Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
  • A61K39/215—Coronaviridae, e.g. avian infectious bronchitis virus
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K39/12—Viral antigens
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
  • A61P31/14—Antivirals for RNA viruses
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
  • A61K2039/53—DNA (RNA) vaccination
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/545—Medicinal preparations containing antigens or antibodies characterised by the dose, timing or administration schedule
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K39/00—Medicinal preparations containing antigens or antibodies
  • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
  • A61K2039/55511—Organic adjuvants
  • A61K2039/55555—Liposomes; Vesicles, e.g. nanoparticles; Spheres, e.g. nanospheres; Polymers
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2770/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssRNA viruses positive-sense
  • C12N2770/00011—Details
  • C12N2770/20011—Coronaviridae
  • C12N2770/20034—Use of virus or viral component as vaccine, e.g. live-attenuated or inactivated virus, VLP, viral protein

Definitions

• SARS-CoV-2 The Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) is a novel virus belonging to the Coronaviridae family of enveloped viruses.
• SARS-CoV-2 is a positive-sense single-stranded RNA virus with genetic similarity to bat coronaviruses and was first isolated in January 2020 from patients in Wuhan, China (Hui et al., International J Infectious Diseases 91: 264-266 (2020)). To date, over 750,000 people have been infected by SARS-CoV-2 and over 35,000 people have died from Coronavirus Disease 2018 (COVID-19), the disease caused by SARS-CoV-2 infection. To reduce the number of SARS CoV-2-related deaths, a vaccine to safely induce immunity against SARS-CoV-2 is needed.
• Lipid nanoparticles comprising mRNA as vaccines have been studied. See, e.g., Reichmuth et al.,
• RNA vaccines have several advantages over traditional modalities. RNA has potent effects on both the innate and adaptive immune system. RNA can act as a toll-like receptor (TLR) agonist for receptors 3, 7, and 8 inducing potent TLR dependent innate immunity. RNA can also stimulate intracellular pathogen recognition receptors (i.e., melanoma differentiation antigen 5 (MDA-5) and retinoic acid inducible gene I (RIG-I)) and culminates in activating both helper-CD4 and cytotoxic CD8 T cell responses.
• TLR toll-like receptor
• MDA-5 melanoma differentiation antigen 5
• RAG-I retinoic acid inducible gene I
• RNA Unlike DNA vaccines mired by having to cross both cellular and nuclear membranes, RNA only requires access to the cytoplasm and carries a significant safety advantage since it cannot be integrated into the host-genome. Unlike many peptide vaccines, which have only been developed for specific HLA haplotypes (i.e., HLA-A2), RNA bypasses MHC class restriction and can be leveraged for the population at large.
• Nanoparticles comprising a positively-charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between a cationic lipid bilayer, mediate peripheral and intratumoral activation of dendritic cells (DCs).
• DCs dendritic cells
• multilamellar RNA nanoparticles of the present disclosure are capable of safely and effectively inducing immunity against SARS-CoV-2.
• the present disclosure provides a nanoparticle comprising a positively-charged surface and an interior comprising (i) a core and (ii) at least two nucleic acid layers, wherein each nucleic acid layer is positioned between a cationic lipid bilayer, wherein the nanoparticle comprises RNA molecules encoding a SARS-CoV-2 protein, optionally, a SARS-CoV-2 Spike (S) protein.
• the nanoparticle comprises at least three nucleic acid layers, each of which is positioned between a cationic lipid bilayer.
• the nanoparticle comprises at least four or five or more nucleic acid layers, each of which is positioned between a cationic lipid bilayer.
• the outermost layer of the nanoparticle comprises a cationic lipid bilayer.
• the surface comprises a plurality of hydrophilic moieties of the cationic lipid of the cationic lipid bilayer.
• the core comprises a cationic lipid bilayer.
• 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 250 nm in diameter, optionally, about 70 nm to about 200 nm in diameter.
• 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.
• 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, such as 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.
• the nucleic acid molecules are RNA molecules, optionally, messenger RNA (mRNA).
• the mRNA is in vitro transcribed mRNA wherein the in vitro transcription template is cDNA made from RNA extracted from a sample.
• the present disclosure also provides a method of making 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, wherein the nanoparticle comprises RNA molecules encoding a SARS-CoV-2 protein, optionally, a SARS-CoV-2 Spike (S) protein, said method comprising: (A) mixing nucleic acid molecules and liposomes at a RNA: liposome ratio of 1 to about 5 to about 1 to about 25, such as about 1 to about 5 to about 1 to about 20, optionally, about 1 to about 15, about 1 to about 10, or about 1 to about 7.5, to obtain a RNA-coated liposomes, wherein 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 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.
• 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.
• sizing the rehydrated lipid mixture comprises sonicating, extruding and/or filtering the rehydrated lipid mixture.
• the RNA molecules are mRNA encoding SARS-CoV-2 protein, optionally mRNA encoding the SARS-CoV-2 S protein.
• a method of inducing or increasing an immune response against a SARS-CoV-2 virus in a subject comprises administering to the subject the pharmaceutical composition of the present disclosure.
• the nucleic acid molecules are mRNA.
• the composition is systemically administered to the subject.
• the composition is administered intravenously.
• the pharmaceutical composition is administered in an amount which is effective to activate dendritic cells (DCs) in the subject.
• the immune response is a T cell-mediated immune response.
• the T cell-mediated immune response comprises activity by tumor infiltrating lymphocytes (TILs).
• the present disclosure also provides a method of delivering RNA molecules to lungs of a subject.
• the present disclosure also provides a method of delivering RNA molecules to a lymph node and/or a reticuloendothelial organ (e.g., spleen or liver).
• the method comprises administering to the subject a presently disclosed pharmaceutical composition.
• a method of treating a subject with a disease or disorder is furthermore provided herein.
• the method comprises administering to the subject a pharmaceutical composition of the present disclosure in an amount effective to treat the disease or disorder in the subject.
• the subject has COVID-19 or is at risk of infection with SARS-CoV-2.
• Figure 1A is a series of illustrations of a lipid bilayer, liposome and a general scheme leading to multilamellar (ML) RNA NPs (boxed).
• ML multilamellar
• Figures 2C-2E are CEM images.
• Figure 2C is a CEM image of uncomplexed NPs;
• Figure 2D is a CEM image of RNA LPXs;
• Figure 2E is a CEM image of ML RNA NPs.
• Figure 2F 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.
• ML RNA-NPs ML RNA-NPs
• RNA LPXs RNA LPXs
• anionic LPXs or of untreated mice.
• Figure 2H 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.
• ML RNA-NPs ML RNA-NPs
• RNA LPXs RNA LPXs
• anionic LPXs or of untreated mice.
• Figure 2I is a graph of the % survival of mice treated with ML RNA NPs (ML RNA-NPs), RNA LPXs, anionic LPXs, or of untreated mice.
• Figure 2J 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.
• ML RNA-NPs ML RNA-NPs
• RNA LPXs RNA LPXs
• anionic LPXs or of untreated mice.
• Figure 3A is a pair of photographs of lungs of mice treated with ML RNA NPs or of untreated mice.
