WO2024129826A2 - Compositions and methods for modulating hsp70 activity - Google Patents

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

GRH-00125 C^{OMPOSITIONS AND METHODS FOR MODULATING HSP70} A_CTIVITY CROSS-REFERENCE TO RELATED APPLICATIONS T^{his application claims the benefit of priority to U.S. Provisional Patent Application serial number 63/528239, filed July 21, 2023, and U.S. Provisional Patent Application serial} _{number 63/432471, filed December 14, 2022, each of which is hereby incorporated by reference in its entirety.} BACKGROUND A^{s 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 type of dementia in the elderly. Epidemiological studies indicate that around 10% of the population over 65 years and 50% of individuals over 90 years suffer from Alzheimer’s disease. Though Alzheimer’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; extracellular amyloid plaques composed of A-β peptide derived from proteolytic processing of the amyloid} _{precursor protein (APP), and the intracellular neurofibrillary tangles (NFT) formed by the aggregation of tau protein. The metabolic cascades of each protein continue to be the target of investigation and debate in the context of Alzheimer’s Disease pathogenesis, post-mortem diagnostic criteria for, and therapeutic intervention. Notably, many neurodegenerative diseases can be characterized by the accumulation/deposition of misfolded and/or insoluble proteins. Advances in molecular biology and neuropathology have allowed for the classification of a plurality of proteinopathies. Yet, despite such advances, the available therapies for subjects suffering from Alzheimer’s disease (and proteinopathies in general) are very limited. Hence, there is an unmet need for new and efficacious treatments.} SUMMARY A^{spects of the invention, as provided herein, include therapeutic compositions comprising an mRNA formulated in a lipid nanoparticle (LNP), wherein the mRNA comprises an open reading frame encoding heat shock protein polypeptide, or a functional fragment thereof. In some} _{embodiments of the invention, the heat shock protein polypeptide, or a functional fragment thereof is HSP100, HSP90, HSP70, HSP60, HSP40, or HSP27. Preferably, the heat shock protein polypeptide is HSP70. In some embodiments, the LNP is a solid lipid nanoparticle (SLN). FH11736604.1} ¹ GRH-00161 I^{n certain aspects of the invention, provided herein are cells comprising the LNPs disclosed herein. In some aspects of the invention, provided herein are methods of treating a} _{neurodegenerative disease in a subject, the method comprising administering the therapeutic compositions disclosed herein. In some such embodiments, the composition comprises the LNP- comprising cells disclosed herein.} ^{BRIEF DESCRIPTION OF FIGURES Figure 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 the mRNA 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 results and produced mRNA for HSP-70 at 2,100 bases long in high concentrations.} ^{DETAILED DESCRIPTION General Heat shock proteins (HSPs) represent a class of molecular chaperones known to be expressed 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 biochemical stressors. HSPs perform chaperone functions by binding and stabilizing new or mis-folded proteins and assisting them to acquire their native structure, thus preventing mis-folding and the aggregation processes. HSPs may be classified into families on the basis of molecular weight, for example HSP100, HSP90, HSP70, HSP60, HSP40, and HSP27, each playing a diverse role in} _{influencing proper protein assembly, folding, and translocation. For instance, HSP70, HSP60, and HSP27 are known to prevent protein aggregation and help protein folding; HSP100 releases proteins from aggregates; and HSP90 plays a role in maturation and activation of a number of proteins. Therefore, HSPs are expected to have strong potential as therapeutic agents in suppressing or treating a range of diseases associated with proteinopathy, including cancer, neurodegeneration, allograft rejection, and infection. In such diseases or disease states, proteins} 2 _FH11736604.1 GRH-00161 ^{fail to fold into their normal configuration and in this mis-folded state the proteins can become toxic 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 of adenosine triphosphatases that represent the most structurally and functionally conserved proteins amongst HSPs. HSP70 is also the most ubiquitous class of chaperone protein, inducing cytoprotective effects under a number of different conditions, primarily in cellular protein quality control (PQC) and degradation systems. Briefly, in humans the HSP70 multigene family acts on nonnative polypeptides, fueled by ATP binding and hydrolysis. The HSP70 chaperone binds to} _{protein 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-terminal nucleotide binding domain (NBD) and a 25 kDa C-terminal substrate binding domain (SBD). These domains undergo reciprocal allosteric interactions induced by ligand binding. The NBD comprises two lobes, forming a cleft that binds ATP with a nucleotide binding cassette that is related to those in actin and hexokinase. The SBD comprises a ^^-sandwich subdomain harboring the substrate binding site, and an ^^-helical lid. Both these domains are critical for chaperone function and are connected by a short flexible linker.}

