Compositions and methods for modulating hsp70 activity

Abstract

In certain aspects, provided herein are therapeutic compositions comprising an mRNA formulated in a lipid nanoparticle (LNP), wherein the mRNA comprises an open reading frame encoding an HSP70 polypeptide, and methods of using the same.

Description

[0001]GRH-00125 COMPOSITIONS AND METHODS FOR MODULATING HSP70ACTIVITYCROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of priority to U.S. Provisional Patent Applicationserial number 63 / 528239, filed July 21, 2023, and U.S. Provisional Patent Application serialnumber 63 / 432471, filed December 14, 2022, each of which is hereby incorporated by referencein its entirety.BACKGROUND As populations age, neurodegenerative diseases become a significant concern and burden,particularly an economic burden associated with medications, hospitalization, and potential high-risk groups. Among such neurodegenerative diseases, Alzheimer’s disease (AD) is the main typeof dementia in the elderly. Epidemiological studies indicate that around 10% of the populationover 65 years and 50% of individuals over 90 years suffer from Alzheimer’s disease. ThoughAlzheimer’s disease is the most common form of dementia, several other clinically relevant, non-Alzheimer’s disease dementias (non-AD dementias) affect around 40% of dementia sufferers.The pathology of Alzheimer’s disease comprises two principal hallmark lesions; extracellularamyloid plaques composed of A-β peptide derived from proteolytic processing of the amyloidprecursor protein (APP), and the intracellular neurofibrillary tangles (NFT) formed by theaggregation of tau protein. The metabolic cascades of each protein continue to be the target ofinvestigation and debate in the context of Alzheimer’s Disease pathogenesis, post-mortemdiagnostic criteria for, and therapeutic intervention. Notably, many neurodegenerative diseasescan be characterized by the accumulation / deposition of misfolded and / or insoluble proteins.Advances in molecular biology and neuropathology have allowed for the classification of aplurality of proteinopathies. Yet, despite such advances, the available therapies for subjectssuffering from Alzheimer’s disease (and proteinopathies in general) are very limited. Hence,there is an unmet need for new and efficacious treatments.SUMMARY Aspects of the invention, as provided herein, include therapeutic compositions comprisingan mRNA formulated in a lipid nanoparticle (LNP), wherein the mRNA comprises an openreading frame encoding heat shock protein polypeptide, or a functional fragment thereof. In someembodiments of the invention, the heat shock protein polypeptide, or a functional fragmentthereof is HSP100, HSP90, HSP70, HSP60, HSP40, or HSP27. Preferably, the heat shock proteinpolypeptide is HSP70. In some embodiments, the LNP is a solid lipid nanoparticle (SLN).FH11736604.11 GRH-00161 In certain aspects of the invention, provided herein are cells comprising the LNPsdisclosed herein.In some aspects of the invention, provided herein are methods of treating aneurodegenerative disease in a subject, the method comprising administering the therapeuticcompositions disclosed herein. In some such embodiments, the composition comprises the LNP-comprising cells disclosed herein.BRIEF DESCRIPTION OF FIGURESFigure 1 shows a data sheet for a single run of Dynamic Light Scattering (DLS).Figure 2 shows a DLS data sheet that includes an overlay of a triplicate test run.Figure 3 shows 24-hour cell viability assay results.Figure 4 shows 48-hour cell viability assay results.Figure 5 depicts data for nucleic acid encapsulation by LNPs. Panel A shows Dose w / Triton X. Panel B, Control w / Triton X; panel C, LNP control with no mRNA.Figure 6 depicts four cDNA synthesis runs of the HSPA1A gene, which codes for themRNA of HSP-70, based on RNA isolated from an HBEC3-KT cell line.Figure 7 depicts six separate HSP-70 mRNA synthesis runs that yielded identical resultsand produced mRNA for HSP-70 at 2,100 bases long in high concentrations.DETAILED DESCRIPTIONGeneralHeat shock proteins (HSPs) represent a class of molecular chaperones known to beexpressed in response to exposure to stressful conditions, such as heat, cold, ultraviolet light,during 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 and theaggregation processes. HSPs may be classified into families on the basis of molecular weight, forexample HSP100, HSP90, HSP70, HSP60, HSP40, and HSP27, each playing a diverse role ininfluencing proper protein assembly, folding, and translocation. For instance, HSP70, HSP60,and HSP27 are known to prevent protein aggregation and help protein folding; HSP100 releasesproteins from aggregates; and HSP90 plays a role in maturation and activation of a number ofproteins.Therefore, HSPs are expected to have strong potential as therapeutic agents insuppressing or treating a range of diseases associated with proteinopathy, including cancer,neurodegeneration, allograft rejection, and infection. In such diseases or disease states, proteins2FH11736604.1 GRH-00161fail to fold into their normal configuration and in this mis-folded state the proteins can becometoxic or lose their normal function (e.g., amyloid plaques and neurofibrillary tangles (NFT)).The 70 kilodalton heat shock proteins (Hsp70s or DnaK) HSP70 are a family ofadenosine triphosphatases that represent the most structurally and functionally conserved proteinsamongst HSPs. HSP70 is also the most ubiquitous class of chaperone protein, inducingcytoprotective effects under a number of different conditions, primarily in cellular protein qualitycontrol (PQC) and degradation systems. Briefly, in humans the HSP70 multigene family acts onnonnative polypeptides, fueled by ATP binding and hydrolysis. The HSP70 chaperone binds toprotein substrates (e.g., nascent or misfolded protein) to assist with folding, re-folding,reactivation, degradation, transport, regulation, and aggregation prevention.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. The NBDcomprises two lobes, forming a cleft that binds ATP with a nucleotide binding cassette that isrelated to those in actin and hexokinase. The SBD comprises a ^^-sandwich subdomain harboringthe substrate binding site, and an ^^-helical lid. Both these domains are critical for chaperonefunction and are connected by a short flexible linker. 3FH11736604.1 GRH-00161 As a molecular chaperone, HSP70 also has multiple responsibilities during normal growth. It isintegral to the folding of newly synthesized proteins, the subcellular transport of proteins andvesicles, the formation and dissociation of complexes, and degradation of unwanted proteins. Incarrying out these diverse functions HSP70 adopts different conformations, e.g., in the absenceof nucleotide, when bound with ADP, or when bound with ATP. In addition, the functions ofHSP70 rely on crosstalk between the SBD and NBD, with ATP influencing substrate binding.The cycle of 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. Thecompositions and methods of the present disclosure rely, at least in part, on the delivery of heatshock protein-encoding nucleic acids (e.g., HSP70-encoding nucleic acids) to cells of a subject inneed thereof (e.g., a gene therapy composition). For example, and without limitation,compositions comprising the heat shock protein-encoding nucleic acids disclosed herein can beused to treat a proteinopathy. In some embodiments, said compositions may be used to treatcancers, neurodegenerative diseases, allograft rejection, and / or infection.Aspects of the invention, as provided herein, include therapeutic compositions comprisingan 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 protein polypeptide,or a functional fragment thereof. In some embodiments, the heat shock protein polypeptide, orfunctional fragment thereof is selected from HSP100, HSP90, HSP70, HSP60, HSP40, orHSP27. Preferably, the heat shock protein polypeptide, or functional fragment thereof is HSP70.In some embodiments, the open reading frame is derived from the nucleic acid sequenceset forth in SEQ ID NO. 2, or a functional fragment thereof. For example, and without limitation,the mRNA 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.4FH11736604.1 GRH-00161 In some aspects, provided herein are nucleic acids encoding the HSP70 polypeptidesdisclosed 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-based amplification techniques). In some such embodiments,the primers are selected from 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 usingany one of the forward and reverse primer pairs set forth in: SEQ ID NOs. 3 and 5, SEQ ID NOs.4 and 5, SEQ ID NOs. 7 and 9, SEQ ID NOs. 8 and 9, SEQ ID NOs. 11 and 13, SEQ ID NOs. 12and 13, SEQ ID NOs. 15 and 17, and SEQ ID NOs. 16 and 17. One or more of the uridinenucleosides in the isolated / amplified mRNA is a pseudouridine, such as, N1-methylpseudouridine. In some such embodiments, all of the uridine nucleosides in the mRNA arepseudouridine, e.g., N1-methylpseudouridine. For example, and without being bound by theoryor methodology, the aforementioned nucleic acids are amplified from whole cell lysate. In someembodiments, the whole transcriptome mRNA is isolated from cell lysate. In some suchembodiments cDNAs of interest, e.g., double-stranded cDNA encoding HSP-70, are synthesizedfrom the isolated mRNA, .e.g., targeted amplification of sequence from forward and reverseprimer pairs disclosed herein, such as in reverse transcription-polymerase chain reaction (RT-PCR). The resultant double-stranded cDNA is used for mRNA synthesis. Without being boundby theory, and for the purpose of exemplification, the mRNAs to be incorporated in the LNPsdisclosed herein may be synthesized by targeted T7 amplification of mRNA sequence from thecDNA encoding HSP-70, e.g., using primers disclosed herein. In some preferred embodiments, aone-step RT-PCR reaction is used, e.g., reverse transcription and PCR are performed in a singlereaction mixture. Alternatively, the mRNA is synthesized from a plasmid template by methodsknown in the art.In other aspects, provided herein are vectors comprising the nucleic acids contemplatedherein. In some such embodiments, the vector is selected from nanoparticles, adenovirus vectors,adeno-associated virus (AAV) vectors, retrovirus vectors, picorna virus 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., theSLNs) disclosed herein. In certain aspects, provided here are cells comprising the vectorsdisclosed herein. In further aspects, provided herein are cells expressing the heat shock protein(e.g., HSP70 polypeptides) disclosed herein. For example and without limitation, the cell is anendothelial cell, epithelial cell, neuronal cell, or hematopoietic cell. In some such embodiments,the hematopoietic cell is an immune cell selected from a lymphocyte, a monocyte, a macrophage,5FH11736604.1 GRH-00161a dendritic cell, a mast cell, a neutrophil, a basophil, or an eosinophil. In certain embodiments,the immune cell is a lymphocyte selected from a an αβT cell, γδT 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 T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, or aregulatory T cell.In certain embodiments, the cells contemplated herein are cells of the central nervoussystem (CNS) or peripheral nervous system (PNS). In other embodiments, the cell is a cell of thebone marrow.In some embodiments, the cell contemplated herein is a cell present in the CNS. In someembodiments, the cell is a neuronal cell. Said neuronal cell may be a sensory neuron, a motorneuron, or an interneuron. In other embodiments, the cell is a non-neuronal cell. In someembodiments, the non-neuronal cell is a glial cell. The glial cell may be an astrocyte cell, anoligodendrocyte cell, an ependymal cell, a radial glial cell, a Schwann cell, a satellite cell, anenteric glial cell, or a microglial cell.In some embodiments, the nucleic acids contemplated herein may refer to a polymericform of nucleotides or nucleosides of any length, such as deoxyribonucleotides orribonucleotides, or analogs thereof. Nucleic acids may have any three-dimensional structure, andmay perform any function. The following are non-limiting examples of Nucleic acids: coding ornon-coding regions of a gene or gene fragment, loci (locus) defined from linkage analysis, 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 acidprobes, and primers. A nucleic acid may comprise modified nucleotides / nucleosides, such asmethylated nucleotides / nucleosides and nucleotide / nucleoside analogs. If present, modificationsto the polynucleotide / polynucleoside structure may be imparted before or after assembly of thepolymer. A polynucleotide / polynucleoside may be further modified, such as by conjugation witha labeling component.Aspects include therapeutic compositions comprising the mRNAs disclosed herein. Insome embodiments, the therapeutic composition comprises a vector selected from nanoparticles,adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, picorna virusvectors, liposomes, cationic lipid systems, and protein / nucleic acid complexes. For example andwithout limitation, the therapeutic composition comprising an mRNA may be formulated in alipid nanoparticle (LNP). In some embodiments of the therapeutic composition, one or more ofthe uridine nucleosides