Compositions and methods for modulating heat shock protein 70 activity for treatment of inflammatory diseases

Abstract

In certain aspects, provided herein compositions and methods for treating inflammation or an inflammatory disease using an mRNA formulated in a lipid nanoparticle (LNP), wherein the mRNA comprises an open reading frame encoding HSP70 polypeptide, and methods of using the same.

Description

[0001] 7504-00202 GRH-00462COMPOSITIONS AND METHODS FOR MODULATING HEAT SHOCK PROTEIN 70ACTIVITY FOR TREATMENT OF INFLAMMATORY DISEASESBACKGROUND Heat shock proteins (HSPs) represent a class of molecular chaperones known to beexpressed in response to exposure to stressful conditions, such as, heat, cold, ultravioletlight, wound healing, tissue remodeling, and a number of other systemic and biochemicalstressors. HSPs perform chaperone functions by binding and stabilizing new or mis-foldedproteins and assisting them to acquire their native structure, thus preventing mis-folding andthe aggregation process, such as stress-induced protein denaturation. What is more, HSPshave been shown to have pro-inflammatory and / or anti-inflammatory effects. Thus, HSPsmay play a role critical to biological pathways for the mediation and resolution ofinflammation, such as under metabolic and proteotoxic stress conditions. In the context ofdegenerative inflammation disorders, particularly those characterized by persistent, low-levelinflammation, a gradual suppression or exhaustion of the heat shock response (HSR) mayoccur. Unfortunately, chronic inflammatory diseases have become epidemic, at least, in part,due to lifestyle choices characterized by high- calorie diet, intestinal dysbiosis, and sedentarybehavior. Conditions such as obesity, diabetes, liver disease, cardiovascular diseases (CVD),and neurodegenerative diseases now collectively account for approximately 74% of globaldeaths. Thus, despite the many advances in various fields of medical science, there remains aneed for safe, well-tolerated treatments for inflammatory disease, and the maintenance oflong-term remission. SUMMARY Aspects of the invention, as provided herein, include therapeutic compositionscomprising an mRNA formulated lipid nanoparticle (LNP), wherein the mRNA comprisesan open reading frame encoding heat shock protein polypeptide, or a functional fragmentthereof. In some embodiments of the invention, the heat shock protein polypeptide, or afunctional fragment thereof, is HSPlO0, HSP90, HSP70, HSP60, HSP40, or HSP27.Preferably, the heat shock protein polypeptide is HSP70. In some embodiments, the LNP is asolid lipid nanoparticle (SLN).In certain aspects of the invention, provided herein are cells comprising the LNPsdisclosed herein.7504-00202 GRH-00462In some aspects of the invention, provided herein are methods of treating aneurodegenerative disease in a subject, the method comprising administering thetherapeutic composition disclosed herein. In some such embodiments, the compositioncomprises the LNP- comprising cells disclosed herein.BRIEF DESCRIPTION OF FIGURESFigure 1 depicts the percent body weight change in BALB / cJ mice. (Prime is thefirst day of dosing.) No significant weight change was observed between HSP70 mRNA-LNPs and control (Empty LNPs). On days 4 and 5, all roads to the vivarium were flooded,preventing the dosing and weighing of the mice. Figure 2 depicts the percent body weight change in MS-NASH mice (Cohort #1).(Prime is the first day of dosing.) Figure 3 depicts the percent body weight change in MS-NASH mice (Cohort #2).(Prime is the first day of dosing.) Figure 4 depicts the percent body weight change in MS-NASH mice (Cohort #3).(Prime is the first day of dosing). Figure 5 depicts the percent body weight change in MS-NASH mice (Cohort #4).(Prime is the first day of dosing). Figure 6 depicts a DNA agarose gel representing four samples of amplified HSP70DNA, each having the expected length of ~2100 base pairs relative to a 1KB DNA ladder(left- most lane). Figure 7 depicts an RNA agarose gel representing four samples of fully cleaned andcapped mRNA, each corresponding Payload 1 used to create the final lipid nanoparticlecontaining HSP70 mRNA product contemplated herein. Figure 8 depicts hematoxylin and eosin (H&E) staining of sectioned liver from MS-NASH mice (Cohort #1), Control #1 (Empty LNP5) (4X magnification). Lipid accumulation,fatty liver, is observed as clear vacuoles (white space) in hepatocytes.Figure 9 depicts hematoxylin and eosin (H&E) staining of sectioned liver from MS-NASH mice (Cohort #1), Dosed #3 (HSP7O-LNPs) (4X magnification) and exhibitssignificantly less lipid accumulation. Figure 10 depicts hematoxylin and eosin (H&E) staining of sectioned liver from MS-NASH mice (Cohort #2), Control #5 (Empty LNP5) (lOX magnification). Lipidaccumulation, fatty liver, is observed as clear vacuoles (white space) in hepatocytes.Figure 11 depicts hematoxylin and eosin (H&E) staining of sectioned liver fromMS- NASH mice (Cohort #2), Dosed #2 (HSP7O-LNPs) (lOX magnification) and exhibits7504-00202 GRH-00462significantly less lipid accumulation. Figure 12 depicts hematoxylin and eosin (H&E) staining of sectioned liver from MS-NASH mice (Cohort #2), Control #5 (Empty LNP5) (2OX magnification). Lipidaccumulation, fatty liver, is observed as clear vacuoles (white space) in hepatocytes.Figure 13 depicts hematoxylin and eosin (H&E) staining of sectioned liver fromMS- NASH mice (Cohort #3), Dosed #1 (HSP7O-LNPs) (2OX magnification) and exhibitssignificantly less lipid accumulation. Figure 14 depicts an Empty LNP Dosed BALB / cJ brain H&E image from HSP7OCohort A at 1OX magnification. Figure 15 depicts an HSP7O mRNA-LNP Dosed BALB / cJ brain H&E image fromHSP7O Cohort A at 1OX magnification. Figure 16 depicts an Empty LNP Dosed BALB / cJ liver H&E image from HSP7OCohort A at 1OX magnification. Figure 17 depicts an HSP7O mRNA-LNP Dosed BALB / cJ liver H&E image fromHSP7O Cohort A at 1OX magnification. Figure 18 depicts immunofluorescent staining of embedded liver sections from BALB / cJmice control (Empty LNPs) (lOX magnification). HSP7O was stained with anti-human HSP7Omonoclonal rabbit antibody (primary) and anti-rabbit secondary antibody conjugated to AlexaFluor™ 568. Nuclei was stained with Hoechst 33342.Figure 19 depicts immunofluorescent staining of embedded liver sections fromBALB / cJ mice dosed with HSP7O-LNPs (lOX magnification), confirming HSP7Ooverexpression. HSP7O was stained with anti-human HSP7O monoclonal rabbit antibody(primary) and anti-rabbit secondary antibody conjugated to Alexa Fluor™ 568. Nuclei wasstained with Hoechst 33342. Figure 20 depicts immunofluorescent staining of embedded liver sections fromMS- NASH mice (Cohort #3) control (Empty LNPs) (20X magnification). HSP70 wasstained with anti-human HSP70 monoclonal rabbit antibody (primary) and anti-rabbitsecondary antibody conjugated to Alexa Fluor™ 568. Nuclei was stained with Hoechst33342. Figure 21 depicts immunofluorescent staining of embedded liver sections from MS-NASH mice (Cohort #3) dosed with HSP70-LNPs (20X magnification), confirming HSP70overexpression. HSP70 was stained with anti-human HSP70 monoclonal rabbit antibody(primary) and anti-rabbit secondary antibody conjugated to Alexa Fluor™ 568. Nuclei wasstained with Hoechst 33342.7504-00202 GRH-00462Figure 22 depicts Dynamic Light Scattering (DLS) particle size distributionanalysis of LNP solutions. At least three measurements were taken of a sample, asillustrated in the overlay of the Z-Average (measure of the average size of a particle sizedistribution) of sample LNP_HSP70_6-27-24. Figure 23 depicts Dynamic Light Scattering (DLS) particle size distribution analysisof LNP solutions. This figure shows a single measurement run of the sample LNP_HSP70_6-27-24. Figure 24 depicts a full liver image of an HSP70-LNP treated MS-NASH Liverfrom Experimental mouse #2 Cohort #1. Figure 25 depicts a full liver image of an HSP70-LNP treated MS-NASH Liverfrom Experimental mouse #1 Cohort #2. Figure 26 depicts a full liver image of an HSP70-LNP treated MS-NASH Liverfrom Experimental mouse #2 Cohort #2. Figure 27 depicts a full liver image of an empty LNP treated MS-NASH Liver fromControl mouse #1 Cohort #1. Figure 28 depicts a full liver image of an empty LNP treated MS-NASH Liver fromControl mouse #4 Cohort #2. Figure 29 depicts a full liver image of an empty LNP treated MS-NASH Liver fromControl mouse #5 Cohort #2.DETAILED DESCRIPTIONGeneral HSPs may be classified into families on the basis of molecular weight. For example,HSPlO0, HSP90, HSP70, HSP60, HSP40, and H5P27, each playing a diverse role ininfluencing proper protein assembly, folding, and translocation. The heat shock proteinsHSP70, HSP60, and H5P27 are known to prevent protein aggregation and help proteinfolding; HSP 100 releases proteins from aggregates; and HSP90 plays a role in maturationand activation of a number of proteins. Recent studies have shown that HSPs may besecreted proteins with pro- and / or anti- inflammatory actions that could be relevant to humandiseases. HSPs have also demonstrated the ability to bind peptides and present them to Tcells to modulate immune responses, which may have implications in a number of diseasesettings. HSPs may also be present in the extracellular and cell-associated compartments,having the capacity to 'escape' from cells and interact with different cell types to elicit arange of biological effects, potentially acting as receptors for inflammatory mediators as anumber of HSPs have been found in the bodily fluids of humans and animals.7504-00202 GRH-00462The 70 kilodalton heat shock proteins (Hsp70s or DnaK) HSP70 are a family ofadenosine triphosphates that represent the most structurally and functionally conservedproteins amongst HSPs. HSP70 is also the most ubiquitous class of chaperone protein,including cytoprotective effects under a number of different conditions, primarily in cellularprotein quality control (PQC) and degradation systems. Briefly, in humans the HSP70multigene family acts on nonnative polypeptides, fueled by ATP binding and hydrolysis. TheHSP70 chaperone binds to protein substrates (e.g., nascent or misfolded protein) to assistwith folding, re-folding, reactivation, degradation, transport, regulation, and aggregationprevention. HSP70 consists of two highly conserved domain structures; a 45 kDa N-terminalnucleotide binding domain (NBD) and a 25 kDa C-terminal substrate binding domain (SBD).These domains undergo reciprocal allosteric interactions induced by ligand binding. TheNBD comprises two lobes, forming a cleft that binds ATP with a nucleotide binding cassettethat is related to those in actin and hexokinase. The SBD comprises a B-sandwich domainharboring the substrate binding site, and an a-helical lid. Both these domains are critical forchaperone function and are connected by a short flexible linker. As a molecular chaperone,HSP70 also has multiple responsibilities during normal growth. It is integral to the folding ofnewly synthesized proteins, the subcellular transport of proteins and vesicles, the formationand dissociation of complexes, and degradation of unwanted proteins. In carrying out thesediverse functions HSP70 adopts different conformations, e.g., in the absence of nucleotide,when bound with ADP, or when bound with ATP. In addition, the functions of HSP70 relyon crosstalk between the SBD and NBD, with ATP influencing substrate binding. The cycleof rapid, controlled, binding and release of substrate promotes unfolding / folding andassembly with partner proteins while preventing aggregation of the substrate proteins. Provided herein are nucleic acids (e.g., mRNAs) encoding heat shock proteins. Thecomposition and methods of the present disclosure rely, at least in part, on the delivery ofheat shock protein-encoding nucleic acids (e.g., HSP70-encoding nucleic acids) to cells of asubject in need thereof (e.g., a gene therapy composition). For example, and withoutlimitation, compositions comprising the heat shock protein-encoding nucleic acids disclosedherein can be used to treat inflammatory conditions. In some embodiments, said compositionmay be used to treat inflammatory disease or dysfunction, and / or infection. Indeed, HSPs,such as HSP70, affect several pro- and anti-inflammatory pathways, including but not limitedto, caspase-dependent pathways, toll-like receptor 2 inflammatory pathways, P13K, p38MAPK, and IL-6 / JAK / STAT3. In particular, HSP70 has been shown to have an inhibitory7504-00202 GRH-00462effect on nuclear factor kappa-light- chain-enhancer of activated B cells (NF-KB), a family oftranscription factor protein complexes that controls transcription of DNA, cytokineproduction and cell survival.Inflammation is a common effect of thousands of diseases affecting billions of peopleevery year. Without being bound by theory, a set of intracellular pathways that signal thepresence of cellular stress, collectively known as the unfolded-protein response (UPR)pathways, can initiate inflammatory signal cascades in response to imbalances in theendoplasmic reticulum (ER), an organelle responsible for the biosynthesis, folding, assemblyand modification of protein in the cell. ER stress, through the UPR, induces an inflammatoryprofile comprising NF- KB signaling and inflammasome activation, while at the same time,the UPR enlists the HSR to monitor protein chaperoning and NF-KB signals. Inflammation,particularly chronic inflammation, may overwhelm the HSR leading to its gradual suppression. As a non-limiting example, to display HSP70s ability to treat inflammation, NASHand NAFLD are used as proof-of-concept. Non-Alcoholic Fatty Liver Disease (NAFLD)affects about 25% of all adults in the United States and causes high amounts of fat in theliver. Approximately 20% of Americans with NAFLD have the more severe Non-AlcoholicSteatohepatitis (NASH), which causes high amounts of fat, inflammation, and liver cell damage.Several lifestyle changes (diet change, increased exercise, decreased alcohol consumption,etc.) are often implemented to reduce fat buildup in the liver. Vitamin E and pioglitazone,treatments for other conditions, have been seen to have some effect for NAFLD and NASHwhen implemented with several lifestyle changes. Yet, despite being a growing global healthproblem, to date, no drug has been approved by regulatory agency specifically to treat NAFLD,while only just recently the drug resmetirom, a thyroid hormone receptor beta-selective agonist,has been approved for treatment of NASH. Clearly, there is an unmet need for safe, tolerable,and efficacious treatment of inflammatory diseases such as NASH and NAFLD.Table 1HSP70 Sequence FeaturesMAKAAAIGIDLGTTYSCVGVFQHGKVEIIANDQGNRTTPSYVAFTDTERLIGDAAKNQVALNPQNTVFDAKRL Amino Acid Sequence:IGRKFGDPVVQSDMKHWPFQVINDGDKPKVQVSYKGETKAFYPEEISSMVLTKMKEIAEAYLGYPVTNAVITV• NBD: aa2-PAYFNDSQRQATKDAGVIAGLNVLRIINEPTAAAIAYGLDRTGKGERNVLIFDLGGGTFDVSILTIDDGIFEV aa386 KATAGDTHLGGEDFDNRLVNHFVEEFKRKHKKDISQNKRAVRRLRTACERAKRTLSSSTQASLEIDSLFEGIDLinker: aa387-FYTSITRARFEELCSDLFRSTLEPVEKALRDAKLDKAQIHDLVLVGGSTRUOKVQKLLQDFFNGRDLNKSINP• aa393DEAVAYGAAVQAAILMGDKSENVQDLLLLDVAPLSLGLETAGGVMTALIKRNSTIPTKOTOIFTTYSDNOPGV GERAMTKDNNLLGRFELSGIPPAPRGVPOIEVTFDIDANGILNVTATDKSTGKANKITITNDKGRLS• SBD: aa394-LIOVYEaa509KEEIERMVQEAEKYKAEDEVQRERVSAKNALESYAFNMKSAVEDEGLKGKISEADKKKVLDKCQEVISWLDAN TLAEKDEFEHRKELEQVCNPIISGLYQGAGGPGPGGFGAQGPKGGSGPTIEEVD(SEQ ID NO. 1)7504-00202 GRH-004621 aacggctagc ctgaggagct gctgcgacag tccactacct ttttcgagag tgtctcccgtnucleotide sequence61 tgtcccaagg cttcccagag cgaacctgtg cggctgcagg caccggcgcg tcgagtttcc (corresponding to 121 ggcgtccgga aggaccgagc tcttctcgcg gatccagtgt tccgtttcca gcccccaatc Accession number 181 tcagagcgga gccgacagag agcagggaac cggcatggcc aaagccgcgg cgatcggcat NM_005345.6,, 241cgacctgggc accacctact cctgcgtggg ggtgttccaa cacggcaagg tggagatcat incorporated herein by301cgccaacgac cagggcaacc gcaccacccc cagctacgtg gccttcacgg acaccgagcgreference): 361gctcatcggg gatgcggcca agaaccaggt ggcgctgaac ccgcagaaca ccgtgtttga421cgcgaagcgg ctgattggcc gcaagttcgg cgacccggtg gtgcagtcgg acatgaagca • NBD: nt218-nt1372481ctggcctttc caggtgatca acgacggaga caagcccaag gtgcaggtga gctacaaggg541gacca agatgaagga • Linker: ntl373-ggagaccaag gcarrcracc ccgaggagat ctcgtccatg gtgct ntl393601gatcgccgag gcgtacctgg gctacccggt gaccaacgcg gtgatcgcgg ggctcaacgtcttcaacgac tcgcagcgcc aggccaccaa ggatgcgg • SBD: nt1394-661gt gtgatcgcgg ggctcaacgt 1741721gctgcggatc atcaacgagc ccacggccgc cgccatcgcc tacggcctgg acagaacggg781caagggggag cgcaacgtgc tcatctttga cctgggcggg ggcaccttcg acgtgtccat841cctgacgatc gacgacggca tcttcgaggt gaaggccacg gccggggaca cccacctggg901tggggaggac tttgacaaca ggctggtgaa ccacttcgtg gaggagttca agagaaaaca961caagaaggac atcagccaga acaagcgagc cgtgaggcgg ctgcgcaccg cctgcgagag1021ggccaagagg accctgtcgt ccagcaccca ggccagcctg gagatcgact ccctgtttga1081gggcatcgac ttctacacgt ccatcaccag ggcgaggttc gaggagctgt gctccgacct1141gttccgaagc accctggagc ccgtggagaa ggctctgcgc gacgccaagc tggacaaggc1201ccagattcac gacctggtcc tggtcggggg ctccacccgc atccccaagg tgcagaagct1261gctgcaggac ttcttcaacg ggcgcgacct gaacaagagc atcaaccccg acgsggctgt1321ggcctacggg gcggcggtgc aggcggccat cctfatgggg gacaagrccg agaacgtgca1381ggacctgctg ctgctggacg tggctcccct gtcgctgggg ctggagacgg ccggaggcgt1441gatgactgcc ctgstcssgc gcaactccac catccccacc aagcagacgc agatcttcac1501 cacctactcc gacaaccaac ccggggtgct gatccaggtg tacgagggcg agagggccct1561 gacgaaagac aacaatctgt tggggcgctt cgagctgagc ggcatccctc cggcccccag1621 gggcgtgccc cagatcgagg tgaccttcga catcgatgcc aacggcatcc tgaacgtcac1681 ggccacggac aagagcaccg gcaaggccaa caagatcacc atcaccaacg acaagggccg1741 cctgagcaag gaggagatcg agcgcatggt gcaggaggcg gagaagtaca aagcggagga1801 cgaggtgcag cgcgagaggg tgtcagccaa gaacgccctg gagtcctacg ccttcaacat1861 gaagagcgcc gtggaggatg aggggctcaa gggcaagatg agcgaggcgg acaagaagaa