3 _FH11736604.1 GRH-00161

^{As a molecular chaperone, HSP70 also has multiple responsibilities during normal growth. It is integral to the folding of newly synthesized proteins, the subcellular transport of proteins and vesicles, the formation and dissociation of complexes, and degradation of unwanted proteins. In carrying out these diverse functions HSP70 adopts different conformations, e.g., in the absence of nucleotide, when bound with ADP, or when bound with ATP. In addition, the functions of HSP70 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 and assembly with partner proteins while preventing aggregation of the substrate proteins. Provided herein are nucleic acids (e.g., mRNAs) encoding heat shock proteins. The compositions and methods of the present disclosure rely, at least in part, on the delivery of heat shock protein-encoding nucleic acids (e.g., HSP70-encoding nucleic acids) to cells of a subject in need thereof (e.g., a gene therapy composition). For example, and without limitation,} _{compositions comprising the heat shock protein-encoding nucleic acids disclosed herein can be used to treat a proteinopathy. In some embodiments, said compositions may be used to treat cancers, neurodegenerative diseases, allograft rejection, and/or infection. Aspects of the invention, as provided herein, include therapeutic compositions comprising an mRNA formulated in a lipid nanoparticle (LNP) (e.g., a solid lipid nanoparticle (SLN)), wherein the mRNA comprises an open reading frame encoding heat shock protein polypeptide, or a functional fragment thereof. In some embodiments, the heat shock protein polypeptide, or functional fragment thereof is selected from HSP100, HSP90, HSP70, HSP60, HSP40, or HSP27. 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 sequence set 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 ID NO. 10, SEQ ID NO. 14, SEQ ID NO. 18, or any functional fragment thereof.} 4 _FH11736604.1 GRH-00161 I^{n some aspects, provided herein are nucleic acids encoding the HSP70 polypeptides disclosed herein. In certain aspects, provided herein are primers for isolating and/or amplifying a nucleic acid sequence encoding a heat shock protein as disclosed herein, using methods known in the 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 using any 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. 12 and 13, SEQ ID NOs. 15 and 17, and SEQ ID NOs. 16 and 17. One or more of the uridine nucleosides in the isolated/amplified mRNA is a pseudouridine, such as, N1- methylpseudouridine. In some such embodiments, all of the uridine nucleosides in the mRNA are pseudouridine, e.g., N1-methylpseudouridine. For example, and without being bound by theory or methodology, the aforementioned nucleic acids are amplified from whole cell lysate. In some embodiments, the whole transcriptome mRNA is isolated from cell lysate. In some such embodiments cDNAs of interest, e.g., double-stranded cDNA encoding HSP-70, are synthesized from the isolated mRNA, .e.g., targeted amplification of sequence from forward and reverse primer 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 bound by theory, and for the purpose of exemplification, the mRNAs to be incorporated in the LNPs disclosed herein may be synthesized by targeted T7 amplification of mRNA sequence from the cDNA encoding HSP-70, e.g., using primers disclosed herein. In some preferred embodiments, a one-step RT-PCR reaction is used, e.g., reverse transcription and PCR are performed in a single reaction mixture. Alternatively, the mRNA is synthesized from a plasmid template by methods known in the art. In other aspects, provided herein are vectors comprising the nucleic acids contemplated herein. 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 acids disclosed herein. Preferred aspects of the invention include cells comprising the LNPs (e.g., the SLNs) disclosed herein. In certain aspects, provided here are cells comprising the vectors disclosed 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 an endothelial 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,} 5 _FH11736604.1 GRH-00161 ^{a 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 a regulatory T cell. In certain embodiments, the cells contemplated herein are cells of the central nervous system (CNS) or peripheral nervous system (PNS). In other embodiments, the cell is a cell of the bone marrow. In some embodiments, the cell contemplated herein is a cell present in the CNS. In some embodiments, the cell is a neuronal cell. Said neuronal cell may be a sensory neuron, a motor neuron, or an interneuron. In other embodiments, the cell is a non-neuronal cell. In some embodiments, the non-neuronal cell is a glial cell. The glial cell may be 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. In some embodiments, the nucleic acids contemplated herein may refer to a polymeric form of nucleotides or nucleosides of any length, such as deoxyribonucleotides or ribonucleotides, or analogs thereof. Nucleic acids may have any three-dimensional structure, and may perform any function. The following are non-limiting examples of Nucleic acids: coding or} _{non-coding regions of a gene or gene fragment, loci (locus) defined from 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 acid probes, and primers. A nucleic acid may comprise modified nucleotides/nucleosides, such as methylated nucleotides/nucleosides and nucleotide/nucleoside analogs. If present, modifications to the polynucleotide/polynucleoside structure may be imparted before or after assembly of the polymer. A polynucleotide/polynucleoside may be further modified, such as by conjugation with a labeling component. Aspects include therapeutic compositions comprising the mRNAs disclosed herein. In some embodiments, the therapeutic composition comprises a vector selected from nanoparticles, adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, picorna virus vectors, liposomes, cationic lipid systems, and protein/nucleic acid complexes. For example and without limitation, the therapeutic composition comprising an mRNA may be formulated in a lipid nanoparticle (LNP). In some embodiments of the therapeutic composition, one or more of the uridine nucleosides in the mRNA are pseudouridine, such as, N1-methylpseudouridine. In some such embodiments, all of the uridine nucleosides in the mRNA are pseudouridine, e.g., N1-} 6 _FH11736604.1 GRH-00161 ^{methylpseudouridine. In some embodiments of the therapeutic composition, the LNP (e.g., the SLN) comprises an ionizable lipid, a structural lipid, a phospholipid, a sterol, a PEG-modified lipid, or any combination thereof. In some aspects, provided herein are methods of treating a cancer in a subject, the method comprising administering an effective amount of a therapeutic composition contemplated herein. In some embodiments, the cancer is selected from: hepatocellular carcinoma, lymphoma, B cell lymphoma, T cell lymphoma, mycosis fungoides, Hodgkin’s Disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of head and neck, kidney cancer, lung cancers such as small cell lung cancer and non-small cell lung cancer, neuroblastoma/glioblastoma, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, melanoma, squamous cell carcinomas of the mouth, throat, larynx, and lung, endometrial cancer, cervical cancer, cervical carcinoma, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, head and neck carcinoma, large bowel cancer, hematopoietic cancers; testicular cancer; colon and rectal cancers,} _{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 an immunotherapy. The immunotherapy may comprise administration of a therapeutic antibody, such as aducanumab. The immunotherapy may comprise administration of an immune checkpoint inhibitor. In some such embodiments, the immune checkpoint inhibitor comprises an antibody or antigen-binding fragment thereof specific for PD-1, PD-L1, or CTLA4. In certain embodiments, 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 a cholinesterase inhibitor, such as, donepezil, rivastigmine, or galantamine. In yet further embodiments, the method further comprises administering to the subject a glutamate regulator, such as memantine.} Definitions F^{or convenience, certain terms employed in the specification, examples, and appended} _{claims are collected here.} 7 _FH11736604.1 GRH-00161 T^{he articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. The term “about” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured 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 of ingredients or conditions such as temperature, these values include the stated value with a variation of 0-10% around the value (X ± 10%). Ranges are stated in shorthand to avoid having to set out at length and describe each and every value within the range. Therefore, when ranges are stated for a value, any appropriate value within the range can be selected, and these values include the upper value and the lower value of the range. For example, a range of 0.1-1.0 represents the terminal values of 0.1 and 1.0, as well as the intermediate values of 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, and all intermediate ranges encompassed within 0.1-1.0, such as 0.2-0.5, 0.2-0.8, 0.7-1.0, etc. “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 for} _{pharmaceutically active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with angiotensin II, its use in the pharmaceutical formulations of the invention is contemplated. In certain embodiments, the pharmaceutically acceptable carrier/excipient is a saline solution. As used herein, the term “administering" means providing a pharmaceutical agent or composition to a subject, and includes, but is not limited to, administering by a medical professional and self-administering. Such an agent can contain, for example, peptide or nucleic acid described herein. As used herein, the term “treatment” refers to clinical intervention designed to alter the natural course of the individual being treated during the course of clinical pathology. Desirable effects of treatment include decreasing the rate of progression, ameliorating or palliating the pathological state, and remission or improved prognosis of a particular disease, disorder, or condition. An individual is successfully “treated,” for example, if one or more symptoms associated 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, when administered to a statistical sample prior to the onset of the disorder or condition, reduces the occurrence of the disorder or condition in the treated sample relative to an untreated control} 8 _FH11736604.1 GRH-00161 ^{sample, or delays the onset or reduces the severity of one or more symptoms of the disorder or condition relative to the untreated control sample. In certain embodiments, agents of the invention may be used alone or conjointly administered with another type of therapeutic agent. As used herein, the phrase “conjoint administration” or “administered conjointly” refers to any form of administration of two or more different therapeutic agents such that the second agent is administered while the previously administered therapeutic agent is still effective in the body (e.g., the two agents are simultaneously effective in the subject, which may include synergistic effects of the two agents). For example, the different therapeutic compositions disclosed herein can be administered either in the same formulation or in separate formulations, either concomitantly or sequentially. In certain embodiments, the different therapeutic agents (e.g., a therapeutic composition comprising an 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 be administered within about one hour, about 12 hours, about 24 hours, about 36 hours, about 48} _{hours, about 72 hours, or about a week of one another. Thus, a subject who receives such treatment can benefit from a combined effect of different therapeutic agents. The terms “polypeptide fragment” or “fragment”, when used in reference to a particular polypeptide, refers to a polypeptide in which amino acid residues are deleted as compared to the reference polypeptide itself, but where the remaining amino acid sequence is usually identical to that 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 about 5, 6, 8 or 10 amino acids long, at least about 14 amino acids long, at least about 20, 30, 40 or 50 amino acids long, at least about 75 amino acids long, or at least about 100, 150, 200, 300, 500 or more amino acids long. A fragment can retain one or more of the biological activities of the reference polypeptide. In various embodiments, a fragment may comprise an enzymatic activity and/or an interaction site of the reference polypeptide. In some embodiments, a fragment may have suppressive, disruptive, or enhancing properties.} Nucleic acids and vectors N^{ucleic acids and vectors disclosed herein include polynucleotides and polynucleotide vectors encoding the disclosed heat shock proteins (e.g., HSP70 polypeptides) that allow expression in the disclosed cells.} N_{ucleic acid sequences contemplated herein can be obtained using recombinant methods known in the art. Alternatively, the sequence of interest can be produced synthetically, rather than cloned.