CTC RNA-loaded nanoparticles and use thereof for the treatment of cancer

Abstract

The disclosure provides a nanoparticle comprising a positively-charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between a cationic lipid bilayer, and nucleic acid molecules in the nucleic acid layers comprise a sequence of a nucleic acid molecule expressed by a circulating tumor cell (CTC). The disclosure further provides a method of treating cancer in a subject need thereof, the method comprising administering to the subject the nanoparticle described herein, optionally wherein two or more administrations of the nanoparticle are provided, wherein each administration comprises a nanoparticle comprising RNA comprising a sequence of RNA expressed in a CTC isolated from the subject at different points in time.

Description

MGB 32917 / 59848 PCCTC RNA-LOADED NANOPARTICLES AND USE THEREOFFOR THE TREATMENT OF CANCER FIELD OF THE INVENTION

[0001] This application relates to use of multilamellar nanoparticles comprising circulating tumor cell RNA and methods of use.CROSS REFERENCE TO RELATED APPLICATIONS AND INCORPORATION BY REFERENCE

[0002] This application claims the benefit of U.S. Provisional Application No. 63 / 570,354, filed on March 27, 2024, the contents of which are hereby incorporated in their entirety by reference. The following applications also are hereby incorporated by reference in their entireties:International Patent Application No. PCT / US20 / 42606, filed July 17, 2020; International Patent Application No. PCT / US21 / 16925, filed February 5, 2021 ; and International Patent Application No. PCT / US21 / 18831, filed February 19, 2021.GRANT FUNDING DISCLOSURE

[0003] This invention was made with government support under Grant Numbers R01 CA266857 and R37 CA251978, awarded by the National Institutes of Health; and Grant Number R01 FD007268, awarded by the United States Food & Drug Administration. The government has certain rights in the invention.BACKGROUND

[0004] Immunotherapy promises to re-direct the host immune system with exquisite precision without toxicity. Immunotherapy relies on the cytotoxic potential of activated T cells, which scavenge to recognize and reject tumor associated or specific antigens (TAAs or TSAs). T cells can be endogenously activated using cancer vaccines. Compared with other vaccine modalities, therapeutic cancer vaccines must induce immunologic response much more rapidly against malignancies (e.g., GBM) that are rapidly evolving. Further, many cancers, including GBMs, are highly invasive and marked by heterogeneous tumors associated with profound systemic / intratumoral suppression that stymies a nascent immunotherapeutic response and limits, if not completely blocks, vaccine effectiveness. Additionally, much of immunotherapy's promise lies in cancers with high mutational burdens. Yet, many childhood cancers like pediatric brain tumors are immunologically bland without a significant burden of mutations.

[0005] There is a need in the art for new immunotherapy options for the treatment of cancer.MGB 32917 / 59848 PCSUMMARY

[0006] The disclosure provides a nanoparticle comprising a positively-charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between a cationic lipid bilayer, and nucleic acid molecules in the nucleic acid layers comprise a sequence of a nucleic acid molecule expressed by a circulating tumor cell (CTC). In exemplary embodiments, the nanoparticle comprises at least three nucleic acid layers (e.g., at least four or at least five nucleic acid layers), each of which is positioned between a cationic lipid bilayer. In various aspects, the outermost layer of the nanoparticle comprises a cationic lipid bilayer. In various instances, the surface comprises a plurality of hydrophilic moieties of the cationic lipid of the cationic lipid bilayer. In exemplary aspects, the core comprises a cationic lipid bilayer. Optionally, the core comprises less than about 0.5 wt% nucleic acid. The diameter of the nanoparticle, in various aspects, is about 50 nm to about 500 nm in diameter, optionally, about 70 nm to about 250 nm in diameter or about 70 nm to about 200 nm in diameter. In exemplary instances, the nanoparticle is characterized by a zeta potential of about +40 mV to about +60 mV, optionally, about +45 mV to about +55 mV. The nanoparticle, in various instances, has a zeta potential of about 50 mV. In some aspects, the nucleic acid molecules are present at a nucleic acid molecule:cationic lipid ratio of about 1 to about 5 to about 1 to about 25, optionally, about 1 to about 15, about 1 to about 10 or about 1 to about 7.5. In various aspects, the nucleic acid molecules are RNA molecules, optionally, messenger RNA (mRNA). In various aspects, the mRNA is in vitro transcribed mRNA wherein the in vitro transcription template is cDNA made from RNA extracted from a tumor cell. In various aspects, the subject is suffering from cancer, such as a glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma, a peripheral tumor with metastatic infiltration into the central nervous system, or osteosarcoma.

[0007] Also provided is a method of treating cancer in a subject need thereof, the method comprising administering to the subject the nanoparticle described herein (e.g., comprising a positively-charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between a cationic lipid bilayer, and nucleic acid molecules in the nucleic acid layers comprise a sequence of a nucleic acid molecule expressed by a CTC. In various aspects, the method comprises two or more administrations of the nanoparticle, wherein each administration comprises a nanoparticle comprising RNA comprising a sequence of RNA expressed in a CTC isolated from the subject at different points in time. In various aspects, the disclosure provides a method comprising (a) isolating aMGB 32917 / 59848 PCcirculating tumor cell (CTC) from a subject, (b) producing a nanoparticle as described herein comprising RNA comprising a sequence of RNA expressed in the CTC, (c) administering the nanoparticle of step (b) to the subject, and (d) repeating steps (a)-(c) one or more times.

[0008] The disclosure further contemplates use of a nanoparticle comprising a positively-charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between a cationic lipid bilayer, and nucleic acid molecules in the nucleic acid layers comprise a sequence of a nucleic acid molecule expressed by a CTC, for treating cancer in a subject. Use of the nanoparticle in the preparation of a medicament for treating cancer in a subject also is contemplated, as is the nanoparticle described herein for use in treating cancer in a subject.

[0009] Additional embodiments and aspects of the presently disclosed nanoparticles, compositions, and methods are provided below. The use of section headings is merely for the convenience of reading; it should be understood that the disclosure should be read as a whole and all combinations of features described herein are contemplated.BRIEF DESCRIPTION OF THE DRAWINGS

[0010] FIG. 1 provides CEM images of DOTAP liposomes (left) and multilamellar RNA nanoparticles (ML RNA-NPs) (right).

[0011] FIG. 2 provides low and high magnification images of ML RNA-NPs.

[0012] FIG. 3A is a graph of the % CD86+ of CD11c+MHC Class II+ splenocytes present in the spleens of mice treated with ML RNA NPs (ML RNA-NPs), RNA LPXs, anionic LPXs, or of untreated mice.

[0013] FIG. 3B is a graph of the % CD44+CD62L+ of CD8+ splenocytes present in the spleens of mice treated with ML RNA NPs (ML RNA-NPs), RNA LPXs, anionic LPXs, or of untreated mice.

[0014] FIG. 3C is a graph of the % CD44+CD62L of CD4+ splenocytes present in the spleens of mice treated with ML RNA NPs (ML RNA-NPs), RNA LPXs, anionic LPXs, or of untreated mice.

[0015] FIG. 3D is a graph of the % survival of mice treated with ML RNA NPs (ML RNA-NPs), RNA LPXs, anionic LPXs, or of untreated mice.

[0016] FIG. 3E is a graph of the amount of IFN-a produced in mice upon treatment with ML RNA NPs (ML RNA-NPs), RNA LPXs, anionic LPXs, or of untreated mice.MGB 32917 / 59848 PC

[0017] FIG. 4 illustrates survival outcomes from osteosarcoma models treated with CTCs-based RNA-NP. The figure provides a Kaplan-Meier survival curve of Balb / c mice (n=5-7 / group; exp) bearing pulmonary K7M2 (osteosarcoma) tumors treated i.v. with CTC-based ML RNA-NPs compared to different control groups for six administrations of NPs, three the first week, and once per week thereafter for three more weeks (totaling six administrations). CTO RNA-NPs significantly enhanced survival compared to non-treated subjects, empty NP, and non-specific RNA-NPs.

[0018] FIG. 5A depicts a schema of personalized mRNA vaccine generation from single cells in peripheral blood.

[0019] FIGs. 5B-5C depict representative cryo electron microscopy images of RNA vaccines from single cells.

[0020] FIG. 5D depicts a graph of the measured zeta potential of mRNA vaccine from CTO versus bulk mRNA.

[0021] FIG. 6A depicts a graph of percent survival for K7M2 cells transduced with EWS-FLI1 fusion protein and inoculated intravenously into Balb / c mice followed by treatment with nonspecific (GFP) mRNA or mRNA encoding for EWS-FLI1 fusion protein.

[0022] FIG. 6B depicts representative timelapse images (left, middle, right) of 011 orf95-RELA murine ependymomas were cultured with splenocytes derived from C11orf95-RELA fusion mRNA vaccinated animals.DETAILED DESCRIPTION

[0023] The disclosure provides materials and methods useful for treating cancer in a subject. In this regard, the disclosure provides a nanoparticle comprising a positively-charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between a cationic lipid bilayer, and nucleic acid molecules in the nucleic acid layers comprise a sequence of a nucleic acid molecule expressed by a circulating tumor cell (CTO). CTOs are cells that have detached from a primary tumor or tumor metastases and circulate in the bloodstream. The method described herein allows for, e.g., personalized therapies for patients which keep pace with tumor evolution, thereby addressing some of the limitations associated with previous cancer therapeutics.MGB 32917 / 59848 PC

[0024] Nanoparticles

[0025] The nanoparticles of the method comprise a cationic lipid and nucleic acids. The term “nanoparticle” refers to a particle that is less than about 1000 nm in diameter. As the nanoparticles of the present disclosure comprise cationic lipids that have been processed to induce liposome formation, the presently disclosed nanoparticles in various aspects comprise liposomes. Liposomes are artificially-prepared vesicles which, in exemplary aspects, are primarily composed of a lipid bilayer. The nanoparticles described herein comprise a multilamellar vesicle (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments. In various aspects, the liposomes of the present disclosure are of different sizes and the composition may comprise one or more multilamellar vesicles (MLV) which may be hundreds of nanometers in diameter and may contain a series of concentric bilayers separated by narrow aqueous compartments (i.e., an onion-like structure). The nanoparticles have, in various aspects, an aggregate structure having portions of concentric bilayers separated by narrow aqueous compartments comprising nucleic acid molecules which envelope or enveloped by larger lipid bilayers.

[0026] In exemplary embodiments, the nanoparticle comprises a surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, optionally, more than two nucleic acid layers. In exemplary instances, each nucleic acid layer is positioned between a lipid layer, e.g., a cationic lipid layer. In exemplary aspects, the nanoparticles are multilamellar comprising alternating layers of nucleic acid and lipid. In exemplary embodiments, the nanoparticle comprises at least three nucleic acid layers, each of which is positioned between a cationic lipid bilayer. In exemplary aspects, the nanoparticle comprises at least four or five nucleic acid layers, each of which is positioned between a cationic lipid bilayer. In exemplary aspects, the nanoparticle comprises at least more than five (e.g., 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) nucleic acid layers, each of which is positioned between a cationic lipid bilayer. As used herein the term “cationic lipid bilayer” is meant a lipid bilayer comprising, consisting essentially of, or consisting of a cationic lipid or a mixture thereof. Suitable cationic lipids are described herein. As used herein the term “nucleic acid layer” is meant a layer of the presently disclosed nanoparticle comprising, consisting essentially of, or consisting of a nucleic acid, e.g., RNA.

[0027] The unique structure of the nanoparticle of the present disclosure results in mechanistic differences in how the multilamellar nanoparticles (ML-NPs) exert a biological effect. Previously described RNA-based nanoparticles exert their effect, at least in part, throughMGB 32917 / 59848 PCthe toll-like receptor 7 (TLR7) pathway. Surprisingly, the multilamellar nanoparticles of the instant disclosure mediate efficacy independent of TLR7. While not wishing to be bound to any particular theory, intracellular pathogen recognition receptors (PRRs), such as MDA-5, appear more relevant to biological activity of the multilamellar nanoparticles than TLRs. This likely allows ML RNA-NPs to stimulate multiple intracellular PRRs (e.g., RIG-1, MDA-5) as opposed to singular TLRs (e.g., TLR7 in the endosome) culminating in greater release of type I interferons and induction of more potent innate immunity. This allows RNA-NPs to demonstrate superior efficacy with long-term survivor benefit.

[0028] In various aspects, the presently disclosed nanoparticle comprises a positively-charged surface. In some instances, the positively-charged surface comprises a lipid layer, e.g., a cationic lipid layer. In various aspects, the outermost layer of the nanoparticle comprises a cationic lipid bilayer. Optionally, the cationic lipid bilayer comprises, consists essentially of, or consists of DOTAP. In various instances, the surface comprises a plurality of hydrophilic moieties of the cationic lipid of the cationic lipid bilayer. In some aspects, the core comprises a cationic lipid bilayer. In various instances, the core lacks nucleic acids, optionally, the core comprises less than about 0.5 wt% nucleic acid.