[0002] 7504-00202 GRH-004621921 ggtgctggac aagtgtcaag aggtcatctc gtggctggac gccaacacct tggccgagaa1981 ggacgagttt gagcacaaga ggaaggagct ggagcaggtg tgtaacccca tcatcagcgg2041 actgtaccag ggtgccggtg gtcccgggcc tgggggcttc ggggctcagg gtcccaaggg2101 agggtctggg tcaggcccca ccattgagga ggtagattag gggcctttcc aagattgctg2161 tttttgtttt ggagcttcaa gactttgcat ttcctagtat ttctgtttgt cagttctcaa2221 tttcctgtgt ttgcaatgtt gaaatttttt ggtgaagtac tgaacttgct ttttttccgg2281 tttctacatg cagagatgaa tttatactgc catcttacga ctatttcttc tttttaatac2341 acttaactca ggccattttt taagttggtt acttcaaagt aaataaactt taaaattcaa(SEQ ID NO. 2) Aspects of the invention, as provided herein, include therapeutic compositionscomprising an mRNA formulated in a lipid nanoparticle (LNP) (e.g., a solid lipid nanoparticle(SLN)), wherein the mRNA comprises an open reading frame encoding heat shock proteinpolypeptide, or a functional fragment thereof. In some embodiments, the heat shock proteinpolypeptide, or functional fragment thereof is selected from HSPlO0, HSP90, HSP70, HSP60,HSP40, or HSP27. Preferably the heat shock protein polypeptide, or functional fragment thereof isHSP70. In some embodiments, the open reading frame is derived from the nucleic acid sequence setforth in SEQ ID NO. 2, or a functional fragment thereof. For example, and without limitation, themRNA comprises the nucleic acid sequence set forth in any one of SEQ ID NO. 6, SEQ ID NO.10, SEQ ID NO. 14, SEQ ID NO. 18, or any functional fragment thereof.In some aspects, provided herein are nucleic acids encoding the HSP70 polypeptides disclosed herein. In certain aspects, provided herein are primers for isolating and / or amplifying anucleic acid sequence encoding a heat shock protein as disclosed herein, using methods known inthe art (e.g., T7 RNA Polymerase techniques). In some such embodiments, the primers are selectedfrom the primer sequences set forth in SEQ ID NOs. 3, 4, 5, 7, 8, 9, 11, 12, 13, 15, 16, and 17.In some embodiments, the nucleic acid is isolated and / or amplified using any one of the forward andreverse primer pairs set forth in: SEQ ID NOs. 3 and 5, SEQ ID NOs. 4 and 5, SEQ ID NOs. 7 and9, SEQ ID NOs. 8 and 9, SEQ ID NOs. 11 and 13, SEQ ID NOs. 12 and 13, SEQ ID NOs.15 and17, and SEQ ID NOs. 16 and 17. One or more of the uridine nucleosides in the amplified mRNAis a pseudouridine, such as, Nl-pseudouridine. In some such embodiments, all of the uridine7504-00202 GRH-00462nucleosides in the mRNA are pseudouridine, e.g., Nl-methylpseudouridine. For example, andwithout being bound by theory or methodology, the aforementioned nucleic acids are amplifiedfrom whole cell lysate. In some embodiments, a DNA template encoding the UTR regions andcoding DNA sequence (CDS) of HSP70 (e.g., cDNA) is used as a template for polymerase chainreaction (PCR) DNA amplification / replication with specifically designed primers. In somepreferred embodiments, the DNA template is a plasmid. The DNA template can then be used as atemplate for mRNA synthesis by methods such as those well known in the art. Alternatively, themRNA may be transcribed from DNA synthesized using RT-PCR and isolated RNA from a celllysate, e.g., by any suitable methods and techniques known in the art.In other aspects, provided herein are vectors comprising the nucleic acidscontemplated herein. In some such embodiment, the vector is selected from nanoparticles,adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, picomavirus vectors, liposomes, cationic lipid systems, and protein / nucleic acid complexes. In other aspects, provided here are cells comprising the LNPs and / or nucleic acidsdisclosed herein. Preferred aspects of the invention include cells comprising the LNPs (e.g.,the SLNs) disclosed herein. In certain aspects, provided here are cells comprising the vectorsdisclosed herein. In further aspects, provided herein are cells expressing the heat shockprotein (e.g., HSP70 polypeptides) disclosed herein. For example and without limitation, thecell is an endothelial cell, epithelial cell, neuronal cell, or hematopoietic cell. In some suchembodiments, the hematopoietic cell is an immune cell selected from a lymphocyte, amonocyte, a macrophage, a dendritic cell, a mast cell, a neutrophil, a basophil, or aneosinophil. In certain embodiments, the immune cell is a lymphocyte selected from a ana T cell, y8T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, a B cell, aninnate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte(CTL), a lymphokine activated killer (LAK) cell, or a regulatory T cell.In certain embodiments, the cell contemplated herein are cells of the central nervoussystem (CNS) or peripheral nervous system (PNS). In other embodiments, the cell is a cell ofthe bone marrow. In some embodiments, the cell contemplated herein is a cell present in the CNS. Insome embodiments, the cell is a neuronal cell. Said neuronal cell may be a sensory neuron, amotor neuron, or an interneuron. In other embodiments, the cell is a non-neuronal cell. Insome embodiments, the non-neuronal cell is a glial cell. The glial cell may be an astrocytecell, an oligodendrocyte, an ependymal cell, a radial glial cell, a Schwann cell, a satellite cell,an enteric glial cell, or a microglial cell.7504-00202 GRH-00462In some embodiments, the nucleic acids contemplated herein may refer to apolymeric form of nucleotides or nucleosides of any length, such as deoxyribonucleotides orribonucleotides, or analogs thereof. Nucleic acids may have any three-dimensional structure,and may perform any function. The following are non-limiting examples of nucleic acids:coding or non-coding regions of a gene or gene fragment, loci (locus) defined from linkageanalysis, exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA,ribozymes, cDNA, recombinant polynucleotides / polynucleosides, branched polynucleotides / polynucleosides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes, and primers. A nucleic acid may comprisemodified nucleotides / nucleosides, such as methylated nucleotides / nucleosides andnucleotide / nucleoside analogs. If present, modifications to the polynucleotide / polynucleosidestructure may be imparted before or after assembly of the polymer. Apolynucleotide / polynucleoside may be further modified, such as by conjugation with alabeling component. Aspects include therapeutic compositions comprising the mRNAs disclosed herein.In some embodiments, the therapeutic composition comprises a vector selected fromnanoparticles, adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors,picornavirus vectors, liposomes, cationic lipid systems, and protein / nucleic acid complexes.For example and without limitation, the therapeutic composition comprising an mRNA maybe formulated in a lipid nanoparticle (LNP). In some embodiments of the therapeuticcomposition, one or more of the uridine nucleosides in the mRNA are pseudouridine, such asNI-methyl pseudouridine. In some embodiments, all of the uridine nucleosides in the mRNAare pseudouridine, e.g., Nl- methylpseudouridine. In some embodiments of the therapeuticcomposition, the LNP (e.g., the SLN) comprises an ionizable lipid, a structural lipid, aphospholipid, a sterol, a PEG-modified lipid, or any combination thereof.In some aspects, provided herein are methods of treating a cancer in a subject, themethod comprising administering an effective amount of a therapeutic compositioncontemplated herein. In some embodiments, the cancer is selected from: hepatocellularcarcinoma, lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin'sDisease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head andneck cancer, squamous cell carcinomas of the mouth, throat, larynx, and lung, endometrialcancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer,genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma,large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers,7504-00202 GRH-00462prostatic cancer, and pancreatic cancer. In some such embodiments, the therapeutic composition is administered intrapleurally,intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intraperitoneally,intracranially, or by direct administration to an organ. In certain embodiments, the method further comprises administering to the subject animmunotherapy. The immunotherapy may comprise administration of a therapeuticantibody, such as aducanumab. The immunotherapy may comprise administration of animmune checkpoint inhibitor. In some such embodiments, the immune checkpoint inhibitorcomprises an antibody or antigen-binding fragment thereof specific for PD-1, PD-Ll, orCTLA4. In certain embodiments, cancer immunotherapy comprises administration of aCAR-T cell or a CAR-NK cell.In other embodiments, the method further comprises administering to the subject acholinesterase inhibitor, such as, donepezil, rivastigmine, or galantamine. In yet furtherembodiments, the method further comprises administering to the subject a glutamateregulator, such as memantine. Definitions For convenience, certain terms employed in the specification, examples, and appendedclaims are collected here. The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to atleast one) of the grammatical object of the article. By way of example, "an element" meansone element or more than one element. The term "about" means within an acceptable error range for the particular value asdetermined by one of ordinary skill in the art, which will depend in part on how the value ismeasured or determined, i.e., the limitations of the measurement system. Where the terms"about" or "approximately" are used in the context of compositions containing amounts ofingredients or conditions such as temperature, these values include the stated value with avariation of 0-10%R around the value (X ± 10%).Ranges are stated in the shorthand to avoid having to set out at length and describeeach and every value within the range. Therefore, when ranges are stated for a value, anyappropriate value of the range. For example, a range of 0.1-1.0 represents the terminalvalues of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8,0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc.7504-00202 GRH-00462"Pharmaceutically acceptable carrier" or "pharmaceutically acceptable excipient"includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents,isotonic and absorption delaying agents and the like. The use of such media and agents forpharmaceutically active substances is well known in the art. Except insofar as anyconventional media or agent is incompatible with angiotensin II, its use in the pharmaceuticalformulations of the invention is contemplated. In certain embodiments, the pharmaceuticallyacceptable carrier / excipient is a saline solution. As used herein, the term "administering" means providing a pharmaceutical; agent orcomposition to a subject, and includes, but is not limited to, administering by a medicalprofessional and self-administering. Such an agent can contain, for example, peptide ornucleic acid described herein. As used herein, the term "treatment" refers to clinical intervention designed to alterthe natural course of the individual being treated during the course of clinical pathology.Desirable effects of treatment include decreasing the rate of progression, ameliorating orpalliating the pathological state, and remission or improved prognosis of a particular disease,disorder, or condition. An individual is successfully "treated," for example, if one or moresymptoms associated with a particular disease, disorder, or condition are mitigated oreliminated. As used herein, a therapeutic that "prevents" a condition refers to a compound that,when administered to a statistical sample prior to the onset of the disorder or condition,reduces the occurrence of the disorder or condition in the treated sample relative to anuntreated control sample, or delays the onset or reduces the severity of one or moresymptoms of the disorder or condition relative to the untreated control sample.In certain embodiments, agents of the invention may be used alone or conjointlyadministered with another type of therapeutic agent. As used herein, the phrase "conjointadministration" or "administered conjointly" refers to any form of administration of two ormore different therapeutic agents such that the second agent is administered while thepreviously administered therapeutic agent is still effective in the body (e.g., the two agentsare simultaneously effective in the subject, which may include synergistic effects of the twoagents). For example, the different therapeutic compositions disclosed herein can beadministered either in the same formulation or in separate formulations, either concomitantlyor sequentially. In certain embodiments, the different therapeutic agents (e.g., a therapeuticcomposition comprising an mRNA disclosed herein and an immunotherapy or standard-of-care treatment (e.g., standard- of-care treatment for a neurodegenerative disease, such as7504-00202 GRH-00462Alzheimer's disease)) can be administered within about one hour, about 12 hours, about 24hours, about 36 hours, about 48 hours, about 72 hours, or about a week of one another. Thus,a subject who receives such treatment can benefit from a combined effect of differenttherapeutic agents. The terms "polypeptide fragment" or "fragment", when used in reference to a particularpolypeptide, refers to a polypeptide in which amino acid residues are deleted as compared tothe reference polypeptide itself, but where the remaining amino acid sequence is usuallyidentical to that of the reference polypeptide. Such deletions may occur at the amino-terminus or carboxy- terminus of the reference polypeptide, or alternatively both. Fragmentstypically are at least about 5, 6, 8 or 10 amino acids long, at least about 14 amino acids long,at least about 20, 30, 40 or 50 amino acids long, at least about 75 amino acids long, or at leastabout 100, 150, 200, 300, 500 or more amino acids long. A fragment can retain one or moreof the biological activities of the reference polypeptide. In various embodiments, a fragmentmay comprise an enzymatic activity and / or an interaction site of the reference polypeptide. Insome embodiments, a fragment may have suppressive, disruptive, or enhancing properties.Nucleic acids and vectorsNucleic acids and vectors disclosed herein include polynucleotides and polynucleotidevectors encoding the disclosed heat shock proteins (e.g., HSP70 polypeptides) that allowexpression in the disclosed cells. Nucleic acid sequences contemplated herein can be obtained using recombinantmethods known in the art. Alternatively, the sequence of interest can be producedsynthetically, rather than cloned.In addition to the polypeptide-encoding sequences, other structural properties asdescribed herein for mRNA constructs (e.g., modified nucleobases, 5' cap, 5' UTR, 3' UTR,miR binding site(s), polyA tail, as described herein). Suitable mRNA construct componentsarc as described herein. In some embodiments, a nucleic acid of the disclosure may bemodified in a coding region (e.g., an open reading frame of an mRNA encoding apolypeptide). In other embodiments, nucleic acid may be modified in regions besides acoding region, such as, 5' cap, a 5'-untranslated region (UTR) and / or a 3'- UTR, polyA tail ofan mRNA, wherein any combination of elements may be independently modified. In someembodiments, such regions may contain one or more different nucleoside modifications. Insuch embodiments, modifications may also be present in the coding region.Examples of nucleoside modifications and combinations thereof that may be present7504-00202 GRH-00462in mRNAs disclosed herein include, but are not limited to, those described in PCT PatentApplication Publications: WO2012045075, WO2014081507, WO2014093924, WO2014164253, WO2014159813, WO2018144775, WO2018081459, each of which areincorporated herein in their entirety.In some embodiments, the mRNAs of the disclosure can include a combination ofmodifications to the sugar, the nucleobase, and / or the intemucleoside linkage. Thesecombinations can include any one or more modifications described herein. As a non-limitingexample, the natural nucleotide uridine may be substituted with a modified nucleosidedescribed herein. In another non-limiting example, the natural nucleoside uridine may bepartially substituted (e.g., about 0.1 %, 1 %, 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40%, 45 %, 50%,55 %, 60 %,65 %,70 %,75 %, 80%, 85%, 90 %,95 % or 99.9 % of the naturaluridines) with at least one of the modified nucleosides disclosed herein, e.g., pseudouridine.Expression of nucleic acids encoding heat shock proteins (e.g., HSP70) is typicallyachieved by operably linking a nucleic acid encoding the HSP70 polypeptide to a promoterand incorporating the construct into an expression vector. Typical cloning vectors containtranscription and translation terminators, initiation sequences, and promoters useful forregulation of the expression of the desired nucleic acid sequence.The disclosed nucleic acids can be cloned into a number of types of vectors. Forexample, the nucleic acid can be cloned into a vector including, but not limited to a plasmid, aphagemid, a phage derivative, an animal virus, and a cosmid. Vectors of particular interestinclude expression vectors, replication vectors, probe generation vectors, and sequencingvectors. Further, the expression vector may be provided to a cell in the form of a viral vector.Viral vector technology is well known in the art and is described, for example, in Sambrook etal. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, NewYork), and in other virology and molecular biology manuals. Viruses, which are useful asvectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses,herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replicationfunctional in at least one organism, a promoter sequence, convenient restriction endonucleasesites, and one or more selectable markers. In some embodiments, the polynucleotide vectorsare lentiviral or retroviral vectors.A number of viral based systems have been developed for gene transfer intomammalian cells. For example, retroviruses and AAVs provide a convenient platform forgene delivery systems. A selected gene can be inserted into a vector and packaged in viral7504-00202 GRH-00462particles using techniques known in the art. The recombinant virus can then be isolated anddelivered to cells of the subject either in vivo or ex vivo.One example of a suitable promoter is the immediate early cytomegalovirus (CMV)promoter sequence. This promoter sequence is a strong constitutive promoter sequencecapable of driving high levels of expression of any polynucleotide sequence operativelylinked thereto. Another example of a suitable promoter is Elongation Growth Factor-la(EF-la). However, other constitutive promoter sequences may also be used, including, but notlimited to the simian virus 40 (SV4O) early promoter, MND (myeloproliferative sarcomavirus) promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus(HIV) long terminal repeat (LTR) promoter, MoMuLV promoter, an avian leukemia viruspromoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter,as well as human gene promoters such as, but not limited to, the actin promoter, the myosinpromoter, the hemoglobin promoter, and the creatine kinase promoter. The promoter canalternatively be an inducible promoter. Examples of inducible promoters include, but arenot limited to a metallothionine promoter, a glucocorticoid promoter, a progesteronepromoter, and a tetracycline promoter. Additional promoter elements, e.g., enhancers, regulate the frequency oftranscriptional initiation. Typically, these are located in the region 30-110 base pairs (bp)upstream of the start site, although a number of promoters have recently been shown tocontain functional elements downstream of the start site as well. The spacing betweenpromoter elements frequently is flexible, so that promoter function is preserved whenelements are inverted or moved relative to one another.In order to assess the expression of a heat shock protein disclosed herein or portionsthereof, the expression vector to be introduced into a cell can also contain either a selectablemarker gene or a reporter gene or both to facilitate identification and selection of expressionexpressing cells from the population of cells sought to be transfected or infected through viralvectors. The selectable marker may be carried on a separate piece of DNA and used in a co-transfection procedure. Both selectable markers and reporter genes may be flanked withappropriate regulatory sequences to enable expression in the host cells. Useful selectablemarkers include, for example, antibiotic-resistance genes. Reporter genes may be used for identifying potentially transfected cells and forevaluating the functionality of regulatory sequences. In general, a reporter gene is a gene thatis not present in or expressed by the recipient organism or tissue and that encodes apolypeptide whose expression is manifested by some easily detectable property, e.g.,7504-00202 GRH-00462enzymatic activity. Expression of the reporter gene is assayed at a suitable time after thenucleic acid has been introduced into the recipient cells. Suitable reporter genes may includegenes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secretedalkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems arewell known and may be prepared using known techniques or obtained commercially. Ingeneral, the construct with the minimal 5' flanking region showing the highest level ofexpression of reporter gene is identified as the promoter. Such promoter regions may belinked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription. Methods of introducing and expressing genes into a cell are known in the art. In thecontext of an expression vector, the vector can be readily introduced into a host cell, e.g.,mammalian, bacterial, yeast, or insect cell by any method in the art. For example, theexpression vector can be transferred into a host cell by physical, chemical, or biologicalmeans. Physical methods for introducing a polynucleotide into a host cell include calciumphosphate precipitation, lipofection, particle bombardment, microinjection, electroporation,and the like. Methods for producing cells comprising vectors and / or exogenous nucleicacids are well-known in the art. See, for example, Sambrook et al. (2001, MolecularCloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Biological methods for introducing a polynucleotide of interest into a host cellinclude the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors,have become the most widely used method for inserting genes into mammalian, e.g., humancells. Chemical means for introducing a polynucleotide into a host cell include colloidaldispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads,and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, andliposomes. An exemplary colloidal system for use as a delivery vehicle in vitro and in vivois a liposome (e.g., an artificial membrane vesicle).In the case where a non-viral delivery system is utilized, an exemplary deliveryvehicle is a liposome. In another aspect, the nucleic acid may be associated with a lipid. Thenucleic acid associated with a lipid may be encapsulated in the aqueous interior of aliposome, interspersed within the lipid bilayer of a liposome, attached to a liposome via alinking molecule that is associated with both the liposome and the oligonucleotide, entrappedin a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed7504-00202 GRH-00462with a lipid, combined with a lipid, contained as a suspension in a lipid, contained orcomplexed with a micelle, or otherwise associated with a lipid. Lipid, lipid / nucleic acid orlipid / expression vector associated compositions are not limited to any particular structure insolution. For example, they may be present in a bilayer structure, as micelles, or with a"collapsed" structure. They may also simply be interspersed in a solution, possibly formingaggregates that are not uniform in size or shape. Lipids are fatty substances, which may benaturally occurring or synthetic lipids. For example, lipids include the fatty droplets thatnaturally occur in the cytoplasm as well as the class of compounds, which contain long-chainaliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, aminoalcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources.For example, dimyristyl phosphatidylcholine ("DMPC") can be obtained from Sigma, St.Louis, Mo.; dicetyl phosphate ("DCP") can be obtained from K & K Laboratories(Plainview, N.Y.); cholesterol ("Choi") can be obtained from Calbiochem-Behring;dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from AvantiPolar Lipids, me, (Birmingham, Ala.). In some embodiments the nucleic acids of the disclosure may be formulated innanoparticles (e.g., lipid nanoparticles) or other delivery vehicles, e.g., to protect them fromdegradation when delivered to a subject. Illustrative nanoparticles are described in Panyam,J. & Labhasetwar, V. Adv. Drug Deliv. Rev. 55, 329-347 (2003) and Peer, D. et al. NatureNanotech. 2,751-760 (2007), W02018144775, and W02018081459, each of which areincorporated herein by reference in their entirety. In certain embodiments, an mRNA of thedisclosure is encapsulated within a nanoparticle. In particular embodiments, a nanoparticle isa particle having at least one dimension (e.g., a diameter) less than or equal to 1000nanometers (nm), less than or equal to 500 nm or less than or equal to 100 nm. Jr particularembodiments, a nanoparticle includes lipids. Lipid nanoparticles (LNPs) include, but are notlimited to, solid lipid nanoparticles (SLNs), liposomes, and micelles. For example, andwithout limitation, the nucleic acids described herein (e.g., mRNAs) are formulated as a solidlipid nanoparticle (SLN), which can be spherical with an average diameter between 10 to1000 nm. In some such embodiments, the SLN possesses a solid lipid core matrix that cansolubilize lipophilic molecules and can be stabilized with surfactants and / or emulsifiers.Exemplary SLN can be those as described in Inti. Pub. No. WO2013105101, hereinincorporated by reference in its entirety. Any of a number of lipids may be present, including cationic and / or ionizable lipids,anionic lipids, neutral lipids, amphipathic lipids, PEGylated lipids, and / or structural lipids.7504-00202 GRH-00462Such lipids can be used alone or in combination. In certain embodiments, a lipid nanoparticlecomprises one or more nucleic acids, e.g., mRNAs, described herein. In certainembodiments, it is desirable to target a nanoparticle, e.g., a lipid nanoparticle, of thedisclosure using a targeting moiety that is specific to a cell type and / or tissue type. In someembodiments, a nanoparticle may be targeted to a particular cell, tissue, and / or organ using atargeting moiety. In particular embodiments, a nanoparticle comprises one or more mRNAdescribed herein and a targeting moiety. Exemplary non-limiting targeting moieties includeligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies (e.g.,full-length antibodies, antibody fragments (e.g., Fv fragments, single chain Fv (scFv)fragments, Fab' fragments, or F(ab')2 fragments), single domain antibodies, camelidantibodies and fragments thereof, human antibodies and fragments thereof, monoclonalantibodies, and multispecific antibodies (e.g.,bispecific antibodies)). In some embodiments,the targeting moiety may be a polypeptide. The targeting moiety may include the entirepolypeptide (e.g., peptide or protein) or fragments thereof. A targeting moiety is typicallypositioned on the outer surface of the nanoparticle in such a manner that the targeting moietyis available for interaction with the target, for example, a cell surface receptor. A variety ofdifferent targeting moieties and methods are known and available in the art, including thosedescribed, e.g., in Sapra et al., Prog. Lipid Res. 42(5):439-62, 2003 and Abra et al., J.Liposome Res. 12:1-3, 2002. For example, the lipid nanoparticle may include a targetingmoiety that targets the lipid nanoparticle to a cell including, but not limited to, hepatocytes,colon cells, epithelial cells, hematopoietic cells, epithelial cells, endothelial cells, lung cells,bone cells, stem cells, mesenchymal cells, neural cells, cardiac cells, adipocytes, vascularsmooth muscle cells, cardiomyocytes, skeletal muscle cells, beta cells, pituitary cells,synovial lining cells, ovarian cells, testicular cells, fibroblasts, B cells, T cells, reticulocytes, leukocytes, granulocytes, and tumor cells (including primary tumor cells and metastatictumor cells). In particular embodiments, the targeting moiety targets the lipid nanoparticle toa hepatocyte. In other embodiments, the targeting moiety targets the lipid nanoparticle to acolon cell. In some embodiments, the targeting moiety targets the lipid nanoparticle to a livercancer cell (e.g., a hepatocellular carcinoma cell) or a colorectal cancer cell (e.g., a primarytumor or a metastasis). In addition to nanoparticle compositions provided herein, also disclosed are methodsof producing lipid nanoparticles, which may include encapsulating a polynucleotide (e.g., anmRNA contemplated herein). Such contemplated methods comprise using any of thecompositions disclosed herein and producing lipid nanoparticles in accordance with methods7504-00202 GRH-00462of production of lipid nanoparticles known in the art, e.g., Wang et al. (2015) "Delivery ofoligonucleotides with lipid nanoparticles" Adv. Drug Deliv. Rev. 87:68-80; Silva et al.(2015) "Delivery Systems for Biopharmaceuticals. Part I: Nanoparticles and Microparticles"Curr. Pharm. Biotechnol. 16: 940-954; Naseri et al. (2015) "Solid Lipid Nanoparticles andNanostructured Lipid Carriers: Structure, Preparation and Application" Adv. Pharm. Bull.5:305- 13; Silva et al. (2015) "Lipid nanoparticles for the delivery of biopharmaceuticals"Curr. Pharm. Biotechnol. 