} 9 _FH11736604.1 GRH-00161 I^{n addition to the polypeptide-encoding sequences, other structural properties as described herein for mRNA constructs (e.g., modified nucleobases, 5' cap, 5' UTR, 3' UTR, miR binding site(s), polyA tail, as described herein). Suitable mRNA construct components are as described herein. In some embodiments, a nucleic acid of the disclosure may be modified in a coding region (e.g., an open reading frame of an mRNA encoding a polypeptide). In other embodiments, nucleic acid may be modified in regions besides a coding region, such as, 5' cap, a 5’-untranslated region (UTR) and/or a 3’- UTR, polyA tail of an mRNA, wherein any combination of elements may be independently modified. In some embodiments, such regions may contain one or more different nucleoside modifications. In such embodiments, modifications may also be present in the coding region. Examples of nucleoside modifications and combinations thereof that may be present in mRNAs disclosed herein include, but are not limited to, those described in PCT Patent Application Publications: WO2012045075, WO2014081507, WO2014093924, WO2014164253, WO2014159813, WO2018144775, WO2018081459, each of which are incorporated herein in their entirety. In some embodiments, the mRNAs of the disclosure can include a combination of modifications to the sugar, the nucleobase, and/or the internucleoside linkage. These combinations can include any one or more modifications described herein. As a non-limiting} _{example, the natural nucleotide uridine may be substituted with a modified nucleoside described herein. In another non-limiting example, the natural nucleoside uridine may be partially substituted (e.g., about 0.1 %, 1 %, 5 %, 10 %, 15 %, 20 %, 25 %, 30 %, 35 %, 40 %, 45 %, 50 %, 55 %, 60 %, 65 %, 70 %, 75 %, 80 %, 85 %, 90 %, 95 % or 99.9 % of the 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 typically achieved by operably linking a nucleic acid encoding the HSP70 polypeptide to a promoter, and incorporating the construct into an expression vector. Typical cloning vectors contain transcription and translation terminators, initiation sequences, and promoters useful for regulation of 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, a phage derivative, an animal virus, and a cosmid. Vectors of particular interest include expression vectors, 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),} 1⁰ _FH11736604.1 GRH-00161 ^{and in other virology and molecular biology manuals. Viruses, which are useful as vectors include, but are not limited to, retroviruses, adenoviruses, adeno-associated viruses, herpes viruses, and lentiviruses. In general, a suitable vector contains an origin of replication functional in at least one organism, a promoter sequence, convenient restriction endonuclease sites, and one or more selectable markers. In some embodiments, the polynucleotide vectors are lentiviral or retroviral vectors. A number of viral based systems have been developed for gene transfer into mammalian cells. For example, retroviruses and AAVs provide a convenient platform for gene delivery systems. A selected gene can be inserted into a vector and packaged in viral particles using techniques known in the art. The recombinant virus can then be isolated and delivered to cells of the subject either in vivo or ex vivo. One example of a suitable promoter is the immediate early cytomegalovirus (CMV) promoter sequence. This promoter sequence is a strong constitutive promoter sequence capable of 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 simian virus 40 (SV40) early promoter, MND (myeloproliferative sarcoma virus) promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat} _{(LTR) promoter, MoMuLV promoter, an avian leukemia virus promoter, an Epstein-Barr virus immediate early promoter, a Rous sarcoma virus promoter, as well as human gene promoters such as, but not limited to, the actin promoter, the 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, a glucocorticoid promoter, a progesterone promoter, and a tetracycline promoter. Additional promoter elements, e.g., enhancers, regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 base pairs (bp) upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In order to assess the expression of a heat shock protein disclosed herein or portions thereof, the expression vector to be introduced into a cell can also contain either a selectable marker gene or a reporter gene or both to facilitate identification and selection of expressing cells from the population of cells sought to be transfected or infected through viral vectors. The selectable marker may be carried on a separate piece of DNA and used in a co-transfection} 1¹ _FH11736604.1 GRH-00161 ^{procedure. Both selectable markers and reporter genes may be flanked with appropriate regulatory sequences to enable expression in the host cells. Useful selectable markers include, for example, antibiotic-resistance genes. Reporter genes may be used for identifying potentially transfected cells and for evaluating the functionality of regulatory sequences. In general, a reporter gene is a gene that is not present in or expressed by the recipient organism or tissue and that encodes a polypeptide whose expression is manifested by some easily detectable property, e.g., enzymatic activity. Expression of the reporter gene is assayed at a suitable time after the nucleic acid has been introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta- galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, or the green fluorescent protein gene. Suitable expression systems are well known and may be prepared using known 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 the promoter. Such promoter regions may be linked to a reporter gene and used to evaluate agents for the ability to modulate promoter-driven transcription. Methods of introducing and expressing genes into a cell are known in the art. In the context 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 expression vector can be transferred into a host cell by physical, chemical, or biological means. Physical methods for introducing a polynucleotide into a host cell include calcium phosphate precipitation, lipofection, particle bombardment, microinjection, electroporation, and the like. Methods for producing cells comprising vectors and/or exogenous nucleic acids are well-known in the art. See, for example, Sambrook et al. (2001, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York). Biological methods for introducing a polynucleotide of interest into a host cell include the use of DNA and RNA vectors. Viral vectors, and especially retroviral vectors, have become the most widely used method for inserting genes into mammalian, e.g., human cells. Chemical means for introducing a polynucleotide into a host cell include colloidal dispersion systems, such as macromolecular complexes, nanocapsules, microspheres, beads, and lipid-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 is a liposome. In another aspect, the nucleic acid may be associated with a lipid. The nucleic acid associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed} 1² _FH11736604.1 GRH-00161 ^{within the lipid bilayer of a liposome, attached to a liposome via a linking molecule that is associated with both the liposome and the oligonucleotide, entrapped in a liposome, complexed with a liposome, dispersed in a solution containing a lipid, mixed with a lipid, combined with a lipid, contained as a suspension in a lipid, contained or complexed with a micelle, or otherwise associated with a lipid. Lipid, lipid/nucleic acid or lipid/expression vector associated compositions are not limited to any particular structure in solution. For example, they may be present in a bilayer structure, as micelles, or with a “collapsed” structure. They may also simply be interspersed in a solution, possibly 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 of compounds, which contain long-chain aliphatic hydrocarbons and their derivatives, such as fatty acids, alcohols, amines, amino alcohols, and aldehydes. Lipids suitable for use can be obtained from commercial sources. For example, dimyristyl phosphatidylcholine (“DMPC”) can be obtained from Sigma, St. Louis, Mo.; dicetyl phosphate (“DCP”) can be obtained from K & K Laboratories (Plainview, N.Y.); cholesterol (“Choi”) can be obtained from Calbiochem-Behring; dimyristyl phosphatidylglycerol (“DMPG”) and other lipids may be obtained from Avanti Polar Lipids, Inc, (Birmingham, Ala.).} I_{n some embodiments the nucleic acids of the disclosure may be formulated in nanoparticles (e.g., lipid nanoparticles) or other delivery vehicles, e.g., to protect them from degradation 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 herein by reference in their entirety. In certain embodiments, an mRNA of the disclosure is encapsulated within a nanoparticle. In particular embodiments, a nanoparticle is a particle having at least one dimension (e.g., a diameter) less than or equal to 1000 nanometers (nm), less than or equal to 500 nm 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 an average diameter between 10 to 1000 nm. In some such embodiments, the SLN possesses a solid lipid core matrix that can solubilize lipophilic molecules and can be stabilized with surfactants and/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. Such} 1³ _FH11736604.1 GRH-00161 ^{lipids can be used alone or in combination. In certain embodiments, a lipid nanoparticle comprises one or more nucleic acids, e.g., mRNAs, described herein. In certain embodiments, it is desirable to target a nanoparticle, e.g., a lipid nanoparticle, of the disclosure using a targeting moiety that is specific to a cell type and/or tissue type. In some embodiments, a nanoparticle may be targeted to a particular cell, tissue, and/or organ using a targeting moiety. In particular embodiments, a nanoparticle comprises one or more mRNA described herein and a targeting moiety. Exemplary non-limiting targeting moieties include ligands, cell surface receptors, glycoproteins, vitamins (e.g., riboflavin) and antibodies (e.g., full-length antibodies, antibody fragments (e.g., Fv fragments, single chain Fv (scFv) fragments, Fab′ fragments, or F(ab′)2 fragments), single domain antibodies, camelid antibodies and fragments thereof, human antibodies and fragments thereof, monoclonal antibodies, and multispecific antibodies (e.g., bispecific antibodies)). In some embodiments, the targeting moiety may be a polypeptide. The targeting moiety may include the entire polypeptide (e.g., peptide or protein) or fragments thereof. A targeting moiety is typically positioned on the outer surface of the nanoparticle in such a manner that the targeting moiety is available for interaction with the target, for example, a cell surface receptor. A variety of different targeting moieties and methods are known and available in the art, including those described, e.g., in Sapra et al., Prog. Lipid Res. 42(5):439-62, 2003 and} _{Abra et al., J. Liposome Res. 12:1-3, 2002. For example, the lipid nanoparticle may include a targeting 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 tumor cells). In particular embodiments, the targeting moiety targets the lipid nanoparticle to a hepatocyte. In other embodiments, the targeting moiety targets the lipid nanoparticle to a colon cell. In some embodiments, the targeting moiety targets the lipid nanoparticle to a liver cancer cell (e.g., a hepatocellular carcinoma cell) or a colorectal cancer cell (e.g., a primary tumor or a metastasis). In addition to nanoparticle compositions provided herein, also disclosed are methods of producing lipid nanoparticles, which may include encapsulating a polynucleotide (e.g., an mRNA contemplated herein). Such contemplated methods comprise using any of the compositions disclosed herein and producing lipid nanoparticles in accordance with methods of production of lipid nanoparticles known in the art, e.g., Wang et al. (2015) “Delivery of oligonucleotides with lipid nanoparticles” Adv. Drug Deliv. Rev. 87:68-80; Silva et al. (2015) “Delivery Systems for} 1⁴ _FH11736604.1 GRH-00161 ^{Biopharmaceuticals. 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) “Lipid nanoparticles for the delivery of biopharmaceuticals” Curr. Pharm. Biotechnol. 