[0029] In exemplary aspects, the nanoparticle has a diameter within the nanometer range and accordingly in certain instances are referred to herein as “nanoliposomes” or “liposomes”. In exemplary aspects, the nanoparticle has a diameter between about 50 nm to about 500 nm, e.g., about 50 nm to about 450 nm, about 50 nm to about 400 nm, about 50 nm to about 350 nm, about 50 nm to about 300 nm, about 50 nm to about 250 nm, about 50 nm to about 200 nm, about 50 nm to about 150 nm, about 50 nm to about 100 nm, about 70 nm to about 250 nm, about 70 nm to about 200 nm, about 100 nm to about 500 nm, about 150 nm to about 500 nm, about 200 nm to about 500 nm, about 250 nm to about 500 nm, about 300 nm to about 500 nm, about 350 nm to about 500nm, or about 400 nm to about 500 nm. In exemplary aspects, the nanoparticle has a diameter between about 50 nm to about 500 nm, e.g., about 100 nm to about 250 nm, about 110 nm ±5 nm, about 115 nm ±5 nm, about 120 nm ±5 nm, about 125 nm ±5 nm, about 130 nm ±5 nm, about 135 nm ±5 nm, about 140 nm ±5 nm, about 145 nm ±5 nm, about 150 nm ±5 nm, about 155 nm ±5 nm, about 160 nm ±5 nm, about 165 nm ±5 nm, about 170 nm ±5 nm, about 175 nm ±5 nm, about 180 nm ±5 nm, about 190 nm ±5 nm, about 200 nm ±5 nm, about 210 nm ±5 nm, about 220 nm ±5 nm, about 230 nm ±5 nm, about 240 nm ±5 nm, about 250 nm ±5 nm, about 260 nm ±5 nm, about 270 nm ±5 nm, about 280 nm ±5 nm, about 290 nm ±5 nm, or about 300 nm ±5 nm. In exemplary aspects, the nanoparticle is about 50 nm to aboutMGB 32917 / 59848 PC250 nm in diameter. In some aspects, the nanoparticle is about 70 nm to about 250 nm in diameter. In some aspects, the nanoparticle is about 70 nm to about 200 nm in diameter.

[0030] In exemplary aspects, the nanoparticle is present in a pharmaceutical composition comprising a heterogeneous mixture of nanoparticles ranging in diameter, e.g., about 50 nm to about 500 nm or about 50 nm to about 250 nm in diameter. Optionally, the pharmaceutical composition comprises a heterogeneous mixture of nanoparticles ranging from about 70 nm to about 250 nm in diameter or from about 70 nm to about 200 nm in diameter.

[0031] In exemplary instances, the nanoparticle is characterized by a zeta potential of about +40 mV to about +60 mV, e.g., about +40 mV to about +55 mV, about +40 mV to about +50 mV, about +40 mV to about +50 mV, about +40 mV to about +45 mV, about +45 mV to about +60 mV, about +50 mV to about +60 mV, about +55 mV to about +60 mV. In exemplary aspects, the nanoparticle has a zeta potential of about +45 mV to about +55 mV. The nanoparticle in various instances, has a zeta potential of about +50 mV. In various aspects, the zeta potential is greater than +30 mV or +35 mV. The zeta potential is one parameter which distinguishes the nanoparticles of the present disclosure and those described in Sayour et al., Oncoimmunology 6(1): e1256527 (2016).

[0032] In exemplary embodiments, the nanoparticles comprise a cationic lipid. In some embodiments, the cationic lipid is a low molecular weight cationic lipid such as those described in U.S. Patent Application Publication No. 20130090372, the contents of which are herein incorporated by reference in their entirety. The cationic lipid in exemplary instances is a cationic fatty acid, a cationic glycerolipid, a cationic glycerophospholipid, a cationic sphingolipid, a cationic sterol lipid, a cationic prenol lipid, a cationic saccharolipid, or a cationic polyketide. In exemplary aspects, the cationic lipid comprises two fatty acyl chains, each chain of which is independently saturated or unsaturated. In some instances, the cationic lipid is a diglyceride. For example, in some instances, the cationic lipid may be a cationic lipid of Formula I or Formula II:MGB 32917 / 59848 PC[Formula II]wherein each of a, b, n, and m is independently an integer between 2 and 12 (e.g., between 3 and 10). In some aspects, the cationic lipid is a cationic lipid of Formula I wherein each of a, b, n, and m is independently an integer selected from 3, 4, 5, 6, 7, 8, 9, and 10. In exemplary instances, the cationic lipid is DOTAP (1,2-dioleoyl-3-trimethylammonium-propane), or a derivative thereof. In exemplary instances, the cationic lipid is DOTMA (1 ,2-di-0-octadecenyl-3-trimethylammonium propane), or a derivative thereof.

[0033] In some embodiments, the nanoparticles comprise liposomes formed from 1 ,2-dioleyloxy-N,N-dimethylaminopropane (DODMA) liposomes, DiLa2 liposomes from Marina Biotech (Bothell, Wash.), 1 ,2-dilinoleyloxy-3-dimethylaminopropane (DLin-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), and MC3 (US20100324120; herein incorporated by reference in its entirety). In some embodiments, the nanoparticles comprise liposomes formed from the synthesis of stabilized plasmid-lipid particles (SPLP) or stabilized nucleic acid lipid particle (SNALP) that have been previously described and shown to be suitable for oligonucleotide delivery in vitro and in vivo. The nanoparticles in some aspects are composed of 3 to 4 lipid components in addition to the nucleic acid molecules. In exemplary aspects, the liposome comprises 55% cholesterol, 20% disteroylphosphatidyl choline (DSPC), 10% PEG-S-DSG, and 15% 1 ,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), as described by Jeffs et al., Pharm Res. 2005; 22(3):362-72. In exemplary instances, the liposome comprises 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where theMGB 32917 / 59848 PCcationic lipid can be 1,2-distearloxy-N,N-dimethylaminopropane (DSDMA), DODMA, DLin-DMA, or 1,2-dilinolenyloxy-3-dimethylaminopropane (DLenDMA), as described by Heyes et aL, J. Control Release, 107(2): 276-87 (2005).

[0034] In some embodiments, the liposomes comprise from about 25.0% cholesterol to about 40.0% cholesterol, from about 30.0% cholesterol to about 45.0% cholesterol, from about 35.0% cholesterol to about 50.0% cholesterol and / or from about 48.5% cholesterol to about 60% cholesterol. In some embodiments, the liposomes may comprise 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 comprise from about 5.0% to about 10.0% DSPC and / or from about 7.0% to about 15.0% DSPC. The disclosure also contemplates nanoparticles which do not comprise cholesterol or neutral lipid.

[0035] 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 20065(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).

[0036] In various instances, the cationic lipid comprises 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 comprises a neutral lipid, a sterol and a molecule capable of reducing particle aggregation, for example, a PEG or PEG-modified lipid.

[0037] The liposome in various aspects comprises 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 aspects, the liposome comprises a cationic lipid such as, but not limited to, DLin-DMA, DLin-D-DMA, DLin-MC3-DMA, DLin-KC2-DMA, DODMA and amino alcohol lipids. The amino alcohol cationic lipid comprises, in some aspects, lipids described in and / or made by the methods described in U.S. Patent Publication No. 20130150625, herein incorporated by reference in its entirety. As a non-limiting example, the cationic lipid in certain aspects is 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-{[(9Z,2Z)-octadeca-9,12-dien-1-yloxy]methyl}propan-1-ol (Compound 1 in US20130150625); 2-amino-3-[(9Z)-octadec-9-en-1-yloxy]-2-{[(9Z)-octadec-9-en-1-yloxy]methyl}propan-1-ol (Compound 2 in US20130150625); 2-amino-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 in US20130150625); and 2-(dimethylamino)-3-[(9Z,12Z)-octadeca-9,12-dien-1 -yloxy]-2-{[(9Z,12Z)-MGB 32917 / 59848 PCoctadeca-9,12-dien-1 -yloxy]methyl}propan-1 -ol (Compound 4 in LIS2O130150625); or any pharmaceutically acceptable salt or stereoisomer thereof.

[0038] In various embodiments, the liposome comprises (i) at least one lipid selected from the group consisting of 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319); (ii) a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM; (iii) a sterol, e.g., cholesterol; and (iv) a PEG-lipid, e.g., PEG-DMG or PEG-cDMA, in a molar ratio of about 20-60% cationic lipid: 5-25% neutral lipid: 25-55% sterol; 0.5-15% PEG-lipid.

[0039] In some embodiments, the liposome comprises from about 25% to about 75% on a molar basis of a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane (DLin-KC2-DMA), dilinoleyl-methyl-4-dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-(dimethylamino)butanoyl)oxy)heptadecanedioate (L319), e.g., from about 35 to about 65%, from about 45 to about 65%, about 60%, about 57.5%, about 50% or about 40% on a molar basis.

[0040] In some embodiments, the liposome comprises from about 0.5% to about 15% on a molar basis of the neutral lipid e.g., from about 3 to about 12%, from 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 aspects, the nanoparticle does not comprise a neutral lipid. In some embodiments, the formulation includes from about 5% to about 50% on a molar basis of the sterol (e.g., 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 includes from about 0.5% to about 20% on a molar basis of the PEG or PEG-modified lipid (e.g., about 0.5 to about 10%, about 0.5 to about 5%, about 1.5%, about 0.5%, about 1.5%, about 3.5%, or about 5% on a molar basis). In some embodiments, the PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da. In other embodiments, the PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of less than 2,000, for example around 1 ,500 Da, around 1,000 Da, or around 500 Da. Examples of PEG-modified lipids include, but are not limited to, PEG-distearoyl glycerol (PEG-DMG) (also referred herein as PEG-C14 or C14-PEG), PEG-cDMA (further discussed in Reyes et al. J. Controlled Release, 107, 276-287 (2005) the contents of which is herein incorporated by reference in its entirety).MGB 32917 / 59848 PC

[0041] In exemplary aspects, the cationic lipid may be selected from (20Z,23Z)— N,N-dimethylnonacosa-20,23-dien-10-amine, (17Z,20Z) — N,N-dimemylhexacosa-17,20-dien-9-amine, (1Z,19Z) — N,N-dimethylpentacosa-1 6, 19-dien-8-amine, (13Z,16Z) — N,N-dimethyldocosa-13,16-dien-5-amine, (12Z,15Z) — N,N-dimethylhenicosa-12,15-dien-4-amine, (14Z,17Z) — N,N-dimethyltricosa-14,17-dien-6-amine, (15Z,18Z) — N,N-dimethyltetracosa-15,18-dien-7-amine, (18Z,21Z) — N,N-dimethylheptacosa-18,21 -dien-10-amine, (15Z,18Z) — N,N-dimethyltetracosa-15,18-dien-5-amine, (14Z,17Z) — N,N-dimethyltricosa-14,17-dien-4-amine, (19Z,22Z) — N,N-dimeihyloctacosa-19,22-dien-9-amine, (18Z,21 Z) — N,N-dimethylheptacosa-18,21 -dien-8-amine, (17Z,20Z) — N,N-dimethylhexacosa-17,20-dien-7-amine, (16Z,19Z) — N,N-dimethylpentacosa-16,19-dien-6-amine, (22Z,25Z) — N,N-dimethylhentriaconta-22,25-dien-10-amine, (21 Z,24Z) — N,N-dimethyltriaconta-21,24-dien-9-amine, (18Z) — N,N-dimetylheptacos-18-en-10-amine, (17Z) — N,N-dimethylhexacos-17-en-9-amine, (19Z,22Z) — N,N-dimethyloctacosa-19,22-dien-7-amine, N,N-dimethylheptacosan-10-amine, (20Z,23Z) — N-ethyl-N-methylnonacosa-20,23-dien-10-amine, 1-[(11Z,14Z)-1-nonylicosa-11,14-dien-1-yl]pyrrolidine, (20Z) — N,N-dimethylheptacos-20-en-10-amine, (15Z) — N,N-dimethyl eptacos-15-en-10-amine, (14Z) — N,N-dimethylnonacos-14-en-10-amine, (17Z) — N,N-dimethylnonacos-17-en-10-amine, (24Z) — N,N-dimethyltritriacont-24-en-10-amine, (20Z) — N,N-dimethylnonacos-20-en-10-amine, (22Z) — N,N-dimethylhentriacont-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]eptadecan-8-amine, 1 -[(1 S,2R)-2-hexylcyclopropyl]-N,N-dimethylnonadecan-10-amine, N,N-dimethyl-1 -[(1 S,2R)-2-octylcyclopropyl]nonadecan-10-amine, N,N-dimethyl-21 -[(1 S,2R)-2-octylcyclopropyl]henicosan-10-amine, N,N-dimethyl-1 -[(1 S,2S)-2-{[(1 R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]nonadecan-10-amine, N,N-dimethyl-1 -[(1 S,2R)-2-octylcyclopropyl]hexadecan-8-amine, N,N-dimethyl-[(1 R,2S)-2-undecylcyclopropyl]tetradecan-5-amine, N,N-dimethyl-3-{7-[(1S,2R)-2-octylcyclopropyl]heptyl}dodecan-1 -amine, 1-[(1 R,2S)-2-heptylcyclopropyl]-N,N-dimethyloctadecan-9-amine, 1 -[(1 S,2R)-2-decylcyclopropyl]-N,N-dimethylpentadecan-6-amine, N,N-dimethyl-1-[(1S,2R)-2-octylcyclopropyl]pentadecan-8-amine, R — N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, S — N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-(octyloxy)propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}pyrrolidine, (2S) — N,N-dimethyl-1-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-3-[(5Z)-oct-5-en-1-yloxy]propan-2-amine, 1-{2-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]-1-[(octyloxy)methyl]ethyl}azetidine, (2S)-1-(hexyloxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2S)-1-(heptyloxy)-N,N-dimethyl-3-MGB 32917 / 59848 PC[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-(nonyloxy)-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1-[(9Z)-octadec-9-en-1-yloxy]-3-(octyloxy)propan-2-amine; (2S) — N,N-dimethyl-1-[(6Z,9Z,12Z)-octadeca-6,9,12-trien-1-yloxy]-3-(octyloxy)propan-2-amine, (2S)-1-[(11Z,14Z)-icosa-11 ,14-dien-1-yloxy]-N,N-dimethyl-3-(pentyloxy)propan-2-amine, (2S)-1-(hexyloxy)-3-[(11Z,14Z)-icosa-11 ,14-dien-1-yloxy]-N,N-dimethylpropan-2-amine, 1-[(11Z,14Z)-icosa-11,14-dien-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1 -[(13Z,16Z)-docosa-13, 16-dien-1 -y loxy]-N , N-di methy I-3-(octyloxy)propan-2-amine, (2S)-1-[(13Z,16Z)-docosa-13,16-dien-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, (2S)-1-[(13Z)-docos-13-en-1-yloxy]-3-(hexyloxy)-N,N-dimethylpropan-2-amine, 1-[(13Z)-docos-13-en-1-yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, 1-[(9Z)-hexadec-9-en-1 -yloxy]-N,N-dimethyl-3-(octyloxy)propan-2-amine, (2R) — N,N-dimethyl-H(1 -metoyloctyl)oxyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, (2R)-1-[(3,7-dimethyloctyl)oxy]-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-2-amine, N,N-dimethyl-1 -(octyloxy)-3-({8-[(1 S,2S)-2-{[(1 R,2R)-2-pentylcyclopropyl]methyl}cyclopropyl]octyl}oxy)propan-2-amine, N,N-dimethyl-1-{[8-(2-oclylcyclopropyl)octyl]oxy}-3-(octyloxy)propan-2-amine and (11 E,20Z,23Z) — N,N-dimethylnonacosa-11 ,20,2-trien-10-amine or a pharmaceutically acceptable salt or stereoisomer thereof.

