Multilayered RNA nanoparticles and method for sensitizing tumors to immune checkpoint inhibitor therapy

Abstract

This invention provides a method for increasing the susceptibility of tumors to treatment with immune checkpoint inhibitors (ICIs) in a given subject. [Solution] The method involves administering a composition comprising nanoparticles having a positively charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, each nucleic acid layer being positioned between cationic lipid bilayers.

Description

[Technical Field]

[0001] The present invention relates to multilayer nanoparticles that sensitize tumors to immune checkpoint inhibitors and their use.

[0002] Disclosure of grants This invention was awarded by the National Institutes of Health, grant number K08 CA199224, and USA Army Medical. This work was carried out with the support of the U.S. Government under grant number W81XWH-17-1-0510, awarded by the Research Acquisition. The Government has certain rights in this invention.

[0003] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 62 / 978,694, filed on 19 February 2020, which is incorporated herein by reference in its entirety.

[0004] The following applications are also incorporated by reference: International Patent Application No. PCT / US20 / 42606, filed on 17 July 2020, and International Patent Application No. PCT / US21 / 16925, filed on 5 February 2021. [Background technology]

[0005] Due to the severe and nonspecific adverse effects of radiation and chemotherapy, targeted therapies that can selectively kill tumor cells in patients with glioblastoma (GBM) are essential. Tumor-specific immunotherapy can be used to eradicate malignant brain tumors with exquisite precision without collateral damage to normal tissue. Immunotherapy relies on the cytotoxic potential of activated T cells, which scavenge to recognize and reject tumor-associated or specific antigens (TAAs or TSAs). Unlike most drugs, activated T cells can cross the blood-brain barrier (BBB) ​​via ICAM / VCAM integrin (i.e., LFA-1, VLA-4) binding. T cells can be activated ex vivo by co-culture with dendritic cells (DCs) presenting TAAs / TSAs, or by transduction with chimeric antigen receptors (CARs). Alternatively, T cells can be intrinsically activated using cancer vaccines, but in a randomized phase III trial in patients with primary GBM, peptide vaccines targeting tumor-specific EGFRVIII surface antigens did not mediate improved survival compared to the control vaccine. The fact that EGFRVIII vaccines do not mediate antitumor effects highlights the challenges of therapeutic cancer vaccines. While prophylactic cancer vaccines work to prevent malignancies (e.g., HPV vaccines to prevent cervical cancer), they require several booster immunizations over months to years to provide protection in immunocompromised patients. Furthermore, therapeutic cancer vaccines need to induce an immune response much more rapidly against rapidly developing malignancies (e.g., GBM). Moreover, GBM is a highly invasive and heterogeneous tumor associated with severe systemic / intratumor suppression, which can interfere with initial immunotherapy responses.

[0006] RNA vaccines offer several advantages over conventional modalities. RNA has a potent effect on both the innate and adaptive immune systems. RNA acts as a Toll-like receptor (TLR) agonist for receptors 3, 7, and 8, inducing potent TLR-dependent innate immunity. RNA also acts as an intracellular pathogen recognition receptor (e.g., melanoma differentiation antigen 5). It can stimulate (MDA-5) and retinoic acid-inducible gene I (RIG-I) and reach peaks in activation of both helper CD4 and cytotoxic CD8 T cell responses. Unlike DNA vaccines, which suffer from the need to cross both the cell membrane and nuclear membrane, RNA requires only cytoplasmic access because it cannot be integrated into the host genome, thus offering a significant safety advantage. Unlike many peptide vaccines developed only for specific HLA haplotypes (e.g., HLA-A2), RNA can bypass MHC class restrictions and be utilized by the general public (28). One drawback of RNA is that its lack of stability makes it difficult to directly administer "naked" RNA to patients. Since cancer vaccines need to localize to antigen-presenting cells (APCs) where 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.

[0007] Advances in cell therapy are accompanied by developmental challenges that make it difficult to produce vaccines for the general public. To circumvent these challenges, nanocarriers have been developed as RNA delivery vehicles, but the unknown biological reactivity of novel nanoparticle (NP) designs has slowed the transition of NPs to human clinical trials. Alternatively, simple biodegradable lipid-NPs have been developed as cationic and anionic cancer vaccine formulations. Cationic formulations have been designed to shield mRNA within the lipid core, while anionic formulations have been designed to tether mRNA to the particle surface. However, cationic formulations suffer from low immunogenicity, and anionic formulations remain hampered by severe intratumor and systemic immunosuppression that can interfere with activated T cell responses.

[0008] Furthermore, while cancer immunotherapy with immune checkpoint inhibitors (ICIs) has shown great promise for malignancies with an immunologically active ("hot") microenvironment, this therapy has failed in clinical trials in patients with immunologically inactive ("cold") tumors. The response to ICIs appears to be based on the presence of intratumoral CD8+PD-1+ cells and activated PD-L1+ host myeloid cells. These cell populations may spontaneously increase in patients with high mutational burdens but are absent in unresponsive patients.

[0009] In light of the above, there is a need for improved RNA lipid nanoparticle (NP) vaccines, methods for using these vaccines to treat tumors or cancers in patients with immune checkpoint inhibitor (ICI)-resistant tumors, and methods for enhancing the response to immunotherapy for immunologically inactive ("cold") tumors. [Overview of the project]

[0010] This disclosure 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 being arranged between cationic lipid bilayers. In exemplary embodiments, the nanoparticle comprises at least three nucleic acid layers, each of which is arranged between cationic lipid bilayers. In exemplary embodiments, the nanoparticle comprises at least four or five or more nucleic acid layers, each of which is arranged between cationic lipid bilayers. In various embodiments, the outermost layer of the nanoparticle comprises a cationic lipid bilayer. In various examples, the surface comprises multiple hydrophilic portions of the cationic lipids in the cationic lipid bilayer. In exemplary embodiments, the core comprises a cationic lipid bilayer. Optionally, the core contains less than about 0.5% by weight of nucleic acid. The diameter of the nanoparticles is about 50 nm to about 250 nm in diameter in various embodiments, and optionally, about 70 nm to about 200 nm in diameter. In exemplary examples, nanoparticles are characterized by zeta potentials of approximately +40mV to +60mV, and optionally, approximately +45mV to +55mV. The zeta potential of nanoparticles in various examples is approximately 50mV. In some embodiments, nucleic acid molecules exist in nucleic acid molecule:cationic lipid ratios of approximately 1:5 to approximately 1:20, optionally, approximately 1:15, approximately 1:10, or approximately 1:7.5. In various embodiments, nucleic acid molecules are RNA molecules The mRNA is, and 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 contain a mixture of RNAs, which are RNA isolated from human tumors, optionally, malignant brain tumors, optionally, glioblastoma, medulloblastoma, diffuse endogenous pontine glioma, or peripheral tumors with metastatic invasion into the central nervous system.

[0011] Methods for increasing the immune response against a tumor in a subject are provided by this disclosure. In exemplary embodiments, the method comprises administering to a subject nanoparticle or pharmaceutical composition of this disclosure. In exemplary embodiments, the nucleic acid molecule is mRNA. Optionally, the composition is administered systemically to the subject. For example, the composition is administered intravenously. In various embodiments, the nanoparticle or 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 activation by tumor-infiltrating lymphocytes (TILs).

[0012] This disclosure provides a method for increasing the susceptibility of a tumor to treatment with an immune checkpoint inhibitor (ICI) in a subject. In an exemplary embodiment, the method involves administering a composition to a subject comprising nanoparticles having a positively charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, each nucleic acid layer being positioned between cationic lipid bilayers, wherein the composition is optionally administered systemically to the subject.

[0013] The disclosure further provides a method for treating subjects having immune checkpoint inhibitor (ICI)-resistant tumors. In exemplary embodiments, the method comprises administering to a subject a composition comprising nanoparticles having a positively charged surface and (i) a core and (ii) an interior comprising at least two nucleic acid layers, each nucleic acid layer being positioned between cationic lipid bilayers, and (ii) an ICI, optionally, the composition being administered systemically to the subject.

[0014] A method for treating a subject having a tumor or cancer is also provided, the method comprising (i) increasing the number of activated plasmacytoid dendritic cells (pDCs) in the subject according to the method described herein, (ii) isolating leukocytes (WBCs) from the subject, (iii) isolating dendritic cells (DCs) from the WBCs, (iv) contacting the DCs with a fusion protein comprising prostatic acid phosphatase (PAP) and GM-CSF, and (v) administering the DCs to the subject.

[0015] Furthermore, the present disclosure provides a method for preparing a dendritic cell vaccine, the method comprising (i) increasing the number of activated plasmacytoid dendritic cells (pDCs) in a subject, (ii) isolating leukocytes (WBCs) from the subject, (iii) isolating dendritic cells (DCs) from the WBCs, and (iv) contacting the DCs with a fusion protein comprising prostatic acid phosphatase (PAP) and GM-CSF.

[0016] Additional embodiments and aspects of the nanoparticles, pharmaceutical compositions, and methods disclosed herein are provided below. [Brief explanation of the drawing]

[0017] [Figure 1A] A series of diagrams illustrating a general scheme leading to lipid bilayers, liposomes, and multilayer (ML) RNA NPs (outlined). [Figure 1B] A pair of CEM images of uncomplexed NPs (left) and ML RNA NPs (right). [Figure 2A] A diagram illustrating a common scheme leading to cationic RNA lipoplexes. [Figure 2B] A diagram illustrating a common scheme leading to cationic RNA lipoplexes. [Figure 2C] CEM image of uncomplexed NPs. [Figure 2D] CEM image of RNA LPX. [Figure 2E] CEM image of ML RNA NPs. [Figure 2F]Graph of the 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 mice. [Figure 2G] Graph of the 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 mice. [Figure 2H] Graph of the 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 mice. [Figure 2I] Graph of the survival rate of mice treated with ML RNA NP (ML RNA-NP), RNA LPX, anionic LPX, or untreated mice. [Figure 2J] 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] Pair of photographs of the lungs of mice treated with ML RNA NP or untreated mice. [Figure 3B] Graph of the percentage of central memory T cells (CD62L+CD44+ of CD3+ cells) present in mice treated with ML RNA NP loaded with tumor-specific RNA or ML RNA NP containing non-specific RNA (GFP RNA), or untreated mice. [Figure 3C] Graph of the survival rate (%) of mice treated with ML RNA NP loaded with tumor-specific RNA or ML RNA NP containing non-specific RNA (GFP RNA), or untreated mice. [Figure 3D] Graph of the survival rate (%) of mice treated with ML RNA NP loaded with tumor-specific RNA or ML RNA NP containing non-specific RNA (GFP RNA), or untreated mice. This model is different from the model used to obtain the data in Figure 3C. [Figure 4A]Graph of CD8 or CD44 and CD8 expression percentages in CD3+ cells, plotted as a function of time after administration of ML RNA NP. [Figure 4B] Graphs of PDL1, MHC II, CD86, or CD80 expression percentages in CD11c+ cells, plotted as a function of time after administration of ML RNA NPs. [Figure 4C] Graphs of CD44 and CD8 expression percentages in CD3+ cells, plotted as a function of time after administration of ML RNA NPs. [Figure 4D] Graph showing the survival rate (%) of dogs treated with ML RNA NP compared to the median survival time (dotted line). [Figure 4E] This graph shows the percentage of lymphocytes induced after administration of ML RNA-NP in a canine model (x-axis) (y-axis). [Figure 4F] This graph shows interferon production (pg / mL; y axis) over time following administration of ML RNA-NP in an α-canine model. [Figure 4G] The increase in CD80+ expression on Cd11c+ cells after administration of ML RNA-NP (expression percentage, y-axis) is shown over time (x-axis). [Figure 4H] The expression of CD8 and CD44+CD8+ cells in dogs after administration of ML RNA-NP is shown over time (x axis). [Figure 5] CEM image of ML RNA NPs and points to an example with several layers. [Figure 6]Schematic diagram of the production of personalized tumor mRNA-loaded NPs: From as few as 100-500 biopsied brain tumor cells, total RNA is extracted, and a cDNA library is generated from this library, from which a large amount of mRNA (representing a personalized tumor-specific transcriptome) can be amplified. Next, the negatively charged tumor mRNA is encapsulated in positively charged lipid NPs. The NPs encapsulate the RNA via electrostatic interactions and are administered intravenously (iv) for uptake by dendritic cells (DCs) in reticuloendothelial organs (i.e., hepatospleen and lymph nodes). The RNA is then translated and processed by intracellular mechanisms of the DCs to present peptides to MHC class I and II molecules that activate CD4 and CD8+ T cells. [Figure 7] Figure 7A: Timeline of long-term survivor treatment. First and second tumor inoculations are shown. Figure 7B: Graphs of animal survival rates after the second tumor inoculation for each of the three mouse groups: two groups treated with ML RNA NPs containing non-specific RNA (RNA not specific to the target tumor; 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. Survival rates for the control group are shown as "untreated". [Figure 8] A series of images illustrating the localization of anionic LPX in mice upon administration. [Figure 9] Graph showing the percentage of surviving mice treated with ML RNA NP alone (RNA-NP) or in combination with PDL1 monoclonal antibody (RNA-NP + PDL1 mAb) as a function of time (days) after tumor transplantation. The control group included untreated mice (untreated), mice treated with ML NP containing no RNA (NP alone), and mice treated with PDL1 monoclonal antibody alone (PDL1 mAb). *p<0.05, Gehan-Breslow-Wilcox. [Figure 10A-10C]Line graphs showing tumor volume (mm3) of melanoma at various days after tumor transplantation (Figure 10A), survival rate in a sarcoma model (Figure 10B), and survival rate in a metastatic lung model (Figure 10C). These figures demonstrate that the ML RNA-NPs of this disclosure mediate an effective antitumor immune response against immunologically cold tumors in vivo. [Figure 11A-11C] The nonspecific ML RNA-NPs of this disclosure mediate a significant antitumor effect that can synergistically interact with ICI, resulting in the demonstration that “off-the-shelf” (i.e., non-individualized) constructs sensitize cancer to ICI. Figure 11A: Tumor volume (mm3) of C57Bl / 6 mice (7-8 mice / group) with subcutaneous B16F0 tumors was either vaccinated with luciferase RNA-NP once a week (x3) or treated with PD-L1-mAb twice a week (x3). Figure 11B: Survival plot (survival %; y axis) of BALB / c mice (8 mice / group) inoculated with K7M2 lung tumors and vaccinated with GFP RNA-NP three times a week (x3) or PD-L1mAb twice a week. Figure 11C: Nonspecific RNA-NPs (luciferase) sensitize the response to ICI in a checkpoint-resistant mouse tumor model (B16F0). The y-axis represents tumor volume (mm³), and the x-axis represents the number of days after tumor transplantation. [Figure 12] A table listing the top 620 genes representative of the slow-cycling cell (SCC) transcriptome. [Modes for carrying out the invention]

[0018] This disclosure relates to nanoparticles comprising cationic lipids and nucleic acids. As used herein, the term “nanoparticles” refers to particles with a diameter of less than approximately 1000 nm. Since the nanoparticles of this disclosure contain cationic lipids treated to induce liposome formation, the nanoparticles disclosed herein in various embodiments include liposomes. Liposomes are artificially prepared vesicles and, in exemplary embodiments, consist 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 this disclosure are of different sizes, and the composition may be (a) a series of concentric groups separated by narrow aqueous compartments, with a diameter of several hundred nanometers. Liposomes may comprise one or more of the following: (b) multilayer vesicles (MLVs) which may include bilayers, (b) small monolayer vesicles (SUVs) which may have a diameter of less than 50 nm, and (c) large monolayer vesicles (LUVs) which may have a diameter of 50–500 nm, for example. In various examples, liposomes are designed to contain opsonins or ligands to improve the adhesion of liposomes to unhealthy tissues or to activate events such as endocytosis (but not limited to this). In exemplary embodiments, liposomes may contain low or high pH to improve the delivery of the formulation. In various examples, liposomes are formulated depending on, but are not limited to, the encapsulated formulation and liposome components, the properties 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, the optimized vesicle size for the intended use, polydispersity and shelf life, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposome products.

[0019] In exemplary embodiments, the nanoparticles include 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 embodiments, each nucleic acid layer is positioned between lipid layers, e.g., cationic lipid layers. In exemplary embodiments, the nanoparticles are multilayers comprising alternating layers of nucleic acids and lipids. In exemplary embodiments, the nanoparticles include at least three nucleic acid layers, each of which is positioned between cationic lipid bilayers. In exemplary embodiments, the nanoparticles include at least four or five nucleic acid layers, each of which is positioned between cationic lipid bilayers. In exemplary embodiments, the nanoparticles include at least five or more (e.g., six, seven, eight, nine, ten, eleven, twelfth, thirteenth, fifteenth, fifteenth, sixteenth, seventeenth, eighteenth, nineteenth, twentyth, or more) nucleic acid layers, each of which is positioned between cationic lipid bilayers. As used herein, the term “cationic lipid bilayer” means a lipid bilayer comprising, essentially consisting of, or composed of cationic lipids or mixtures thereof. Suitable cationic lipids are described herein. As used herein, the term “nucleic acid layer” means a layer of nanoparticles disclosed herein, containing, essentially having, or consisting of, nucleic acids, such as RNA.

[0020] The unique structure of the nanoparticles in this disclosure leads to a difference in the mechanism by which multilayer nanoparticles (ML-NPs) exert their biological effects. Previously described RNA-based nanoparticles exert their effects, at least partially, via the Toll-like receptor 7 (TLR7) pathway. Surprisingly, the multilayer nanoparticles in this disclosure mediate efficacy independently of TLR7. While we do not wish to be bound by any particular theory, intracellular pathogen recognition receptors (PRRs), such as MDA-5, appear to be more relevant to the bioactivity of multilayer nanoparticles than TLRs. This suggests that ML RNA-NPs may be able to stimulate multiple intracellular PRRs (e.g., RIG-I, MDA-5), as opposed to a single TLR (e.g., TLR7 in endosomes), to achieve greater release of type I interferon and induction of a more potent innate immune response. This could allow RNA-NPs to demonstrate superior efficacy with long-term survivor benefits.

[0021] In various embodiments, the nanoparticles disclosed herein include a positively charged surface. In some examples, the positively charged surface includes a lipid layer, such as a cationic lipid layer. In various embodiments, the outermost layer of the nanoparticle includes a cationic lipid bilayer. Optionally, the cationic lipid bilayer includes, essentially consists of, or comprises DOTAP. In various examples, the surface includes multiple hydrophilic portions of the cationic lipids in the cationic lipid bilayer. In some embodiments, the core includes a cationic lipid bilayer. In various examples, the core is nucleic acid-free, and optionally, the core contains less than about 0.5% by weight of nucleic acid.