• Figure 3C is a graph of the % survival of mice treated with ML RNA NPs loaded with tumor specific RNA or with ML RNA NPs with non-specific RNA (GFP RNA) or of untreated mice.
• Figure 5 is a CEM image of ML RNA NPs with arrows pointing to examples with several layers.
• Figure 6 is a cartoon delineating the generation of personalized ML RNA NP, using tumor mRNA loaded NPs as an illustrative example. From as few as 100-500 biopsied brain tumor cells, total RNA is extracted and a cDNA library is generated from which copious amounts of mRNA (representing a personalized tumor specific transcriptome) can be amplified. Negatively charged mRNA is then encapsulated into positively charged lipid NPs. NPs encapsulate RNA through electrostatic interaction and are administered intravenously (iv) for uptake by dendritic cells (DCs) in reticuloendothelial organs (i.e., liver spleen and lymph nodes). The RNA is then translated and processed by a DC's intracellular machinery for presentation of peptides onto MHC Class I and II molecules, which activate CD4 and CD8+T cells.
• DCs dendritic cells
• Figure 10 demonstrates multi-lamellar RNA NPs form complex structures that coil mRNA into multi- lamellar vesicles enhancing payload delivery.
• the bar graph illustrates gene expression (luminescence) for anionic RNA-LPS (first bar on left), RNA-lipoplex (second bar), RNA-NPs (lo) (third bar), and RNA-NPs (high) (fourth bar).
• Figure 11 demonstrates multi-lamellar RNA NPs mediate increased DC activation and IFN-a release.
• RNA/anionic lipoplex (LPX) or RNA-NPs were i.v. (intravenously) administered once weekly (x3) to C57BI/6 mice, and spleens were harvested one week later for assessment of activated DCs (left). Serum was drawn 6h after the initial treatment for IFN-a assessment by ELISA (right).
• Figure 12 demonstrates multi-lamellar RNA-NPs are superior to LPX and peptide based vaccines in eliciting antigen specific T cells.
• Figure 13 demonstrates RNA-NPs induce memory re-stimulation response against CMV matrix protein pp65.
• Weekly pp65 RNA-NPs (x3) were administered to naive C57/BI/6 mice, and splenocytes were harvested one week later for culture with overlapping pp65 peptide pool and assessment of IFN-g (*p ⁇ 0.05, **p ⁇ 0.01, Mann Whitney).
• Figure 14 demonstrates multi-lamellar tumor specific mRNA-NPs mediate superior efficacy.
• K7M2 therapeutic lung cancer model
• Each vaccine was iv administered weekly (x3), **p ⁇ 0.01, Gehan-Wilcoxon test.
• Figure 15 demonstrates RNA-NPs elicit near immediate IFN-a surge, PBMC margination, and peripheral DC activation in canines.
• Figure 16 demonstrates RNA-NPs improve median survival in canines with terminal gliomas, demonstrating the ability of the composition described herein to elicit a clinically meaningful immune response in vivo.
• Survival of canines boxer breed
• tumor RNA-NPs x3
• Median survival shown as dotted line 29 days
• Mean survival from separate studies ranges between 60-80 days.
• Median one-sided exact log rank test p-value distribution with bootstrapping to account for uncertainty: *p 0.021.
• Figure 17 demonstrates GLP Toxicity study results of tumor specific RNA-NPs against KR158b. Intravenous injection into the tail vein of mice at 1.0 mg/kg KR158 and pp65 mRNAs + 15.0 mg/kg lipid NP on Study Days 0, 14 and 28 resulted in no gross or microscopic test article related findings.
• Figures 18A-18D demonstrate charge modified RNA-NPs can be directed to, e.g., the lung or the spleen.
• Figure 18A Cationic ( ⁇ +40mV, left) versus anionic ( ⁇ -30mV, right) RNA-NPs encoding for luciferase were administered to Balb/c mice by i.v. tail vein injection. Mice were injected i.p. with luciferin 6 h after RNA- NPs and imaged for bioluminescence by MS imaging. The ability to manipulate RNA-NPs for lung localization is especially relevant for COVID-19 protection.
• Reticuloendothelial organs were harvested within 24 h for assessment of CD11c cells expressing activation marker CD86 (*p ⁇ 0.05, **p ⁇ 0.01, Mann-Whitney test) from lymph nodes ( Figure 18B), splenocytes (Figure 18C), or liver cells ( Figure 18D).
• the data establish that the constructs of the disclosure can be leveraged for near immediate pulmonary protection against, e.g., viral pathogens (such as coronavirus) with only a single administration.
• Figure 19 is an illustration of an exemplary mRNA comprising a nucleotide sequence encoding a SARS-CoV-2 protein (e.g., spike, membrane, envelope, nucleocapsid) and a poly(A) tail.
• SARS-CoV-2 protein e.g., spike, membrane, envelope, nucleocapsid
• poly(A) tail e.g., poly(A) tail
• Figure 20 is a table of sequences provided in the Sequence Listing.
• Figure 21 A and 21 B are graphs illustrating the results of the study of Example 11.
• Figure 21 A is a graph of the number of effector memory T cells (CD3+CD8+CD44+ cells per well) upon re-stimulation with SARS-CoV-2 Spike peptide (Spike peptide restim - empty circles) or without re-stimulation (unstimulated, gray filled circles).
• Mice were administered multilamellar (ML) RNA nanoparticles (NPs) loaded with mRNA encoding the full-length Spike protein of SARS-CoV-2. The mice received a total of 3 vaccinations within a week.
• ML multilamellar
• NPs RNA nanoparticles
• Figure 21 B is a graph illustrating MIP1-a released (pg/mL) from unstimulated cells (empty bar) and cells re-stimulated with spike peptide (solid bar).
• Figures 22A-C provide sequences of the SARS-CoV-2 Spike protein relevant to the nanoparticles of the present disclosure ( Figures 22A and 22C) as well as a vector map ( Figure 22B).
• Figure 23 is inclusion criteria for subjects in the study of Example 14.
• Figure 24 is exclusion criteria for subjects in the study of Example 14.
• Figure 25 is a calendar for patient assessments referenced in the Examples.
• Figure 26 is a bar graph comparing SARS-CoV-2 IgG (absorbance) (y-axis) of untreated subjects and RNA-NP-treated subjects (x-axis) C57BI/6 mice were vaccinated with weekly RNA-NPs encoding full-length SARS-CoV-2 spike mRNA. Serum was harvested at one month for assessment of spike specific antibodies by ELISA.
• Figures 27A-27B are bar graphs illustrating effector memory T cell expansion (Figure 27A) and MIP-1 a production ( Figure 27B) following spike peptide restimulation.
• Mice receiving SARS-CoV-2 spike RNA-NPs had more effector memory T cells after vaccination with significant memory recall expansion.
• Figure 28 is a bar graph illustrating IFN-g production following spike protein restimulation.
• Mice receiving SARS-CoV-2 spike RNA-NPs demonstrate memory recall response ex vivo.
• PBMCs were left unstimulated or re-stimulated with cell line of DC2.4s electroporated with SARS-CoV-2 spike mRNA and compared with PBMCs from untreated mice. Supernatants were collected for IFN-g analysis (*p ⁇ 0.05, Mann-Whitney).