in the mRNA are pseudouridine, such as, N1-methylpseudouridine. Insome such embodiments, all of the uridine nucleosides in the mRNA are pseudouridine, e.g., N1-6FH11736604.1 GRH-00161methylpseudouridine. In some embodiments of the therapeutic composition, the LNP (e.g., theSLN) comprises an ionizable lipid, a structural lipid, a phospholipid, a sterol, a PEG-modifiedlipid, or any combination thereof.In some aspects, provided herein are methods of treating a cancer in a subject, the methodcomprising administering an effective amount of a therapeutic composition contemplated herein.In some embodiments, the cancer is selected from: hepatocellular carcinoma, lymphoma, B celllymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladdercancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma ofhead and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small celllung cancer, neuroblastoma / glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skincancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung,endometrial cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renalcancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neckcarcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers,prostatic 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 therapeutic antibody,such as aducanumab. The immunotherapy may comprise administration of an immunecheckpoint inhibitor. In some such embodiments, the immune checkpoint inhibitor comprises anantibody or antigen-binding fragment thereof specific for PD-1, PD-L1, or CTLA4. In certainembodiments, the cancer immunotherapy comprises administration of a CAR-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 glutamate regulator,such as memantine.Definitions For convenience, certain terms employed in the specification, examples, and appendedclaims are collected here.7FH11736604.1 GRH-00161 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” means oneelement 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% around the value (X ± 10%).Ranges are stated in shorthand to avoid having to set out at length and describe each andevery value within the range. Therefore, when ranges are stated for a value, any appropriate valuewithin the range can be selected, and these values include the upper value and the lower value ofthe range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as wellas the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate rangesencompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc.“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 any conventionalmedia or agent is incompatible with angiotensin II, its use in the pharmaceutical formulations ofthe invention is contemplated. In certain embodiments, the pharmaceutically acceptablecarrier / 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 or nucleicacid described herein.As used herein, the term “treatment” refers to clinical intervention designed to alter thenatural course of the individual being treated during the course of clinical pathology. Desirableeffects of treatment include decreasing the rate of progression, ameliorating or palliating thepathological state, and remission or improved prognosis of a particular disease, disorder, orcondition. An individual is successfully “treated,” for example, if one or more symptomsassociated with a particular disease, disorder, or condition are mitigated or eliminated.As used herein, a therapeutic that “prevents” a condition refers to a compound that, whenadministered to a statistical sample prior to the onset of the disorder or condition, reduces theoccurrence of the disorder or condition in the treated sample relative to an untreated control8FH11736604.1 GRH-00161sample, or delays the onset or reduces the severity of one or more symptoms of the disorder orcondition 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 or moredifferent therapeutic agents such that the second agent is administered while the previouslyadministered therapeutic agent is still effective in the body (e.g., the two agents aresimultaneously effective in the subject, which may include synergistic effects of the two agents).For example, the different therapeutic compositions disclosed herein can be administered eitherin the same formulation or in separate formulations, either concomitantly or sequentially. Incertain embodiments, the different therapeutic agents (e.g., a therapeutic composition comprisingan mRNA disclosed herein and an immunotherapy or standard-of-care treatment (e.g., standard-of-care treatment for a neurodegenerative disease, such as Alzheimer’s disease)) can beadministered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48hours, about 72 hours, or about a week of one another. Thus, a subject who receives suchtreatment can benefit from a combined effect of different therapeutic 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 to thereference polypeptide itself, but where the remaining amino acid sequence is usually identical tothat of the reference polypeptide. Such deletions may occur at the amino-terminus or carboxy-terminus of the reference polypeptide, or alternatively both. Fragments typically are at least about5, 6, 8 or 10 amino acids long, at least about 14 amino acids long, at least about 20, 30, 40 or 50amino acids long, at least about 75 amino acids long, or at least about 100, 150, 200, 300, 500 ormore amino acids long. A fragment can retain one or more of the biological activities of thereference polypeptide. In various embodiments, a fragment may comprise an enzymatic activityand / or an interaction site of the reference polypeptide. In some embodiments, a fragment mayhave suppressive, disruptive, or enhancing properties.Nucleic acids and vectors Nucleic 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 recombinant methodsknown in the art. Alternatively, the sequence of interest can be produced synthetically, ratherthan cloned.9FH11736604.1 GRH-00161 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, miRbinding site(s), polyA tail, as described herein). Suitable mRNA construct components are asdescribed herein. In some embodiments, a nucleic acid of the disclosure may be modified in acoding region (e.g., an open reading frame of an mRNA encoding a polypeptide). In otherembodiments, nucleic acid may be modified in regions besides a coding region, such as, 5' cap, a5’-untranslated region (UTR) and / or a 3’- UTR, polyA tail of an mRNA, wherein anycombination of elements may be independently modified. In some embodiments, such regionsmay contain one or more different nucleoside modifications. In such embodiments, modificationsmay also be present in the coding region.Examples of nucleoside modifications and combinations thereof that may be present inmRNAs disclosed herein include, but are not limited to, those described in PCT PatentApplication Publications: WO2012045075, WO2014081507, WO2014093924, WO2014164253,WO2014159813, WO2018144775, WO2018081459, each of which are incorporated herein intheir entirety.In some embodiments, the mRNAs of the disclosure can include a combination ofmodifications to the sugar, the nucleobase, and / or the internucleoside 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 nucleoside describedherein. In another non-limiting example, the natural nucleoside uridine may be partiallysubstituted (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 natural uridines)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 promoter, andincorporating the construct into an expression vector. Typical cloning vectors containtranscription and translation terminators, initiation sequences, and promoters useful for regulationof the expression of the desired nucleic acid sequence.The disclosed nucleic acids can be cloned into a number of types of vectors. For example,the nucleic acid can be cloned into a vector including, but not limited to a plasmid, a phagemid, aphage derivative, an animal virus, and a cosmid. Vectors of particular interest include expressionvectors, replication vectors, probe generation vectors, and sequencing vectors.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 et al.(2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York),10FH11736604.1 GRH-00161and in other virology and molecular biology manuals. Viruses, which are useful as vectorsinclude, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpesviruses, and lentiviruses. In general, a suitable vector contains an origin of replication functionalin at least one organism, a promoter sequence, convenient restriction endonuclease sites, and oneor more selectable markers. In some embodiments, the polynucleotide vectors are lentiviral orretroviral vectors.A number of viral based systems have been developed for gene transfer into mammaliancells. For example, retroviruses and AAVs provide a convenient platform for gene deliverysystems. A selected gene can be inserted into a vector and packaged in viral particles usingtechniques known in the art. The recombinant virus can then be isolated and delivered to cells ofthe 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 sequence capableof driving high levels of expression of any polynucleotide sequence operatively linked thereto.Another example of a suitable promoter is Elongation Growth Factor-1α (EF-1α). However,other constitutive promoter sequences may also be used, including, but not limited to the simianvirus 40 (SV40) early promoter, MND (myeloproliferative sarcoma virus) promoter, mousemammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat(LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virusimmediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoterssuch as, but not limited to, the actin promoter, the myosin promoter, the hemoglobin promoter,and the creatine kinase promoter. The promoter can alternatively be an inducible promoter.Examples of inducible promoters include, but are not limited to a metallothionine promoter, aglucocorticoid promoter, a progesterone promoter, and a tetracycline promoter.Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptionalinitiation. Typically, these are located in the region 30-110 base pairs (bp) upstream of the startsite, although a number of promoters have recently been shown to contain functional elementsdownstream of the start site as well. The spacing between promoter elements frequently isflexible, so that promoter function is preserved when elements are inverted or moved relative toone 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 expressing cellsfrom the population of cells sought to be transfected or infected through viral vectors. Theselectable marker may be carried on a separate piece of DNA and used in a co-transfection11FH11736604.1 GRH-00161procedure. Both selectable markers and reporter genes may be flanked with appropriateregulatory sequences to enable expression in the host cells. Useful selectable markers include, forexample, antibiotic-resistance genes.Reporter genes may be used for identifying potentially transfected cells and for evaluatingthe functionality of regulatory sequences. In general, a reporter gene is a gene that is not presentin or expressed by the recipient organism or tissue and that encodes a polypeptide whoseexpression is manifested by some easily detectable property, e.g., enzymatic activity. Expressionof the reporter gene is assayed at a suitable time after the nucleic acid has been introduced intothe recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the greenfluorescent protein gene. Suitable expression systems are well known and may be prepared usingknown techniques or obtained commercially. In general, the construct with the minimal 5′flanking region showing the highest level of expression of reporter gene is identified as thepromoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents forthe 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, the expressionvector can be transferred into a host cell by physical, chemical, or biological means.Physical methods for introducing a polynucleotide into a host cell include calciumphosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, andthe like. Methods for producing cells comprising vectors and / or exogenous nucleic acids arewell-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: ALaboratory Manual, Cold Spring Harbor Laboratory, New York).Biological methods for introducing a polynucleotide of interest into a host cell include theuse of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become themost widely used method for inserting genes into mammalian, e.g., human cells.Chemical means for introducing a polynucleotide into a host cell include colloidaldispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes.An exemplary colloidal system for use as a delivery vehicle in vitro and in vivo is a liposome(e.g., an artificial membrane vesicle).In the case where a non-viral delivery system is utilized, an exemplary delivery vehicle isa liposome. In another aspect, the nucleic acid may be associated with a lipid. The nucleic acidassociated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed12FH11736604.1 GRH-00161within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that isassociated with both the liposome and the oligonucleotide, entrapped in a liposome, complexedwith