[0022] In exemplary embodiments, the nanoparticles have a diameter in the nanometer range, and therefore, specific In this example, the terms "nanoliposome" or "liposome" are used herein. In exemplary embodiments, the nanoparticles have a diameter of approximately 50 nm to approximately 500 nm, for example, approximately 50 nm to approximately 450 nm, approximately 50 nm to approximately 400 nm, approximately 50 nm to approximately 350 nm, approximately 50 nm to approximately 300 nm, approximately 50 nm to approximately 250 nm, approximately 50 nm to approximately 200 nm, approximately 50 nm to approximately 150 nm, approximately 50 nm to approximately 100 nm, approximately 100 nm to approximately 500 nm, approximately 150 nm to approximately 500 nm, approximately 200 nm to approximately 500 nm, approximately 250 nm to approximately 500 nm, approximately 300 nm to approximately 500 nm, approximately 350 nm to approximately 500 nm, and approximately 400 nm to approximately 500 nm. In exemplary embodiments, liposomes are approximately 50 nm to 300 nm, for example, approximately 100 nm to 250 nm, approximately 110 nm ± 5 nm, approximately 115 nm ± 5 nm, approximately 120 nm ± 5 nm, approximately 125 nm ± 5 nm, approximately 130 nm ± 5 nm, approximately 135 nm ± 5 nm, approximately 140 nm ± 5 nm, approximately 145 nm ± 5 nm, approximately 150 nm ± 5 nm, approximately 155 nm ± 5 nm, approximately 160 nm ± 5 nm, approximately 165 nm ± The nanoparticles have diameters of 5 nm, approximately 170 nm ± 5 nm, approximately 175 nm ± 5 nm, approximately 180 nm ± 5 nm, approximately 190 nm ± 5 nm, approximately 200 nm ± 5 nm, approximately 210 nm ± 5 nm, approximately 220 nm ± 5 nm, approximately 230 nm ± 5 nm, approximately 240 nm ± 5 nm, approximately 250 nm ± 5 nm, approximately 260 nm ± 5 nm, approximately 270 nm ± 5 nm, approximately 280 nm ± 5 nm, approximately 290 nm ± 5 nm, and approximately 300 nm ± 5 nm. In exemplary embodiments, the nanoparticles have a diameter of approximately 50 nm to approximately 250 nm. In some embodiments, the nanoparticles have a diameter of approximately 70 nm to approximately 200 nm.

[0023] In exemplary embodiments, the nanoparticles are present in a pharmaceutical composition comprising a heterogeneous mixture of nanoparticles with diameters ranging, for example, from about 50 nm to about 500 nm or from about 50 nm to about 250 nm. Optionally, the pharmaceutical composition comprises a heterogeneous mixture of nanoparticles with diameters ranging from about 70 nm to about 200 nm.

[0024] In exemplary examples, nanoparticles are characterized by zeta potentials of approximately +40mV to approximately +60mV, for example, approximately +40mV to approximately +55mV, approximately +40mV to approximately +50mV, approximately +40mV to approximately +50mV, approximately +40mV to approximately +45mV, approximately +45mV to approximately +60mV, approximately +50mV to approximately +60mV, and approximately +55mV to approximately +60mV. In exemplary embodiments, nanoparticles have a zeta potential of approximately +45mV to approximately +55mV. The zeta potential of nanoparticles in various examples is approximately +50mV. In various embodiments, the zeta potential is greater than +30mV or +35mV. The zeta potential of nanoparticles in this disclosure and Sayour et al. This is one parameter that distinguishes it from the nanoparticles described in al., Oncoimmunology 6(1):e1256527(2016).

[0025] In exemplary embodiments, the nanoparticles include 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). Cationic lipids in exemplary examples include cationic fatty acids, cationic glycerophospholipids, cationic sphingolipids, cationic sterol lipids, cationic prenolipids, cationic glycolipids, or cationic polyketides. In exemplary embodiments, the cationic lipids include 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 may be cationic lipids of formula I or formula II.

[0026] [ka]

[0027] Here, each of a, b, n, and m is independently an integer between 2 and 12 (e.g., 3 and 10). In some embodiments, the cationic lipid is the cationic lipid of formula I, where each of a, b, n, and m is independently an integer selected from 3, 4, 5, 6, 7, 8, 9, and 10. In exemplary examples, the cationic lipid is DOTAP (1,2-dioleoyl-3-trimethylammonium-propane) or a derivative thereof. In exemplary examples, the cationic lipid is DOTMA (1,2-di-O-octadecenyl-3-trimethylammoniumpropane) or a derivative thereof.

[0028] In some embodiments, the nanoparticles include 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 (US2010 / 0324120, the whole of which is incorporated herein by reference). In some embodiments, the nanoparticles include liposomes formed from the synthesis of stabilized plasmid lipid particles (SPLPs) or stabilized nucleic acid lipid particles (SNALPs), which have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo. In some embodiments, the nanoparticles consist of 3 to 4 lipid components in addition to nucleic acid molecules. In exemplary embodiments, liposomes contain 55% cholesterol, 20% distearoylphosphatidylcholine (DSPC), 10% PEG-S-DSG, and 15% 1,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as reported by Jeffs et al., Pharm Res. 2005;22(3):362-72. In exemplary examples, liposomes contain 48% cholesterol, 20% DSPC, 2% PEG-C-DMA, and 30% cationic lipids, as reported by Heyes et al., J. Control Release 2005;107(2):276-87, where the cationic lipids may be 1,2-distearoyloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA).

[0029] In some embodiments, the liposomes contain approximately 25.0% to 40.0% cholesterol, approximately 30.0% to 45.0% cholesterol, approximately 35.0% to 50.0% cholesterol, and / or approximately 48.5% to 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 approximately 5.0% to 10.0% DSPC and / or approximately 7.0% to 15.0% DSPC.

[0030] 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), the whole of which is incorporated herein by reference), and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

[0031] In various examples, cationic lipids include 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), and further include neutral lipids, sterols, and molecules that can reduce particle aggregation, such as PEG or PEG-modified lipids.

[0032] Liposomes in various embodiments include DLin-DMA, DLin-K-DMA, 98N12-5, C12-200, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, PLGA, PEG, PEG-DMG, PEGylated lipids, and amino alcohol lipids. In some embodiments, liposomes include, but are not limited to, cationic lipids such as DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA, and amino alcohol lipids. In some embodiments, amino alcohol cationic lipids include lipids described in U.S. Patent Publication 2013 / 0150625, which is incorporated in whole herein by reference, and / or lipids prepared by the method described therein. As a non-limiting example, cationic lipids in certain embodiments include 2-amino-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-diene-1-yloxy]methyl}propan-1-ol (compound 1 in US2013 / 0150625), 2-amino-3-[(9Z)-octadeca-9-en-1-yloxy]-2-{[(9Z)-octadeca-9-en-1-yloxy]methyl}propan-1-ol (compound 2 in US2013 / 0150625), 2- The compounds are amino-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (compound 3 in US2013 / 0150625) and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-2-{[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]methyl}propan-1-ol (compound 4 in US2013 / 0150625), or any pharmaceutically acceptable salt or stereoisomer thereof.

[0033] In various embodiments, the liposomes contain (i) 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z) The present invention comprises (ii) at least one lipid selected from the group consisting of (-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, e.g., cholesterol, and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA in a molar ratio of approximately 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol: 0.5-15% PEG lipid.

[0034] In some embodiments, the liposomes contain, on a molar basis, about 25% to about 75% of cationic lipids 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), for example, about 35 to about 65%, about 45 to about 65%, about 60%, about 57.5%, about 50%, or about 40% on a molar basis.

[0035] In some embodiments, liposomes contain about 0.5% to about 15% of neutral lipids on a molar basis, for example, 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 various embodiments, nanoparticles do not contain neutral lipids. In some embodiments, formulations contain about 5% to about 50% of sterols on a molar basis (for example, about 15% to about 45%, about 20% to about 40%, about 40%, about 38.5%, about 35%, or about 31% on a molar basis). An exemplary sterol is cholesterol. In some embodiments, the formulation contains PEG or PEG-modified lipids in a molar amount of about 0.5% to about 20% (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%). In some embodiments, the PEG or PEG-modified lipids contain PEG molecules with an average molecular weight of 2,000 Da. In other embodiments, the PEG or PEG-modified lipids contain PEG molecules with an average molecular weight of less than 2,000, e.g., 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) and PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005), the entirety of which is incorporated herein by reference).

[0036] In exemplary embodiments, cationic lipids include (20Z,23Z)-N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z)-N,N-dimethylhexacosa-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, and (14Z,17Z)-N,N-dimethyl Tyltricosa-14,17-diene-6-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene-7-amine, (18Z,21Z)-N,N-dimethylheptacosa-18,21-diene-10-amine, (15Z,18Z)-N,N-dimethyltetracosa-15,18-diene-5-amine, (14Z,17Z)-N,N-dimethyltricosa-14,17-diene-4-amine, (19Z,22Z)-N,N-dimethylheptacosa-19,22-diene-9-amine, (18Z,21 (Z)-N,N-dimethylheptacosa-18,21-diene-8-amine, (17Z,20Z)-N,N-dimethylhexacosa-17,20-diene-7-amine, (16Z,19Z)-N,N-dimethylpentacosa-16,19-diene-6-amine, (22Z,25Z)-N,N-dimethylhenthacosa- Nta-22,25-dien-10-amine, (21 Z,24 Z)-N,N-dimethyltriaconta-21,24-dien-9-amine, (18 Z)-N,N-dimethylheptacos-18-en-10-amine, (17 Z)-N,N-dimethylhexacos-17-en-9-amine, (19 Z,22 Z)-N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosa-10-amine, (20 Z,23 Z)-N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11 Z,14 Z)-1-nonylicosa-11,14-dien-1-yl] Pyrrolidine, (20Z)-N,N-dimethylheptacos-20-en-10-amine, (15Z)-N,N-dimethyleptacos-15-en-10-amine, (14Z)-N,N-dimethylnonacos-14-en-10-amine, (17Z)-N,N-dimethylnonacos-17-en-10-amine, (24Z)-N,N-dimethyltriaconto-24-en-10-amine, (20Z)-N,N-dimethylnonacos-20-en-10-amine, (22Z)-N,N-dimethylhentriaconto-22-en-10-amine, (16Z )-N,N-dimethylpentacos-16-en-8-amine, (12Z,15Z)-N,N-dimethyl-2-nonylhenicosa-12,15-dien-1-amine, (13Z,16Z)-N,N-dimethyl-3-nonyldocosa-13,16-dien-1-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]eptadecane-8-amine, 1-[(1S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecane-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]tetradecane-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecane-1-amine, 1-[(1R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecane-9-amine, 1-[(1S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecane-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecane-8-amine, RN,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-(octyloxy)propane-2-amine, SN,N-dimethyl Tyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S)-N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]-3-[(5Z)-octa-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy] -1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-2-amine, N,N-dimethyl-1-[ (9Z)-Octadeca-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S)-N,N-dimethyl-1-[(6Z,9Z,12Z)-Octadeca-6,9,12-triene-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11,14-diene-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11,14-diene-1-yloxy], -N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-diene-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(13Z,16Z)-docosa-13,16-diene-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-diene-1-yloxy]-3-(hexyl (Hexyloxy)-N,N-dimethylpropane-2-amine, (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropane-2-amine, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane-2-amine, 1-[(9Z)-hexadeca-9-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propane- 2-amine, (2R)-N,N-dimethyl-H(1-methoyloctyl)oxyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-diene-1-yloxy]propan-2-amine, N,N-dimethyl-1-(octyloxy)-3-({8-[(1S,2S) -2-{[(1R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[8-(2-ocrylcyclopropyl)octyl]oxy}3-(octyloxy)propan-2-amine and (11E,20Z,23Z)-N,N-dimethylnonacosa-11,20,2-triene-10-amine or their pharmaceutically acceptable salts or stereoisomers may be selected.

[0037] In some embodiments, the nanoparticles comprise lipid-polycation complexes. The 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 2012 / 0178702 (which is incorporated herein in its entirety by reference). In non-limiting examples, the polycation may comprise cationic peptides or polypeptides, such as, but not limited to, polylysine, polyornithine, and / or polyarginine. In some embodiments, the composition may comprise a lipid-polycation complex, which may further comprise non-cationic lipids, such as, but not limited to, cholesterol or dioleoylphosphatidylethanolamine (DOPE).

[0038] In various embodiments, cationic liposomes are optionally free of non-cationic lipids. Neutral molecules may, in some embodiments, interfere with the coiling / condensation of multilayer nanoparticles, resulting in RNA-loaded liposomes exceeding 200 nm in size. Cationic liposomes produced without helper molecules may range in size from approximately 70–200 nm (or less). These constructs essentially consist of cationic lipids with negatively charged nucleic acids and can be formulated in a sealed rotary vacuum evaporator to prevent oxidation of the particles (if exposed to the ambient environment). In this embodiment, the absence of helper lipids optimizes mRNA coil formation into densely packaged multilayer NPs, where each NP contains a greater amount of nucleic acid per particle. Due to the increased nucleic acid payload per particle, these multilayer RNA nanoparticles drive significantly larger innate immune responses, which are a key predictor of efficacy for modulating the immune system.

[0039] In some embodiments, nucleic acid molecules are present in a nucleic acid molecule:cationic lipid ratio of approximately 1:5 to approximately 1:25. In some embodiments, nucleic acid molecules are present in a nucleic acid molecule:cationic lipid ratio of approximately 1:5 to approximately 1:20, optionally approximately 1:15, approximately 1:10, or approximately 1:7.5. As used herein, the term “nucleic acid molecule:cationic lipid ratio” means a mass ratio, where the mass of the nucleic acid molecule is relative to the mass of the cationic lipid. Also, in exemplary embodiments, the term “nucleic acid molecule:cationic lipid ratio” is used herein. The term "ML RNA NP" refers to the ratio of the mass of nucleic acid molecules, e.g., RNA, added to cationic lipid-containing liposomes during the process of producing ML RNA NPs. In exemplary embodiments, nanoparticles contain less than about 10 μg or about 10 μg of RNA molecules per 150 μg of lipid mixture. In exemplary embodiments, nanoparticles are produced by incubating about 10 μg of RNA with about 150 μg of liposomes. In other embodiments, nanoparticles contain more RNA molecules per unit mass of lipid mixture. For example, nanoparticles may contain more than 10 μg of RNA molecules per 150 μg of liposomes. In some examples, nanoparticles contain more than 15 μg of RNA molecules per 150 μg of liposomes or lipid mixture.

[0040] In various embodiments, nucleic acid molecules are RNA molecules, such as transfer RNA (tRNA), ribosomal RNA (rRNA), and messenger RNA (mRNA). In various embodiments, RNA molecules include tRNA, rRNA, mRNA, or combinations thereof. In various embodiments, RNA is total RNA isolated from a cell. In exemplary embodiments, RNA is 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.

[0041] In exemplary examples, the RNA molecule is mRNA. In various embodiments, the mRNA is in vitro transcribed mRNA. In various embodiments, the mRNA molecule is produced by in vitro transcription (IVT). Preferred techniques for performing IVT are known in the art. In exemplary embodiments, an IVT kit is used. In exemplary embodiments, the kit comprises 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 comprise a prokaryotic phage RNA polymerase and promoter (T7, T3, or SP6) with a eukaryotic or prokaryotic extract for synthesizing a protein from an exogenous DNA template. Optionally, the RNA is in vitro transcribed mRNA, and the in vitro transcription template is cDNA prepared from RNA extracted from tumor cells. In various embodiments, the nanoparticles comprise a mixture of RNAs, which are RNAs isolated from human tumors, optionally malignant brain tumors, optionally glioblastoma, medulloblastoma, diffuse endogenous pontine glioma, or peripheral tumors with metastatic invasion of the central nervous system. In various embodiments, the RNAs comprise a sequence encoding a poly(A) tail, and as a result, the in vitro transcribed RNA molecules contain a poly(A) tail at their 3' end. In various embodiments, the method for producing the nanoparticles comprises additional processing steps, such as capping the in vitro transcribed RNA molecules.

[0042] In exemplary embodiments, RNA (e.g., mRNA) encodes a protein. Optionally, the protein is selected from the group consisting of tumor antigens, cytokines, or costimulatory molecules. In practice, in some embodiments, the protein is selected from the group consisting of tumor antigens, costimulatory molecules, cytokines, growth factors, lymphokines, for example, cytokines and growth factors effective in inhibiting tumor metastasis, or cytokines or growth factors shown to have antiproliferative effects on at least one cell population. Such cytokines, lymphokines, growth factors, or other hematopoietic factors include, but are not limited to, 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 factors, and erythropoietin. Additional growth factors for use herein include, but are not limited to, angiogenin, morphogenetic protein-1, morphogenetic protein-2, morphogenetic protein-3, morphogenetic protein-4, morphogenetic protein-5, morphogenetic protein-6, morphogenetic protein-7, morphogenetic protein-1 Protein-8, Bone Morphogenesis Protein-9, Bone Morphogenesis Protein-10, Bone Morphogenesis Protein-11, Bone Morphogenesis Protein-12, Bone Morphogenesis Protein-13, Bone Morphogenesis Protein-14, Bone Morphogenesis Protein-15, Bone Morphogenesis Protein Receptor IA, Bone Morphogenesis Protein Receptor IB, Brain-derived Neurotrophic Factor, Ciliary Neurotrophic Factor, Ciliary Neurotrophic Factor Receptor α, Cytokine-induced Neutrophil Chematologist 1, Cytokine-induced Neutrophil, Chematologist 2α, Cytokine-induced Neutrophil Chematologist 2β, β-Endothelial Growth Factor, Endothelin 1, Epithelial Neutrophil Attractant, Glial Cell Line-derived Neurotrophic Factor Receptor α1, Glial Cell Line-derived Neurotrophic Factor Receptor α2, Growth-related Protein, Growth-related Protein α, Growth-related Protein β, Growth-related Protein γ, Heparin-binding Epithelial 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 Examples include growth factor-binding proteins, keratinocyte growth factor, leukemia suppressor, leukemia suppressor receptor α, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulant, stem cell factor, stem cell factor receptor, transforming growth factor α, transforming growth factor β, transforming growth factor β1, transforming growth factor β1.2, transforming growth factor β2, transforming growth factor β3, transforming growth factor β5, latent transforming growth factor β1, transforming growth factor β-binding protein I, transforming growth factor β-binding protein II, transforming growth factor β-binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and their biologically or immunologically active fragments. In exemplary embodiments, tumor antigens are antigens derived from viral proteins, antigens derived from point mutations, or antigens encoded by onco-germline genes.In exemplary embodiments, tumor antigens include 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, and WT1. In exemplary embodiments, the co-stimulatory 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 associated with tumor antigens or cancer antigens. In some embodiments, the protein is nonspecific to tumors or cancer. For example, a nonspecific protein may be green fluorescent protein (GFP) or ovalbumin (OVA).

[0043] In various embodiments, the nucleic acid layer contains a sequence of nucleic acid molecules expressed by slow-cycling cells (SCCs). The term “slow-cycling cells” or “SCC” refers to tumor or cancer cells that grow slowly. In exemplary embodiments, SCCs have a doubling time of at least about 50 hours. SCCs have been identified in numerous cancer tissues, including melanoma, ovarian cancer, pancreatic adenocarcinoma, breast cancer, glioblastoma, and colon cancer. As taught in Deleyrolle et al., Brain 134(5):1331-1343 (2011) (which is incorporated herein by reference in particular with respect to the description of SCCs), SCCs exhibit increased tumor-initiating characteristics and are stem cell-like. Due to their slow growth rate, SCCs also refer to label-retaining cells (LRCs). In exemplary examples, the nucleic acid molecule is RNA extracted from isolated SCCs or a nucleic acid molecule that hybridizes to RNA extracted from isolated SCCs. Optionally, SCCs are isolated from mixed tumor cell populations obtained from subjects having tumors (e.g., glioblastoma). As used herein, the term “mixed tumor cell population” refers to a heterogeneous population of tumor cells comprising different subtypes, including slow-cycling cells and at least one other tumor cell type, e.g., fast-cycling cells (FCCs).