• Figures 29A and 29B illustrate that 5' and 3' UTRs for alpha globin enhance percentage of tetramer+ T cells and long-term survival benefit.
• RNA-NPs encoding for OVA mRNA with 5' and 3' UTRs for alpha-globin were administered i.v. to naive C57BI/6 mice with OT-I T cells, and spleens were harvested a week later for assessment of tetramer+ T cells.
• alpha globin modified RNA-NPs increase percentage of antigen specific T cells.
• Figures 32A and 32B are graphs illustrating % OVA specific Tetramer+ CD8 cells in subjects administered NP alone and RNA-NP in MDAS knock-out subjects.
• nanoparticles comprising a cationic lipid and nucleic acids.
• nanoparticle refers to a particle that is less than about 1000 nm in diameter.
• 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. Liposomes in various instances are used as a delivery vehicle for the administration of nutrients and pharmaceutical agents.
• liposomes are formulated depending on the physicochemical characteristics such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of the vesicles for the intended application, and the batch-to-batch reproducibility and possibility of large-scale production of safe and efficient liposomal products.
• the pharmaceutical formulation entrapped and the liposomal ingredients such as, but not limited to, the pharmaceutical formulation entrapped and the liposomal ingredients, the nature of the medium in which the lipid vesicles are dispersed, the effective concentration of the entrapped substance and its potential toxicity, any additional processes involved during the application and/or delivery of the vesicles, the optimization size, polydispersity and the shelf-life of
• 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.
• each nucleic acid layer is positioned between a lipid layer, e.g., a cationic lipid layer.
• the nanoparticles are multilamellar comprising alternating layers of nucleic acid and lipid.
• the nanoparticle comprises at least three nucleic acid layers, each of which is positioned between a cationic lipid bilayer.
• 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.
• 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.
• 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.
• RNA-NPs demonstrate superior efficacy with long-term survivor benefit.
• the presently disclosed nanoparticle comprises a positively-charged surface.
• the positively-charged surface comprises a lipid layer, e.g., a cationic lipid layer.
• the outermost layer of the nanoparticle comprises a cationic lipid bilayer.
• the cationic lipid bilayer comprises DOTAP.
• the surface comprises a plurality of hydrophilic moieties of the cationic lipid of the cationic lipid bilayer.
• the core comprises a cationic lipid bilayer.
• the outermost region of the core comprise a cationic lipid bilayer comprising DOTAP.
• the core lacks nucleic acids, optionally, the core comprises less than about 0.5 wt% nucleic acid.
• the nanoparticle has a diameter within the nanometer range and accordingly in certain instances are referred to herein as "nanoliposomes” or "liposomes.”
• 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 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
• the nanoparticle has a diameter between about 50 nm to about 300 nm, e.g., about 100 nm to about 250 nm, about 110 nm ⁇ 5 nm, about 115 nm ⁇ 5 nm, about 120 nm ⁇ 5 nm, about 125 nm ⁇ 5 nm, about 130 nm ⁇ 5 nm, about 135 nm ⁇ 5 nm, about 140 nm ⁇ 5 nm, about 145 nm ⁇ 5 nm, about 150 nm ⁇ 5 nm, about
• the nanoparticle is about 50 nm to about 250 nm in diameter. In some aspects, the nanoparticle is about 70 nm to about 200 nm in diameter.
• 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.
• the pharmaceutical composition comprises a heterogeneous mixture of nanoparticles ranging from about 70 nm to about 200 nm in diameter.
• 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, or about +55 mV to about +60 mV.
• 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).
• the cationic lipid comprises two fatty acyl chains, each chain of which is independently saturated or unsaturated.
• the cationic lipid is a diglyceride.
• the cationic lipid may be a cationic lipid of Formula I or Formula II:
• 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.
• the cationic lipid is DOTAP (1,2-dioleoyl- 3-trimethylammonium-propane), or a derivative thereof.
• the cationic lipid is DOTMA (1,2-di-0-octadecenyl-3-trimethylammonium propane), or a derivative thereof.
• 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).
• DODMA 1,2-dioleyloxy-N,N- dimethylaminopropane
• DiLa2 liposomes from Marina Biotech (Bothell, Wash.)
• DLin-DMA 1,2- dilinoleyloxy-3-dimethylaminopropane
• DLin- KC2-DMA 2,2-dilinoleyl-4
• 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.
• 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.
• the liposome comprises 48% cholesterol, 20% DSPC, 2% PEG-c-DMA, and 30% cationic lipid, where the cationic 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 2005; 107(2): 276-87.
• DSDMA 1,2-distearloxy-N,N-dimethylaminopropane
• DODMA 1,2-dilinolenyloxy-3-dimethylaminopropane
• 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 liposomes are DiLa2 liposomes (Marina Biotech, Bothell, Wash.), SMARTICLES® (Marina Biotech, Bothell, Wash.), neutral DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) based liposomes (e.g., siRNA delivery for ovarian cancer (Landen et al. Cancer Biology & Therapy 2006 5(12)1708-1713); herein incorporated by reference in its entirety) and hyaluronan-coated liposomes (Quiet Therapeutics, Israel).
• DiLa2 liposomes Marina Biotech, Bothell, Wash.
• SMARTICLES® Marina Biotech, Bothell, Wash.
• neutral DOPC 1,2-dioleoyl-sn-glycero-3-phosphocholine
• 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.
• 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.
• 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-ami no-3-[(9Z,12Z)-octadeca-9, 12- dien-1-yloxy]-2-[(octyloxy)methyl]propan-1-ol (Compound 3 in US20130150625); and 2-(
• 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-KC2-DMA), dilinoleyl-methyl-4- dimethylaminobutyrate (DLin-MC3-DMA), and di((Z)-non-2-en-1-yl) 9-((4-)
• a neutral lipid selected from DSPC, DPPC, POPC, DOPE and SM;
• a sterol e.g., cholesterol;
• 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.
• 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.
• a cationic lipid selected from 2,2-dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane (DLin-KC2-DMA), d
• 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.
• neutral lipids include, but are not limited to, DSPC, POPC, DPPC, DOPE and SM.
• the nanoparticle does not comprise a neutral lipid.
• 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.
• the nanoparticle does not comprise a sterol, such as cholesterol.
• 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).
• the PEG or PEG modified lipid comprises a PEG molecule of an average molecular weight of 2,000 Da.
• 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.
• 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).
• 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,
• the cationic liposomes optionally do not comprise a non-cationic lipid.
• Neutral molecules may interfere with coiling/condensation of multi-lamellar NPs resulting in RNA/DNA loaded liposomes greater than 200 nm in size.
• Cationic liposomes generated without helper molecules can comprise a size of about 70-200 nm (or less).
• These constructs consist 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 (when exposed to the ambient environment).
• 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 multi-lamellar RNA-NPs drive significantly greater innate immune responses, which are a significant predictor of efficacy for modulating the immune system.
• the presently disclosed nanoparticles comprise 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 nucleic acid layers comprise nucleic acid molecules.