16:291-302, and references cited therein, all of which areincorporated herein by reference in their entirety. In certain embodiments, lipid nanoparticles(LNP5) comprise lipids including an ionizable lipid, a structural lipid, a phospholipid, astabilizing lipid, and one or more mRNAs. For example, without being bound by theory ormethodology, a solid lipid nanoparticle (SLN) may include one or more mRNAs. Thus, eachof the LNPs described herein may be used in a formulation comprising the mRNA describedherein. In one embodiment, a lipid nanoparticle comprises an ionizable lipid, a structurallipid, a phospholipid, a PEG-modified lipid, a sterol and a phospholipid. In someembodiments, the LNP has a molar ratio about 20- 60% ionizable lipid:about 5-25%phospholipid:about 25-55% sterol: and about 0.5-15% PEG- modified lipid, about 38.5%cholesterol and about 10% phospholipid. In some embodiments, the LNP comprises a molarratio of about 55% ionizable lipid, about 2.5% PEG lipid, about 32.5% cholesterol and about10% phospholipid, In some embodiments, the ionizable lipid is an ionizable amino orcationic lipid and the neutral lipid is a phospholipid, and the sterol is a cholesterol.The ionizable lipids contemplated herein include cationic and / or ionizable lipids.Such cationic and / or ionizable lipids include, but are not limited to, SM-102, 9-Heptadecanyl 8-{(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, 3-(didodecylamino)-Nl,N4,N4- tridodecyk-1-piperazineethanamine (KLlO), Nl-[2-(didodecylamino)ethyl]-Nl,N4,N4- tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza- octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 2-({8-[(3 )-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA),(2R)-2-({8-[(3 )-cholest-5-en-3-yloxy]octyl }oxy)-N,N-dimethyl-3-[(9Z, 12Z)-octadeca-9,12-dien-1-yloxy]propan-(Octyl-CLinDMA (2R)), (2S)-2-({8-[(3 )-cholest-5- en-3-yloxyl]octyl} oxy)-N,N-dimethyl-3 -[(9Z,12Z)-octadeca- 9,12-dien-1-yloxylpropan-1-7504-00202 GRH-00462amine (Octyl-CLinDMA (2S)). N,N-dioleyl-N,N-dimethylammonium chloride("DODAC"); N- (2,3-dioleyloxy)propyl-N,N--N-triethylammonium chloride("DOTMA"); N,N-distearyl-N,N- dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethy!ammonium chloride ("DOTAP"); 1,2-Dioleyloxy-3-trirnethylarninopropane chloride salt ("DOTAP.01"); 3- -(N- (N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-ammonium trifluoracetate ("DOSPA"),dioctadecylamidoglycyl carboxyspermine ("DOGS"), 1,2-dioleoyl-3-35dimethylammonium propane ("DODAP"), N,N-dimethyl-2,3-dioleyloxy)propylamine("DODMA"), and N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethylammonium bromide ("DMRIE"). Additionally, a number of commercial preparations ofcationic and / or ionizable lipids can be used, such as, e.g., LIPOFECTIN® (includingDOTMA and DOPE, available from GIBCO / BRL), and LIPOFECTAMINE® (includingDOSPA and DOPE, available from GIBCO / BRL). KLlO, KL22, and KL25 are described,for example, in U.S. Pat. No. 8,691,750, which is incorporated herein by reference in itsentirety. In particular embodiments, the lipid is DLin-MC3-DMA, DLin-KC2-DMA, or ALC-0159. The phospholipids provided herein may, for example, be one or more saturated or(poly)unsaturated phospholipids or a combination thereof. In general, phospholipidscomprise a phospholipid moiety and one or more fatty acid moieties.A phospholipid moiety can be selected, for example, from the non-limiting groupconsisting of phosphatidyl choline, phosphatidyl ethanolamine, phosphatidyl glycerol,phosphatidyl serine, phosphatidic acid, 2-lysophosphatidyl choline, and a sphingomyelin.A fatty acid moiety can be selected, for example, from the non-limiting groupconsisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid,stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid,arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Particular phospholipids can facilitate fusion to a membrane. For example, a cationicphospholipid can interact with one or more negatively charged phospholipids of a membrane(e.g., a cellular or intracellular membrane). Fusion of a phospholipid to a membrane canallow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g.,LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements toa target tissue.7504-00202 GRH-00462Non-natural phospholipid species including natural species with modifications andsubstitutions including branches, oxidation, cyclization, and alkynes are also contemplated.For example, a phospholipid can be functionalized with or cross-linked to one or morealkynes (e.g., an alkenyl group in which one or more double bonds is replaced with a triplebond). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful infunctionalizing the surface (e.g., the lipid monolayer or bilayer) of a nanoparticlecomposition to a useful component such as a targeting or imaging moiety (e.g., a dye).Phospholipids include, but are not limited to, glycerophospholipids such asphosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols,phosphatidyl glycerols, and phosphatidic acids. In some embodiments, the phospholipid isdistearoylphosphatidylcholine (DSPC). Phospholipids also include phosphosphingolipid, such assphingomyelin. The lipid composition of a pharmaceutical composition disclosed herein cancompromise one or more structural lipids. As used herein, the term "structural lipid" refers tosterols and also to lipids containing sterol moieties.Incorporation of structural lipids in the lipid nanoparticle may help mitigateaggregation of other lipids in the particle. Structural lipids can be selected from the groupincluding but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol,stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids,phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is asterol. As defined herein, "sterols" are a subgroup of steroids consisting of steroid alcohols.In certain embodiments, the structural lipid is a steroid. In some embodiments, the structurallipid is cholesterol. In certain embodiments, the structural lipid is an analog of cholesterol.The term "PEG-modified lipid" may refer to polyethylene glycol (PEG)-modifiedlipids / Non-limiting examples of PEG-lipids include PEG-modifiedphosphatidylethanolamine and phosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20)), PEG- modified dialkylamines and PEG-modified 1,2-diacyloxpropan-3-amines. Such lipids are also referred to as PEGylated lipids. For example,a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or aPEG-DSPE lipid. In some embodiments, the PEG-lipid includes, but is not limited to 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl7504-00202 GRH-00462glycerol (PEG-DSG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2- dimyristyloxlpropyl-3-amine (PEG-c-DMA). Preferably, the PEG-modified lipid is1,2- Dimyristoyl-sn-glycero-3-methoxypolyethylene glycol-2000 (DMG-PEG 2000).In some embodiments, the PEG-lipid is selected from the group consisting of a PEG-modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modifiedceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modifieddialkylglycerol, and mixture thereof. In some embodiments, the lipid moiety of the PEG-lipids includes those havinglengths of from about C14 to about C22, preferably from about C14 to about C16. In someembodiments, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000,5000, 10,000, 15,000 or 20,000 daltons. In some embodiments, the PEG-lipid is PEG2k-DMG. In certain embodiments, the lipid nanoparticles described herein can comprise aPEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGsinclude PEG-DSG and PEG-DSPE. PEG-lipids are known in the art, such as those described in U.S. Pat. No, 8,158,601 andInternational Publ. No. WO2015 / 130584 A2, which are incorporated herein by reference in theirentirety. In general, some of the other lipid components (e.g., PEG lipids) of various formulae,described herein may be synthesized as described in International Patent Application No.PCT / US2016 / 000129, filed Dec.10, 2016, entitled "Compositions and methods for Delivery ofTherapeutic Agents," which is now incorporated herein by reference in its entirety.The lipid component of a lipid nanoparticle composition may include one or moremolecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Suchspecies may be alternatively referred to as PEGylated lipids. A PEG lipid is a lipidmodified with polyethylene glycol. A PEG lipid may be selected from the non-limitinggroup including PEG- modified phosphatidylethanolamines, PEG-modified phosphatidicacids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modifieddiacylglycerols, PEG-modified dialkylglycerols, and mixtures thereof. For example, aPEG lipid may be DMG-PEG 2000, PEG0c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.In some embodiments the PEG-modified lipids are a modified form of PEG DMG,including DMG-PEG 2000. In some embodiments, the LNPs comprise ALC-0159, a PEGylated lipid; the N,N-7504-00202 GRH-00462dimyristylamide of 2-hydroxyacetic acid, O-pegylated to a PEG chain mass of about 2kilodaltons. In certain embodiments, the LNPs comprise of ALC-0315, a synthetic ionizable cationicamino lipid. "Stabilizing lipids," as used herein, may include, but is not limited to, lipids thatcontain surface stabilizing polymers conjugated to the lipid headgroup. In someembodiments, the polymer conjugated to the lipid headgroup is hydrophilic. Thehydrophilic polymer-conjugated lipid may be a polyethyleneglycol (PEG)-conjugated lipid.In other embodiments, the polymer making up the polymer-lipid conjugate can be apolymer that contains a backbone that allows it to associate with the core of the particlethereby enhancing the stability of the delivery vehicle (e.g., poly(vinyl alcohol) conjugatedto a lipid). PEG lipids may be used to stabilize the nanoparticle, e.g., in terms of making itinvisible to the immune system. Without being bound by theory, hydrophilic polymer PEGFon the outer surface of the nanoparticle induces steric stabilization due to the local surfaceconcentration of highly hydrated PEG groups. This attracts a water shell that surrounds thenanoparticle that acts as a barrier against certain interactions in the biological environment,e.g., making the nanoparticle less detectable by, or otherwise invisible to, the immunesystem, including inhibition of adsorption and opsonization of the nanoparticle and itscontents. Such nanoparticles may have reduced detection and destruction in the biologicalenvironment and can lead to extended blood circulation time and a preferential accumulationat target sites. Stabilizing lipids may include some lipids that are not conjugated to a stabilizingpolymer. Such lipids contain a negatively charged phosphate group shielded by a hydrophilicneutral moiety such as phosphatidylglycerol (PG) and phosphatidylinositol (Pl).In some embodiments, the LNP has a molar ratio of 50:38.5:10:1.5 of ionizablelipid:structural lipid:phospholipid:PEG-modified lipid. Preferably the LNPO has a molarratio of 50:38.5:10:1.5 of SM-102:cholesterolLDSPC (Distearoylphosphatidylcholine):DMG-PEG 2000. In some such embodiments, the LNP is a solid lipid nanoparticle (SLN).Compositions In some aspects, provided herein is a composition (e.g., a pharmaceuticalcomposition, such as a therapeutic or vaccine composition), containing the nucleic aciddisclosed herein, formulated together with a pharmaceutically acceptable carrier, (e.g., acomposition of the nanoparticles disclosed herein) as well as methods of administering7504-00202 GRH-00462such pharmaceutical compositions. In some embodiments, the nucleic acids, polypeptides, or compositions providedherein are used as an adjuvant. As used, herein, the term "adjuvant" broadly refers to anagent that affects an immunological or physiological response in a patient or subject. Forexample and without limitation , when used as an adjuvant the polypeptides or compositionsprovided herein may increase the presence of an antigen over time or to an area of interestlike a tumor, facilitate absorption of a presented antigen, activate macrophages andlymphocytes, and / or support the production of cytokines. By changing an immune response,the adjuvant might permit a smaller dose of an immune interacting agent to increase theeffectiveness or safety of a particular dose of the immune interacting agent. For example, theadjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of aparticular immune interacting agent. Compositions contemplated herein may be administered intrapleurally,intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intraperitoneally,intracranially, or by direct administration to an organ. Said compositions may comprise oneor more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solution,dispersions, suspensions, or emulsions, or sterile powders which may be reconstituted intosterile injectable solutions or dispersions just prior to use, which may contain sugars,alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonicwith the blood of the intended recipient or suspending thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed inthe pharmaceutical compositions include water, ethanol, polyols (such as glycerol, propyleneglycol, polyethylene glycol, and the like), and suitable mixtures thereof vegetable oils, suchas olive oil, and injectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such as lecithin, by themaintenance of the required particle size in the case of dispersions, and by the use ofsurfactants. In some embodiments, the administered dose size is about 100 μL to about 50 mL(50,000 μL) or any intermediate value encompassed therein, particularly, about: 100 μL, 200μL, 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1000 μL (1 mL), 2 mL, 3mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28mL, 29 mL, 30mL, 31 mL, 32 mL, 33 mL, 34 mL, 35 mL, 36 mL, 37 mL, 38 mL, 39 mL, 40mL, 41 mL, 42mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL, or 50 mL. In some7504-00202 GRH-00462preferred embodiments, the dose size is 10 mL. In some such embodiments, the dosecomprises an mRNA amount of about 50 μg to about 2,500 μg or any intermediate valueencompassed therein, particularly, about: 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg,400 μg, 450 μg, 500 μg, 550μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950μg, 1,000 μg, 1,050 μg, 1,100μg, 1,150 μg, 1,200 μg, 1,250 μg, 1,300 μg, 1,350 μg, 1,400 μg,1,450 μg, 1,500 μg, 1,550 μg, 1,600 μg, 1,650 μg, 1,700 μg, 1,750 μg, 1,800 μg, 1,850 μg,1,900 μg, 1,950 μg, 2,000 μg, 2,050 μg, 2,100 μg, 2,150 μg, 2,200 μg, 2,250 μg, 2,300 μg,2,350 μg, 2,400 μg, or 2,450 μg. In some such embodiments, the dose administered may beat a concentration of about 0.002 mg / kg to about 0.03 mg / kg mRNA or any intermediatevalue encompassed therein, such as about: 0.003 mg / kg, 0.008mg / kg, 0.01 mg / kg, 0.012 mg / kg, 0.015 mg / kg, 0.016mg / kg, 0.02 mg / kg, 0.021 mg / kg, 0.022mg / kg, 0.023 mg / kg, or0.025 mg / kg. For example, the dose may comprise an mRNA concentration of 0.33 μg / μL.Accordingly, without being bound by any particular theory or methodology, the dose sizemay be 1 mL, comprising 660 μL of mRNA (concentration of 500 μg / mL, i.e., 330 μg) and340 μL of ethanol lipid nanoparticle solution. The ethanol may be removed via dialysis afterparticle formation, leaving the lipid nanoparticles containing mRNA in saline buffer alone.This may then be stored at 4°C until use. Thus, in some embodiments, the administered dosemay comprise an mRNA concentration of 0.025 μg / μL - 0.33 μg / μL for the suspension ofLNPs in saline buffer (preferably phosphate buffered saline) or any intermediate valueencompassed therein, particularly, about: 0.03 μg / μL, 0.033 μg / μL, 0.035 μg / μL, 0.04μg / μL, 0.045 μg / μL, 0.05 μg / μL, 0.055 μg / μL, 0.06 μg / μL, 0.065 μg / μL 0.07 μg / μL, 0.075μg / μL, 0.08 μg / μL, 0.085 μg / μL, 0.09 μg / μL, 0.095, μg / μL, 0.1 μg / μL, 0.15 μg / μL, 0.2μg / μL, 0.25 μg / μL, or 0.3 μg / μL.In other aspects, provided herein is a composition containing the cells comprisingthe LNPs (e.g., the SLNs) disclosed herein, carrying the nucleic acids encoding the heatshock protein (e.g., HSP70 polypeptides) contemplated herein. The person of skill in therelevant art will appreciate that such cells may be administered via the adoptive transfer ofsaid cells to a recipient subject in need thereof, as is known in the art. Briefly, and withoutbeing limited by theory, cells (selected from a third-party donor or cells derived from therecipient subject) may be brought into contact with the LNPs provided herein (e.g., in vitroor ex vivo), and administered to the subject in need, by means known in the art.Therapeutic MethodsIn certain embodiments, provided herein are methods of treating a subject,comprising administering to the subject a therapeutic composition provided herein. The7504-00202 GRH-00462subject may be an adult (e.g., 18 years old or older). The subject may be a fetus (e.g., adeveloping fetus within a pregnant person). In some embodiments, the subject is no morethan 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 years of age. In someembodiments, the condition disclosed herein may be a condition in children. Thus, thecondition may be a pediatric condition. The child may be less than about 1 week old. Thechild may be less than about 1 month old. The child may be less than about 6 months old.The child may be less than about 12 months old. The child may be less than about 2 yearsold. The child may be less than about 3 years old. The child may be less than about 4 yearsold. The child may be less than about 5 years old. The child may be less than about 6 yearsold. The child may be less than about 7 years old. The child may be less than about 8 yearsold. The child may be less than about 9 years old. The child may be less than about 10 yearsold. The child may be less than about 12 years old.In some embodiments, the methods provided herein are used to treat or prevent aninflammatory disease or disorder. In some embodiments, the compositions and methodsprovided herein are useful for the treatment of inflammation. In certain embodiments, theinflammation is of any the tissues and organs of the body, including musculoskeletalinflammation, vascular inflammation, neural inflammation, digestive system inflammation,ocular inflammation, hepatic inflammation, inflammation of the reproductive system, andother inflammation. Without being bound by theory, inflammatory diseases may include:inflammatory diseases associated with autoimmune diseases, central nervous system (CNS)inflammatory diseases, arthritic diseases, inflammatory gastrointestinal diseases,inflammatory skin diseases, and other inflammatory diseases associated with epithelial cellssuch as bronchitis, inflammation associated with cancers (e.g., colon and lung cancer),inflammation associated with stimulation / irritation (e.g., by ingestion and / or exposure to adrug or chemical), and inflammation associated with injury.In some such embodiments, the inflammatory disorder is selected from ocularallergy, conjunctivitis, keratoconjunctivitis sicca, vernal conjunctivitis, allergic rhinitis,autoimmune hematological disorders (e.g., hemolytic anemia, aplastic anemia, pure red cellanemia and idiopathic thrombocytopenia), systemic lupus erythematosus, rheumatoidarthritis, polychondritis, scleroderma, Wegener granulomatosis, dermatomyositis, chronicactive hepatitis, myasthenia gravis, Steven-Johnson syndrome, idiopathic sprue,autoimmune inflammatory bowel disease (e.g., ulcerative colitis and Crohn's disease),7504-00202 GRH-00462irritable bowel syndrome, celiac disease, periodontitis, hyaline membrane disease, kidneydisease, glomerular disease, liver disease (e.g., hepatitis, alcoholic liver disease, NAFLD,and / or NASH), multiple sclerosis, endocrine ophthalmopathy, Grave's disease, sarcoidosis,alveolitis, chronic hypersensitivity pneumonitis, primary biliary cirrhosis, uveitis (anteriorand posterior), Sjogren's syndrome, interstitial lung fibrosis, psoriatic arthritis, systemicjuvenile idiopathic arthritis, nephritis, vasculitis, diverticulitis, interstitial cystitis,glomerulonephritis (e.g. including idiopathic nephrotic syndrome or minimal changenephropathy), chronic granulomatous disease, endometriosis, leptospirosis renal disease,glaucoma, retinal disease, headache, pain, complex regional pain syndrome, cardiachypertrophy, muscle wasting, catabolic disorders, obesity, fetal growth retardation,hypercholesterolemia, heart disease, chronic heart failure, mesothelioma, anhidroticectodermal dysplasia, Behcet's disease, incontinentia pigmenti, Paget's disease, pancreatitis,hereditary periodic fever syndrome, asthma, acute lung injury, acute respiratory distresssyndrome, eosinophilia, hypersensitivities, anaphylaxis, fibrositis, gastritis, gastroenteritis,nasal sinusitis, ocular allergy, silica induced diseases, chronic obstructive pulmonarydisease (COPD), cystic fibrosis, acid-induced lung injury, pulmonary hypertension,polyneuropathy, cataracts, muscle inflammation in conjunction with systemic sclerosis,inclusion body myositis, myasthenia gravis, thyroiditis, Addison's disease, lichen planus,appendicitis, atopic dermatitis, asthma, allergy, blepharitis, bronchiolitis, bronchitis,bursitis, cervicitis, cholangitis, cholecystitis, chronic graft rejection, colitis, conjunctivitis,cystitis, dacryoadenitis, dermatitis, eczema, juvenile rheumatoid arthritis, dermatomyositis,encephalitis, endocarditis, endometritis, enteritis, enterocolitis, epicondylitis, epididymitis,fasciitis, Henoch-Schonlein purpura, hepatitis, hidradenitis suppurativa, immunoglobulin Anephropathy, interstitial lung disease, laryngitis, mastitis, meningitis, myelitis myocarditis,myositis, nephritis, oophoritis, orchitis, osteitis, otitis, pancreatitis, parotitis, pericarditis,peritonitis, pharyngitis, pleuritis, phlebitis, pneumonitis, pneumonia, polymyositis,proctitis, prostatitis, pyelonephritis, rhinitis, salpingitis, sinusitis, stomatitis, synovitis,tendonitis, tonsillitis, ulcerative colitis, vasculitis, vulvitis, alopecia areata, erythemamultiforme, dermatitis herpetiformis, scleroderma, vitiligo, hypersensitivity angiitis,urticaria, bullous pemphigoid, pemphigus vulgaris, pemphigus foliaceus, paraneoplasticpemphigus, epidermolysis bullosa acquisita, acute and chronic gout, chronic gouty arthritis,ankylosing spondylitis, psoriasis, psoriatic arthritis, rheumatoid arthritis, CryopyrinAssociated Periodic Syndrome (CAPS), atherosclerosis, and osteoarthritis.In some preferred embodiments, the methods provided herein are used to treat or prevent7504-00202 GRH-00462inflammation of the liver, preferably Non-Alcoholic Fatty Liver Disease (NAFLD) and Non-Alcoholic Steatohepatitis (NASH). Actual dosage levels of the active ingredients in the pharmaceutical compositionsprovided herein may be varied so as to obtain an amount of the active ingredient which iseffective to achieve the desired therapeutic response for a particular patient, composition, andmode of administration, without being toxic to the patient.The selected dosage level will depend upon a variety of factors including the activityof the particular agent employed, the route of administration, the time of administration, therate of excretion or metabolism of the particular compound being employed, the duration ofthe treatment, other drugs, compounds and / or materials used in combination with theparticular compound employed, the age, sex, weight, condition, general health, and priormedical history of the patient being treated, and like factors well known in the medical arts.In some embodiments, the methods provided herein further comprise treating theidentified subject using a therapeutic method provided herein (e.g., by administering to thesubject a composition provided herein). The administration of the disclosed compositions may be carried out in anyconvenient manner, including by injection, transfusion, or implantation, the compositionsdescribed herein may be administered to a patient subcutaneously, intradermally,intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection,or intraperitoneally. In some embodiments, the disclosed compositions are administered to apatient by intradermal or subcutaneous injection. In some embodiments, the disclosedcompositions are administered by intravenous (i.v.) injection. The compositions may also beinjected directly into a tumor, lymph node, organ, or site of disease or disorder (e.g.,inflammation). In certain embodiments, the disclosed compositions are administered to apatient in conjunction with (e.g., before, simultaneously or following) any number ofrelevant treatment modalities, including but not limited to standard-of-care treatment for adisease or condition contemplated herein. In some embodiments, the disclosed compositionsare conjointly administered with an additional therapy. Such therapies may comprise animmunosuppressive agent, a DMARD, a pain-control drug, a steroid, a non-steroidal anti-inflammatory drug (NSAID), or a cytokine antagonist, and combinations thereof.In further embodiments, the compositions may be used in combination withchemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine,methotrexate, mycophenolate, FK506, antibodies, or other immunoablative agents such asCAMPATH (Alemtuzumab), anti-CD3 antibodies or other antibody therapies, cytotoxin,7504-00202 GRH-00462fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228,cytokines, and irradiation. In some embodiments, the said compositions are administered toa patient in conjunction with (e.g., before simultaneously or following) bone marrowtransplantation, T cell ablative therapy using either chemotherapy agents such as fludarabine,external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 orCAMPATH. In other embodiments, the compositions disclosed herein are administeredfollowing B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. Forexample, in some embodiments, subjects may undergo standard treatment with high dosechemotherapy followed by peripheral blood stem cell transplantation. In certainembodiments, following the transplant, subjects receive an infusion of the cells and / or LNPsdisclosed herein. In additional embodiments, expanded cells are administered before orfollowing surgery. Thus, without limitation, the compositions and methods contemplated herein may beused in combination with additional therapeutic procedures and agents selected from thefollowing representative list: cyclosporin, retinoids, corticosteroids, propionic acidderivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2inhibitors, lumiracoxib, ibuprophen, choline magnesium salicylate, fenoprofen, salsalate,difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac,ketorolac, nabumetone, naproxen, valdecoxib, etoricoxib, MK0966; rofecoxib,acetominophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam, tenoxicam,droxicam, lomoxicam, isoxicam, mefanamic acid, meclofenamic acid, flufenamic acid,tolfenamic, valdecoxib, parecoxib, etodolac, indomethacin, aspirin, ibuprophen, firocoxib,methotrexate (MTX), antimalarial drugs (e.g., hydroxychloroquine and chloroquine),sulfasalazine, Leflunomide, azathioprine, cyclosporin, gold salts, minocycline,cyclophosphamide, D-penicillamine, minocycline, auranofin, tacrolimus, myocrisin,chlorambucil, TNF alpha antagonists (e.g., TNF alpha antagonists or TNF alpha receptorantagonists), e.g., ADALIMUMAB (Humira®), ETANERCEPT (Enbrel®), INFL]IXIMAB(Remicade®; TA-650), CERTOLIZUMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB(Simponi®; CNTO 148), ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; MabThera®),ABATACEPT (Orencia®), TOCILIZUMAB (RoActemra / Actemra®), integrin antagonists(TYSABRI® (natalizumab)), IL-i antagonists (ACZ885 (Haris)), Anakinra (Kineret®)),CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6 antagonists, BLyS antagonists(e.g., Atacicept, Benlysta®, LymphoStat-B® (belimumab)), p38 Inhibitors, CD20antagonists (Ocrelizumab, Ofatumumab (Arzerra®)), interferon gamma antagonists7504-00202 GRH-00462(Fontolizumab), prednisolone, Prednisone, dexamethasone, Cortisol, cortisone,hydrocortisone, methylprednisolone, betamethasone, triamcinolone, beclometasome,fludrocortisone, deoxycorticosterone, aldosterone, Doxycycline, vancomycin, pioglitazone,SBI-087, SCIO-469, Cura-100, Oncoxin + Viusid, TwHF, Methoxsalen, Vitamin D -ergocalciferol, Milnacipran, Paclitaxel, rosig tazone, Tacrolimus (Prograf®), RAD00l,rapamune, rapamycin, fostamatinib, Fentanyl, XOMA 052, Fostamatinib disodium,rosiglitazone, Curcumin (Longvida™), Rosuvastatin, Maraviroc, ramipril, Milnacipran,Cobiprostone, somatropin, tgAAC94 gene therapy vector, MK0359, GW856553,esomeprazole, everolimus, trastuzumab, JAKl and JAK2 inhibitors, pan JAK inhibitors, e.g.,tetracyclic pyridone 6 (P6), 325, PF-956980, denosumab, IL- 6 antagonists, CD2Oantagonists, CTLA4 antagonists, IL-8 antagonists, IL-21 antagonists, IL-22 antagonist,integrin antagonists (Tysabri® (natalizumab)), VGEF antagonists, CXCL antagonists, MMPantagonists, defensin antagonists, IL-1 antagonists (including IL-1 beta antagonists), and IL-23 antagonists (e.g., receptor decoys, antagonistic antibodies, etc.).EXAMPLES The composition contemplated herein may comprise four primary parts, an ionizablelipid, a phospholipid, a sterol, and a PEG-modified lipid. The LNPs are prepared by mixingan ethanolic lipid mixture with an acidic aqueous buffer containing the oligonucleotides ofinterest. A 1:3 ratio of ethanolic lipid mixture to aqueous buffer is generally used.For Example, the composition comprises,1. the ionizable lipid SM-102,2. the phospholipid DSPC (distearoylphosphatidylcholine),3. cholesterol, and4. DMG-PEG 2000.Optionally, a secondary ionizable lipid may be used, such as ALC-0159 or ALC-0315, whichhas a PEG-lipid conjugate. Adose size of 500 μL was composed of 330 μL of mRNA and 170 μL ofethanol / lipid nanoparticle solution. For example, an initial mRNA payload concentration of500 μg / mL was diluted to achieve a desired mass ratio. In some embodiments, the mRNA tolipid mass-to-mass ratio is about 1:10. In some embodiments, the ethanol-to-water ratio maybe about 1:3. The ethanol may be removed after the particle formation via dialysis leavingthe lipid nanoparticles containing mRNA in PBS (phosphate buffered saline). This can then7504-00202 GRH-00462be run through a 220 nm filter to remove any aggregation that may have occurred during thedialysis process.Ethanolic Lipid MixturesThe four primary components comprise a molar ratio of about:50 (SM-102):10 (DSPC):38.5 (Cholesterol):1.5 (PEG-Lipid)The molar ratio may be adjusted to optimally deliver the payload based on this starting ratio. Forexample, •the ionizable lipid rage can be adjusted± 5,• the DSPC can be adjusted± 3,• the cholesterol can be adjusted± 5, and• the DMG-PEG 2000 can be adjusted± 0.5.Individual lipid stock solutions for each of the lipids (i.e., SM-102, DSPC,Cholesterol, and PEG-Lipid) in absolute ethanol were brought to room temperature prior touse, and the lipid mixture prepared as described in Table 2, which yielded 10 syntheses at atotal volume of 5, 0 mL. Amounts were calculated such that the ratio was kept at50:10:38.5:1.5 molar ratio for ionizable lipid, DSPC, cholesterol, and PEG-lipid. Ethanolwas used to dilute to the final volume. For example, 10.0 mg or DSPC was mixed with 400μL ethanol, and so on, in accordance with Table 2 below. The appropriate volume of eachlipid mixture component, as listed in Table 2, was then transferred to a single tube to preparethe ethanolic lipid mixture and pipetted several times to ensure mixing and avoidprecipitation and cloudiness.