[0044] In an exemplary example, nanoparticles are a mixture of different RNA molecules expressed by SCC. This includes a mixture of multiple RNA molecules. In certain examples, the mixture or multiple includes at least 10 (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90) different RNA molecules expressed by SCC. In some embodiments, the mixture or multiple includes at least 100 (e.g., at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, or more (e.g., at least 700, at least 800, at least 900)) different RNA molecules expressed by SCC. In embodiments, the nanoparticles include a mixture or multiple RNA molecules that at least partially represent the transcriptome of SCC. The term "transcriptome" refers to the sum of all messenger RNA molecules expressed from the genes of an organism. The term "SCC transcriptome" refers to the sum of all mRNA molecules expressed by SCC. In a specific example, the SCC transcriptome is first generated by isolating total RNA from tumor cells, and then the sum of that RNA is used to generate cDNA by RT-PCR using a conventional method. Using the cDNA, protected mRNA transcripts (e.g., 7-methylguanosine-capped RNA) can be synthesized, for example, using the Ambion® mMESSAGE mMACHINE® transcription kit. In an exemplary embodiment, the SCC transcriptome is the sum of all mRNA expressed from the genes listed in Figure 12. In an alternative or additional embodiment, the nucleic acid molecules of the nanoparticles, e.g., RNA, are at least two (e.g., at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine) of pre-synthesized RNAs encoded by different genes listed in Figure 12.In exemplary cases, the nucleic acid molecules are at least 10 (e.g., at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90) RNAs encoded by different genes listed in Figure 12. In some embodiments, the nucleic acid molecules are at least 100 (e.g., at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 450, at least 500, at least 550, at least 600, or more (e.g., at least 700, at least 800, at least 900)) RNAs encoded by different genes listed in Figure 12. Exemplary methods for isolating SCCs are described in the examples.

[0045] In various examples, the RNA molecule may be an antisense molecule, optionally siRNA, shRNA, miRNA, or any combination thereof. The antisense molecule may mediate RNA interference (RNAi). As is 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 by sequence-specific means (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, known as small interfering RNAs (siRNA, also known as short interfering RNAs) (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)). siRNA is incorporated into a large protein complex that recognizes and cleaves target mRNA (Nykanen et al., Cell, 107, 309-321 (2001)). Introduction of dsRNA into mammalian cells is effective. It has been reported that this does not lead to the rate of 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 need for Dicer in siRNA maturation in cells can be bypassed by introducing synthetic 21-nucleotide siRNA double helix that inhibits the expression of transfected and endogenous genes in various mammalian cells (Elbashir et al., Nature, 411:494-498 (2001)).

[0046] In this regard, the RNA molecule in some embodiments is an siRNA molecule that mediates RNAi and, in some embodiments, is specific to inhibit protein expression. As used herein, the term “siRNA” refers to RNA (or RNA analogues) containing about 10 to about 50 nucleotides (or nucleotide analogues) that can induce or mediate RNAi. In exemplary embodiments, the siRNA molecule contains about 15 to about 30 nucleotides (or nucleotide analogues) or about 20 to about 25 nucleotides (or nucleotide analogues), for example, 21 to 23 nucleotides (or nucleotide analogues). The siRNA may be double-stranded or single-stranded, preferably double-stranded.

[0047] In an alternative embodiment, the RNA molecule is, alternatively, a short hairpin RNA (shRNA) molecule that is specific to inhibit protein expression. As used herein, the term “shRNA” refers to a molecule of about 20 or more base pairs in which single-stranded RNA partially contains a palindromic base sequence, in which it forms a double-stranded structure (i.e., a hairpin structure). shRNA may be siRNA (or an siRNA analog) folded into a hairpin structure. shRNA typically contains about 45 to about 60 nucleotides and includes an antisense and sense portion of about 21 nucleotides of the hairpin, an optional overhang on the non-loop side of about 2 to about 6 nucleotides in length, and a loop portion which may be, for example, about 3 to 10 nucleotides in length. shRNA can be chemically synthesized. Alternatively, shRNA can be produced by ligating the sense and antisense strands of a DNA sequence in reverse and synthesizing the RNA in vitro using T7 RNA polymerase with the DNA as a template.

[0048] While we do not wish to be bound by any theory or mechanism, after shRNA is introduced into a cell, it is broken down into lengths of approximately 20 bases or more (e.g., typically 21, 22, or 23 bases), inducing RNAi and resulting in an inhibitory effect. Therefore, shRNA can induce RNAi and thus be used as an effective component of this disclosure. The shRNA may preferably have a 3' overhang. The length of the double-stranded portion is not particularly limited, but is preferably 10 nucleotides or more, more preferably 20 nucleotides or more. Here, the 3'-overhang is preferably DNA, more preferably DNA of at least 2 nucleotides in length, and even more preferably DNA of 2 to 4 nucleotides in length.

[0049] In exemplary embodiments, the antisense molecule is a microRNA (miRNA). As used herein, the term “microRNA” refers to a small (e.g., 15-22 nucleotides) non-coding RNA molecule that forms base pairs with an mRNA molecule to silence gene expression through translational repression or targeted degradation. MicroRNAs and their therapeutic potential have been described in the art. For example, Mulligan, *MicroRNA: Expression, Detection, and Therapeutic Strategies*, Nova Science Publishers, Inc., Hauppauge, NY, 2011; Bader and Lammers, “The Therapeutic Potential of microRNAs” In See Novations in Pharmaceutical Technology, pages 52-55 (March 2011).

[0050] In certain cases, the RNA molecule is an antisense molecule, optionally siRNA, shRNA, or miRNA, that targets and reduces the expression of proteins in the immune checkpoint pathway. In various embodiments, the proteins in the immune checkpoint pathway are CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, TIGIT, LAG3, CD112, TIM3, BTLA, or the costimulatory receptors ICOS, OX40, 41BB, or GITR. In certain cases, the proteins in the immune checkpoint pathway are CTLA4, PD-1, PD-L1, B7-H3, B7H4, or TIM3. The immune checkpoint signaling pathway is outlined in Pardoll, Nature Rev Cancer 12(4):252-264 (2012).

[0051] In exemplary embodiments, the NP of this disclosure comprises a mixture of RNA molecules. In exemplary embodiments, the mixture of RNA molecules is RNA isolated from cells from a human, and optionally, the human has a tumor. In some embodiments, the mixture of RNA is RNA isolated from a human tumor. In exemplary embodiments, the human has cancer, optionally, any cancer described herein. Optionally, the tumor from which the RNA is isolated is selected from the group consisting of gliomas (including, but not limited to, glioblastomas), medulloblastomas, diffuse endogenous pontine gliomas, or peripheral tumors with metastatic invasion into the central nervous system (e.g., melanoma or breast cancer). In exemplary embodiments, the tumor from which the RNA is isolated is a cancerous tumor, e.g., any of these cancers described herein.

[0052] In various embodiments, the nanoparticles comprise a nucleic acid molecule (e.g., an RNA molecule) containing a nucleic acid sequence encoding a chimeric protein that includes a LAMP protein. In certain embodiments, the LAMP protein is LAMP1, LAMP2, LAMP3, LAMP4, or LAMP5 protein.

[0053] Manufacturing method The disclosure also provides a method for producing nanoparticles comprising a positively charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid (e.g., RNA) layers, wherein each nucleic acid layer is positioned between cationic lipid bilayers, the method comprising (A) mixing nucleic acid molecules and liposomes in a nucleic acid (e.g., RNA):liposome ratio of about 1:15, optionally, such as about 1:5 to about 1:20. The liposomes are produced by a process for producing liposomes, which comprises 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.

[0054] In exemplary embodiments, nanoparticles prepared by the methods disclosed herein are consistent with the descriptions of nanoparticles described herein. For example, nanoparticles prepared 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 prepared by the methods disclosed herein is about +50 mV. In various embodiments, the core of nanoparticles prepared by the methods of this disclosure comprises less than about 0.5 wt% 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 multiple hydrophilic portions of the cationic lipids in the cationic lipid bilayer.

[0055] In an exemplary embodiment, the lipid mixture contains a cationic lipid and an organic solvent, and 1 mL of organic solvent The mixture contains approximately 40 mg of cationic lipid per medium to approximately 60 mg of cationic lipid per 1 mL of organic solvent, or optionally, approximately 50 mg of cationic lipid per 1 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, and then stirring, pausing, and sizing the rehydrated lipid mixture. Optionally, the sizing of the rehydrated lipid mixture includes sonication, extrusion, and / or filtration of the rehydrated lipid mixture.

[0056] An exemplary method for producing 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 positioned between cationic lipid bilayers, is described herein, in Example 1. One or more of the steps described in Example 1 can be included in the method disclosed herein. For example, in some embodiments, the method includes one or more steps required to prepare RNA before it forms a complex with liposomes. In exemplary embodiments, the method includes downstream steps for preparing nanoparticles for administration to a subject, e.g., humans. In exemplary embodiments, the method includes formulating NPs for intravenous injection. In various embodiments, the method includes adding one or more pharmaceutically acceptable carriers, diluents, or excipients, and optionally packaging the resulting composition into a container, e.g., a vial, syringe, bag, ampoule, etc. The container in some embodiments is a ready-to-use container and optionally is disposable.

[0057] This specification further provides nanoparticles prepared by the nanoparticle preparation methods disclosed herein.

[0058] Cells and their populations In addition, this specification provides cells (e.g., isolated cells or ex vivo cells) containing (e.g., transfected with) the nanoparticles of the Disclosure. In exemplary embodiments, the cells are any type of cell that may contain the nanoparticles of the Disclosure. In some embodiments, the cells are eukaryotic cells, e.g., plants, animals, fungi, or algae. In alternative embodiments, the cells are prokaryotic cells, e.g., bacteria or protists. In exemplary embodiments, the cells are cultured cells. In alternative embodiments, the cells are primary cells, i.e., directly isolated from an organism (e.g., human). The cells may be adherent cells or suspension cells, i.e., cells that grow in a suspension state. In exemplary embodiments, the cells are mammalian cells. Most preferably, the cells are human cells. The cells may be any cell type, may originate from any type of tissue, and may be at any developmental stage. In exemplary embodiments, the cells 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 an antigen for recognition by a specific T cell. In exemplary embodiments, an APC is a dendritic cell, macrophage, Langerhans cell, or B cell. In exemplary embodiments, an APC is a dendritic cell (DC). In exemplary embodiments, if the cells are administered to a subject, e.g., a human, the cells are of autologous origin to the subject. In exemplary embodiments, the immune cell is a tumor-associated macrophage (TAM).

[0059] Furthermore, the Disclosure provides a population of cells in which at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the population are cells containing (e.g., transfected with) the nanoparticles of the Disclosure. In some embodiments, the population of cells is a heterogeneous population of cells, or alternatively, in some embodiments, a substantially homogeneous population, which primarily comprises cells containing the nanoparticles of the Disclosure. When the cells are intended to be administered to a subject, the cells may be autologous or allogeneic with respect to the subject being treated.

[0060] Pharmaceutical composition This specification provides compositions comprising nanoparticles of the Disclosure, cells containing nanoparticles of the Disclosure, populations of cells of the Disclosure, or any combination thereof, and pharmaceutically acceptable carriers, excipients, or diluents. In exemplary embodiments, the composition comprises a plurality of nanoparticles of the Disclosure and a pharmaceutically acceptable carrier, diluent, or excipient, and is a pharmaceutical composition intended for administration to humans. In exemplary embodiments, the composition is a sterile composition. In exemplary examples, the composition comprises a plurality of nanoparticles of the Disclosure. Optionally, at least 50% of the plurality of nanoparticles have a diameter of about 100 nm to about 250 nm. In various embodiments, the composition contains about 10 per mL 10 Nanoparticles ~ approximately 10 per 1 mL 15 Nanoparticles, optionally selected, approximately 10 per 1 mL 12 Contains ±10% nanoparticles.

[0061] In exemplary embodiments, the compositions of the present disclosure may include additional components other than nanoparticles, cells containing nanoparticles, or populations of cells. In various embodiments, the compositions may include, for example, acidifying agents, additives, adsorbents, aerosol propellants, air replacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, preservatives, bases, binders, buffers, chelating agents, coating agents, colorants, drying agents, cleaning agents, diluents, disinfectants, disintegrants, dispersants, dissolution accelerators, pigments, softeners, emulsifiers, emulsifying stabilizers, fillers, film-forming agents, flavor enhancers, flavoring agents, and flow enhancers. Contains any pharmaceutically acceptable ingredients, including agents, gelling agents, granulating agents, humectants, lubricants, mucosal adhesives, ointment bases, ointments, oily media, organic bases, pastel bases, pigments, plasticizers, abrasives, preservatives, chelating agents, skin penetration agents, solubilizers, solvents, stabilizers, suppository bases, surfactants, suspending agents, sweeteners, therapeutic agents, thickeners, tonics, toxicants, water absorbents, water-miscible cosolvents, water softeners, or wetting agents. See, for example, the Handbook of Pharmaceutical Excipients, Third Edition, AH Kibbe (Pharmaceutical Press, London, UK, 2000) (in its entirety incorporated by reference) and Remington's Pharmaceutical Sciences, Sixteenth Edition, EW Martin (Mack Publishing Co., Easton, Pa., 1980) (in its entirety incorporated by reference).

[0062] The compositions of this disclosure may be suitable for administration by any acceptable route, including parenteral and subcutaneous. Other routes include, for example, intravenous, intradermal, intramuscular, intraperitoneal, intranodal, and intrasplenic administration. In exemplary embodiments, if the composition comprises liposomes (but not cells containing liposomes), the composition is suitable for systemic (e.g., intravenous) administration.

[0063] If the composition is intended for administration to a subject, it may be isotonic with respect to the intended administration site. For example, if the solution is intended for parenteral administration, it may be isotonic with blood. The composition is typically sterile. In certain embodiments, this may be achieved by filtration through a sterile filtration membrane. In certain embodiments, parenteral compositions are generally placed in containers with a sterile access port, such as intravenous solution bags, or vials or pre-filled syringes with stoppers that can be punctured by a subcutaneous needle. In certain embodiments, the composition may be stored either in a prepared form or in a form that is reconstituted or diluted before administration (e.g., lyophilized).

[0064] use Without being bound by any particular theory, the data provided herein support the use of RNA NPs of this disclosure to increase the immune response against a tumor in a subject. Thus, a method for increasing the immune response against a tumor in a subject is provided by this disclosure. In exemplary embodiments, the method comprises administering the pharmaceutical composition of this disclosure to a subject. In exemplary embodiments, the nucleic acid molecule is mRNA. Optionally, the composition is administered systemically to the subject. For example, the composition is administered intravenously. In various embodiments, the pharmaceutical composition is The drug is administered in an effective dose to activate the target dendritic cells (DCs). In various cases, the immune response is a T cell-mediated immune response. Optionally, the T cell-mediated immune response includes activation by tumor-infiltrating lymphocytes (TILs). In exemplary embodiments, the immune response is an innate immune response.

[0065] In various embodiments, tumors are refractory to immune checkpoint therapy prior to administration of compositions containing RNA-LPs; that is, one or more ICIs have reduced efficacy in inducing an immune response against the tumor. Alternatively, although the tumor is not refractory, this method further enhances sensitivity to the immune response so that enhanced tumor cell death is achieved.

[0066] The data provided herein also support the use of RNA NPs of this disclosure to increase dendritic cell (DC) activation in a subject. Thus, methods for activating or increasing DC activation in a subject are further provided. In exemplary embodiments, this method involves administering the pharmaceutical composition of this disclosure to a subject. In exemplary embodiments, the nucleic acid molecule is mRNA. Optionally, the composition is administered systemically to the subject. For example, the composition is administered intravenously. In various embodiments, the pharmaceutical composition is administered in an amount effective in increasing the immune response against the tumor of 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 exemplary embodiments, the immune response is an innate immune response.

[0067] This disclosure also provides a method for increasing the susceptibility of tumors to treatment with immune checkpoint inhibitors (ICIs) in a subject. In exemplary embodiments, the method involves administering a composition to a subject comprising nanoparticles described herein, for example, 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 being positioned between cationic lipid bilayers, wherein the composition is optionally administered systemically to the subject.

[0068] This disclosure further provides a method for treating subjects having immune checkpoint inhibitor (ICI)-resistant tumors. In exemplary embodiments, the method comprises administering to a subject (a) a composition comprising nanoparticles as described herein, for example, 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 being positioned between cationic lipid bilayers, and (b) an ICI. Optionally, the composition is administered systemically to the subject.

[0069] As used herein, “immune checkpoint inhibitor” or “ICI” is any agent (e.g., a compound or molecule) that reduces, blocks, inhibits, suppresses, or interferes with the function of proteins in the immune checkpoint pathway. Proteins in the immune checkpoint pathway modulate the immune response and, in some examples, prevent T cells from attacking cancer cells. In various embodiments, proteins in the immune checkpoint pathway are, for example, CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, TIGIT, VISTA, LAG3, CD112, TIM3, BTLA, or the costimulatory receptors ICOS, OX40, 41BB, or GITR. In various embodiments, an ICI is a small molecule, an inhibitory nucleic acid, or an inhibitory polypeptide. In various embodiments, an ICI is an antibody, an antigen-binding antibody fragment, or an antibody-protein product that binds to and inhibits the function of proteins in the immune checkpoint pathway. Preferred ICIs, which are antibodies, antigen-binding antibody fragments, or antibody protein products, are known in the art and are not limited to, but include ipilimumab (CTLA-4; Bristol Meyers Squibb), nivolumab (PD-1; Bristol Meyers Squibb), pembrolizumab (PD-1; Merck), atezolizumab (PD-L1; Genentech), avelumab (PD-L1; Merck), and durvalumab (PD-L1; Medimmune) (Wei et al.). This includes Cancer Discovery 8:1069-1086 (2018). Other examples of ICIs include, but are not limited to, IMP321 (LAG3: Immuntep), BMS-986016 (LAG3; Bristol Meyers Squibb), IPH2101 (KIR; Innate Pharma), tremelimumab (CTLA-4; Medimmune), pidilizumab (PD-1; Medivation), MPDL3280A (PD-L1; Roche), MEDI4736 (PD-L1; AstraZeneca), MSB0010718C (PD-L1; EMD Serono), AUNP12 (PD-1; Aurigene), MGA271 (B7-H3: MacroGenics), and TSR-022 (TIM3; Tesaro).

[0070] In various embodiments, ICIs are PD-L1 inhibitors. Programmed death ligand 1 (PD-L1; also known as differentiation cluster 274 (CD274) or B7 homolog 1 (B7-H1)) is a transmembrane protein that functions to suppress the immune system, for example, in pregnancy, tissue allografts, and autoimmune diseases. When PD-L1 binds to its receptor PD-1, it transmits a repressive signal that can reduce T cell proliferation and function and induce apoptosis. For example, PD-L1 inhibitors bind to PD-L1 and inhibit its function. In various embodiments, PD-L1 inhibitors are anti-PD-L1 antibodies, antigen-binding antibody fragments, or antibody-like molecules.

[0071] In various embodiments, ICIs are PD-1 inhibitors. "Programmed Death-1" (PD-1), also known as differentiation cluster 279 (CD279), refers to an immunosuppressive receptor belonging to the CD28 family. PD-1 is expressed in vivo on previously activated T cells and binds to two ligands, PD-L1 and PD-L2. The human PD-1 sequence can be found under GenBank Accession number U64863. For example, PD-1 inhibitors bind to PD-1, e.g., anti-PD-1 antibodies, antigen-binding antibody fragments, or antibody-like molecules, and inhibit its function. In various embodiments, PD-1 inhibitors are durvalumab, atezolizumab, or avelumab. In various embodiments, ICIs are PD-L2 inhibitors. For example, PD-L2 inhibitors bind to PD-L2, e.g., anti-PD-L2 antibodies, antigen-binding antibody fragments, or antibody-like molecules, and inhibit its function.

[0072] Examples of PD-1 inhibitors and PD-L1 inhibitors are described, for example, in U.S. Patents 7,488,802, 7,943,743, 8,008,449, 8,168,757, 8,217,149, and PCT Publications 03 / 042402, 2008 / 156712, 2010 / 089411, 2010 / 036959, 2011 / 066342, 2011 / 159877, 2011 / 082400, and 2011 / 161699, which are incorporated herein by reference in their entirety.