a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with alipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwiseassociated with a lipid. Lipid, lipid / nucleic acid or lipid / expression vector associatedcompositions are not limited to any particular structure in solution. For example, they may bepresent in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simplybe interspersed in a solution, possibly forming aggregates that are not uniform in size or shape.Lipids are fatty substances, which may be naturally occurring or synthetic lipids. For example,lipids include the fatty droplets that naturally occur in the cytoplasm as well as the class ofcompounds, which contain long-chain aliphatic hydrocarbons and their derivatives, such as fattyacids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtainedfrom commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can beobtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & KLaboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring;dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti PolarLipids, Inc, (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. Nature Nanotech.2, 751-760 (2007), WO2018144775, and WO2018081459, each of which are incorporated hereinby reference in their entirety. In certain embodiments, an mRNA of the disclosure is encapsulatedwithin a nanoparticle. In particular embodiments, a nanoparticle is a particle having at least onedimension (e.g., a diameter) less than or equal to 1000 nanometers (nm), less than or equal to 500nm or less than or equal to 100 nm. In particular embodiments, a nanoparticle includes lipids.Lipid nanoparticles (LNPs) include, but are not limited to, solid lipid nanoparticles (SLNs),liposomes, and micelles. For example, and without limitation, the nucleic acids described herein(e.g., mRNAs) are formulated as a solid lipid nanoparticle (SLN), which can be spherical with anaverage diameter between 10 to 1000 nm. In some such embodiments, the SLN possesses a solidlipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactantsand / or emulsifiers. Exemplary SLN can be those as described in Inti. Pub. No. WO2013105101,herein incorporated 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. Such13FH11736604.1 GRH-00161lipids can be used alone or in combination. In certain embodiments, a lipid nanoparticlecomprises one or more nucleic acids, e.g., mRNAs, described herein. In certain embodiments, itis desirable to target a nanoparticle, e.g., a lipid nanoparticle, of the disclosure using a targetingmoiety that is specific to a cell type and / or tissue type. In some embodiments, a nanoparticle maybe targeted to a particular cell, tissue, and / or organ using a targeting moiety. In particularembodiments, a nanoparticle comprises one or more mRNA described herein and a targetingmoiety. Exemplary non-limiting targeting moieties include ligands, cell surface receptors,glycoproteins, vitamins (e.g., riboflavin) and antibodies (e.g., full-length antibodies, antibodyfragments (e.g., Fv fragments, single chain Fv (scFv) fragments, Fab′ fragments, or F(ab′)2fragments), single domain antibodies, camelid antibodies and fragments thereof, humanantibodies and fragments thereof, monoclonal antibodies, and multispecific antibodies (e.g.,bispecific antibodies)). In some embodiments, the targeting moiety may be a polypeptide. Thetargeting moiety may include the entire polypeptide (e.g., peptide or protein) or fragmentsthereof. A targeting moiety is typically positioned on the outer surface of the nanoparticle in sucha manner that the targeting moiety is available for interaction with the target, for example, a cellsurface receptor. A variety of different targeting moieties and methods are known and availablein the art, including those described, e.g., in Sapra et al., Prog. Lipid Res. 42(5):439-62, 2003 andAbra et al., J. Liposome Res. 12:1-3, 2002. For example, the lipid nanoparticle may include atargeting moiety 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,vascular smooth 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 metastatic tumorcells). In particular embodiments, the targeting moiety targets the lipid nanoparticle to ahepatocyte. In other embodiments, the targeting moiety targets the lipid nanoparticle to a coloncell. In some embodiments, the targeting moiety targets the lipid nanoparticle to a liver cancercell (e.g., a hepatocellular carcinoma cell) or a colorectal cancer cell (e.g., a primary tumor or ametastasis).In addition to nanoparticle compositions provided herein, also disclosed are methods ofproducing lipid nanoparticles, which may include encapsulating a polynucleotide (e.g., an mRNAcontemplated herein). Such contemplated methods comprise using any of the compositionsdisclosed herein and producing lipid nanoparticles in accordance with methods of production oflipid nanoparticles known in the art, e.g., Wang et al. (2015) “Delivery of oligonucleotides withlipid nanoparticles” Adv. Drug Deliv. Rev. 87:68-80; Silva et al. (2015) “Delivery Systems for14FH11736604.1 GRH-00161Biopharmaceuticals. Part I: Nanoparticles and Microparticles” Curr. Pharm. Technol. 16: 940-954; Naseri et al. (2015) “Solid Lipid Nanoparticles and Nanostructured Lipid Carriers:Structure, Preparation and Application” Adv. Pharm. Bull. 5:305-13; Silva et al. (2015) “Lipidnanoparticles for the delivery of biopharmaceuticals” Curr. Pharm. Biotechnol. 16:291-302, andreferences cited therein, all of which are incorporated herein by reference in their entirety. Incertain embodiments, lipid nanoparticles (LNPs) comprise lipids including an ionizable lipid, astructural lipid, a phospholipid, a stabilizing lipid, and one or more mRNAs. For example,without being bound by theory or methodology, a solid lipid nanoparticle (SLN) may include oneor more mRNAs. Thus, each of the LNPs described herein may be used in a formulationcomprising the mRNA described herein. In one embodiment, a lipid nanoparticle comprises anionizable lipid, a structural lipid, a phospholipid, a PEG-modified lipid and one or more mRNAs.In some embodiments, the LNP comprises an ionizable lipid, a PEG-modified lipid, a sterol and aphospholipid. In some embodiments, the LNP has a molar ratio of about 20-60% ionizablelipid:about 5-25% phospholipid:about 25-55% sterol; and about 0.5-15% PEG-modified lipid. Insome embodiments, the LNP comprises a molar ratio of about 50% ionizable lipid, about 1.5%PEG-modified lipid, about 38.5% cholesterol and about 10% phospholipid. In someembodiments, the LNP comprises a molar ratio of about 55% ionizable lipid, about 2.5% PEGlipid, about 32.5% cholesterol and about 10% phospholipid. In some embodiments, the ionizablelipid is an ionizable amino or cationic lipid and the neutral lipid is a phospholipid, and the sterolis a cholesterol.The ionizable lipids contemplated herein include cationic and / or ionizable lipids. Suchcationic 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)-N1,N1,4-tridodecyl-1-piperazineethanamine (KL10), N1-[2-(didodecylamino)ethyl]-N1,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-[1,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31-tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2-dimethylaminoethyl)-[1,3]-dioxolane (DLin-KC2-DMA), 2-({8-[(3β)-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-[(3β)-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-[(3β)-cholest-5-en-3-yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine(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-15FH11736604.1 GRH-00161dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (“DOTAP”); 1,2-Dioleyloxy-3-trimethylaminopropane chloridesalt (“DOTAP.C1”); 3-β-(N—(N′,N′-dimethylaminoethane)-carbamoyl)cholesterol (“DC-Chol”),N-(1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-ammoniumtrifluoracetate (“DOSPA”), dioctadecylamidoglycyl carboxyspermine (“DOGS”), 1,2-dioleoyl-3-dimethylammonium propane (“DODAP”), N,N-dimethyl-2,3-dioleyloxy)propylamine(“DODMA”), and N-(1,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammoniumbromide (“DMRIE”). Additionally, a number of commercial preparations of cationic and / orionizable lipids can be used, such as, e.g., LIPOFECTIN® (including DOTMA and DOPE,available from GIBCO / BRL), and LIPOFECTAMINE® (including DOSPA and DOPE,available from GIBCO / BRL). KL10, KL22, and KL25 are described, for example, in U.S. Pat.No. 8,691,750, which is incorporated herein by reference in its entirety. In particularembodiments, 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, phospholipids comprise aphospholipid 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 group consistingof lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleicacid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonicacid, 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 can allowone or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) topass through the membrane permitting, e.g., delivery of the one or more elements to a targettissue.Non-natural phospholipid species including natural species with modifications andsubstitutions including branching, oxidation, cyclization, and alkynes are also contemplated. Forexample, a phospholipid can be functionalized with or cross-linked to one or more alkynes (e.g.,an alkenyl group in which one or more double bonds is replaced with a triple bond). Underappropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloadditionupon exposure to an azide. Such reactions can be useful in functionalizing the surface (e.g., the16FH11736604.1 GRH-00161lipid monolayer or bilayer) of a nanoparticle composition to facilitate membrane permeation orcellular recognition or in conjugating a nanoparticle composition to a useful component such as atargeting 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 can compriseone or more structural lipids. As used herein, the term “structural lipid” refers to sterols and alsoto lipids containing sterol moieties.Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregationof other lipids in the particle. Structural lipids can be selected from the group including but notlimited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol,tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, andmixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein,“sterols” are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, thestructural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In certainembodiments, the structural lipid is an analog of cholesterol.The term “PEG-modified lipid” may refer to polyethylene glycol (PEG)-modified lipids.Non-limiting examples of PEG-lipids include PEG-modified phosphatidylethanolamine andphosphatidic acid, PEG-ceramide conjugates (e.g., PEG-CerC14 or PEG-CerC20), PEG-modified dialkylamines and PEG-modified 1,2-diacyloxypropan-3-amines. Such lipids are alsoreferred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG,PEG-DLPE, PEG-DMPE, PEG-DPPC, or a PEG-DSPE lipid.In some embodiments, the PEG-lipid includes, but 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-disteryl glycerol(PEG-DSG), PEG-dipalmetoleyl, PEG-dioleyl, PEG-distearyl, PEG-diacylglycamide (PEG-DAG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2-dimyristyloxlpropyl-3-amine (PEG-c-DMA). Preferably, the PEG-modified lipid is 1,2-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-modified17FH11736604.1 GRH-00161ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modifieddialkylglycerol, and mixtures thereof.In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths offrom about C14 to about C22, preferably from about C14 to about C16. In some embodiments, aPEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000or 20,000 daltons. In some embodiments, the PEG-lipid is PEG2k-DMG.In certain embodiments, the lipid nanoparticles described herein can comprise a PEGlipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs includePEG-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. WO 2015 / 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 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. Such speciesmay be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified withpolyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG-modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modifiedceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modifieddialkylglycerols, and mixtures thereof. For example, a PEG lipid may be DMG-PEG 2000, PEG-c-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-dimyristylamide of 2-hydroxyacetic acid, O-pegylated to a PEG chain mass of about 2kilodaltons.In certain embodiments, the LNPs comprise ALC-0315, a synthetic ionizable cationicamino lipid.