[0003] 7504-00202 GRH-00462Table 2Component MolarMolarConcentration of mg neededVolumes each, Volumes each, Mass Ratiostock in ethanol in 5 mL for diluted to 5 mL diluted to 5 mL1.5 mg / mL in total solution in total solutionfor 1.5 g / L for 2.0 g / L SM-102 710.2 50 50 mg in 500 μL 4.27 mg 43 μL 57 μL(100 mg / mL)DSPC 790.145 10 10 mg in 400 μL 0.95 mg 38 μL 51 μLat 25 mg / mLCholesterol 386.65 38.5 5.0 mg in 1000 μL 1.79 mg 358 μL 477 μLat5 mg / mLPEG-lipid 2509.2 1.5 5.0 mg in 1000 μL 0.45 mg 90 μL 120 μLat5 mg / mLTotal mass lipids 7.46 mg in 5 mL 9.95 mg in 5 mLlipid mixture lipid mixture stock solution stock solutionAqueous mRNA Solution:A lipid:mRNA (w:w) ratio of 10:1 and an ethanol:aqueous ratio of 1:3 was used.Utilizing 0.50 mL from the 5.0 mL lipid mixture stock solution (e.g., for 1.5 g / L above), 75 μgmRNA was added to a separate tube and adjusted to a volume of 1.5 mL with 50 mM sodiumacetate, pH 5.0. (e.g., 7.46 mg / 10= 0.75 mg; for a ratio of 10:1, 0.75 mg / 10 = 0.075 mg= 75 μg).The ethanolic lipid mixture was mixed with mRNA in a microfluidic mixer. The output,comprising the LNPs, were collected and injected into a dialysis cartridge. The cartridge wasdialyzed in fresh PBS buffer to remove ethanol and loose lipids. The buffer was exchanged threetimes and the final LNP product was extracted and placed into a vessel for storage.Example 2: Payload IAn intended payload for the LNPs was an mRNA strand which codes for the proteinHsp70 which is located on the gene HSPAlA. Utilizing specifically designed primers thefollowing sequence was amplified and applied to the lipid nanoparticles disclosed herein.Table 3HSP70 sequence FeaturesGTGACTCCCGTTGTCCCAAG (SEQ ID NO. 3) Forward Primers:• T7 RNA 12olymeraseTAATACGACTCACTATAGGTGACTCCCGTTGTCCCAAG (SEQ ID NO. 4) 12romoter7504-00202 GRH-00462AAGCTCCAAAACAAAAACAGCAA(SEQ ID NO. 5) Reverse Primer1 aacggctagc ctgaggagct gctgcgacag tccactacct ttttcgagag tgtctcccgt HSP70 nucleotide sequence61 tgtcccaagg cttcccagag cgaacctgtg cggctgcagg caccggcgcg tcgagtttccdepicting complementary121 ggcgtccgga aggaccgagc tcttctcgcg gatccagtgt tccgtttcca gcccccaatc181 tcagagcgga gccgacagag agcagggaac cggcatggcc aaagccgcgg cgatcggcatprimer sites:241 cgacctgggc accacctact cctgcgtggg ggtgttccaa cacggcaagg tggagatcat • forward primer301 cgccaacgac cagggcaacc gcaccacccc cagctacgtg gccttcacgg acaccgagcg • reverse primer361 gctcatcggg gatgcggcca agaaccaggt ggcgctgaac ccgcagaaca ccgtgtttga • coding region421 cgcgaagcgg ctgattggcc gcaagttcgg cgacccggtg gtgcagtcgg acatgaagca481 ctggcctttc caggtgatca acgacggaga caagcccaag gtgcaggtga gctacaaggg541 ggagaccaag gcarrcracc ccgaggagat ctcgtccatg gtgctgacca agatgaagga601 gatcgccgag gcgtacctgg gctacccggt gaccaacgcg gtgatcgcgg ggctcaacgt661 cttcaacgac tcgcagcgcc aggccaccaa ggatgcgggt gtgatcgcgg ggctcaacgt721 gctgcggatc atcaacgagc ccacggccgc cgccatcgcc tacggcctgg acagaacggg781 caagggggag cgcaacgtgc tcatctttga cctgggcggg ggcaccttcg acgtgtccat841 cctgacgatc gacgacggca tcttcgaggt gaaggccacg gccggggaca cccacctggg901 tggggaggac tttgacaaca ggctggtgaa ccacttcgtg gaggagttca agagaaaaca961 caagaaggac atcagccaga acaagcgagc cgtgaggcgg ctgcgcaccg cctgcgagag1021 ggccaagagg accctgtcgt ccagcaccca ggccagcctg gagatcgact ccctgtttga1081 gggcatcgac ttctacacgt ccatcaccag ggcgaggttc gaggagctgt gctccgacct1141 gttccgaagc accctggagc ccgtggagaa ggctctgcgc gacgccaagc tggacaaggc1201 ccagattcac gacctggtcc tggtcggggg ctccacccgc atccccaagg tgcagaagct1261 gctgcaggac ttcttcaacg ggcgcgacct gaacaagagc atcaaccccg acgsggctgt1321 ggcctacggg gcggcggtgc aggcggccat cctfatgggg gacaagrccg agaacgtgca1381 ggacctgctg ctgctggacg tggctcccct gtcgctgggg ctggagacgg ccggaggcgt1441 gatgactgcc ctgstcssgc gcaactccac catccccacc aagcagacgc agatcttcac1501 cacctactcc gacaaccaac ccggggtgct gatccaggtg tacgagggcg agagggccct1561 gacgaaagac aacaatctgt tggggcgctt cgagctgagc ggcatccctc cggcccccag1621 gggcgtgccc cagatcgagg tgaccttcga catcgatgcc aacggcatcc tgaacgtcac1681 ggccacggac aagagcaccg gcaaggccaa caagatcacc atcaccaacg acaagggccg1741 cctgagcaag gaggagatcg agcgcatggt gcaggaggcg gagaagtaca aagcggagga1801 cgaggtgcag cgcgagaggg tgtcagccaa gaacgccctg gagtcctacg ccttcaacat1861 gaagagcgcc gtggaggatg aggggctcaa gggcaagatg agcgaggcgg acaagaagaa1921 ggtgctggac aagtgtcaag aggtcatctc gtggctggac gccaacacct tggccgagaa1981 ggacgagttt gagcacaaga ggaaggagct ggagcaggtg tgtaacccca tcatcagcgg2041 actgtaccag ggtgccggtg gtcccgggcc tgggggcttc ggggctcagg gtcccaaggg2101 agggtctggg tcaggcccca ccattgagga ggtagattag gggcctttcc aagattgctg2161 tttttgtttt ggagcttcaa gactttgcat ttcctagtat ttctgtttgt cagttctcaa2221 tttcctgtgt ttgcaatgtt gaaatttttt ggtgaagtac tgaacttgct ttttttccgg2281 tttctacatg cagagatgaa tttatactgc catcttacga ctatttcttc tttttaatac2341 acttaactca ggccattttt taagttggtt acttcaaagt aaataaactt taaaattcaa(SEQ ID NO. 2)g ugacucccgu uguccaag cuucccagag cgaaccugug cggcugcagg caccggcgcg Amplified mRNA sequence.ucgaguuucc ggcguccgga aggaccgagc ucuucucgcg gauccagugu uccguuuccagcccccaauc ucagagcgga gccgacagag agcagggaac cggcauggcc aaagccgcgg • coding regioncgaucggcau cgaccugggc accaccuacu ccugcguggg gguguuccaa cacggcaagguggagaucau cgccaacgac cagggcaacc gcaccacccc cagcuacgug gccuucacggacaccgagcg gcucaucggg gaugcggcca agaaccaggu ggcgcugaac ccgcagaacaccguguuuga cgcgaagcgg cugauuggcc gcaaguucgg cgacccggug gugcagucggacaugaagca cuggccuuuc caggugauca acgacggaga caagcccaag gugcaggugagcuacaaggg ggagaccaag gcauucuacc ccgaggagau cucguccaug gugcugaccaagaugaagga gaucgccgag gcguaccugg gcuacccggu gaccaacgcg gugaucaccgugccggccua cuucaacgac ucgcagcgcc aggccaccaa ggaugcgggu gugaucgcggggcucaacgu gcugcggauc aucaacgagc ccacggccgc cgccaucgcc uacggccuggacagaacggg caagggggag cgcaacgugc ucaucuuuga ccugggcggg ggcaccuucgacguguccau ccugacgauc gacgacggca ucuucgaggu gaaggccacg gccggggacacccaccuggg uggggaggac uuugacaaca ggcuggugaa ccacuucgug gaggaguucaagagaaaaca caagaaggac aucagccaga acaagcgagc cgugaggugg cugcgcaccgccugcgagag ggccaagagg acccugucgu ccagcaccca ggccagccug gagaucgacucccuguuuga gggcaucgac uucuacacgu ccaucaccag ggcgagguuc gaggagcugugcuccgaccu guuccgaagc acccuggagc ccguggagaa ggcucugcgc gacgccaagcuggacaaggc ccagauucac gaccuggucc uggucggggg cuccacccgc auccccaaggugcagaagcu gcugcaggac uucuucaacg ggcgcgaccu gaacaagagc aucaaccccgacgaggcugu ggccuacggg gcggcggugc aggcggccau ccugaugggg gacaaguccgagaacgugca ggaccugcug cugcuggacg uggcuccccu gucgcugggg cuggagacggccggaggcgu gaugacugcc cugaucaagc gcaacuccac cauccccacc aagcagacgcagaucuucac caccuacucc gacaaccaac ccggggugcu gauccaggug uacgagggcg7504-00202 GRH-00462agagggccau gacgaaagac aacaaucugu uggggcgcuu cgagcugagc ggcaucccuccggcccccag gggcgugccc cagaucgagg ugaccuucga caucgaugcc aacggcauccugaacgucac ggccacggac aagagcaccg gcaaggccaa caagaucacc aucaccaacgacaagggccg ccugagcaag gaggagaucg agcgcauggu gcaggaggcg gagaaguacaaagcggagga cgaggugcag cgcgagaggg ugucagccaa gaacgcccug gaguccuacgccuucaacau gaagagcgcc guggaggaug aggggcucaa gggcaagauc agcgaggcggacaagaagaa ggugcuggac aagugucaag aggucaucuc guggcuggac gccaacaccuuggccgagaa ggacgaguuu gagcacaaga ggaaggagcu ggagcaggug uguaaccccaucaucagcgg acuguaccag ggugccggug gucccgggcc ugggggcucc ggggcucagggucccaaggg agggucuggg ucaggcccca ccauugagga gguagauuag gggccuuuccaagauugcug uuuuuguuuu ggagcuu (SEQ ID NO. 6)The amplified mRNA sequence was inserted into the lipid nanoparticles (LNPs) of theinvention. Such mRNA sequences comprised modifications to the 5' cap, and optionallysubstituting some or all of the uridines with pseudouridines, as disclosed herein. Afurther intended payload will be an mRNA strand encoding the protein Hsp70specified by the following primer pairs.Table 4HSP70 sequence FeaturesGGAAGGACCGAGCTCTTCTC (SEQ ID NO. 7) Forward Primers:• T7 RNA 12olymeraseTAATACGACTCACTATAGGGAAGGACCGAGCTCTTCTC (SEQ ID NO. 8)12romoter GGAAATGCAAAGTCTTGAAGCTC (SEQ ID NO. 9) Reverse Primer1 aacggctagc ctgaggagct gctgcgacag tccactacct ttttcgagag tgtctcccgt HSP70 nucleotide sequence61 tgtcccaagg cttcccagag cgaacctgtg cggctgcagg caccggcgcg tcgagtttccdepicting complementary 121 ggcgtccgga aggaccgagc tcttctcgcg gatccagtgt tccgtttcca gcccccaatc181 tcagagcgga gccgacagag agcagggaac cggcatggcc aaagccgcgg cgatcggcatprimer sites: 241 cgacctgggc accacctact cctgcgtggg ggtgttccaa cacggcaagg tggagatcat • forward primer301 cgccaacgac cagggcaacc gcaccacccc cagctacgtg gccttcacgg acaccgagcg • reyerse Qrimer361 gctcatcggg gatgcggcca agaaccaggt ggcgctgaac ccgcagaaca ccgtgtttga • coding region421 cgcgaagcgg ctgattggcc gcaagttcgg cgacccggtg gtgcagtcgg acatgaagca481 ctggcctttc caggtgatca acgacggaga caagcccaag gtgcaggtga gctacaaggg541 ggagaccaag gcarrcracc ccgaggagat ctcgtccatg gtgctgacca agatgaagga601 gatcgccgag gcgtacctgg gctacccggt gaccaacgcg gtgatcgcgg ggctcaacgt661 cttcaacgac tcgcagcgcc aggccaccaa ggatgcgggt gtgatcgcgg ggctcaacgt721 gctgcggatc atcaacgagc ccacggccgc cgccatcgcc tacggcctgg acagaacggg781 caagggggag cgcaacgtgc tcatctttga cctgggcggg ggcaccttcg acgtgtccat841 cctgacgatc gacgacggca tcttcgaggt gaaggccacg gccggggaca cccacctggg901 tggggaggac tttgacaaca ggctggtgaa ccacttcgtg gaggagttca agagaaaaca961 caagaaggac atcagccaga acaagcgagc cgtgaggcgg ctgcgcaccg cctgcgagag1021 ggccaagagg accctgtcgt ccagcaccca ggccagcctg gagatcgact ccctgtttga1081 gggcatcgac ttctacacgt ccatcaccag ggcgaggttc gaggagctgt gctccgacct1141 gttccgaagc accctggagc ccgtggagaa ggctctgcgc gacgccaagc tggacaaggc1201 ccagattcac gacctggtcc tggtcggggg ctccacccgc atccccaagg tgcagaagct1261 gctgcaggac ttcttcaacg ggcgcgacct gaacaagagc atcaaccccg acgsggctgt1321 ggcctacggg gcggcggtgc aggcggccat cctfatgggg gacaagrccg agaacgtgca1381 ggacctgctg ctgctggacg tggctcccct gtcgctgggg ctggagacgg ccggaggcgt1441 gatgactgcc ctgstcssgc gcaactccac catccccacc aagcagacgc agatcttcac1501 cacctactcc gacaaccaac ccggggtgct gatccaggtg tacgagggcg agagggccct1561 gacgaaagac aacaatctgt tggggcgctt cgagctgagc ggcatccctc cggcccccag1621 gggcgtgccc cagatcgagg tgaccttcga catcgatgcc aacggcatcc tgaacgtcac7504-00202 GRH-004621681 ggccacggac aagagcaccg gcaaggccaa caagatcacc atcaccaacg acaagggccg1 741 cctgagcaag gaggagatcg agcgcatggt gcaggaggcg gagaagtaca aagcggagga1801 cgaggtgcag cgcgagaggg tgtcagccaa gaacgccctg gagtcctacg ccttcaacat1861 gaagagcgcc gtggaggatg aggggctcaa gggcaagatg agcgaggcgg acaagaagaa1921 ggtgctggac aagtgtcaag aggtcatctc gtggctggac gccaacacct tggccgagaa1981 ggacgagttt gagcacaaga ggaaggagct ggagcaggtg tgtaacccca tcatcagcgg2041 actgtaccag ggtgccggtg gtcccgggcc tgggggcttc ggggctcagg gtcccaaggg2101 agggtctggg tcaggcccca ccattgagga ggtagattag gggcctttcc aagattgctg2161 tttttgtttt ggagcttcaa gactttgcat ttcctagtat ttctgtttgt cagttctcaa2221 tttcctgtgt ttgcaatgtt gaaatttttt ggtgaagtac tgaacttgct ttttttccgg2281 tttctacatg cagagatgaa tttatactgc catcttacga ctatttcttc tttttaatac2341 acttaactca ggccattttt taagttggtt acttcaaagt aaataaactt taaaattcaa(SEQ ID NO. 2)gga aggaccgagc ucuucucgcg gauccagugu uccguuucca gcccccaauc ucagagcgga Amplified mRNA sequence.gccgacagag agcagggaac cggcauggcc aaagccgcgg cgaucggcau cgaccugggc accaccuacu ccugcguggg gguguuccaa cacggcaagg uggagaucau cgccaacgac• coding regioncagggcaacc gcaccacccc cagcuacgug gccuucacgg acaccgagcg gcucaucggg gaugcggcca agaaccaggu ggcgcugaac ccgcagaaca ccguguuuga cgcgaagcgg cugauuggcc gcaaguucgg cgacccggug gugcagucgg acaugaagca cuggccuuuc caggugauca acgacggaga caagcccaag gugcagguga gcuacaaggg ggagaccaag gcauucuacc ccgaggagau cucguccaug gugcugacca agaugaagga gaucgccgag gcguaccugg gcuacccggu gaccaacgcg gugaucaccg ugccggccua cuucaacgac ucgcagcgcc aggccaccaa ggaugcgggu gugaucgcgg ggcucaacgu gcugcggauc aucaacgagc ccacggccgc cgccaucgcc uacggccugg acagaacggg caagggggag cgcaacgugc ucaucuuuga ccugggcggg ggcaccuucg acguguccau ccugacgauc gacgacggca ucuucgaggu gaaggccacg gccggggaca cccaccuggg uggggaggac uuugacaaca ggcuggugaa ccacuucgug gaggaguuca agagaaaaca caagaaggac aucagccaga acaagcgagc cgugaggugg cugcgcaccg ccugcgagag ggccaagagg acccugucgu ccagcaccca ggccagccug gagaucgacu cccuguuuga gggcaucgac uucuacacgu ccaucaccag ggcgagguuc gaggagcugu gcuccgaccu guuccgaagc acccuggagc ccguggagaa ggcucugcgc gacgccaagc uggacaaggc ccagauucac gaccuggucc uggucggggg cuccacccgc auccccaagg ugcagaagcu gcugcaggac uucuucaacg ggcgcgaccu gaacaagagc aucaaccccg acgaggcugu ggccuacggg gcggcggugc aggcggccau ccugaugggg gacaaguccg agaacgugca ggaccugcug cugcuggacg uggcuccccu gucgcugggg cuggagacgg ccggaggcgu gaugacugcc cugaucaagc gcaacuccac cauccccacc aagcagacgc agaucuucac caccuacucc gacaaccaac ccggggugcu gauccaggug uacgagggcg agagggccau gacgaaagac aacaaucugu uggggcgcuu cgagcugagc ggcaucccuc cggcccccag gggcgugccc cagaucgagg ugaccuucga caucgaugcc aacggcaucc ugaacgucac ggccacggac aagagcaccg gcaaggccaa caagaucacc aucaccaacg acaagggccg ccugagcaag gaggagaucg agcgcauggu gcaggaggcg gagaaguaca aagcggagga cgaggugcag cgcgagaggg ugucagccaa gaacgcccug gaguccuacg ccuucaacau gaagagcgcc guggaggaug aggggcucaa gggcaagauc agcgaggcgg acaagaagaa ggugcuggac aagugucaag aggucaucuc guggcuggac gccaacaccu uggccgagaa ggacgaguuu gagcacaaga ggaaggagcu ggagcaggug uguaacccca ucaucagcgg acuguaccag ggugccggug gucccgggcc ugggggcucc ggggcucagg gucccaaggg agggucuggg ucaggcccca ccauugagga gguagauuag gggccuuucc aagauugcug uuuuuguuuu ggagcuucaa gacuuugcau uucc (SEQ ID NO. 10)The amplified mRNA sequence will be inserted into the lipid nanoparticles (LNPs) of theinvention. Such mRNA sequences will comprise modifications to the 5' cap, and, optionally substitutingsome or all of the uridines with pseudouridines, as disclosed herein.Example 4: Payload IIIAn additional payload contemplated herein is an mRNA strand encoding the proteinHsp70 specified by the following primer pairs.Table 57504-00202 GRH-00462HSP70 sequence FeaturesCTGCTGCGACAGTCCACTAC (SEQ ID NO. 11) Forward Primers:• T7 RNA 12olymeraseTAATACGACTCACTATAGCTGCTGCGACAGTCCACTAC (SEQ ID NO. 12) momoterGGAAATGCAAAGTCTTGAAGCTC (SEQ ID NO. 13) Reverse Primer1 aacggctagc ctgaggagct g:ctg:cg:acag: tccactacct ttttcgagag tgtctcccgt HSP70 nucleotide sequence61 tgtcccaagg cttcccagag cgaacctgtg cggctgcagg caccggcgcg tcgagtttcc depicting complementary121 ggcgtccgga aggaccgagc tcttctcgcg gatccagtgt tccgtttcca gcccccaatc181 tcagagcgga gccgacagag agcagggaac cggcatggcc aaagccgcgg cgatcggcat primer sites:241 cgacctgggc accacctact cctgcgtggg ggtgttccaa cacggcaagg tggagatcat • forward primer301 cgccaacgac cagggcaacc gcaccacccc cagctacgtg gccttcacgg acaccgagcg • reyerse Qrimer361 gctcatcggg gatgcggcca agaaccaggt ggcgctgaac ccgcagaaca ccgtgtttga • coding region421 cgcgaagcgg ctgattggcc gcaagttcgg cgacccggtg gtgcagtcgg acatgaagca481 ctggcctttc caggtgatca acgacggaga caagcccaag gtgcaggtga gctacaaggg541 ggagaccaag gcarrcracc ccgaggagat ctcgtccatg gtgctgacca agatgaagga601 gatcgccgag gcgtacctgg gctacccggt gaccaacgcg gtgatcgcgg ggctcaacgt661 cttcaacgac tcgcagcgcc aggccaccaa ggatgcgggt gtgatcgcgg ggctcaacgt721 gctgcggatc atcaacgagc ccacggccgc cgccatcgcc tacggcctgg acagaacggg781 caagggggag cgcaacgtgc tcatctttga cctgggcggg ggcaccttcg acgtgtccat841 cctgacgatc gacgacggca tcttcgaggt gaaggccacg gccggggaca cccacctggg901 tggggaggac tttgacaaca ggctggtgaa ccacttcgtg gaggagttca agagaaaaca961 caagaaggac atcagccaga acaagcgagc cgtgaggcgg ctgcgcaccg cctgcgagag1021 ggccaagagg accctgtcgt ccagcaccca ggccagcctg gagatcgact ccctgtttga1081 gggcatcgac ttctacacgt ccatcaccag ggcgaggttc gaggagctgt gctccgacct1141 gttccgaagc accctggagc ccgtggagaa ggctctgcgc gacgccaagc tggacaaggc1201 ccagattcac gacctggtcc tggtcggggg ctccacccgc atccccaagg tgcagaagct1261 gctgcaggac ttcttcaacg ggcgcgacct gaacaagagc atcaaccccg acgsggctgt1321 ggcctacggg gcggcggtgc aggcggccat cctfatgggg gacaagrccg agaacgtgca1381 ggacctgctg ctgctggacg tggctcccct gtcgctgggg ctggagacgg ccggaggcgt1441 gatgactgcc ctgstcssgc gcaactccac catccccacc aagcagacgc agatcttcac1501 cacctactcc gacaaccaac ccggggtgct gatccaggtg tacgagggcg agagggccct1561 gacgaaagac aacaatctgt tggggcgctt cgagctgagc ggcatccctc cggcccccag1621 gggcgtgccc cagatcgagg tgaccttcga catcgatgcc aacggcatcc tgaacgtcac1681 ggccacggac aagagcaccg