[0073] Cytotoxic T lymphocyte-associated protein 4 (CTLA-4, also known as CD152) is a membrane protein expressed on T cells and regulatory T cells (Tregs). CTLA-4 binds to B7-1 (CD80) and B7-2 (CD86) on antigen-presenting cells (APCs) and inhibits the adaptive immune response. In humans, CTLA-4 is encoded in various isoforms, and an exemplary amino acid sequence is available as GenBank Accession number NP_001032720. A representative anti-CTLA-4 antibody is ipilimumab (YERVOY®, Bristol-Myers Squibb).

[0074] As used herein, the term “antibody” includes heavy and light chains and variable region This refers to proteins having a conventional immunoglobulin form, including a constant region. For example, an antibody can be an IgG, which is a "Y-type" structure of two identical pairs of polypeptide chains, each pair having one "light" chain (typically having a molecular weight of about 25 kDa) and one "heavy" chain (typically having a molecular weight of about 50–70 kDa). Antibodies can be cleaved into fragments by enzymes such as papain and pepsin. Papain cleaves the antibody to produce two Fab fragments and one Fc fragment. Pepsin cleaves the antibody to produce an F(ab')2 fragment and a pFc' fragment. In exemplary embodiments, ICI is an antigen-binding antibody fragment, e.g., Fab, Fc, F(ab')2, or pFc'. Antibody architectures have been utilized to generate a growing range of alternative antibody formats spanning a molecular weight range of at least or about 12–150 kDa, from monomers (n=1), dimers (n=2), and trimers (n=3) to tetramers (n=4) and potentially more, such alternative antibody formats are referred to herein as “antibody-like molecules.” Antibody-like molecules may be antigen-binding formats based on antibody fragments that retain full antigen-binding ability, e.g., scFv, Fab, and VHH / VH. The smallest antigen-binding fragment that retains full antigen-binding sites is the Fv fragment, which consists only of a variable (V) region. A soluble and flexible amino acid peptide linker is used to annex the V region to the scFv fragment (single-chain fragment variable) for molecular stabilization, or to add a constant (C) domain to the V region to produce a Fab fragment [fragment, antigen-binding]. Both scFv and Fab are widely used fragments that can be readily produced in prokaryotic hosts. Other antibody-like molecules include various formats consisting of scFv linked to oligomerized domains, such as disulfide-stabilized scFv (ds-scFv), single-chain Fab (scFab), and dimer and multimer antibody formats such as diabodies, triabodies, tetrabodies, or minibodies (miniAb). The smallest fragments are the VHH / VH and single-domain Abs (sdAb) of camel heavy-chain Abs.The most frequently used building blocks for creating new antibody fragments are single-chain variable (V)-domain antibody fragments (scFv) containing V domains (VH and VL domains) derived from heavy and light chains linked by a peptide linker of approximately 15 amino acid residues. Peptibodies, or peptide-Fc fusions, are yet another antibody-like molecule. The structure of a peptibody consists of a biologically active peptide transplanted into an Fc domain. Peptibodies have been well described in the art; see, for example, Shimamoto et al., mAbs 4(5):586-591 (2012). Other antibody-like molecules include single-chain antibodies (SCAs), diabodies, triabodies, tetrabodies, and bispecific or trispecific antibodies. Bispecific antibodies can be classified into five main classes: BsIgG, IgG adducts, BsAb fragments, bispecific fusion proteins, and BsAb conjugates. See, for example, Spiess et al., Molecular Immunology 67(2) Part A:97-106 (2015). In exemplary embodiments, an antibody-like molecule comprises any one of these antibody-like molecules (e.g., scFv, Fab VHH / VH, Fv fragment, ds-scFv, scFab, dimeric antibodies, multimeric antibodies (e.g., diabody, triabody, tetrabody), miniAb, peptibody VHH / VH of camel heavy chain antibody, sdAb, diabody, triabody, tetrabody, bispecific or triplicate antibodies, BsIgG, IgG adduct, BsAb fragment, bispecific fusion protein, and BsAb conjugate).

[0075] As used herein, the term “inhibit” and any derived terms do not require 100% or complete inhibition or suppression. Rather, there are various degrees of inhibition that a person skilled in the art would recognize as having a potential benefit or therapeutic effect. ICI 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 this 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). This includes 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, and at least or about 98% inhibition.

[0076] As used herein, “sensitivity” refers to the manner in which a tumor responds to a drug / compound, e.g., an ICI inhibitor (e.g., a PD-L1 inhibitor). In exemplary embodiments, “sensitivity” means “responsiveness to treatment,” and the concepts of “sensitivity” and “responsiveness” are positively related in that a tumor or cancer cell that responds to drug / compound treatment is said to be sensitive to that drug. In exemplary embodiments, “sensitivity” is defined, according to the Pelikan, Edward Glossary of Terms and Symbols used in Pharmacology (Pharmacology and Experimental Therapeutics Department Glossary at Boston University School of Medicine), as the ability of a group, individual, or organization to respond qualitatively normal to a particular drug dose, compared to the ability of others. The lower the dose required to produce an effect, the more sensitive the response system becomes. “Sensitivity” may be quantitatively measured or described in relation to the intersection of a dose-effect curve and the x-axis value axis or a line parallel thereto, where such a point corresponds to the dose just required to produce a given degree of effect. Similarly, the “sensitivity” of a measurement system is defined as the minimum input (minimum dose) required to produce a given degree of output (effect). In exemplary embodiments, “sensitivity” is the opposite of “resistance,” and the concept of “resistance” is negatively related to “sensitivity.” For example, a tumor resistant to drug therapy is neither sensitive to nor responsive to the drug, and the drug is not an effective treatment for that tumor or cancer cells. In relation to ICIs, an ICI-insensitive tumor is one that does not respond to ICI treatment in a clinically significant manner. Improving the susceptibility of a tumor to ICIs encompasses, for example, any improvement in clinical responsiveness to ICI therapy, which may be detected by a reduction in tumor volume or an increase in tumor cell death, a reduction in the dose of ICI required to achieve a clinically detectable response, or an increase in the time interval between ICI administrations (requiring less frequent administration) while maintaining a clinically detectable response. “Sensitivity” is also used herein in relation to the host immune response.In this respect, tumors that evade the host's immune response are "resistant" (or refractory). Tumors that are "sensitive" to the host's immune response are recognized by the host's immune system and attacked by immune effector cells. Tumors that are "sensitive" to the host's immune response are recognized by the host's immune system and attacked by immune effector cells.

[0077] In the context of this disclosure, administration of the RNA-LPs of this disclosure sensitizes the tumor to ICIs, and the two activators together enhance the tumor's sensitivity to the host immune response. Notably, the RNA-LPs of this disclosure can transform immunologically “cold” tumors, e.g., tumors lacking invasive T cells and / or unrecognized by the immune system, into immunologically “hot” tumors, i.e., tumors exhibiting lymphocyte infiltration and interferon-gamma production in the tumor microenvironment. As described herein, immunological treatment of “cold” tumors presents significant challenges, at least in part, due to the absence of an adaptive immune response. Cancers prone to becoming immunologically “cold” tumors include, but are not limited to, glioblastoma, ovarian cancer, prostate cancer, pancreatic cancer, and many breast cancers. However, “cold” tumors are limited to these types of cancers, and as cancer progresses in the target, some cancers develop resistance mechanisms that allow them to evade the immune system. Remarkably, the nanoparticles of this disclosure “reprogram” tumors so that they are recognized by the host immune system. The materials and methods of this disclosure generally represent a significant advance in the art by providing means to expand the patient population that responds to ICIs and immunotherapies.

[0078] The increased sensitivity provided by the methods of this disclosure may be at least or about 1% to about 10% compared to a control (e.g., at least or about 1%, at least or about 2%, at least or about 3%, at least or about 4%, at least or about 5%, at least or about 6%, at least or about 7%, at least or about 8%, at least or about 9%, at least or about 9.5%, at least or about 9.8%, at least or about 10%). The increased sensitivity provided by the methods of this disclosure may be at least or about 10% to more than about 95% compared to a control (e.g., at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 95%, at least or about 98%, at least or about 100%). In exemplary embodiments, the control is a subject or population of subjects that has not been treated with the pharmaceutical composition of the Disclosure, or a population of subjects that has been treated with a placebo. In some embodiments, the "control" is a subject's tumor or cancer prior to FNA-LP treatment.

[0079] Increased sensitivity to ICI or increased sensitivity to the host immune response can be determined by one of many methods. For example, administration of RNA-LP and ICI may increase the number of cytotoxic T cells in tumors and / or enhance cytotoxic T cell activity. For example, in various embodiments, the method may increase the production of perforin, IFN-gamma, and / or granzymes by cytotoxic T cells and increase cytolytic activity. Furthermore, the methods described herein may enhance T cell survival, accelerate T cell lifespan, and / or limit the loss of replication capacity. Methods for measuring T cell activity and immune responses are known in the art. T cell activity can be measured by cytotoxic assays, such as those described in Fu et al., PLoS ONE 5(7):e11867 (2010). Other T cell activity assays are described in Bercovici et al., Clin Diagn Lab Immunol. 7(6):859-864 (2000). Methods for measuring the immune response include, for example, Macatangay et al. This is described in Clin Vaccine Immunol 17(9):1452-1459 (2010) and Clay et al., Clin Cancer Res. 7(5):1127-35 (2001). In various embodiments, the methods of this disclosure enhance cytotoxic T cell-mediated killing of cancer cells within tumors.

[0080] The methods of the present disclosure may include the above steps alone or in combination with other steps. The methods may include repeating any one of the above steps and / or including additional steps separate from those described above. For example, the currently disclosed methods may further include steps for producing or preparing the nanoparticles or compositions of the present disclosure. For example, the currently disclosed methods may further include, optionally, obtaining a sample of the tumor of interest by biopsy. The methods may also further include isolating total RNA from the tumor cells, generating cDNA from the total RNA by reverse transcription, and amplifying mRNA from the cDNA. In some embodiments, the methods of the present disclosure may also further include mixing mRNA and cationic lipids in an RNA:cationic lipid ratio of about 1 to about 10 to about 1 to about 20 (e.g., about 1 to about 19, about 1 to about 18, about 1 to about 17, about 1 to about 16, about 1 to about 15, about 1 to about 14, about 1 to about 13, about 1 to about 12, about 1 to about 11). In an exemplary example, the currently disclosed method further includes mixing mRNA with cationic lipids in an RNA:cationic lipid ratio of about 1 to about 15.

[0081] In exemplary embodiments, the method includes administering an ICI to a subject. In this regard, the present disclosure describes a method for treating a subject having an immune checkpoint inhibitor (ICI)-resistant tumor. The following is provided. In an exemplary embodiment, the method comprises (a) a composition comprising liposomes comprising cationic lipids and nucleic acid molecules, and (b) administering a PD-L1 inhibitor to a subject, the liposomes being systemically administered to the subject. The composition and liposomes may be any of those described herein. For example, the liposomes (liposome nanoparticles) may comprise DOTAP, and the nucleic acid molecules may be a mixture of mRNAs expressed by the tumor of the subject. In an exemplary embodiment, the composition comprising liposomes comprises a heterogeneous mixture of liposomes of different sizes, having a diameter in the range of 50 nm to about 250 nm. In an exemplary embodiment, the liposomes have a zeta potential of about 30 mV to about 60 mV, optionally, about 40 mV to about 50 mV. In an exemplary embodiment, the PD-L1 inhibitor is a PD-L1 antibody. PD-L1 inhibitors are known in the art and include, but are not limited to, atezolizumab, avelumab, and durvalumab.

[0082] This disclosure further provides a method for increasing activated plasma cell-like (pDC) populations in a subject, comprising administering the subject to the nanoparticles (or compositions comprising the nanoparticles) described herein. The disclosed method is useful, for example, in settings related to the treatment and preparation of dendritic cell (DC) vaccines. DC vaccines are outlined in Pyzer et al., Hum Vaccin Immunother 10(11):3125-3131 (2014). In exemplary embodiments, the currently disclosed method for increasing activated pDCs in a subject may further include isolating pDCs from the subject. pDCs are distinguished from other DC subsets by the expression of the surface markers CD303 (BDCA2), CD304 (BDCA4), CD123 (IL-3R), and CD45RA in humans. Musumeci et al., Front.Immunol.2019, Vol.10, Article1222. Methods for obtaining pDCs from a subject are known in the art and include, for example, leukocytapheresis. The pDCs obtained from the subject in this manner can be cultured and primed for antigen presentation. Thus, the pDCs can be loaded with antigens, for example, by pulsing the cells with antigen peptides or whole tumor cells as an antigen source. Alternatively, or further, the pDCs can be primed or activated by culturing them with a fusion protein containing prostatic acid phosphatase (PAP) and GM-CSF. The fusion protein may be the same as that found in PROVENGE® (Cyproisel-T). The pDCs, once primed, may then be administered to the subject from which they were obtained. In exemplary embodiments, the pDCs are administered intradermally or subcutaneously to the subject.

[0083] Accordingly, the Disclosure also provides a method for treating a subject having a tumor or cancer. In an exemplary embodiment, the method includes (i) increasing the number of activated plasmacytoid dendritic cells (pDCs) in a subject according to the method of the Disclosure, (ii) isolating leukocytes (WBCs) from the subject, (iii) isolating dendritic cells (DCs) from the WBCs, (iv) contacting the DCs with a fusion protein comprising prostatic acid phosphatase (PAP) and GM-CSF, and (v) administering the DCs to the subject. The Disclosure also provides a method for preparing a dendritic cell vaccine. In an exemplary embodiment, the method includes (i) increasing the number of activated plasmacytoid dendritic cells (pDCs) in a subject according to the method of the Disclosure, (ii) isolating leukocytes (WBCs) from the subject, (iii) isolating dendritic cells (DCs) from the WBCs, and (iv) contacting the DCs with a fusion protein comprising prostatic acid phosphatase (PAP) and GM-CSF. In exemplary embodiments, DCs are genetically engineered to express a protein. In some embodiments, the protein is a tumor antigen. In other embodiments, the protein is an antigen-presenting molecule fused to a peptide, such as MHC. WBCs can be isolated by known techniques, for example, leukocyte apheresis. Isolation of DCs from WBCs can be achieved by methods known in the art, such as fluorescence-activated cell sorting (FACS). When used herein, "prostatic acid phosphatidyl" The term "PAP" refers to a glycoprotein synthesized by the prostate gland that functions as an acid phosphatase, hydrolyzing phosphate esters in an acidic medium. PAP was identified more than 50 years ago as a marker for prostate cancer.

[0084] With respect to the methods disclosed herein, the nanoparticles, in various embodiments, comprise at least three (e.g., at least four or at least five or more) nucleic acid layers, each arranged between cationic lipid bilayers. In various examples, the outermost layer of the nanoparticle comprises a cationic lipid bilayer. In exemplary embodiments, the surface comprises multiple hydrophilic portions of the cationic lipids in the cationic lipid bilayer. Optionally, the core comprises a cationic lipid bilayer. In various examples, the core comprises less than about 0.5% by weight of nucleic acid. In exemplary embodiments, the diameter of the nanoparticles is about 50 nm to about 250 nm in diameter, and optionally, about 70 nm to about 200 nm in diameter. In various embodiments, the nanoparticles have a zeta potential of about 40 mV to about 60 mV, and optionally, about 45 mV to about 55 mV. Optionally, the nanoparticles have a zeta potential of about 50 mV. In exemplary embodiments, nanoparticles contain nucleic acid molecules and cationic lipids in a ratio of approximately 1:5 to approximately 1:20, optionally, approximately 1:15 or approximately 1:7.5. In various embodiments, the cationic lipid is DOTAP or DOTMA. Optionally, the nucleic acid molecule is an RNA molecule. In various examples, the RNA molecule is mRNA. In certain embodiments, the mRNA is in vitro transcribed mRNA, and the in vitro transcription template is a cDNA made from RNA extracted from tumor cells. In various embodiments, the mRNA encodes a protein. In some examples, the protein is selected from a group consisting of tumor antigens, cytokines, or costimulatory molecules. Optionally, the protein is not expressed by tumor cells or humans. In other examples, the RNA molecule is an antisense molecule, optionally, siRNA, shRNA, miRNA, or any combination thereof. Optionally, the nanoparticles contain a mixture of RNA molecules. In various examples, the mixture of RNA molecules contains RNA isolated from human-derived cells. In specific cases, a human has a tumor, and the RNA mixture is RNA isolated from human tumors, and the tumor is, at random selection, a malignant brain tumor, and at random selection, a glioblastoma, medulloblastoma, diffuse endogenous pontine glioma, or a peripheral tumor with metastatic invasion into the central nervous system.Optionally, nanoparticles are prepared by mixing nucleic acid molecules and cationic lipids in an RNA:cationic lipid ratio of approximately 1:5 to approximately 1:20, or optionally, approximately 1:15. In exemplary embodiments, the composition is administered systemically via parenteral administration, or optionally, intravenous administration. In exemplary embodiments, the subjects have immune checkpoint inhibitor (ICI) resistant tumors. Optionally, the pDC is PD-L1. + / CD86 + It is pDC.

[0085] As used herein, the term “increase” and any derived terms may not mean a 100% or complete increase. Rather, there are varying degrees of increase that a person skilled in the art would recognize as having a potential benefit or therapeutic effect. In exemplary embodiments, the increase provided by this method is an increase of at least or about 10% (e.g., at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 95%, at least or about 98%). In various embodiments, “increase” is related to baseline measurements (e.g., baseline immunity, sensitivity, or activation) before administration of the nanoparticles of this disclosure (e.g., before administration).

[0086] This disclosure also provides a method for delivering RNA molecules to the tumor microenvironment, lymph nodes, and / or reticuloendothelial organs. In exemplary embodiments, this method involves administering the pharmaceutical composition disclosed herein to a target. If necessary, the reticuloendothelial organ is the spleen or liver. This specification provides a method for delivering RNA to cells of a tumor, for example, a brain tumor, comprising intravenous administration of the composition of this disclosure, wherein the composition comprises nanoparticles. Furthermore, this specification also provides a method for delivering RNA to cells in the microenvironment of a tumor, optionally a brain tumor. In exemplary embodiments, this method comprises administering a composition of the Disclosure systemically (e.g., intravenously), the composition comprising nanoparticles. In some embodiments, the nanoparticles comprise proteins of an immune checkpoint pathway, optionally siRNA targeting PDL1. In various embodiments, the cells in the microenvironment are antigen-presenting cells (APCs), optionally tumor-associated macrophages. This disclosure also provides a method for activating antigen-presenting cells in the brain tumor microenvironment. In exemplary embodiments, this method comprises administering a composition of the Disclosed herein systemically (e.g., intravenously), the composition comprising NPs.

[0087] This disclosure provides a method for delivering RNA molecules to cells. In exemplary embodiments, the method includes incubating cells with NPs of this disclosure. In exemplary embodiments, the cells are antigen-presenting cells (APCs), optionally dendritic cells (DCs). In various embodiments, the APCs (e.g., DCs) are obtained from a subject. In certain embodiments, the RNA molecule is isolated from a subject, e.g., tumor cells obtained from a human. In certain embodiments, the RNA molecule is an antisense molecule that targets and reduces the expression of a protein of interest. In exemplary embodiments, the RNA molecule is an siRNA molecule that targets a protein in the immune checkpoint pathway. Suitable proteins in the immune checkpoint pathway are known in the art and are described herein. In various embodiments, the siRNA targets PDL1.