“Stabilizing lipids”, as used herein, may include, but is not limited to, lipids that containsurface stabilizing polymers conjugated to the lipid headgroup. In some embodiments, thepolymer conjugated to the lipid headgroup is hydrophilic. The hydrophilic polymer-conjugatedlipid may be a polyethyleneglycol (PEG)-conjugated lipid. In other embodiments, the polymer18FH11736604.1 GRH-00161making up the polymer-lipid conjugate can be a polymer that contains a backbone that allows itto associate with the core of the particle thereby enhancing the stability of the delivery vehicle(e.g., poly(vinyl alcohol) conjugated to a lipid).PEG lipids may be used to stabilize the nanoparticle, e.g., in terms of making it invisibleto the immune system. Without being bound by theory, hydrophilic polymer PEG on the outersurface of the nanoparticle induces steric stabilization due to the local surface concentration ofhighly hydrated PEG groups. This attracts a water shell that surrounds the nanoparticle that actsas a barrier against certain interactions in the biological environment, e.g., making thenanoparticle less detectable by, or otherwise invisible to, the immune system, includinginhibition of adsorption and opsonization of the nanoparticle and its contents. Such nanoparticlesmay have reduced detection and destruction in the biological environment, and can lead toextended blood circulation time and a preferential accumulation at 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 (PI).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 LNP has a molar ratio of50:38.5:10:1.5 of SM-102:cholesterol:DSPC (Distearoylphosphatidylcholine): DMG-PEG 2000.In some such embodiments, the LNP is a solid lipid nanoparticle (SLN).CompositionsIn some aspects, provided herein is a composition (e.g., a pharmaceutical composition,such as a therapeutic or vaccine composition), containing the nucleic acids disclosed herein,formulated together with a pharmaceutically acceptable carrier, (e.g., a composition comprisingthe nanoparticles disclosed herein) as well as methods of administering such pharmaceuticalcompositions.In some embodiments, the nucleic acids, polypeptides, or compositions provided hereinare used as an adjuvant. As used herein, the term “adjuvant” broadly refers to an agent thataffects an immunological or physiological response in a patient or subject. For example andwithout limitation, when used as an adjuvant the polypeptides or compositions provided hereinmay increase the presence of an antigen over time or to an area of interest like a tumor, facilitateabsorption of a presented antigen, activate macrophages and lymphocytes, and / or support theproduction of cytokines. By changing an immune response, the adjuvant might permit a smallerdose of an immune interacting agent to increase the effectiveness or safety of a particular dose ofthe immune interacting agent. For example, the adjuvant might prevent T cell exhaustion andthus increase the effectiveness or safety of a particular immune interacting agent.19FH11736604.1 GRH-00161 Compositions contemplated herein may be administered intrapleurally, intravenously,subcutaneously, intranodally, intratumorally, intrathecally, intraperitoneally, intracranially, or bydirect administration to an organ. Said compositions may comprise one or more pharmaceuticallyacceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterile injectable solutions ordispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers,bacteriostats, solutes which render the formulation isotonic with the blood of the intendedrecipient or suspending or thickening agents.Examples of suitable aqueous and nonaqueous carriers which may be employed in thepharmaceutical compositions include water, ethanol, polyols (such as glycerol, propylene glycol,polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils, such as olive oil,and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example,by the use of coating materials, such as lecithin, by the maintenance of the required particle sizein the case of dispersions, and by the use of surfactants.In some embodiments, the administered dose size is about 100 µL to about 1000 µL orany intermediate value encompassed therein, particularly, about: 100 µL, 200 µL, 300 µL, 400µL, 500 µL, 600 µL, 700 µL, 800 µL, 900 µL, or 1000 µL. In some preferred embodiments, thedose size is 500 µL. In some such embodiments, the dose comprises an mRNA concentration ofabout 0.10 µg / µL to about 0.50 µg / µL or any intermediate value encompassed therein,particularly, about: 0.10 µg / µL, 0.20 µg / µL, 0.30 µg / µL, 0.40 µg / µL, or 0.50 µg / µL. Inpreferred embodiments, the dose comprises an mRNA concentration of 0.33 µg / µL. Forexample, without being bound by any particular theory or methodology, the dose size is 500 µL,composed of 330 µL of mRNA (concentration of 500 µg / mL, i.e., 165 µg) and 170 µL of ethanollipid nanoparticle solution. The ethanol may be removed after the particle formation via dialysisleaving the lipid nanoparticles containing mRNA in saline buffer alone. This may then be storedat -20°C until use.In other aspects, provided herein is a composition containing the cells comprising theLNPs (e.g., the SLNs) disclosed herein, carrying the nucleic acids encoding the heat shockproteins (e.g., HSP70 polypeptides) contemplated herein. The person of skill in the relevant artwill appreciate that such cells may be administered via the adoptive transfer of said cells to arecipient subject in need thereof, as is known in the art. Briefly, and without being limited bytheory, cells (selected from a third-party donor or cells derived from the recipient subject) may bebrought into contact with the LNPs provided herein (e.g., in vitro or ex vivo), and administered tothe subject in need, by means known in the art.Therapeutic Methods 20FH11736604.1 GRH-00161 In certain embodiments, provided herein are methods of treating a subject, comprisingadministering to the subject a therapeutic composition provided herein.In some embodiments, the methods provided herein are used to treat or prevent aneurodegenerative disease, e.g., a disease associated with the progressive loss of structure orfunction of neurons. Such diseases may include, without limitation, amyotrophic lateral sclerosis,multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiplesystem atrophy, and prion diseases (e.g., Creutzfeldt–Jakob disease (CJD)). Notably, severalneurodegenerative diseases are classified in the art as proteinopathies, being associated with theaggregation of misfolded proteins. A key mechanism of action in many neurodegenerativediseases is protein toxicity.Without being bound by theory, alpha-synuclein may aggregate into insoluble fibrilsresulting in Lewy bodies observed in Parkinson's disease, dementia with Lewy bodies, andmultiple system atrophy and may contribute to the amyloid plaques in Alzheimer's disease. Theterm synucleinopathies (a / k / a α-synucleinopathies) describes neurodegenerative diseases anddisorders characterized by the abnormal accumulation of aggregates of α-synuclein protein inneurons, nerve fibres, or glial cells. These diseases / disorders include Shy-Drager syndrome,striatonigral degeneration, olivopontocerebellar atrophy, neurodegeneration with brain ironaccumulation, type I (a / k / a neuroaxonal dystrophy or Hallervorden-Spatz syndrome), axonallesions after traumatic brain injury, Pick’s disease, and amyotrophic lateral sclerosis.Tau protein is the main component of neurofibrillary tangles in Alzheimer's disease andof Pick bodies found in behavioral variant frontotemporal dementia. Indeed, tauopathy describesa class of neurodegenerative diseases involving the aggregation of tau protein into neurofibrillaryor gliofibrillary tangles in the human brain, which includes primary age-related tauopathy(PART) dementia, chronic traumatic encephalopathy (CTE), progressive supranuclear palsy(PSP), corticobasal degeneration (CBD), frontotemporal dementia and parkinsonism linked tochromosome 17 (FTDP-17), vacuolar tauopathy, lytico-bodig disease (Parkinson-dementiacomplex of Guam), Ganglioglioma and gangliocytoma, meningioangiomatosis, postencephaliticparkinsonism, subacute sclerosing panencephalitis (SSPE), lead encephalopathy, tuberoussclerosis, pantothenate kinase-associated neurodegeneration, lipofuscinosis, Pick's disease,corticobasal degeneration, and argyrophilic grain disease (AGD).Those of skill in the art will recognize that tauopathies are often overlapped withsynucleinopathies.Misfolded Amyloid β and Aβ oligomers are major components of amyloid plaques inAlzheimer's disease. Misfolded PRNP proteins are the main component of prion diseases andtransmissible spongiform encephalopathy. In some embodiments, the disease is characterized by21FH11736604.1 GRH-00161disruption or dysregulation of protein degradation pathways (e.g., ubiquitin–proteasomepathways and autophagy–lysosome pathways), by membrane damage (e.g., tubulation andvesiculation as induced by alpha-synuclein), by mitochondrial dysfunction (e.g., the generationof reactive oxygen species (ROS), perturbation of calcium homeostasis, programmed cell death(PCD), mitochondrial fission and fusion, lipid concentration of mitochondrial membranes, andmitochondrial permeability transition).In some embodiments, the compositions and methods provided herein can be used to treatan autoimmune disease. Examples of autoimmune diseases include, for example, glomerularnephritis, arthritis, dilated cardiomyopathy-like disease, ulcerous colitis, Sjogren syndrome,Crohn disease, systemic erythematosus, chronic rheumatoid arthritis, juvenile rheumatoidarthritis, Still’s disease, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis,pachyderma, periarteritis nodosa, rheumatic fever, vitiligo vulgaris, Behcet disease, Hashimotodisease, Addison disease, dermatomyositis, myasthenia gravis, Reiter syndrome, Graves' disease,anaemia perniciosa, sterility disease, pemphigus, autoimmune thrombopenic purpura,autoimmune hemolytic anemia, active chronic hepatitis, Addison's disease, anti-phospholipidsyndrome, atopic allergy, autoimmune atrophic gastritis, achlorhydria autoimmune, celiacdisease, Cushing’s syndrome, dermatomyositis, discoid lupus erythematosus, Goodpasture'ssyndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia,insulin-dependent diabetes, Lambert-Eaton syndrome, lupoid hepatitis, lymphopenia, mixedconnective tissue disease, pemphigoid, pemphigus vulgaris, pernicious anemia, phacogenicuveitis, polyarteritis nodosa, polyglandular autosyndromes, primary biliary cirrhosis, primarysclerosing cholangitis, Raynaud’s syndrome, relapsing polychondritis, Schmidt's syndrome,limited scleroderma (or crest syndrome), sympathetic ophthalmia, systemic lupus erythematosus,Takayasu's arteritis, temporal arteritis, thyrotoxicosis, type b insulin resistance, type I diabetes,ulcerative colitis and Wegener's granulomatosis.In some embodiments, the methods provided herein are used to treat multiple sclerosis(MS). In some embodiments, the MS is relapsing-remitting MS, secondary progressive MS,primary progressive MS or progressively relapsing MS.In certain embodiments, the methods provided herein are used to treat rheumatoidarthritis, systemic lupus erythematosus and / or Sjögren’s syndrome.In some embodiments, the methods provided herein are used to treat inflammatory boweldiseases (IBDs). For example, in certain embodiments the methods provided herein are used totreat Crohn's disease (regional bowel disease, e.g., inactive and active forms), celiac disease (e.g.,inactive or active forms) and / or ulcerative colitis (e.g., inactive and active forms). In someembodiments, the methods provided herein are used to treat irritable bowel syndrome,22FH11736604.1 GRH-00161microscopic colitis, lymphocytic-plasmocytic enteritis, coeliac disease, collagenous colitis,lymphocytic colitis, eosinophilic enterocolitis, indeterminate colitis, infectious colitis (viral,bacterial or protozoan, e.g. amoebic colitis) (e.g., clostridium difficile colitis),pseudomembranous colitis (necrotizing colitis), ischemic inflammatory bowel disease, Behcet’sdisease, sarcoidosis, scleroderma, IBD-associated dysplasia, dysplasia associated masses orlesions, and / or primary sclerosing cholangitis.In some embodiments, the methods provided herein are used to treat a disease, disorder,condition, and / or illness associated with inflammation can include, but not limited to, septicshock, obesity-related inflammation, Parkinson's Disease, Crohn's Disease, Alzheimer's Disease(AD), cardiovascular disease (CVD), inflammatory bowel disease (IBD), chronic obstructivepulmonary disease, an allergic reaction, an autoimmune disease, blood inflammation, jointinflammation, arthritis, asthma, ulcerative colitis, hepatitis, psoriasis, atopic dermatitis,pemphigus, glomerulonephritis, atherosclerosis, sarcoidosis, rheumatoid arthritis, psoriaticarthritis, ankylosing spondylitis, Wegner's syndrome, Goodpasture's syndrome, giant cellarteritis, polyarteritis nodosa, idiopathic pulmonary fibrosis, acute lung injury, post-influenzapneumonia, SARS, tuberculosis, malaria, sepsis, cerebral malaria, Chagas disease,schistosomiasis, bacteria and viral meningitis, cystic fibrosis, multiple sclerosis,encephalomyelitis, sickle cell anemia, pancreatitis, transplantation, systemic lupuserythematosus, autoimmune diabetes, thyroiditis, and radiation pneumonitis, respiratoryinflammation, or pulmonary inflammation.In some embodiments, provided herein is a method of treating a viral or bacterialinfection in a subject. In some embodiments, the subject treated is immunocompromised. Forexample, in some embodiments, the subject has a T cell deficiency. In some embodiments, thesubject has leukemia, lymphoma or multiple myeloma. In some embodiments, the subject isinfected with HIV and / or has AIDS. In some embodiments, the subject has undergone a tissue,organ and / or bone marrow transplant. In some