises a cationic lipid bilayer, and / or the surface of the nanoparticle comprises a plurality of hydrophilic moieties of the cationic lipids of the cationic lipid bilayer.

[0066] In exemplary embodiments, the lipid mixture comprises cationic lipid and organic solvent in a ratio of about 40 mg cationic lipid per mL of organic solvent to about 60 mg cationic lipid per mL of organic solvent, optionally about 50 mg cationic lipid per mL of organic solvent. In various examples, the process for producing liposomes further comprises rehydrating the lipid mixture with a rehydration solution to form a rehydrated lipid mixture, then stirring, resting, and sizing the rehydrated lipid mixture. Optionally, sizing the rehydrated lipid mixture comprises sonicating, extruding, and / or filtering the rehydrated lipid mixture.

[0067] An exemplary method for making nanoparticles comprising a positively charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, each nucleic acid layer disposed between cationic lipid bilayers, is described herein in Example 1. Any one or more of the steps described in Example 1 can be included in the methods disclosed herein. For example, in some embodiments, the method includes one or more steps required to prepare the RNA prior to complexing with liposomes. In exemplary aspects, the method includes downstream steps for preparing the nanoparticles for administration to a subject, e.g., a human. In an exemplary example, the method includes formulating the NPs for intravenous injection. In various aspects, the method includes adding one or more pharmaceutically acceptable carriers, diluents, or excipients, and optionally packaging the resulting composition in a container, e.g., a vial, syringe, bag, ampoule, or the like. The container in some aspects is a ready-to-use container, optionally disposable.

[0068] Further provided herein are nanoparticles made by the methods of making nanoparticles disclosed herein.

[0069] Cells and their populations Further provided herein are cells comprising (e.g., transfected with) nanoparticles of the present disclosure. In exemplary embodiments, the cells are any type of cells capable of containing nanoparticles of the present disclosure. In some embodiments, the cells are eukaryotic cells, such as plants, animals, fungi, or algae. In alternative embodiments, the cells are prokaryotic cells, such as bacteria or protozoa. In exemplary embodiments, the cells are cultured cells. In alternative embodiments, the cells are primary cells, i.e., primary cells isolated directly from an organism (e.g., a human). The cells may be adherent cells or suspension cells, i.e., cells that grow in suspension. In exemplary embodiments, the cells are mammalian cells. Most preferably, the cells are human cells. The cells may be of any cell type, derived from any type of tissue, and at any developmental stage. In exemplary embodiments, the cells comprising liposomes are antigen-presenting cells (APCs). As used herein, "antigen-presenting cell" or "APC" refers to an immune cell that mediates a cellular immune response by processing and presenting antigens for recognition by specific T cells. In exemplary embodiments, the APC is a dendritic cell, macrophage, Langerhans cell, or B cell. In exemplary embodiments, the APC is a dendritic cell (DC). In exemplary embodiments, when the cells are administered to a subject, e.g., a human, the cells are delivered to the subject. In an illustrative example, the immune cells are tumor-associated macrophages (TAMs).

[0070] The present disclosure also provides a population of cells, wherein at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the population are cells comprising (e.g., transfected with) nanoparticles of the present disclosure. In some embodiments, the population of cells is a heterogeneous cell population, or in some embodiments, a substantially homogeneous population, wherein the population primarily comprises cells comprising nanoparticles of the present disclosure.

[0071] Pharmaceutical Composition Provided herein are compositions comprising nanoparticles of the present disclosure, cells comprising nanoparticles of the present disclosure, populations of cells of the present disclosure, or any combination thereof, and a pharmaceutically acceptable carrier, excipient, or diluent. In exemplary embodiments, the composition is a pharmaceutical composition intended for administration to a human, comprising a plurality of nanoparticles according to the present disclosure and a pharmaceutically acceptable carrier, diluent, or excipient. In exemplary embodiments, the composition is a sterile composition. In exemplary examples, the composition comprises a plurality of nanoparticles of the present disclosure. Optionally, at least 50% of the nanoparticles of the plurality have a diameter of about 100 nm to about 250 nm. In various embodiments, the composition comprises a concentration of about 10 nanoparticles per mL. 10 Nanoparticles ~ approx. 10 per mL 15 Nanoparticles, optionally about 10 per mL 12 Contains nanoparticles ±10%.

[0072] In exemplary embodiments, the compositions of the present disclosure may include additional components other than nanoparticles, cells comprising nanoparticles, or populations of cells. In various embodiments, the compositions may include, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacing agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, preservatives, bases, binders, buffers, chelating agents, coating agents, colorants, desiccants, detergents, diluents, disinfectants, disintegrants, dispersants, solubility enhancers, dyes, softeners, emulsifiers, emulsion stabilizers, fillers, film-forming agents, flavor enhancers, flavorings, glidants, etc. The composition may contain any pharmaceutically acceptable ingredients, including, for example, gelling agents, granulating agents, moisturizing agents, lubricants, mucoadhesives, ointment bases, ointments, oily vehicles, organic bases, pastille bases, pigments, plasticizers, abrasives, preservatives, sequestering agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, surface active agents, suspending agents, sweeteners, therapeutic agents, thickening agents, tonicity agents, toxicological agents, thickening agents, water-absorbing agents, water-miscible cosolvents, water softeners, or humectants. See, for example, the Handbook of Pharmaceutical Excipients, Third Edition, A.H. Kibbe (Pharmaceutical Press, London, UK, 2000) (incorporated by reference in its entirety), and Remington's Pharmaceutical Sciences, Sixteenth Edition, E.W. Martin (Mack Publishing Co., Easton, Pa., 1980) (incorporated by reference in its entirety).

[0073] The compositions of the present disclosure can be suitable for administration by any acceptable route, including parenteral and subcutaneous.Other routes include, for example, intravenous, intradermal, intramuscular, intraperitoneal, intranodal and intrasplenic.In an exemplary embodiment, when the composition comprises liposomes (not cells containing liposomes), the composition is suitable for systemic (e.g., intravenous) administration.

[0074] When the composition is in a form intended for administration to a subject, it can be made isotonic with the intended administration site.For example, when a solution is in a form intended for parenteral administration, it can be isotonic with blood.The composition is typically sterile.In certain embodiments, this can be achieved by filtration through a sterile filtration membrane.In certain embodiments, parenteral compositions are generally placed in a container with a sterile access port, for example, an intravenous solution bag, or a vial with a stopper that can be pierced by a hypodermic injection needle, or a pre-filled syringe. In certain embodiments, the compositions may be stored either in a prepared form or in a form (e.g., lyophilized) that is reconstituted or diluted prior to administration.

[0075] use Without being bound by any particular theory, data disclosed herein for the first time support the use of the RNA NPs disclosed herein to increase immune responses, including inducing an immune response against a tumor in a subject. Accordingly, the present disclosure provides a method for increasing an immune response against a tumor in a subject. In an exemplary embodiment, the method comprises administering a pharmaceutical composition of the present disclosure to a subject. In an exemplary aspect, the nucleic acid molecule is mRNA. Optionally, the composition is administered systemically to the subject. For example, the composition is administered intravenously. In various aspects, the pharmaceutical composition is administered in an amount effective to activate dendritic cells (DCs) in the subject. In various examples, the immune response is a T cell-mediated immune response. Optionally, the T cell-mediated immune response includes activity by tumor-infiltrating lymphocytes (TILs). In an exemplary aspect, the immune response is an innate immune response.

[0076] Additionally, data provided herein for the first time support the use of the presently disclosed RNA NPs to increase dendritic cell (DC) activation in a subject. Accordingly, a method of activating DCs or increasing DC activation in a subject is further provided. In an exemplary embodiment, the method comprises administering a pharmaceutical composition of the present disclosure to a subject. In an exemplary aspect, the nucleic acid molecule is mRNA. Optionally, the composition is administered systemically to the subject. For example, the composition is administered intravenously. In various aspects, the pharmaceutical composition is administered in an amount effective to increase an immune response against a tumor in the subject. In various examples, the immune response is a T cell-mediated immune response. Optionally, the T cell-mediated immune response includes activity by tumor-infiltrating lymphocytes (TILs). In an exemplary aspect, the immune response is an innate immune response.

[0077] As used herein, the term "increase" and derivatives thereof may not be a 100% or complete increase. Rather, there are various degrees of increase that those skilled in the art will recognize as having potential benefits or therapeutic effects. In exemplary embodiments, the increase provided by this method is at least or about a 10% increase (e.g., at least or about a 20% increase, at least or about a 30% increase, at least or about a 40% increase, at least or about a 50% increase, at least or about a 60% increase, at least or about a 70% increase, at least or about a 80% increase, at least or about a 90% increase, at least or about a 95% increase, at least or about a 98% increase).

[0078] The present disclosure also provides a method for delivering RNA molecules to a tumor microenvironment, lymph nodes, and / or reticuloendothelial organs. In an exemplary embodiment, the method comprises administering a pharmaceutical composition disclosed herein to a subject. Optionally, the reticuloendothelial organ is the spleen or liver. Provided herein is a method for delivering RNA to cells in a tumor, e.g., a brain tumor, comprising intravenously administering a composition of the present disclosure, wherein the composition comprises nanoparticles. Also provided herein is a method for delivering RNA to cells in the microenvironment of a tumor, optionally a brain tumor. In an exemplary embodiment, the method comprises systemically (e.g., intravenously) administering a composition of the present disclosure, wherein the composition comprises nanoparticles. In some aspects, the nanoparticles comprise siRNA targeting a protein in an immune checkpoint pathway, optionally PD-L1. In various aspects, the cells in the microenvironment are antigen-presenting cells (APCs), optionally tumor-associated macrophages. The present disclosure also provides a method for activating antigen-presenting cells in a brain tumor microenvironment. In an exemplary embodiment, the method comprises systemically (eg, intravenously) administering a composition disclosed herein, wherein the composition comprises a NP.