• 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, such as about 1 to about 5 to about 1 to about 20, optionally, about 1 to about 18, about 1 to about 17, about 1 to about 15, about 1 to about 10, or about 1 to about 7.5.
• 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.
• 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.
• the nanoparticle comprises less than or about 10 g RNA molecules per 150 g lipid mixture.
• the nanoparticle is made by incubating about 10 g RNA with about 150 g liposomes.
• the nanoparticle comprises more RNA molecules per mass of lipid mixture.
• the nanoparticle may comprise more than 10 g RNA molecules per 150 g liposomes.
• the nanoparticle in some instances comprises more than 15 g RNA molecules per 150 g liposomes or lipid mixture.
• the nucleic acid molecules are RNA molecules, e.g., transfer RNA (tRNA), ribosomal RNA (rRNA), and/or messenger RNA (mRNA).
• the RNA molecules comprise tRNA, rRNA, mRNA, or a combination thereof.
• the RNA is total RNA isolated from a cell.
• the RNA is total RNA isolated from a diseased cell, such as, for example, a tumor cell or a cancer cell or an infected cell. Methods of obtaining total tumor RNA is known in the art and described herein at Example 1, and represents an example of methods for obtaining nucleic acid for incorporation into nanoparticles.
• the RNA molecules are mRNA.
• mRNA is in vitro transcribed mRNA.
• the mRNA molecules are produced by in vitro transcription (IVT). Suitable techniques of carrying out IVT are known in the art.
• an IVT kit is employed.
• the kit comprises one or more IVT reaction reagents.
• IVT reaction reagent refers to any molecule, compound, factor, or salt, which functions in an IVT reaction.
• the kit may comprise prokaryotic phage RNA polymerase and promoter (T7, T3, or SP6) with eukaryotic or prokaryotic extracts to synthesize proteins from exogenous DNA templates.
• the RNA is in vitro transcribed mRNA, wherein the in vitro transcription template is cDNA made from RNA extracted from a virus or infected cell.
• the nanoparticles of the disclosure are useful, in various aspects, in the treatment of cancer, and in these embodiments the nanoparticle optionally comprises a mixture of RNA which is RNA isolated from a tumor of a human, optionally, a malignant brain tumor, optionally, a glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma, or a peripheral tumor with metastatic infiltration into the central nervous system.
• RNA is RNA isolated from a tumor of a human, optionally, a malignant brain tumor, optionally, a glioblastoma, medulloblastoma, diffuse intrinsic pontine glioma, or a peripheral tumor with metastatic infiltration into the central nervous system.
• 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.
• the method of making a nanoparticle comprises additional processing steps, such as, for example, capping the in vitro transcribed RNA molecules.
• the viral protein is expressed by the species SARS-CoV-2.
• SARS-CoV-2 is a positive-sense single-stranded RNA virus with genetic similarity to bat coronaviruses and was first isolated in January 2020 from patients in Wuhan, China (Hui et al., International J Infectious Diseases 91: 264-266 (2020)).
• the viral protein is one encoded and expressed by a SARS CoV-2 virus, e.g., a SARS CoV- 2 protein.
• SARS-CoV-2 virus e.g., a SARS CoV- 2 protein.
• Complete genome sequences of SARS-CoV-2 are publicly available online at the GenBank database of the National Center for Biotechnology Information (NCBI) website (ncbi.nlm.nih.gov). Exemplary genome sequences include those having the following GenBank Accession numbers: MN988668, NC_045512), MN938384.1, MN975262.1, MN985325.1, MN988713.1, MN994467.1, MN994468.1, MN997409.1, MN988668. The complete genome sequence of GenBank Accession No.
• the SARS-CoV-2 protein is a spike protein, membrane protein, envelope protein, or nucleocapsid protein.
• the SARS CoV-2 protein is a SARS-CoV-2 Spike (S) protein or a fragment thereof.
• the NPs of the present disclosure comprise a mixture of RNA molecules.
• the mixture of RNA molecules comprises mRNAs encoding the S protein or a portion thereof.
• the mixture of RNA molecules comprises mRNAs encoding other proteins encoded by the SARS CoV-2 genome.
• SARS CoV-2 proteins include but are not limited to ORFlab polyprotein (QIQ50091.1), ORF3a protein (QIQ50093.1), envelope protein (QIQ50094.1), membrane glycoprotein (QIQ50095.1), ORF6 protein (QIQ50096.1), ORF7a protein (QIQ50097.1), ORF8 protein (QIQ50098.1), nucleocapsid protein (QIQ50099.1), and ORF10 protein (QIQ50100.1).
• ORFlab polyprotein QIQ50091.1
• ORF3a protein QIQ50093.1
• envelope protein QIQ50094.1
• membrane glycoprotein QIQ50095.1
• ORF6 protein QIQ50096.1
• ORF7a protein QIQ50097.1
• ORF8 protein QIQ50098.1
• nucleocapsid protein QIQ50099.1
• the mRNA comprises a nucleotide sequence encoding an amino acid sequence set forth in the Sequence Listing.
• the nucleotide sequence encodes an amino acid sequence which has at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90% or has greater than 90% sequence identity (e.g., about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%) to an amino acid sequence set forth in the Sequence Listing.
• the nucleotide sequence encodes an amino acid sequence set forth in the Sequence Listing with 1 to 10 amino acid substitutions, e.g., about 1 to 9, about 1 to 8, about 1 to 7, about 1 to 6, about 1 to 5, about 1 to 4, about 1 to 3, about 1 to 2 amino acid substitutions or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 amino acid substitutions.
• the mRNA comprises a nucleotide sequence provided in the Sequence Listing.
• the nanoparticle comprises RNA molecules comprising a nucleotide sequence encoding a viral protein, or a mixture of viral proteins, and a poly(A) tail at the 3' end.
• the nanoparticle comprises RNA molecules as essentially depicted in Figure 19, optionally comprising the sequences referenced in Figure 20.
• the viral protein encoded by the RNA (e.g., mRNA) of the presently disclosed nanoparticles is a full-length spike (S) protein of SARS-CoV-2.
• the viral protein comprises the sequence of SEQ ID NO: 3 or a sequence which is at least 75% identical to SEQ ID NO: 3 (e.g., at least 80% identical to SEQ ID NO: 3, at least 85% identical to SEQ ID NO: 3, at least 90% identical to SEQ ID NO: 3, at least 95% identical to SEQ ID NO: 3, at least 98% identical to SEQ ID NO: 3, or more).
• the mRNA is a product of in vitro transcription of a cDNA sequence encoding the full-length spike (S) protein of SARS-CoV-2.
• the mRNA is a product of in vitro transcription of a cDNA sequence encoding the viral protein having the sequence of SEQ ID NO: 3 or a sequence which is at least 75% identical to SEQ ID NO: 3 (e.g., at least 80% identical to SEQ ID NO: 3, at least 85% identical to SEQ ID NO: 3, at least 90% identical to SEQ ID NO: 3, at least 95% identical to SEQ ID NO: 3, at least 98% identical to SEQ ID NO: 3, or more).