16:291-302, and references cited therein, all of which are incorporated herein by reference in their entirety. In certain embodiments, lipid nanoparticles (LNPs) comprise lipids including an ionizable lipid, a structural 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 one or more mRNAs. Thus, each of the LNPs described herein may be used in a formulation comprising the mRNA described herein. In one embodiment, a lipid nanoparticle comprises an ionizable 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 a phospholipid. In some embodiments, the LNP has a molar ratio of about 20-60% ionizable lipid:about 5-25% phospholipid:about 25-55% sterol; and about 0.5-15% PEG-modified lipid. In some 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 some embodiments, the LNP comprises a molar ratio of about 55% ionizable lipid, about 2.5% PEG} _{lipid, about 32.5% cholesterol and about 10% phospholipid. In some embodiments, the ionizable lipid is an ionizable amino or cationic lipid and the neutral lipid is a phospholipid, and the sterol is a cholesterol. The ionizable lipids contemplated herein include cationic and/or ionizable lipids. Such cationic and/or ionizable lipids include, but are not limited to, SM-102, 9-Heptadecanyl 8-{(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino}octanoate, 3-(didodecylamino)-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-} 1⁵ _FH11736604.1 GRH-00161 ^{dimethylammonium bromide (“DDAB”); N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethylammonium chloride (“DOTAP”); 1,2-Dioleyloxy-3-trimethylaminopropane chloride salt (“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-ammonium trifluoracetate (“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 ammonium bromide (“DMRIE”). Additionally, a number of commercial preparations of cationic and/or ionizable 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 particular embodiments, the lipid is DLin-MC3-DMA, DLin-KC2-DMA, or ALC-0159. The phospholipids provided herein may, for example, be one or more saturated or (poly)unsaturated phospholipids or a combination thereof. In general, phospholipids comprise a phospholipid moiety and one or more fatty acid moieties. A phospholipid moiety can be selected, for example, from the non-limiting group consisting 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 consisting of lauric acid, myristic acid, myristoleic acid, palmitic acid, palmitoleic acid, stearic acid, oleic acid, linoleic acid, alpha-linolenic acid, erucic acid, phytanoic acid, arachidic acid, arachidonic acid, eicosapentaenoic acid, behenic acid, docosapentaenoic acid, and docosahexaenoic acid. Particular phospholipids can facilitate fusion to a membrane. For example, a cationic phospholipid 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 allow one or more elements (e.g., a therapeutic agent) of a lipid-containing composition (e.g., LNPs) to pass through the membrane permitting, e.g., delivery of the one or more elements to a target tissue. Non-natural phospholipid species including natural species with modifications and substitutions including branching, oxidation, cyclization, and alkynes are also contemplated. For example, 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). Under appropriate reaction conditions, an alkyne group can undergo a copper-catalyzed cycloaddition upon exposure to an azide. Such reactions can be useful in functionalizing the surface (e.g., the} 1⁶ _FH11736604.1 GRH-00161 ^{lipid monolayer or bilayer) of a nanoparticle composition to facilitate membrane permeation or cellular recognition or in conjugating a nanoparticle composition to a useful component such as a targeting or imaging moiety (e.g., a dye). Phospholipids include, but are not limited to, glycerophospholipids such as phosphatidylcholines, phosphatidylethanolamines, phosphatidylserines, phosphatidylinositols, phosphatidyl glycerols, and phosphatidic acids. In some embodiments, the phospholipid is distearoylphosphatidylcholine (DSPC). Phospholipids also include phosphosphingolipid, such as sphingomyelin. The lipid composition of a pharmaceutical composition disclosed herein can comprise one or more structural lipids. As used herein, the term “structural lipid” refers to sterols and also to lipids containing sterol moieties. Incorporation of structural lipids in the lipid nanoparticle may help mitigate aggregation of other lipids in the particle. Structural lipids can be selected from the group including but not limited to, cholesterol, fecosterol, sitosterol, ergosterol, campesterol, stigmasterol, brassicasterol, tomatidine, tomatine, ursolic acid, alpha-tocopherol, hopanoids, phytosterols, steroids, and mixtures thereof. In some embodiments, the structural lipid is a sterol. As defined herein, “sterols” are a subgroup of steroids consisting of steroid alcohols. In certain embodiments, the} _{structural lipid is a steroid. In some embodiments, the structural lipid is cholesterol. In certain embodiments, 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 and phosphatidic 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 also referred to as PEGylated lipids. For example, a PEG lipid can be PEG-c-DOMG, PEG-DMG, PEG-DLPE, PEG-DMPE, PEG-DPPC, or 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-modified} 1⁷ _FH11736604.1 GRH-00161 ^{ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixtures thereof. In some embodiments, the lipid moiety of the PEG-lipids includes those having lengths of from about C14 to about C22, preferably from about C14 to about C16. In some embodiments, a PEG moiety, for example an mPEG-NH2, has a size of about 1000, 2000, 5000, 10,000, 15,000 or 20,000 daltons. In some embodiments, the PEG-lipid is PEG2k-DMG. In certain embodiments, the lipid nanoparticles described herein can comprise a PEG lipid which is a non-diffusible PEG. Non-limiting examples of non-diffusible PEGs include PEG-DSG and PEG-DSPE. PEG-lipids are known in the art, such as those described in U.S. Pat. No. 8,158,601 and International Publ. No. WO 2015/130584 A2, which are incorporated herein by reference in their entirety. 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 of Therapeutic Agents,” which is incorporated herein by reference in its entirety. The lipid component of a lipid nanoparticle composition may include one or more} _{molecules comprising polyethylene glycol, such as PEG or PEG-modified lipids. Such species may be alternately referred to as PEGylated lipids. A PEG lipid is a lipid modified with polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified ceramides, PEG-modified dialkylamines, PEG-modified diacylglycerols, PEG-modified dialkylglycerols, 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 2 kilodaltons. In certain embodiments, the LNPs comprise ALC-0315, a synthetic ionizable cationic amino lipid. “Stabilizing lipids”, as used herein, may include, but is not limited to, lipids that contain surface stabilizing polymers conjugated to the lipid headgroup. In some embodiments, the polymer conjugated to the lipid headgroup is hydrophilic. The hydrophilic polymer-conjugated lipid may be a polyethyleneglycol (PEG)-conjugated lipid. In other embodiments, the polymer} 1⁸ _FH11736604.1 GRH-00161 ^{making up the polymer-lipid conjugate can be a polymer that contains a backbone that allows it to 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 invisible to the immune system. Without being bound by theory, hydrophilic polymer PEG on the outer surface of the nanoparticle induces steric stabilization due to the local surface concentration of highly hydrated PEG groups. This attracts a water shell that surrounds the nanoparticle that acts as a barrier against certain interactions in the biological environment, e.g., making the nanoparticle less detectable by, or otherwise invisible to, the immune system, including} _{inhibition of adsorption and opsonization of the nanoparticle and its contents. Such nanoparticles may have reduced detection and destruction in the biological environment, and can lead to extended blood circulation time and a preferential accumulation at target sites. Stabilizing lipids may include some lipids that are not conjugated to a stabilizing polymer. Such lipids contain a negatively charged phosphate group shielded by a hydrophilic neutral 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 ionizable lipid:structural lipid:phospholipid:PEG-modified lipid. Preferably, the LNP has a molar ratio of 50:38.5:10:1.5 of SM-102:cholesterol:DSPC (Distearoylphosphatidylcholine): DMG-PEG 2000. In some such embodiments, the LNP is a solid lipid nanoparticle (SLN).} C^{ompositions In 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 comprising the nanoparticles disclosed herein) as well as methods of administering such pharmaceutical compositions. In some embodiments, the nucleic acids, polypeptides, or compositions provided herein are used as an adjuvant. As used herein, the term “adjuvant” broadly refers to an agent that affects an immunological or physiological response in a patient or subject. For example and} _{without limitation, when used as an adjuvant the polypeptides or compositions provided herein may increase the presence of an antigen over time or to an area of interest like a tumor, facilitate absorption of a presented antigen, activate macrophages and lymphocytes, and/or support the production of cytokines. By changing an immune response, the adjuvant might permit a smaller dose of an immune interacting agent to increase the effectiveness or safety of a particular dose of the immune interacting agent. For example, the adjuvant might prevent T cell exhaustion and thus increase the effectiveness or safety of a particular immune interacting agent.} 1⁹ _FH11736604.1 GRH-00161 C^{ompositions contemplated herein may be administered intrapleurally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intraperitoneally, intracranially, or by direct administration to an organ. Said compositions may comprise one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solutions, dispersions, suspensions or emulsions, or sterile powders which may be reconstituted into sterile injectable solutions or dispersions just prior to use, which may contain sugars, alcohols, antioxidants, buffers, bacteriostats, solutes which render the formulation isotonic with the blood of the intended recipient or suspending or thickening agents. Examples of suitable aqueous and nonaqueous carriers which may be employed in the pharmaceutical 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 size in 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 or any intermediate value encompassed therein, particularly, about: 100 µL, 200 µL, 300 µL, 400 µL, 500 µL, 600 µL, 700 µL, 800 µL, 900 µL, or 1000 µL. In some preferred embodiments, the} _{dose size is 500 µL. In some such embodiments, the dose comprises an mRNA concentration of about 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. In preferred embodiments, the dose comprises an mRNA concentration of 0.33 µg/ µL. For example, 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 ethanol lipid nanoparticle solution. The ethanol may be removed after the particle formation via dialysis leaving the lipid nanoparticles containing mRNA in saline buffer alone. This may then be stored at -20°C until use. In other aspects, provided herein is a composition containing the cells comprising the LNPs (e.g., the SLNs) disclosed herein, carrying the nucleic acids encoding the heat shock proteins (e.g., HSP70 polypeptides) contemplated herein. The person of skill in the relevant art will appreciate that such cells may be administered via the adoptive transfer of said cells to a recipient subject in need thereof, as is known in the art. Briefly, and without being limited by theory, cells (selected from a third-party donor or cells derived from the recipient subject) may be brought into contact with the LNPs provided herein (e.g., in vitro or ex vivo), and administered to the subject in need, by means known in the art.