[0042] In some embodiments, the nanoparticle comprises a lipid-polycation complex. The formation of the lipid-polycation complex may be accomplished by methods known in the art and / or as described in U.S. Patent Publication No. 20120178702, herein incorporated by reference in its entirety. As a non-limiting example, the polycation may include a cationic peptide or a polypeptide 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 include a non-cationic lipid such as, but not limited to, cholesterol or dioleoyl phosphatidylethanolamine (DOPE).

[0043] In various aspects, the cationic liposomes optionally do not comprise a non-cationic lipid (i.e. , a neutral lipid). Neutral molecules, in some aspects, may interfere with coiling / condensation of multilamellar nanoparticles resulting in RNA loaded liposomes greater than 200 nm in size. Cationic liposomes generated without helper molecules can comprise a size of about 50-500 nm, such as about 70-250 nm or 70-200 nm (or less). These constructs consist essentially of a cationic lipid with negatively charged nucleic acid, and may be formulated in a sealed rotary vacuum evaporator which prevents oxidation of the particles (whenMGB 32917 / 59848 PCexposed to the ambient environment). In this aspect, the absence of a helper lipid optimizes mRNA coiling into tightly packaged multilamellar NPs where each NP contains a greater amount of nucleic acid per particle. Due to increased nucleic acid payload per particle, these multilamellar RNA nanoparticles drive significantly greater innate immune responses, which are a significant predictor of efficacy for modulating the immune system.

[0044] In some aspects, the nucleic acid molecules are present at a nucleic acid molecule: cationic lipid ratio of about 1 to about 5 to about 1 to about 25. In some aspects, the nucleic acid molecules are present at a nucleic acid molecule: cationic lipid ratio of about 1 to about 5 to about 1 to about 20, optionally, about 1 to about 15, about 1 to about 10, or about 1 to about 7.5. As used herein, the term “nucleic acid molecule: cationic lipid ratio” is meant a mass ratio, where the mass of the nucleic acid molecule is relative to the mass of the cationic lipid. Also, in exemplary aspects, the term “nucleic acid molecule: cationic lipid ratio” is meant the ratio of the mass of the nucleic acid molecule, e.g., RNA, added to the liposomes comprising cationic lipids during the process of manufacturing the ML RNA NPs of the present disclosure. In exemplary aspects, the nanoparticle comprises less than or about 10 pg RNA molecules per 150 pg lipid mixture. In exemplary aspects, the nanoparticle is made by incubating about 10 pg RNA with about 150 pg liposomes. In alternative aspects, the nanoparticle comprises more RNA molecules per mass of lipid mixture. For example, the nanoparticle may comprise more than 10 pg RNA molecules per 150 pg liposomes. The nanoparticle in some instances comprises more than 15 pg RNA molecules per 150 pg liposomes or lipid mixture.

[0045] The nucleic acid molecules incorporated into the nanoparticles comprise a sequence of a nucleic acid molecule expressed by a CTC. In this respect, the nucleic acid molecules may be a pool of nucleic acid molecules comprising the sequences of one or more nucleic molecules expressed by a CTC, such that the nanoparticles comprise a nucleic acid payload that is similar to (e.g., is representative of) the nucleic acid content of one or more CTCs. For instance, the RNA in the nanoparticle may comprise RNA which comprises, consists essentially of, or consists of CTC transcriptome RNA (e.g., all or part of the RNA transcriptome of CTCs). In various aspects, the nanoparticle comprises a mixture of RNA which is RNA isolated from a tumor of a human. The nucleic acid molecules of the nanoparticles need not be directly isolated from CTCs (i.e., purified from CTCs and directly incorporated into nanoparticles), although this is contemplated. The nucleic acid molecules may be generated from nucleic acid molecules expressed by one or more CTCs (e.g., via in vitro transcription), as described further below, which is also referenced herein as “isolated” from tumor cells.MGB 32917 / 59848 PC

[0046] The CTC-based RNA in the nanoparticles may encode an epitope of a nucleic acid encoding a fusion protein expressed by the CTC. In exemplary aspects, the epitope comprises a junction of the nucleic acid encoding the fusion protein. In various aspects, the epitope encodes an amino acid sequence which binds to an MHC Class II. By way of example, the fusion protein in various instances is a C11 orf95-RELA fusion protein or a fusion protein described herein or in Parker and Zhang, Chin J Cancer 32(11): 594-603 (2013); Ding et al., In J Mol Sci 19(1): 177 (2018); Wener et al., Molecular Cancer 17, article number 28 (2018); or Yu et al., Scientific Reports 9, article number 1074 (2019). See Figures 25-28 of International Patent Application No. PCT / US21 / 16925, hereby incorporated by reference. In exemplary aspects, the fusion protein is a fusion protein that comprises at least a portion of two of Erdrl , Midi , Ppp1r13b, or CKB. In exemplary aspects, the fusion protein is a fusion protein comprising at least a portion of Erdrl and at least a portion of Midi (e.g., Erdrl / Midi or Midi / Erdrl ) or at least a portion of Ppp1r13b and at least a portion of CKB (e.g., Ppp1r13b / CKB or CKB / Ppp1r13b). In exemplary aspects, the fusion protein is a fusion protein comprising at least a portion of two of EWSR1 , FUSR1 , FOXO1 , SS18, FLI1 , ERG, ETV1 , ETV4, FEV, SSX1. In exemplary aspects, the fusion protein comprises at least a portion of EWSR1 , FUSR1 , FOXO1 , or SS18 and at least a portion of FLI1 , ERG, ETV1 , ETV4, FEV, or SSX1 (e.g., EWSR1 / FLI1, FLI1 / EWSR1, EWSR1 / ERG, ERG / EWSR1, EWSR1 / ETV1 or ETV1 / EWSR1, EWSR1 / ETV4, ETV4 / EWSR1, EWSR1 / FEV, FEV / EWSR1, FUSR1 / FEV, FEV / FUSR1, FUSR1 / ERG, ERG / FUSR1, FOXO1 / PAX3, PAX3 / FOXO1, FOXO1 / PAX7, PAX7 / FOXO1, SS18 / SSX1 , or SSX1 / SS18). In exemplary aspects, the fusion protein is expressed by a sarcoma tumor. In various aspects, the fusion protein comprises at least a portion of two of YAP1, FAM118B, MAMLD1 , C11or95, RELA, EPN, MTOR, CASZ1, TP53, DEK, FXR2, BRAF, KIAA1549, or EML4. In exemplary aspects, the fusion protein comprises at least a portion of YAP1 , C11or95, MTOR, TP53 or BRAF and at least a portion of FAM118B, MAMLD1, RELA, C11orf95, EPN, CASZ1, DEK, FXR2, KIAA1549, or EML4 (e.g., YAP1 / FAM118B, FAM118B / YAP1, YAP1 / MAMLD1, MAMLD1 / YAP1, YAP1 / C11orf95, c11orf95 / YAP1 , C11orf95-RELA, c11orf95 / RELA, EPN-YAP1, EPN-YAP1, MTOR / MTOR, MTOR / CASZ1 , CASZ1 / MTOR, TP53 / TP53, TP53 / DEK, DEK / TP53, TP53 / FXR2, FXR2 / TP53, BRAF / KIAA1549,KIAA1549 / BRAF, BRAF / EML4, or EML4 / BRAF). In exemplary aspects, the fusion protein is expressed by a neuro-tumor. The fusion protein may be any one of those described at the website for the Catalog of Somatic Mutations in Cancer (COSMIC) at cancer.sanger.ac.uk / cosmic / fusion or at the website for the Atlas of Genetics and Cytogenetics in Oncology and Haematology atMGB 32917 / 59848 PCatlasgeneticsoncology.org / Deep / Cancer_CytogenomicslD20145.html. Nanoparticles comprising RNA molecules bind to or encode an epitope of a nucleic acid encoding a fusion protein expressed by a tumor are further described in International Patent Application No.PCT / US21 / 16925, which is hereby incorporated by reference in its entirety.

[0047] Methods of isolating CTCs from a biological sample include, but are not limited to, immunomagnetic bead-based capture (e.g., using antibodies to EpCAM conjugated to magnetic particles, followed by separation of tagged cells in a magnetic field). See, e.g., Allard et al., 2004: Clin Cancer Res 10, 6897-6904. CTCs may also be isolated from a subject’s biological sample (e.g., blood) via a microfluidics-based CTC capture device. See, e.g., Nagrath et al., 2007: Nature 450, 1235-1239. Another tool for isolating CTCs is using the CellSearch® Circulating Tumor Cell (CTC) Test from Veridex, LLC (Raritan, N.J.), a blood test for enumerating CTC in blood based on immunomagnetic labeling and automated digital microscopy. It will be appreciated that other methods of CTC isolation are known in the art and suitable for use in the context of the disclosure.

[0048] In various aspects, the nucleic acid molecules are RNA molecules, e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), or messenger RNA (mRNA). In various aspects, the RNA molecules comprise tRNA, rRNA, mRNA, or a combination thereof. In various aspects, the RNA is total RNA isolated from a cell. In exemplary aspects, the RNA is total RNA isolated from a diseased cell, such as, for example, a tumor cell or a cancer cell.

[0049] In exemplary instances, the RNA molecules are mRNA. In various aspects of the disclosure, a nanoparticle is used which comprises total RNA isolated from a cell. In various aspects, mRNA is in vitro transcribed mRNA. In various instances, the mRNA molecules are produced by in vitro transcription (IVT). Suitable techniques of carrying out IVT are known in the art. Optionally, the RNA is in vitro transcribed mRNA, wherein the in vitro transcription template is cDNA made from RNA extracted from a circulating tumor cell (or, in the context of nanoparticles used in co-therapy, another tumor cell). In various aspects, the RNA comprises a sequence encoding a poly(A) tail so that the in vitro transcribed RNA molecule comprises a poly(A) tail at the 3’ end. In various aspects, the method of making a nanoparticle comprises additional processing steps, such as, for example, capping the in vitro transcribed RNA molecules.

[0050] The CTC RNA-NP may be used with other RNA-NPs as described herein but which contain non-CTC RNA. In this regard, the CTC RNP-NP may be administered as part of a therapeutic regimen that comprises administration of one or more “second" nanoparticles, i.e.,MGB 32917 / 59848 PCnanoparticles that do not comprise nucleic acid molecules isolated from CTCs. For instance, a second nanoparticle may be used in a therapeutic regimen that comprises a nucleic acid that encodes a tumor antigen which is not isolated from a CTC. A number of cancer antigens are known and described in e.g., U.S. Patent No. 10,688,166 (incorporated by reference in its entirety, and particularly with respect to the disclosure of tumor antigens). The antigen may be, for example, a claudin, CD19, CD20, CD22, CD33, CD166, CD70, CD123, CEA, c-Met, PSMA, GD2, GD3, FRoc, CAIX, CD171, EGFRVIII, HER2, mesothelin, CD133, CEACAM5, EGFR, GPC3, PSMA, ROR1, VEGFR2, B7-H3, IL-13Ra, PD-L1, IL-11Ra, EphA2, MAGE, MCAM, NKG2D ligands, TEM1 , FAP, GAGE, MUC1 , or NY-ESO-1. In various aspects, the surface antigen is CD70. The sequence of human CD70 is known in the art. See, e.g., UniProtKB No. P32970. The second nanoparticle may comprise RNA which does not encode a tumor antigen. The second nanoparticle may comprise RNA which, for instance, encodes a viral antigen.