embodiments, the subject is being administeredimmunosuppressive drugs. In some embodiments, the subject has undergone and / or isundergoing a chemotherapy. In some embodiments, the subject has undergone and / or isundergoing radiation therapy.In some embodiments, the subject is also administered an antiviral drug that inhibits viralreplication. For example, in some embodiments, the subject is administered ganciclovir,valganciclovir, foscarnet, cidofovir, acyclovir, fomivirsen, maribavir, BAY 38-4766 orGW275175X.In some embodiments, the subject has cancer. In some embodiments, the methodsdescribed herein may be used to treat any cancerous or pre-cancerous tumor. In some23FH11736604.1 GRH-00161embodiments, the cancer includes a solid tumor. Cancers that may be treated by methods andcompositions provided herein include, but are not limited to, cancer cells from the bladder, blood,bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gum, head, kidney, liver,lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus. In addition, thecancer may specifically be of the following histological type, though it is not limited to these:neoplasm, malignant; carcinoma; carcinoma, undifferentiated; giant and spindle cell carcinoma;small cell carcinoma; papillary carcinoma; squamous cell carcinoma; lymphoepithelialcarcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillarytransitional cell carcinoma; adenocarcinoma; gastrinoma, malignant; cholangiocarcinoma;hepatocellular carcinoma; combined hepatocellular carcinoma and cholangiocarcinoma;trabecular adenocarcinoma; adenoid cystic carcinoma; adenocarcinoma in adenomatous polyp;adenocarcinoma, familial polyposis coli; solid carcinoma; carcinoid tumor, malignant;branchiolo-alveolar adenocarcinoma; papillary adenocarcinoma; chromophobe carcinoma;acidophil carcinoma; oxyphilic adenocarcinoma; basophil carcinoma; clear cell adenocarcinoma;granular cell carcinoma; follicular adenocarcinoma; papillary and follicular adenocarcinoma;non-encapsulating sclerosing carcinoma; adrenal cortical carcinoma; endometrioid carcinoma;skin appendage carcinoma; apocrine adenocarcinoma; sebaceous adenocarcinoma; ceruminousadenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillarycystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma;mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullarycarcinoma; lobular carcinoma; inflammatory carcinoma; mammary Paget’s disease; acinar cellcarcinoma; adenosquamous carcinoma; adenocarcinoma w / squamous metaplasia; malignantthymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor;and malignant neuroblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignantlipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma;pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficialspreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma;malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma;liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolarrhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor;nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignantBrenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma;dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii;choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignanthemangioendothelioma; Kaposi’s sarcoma; malignant hemangiopericytoma;24FH11736604.1 GRH-00161lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignantchondroblastoma; mesenchymal chondrosarcoma; giant cell tumor of bone; Ewing’s sarcoma;malignant odontogenic tumor; ameloblastic odontosarcoma; malignant ameloblastoma;ameloblastic fibrosarcoma; malignant pinealoma; chordoma; malignant glioma; ependymoma;astrocytoma; protoplasmic astrocytoma; fibrillary astrocytoma; astroblastoma; glioblastoma;oligodendroglioma; oligodendroblastoma; primitive neuroectodermal; cerebellar sarcoma;ganglioneuroblastoma; neuroblastoma; retinoblastoma; olfactory neurogenic tumor; malignantmeningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor;malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; smalllymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignantlymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignanthistiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease;leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cellleukemia; myeloid leukemia; basophilic leukemia; eosinophilic leukemia; monocytic leukemia;mast cell leukemia; megakaryoblastic leukemia; myeloid sarcoma; and hairy cell leukemia.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 activity ofthe particular agent employed, the route of administration, the time of administration, the rate ofexcretion or metabolism of the particular compound being employed, the duration of thetreatment, other drugs, compounds and / or materials used in combination with the particularcompound employed, the age, sex, weight, condition, general health and prior medical history ofthe 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 any convenientmanner, including by injection, transfusion, or implantation. The compositions described hereinmay be administered to a patient subcutaneously, intradermally, intratumorally, intranodally,intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In someembodiments, the disclosed compositions are administered to a patient by intradermal orsubcutaneous injection. In some embodiments, the disclosed compositions are administered byi.v. injection. The compositions may also be injected directly into a tumor, lymph node, organ, or 25FH11736604.1 GRH-00161site of disease or disorder. In certain embodiments, the disclosed compositions are administeredto a patient 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 compositions areconjointly administered with an immunotherapy. Such immunotherapies may comprise plaque-binding antibodies such as aducanumab. In some embodiments, the composition is conjointlyadministered to the subject with a cholinesterase inhibitor. The cholinesterase inhibitor may be,for example, donepezil, rivastigmine, or galantamine. In some embodiments, a glutamateregulator is administered conjointly with the compositions of the invention, such as the glutamateregulator memantine.In further embodiments, the compositions may be used in combination withchemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine,methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such asCAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin,FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In someembodiments, the said compositions are administered to a patient in conjunction with (e.g.,before, simultaneously or following) bone marrow transplantation, T cell ablative therapy usingeither chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT),cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In other embodiments, thecompositions disclosed herein are administered following B-cell ablative therapy such as agentsthat react with CD20, e.g., Rituxan. For example, in some embodiments, subjects may undergostandard treatment with high dose chemotherapy followed by peripheral blood stem celltransplantation. In certain embodiments, following the transplant, subjects receive an infusion ofthe cells disclosed herein. In additional embodiments, expanded cells are administered before orfollowing surgery.EXAMPLES Example 1: Composition of Lipid Nanoparticle The compositions contemplated herein may comprise four primary parts, an ionizablelipid, a phospholipid, a sterol, and a PEG-modified lipid. The LNPs are prepared by mixing anethanolic lipid mixture with an acidic aqueous buffer containing the oligonucleotides of interest.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),26FH11736604.1 GRH-00161 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.A dose size of 500 µL was composed of 330 µL of mRNA and 170 µL of ethanol / lipidnanoparticle solution. For example, an initial mRNA payload concentration of 500 µg / mL wasdiluted to achieve a desired mass ratio. In some embodiments, the mRNA to lipid mass-to-massratio is about 1:10. In some embodiments, the ethanol-to-water ratio may be about 1:3. Theethanol may be removed after the particle formation via dialysis leaving the lipid nanoparticlescontaining mRNA in PBS (phosphate buffered saline). This can then be run through a 220 nmfilter to remove any aggregation that may have occurred during the dialysis process.Ethanolic Lipid Mixtures The 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 range 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 to use, and the lipidmixture prepared as described in Table 1, which yielded 5 syntheses at a total volume of 4.0 mL.Amounts were calculated such that the ratio was kept at 50:10:38.5:1.5 molar ratio for ionizablelipid, DSPC, cholesterol, and PEG-lipid. Ethanol was used to dilute to the final volume. Forexample, 10.0 mg or DPSC was mixed with 400 µL ethanol, and so on, in accordance with Table1 below. The appropriate volume of each lipid mixture component, as listed in Table 1, was thentransferred to a single tube to prepare the ethanolic lipid mixture and pipetted several times toensure mixing and avoid precipitate and cloudiness.Table 1 27FH11736604.1 GRH-00161 Aqueous mRNA Solution:A lipid:mRNA (w:w) ratio of 10:1 and an ethanol:aqueous ratio of 1:3 was used. Utilizing0.50 mL from the 5.0 mL lipid mixture stock solution (e.g., for 1.5 g / L above), 75 μg mRNA wasadded to a separate tube and adjusted to a volume of 1.5 ml with 50 mM sodium acetate, 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 An intended payload for the LNPs was an mRNA strand which codes for the proteinHsp70 which is located on the gene HSPA1A. Utilizing specifically designed primers thefollowing sequence was amplified and applied to the lipid nanoparticles disclosed herein. 28FH11736604.1 GRH-00161 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.Example 3: Payload Afurther intended payload will be an mRNA strand encoding the protein Hsp70 specifiedby the following primer pairs. 29FH11736604.1 GRH-00161 30FH11736604.1 GRH-00161 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, optionallysubstituting some or all of the uridines with pseudouridines, as disclosed herein.Example 4: Payload III An additional payload contemplated herein is an mRNA strand encoding the proteinHsp70 specified by the following primer pairs. 31FH11736604.1 GRH-00161 The amplified mRNA sequence will be inserted into the LNPs of the invention. SuchmRNA sequences will comprise modifications to the 5’ cap, and, optionally substituting some orall of the uridines with pseudouridines, as disclosed herein.Example 5: Payload IV Another intended payload will be an mRNA strand encoding the protein Hsp70 specifiedby the following primer pairs. 32FH11736604.1 GRH-00161 The amplified mRNA sequence will be inserted into the LNPs of the invention. SuchmRNA sequences will comprise modifications to the 5’ cap, and, optionally substituting some orall of the uridines with pseudouridines, as disclosed herein.Example 6: Dynamic Light Scattering (DLS) In order for the LNP formulations disclosed herein to be used as an Alzheimer’streatment, each particle must be small enough to pass through the blood brain barrier. Particlesthat are less than 200 nm (and preferably about 100 nm) are able to efficiently pass through the33FH11736604.1 GRH-00161blood brain barrier (Ceña & Jávita, 2018). Doses of LNP-encapsulated mRNAs were tested witha Dynamic Light Scattering (DLS) machine. In DLS, when laser light encountersmacromolecules in a solution the incident light scatters in all directions and scattering intensity isrecorded by a detector. The rate of fluctuations in scattered light is directly related to the rate ofdiffusion of the particle through the solvent, which is related in turn to the particles'hydrodynamic radii. Smaller particles diffuse faster, causing more rapid fluctuations in theintensity than larger particles. Therefore, the fluctuation in light intensity contains informationabout the diffusion of the molecules and can be used to extract a diffusion coefficient andcalculate a particle size.Continuous DLS data collection showed that the LNP formulations disclosed hereinconsistently comprised particles with a diameter of 106 nm or smaller. In a typical DLS analysisthe Z-Average is the estimated average size of the particles being measured. The D50 shows that50% of the particles are the reported size or below. For example, Figure 1 shows that 50% of theparticles are estimated to be 98.4 nm or below in size (see Histogram Operations: % Cumulative(6), the aforementioned D50). The Count Rate (measured in kilo counts per second (kCPS))correlates with the concentration of the sample being measured. A Count Rate above 1,500 kCPSis acceptable for the particles of the invention disclosed herein. Higher Count Rates can indicatea more concentrated dose, i.e., that there are more particles containing the HSP-70 mRNA. Thus,without being bound by theory or methodology the higher the concentration, the more effectivethat 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 (a measure of the average size of a particle sizedistribution) depicted in Figure 2. Notably, there is little variance among these measurementsresulting in a single visible peak. (93.0 nm to 94.6 nm, (all three replicates