} Therapeutic Methods 2⁰ _FH11736604.1 GRH-00161 I^{n certain embodiments, provided herein are methods of treating a subject, comprising administering to the subject a therapeutic composition provided herein. In some embodiments, the methods provided herein are used to treat or prevent a neurodegenerative disease, e.g., a disease associated with the progressive loss of structure or function of neurons. Such diseases may include, without limitation, amyotrophic lateral sclerosis, multiple sclerosis, Parkinson's disease, Alzheimer's disease, Huntington's disease, multiple system atrophy, and prion diseases (e.g., Creutzfeldt–Jakob disease (CJD)). Notably, several neurodegenerative diseases are classified in the art as proteinopathies, being associated with the aggregation of misfolded proteins. A key mechanism of action in many neurodegenerative diseases is protein toxicity. Without being bound by theory, alpha-synuclein may aggregate into insoluble fibrils resulting in Lewy bodies observed in Parkinson's disease, dementia with Lewy bodies, and multiple system atrophy and may contribute to the amyloid plaques in Alzheimer's disease. The term synucleinopathies (a/k/a α-synucleinopathies) describes neurodegenerative diseases and disorders characterized by the abnormal accumulation of aggregates of α-synuclein protein in neurons, nerve fibres, or glial cells. These diseases/disorders include Shy-Drager syndrome, striatonigral degeneration, olivopontocerebellar atrophy, neurodegeneration with brain iron} _{accumulation, type I (a/k/a neuroaxonal dystrophy or Hallervorden-Spatz syndrome), axonal lesions after traumatic brain injury, Pick’s disease, and amyotrophic lateral sclerosis. Tau protein is the main component of neurofibrillary tangles in Alzheimer's disease and of Pick bodies found in behavioral variant frontotemporal dementia. Indeed, tauopathy describes a class of neurodegenerative diseases involving the aggregation of tau protein into neurofibrillary or 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 to chromosome 17 (FTDP-17), vacuolar tauopathy, lytico-bodig disease (Parkinson-dementia complex of Guam), Ganglioglioma and gangliocytoma, meningioangiomatosis, postencephalitic parkinsonism, subacute sclerosing panencephalitis (SSPE), lead encephalopathy, tuberous sclerosis, 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 with synucleinopathies. Misfolded Amyloid β and Aβ oligomers are major components of amyloid plaques in Alzheimer's disease. Misfolded PRNP proteins are the main component of prion diseases and transmissible spongiform encephalopathy. In some embodiments, the disease is characterized by} 2¹ _FH11736604.1 GRH-00161 ^{disruption or dysregulation of protein degradation pathways (e.g., ubiquitin–proteasome pathways and autophagy–lysosome pathways), by membrane damage (e.g., tubulation and vesiculation as induced by alpha-synuclein), by mitochondrial dysfunction (e.g., the generation of reactive oxygen species (ROS), perturbation of calcium homeostasis, programmed cell death (PCD), mitochondrial fission and fusion, lipid concentration of mitochondrial membranes, and mitochondrial permeability transition). In some embodiments, the compositions and methods provided herein can be used to treat an autoimmune disease. Examples of autoimmune diseases include, for example, glomerular nephritis, arthritis, dilated cardiomyopathy-like disease, ulcerous colitis, Sjogren syndrome, Crohn disease, systemic erythematosus, chronic rheumatoid arthritis, juvenile rheumatoid arthritis, Still’s disease, multiple sclerosis, psoriasis, allergic contact dermatitis, polymyositis, pachyderma, periarteritis nodosa, rheumatic fever, vitiligo vulgaris, Behcet disease, Hashimoto disease, 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-phospholipid syndrome, atopic allergy, autoimmune atrophic gastritis, achlorhydria autoimmune, celiac disease, Cushing’s syndrome, dermatomyositis, discoid lupus erythematosus, Goodpasture's} _{syndrome, Hashimoto's thyroiditis, idiopathic adrenal atrophy, idiopathic thrombocytopenia, insulin-dependent diabetes, Lambert-Eaton syndrome, lupoid hepatitis, lymphopenia, mixed connective tissue disease, pemphigoid, pemphigus vulgaris, pernicious anemia, phacogenic uveitis, polyarteritis nodosa, polyglandular autosyndromes, primary biliary cirrhosis, primary sclerosing 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 rheumatoid arthritis, systemic lupus erythematosus and/or Sjögren’s syndrome. In some embodiments, the methods provided herein are used to treat inflammatory bowel diseases (IBDs). For example, in certain embodiments the methods provided herein are used to treat 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 some embodiments, the methods provided herein are used to treat irritable bowel syndrome,} 2² _FH11736604.1 GRH-00161 ^{microscopic 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’s disease, sarcoidosis, scleroderma, IBD-associated dysplasia, dysplasia associated masses or lesions, 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, septic shock, obesity-related inflammation, Parkinson's Disease, Crohn's Disease, Alzheimer's Disease (AD), cardiovascular disease (CVD), inflammatory bowel disease (IBD), chronic obstructive pulmonary disease, an allergic reaction, an autoimmune disease, blood inflammation, joint inflammation, arthritis, asthma, ulcerative colitis, hepatitis, psoriasis, atopic dermatitis, pemphigus, glomerulonephritis, atherosclerosis, sarcoidosis, rheumatoid arthritis, psoriatic arthritis, ankylosing spondylitis, Wegner's syndrome, Goodpasture's syndrome, giant cell arteritis, polyarteritis nodosa, idiopathic pulmonary fibrosis, acute lung injury, post-influenza pneumonia, SARS, tuberculosis, malaria, sepsis, cerebral malaria, Chagas disease, schistosomiasis, bacteria and viral meningitis, cystic fibrosis, multiple sclerosis,} _{encephalomyelitis, sickle cell anemia, pancreatitis, transplantation, systemic lupus erythematosus, autoimmune diabetes, thyroiditis, and radiation pneumonitis, respiratory inflammation, or pulmonary inflammation. In some embodiments, provided herein is a method of treating a viral or bacterial infection in a subject. In some embodiments, the subject treated is immunocompromised. For example, in some embodiments, the subject has a T cell deficiency. In some embodiments, the subject has leukemia, lymphoma or multiple myeloma. In some embodiments, the subject is infected 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 administered immunosuppressive drugs. In some embodiments, the subject has undergone and/or is undergoing a chemotherapy. In some embodiments, the subject has undergone and/or is undergoing radiation therapy. In some embodiments, the subject is also administered an antiviral drug that inhibits viral replication. For example, in some embodiments, the subject is administered ganciclovir, valganciclovir, foscarnet, cidofovir, acyclovir, fomivirsen, maribavir, BAY 38-4766 or GW275175X. In some embodiments, the subject has cancer. In some embodiments, the methods described herein may be used to treat any cancerous or pre-cancerous tumor. In some} 2³ _FH11736604.1 GRH-00161 ^{embodiments, the cancer includes a solid tumor. Cancers that may be treated by methods and compositions 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, the cancer 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; lymphoepithelial carcinoma; basal cell carcinoma; pilomatrix carcinoma; transitional cell carcinoma; papillary transitional 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; ceruminous} _{adenocarcinoma; mucoepidermoid carcinoma; cystadenocarcinoma; papillary cystadenocarcinoma; papillary serous cystadenocarcinoma; mucinous cystadenocarcinoma; mucinous adenocarcinoma; signet ring cell carcinoma; infiltrating duct carcinoma; medullary carcinoma; lobular carcinoma; inflammatory carcinoma; mammary Paget’s disease; acinar cell carcinoma; adenosquamous carcinoma; adenocarcinoma w/squamous metaplasia; malignant thymoma; malignant ovarian stromal tumor; malignant thecoma; malignant granulosa cell tumor; and malignant neuroblastoma; Sertoli cell carcinoma; malignant Leydig cell tumor; malignant lipid cell tumor; malignant paraganglioma; malignant extra-mammary paraganglioma; pheochromocytoma; glomangiosarcoma; malignant melanoma; amelanotic melanoma; superficial spreading melanoma; malignant melanoma in giant pigmented nevus; epithelioid cell melanoma; malignant blue nevus; sarcoma; fibrosarcoma; malignant fibrous histiocytoma; myxosarcoma; liposarcoma; leiomyosarcoma; rhabdomyosarcoma; embryonal rhabdomyosarcoma; alveolar rhabdomyosarcoma; stromal sarcoma; malignant mixed tumor; mullerian mixed tumor; nephroblastoma; hepatoblastoma; carcinosarcoma; malignant mesenchymoma; malignant Brenner tumor; malignant phyllodes tumor; synovial sarcoma; malignant mesothelioma; dysgerminoma; embryonal carcinoma; malignant teratoma; malignant struma ovarii; choriocarcinoma; malignant mesonephroma; hemangiosarcoma; malignant hemangioendothelioma; Kaposi’s sarcoma; malignant hemangiopericytoma;} 2⁴ _FH11736604.1 GRH-00161 ^{lymphangiosarcoma; osteosarcoma; juxtacortical osteosarcoma; chondrosarcoma; malignant chondroblastoma; 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; malignant meningioma; neurofibrosarcoma; malignant neurilemmoma; malignant granular cell tumor; malignant lymphoma; Hodgkin's disease; Hodgkin's lymphoma; paragranuloma; small lymphocytic malignant lymphoma; diffuse large cell malignant lymphoma; follicular malignant lymphoma; mycosis fungoides; other specified non-Hodgkin's lymphomas; malignant histiocytosis; multiple myeloma; mast cell sarcoma; immunoproliferative small intestinal disease; leukemia; lymphoid leukemia; plasma cell leukemia; erythroleukemia; lymphosarcoma cell leukemia; 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 compositions provided herein may be varied so as to obtain an amount of the active ingredient which is} _{effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level will depend upon a variety of factors including the activity of the particular agent employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. In some embodiments, the methods provided herein further comprise treating the identified subject using a therapeutic method provided herein (e.g., by administering to the subject a composition provided herein). The administration of the disclosed compositions may be carried out in any convenient manner, including by injection, transfusion, or implantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intramedullary, intramuscularly, by intravenous (i.v.) injection, or intraperitoneally. In some embodiments, the disclosed compositions are administered to a patient by intradermal or subcutaneous injection. In some embodiments, the disclosed compositions are administered by} i.v. injection. The compositions may also be injected directly into a tumor, lymph node, organ, or 2⁵ _FH11736604.1 GRH-00161 ^{site of disease or disorder. In certain embodiments, the disclosed compositions are administered to a patient in conjunction with (e.g., before, simultaneously or following) any number of relevant treatment modalities, including but not limited to standard-of-care treatment for a disease or condition contemplated herein. In some embodiments, the disclosed compositions are conjointly administered with an immunotherapy. Such immunotherapies may comprise plaque- binding antibodies such as aducanumab. In some embodiments, the composition is conjointly administered to the subject with a cholinesterase inhibitor. The cholinesterase inhibitor may be, for example, donepezil, rivastigmine, or galantamine. In some embodiments, a glutamate regulator is administered conjointly with the compositions of the invention, such as the glutamate regulator memantine. In further embodiments, the compositions may be used in combination with chemotherapy, radiation, immunosuppressive agents, such as cyclosporine, azathioprine,} _{methotrexate, mycophenolate, and FK506, antibodies, or other immunoablative agents such as CAM PATH, anti-CD3 antibodies or other antibody therapies, cytoxin, fludarabine, cyclosporin, FK506, rapamycin, mycophenolic acid, steroids, FR901228, cytokines, and irradiation. In some embodiments, the said compositions are administered to a patient in conjunction with (e.g., before, simultaneously or following) bone marrow transplantation, T cell ablative therapy using either chemotherapy agents such as, fludarabine, external-beam radiation therapy (XRT), cyclophosphamide, or antibodies such as OKT3 or CAMPATH. In other embodiments, the compositions disclosed herein are administered following B-cell ablative therapy such as agents that react with CD20, e.g., Rituxan. For example, in some embodiments, subjects may undergo standard treatment with high dose chemotherapy followed by peripheral blood stem cell transplantation. In certain embodiments, following the transplant, subjects receive an infusion of the cells disclosed herein. In additional embodiments, expanded cells are administered before or following surgery.} EXAMPLES Example 1: Composition of Lipid Nanoparticle T^{he compositions contemplated herein may comprise four primary parts, an ionizable lipid, a phospholipid, a sterol, and a PEG-modified lipid. The LNPs are prepared by mixing an ethanolic 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),} 2⁶ _FH11736604.1 GRH-00161 3^{. cholesterol, and 4. DMG-PEG 2000. Optionally, a secondary ionizable lipid may be used, such as, ALC-0159 or ALC-0315, which has a PEG-lipid conjugate. A dose size of 500 µL was composed of 330 µL of mRNA and 170 µL of ethanol/lipid nanoparticle solution. For example, an initial mRNA payload concentration of 500 µg/mL was} _{diluted to achieve a desired mass ratio. In some embodiments, the mRNA to lipid mass-to-mass ratio is about 1:10. In some embodiments, the ethanol-to-water ratio may be about 1:3. The ethanol may be removed after the particle formation via dialysis leaving the lipid nanoparticles containing mRNA in PBS (phosphate buffered saline). This can then be run through a 220 nm filter to remove any aggregation that may have occurred during the dialysis process.} Ethanolic Lipid Mixtures T^{he 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. For example,} ^ 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 lipid mixture 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 ionizable} _{lipid, DSPC, cholesterol, and PEG-lipid. Ethanol was used to dilute to the final volume. For example, 10.0 mg or DPSC was mixed with 400 µL ethanol, and so on, in accordance with Table 1 below. The appropriate volume of each lipid mixture component, as listed in Table 1, was then transferred to a single tube to prepare the ethanolic lipid mixture and pipetted several times to ensure mixing and avoid precipitate and cloudiness.} Table 1