[0088] Once RNA is delivered to cells, the cells can be administered to a subject for the treatment of a disease. Therefore, this disclosure provides a method for treating a subject having a disease. In exemplary embodiments, the method includes delivering RNA molecules to cells of a subject according to the above-described method for delivering RNA molecules to cells. In some embodiments, the RNA molecules are delivered to cells ex vivo, and the cells are administered to the subject. Alternatively, the method includes directly administering liposomes to the subject. In exemplary embodiments, a method for treating a subject having a disease includes administering a composition of the disclosure in the subject in an amount effective to treat the disease. In exemplary embodiments, the disease is cancer, and in some embodiments, the cancer is located across the blood-brain barrier and / or the subject has a tumor located in the brain. In some embodiments, the tumor is a glioma, a low-grade glioma, or a high-grade glioma, specifically a grade III astrocytoma or glioblastoma. Alternatively, the tumor may be a medulloblastoma or diffuse endogenous pontine glioma. In another example, the tumor may be a non-central nervous system tumor, such as a metastatic invasion from 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 groin of the subject. In an exemplary embodiment, DCs are isolated from leukocytes (WBCs) obtained from the subject, and optionally, WBCs are obtained via leukocyte depletion. In some embodiments, RNA molecules encode tumor antigens. In some embodiments, RNA molecules are isolated from tumor cells, for example, tumor cells are cells of the tumor of the subject. Thus, methods for treating subjects having a disease are further provided herein. In exemplary embodiments, the method includes delivering the RNA molecule to cells of interest by a method disclosed herein that delivers the RNA molecule to the tumor microenvironment, lymph nodes, and / or retinoendothelial organs. In various embodiments, the RNA molecule is delivered to the cells ex vivo, and the cells are administered to the subject.In exemplary embodiments, the method comprises administering to a subject an amount effective to treat the disease of interest. In various examples, the subject has cancer or tumors, optionally, malignant brain tumors, optionally, glioblastoma, medulloblastoma, diffuse endogenous pontine glioma, or peripheral tumors with metastatic invasion of the central nervous system.

[0089] As used herein, the term “to treat” and related terms do not necessarily mean 100% or complete treatment. Rather, there are various degrees of treatment that a person skilled in the art would recognize as having a potential benefit or therapeutic effect. In this regard, the methods for treating cancer of this disclosure may provide any amount or any level of treatment. Furthermore, the treatment provided by the methods may include treatment of one or more conditions, symptoms or signs of the disease being treated. For example, the treatment methods of this disclosure may suppress one or more symptoms of the disease. Also, the treatment provided by the methods of this disclosure may include slowing the progression of the disease. For example, the methods may treat cancer by reducing tumor or cancer growth, reducing metastasis of tumor cells, or increasing cell death of tumor or cancer cells.

[0090] The term “treat” also encompasses preventive treatment of a disease. Therefore, treatment provided by the methods of this disclosure may delay the onset or recurrence of a disease being treated preventively. In exemplary embodiments, the methods delay the onset of a disease by 1, 2, 4, 6, 8, 10, 15, 30 days, 2 months, 4 months, 6 months, 1 year, 2 years, 4 years, or more. Preventive treatment encompasses reducing the risk of a disease being treated. In exemplary embodiments, the methods reduce the risk of a disease by 2, 5, 10, 20, 50, 100, or more.

[0091] In certain embodiments, a method for treating a disease may be considered a method for inhibiting the disease or its symptoms. As used herein, the term “inhibit” and any derived terms may not mean 100% or complete inhibition. Rather, there are varying degrees of inhibition that a person skilled in the art would recognize as having a potential benefit or therapeutic effect. The methods disclosed herein may 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 disclosed herein 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).

[0092] The susceptibility of a tumor to an immune response (or ICI), or in other words, the effectiveness of an immune response (or ICI) against a tumor, can be determined in various ways. Similarly, the treatment of a subject for cancer may be determined by any of a number of methods. Any improvement in the subject's health status is intended (e.g., a reduction of at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 95%). For example, a therapeutic response refers to one or more of the following improvements in the disease: (1) a reduction in the number of neoplastic cells, (2) an increase in neoplastic cell death, (3) an inhibition of neoplastic cell survival, (5) an inhibition of tumor growth or the appearance of new lesions (i.e., a degree of delay, preferably cessation), (6) a reduction in tumor size or burden, (7) the absence of clinically detectable disease, (8) a decrease in cancer marker levels, (9) an increase in patient survival rate, and / or (10) some relief of one or more symptoms associated with the disease or condition (e.g., pain). For example, the effectiveness of treatment may be determined by detecting changes in tumor volume and / or tumor size after treatment. Tumor size may be compared to initial size and dimensions measured by CT, PET, mammography, ultrasound, or palpation, as well as by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. The response may be, for example, a percentage change in tumor volume. The response may be quantitatively characterized using changes in duration (for example, the method of this disclosure results in a reduction of at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% of tumor volume). Alternatively, the tumor response or cancer response may be characterized qualitatively by criteria such as “pathological complete response” (pCR), “clinical complete remission” (cCR), “clinical partial remission” (cPR), “clinically stable disease” (cSD), “clinically progressive disease” (cPD), or other qualitative criteria. Furthermore, the effectiveness of the treatment may be characterized in terms of responsiveness to other immunotherapies or chemotherapy. In various embodiments, the method of this disclosure further includes monitoring the treatment in the subject.

[0093] With respect to the methods described above, the NP or a composition containing it is administered systemically to a subject in some embodiments. Optionally, the method includes parenteral administration of the liposome or composition.

[0094] Parenteral dosage forms of any drug described herein may be administered to a subject by a variety of routes, including, but not limited to, epidural, intracerebral, intraventricular, intracutaneous, intra-arterial, intra-articular, intracardiac, intracavernosal injection, intradermal, intrafocal, intramuscular, intraocular, intraosseous, intraperitoneal, intrathecal, intrauterine, intravaginal administration, intravenous, intravesical, intravitreous, subcutaneous, transdermal, perivascular, or transmucosal administration. For administration to the brain, the pharmaceutical composition may be introduced into tumor tissue using an intratumoral delivery catheter, a ventricular shunt catheter attached to a reservoir (e.g., an Omaya reservoir), an infusion pump, or into a tumor resection cavity (Gliasite, Proxima Therapeutics). Tumor tissue in the brain may also be in contact with the pharmaceutical composition by administering it via convection using a continuous infusion catheter or via cerebrospinal fluid. In various examples, liposomes or compositions are administered intravenously to the subject.

[0095] For the purposes of this disclosure, the amount or dose of the activator administered (i.e., the “effective dose”) must be sufficient to achieve the desired biological effect, e.g., a therapeutic or prophylactic response, in a subject over a reasonable time frame. For example, one or more doses of the nanoparticles and ICIs described herein must be sufficient to sensitize a tumor to an immune response (and optionally treat cancer) over a clinically acceptable period, e.g., 1 to 20 weeks or longer from the first dose. In certain embodiments, the period may be longer. As an example and not intended to limit this disclosure, doses of the activators of this disclosure may be about 0.0001 to about 1 g / kg of body weight / day of the subject being treated, about 0.0001 to about 0.001 g / kg of body weight / day, or about 0.01 mg to about 1 g / kg of body weight / day.

[0096] Optionally, the composition may be systemically administered in amounts effective to increase the number of PD-L1+ / CD86+ myeloid antigen-presenting cells (APCs) around tumors and / or in reticuloendothelial organs, increase PD-L1 / CD86 expression by plasmacytoid dendritic cells (pDCs) and CD11c+ myeloid cells, increase type I interferon release by pDCs, activate T cell responses, or a combination thereof.

[0097] In examples where the method involves administering the nanoparticles and ICI of the Disclosure to a subject, the nanoparticle composition and ICI may be administered together (in the same formulation or in separate formulations administered within a time period) or sequentially (i.e., the nanoparticle composition may be administered and the ICI may be administered separately at different times (e.g., at intervals of several hours or several days). In this regard, the nanoparticle composition of the Disclosure may be administered prior to the ICI, for example, at least about 6 hours, at least about 12 hours, at least about 18 hours, or at least about 24 hours before the administration of the ICI. In this regard, the nanoparticles may be administered at least about 3 days, 1 week, 2 weeks, 3 weeks, 4 weeks (i.e., 1 month), 2 months, or 3 months before the administration of the ICI. This is possible. For example, the method may, in various cases, include a first period of nanoparticle therapy followed by a second period of ICI therapy. The second period of ICI therapy may also be accompanied by nanoparticle therapy to enhance the immune response (for example, the second period may include both ICI administration and nanoparticle administration). The first period of nanoparticle administration may involve multiple doses of nanoparticles administered over a treatment period of one, two, three, four, five, or six weeks prior to ICI administration, e.g., two, three, four, five, or more doses administered to the subject. For example, in an exemplary regimen, three doses of RNA-NP are administered to the subject over one month, after which the subject is treated with ICI (optionally, in combination with RNA-NP therapy).

[0098] In various embodiments, the NP or composition is administered according to any regimen, including, for example, daily (once, twice, three times, four times, five times, 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 the subject once a week.

[0099] This disclosure further provides kits containing immune checkpoint inhibitors (e.g., PD-1 antigen-binding proteins such as anti-PD-1 antibodies) and nanoparticle compositions in containers with instructions for use. In exemplary embodiments, the checkpoint inhibitors and nanoparticle compositions are provided in the kit as unit doses. "Unit dose" refers to individual amounts dispersed in a suitable carrier. In exemplary embodiments, the unit dose is an amount sufficient to produce a desired effect on a subject, e.g., cancer cell death. In exemplary embodiments, the kit includes several unit doses, e.g., weekly or monthly supplies of unit doses, each optionally packaged individually or otherwise separated from other unit doses. In some embodiments, the components of the kit / unit dose are packaged with instructions for administration to a patient. In some embodiments, the kit includes one or more devices for administration to a patient, e.g., needles and syringes. In some embodiments, the components of the kit are pre-packaged in ready-to-use forms, e.g., syringes, intravenous bags, etc. In exemplary embodiments, the ready-to-use forms are single-use. In exemplary embodiments, the kit includes multiple single-use, ready-to-use forms of the components. In some embodiments, the kit further comprises other therapeutic or diagnostic agents or pharmaceutically acceptable carriers (e.g., solvents, buffers, diluents, etc.) including any of those described herein.

[0100] subject The subjects are mammals including, but not limited to, rodents such as mice and hamsters, lagomorphs such as rabbits, carnivores including felines (cats) and canines, artiodactyls including cats and pigs, or odd-toed ungulates including horses. In some embodiments, the mammals belong to the order Primates, Ceboids, or Simoids (monkeys), or to the suborder Haplorhini (humans and apes). In typical embodiments, the mammal is a human. In some embodiments, the human is an adult aged 18 years or older. In some embodiments, the human is a child aged 17 years or younger. In exemplary embodiments, the subject has DMG. In various examples, DMG is diffuse endogenous pontine glioma (DIPG).

[0101] In some embodiments, subjects may be subjects who have been previously diagnosed or identified as having or having a condition requiring treatment (e.g., cancer) or one or more complications related to such a condition, and who are optionally already receiving treatment for that condition or one or more complications related to that condition. Alternatively, subjects may also be subjects who have not been previously diagnosed with such a condition or related complications. For example, subjects The subject may have one or more risk factors for the condition, or one or more complications associated with the condition. The subject may have previously received treatment or therapy for the condition in various ways (e.g., previously received anti-cancer therapy).

[0102] cancer Cancers treatable by the methods disclosed herein may 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.

[0103] Cancer in some forms includes acute lymphoblastic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer (glioma), breast cancer (triple-negative breast cancer), anal, anal canal or anorectal cancer, eye cancer, intrahepatic cholangiocarcinoma, joint cancer, head, neck, gallbladder or pleura cancer, nose, nasal cavity or middle ear cancer, oral cancer, vulvar cancer, chronic lymphoblastic leukemia, chronic bone marrow cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer (e.g., gastrointestinal carcinoid tumor), Hodgkin lymphoma, and uterine cancer. The cancers are selected from the group consisting of endometrial cancer or hepatocellular carcinoma, hypopharyngeal cancer, renal cancer, laryngeal cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer, bronchioloalveolar carcinoma), malignant mesothelioma, melanoma, multiple myeloma, nasopharyngeal cancer, non-Hodgkin 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, gastric cancer, testicular cancer, thyroid cancer, ureteral cancer, and bladder cancer. In certain embodiments, the cancers are selected from the group consisting of head and neck, ovarian, cervical, bladder and esophageal cancers, pancreatic cancer, gastrointestinal cancer, stomach, breast, endometrial and colorectal cancers, hepatocellular carcinoma, glioblastoma, bladder and lung cancer (e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma). In various embodiments, the subjects have solid tumors. Participants are randomly selected and have malignant brain tumors such as glioblastoma, medulloblastoma, diffuse endogenous pontine glioma, or peripheral tumors with metastatic invasion to the central nervous system.

[0104] In some embodiments, the methods described herein further include the administration of one or more other therapeutic agents. In some embodiments, the other therapeutic agents are intended to treat or prevent cancer. In some embodiments, the other therapeutic agents are chemotherapeutic agents. Common chemotherapeutic agents include adriamycin, asparaginase, bleomycin, busulfan, cisplatin, carboplatin, carmustine, capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide, phloxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, Examples of therapeutic agents include, but are not limited to, irinotecan, lomustine, mechloretamine, mercaptopurine, mepralan, methotrexate, mitomycin, mitotane, mitoxantrone, nitrourea, paclitaxel, pamidronate, pentostatin, pricamycin, procarbazine, rituximab, streptozocin, teniposide, thioguanine, thiotepa, vinblastine, vincristine, vinorelbine, taxol, transplatinum, and 5-fluorouracil. In some embodiments, other therapeutic agents are drugs used in radiotherapy for the treatment of cancer, and in fact, in some embodiments, this method is part of a treatment regimen that includes radiotherapy. Furthermore, the method of this disclosure may be performed in connection with the surgical resection of tumors such as gliomas (e.g., glioblastomas).

[0105] The following embodiments are provided solely to illustrate the present invention and not to limit its scope in any way. [Examples]

[0106] Example 1 This example illustrates a method for producing the nanoparticles of the present disclosure.

[0107] Preparation of DOTAP liposomes On day 1, the following steps were performed in the fume hood: Water was added to the rotor vapor bath. Chloroform (20 mL) was poured into a sterile glass graduated cylinder. After opening the vial containing 1 g of DOTAP, 5 mL of chloroform was added to the DOTAP vial using a glass pipette. Next, a certain volume of chloroform and DOTAP was transferred to a 1 L evaporating flask. A second volume of 5 mL of chloroform was added to the DOTAP vial to dissolve any remaining DOTAP in the vial, and this amount of chloroform was transferred from the DOTAP vial to the evaporating flask to wash the DOTAP vial. This washing process was repeated two more times until all the chloroform in the graduated cylinder was used. Next, the evaporating flask was placed in the Buchi rotor vapor bath. The water bath was turned on and adjusted to 25°C. The evaporating flask was moved downwards until it touched the water bath. The rotor vapor bath rotation speed was adjusted to 2. The vacuum system was turned on and adjusted to 40 mbar. After 10 minutes, the vacuum system was switched off and the chloroform was collected from the collector flask. The amount of chloroform collected was measured. The collector flask was then moved, the vacuum was switched back on, and the contents of the evaporation flask were allowed to dry overnight until the chloroform had completely evaporated.

[0108] On the second day, using a sterile graduated cylinder, 200 mL of PBS was added to a new, sterile 500 mL PBS bottle maintained at room temperature. A second 500 mL PBS bottle was prepared for collecting DOTAP. The Buchi RotaVapor water bath was set to 50°C. 50 mL of PBS was added to the evaporating flask using a 25 mL disposable serum pipette. The evaporating flask was placed on the Buchi RotaVapor and moved downwards until one-third of the flask was submerged in the water bath. The RotaVapor rotation speed was set to 2 and rotated for 10 minutes, after which the rotation was stopped. 50 mL of PBS containing DOTAP from the evaporating flask was transferred to the second 500 mL PBS bottle. This process was repeated (3 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. During the 1-hour incubation period, the bottle was vortexed every 10 minutes. The second 500 mL PBS bottle was left at room temperature overnight.

[0109] On the third day, PBS (200 mL) was added to a second 500 mL PBS bottle containing DOTAP and PBS. The second 500 mL PBS bottle was placed in an ultrasonic bath. Water was added to the ultrasonic bath and the second 500 mL PBS bottle was ultrasonically treated for 5 minutes. The extruder was washed with PBS (100 mL) 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 inside the extruder's output tube. In a biological safety cabinet, the DOTAP-PBS mixture was loaded into the extruder until the third PBS bottle was approximately 70% full. The extruder was then powered on and the DOTAP-PBS mixture was added until all of the mixture had passed through the extruder. Subsequently, a 0.22 μm pore filter was attached to the filtration unit and a new (third) 500 mL PBS bottle was placed inside the extruder's output tube. The previously filtered DOTAP-PBS mixture was loaded and the process was run again throughout. Next, samples containing DOTAP lipid nanoparticles (NPs) in PBS were stored at 4°C.

[0110] RNA preparation Before integration into NPs, RNA was prepared using 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 prepared in-house or from suppliers. I bought it.

[0111] Total tumor RNA: Total tumor-derived RNA (e.g., B16F0, B16F10, and KR158-luc) from tumor cells was isolated using a commercially available RNeasy mini-kit (Qiagen) according to the manufacturer's instructions.

[0112] In vitro transcribed mRNA: Briefly, RNA was isolated using a commercially available RNeasy mini-kit (Qiagen) according to the manufacturer's instructions, and the cDNA library was generated by RT-PCR. Reverse transcriptase reactions by PCR were performed on all tumor RNA to generate the 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. The cDNA was purified using the Qiagen PCR purification kit according to the manufacturer's instructions. In vitro transcription was performed overnight on the cDNA library using the mMESAGE mMACHINE (Invitrogen) kit with T7 enzyme mix to isolate sufficient mRNA for use in each RNA nanoparticle vaccine. Housekeeping genes were evaluated to ensure transcriptional fidelity. The resulting mRNA was then purified using the Qiagen RNeasy Maxi kit to obtain the final mRNA product.

[0113] Tumor antigen-specific and non-specific mRNA: Plasmids containing DNA encoding tumor antigen-specific RNA (e.g., pp65, OVA-encoding RNA) and non-specific RNA (e.g., green fluorescent protein (GFP), luciferase-encoding RNA) are linearized using restriction enzymes (i.e., SpeI) and purified using the Qiagen PCR MiniElute kit. Subsequently, the linearized DNA is transcribed using the mmRNA in vitro transcription kit (Life Technologies, Invitrogen) and cleaned up using the RNA Maxi kit (Qiagen). Alternatively, non-specific RNA can be purchased from Trilink Biotechnologies (San Diego, CA).

[0114] Preparation of multilayer RNA nanoparticles (NPs) DOTAP lipid NPs were complexed with RNA to create multilayer RNA-NPs designed to have several layers of mRNA contained within densely coiled liposomes with a positively charged surface and an empty core (Figure 1A). Briefly, in a safety cabinet, the RNA was thawed from -80°C and then placed on ice, while samples containing PBS and DOTAP (such as DOTAP lipid NPs) were allowed to return to room temperature. Once the components were prepared, the desired amount of RNA was mixed with PBS in a sterile tube. To the sterile tube containing the RNA and PBS mixture, an appropriate amount of DOTAP lipid NPs was added without physical mixing (e.g., without inverting the tube, without vortexing, without stirring). The RNA, PBS, and DOTAP mixture was incubated for approximately 15 minutes to form multilayer RNA-NPs. 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.

[0115] The amounts of RNA and DOTAP lipid NPs (liposomes) used in the above preparations are determined or selected in advance. In some examples, a ratio of approximately 15 μg of liposomes per approximately 1 μg of RNA was used. For example, approximately 75 μg of liposomes per approximately 5 μg of RNA was used, or approximately 375 μg of liposomes per approximately 25 μg of RNA was used. In other examples, approximately 7.5 μg of liposomes per 1 μg of RNA was used. Therefore, in the exemplary examples, approximately 1 μg to approximately 20 μg of liposomes were used for all μg of RNA used. Liposomes are used.

[0116] Example 2 This example illustrates the characterization of the nanoparticles of this disclosure.