[0051] The RNA (e.g., mRNAs) in exemplary aspects encodes a protein. In the context of a second RNA-NP for use with the CTC RNA-NP of the disclosure, the protein is optionally selected from the group consisting of a tumor antigen, a cytokine, and a co-stimulatory molecule. Indeed, the protein is, in some aspects, selected from the group consisting of a tumor antigen, a co-stimulatory molecule, a cytokine, a growth factor, a hematopoietic factor, or a lymphokine, including, e.g., cytokines and growth factors that are effective in inhibiting tumor metastasis, and cytokines or growth factors that have been shown to have an antiproliferative effect 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, TNFa, TNF1 , TNF2, G-CSF, Meg-CSF, GM-CSF, thrombopoietin, stem cell factor, and erythropoietin. Additional growth factors for use herein include angiogenin, bone morphogenic protein-1, bone morphogenic protein-2, bone morphogenic protein-3, bone morphogenic protein-4, bone morphogenic protein-5, bone morphogenic protein-6, bone morphogenic protein-7, bone morphogenic protein-8, bone morphogenic protein-9, bone morphogenic protein-10, bone morphogenic protein-11 , bone morphogenic protein-12, bone morphogenic protein-13, bone morphogenic protein-14, bone morphogenic protein-15, bone morphogenic protein receptor IA, bone morphogenic protein receptor IB, brain derived neurotrophic factor, ciliary neurotrophic factor, ciliary neurotrophic factor receptor a, cytokine-induced neutrophil chemotactic factor 1 , cytokine-induced neutrophil, chemotactic factor 2 a, cytokine-induced neutrophil chemotactic factor 2 p, p endothelial cell growth factor, endothelin 1, epithelial-derived neutrophil attractant, glial cell line-derived neurotrophic factor receptor a 1 , glial cell line-MGB 32917 / 59848 PCderived neurotrophic factor receptor a 2, growth related protein, growth related protein a, growth related protein p, growth related protein y, heparin binding epidermal growth factor, hepatocyte growth factor, hepatocyte growth factor receptor, insulin-like growth factor I, insulin-like growth factor receptor, insulin-like growth factor II, insulin-like growth factor binding protein, keratinocyte growth factor, leukemia inhibitory factor, leukemia inhibitory factor receptor a, nerve growth factor nerve growth factor receptor, neurotrophin-3, neurotrophin-4, pre-B cell growth stimulating factor, stem cell factor, stem cell factor receptor, transforming growth factor a, transforming growth factor p, transforming growth factor pi , transforming growth factor pi .2, transforming growth factor p2, transforming growth factor P3, transforming growth factor P5, latent transforming growth factor pi , transforming growth factor p binding protein I, transforming growth factor p binding protein II, transforming growth factor p binding protein III, tumor necrosis factor receptor type I, tumor necrosis factor receptor type II, urokinase-type plasminogen activator receptor, and chimeric proteins and biologically or immunologically active fragments thereof. In exemplary aspects, the tumor antigen is an antigen derived from a viral protein, an antigen derived from point mutations, or an antigen encoded by a cancer-germline gene. In exemplary aspects, the tumor antigen is pp65, p53, KRAS, NRAS, MAGEA, MAGEB, MAGEC, BAGE, GAGE, LAGE / NY-ESO1 , SSX, tyrosinase, gp100 / pmel17, Melan-A / MART-1, gp75 / TRP1 , TRP2, CEA, RAGE-1 , HER2 / NEU, or WT 1 , or any other tumor antigens described herein. In exemplary aspects, the co-stimulatory molecule is selected from the group consisting of CD80 and CD86.

[0052] The methods of the disclosure may further comprise use of a second nanoparticle comprising nucleic acid that is not mRNA isolated from a tumor (i.e., not tumor mRNA), or is not a nucleic acid that encodes a protein expressed by a tumor cell or by a human (i.e., the protein is not related to a tumor antigen or cancer antigen).

[0053] In various aspects, the disclosure contemplates use of second nanoparticles wherein the nucleic acid layers comprise nucleic acid molecules having a nucleic acid sequence of nucleic acid molecules expressed by slow-cycling cells (SCCs). For instance, the second nanoparticle may comprise RNA which comprises, consists essentially of, or consists of RNA having the nucleic acid sequence of SCO transcriptome RNA. The term "slow-cycling cells" or "SCCs" refers to tumor or cancer cells that proliferate at a slow rate. In exemplary aspects, the 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): 1331-1343MGB 32917 / 59848 PC(2011) (incorporated by reference herein, particularly with respect to the description of SCCs), SCCs display increased tumor-initiation properties and are stem cell like. Because of their slow proliferation rate, SCCs are also referred to as label-retaining cells (LRCs). In exemplary instances, the nucleic acid molecules are RNA extracted from isolated SCCs or are nucleic acid molecules which hybridize to RNA extracted from isolated SCCs. Optionally, the SCCs are isolated from a mixed tumor cell population obtained from a subject with a tumor (e.g., a glioblastoma). As used herein, the term "mixed tumor cell population" refers to a heterogeneous cell population comprising tumor cells of different sub-types and comprising slow-cycling cells and at least one other tumor cell type, e.g., fast-cycling cells (FCCs). NP comprising nucleic acid layers comprising a sequence of a nucleic acid molecule expressed by slow-cycling cells (SCCs) are further described in International Patent Application No.PCT / US21 / 16925 (WO 2021 / 158996), which is hereby incorporated by reference in its entirety, particularly with respect to Figure 12.

[0054] Optionally, various aspects of the disclosure may involve use of a second nanoparticle comprising RNA molecules that bind to or encode an epitope of a nucleic acid encoding a fusion protein expressed by a tumor (optionally not expressed by a CTC). In exemplary aspects, the epitope comprises a junction of the nucleic acid encoding the fusion protein. In various aspects, the epitope encodes an amino acid sequence which binds to an MHC Class II. By way of example, the fusion protein in various instances is a C11orf95-RELA fusion protein or a fusion protein described herein or in Parker and Zhang, Chin J Cancer 32(11 ): 594-603 (2013); Ding et al., In J Mol Sci 19(1): 177 (2018); Wener et al., Molecular Cancer 17, article number 28 (2018); or Yu et al., Scientific Reports 9, article number 1074 (2019). See Figures 25-28 of International Patent Application No. PCT / US21 / 16925, hereby incorporated by reference. In exemplary aspects, the fusion protein is a fusion protein that comprises at least a portion of two of Erdrl , Midi, Ppp1r13b, or CKB. In exemplary aspects, the fusion protein is a fusion protein comprising at least a portion of Erdrl and at least a portion of Midi (e.g., Erdrl / Midi or Midi / Erdrl ) or at least a portion of Ppp1r13b and at least a portion of CKB (e.g., Ppp1 r13b / CKB or CKB / Ppp1 r13b). In various aspects, the fusion protein is expressed by a murine model of a brain tumor. In exemplary aspects, the fusion protein is a fusion protein comprising at least a portion of two of EWSR1 , FUSR1 , FOXO1 , SS18, FLI1 , ERG, ETV1 , ETV4, FEV, SSX1 . In exemplary aspects, the fusion protein comprises at least a portion of EWSR1 , FUSR1 , FOXO1 , or SS18 and at least a portion of FLI1, ERG, ETV1, ETV4, FEV, or SSXI (e.g., EWSR1 / FLI1, FLI1 / EWSR1, EWSR1 / ERG, ERG / EWSR1, EWSR1 / ETV1 or ETV1 / EWSR1 , EWSR1 / ETV4, ETV4 / EWSR1, EWSR1 / FEV, FEV / EWSR1, FUSR1 / FEV, FEV / FUSR1, FUSR1 / ERG,MGB 32917 / 59848 PCERG / FUSR1, F0X01 / PAX3, PAX3 / F0X01, F0X01 / PAX7, PAX7 / F0X01, SS18 / SSX1, or SSX1 / SS18). In exemplary aspects, the fusion protein is expressed by a sarcoma tumor. In various aspects, the fusion protein comprises at least a portion of two of YAP1, FAM118B, MAMLD1, C11or95, RELA, EPN, MTOR, CASZ1, TP53, DEK, FXR2, BRAF, KIAA1549, or EML4. In exemplary aspects, the fusion protein comprises at least a portion of YAP1, C11or95, MTOR, TP53 or BRAF and at least a portion of FAM118B, MAMLD1 , RELA, 011 orf95, EPN, CASZ1, DEK, FXR2, KIAA1549, or EML4 (e.g., YAP1 / FAM118B, FAM118B / YAP1, YAP1 / MAMLD1, MAMLD1 / YAP1, YAP1 / C11orf95, c11orf95 / YAP1 , C11orf95-RELA, c11orf95 / RELA, EPN-YAP1, EPN-YAP1, MTOR / MTOR, MTOR / CASZ1 , CASZ1 / MTOR, TP53 / TP53, TP53 / DEK, DEK / TP53, TP53 / FXR2, FXR2 / TP53, BRAF / KIAA1549,KIAA1549 / BRAF, BRAF / EML4, or EML4 / BRAF). In exemplary aspects, the fusion protein is expressed by a neuro-tumor. The fusion protein may be any one of those described at the website for the Catalog of Somatic Mutations in Cancer (COSMIC) at cancer.sanger.ac.uk / cosmic / fusion or at the website for the Atlas of Genetics and Cytogenetics in Oncology and Haematology at atlasgeneticsoncology.org / Deep / Cancer_CytogenomicslD20145.html. Nanoparticles comprising RNA molecules bind to or encode an epitope of a nucleic acid encoding a fusion protein expressed by a tumor are further described in International Patent Application No.PCT / US21 / 16925, which is hereby incorporated by reference in its entirety.

[0055] In various instances, aspects of the disclosure may involve use of nanoparticles comprising RNA molecules that are antisense molecules, optionally siRNA, shRNA, miRNA (microRNA), or any combination thereof. The antisense molecule can be one which mediates RNA interference (RNAi). As known by one of ordinary skill in the art, RNAi is a ubiquitous mechanism of gene regulation in plants and animals in which target mRNAs are degraded in a sequence-specific manner (Sharp, Genes Dev., 15, 485-490 (2001); Hutvagner et aL, Curr. Opin. Genet. Dev., 12, 225-232 (2002); Fire et aL, Nature, 391, 806-811 (1998); Zamore et aL, Cell, 101 , 25-33 (2000)). The natural RNA degradation process is initiated by the dsRNA-specific endonuclease Dicer, which promotes cleavage of long dsRNA precursors into doublestranded fragments between 21 and 25 nucleotides long, termed small interfering RNA (siRNA; also known as short interfering RNA) (Zamore et aL, Cell., 101 , 25-33 (2000); Elbashir et aL, Genes Dev., 15, 188-200 (2001); Hammond et aL, Nature, 404, 293-296 (2000); Bernstein et aL, Nature, 409, 363-366 (2001)). siRNAs are incorporated into a large protein complex that recognizes and cleaves target mRNAs (Nykanen et aL, Cell, 107, 309-321 (2001)). It has been reported that introduction of dsRNA into mammalian cells does not result in efficient Dicer-MGB 32917 / 59848 PCmediated generation of siRNA and therefore does not induce RNAi (Caplen et aL, Gene, 252, 95-105 (2000); Ui-Tei et aL, FEES Lett, 479, 79-82 (2000)). The requirement for Dicer in maturation of siRNAs in cells can be bypassed by introducing synthetic 21 -nucleotide siRNA duplexes, which inhibit expression of transfected and endogenous genes in a variety of mammalian cells (Elbashir et al., Nature, 411 : 494-498 (2001 )). Aspects of the disclosure may involve use of nanoparticles comprising RNA molecules that mediate RNAi, and in some aspects the RNA is a siRNA molecule specific for inhibiting the expression of a protein. The term "siRNA" as used herein refers to an RNA (or RNA analog) comprising from about 10 to about 50 nucleotides (or nucleotide analogs) which is capable of directing or mediating RNAi. In exemplary embodiments, an siRNA molecule comprises about 15 to about 30 nucleotides (or nucleotide analogs) or about 20 to about 25 nucleotides (or nucleotide analogs), e.g., 21-23 nucleotides (or nucleotide analogs). The siRNA can be double or single stranded, preferably double-stranded.

[0056] In alternative aspects, the disclosure contemplates use of a nanoparticle comprising RNA molecules that are short hairpin RNA (shRNA) molecules specific for inhibiting the expression of a protein. The term "shRNA" as used herein refers to a molecule of about 20 or more base pairs in which a single-stranded RNA partially contains a palindromic base sequence and forms a double-strand structure therein (i.e., a hairpin structure). An shRNA can be an siRNA (or siRNA analog) which is folded into a hairpin structure. shRNAs typically comprise about 45 to about 60 nucleotides, including the approximately 21 nucleotide antisense and sense portions of the hairpin, optional overhangs on the non-loop side of about 2 to about 6 nucleotides long, and the loop portion that can be, e.g., about 3 to 10 nucleotides long.

[0057] In exemplary aspects, disclosure contemplates use of nanoparticles comprising an antisense molecule which is a microRNA (miRNA). As used herein the term “microRNA” refers to a small (e.g., 15-22 nucleotides), non-coding RNA molecule which base pairs with mRNA molecules to silence gene expression via translational repression or target degradation. microRNA and the therapeutic potential thereof are described in the art. See, e.g., Mulligan, MicroRNA: Expression, Detection, and Therapeutic Strategies, Nova Science Publishers, Inc., Hauppauge, NY, 2011 ; Bader and Lammers, “The Therapeutic Potential of microRNAs” Innovations in Pharmaceutical Technology, pages 52-55 (March 2011 ).

[0058] In certain instances, the RNA molecule is an antisense molecule, optionally, an siRNA, shRNA, or miRNA, which targets a protein of an immune checkpoint pathway for reduced expression. In various aspects, the protein of the immune checkpoint pathway isMGB 32917 / 59848 PCCTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, TIGIT, LAG3, CD112 TIM3, BTLA, or costimulatory receptor ICOS, 0X40, 41 BB, or GITR. The protein of the immune-checkpoint pathway in certain instances is CTLA4, PD-1, PD-L1, B7-H3, B7H4, orTIM3. Immune checkpoint signaling pathways are reviewed in Pardoll, Nature Rev Cancer, 12(4): 252-264 (2012). Inhibitors of immune checkpoint molecules are contemplated herein.

[0059] In exemplary embodiments, the nanoparticles of the present disclosure comprise a mixture of RNA molecules. In exemplary aspects, the mixture of RNA molecules is RNA isolated from cells from a human and optionally, the human has a tumor. In some aspects, the mixture of RNA is RNA isolated from the tumor cells of the human. In exemplary aspects, the human has cancer, optionally, any cancer described herein. Optionally, the tumor cells from which RNA is isolated is selected from the group consisting of a glioma cells (including, but not limited to, a glioblastoma), medulloblastoma cells, a diffuse intrinsic pontine glioma cells, or a cells of a peripheral tumor with metastatic infiltration into the central nervous system (e.g., melanoma or breast cancer). Optionally, the tumor cells from which RNA is isolated is osteosarcoma.