2⁷ _FH11736604.1 GRH-00161

^{Aqueous mRNA Solution: A lipid:mRNA (w:w) ratio of 10:1 and an ethanol:aqueous ratio of 1:3 was used. Utilizing 0.50 mL from the 5.0 mL lipid mixture stock solution (e.g., for 1.5 g/L above), 75 μg mRNA was added 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).} T_{he 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 was dialyzed in fresh PBS buffer to remove ethanol and loose lipids. The buffer was exchanged three times and the final LNP product was extracted and placed into a vessel for storage.} Example 2: Payload

A^{n intended payload for the LNPs was an mRNA strand which codes for the protein Hsp70 which is located on the gene HSPA1A. Utilizing specifically designed primers the} _{following sequence was amplified and applied to the lipid nanoparticles disclosed herein.}

2⁸ _FH11736604.1 GRH-00161

T^{he amplified mRNA sequence was inserted into the lipid nanoparticles (LNPs) of the invention. Such mRNA sequences comprised modifications to the 5’ cap, and, optionally} _{substituting some or all of the uridines with pseudouridines, as disclosed herein.} Example 3: Payload

A ^{further intended payload will be an mRNA strand encoding the protein Hsp70 specified} _{by the following primer pairs.}

2⁹ _FH11736604.1 GRH-00161

³⁰ _FH11736604.1 GRH-00161 T^{he amplified mRNA sequence will be inserted into the lipid nanoparticles (LNPs) of the} _{invention. Such mRNA sequences will comprise modifications to the 5’ cap, and, optionally substituting some or all of the uridines with pseudouridines, as disclosed herein.} Example 4: Payload III A^{n additional payload contemplated herein is an mRNA strand encoding the protein} _{Hsp70 specified by the following primer pairs.}

3¹ _FH11736604.1 GRH-00161

T^{he amplified mRNA sequence will be inserted into the LNPs of the invention. Such} _{mRNA sequences will comprise modifications to the 5’ cap, and, optionally substituting some or all of the uridines with pseudouridines, as disclosed herein.} Example 5: Payload IV A^{nother intended payload will be an mRNA strand encoding the protein Hsp70 specified} _{by the following primer pairs.}

3² _FH11736604.1 GRH-00161

T^{he amplified mRNA sequence will be inserted into the LNPs of the invention. Such} _{mRNA sequences will comprise modifications to the 5’ cap, and, optionally substituting some or all of the uridines with pseudouridines, as disclosed herein.} Example 6: Dynamic Light Scattering (DLS) I^{n order for the LNP formulations disclosed herein to be used as an Alzheimer’s treatment, each particle must be small enough to pass through the blood brain barrier. Particles} _{that are less than 200 nm (and preferably about 100 nm) are able to efficiently pass through the} 3³ _FH11736604.1 GRH-00161 ^{blood brain barrier (Ceña & Jávita, 2018). Doses of LNP-encapsulated mRNAs were tested with a Dynamic Light Scattering (DLS) machine. In DLS, when laser light encounters macromolecules in a solution the incident light scatters in all directions and scattering intensity is recorded by a detector. The rate of fluctuations in scattered light is directly related to the rate of diffusion 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 the intensity than larger particles. Therefore, the fluctuation in light intensity contains information about the diffusion of the molecules and can be used to extract a diffusion coefficient and calculate a particle size. Continuous DLS data collection showed that the LNP formulations disclosed herein consistently comprised particles with a diameter of 106 nm or smaller. In a typical DLS analysis the Z-Average is the estimated average size of the particles being measured. The D50 shows that 50% of the particles are the reported size or below. For example, Figure 1 shows that 50% of the particles 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 kCPS is acceptable for the particles of the invention disclosed herein. Higher Count Rates can indicate a 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 effective that dose can be. LNP solutions were filter sterilized with a 0.22 μm filter and stored at 4°C until use. Optionally, the LNPs can be lyophilized and stored at -80°C for long-term storage. DLS tests were run at least in triplicate. Thus, at least three measurements were taken of a sample, as illustrated in the overlay of the Z-Average (a measure of the average size of a particle size distribution) depicted in Figure 2. Notably, there is little variance among these measurements resulting in a single visible peak. (93.0 nm to 94.6 nm, (all three replicates laid out over each other). This data does not reflect the average kCPS of all three measurements as this output was used to compare triplicate measurements to each other. In addition to testing each batch of doses made, doses were saved to measure the size change over time. Such doses were used to measure if the LNPs encapsulating mRNA were aggregating. Even minor aggregation would be detectable. Generally, for DLS measures in intensity, one larger particle can block out many more smaller particles and skew the data to show a larger Z-Average and D50 than is actually true. The test sample was prepared and stored at 4ºC (the temperature at which all tested doses are stored). Particle size was measured at several different time points following completion of initial dialysis and filtration. To re-measure, at each} 3⁴ _FH11736604.1 GRH-00161 ^{time point a portion of sample was applied to a cuvette and read on the DLS machine and returned, e.g., to a 15 mL conical tube for storage at 4ºC again. Results showed that even after 87 days in storage, there was no aggregation of particles. Thus, the LNPs of the invention are stable at 4ºC for long periods of time without changing size.} I_{n summary, the DLS data shows that particles at 106 nm or smaller can be made consistently. This will allow LNPs to cross the blood-brain barrier to deliver HSP-70 mRNA to the cells of the brain. Said particles do not aggregate together or change in size over time, and can be stored at 4ºC with no degradation or aggregation over 60 days of repeated testing.} Example 7: ELISA Data E^{nzyme-linked immunoassay (ELISAs) are currently considered the preferred measurement of protein concentration within a solution. The ELISA employed herein was a sandwich ELISA, meaning that the analyte to be measured (HSP-70) was bound between two primary antibodies, each detecting a different epitope of the antigen– the capture antibody and the detection antibody. Thus, the measured protein, HSP-70, was initially bound to capture antibody adsorbed/linked to the bottom of wells of a microplate. After this initial binding a biotin conjugated anti-HSP-70 was attached to the bound HSP-70. Streptavidin-HRP (horseradish peroxidase) was complexed with the bound biotin. Finally, TMB (3, 3', 5, 5'- tetramethylbenzidine) was used as a colorimetric substrate, activated by the HRP detection antibody. The multiple washes and specific antibodies ensure that everything except for the desired protein is washed away. This test was conducted in order to ensure that the HSP-70 protein was being overexpressed with application of the LNP-encapsulated mRNA.} B_{riefly, 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 (250,000 cells per well) and left to adhere and grow overnight for 12 hours at 37 °C/ 5.0% CO2. After the 12-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 of HSP-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 in the incubator for 30 minutes. From each group, at least 500,000 cells were harvested for ELISA analysis of HSP-70 concentration. Microplates were loaded as described in Tables 2 and 3 and ELISAs were run using a Crocodile ELISA MiniWorkstation (Berthold Technologies, Germany). Following Biotin- Conjugate, Streptavidin-HRP, and washing steps, TMB solution was used as the colorimetric substrate. Color development of samples was monitored and the absorbance of each microwell} 3⁵ _FH11736604.1 GRH-00161 ^{was read on a spectro-photometer using 630 nm as the primary wavelength (optionally 450 nm as the reference wavelength; 610 nm to 650 nm is acceptable). Two columns were observed for each 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 compared to the standard columns which were made by a serial dilution of a known amount of HSP70 protein. See Tables 2 and 3.} Table 2