[0117] Cryogenic electron microscope (CEM) The structures of multilayer RNA-NPs prepared as described in Example 1, as well as RNA-free control NPs (uncomplexed NPs) prepared according to all steps of Example 1 except for the "RNA preparation" and "Preparation of multilayer RNA nanoparticles (NPs)" steps, were analyzed using CEM. CEM was performed as essentially described in Sayour et al., Nano Lett 17(3)1326-1335 (2016). Briefly, samples containing multilayer RNA-NPs or control NPs were held on ice before flash freezing by loading them into a Vitrobot (and an automated plunge freezer for cryoTEM, which freezes samples without ice crystal formation by controlling temperature, relative humidity, blotting conditions and freezing rate). The samples were then scanned using a Gatan UltraScan. Tecnai G2 F20 TWIN with 4000 (4k x 4k) CCD camera Images were obtained using a 200kV / FEG transmission electron microscope. The resulting CEM images are shown in Figure 1B. The right panel shows CEM images of multilayer RNA-NPs, and the left panel shows CEM images of control NPs (uncomplexed NPs). As shown in Figure 1B, the control NPs contained a maximum of two layers, while the multilayer RNA NPs contained multiple layers. Figure 5 shows another CEM image of an exemplary multilayer RNA NP. Here, the multiple layers of RNA alternating with lipid layers are particularly evident.

[0118] Zeta potential The zeta potential of multilayer RNA NPs is described by Sayour et al., Nano Lett. Measurements were performed by phase-analytic light scattering (PALS) using a Brookhaven ZetaPlus instrument (Brookhaven Instruments Corporation, Holtsville, NY), as essentially described in 17(3)1326-1335(2016). 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, for 25 cycles in each run, using the Smoluchowski model.

[0119] The zeta potential of the multilayer RNA NPs prepared as described in Example 1 was measured at approximately +50 mV. Interestingly, this zeta potential of the multilayer RNA NPs was much higher than that measured at around +27 mV as described in Sayour et al., Oncoimmunology 6(1):e1256527 (2016). Without being bound by any particular theory, a method for preparing DOTAP lipid NPs for use in the preparation of multilayer RNA NPs (Example 1), including a vacuum sealing method for chloroform evaporation, may result in less environmental oxidation of the DOTAP lipid NPs, which may allow more RNA to complex with the DOTAP NPs and / or increase the uptake of RNA into the DOTAP lipid NPs.

[0120] RNA uptake by gel electrophoresis: Gel electrophoresis was performed to measure the amount of RNA incorporated into ML liposomes. Based on this experiment, it was qualitatively shown that almost all, if not all, of the RNA used in the procedure described in Example 1 was incorporated into the DOTAP lipid NPs. Additional experiments to characterize the degree of RNA incorporation are performed by measuring the RNA-NP density and comparing this parameter with the parameter of the lipoplex.

[0121] Example 3 This example demonstrates that the in vivo localization site of RNA-NPs after systemic administration and that these RNA-NPs mediate peripheral and intratumoral activation of DCs.

[0122] DOTAP lipid NPs, prepared essentially as described in Example 1, are complexed with mRNA encoding Cre recombinase to produce Cre-encoding RNA-NPs. These multilayer RNA-NPs are administered to Ai14 transgenic mice equipped with a loxP-adjacent STOP cassette. The STOP cassette prevents tdTomato transcription until Cre-recombinase is expressed. One week after RNA-NP administration, lymph nodes, spleen, and liver are harvested, sectioned, and stained with DAPI from the transgenic mice. tdTomato expression is analyzed by fluorescence microscopy according to the procedure essentially 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.

[0123] DOTAP lipid NPs, prepared essentially as described in Example 1, were complexed with nonspecific RNA (e.g., RNA not specific to tumor antigens, ovalbumin (OVA) mRNA) and intravenously injected into C57Bl / 6 mice (n=3-4 / group) carrying subcutaneous B16F10 tumors. Lymph nodes, spleen, liver, bone marrow, and tumors were collected within 24 hours, and the expression of the dendritic cell (DC) activation marker CD86 was analyzed by CD11c cells (*p<0.05 Mann-Whitney test). OVA mRNA-NPs showed broad in vivo localization to lymph nodes, spleen, liver, bone marrow, and tumors, and were expected to activate DCs within them (indicated by increased expression of the activation marker CD86 on CD11c+ cells). Since activated DCs prepare antigen-specific T cell responses and lead to antitumor efficiency (with increased TILs) in several tumor models, we tested the antitumor effect of multilayer RNA NPs.

[0124] Example 4 This example describes a comparison of the nanoparticles of this disclosure with cationic RNA lipoplexes and anionic RNA lipoplexes.

[0125] Cationic lipoplexes (LPXs) were initially developed using mRNA from a lipid core shielded by a net positive charge on its outer surface (Figure 2A). Anionic RNA lipoplexes (Figure 2B) were developed using excess RNA tethered to the surface of bilayer liposomes. RNA-LPXs were prepared by mixing RNA and lipid NPs in a ratio that equalizes the charge. Anionic RNA-NPs were prepared by mixing RNA and lipid NPs in a ratio that supersaturates the negatively charged lipid NPs. Next, various embodiments of RNA-LPXs and anionic RNA LPXs were compared with the multilayer RNA NPs described in the above examples.

[0126] Cryo-electron microscopy (CEM) was used to compare the structures of RNA LPX and multilayer RNA-NPs prepared as described in Example 1. Uncomplexed NPs were used as a control. CEM was performed as essentially described in Example 2. Figure 2C is a CEM image of uncomplexed NPs, Figure 2D is a CEM image of RNA LPX (its mass ratio to RNA in liposomes is 3.75:1), and Figure 2E is a CEM image of multilayer RNA-NPs (its mass ratio to RNA in liposomes is 15:1). These data support that more RNA is retained by ML RNA-NPs. Additional data show that concentrated droplets with more ML RNA-NP complexing than RNA LPX are not observed by simply mixing equal amounts of RNA and lipid NPs by mass or charge (i.e., RNA-LPX and anionic RNA-LPX, respectively) with ML R This demonstrates the formation of multiple layers of NA NPs. This supports the idea that more RNA is "retained" by the ML RNA-NPs described herein.

[0127] Furthermore, experiments were conducted to determine where anionic LPX localizes to mice upon administration. As shown in Figure 8, anionic LPX localizes to the spleen of the animals upon administration, which is consistent with previous studies (Krantz et al, Nature 534:396-401 (2016)).

[0128] Mice were administered RNA LPX, anionic lipoplex (LPX), or multilayer RNA-NPs, and their spleens were collected one week later to evaluate activated DCs (*p<0.05, unpaired t-test). The RNA used in this experiment was tumor-derived mRNA from the K7M2 tumor osteosarcoma cell line. As shown in Figure 2F, mice treated with multilayer RNA-NPs showed the highest levels of activated DCs.

[0129] Anionic tumor mRNA-lipoplex, tumor mRNA-lipoplex, and multilayer tumor mRNA-loaded NPs were compared in a therapeutic lung cancer model (K7M2) (n=5-8 / group). Each vaccine was administered intravenously weekly (×3) (**p<0.01, Mann-Whitney). %CD44+CD62L+ in CD8+ splenocytes is shown in Figure 2G, and %CD44+CD62L+ in CD4+ splenocytes is shown in Figure 2H. Figure 2J also shows that multilayer (ML)RNA-NPs mediate substantially increased IFN-α, a natural 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. These data also indirectly support that ML RNA-NPs increase the number of activated plasmacytoid dendritic cells (pDCs), the most important IFN-α producing strains. In summary, these data demonstrate the superior efficacy of multilayer tumor-specific RNA-NPs compared to anionic LPX and RNA LPX.

[0130] Anionic tumor mRNA-lipoplexes, cationic tumor mRNA-lipoplexes, and multilayer tumor mRNA-loaded NPs were compared in a therapeutic lung cancer model (K7M2) (n=8 / group). Each vaccine was administered intravenously weekly (×3), *p<0.05, Gehan-Breslow-Wilcoxon test. Survival rates were measured by Kaplan-Meier curve analysis. As shown in Figure 2I, multilayer tumor-specific RNA-NPs mediated superior efficacy in enhancing survival compared to cationic RNA lipoplexes and anionic RNA lipoplexes.

[0131] We also investigated the ability of multilayer RNA-NPs to activate the innate immune response in vivo within the glioma tumor microenvironment.

[0132] RNA-NPs localize to the perivascular region of tumors and reprogram the tumor mesenteric cell microenvironment (TME) for activated bone marrow cells. Animals with K-luc (n=5 / group) were inoculated with tumor RNA-NPs or NPs alone. Tumors were harvested 48 hours later for RNA-seq analysis. Significant upregulation of gene signatures for BATF3, IRF, and IFN response genes was observed in animals administered RNA-NPs. In particular, the RNA-NPs of this invention significantly upregulated the expression of BATF3 (associated with effector dendritic cell phenotype), IRF5 and IRF7 (interferon regulators), and ISG15 and IFITM3 (interferon response genes). These genes have been shown to be essential for sensitizing to immunotherapy responses. Therefore, RNA-NPs upregulate key innate immune gene signatures in the glioma tumor microenvironment related to effector immune responses, effectively transforming tumors from "cold" to "hot" and in areas where immune checkpoint inhibitors were ineffective before RNA-NP treatment. To enable activation.

[0133] Here, multilayer RNA-NP formulations targeting physiologically relevant tumor antigens have been shown to be more immunogenic (Figures 2F-2H, 2J) and significantly more effective (Figure 2I) compared to anionic LPX and RNA LPX. Without being bound by any particular theory, a novel RNA-NP design consisting of a multilayer ring of tightly wound mRNA was developed by altering the RNA-lipid ratio and increasing the zeta potential (Figure 1C). This multilayer design is thought to promote increased NP uptake of mRNA (enriched by alternating positive / negative charges) to enhance the immunogenicity of the particles and extend in vivo localization to the peripheral and tumor microenvironment (TME). Systemic administration of these multilayer RNA-NPs localizes to lymph nodes, reticuloendothelial organs (i.e., spleen and liver), and the TME, where it activates DCs (based on increased expression of the activation marker CD86 in CD11c+ cells). These activated DCs prepare the antigen-specific T cell response, resulting in antitumor effects (with increased TILs) in several tumor models.

[0134] Example 5 This embodiment demonstrates the ability of multilayer RNA-NPs to systemically activate DCs, induce antigen-specific immunity, and elicit antitumor effects.

[0135] The effects of multilayer RNA NPs were tested in a second model. Here, BALB / c mice (8 mice per group) inoculated with K7M2 lung tumors were vaccinated with multilayer RNA NPs three times a week. A control group of mice remained 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 pairs 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+ CD3+ cells) in harvested lungs from untreated mice, mice treated with multilayer RNA NPs with GFP RNA, and mice treated with multilayer RNA NPs with tumor-specific RNA.

[0136] Furthermore, BALB / c mice or BALB / c SCID (Fox Chase) mice (8 mice per group) were inoculated with K7M2 lung tumors and intravenously vaccinated three times a week with multilayer RNA NPs containing GFP RNA or tumor-specific RNA. The control group of mice was untreated. % survival rates were plotted on Kaplan-Meier curves (***p<0.0001, Gehen-Breslow-Wilcox). As shown in Figure 3C, the survival rate of BALB / c mice treated with multilayer 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 multilayer RNA NPs containing GFP RNA was almost the same as that of mice treated with multilayer RNA NPs containing tumor-specific RNA (Figure 3D).

[0137] In summary, 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 immune-responsive animals and SCID mice. In BALB / c mice with metastatic lung tumors (Figures 3A–3D), both GFP (control) and tumor-specific RNA-NPs mediate innate immunity 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). Antitumor activity of RNA-NPs was also demonstrated in mice with intracranial malignancies (data not shown).

[0138] These data suggest that multilayer RNA-NPs activate dendritic cells (DCs) systemically and induce antigen-specific immunity. This demonstrates that the RNA-NPs induce an antitumor effect. Figures 3A-3D show that control RNA-NPs induce a spontaneous response with some efficacy, while tumor-specific RNA-NPs induce a stronger response. No effect of uncomplexed NPs was observed compared to untreated mice, but when incorporated into multilayer RNA NPs, both non-specific (GFP RNA) and tumor-specific RNAs mediate innate immunity; however, only tumor-specific RNA-NPs induce adaptive immunity that provides long-term survival benefits (Figures 3A-3D).

[0139] Example 6 This example demonstrates that personalized tumor RNA-NPs are active in a translational canine model.

[0140] The safety and activity of multilayer RNA-NPs were evaluated in client-owned dogs (pet dogs) diagnosed with malignant glioma or osteosarcoma. Canine malignant gliomas or osteosarcomas were initially biopsied for the production of personalized tumor RNA-NP vaccines.

[0141] To generate personalized multilayer RNA NPs, total RNA material was extracted from each patient's biopsy. Next, a cDNA library was prepared from the extracted total RNA, and then mRNA was amplified from the cDNA library. The mRNA was then complexed with DOTAP lipid NPs to form the multilayer RNA-NPs substantially described in Example 1. For the evaluation of 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, MHCII, PD-L1 / CD80, and PD-L1 / CD86 was plotted over time during the dogs' initial observation period. CD3+ cells were analyzed over time for the proportion of CD4 and CD8 during the dogs' initial observation period, and these subsets were evaluated for the expression of an activation marker (i.e., CD44). From these data, it was concluded that the multilayer RNA-NPs 1) indicate CD11c activation in peripheral DCs. +It was shown to induce an increase in CD80 and MHCII on peripheral blood cells, as well as an increase in activated T cells.

[0142] Interestingly, within hours of administration, tumor-specific RNA-NPs induced periphery of peripheral blood mononuclear cells, and this periphery increased over several days and weeks post-treatment, suggesting that RNA-NPs mediate lymphocyte honing in immune cell populations prior to release.

[0143] These data demonstrate that personalized mRNA-NPs are safe and active in translational canine disease models.

[0144] Specific data from dogs evaluated using this method are shown. A 31kg male Irish Setter was enrolled in the study with the owner's consent to receive multilayer RNA-NP. After tumor biopsy, tumor mRNA was successfully extracted and amplified. The immune response was plotted against the initial vaccine. The data shows an increase in activation markers over time in CD11c+ cells (DCs) (Figure 4A). The data shows an increase in CD8+ cells (CD44+CD8+ cells) activated within the first few hours after RNA-NP vaccination. These data support the immunological activity of multilayer RNA-NP in male Irish Setters. A male Boxer diagnosed with malignant glioma was enrolled in the study with the owner's consent to receive RNA-NP. After tumor biopsy, tumor mRNA was successfully extracted and amplified. The immune response was plotted against the initial vaccine (Figure 4B). The data shows an increase in activation markers over time in CD11c+ cells (DCs). 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 support the immunological activity of multilayer RNA-NP in male Boxer dogs. Additional observations from the treatment of dogs with spontaneously occurring gliomas. The findings are shown in Figures 4E-4H. Figure 4E shows the percentage of lymphocytes induced on the day after vaccination, which suggests a marginal trend for antigen education before release. Figure 4F shows a spike in interferon-α production, and Figure 4G shows increased CD80 expression in CD11c+ cells several hours after administration of ML RNA-NP. Figure 4H shows the expression of CD8+ and CD44+CD8+ cells, focusing on the transition to an immunologically more "active" environment. This data supports the use of ML RNA-NP to transition to an immune environment that is more responsive to immunotherapy.

[0145] Dogs diagnosed with malignant glioma followed a steady course after weekly administration of RNA-NP (x3). Post-vaccination MRI showed a stable tumor burden with increased swelling and enhancement (in some cases), which may be more consistent with pseudoprogression from immunotherapy response in asymptomatic dogs. Survival rates for dogs diagnosed with malignant glioma receiving only supportive care and tumor-specific RNA-NP (after tumor biopsy without resection) are shown in Figure 4D. In Figure 4D, the median survival time (shown by the dotted line) was approximately 65 days, which was reported from a meta-analysis of canine brain tumor patients receiving only symptomatic care. Previous studies have reported a median overall survival of 77 days for canine astrocytoma. Personalized multilayer RNA NP enabled survival beyond 200 days.

[0146] Except for a sharp increase in low-grade fever 6 hours after the first day of vaccination, personalized tumor RNA-NP(1×) showed good tolerability in stable blood cell counts, differential counts, renal function, and hepatic function tests. To date, we have treated four dogs diagnosed with malignant brain tumors. It is important to emphasize that these dogs had not received any other therapeutic interventions for the malignant tumors (i.e., surgery, radiation, or chemotherapy), and all patients evaluated were assessed for the development of an immune response with pseudo-progression or stable / smaller tumors. One dog was necropsied after RNA-NP vaccination. This patient showed no toxicity thought to be related to the intervention.

[0147] These results suggest the safety and activity of tumor-specific RNA-NPs in client-owned dogs with malignant brain tumors, in subjects not receiving other antitumor therapeutic interventions.

[0148] Example 7 This example demonstrates toxicity studies of mouse glioma mRNA and pp65 mRNA encapsulated in DOTAP liposomes after intravenous delivery to C57BL / 6 mice.

[0149] The objective of this study was to evaluate the safety of pp65 mRNA encapsulated by DOTAP liposomes when delivered intravenously to C57BL / 6 mice. Applicable experimental procedures for pathological investigation are summarized in Table 1. All intermediate stage animals were submitted for necropsy at 35±1 days. Necropsies were performed by University of Florida staff. Tissue samples listed in Table 2 were collected and fixed in 10% neutral buffered formalin, except for eye and testicular tissues, then fixed in Davidson's solution, and tissues from animals that died early were fixed in 10% neutral buffered formalin.

[0150] [Table 1]

[0151] [Table 2-1]

[0152] [Table 2-2]

[0153] The tissue required for microscopic evaluation is from Charles River Laboratories. The tissues were trimmed, regularly treated, embedded in paraffin, and stained with hematoxylin and eosin by Skokie, Illinois, Inc. Light microscopy evaluation was performed by a board-certified veterinary pathologist contributor on all animals in groups 1 and 4, and all tissues specified in the protocol from animals that died early.

[0154] Tissues that were supposed to be evaluated under a microscope for each protocol but were not available on the slides (and therefore not evaluated) are listed as "not present" in the "Individual Animal Data" section of the pathology report. Since the number of tissues examined from each treatment group was sufficient for interpretation, these missing tissues did not affect the results or interpretation of the pathology portion of the study.

[0155] Macroscopic pathology: No macroscopic findings related to the test substance were observed. The observed macroscopic findings were considered to be associated with characteristics commonly observed in this strain and at the age of the mice, and / or were observed at similar incidences in control and treated animals, and were therefore considered unrelated to the administration of pp65 mRNA and KR158 mRNA in a 1:1 ratio in DOTAP liposomes.

[0156] Histopathology: No microscopic findings related to the test substance were observed. A few animals had inflammatory cell infiltration at the injection site, but this finding is common at injection sites and was considered ambiguous at this point in the study. The observed microscopic findings were considered to be associated with characteristics commonly observed in this strain and at the age of the mice, and / or were similar in incidence and severity in control and treated animals, and were therefore considered unrelated to the administration of pp65 mRNA and KR158 mRNA in a 1:1 ratio in DOTAP liposomes.

[0157] Intravenous injection of 1.0 mg / kg KR158 and pp65 mRNA + 15.0 mg / kg DOTAP liposomes into the tail vein of mice on days 0, 14, and 28 of the study resulted in no macroscopic or microscopic findings related to the study at day 35 ± 1. A small amount of inflammatory cell infiltration was observed at the injection site, which is a common finding at injection sites. This finding was ambiguous.

[0158] Example 8 This example describes a study aimed at determining the effect of multilayer RNA-NP-transfected pDCs on antigen-specific T cell priming.