[0079] The present disclosure provides methods for delivering RNA molecules to cells. In exemplary embodiments, the methods The method includes incubating cells with the NPs of the present disclosure. In exemplary examples, the cells are antigen-presenting cells (APCs), optionally dendritic cells (DCs). In various examples, the APCs (e.g., DCs) are obtained from a subject. In certain embodiments, the RNA molecules are isolated from tumor cells obtained from a subject, e.g., a human. In certain embodiments, the RNA molecules are antisense molecules that target and reduce the expression of a protein of interest. In exemplary embodiments, the RNA molecules are siRNA molecules that target a protein in an immune checkpoint pathway. Suitable proteins in an immune checkpoint pathway are known in the art and described herein. In various examples, the siRNA targets PD-L1.

[0080] Once the RNA is delivered to the cells, whether in vitro or ex vivo, the cells can be administered to a subject for disease treatment. Accordingly, the present disclosure provides a method for treating a subject with a disease. In an exemplary embodiment, the method comprises delivering an RNA molecule to a subject's cells according to the above-described method for delivering an RNA molecule to a cell. In some aspects, the RNA molecule is delivered to the cell ex vivo, and the cell is administered to the subject. Alternatively, the method comprises directly administering liposomes to the subject. In an exemplary embodiment, a method for treating a subject with a disease comprises administering a composition of the present disclosure in an amount effective to treat the disease in the subject. In an exemplary aspect, the disease is cancer, and in some aspects, the cancer is located across the blood-brain barrier and / or the subject has a tumor located in the brain. In some aspects, the tumor is a glioma, a low-grade glioma, or a high-grade glioma, specifically a grade III astrocytoma or glioblastoma. Alternatively, the tumor can be a medulloblastoma or a diffuse intrinsic pontine glioma. In another example, the tumor may be a metastatic infiltration from a non-CNS tumor, e.g., breast cancer, melanoma, or lung cancer. In an exemplary embodiment, the composition comprises liposomes, and optionally, the composition comprising liposomes is administered intravenously to the subject. In an alternative embodiment, the composition comprises cells transfected with liposomes. Optionally, the cells of the composition are APCs, and optionally, DCs. In an exemplary embodiment, the composition comprising cells comprising liposomes is administered intradermally to the subject, and optionally, the composition is administered intradermally to the subject's groin. In an exemplary example, DCs are isolated from white blood cells (WBCs) obtained from the subject, and optionally, the WBCs are obtained via leukapheresis. In some embodiments, the RNA molecule encodes a tumor antigen. In some embodiments, the RNA molecule is isolated from tumor cells, e.g., the tumor cells are cells of a tumor in the subject. Accordingly, further provided herein are methods of treating a subject with a disease. In exemplary embodiments, the method includes delivering an RNA molecule to cells of a subject by a method disclosed herein that delivers the RNA molecule to the tumor microenvironment, lymph nodes, and / or reticuloendothelial organs. In various aspects, the RNA molecule is delivered to the cells ex vivo and the cells are administered to the subject.In exemplary embodiments, the method includes administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the disease in the subject. In various examples, the subject has a cancer or tumor, optionally a malignant brain tumor, optionally a glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma, or a peripheral tumor with metastatic infiltration into the central nervous system.

[0081] As used herein, the term "treat," as well as related terms, does not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment that those skilled in the art recognize as having potential benefit or therapeutic effect. In this regard, the disclosed methods of treating cancer can provide any amount or level of treatment. Furthermore, the treatment provided by the methods can include treatment of one or more conditions or symptoms or signs of the disease being treated. For example, the disclosed methods of treatment can suppress one or more symptoms of the disease. The treatment provided by the disclosed methods can also encompass delaying the progression of the disease. The term "treat" also encompasses prophylactic treatment of a disease. Thus, the treatment provided by the disclosed methods can delay the onset or recurrence of the disease being prophylactically treated. In exemplary embodiments, the methods delay the onset of the disease by 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, 2 months, 4 months, 6 months, 1 year, 2 years, 4 years, or more. Prophylactic treatment includes reducing the risk of the disease being treated. In exemplary embodiments, the method reduces the risk of disease by 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more.

[0082] In certain embodiments, a method of treating a disease can be considered a method of inhibiting a disease or its symptoms. As used herein, the term "inhibit" and derivatives thereof may not be 100% or complete inhibition. Rather, there are various degrees of inhibition that those skilled in the art recognize as having potential benefits or therapeutic effects. The methods disclosed herein can inhibit the onset or recurrence of a disease or its symptoms to any amount or level. In exemplary embodiments, the inhibition provided by the methods of the present disclosure is at least or about 10% inhibition (e.g., at least or about 20% inhibition, at least or about 30% inhibition, at least or about 40% inhibition, at least or about 50% inhibition, at least or about 60% inhibition, at least or about 70% inhibition, at least or about 80% inhibition, at least or about 90% inhibition, at least or about 95% inhibition, at least or about 98% inhibition).

[0083] In the above-mentioned method, in some embodiments, the NP or a composition comprising the same is administered systemically to the subject. Optionally, the method includes administering the liposome or composition by parenteral administration. In various examples, the liposome or composition is administered intravenously to the subject.

[0084] In various embodiments, the NP or composition is administered according to any regimen, including, for example, daily (once a day, twice a day, three times a day, four times a day, five times a day, six times a day), three times a week, twice a week, every two days, every three days, every four days, every five days, every six days, weekly, every other week, every three weeks, monthly, or every other month. In various embodiments, the liposome or composition is administered to a subject once a week.

[0085] subject The subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Lagomorpha, such as rabbits; mammals of the order Carnivora, including Felines (cats) and Canines (dogs); mammals of the order Artiodactyla, including Bovidae (cows) and Suidae (pigs); or mammals of the order Perissodactyla, including Equidae (horses). In some embodiments, the mammal belongs to the order Primates, Ceboids, or Simoids (monkeys), or to the suborder Anthropoides (humans and apes). In representative embodiments, the mammal is a human. In some embodiments, the human is an adult aged 18 or older. In some embodiments, the human is a child aged 17 or younger. In exemplary embodiments, the subject has DMG. In various examples, the DMG is diffuse intrinsic pontine glioma (DIPG).

[0086] cancer Cancer treatable by the methods disclosed herein can be any cancer, for example, any malignant growth or tumor caused by abnormal and uncontrolled cell division that can spread to other parts of the body through the lymphatic system or bloodstream.

[0087] In some embodiments, the cancer is acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer, breast cancer, anal cancer, anal canal cancer, or anorectal cancer, eye cancer, intrahepatic bile duct cancer, cancer of the joints, cancer of the neck, gallbladder cancer, or cancer of the pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic bone marrow cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal carcinoid tumors, Hodgkin's lymphoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer, malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin's lymphoma, ovarian cancer, pancreatic cancer, peritoneal, omental, and mesenteric cancer, pharyngeal cancer, prostate cancer, rectal cancer, kidney cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and bladder cancer. In certain embodiments, the cancer is selected from the group consisting of head and neck cancer, ovarian cancer, cervical cancer, bladder and esophageal cancer, pancreatic cancer, gastrointestinal cancer, stomach cancer, breast cancer, endometrial and colorectal cancer, hepatocellular carcinoma, glioblastoma, bladder cancer, lung cancer, e.g., non-small cell lung cancer (NSCLC), or bronchioloalveolar carcinoma.

[0088] The following examples are provided merely to illustrate the present invention and not to limit its scope in any way. [Example]

[0089] Example 1 This example illustrates a method for making nanoparticles of the present disclosure.

[0090] Preparation of DOTAP liposomes On day 1, the following procedure was performed in a fume hood. Water was added to the rotavapor bath. Chloroform (20 mL) was poured into a sterile glass graduated cylinder. After opening a vial containing 1 g of DOTAP, 5 mL of chloroform was added to the DOTAP vial using a glass pipette. Next, an amount of chloroform and DOTAP was transferred to a 1 L evaporating flask. The DOTAP vial was washed by adding a second 5 mL volume of chloroform to the DOTAP vial to dissolve any remaining DOTAP in the vial and transferring this amount of chloroform from the DOTAP vial to the evaporating flask. This washing step was repeated two more times until all of the chloroform in the graduated cylinder was used. Next, the evaporating flask was placed in a Buchi rotavapor. The water bath was turned on and adjusted to 25 °C. The evaporating flask was moved down until it touched the water bath. The rotavapor rotation speed was adjusted to 2. The vacuum system was turned on and adjusted to 40 mbar. After 10 minutes, the vacuum system was turned off and the chloroform was collected from the collector flask. The amount of chloroform collected was measured. Once the collector flask was repositioned, the vacuum was turned back on and the contents of the evaporation flask were allowed to dry overnight until the chloroform had completely evaporated.

