Compositions and methods for modulating native human mRNA expression

Abstract

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

Description

[0001] GRH-00361 COMPOSITIONS AND METHODS FOR MODULATING NATIVE HUMAN mRNA EXPRESSION 5 BACKGROUND In recent years, the development of mRNA-based therapeutics has emerged as a promising avenue for treating a wide range of diseases including cancer, genetic, metabolic and 10 neurological disorders, as well as infectious diseases. However, achieving successful delivery of therapeutic mRNA to target cells while minimizing degradation and off-target effects has been considered challenging. For example, mRNAs are typically large in size, negatively charged, unstable, and hydrophilic. Moreover, the success of mRNA-based therapeutics in vivo is also dependent on the ability to overcome extra- and intracellular barriers to expression in the target 15 cells. Lipid Nanoparticles (LNPs), particularly solid lipid nanoparticles (SLNs), have garnered considerable attention as promising delivery vehicles for mRNA-based therapeutics due to their biocompatibility, stability, and ability to encapsulate and protect RNA payloads. However, to overcome obstacles and limitations, current mRNA-SLN based therapeutics focus on either creating non-native proteins to induce an immune response against a particular 20 foreign antigen or use heavily modified mRNA to create a non-native human protein, resulting in unforeseen immunological or other negative responses. Accordingly, there remains an unmet need for safer, more reliable strategies and methodologies for the efficacious delivery of mRNA- LNP-based therapeutics. SUMMARY 25 Aspects of the invention, as provided herein, include therapeutic compositions comprising native human mRNA formulated lipid nanoparticles (LNPs), wherein the mRNA comprises an open reading frame encoding wild-type mRNA or a functional fragment thereof of any human protein. As used herein, the term "native human mRNA" means mRNA corresponding to an 30 mRNA molecule that is expressed naturally in unmodified humans. While conventional 1 GRH-00361 therapeutic mRNA molecules often include many types of modification relative to their naturally occurring counterparts, native human mRNA molecules lack the following: • Synthetic codon optimization or inclusion of synthetically modified nucleotides such as but not limited to: 5 o pseudouracil (p) o 4-acetylcytidine (ac4c) o 5-(carboxy hydroxymethyl)uridine (chm5u) o 2'-O-methylcytidine (cm) o 5-carboxymethylaminomethyl-2-thiouridine (cmnm5s2u) 10 o 5-carboxymethylaminomethyluridine (cmnm5u) o Dihydrouridine (d) o 2' -O-methylpseudouridine (fm) o Beta D-galactosylqueuosine (gal q) o 2'-O-methylguanosine (gm) 15 o Inosine (i) o N6-isopentenyladenosine (i6a) o 1-methyladenosine (mla) o 1-methylpseudouridine (mlf) o 1-methylguanosine (mlg) 20 o 1-methylinosine (mli) o 2,2-dimethylguanosine (m22g) o 2-methyladenosine (m2a) o 2-methylguanosine (m2g) o 3-methylcytidine (m3c) 25 o 5-methylcytidine (m5c) o N6-methyladenosine (m6a) o 5-methylaminomethyluridine (mam5u) o 5-methoxyaminomethyl-2-thiouridine (mam5s2u) o Beta D-mannosylqueuosine (man q) 30 o 5-methoxycarbonylmethyl-2-thiouridine (mcm5s2u) o 5-methoxycarbonylmethyluridine (mcm5u) o 5-methoxyuridine (mo5u) o 2-methylthio-N6-isopentenyladenosine (ms2i6a) o N-((9-beta-D-ribofuranosy1-2-methylthiopurine-6-yl)carbamoy!)threonine 35 (ms2t6a) o N-((9-beta-D-ribofuranosylpurine-6-yl)N-methylcarbamoyl)threonine (mt6a) o Uridine-5-oxyacetic acid-methylester (mv) o Uridine-5-oxyacetic acid (o5u) o Wybutoxosine (osyw) GRH-00361 o Queuosine (q) o 2-thiocytidine (s2c) o 5-methyl-2-thiouridine (s2t) o 2-thiouridine (s2u) 5 o 4-thiouridine (s4u) o 5-methyluridine (t) o N-((9-beta-D-ribofuranosylpurine-6-yl)-carbamoyl)threonine (t6a) o 2'-O-methyl-5-methyluridine (tm) o 2'-O-methyluridine (um), wybutosine (yw) 10 o 3-(3-amino-3-carboxy-propyl)uridine, (acp3)u (x) • Coding Sequence (CDS) Modifications: o Deviations from the nucleotide sequence of the coding sequence of the native humanmRNA. o Codon Optimization 15 • Synthetic 5' Cap modifications such as but not limited to: o ARCACap o Two Headed Cap o S Cap o 2S Cap 20 • Synthetic Poly-A Tail Modifications as listed below but not limited to: o Any synthetic adenine modifications as listed above. • 5' UTR Synthetic Modifications: o Any insertions of additional nucleotides in the 5' UTR of the mRNA which would change the 5' UTRs native human sequence. 