[0159] pDCs are well-known stimulants of innate immunity and type I IFN, but they also mediate significant effects on intratumor adaptive immunity. They can 1) directly present antigens for tumor-specific T cell priming, 2) support adaptive responses via chemokine supplementation of other DC subtypes (via chemokines CCL3, CCL4, and CXCL10), 3) polarize Th1 immunity via IL-12 secretion, and / or 4) mediate the release of tumor antigens (via cytokines, TRAIL, or granzyme B) for DC loading and T cell priming. Despite these effector functions, pDCs can also attenuate immunity by releasing immunomodulatory molecules (IL-10, TGF-β, and IDO) and promoting regulatory T cells (Treg). The aim of this study was to elucidate the effects of RNA-NP-transfected pDCs on adaptive immunity and antigen-specific T cell priming. RNA-NP-activated pDCs were hypothesized to function as direct primers for antigen-specific immunity, supporting classical DCs (cDCs) and / or myeloid-derived DCs (mDCs) in enhancing effector T cell responses. These experiments shed new light on the activation state of pDCs required for RNA-NP-mediated immunity and their depletion over time, which may be employed to enhance the effects of immunotherapy.

[0160] statistical analysis In the Example 9.1 study where survival rate is critical, we use a log-rank test to compare Kaplan-Meier survival curves between the treated and control groups. Our experience with tumor models shows 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 each in two tumor-bearing groups (treated and untreated), a comparison of survival curves using a one-sided log-rank test assessed at significance of 0.05 has at least 80% power to detect an improvement in median 8-day survival in the treated group compared to the untreated group. This effect size was determined by simulating a Weibull-distributed survival dataset of 1000 mice with shape parameter k=6 under the alternative effect size, and we observed the proportion of log-rank tests for these datasets that were significant at p<0.05. In the studies of Examples 9.2–9.4, responses observed at different times were analyzed using a bidirectional ANOVA model with mutually exclusive groups dispersed between treatment and observation times, and changes in immune response parameters over time were evaluated using a generalized linear mixed-effects model (GLMM). Response variables for experiments that were fully replicated at least once were analyzed using the GLMM. Experimental replication was modeled as a random effect to account for “batch” or “experimental day” variability. The treatment and control groups were modeled as fixed effects and compared using an ANOVA-type design nested within a mixed-effects modeling framework.

[0161] Example 8.1 This example describes an experiment designed to measure the antitumor effect of RNA-NPs in wild-type and pDC KO mice.

[0162] The tumorigenic potential of KR158b-luc, GL261-luc, and mouse H3.3K27M mutant cell lines has been established. Since both KR158b-luc and GL261-luc are transfected with luciferase, tumor growth can be monitored using bioluminescence imaging. The tumorigenic dose of the KR158b-luc and H3K27M mutant lines is 1 × 10⁻⁶. 4It is a cell. The tumorigenic dose of GL261-luc is 1 × 10⁻⁶ 5 These are individual cells. GL261 and KR158 are injected into the cerebral cortex of C57Bl / 6 (2 mm to the right of the previous section, at a depth of 3 mm in the brain). H3K27M glioma cells are injected along the midline. Tumor mRNA is conjugated with the inventors' custom lipid-NP preparation (per mouse) and 25 μg of tumor-specific mRNA is injected intravenously (iv) for a vaccine formulation consisting of a parent cell line (i.e., luciferase-free KR158b). Extracts are taken from the NPs. These are compared simultaneously with 10 negative control mice administered with NPs only and nonspecific (i.e., pp65 mRNA) RNA-NPs. Mice are vaccinated three times at 7-day intervals starting 5 days after tumor transplantation. IFN-α levels are assessed at consecutive time points (5, 12, and 19 days) from the serum of wild-type and pDC KO mice. In wild-type mice that respond to treatment but succumb to disease, immunological evasion mechanisms in the tumor (i.e., checkpoint ligand expression, IDO, downregulation of MHC class I) and within the tumor microenvironment (i.e., MDSCs, Tregs, and TAMs) are investigated.

[0163] Based on preclinical data demonstrating the antitumor activity of RNA-NPs in these models, it is expected that the antitumor activity will be ineffective in pDC KO mice.

[0164] Example 8.2 This example describes an experiment designed to determine the phenotype and function of pDCs after activation by RNA-NPs.

[0165] To evaluate the pDC phenotype, C57Bl / 6 mice carrying KR158b are vaccinated with TTRNA-NPs consisting of 375 μg of FITC-labeled DOTAP (Avanti) and 25 μg of TTRNA (derived from KR158b and delivered intravenously). Twenty-four hours after vaccination, recipient mice are euthanized (humanely killed with CO2) for collection of the spleen, tumor dissipation lymph nodes (tdLNs), and tumor. The organs are digested into single-cell suspensions and RBCs are lysed (PharmLyse, BD Bioscience) before incubation at 37°C for 5 minutes. WBCs are separated from parenchymal cells using a Ficoll gradient. Interfacial cells are collected, washed, and analyzed. pDCs are stained for CD11c, B220, and Gr-1 (ebioscience). Different pDC subsets are identified by differential staining for CCR9, SCA1, and Ly49q. The activation state is assessed based on the expression of co-stimulatory molecules (e.g., CD40, CD80, CD86), chemokines (e.g., CCL3, CCL4, CXCL10), and chemokine receptors (e.g., CCR2, CCR5, CCR7). The detection secondary antibody is rabbit IgG conjugated with AlexaFlour® 488 (ThermoFisher Scientific) for FITC detection. Effector vs. regulatory function is determined via intracellular staining for effectors (e.g., IFN-I, IL-12) vs. regulatory cytokines (e.g., TGF-β, IL-10). Analysis will be performed by multi-parameter flow cytometry (LSR, BD Bioscience) and immunohistochemistry (IHC).

[0166] Based on our preliminary data showing a substantial increase in pDCs in peripheral and intratumor organs, we expect to identify FITC-positive pDCs in the spleen, tdLN, and intracranial tumors.

[0167] Example 8.3 This embodiment describes an experiment designed to determine whether RNA-NP-transfected pDCs mediate the direct or indirect activation of antigen-specific T cells.

[0168] pDCs are well-known stimulants of innate immunity and type I IFNs, but their cumulative effect on antigen-specific responses remains unclear. Because they express MHC class II, they possess APC capabilities, but compared to their cDC counterparts, they are considered weak direct primers for antigen-specific immunity. This experiment aims to provide a better understanding of pDCs as either direct primers or enhancers of antigen-specific immunity from the perspective of RNA-NPs. The effect of pDCs on antigen-specific T cells will be determined. To this end, KR158b-possessing mice are vaccinated with FITC-labeled NPs (Avanti) derived from the spleen, tdLN, and intracranial tumors (as described above), and TTRNA (derived from the mouse glioma strain KR158b) encapsulated in FACSort (BD Aria II)-associated FITC+ pDCs. Next, RNA-NP-transfected pDCs are co-cultured with magnetically isolated 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 are evaluated by ex vivo co-culture with TTRNA-loaded DCs (matured ex vivo from mouse bone marrow) that possess naive CD4 and CD8 T cells. Ex vivo co-culture is performed three times for 7 days in 96-well plates containing naive T cells labeled with CFSE (Celltrace, Life Technologies) (400,000 T cells containing 40,000 RNA-NP transfected pDCs). T cell proliferation is determined by measuring CFSE dilution by flow cytometry. The phenotype of the effector and central memory population is determined by differential staining for CD44 and CD62L. These T cells are re-stimulated for a total of two cycles before the supernatant is collected 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 a control tumor (B16F10-GFP) and evaluated for their ability to induce cytotoxicity. The amount of GFP in each co-culture, as a surrogate for viable tumor cells, is quantitatively measured by flow cytometry.

[0169] The in vivo effect of FACSort-transfected RNA-NP pDCs is determined by adopting these cells (250,000 cells / mouse) into tumor-bearing mice (3 times weekly) and collecting the spleen, tdLN, and tumor after 1 week to assess antigen-specific T cells by YFP expression in IFN-γ reporter mice (GREAT mice, B6 transgenic, containing an IFN-γ promoter with an IRES-eYFP reporter, Jackson labs). In another experiment, IFN-γ reporter mice are vaccinated with TTRNA-NP with or without a pDC-depleting mAb before collecting the spleen, tdLN, and intracranial tumor after 1 week to measure antigen-specific T cells by YFP expression. T cell function assays are performed as described above.

[0170] These pDCs are expected to be necessary for priming antigen-specific T cells, either directly or indirectly.

[0171] Example 8.4 This embodiment describes an experiment designed to determine whether RNA-NP-activated pDCs promote antigen-specific T cell priming from cDCs and / or mDCs.

[0172] While IFN-I release from pDCs is known to increase activation markers in cDCs and mDCs, the role of pDCs in direct T cell priming from cDCs / mDCs is not well understood. 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, C57Bl / 6 and pDC knockout (KO) mice (BDCA2-DTR, B6 transgenic mice, Jackson labs) with KR158b are vaccinated, and T cell priming from cDCs and mDCs is evaluated. The FITC+ cDC and mDC populations are classified via FACSort within 24 hours 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. 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, FACSort-treated cDCs and mDCs from TTRNA-NP-vaccinated C57Bl / 6 mice or pDC KO mice are adopted into tumor-bearing mice (250,000 cells / mouse) one week later, before harvesting the spleen, tdLN, and intracranial tumors to evaluate antigen-specific T cells by YFP expression in IFN-γ reporter mice. Proliferation, function, and cytotoxicity assays are performed.

[0173] ML RNA-NPs are expected to enhance the activating phenotype and activate pDCs that facilitate direct priming of T cells from cDCs and mDCs.

[0174] In the absence of indirect effects from pDCs on cDCs and / or mDCs, the effects of pDCs on NK cells are evaluated, including their activation state, function, and cytotoxicity.

[0175] Example 8.5 This example describes an experiment designed to determine how pDCs influence effector / regulatory T cells over time within the tumor microenvironment.

[0176] pDC recruitment to tumors is typically associated with a regulatory phenotype characterized by increased IDO, FoxP3+ Tregs, and secretion of immunomodulatory cytokines. This experiment aims to determine whether RNA-NP-activated pDCs function distinctly by activating T cells over time in the tumor microenvironment. To determine the intratumoral effects of pDCs, TTRNA-NP is administered to KR158b mice with IFN-γ reporters, with or without pDC-depleting mAb (Bioxcell). Activated and regulatory T cells are evaluated over time in the tumor microenvironment at consecutive time points (6 hours, 1 day, 7 days, and 21 days). Effector T cells are characterized, and Tregs are phenotypic determined through the expression of FoxP3, CD25, and CD4. pDCs from non-depleted animals are FACSortized from these sites, and their phenotype is determined for cytokines, chemokines, activation markers (e.g., CD80, CD86, CD40), cytolysis markers (e.g., TRAIL, granzyme b), and regulatory markers (e.g., IL-10, TGF-β, IDO). Immunophenotypic changes by tumor cells are also evaluated over time (i.e., MHC-I, PD-L1, SIRPα).

[0177] Example 9 This example describes a study aimed at evaluating the role of type I interferon in the efflux, transport, and function of RNA-NP-activated T cells.

[0178] Statistical Analysis: Mice with tumors are randomized before receiving interventional treatment. Selecting 10 animals per group should provide sufficient power to detect the effect of interest. As an example, in an ANOVA design with seven treatment groups observed at specific time points, 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 bidirectional ANOVA design where mutually exclusive groups are distributed over treatment and observation times. Response variables for experiments that have been fully replicated at least once are analyzed using GLMM. Experimental replication is referred to as "batch" or "actual". The variability of the "test date" is modeled as a random effect. The treatment group and control group are modeled as fixed effects and compared using an ANOVA-type design nested within a mixed-effects modeling framework.

[0179] Example 9.1 This example describes an experiment designed to determine the expression profiles of the chemokine receptors S1P1 and VLA-4 / LFA-1 on antigen-specific T cells after RNA-NP vaccination.

[0180] The effects of IFN-I on sphingosine-1-phosphate receptor 1 (S1P1), which is necessary for T cell exit from lymphoid organs, and on integrins (i.e., VLA-4, LFA-1) necessary for T cell migration across the blood-brain barrier (BBB) ​​will be evaluated. TTRNA-NPs will be transplanted into KR158b mice containing IFN-γ reporter mice, or IFN-γ reporter mice administered with an IFNAR1 blocking mAb (Bioxcell). RNA-NPs consisting of 375 μg of DOTAP (Avanti) containing 25 μg of TTRNA (extracted from KR158b and administered intravenously) will be administered once a week (x3), starting 5 days after transplantation. One week after the last vaccination, recipient mice will be euthanized (humanely killed with CO2), and the spleen, tdLN, bone marrow, and intracranial tumors will be collected. Organs are digested, and antigen-specific T cells from the spleen, lymph nodes, bone marrow, and tumors are identified at consecutive time points (days 7, 14, and 21) by differential staining for YFP expression and effector and central memory T cells (i.e., CD62L and CD44). Th1-related 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 evaluated by multi-parameter flow cytometry and IHC.

[0181] LFA-1 and CCR2 are expected to be expressed in activated T cells after RNA-NP administration. If there are no changes in the chemokine expression pattern, S1P1, or integrins of activated T cells after IFNAR1 mAb administration, RNA-seq analysis will be performed on FACS-sorted T cells (YFP+ cells) from mice treated with or without IFNAR1 mAb to evaluate changes in immune-related genes.

[0182] Example 9.2 This example describes an experiment designed to determine the effects of IFN-I on the in vitro and in vivo migration of RNA-NP-activated T cells.

[0183] Based on our data showing that antigen-specific T cells in peripheral organs increased after IFNAR1 blockade but lacked an antitumor effect, we determine the effect of IFN-I on RNA-NP-activated T cell migration. IFN-γ reporter mice with KR158b, or IFN-γ reporter mice administered with IFNAR1, LFA-1, or CCR2 blocking antibodies, are vaccinated intravenously with TTRNA-NP once a week (×3). In vivo crossing of the BBB is evaluated from the percentage and absolute number of T cells in intracranial tumors (compared with spleen, lymph nodes, and bone marrow) at consecutive time points (5, 10, 15, 20 days after RNA-NP).

[0184] The migratory ability of T cells is also analyzed via in vitro culture. Naïve, INFAR1, LFA-1, or CCR2 KO animals (B6 transgenic, Jackson) with KR158b tumors are vaccinated intravenously with TTRNA-NP. T cells are FACSorted into 50 - 100% FBS solution via a BD Aria II Cell Sorter. These T cells are evaluated for migratory ability in a transwell assay (ThermoFi sher Scientific). Briefly, T cells are placed on the upper layer of a cell culture insert with a permeable membrane between layers of KR158b-GFP tumor cells. Migration is evaluated by the number of cells moving between layers. T cells are plated in T cell medium at a concentration of 4×10 6 per mL with or without IL-2 (1 microgram / mL) for co-culture with tumor cells (4×10 6 / mL) (×48 hours). As a surrogate for viable tumor cells, the amount of GFP in each co-culture is quantitatively measured by flow cytometry analysis.

[0185] Type I IFN is expected to be required for the transport of activated T cells across the BBB. If antigen-specific T cells cannot be adequately defined, we track the response to pp65 (spiked in our tumor mRNA cohort), a physiologically relevant GBM antigen, in HLA-A2 transgenic mice by analyzing the pp65-HLA-A2 restriction epitope NTUDGDDNNDV by tetramer staining of CD8+ cells in the spleen, tdLN, and intracranial tumors, using a duplicate peptide pool restimulation assay.

[0186] Example 9.3 This example illustrates the contribution of IFN-I to antigen-specific T cell function after RNA-NP.

[0187] IFN-I has been shown to promote Treg cells and modulate effector and memory CD8+ cells (56), and is also essential for enhancing the activated T cell response after RNA-NP vaccination. These clear effects determine the contribution of IFN-I to antigen-specific T cell function after RNA-NP vaccination. IFN-γ reporter mice possessing KR158b, or IFN-γ reporter mice administered with IFNAR1 mAb, are vaccinated with intravenously administered TTRNA-NP once a week (x3). Antigen-specific T cells are evaluated using YFP+ cells. YFP+ T cells from the spleen, lymph nodes, bone marrow, and tumors will be evaluated for their activation status (i.e., CD107a, perforin, granzyme), proliferation (by fluorescence dilution of adoptive transfer cells labeled with CellTrace Violet), differentiation (to effector and central memory subsets), and cytotoxicity. The cytotoxicity of T cells will be determined in the presence of KR158b (stably transfected with GFP) or a control tumor (B16F10). Type I IFN is also expected to enhance T cell proliferation and function within the tumor microenvironment.

[0188] If there is no change in the migratory capacity or function of antigen-specific T cells after blockade of type I IFN, the effect of type I IFN on regulating T cell depletion will be evaluated. The effect of type I IFN on the expression of their ligands to immune checkpoints (i.e., PD-1, TIM-3, LAG-3) and tumor cells and APCs (i.e., PD-L1, galectin-9) will also be evaluated.

[0189] Example 10 This embodiment demonstrates that non-antigen-specific multilayer (ML)RNA NPs mediate antigen-specific immunity of sufficient length to provide memory and evade tumor rechallenge.

[0190] The experiment was conducted using long-surviving mice (e.g., mice that survived for approximately 100 days) that were challenged a total of two times by tumor inoculation, but treated only once a week (x3) with ML RNA NPs containing GFP RNA or pp65 RNA (each non-specific to the tumor), or with ML RNA NPs containing tumor-specific RNA. Treatment was performed immediately after the first tumor inoculation and approximately 100 days before the second tumor inoculation. Since none of the control mice (untreated mice) survived to 100 days, it was determined that the same type of mouse could be inoculated with the K7M2 tumor. A new control group of mice was created. Similar to the original control mice, the new control group received no treatment. Long-term survivors also received no treatment after the second tumor inoculation. The timeline of events in this experiment is shown in Figure 7A.

[0191] Notably, all three mouse groups included long-term survivors who survived a second tumor challenge. As shown in Figure 7B (showing only the period after the second inoculation), all three mouse groups included long-term survivors who survived up to 40 days after tumor transplantation (examples of second tumor inoculation). Interestingly, nonspecific RNA (GFP RNA or pp65) was also present. The percentage of long-term surviving mice previously treated with ML RNA NPs containing tumor-specific RNA (administered before the second tumor challenge) was comparable to that of the group treated with ML RNA NPs containing tumor-specific RNA (administered before the second tumor challenge), surviving up to 40 days after the second tumor inoculation.

[0192] These data support the finding that ML RNA NPs containing tumor-nonspecific RNA provide a therapeutic effect on tumors comparable to that offered by ML RNA NPs containing tumor-specific RNA, leading to a percentage increase in animal survival rates.

[0193] Example 11 This example demonstrates that administering ML RNA NPs in combination with ICI leads to a significant increase in the survival rate of subjects with tumors.

[0194] To test the effects of ML RNA NPs in combination with ICI, C57Bl / 6 mice with tumors were treated with ML RNA NPs alone (RNA NPs) or in combination with an anti-PDL1 monoclonal antibody (PDL1 mAb). The control group included untreated mice, mice treated with nanoparticles without any RNA loaded (NPs only), or mice treated with PDL1 mAb only. For tumor transplantation, approximately 200,000 MOC-1 cells, which are mouse oral squamous cell carcinoma (OSCC) cells, were subcutaneously transplanted into C57Bl / 6 mice. For mice treated with either mAb or NP alone, NP was administered intravenously within 24 hours of tumor transplantation, followed by two more weekly injections. For mice treated with ICI (ML RNA NP + PDL1 mAb or PDL1 mAb alone), PD-L1 mAb (400 μg) was administered intraperitoneally, followed by weekly injections of 200 μg of NP until the third dose. Surviving mice in each group were monitored over a study period of approximately 100 days, and the percentage of surviving mice in each group was plotted as a function of time after tumor transplantation. The results are shown in Figure 9. As shown in this figure, the percentage of surviving mice treated with ML RNA NP in combination with ICI was much higher than that of mice treated with either treatment alone.