[0091] On day 2, 200 mL of PBS was added to a new, sterile 500 mL PBS bottle maintained at room temperature using a sterile graduated cylinder. A second 500 mL PBS bottle was prepared to collect DOTAP. A Buchi rotavapor water bath was set to 50 °C. 50 mL of PBS was added to the evaporation flask using a 25 mL disposable serological pipette. The evaporation flask was placed in a Buchi rotavapor and moved downward until one-third of the flask was submerged in the water bath. The rotavapor was set to a rotation speed of 2 and rotated for 10 minutes, after which the rotation was stopped. The 50 mL of PBS containing DOTAP from the evaporation flask was transferred to a second 500 mL PBS bottle. This process was repeated (three times) until the entire volume of PBS in the PBS bottle was used. The final volume of the second 500 mL PBS bottle was 400 mL. The lipid solution in the second 500 mL PBS bottle was vortexed for 30 seconds and then incubated at 50 °C for 1 hour. The bottle was vortexed every 10 minutes during the 1 hour incubation. The second 500 mL PBS bottle was left at room temperature overnight.

[0092] On the third day, 200 mL of PBS was added to the second 500 mL PBS bottle containing DOTAP and PBS. The second 500 mL PBS bottle was placed in an ultrasonic bath. The ultrasonic bath was filled with water, and the second 500 mL PBS bottle was sonicated for 5 minutes. The extruder was washed with 100 mL of PBS, and this washing process was repeated. A 0.45 μm pore filter was attached to the filtration unit, and a new (third) 500 mL PBS bottle was placed in the output tubing of the extruder. In a biological safety cabinet, the DOTAP-PBS mixture was loaded into the extruder until approximately 70% of the third PBS bottle was filled. The extruder was then turned on, and the DOTAP-PBS mixture was added until all of the mixture had passed through the extruder. The mixture was added. A 0.22 μm pore filter was then attached to the filtration unit, and a new (third) 500 mL PBS bottle was placed in the output tube of the extruder. The previously filtered DOTAP-PBS mixture was loaded and run again throughout. The sample containing DOTAP lipid nanoparticles (NPs) in PBS was then stored at 4 °C.

[0093] RNA preparation Prior to incorporation into NPs, RNA was prepared by one of several methods. Total tumor RNA was prepared by isolating total RNA (including rRNA, tRNA, and mRNA) from tumor cells. In vitro transcribed mRNA was prepared by performing an in vitro transcription reaction using a cDNA template generated by reverse transcription of total tumor RNA. Tumor antigen-specific and non-specific RNA were either produced in-house or purchased from commercial vendors.

[0094] Total tumor RNA: Total tumor-derived RNA from tumor cells (eg, B16F0, B16F10, and KR158-luc) was isolated using a commercially available RNeasy mini kit (Qiagen) according to the manufacturer's instructions.

[0095] In vitro transcribed mRNA: Briefly, RNA was isolated using a commercially available RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions, and cDNA libraries were generated by RT-PCR. A PCR-based reverse transcriptase reaction was performed on total tumor RNA to generate a cDNA library using the SMARTScribe Reverse Transcriptase Kit (Takara). The resulting cDNA was then amplified using the Takara Advantage 2 Polymerase mix containing T7 / SMART and CDS III primers, and the total number of amplification cycles was determined by gel electrophoresis. Purification of the cDNA was performed using a Qiagen PCR purification kit according to the manufacturer's instructions. To isolate sufficient mRNA for use in each RNA nanoparticle vaccine, overnight in vitro transcription was performed on the cDNA library using the mMESAGE mMACHINE (Invitrogen) kit with T7 enzyme mix. Housekeeping genes were evaluated to ensure transcription fidelity. The resulting mRNA was then purified using the Qiagen RNeasy Maxi Kit to obtain the final mRNA product.

[0096] Tumor antigen-specific and non-specific mRNA: Plasmids containing DNA encoding tumor antigen-specific RNA (e.g., RNA encoding pp65 or OVA) and nonspecific RNA (e.g., RNA encoding Green Fluorescent Protein (GFP) or luciferase) were linearized with a restriction enzyme (i.e., SpeI) and purified with a Qiagen PCR MiniElute kit. The linearized DNA was then transcribed using an mmRNA in vitro transcription kit (Life Technologies, Invitrogen) and cleaned up using an RNA Maxi kit (Qiagen). Alternatively, nonspecific RNA was purchased from Trilink Biotechnologies (San Diego, CA).

[0097] Preparation of multilayered RNA nanoparticles (NPs) DOTAP lipid NPs were complexed with RNA to create multilayered RNA-NPs designed to have several layers of mRNA contained within tightly coiled liposomes with positively charged surfaces and empty cores (Figure 1A). Briefly, in a safety cabinet, the RNA was thawed from -80 °C and then placed on ice, while the PBS and DOTAP-containing samples (e.g., DOTAP lipid NPs) were allowed to warm to room temperature. Once the components were prepared, they were mixed in a sterile tube. The desired amount of RNA was mixed with PBS. An appropriate amount of DOTAP lipid NPs was added to the sterile tube containing the RNA and PBS mixture without physical mixing (e.g., without inverting the tube, vortexing, or stirring). The mixture of RNA, PBS, and DOTAP was incubated for approximately 15 minutes to allow multilayered RNA-NPs to form. After 15 minutes, the mixture was gently mixed by repeatedly inverting the tube. The mixture was then considered ready for systemic (i.e., intravenous) administration.

[0098] The amounts of RNA and DOTAP lipid NPs (liposomes) used in the above preparation are determined or selected in advance. In some examples, a ratio of about 15 μg of liposomes per about 1 μg of RNA is used. For example, about 75 μg of liposomes per about 5 μg of RNA is used, or about 375 μg of liposomes per about 25 μg of RNA is used. In other examples, about 7.5 μg of liposomes per 1 μg of RNA is used. Thus, in illustrative examples, about 1 μg to about 20 μg of liposomes are used for every μg of RNA used.

[0099] Example 2 This example illustrates the characterization of nanoparticles of the present disclosure.

[0100] Cryogenic Electron Microscopy (CEM) The structures of multilayered RNA-NPs prepared as described in Example 1 and control NPs (uncomplexed NPs) without RNA, which were produced according to all steps of Example 1 except for the "RNA Preparation" and "Preparation of Multilayered RNA Nanoparticles (NPs)" steps, were analyzed using CEM. CEM was performed essentially as described in Sayour et al., Nano Lett 17(3)1326-1335 (2016). Briefly, samples containing multilayered RNA-NPs or control NPs were kept on ice before being loaded and flash-frozen into a Vitrobot (and automated plunge freezer for cryo-TEM, which freezes samples without ice crystal formation by controlling temperature, relative humidity, blotting conditions, and freezing rate). The samples were then flash-frozen using a Tecnai G2 F20 microscope equipped with a Gatan UltraScan 4000 (4k × 4k) CCD camera. Imaged with a TWIN 200 kV / FEG transmission electron microscope. The resulting CEM image is shown in Figure 1B. The right panel is a CEM image of the multilayered RNA-NPs, and the left panel is a CEM image of the control NPs (uncomplexed NPs). As shown in Figure 1B, the control NPs contained a maximum of two layers, while the multilayered RNA NPs contained multiple layers. Figure 5 shows another CEM image of an exemplary multilayered RNA NP. Here, the multiple layers of RNA alternating with lipid layers are particularly evident.

[0101] Zeta potential The zeta potential of multilayered RNA NPs was determined by Sayour et al., Nano Lett 17(3)1326-1335 (2016) using a Brookhaven ZetaPlus instrument (Brookhaven Instruments). Phase analysis light scattering (PALS) was measured using a fluoroscopy system (Microfluidics Laboratory, Holtsville, NY). Briefly, uncomplexed NPs or RNA-NPs (200 μL) were resuspended in PBS (1.2 mL) and loaded into the instrument. Samples were run five times per sample, with 25 cycles per run, using the Smoluchowski model.

[0102] The zeta potential of the multilayered RNA NPs prepared as described in Example 1 was measured at approximately +50 mV. Interestingly, this zeta potential of the multilayered RNA NPs was much higher than that measured around +27 mV as described by Sayour et al., Oncoimmunology 6(1):e1256527 (2016). Without being bound by theory, the method of preparing DOTAP lipid NPs for use in making multilayered RNA NPs (Example 1), which includes a vacuum-sealing method to evaporate chloroform, may result in less environmental oxidation of the DOTAP lipid NPs, which may allow for greater amounts of RNA to form complexes with the DOTAP NPs and / or greater incorporation of RNA into the DOTAP lipid NPs.

[0103] RNA capture by gel electrophoresis: Gel electrophoresis experiments were performed to measure the amount of RNA incorporated into ML liposomes. Based on this experiment, it was qualitatively demonstrated that nearly all, if not all, of the RNA used in the procedure described in Example 1 was incorporated into DOTAP lipid NPs. Additional experiments to characterize the extent of RNA incorporation will be performed by measuring RNA-NP density and comparing this parameter to that of lipoplexes.

[0104] Example 3 This example demonstrates the in vivo localization site of RNA-NPs upon systemic administration and that RNA NPs mediate peripheral and intratumoral activation of DCs.