25 o Any changes in the nucleotide sequence of the 5' UTR not including deleting sections of the 5' UTR native human sequence. • 3' UTR Synthetic Modifications: o Any insertions of additional nucleotides in the 3' UTR of the mRNA which would change the 3' UTRs native human sequence. 30 o Any changes in the nucleotide sequence of the 3' UTR not including deleting sections of the 3' UTRs native human sequence. The mRNA payload of the mRNA-LNPs of the invention disclosed herein comprises the following: • Nitrogenous Bases / Ribonucleosides: 35 o Adenine / Adenosine o Uracil / Uridine o Cytosine / Cytidine o Guanine / Guanosine GRH-00361 • Coding Sequence o Native human mRNA with the exact coding sequence as found in humans without modification or mutation. • 5' Cap: 5 o 7-methylguanosine (m7G) Cap-0 (m7Gppp-) o Cap-1 (m7GpppNm-) added by 2'-O-methyltransferase to the 2'0 position of the initiating nucleotide of the native human mRNA sequence. • Poly-A Tail: o Optional sequence of unmodified adenines at the 3' end of the native human 10 mRNA. o In some embodiments, a Poly-A Tail may not be absent. • 5' UTR o The native human mRNA will include most of the 5' UTR of the native human mRNA being synthesized. 15 o The only permitted modification to the native 5' UTR is deletion from the front end (upstream end) of the 5' UTR sequence. In some embodiments, 2-90% from the upstream portion of the 5' UTR has been deleted. • 3' UTR o The native human mRNA will include most of the 3' UTR of the native human 20 mRNA being synthesized. o The only permitted modification to the native 3' UTR is deletion from the back end (downstream end) of the 3' UTR sequence. In some embodiments, 2-90% from the upstream portion of the 3' UTR has been deleted. In some aspects of the invention provided herein, the native human mRNA encapsulated 25 in and delivered by SLNs can be used for protein replacement therapy. For example, such native human mRNA-LNPs may be designed to over-express or re-express a lost or down-regulated protein, the loss of which results in human disease. In some embodiments of the invention, the native human mRNA encodes at least one native protein expressed by healthy (i.e., wild-type) Homo sapiens. 30 In certain aspects of the invention, provided herein are cells comprising the mRNA-LNPs disclosed herein. GRH-00361 In certain aspects of the invention, provided herein are methods of inducing expression of a native protein in a cell (e.g., a human cell), comprising contacting the cell with the mRNA- LNPs disclosed herein. In some aspects of the invention, provided herein are methods of treating diseases caused 5 by genetic mutations, e.g., comprising administering the mRNA-LNPs disclosed herein to a subject. Embodiments of said invention may include methods of treating disease caused by lowered or elevated native human protein levels, e.g., comprising administering the mRNA- LNPs disclosed herein to a subject. In preferred such embodiments, the mRNA-LNPs comprise a native human mRNA encoding a protein that is under-expressed in the subject. In other 10 embodiments, the mRNA-LNPs comprise a native human mRNA encoding a protein that is a native antagonist or enzyme that reduces or inhibits the activity or concentration / accumulation of the disease-inducing protein. BRIEF DESCRIPTION OF FIGURES Figure 1 depicts a data sheet for a single run of Dynamic Light Scattering (DLS). 15 Figure 2 depicts a DLS data sheet that includes an overlay of a triplicate test run. Figure 3 depicts the HSP70 mRNA-LNP 24-hour cell viability assay results. Figure 4 depicts the HSP70 mRNA-LNP 48-hour cell viability assay results. Figure 5 depicts the HSP70 mRNA-LNP encapsulation by LNPs data. Panel A shows HSP70 mRNA-LNP w / Triton X. Panel B shows PBS w / Triton X. Panel C shows Empty LNP 20 w / Triton X. Figure 6 depicts an HSP70 DNA agarose gel. Figure 7 depicts an HSP70 RNA agarose gel. Figure 8 depicts the MS-NASH mice weights from HSP70 Cohort 1. Figure 9 depicts the MS-NASH mice weights from HSP70 Cohort 2. 