laid out over eachother). This data does not reflect the average kCPS of all three measurements as this output wasused 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 wereaggregating. Even minor aggregation would be detectable. Generally, for DLS measures inintensity, one larger particle can block out many more smaller particles and skew the data toshow a larger Z-Average and D50 than is actually true. The test sample was prepared and storedat 4ºC (the temperature at which all tested doses are stored). Particle size was measured at severaldifferent time points following completion of initial dialysis and filtration. To re-measure, at each34FH11736604.1 GRH-00161time point a portion of sample was applied to a cuvette and read on the DLS machine andreturned, e.g., to a 15 mL conical tube for storage at 4ºC again. Results showed that even after 87days in storage, there was no aggregation of particles. Thus, the LNPs of the invention are stableat 4ºC for long periods of time without changing size.In summary, the DLS data shows that particles at 106 nm or smaller can be madeconsistently. This will allow LNPs to cross the blood-brain barrier to deliver HSP-70 mRNA tothe cells of the brain. Said particles do not aggregate together or change in size over time, andcan be stored at 4ºC with no degradation or aggregation over 60 days of repeated testing.Example 7: ELISA Data Enzyme-linked immunoassay (ELISAs) are currently considered the preferredmeasurement of protein concentration within a solution. The ELISA employed herein was asandwich ELISA, meaning that the analyte to be measured (HSP-70) was bound between twoprimary antibodies, each detecting a different epitope of the antigen– the capture antibody andthe detection antibody. Thus, the measured protein, HSP-70, was initially bound to captureantibody adsorbed / linked to the bottom of wells of a microplate. After this initial binding a biotinconjugated anti-HSP-70 was attached to the bound HSP-70. Streptavidin-HRP (horseradishperoxidase) was complexed with the bound biotin. Finally, TMB (3, 3', 5, 5'-tetramethylbenzidine) was used as a colorimetric substrate, activated by the HRP detectionantibody. The multiple washes and specific antibodies ensure that everything except for thedesired protein is washed away. This test was conducted in order to ensure that the HSP-70protein was being overexpressed with application of the LNP-encapsulated mRNA.Briefly, SH-SY5Y Neuroblastoma cells were grown in DMEM media containing 9.1%FBS in 75 mL tissue culture flasks for 3 days. Cells were then split into 12 well plates (250,000cells per well) and left to adhere and grow overnight for 12 hours at 37 °C / 5.0% CO2. After the12-hour period, HSP-70 mRNA-LNPs were added to wells 1-6 and nothing was added to wells 7-12. The cells were returned to the incubator for 4 hours. Cells then received an additional dose ofHSP-70 mRNA-LNPs and were returned to the incubator for 12 hours (overnight). After the 12-hour hold, cells in wells 1-6 received a further dose of HSP-70 mRNA-LNPs and were placed inthe incubator for 30 minutes. From each group, at least 500,000 cells were harvested for ELISAanalysis of HSP-70 concentration.Microplates were loaded as described in Tables 2 and 3 and ELISAs were run using aCrocodile ELISA MiniWorkstation (Berthold Technologies, Germany). Following Biotin-Conjugate, Streptavidin-HRP, and washing steps, TMB solution was used as the colorimetricsubstrate. Color development of samples was monitored and the absorbance of each microwell35FH11736604.1 GRH-00161was read on a spectro-photometer using 630 nm as the primary wavelength (optionally 450 nm asthe reference wavelength; 610 nm to 650 nm is acceptable). Two columns were observed foreach of “Sample” (collected from cells dosed with HSP-70 mRNA-LNPs) and “Control”(collected from cells that did not receive HSP-70 mRNA-LNPs). These columns were comparedto the standard columns which were made by a serial dilution of a known amount of HSP70protein. See Tables 2 and 3.Table 2 36FH11736604.1 GRH-00161 Table 3 Overall, the fluorescence readings were shown to be higher in the sample columns thanthe control columns, which indicates that HSP-70 was overexpressed following application of theLNP particles.Using this data, and the data from the standard wells, the average concentration of HSP-70 per well and the total amount of HSP-70 present in the original 850 µL sample was calculated.All three of the trials showed higher average fluorescence, average HSP-70 in ng / mL per well,and total HSP-70 concentration in the original 850 µL sample for samples dosed with HSP-70mRNA-LNPs. See Table 4.Table 4 37FH11736604.1 GRH-00161 The ELISA data confirms that the HSP-70 mRNA-LNPs can induce an overproduction ofthe HSP-70 protein in human cells. Repeated tests showed that cells that have been dosed withHSP-70 mRNA-LNPs produce significantly more HSP-70 protein than cells that have not beendosed.Example 8: Cell line Data In the human body, HSP-70 is a chaperone protein that is used to help refold misfoldedproteins. Current research indicates that Alzheimer’s disease is caused through proteinaggregation of amyloid beta and tau proteins, among others. This protein aggregation preventsneurons from communicating and can result in memory loss and overall loss in neurologicalfunctions over time due to neuronal death. Therefore, experiments in human-derived cell lineswere designed to cause protein aggregation and observe said cells for reversal of the effects fromprotein aggregation following introduction of HSP-70 mRNA-LNPs.In order to induce protein aggregation, SH-SY5Y neuroblastoma cells were treated with80mM lead acetate. Notably, any type of lead solution or metal ion solution can lead to proteinmisfolding / malfunction, hence why lead poisoning can be extremely fatal.Experiments were run in 24-hour and 48-hour groups. This was done to observe howHSP-70 mRNA-LNPs would affect lead-treated cells over different periods of time. In eachgroup, two 12-well plates were used, one plate as a control and the other as experimental (lead-poisoned).Cells treated with HSP-70 mRNA-LNPs had between a 15%-40% higher viability thanthe cells without HSP-70 mRNA-LNPs. The control plate also showed that HSP-70 mRNA-LNPs was not damaging or killing the SH-SY5Y cell lines, thus demonstrating that HSP-70mRNA-LNPs is not toxic to human cells. This data suggests that HSP-70 mRNA-LNPs providesa viable strategy for the treatment of Alzheimer’s disease, as it is successful in protecting cellsfrom the effects of protein misfolding, particularly, neurons.24 Hour Cell Count: This cell count consisted of running two 12-well plates. The testwell plate consisted of 6 wells of cells that were treated with lead acetate and HSP-70 mRNA-LNPs and 6 wells with just cells treated with lead acetate. The purpose of creating this plate wasto compare cells treated with lead acetate either with or without HSP-70 mRNA-LNPs. Thecontrol well plate consisted of 3 wells treated with lead acetate, 3 wells treated with HSP-70mRNA-LNPs and 3 wells treated with PBS. Three wells were treated with lead acetate as acontrol to ensure that lead acetate alone is killing the cells and to determine the viability of cellswhen treated with just lead acetate. Another 3 wells were treated with just HSP-70 mRNA-LNPs38FH11736604.1 GRH-00161to ensure that the drug itself is not harming or damaging the cells in any way that would lead tocell death or a decrease in cell viability. The last 3 wells were treated with just PBS to ensure thesolution the HSP-70 mRNA-LNPs is in, or PBS, is not harming the cells and causing a decreasein viability. The test well plate showed that HSP-70 mRNA-LNPs were able to slow down / stopthe effects of lead acetate on the cells.Briefly, SH-SY5Y Neuroblastoma cells were grown in DMEM media containing 9.1%FBS in 75 mL tissue culture flasks for 3 days. Cells were transferred to 12-well plates using250,000 cells per well, and returned to incubation at 37 °C / 5.0% CO2 to allow cells to adhereand grow for at least 12 hours. In the first plate (test well plate) cells in wells 1-6 received 17 µLof HSP-70 mRNA-LNPs and cells in wells 7-12 received 17 µL of PBS. The 12-well plate wasreturned to the incubator for 15 minutes, and then 50 µL of 80 mM lead acetate was added toeach well.Table 5: Test well plate In the second plate (control well plate), after the 12-hour incubation, cells in wells 1, 5,and 9 received 50 μL of 80 mM lead acetate; cells in wells 2, 6, and 10 received 17 µL of HSP-70 mRNA-LNPs; cells in wells 3, 7, and 11 received 17 µL of PBS; and cells in wells 4, 8, and12 did not receive anything.Table 6: Control well plate Both 12-well plates were then set in an incubator for 4 hours at 37 °C / 5.0% CO2. Afterthe 4-hour period, cells in wells 1-6 in the first plate received 17 µL of HSP-70 mRNA-LNPs andcells in wells 7-12 received 17 µL of PBS. In the second plate, cells in wells 1, 5, and 9 weredosed with 50 µL of 80 mM lead acetate, cells in wells 2, 6, and 10 were dosed with 17 µL ofHSP-70 mRNA-LNPs, cells in wells 3, 7, and 11 received 17 µL of PBS, and cells in wells 4, 8,and 12 did not receive anything.39FH11736604.1 GRH-00161 Both plates were returned to the incubator for an additional 4 hours at 37 °C / 5.0% CO2.After the second 4-hour incubation, cell viability in each well was assessed by microscopy on ahemocytometer using trypan blue staining.Data showed that there was an average of a 15% increase in cell viability (Dose + Lead)compared to the cells with the addition of lead acetate, i.e., Lead Only and PBS + Lead (Figure3). The data from the dose only had a 1.3% difference in viability from the wells with only cellsand media (Nothing) showing that HSP-70 mRNA-LNPs do not negatively affect the humanneuroblastoma cells. Compared to the PBS Only wells, there was approximately a 2% differencein viability, further showing that the solution the dose is in also does not negatively affect theneuroblastoma cells’ viability.48 Hour Cell Count: Similar to the above assay, this cell count consisted of running two12-well plates with increased exposure time to lead acetate. The test well plate consisted of 6wells of cells that were treated with lead acetate and HSP-70 mRNA-LNPs and 6 wells with cellstreated with just lead acetate. The purpose of creating this plate was to compare cells treated withlead acetate either with or without HSP-70 mRNA-LNPs. The control well plate consisted of 3wells treated with lead acetate, 3 wells treated with HSP-70 mRNA-LNPs and 3 wells treatedwith PBS. The three wells treated with lead acetate act as a control to ensure that lead acetatealone is killing the cells and to determine the viability of cells when treated with just lead acetate.The 3 wells treated with just HSP-70 mRNA-LNPs confirm that the drug itself is not harming ordamaging the cells in any way that would lead to cell death or a decrease in cell viability. Thelast 3 wells treated with just PBS confirm that the solution comprising the HSP-70 mRNA-LNPsis not harming the cells and causing a decrease in viability.Briefly, after overnight incubation (12 hours) at 37 °C / 5.0% CO2 to allow cells to adhereand grow, the first plate (test well plate) received 17 µL of HSP-70 mRNA-LNPs per well or 17μL of PBS and returned to the incubator for 3 hours, and then 50 µL of 80 mM lead acetate wasadded to each well, and the plates returned to the incubator for 8 hours. The control well plate,after the overnight incubation, received 50 µL of 80 mM lead acetate in wells 1, 5, and 9; 17 µLof HSP-70 mRNA-LNPs in wells 2, 6, and 10; 17 µL of PBS in wells 3, 7, and 11; and nothingfurther in wells 4, 8, and 12, before returning to the incubator for 8 hours.After the first 4-hour period, wells 1-6 of the test plate received 17 µL of HSP-70 mRNA-LNPs and wells 7-12 received 17 µL of PBS. In the control plate wells 1, 5, and 9 were dosedwith 50 µL of 80 mM lead acetate; wells 2, 6, and 10 were dosed with 17 µL of HSP-70 mRNA-LNPs; wells 3, 7, and 11 received 17 µL of PBS. Wells 4, 8, and 12 did not receive anything.Plates were then returned to the incubator for an additional 8 hours before cell viability wasassessed as described above.40FH11736604.1 GRH-00161 Data from the test well plate showed that HSP-70 mRNA-LNPs were able to slowdown / stop the effects of lead acetate on the cells. The data showed that there was an average of a21% increase in cell viability compared to the cells with just lead acetate.Data from the wells containing cells and HSP-70 mRNA-LNPs (Dose Only) had anaverage viability of 87%, which displays that the HSP-70 mRNA-LNPs do not negatively affecthuman neuroblastoma cells. The Dose + Lead wells showed a 28% cell viability while Lead Onlyshowed a 7% cell viability. The PBS + Lead wells showed a 2% cell viability. This data showsthat the HSP-70 mRNA-LNPs increased the viability of human neuroblastoma cells by about21% when both were dosed with 80mM lead acetate (Figure 4).Overall, the data from the 24- and 48-hour cell counts show that HSP-70 mRNA-LNPsincrease the viability of lead poisoned cells for the first time, i.e., exposure to a 50 µL dose of 80mM lead acetate solution. The SH-SY5Y cells dosed with HSP-70 mRNA-LNPs and lead acetatehad significantly higher viability than