• Plasmids e.g., a modified psP73 vector (Promega) containing a T7 RNA polymerase promoter and a poly-A tail that is 64 nucleotides long (pSP73-Sph/A64) protecting RNA from degradation, were obtained and made to comprise cDNA encoding mRNA encoding the full length SARS CoV-2 Spike protein.
• the plasmids were linearized using restriction enzymes (i.e., Spel) and purified with Qiagen PCR MiniElute kits.
• the linearized DNA was subsequently in vitro transcribed using the mMESSAGE mMACHINE T7TM transcription kit (Invitrogen) per manufacturer's protocol. The transcribed mRNA was then cleaned up using RNA Maxi kits (Qiagen).
• mMESAGE mMACFIINE Invitrogen kits with T7 enzyme mix were used to 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.
• 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 mmRNA 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).
• RNA-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 (Figure 1 A). 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.
• DOTAP DOTAP
• DOTAP lipid NPs 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.
• systemic i.e., intravenous
• RNA and DOTAP lipid NPs used in the above preparation is pre determined or pre-selected. In some instances, a ratio of about 15 g liposomes per about 1 g RNA were used. For instance, about 75 g liposomes are used per ⁇ 5 g RNA or about 375 g liposomes are used per ⁇ 25 g RNA. In other instances, about 7.5 g liposomes were used per 1 g RNA. Thus, in exemplary instances, about 1 g to about 20 g liposomes are used for every g RNA used.
• 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).
• samples comprising multilamellar RNA-NPs or control NPs were kept on ice prior to being loaded in a snap-freezed 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 / FEG transmission electron microscope with a Gatan UltraScan 4000 (4k x4k) CCD camera.
• the resulting CEM images are shown in Figure 1 B.
• the right panel is a CEM image of multilamellar RNA-NPs and the left panel is a CEM image of control NPs (uncomplexed NPs).
• control NPs contained at most 2 layers, whereas multilamellar RNA NPs contained several layers.
• Figure 5 provides another CEM image of exemplary multilamellar RNA NPs. Here, the multiple layers of RNA layers alternating with lipid layers are especially evident.
• RNA NPs mediate peripheral and intratumoral activation of DCs.
• DOTAP lipid NPs made as essentially described in Example 1 are complexed with Ore recombinase- encoding mRNA to make Cre-encoding RNA-NPs. These multilamellar RNA-NPs are administered to Ai14 transgenic mice, which carry a STOP cassette flanked by loxP. The STOP cassette prevents the transcription of tdTomato until Cre-recombinase is expressed. A week after RNA-NPs are administered, the lymph nodes, spleens and livers of the transgenic mice are harvested, sectioned and stained with DAPI. The expression of tdTomato is analyzed by fluorescent microscopy following the procedures as essentially described in Sayour et al, Nano Letters 2018. It is expected that the Cre-mRNA-NPs localize in vivo to lymphoid organs, including liver, spleen, and lymph nodes.
• DC Dendritic Cell
• This example describes a comparison of the nanoparticles of the present disclosure to cationic RNA lipoplexes and anionic RNA lipoplexes.
• CEM Cryo-Electron Microscopy
• anionic LPXs localize upon administration to mice. As shown in Figure 8, anionic LPXs localized to the spleens of animals upon administration, consistent with previous studies (Krantz et al, Nature 534: 396-401 (2016)).
• 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.
• mice treated with multilamellar RNA NPs exhibited the highest levels of activated DCs.
• ML RNA-NPs allow for substantially greater innate immunity which is enough to drive efficacy from even non-antigen specific ML RNA-NPs.
• these figures demonstrate the superior efficacy of multilamellar tumor specific RNA-NPs, relative to anionic LPX and RNA LPX.
• RNA-NP formulation targeting physiologically relevant tumor antigens is more immunogenic ( Figures 2F-2H, 2J) and significantly more efficacious (Figure 2I) compared with anionic LPX and RNA LPX.
• Figure 1C a novel RNA-NP design composed of multi-lamellar rings of tightly coiled mRNA has been developed ( Figure 1C), 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).
• 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).
• 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 anti-tumor efficacy (with increased TILs) in several tumor models.
• This example demonstrates the ability of multilamellar RNA-NPs to systemically activate DCs, induce antigen specific immunity and elicit anti-tumor efficacy.
• Figure 3B is a graph of the % central memory T cells (CD62L+CD44+ of CD3+ cells) in the harvested lungs of untreated mice, mice treated multilamellar RNA NPs with GFP RNA, and mice treated multilamellar RNA NPs with tumor-specific RNA.
• BALB/c mice or BALB/c SCID (Fox Chase) mice (8 mice per group) were inoculated with K7M2 lung tumors and vaccinated intravenously thrice-weekly with multilamellar RNA-NPs comprising GFP RNA or tumor-specific RNA.
• a control group of mice was untreated.
• % survival was plotted on a Kaplan-Meier curve (***p ⁇ 0.0001, Gehen-Breslow-Wilcox).
• Figure 3C the percent survival of BALB/c mice treated with multilamellar RNA NPs with tumor-specific RNA was highest among the three groups.
• RNA-NPs comprising GFP RNA or tumor-specific RNA
• both GFP (control) and tumor specific RNA-NPs mediate innate immunity and anti-tumor activity; however, only tumor specific RNA-NPs mediate increases in intratumoral memory T cells and long-term survivor outcome ( Figures 3A-3D).
• Anti-tumor activity of RNA-NPs in mice bearing intracranial malignancies was also demonstrated (data not shown).
• RNA-NP vaccines The safety and activity of multilamellar RNA-NPs was evaluated in client-owned canines (pet dogs) diagnosed with malignant gliomas or osteosarcomas. The malignant gliomas or osteosarcomas from dogs were first biopsied for generation of personalized tumor RNA-NP vaccines.
• RNA materials was extracted from each patient's biopsy.
• a cDNA library was then prepared from the extracted total RNA, and then mRNA was amplified from the cDNA library.
• mRNA was then complexed with DOTAP lipid NPs, into multilamellar RNA-NPs as essentially described in Example 1.
• Blood was drawn at baseline, then 2 hours and 6 hours post-vaccination for assessment of PD-L1, MHCII, CD80, and CD86 on CD11c+ cells.
• CD11c expression of PD-L1, MHC-II, PDL1/CD80, and PD-L1/CD86 is plotted over time during the canine's initial observation period.
• CD3+ cells were analyzed over time during the canine's initial observation period for percent CD4 and CD8, and these subsets were assessed for expression of activation markers (i.e. CD44). From these data, it was shown that multilamellar RNA-NPs elicited an increase in 1) CD80 and MHCII on CD11c + peripheral blood cells demonstrating activation of peripheral DCs; and 2) an increase in activated T cells
• RNA-NPs mediate lymphoid honing of immune cell populations before egress.
• RNA-NPs A male boxer diagnosed with a malignant glioma was enrolled on study per owner's consent to receive RNA-NPs. Tumor mRNA was successfully extracted and amplified after tumor biopsy. Immunologic response is plotted in response to 1 st vaccine ( Figure 4B). The data show increased activation markers over time on CD11c+ cells (DCs). As shown in Figure 4C, an increase in activated T cells (CD44+CD8+ cells) was observed within the first few hours post RNA-NP vaccine. These data support that the multilamellar RNA-NPs are immunologically active in a male canine boxer.