25 Figure 10 depicts the MS-NASH mice weights from HSP70 Cohort 3. Figure 11 depicts the MS-NASH mice weights from HSP70 Cohort 4. Figure 12 depicts the BALB / cJ mice weights from HSP70 Cohort A. Figure 13 depicts an Empty LNP dosed MS-NASH mouse liver H&E image at 4X magnification. 30 Figure 14 depicts an HSP70 mRNA-LNP dosed MS-NASH mouse liver H&E image at 4X magnification. GRH-OO361 Figure 15 depicts an Empty LNP dosed MS-NASH mouse liver H&E image at lOX magnification. Figure 16 depicts an HSP7O mRNA-LNP dosed MS-NASH mouse liver H&E image at 1OX magnification. 5 Figure 17 depicts an Empty LNP dosed MS-NASH mouse liver H&E image at 2OX magnification. Figure 18 depicts an HSP7O mRNA-LNP dosed MS-NASH mouse liver H&E image at 2OX magnification. Figure 19 depicts an Empty LNP Dosed BALB / cJ brain H&E image from HSP7O Cohort 10 A at 1OX magnification. Figure 20 depicts an HSP7O mRNA-LNP Dosed BALB / cJ brain H&E image from HSP7O Cohort A at 1OX magnification. Figure 21 depicts an Empty LNP Dosed BALB / cJ liver H&E image from HSP7O Cohort A at 1OX magnification. 15 Figure 22 depicts an HSP7O mRNA-LNP Dosed BALB / cJ liver H&E image from HSP7O Cohort A at 1OX magnification. Figure 23 depicts a full liver image of an HSP7O mRNA-LNP treated MS-NASH Liver from Experimental mouse #2 HSP7O Cohort 1. Figure 24 depicts a full liver image of an HSP7O mRNA-LNP treated MS-NASH Liver 20 from Experimental mouse #1 HSP7O Cohort 2. Figure 25 depicts a full liver image of an HSP7O mRNA-LNP treated MS-NASH Liver from Experimental mouse #2 HSP7O Cohort 2. Figure 26 depicts a full liver image of an Empty LNP treated MS-NASH Liver from Control mouse #1 HSP7O Cohort 1. 25 Figure 27 depicts a full liver image of an Empty LNP treated MS-NASH Liver from Control mouse #4 HSP7O Cohort 2. Figure 28 depicts a full liver image of an Empty LNP treated MS-NASH Liver from Control mouse #5 HSP7O Cohort 2. Figure 29 depicts the green and blue fluorescence areas found during an IF imaging of 30 SK-N-FI cells treated with MECP2 mRNA-LNPs. Panel A depicts the green fluorescence area. Panel B depicts the blue fluorescence area. GRH-00361 Figure 30 depicts the green and blue fluorescence areas found during an IF imaging of SK-N-FI cells treated with Empty LNPs. Panel A depicts the green fluorescence area. Panel B depicts the blue fluorescence area. Figure 31 depicts a MECP2 quantitative immunofluorescence graph over time. 5Figure 32 depicts the B6. l29P2(C)-Mecp2tml.IBird / J mice weights from MECP2 Cohort 1.Figure 33 depicts the B6. l29P2(C)-Mecp2tml.IBird / J mice weights from MECP2 Cohort 2.Figure 34 depicts the average hindlimb clasping scores of all B6.l29P2(C)- Mecp2tmI.IBird / J Mice. Figure 35 depicts the BALB / cJ mice weights from MECP2 Cohort A. 10 Figure 36 depicts the BALB / cJ mice weights from MECP2 Cohort B. Figure 37 depicts a full liver image of a MECP2 mRNA-LNP treated B6.l29P2(C)- Mecp2tmI.IBird / J liver. Figure 38 depicts a full liver image of an Empty LNP treated B6. l29P2(C)- Mecp2tmI.IBird / J liver. 15 Figure 39 depicts a MECP2 mRNA-LNP dosed BALB / cJ brain at 4X magnification. Figure 40 depicts a MECP2 mRNA-LNP dosed BALB / cJ brain at lOX magnification. Figure 41 depicts a MECP2 mRNA-LNP dosed BALB / cJ liver at 4X magnification. Figure 42 depicts a MECP2 mRNA-LNP dosed BALB / cJ liver at lOX magnification. Figure 43 depicts an Empty LNP dosed BALB / cJ brain at 4X magnification. 20 Figure 44 depicts an Empty LNP dosed BALB / cJ brain at lOX magnification. Figure 45 depicts an Empty LNP dosed BALB / cJ liver at 4X magnification. Figure 46 depicts an Empty LNP dosed BALB / cJ liver at lOX magnification. Figure 47 depicts a MECP2 mRNA-LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J brain at 4Xmagnification.25 Figure 48 depicts a MECP2 mRNA-LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J brain at1OX magnification. Figure 49 depicts a MECP2 mRNA-LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J brain at20X magnification. Figure 50 depicts a MECP2 mRNA-LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J liver at 4X30 magnification. GRH-00361 Figure 51 depicts a MECP2 mRNA-LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J liver at1OX magnification. Figure 52 depicts a MECP2 mRNA-LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J liver at20X magnification. 