[0060] In various aspects, the nanoparticles comprise a nucleic acid molecule (e.g., RNA molecule) comprising a nucleotide sequence encoding a chimeric protein comprising a LAMP protein. In certain aspects, the LAMP protein is a LAMP1 , LAMP 2, LAMP3, LAMP4, or LAMP5 protein.

[0061] Use

[0062] The disclosure further provides a method of treating cancer in a subject, the method comprising administering to a subject in need thereof a nanoparticle comprising a positively-charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between a cationic lipid bilayer and the nucleic acid molecules in the nucleic acid layers comprise a sequence of a nucleic acid molecule expressed by a CTC. Optionally, the nanoparticles are systemically administered to the subject. For example, the nanoparticles are administered intravenously. Optionally, nanoparticles are administered intramuscularly.

[0063] The method of the disclosure may comprise multiple administrations of a CTC nanoparticle as described herein. For instance, the method may comprise two or more administrations of a nanoparticle, wherein each administration comprises a nanoparticle comprising RNA comprising a sequence of RNA expressed in a CTC isolated from the subjectMGB 32917 / 59848 PCat different points in time. Merely to illustrate, a first administration of a CTC nanoparticle may comprise administering a nanoparticle comprising nucleic acid molecules comprising a sequence (i.e., one or more sequences) of a nucleic acid molecule(s) expressed by a CTC isolated from a subject at a first time point. A second administration of a CTC nanoparticle to the subject may comprise administration of the same CTC nanoparticle as administered in the first administration; however, the second administration may alternatively comprise administering a nanoparticle comprising nucleic acid molecules comprising a sequence (i.e., one or more sequences) of a nucleic acid molecule(s) expressed by a CTC isolated from a subject at a second time point (which is different than the first time point). This aspect of the disclosure allows the therapeutic regimen to follow the potential evolution of a cancer in the subject; if the cancer mutates, the nanoparticles of the subsequent administrations comprise nucleic acid molecules which more closely reflect the mutated cancer cells.

[0064] In various aspects, the disclosure provides a method comprising (a) isolating a circulating tumor cell (CTC) from a subject, (b) producing a nanoparticle as described herein comprising RNA comprising a sequence of RNA expressed in the CTC, (c) administering the nanoparticle of step (b) to the subject, and (d) repeating steps (a)-(c) one or more times.

[0065] In various aspects, the method further comprises administering an additional (a second or third) nanoparticle comprising a positively-charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between a cationic lipid bilayer. The additional (i.e., second) nanoparticles comprise different nucleic acids compared to the nanoparticles produced using CTC nucleic acid molecules. In various aspects, the nucleic acid incorporated into the additional (i.e., second) nanoparticles is tumor mRNA, such as mRNA that is in vitro transcribed mRNA wherein the in vitro transcription template is cDNA made from RNA extracted from a tumor cell.

[0066] The materials and methods described herein are useful, e.g., for treating a subject for a disease or disorder, such as cancer (e.g., a solid tumor). As used herein, the term “treat,” as well as words related thereto, does not necessarily imply 100% or complete treatment or remission. Rather, there are varying degrees of treatment of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. In this respect, the methods of treating a disease or disorder can provide any amount or any level of treatment. Furthermore, the treatment provided by the method may include treatment of one or more conditions or symptoms or signs of the disease being treated. For instance, the treatment method of the present disclosure may inhibit one or more symptoms of the disease. Also, the treatmentMGB 32917 / 59848 PCprovided by the methods of the present disclosure may encompass slowing the progression of the disease.

[0067] The term “treat” also encompasses prophylactic treatment of the disease.Accordingly, the treatment provided by the presently disclosed method may delay the onset or reoccurrence / relapse of the disease being prophylactically treated. In exemplary aspects, the method delays the onset of the disease by 1 day, 2 days, 4 days, 6 days, 8 days, 10 days, 15 days, 30 days, two months, 4 months, 6 months, 1 year, 2 years, 4 years, or more. The prophylactic treatment encompasses reducing the risk of the disease being treated. In exemplary aspects, the method reduces the risk of the disease 2-fold, 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or more.

[0068] In certain aspects, the method of treating the disease may be regarded as a method of inhibiting the disease or a symptom thereof. As used herein, the term “inhibit” and words stemming therefrom may not be a 100% or complete inhibition or abrogation. Rather, there are varying degrees of inhibition of which one of ordinary skill in the art recognizes as having a potential benefit or therapeutic effect. The presently disclosed methods may inhibit the onset or re-occurrence of the disease or a symptom thereof to any amount or level. In exemplary embodiments, the inhibition provided by the methods is at least or about a 10% inhibition (e.g., at least or about a 20% inhibition, at least or about a 30% inhibition, at least or about a 40% inhibition, at least or about a 50% inhibition, at least or about a 60% inhibition, at least or about a 70% inhibition, at least or about an 80% inhibition, at least or about a 90% inhibition, at least or about a 95% inhibition, at least or about a 98% inhibition). The materials and methods may inhibit the spread or growth of a tumor in any amount or level.

[0069] Treatment for cancer (e.g., a solid tumor) may be determined by any of a number of ways. Any improvement in the subject's wellbeing is contemplated (e.g., at least or about a 10% reduction, at least or about a 20% reduction, at least or about a 30% reduction, at least or about a 40% reduction, at least or about a 50% reduction, at least or about a 60% reduction, at least or about a 70% reduction, at least or about an 80% reduction, at least or about a 90% reduction, or at least or about a 95% reduction of any parameter described herein). For example, a therapeutic response would refer 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) inhibition of neoplastic cell survival; (4) inhibition (i.e., slowing to some extent, preferably halting) of tumor growth or appearance of new lesions; (5) decrease in tumor size or burden; (6) absence of clinically detectable disease, (7) decrease in levels of cancer markers;MGB 32917 / 59848 PC(8) an increased patient survival rate; and / or (9) some relief from one or more symptoms associated with the disease or condition (e.g., pain). For example, the efficacy of treatment may be determined by detecting a change in tumor mass and / or volume after treatment. The size of a tumor may be compared to the initial size and dimensions as measured by CT, PET, mammogram, ultrasound, or palpation, as well as by caliper measurement or pathological examination of the tumor after biopsy or surgical resection. Response may be characterized quantitatively using, e.g., percentage change in tumor volume (e.g., the method of the disclosure results in a reduction of tumor volume by 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%).Alternatively, tumor response or cancer response may be characterized in a qualitative fashion like "pathological complete response" (pCR), "clinical complete remission" (cCR), "clinical partial remission" (cPR), "clinical stable disease" (cSD), "clinical progressive disease" (cPD), or other qualitative criteria. In addition, treatment efficacy also can be characterized in terms of responsiveness to other immunotherapy treatment or chemotherapy. In various aspects, the methods of the disclosure further comprise monitoring treatment in the subject.

[0070] With regard to the foregoing methods, the composition comprising the nanoparticles, in some aspects, is systemically administered to the subject. Optionally, the method comprises administration of any of the nanoparticles and compositions described herein by way of parenteral administration. Parenteral dosage forms of any agent described herein can be administered to a subject by various routes, including, but not limited to, epidural, intracerebral, intracerebroventricular, epicutaneous, intraarterial, intraarticular, intracardiac, intracavernous injection, intradermal, intralesional, intramuscular, intraocular, intraosseous infusion, intraperitoneal, intrathecal, intrauterine, intravaginal administration, intravenous, intravesical, intravitreal, subcutaneous, transdermal, perivascular administration, or transmucosal. For administration to the brain, a pharmaceutical composition can be introduced into tumor tissue using an intratumoral delivery catheter, ventricular shunt catheter attached to a reservoir (e.g., Omaya reservoir), infusion pump, or introduced into a tumor resection cavity (such as Gliasite, Proxima Therapeutics). Tumor tissue in the brain also can be contacted by administering a pharmaceutical composition via convection using continuous infusion catheter or through cerebrospinal fluid. In various instances, the composition is administered to the subject intravenously.

[0071] The amount or dose of an active agent (i.e., the "effective amount") administered should be sufficient to achieve a desired biological effect, e.g., a therapeutic or prophylacticMGB 32917 / 59848 PCresponse, in the subject over a reasonable time frame. By way of example and not intending to limit the present disclosure, the dose of the active agents of the present disclosure can be about 0.0001 to about 1 g / kg body weight of the subject being treated / day, from about 0.0001 to about 0.001 g / kg body weight, or about 0.01 mg to about 1 g / kg body weight.

[0072] In various aspects, the nanoparticle composition is administered according to any regimen including, for example, daily (1 time per day, 2 times per day, 3 times per day, 4 times per day, 5 times per day, 6 times per day), three times a week, twice a week, every two days, every three days, every four days, every five days, every six days, weekly, bi-weekly, etc. In various aspects, the composition is administered to the subject once a week. An administration regimen for the CTC nanoparticle of the disclosure and an additional nanoparticle composition may be the same or may be different. For instance, the additional nanoparticle (also referenced herein as “second” nanoparticle) may be administered as a first line of treatment. To illustrate, the additional (i.e., second) nanoparticle may comprise nucleic acid molecules comprising a sequence (or sequences) of nucleic acid molecules expressed by a tumor which is biopsied in the patient. The CTC nanoparticle of the disclosure may then be administered as a follow-on treatment, following the first line of treatment based on biopsied tumor. The first line of treatment and the follow-on treatment may comprise independent administration regimens (i.e., the dosing, timing, and route of administration of a first line of treatment and the follow-on treatment may be the same or different).

[0073] The methods of the present disclosure may comprise the above described step(s) alone or in combination with other steps. The methods may comprise repeating any one of the above-described step(s) and / or may comprise additional steps, aside from those described above. For example, the presently disclosed methods may further comprise steps for making or preparing the nanoparticles or compositions of the present disclosure. For instance, the presently disclosed methods further comprise obtaining a sample of CTCs from the subject, optionally, via blood draw. The methods also may further comprise isolating total RNA from the cells of the tumor cells, generating cDNA from the total RNA via reverse transcription, and amplifying mRNA from the cDNA. The presently disclosed methods also in some aspects further comprise mixing the mRNA and the cationic lipid at a 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 exemplary instances, the presently disclosedMGB 32917 / 59848 PCmethods further comprise mixing the mRNA and the cationic lipid at a RNA: cationic lipid ratio of about 1 to about 15.

[0074] Subjects

[0075] The subject is a mammal, including, but not limited to, mammals of the order Rodentia, such as mice and hamsters, and mammals of the order Logomorpha, such as rabbits, mammals from the order Carnivora, including Felines (cats) and Canines (dogs), mammals from the order Artiodactyla, including Bovines (cows) and Swines (pigs) or of the order Perssodactyla, including Equines (horses). In some aspects, the mammals are of the order Primates, Ceboids, or Simoids (monkeys) or of the order Anthropoids (humans and apes). In some aspects, the mammal is a human. In some aspects, the human is an adult aged 18 years or older. In some aspects, the human is a child aged 17 years or less.

[0076] Cancer

[0077] The cancer treatable by the methods disclosed herein may be any cancer, e.g., any malignant growth or tumor caused by abnormal and uncontrolled cell division that optionally may spread to other parts of the body through the lymphatic system or the blood stream. In various aspects, the subject has a solid tumor. The cancer in some aspects is one selected from the group consisting of acute lymphocytic cancer, acute myeloid leukemia, alveolar rhabdomyosarcoma, bone cancer, brain cancer (e.g., glioma), breast cancer (e.g., triple negative breast cancer), cancer of the anus, cancer of the anal canal, cancer of the anorectum, cancer of the eye, cancer of the intrahepatic bile duct, cancer of the joints, cancer of the head, neck, gallbladder, or pleura, cancer of the nose, nasal cavity, or middle ear, cancer of the oral cavity, cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid cancer, colon cancer, esophageal cancer, cervical cancer, gastrointestinal cancer (e.g., gastrointestinal carcinoid tumor), Hodgkin lymphoma, endometrial or hepatocellular carcinoma, hypopharynx cancer, kidney cancer, larynx cancer, liver cancer, lung cancer (e.g., non-small cell lung cancer, bronchioloalveolar carcinoma), malignant mesothelioma, melanoma, multiple myeloma, nasopharynx cancer, non-Hodgkin lymphoma, ovarian cancer, osteosarcoma, pancreatic cancer, cancer of the peritoneum, cancer of the omentum, mesentery cancer, pharynx cancer, prostate cancer, rectal cancer, renal cancer (e.g., renal cell carcinoma (RCC)), small intestine cancer, soft tissue cancer, stomach cancer, testicular cancer, thyroid cancer, ureter cancer, and urinary bladder cancer. In particular aspects, the cancer is selected from the group consisting of head and neck, ovarian, cervical, bladder and oesophageal cancers, pancreatic, gastrointestinal cancer, gastric, breast, endometrial and colorectal cancers, hepatocellularMGB 32917 / 59848 PCcarcinoma, glioblastoma, bladder and lung cancer (e.g., non-small cell lung cancer (NSCLC), bronchioloalveolar carcinoma). Optionally, the subject suffers from a malignant brain tumor, such as a glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma, or a peripheral tumor with metastatic infiltration into the central nervous system. Optionally, the subject suffers from osteosarcoma, such as recurrent or metastatic osteosarcoma.

[0078] In various aspects, the subject is suffering from a refractory malignancy. In this respect, a tumor which evades a particular therapy or host immune response is “refractory” (or resistant). A tumor that is “sensitive” to a therapy demonstrates a beneficial clinical response to treatment. A tumor that is “sensitive” to a host immune response is recognized by the host immune system and subject to attack by immune effector cells. In various aspects, the cancer is refractory or resistant to immune checkpoint inhibitors or a first line treatment for the particular cancer.