gcaaggccaa caagatcacc atcaccaacg acaagggccg1 741 cctgagcaag gaggagatcg agcgcatggt gcaggaggcg gagaagtaca aagcggagga1801 cgaggtgcag cgcgagaggg tgtcagccaa gaacgccctg gagtcctacg ccttcaacat1861 gaagagcgcc gtggaggatg aggggctcaa gggcaagatg agcgaggcgg acaagaagaa1921 ggtgctggac aagtgtcaag aggtcatctc gtggctggac gccaacacct tggccgagaa1981 ggacgagttt gagcacaaga ggaaggagct ggagcaggtg tgtaacccca tcatcagcgg2041 actgtaccag ggtgccggtg gtcccgggcc tgggggcttc ggggctcagg gtcccaaggg2101 agggtctggg tcaggcccca ccattgagga ggtagattag gggcctttcc aagattgctg2161 tttttgtttt ggagcttcaa gactttgcat ttcctagtat ttctgtttgt Cs!, tctCs!,s!, 2221 tttcct ttgcaatgtt gaaatttttt ggtgaagtac tgaacttgctttttttccgg 2281 tttctacatg cagagatgaa tttatactgc catcttacga ctatttcttctttttaatac 2341 acttaactca ggccattttt taagttggtt acttcaaagt aaataaactttaaaattcaa (SEQ ID NO. 2)cu gcugcgacag uccacuaccu uuuucgagag ugacucccgu ugucccaagg cuucccagag Amplified mRNA sequence.cgaaccugug cggcugcagg caccggcgcg ucgaguuucc ggcguccgga aggaccgagc • coding regionucuucucgcg gauccagugu uccguuucca gcccccaauc ucagagcgga gccgacagagagcagggaac cggcauggcc aaagccgcgg cgaucggcau cgaccugggc accaccuacuccugcguggg gguguuccaa cacggcaagg uggagaucau cgccaacgac cagggcaaccgcaccacccc cagcuacgug gccuucacgg acaccgagcg gcucaucggg gaugcggccaagaaccaggu ggcgcugaac ccgcagaaca ccguguuuga cgcgaagcgg cugauuggccgcaaguucgg cgacccggug gugcagucgg acaugaagca cuggccuuuc caggugaucaacgacggaga caagcccaag gugcagguga gcuacaaggg ggagaccaag gcauucuaccccgaggagau cucguccaug gugcugacca agaugaagga gaucgccgag gcguaccugggcuacccggu gaccaacgcg gugaucaccg ugccggccua cuucaacgac ucgcagcgccaggccaccaa ggaugcgggu gugaucgcgg ggcucaacgu gcugcggauc aucaacgagcccacggccgc cgccaucgcc uacggccugg acagaacggg caagggggag cgcaacgugcucaucuuuga ccugggcggg ggcaccuucg acguguccau ccugacgauc gacgacggcaucuucgaggu gaaggccacg gccggggaca cccaccuggg uggggaggac uuugacaacaggcuggugaa ccacuucgug gaggaguuca agagaaaaca caagaaggac aucagccagaacaagcgagc cgugaggugg cugcgcaccg ccugcgagag ggccaagagg acccugucguccagcaccca ggccagccug gagaucgacu cccuguuuga gggcaucgac uucuacacgu7504-00202 GRH-00462ccaucaccag ggcgagguuc gaggagcugu gcuccgaccu guuccgaagc acccuggagcccguggagaa ggcucugcgc gacgccaagc uggacaaggc ccagauucac gaccugguccuggucggggg cuccacccgc auccccaagg ugcagaagcu gcugcaggac uucuucaacgggcgcgaccu gaacaagagc aucaaccccg acgaggcugu ggccuacggg gcggcggugcaggcggccau ccugaugggg gacaaguccg agaacgugca ggaccugcug cugcuggacguggcuccccu gucgcugggg cuggagacgg ccggaggcgu gaugacugcc cugaucaagcgcaacuccac cauccccacc aagcagacgc agaucuucac caccuacucc gacaaccaacccggggugcu gauccaggug uacgagggcg agagggccau gacgaaagac aacaaucuguuggggcgcuu cgagcugagc ggcaucccuc cggcccccag gggcgugccc cagaucgaggugaccuucga caucgaugcc aacggcaucc ugaacgucac ggccacggac aagagcaccggcaaggccaa caagaucacc aucaccaacg acaagggccg ccugagcaag gaggagaucgagcgcauggu gcaggaggcg gagaaguaca aagcggagga cgaggugcag cgcgagagggugucagccaa gaacgcccug gaguccuacg ccuucaacau gaagagcgcc guggaggaugaggggcucaa gggcaagauc agcgaggcgg acaagaagaa ggugcuggac aagugucaagaggucaucuc guggcuggac gccaacaccu uggccgagaa ggacgaguuu gagcacaagaggaaggagcu ggagcaggug uguaacccca ucaucagcgg acuguaccag ggugccgguggucccgggcc ugggggcucc ggggcucagg gucccaaggg agggucuggg ucaggccccaccauugagga gguagauuag gggccuuucc aagauugcug uuuuuguuuu ggagcuucaagacuuugcau uuccuaguau uucuguuugu caguucucaa uuuccugugu uugca (SEQ IDNO. 14)The amplified mRNA sequence will be inserted into the lipid nanoparticles (LNPs) of theinvention. Such mRNA sequences will comprise modifications to the 5' cap, and, optionally substitutingsome or all of the uridines with pseudouridines, as disclosed herein. Another intended payload will be an mRNA strand encoding the protein Hsp70specified by the following primer pairs.Table 6HSP70 sequence FeaturesGAAGGACCGAGCTCTTCTC (SEQ ID NO. 15) Forward Primers:• T7 RNA 12olymeraseTAATACGACTCACTATAGGAAGGACCGAGCTCTTCTC (SEQ ID NO. 16)12romoter AAGCTCCAAAACAAAACAGCAA (SEQ ID NO. 17) Reverse Primer1 aacggctagc ctgaggagct g:ctg:cg:acag: tccactacct ttttcgagag tgtctcccgt HSP70 nucleotide sequence61 tgtcccaagg cttcccagag cgaacctgtg cggctgcagg caccggcgcg tcgagtttcc121 ggcgtccgga aggaccgagc tcttctcgcg gatccagtgt tccgtttcca gcccccaatcdepicting complementary 181 tcagagcgga gccgacagag agcagggaac cggcatggcc aaagccgcgg cgatcggcatprimer sites: 241 cgacctgggc accacctact cctgcgtggg ggtgttccaa cacggcaagg tggagatcat • forward primer301 cgccaacgac cagggcaacc gcaccacccc cagctacgtg gccttcacgg acaccgagcg • reyerse Qrimer361 gctcatcggg gatgcggcca agaaccaggt ggcgctgaac ccgcagaaca ccgtgtttga • coding region421 cgcgaagcgg ctgattggcc gcaagttcgg cgacccggtg gtgcagtcgg acatgaagca481 ctggcctttc caggtgatca acgacggaga caagcccaag gtgcaggtga gctacaaggg541 ggagaccaag gcarrcracc ccgaggagat ctcgtccatg gtgctgacca agatgaagga601 gatcgccgag gcgtacctgg gctacccggt gaccaacgcg gtgatcgcgg ggctcaacgt661 cttcaacgac tcgcagcgcc aggccaccaa ggatgcgggt gtgatcgcgg ggctcaacgt721 gctgcggatc atcaacgagc ccacggccgc cgccatcgcc tacggcctgg acagaacggg781 caagggggag cgcaacgtgc tcatctttga cctgggcggg ggcaccttcg acgtgtccat841 cctgacgatc gacgacggca tcttcgaggt gaaggccacg gccggggaca cccacctggg901 tggggaggac tttgacaaca ggctggtgaa ccacttcgtg gaggagttca agagaaaaca961 caagaaggac atcagccaga acaagcgagc cgtgaggcgg ctgcgcaccg cctgcgagag1021 ggccaagagg accctgtcgt ccagcaccca ggccagcctg gagatcgact ccctgtttga1081 gggcatcgac ttctacacgt ccatcaccag ggcgaggttc gaggagctgt gctccgacct7504-00202 GRH-004621141 gttccgaagc accctggagc ccgtggagaa ggctctgcgc gacgccaagc tggacaaggc1201 ccagattcac gacctggtcc tggtcggggg ctccacccgc atccccaagg tgcagaagct1261 gctgcaggac ttcttcaacg ggcgcgacct gaacaagagc atcaaccccg acgsggctgt1321 ggcctacggg gcggcggtgc aggcggccat cctfatgggg gacaagrccg agaacgtgca1381 ggacctgctg ctgctggacg tggctcccct gtcgctgggg ctggagacgg ccggaggcgt1441 gatgactgcc ctgstcssgc gcaactccac catccccacc aagcagacgc agatcttcac1501 cacctactcc gacaaccaac ccggggtgct gatccaggtg tacgagggcg agagggccct1561 gacgaaagac aacaatctgt tggggcgctt cgagctgagc ggcatccctc cggcccccag1621 gggcgtgccc cagatcgagg tgaccttcga catcgatgcc aacggcatcc tgaacgtcac1681 ggccacggac aagagcaccg gcaaggccaa caagatcacc atcaccaacg acaagggccg1 741 cctgagcaag gaggagatcg agcgcatggt gcaggaggcg gagaagtaca aagcggagga1801 cgaggtgcag cgcgagaggg tgtcagccaa gaacgccctg gagtcctacg ccttcaacat1861 gaagagcgcc gtggaggatg aggggctcaa gggcaagatg agcgaggcgg acaagaagaa1921 ggtgctggac aagtgtcaag aggtcatctc gtggctggac gccaacacct tggccgagaa1981 ggacgagttt gagcacaaga ggaaggagct ggagcaggtg tgtaacccca tcatcagcgg2041 actgtaccag ggtgccggtg gtcccgggcc tgggggcttc ggggctcagg gtcccaaggg2101 agggtctggg tcaggcccca ccattgagga ggtagattag gggcctttcc aagattgctg2161 ttttt ttt iiSicttcaa gactttgcat ttcctagtat ttctgtttgt cagttctcaa2221 tttcctgtgt ttgcaatgtt gaaatttttt ggtgaagtac tgaacttgct ttttttccgg2281 tttctacatg cagagatgaa tttatactgc catcttacga ctatttcttc tttttaatac2341 acttaactca ggccattttt taagttggtt acttcaaagt aaataaactt taaaattcaa(SEQ ID NO. 2)ga aggaccgagc ucuucucgcg gauccagugu uccguuucca gcccccaauc ucagagcgga Amplified mRNA sequence.gccgacagag agcagggaac cggcauggcc aaagccgcgg cgaucggcau cgaccugggc accaccuacu ccugcguggg gguguuccaa cacggcaagg uggagaucau cgccaacgac• coding regioncagggcaacc gcaccacccc cagcuacgug gccuucacgg acaccgagcg gcucaucggg gaugcggcca agaaccaggu ggcgcugaac ccgcagaaca ccguguuuga cgcgaagcgg cugauuggcc gcaaguucgg cgacccggug gugcagucgg acaugaagca cuggccuuuc caggugauca acgacggaga caagcccaag gugcagguga gcuacaaggg ggagaccaag gcauucuacc ccgaggagau cucguccaug gugcugacca agaugaagga gaucgccgag gcguaccugg gcuacccggu gaccaacgcg gugaucaccg ugccggccua cuucaacgac ucgcagcgcc aggccaccaa ggaugcgggu gugaucgcgg ggcucaacgu gcugcggauc aucaacgagc ccacggccgc cgccaucgcc uacggccugg acagaacggg caagggggag cgcaacgugc ucaucuuuga ccugggcggg ggcaccuucg acguguccau ccugacgauc gacgacggca ucuucgaggu gaaggccacg gccggggaca cccaccuggg uggggaggac uuugacaaca ggcuggugaa ccacuucgug gaggaguuca agagaaaaca caagaaggac aucagccaga acaagcgagc cgugaggugg cugcgcaccg ccugcgagag ggccaagagg acccugucgu ccagcaccca ggccagccug gagaucgacu cccuguuuga gggcaucgac uucuacacgu ccaucaccag ggcgagguuc gaggagcugu gcuccgaccu guuccgaagc acccuggagc ccguggagaa ggcucugcgc gacgccaagc uggacaaggc ccagauucac gaccuggucc uggucggggg cuccacccgc auccccaagg ugcagaagcu gcugcaggac uucuucaacg ggcgcgaccu gaacaagagc aucaaccccg acgaggcugu ggccuacggg gcggcggugc aggcggccau ccugaugggg gacaaguccg agaacgugca ggaccugcug cugcuggacg uggcuccccu gucgcugggg cuggagacgg ccggaggcgu gaugacugcc cugaucaagc gcaacuccac cauccccacc aagcagacgc agaucuucac caccuacucc gacaaccaac ccggggugcu gauccaggug uacgagggcg agagggccau gacgaaagac aacaaucugu uggggcgcuu cgagcugagc ggcaucccuc cggcccccag gggcgugccc cagaucgagg ugaccuucga caucgaugcc aacggcaucc ugaacgucac ggccacggac aagagcaccg gcaaggccaa caagaucacc aucaccaacg acaagggccg ccugagcaag gaggagaucg agcgcauggu gcaggaggcg gagaaguaca aagcggagga cgaggugcag cgcgagaggg ugucagccaa gaacgcccug gaguccuacg ccuucaacau gaagagcgcc guggaggaug aggggcucaa gggcaagauc agcgaggcgg acaagaagaa ggugcuggac aagugucaag aggucaucuc guggcuggac gccaacaccu uggccgagaa ggacgaguuu gagcacaaga ggaaggagcu ggagcaggug uguaacccca ucaucagcgg acuguaccag ggugccggug gucccgggcc ugggggcucc ggggcucagg gucccaaggg agggucuggg ucaggcccca ccauugagga gguagauuag gggccuuucc aagauugcug uuuuuguuuu ggagcuu (SEQ ID NO. 18)The amplified mRNA sequence will be inserted into the lipid nanoparticles (LNPs) of theinvention. Such mRNA sequences will comprise modifications to the 5' cap, and, optionally substitutingsome or all of the uridines with pseudouridines, as disclosed herein.Example 6: Dynamic Light Scattering7504-00202 GRH-00462In order for the LNP formulations disclosed herein to be used as an inflammatory,obesity, or NAFLD treatment, each therapeutic particle average must be stable and be equalto or smaller than 120 nm. Particles over 120 nm have a high likelihood of contaminationand / or error and will therefore be discarded. Each DLS reading is performed in triplicate andeach particle size average must be within 5 nm of each reading. Doses of LNP-encapsulatedmRNAs were tested with a Dynamic Light Scattering (DLS) machine. In DLS, when laserlight encounters macromolecules in a solution the incident light scatters in all directions andscattering intensity is recorded by a detector. The rate of fluctuations in scattered light isdirectly related to the rate of diffusion of the particle through the solvent, which is related inturn to the particles' hydrodynamic radii. Smaller particles diffuse faster, causing more rapidfluctuations in the intensity than larger particles. Therefore, the fluctuation in light intensitycontains information about the diffusion of the molecules and can be used to extract adiffusion coefficient and calculate a particle size. Continuous DLS data collection showed that the LNP formulations disclosed hereinconsistently comprised particles with a diameter of 110 nm or smaller. In a typical DLSanalysis the Z-Average is the estimated average size of the particles being measured. TheD50 showed that 50% of the particles are the reported size or below. For example, Figure 23shows that 50% of the particles are estimated to be 89.7 nm or below in size (see HistogramOperations: % Cumulative (6), the aforementioned D50). The Count Rate (measured in kilocounts per second (kCPS)) correlates with the concentration of the sample being measured.A Count Rate above 400 kCPS is acceptable for the particles of the invention disclosedherein. Higher Count Rates can indicate a more concentrated dose, i.e., that there are moreparticles containing the MECP2 mRNA. This without being bound by theory ormethodology the higher the concentration, the more effective that dose can be.LNP solutions were filter sterilized with a 0.22 μm filter and stored at 4°C until use.Optionally, the LNPs can be lyophilized and stored at -80°C for long-term storage. DLS testswere run at least in triplicate. Thus, at least three measurements were taken of a sample, asillustrated in the overlay of the Z-Average (measure of the average size of a particle sizedistribution) depicted in Figure 22. Notably, there is little variance among thesemeasurements resulting in a single visible peak. (88.1 nm to 89.7 nm, (all three replicateslaid out over each other). This data does not reflect the average kCPS of all threemeasurements as this output was used to compare triplicate measurements to each other.In addition to testing each batch of doses made, doses were saved to measure the sizechange over time. Such doses were used to measure if the LNPs encapsulating mRNA were7504-00202 GRH-00462aggregating. Even minor aggregation would be detectable. Generally, for DLS measure inintensity, one larger particle can block out many smaller particles and skew the data to showa larger Z-Average and D50 than is actually true. The test sample was prepared and stored at4°C (the temperature at which all tested doses are stored). Particle size was measured atseveral different time points following completion of initial dialysis and filtration. To re-measure, at each time point a portion of sample was applied to a cuvette and read on the DLSmachine and retired, e.g., to a 15 mL conical tube for storage at 4°C again. Results showedthat even after 145 days in storage, there was no aggregation of particles. Thus, the LNPs ofthe invention are stable at 4°C for long periods of time without changing size.In summary, the DLS data showed that particles at 110 nm or smaller can be madeconsistently and are stable over long periods of time. Said particles do not aggregatetogether or change in size over time and can be stored at 4°C with no degradation oraggregation over 145 days of repeated testing. Toxicology mouse testing began with BALB / cJ mice. An experimental group dosedwith 75 μL of LNPs containing HSP70 mRNA and a control group dosed with 75 μL ofEmpty LNPs. The experimental group consisted of 3 BALB / cJ mice that were dosed vialateral tail vein injection every 48 hours. The control group consisted of 2 BALB / cJ micethat were dosed via lateral tail vein injection every 48 hours. All 5 mice were kept in thesame cage, and each mouse was assigned a mouse number. The mice were weighedeveryday, regardless of injections (see table 7).Table 7Day Mouse Weight (g) Weight change Weight change fromfrom 1st day (g)previous day (g) 0Experimental (#1) 26.9 NIA NIA0 Experimental (#2) 23.5 NIA NIA7504-00202 GRH-004620 Experimental (#3) 26.9 NIA NIA0 Control (#4) 26.6 NIA NIA0 Control (#5) 25.8 NIA NIA1 Experimental (#1) 26 -0.9 -0.91 Experimental (#2) 21.8 -1.7 -1.71 Experimental (#3) 27.1 0.2 0.21 Control (#4) 26.5 -0.1 -0.11 Control (#5) 25.7 -0.1 -0.12 Experimental (#1) 26.1 -0.8 0.12 Experimental (#2) 22.8 -0.7 12 Experimental (#3) 26.9 0 -0.22 Control (#4) 26.4 -0.2 -0.12 Control (#5) 25.5 -0.3 -0.23 Experimental (#1) 26 -0.9 -0.13 Experimental (#2) 22.9 -0.6 0.13 Experimental (#3) 26.2 -0.7 -0.73 Control (#4)26.7 0.1 0.33 Control (#5) 25.8 0 0.34 Experimental (#1) NIA NIA NIA4 Experimental (#2) NIA NIA NIA4 Experimental (#3) NIA NIA NIA4 Control (#4) NIA NIA NIA4 Control (#5) NIA NIA NIA5 Experimental (#1) NIA NIA NIA5 Experimental (#2) NIA NIA NIA5 Experimental (#3) NIA NIA NIA7504-00202 GRH-004625 Control (#4) NIA NIA NIA5 Control (#5) NIA NIA NIA6 Experimental (#1) 26.8 -0.1 NIA6 Experimental (#2) 24.2 0.7 NIA6 Experimental (#3) 27 0.1 NIA6 Control (#4) 26.9 0.3 NIA6 Control (#5) 25.7 -0.1 NIA7 Experimental (#1) 26.8 -0.1 07 Experimental (#2) 24.1 0.6 -0.17 Experimental (#3) 27.1 0.2 0.17 Control (#4) 27.3 0.7 0.47 Control (#5) 26.3 0.5 0.68 Experimental (#1) 26.7 -0.2 -0.18 Experimental (#2) 23.8 0.3 -0.38 Experimental (#3) 27 0.1 -0.18 Control (#4) 27.2 0.6 -0.18 Control (#5) 26.6 0.8 0.39 Experimental (#1) 26.5 -0.4 -0.29 Experimental (#2) 23.8 0.3 09 Experimental (#3) 27.3 0.4 0.39 Control (#4) 27.6 1 0.49 Control (#5) 26.6 0.8 010 Experimental (#1) 27.2 0.3 0.710 Experimental (#2) 24.2 0.7 0.410 Experimental (#3) 27.4 0.5 0.110 Control (#4) 27 0.4 -0.610 Control (#5) 26.3 0.5 -0.37504-00202 GRH-0046211 Experimental (#1) 26.3 -0.6 -0.911 Experimental (#2) 23.3 -0.2 -0.911 Experimental (#3) 26.7 -0.2 -0.711 Control (#4) 27.2 0.6 0.211 Control (#5) 26.6 0.8 0.312 Experimental (#1) 26.6 -0.3 0.312 Experimental (#2) 23.6 0.1 0.312 Experimental (#3) 27 0.1 0.312 Control (#4) 27 0.4 -0.212 Control (#5) 26.5 0.7 -0.113 Experimental (#1) 26.7 -0.2 0.113 Experimental (#2) 23.5 0 -0.113 Experimental (#3) 27.2 0.3 0.213 Control (#4) 27.4 0.8 0.413 Control (#5) 27.2 1.4 0.714 Experimental (#1) 26.8 -0.1 0.114 Experimental (#2) 23.4 -0.1 -0.114 Experimental (#3) 27.5 0.6 0.314 Control (#4) 27.7 1.1 0.314 Control (#5) 27.4 1.6 0.2*On 03-30-24 and 03-31-24 (Days 4 and 5), all roads to the vivarium were flooded,preventing the dosing and weighing of the mice. After 2 weeks (14 days) of injection, all 5 mice were euthanized via CO2 inhalation.This was followed by a secondary method of cervical dislocation to ensure the mice's death.Following the euthanasia of the mice, a complete craniotomy and hepatectomy was performed oneach mouse. Following the surgery, the brains and livers of the BALB / cJ mice were placed inseparate 15 mL conical tubes with 7 mL of 10% Neutral Buffered Formalin. These samples were7504-00202 GRH-00462subsequently processed for paraffin-embedded sectioning as well as hematoxylin and eosin(H&E) staining. Using the inbred mouse line MS-NASH, an experimental group dosed with 75 μL ofLNPs containing HSP70 mRNA and a control group dosed with 75 μL of Empty LNPs. Theexperimental group consisted of 3 MS-NASH mice that were dosed via lateral tail veininjection every 24 hours. The control group consisted of 3 MS-NASH mice that were dosedvia lateral tail vein injection every 24 hours. The experimental and control mice were kept inseparate cages and assigned a number within each cage. The mice were weighed everyday(see table 8).Table 8Day Mouse Weight (g) Weight change Weight change fromfrom 1st day (g)previous day (g) 0Experimental 1 36.3 NA NA0 Experimental 2 41.5 NA NA0 Experimental 3 36.3 NA NA0 Control I 46.2 NA NA0 Control 2 38.7 NA NA0 Control 3 37.3 NA NA1 Experimental 1 36 -0.3 -0.31 Experimental 2 41.7 0.2 0.21 Experimental 3 36 -0.3 -0.31 Control I 46.2 0 01 Control 2 38.3 -0.4 -0.41 Control 3 37.3 0 02 Experimental 1 35.1 -1.2 -0.92 Experimental 2 40.3 -1.2 -1.42 Experimental 3 34.6 -1.7 -1.47504-00202 GRH-004622 Control I 46.7 0.5 0.52 Control 2 38.7 0 0.42 Control 3 37 -0.3 -0.33 Experimental 1 34.1 -2.2 -13 Experimental 2 39.7 -1.8 -0.63 Experimental 3 34.3 -2 -0.33 Control I 46.7 0.5 03 Control 2 38.7 0 03 Control 3 37.3 0 0.34 Experimental 1 34.1 -2.2 04 Experimental 2 39.2 -2.3 -0.54 Experimental 3 34.2 -2.1 -0.14 Control I 46.8 0.6 0.14 Control 2 38.9 0.2 0.24 Control 3 37.5 0.2 0.25 Experimental 1 33.9 -2.4 -0.25 Experimental 2 39 -2.5 -0.25 Experimental 3 34.5 -1.8 0.35 Control I 47.8 1.6 15 Control 2 39.2 0.5 0.35 Control 3 37.7 0.4 0.26 Experimental 1 34.5 -1.8 0.66 Experimental 2 39 -2.5 06 Experimental 3 34.3 -2 -0.26 Control I 46.7 0.5 -1.17504-00202 GRH-004626 Control 2 38.8 0.1 -0.46 Control 3 37.5 0.2 -0.27 Experimental 1 33.9 -2.4 -0.67 Experimental 2 38.7 -2.8 -0.37 Experimental 3 34 -2.3 -0.37 Control I 45.5 -0.7 -1.27 Control 2 38.6 -0.1 -0.27 Control 3 37.4 1.1 -0.18 Experimental 1 34.4 -1.9 0.58 Experimental 2 38.2 -3.3 -0.58 Experimental 3 33.9 -2.4 -0.18 Control I 46.6 0.4 1.18 Control 2 39.2 0.5 0.68 Control 3 37.7 0.4 0.39 Experimental 1 33.4 -2.9 -19 Experimental 2 37.4 -4.1 -0.89 Experimental 3 33 -3.3 -0.99 Control I 45.9 -0.3 -0.79 Control 2 38.8 0.1 -0.49 Control 3 37.3 0 -0.410 Experimental 1 33.7 -2.6 0.310 Experimental 2 37.2 -4.3 -0.210 Experimental 3 33 -3.3 010 Control I 46.4 0.2 0.510 Control 2 38.7 0 -0.17504-00202 GRH-0046210 Control 3 37.4 0.1 0.111 Experimental 1 33.9 -2.4 0.211 Experimental 2 38.2 -3.3 111 Experimental 3 33 -3.3 011 Control I 46.1 -0.1 -0.311 Control 2 39 0.3 0.311 Control 3 38.1 0.8 0.712 Experimental 1 34 -2.3 0.112 Experimental 2 37.7 -3.8 -0.512 Experimental 3 33.4 -2.9 0.412 Control I 46.5 0.3 0.412 Control 2 39.5 0.8 0.512 Control 3 38 0.7 -0.113 Experimental 1 32.9 -3.4 -1.113 Experimental 2 37.2 -4.3 -0.513 Experimental 3 33.1 -3.2 -0.313 Control I 45.9 -0.3 -0.613 Control 2 39.1 0.4 -0.413 Control 3 37.5 0.2 -0.514 Experimental 1 33.1 -3.2 0.214 Experimental 2 37.8 -3.7 0.614 Experimental 3 33.7 -2.6 0.614 Control I 47 0.8 1.114 Control 2 38.1 -0.6 -114 Control 3 38.1 0.8 0.67504-00202 GRH-00462After 2 weeks (14 days) of injection, all 6 mice were euthanized as described above.Also as described above, liver and brain samples were processed for paraffin-embeddedsectioning and H&E staining.Example 9: MS-NASH Mouse Weight Data (HSP70 Cohort #2)The same MS-NASH procedure from example 8 was repeated, but with 2 controlMS- NASH mice instead of 3, i.e., 5 total MS-Mice used for the second trial of efficacytesting. All 5 mice were kept in the same cage, and each mouse was assigned a mousenumber. The mice were weighed every day (see table 9).Table 9Day Mouse (Mouse Weight (g) Weight change Weight change fromNumber)from 1st day (g)previous day (g) 0Experimental (#1) 42.6 NIA NIA0 Experimental (#2) 39.6 NIA NIA0 Experimental (#3) 35.6 NIA NIA0 Control (#4) 39.5 NIA NIA0 Control (#5) 38.8 NIA NIA1 Experimental (#1) 41.3 -1.3 -1.31 Experimental (#2) 38.3 -1.3 -1.31 Experimental (#3) 34.3 -1.3 -1.31 Control (#4) 39.6 0.1 0.11 Control (#5) 38.3 -0.5 -0.52 Experimental (#1) 40.7 -1.9 -0.62 Experimental (#2) 37.5 -2.1 -0.82 Experimental (#3) 33.2 -2.4 -1.12 Control (#4)39.7 0.2 0.17504-00202 GRH-004622 Control (#5) 38.6 -0.2 0.33 Experimental (#1) 39.7 -2.9 -13 Experimental (#2) 36.9 -2.7 -0.63 Experimental (#3) 32.2 -2.4 -13 Control (#4) 39.5 0 -0.23 Control (#5) 38.6 -0.2 04 Experimental (#1) 39.7 -2.9 04 Experimental (#2) 36.9 -2.7 04 Experimental (#3) 32.8 -2.8 0.64 Control (#4) 39 -0.5 -0.54 Control (#5) 38.7 -0.1 0.15 Experimental (#1) 39.5 -3.1 -0.25 Experimental (#2) 36.8 -2.8 -0.15 Experimental (#3) 32.7 -2.9 -0.15 Control (#4) 39.4 -0.1 0.45 Control (#5) 38.6 -0.2 -0.16 Experimental (#1) 39 -3.6 -0.56 Experimental (#2) 36.2 -3.4 -0.66 Experimental (#3) 32.8 -2.8 0.16 Control (#4) 39 -0.5 -0.46 Control (#5) 38.7 -0.1 0.17 Experimental (#1) 38.7 -3.9 -0.37 Experimental (#2) 35.6 -4 -0.67 Experimental (#3) 32.3 -3.3 -0.57 Control (#4) 39.3 -0.2 0.37504-00202 GRH-004627 Control (#5) 38.9 0.1 0.28 Experimental (#1) 38.4 -4.2 -0.38 Experimental (#2) 35.7 -3.9 0.18 Experimental (#3) 32.1 -3.5 -0.28 Control (#4) 39.4 -0.1 0.18 Control (#5) 38.7 -0.1 -0.29 Experimental (#1) 38.9 -3.7 0.59 Experimental (#2) 36 -3.6 0.39 Experimental (#3) 32.4 -3.2 0.39 Control (#4) 40 0.5 0.69 Control (#5) 39.4 0.6 0.710 Experimental (#1) 38.8 -3.8 -0.110 Experimental (#2) 35.3 -4.3 -0.710 Experimental (#3) 32.2 -3.4 -0.210 Control (#4) 39.8 0.3 -0.210 Control (#5) 38.9 0.1 -0.511 Experimental (#1) 38.6 -4 -0.211 Experimental (#2) 35.4 -4.2 0.111 Experimental (#3) 32.5 -3.1 0.311 Control (#4) 40.1 0.6 0.311 Control (#5) 39.3 0.5 0.412 Experimental (#1) 38.7 -3.9 0.112 Experimental (#2) 35.6 -4 0.212 Experimental (#3) 32.5 -3.1 012 Control (#4) 40.3 0.8 0.27504-00202 GRH-0046212 Control (#5) 39.2 0.4 -0.113 Experimental (#1) 38.9 -3.7 0.213 Experimental (#2) 35.1 -4.5 -0.513 Experimental (#3) 32.5 -3.1 013 Control (#4) 40.1 0.6 -0.213 Control (#5) 39.1 0.3 -0.114 Experimental (#1) 38.9 -3.7 014 Experimental (#2) 35.4 -4.2 0.314 Experimental (#3) 32.6 -3 0.114 Control (#4) 40.1 0.6 014 Control (#5) 39.7 0.9 0.615 Experimental (#1) 38.9 -3.7 015 Experimental (#2) 36.3 -3.3 0.915 Experimental (#3) 33.2 -2.4 0.615 Control (#4) 40.8 1.3 0.715 Control (#5) 39.5 0.7 -0.2After 2 weeks (15 days) of injection, all 5 mice were euthanized, and brain and liversamples were processed for paraffin-embedded section and H&E staining as described above. The same MS-NASH procedure from example 9 was repeated. All 5 mice were keptin the same cage, and each mouse was assigned a mouse number. The mice were weighedevery day (see table 10).Table 10Day Mouse (Mouse Weight (g) Weight change Weight change fromNumber)from 1st day (g)previous day (g) 0Experimental (#1) 40.1 NIA NIA7504-00202 GRH-004620 Experimental (#2) 35.8 NIA NIA0 Experimental (#3) 41.2 NIA NIA0 Control (#4) 40.1 NIA NIA0 Control (#5) 41.1 NIA NIA1 Experimental (#1)38 -2.1 -2.11 Experimental (#2) 34.4 -1.4 -1.41 Experimental (#3) 39.5 -1.7 -1.71 Control (#4) 40 -0.1 -0.11 Control (#5) 40.4 -0.7 -0.72 Experimental (#1) 37.5 -2.6 -0.52 Experimental (#2) 33.8 -2 -0.62 Experimental (#3) 38.7 -2.5 -0.82 Control (#4)40.3 0.2 0.32 Control (#5) 40 -1.1 -0.43 Experimental (#1) 37.4 -2.7 -0.13 Experimental (#2) 33.3 -2.5 -0.53 Experimental (#3) 38.2 -3 -0.53 Control (#4) 40.1 0 -0.23 Control (#5) 40.7 -0.4 0.74 Experimental (#1) 37 -3.1 -0.44 Experimental (#2) 33.2 -2.6 -0.14 Experimental (#3) 38.5 -2.7 0.34 Control (#4) 39.6 -0.5 -0.54 Control (#5) 40.5 -0.6 -0.27504-00202 GRH-004625 Experimental (#1) 36.4 -3.7 -0.65 Experimental (#2) 33 -2.8 -0.25 Experimental (#3) 37.9 -3.3 -0.65 Control (#4) 39.5 -0.6 -0.15 Control (#5) 40.1 -1 -0.46 Experimental (#1) 36.5 -3.6 0.16 Experimental (#2) 32.5 -3.3 -0.56 Experimental (#3) 37 -4.2 -0.96 Control (#4) 39.4 -0.7 -0.16 Control (#5) 39.3 -1.8 -0.87 Experimental (#1) 36.6 -3.5 0.17 Experimental (#2) 32.5 -3.3 07 Experimental (#3) 36.4 -4.8 -0.67 Control (#4) 39.9 -0.2 0.57 Control (#5) 39.6 -1.5 0.38 Experimental (#1) 36.6 -3.5 08 Experimental (#2) 31.8 -4 -0.78 Experimental (#3) 35.8 -5.4 -0.68 Control (#4) 39.2 -0.9 -0.78 Control (#5) 39.5 -1.6 -0.19 Experimental (#1) 37.1 -3 0.59 Experimental (#2) 32 -3.8 0.29 Experimental (#3) 36.9 -4.3 1.19 Control (#4) 40.2 0.1 19 Control (#5) 40 -1.1 0.57504-00202 GRH-0046210 Experimental (#1) 36.3 -3.8 -0.810 Experimental (#2) 32.1 -3.7 0.110 Experimental (#3) 36.1 -5.1 -0.810 Control (#4) 39 -1.1 -1.210 Control (#5) 39.8 -1.3 -0.211 Experimental (#1) 36.8 -3.3 0.511 Experimental (#2) 31.9 -3.9 -0.211 Experimental (#3) 36.2 -5 0.111 Control (#4) 39.6 -0.5 0.611 Control (#5) 39.5 -1.6 -0.312 Experimental (#1) 36.6 -3.5 -0.212 Experimental (#2) 31.2 -4.6 -0.712 Experimental (#3) 35.4 -5.8 -0.812 Control (#4) 39.6 -0.5 012 Control (#5) 39.8 -1.3 0.313 Experimental (#1) 35.5 -4.6 -1.113 Experimental (#2) 31.5 -4.3 0.313 Experimental (#3) 35 -6.2 -0.413 Control (#4) 39.7 -0.4 0.113 Control (#5) 39.8 -1.3 014 Experimental (#1) 35.7 -4.4 0.214 Experimental (#2) 31.7 -4.1 0.214 Experimental (#3) 35.1 -6.1 0.114 Control (#4) 40.2 0.1 0.514 Control (#5) 41.1 0 1.37504-00202 GRH-00462After 2 weeks (14 days) of injection, all 5 mice were euthanized, and brain and liversamples were processed for paraffin-embedded section and H&E staining as described abovein Example 8.Example 11: MS-NASH Mouse Weight Data (Cohort #4)4 MS-NASH mice were dosed every 24 hours with various amounts of HSP70-LNPsfor 2 weeks (14 days). All 4 mice were kept in the same cage, and each mouse was assigneda number. Mouse #1 received a dose of 30 μL. Mouse #2 received a dose of 150 μL. Mouse#3 received a dose of 250 μL. Mouse #4 received a dose equal to 1% of its body weight involume (i.e., a 39.5g mouse would receive a 395 μL dose). The mice were weighed everyday (see table 11).Table 11Day Mouse (Mouse Weight (g) Weight change Weight change fromNumber)from 1st day (g)previous day (g) 030 μL Dose (#1) 39.3 N / A N / A0 150 μL Dose (#2) 39.2 N / A N / A0 250 μL Dose (#3) 39 N / A N / A0 1 % Body Weight N / A N / ADose (#4) 401 30 μL Dose (#1) 38.8 -0.5 -0.51 150 μL Dose (#2) 38 -1.2 -1.21 250 μL Dose (#3) 37.9 -1.1 -1.11 1% Body WeightDose (#4) 39.2 -0.8 -0.82 30 μL Dose (#1) 37.7 -1.6 -1.12 150 μL Dose (#2) 36.7 -2.5 -1.32 250 μL Dose (#3) 36.7 -2.3 -1.22 1% Body WeightDose (#4) 37.5 -2.5 -1.77504-00202 GRH-004623 30 μL Dose (#1) 37 -2.3 -0.73 150 μL Dose (#2) 35.9 -3.3 -0.83 250 μL Dose (#3) 36 -3 -0.73 1% Body WeightDose (#4) 36.3 -3.7 -1.24 30 μL Dose (#1) 36.7 -2.6 -0.34 150 μL Dose (#2) 35.4 -3.8 -0.54 250 μL Dose (#3) 34.8 -4.2 -1.24 1% Body WeightDose (#4) 35.8 -4.2 -0.55 30 μL Dose (#1) 36.7 -2.6 05 150 μL Dose (#2) 35 -4.2 -0.45 250 μL Dose (#3) 34.6 -4.4 -0.25 1% Body WeightDose (#4) 35.5 -4.5 -0.36 30 μL Dose (#1) 36.4 -2.9 -0.36 150 μL Dose (#2) 34.5 -4.7 -0.56 250 μL Dose (#3) 34.5 -4.5 -0.16 1% Body WeightDose (#4) 35.8 -4.2 0.37 30 μL Dose (#1) 35.5 -3.8 -0.97 150 μL Dose (#2) 33.4 -5.8 -1.17 250 μL Dose (#3) 33.4 -5.6 -1.17 1% Body WeightDose (#4) 35.1 -4.9 -0.78 30 μL Dose (#1) 35.4 -3.9 -0.18 150 μL Dose (#2) 32.9 -6.3 -0.58 250 μL Dose (#3) 32.9 -6.1 -0.57504-00202 GRH-004628 1% Body WeightDose (#4) 33.9 -6.1 -1.29 30 μL Dose (#1) 35.3 -4 -0.19 150 μL Dose (#2) 32.4 -6.8 -0.59 250 μL Dose (#3) 32.3 -6.7 -0.69 1% Body WeightDose (#4) 33.2 -6.8 -0.710 30 μL Dose (#1) 35.5 -3.8 0.210 150 μL Dose (#2) 32.4 -6.8 010 250 μL Dose (#3) 31.8 -7.2 -0.510 1% Body WeightDose (#4) 33 -7 -0.211 30 μL Dose (#1) 35.8 -3.5 0.311 150 μL Dose (#2) 32.4 -6.8 011 250 μL Dose (#3) 31.2 -7.8 -0.611 1% Body WeightDose (#4) 32.8 -7.2 -0.212 30 μL Dose (#1) 35.8 -3.5 012 150 μL Dose (#2) 31.9 -7.3 -0.512 250 μL Dose (#3) 31.2 -7.8 012 1% Body WeightDose (#4) 32.7 -7.3 -0.113 30 μL Dose (#1) 35.6 -3.7 -0.213 150 μL Dose (#2) 31.8 -7.4 -0.113 250 μL Dose (#3) 31 -8 -0.213 1% Body WeightDose (#4) 32.8 -7.2 0.114 30 μL Dose (#1) 35.5 -3.8 -0.17504-00202 GRH-0046214 150 μL Dose (#2) 31.8 -7.4 014 250 μL Dose (#3) 31.6 -7.4 0.614 1% Body WeightDose (#4) 32.5 -7.5 -0.3After 2 weeks (14 days) of injection, all 4 mice were euthanized, and brain and liversamples were processed for paraffin-embedded section and H&E staining as described abovein Example 8. 