5Figure 53 depicts an Empty LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J brain at 4Xmagnification. Figure 54 depicts an Empty LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J brain at lOXmagnification. Figure 55 depicts an Empty LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J brain at 20X10 magnification. Figure 56 depicts an Empty LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J liver at 4Xmagnification. Figure 57 depicts an Empty LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J liver at lOXmagnification.15 Figure 58 depicts an Empty LNP dosed B6.l29P2(C)-Mecp2tml.IBird / J liver at 20Xmagnification. Figure 59 depicts a MECP2 DNA agarose gel. Figure 60 depicts a MECP2 RNA agarose gel. Figure 61 depicts the MECP2 expression in the livers of female B6.129P2(C)- 20 Mecp2tml. lBird / J and female BALB / cJ mice. Figure 62 depicts the MECP2 expression in the lungs of female B6.129P2(C)- Mecp2tml. lBird / J and female BALB / cJ mice. Figure 63 depicts the MECP2 expression in the hearts of female B6.129P2(C)- Mecp2tml. lBird / J and female BALB / cJ mice. 25 Figure 64 depicts the MECP2 expression in the kidneys of female B6.129P2(C)- Mecp2tml. lBird / J and female BALB / cJ mice. Figure 65 depicts the MECP2 expression in the pancreas of female B6.129P2(C)- Mecp2tml. lBird / J and female BALB / cJ mice. Figure 66 depicts the MECP2 expression in the spleens of female B6.129P2(C)- 30 Mecp2tml. lBird / J and female BALB / cJ mice. GRH-00361 Figure 67 depicts the MECP2 expression in the brains of female B6.129P2(C)- Mecp2tml. lBird / J and female BALB / cJ mice. Figure 68 depicts the MECP2 expression in the gonads of female B6.129P2(C)- Mecp2tml. lBird / J and female BALB / cJ mice. 5Figure 69 shows the PEG lipid concentration in female B6. l29P2(C)-Mecp2tml.IBirdlJmice after MECP2 mRNA-LNP administration. Figure 70 depicts the p53 mRNA-LNP 24-hour cell viability assay results for SW-1417 cells. 20 Figure 71 depicts the p53 mRNA-LNP 24-hour cell total average cell count for SW-1417 10 cells. Figure 72 depicts the p53 mRNA-LNP 24-hour cell viability assay results for SK-N-FI 25 cells. 15 Figure 73 depicts the p53 mRNA-LNP 24-hour cell total average cell count for SK-N-FI cells. 30 Figure 74 depicts the p53 mRNA-LNP 48-hour (single dosing) cell viability assay results for SW-1417 cells. Figure 75 depicts the p53 mRNA-LNP 48-hour (single dosing) cell total average cell count for SW-1417 cells. Figure 76 depicts the p53 mRNA-LNP 48-hour (dosing every 24 hours) cell viability 35 assay results for SW-1417 cells. Figure 77 depicts the p53 mRNA-LNP 48-hour (dosing every 24 hours) cell total average cell count for SW-1417 cells. Figure 78 depicts the p53 mRNA-LNP 48-hour (dosing every 24 hours) cell viability assay results for SK-N-FI cells. 40 Figure 79 depicts the p53 mRNA-LNP 48-hour (dosing every 24 hours) cell total average cell count for SK-N-FI cells. Figure 80 depicts a p53 DNA agarose gel. Figure 81 depicts a p53 RNA agarose gel. Figure 82 depicts immunofluorescent staining of embedded liver sections from BALB / cJ 45 mice control (Empty LNPs) (l0X magnification). HSP70 was stained with anti-human HSP70 GRH-00361 monoclonal rabbit antibody (primary) and anti-rabbit secondary antibody conjugated to Alexa Fluor™ 568. Nuclei was stained with Hoechst 33342. Figure 83 depicts immunofluorescent staining of embedded liver sections from BALB / cJ mice dosed with HSP70-LNPs (l0X magnification), confirming HSP70 overexpression. HSP70 5 was stained with anti-human HSP70 monoclonal rabbit antibody (primary) and anti-rabbit secondary antibody conjugated to Alexa Fluor™ 568. Nuclei was stained with Hoechst 33342. Figure 84 depicts immunofluorescent staining of embedded liver sections from MS- NASH mice (Cohort #3) control (Empty LNPs) (20X magnification). HSP70 was stained with anti-human HSP70 monoclonal rabbit antibody (primary) and anti-rabbit secondary antibody 10 conjugated to Alexa Fluor™ 568. Nuclei was stained with Hoechst 