[0079] In some embodiments, the method described herein further comprises administration of one or more other therapeutic agents. In some aspects, the other therapeutic agent aims to treat or prevent cancer. In some embodiments, the other therapeutic is a chemotherapeutic agent. Common chemotherapeutics include, but are not limited to, adriamycin, asparaginase, bleomycin, busulphan, cisplatin, carboplatin, carmustine, capecitabine, chlorambucil, cytarabine, cyclophosphamide, camptothecin, dacarbazine, dactinomycin, daunorubicin, dexrazoxane, docetaxel, doxorubicin, etoposide, floxuridine, fludarabine, fluorouracil, gemcitabine, hydroxyurea, idarubicin, ifosfamide, irinotecan, lomustine, mechlorethamine, mercaptopurine, meplhalan, methotrexate, mitomycin, mitotane, mitoxantrone, nitrosurea, paclitaxel, pamidronate, pentostatin, plicamycin, procarbazine, rituximab, streptozocin, teniposide, thioguanine, thiotepa, vinblastine, vincristine, vinorelbine, taxol, transplatinum, 5-fluorouracil, and the like.

[0080] In some embodiments, the other therapeutic is an agent used in radiation therapy for the treatment of cancer; indeed, in some embodiments, the method is part of a treatment regimen that includes radiation therapy. Further, the method of the disclosure can be performed in connection with surgical resection of a tumor, such as a glioma (e.g., glioblastoma).

[0081] In exemplary aspects, the method comprises administering an immune checkpoint inhibitor (ICI) to the subject. An “immune checkpoint inhibitor” or “ICI” is any agent (e.g., compound or molecule) that that decreases, blocks, inhibits, abrogates or interferes with the function of a protein of an immune checkpoint pathway. Proteins of the immune checkpoint pathway regulate immune responses and, in some instances, prevent T cells from attackingMGB 32917 / 59848 PCcancer cells. In various aspects, the protein of the immune checkpoint pathway is, for example, CTLA-4, PD-1, PD-L1, PD-L2, B7-H3, B7-H4, TIGIT, VISTA, LAG3, CD112 TIM3, BTLA, or costimulatory receptor ICOS, 0X40, 41 BB, or GITR. In various aspects, the ICI is a small molecule, an inhibitory nucleic acid, or an inhibitor polypeptide. In various aspects, the ICI is an antibody, antigen-binding antibody fragment, or an antibody protein product, that binds to and inhibits the function of the protein of the immune checkpoint pathway. Suitable ICIs which are antibodies, antigen-binding antibody fragments, or an antibody protein products are known in the art and include, but are not limited to, 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., 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).

[0082] Methods of Nanoparticle Manufacture

[0083] The nanoparticle 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 a cationic lipid bilayer, may be manufactured by a method comprising (A) mixing nucleic acid molecules and liposomes at a nucleic acid (e.g., RNA): liposome ratio of about 1 to about 5 to about 1 to about 25, such as about 1 to 5 to about 1 to about 20, optionally, about 1 to about 15, to obtain nucleic acid- (e.g., RNA-) coated liposomes. The liposomes are made by a process of making liposomes comprising drying a lipid mixture comprising a cationic lipid and an organic solvent by evaporating the organic solvent under a vacuum. The method further comprises (B) mixing the RNA-coated liposomes with a surplus amount of liposomes. In exemplary aspects, the nanoparticle made by the presently disclosed method accords with the descriptions of the nanoparticles described herein. For example, the nanoparticle made by the presently disclosed methods has a zeta potential of about +40 mV to about +60 mV, optionally, about +45 mV to about +55 mV. Optionally, the zeta potential of the nanoparticle made by the presently disclosed methods is about +50 mV. In various aspects, the core of the nanoparticle made by the presently disclosed methods comprises less than about 0.5 wt% nucleic acid and / or the core comprises a cationic lipid bilayer and / or the outermostMGB 32917 / 59848 PClayer of the nanoparticle comprises a cationic lipid bilayer and / or the surface of the nanoparticle comprises a plurality of hydrophilic moieties of the cationic lipid of the cationic lipid bilayer.

[0084] In exemplary aspects, the lipid mixture comprises the cationic lipid and the organic solvent at a ratio of about 40 mg cationic lipid per ml_ organic solvent to about 60 mg cationic lipid per mL organic solvent, optionally, at a ratio of about 50 mg cationic lipid per mL organic solvent. In various instances, the process of making liposomes further comprises rehydrating the lipid mixture with a rehydration solution to form a rehydrated lipid mixture and then agitating, resting, and sizing the rehydrated lipid mixture. Optionally, sizing the rehydrated lipid mixture comprises sonicating, extruding, and / or filtering the rehydrated lipid mixture.

[0085] A description of an exemplary method of making a nanoparticle is provided herein at Example 1 . It will be appreciated that any one or more of the steps described in Example 1 may be adjusted as needed. For instance, in some embodiments, the method comprises one or more steps required for preparing the RNA prior to being complexed with the liposomes. In exemplary aspects, downstream steps are included to prepare the nanoparticles for administration to a subject, e.g., a human. In exemplary instances, the method comprises formulating the NP for intravenous injection. The method comprises in various aspects adding one or more pharmaceutically acceptable carriers, diluents, or excipients, and optionally comprises packaging the resulting composition in a container, e.g., a vial, a syringe, a bag, an ampoule, and the like. The container in some aspects is a ready-to-use container and optionally is for single-use.

[0086] Pharmaceutical Compositions

[0087] Provided herein are compositions comprising a nanoparticle of the present disclosure and a pharmaceutically acceptable carrier, excipient or diluent. In exemplary aspects, the composition is a sterile composition. In exemplary instances, the composition comprises a plurality of nanoparticles of the present disclosure. Optionally, at least 50% of the nanoparticles of the plurality have a diameter between about 50 nm and about 500 nm (e.g., between about 50 nm and 450 nm, between about 50 nm and 400 nm, between about 50 nm and 350 nm, between about 50 nm and 300 nm, between about 100 nm to about 250 nm, between about 70 nm and about 250 nm, or between about 70 nm and about 200 nm). In various aspects, the composition comprises about 1010nanoparticles per mLto about 1015nanoparticles per mL, optionally about 1012nanoparticles ± 10% per mL.MGB 32917 / 59848 PC

[0088] In exemplary aspects, the composition of the present disclosure may comprise additional components other than the nanoparticle. The composition, in various aspects, comprises any pharmaceutically acceptable ingredient, including, for example, acidifying agents, additives, adsorbents, aerosol propellants, air displacement agents, alkalizing agents, anticaking agents, anticoagulants, antimicrobial preservatives, antioxidants, antiseptics, bases, binders, buffering agents, chelating agents, coating agents, coloring agents, desiccants, detergents, diluents, disinfectants, disintegrants, dispersing agents, dissolution enhancing agents, dyes, emollients, emulsifying agents, emulsion stabilizers, fillers, film forming agents, flavor enhancers, flavoring agents, flow enhancers, gelling agents, granulating agents, humectants, lubricants, mucoadhesives, ointment bases, ointments, oleaginous vehicles, organic bases, pastille bases, pigments, plasticizers, polishing agents, preservatives, sequestering agents, skin penetrants, solubilizing agents, solvents, stabilizing agents, suppository bases, surface active agents, surfactants, suspending agents, sweetening agents, therapeutic agents, thickening agents, tonicity agents, toxicity agents, viscosity-increasing agents, water-absorbing agents, water-miscible cosolvents, water softeners, or wetting agents. See, e.g., the Handbook of Pharmaceutical Excipients, Third Edition, A. H. Kibbe (Pharmaceutical Press, London, UK, 2000), which is incorporated by reference in its entirety. Remington’s Pharmaceutical Sciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton, Pa., 1980), which is incorporated by reference in its entirety.

[0089] Compositions of the present disclosure can be suitable for administration by any acceptable route, including parenteral and subcutaneous. Other routes include intravenous, intradermal, intramuscular, intraperitoneal, intranodal and intrasplenic, for example. In exemplary aspects, the composition is suitable for systemic (e.g., intravenous) administration. If the composition is in a form intended for administration to a subject, it can be made to be isotonic with the intended site of administration. For example, if the solution is in a form intended for administration parenterally, it can be isotonic with blood. The composition typically is sterile. In certain embodiments, this may be accomplished by filtration through sterile filtration membranes. In certain embodiments, parenteral compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag, or vial having a stopper pierceable by a hypodermic injection needle, or a prefilled syringe. In certain embodiments, the composition may be stored either in a ready-to-use form or in a form (e.g., lyophilized) that is reconstituted or diluted prior to administration.MGB 32917 / 59848 PCEXAMPLES

[0090] The following examples are given merely to illustrate the present invention and not in any way to limit its scope.EXAMPLE 1

[0091] This example describes a method of making nanoparticles of the present disclosure.

[0092] Preparation of DOTAP Liposomes

[0093] On Day 1 , the following steps were carried out in the fume hood. Water was added to a rotavapor bath. Chloroform (20 mL) was poured into a sterile, glass graduated cylinder. After opening a vial containing 1 g of DOTAP, 5 mL chloroform was added to the DOTAP vial using a glass pipette. The volume of chloroform and DOTAP was then transferred into a 1-L evaporating flask. The DOTAP vial was washed by adding a second 5-mL volume of chloroform to the DOTAP vial to dissolve any remaining DOTAP in the vial and then transferring this volume of chloroform from the DOTAP vial to the evaporating flask. This washing step was repeated 2 more times until all the chloroform in the graduated cylinder was used. The evaporating flask was then placed into the Buchi rotavapor. The water bath was turned on and adjusted to 25 °C. The evaporating flask was moved downward until it touched the water bath. The rotation speed of the rotavapor was adjusted to 2. The vacuum system was turned on and adjusted to 40 mbar. After 10 minutes, the vacuum system was turned off and the chloroform was collected from the collector flask. The amount of chloroform collected was measured. Once the collector flask is repositioned, the vacuum was turned on again and the contents in the evaporating flask was allowed to dry overnight until the chloroform was completely evaporated.

[0094] On Day 2, using a sterile graduated cylinder, PBS (200 mL) 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 50eC. PBS (50 mL) was added into the evaporating flask using a 25-mL disposable serological pipette. The evaporating flask was positioned in the Buchi rotavapor and moved downward until 1 / 3 of the flask was submerged into the water bath. The rotation speed of the rotavapor was set to 2, allowed to rotate for 10 min, and then rotation was turned off. A 50-mL volume of PBS with DOTAP from the evaporating flask was transferred to the second 500 mL PBS bottle. The steps were 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 s and then incubated at 50eC for 1 hour. During the 1 hourMGB 32917 / 59848 PCincubation, the bottle was vortexed every 10 min. The second 500 mL PBS bottle was allowed to rest on overnight at room temperature.

[0095] On Day 3, PBS (200 mL) was added to the second 500 mL PBS bottle containing DOTAP and PBS. The second 500 mL PBS bottle was placed into an ultrasonic bath. Water was filled in the ultrasonic bath and the second 500 mL PBS bottle was sonicated for 5 min. The extruder was washed with PBS (100 mL) and this wash step was repeated. A 0.45 pm pore filter was assembled into a filtration unit and a new (third) 500 mL PBS bottle was positioned into the output tube of the extruder. In a biological safety cabinet, the DOTAP-PBS mixture was loaded into the extruder, until about 70% of the third PBS bottle was filled. The extruder was then turned on and the DOTAP PBS mixture was added until all the mixture was run through the extruder. Subsequently, a 0.22 pm pore filter was assembled into the filtration unit and a new (third) 500 mL PBS bottle was positioned into the output tube of the extruder. The previously filtered DOTAP-PBS mixture was loaded and run again throughout. The samples comprising DOTAP lipid nanoparticles (NPs) in PBS were then stored at 4eC.

[0096] RNA Preparation

[0097] Prior to incorporation into NPs, RNA was prepared in one of a few ways. Total tumor RNA was prepared by isolating total RNA (including rRNA, tRNA, and mRNA) from tumor cells. In vitro transcribed mRNA was prepared by carrying out in vitro transcription reactions using cDNA templates produced by reverse transcription of total tumor RNA. Tumor antigen-specific and Non-specific RNAs were either made in-house or purchased from a vendor.

[0098] Total Tumor RNA-. Total tumor-derived RNA from tumor cells (e.g., B16F0, B16F10, and KR158-luc) is isolated using commercially available RNeasy mini kits (Qiagen) based on manufacturer instructions.

[0099] In vitro transcribed mRNA-. Briefly, RNA is isolated using commercially available RNeasy mini kits (Qiagen) per manufacturer’s instructions and cDNA libraries were generated by RT-PCR. Using a SMARTScribe Reverse Transcriptase kit (Takara), a reverse transcriptase reaction by PCR was performed on the total tumor RNA in order to generate cDNA libraries. The resulting cDNA was then amplified using Takara Advantage 2 Polymerase mix with T7 / SMART and CDS III primers, with the total number of amplification cycles determined by gel electrophoresis. Purification of the cDNA was performed using a Qiagen PCR purification kit per manufacturer’s instructions. In order to isolate sufficient mRNA for use in each RNA-nanoparticle vaccine, mMESAGE mMACHINE (Invitrogen) kits with T7 enzyme mix were usedMGB 32917 / 59848 PCto perform overnight in vitro transcription on the cDNA libraries. Housekeeping genes were assessed to ensure fidelity of transcription. The resulting mRNA was then purified with a Qiagen RNeasy Maxi kit to obtain the final mRNA product.