[0195] Example 12 This embodiment demonstrates that the ML RNA-NPs of this disclosure mediate an antitumor immune response against immunologically "cold" tumors, i.e., tumors that did not respond to ICIs. As shown in Figures 10A–10C, administration of the ML RNA-NPs of this disclosure together with an immune checkpoint inhibitor (here, an anti-PD-L1 antibody) resulted in a reduction in tumor volume in a melanoma model compared to administration of RNA-NPs alone and the checkpoint inhibitor alone. Administration of ML RNA-NPs also resulted in enhanced survival of subjects in sarcoma and metastatic lung models. These data establish that ML RNA-NPs reprogram immunologically "cold" tumors and demonstrate the efficacy of ML RNA-NPs across cancer and tumor types.

[0196] Example 13 This embodiment describes an exemplary method for isolating slow-cycle cells.

[0197] An exemplary method includes (a) contacting a mixed tumor cell population with a cell growth dye or mitochondrial dye (e.g., MitoTracker®) that binds to the cells of the mixed tumor cell population (e.g., to the surface or interior of the cells); (b) separating the stained cells into subpopulations based on the intensity of fluorescence emitted by the cell growth dye or mitochondrial dye; and (c) selecting and isolating subpopulations exhibiting the top 1–20% of fluorescence intensity, or removing subpopulations exhibiting the bottom 80% of fluorescence intensity, thereby isolating SCC from the mixed tumor cell population.

[0198] Cell growth pigments or mitochondrial pigments may contain thiol-reactive chloromethyl groups or amine-reactive groups. Cell growth pigments can bind to the inside of cells and may contain carboxyfluorescein succinimidyl ester (CFSE), optionally CellTrace® CFSE, CFDA-SE, CFDA, CellTrace® Violet, Blue, Yellow, Far Red, or any wavelength of the color spectrum. In exemplary embodiments, cell growth pigments are cell surface-bound pigments such as CellVue Claret pigment, PKH26, and e-Fluor growth pigment. In exemplary embodiments, mitochondrial pigments are cell mitochondrial pigments including rosamine-based mitochondrial probes (orange CMTMRos, orange CM-H2TMRos, red CMXRos, red CM-H2XRos, deep red CMXRos, deep red CM-H2XRos) and carbocyanine-based mitochondrial probes (green FM, orange FM, red FM, deep red FM).

[0199] Additional dyes that can be used in the method for separating SCCs include, but are not limited to, CellTrace growth dyes (blue, violet, CFSE, yellow, far-infrared), CFDA, CFDA-SE, CellVue Claret dye, PKH26, and e-Fluor growth dye. Dye concentrations can vary from 0.1 μM to 50 μM, and labeling times can vary from 1 minute to 1 hour. The labeling solution can be PBS or any serum-free or protein-free medium. Cell densities for labeling can range from 100,000 cells per ml of labeling solution to 20 million cells per ml of labeling solution. A follow-up period may need to be performed after labeling. After this follow-up period, which varies between 2 days and 8 weeks, the labeling intensity is quantified by flow cytometry.

[0200] The method may include one or more combinations of the aforementioned dyes. For example, the method may include contacting a mixed tumor cell population with at least two cell growth dyes or mitochondrial dyes, optionally, at least three, at least four, at least five, at least six, or more cell growth dyes or mitochondrial dyes.

[0201] Selected SCCs may be the cells that exhibit the most fluorescence. In an exemplary embodiment, SCCs represent the top 1–20% of cells with the highest fluorescence intensity. In an exemplary embodiment, FCCs (fast-cycling cells) may be the cells that exhibit the least fluorescence. In an exemplary embodiment, FCCs represent the bottom 1–20% of cells with the lowest fluorescence intensity. Therefore, a method for isolating SCCs may involve selecting and isolating a subpopulation of cells exhibiting the top 1–20% of fluorescence intensity. For example, a method may involve selecting and isolating a subpopulation exhibiting the top 1%, top 2%, top 3%, top 4%, top 5%, top 6%, top 7%, top 8%, top 9%, top 10%, top 11%, top 12%, top 13%, top 14%, top 15%, top 16%, top 17%, top 18%, top 19%, or top 20% of fluorescence intensity. Cell selection based on fluorescence intensity can be achieved by flow cytometry and cell sorting techniques, such as fluorescence-activated cell sorting (FACS). It is understood that larger isolated fractions may have lower efficacy, and smaller fractions may have lower efficiency. SCC and FCC are respectively They are identified based on their staining ability and label retention ability.

[0202] Optionally, dead cells can be removed from a mixed tumor cell population. In some embodiments, the method involves contacting cells of a mixed tumor cell population with a dead cell stain, including, but not limited to, propidium iodide (PI), unfixed SYTOX DNA-binding dyes (e.g., SYTOX AADvanced, SYTOX blue, SYTOX orange, SYTOX red, or SYTOX green), and live / dead fixable dyes (e.g., LIVE / DEAD Fixable Dead Cell Stain Blue, Aqua, Yellow, Green, Red, Far Red, Near-IR). The dead cell stain is a dye that enters dead cells but cannot penetrate living cells.

[0203] Isolation of SCCs from a mixed tumor population can be carried out by one of the following methods. In the first method, SCCs are isolated from a mixed population of tumor cells based on their growth rate. In exemplary embodiments, SCCs are isolated based on their ability to retain CellTrace dyes (carboxyfluorescein succinimimidyl ester - CFSE or Cell Trace Violet - CTV, Invitrogen). SCCs and FCCs are grouped as the top 10% of CFSE / Violethigh and the bottom 10% of CFSE / Violetlow, respectively, or FCCs are isolated as the bottom 85% of CFSElow in some embodiments (Deleyrolle LP, et al. (2011) Brain 134:1331-43). Thus, SCCs are isolated in some embodiments by selecting cells grouped as the top 10% of CFSE / Violethigh or by removing the bottom 85% of CFSElow (FCCs). In the second method, SCCs are isolated based on their mitochondrial content. In various examples, cell-permeable MitoTracker™ (ThermoFisher), which contains a mild thiol-reactive chloromethyl moiety for labeling mitochondria, is used. Alternatively, SCCs can be identified and isolated using probes (Scientific, Waltham, MA). In alternative or additional embodiments, the following dyes are used to label living cells: Rosamine-based MitoTracker dyes, including MitoTracker Orange CMTMRos, a derivative of tetramethylrosamine, and MitoTracker Red CMXRos, a derivative of X-rosamine. Reduced MitoTracker dyes, MitoTracker Orange CM-H2TMRos and MitoTracker Red CM-H2XRos, which are derivatives of dihydrotetramethylrosamine and dihydro-X-rosamine, respectively, are also used in various examples. MitoTracker Red FM, MitoTracker Carbocyanin-based MitoTracker dyes, such as Green FM dye and MitoTracker® Deep Red FM, are suitable additional dyes for use in staining mitochondria to identify SCCs. MitoProbe® DiIC1(5) (1,1”,3,3,3'3”-hexamethylindodicarbocyanine iodide) can be used to permeate the cytosol of eukaryotic cells and accumulate mainly in mitochondria with active membrane potentials of less than 100 nM, and to identify and isolate SCCs that exhibit larger mitochondrial membrane potentials. Cell labeling is performed at 1 nM to 100 nM for 5 minutes to 12 hours. At this time, SCCs may be identified by the top 50% of the brightest cells. In a third method, SCCs are isolated based on lipid content. In exemplary embodiments, LipidSpot is used. Live or fixed cells are incubated with LipidSpot dyes, including but not limited to LipidSpot 610 and LipidSpot 488. In other exemplary embodiments, LipidTox is used. Fixed cells are incubated with LipidTox dyes, including but not limited to LipidTOX Green neutral lipid stain, LipidTOX Red neutral lipid stain, or LipidTOX Deep Red neutral lipid stain.

[0204] The dilution of the dye can vary from 1 / 10 to 1 / 5000 (e.g., approximately 1 / 10, 1 / 50, 1 / 100, 1 / 250, 1 / 500, 1 / 750, 1 / 1000, 1 / 2000, 1 / 3000, 1 / 4000, 1 / 5000). In certain embodiments, the dye concentration is in the range of approximately 5 nM to 1000 nM. In various embodiments, the labeling time is in the range of approximately 1 minute to approximately 24 hours. The labeling solution may contain PBS or any buffer. Optionally, the buffer does not contain surfactants. In various embodiments, the buffer is at a neutral pH. The cell density for labeling can be approximately 100,000 cells per ml of labeling solution to approximately 20 million cells per ml of labeling solution.

[0205] All references incorporated herein, including publications, patent applications, and patents, are incorporated by reference to the same extent that each reference is individually and specifically indicated and incorporated herein to the same extent that the whole is included herein.

[0206] In the context describing this disclosure (in particular in the context of the following claims), the terms “a,” “an,” and “the,” as well as similar references, should be interpreted as encompassing both singular and plural unless otherwise stated herein or unless it is clearly inconsistent with the context. The terms “comprising,” “having,” “including,” and “containing” should be interpreted as open terminology unless otherwise noted (i.e., “including but not limited to”). Where an aspect of the invention is described as “comprising” a feature, the embodiment is also intended to “consist of” or “essentially comprise of” the feature.

[0207] Unless otherwise specified herein, the descriptions of value ranges herein are intended solely as abbreviations to refer individually to each distinct value that falls within that range and each endpoint, and each distinct value and each endpoint is incorporated herein as if it were individually described herein. Except for examples of operation or unless otherwise shown, all figures used herein to represent amounts of ingredients or reaction conditions should be understood to be modified in all cases by the term “approximately,” since the term “approximately” is interpreted by those skilled in the art in the relevant field.

[0208] All methods described herein may be performed in any preferred order, unless otherwise specified herein or unless the context clearly contradicts it. Unless otherwise requested, any use of any examples or exemplary language (e.g., "etc.") provided herein is intended solely to better illustrate the disclosure and not to limit its scope. The language herein should not be construed as indicating any non-claimed element essential to the implementation of the disclosure.

[0209] Preferred embodiments of the Disclosure, including the best mode known to the inventors for carrying out the Disclosure, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art by reading the preceding description. The inventors expect that those skilled in the art will appropriately use such variations, and the inventors intend that the Disclosure will be carried out in ways other than those specifically described herein. Accordingly, the Disclosure includes all modifications and equivalents of the subject matter enumerated in the claims appended herein, as permitted by applicable law. Furthermore, unless otherwise specifically indicated herein, or unless clearly inconsistent with the context, any combination of the above elements in all possible variations is incorporated herein.

Claims

1. A method for increasing the susceptibility of a tumor to treatment with an immune checkpoint inhibitor (ICI) in a subject, the method comprising administering to the subject a composition comprising nanoparticles having a positively charged surface and an interior comprising at least two nucleic acid layers, each nucleic acid layer being positioned between cationic lipid bilayers, wherein the composition is optionally administered systemically to the subject.

2. A method for treating a subject having an immune checkpoint inhibitor (ICI)-resistant tumor, comprising administering to the subject a composition comprising nanoparticles including (i) a positively charged surface and (i) a core and (ii) an interior comprising at least two nucleic acid layers, each nucleic acid layer being positioned between cationic lipid bilayers, and (ii) an ICI, wherein optionally, the composition is systemically administered to the subject.

3. The method according to claim 1 or 2, wherein the ICI is a PD-L1 inhibitor.

4. The method according to claim 3, wherein the PD-L1 inhibitor is a PD-L1 antibody.

5. The method according to any one of claims 1 to 4, wherein the nanoparticles comprise at least three nucleic acid layers, each of which is arranged between cationic lipid bilayers.

6. The method according to claim 5, wherein the nanoparticles comprise at least four nucleic acid layers, each of which is arranged between cationic lipid bilayers.

7. The method according to claim 6, wherein the nanoparticles comprise five or more nucleic acid layers, each of which is arranged between cationic lipid bilayers.

8. The method according to any one of claims 1 to 7, wherein the outermost layer of the nanoparticles comprises a cationic lipid bilayer.

9. The method according to any one of claims 1 to 8, wherein the surface includes a plurality of hydrophilic portions of the cationic lipid in the cationic lipid bilayer.

10. The method according to any one of claims 1 to 9, wherein the core comprises a cationic lipid bilayer.

11. The method according to any one of claims 1 to 10, wherein the core contains less than about 0.5% by weight of nucleic acid.

12. The method according to any one of claims 1 to 11, wherein the diameter of the nanoparticles is approximately 50 nm to approximately 250 nm in diameter, and optionally approximately 70 nm to approximately 200 nm in diameter.

13. The method according to any one of claims 1 to 12, wherein the nanoparticles have a zeta potential of about 40 mV to about 60 mV, and optionally, about 45 mV to about 55 mV.

14. The method according to claim 13, wherein the nanoparticles have a zeta potential of about 50 mV.

15. The method according to any one of claims 1 to 14, wherein the nanoparticles contain nucleic acid molecules and cationic lipids in a ratio of about 1:5 to about 1:20, optionally, about 1:15 or about 1:7.

5.

16. The method according to any one of claims 1 to 15, wherein the cationic lipid is DOTAP or DOTMA.

17. The method according to any one of claims 1 to 16, wherein the nucleic acid molecule is an RNA molecule.

18. The method according to claim 17, wherein the RNA molecule is mRNA.

19. The method according to claim 18, wherein the mRNA is an in vitro transcription mRNA, and the in vitro transcription template is a cDNA prepared from RNA extracted from tumor cells.

20. The method according to claim 18 or 19, wherein the mRNA encodes a protein.

21. The method according to claim 20, wherein the protein is selected from the group consisting of tumor antigens, cytokines, and costimulatory molecules.

22. The method according to claim 20, wherein the protein is not expressed by tumor cells or by humans.

23. The method according to claim 17, wherein the RNA molecule is an antisense molecule, optionally siRNA, shRNA, miRNA, or any combination thereof.

24. The method according to claim 17, wherein the nanoparticles include a mixture of RNA molecules.

25. The method according to claim 24, wherein the mixture of RNA molecules is RNA isolated from human cells.

26. The method according to claim 25, wherein the human has a tumor, the RNA mixture is RNA isolated from the tumor of the human, and optionally the tumor is a malignant brain tumor, optionally glioblastoma, medulloblastoma, diffuse endogenous pontine glioma, or peripheral tumor with metastatic invasion to the central nervous system.

27. The method according to any one of claims 1 to 26, wherein the nanoparticles are prepared by mixing the nucleic acid molecule and the cationic lipid in an RNA:cationic lipid ratio of about 1:5 to about 1:20, or optionally, about 1:

15.

28. The method according to any one of claims 1 to 27, wherein the composition is administered systemically via parenteral administration, or optionally via intravenous administration.

29. The method according to any one of claims 1 to 28, wherein the composition is administered systemically in an amount effective to increase the number of PD-L1+ / CD86+ myeloid antigen-presenting cells (APCs) around a tumor and / or in reticuloendothelial organs, increase PD-L1 / CD86 expression by plasmacytoid dendritic cells (pDCs) and CD11c+ myeloid cells, increase type I interferon release by pDCs, activate a T cell response, or a combination thereof.

30. A method for increasing the number of activated plasmacytoid dendritic cells (pDCs) in a subject, comprising administering to the subject a composition comprising 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 being positioned between cationic lipid bilayers, wherein the nanoparticles are optionally administered systemically to the subject.

31. The method according to claim 30, wherein the nanoparticles comprise at least three nucleic acid layers, each of which is arranged between cationic lipid bilayers.

32. The method according to claim 31, wherein the nanoparticles comprise at least four nucleic acid layers, each of which is arranged between cationic lipid bilayers.

33. The method according to claim 32, wherein the nanoparticles comprise five or more nucleic acid layers, each of which is arranged between cationic lipid bilayers.

34. The method according to any one of claims 30 to 33, wherein the outermost layer of the nanoparticles comprises a cationic lipid bilayer.

35. The method according to any one of claims 30 to 34, wherein the surface includes a plurality of hydrophilic portions of the cationic lipid in the cationic lipid bilayer.

36. The method according to any one of claims 30 to 35, wherein the core comprises a cationic lipid bilayer.

37. The method according to any one of claims 30 to 36, wherein the core contains less than about 0.5% by weight of nucleic acid.

38. The method according to any one of claims 30 to 37, wherein the diameter of the nanoparticles is approximately 50 nm to approximately 250 nm in diameter, and optionally approximately 70 nm to approximately 200 nm in diameter.

39. The method according to any one of claims 30 to 38, wherein the nanoparticles include a zeta potential of about 40 mV to about 60 mV, and optionally, about 45 mV to about 55 mV.

40. The method according to claim 39, wherein the nanoparticles have a zeta potential of about 50 mV.

41. The method according to any one of claims 30 to 40, wherein the nanoparticles contain nucleic acid molecules and cationic lipids in a ratio of about 1:5 to about 1:20, optionally, about 1:15 or about 1:7.

5.

42. The method according to any one of claims 30 to 41, wherein the cationic lipid is DOTAP or DOTMA.

43. The method according to any one of claims 30 to 42, wherein the nucleic acid molecule is an RNA molecule.

44. The method according to claim 43, wherein the RNA molecule is mRNA.

45. The method according to claim 44, wherein the mRNA is an in vitro transcription mRNA, and the in vitro transcription template is a cDNA prepared from RNA extracted from tumor cells.

46. The method according to claim 44 or 45, wherein the mRNA encodes a protein.

47. The method according to claim 46, wherein the protein is selected from the group consisting of tumor antigens, cytokines, or costimulatory molecules.

48. Claim 46, wherein the protein is not expressed by tumor cells or by humans. Method of description.

49. The method according to claim 43, wherein the RNA molecule is an antisense molecule, optionally siRNA, shRNA, miRNA, or any combination thereof.

50. The method according to claim 43, wherein the nanoparticles include a mixture of RNA molecules.

51. The method according to claim 50, wherein the mixture of RNA molecules is RNA isolated from human cells.

52. The method according to claim 51, wherein the human has a tumor, the RNA mixture is RNA isolated from the tumor of the human, and optionally the tumor is a malignant brain tumor, optionally glioblastoma, medulloblastoma, diffuse endogenous pontine glioma, or peripheral tumor with metastatic invasion to the central nervous system.

53. The method according to any one of claims 30 to 52, wherein the nanoparticles are prepared by mixing the nucleic acid molecule and the cationic lipid in an RNA:cationic lipid ratio of about 1:5 to about 1:20, or optionally, about 1:

15.

54. The method according to any one of claims 30 to 53, wherein the composition is administered systemically via parenteral administration, or optionally via intravenous administration.

55. The method according to any one of claims 30 to 54, wherein the subject has an immune checkpoint inhibitor (ICI) resistant tumor.

56. The aforementioned pDC is PD-L1 ＋ / CD86 ＋ The method according to any one of claims 30 to 55, wherein pDC.

57. A method for treating a subject having a tumor or cancer, the method comprising: (i) increasing the number of activated plasmacytoid dendritic cells (pDCs) in the subject according to the method of any one of claims 30 to 56; (ii) isolating leukocytes (WBCs) from the subject; (iii) isolating dendritic cells (DCs) from the WBCs; (iv) contacting the DCs with a fusion protein comprising prostatic acid phosphatase (PAP) and GM-CSF; and (v) administering the DCs to the subject.

58. A method for preparing a dendritic cell vaccine, the method comprising: (i) increasing the number of activated plasmacytoid dendritic cells (pDCs) in a subject according to the method of any one of claims 30 to 56; (ii) isolating leukocytes (WBCs) from the subject; (iii) isolating dendritic cells (DCs) from the WBCs; and (iv) contacting the DCs with a fusion protein comprising prostatic acid phosphatase (PAP) and GM-CSF.

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