33342. Figure 85 depicts immunofluorescent staining of embedded liver sections from MS- NASH mice (Cohort #3) dosed with HSP70-LNPs (20X magnification), confirming HSP70 overexpression. HSP70 was stained with anti-human HSP70 monoclonal rabbit antibody (primary) and anti-rabbit secondary antibody conjugated to Alexa Fluor™ 568. Nuclei was 15 stained with Hoechst 33342. DETAILED DESCRIPTION In many genetic diseases and disorders, mutated proteins are translated from mutated RNA, which arises from the transcription of DNA encoding any number of sequence mutations, ultimately resulting in nonfunctional or even harmful proteins. Therapies seeking to deliver 20 therapeutically beneficial mRNA, e.g., for translation of functional protein, rely on dogmatic design-and manufacture strategies to obtain highly biologically active mRNA. For example, without being bound by theory or methodology, when mRNA is translate in a ribosome to produce protein, there typically exists a balance between the processes of translation and mRNA decay, which is affected by the structural elements of the mRNA itself, namely the 5' cap, 3' 25 poly(A) tail, protein-coding sequence, and 5' and 3' untranslated regions (UTRs). Thus, strategies arose, and are often considered to be essential, for developing mRNA-based therapeutics. Typically, 5' cap analogs are used, which are designed and / or chemically modified to have high affinity for eukaryotic initiation factor 4F (e1F4F) and resistance to de-capping enzymes; the length and composition of the poly(A) tail are modified to stabilize the mRNA and 30 increase protein expression; the 5' and 3' UTRs are engineered to increase half-life and translational efficiency; codon optimization of the protein-coding sequence is used to modify GRH-00361 secondary structure and improve translational efficiency; chemically modified nucleosides are used to decrease immunogenicity and increase translation; and RNA can be circularized to resist nuclease-mediated degradation. Until now, native human mRNA-LNP-based therapeutics were not considered feasible 5 for transfection in human cells for translation of non-mutated protein. The native wildtype mRNA provided herein closely adheres to the structure corresponding naturally occurring mRNA for optimal transfection and reduced immunological response. As disclosed herein, the introduction of unmodified. Native human mRNA into human cells allows for advancements in dose-dependent protein-replacement therapeutics for a wide range of conditions. Notably, and in 10 contrast to the understanding in the relevant art, mRNA stability / resistance to degradation appears to be preserved over time following administration of the native mRNA-LNPs provided herein. Utilizing native human mRNA does not require the coding sequence of the mRNA to be changed either through nucleotide modification or codon optimization. By leveraging the unique properties of SLNs as provided herein, this innovative composition and method for modulation 15 in vivo native human mRNA translation offers new avenues for therapeutic intervention and elucidates fundamental insights into mRNA biology. Without being bound by theory or methodology, a DNA template encoding the UTR regions and coding DNA sequence (CDS) of HSP70 (e.g., cDNA) may be used as a template for polymerase chain reaction (PCR) DNA amplification / replication with specifically designed 20 primers. In some preferred embodiments, the DNA template is a plasmid. The DNA template can then be used as a template for mRNA synthesis by methods such as those well known in the art. Alternatively, the mRNA may be transcribed from DNA synthesized using RT-PCR and isolated RNA from a cell lysate, e.g., by any suitable methods and techniques known in the art. In other aspects, provided herein are vectors comprising the nucleic acids contemplated 25 herein. In some such embodiment, the vector is selected from nanoparticles, adenovirus vectors, adeno-associated virus (AAV) vectors, retrovirus vectors, picomavirus vectors, liposomes, cationic lipid systems, and protein / nucleic acid complexes. As provided herein, native human mRNA-LNP creation begins with polymerase chain reaction (PCR) DNA amplification