[0105] DOTAP lipid NPs, prepared essentially as described in Example 1, were complexed with mRNA encoding Cre recombinase to generate Cre-encoding RNA-NPs. These multilayered RNA-NPs were administered to Ai14 transgenic mice carrying a loxP-flanked STOP cassette. The STOP cassette prevents tdTomato transcription until Cre recombinase expression. One week after RNA-NP administration, the lymph nodes, spleen, and liver of the transgenic mice were harvested, sectioned, and stained with DAPI. tdTomato expression was analyzed by fluorescence microscopy, following procedures essentially as described in Sayour et al., Nano Letters 2018. Cre-mRNA-NPs are expected to localize in vivo to lymphoid organs such as the liver, spleen, and lymph nodes.

[0106] DOTAP lipid NPs, prepared essentially as described in Example 1, were complexed with nonspecific RNA (e.g., ovalbumin (OVA) mRNA, an RNA not specific for a tumor antigen) and intravenously injected into C57Bl / 6 mice (n = 3-4 per group) bearing subcutaneous B16F10 tumors. Lymph nodes, spleen, liver, bone marrow, and tumors were harvested within 24 hours and analyzed for the expression of the dendritic cell (DC) activation marker CD86 by CD11c cells (*p < 0.05 Mann-Whitney test). OVA mRNA-NPs showed widespread in vivo localization to lymph nodes, spleen, liver, bone marrow, and tumors, and were expected to activate DCs therein (as indicated by increased expression of the activation marker CD86 on CD11c+ cells). Because activated DCs prime antigen-specific T cell responses and lead to antitumor efficacy (with increased TILs) in several tumor models, we tested the antitumor efficacy of multilayered RNA NPs.

[0107] Example 4 This example describes a comparison of nanoparticles of the present disclosure with cationic and anionic RNA lipoplexes.

[0108] Cationic lipoplexes (LPXs) were first developed using mRNA in a lipid core shielded by a net positive charge on the outer surface (Figure 2A). Anionic RNA lipoplexes (Figure 2B) were developed using excess RNA tethered to the surface of bilayer liposomes. RNA-LPXs were created by mixing RNA and lipid NPs in a charge-equivalent ratio. Anionic RNA-NPs were created by mixing RNA and lipid NPs in a ratio that supersaturated the negatively charged lipid NPs. Next, RNA-LPXs were synthesized. Various aspects of X and anionic RNA LPX can be used in combination with the multi-layered RNAs described in the examples above. Compared with NP.

[0109] Cryo-electron microscopy (CEM) was used to compare the structures of RNA LPX and multilayered RNA-NPs prepared as described in Example 1. Uncomplexed NPs were used as a control. CEM was performed essentially as described in Example 2. Figure 2C shows a CEM image of uncomplexed NPs, Figure 2D shows a CEM image of RNA LPX (liposome to RNA mass ratio of 3.75:1), and Figure 2E shows a CEM image of multilayered RNA-NPs (liposome to RNA mass ratio of 15:1). These data support the retention of more RNA by ML RNA-NPs. Additional data show that ML RNA-NP complexation results in a greater decrease in concentration relative to RNA-LPX, supporting the formation of multilayered RNA NPs that cannot be observed by simply mixing equal amounts of RNA and lipid NPs by mass or charge (i.e., RNA-LPX and anionic RNA-LPX, respectively). This supports the retention of more RNA by ML RNA-NPs.

[0110] We also performed experiments to determine where anionic LPX localizes upon administration to mice. As shown in Figure 8, anionic LPX localizes to the spleen of the animals upon administration, consistent with previous studies (Krantz et al., Nature 534:396-401 (2016)).

[0111] Mice were administered RNA LPX, anionic lipoplexes (LPX), or multilayered RNA-NPs, and spleens were harvested one week later for evaluation of activated DCs (*p<0.05 unpaired t-test). The RNA used in this experiment was tumor-derived mRNA from the K7M2 osteosarcoma cell line. As shown in Figure 2F, mice treated with multilayered RNA-NPs exhibited the highest levels of activated DCs.

[0112] Anionic tumor mRNA-lipoplexes, tumor mRNA-lipoplexes, and multilayered tumor mRNA-loaded NPs were compared in a therapeutic lung cancer model (K7M2) (n = 5–8 / group). Each vaccine was administered intravenously weekly (x3) (**p < 0.01, Mann-Whitney). Figure 2G shows the %CD44+CD62L+ of CD8+ splenocytes, and Figure 2H shows the %CD44+CD62L+ of CD4+ splenocytes. Figure 2J also shows that multilayered (ML) RNA-NPs mediate substantially increased IFN-α, an innate antiviral cytokine. This indicates that ML RNA-NPs enable substantially greater innate immunity, sufficient to promote efficacy even from non-antigen-specific ML RNA-NPs. Collectively, these figures demonstrate the superior efficacy of multilayered tumor-specific RNA-NPs compared to anionic LPX and RNA LPX.

[0113] Anionic tumor mRNA-lipoplexes, cationic tumor mRNA-lipoplexes, and multilayered tumor mRNA-loaded NPs were compared in a therapeutic lung cancer model (K7M2) (n = 8 / group). Each vaccine was administered intravenously weekly (x3). *p < 0.05, Gehan Breslow-Wilcoxon test. Survival rates were measured by Kaplan-Meier curve analysis. As shown in Figure 2I, multilayered tumor-specific RNA-NPs mediated superior efficacy in enhancing survival compared to cationic and anionic RNA lipoplexes.

[0114] Here, we show that multilayered RNA-NP formulations targeting physiologically relevant tumor antigens are more immunogenic (Figures 2F-2H, 2J) and significantly more efficacious (Figure 2I) compared to anionic LPX and RNA LPX. Without being bound by any particular theory, we hypothesize that by altering the RNA-lipid ratio and increasing the zeta potential, tightly wound RNA-NPs can be effectively mobilized. A novel RNA-NP design, consisting of multilayered rings of RNA (Figure 1C), was developed. This multilayered design is believed to enhance particle immunogenicity and promote increased NP uptake of mRNA (enriched by alternating positive and negative charges) to extend in vivo localization to the periphery and tumor microenvironment (TME). Systemic administration of these multilayered RNA-NPs localizes to lymph nodes, reticuloendothelial organs (i.e., spleen and liver), and the TME, where they activate DCs (based on increased expression of the activation marker CD86 on CD11c+ cells). These activated DCs prime antigen-specific T cell responses, resulting in antitumor effects (accompanied by increased TILs) in several tumor models.

[0115] Example 5 This example demonstrates the ability of multilayered RNA-NPs to systemically activate DCs, induce antigen-specific immunity, and elicit antitumor effects.

[0116] The efficacy of multilayered RNA NPs was tested in a second model. Here, BALB / c mice (8 mice per group) inoculated with K7M2 lung tumors were vaccinated with multilayered RNA-NPs three times a week. A control group of mice was untreated. Lungs were harvested one week after the third vaccination for analysis of intratumoral memory T cells ( *** p<0.001, Mann-Whitney test). Figure 3A provides a pair of photographs of lungs treated with RNA-NPs (left) and untreated lungs (right). Figure 3B is a graph of % central memory T cells (CD62L+CD44+ of CD3+ cells) in harvested lungs from untreated mice, mice treated with multi-layered RNA NPs with GFP RNA, and mice treated with multi-layered RNA NPs with tumor-specific RNA.

[0117] BALB / c mice or BALB / c SCID (Fox Chase) mice (8 mice per group) were inoculated with K7M2 lung tumors and intravenously vaccinated with multilayered RNA-NPs containing GFP RNA or tumor-specific RNA three times a week. The control group of mice was untreated. The % survival rate was plotted on a Kaplan-Meier curve ( *** p<0.0001, Gehen-Breslow-Wilcox). As shown in Figure 3C, the survival rate of BALB / c mice treated with multilayered RNA NPs containing tumor-specific RNA was the highest among the three groups. Interestingly, the survival rate of BALB / c SCID (Fox Chase) mice treated with multilayered RNA NPs containing GFP RNA was almost the same as that of mice treated with multilayered RNA NPs containing tumor-specific RNA (Figure 3D).

[0118] Taken together, the data in Figures 3A-3D demonstrate that monotherapy with RNA-NPs containing GFP RNA or tumor-specific RNA mediates significant antitumor effects against metastatic lung tumors in immunocompetent animals and SCID mice. In BALB / c mice bearing metastatic lung tumors (Figures 3A-3D), both GFP (control) and tumor-specific RNA-NPs mediate innate immune and antitumor activity. However, only tumor-specific RNA-NPs mediate an increase in intratumoral memory T cells and long-term survivor outcomes (Figures 3A-3D). The antitumor activity of RNA-NPs in mice bearing intracranial malignant tumors was also demonstrated (data not shown).

[0119] These data indicate that multi-layered RNA-NPs systemically activate DCs, induce antigen-specific immunity, and trigger antitumor effects. Figures 3A-3D show that control RNA-NPs induce innate responses with some efficacy that is not as robust as tumor-specific RNA-NPs. Compared to untreated mice, no effect of uncomplexed NPs was observed, whereas multi-layered RNA-NPs When incorporated into NPs, both nonspecific (GFP RNA) and tumor-specific RNA mediate innate immunity, however, only tumor-specific RNA-NPs induce adaptive immunity and confer long-term survival benefit (Figure 3A-3D).

[0120] Example 6 This example demonstrates that personalized tumor RNA-NPs are active in a translational canine model.