• RNA-NPs After receiving weekly RNA-NPs (x3), the canines diagnosed with malignant gliomas had a steady course. Post vaccination MRI showed stable tumor burdens, with increased swelling and enhancement (in some cases), which may be more consistent with pseudoprogression from an immunotherapeutic response in otherwise asymptomatic canines. Survival of canines diagnosed with malignant gliomas receiving only supportive care and tumor specific RNA-NPs (following tumor biopsy without resection) is shown in Figure 4D. In Figure 4D, the median survival (shown as dotted line) was about 65 days and was reported from a meta-analysis of canine brain tumor patients receiving only symptomatic management. In a previous study, cerebral astrocytomas in canines has been reported to have a median overall survival of 77 days. The personalized, multilamellar RNA NPs allowed for survival past 200 days.
• RNA-NPs (1x) were well tolerated with stable blood counts, differentials, renal and liver function tests.
• One canine was autopsied after RNA-NP vaccines. In this patient, there were no toxicities believed to be related to the interventional agent.
• Tissues required for microscopic evaluation were trimmed, processed routinely, embedded in paraffin, and stained with hematoxylin and eosin by Charles River Laboratories Inc., Skokie, Illinois. Light microscopic evaluation was conducted by the Contributing Engineer, a board-certified veterinary pathologist on all protocol- specified tissues from all animals in Groups 1 and 4, and any early death animals.
• This example describes a study aimed at determining the impact of pDCs transfected with multilamellar RNA-NPs on antigen specific T-cell priming.
• pDCs are well-known stimulators of innate immunity and type I IFN, they also mediate profound effects on intratumoral adaptive immunity. They can: 1) directly present antigen for priming of tumor specific T cells; 2) assist adaptive response through chemokine recruitment of other DC subtypes (via chemokines CCL3, CCL4, CXCL10); 3) polarize Th1 immunity through IL-12 secretion; and/or 4) mediate tumor antigen shedding (through cytokine, TRAIL or granzyme B) for DC loading and T cell priming.
• RNA-NP activated pDCs serve as direct primers of antigen specific immunity and assist classical DCs (cDCs) and/or myeloid-derived DCs (mDCs) in promoting effector T-cell response.
• responses observed at different times are analyzed using a two-way ANOVA model with mutually exclusive groups distributed among treatments and observation times, change in immune response parameters over time are assessed using generalized linear mixed effect models (GLMMs).
• Response variables for experiments that are completely replicated at least once are analyzed using GLMMs.
• Experimental replication is modeled as a random effect to account for "batch" or "laboratory day” variability.
• Treatment and control groups are modeled as fixed effects and compared using ANOVA-type designs nested within the mixed effect modeling framework.
• Tumorgenicities for KR158b-luc, GL261 -luc and a murine H3.3K27M mutant cell line have been set up.
• KR158b-luc and GL261-luc are both transfected with luciferase so that tumors can be monitored for growth using bioluminescent imaging.
• Tumorigenic dose of KR158b-luc and the H3K27M mutant line is 1x10 4 cells.
• Tumorigenic dose of GL261 -luc is 1x10 5 cells.
• GL261 and KR158 are injected into the cerebral cortex of C57BI/6 (3 mm deep into the brain at a site 2 mm to the right of the bregma); H3K27M glioma cells are injected midline.
• This example describes an experiment designed to determine the pDC phenotype and function following activation by RNA-NPs.
• TTRNA-NPs composed from 375 g of FITC labeled DOTAP (Avanti) with 25 g of TTRNA (derived from KR158b and delivered iv). Twenty-four hours after vaccination recipient mice are euthanized (humanely killed with C02) for collection of spleens, tumor draining lymph nodes (tdLNs) and tumors. Organs are digested into a single cell suspension, undergo RBC lysis (PharmLyse, BD Bioscience) before incubation at 37°C for 5 minutes. Ficoll gradients are used to separate WBCs from parenchymal cells.
• pDCs are stained for CD11c, B220 and Gr-1 (ebioscience). Distinct pDC subsets are identified by differential staining for CCR9, SCA1, and Ly49q.
• Activation state is assessed based on expression of co stimulatory molecules (i.e. CD40, CD80, CD86) chemokines (i.e. CCL3, CCL4, CXCL10) and chemokine receptors (i.e. CCR2, CCR5, CCR7).
• Detection secondary antibody is rabbit IgG conjugated with AlexaFlour®488 (ThermoFisher Scientific) for FITC detection.
• Effector versus regulatory function is determined through intracellular staining for effector (i.e. IFN-I, IL-12) versus regulatory cytokines (i.e. TGF-b, IL-10). Analyses will be conducted by multi-parameter flow cytometry (LSR, BD Bioscience) and immunohistochemistry (IHC).
• effector i.e. IFN-I, IL-12
• regulatory cytokines i.e. TGF-b, IL-10
• This example describes an experiment designed to determine whether RNA-NP transfected pDCs mediate direct or indirect activation of antigen specific T cells.
• pDCs are well known stimulators of innate immunity and type I IFN, their cumulative effects on antigen specific responses are still being uncovered. Since they express MHC class II, they have APC capacity, but compared to their cDC counterparts, they are believed to be poor direct primers of antigen specific immunity. This experiment is aimed at yielding a better understanding of pDCs, in the context of RNA-NPs, as either direct primers or facilitators of antigens specific immunity.
• RNA-NP transfected pDCs are then co-cultured with naive magnetically separated CD4 and CD8 T cells, and T cells are assessed for proliferation, phenotype (effector vs central memory), function and cytotoxicity.
• Indirect effects from pDCs are assessed via ex vivo co-cultures with TTRNA- loaded DCs (matured ex vivo from murine bone marrow) with naive CD4 and CD8 T cells.
• Ex vivo co-cultures will be performed in triplicate, for 7 days in a 96 well plate with naive T cells (40,000 RNA-NP transfected pDCs with 400,000 T cells) labeled with CFSE (Celltrace, Life Technologies).
• T cell proliferation is determined by measuring CFSE dilution by flow cytometry. Phenotype for effector and central memory populations is determined through differential staining for CD44 and CD62L.
• T cells are re-stimulated for a total of two cycles before supernatants are harvested for detection of Th1 cytokines (i.e. IL-2, TNF-a, and IFN-g) by bead array (BD Biosciences). Stimulated T cells are also incubated in the presence of KR158b (stably transfected with GFP) or control tumor (B16F10-GFP) and assessed for their ability to induce cytotoxicity. Amount of GFP in each co culture, as a surrogate for living tumor cells, are quantitatively measured by flow cytometry.