for a desired native human gene. This sequence may be 30 obtained from a DNA plasmid engineered to comprise the nucleotide sequence for the desired human protein, e.g., selected from a plasmid library comprising human genes or fragments GRH-00361 thereof. Commercially available PCR kits, such as the LongAmp® Taq PCR kit from New England Biolabs®, may be used along with specific primers to amplify the sequence of interest as double stranded DNA. Said primers are designed and / or selected for low G-C percentage and low self-complementarity. In some such embodiments, the forward primer contains the T7 RNA 5 polymerase promoter sequence on the 3' end, with the actual primer sequence being on the 5' end. Following the PCR amplification of the double stranded DNA, RNA polymerase, e.g., from a commercially available kit, such as the HiScribe® T7 Quick High Yield RNA Synthesis Kit, may be used to transcribe the DNA sequence into the RNA sequence of interest. In some 10 embodiments of the invention, a 5' cap is added to the RNA sequence (e.g., using the Faustovirus Capping Enzyme and Cap 2' -O-methyltransferase). In some such embodiments, the RNA is cleaned using a commercially available kit, such as, the Monarch® RNA Cleanup Kit, to remove any excess nucleotides, enzymes, and template DNA. The RNA concentration may be determined by any means known in the art, such as by using a Nanodrop™ spectrophotometer. 15 With a known concentration of mRNA, the LNPs may be prepared by to deliver a predetermined payload of mRNA sequence. For example, and without limitation, a hydrofluidic (e.g., microfluidic) mixer may be used to mix a prepared LNP mixture with an aqueous mRNA solution (e.g., mRNA in sodium acetate solution). In some embodiments, the lipid mixture may be prepared using stock solutions of the various LNP components (cholesterol, DSPC, DMG- 20 PEG 2000, and SM-102) and ethanol, as provided herein. In some exemplary embodiments, 75 μg of RNA is added to 50 mM sodium acetate buffer, and this is adjusted to a final volume of 1.5 mL with the 50 mM sodium acetate buffer. The microfluidic mixer produces mRNA encapsulated by an LNP, suspended in a mixture of ethanol and sodium acetate buffer. The resultant mRNA-LNPs suspension may then be dialyzed against phosphate buffered 25 saline (PBS), e.g., using a dialysis cassette with a 3.5K - lOK molecular weight cut-off put in a volume of PNS 200-300x the volume of the suspended RNA-LNP solution. This dialysis process replaces the ethanol and sodium acetate buffer with PBS and removes any excess lipids and nucleic acids that were not consumed in generating the mRNA-LNPs. After dialysis, the RNA- LNPs are analyzed for size and purity on a dynamic light scattering (DLS) machine. After this 30 DSLS analysis, the RNA-LNPs may be administered or stored at 4°C until needed. GRH-00361 For example, without being bound by theory or technology, heat shock proteins (HSPs) represent a class of molecular chaperones that have multiple responsibilities during normal cell growth and are integral to the folding of newly synthesized protein, the subcellular transport of protein and vesicles, the formation and dissociation of complexes, and degradation of unwanted 5 proteins. HSPs may be classified into families on the basis of molecular weight, for example HSPl00, HSP90, HSP70, HSP60, HSP40, and HSP27, with each playing a role in influencing protein assembly, folding, and translocation. HSP70, HSP60, and HSP27 are known to prevent protein aggregation and help protein folding, while HSPlO0 releases proteins from aggregates and HSP90 plays a role in maturation and activation of a number of proteins. In carrying out 10 these diverse functions HSPs, such as HSP70, undergo a cycle of rapid, controlled, binding and release of substrate protein that promotes unfolding / folding and assembly while preventing aggregation of the substrate proteins. As such, HSPs may have particular utility in proteinopathies. Accordingly, for exemplary purposes, contemplated herein are nucleic acids (e.g., mRNAs) encoding HSP70 as follows. 15 Table 1