[0121] The safety and activity of multilayered RNA-NPs were evaluated in client-owned pet dogs diagnosed with malignant glioma or osteosarcoma. Canine malignant glioma or osteosarcoma were initially biopsied for the generation of personalized tumor RNA-NP vaccines.

[0122] To generate personalized multilayered RNA NPs, total RNA material was extracted from each patient's biopsy. A cDNA library was then created from the extracted total RNA, and mRNA was then amplified from the cDNA library. The mRNA was then complexed with DOTAP lipid NPs to form multilayered RNA-NPs essentially as described in Example 1. To evaluate PD-L1, MHCII, CD80, and CD86 on CD11c+ cells, blood was collected at baseline and then 2 and 6 hours after vaccination. CD11c expression of PD-L1, MHC-II, PDL1 / CD80, and PD-L1 / CD86 is plotted over time during the dog's initial observation period. CD3+ cells were analyzed over time for the proportion of CD4 and CD8 cells, and these subsets were evaluated for expression of activation markers (i.e., CD44). These data suggest that multilayered RNA-NPs: 1) increased CD11c expression, indicating peripheral DC activation; + It has been shown to induce 1) an increase in CD80 and MHCII on peripheral blood cells, and 2) an increase in activated T cells.

[0123] Interestingly, within hours of administration, tumor-specific RNA-NPs induced peripheral blood mononuclear cell margination, which increased in the days and weeks following treatment, suggesting that RNA-NPs mediate lymphocyte honing of immune cell populations prior to their egress.

[0124] These data demonstrate that personalized mRNA-NPs are safe and active in a translational canine disease model.

[0125] Specific data from dogs evaluated with this method are shown. A 31 kg male Irish Setter was enrolled in the study with owner consent to receive multilayered RNA-NP. After tumor biopsy, tumor mRNA was successfully extracted and amplified. The immune response was plotted against the initial vaccine. The data show an increase in activation markers in CD11c+ cells (DCs) over time (Figure 4A). The data show an increase in activated CD8+ cells (CD44+CD8+ cells) within the first few hours after RNA-NP vaccination. These data confirm that multilayered RNA-NP is immunologically active in male Irish Setters. A male Boxer diagnosed with malignant glioma was enrolled in the study with owner consent to receive RNA-NP. After tumor biopsy, tumor mRNA was successfully extracted and amplified. The immune response is plotted against the initial vaccine (Figure 4B). The data show an increase in activation markers in CD11c+ cells (DCs) over time. As shown in Figure 4C, an increase in activated T cells (CD44+CD8+ cells) was observed within the first few hours after RNA-NP vaccination. These data confirm that multilayered RNA-NPs are immunologically active in male Boxer dogs.

[0126] After receiving weekly RNA-NP (x3), dogs diagnosed with malignant gliomas experienced a steady progression. Post-vaccination MRI showed stable tumor burden with increased swelling and enhancement (in some cases), which may be more consistent with pseudoprogression from an immunotherapy response in asymptomatic dogs. The survival rates of dogs diagnosed with malignant gliomas receiving only supportive care and tumor-specific RNA-NP (after tumor biopsy without resection) are shown in Figure 4D. In 4D, the median survival time (shown by the dotted line) was approximately 65 days, as reported in a meta-analysis of canine brain tumor patients receiving only supportive care. Previous studies have reported that canine brain astrocytomas have a median overall survival time of 77 days. Personalized multilayered RNA NPs enabled survival of over 200 days.

[0127] With the exception of a slight fever that spiked 6 hours after vaccination on the first day, personalized tumor RNA-NP (1x) was well tolerated, with stable blood counts, differential, renal, and liver function tests. To date, we have treated four client-owned dogs diagnosed with malignant brain tumors. It is important to emphasize that these dogs had not received other therapeutic interventions (i.e., surgery, radiation, or chemotherapy) for their malignant tumors, and all patients evaluated experienced either pseudoprogression or the development of an immune response accompanied by stable or smaller tumors. One dog was necropsied after RNA-NP vaccination. No toxicity was considered related to the intervention in this patient.

[0128] These results suggest the safety and activity of tumor-specific RNA-NP in client-owned dogs with malignant brain tumors in subjects not receiving other antitumor therapeutic interventions.

[0129] Example 7 This example demonstrates a toxicity study of mouse glioma mRNA and pp65 mRNA encapsulated in DOTAP liposomes after intravenous delivery to C57BL / 6 mice.

[0130] The purpose of this study was to evaluate the safety of pp65 mRNA encapsulated by DOTAP liposomes when delivered intravenously to C57BL / 6 mice. The experimental procedures applicable to pathology investigations are summarized in Table 1. All intermediate animals were submitted for necropsy on day 35 ± 1. Necropsies were performed by University of Florida personnel. Tissue samples listed in Table 2 were collected and fixed in 10% neutral-buffered formalin unless otherwise noted. Tissues from animals that died early were fixed in 10% neutral-buffered formalin.

[0131] [Table 1]

[0132] [Table 2]

[0133] Tissues required for microscopic evaluation were obtained from Charles River Laboratories Trimmed and regularly treated by Inc., Skokie, Illinois The tissues were then processed, embedded in paraffin, and stained with hematoxylin and eosin. Light microscopic evaluation was performed by a contributing board-certified veterinary pathologist on all tissues specified in the protocol from all animals in groups 1 and 4, and from animals that died prematurely.

[0134] Tissues that were to be evaluated microscopically per protocol but were not available on slides (and therefore not evaluated) are listed as "not present" in the "Individual Animal Data" section of the pathology report. These missing tissues did not affect the results or interpretation of the pathology portion of the study, as the number of tissues examined from each treatment group was sufficient for interpretation.

[0135] Gross Pathology: No test article-related gross findings were observed. Any gross findings observed were considered to be incidental to those typically observed in this strain and age of mice and / or were considered to be unrelated to administration of a 1:1 ratio of pp65 mRNA and KR158 mRNA in DOTAP liposomes, as they occurred at similar incidence in control and treated animals.

[0136] Histopathology: No test article-related microscopic findings were observed. Although a few animals had inflammatory cell infiltrates at the injection site, this finding was general to injection sites and considered equivocal at this point in the study. The observed microscopic findings were considered incidental to those generally observed in this strain and age of mice and / or were of similar incidence and severity in control and treated animals and therefore unrelated to the administration of a 1:1 ratio of pp65 mRNA and KR158 mRNA in DOTAP liposomes.

[0137] It was concluded that intravenous injection of 1.0 mg / kg KR158 and pp65 mRNA + 15.0 mg / kg DOTAP liposomes into the tail vein of mice on study days 0, 14, and 28 resulted in no gross or microscopic test article-related findings on day 35 ± 1. There was a small amount of inflammatory cell infiltration at the injection site, which is a common finding at injection sites. This finding was equivocal.

[0138] Example 8 In this example, we describe a study aimed at determining the impact of pDCs transfected with multilayered RNA-NPs on antigen-specific T cell priming.

[0139] Although pDCs are well-known stimulators of innate immunity and type I IFN, they also mediate significant effects on intratumor adaptive immunity. They can 1) directly present antigens for priming tumor-specific T cells, 2) support adaptive responses through chemokine recruitment of other DC subtypes (via the chemokines CCL3, CCL4, and CXCL10), 3) polarize Th1 immunity via IL-12 secretion, and / or 4) mediate the release of tumor antigens (via cytokines such as TRAIL or granzyme B) for DC loading and T cell priming. Despite these effector functions, pDCs can also attenuate immunity by releasing immunoregulatory molecules (IL-10, TGF-β, and IDO) and promoting regulatory T cells (Tregs). The purpose of this study is to elucidate the effects of RNA-NP-transfected pDCs on adaptive immunity and antigen-specific T cell priming. RNA-NP-activated pDCs are hypothesized to act as direct primers for antigen-specific immunity and support classical DCs (cDCs) and / or bone marrow-derived DCs (mDCs) in promoting effector T cell responses. These experiments shed new light on the activation state of pDCs required for RNA-NP-mediated immunity and the sequential depletion that can be employed to enhance immunotherapeutic efficacy.

[0140] statistical analysis In the study in Example 9.1, where survival is important, the log-rank test is used to compare Kaplan-Meier survival curves between treatment and control groups. Experience with our tumor models indicates that the median overall survival of untreated control mice is approximately 30 days, and survival follows a Weibull distribution with shape parameter k = 6. As an example, using 10 mice in each of two tumor-bearing groups (treated and untreated), comparison of survival curves using a one-sided log-rank test assessed at a significance level of 0.05 has at least 80% power to detect an 8-day improvement in median survival in the treated group compared to the untreated group. This effect size was determined by simulating 1,000 Weibull-distributed survival data sets with shape parameter k = 6 under the alternative hypothesis effect size, and observing the proportion of log-rank tests for these data sets that were significant at p < 0.05. In the studies in Examples 9.2-9.4, responses observed at different times are analyzed using a two-way ANOVA model with mutually exclusive groups distributed across treatment and observation times. Changes in immune response parameters over time are assessed using generalized linear mixed-effects models (GLMMs). Response variables from experiments that were fully replicated at least once are analyzed using GLMMs. Experimental replicates are modeled as random effects to account for "batch" or "experimental day" variability. Treatment and control groups are modeled as fixed effects and compared using a nested ANOVA-type design within a mixed-effects modeling framework.

[0141] Example 8.1 This example describes experiments designed to measure the antitumor efficacy of RNA-NPs in wild-type and pDC KO mice.