cells with just lead acetate or lead acetate and PBS(Phosphate Buffered Saline). Lead acetate was used as it misfolds proteins to a high degree andthe particles overexpress HSP70 which refolds misfolded proteins. The viability of cellsmaintained with no added dose or PBS was compared to cells that were treated with just leadacetate, just HSP-70 mRNA-LNPs, just PBS, and a combination of PBS and lead acetate. Thisdata showed that the cells treated with just lead acetate solution and cells with the combination oflead acetate and PBS had a very low viability. Cells treated with just PBS or just HSP-70 mRNA-LNPs had viabilities that were almost identical to cells with no treatments added. These cellularstudies suggest that when HSP70 is overexpressed by introduction of HSP-70 mRNA-LNPs, theHSP70 has the capability to save cells from high levels of toxicity from lead poisoning. Thisfurther suggests that overexpression of HSP70 via the methods and nanoparticles disclosedherein has the capability to treat the protein misfolding caused by Alzheimer’s disease.Example 9: Encapsulation DataThese experiments were performed to ensure that mRNA coding for HSP-70 was beingsuccessfully inserted via hydro-fluidic mixing into the solid lipid nanoparticles to successfullyform the HSP-70 mRNA-LNPs of the invention. Using ethidium bromide, which is a DNA andRNA intercalator, particles were confirmed to contain fully formed mRNA strands coding forHSP-70 as the fluorescence in said particles was more than double the fluorescence of a controlsolution and control solid lipid nanoparticles. Ethidium bromide fluoresces significantly morewhen it is bound to RNA and DNA strands than it does when unbound. Although this test is evenstronger for double-stranded nucleotides, a less pronounced yet still significant increase influorescence can be measured for single-stranded RNA or DNA. Further, ethidium bromide bindsto intact strands much more readily than it does to single nucleotides. Essentially ethidium41FH11736604.1 GRH-00161bromide fluoresces strongly when there are RNA or DNA strands present, but does so weaklywhen there are only single nucleotides, or no RNA or DNA strands present.Briefly, a minimum of three 200 µL samples of HSP-70 mRNA-LNPs was prepared. A5% triton X-100 was made using Rnase and Dnase free H2O. A 15 mL conical tube of ethidiumbromide solution using 15 mL of DI water and 5 ug of ethidium bromide was made. Test solutionwas prepared from 200 µL of HSP-70 mRNA-LNPs, 800 µL of 5% Triton X-100, and 50 µL ofethidium bromide solution. Control solution not containing solid lipid nanoparticles comprised200 µL of PBS, 800 µL of 5% Triton X-100, and 50 µL of ethidium bromide solution. Controlsolution containing solid lipid nanoparticles comprised 200 µL of PBS containing solid lipidnanoparticles lacking mRNA, 800 µL of 5% Triton X-100, and 50 µL of ethidium bromidesolution. Samples were transferred, one at a time, to a 1 mL cuvette and inserted into afluorometer where each sample was read at an emission wavelength of 540 nm to 750 nm.Figure 5A displays the fluorescence from ethidium bromide when solid lipidnanoparticles containing mRNA coding for HSP-70 are broken apart using triton X. The finalsolution read for this test sample was composed of solid lipid nanoparticles containing mRNAcoding for HSP-70, ethidium bromide solution, and triton X. The S1c curve (the corrected S1curve) has a peak fluorescence of 4.34 x 10^6 CPS for this test sample. The first control curvedepicted in Figure 5B displays the fluorescence of ethidium bromide without particles containingmRNA being present. The solution being measured was ethidium bromide, triton X and PBS.The fluorescence peak of this control sample was 1.75 x 10^6 CPS. The second control sampledepicted in 5C consisted of solid lipid nanoparticles lacking any mRNA. These particles werelysed using triton X and then exposed to ethidium bromide. The final solution was composed oflysed solid lipid nanoparticles, ethidium bromide solution, and triton X. This solution had ameasured fluorescence of 2.14 x 10^6 CPS. This data displays that our solid lipid nanoparticlessuccessfully encapsulated the HSP70 mRNA as the fluorescence in our test samples was over 2million CPS higher than either of our control samples. Figure 5A also displayed a different peakwavelength than our controls with the peak being read at 607 nm compared to Figure 5B at apeak wavelength of 614 nm and Figure 5C with a peak wavelength of 613 nm. This shift in thefluorescence peak wavelength confirms that ethidium bromide is intercalating betweennucleotides as the known excitation wavelength when bound is 605-608 nm, which is where thetest samples peak wavelength was observed (607 nm).Example 10: HSP-70 mRNA synthesisRNA Isolation ProcedureTissue culture cells were harvested and centrifuged in to obtain a pellet, which could be42FH11736604.1 GRH-00161stored at -20ºC. Cell pellets were thawed or otherwise broken up by mechanical agitation of thesample tube prior to employing a commercial RNA isolation kit. Briefly, for a kit comprising aspin column, an amount of the appropriate lysis buffer was added to the tube (e.g., 350 μL forless than 5 x 106 cells; 700 μL for 5 x 106 - 1 x 107 cells), and the solution mixed by vortexingand / or pipetting, resulting in homogenized cell lysate. A matched volume of 70% ethanol wasadded to the cell lysate (e.g., for 350 μL of lysis buffer used to homogenize the pellet, 350 μL of70% ethanol was added) and pipetted up and down for 15-30 seconds. A volume of 700 μL wastransferred to the spin column, comprising a collection tube, and centrifuged for 15 seconds at11,400 rpm. The liquid flowthrough was discarded and 700 μL of appropriate wash buffer wasadded to the spin column. This was centrifuged for 15 seconds at 11,400 rpm, and the liquidflowthrough in the collection tube was discarded. The washing was repeated at least two moretimes with appropriate volumes of the appropriate wash buffer. The spin column was placed intoa new 1.5 mL collection tube and 50 μL of RNase-free water was added to the spin column. Thespin column was centrifuged for 1 minute at 11,400 rpm and the eluted RNA in the collectiontube was used for cDNA synthesis, optionally stored at -20ºC.RT-PCR (double-stranded cDNA synthesis)Isolated RNA (see RNA Isolation Procedure) was used in the synthesis of cDNA.Commercially available kits, such as the SuperscriptTM III or IV Reverse Transcriptase Kit, wereused. In a single reaction mixture, the enzymes Superscript III or IV were used to create cDNAfrom the extracted RNA with Platinum SuperFi DNA Polymerase to synthesize double-strandedcDNA coding for the HSPA1A gene with the forward and reverse primer pairs disclosed herein(e.g., SEQ ID NOs. 4 and 5, 8 and 9, 12 and 13, and 16 and 17).Synthesis reactions were run with the following thermal cycler parameters: After the cycler finished running, reaction tubes could be stored at -20ºC freezer,otherwise the resultant double-stranded cDNA was used immediately for mRNA creation.cDNA Agarose Gel Analysis:Samples were prepared for loading onto agarose gel by taking 1 μL of cDNA sample with2 μL of 1X loading dye (3 μL total), pipetted up and down slowly 3-5 times to mix. Another 1 μLof cDNA sample was diluted with 4 μL of nuclease free water (5 μL total) and 1 μL aliquots ofthis diluted cDNA sample was mixed with 2 μL of 1X loading dye to make five 3 μL running43FH11736604.1 GRH-00161samples.A 1KB DNA ladder was applied to the first well and the 3 μL samples were each added toa separate well (i.e., undiluted sample well followed by 5 diluted sample wells). Samples wereelectrophoresed for 1 hour at 100 mV, 400 mA (0.4 A). The expected length of the sequenceshould be ~2100 base pairs which was observed in all four samples shown in the HSPA1A GeneDNA Agarose Gel of Figure 6. This double-stranded cDNA coding for the HSPA1A gene wasthen used in the synthesis of mRNA seen in Figure 7. The nucleotide sequence of the double-stranded cDNA was assessed and confirmed to be the intended target sequence by Sangersequencing (i.e., chain termination method) analysis.mRNA SynthesisThe prepared cDNA was used in the synthesis of HSP-70 mRNA. Commerciallyavailable kits, such as the HiScribe® T7 Quick Yield RNA Synthesis Kit, were used. Thermalcycle reactions were run according to kit manufacture protocols.Once thermal cycling was complete, 30 μL of Nuclease-Free Water was added to eachreaction tube containing the newly synthesized mRNA, followed by 2 μL of DNase for a total of52 μL. Once sufficiently mixed, each reaction tube was returned to the thermal cycler and set fora 15-minute hold at 37ºC followed by an indefinite 4ºC hold.Following the DNase treatment, the mRNA was capped using a Fausto virus cappingenzyme (FCE). For each tube, 6.6 µL of FCE capping buffer, 3.3 µL of Guanosine triphosphate(GTP), 3.3 µL of S-Adenosyl methionine (SAM), and 2.6 µL of the FCE enzyme was added inthat order, giving a final volume of 67.8 µL. The tubes were then incubated in a thermal cyclerand set for a 30-minute hold at 37ºC followed by an indefinite 4ºC hold.RNA purification and concentration following enzymatic reaction was performed usingcommercially available kits, such as the Monarch® RNA Clean up Kit. Briefly, 100 μL of RNABinding Buffer was added to the 67.8 μL mRNA synthesis sample and mixed by pipetting. Thetotal 167.8 μL of solution was added to spin columns provided with 167.8 μL of 100% ethanoland carefully pipetted to mix. Spin columns (with collection tubes) were centrifuged for 1 minuteat 13,000 rpm. Liquid flowthrough (liquid in collection tube) was removed and 500 μl ofappropriate RNA wash buffer was added to the spin column. Following centrifugation for 1minute at 13,000 rpm, the liquid flowthrough was removed, and the wash repeated at least oncemore.Following washing, 50 μL of nuclease free water was added to the spin column (withempty collection tube) and centrifuged for 1 minute at 13,000 rpm. The collection tubecontaining the resultant flowthrough was held at 4ºC. The purified RNA from multiple collectiontubes were combined and mixed via pipetting. Purified HSP-70 RNA could be stored at -20ºC44FH11736604.1 GRH-00161 until use. mRNA Agarose Gel Analysis:Samples were prepared for loading onto a 1% agarose gel by taking 1 μL of mRNAsample and adding 9 μL of nuclease free water (10 μL total), pipetting up and down slowly 3-5times to mix. The diluted mRNA sample was divided into 1 μL-aliquots and mixed with 3 μL of1X loading dye (4 μL total for each of 10 running samples).A 1KB RNA ladder was applied to the first well and the 4 μL samples were each added toa subsequent well (i.e., undiluted sample well followed by 5 diluted sample wells). Samples wereelectrophoresed for 1 hour at 100 mV, 400 mA (0.4 A). Six separate HSP-70 mRNA synthesisruns yielded identical results and produced mRNA for HSP-70 at 2100 bases long in highconcentrations. (See figure 7.) This mRNA represents the sequence (or payload) disclosed hereinand incorporated into the LNPs of the invention.In summary, HSP-70 cDNA was created from RNA isolated from tissue culture (e.g.,HBEC3-KT cell pellet). The reverse transcriptase enzymes Superscript III or IV and the DNApolymerase PlatinumTM SuperFiTM were added to the extracted RNA to create double-strandedcDNA coding for the HSPA1A gene using the appropriate primers. This 2128 base pair longstrand was consistently synthesized as shown in Figure 6. The double-stranded cDNA coding forthe HSPA1A gene was then used to create mRNA, as the forward primer used to synthesizecDNA contained a promoter for T7 RNA polymerase. After the mRNA synthesis, the resultantsequence was treated with DNase and then cleaned to remove any impurities such as free-floatingnucleotides, enzymes, etc. The cleaned mRNA was then capped with a Faustovirus CappingEnzyme (FCE). The fully cleaned and capped mRNA is shown in figure 7 and represents Payload1 used to create a final lipid nanoparticle containing HSP-70 mRNA product contemplatedherein.INCORPORATION BY REFERENCEAll publications and patents mentioned herein are hereby incorporated by reference intheir entirety as if each individual publication or patent was specifically and individuallyindicated to be incorporated by reference. In case of conflict, the present specification, includingits specific definitions, will control. While specific aspects of the patient matter have beendiscussed, the above specification is illustrative and not restrictive. Many variations will becomeapparent to those skilled in the art upon review of this specification and the claims below. Thefull scope should be determined by reference to the claims, along with their full scope ofequivalents, and the specification, along with such variations.45FH11736604.1 GRH-00161EQUIVALENTSThe present invention has been described in connection with what are presentlyconsidered to be the most practical and preferred embodiments. However, the invention has beenpresented by way of illustration and is not intended to be limited to the disclosed embodiments.Accordingly, one of skill in the art will realize that the invention is intended to encompass allmodifications and alternative arrangements within the spirit and scope as set forth in theappended claims.46FH11736604.1