[0002] GRH-00361 Intended payloads of the LNPS provided herein may be derived from the HSPAlA gene utilizing specifically designed primers as follows: Table 2 GRH-00361 gt cc aa tg ac tg ag tg cg gt cc gg cg tg gg tg tc gc gc gc gg gg cc tg gc cc cc cg tg tg ac tg tc ta at ac ga gt gc uc gc ug gc cg ac ag cg cc au cc ug ag ac ug cc cg cu cc 15 GRH-00361 Table 3 GRH-00361 Table 4 GRH-00361 GRH-00361 GRH-00361 Thus, HSP70 mRNAs having a sequence as disclosed above, or encoding an HSP70 peptide as disclosed above, can be utilized in native mRNA compositions and methods as disclosed herein. However, in certain preferred embodiments, the intended mRNA payload of the 5 LNPs (e.g., the solid lipid nanoparticles (SLNs) of the invention disclosed herein, does not comprise an open reading frame encoding a heat shock protein polypeptide, such as an HSPlO0, HSP90, HSP70, HSP 60, HSP40, or HSP27 polypeptide (e.g., an HSP70 polypeptide as GRH-00361 disclosed above), or any functional fragment thereof. In particular such embodiments, the LNPs of the invention do not comprise a nucleic acid sequence as 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, nor do they comprise a nucleic acid isolated and / or amplified using any one of the forward and reverse 5 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. As a further non-limiting example, the methyl-CpG-binding domain (MBD) family consists of 11 chromosomal proteins and their purpose is to regulate surrounding chromatin in 10 the cell by silencing transcription. These proteins are associated with various other protein partners to aid in establishing the link between DNA methylation and chromatin modification. All known 11 proteins (e.g., MeCP2, MBDl, MBD2, MBD3, MBD4, MBD5, MBD6, SETDBl, SETDB2, TIP5 / BAZ2A, and BAZ2B) play crucial roles in histone and chromatin regulation and are characterized by their presence of a MBD domain. MBDs have a strong potential as 15 therapeutic targets suppressing or treating a range of diseases associated with proteinopathy, including neurodevelopmental disease, such as Rett Syndrome, neurodegeneration, and cancer. In such disease, proteins fail to maintain integrity within chromatin structure and various membranes within the cell, such as nuclear membranes. The 52 kDa methyl-CpG-binding protein 2 (MECP2) represents one of the most essential 20 proteins within the MDB family, regulating higher order / long-range chromatin remodeling and silencing, with role in heterochromatin formation and chromatin organization. MECP2 is also crucial in mediating the translation of intragenic methylation into alternative splicing. MECP2 is also one of the most prevalent proteins within the MBD family; its mutation is the leading cause of Rett Syndrome. Mutation within MECP2 typically affects the MBD / TRD domain, which 25 prevents it from appropriately interacting with DNA or any other crucial binding partners needed for DNA or chromatin regulation. MECP2 expression also allows for nerve maturation and is highly expressed in mature neurons. When mutated, MECP2 can lead to the decline or absence of nerve maturation leading to cognitive impairment and decline. The MECP2 protein is an intrinsically disordered nuclear protein (IDP), with a predicted 30 molecular mass of 53 kDa and subject to post-translational modification (e.g., acetylation, ubiquitination, sumoylation, and phosphorylation). It comprises several highly conserved domain GRH-00361 structures: a N-terminal domain (NTD); the methyl CpG binding domain (MBD); an intervening domain (ID); a transcription repression domain (TRD) comprising nuclear localization sequence and the NCoR / SMRT interaction domain (NID); and C-terminal domain (CTD) comprising an alpha and beta domain. The MBD comprises an a / B sandwich fold composed of four B-strands 5 and an a-helix. This functional domain attaches to methylated CpG sites on DNA strands and is used to recruit histone deacetylases (H+DACs) in order to regulate gene expression. Thus, exemplified herein are nucleic acids (e.g., mRNAs) encoding the MBD family member MECP2 as follows: 10 GRH-00361 5 10 15 20 25 30 35 40 45 50 55 60 GRH-00361 5 10 15 20 25 30 35 40 45 50 55 60 GRH-00361 An exemplary payload for the LNPs contemplated herein is an mRNA strand derived 5 from an MBD family member gene. Provided