[0142] The tumorigenicity of KR158b-luc, GL261-luc, and mouse H3.3K27M mutant cell lines was established. Because KR158b-luc and GL261-luc are both transfected with luciferase, tumor growth can be monitored using bioluminescence imaging. The tumorigenic dose of KR158b-luc and H3K27M mutant lines was 1 × 10 4The tumorigenic dose of GL261-LUC was 1 × 10 5 GL261 and KR158 are injected into the cerebral cortex of C57Bl / 6 mice (2 mm to the right of the previous section, 3 mm deep in the brain). H3K27M glioma cells are injected at the midline. Tumor mRNA is extracted from the parental cell line (i.e., luciferase-free KR158b) and used in a vaccine formulation consisting of an intravenous (iv) injection of 25 μg of tumor-specific mRNA complexed with 375 μg of our custom lipid-NP formulation (per mouse). These are compared simultaneously with 10 negative control mice receiving NPs only and nonspecific (i.e., pp65 mRNA) RNA-NPs. Mice are vaccinated three times at 7-day intervals, starting 5 days after tumor implantation. IFN-α levels are assessed in the serum of wild-type and pDC KO mice at consecutive time points (days 5, 12, and 19). In wild-type mice that respond to treatment but succumb to disease, immunological escape mechanisms in the tumor (i.e., checkpoint ligand expression, IDO, MHC class I downregulation) and within the tumor microenvironment (i.e., MDSCs, Tregs, and TAMs) will be investigated.

[0143] Based on preclinical data demonstrating the antitumor activity of RNA-NPs in these models, it is expected that antitumor activity will be abrogated in pDC KO mice.

[0144] Example 8.2 This example describes experiments designed to determine pDC phenotype and function after activation with RNA-NP.

[0145] To assess pDC phenotype, KR158b bearing C57Bl / 6 mice were transfected with 375 μg of FITC-labeled DOTAP (Avanti) and 25 μg of TTRNA (KR The recipient mice were vaccinated with TTRNA-NPs composed of 158b-derived TTRNA-NPs (delivered intravenously). 24 hours after vaccination, recipient mice were euthanized (humanely killed with CO2) for collection of spleen, tumor-draining lymph nodes (tdLN), and tumor. Organs were digested into single-cell suspensions and subjected to RBC lysis (PharmLyse, BD Bioscience) before being incubated at 37°C for 5 minutes. WBCs were separated from parenchymal cells using a Ficoll gradient. Cells at the interface were collected, washed, and analyzed. pDCs were stained with CD11c, B220, and Gr-1 (BD Bioscience). Different pDC subsets were identified by differential staining for CCR9, SCA1, and Ly49q. Activation status will be assessed based on the expression of costimulatory molecules (i.e., CD40, CD80, CD86), chemokines (i.e., CCL3, CCL4, CXCL10), and chemokine receptors (i.e., CCR2, CCR5, CCR7). The secondary detection antibody will be rabbit IgG conjugated with AlexaFlour® 488 (ThermoFisher Scientific) for FITC detection. Effector versus regulatory function will be determined by intracellular staining of effector (i.e., IFN-I, IL-12) versus regulatory cytokines (i.e., TGF-β, IL-10). Analysis will be performed by multiparameter flow cytometry (LSR, BD Bioscience) and immunohistochemistry (IHC).

[0146] Based on our preliminary data showing a substantial increase in pDCs in peripheral and intratumoral organs, we expect to identify FITC-positive pDCs in the spleen, tdLN, and intracranial tumors.

[0147] Example 8.3 This example describes experiments designed to determine whether RNA-NP-transfected pDCs mediate direct or indirect activation of antigen-specific T cells.

[0148] pDCs are well-known stimulators of innate immunity and type I IFN, but their cumulative impact on antigen-specific responses has yet to be elucidated. Because they express MHC class II, they possess APC capabilities; however, compared to their cDC counterparts, they are considered weak direct primers of antigen-specific immunity. This experiment aims to generate a better understanding of pDCs as either direct primers or promoters of antigen-specific immunity in the context of RNA-NPs. To determine the effect of pDCs on antigen-specific T cells, KR158b-bearing mice are vaccinated with FITC-labeled NPs (Avanti) derived from spleen, tdLN, and intracranial tumors (as described above), and TTRNA (derived from the mouse glioma line KR158b) encapsulated in FACSort (BD Aria II)-associated FITC+ pDCs. RNA-NP-transfected pDCs are then cocultured with magnetically separated naive CD4 and CD8 T cells, and the T cells are evaluated for proliferation, phenotype (effector vs. central memory), function, and cytotoxicity. Indirect effects from pDCs were assessed by ex vivo coculture of naive CD4 and CD8 T cells with TTRNA-loaded DCs (matured ex vivo from mouse bone marrow). Ex vivo cocultures were performed in triplicate for 7 days in 96-well plates containing naive T cells (40,000 RNA-NP-transfected pDCs containing 400,000 T cells) labeled with CFSE (Celltrace, Life Technologies). T cell proliferation was determined by measuring CFSE dilution by flow cytometry. The phenotypes of effector and central memory populations were determined by differential staining for CD44 and CD62L. These T cells were restimulated for a total of two cycles before harvesting supernatants for detection of Th1 cytokines (i.e., IL-2, TNF-α, and IFN-γ) by bead array (BD Biosciences). Stimulated T cells are also incubated in the presence of KR158b (stably transfected with GFP) or control tumor (B16F10-GFP) and assessed for their ability to induce cytotoxicity. The amount of GFP in each co-culture, as a surrogate for surviving tumor cells, is quantitatively measured by flow cytometry.

[0149] The in vivo efficacy of FACS sorted RNA-NP-transfected pDCs was determined by adoptively transferring these cells (250,000 cells / mouse) into tumor-bearing mice (weekly x3). One week later, the spleen, tdLN, and tumor were harvested to assess antigen-specific T cell activity by YFP expression in IFN-γ reporter mice (GREAT mice, B6 transgenic, containing the IFN-γ promoter with an IRES-eYFP reporter, Jackson Labs). In a separate experiment, the IFN-γ reporter mice were vaccinated with TTRNA-NPs with or without pDC-depleting mAb before harvesting the spleen, tdLN, and intracranial tumor to measure antigen-specific T cell activity by YFP expression one week later. T cell functional assays were performed as described above.

[0150] These pDCs are expected to be required for priming antigen-specific T cells through either direct and / or indirect means.

[0151] Example 8.4 This example describes experiments designed to determine whether RNA-NP-activated pDCs promote antigen-specific T cell priming from cDCs and / or mDCs.

[0152] Although IFN-I release from pDCs is known to increase activation markers of cDCs and mDCs, the role of pDCs in direct T cell priming from cDCs / mDCs is less clear. This experiment aims to elucidate the ability of RNA-transfected cDCs and mDCs to prime antigen-specific T cells in the presence or absence of activated pDCs. To determine the effect of pDCs on other DC subsets, we vaccinated KR158b-bearing C57Bl / 6 and pDC knockout (KO) mice (BDCA2-DTR, B6 transgenic mice, Jackson Labs) with the vaccine and evaluated T cell priming from cDCs and mDCs. FITC+ cDC and mDC populations were sorted via FACSort within 24 h of intravenous TTRNA-NP (FITC-labeled) and evaluated for their ability to prime naive T cell responses in vitro based on proliferation, function, and cytotoxicity assays. Resident and migratory cDCs are identified by CD11c+CD103+MHCII+ cells and CD11c+CD11b+MHCII+ cells, respectively, and mDCs are identified by CD11c+CD14+MHCII+ cells. Cytokines, chemokines, and activation markers are analyzed as described in Example 9.1. The in vivo effects of these cDCs / mDCs are performed in cell migration experiments as described in Example 9.2. Briefly, FACSorted cDCs and mDCs from TTRNA-NP-vaccinated C57Bl / 6 mice or pDC KO mice are adoptively transferred (250,000 cells / mouse) into tumor-bearing mice (once a week x 3) before harvesting spleens, tdLNs, and intracranial tumors to assess antigen-specific T cells by YFP expression in IFN-γ reporter mice one week later. Proliferation, functional, and cytotoxicity assays are performed.

[0153] ML RNA-NPs are expected to activate pDCs, enhancing their activation phenotype and promoting the direct priming of T cells from cDCs and mDCs.

[0154] In the absence of indirect effects from pDCs on cDCs and / or mDCs, the effects of pDCs on NK cells will be assessed, including their activation state, function, and cytotoxicity.

[0155] Example 8.5 This example describes experiments designed to determine how pDCs influence effector / regulatory T cells over time within the tumor microenvironment.

[0156] Recruitment of pDCs to tumors is typically associated with a regulatory phenotype characterized by increased IDO and FoxP3+ Tregs and secretion of immunoregulatory cytokines. This experiment determines whether RNA-NP-activated pDCs specifically function by activating T cells over time in the tumor microenvironment. To determine the intratumoral effects of pDCs, TTRNA-NPs are administered to KR158b-bearing IFN-γ reporter mice with or without a pDC-depleting mAb (Bioxcell). Activated and regulatory T cells are assessed over time in the intratumoral microenvironment at consecutive time points (6 hours, 1 day, 7 days, and 21 days). Effector T cells are characterized, and Tregs are phenotyped by expression of FoxP3, CD25, and CD4. pDCs from non-depleted animals will be FACSorted from these sites and phenotyped for expression of cytokines, chemokines, activation markers (i.e., CD80, CD86, CD40), cytolytic markers (i.e., TRAIL, granzyme b), and regulatory markers (i.e., IL-10, TGF-β, IDO). Tumor cell-induced immunophenotypic changes will also be assessed over time (i.e., MHC-I, PD-L1, SIRPα).