• Th1 cytokines i.e. IL-2, TNF-a, and IFN-g
• Stimulated T cells are also incubated in the presence of KR158b (stably transfected with GFP) or control tumor (B16F10-GFP) and assessed for their ability to induce cytotoxicity. Amount of GFP in each co culture, as a surrogate for living tumor cells, are quantitatively measured by flow
• RNA-NP transfected pDCs The in vivo effects of FACSorted RNA-NP transfected pDCs are determined by adoptively transferring these cells (250,000 cells/mouse) to tumor-bearing mice (weekly x3) and harvesting spleens, tdLNs, and tumors one week later for assessment of antigen specific T cells by YFP expression in IFN-g reporter mice (GREAT mice, B6 transgenic, containing IFN-g promotor with IRES-eYFP reporter, Jackson labs).
• HEK293T cells are co-transfected with a SARS-CoV-2 spike encoding- plasmid, an MLV Gag-Pol packaging construct, and a MLV transfer vector encoding a luciferase reporter. Cells are incubated for 5 hours at 37°C with transfection medium. Cells are then washed with DMEM two times, and DMEM containing 10% FBS is added for 60 hours. The supernatants are harvested and filtered through 0.45- mm membranes, concentrated with a 30kDa membrane for 10 min at 3,000 rpm, and then frozen at -80°C.
• Target volume definition (a) Gross Tumor Volume (GTV): The GTV includes all gross residual tumor and/or the tumor bed as defined by MR imaging and operative report. The GTV in many cases will involve a contracted or collapsed tumor bed. Tissue defects resulting from surgical approaches will not be included as part of the GTV when not previously involved by tumor, (b) Clinical Target Volume (CTV): The CTV is meant to treat subclinical microscopic disease and will be an anatomically constrained 5-10 mm margin on the GTV, with additional expansion as necessary to encompass areas of T2/FLAIR change suspicious for tumor involvement prior to surgery.
• GTV Gross Tumor Volume
• CTV Clinical Target Volume
• Planning target volume 1 (PTV1): CTV + 3 mm;
• Planning target volume 2 (PTV2): GTV + 3 mm.
• Temozolomide dosing should be performed following institutional practice (e.g. +/- 5 to 10% of the calculated dose). It is recommended that antiemetics be given 30 minutes prior to each temozolomide dose. If emesis occurs within 20 minutes of taking a given dose, then the dose may be repeated once. If emesis occurs after 20 minutes, the dose should not be repeated. Oral suspension may be compounded if unable to swallow capsules. While receiving TMZ, subjects should receive POP prophylaxis per institutional guidelines.
• the 42 days of temozolomide should be given regardless of the end date of RT. If a dose of temozolomide is given and radiation therapy is NOT administered due to sedation or technical issues, the temozolomide doses should not be made up (i.e., no more than 42 doses of temozolomide should be given).
• Supportive care may include, but is not limited to, antibiotics, antiemetics, antidiarrheals, topical treatments, blood products, intravenous or oral fluids, electrolyte repletion, and will be used as clinically indicated.
• antibiotics antibiotics, antiemetics, antidiarrheals, topical treatments, blood products, intravenous or oral fluids, electrolyte repletion, and will be used as clinically indicated.
• bevacizumab per institutional guidelines is allowed to minimize swelling.
• Standard of Care Radiation (to begin 4 weeks (+/- 2 week after surgery) or sooner based on institutional preference): Administration of TMZ, Radiation per protocol or institutional guidelines for GBM
• RNA-LP Administrations #4-15 administration on Day 1 of each cycle following initial 3 administrations) (to begin 4 weeks after administration #3 once hematologic recovery is observed (ANC> 1500/DL, pi atelets> 150/DL): Administration of RNA-LP vaccine, Physical & Neurological exam, Vital signs (including height & weight), Adverse event assessment, Concomitant medications, Performance Status, Brain MRI (within 2 weeks of administration #4, 6, 810, 12, 14), Laboratory procedures (CBC with differential and platelet count, Complete metabolic panel (CMP) including AST/ALT, BUN, Total bilirubin and creatinine, CD4/CD8 panel and ratio (administration #5, 10 and 15), Peripheral blood for immune-monitoring within 24 hours prior to vaccination, 3 and 6 hours post vaccination, Blood for circulating tumor DNA studies)
• RNA-LP vaccines are generated but do not meet release criteria or targeted dose then the patient may remain on study and receive the qualified product but will be replaced for the purposes of safety assessment
• Subjects receiving less than 3 vaccines without toxicity will be replaced for safety assessments.
• RNA-NP technology can address each of these concerns by, e.g., targeting full-length SARS-CoV-2 structural genes and overcoming mutational heterogeneity; inducing bidirectional adaptive immunity; and eliciting near immediate immune activation (within hours).
• Mice receiving SARS-CoV-2 full-length spike RNA-NPs had more SARS-CoV-2 specific antibodies (Figure 26), and effector T cells after vaccination with significant memory recall expansion after in vitro re-stimulation with overlapping SARS-CoV-2 spike peptide mix ( Figures 27A, 28B, and 28).
• RNA-NP activity is associated with IFN-g dependent innate immunity from plasmacytoid DCs (pDCs) in a non-TLR7 dependent manner.
• HEK293T cells stably transfected with ACE2 (creative-biogene.com) are infected with prepared pseudovirus in the presence of murine serum.
• ACE2 expressing 293T cells are seeded to 24-well plates at 2.5 x 10 5 cells/well until confluent and inoculate wells with 200 pi of pseudotyped virus solution or non- infected control conditions (200 pi DMEM-C solution per well) with and without serum from vaccinated animals. Incubation occurs at 37°C 5% CO2 for 1-2 h before adding 300 mI DMEM-C for 72 h incubation.
• DCs are co-cultured with magnetically separated CD4 and CD8 T cells from vaccinated mice, and assessment of T cells is performed for proliferation, phenotype (effector versus central memory by differential staining for CD44 and CD62L), function and cytotoxicity.
• Ex vivo co-cultures are performed in triplicate, for 48 h in a 96 well plate (4 x10 4 DCs will be ex vivo co-cultured with 4x10 5 T cells per well).
• T cells are labeled with CFSE (Celltrace) for assessment of proliferation by flow cytometry, and re-stimulated in culture before supernatants and cells are harvested.
• CFSE Celltrace
• T cells are incubated in the presence of ACE2 expressing 293T cells containing a luciferase bioreporter (infected with pseudovirus as described above) or control cells cultured in a 10:1 effector/target ratio x 48h and assessed for bioluminescence. Bioluminescence from each co-culture, as a surrogate for living cells, is quantitatively measured by MS imaging.
• RNA-NPs Activity from RNA-NPs is dependent on IFN-g (type I interferon) from plasmacytoid DCs (pDCs). Interestingly, the innate immunomodulating effects of RNA-NPs do not appear to be driven by toll-like receptor (TLR) stimulation, as efficacy is intact in TLR7 KO mice.
• TLR toll-like receptor
• SARS-CoV-2 specific RNA-NPs elicit bidirectional adaptive responses in an IFN-g dependent manner, and identifies cell types and signaling pathways involved.
• RNA-NPs may reset IFNAR1 signaling through non-TLR dependent intracellular PRRs from pDCs. RIG-I, MDA-5 or STING may be involved in IFN-g signaling.
• SARS-Co-V-2 specific RNA-NPs, pp65 RNA-NPs or NPs alone are administered to mice as described above.
• Vaccines are administered once weekly (x3).