12: HSP70 mRNA synthesis PCR (DNA amplification and synthesis):Plasmids containing the nucleotide sequence for HSP70 were used in the synthesisand amplification of double stranded DNA containing the nucleotide sequence of the HSP70protein. Commercially available kits, such as the LongAmp® Taq PCR Kit, were used. In asingle reaction mixture, the enzyme LongAmp® Taq DNA polymerase was used to createDNA from a HSP70 plasmid template with the forward and reverse primer pairs disclosedherein (e.g., SEQ ID NOs. 4 and 5, 8 and 9, 12 and 13, and 16 and 17). This double strandedDNA is coded for the HSPAlA gene.Table 12: Synthesis reactions were run with the following thermal cycler parameters.InitialDenature Anneal Extend Final Final HoldDenaturation Extension 1cycle 30 cycles 1 cycle 1 cycle94°c 94°c 52.5°C 65°C 65°C 4°c30 seconds 20 seconds 35 seconds 2 minutes and 10 minutes indefinite15 secondsAfter the cycler finished running, the reaction tubes could be stored at -20°C,otherwise the resultant double-stranded DNA was used immediately for mRNA creation.DNA Agarose Analysis:Samples were prepared for loading onto agarose gel by taking 1 μL of DNA samplewith 3 μL of lX loading dye (4 μL total), pipetted up and down slowly 3-5 times to mix.This was done with each sample run on an agarose gel. A1KB DNA ladder was applied to the first well and the 4 μL samples were added toseparate wells (i.e., up to 9 DNA samples in each agarose gel). Samples were electrophoresed7504-00202 GRH-00462for 1 hour at a constant 100 V. The expected length of the sequence should be ~2100 basepairs which was observed for all 4 samples shown in the HSP70 DNA Agarose Gel of Figure6. The nucleotide sequence of the double-stranded DNA was assessed and confirmed to bethe intended target sequence by Sanger sequencing (i.e., chain termination method) analysis.mRNA SynthesisThe prepared DNA was used in the synthesis of HSP70 mRNA. Commerciallyavailable kits, such as the HiScribe® T7 Quick Yield RNA Synthesis Kit, were used.Thermal cycler reactions were run according to kit manufacture protocols. Once thermal cycling was complete, the mRNA was capped using a Fausto viruscapping enzyme (FCE) and Cap 2'-O-methyltransferase. For each tube 24.2 μL of Nuclease-Free Water was added, followed by 6.5 μL of FCE capping buffer, 3.25 μL of S-Adenosylmethionine (SAM) (32 mM), 3.25 μL of Guanosine triphosphate (GTP) (10 mM), 2.6 μL ofthe FCE enzyme (25, 000 units / mL), and 5.2 μL of Cap 2'-O-methyltransferase (50, 000units / mL) was added in that order, giving a final volume of 65 μL. The tubes were thenincubated in a thermal cycler and set for a 1-hour hold at 37°C followed by an indefinite 4°Chold. RNA purification and concentration following enzymatic reaction was performedusing commercially available kits such as the Monarch® RNA Clean up Kit. Briefly, 100 μLof RNA Binding Buffer was added to the 65 μL mRNA synthesis sample and mixed bypipetting. The total 165 μL of solution was added to the spin column provided with 165 μLof 100% ethanol and carefully pipetted to mix. Spin columns (with collection tubes) werecentrifuged for 1 minute at 13, 000 rpm. Liquid flowthrough (liquid in collection tube) wasremoved and 500 μL of appropriate RNA wash buffer was added to the spin column.Following centrifugation for 1 minute at 13, 000 rpm, the liquid flow through was removed,and the wash repeated at least once more.Following washing, 50-100 μL of nuclease free water was added to the spin column(with a new empty collection tube) and incubated at room temperature for 5 minutes. Afterthe 5- minute incubation, the spin column (with empty collection tube) was centrifuged for 1minute at 13, 000 rpm. The collection tube containing the resultant flowthrough was held at4°C. The purified mRNA from multiple collection tubes were combined and mixed via pipetting.Following collection of purified RNA, the concentration of the combined RNA wasdetermined by using a Nanophotometer. Purified HSP70 RNA could be stored at 4°C until use.7504-00202 GRH-00462mRNA Agarose Gel Analysis:Samples were prepared for loading onto a 1% agarose gel by diluting 1 μL of mRNAto a concentration of about 300-400 μg / mL in nuclease free water. This diluted mRNA wasmixed by slowly pipetting up and down 3-5 times. 1 μL of diluted mRNA sample was addedto 3 μL of 2X RNA loading dye, pipetting up and down slowly 3-5 times to mix.A ssRNA ladder was applied to the first well and the 4 μL sample(s) were added toeach subsequent well. (i.e., synthesized mRNA from a day followed by synthesized mRNAfrom another day). Samples were electrophoresed for 1 hour at a constant 100 V. Multipleseparate HSP70 mRNA synthesis runs yielded identical results and produced mRNA forHSP70 at 2100 bases long in high concentrations (see Figure 7). This mRNA represents thesequence (or payload) disclosed herein and incorporated into the LNPs of the invention. In summary, HSP70 DNA was created from a HSP70 plasmid template. The DNApolymerase enzyme LongAmp® Taq DNA Polymerase was added to the HSP70 plasmidtemplate to create double stranded DNA coding for the HSP70 gene using the appropriateprimers. This 2128 base pair long strand was consistently synthesized as shown in Figure 6.The double-stranded DNA coding for the HSP70 gene was then used to create mRNA, as theforward primer used to synthesize DNA contained a promoter for T7 RNA polymerase. Afterthe mRNA synthesis, a 5' cap was added with a Faustovirus Capping Enzyme (FCE) andCap 2' -0- methyltransferase. After the cap was added, the resultant sequence was thencleaned to remove any impurities such as free-floating nucleotides, enzymes, DNA, etc. Thefully cleaned and capped mRNA is shown in Figure 7 and represents Payload 1 used tocreate a final lipid nanoparticle containing HSP70 mRNA product contemplated herein. H&E-stained slides of sectioned liver from MS-NASH mice dosed with HSP70-LNPs were compared to MS-NASH mice dosed with Empty LNPs. H&E staining ofsectioned liver can reveal lipid accumulation as clear vacuoles in hepatocytes and theseimages illustrated major differences between the two groups of mice. The HSP70-LNP-dosed mice had significantly less steatosis (fat buildup) and hepatocyte ballooning in theliver, as indicated by the significantly lower amount of white space (see Figures 9, 11, and13) relative to the Empty-LNP-dosed mice (control group). (See Figures 8, 10, and 12). Thebrain sections were also compared, but showed no noticeable or significant differencesbetween the two groups. Whole brains and livers were sectioned at 5-micron thick slices and stained with7504-00202 GRH-00462H&E. Slides were imaged on a fluorescent microscope and BALB / cJ mice dosed withHSP70-LNPs (Figures 15 & 17). were compared to BALB / cJ mice dosed with Empty LNPs(Figures 14 & 16). No significant or notable differences between the two groups of mice.Example 15: IHC ImagingTo see how much the HSP70-LNP is increasing HSP70 expression in the liver of theBALB / cJ and MS-NASH mice, immunohistochemistry (IHC) was performed on paraffinembedded tissue sections. Slides of the embedded tissue sections were heated in a dry ovenfor 20 minutes at about 55°C. After this, the slides were placed in a tray and covered with anonpolar solvent, such as Risto-Clear II, and allowed to sit for 5 minutes. After this 5-minutewash, the slides were removed and washed again in the same manner for a total of threewashes. Slides were then subjected to the following wash cycle: 100% Ethanol (2 washes for5 minutes each), 90% Ethanol (1 wash for 5 minutes), 70% Ethanol (1 wash for 5 minutes),and finally DI H20 (1 wash for 5 minutes). After the wash cycle, each slide was placed in a500 mL beaker with enough Tris-EDTA antigen Retrieval solution to comfortably cover eachslide (about 40-80 mL). The slides in the Tris- EDTA solution were then microwaved untilboiling (about 30-45 seconds) and then placed on a preheated hotplate set to 95°C- 100°Cand allowed to sit for 15 minutes.After the 15-minute heating period, the beakers were removed from the hot plate andallowed to come down to room temperature (about 15-25 minutes). After cooling, the slideswere placed in PBS and on a shaker set to 40 rpm at room temperature for 5 minutes.Following this PBS wash, the edges of the slides were dried and nail polish, or a hydrophobicbarrier pen, was used along the edges of the slide to create a barrier. The slides were thenplaced on a wet paper towel in a tray and 600-900 μL of 2% PBS in PBS solution) was thencarefully added to each slide. The slides were then covered and protected from light andallowed to incubate at room temperature for 1 hour. After the incubation period, the 2% PBSsolution was removed, and the slides were placed in PBS and put on a shaker set to 40 rpmfor 5 minutes. The PBS was replaced, and the slides were returned to the shaker for another 5minutes. During these washes, a primary antibody solution was made with an anti-humanHSP70 monoclonal rabbit antibody at a dilution of 1:2, 000 in 0.1% PBS. The slides wereremoved from the PBS wash and placed on a wet paper towel in a tray and 600-900 μL of theprimary antibody solution was carefully pipetted onto each slide. The slides were coveredand protected from light and were incubated at 4°C overnight.After the overnight incubation the slides underwent the same PBS washing procedure7504-00202 GRH-00462for a total of 3 washes. During the PBS washes, a secondary antibody solution was madewith an anti- rabbit secondary antibody tagged with Alexa FluorTM 568 and Hoechst 33342.Both components were diluted in the same 0.1% PBS / PBS solution at a concentration of 1:2,000. After the 3 PBS washes, the slides were placed on a wet paper towel-lined tray andmoved to a dark area and 600-900 μL of the secondary antibody solution was carefullypipetted onto each slide. The slides were covered and protected from light while theyincubated at room temperature for 1 hour. The secondary antibody solution was removed,and the slides were stored in PBS, protected from light, at 4°C until ready for analysis on afluorescent microscope. Prior to imaging, the slides were removed from PBS and allowed todry in a light protected space. Said imaging confirmed HSP70 overexpression (Figures 18-21), which was further confirmed using enzyme-linked immunosorbent assay (ELISA).Example 16: MS-NASH Bloodwork Data Overview:In order to determine the altered levels of relevant components within the blood fromthe MS-NASH mice, blood samples were collected and analyzed using IDEXX'sHematology for Veterinary Diagnostics service. A post-euthanasia cardiac punch wasperformed on the MS- NASH mice for blood collection before being sent off for analysis.The two types of hematology diagnostics that were used were complete blood count (CBC)and expanded toxicology. The CBC test is a method of analysis that evaluates the quantity ofa variety of cells and components in the whole blood. It assists in the diagnosis of certainmedical conditions as it provides data on red blood cell count, platelets, hemoglobin, and therelative concentration of the different typesof white blood cells. The expanded toxicology test provides insight on the varying levels ofrelevant enzymes present in the serum component of the blood. It also assists in the diagnosisof medical conditions due to the insight it provides on liver enzymes, glucose, andcholesterol levels.Table 13LOR: Low (Outside of Range) LWR: Low (Within Range)Average:(Near Mean) HWR: High (Within Range) HOR: High (Outside of Range)Complete Blood Count C57BL / 6, I Average HSP70- Average Em:uty(CBC)Average Levels LNP Dosed LNPDosedEnd:uoint End:uointNeutrophil (K / μL) .98 - 1.44 1.31 - Average 1.1 - Average7504-00202 GRH-00462WBC (K / μL) 6.7 - 8.9 4.1-LOR 5.8 -LORRBC (M / μL) 8.31 - 9.48 8.77 - Average 10.12- HORHGB (g / dL) 12.4 - 14.2 12.4- LWR 13.4 - AverageLymphocyte (K / μL) 5.10 - 6.87 2.814- LOR 4.2-LORPlatelet Count (K / μL) 1094- 1347 471- LOR 534-LORMCHC (g / dL) 27.4 - 30.2 28.15 - Average 26.5 - AverageThe above CBC blood work data displays several key points. Primarily, MS-NASHmice treated with HSP70 mRNA-LNPs display healthy levels of red blood cells as well asaverage hemoglobin concentrations. Control MS-NASH mice, receiving empty LNP doses,had higher than average red blood cell counts but average hemoglobin concentrations.Normal MS-NASH mice have oxygen transport issues due to their difficulty breathing andhigh blood pressure from the symptoms induced by obesity (Kosmalski et al, 2018). Thisshows the broad impact and improvement of the experimental group of MS-NASH micetreated with HSP70 mRNA-LNPs as their RBC and HGB levels were significantly lowered incomparison to the control group. The NASH & NAFLD diseases have been linked tocompromised immune systems (Kosmalski et al, 2018). Low WBC, lymphocytes, andplatelet counts are all factors associated with hepatitis and cirrhosis of the liver. Thereforeboth experimental and control groups had decreased levels of lymphocytes and other immunesystem related blood components. Over the short treatment period, these numbers did notchange. In further studies, we predict HSP70 will begin to repair these numbers as the liverheals.Table 14LOR: Low (Outside of Range) LWR: Low (Within Range)Average:(Near Mean) HWR: High (Within Range) HOR: High (Outside of Range)ExpandedC57BL / 6, I Average Average HSP70- Average EmptyToxicologyLevels LNP Dosed LNPDosedEndpoint EndpointALP (U / L) 111-275 69-LOR 81 - LORAST (U / L) 46-392 202 - Average 71 - LWRALT (U / L) 28-129 54 -Average 36.5 - LWRCholesterol (mg / dL) 69-169 130 - Average 162.5 -HWR7504-00202 GRH-00462Glucose (mg / dL) 172-372 372-HWR 543.5 -HORHDL (mg / dL) 57-75 74-HWR 84.5 -HORLDL (mg / dL) 2.5-4.1 8-HOR 15 -HORThe above blood work data displays the ability of HSP70-LNPs to rescue liverfunction inside of NASH affected mice. HSP70-LNPs successfully brought AST and ALTlevels back to normal ranges as well as massively lowering glucose and cholesterol in thesemice. This suggests that HSP70-LNPs anti-inflammatory abilities are able to revitalize theliver and return it to its normal functioning levels.Example 17: MS-NASH Complete Liver Images:Following the full hepatectomy of the HSP70-LNP treated MS-NASH mice and theEmpty LNP treated MS-NASH mice, full liver images were taken to display changes in fatbuild up in the livers. HSP70-LNP treated MS-NASH livers lacked the liver yellowing andfat spots clearly seen on the control empty LNP treated MS-NASH livers. Figures 24-26display the experimental HSP70-LNP treated livers and Figures 27-29 display the controlEmpty LNP treated livers.Example 18: MS-NASH Liver ELISA DataIn order to determine how much HSP70 mRNA-LNPs were increasing HSP70expression in the livers of mice, an ELISA was run on the livers of MS-NASH mice. Thisassay was run with a commercially available HSP70 sandwich ELISA kit from ThermoFisher Scientific. The kit recommended procedure was used for this assay. Tissue sampleswere prepped by first removing the livers of MS-NASH mice (both mice dosed with HSP70mRNA-LNPs and empty LNPs) and storing them at -80°C until the ELISA was run. Thelivers were then placed in a flat bottom conical tube and 1 mL of PBS was added. Then atissue homogenizer was used to blend the organs and PBS for at least 30 seconds, or until novisible tissue chunks were left (the organ was uniformly blended and mixed with the 1 mL ofadded PBS). The tubes were then spun in a centrifuge set to 2, 000-2, 500xg for 5-7 minutes.After the centrifugation, the tubes were inspected to see a visual separation of organ solidsand supernatant. 50 μL of the supernatant was added to 50 μL of antibody diluent in thecorrect wells of a 96 well plate coated with an anti- human HSP70 antibody. As the tissuesamples are added to the 96 well plate, the HSP70 standards are also added to the 96 wellplate. After all samples and standards were added, the ELISA was run according to kitspecifications. See table 15 for results.7504-00202 GRH-00462Table 15Sample Average HSP70 Concentration (ng / mL)HSP70 mRNA-LNP Dosed 9.45Empty LNP Dosed 4.8The ELISA data above shows that HSP70 mRNA-LNPs can almost double theconcentration of HSP70 found in the livers of MS-NASH mice. The concentration pairedwith the whole organ images and H&E stained images suggests that the cause of the liver'shealing and returning to normal functioning is the increased expression of HSP70 in the liverscaused by the HSP70 mRNA-LNPs.INCORPORATION BY REFERENCEAll publications and patents mentioned herein are hereby incorporated by referencein their entirety as if each individual publication or patent was specifically and individuallyindicated to be incorporated by reference. In case of conflict, the present specification,including its specific definitions, will control. While specific aspects of the patient matterhave been discussed, the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon review of this specificationand the claims below. The full scope should be determined by reference to the claims, alongwith their full scope of equivalents, and the specification, along with such variations.EQUIVALENTS The present invention has been described in connection with what are presentlyconsidered to be the most practical and preferred embodiments. However, the invention hasbeen presented by way of illustration and is not intended to be limited to the disclosedembodiments. Accordingly, one of skill in the art will realize that the invention is intended toencompass all modification and alternative arrangements within the spirit and scope as setforth in the appended claims.