[0157] Example 9 This example describes a study aimed at assessing the role of type I interferon on the egress, trafficking, and function of RNA-NP-activated T cells.

[0158] Statistical Analysis: Tumor-bearing mice are randomized before receiving intervention treatment. Selecting 10 animals per group should provide sufficient power to detect effects of interest. As an example, within an ANOVA design with seven treatment groups observed at a specific time, pairwise comparisons performed within the ANOVA framework may detect an effect size equal to 1.27 SD units with 80% power at a two-sided significance level of 0.05. Immune parameter responses observed in experimental groups at several observation times are analyzed using a generalized linear model (GLM) with normal or negative binomial response errors. Responses are organized in a two-way ANOVA design with mutually exclusive groups distributed across treatment and observation time. Response variables from experiments replicated at least once are analyzed using a GLMM. Experimental replicates are modeled as random effects to account for variation in "batch" or "experimental day." Treatment and control groups are modeled as fixed effects and compared using a nested ANOVA-type design within a mixed-effects modeling framework.

[0159] Example 9.1 This example describes experiments designed to determine the chemokine receptor, S1P1, and VLA-4 / LFA-1 expression profiles of antigen-specific T cells following RNA-NP vaccination.

[0160] The effects of IFN-I on sphingosine-1-phosphate receptor 1 (S1P1), required for T cell egress from lymphoid organs, and integrins (i.e., VLA-4, LFA-1), required for T cell migration across the BBB, were evaluated. IFN-γ reporter mice bearing KR158b or IFN-γ reporter mice receiving an IFNAR1-blocking mAb (Bioxcell) were implanted with TTRNA-NPs. RNA-NPs consisting of 375 μg of DOTAP (Avanti) (extracted from KR158b and administered intravenously) with 25 μg of TTRNA were administered once a week (×3), starting 5 days after implantation. One week after the last vaccination, recipient mice were euthanized (humanely killed with CO2) and the spleen, tdLN, bone marrow, and intracranial tumor were harvested. Organs were then digested. Antigen-specific T cells from spleen, lymph nodes, bone marrow, and tumors are identified by differential staining for YFP expression and sequential staining for effector and central memory T cells (i.e., CD62L and CD44) (days 7, 14, and 21). Th1-associated chemokine receptors (i.e., CCR2, CCR5, CCR7, and CXCR3), S1P1 expression, VLA-4, and LFA-1 expression (Ebioscience) from CD4 and CD8 T cells are assessed by multiparameter flow cytometry and IHC.

[0161] LFA-1 and CCR2 are expected to be expressed on activated T cells after RNA-NP administration. Given that there are no changes in the chemokine expression patterns, S1P1, and integrins of activated T cells after IFNAR1 mAb, we will perform RNA-seq analysis on FACS-sorted T cells (YFP+ cells) from mice treated with or without IFNAR1 mAb to evaluate changes in immune-related genes.

[0162] Example 9.2 This example describes experiments designed to determine the effect of IFN-I on the in vitro and in vivo migration of RNA-NP-activated T cells.

[0163] Based on our data demonstrating an increase in antigen-specific T cells in peripheral organs after IFNAR1 blockade but a lack of antitumor effects, we determined the effect of IFN-I on RNA-NP-activated T cell migration. IFN-γ reporter mice bearing KR158b or receiving IFNAR1, LFA-1, or CCR2-blocking antibodies were vaccinated intravenously with TTRNA-NP once a week (x3). In vivo crossing of the BBB was assessed by the percentage and absolute number of T cells in the intracranial tumor (relative to the spleen, lymph nodes, and bone marrow) at consecutive time points (5, 10, 15, and 20 days after RNA-NP).

[0164] The migratory capacity of T cells is also analyzed in in vitro culture. Naive, INFAR1, LFA-1, or CCR2 KO animals (B6 transgenic, Jackson) bearing KR158b tumors are vaccinated intravenously with TTRNA-NP. T cells are FACSorted into 50–100% FBS solution using a BD Aria II Cell Sorter. These T cells are assessed for their migratory capacity using a transwell assay (ThermoFisher Scientific). Briefly, T cells are placed in the upper layer of a cell culture insert with a permeable membrane between layers of KR158b-GFP tumor cells. Migration is assessed by the number of cells migrating between the layers. T cells are then incubated with tumor cells (4 × 10 cells) before IFN-γ is determined by ELISA (ebioscience). 6 4 × 10 per mL, with or without IL-2 (1 microgram / mL) for coculture with IL-2 (1 microgram / mL) (× 48 h) 6 T cells are plated in culture medium at a concentration of 1000 μg / ml. The amount of GFP in each co-culture, as a surrogate for viable tumor cells, is quantitatively measured by flow cytometry analysis.

[0165] Type I IFN is expected to be required for the transport of activated T cells across the BBB. In the absence of adequate definition of antigen-specific T cells, responses to a physiologically relevant GBM antigen, pp65 (spiked into our tumor mRNA cohort), will be tracked by analysis of the pp65-HLA-A2-restricted epitope NTUDGDDNNDV by tetramer staining of CD8+ cells in spleen, tdLN, and intracranial tumors, and by overlapping peptide pool restimulation assays in HLA-A2 transgenic mice.

[0166] Example 9.3 This example illustrates the contribution of IFN-I to antigen-specific T cell function after RNA-NP.

[0167] IFN-I has been shown to promote Tregs and regulate effector and memory CD8+ cells, and is also essential for promoting activated T cell responses after RNA-NP vaccination. These distinct effects will determine the contribution of IFN-I to antigen-specific T cell function after RNA-NP vaccination. IFN-γ reporter mice bearing KR158b or IFN-γ reporter mice receiving IFNAR1 mAb will be vaccinated with intravenous TTRNA-NP once a week (x3). Antigen-specific T cells will be assessed by YFP+ cells. YFP+ T cells from spleen, lymph nodes, bone marrow, and tumors are assessed for their activation status (i.e., CD107a, perforin, granzymes), proliferation (by fluorescent dilution of adoptively transferred cells labeled with CellTrace Violet), differentiation (into effector and central memory subsets), and cytotoxicity. T cell cytotoxicity is determined in the presence of KR158b (stably transfected with GFP) or control tumor (B16F10). Type I IFN is also expected to enhance T cell proliferation and function within the tumor microenvironment.

[0168] If there is no change in the migratory capacity or function of antigen-specific T cells after type I IFN blockade, we will evaluate the effect of type I IFN on regulating T cell exhaustion. We will also evaluate the effect of type I IFN on the expression of immune checkpoints (i.e., PD-1, TIM-3, LAG-3) and their ligands on tumor cells and APCs (i.e., PD-L1, galectin-9).

[0169] Example 10 This example demonstrates that non-antigen-specific multilayered (ML) RNA NPs confer memory and mediate antigen-specific immunity long enough to fend off tumor rechallenge.

[0170] Experiments were performed using long-term survivors (e.g., mice that survived approximately 100 days) who were challenged twice with tumor inoculation but treated only once weekly (×3) with ML RNA NPs containing GFP RNA or pp65 RNA (non-tumor specific), or with ML RNA NPs containing tumor-specific RNA. Treatment occurred immediately after the first tumor inoculation and approximately 100 days before the second tumor inoculation. Because none of the control mice (untreated mice) survived to 100 days, a new control group of mice was created by inoculating the same type of mice with K7M2 tumors. Like the original control mice, the new control group received no treatment. The long-term survivors also received no treatment after the second tumor inoculation. The timeline of events in this experiment is shown in Figure 7A.

[0171] Notably, all three groups of mice contained long-term survivors who survived the second tumor challenge. As shown in Figure 7B (showing only the period after the second inoculation), all three groups of mice contained long-term survivors who survived up to 40 days after tumor inoculation (the second tumor inoculation). Interestingly, the expression of nonspecific RNA (GFP RNA or pp65 The percentage of long-term surviving mice previously treated with ML RNA NPs containing tumor-specific RNA (treated before the second tumor challenge) survived up to 40 days after the second tumor inoculation, comparable to the group treated with ML RNA NPs containing tumor-specific RNA (treated before the second tumor challenge).

[0172] These data confirm that ML RNA NPs containing RNA non-specific to the tumor of interest provide therapeutic treatment to tumors comparable to that provided by ML RNA NPs containing tumor-specific RNA, leading to an increased percentage of animal survival.

[0173] Example 11 This example demonstrates an exemplary method for making DOTAP-coated iron oxide particles.

[0174] DOTAP-coated iron oxide particles (IONPs) were synthesized for incorporation into multilayered RNA NPs. Briefly, a stock solution of DOTAP (approximately 2 to 4 mg / ml) was prepared by dissolving DOTAP in ethanol. The DOTAP stock solution was probe sonicated in a Q Sonica (model: Q500) using 38% amplitude for a total sonication time of 30 seconds. The appropriate amount of DOTAP was slowly removed stepwise into the aqueous phase by first dissolving an equal volume of sonicated DOTAP stock in an equal volume of water. The resulting solution was further dissolved in water to a final volume of 10 ml. Hereafter, this solution containing water and DOTAP is referred to as the "DOTAP aqueous solution."