Claims

What is claimed is:

1. A therapeutic composition comprising a nucleic acid formulated in a lipid nanoparticle (LNP), wherein the nucleic acid comprises an open reading frame encoding heat shock protein polypeptide, or a functional fragment thereof.

2. The therapeutic composition of claim 1, wherein the heat shock protein polypeptide is HSP100, HSP90, HSP70, HSP60, HSP40, or HSP27.

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

4. The therapeutic composition of claim 1, wherein the nucleic acid is mRNA, optionally wherein the mRNA 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.

5. The therapeutic composition of any one of claims 1-4, wherein one or more uridine nucleosides in the nucleic acid are pseudouridine.

6. The therapeutic composition of claim 5, wherein the pseudouridine is N1- methylpseudouridine.

7. The therapeutic composition of any one of claims 1-6, wherein the LNP comprises an ionizable lipid, a phospholipid, a sterol, a PEG-modified lipid, or any combination thereof.

8. The therapeutic composition of any one of claims 1-6, wherein the LNP consists essentially of an ionizable lipid, a phospholipid, a sterol, a PEG-modified lipid, or any combination thereof.

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

10. The therapeutic composition of any one of claims 7-9, wherein the phospholipid is distearoylphosphatidylcholine (DSPC).

11. The therapeutic composition of any one of claims 7-10, wherein the sterol is cholesterol.

12. The therapeutic composition of any one of claims 7-11, wherein the PEG-modified lipid is DMG-PEG 2000.

13. A cell comprising the LNP of any one of claims 1-12.

14. The cell of claim 13, expressing the HSP70 polypeptide encoded by an mRNA.

15. The cell of claim 14, wherein the HSP70 polypeptide comprises the amino acid sequence set forth in SEQ ID NO. 1, or a functional fragment thereof.

16. The cell 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 cell of claim 16, wherein the haematopoietic cell is an immune cell selected from a lymphocyte, a monocyte, a macrophage, a dendritic cell, a mast cell, a neutrophil, a basophil, or an eosinophil.

18. The cell of claim 17, wherein the immune cell is a lymphocyte selected from an αβT cell, γδT 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 T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, or a regulatory T cell.

19. The cell of any one of claims 13-18, wherein the cell is a cell derived from bone marrow.

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

21. The cell of any one of claims 13-20, wherein the cell is a cell present in the CNS.

22. The cell of claim 20 or 21, wherein the cell is a neuronal cell.

23. The cell of claim 22, wherein the neuronal cell is a sensory neuron, a motor neuron, or an interneuron.

24. The cell of claim 20 or 21, wherein the cell is a non-neuronal cell.

25. The cell of claim 24, wherein the non-neuronal cell is a glial cell.

26. The cell of claim 25, wherein the glial cell is an astrocyte cell, an oligodendrocyte cell, an ependymal cell, a radial glial cell, a Schwann cell, a satellite cell, an enteric glial cell, or a microglial cell.

27. A method of treating a neurodegenerative disease in a subject, the method comprising administering the therapeutic composition of any one of claims 1-12.

28. A method of treating a neurodegenerative disease in a subject, the method comprising administering a composition comprising the cells of any one of claims 13-26.

29. The method of claim 27 or 28, wherein the neurodegenerative disease is characterized by a proteinopathy.

30. The method of any one of claims 27-29, wherein the neurodegenerative is a tauopathy.

31. The method of any one of claims 27-29, wherein the neurodegenerative is a synucleinopathy.

32. The method of any one of claims 27-31, wherein the neurodegenerative is Alzheimer’s disease.

33. The method of any one of claims 27-32, wherein the therapeutic composition is administered intrapleurally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intraperitoneally, intracranially, or by direct administration to an organ.

34. The method of any one of claims 27-33, further comprising conjointly administering to the subject an immunotherapy.

35. The method of claim 31, wherein the immunotherapy comprises a plaque-binding antibody.

36. The method of claim 35, wherein the plaque-binding antibody is aducanumab.

37. The method of any one of claims 27-36, further comprising conjointly administering to the subject a cholinesterase inhibitor.

38. The method of claim 37, wherein the cholinesterase inhibitor is donepezil, rivastigmine, or galantamine.

39. The method of any one of claims 27-38, further comprising conjointly administering to the subject a glutamate regulator.

40. The method of claim 39, wherein the glutamate regulator memantine.

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