[0100] Tumor Antigen-Specific and Non-Specific mRNA: Plasmids comprising DNA encoding tumor antigen-specific RNA (RNA encoding, e.g., pp65, OVA) and non-specific RNA (RNA encoding, e.g., Green Fluorescent Protein (GFP), luciferase) are linearized using restriction enzymes (i.e. , Spel) and purified with Qiagen PCR MiniElute kits. Linearized DNA is subsequently transcribed using the mm RNA in vitro transcription kit (Life technologies, Invitrogen) and cleaned up using RNA Maxi kits (Qiagen). In alternative methods, non-specific RNA is purchased from Trilink Biotechnologies (San Diego, CA).

[0101] Preparation of Multilamellar RNA nanoparticles (NPs)

[0102] The DOTAP lipid NPs were complexed with RNA to make multilamellar RNA-NPs which were designed to have several layers of mRNA contained inside a tightly coiled liposome with a positively charged surface and an empty core. Briefly, in a safety cabinet, RNA was thawed from -80 °C and then placed on ice, and samples comprising PBS and DOTAP (e.g., DOTAP lipid NPs) were brought up to room temperature. Once components were prepared, the desired amount of RNA was mixed with PBS in a sterile tube. To the sterile tube containing the mixture of RNA and PBS, the appropriate amount of DOTAP lipid NPs was added without any physical mixing (without e.g., inversion of the tube, without vortexing, without agitation). The mixture of RNA, PBS, and DOTAP was incubated for about 15 minutes to allow multilamellar RNA-NP formation. After 15 min, the mixture was gently mixed by repeatedly inverting the tube. The mixture was then considered ready for systemic (i.e. intravenous) administration.

[0103] The amount of RNA and DOTAP lipid NPs (liposomes) used in the above preparation is pre-determined or pre-selected. In some instances, a ratio of about 15 pg liposomes per about 1 pg RNA were used. For instance, about 75 pg liposomes are used per ~5 pg RNA or about 375 pg liposomes are used per ~25 pg RNA. In other instances, about 7.5 pg liposomes were used per 1 pg RNA. Thus, in exemplary instances, about 1 pg to about 20 pg liposomes are used for every pg RNA used.EXAMPLE 2

[0104] This example describes the characterization of the nanoparticles of the present disclosure.

[0105] Cryo-Electron Microscopy (CEM)MGB 32917 / 59848 PC

[0106] CEM was used to analyze the structure of multilamellar RNA-NPs prepared as described in Example 1 and control NPs devoid of RNA (uncomplexed NPs) which were made by following all the steps of Example 1 , except for the steps under “RNA Preparation” and “Preparation of Multilamellar RNA nanoparticles (NPs)”. CEM was carried out as essentially described in Sayour et aL, Nano Lett 17(3) 1326-1335 (2016). Briefly, samples comprising multilamellar RNA-NPs or control NPs were kept on ice prior to being loaded in a snap-frozen in Vitrobot (and automated plunge-freezer for cryoTEM, that freezes samples without ice crystal formation, by controlling temperature, relative humidity, blotting conditions and freezing velocity). Samples were then imaged in a Tecnai G2 F20 TWIN 200 kV / PEG transmission electron microscope with a Gatan UltraScan 4000 (4k x4k) CCD camera.

[0107] Representative CEM images are shown in Figure 1. The right panels are CEM images of multilamellar RNA-NPs and the left panel is a CEM image of DOTAP liposomes (uncomplexed NPs). As shown in Figure 1 and Figure 2, the uncomplexed NPs are visibly distinct from the multilamellar RNA NPs; the ML RNA-NPs contained several layers with clustering.

[0108] Zeta Potentials

[0109] Zeta potentials of multilamellar RNA NPs were measured by phase analysis light scattering (PALS) using a Brookhaven ZetaPlus instrument (Brookhaven Instruments Corporation, Holtsville, NY), as essentially described in Sayour et aL, Nano Lett 17(3) 1326-1335 (2016). Briefly, uncomplexed NPs or RNA-NPs (200 pL) were resuspended in PBS (1.2 mL) and loaded in the instrument. The samples were run at 5 runs per sample, 25 cycles each run, and using the Smoluchowski model.

[0110] The zeta potential of the multilamellar RNA NPs prepared as described in Example 1 was measured at about +50 mV. Interestingly, this zeta potential of the multilamellar RNA NPs was much higher than those described in Sayour et al., Oncoimmunology 6(1): e1256527 (2016), which measured at around +27 mV. Without being bound to any particular theory, the way in which the DOTAP lipid NPs are made for use in making the multilamellar RNA NPs (Example 1) involving a vacuum-seal method for evaporating off chloroform leads to less environmental oxidation of the DOTAP lipid NPs, which, in turn, may allow for a greater amount of RNA to complex with the DOTAP NPs and / or greater incorporation of RNA into the DOTAP lipid NPs.

[0111] RNA Incorporation by Gel Electrophoresis:MGB 32917 / 59848 PC

[0112] A gel electrophoresis experiment was conducted to measure the amount of RNA incorporated into ML liposomes. Based on this experiment, it was qualitatively shown that nearly 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 extent of RNA incorporation are carried out by measuring RNA-NP density and comparing this parameter to that of lipoplexes.EXAMPLE 3

[0113] This example describes a comparison of the nanoparticles of the present disclosure to cationic RNA lipoplexes and anionic RNA lipoplexes.

[0114] Cationic lipoplexes (LPX) were first developed with mRNA in the lipid core shielded by a net positive charge located on the outer surface. Anionic RNA lipoplexes have been developed with an excess of RNA tethered to the surface of bi-lamellar liposomes. RNA-LPX were made by mixing RNA and lipid NP at ratios to equalize charge. Anionic RNA-NPs were made by mixing RNA and lipid NP at ratios to oversaturate lipid NPs with negative charge. Various aspects of the RNA-LPX and anionic RNA LPX were then compared to the multilamellar RNA NPs described in the above examples.

[0115] Cryo-Electron Microscopy (CEM) was used to compare the structures of the RNA LPX and the multilamellar RNA-NPs prepared as described in Example 1. Uncomplexed NPs were used as a control. CEM was carried out as essentially described in Example 2. These data showed that more RNA is held by the ML RNA-NPs. Additional data showed that the concentration drops more with ML RNA-NP complexation versus RNA LPX supporting multilamellar formation of ML RNA-NPs not observed by simple mixing of equivalent amounts of RNA and lipid NPs by mass or charge (i.e. RNA-LPX and anionic RNA-LPX respectively). This supports that more RNA is “held” by ML RNA-NPs described herein.

[0116] Also, an experiment was conducted to determine where the anionic LPXs localize upon administration to mice. Anionic LPXs localized to the spleens of animals upon administration.

[0117] RNA LPX, anionic lipoplex (LPX) or multilamellar RNA-NPs were administered to mice and spleens were harvested one week later for assessment of 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 3A, mice treated with multilamellar RNA NPs exhibited the highest levels of activated DCs.MGB 32917 / 59848 PC

[0118] Anionic tumor mRNA-lipoplexes, tumor mRNA-lipoplexes, and multilamellar tumor mRNA loaded NPs were compared in a therapeutic lung cancer model (K7M2) (n=5-8 / group). Each vaccine was intravenously administered weekly (x3) (**p<0.01 , Mann Whitney). The % CD44+CD62L+of CD8+ splenocytes is shown in Figure 3B and the % CD44+CD62L+of CD4+ splenocytes is shown in Figure 3C. Also, Figure 3E shows that multilamellar (ML) RNA-NPs mediate substantially increased IFN-alpha, which is an innate anti-viral cytokine. This demonstrates that ML RNA-NPs allow for substantially greater innate immunity which is enough to drive efficacy from even non-antigen specific ML RNA-NPs. These data also indirectly support that ML RNA-NPs increase the number of activated plasmacytoid dendritic cells (pDCs) which cells are the most important producers of IFN-alpha. Taken together, the data demonstrates the superior efficacy of multilamellar tumor specific RNA-NPs, relative to anionic LPX and RNA LPX.

[0119] Anionic tumor mRNA-lipoplexes, cationic tumor mRNA-lipoplexes and multilamellar tumor mRNA loaded NPs were compared in a therapeutic lung cancer model (K7M2) (n=8 / group). Each vaccine was iv administered weekly (x3), *p<0.05, Gehan Breslow-Wilcoxon test. The percent survival was measured by Kaplan-Meier Curve analysis. As shown in Figure 3D, multilamellar tumor specific RNA-NPs mediated superior efficacy, compared to cationic RNA lipoplexes and anionic RNA lipoplexes, for increasing survival.

[0120] The ability of multilamellar RNA-NP to activate the innate immune response in vivo also was examined in the glioma tumor microenvironment.

[0121] RNA-NPs localize to perivascular regions of tumors and reprogram the TME in favor of activated myeloid cells. K-luc bearing animals (n=5 / group) were vaccinated with tumor RNA-NPs or NPs alone. Tumors were harvested 48h later for RNA-seq analysis. In animals receiving RNA-NPs, a significant upregulation of gene signatures for BATF3, IRFs, and IFN response genes was observed. In particular, the RNA-NP of the invention significantly upregulated expression of BATF3 (associated with effector dendritic cell phenotype), IRF5 and IRF7 (interferon regulatory factors), and ISG15 and IFITM3 (interferon response genes). These genes have been shown to be essential for sensitizing immunotherapeutic responses. As such, the RNA-NPs upregulate critical innate immune gene signatures in the glioma tumor microenvironment that associated with effector immune response, in effect turning tumors from “cold” to “hot,” allowing immune checkpoint inhibitors to be active where they were previously ineffective prior to RNA-NP treatment.MGB 32917 / 59848 PC

[0122] Herein it is demonstrated that the multilamellar RNA-NP formulation targeting physiologically relevant tumor antigens is more immunogenic and significantly more efficacious compared with anionic LPX and RNA LPX. Without being bound to any particular theory, by altering RNA-lipid ratios and increasing the zeta potential, a novel RNA-NP design composed of multi-lamellar rings of tightly coiled mRNA has been developed, which multi-lamellar design is thought to facilitate increased NP uptake of mRNA (condensed by alternating positive / negative charge) for enhanced particle immunogenicity and widespread in vivo localization to the periphery and tumor microenvironment (TME). Systemic administration of these multi-lamellar RNA-NPs localize to lymph nodes, reticuloendothelial organs (i.e. spleen and liver) and to the TME, activating DCs therein (based on increased expression of the activation marker CD86 on CD11c+ cells). These activated DCs prime antigen specific T cell responses, which lead to antitumor efficacy (with increased TILs) in several tumor models.EXAMPLE 4

[0123] This example describes a study assessing the efficiency of CTC-based RNA-NPs in comparison with RNA-NPs prepared from lysate from an entire tumor (referred herein as “bulk RNA-NPs” or “bulk tumor”).

[0124] Tumor-specific mRNA-NP were generated from CTCs isolated from peripheral blood and administered to a mouse model (e.g., 25 ug / mouse) to inhibit cancer growth. The tumorigenic dose of K7M2 (osteosarcoma mouse cell line) is 1 .25x106cells, which were injected intravenously into the tail vein of Balb / c (with a loading dose of 200pL). For each vaccine (per mouse), 375 pg of custom lipid-NP formulation was complexed with 25 pg of CTCs mRNA, 25 pg of bulk tumor mRNA 25 pg, or control (non-specific) mRNA. The different RNA-NPs were compared simultaneously to 5-7 negative control mice receiving NPs alone. Mice were i.v. vaccinated six times beginning one day after tumor implantation. Three administrations were performed in the first week and one administration was performed per week thereafter for a total of three additional administrations. The control for this experiment was the standard tumorspecific generated mRNA-NPs.

[0125] The results of this experiment, illustrated in Figure 4, demonstrate a significant difference between the RNA-NP treatment using circulating tumor cells (CTCs) RNA compared to the NP alone group. Additionally, the CTC-based RNA-NP treatment paralleled the results observed in the K7M2 bulk RNA-NPs group. These findings demonstrate that CTCs can be utilized instead of an initial biopsy for RNA-NP treatment. An advantage of the CTC approach is its capacity for repeated customization based on serial patient blood samples, collected duringMGB 32917 / 59848 PCroutine clinical visits. This novel strategy not only aligns with the dynamic nature of tumor evolution but also addresses a challenge of therapeutic resistance. The periodic adjustment of the RNA-NP composition in response to the evolving tumor phenotype can significantly enhance treatment efficacy. Furthermore, this non-invasive procedure has the transformative potential of leveraging CTCs for the development of personalized RNA-NP cancer vaccines, allowing a shift toward more adaptive, patient-centric therapeutic paradigms.EXAMPLE 5

[0126] This example describes generation of nanoparticles comprising RNA based on CTC transcriptome which demonstrated significant activity in clinically-relevant in vivo models.Materials and methods:Mice Experiments

[0127] Balb / c mice used in K7M2 experiments were acquired from The Jackson Laboratory. Each mouse was intravenously given a 25-pg dose of RNA nanoparticles, and unless otherwise noted, the mass ratio of RNA to liposome was maintained at 1 :15. A total of six vaccines were administered for each experimental group.Cell Lines and Injections

[0128] The K7M2 cell line was purchased from ATCC (catalog no. CRL-2836) and was introduced into the mice through intravenous tail vein injections, each containing 1.25 million cells to induce lung tumors. The cell lines used for this study were cultured according to the supplier's recommended guidelines.Blood Collection

[0129] Blood was collected from K7M2-bearing BALB / c mice at a pre-defined endpoint. Animals were anesthetized using isoflurane vaporized to a surgical plane and approximately 750 pL to 1 mL of blood was drawn into EDTA-coated tubes to prevent coagulation.Immediately following collection, the blood was processed to isolate circulating tumor cells (CTCs).Circulating Tumor Cell (CTC) Isolation