[0175] IONPs were synthesized by pyrolysis, coated with oleic acid, magnetically separated to remove free oleic acid, and finally suspended in chloroform.

[0176] One milliliter of the appropriate concentration of IONP in chloroform was added to 10 milliliters of aqueous DOTAP solution. The expected ratio of DOTAP:iron oxide particles was 0.1:0.5, requiring 0.1 mg of DOTAP to coat 0.5 mg of iron oxide particles (IONP). This solution was probe sonicated at 38% amplitude, 59-second pulses, 10-second pulses, and 2000 J intensity (Q Sonica model: Q500). The solution was left overnight in a fume hood with constant stirring to allow the organic solvent to evaporate.

[0177] This method produced iron oxide nanoparticles bound by a lipid coating of DOTAP. The resulting particles were analyzed by transmission electron microscopy (TEM). Figure 9 shows an image of IONPs bound by a DOTAP coating.

[0178] Example 12 This example demonstrates how to create multilayered RNA NPs loaded with iron oxide nanoparticles.

[0179] Example 11 describes a method for producing oleic acid-coated IONPs held together by a coating of DOTAP, which provides the core of the multilayered RNA NPs. The IONP cores are layered with a negative charge using iron-free free DOTAP before encapsulation into the multilayered structure. Briefly, rotary vacuum evaporation is used to remove organic solvent from the DOTAP / chloroform mixture before resuspension in aqueous solution for rotary heating, bath sonication, extrusion, and layering with tumor mRNA at a specific mass ratio of 1:15 (µg dose, RNA to NPs). The multilayered charge is maintained by performing the procedure in a vacuum-sealed container to prevent oxidation from the ambient environment. Iron oxide nanoparticle-loaded multilayered RNA NPs are characterized by CEM in terms of zeta potential and RNA uptake, as described above. Complexes are verified by Nanosight measurements of size and concentration, and layers are visualized by cryo-electron microscopy (CEM). In vitro transfection is demonstrated using GFP mRNA multilayered particles, and in vivo immunogenicity is performed using OVA mRNA. The transfection efficiency of iron oxide nanoparticle-loaded multilayered RNA NPs was determined. Dendritic cells were transfected with multilayered RNA NPs containing GFP RNA with or without iron oxide loading, and GFP-positive cells were measured by flow cytometry. Bright-field and fluorescent images of transfected DCs were taken.

[0180] Example 13 This example demonstrates the effect of a magnetic field on multilayered RNA NPs loaded with iron oxide nanoparticles.

[0181] Testing the effect of a 101mT magnetic field on the ability of magnetic liposomes to deliver RNA to cells IONP-loaded multilayered RNA NPs containing GFP RNA are prepared essentially as described in Example 12. The IONP-loaded multilayered RNA NPs are incubated with DC2.4 dendritic cells for 30 minutes in the presence or absence of a magnetic field. For one set of cells, RNA-loaded magnetic liposomes are incubated with DC2.4 dendritic cells overnight in the absence of a magnetic field generated by a MagneFect-Nano II 24-well magnet array. After 30 minutes, the particle-containing medium is removed and replaced with fresh medium. Gene delivery is assessed by flow cytometry at 24 hours as GFP expression. It is expected that the number of GFP+DCs will be higher in the presence of a magnetic field compared to that in the absence of a magnetic field.

[0182] All references, including publications, patent applications, and patents, cited herein are incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and set forth in its entirety herein.

[0183] As used in the context of describing this disclosure (particularly in the context of the claims that follow), the terms "a," "an," and "the" and similar referents should be construed to encompass both the singular and the plural, unless stated otherwise herein or clearly contradicted by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terminology (i.e., meaning "including, but not limited to"), unless otherwise noted.

[0184] Unless otherwise stated herein, the recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within that range, and each endpoint, and each separate value and each endpoint is incorporated herein as if it were individually set forth herein.

[0185] All methods described herein can be performed in any suitable order unless otherwise specified herein or clearly contradicted by context. Unless otherwise required, any examples provided herein, or the use of exemplary language (e.g., "etc."), are intended merely to better explain the disclosure and do not pose a limitation on the scope of the disclosure. No language in the specification should be construed as indicating any non-claimed element essential to the practice of the disclosure.

[0186] Preferred embodiments of the present disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of these preferred embodiments may become apparent to those skilled in the art upon reading the foregoing description. The inventors expect those skilled in the art to utilize such variations as appropriate, and the inventors intend the present disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Furthermore, the present disclosure encompasses any combination of the above-described elements in all possible variations thereof unless otherwise indicated herein or clearly contradicted by context.

Claims

Claim 1: A method for making nanoparticles comprising a positively charged surface and an interior comprising (i) a core and (ii) at least two layers of nucleic acid, each layer of nucleic acid disposed between cationic lipid bilayers, comprising: (A) adding mRNA molecules and liposomes without physical mixing at an RNA:liposome mass ratio of 1:5 to 1:20 to obtain RNA-coated liposomes, wherein the liposomes are produced by a process for producing liposomes comprising drying a lipid mixture comprising a cationic lipid and an organic solvent by evaporating the organic solvent under vacuum; (B) mixing the RNA-coated liposomes with an excess amount of liposomes.

2. The method of claim 1, wherein the lipid mixture comprises the cationic lipid and the organic solvent in a ratio of 40 mg of cationic lipid per mL of organic solvent to 60 mg of cationic lipid per mL of organic solvent.

3. The method described in claim 1 or 2, wherein the process of producing the liposomes further comprises rehydrating the lipid mixture with a rehydration solution to form a rehydrated lipid mixture, and then agitating, resting, and sizing the rehydrated lipid mixture.

4. The method of claim 3, wherein sizing the rehydrated lipid mixture comprises sonicating, extruding and / or filtering the rehydrated lipid mixture.

5. The method described in claim 1 or 2, wherein the nanoparticles have a zeta potential of 40 mV to 60 mV.

6. The method described in claim 1 or 2, wherein the core of the nanoparticle contains less than 0.5% by weight of nucleic acid and / or the core contains a cationic lipid bilayer.

7. The method described in claim 1 or 2, wherein the outermost layer of the nanoparticle comprises a cationic lipid bilayer and / or the surface of the nanoparticle comprises multiple hydrophilic portions of the cationic lipids of the cationic lipid bilayer.

8. The method described in claim 1 or 2, wherein the mRNA molecules are added to the liposomes without physical mixing at an RNA:liposome mass ratio of 1:

15.

9. A nanoparticle comprising a positively charged surface and an interior comprising (i) a core and (ii) at least two layers of nucleic acid, each nucleic acid layer being disposed between cationic lipid bilayers, the core comprising either a cationic lipid bilayer or less than 0.5% by weight of nucleic acid, the nanoparticle comprising a zeta potential of 40 mV to 60 mV and having a diameter of 200 nm to 500 nm.

10. The nanoparticle of claim 9, comprising at least three nucleic acid layers, each of which is disposed between cationic lipid bilayers.

11. The nanoparticle of claim 10, comprising at least four nucleic acid layers, each of which is disposed between cationic lipid bilayers.

12. The nanoparticle described in claim 10, comprising five or more nucleic acid layers, each of which is disposed between cationic lipid bilayers.

13. A nanoparticle described in any one of claims 9 to 12, wherein the outermost layer of the nanoparticle comprises a cationic lipid bilayer.

14. A nanoparticle described in any one of claims 9 to 12, wherein the surface comprises multiple hydrophilic moieties of the cationic lipids of the cationic lipid bilayer.

15. The nanoparticles of any one of claims 9 to 12, having a zeta potential of 45 mV to 55 mV.

16. The nanoparticles of claim 15, comprising a zeta potential of 50 mV.

17. A nanoparticle described in any one of claims 9 to 12, comprising mRNA molecules and cationic lipids in a mass ratio of 1:5 to 1:

20.

18. The nanoparticles described in claim 17, comprising mRNA molecules and cationic lipids in a mass ratio of 1:15 or 1:7.

5.

19. A nanoparticle described in any one of claims 9 to 12, wherein the nucleic acid comprises mRNA encoding an antigen derived from a viral protein.

20. A pharmaceutical composition comprising a plurality of nanoparticles according to any one of claims 9 to 12, and a pharmaceutically acceptable carrier, diluent, or excipient.

21. The pharmaceutical composition of claim 20, wherein the composition comprises between 10 10 nanoparticles per mL and 10 15 nanoparticles per mL.

22. The pharmaceutical composition of claim 20 for use in increasing an immune response to a tumor in a subject.

23. The pharmaceutical composition for use according to claim 22, wherein the composition is administered systemically to the subject.

24. The pharmaceutical composition for use according to claim 23, wherein the composition is administered intravenously.

25. The pharmaceutical composition of claim 20 for use in treating a subject having a disease.

26. The pharmaceutical composition for use according to claim 25, wherein the subject has cancer or a tumor.

27. The pharmaceutical composition for use according to claim 26, wherein the tumor is a malignant brain tumor.

28. The pharmaceutical composition for use according to claim 27, wherein the tumor is a glioblastoma, a medulloblastoma, a diffuse intrinsic pontine glioma, or a peripheral tumor with metastatic infiltration into the central nervous system.

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