3⁶ _FH11736604.1 GRH-00161 Table 3

O^{verall, the fluorescence readings were shown to be higher in the sample columns than the control columns, which indicates that HSP-70 was overexpressed following application of the LNP 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-70 mRNA-LNPs. See Table 4.} ^{Table 4}

3⁷ _FH11736604.1 GRH-00161 T^{he ELISA data confirms that the HSP-70 mRNA-LNPs can induce an overproduction of the HSP-70 protein in human cells. Repeated tests showed that cells that have been dosed with} _{HSP-70 mRNA-LNPs produce significantly more HSP-70 protein than cells that have not been dosed.} Example 8: Cell line Data I^{n the human body, HSP-70 is a chaperone protein that is used to help refold misfolded proteins. Current research indicates that Alzheimer’s disease is caused through protein aggregation of amyloid beta and tau proteins, among others. This protein aggregation prevents neurons from communicating and can result in memory loss and overall loss in neurological functions over time due to neuronal death. Therefore, experiments in human-derived cell lines were designed to cause protein aggregation and observe said cells for reversal of the effects from protein aggregation following introduction of HSP-70 mRNA-LNPs. In order to induce protein aggregation, SH-SY5Y neuroblastoma cells were treated with 80mM lead acetate. Notably, any type of lead solution or metal ion solution can lead to protein misfolding/malfunction, hence why lead poisoning can be extremely fatal.} E_{xperiments were run in 24-hour and 48-hour groups. This was done to observe how HSP-70 mRNA-LNPs would affect lead-treated cells over different periods of time. In each group, 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 than the 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-70 mRNA-LNPs is not toxic to human cells. This data suggests that HSP-70 mRNA-LNPs provides a viable strategy for the treatment of Alzheimer’s disease, as it is successful in protecting cells from the effects of protein misfolding, particularly, neurons.} 2^{4 Hour Cell Count: This cell count consisted of running two 12-well plates. The test well 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 was to compare cells treated with lead acetate either with or without HSP-70 mRNA-LNPs. The} _{control well plate consisted of 3 wells treated with lead acetate, 3 wells treated with HSP-70 mRNA-LNPs and 3 wells treated with PBS. Three wells were treated with lead acetate as a control to ensure that lead acetate alone is killing the cells and to determine the viability of cells when treated with just lead acetate. Another 3 wells were treated with just HSP-70 mRNA-LNPs} 3⁸ _FH11736604.1 GRH-00161 ^{to ensure that the drug itself is not harming or damaging the cells in any way that would lead to cell death or a decrease in cell viability. The last 3 wells were treated with just PBS to ensure the solution the HSP-70 mRNA-LNPs is in, or PBS, is not harming the cells and causing a decrease in viability. The test well plate showed that HSP-70 mRNA-LNPs were able to slow down/stop the 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 using 250,000 cells per well, and returned to incubation at 37 °C/ 5.0% CO2 to allow cells to adhere and grow for at least 12 hours. In the first plate (test well plate) cells in wells 1-6 received 17 µL of HSP-70 mRNA-LNPs and cells in wells 7-12 received 17 µL of PBS. The 12-well plate was returned to the incubator for 15 minutes, and then 50 µL of 80 mM lead acetate was added to each well.} ^{Table 5: Test well plate}

I_{n 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, and 12 did not receive anything.} ^{Table 6: Control well plate}

B^{oth 12-well plates were then set in an incubator for 4 hours at 37 °C/ 5.0% CO2. After} _{the 4-hour period, cells in wells 1-6 in the first plate received 17 µL of HSP-70 mRNA-LNPs and cells in wells 7-12 received 17 µL of PBS. In the second plate, cells in wells 1, 5, and 9 were dosed with 50 µL of 80 mM lead acetate, cells in wells 2, 6, and 10 were dosed with 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, and 12 did not receive anything.} 3⁹ _FH11736604.1 GRH-00161 B^{oth 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 a hemocytometer 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 (Figure} _{3). The data from the dose only had a 1.3% difference in viability from the wells with only cells and media (Nothing) showing that HSP-70 mRNA-LNPs do not negatively affect the human neuroblastoma cells. Compared to the PBS Only wells, there was approximately a 2% difference in viability, further showing that the solution the dose is in also does not negatively affect the neuroblastoma cells’ viability.} 4^{8 Hour Cell Count: Similar to the above assay, this cell count consisted of running two 12-well plates with increased exposure time to lead acetate. The test well plate consisted of 6 wells of cells that were treated with lead acetate and HSP-70 mRNA-LNPs and 6 wells with cells treated with just lead acetate. The purpose of creating this plate was to compare cells treated with lead acetate either with or without HSP-70 mRNA-LNPs. The control well plate consisted of 3 wells treated with lead acetate, 3 wells treated with HSP-70 mRNA-LNPs and 3 wells treated with PBS. The three wells treated with lead acetate act as a control to ensure that lead acetate alone 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 or damaging the cells in any way that would lead to cell death or a decrease in cell viability. The last 3 wells treated with just PBS confirm that the solution comprising the HSP-70 mRNA-LNPs is not harming the cells and causing a decrease in viability.} B_{riefly, after overnight incubation (12 hours) at 37 °C/ 5.0% CO2 to allow cells to adhere and grow, the first plate (test well plate) received 17 µL of HSP-70 mRNA-LNPs per well or 17 μL of PBS and returned to the incubator for 3 hours, and then 50 µL of 80 mM lead acetate was added 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 µL of HSP-70 mRNA-LNPs in wells 2, 6, and 10; 17 µL of PBS in wells 3, 7, and 11; and nothing further 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 dosed with 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 was assessed as described above.} 4⁰ _FH11736604.1 GRH-00161 D^{ata from the test well plate showed that HSP-70 mRNA-LNPs were able to slow down/stop the effects of lead acetate on the cells. The data showed that there was an average of a 21% 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 an average viability of 87%, which displays that the HSP-70 mRNA-LNPs do not negatively affect human neuroblastoma cells. The Dose + Lead wells showed a 28% cell viability while Lead Only showed a 7% cell viability. The PBS + Lead wells showed a 2% cell viability. This data shows that the HSP-70 mRNA-LNPs increased the viability of human neuroblastoma cells by about 21% 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-LNPs increase the viability of lead poisoned cells for the first time, i.e., exposure to a 50 µL dose of 80 mM lead acetate solution. The SH-SY5Y cells dosed with HSP-70 mRNA-LNPs and lead acetate} _{had 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 and the particles overexpress HSP70 which refolds misfolded proteins. The viability of cells maintained with no added dose or PBS was compared to cells that were treated with just lead acetate, just HSP-70 mRNA-LNPs, just PBS, and a combination of PBS and lead acetate. This data showed that the cells treated with just lead acetate solution and cells with the combination of lead 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 cellular studies suggest that when HSP70 is overexpressed by introduction of HSP-70 mRNA-LNPs, the HSP70 has the capability to save cells from high levels of toxicity from lead poisoning. This further suggests that overexpression of HSP70 via the methods and nanoparticles disclosed herein has the capability to treat the protein misfolding caused by Alzheimer’s disease.} E^{xample 9: Encapsulation Data These experiments were performed to ensure that mRNA coding for HSP-70 was being successfully inserted via hydro-fluidic mixing into the solid lipid nanoparticles to successfully form the HSP-70 mRNA-LNPs of the invention. Using ethidium bromide, which is a DNA and RNA intercalator, particles were confirmed to contain fully formed mRNA strands coding for HSP-70 as the fluorescence in said particles was more than double the fluorescence of a control} _{solution and control solid lipid nanoparticles. Ethidium bromide fluoresces significantly more when it is bound to RNA and DNA strands than it does when unbound. Although this test is even stronger for double-stranded nucleotides, a less pronounced yet still significant increase in fluorescence can be measured for single-stranded RNA or DNA. Further, ethidium bromide binds to intact strands much more readily than it does to single nucleotides. Essentially ethidium} 4¹ _FH11736604.1 GRH-00161 ^{bromide fluoresces strongly when there are RNA or DNA strands present, but does so weakly when 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. A 5% triton X-100 was made using Rnase and Dnase free H2O. A 15 mL conical tube of ethidium bromide solution using 15 mL of DI water and 5 ug of ethidium bromide was made. Test solution was prepared from 200 µL of HSP-70 mRNA-LNPs, 800 µL of 5% Triton X-100, and 50 µL of ethidium bromide solution. Control solution not containing solid lipid nanoparticles comprised 200 µL of PBS, 800 µL of 5% Triton X-100, and 50 µL of ethidium bromide solution. Control solution containing solid lipid nanoparticles comprised 200 µL of PBS containing solid lipid nanoparticles lacking mRNA, 800 µL of 5% Triton X-100, and 50 µL of ethidium bromide solution. Samples were transferred, one at a time, to a 1 mL cuvette and inserted into a fluorometer 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 lipid nanoparticles containing mRNA coding for HSP-70 are broken apart using triton X. The final solution read for this test sample was composed of solid lipid nanoparticles containing mRNA coding for HSP-70, ethidium bromide solution, and triton X. The S1c curve (the corrected S1} _{curve) has a peak fluorescence of 4.34 x 10^6 CPS for this test sample. The first control curve depicted in Figure 5B displays the fluorescence of ethidium bromide without particles containing mRNA 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 sample depicted in 5C consisted of solid lipid nanoparticles lacking any mRNA. These particles were lysed using triton X and then exposed to ethidium bromide. The final solution was composed of lysed solid lipid nanoparticles, ethidium bromide solution, and triton X. This solution had a measured fluorescence of 2.14 x 10^6 CPS. This data displays that our solid lipid nanoparticles successfully encapsulated the HSP70 mRNA as the fluorescence in our test samples was over 2 million CPS higher than either of our control samples. Figure 5A also displayed a different peak wavelength than our controls with the peak being read at 607 nm compared to Figure 5B at a peak wavelength of 614 nm and Figure 5C with a peak wavelength of 613 nm. This shift in the fluorescence peak wavelength confirms that ethidium bromide is intercalating between nucleotides as the known excitation wavelength when bound is 605-608 nm, which is where the test samples peak wavelength was observed (607 nm).} Example 10: HSP-70 mRNA synthesis ^{RNA Isolation Procedure} T_{issue culture cells were harvested and centrifuged in to obtain a pellet, which could be} 4² _FH11736604.1 GRH-00161 ^{stored at -20ºC. Cell pellets were thawed or otherwise broken up by mechanical agitation of the sample tube prior to employing a commercial RNA isolation kit. Briefly, for a kit comprising a spin column, an amount of the appropriate lysis buffer was added to the tube (e.g., 350 μL for less than 5 x 106 cells; 700 μL for 5 x 106 - 1 x 107 cells), and the solution mixed by vortexing and/or pipetting, resulting in homogenized cell lysate. A matched volume of 70% ethanol was added to the cell lysate (e.g., for 350 μL of lysis buffer used to homogenize the pellet, 350 μL of 70% ethanol was added) and pipetted up and down for 15-30 seconds. A volume of 700 μL was} _{transferred to the spin column, comprising a collection tube, and centrifuged for 15 seconds at 11,400 rpm. The liquid flowthrough was discarded and 700 μL of appropriate wash buffer was added to the spin column. This was centrifuged for 15 seconds at 11,400 rpm, and the liquid flowthrough in the collection tube was discarded. The washing was repeated at least two more times with appropriate volumes of the appropriate wash buffer. The spin column was placed into a new 1.5 mL collection tube and 50 μL of RNase-free water was added to the spin column. The spin column was centrifuged for 1 minute at 11,400 rpm and the eluted RNA in the collection tube 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, were used. In a single reaction mixture, the enzymes Superscript III or IV were used to create cDNA} _{from the extracted RNA with Platinum SuperFi DNA Polymerase to synthesize double-stranded cDNA 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:}