[0130] Following the collection of blood from K7M2-bearing BALB / c mice, circulating tumor cells (CTCs) were isolated using Miltenyi Biotec mouse tumor isolation kit (Catalog number 130-110-187), in accordance with the manufacturer's guidelines, with some minor modifications. The collected blood samples were first diluted with processing buffer (0.5% BSA in 1xPBS) andMGB 32917 / 59848 PCsubjected to red blood cell (RBC) lysis using an RBC lysis buffer, as specified in the kit manual. After RBC removal, the cells were washed and centrifuged to collect a pellet containing white blood cells, including the CTCs of interest. This pellet was then resuspended in the processing buffer, and a Non-Tumor Cell Depletion Cocktail from the kit was added to label non-tumor cells. Rather than using the LS column recommended by the manufacturer, an in-house technique was employed using flow tubes in conjunction with an EasySep magnet for magnetic separation. Following incubation with the depletion cocktail, the tube was placed in the EasySep magnet to isolate the magnetically labeled non-tumor cells. The flow-through, containing the desired CTCs, was carefully collected. Cells in the flow-through were washed using washing buffer and then subjected to a second round of magnetic separation to ensure maximal removal of non-tumor cells. Quality control steps to check for cell viability and microscopic verification were carried out to confirm the quality of the cells. All steps were conducted at4°C to maintain cell integrity.Single-Cell Immunolabeling

[0131] For each tumor type, a customized cocktail of antibodies was devised. This mix consisted of markers for positive selection, a nuclear stain, and a marker to negatively identify white blood cells (WBCs). Prior to combining them, each individual antibody was validated by testing it on a compatible tumor cell line. The efficacy of the cocktail was also verified by testing it both in duplicates and as a unified mixture.Single cell isolation

[0132] After the enrichment process, the isolated cells were carefully dispersed at a low concentration into a 6-well plate for further examination. Each well was prepared with 2 ml_ of a carefully prepared buffer solution containing 0.5% Bovine Serum Albumin (BSA) mixed with 1x Phosphate-Buffered Saline (PBS) to maintain cell viability and prevent nonspecific adhesion. CellCelector™ was utilized, an automated single cell picking system to perform a comprehensive scan of each well. Prior to scanning, the machine was pre-programmed with parameters designed to identify cells exhibiting specific morphological traits and sizes, as well as cells expressing pre-defined marker proteins. These parameters were rigorously tested to ensure high specificity and sensitivity in cell selection. Upon completion of the automated scanning process, the CellCelector™ generated a digital gallery showcasing all potential circulating tumor cells (CTCs), referred to as ’’positive hits.” Each ’’hit” in this gallery was then meticulously reviewed by a trained team member specializing in cytology, who handpicked the cells that met the set criteria for further analysis. For every positively identified cell, theMGB 32917 / 59848 PCCellCelector™'s software recorded the X-Y-Z coordinates, allowing the ability to pinpoint the exact spatial location of the cell within the well. This data was helpful for the subsequent isolation process. Using the highly accurate robotic arm of the CellCelector™, each chosen cell was delicately picked up with a minute volume of 0.7uL PBS buffer. The cell was then transferred into a sterile PCR tube that had been pre-filled with 1 ,3uL of buffer. This resulted in each PCR tube containing a total volume of 2 uL and precisely one cell. These tubes were immediately placed in a cool environment to preserve the integrity of the cells until they could be processed further in downstream applications. Through this detailed and rigorous process, it was possible to ensure the highly selective and accurate isolation of single cells for further analytical studies.Amplification

[0133] The SMART-Seq® Single Cell Kit from Takara Bio (catalog number 634471) was used to amplify the full-length transcriptome. The manufacturer's protocol was followed, with a starting sample volume of 2 pL. The kit's primers were substituted with in-house primers for further downstream modification. After amplification, the PCR product was purified using the QIAquick PCR Purification Kit from QIAGEN (catalog number 28104). In vitro transcription (IVT) was subsequently carried out with a commercial kit from ThermoFisher Scientific (cat. AM1344). Finally, RNA cleanup was performed using the RNeasy Mini Kit from QIAGEN (catalog number 74104).RNA-nanoparticle Generation

[0134] Freeze-dried DOTAP material was purchased from Avanti Polar Lipids. To make clean liposomes and to minimize damage from air, a rota-evaporator was used to remove unwanted solvents and quickly dissolve the lipids into a water-based solution. In short, chloroform was added to the dried DOTAP and mixed while under a vacuum to evaporate the chloroform. Afterward, the remaining lipid film was re-suspended in PBS. To complete the process, this mixture was heated in a water bath, shook with sound waves (sonication), and pushed through filters that were 0.45 pm and 0.2 pm in size. This helped form positively charged double-layered liposomes (LPs). Upon forming these LPs, negatively charged synthetic mRNAs or tumor-amplified mRNAwas introduced to facilitate LP aggregation. A specific RNA to LP weight ratio of 1 :15 was used. To confirm the quality of the resulting RNA-nanoparticles, their concentration and size was assessed using Nanosight NS300 (Malvern Panalytical), their electrical charge (zeta potential) using Zetasizer Ultra (Malvern Panalytical),MGB 32917 / 59848 PCand the effectiveness of RNA encapsulation through gel-electrophoresis (Mendez-Gomez et al., Cell 187, 2521-2535 e2521 (2024))Cryo-Electron Microscopy of LP and RNA-LPA

[0135] Cryo-transmission electron microscopy (Cryo-TEM) images were taken using core instruments at the Interdisciplinary Center for Biotechnology Research at the University of Florida. Thin films were prepared by placing 3 pL of sample suspension onto holey carbon grids (C-Flats; Protochips, Inc.); these were vitrified by plunging into nitrogen cooled ethane, using a Vitrobot™ Mark IV freezing device (FEI Co.). The frozen grids were then imaged using a 16-megapixel CCD camera (Gatan, Inc.) in aTecnai G2 F20-TWIN Transmission Electron Microscope (FEI), which was operated at a voltage of 200 kV using low-dose conditions (~20 e / A2). Both LP and RNA-nanoparticle samples were analyzed following previously set protocols (Sayour et al., Nature reviews. Clinical Oncology 21 , 489-500 (2024), Sayour et al., Oncolmmunology, 6(1), e1256527 (2016), Sayour et al., Nano Letters, 18(10), 6195-6206 (2018)).Results:

[0136] The CellCelector™ system was utilized to isolate single tumor cells. A representative method for isolating CTCs, extracting RNA, and generating nanoparticles comprising CTC RNA is described above and illustrated in FIG. 5A. The ability to extract and amplify whole transcriptome specific mRNA from single cells for loading into multilamellar cationic mRNA vaccines was demonstrated (FIGS. 5B-5D), creating a noninvasive renewable resource for rapid and repeatable personalized mRNA-based stimulation of the immune system. Following magnetic filtration of peripheral blood cells, suspicion of a cancer’s genealogy allows for customized antibody selection of CTCs. Osteosarcoma cells inoculated into peripheral blood of BALB / c mice were picked using fluorescent detection of c-kit (CD117). CD117 expression is expected on osteoid or sarcoma cells in peripheral blood, but not normal blood cells. From these peripheral blood isolated CTCs, the ability to extract RNA, synthesize a cDNA library and amplify mRNA specific to the CTCs was demonstrated. Importantly, K7M2 CTC mRNA extracted from peripheral blood of animals inoculated with K7M2 sarcomas was able to elicit significantly enhanced survivorship, as described above and illustrated in FIG. 4.

[0137] While the results described above demonstrated the ability to create personalized mRNA libraries from individual tumor cells, many of these cells may be amenable to targeting unique drivers. For example, In inaccessible central nervous system (CNS) malignanciesMGB 32917 / 59848 PC(including primitive neuroectodermal tumors and ependymoma), diagnosis, recurrence, and treatment paradigms can be created through identification of known fusions in these cancers. PNET tumor cells were picked through differential staining of CD99 and EPCAM. These tumor cells were believed to express canonic EWS-FLI1 fusion protein. Following gel electrophoresis, the presence of EWS-FLI1 fusion proteins was confirmed in CTCs. Rapid noninvasive identification of these fusion proteins enables customization of RNA nanoparticles for immune system stimulation. RNA nanoparticles were synthesized against EWS-FLI-1 protein, and were administered to immunocompetent Balb / c mice inoculated with EWS-FLI1 transduced K7M2 murine tumors. Fusion protein specific RNA nanoparticles elicited therapeutic activity evidenced by enhancement of long-term survival outcome (FIG. 6A). To extend these observations, another fusion protein specific RNA nanoparticle was synthesized wherein the RNA payload encoded C11orf95-RELA fusion proteins expressed in supratentorial ependymomas. Following administration of RNA nanoparticles targeted against 011 orf95-RELA fusions in tumor bearing C57BI / 6 animals, splenocytes were harvested and co-cultured cells with C11 orf95-RELA fusion expression tumoroids. Following co-culture with C11 orf95-RELA fusion vaccinated splenocytes, there was significant cytotoxic killing activity over time (FIG. 6B) that was not observed in animals receiving unvaccinated splenocytes. There was also an increase in tumor associated mononuclear cells from 011orf95-RELA murine ependymomas cultured with splenocytes derived from 011orf95-RELA fusion mRNA vaccinated versus unvaccinated animals. Taken together, these data highlight the ability to rapidly pick CTCs and identify unique molecular fingerprints amenable to rapid preparation of RNA nanoparticles in the absence of a tumor specific biopsy and which demonstrate anti-cancer effects in vivo.

[0138] A complication associated with development of anti-tumor immunotherapy is the antigenic heterogeneity intrinsic to cancer and malignant evolution. CTCs represent disease burden and metastatic potential of resistant subclones, offering a replenishable source of tumor antigen that reflects the state of the cancer over time. CTO-based nanoparticles are useful in overcoming malignant cancer spread, allowing immunotherapeutic responses to be directed at evolving disease burden. The example described above describes the development of an RNA nanoparticle manufactured from CTCs, which serves as a rapid and personalized approach to elicit sustained anti-tumor activity that overcomes malignant cancer evolution. The data shows the feasibility of using a non-invasive CTC-based mRNA cancer vaccine to treat patients with limited tumor access, and the ability to alter the treatment based on tumor clonal evolution.MGB 32917 / 59848 PC

[0139] All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

[0140] The use of the terms “a” and “an” and “the” and similar referents in the context of describing the disclosure (especially in the context of the following claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning “including, but not limited to,”) unless otherwise noted. If aspects of the invention are described as "comprising" a feature, embodiments also are contemplated "consisting of" or "consisting essentially of" the feature.

[0141] Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range and each endpoint, unless otherwise indicated herein, and each separate value and endpoint is incorporated into the specification as if it were individually recited herein. Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein should be understood as modified in all instances by the term "about" as that term would be interpreted by the person skilled in the relevant art.

[0142] All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the disclosure and does not pose a limitation on the scope of the disclosure unless otherwise claimed. No language in the specification should be construed as indicating any nonclaimed element as essential to the practice of the disclosure.

[0143] Preferred embodiments of this disclosure are described herein, including the best mode known to the inventors for carrying out the disclosure. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the disclosure to be practiced otherwise than as specifically described herein. Accordingly, this disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possibleMGB 32917 / 59848 PCvariations thereof is encompassed by the disclosure unless otherwise indicated herein or otherwise clearly contradicted by context.

Claims

MGB 32917 / 59848 PCWHAT IS CLAIMED:

1. A nanoparticle comprising a positively-charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between a cationic lipid bilayer, and nucleic acid molecules in the nucleic acid layers comprise a sequence of a nucleic acid molecule expressed by a circulating tumor cell (CTC).

2. The nanoparticle of claim 1 , comprising at least three nucleic acid layers, each of which is positioned between a cationic lipid bilayer.

3. The nanoparticle of claim 1 , comprising at least four nucleic acid layers, each of which is positioned between a cationic lipid bilayer.

4. The nanoparticle of claim 1 , comprising five or more nucleic acid layers, each of which is positioned between a cationic lipid bilayer.

5. The nanoparticle of any one of claims 1 -4, wherein the outermost layer of the nanoparticle comprises a cationic lipid bilayer.

6. The nanoparticle of any one of claims 1 -5, wherein the core comprises a cationic lipid bilayer.

7. The nanoparticle of any one of claims 1 -6, wherein the diameter of the nanoparticle is about 50 nm to about 500 nm in diameter, optionally, about 70 nm to about 250 nm in diameter.

8. The nanoparticle of any one of claims 1 -7, comprising a zeta potential of about 40 mV to about 60 mV, optionally, about 45 mV to about 55 mV.

9. The nanoparticle of claim 8, comprising a zeta potential of about 50 mV.

10. The nanoparticle of any one of claims 1 -9, comprising nucleic acid molecules and cationic lipid at a ratio of about 1 to about 5 to about 1 to about 20, optionally, about 1 to about 15 or about 1 to about 7.5.

11. The nanoparticle of any one of claims 1-10, wherein the cationic lipid is DOTAP or DOTMA.

12. The nanoparticle of any one of claims 1-11 , wherein the nanoparticle does not comprise a neutral lipid.

13. The nanoparticle of any one of claims 1-12, wherein the nucleic acid molecules are RNA molecules.MGB 32917 / 59848 PC14. The nanoparticle of claim 13, wherein the RNA molecules are mRNA.

15. A composition comprising the nanoparticle of any one of claims 1-14 and a pharmaceutically acceptable carrier.

16. A method of treating cancer in a subject need thereof, the method comprising administering to the subject the nanoparticle of any one of claims 1-14.

17. The method of claim 16, wherein the method comprises two or more administrations of a nanoparticle of any one of claims 1-14, wherein each administration comprises a nanoparticle comprising RNA comprising a sequence of RNA expressed in a CTC isolated from the subject at different points in time.

18. A method comprising(a) isolating a circulating tumor cell (CTC) from a subject,(b) producing a nanoparticle of any one of claims 1-14 comprising RNA comprising a sequence of RNA expressed in the CTC,(c) administering the nanoparticle of step (b) to the subject, and(d) repeating steps (a)-(c) one or more times.

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