• Mice are vaccinated 3 times at 7-day intervals.
• Blood is collected and assessed for DC subpopulations.
• pDCs are stained for CD11c, B220 and Gr-1 (ebioscience); distinct pDC subsets are identified by differential staining for CCR9, SCA1, and Ly49q.
• Resident/migratory classical DCs are identified by CD11c+CD103+MHCII+cells and CD11c+CD11b+MHCII+cells; myeloid DCs (mDCs) are identified by CD11c+CD14+MHCII+cells.
• Activation state is assessed based on expression of cytokines (i.e., IFN-I/II, IL-12) and co-stimulatory molecules (i.e., CD40, CD80, CD86).
• cytokines i.e., IFN-I/II, IL-12
• co-stimulatory molecules i.e., CD40, CD80, CD86.
• Analyses are conducted by multi-parameter flow cytometry (LSR, BD Biosciences). Innate pathways (i.e., RIG-I, MDA-5 and STING) of interest are explored in these cell subsets by PCR.
• Results may be correlated with SARS-CoV-2 neutralization and adaptive recall as described above.
• IFN-I i.e., IFN-a/IFN-b
• wild-type and knock-out (KO) mice are vaccinated for pathways of interest (i.e., IFNAR1, RIG- 1, MDA-5 and STING, all available through Jackson labs).
• IFN-I i.e. IFN-a/IFN-b
• levels are measured from serum by ELISA 24h post-vaccine and compared with results with anti-SARS-CoV-2 immunogenicity testing as described above.
• the 5' internal ribosomal entry site (IRES) of hepatitis C virus (HCV) leads to cap-independent translation and very rapid and extensive translation of the RNA genome while its 3' polyUUU tail is recognized by PRRs including RIG-1 and MDA5 pathways lead to intense triggering of the innate immune system and production of type I interferons.
• IRS internal ribosomal entry site
• HCV hepatitis C virus
• RNA templates are made using PCR products isolated via gel electrophoresis and purified using the QIAquick Gel Extraction Kit.
• the resulting PCR fragments are digested with the restriction enzymes Hindlll and EcoRI and cloned into the Hindll and EcoRI sites of the plasmid which has a T7 promoter and 64T nucleotides that allow for the production of in vitro transcribed RNA with a polyA tail of 64 residues.
• the plasmids are linearized with Spel, in vitro transcribed (IVT) using mMessage mMachine T7 (Ambion), purified with RNeasy kit (Qiagen), quantified by spectrophotometry, and analyzed by an Agilent Bioanalyzer to confirm quality and synthesis of full-length mRNA.
• LAMP-1 and DC-LAMP constructs are made by inserting the SARS-CoV-2 specific genes between the luminal domain and the transmembrane domain and cytoplasmic tail of LAMP before cloning into pGEM-4z vector.
• the immunologic effects of LAMP modified is compared to unmodified RNA-NPs in: 1) control RNA-NPs; 2) RNA-NPs incorporating LAMP-1 targeted SARS-CoV-2antigens; 3) RNA-NPs incorporating DC-LAMP targeted SARS-CoV-2 antigens; and 4) RNA-NPs incorporating the combination of DC-LAMP and LAMP-1 targeted antigens (equimolar ratio) using the assays described above.
• SARS-CoV-2 structural mRNAs are compared with unmodified targets.
• Unmodified SARS-CoV-2 RNA-NPs are compared with UTR modified RNA-NPs, LAMP modified RNA-nanoparticle vaccine, and UTR + LAMP modified RNA-NPs.
• the effects of smaller and less frequent dosing is examined with respect to anti- SARS-CoV-2 adaptive responses. We will compare effects of decreasing doses (i.e., 10 g, 5 g 2.5 g, 1 g) versus standard 25 g dose given as a single vaccine or given as multiple weekly vaccines (x3).
• RNA-NPs are re-suspended in chloroform before being evaporated off until a thin lipid layer remains.
• SARS-CoV-2 specific mRNA is added to DOTAP NPs at a multilamellar ratio of 1:15 mg. This mixture is kept at room temperature for 15-20 minutes to allow RNA-NP complexes to form. After multi lamellar complexation, contents are mixed with tertiary butanol, vortexed and frozen at -80°C overnight before being lyophilized (Labconco) and stored at -20°C. Validation of lyophilized formulation is assessed based on anti-SARS-CoV-2 mediated humoral and cellular responses compared with freshly produced RNA-NPs.
• Particles are validated by zeta-potential measurements and size measurements.
• RNA-NPs are injected i.v. to define safety and toxicity.
• Starting (low) dose of RNA-NPs involves: RNA (0.05 mg/kg) encapsulated with 0.75 mg/kg of DOTAP nanoliposome. These are compared to medium doses (100% increase) of 0.1 mg/kg of RNA encapsulated with 1.5 mg/kg of DOTAP nanoliposome, which is the feline equivalent dose (based on body surface area) of the dosing in mice (1 mg/kg).
• High dose administrations (100% increase) will involve 0.2 mg/kg of RNA encapsulated with 3 mg/kg of DOTAP nanoliposome.
• Feasibility of SARS-CoV-2 specific mRNA production is determined based on RNA quality, concentration and integrity by gel electrophoresis, nanodrop spectrophotometry and bioanalysis.
• RNA-NPs Animals randomized to receive multiple dosing receive three RNA-NPs one week apart. Peripheral labs are monitored from felines at enrollment, immediately before each RNA-NP, and during 1 -month post-treatment follow-ups. Complete blood counts (with differentials), comprehensive chemistries, lipid panels and renal/liver function tests are monitored. Serum cytokines will be obtained post-infusion to assess for symptoms of cytokine release syndrome (i.e., IL-1, IL-6, TNF-a). Increasing doses of i.v. RNA-NPs are injected to define dose limiting toxicities (DLTs), such as grade 3-4 adverse events and maximal tolerated dose (MTD) in a standard 3+3 study design.
• DLTs dose limiting toxicities
• MTD maximal tolerated dose
• RNA-NPs There are three groups (three felines/group). If DLT attributable to RNA-NPs is experienced in 1 of 3 felines, an additional 3 felines will be enrolled at the same dose. If none of 3 felines or 1 of 6 felines experiences DLT, dose will be escalated in subsequently accrued felines. If >2 felines experience DLT attributable to the RNA-NPs, the accrual to that group will be halted and the immediate lower dose level will be considered the MTD. Based on immunologic response across subjects from 3+3 study across each arm, the lowest effective dose of SARS-CoV-2 RNA-NPs is selected to advance into an expansion cohort (up to 20 subjects per arm).
• PBMCs electroporated with SARS-CoV-2-specific mRNA are co-cultured with CD3 selected T cells before assessment of IFN-y and MIP-1-a (i.e., CCL3) from supernatants by ELISA.
• T cell cytotoxicity against SARS- CoV-2 is assessed. Serum from animals is drawn one, six and 12 months after the last vaccine for assessment of neutralization in 3D pulmonary explants. Neutralization against SARS-CoV-2 and related coronaviruses such as HcoV-OC43 in 3D is assessed in pulmonary explants.

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