A_{fter the cycler finished running, reaction tubes could be stored at -20ºC freezer, otherwise the resultant double-stranded cDNA was used immediately for mRNA creation.} c^{DNA Agarose Gel Analysis: Samples were prepared for loading onto agarose gel by taking 1 μL of cDNA sample with 2 μL of 1X loading dye (3 μL total), pipetted up and down slowly 3-5 times to mix. Another 1 μL} _{of cDNA sample was diluted with 4 μL of nuclease free water (5 μL total) and 1 μL aliquots of this diluted cDNA sample was mixed with 2 μL of 1X loading dye to make five 3 μL running} 4³ _FH11736604.1 GRH-00161 ^{samples. A 1KB DNA ladder was applied to the first well and the 3 μL samples were each added to a separate well (i.e., undiluted sample well followed by 5 diluted sample wells). Samples were electrophoresed for 1 hour at 100 mV, 400 mA (0.4 A). The expected length of the sequence should be ~2100 base pairs which was observed in all four samples shown in the HSPA1A Gene} _{DNA Agarose Gel of Figure 6. This double-stranded cDNA coding for the HSPA1A gene was then 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 Sanger sequencing (i.e., chain termination method) analysis.} m^{RNA Synthesis The prepared cDNA was used in the synthesis of HSP-70 mRNA. Commercially available kits, such as the HiScribe® T7 Quick Yield RNA Synthesis Kit, were used. Thermal cycle reactions were run according to kit manufacture protocols. Once thermal cycling was complete, 30 μL of Nuclease-Free Water was added to each reaction tube containing the newly synthesized mRNA, followed by 2 μL of DNase for a total of 52 μL. Once sufficiently mixed, each reaction tube was returned to the thermal cycler and set for a 15-minute hold at 37ºC followed by an indefinite 4ºC hold. Following the DNase treatment, the mRNA was capped using a Fausto virus capping enzyme (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 in that order, giving a final volume of 67.8 µL. The tubes were then incubated in a thermal cycler and set for a 30-minute hold at 37ºC followed by an indefinite 4ºC hold.} R_{NA purification and concentration following enzymatic reaction was performed using commercially available kits, such as the Monarch® RNA Clean up Kit. Briefly, 100 μL of RNA Binding Buffer was added to the 67.8 μL mRNA synthesis sample and mixed by pipetting. The total 167.8 μL of solution was added to spin columns provided with 167.8 μL of 100% ethanol and carefully pipetted to mix. Spin columns (with collection tubes) were centrifuged for 1 minute at 13,000 rpm. Liquid flowthrough (liquid in collection tube) was removed and 500 μl of appropriate RNA wash buffer was added to the spin column. Following centrifugation for 1 minute at 13,000 rpm, the liquid flowthrough was removed, and the wash repeated at least once more. Following washing, 50 μL of nuclease free water was added to the spin column (with empty collection tube) and centrifuged for 1 minute at 13,000 rpm. The collection tube containing the resultant flowthrough was held at 4ºC. The purified RNA from multiple collection tubes were combined and mixed via pipetting. Purified HSP-70 RNA could be stored at -20ºC} 4⁴ _FH11736604.1 GRH-00161 until use. m^{RNA Agarose Gel Analysis: Samples were prepared for loading onto a 1% agarose gel by taking 1 μL of mRNA sample and adding 9 μL of nuclease free water (10 μL total), pipetting up and down slowly 3-5 times to mix. The diluted mRNA sample was divided into 1 μL-aliquots and mixed with 3 μL of 1X 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 to a subsequent well (i.e., undiluted sample well followed by 5 diluted sample wells). Samples were electrophoresed for 1 hour at 100 mV, 400 mA (0.4 A). Six separate HSP-70 mRNA synthesis runs yielded identical results and produced mRNA for HSP-70 at 2100 bases long in high concentrations. (See figure 7.) This mRNA represents the sequence (or payload) disclosed herein and 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 DNA polymerase PlatinumTM SuperFiTM were added to the extracted RNA to create double-stranded cDNA coding for the HSPA1A gene using the appropriate primers. This 2128 base pair long strand was consistently synthesized as shown in Figure 6. The double-stranded cDNA coding for the HSPA1A gene was then used to create mRNA, as the forward primer used to synthesize cDNA contained a promoter for T7 RNA polymerase. After the mRNA synthesis, the resultant sequence was treated with DNase and then cleaned to remove any impurities such as free-floating nucleotides, enzymes, etc. The cleaned mRNA was then capped with a Faustovirus Capping Enzyme (FCE). The fully cleaned and capped mRNA is shown in figure 7 and represents Payload 1 used to create a final lipid nanoparticle containing HSP-70 mRNA product contemplated herein.} ^{INCORPORATION BY REFERENCE All publications and patents mentioned herein are hereby incorporated by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In case of conflict, the present specification, including its specific definitions, will control. While specific aspects of the patient matter have been} _{discussed, the above specification is illustrative and not restrictive. Many variations will become apparent to those skilled in the art upon review of this specification and the claims below. The full scope should be determined by reference to the claims, along with their full scope of equivalents, and the specification, along with such variations.} 4⁵ _FH11736604.1 GRH-00161 ^{EQUIVALENTS The present invention has been described in connection with what are presently considered to be the most practical and preferred embodiments. However, the invention has been presented 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 all modifications and alternative arrangements within the spirit and scope as set forth in the appended claims.} 4⁶ _FH11736604.1

Claims

GRH-00161 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. 4⁷ _FH11736604.1 GRH-00161 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.} 4⁸ _FH11736604.1 GRH-00161 ^{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. 4⁹ _FH11736604.1