Claims

7504-00202 GRH-00462What is claimed is:

1. A method of treating inflammation or an inflammatory disease in a subject,comprising administering to the subject a composition comprising a nucleic acidformulated in a lipid nanoparticle (LNP), wherein the nucleic acid comprises an openreading frame encoding heat shock protein polypeptide, or a functional fragment thereof.

2. The method of claim 1, where in the heat shock protein polypeptide is HSP 100,HSP9O, HSP70, HSP60, HSP40, or HSP27.

3. The method of claim 1, wherein the open reading frame is derived from the nucleicacid sequence set forth in SEQ ID NO. 2, or a functional fragment thereof.

4. The method of claim 1, wherein the nucleic acid is mRNA, optionally wherein themRNA comprises the nucleic acid sequence set forth in any one of SEQ ID NO. 6, SEQ IDNO. 10, SEQ ID NO. 14, SEQ ID NO. 18, or any functional fragment thereof.

5. The method of any one of claims 1-4, wherein one or more uridine nucleosides inthe nucleic acid are pseudouridine.

6. The method of claim 5, wherein pseudouridine is Nl- methylpseudouridine.

7. The method of any one of claims i-6, wherein the LNP comprises an ionizablelipid, a phospholipid, a sterol, a PEG-modified lipid, or any combination thereof.

8. The method of any one of claims 1-6, wherein the LNP consists essentially ofan ionizable lipid, a phospholipid, a sterol, a PEG-modified lipid, or any combinationthereof.

9. The method of claim 7 or 8, wherein the ionizable lipid is SM-102.

10. The method of any one of claims 7-9, wherein thephospholipid is distearoylphosphatidy!choline (DSPC).

11. The method of any one of claims 7-10, wherein the sterol is cholesterol.7504-00202 GRH-0046212. The method of any one of claims 7-11, wherein the PEG-modified lipid is DMG-PEG 2000.

13. The method of any preceding claim, wherein the method comprises administering acell comprising the LNP.

14. The method of claim 13, wherein the cell expresses the HSP70 polypeptide encodedby anmRNA.

15. The method of claim 14, wherein the HSP70 polypeptide comprises the aminoacid sequence set for in SEQ ID NO. 1, or a functional fragment thereof.

16. The method of any one of claims 13-15, wherein the cell is an endothelial cell,epithelial cell, neuronal cell, non-neuronal cell, or haematopoietic cell.

17. The method of claim 16, wherein the epithelial cell is a parenchymal or non-parenchymal cells of an organ.

18. The method of claim 17, wherein the epithelial cell is a hepatocyte.

19. The method of claim 16, wherein the haematopoietic cell is an immune cell selectedfrom a lymphocyte, a monocyte, a dendritic cell, a mast cell, a neutrophil, a basophil, or aneosinophil.

20. The method of claim 19, wherein the immune cell is lymphocyte selected from aaBT cell, y8T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, a B cell,an innate lymphoid cell (ILC), a cytokine induced killer (CIK) cell, a cytotoxic Tlymphocyte (CTL), a lymphokine activated killer (LAK) cell, or a regulatory T cell.

21. The method of any one of claims 13-20, wherein the cell is a cell derived frombone marrow.

22. The method of any one of claims 13-16, wherein the cell is a cell of the centralnervous system (CNS) or peripheral nervous system (PNS).

23. The method of any one of claims 13-22, wherein the cell is a cell present in the CNS.7504-00202 GRH-0046224. The method of 22 or 23, wherein the cell is a neuronal cell.

25. The method of claim 24, wherein the nerve cell is a sensory neuron, a motorneuron, or an interneuron.

26. The method of claim 22 or 23, wherein the cell is a non-neuronal cell.

27. The method of claim 26, wherein the non-neuronal cell is a glial cell.

28. The method of claim 27, wherein the glial cell is an astrocyte cell, anoligodendrocyte. cell, an ependymal cell, a radial glial cell, a Schwann cell, a satellite cell,an enteric glial cell, or a microglial cell.

29. The method of any preceding claim, wherein the inflammatory disease ischaracterized by the inflammation of a tissue and / or organ of the body, includingmusculoskeletal inflammation, vascular inflammation, neural inflammation, digestive systeminflammation, ocular inflammation, hepatic inflammation, inflammation of the reproductivesystem, and other inflammation.

30. The method of claim 29, wherein the inflammatory disease is characterized byhepatic inflammation.

31. The method of claim 30, wherein the inflammatory disease is Non-Alcoholic FattyLiver Disease (NAFLD) or Non-Alcoholic Steatohepatitis (NASH).

32. The method of any one of the preceding claims, wherein the composition isadministered systemically or directly to a tissue or organ of the subject.

33. The method of claim 32, wherein the tissue or organ is musculoskeletal, vascular,nervous, gastrointestinal, ocular, hepatic, reproductive.

34. The method of claim 32 or 33, wherein the organ is the liver.

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