herein is mRNA sequence which codes for the protein MeCP2 which is located on the gene MECP2. Utilizing specifically designed primers, the following sequence was amplified and applied to the lipid nanoparticles disclosed herein. Table 7 10 GRH-00361 GRH-00361 GRH-00361 GRH-00361 Thus, MBD family proteins (e.g., MECP2) encoded by mRNAs having a sequence as disclosed above or encoding an MBD peptide, or functional fragment thereof, as provided above, 5 can be utilized in native mRNA compositions and methods disclosed herein. However, in certain preferred embodiments, the intended mRNA payload of the LNPs (e.g., the solid lipid nanoparticles (SLNs) of the invention disclosed herein, does not comprise an open reading frame encoding a MBD family protein, such as an MeCP2, MBDl, MBD2, MBD3, MBD4, MBD 5, MBD6, SETDBl, SETDB2, TIP5 / BAZ2A,or BAZ2B polypeptide (e.g., MeCP2 polypeptide as 10 disclosed above), or any functional fragment thereof, or any variant thereof (e.g., MECP2 isoform 1, MECP2 isoform 2, MECP2 isoform 3, MECP2 isoform 4, MECP2 transcript variant 1, MECP2 transcript variant 2, MECP2 transcript variant 3, MECP2 transcript variant 4, MECP2 transcript variant 5, MECP2 transcript variant 6, or MECP2 transcript variant 7. Preferably, the MECP2 polypeptide is MECP2 isoform 2 or MECP2 transcript variant 2). In particular such 15 embodiments, the LNPs of the invention do not comprise a nucleic acid sequence as set forth in SEQ ID NO.24, or any functional fragment thereof; derived from SEQ ID NO. / 20; nor do they comprise a nucleic acid isolated and / or amplified using the forward and reverse primer pair set forth in: SEQ ID NOs. 21, 22, and 23. As a further non-limiting example, native p53 mRNA was inserted into LNPs for the use 20 of treating cancer such as but not limited to: Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia (AML), Adrenocortical Carcinoma, AIDS-Related Cancers (Kaposi Sarcoma (Soft Tissue Sarcoma), AIDS-Related Lymphoma (Lymphoma), Primary CNS Lymphoma (Lymphoma)), Anal Cancer, Appendix Cancer, Astrocytomas, Atypical Teratoid / Rhabdoid Tumor, Basal Cell Carcinoma of the Skin, Bile Duct Cancer, Bladder Cancer, Bone Cancer 25 (includes Ewing Sarcoma and Osteosarcoma and Malignant Fibrous Histiocytoma), Brain Tumors, Breast Cancer, Bronchial Tumors (Lung Cancer), Burkitt Lymphoma, Carcinoma of Unknown Primary, Brain Cancer, Medulloblastoma and Other CNS Embryonal Tumors, Gemi Cell Tumor, Primary CNS Lymphoma, Cervical Cancer, Childhood Cancers, Childhood Cardiac GRH-00361 Tumors, Rare childhood cancers (Nasopharyngeal Cancer, Esthesioneuroblastoma, Thyroid Cancer, Oral Cavity Cancer, Salivary Gland Tumors, Laryngeal Cancer and Papillomatosis, Midline Tract Cancer with NUT Gene Changes (NUT Midline Carcinoma)), Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous 5 Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Ductal Carcinoma In Situ (DCIS), Diffuse Intrinsic Pontine Glioma (DIPG), Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Ewing Sarcoma, Eye Cancer (including Intraocular Melanoma and Retinoblastoma), Fallopian Tube Cancer, Gallbladder Cancer, Gastric Cancer, Gastrointestinal 10 Stromal Tumors (GIST), Germ Cell Tumors (including Extracranial Germ Cell Tumors, Extragonadal Germ Cell Tumors, Ovarian Germ Cell Tumors, and Testicular Cancer), Gestational Trophoblastic Disease, Hairy Cell Leukemia, Head and Neck Cancer, Heart Tumors, Hepatocellular Cancer, Langerhans Cell Histiocytosis, Hodgkin Lymphoma, Hypopharyngeal Cancer, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kidney or Renal Cell Cancer, 15 Laryngeal Cancer, Leukemia, Lip and Oral Cavity Cancer, Liver Cancer, Lung Cancer (including (Non-Small Cell, Small Cell, Pleuropulmonary Blastoma, Pulmonary Inflammatory Myofibroblastic Tumors, and Tracheobronchial Tumors), Lymphoma, Male Breast Cancer, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Cancer, Metastatic Squamous Neck Cancer with Occult Primary, Mouth Cancer, Multiple Endocrine Neoplasia Syndromes, 20 Multiple Myeloma / Plasma Cell Neoplasms, Mycosis Fungoides, Myelodysplastic Syndromes, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Neuroendocrine Tumors, Non-Hodgkin Lymphoma, Oral Cancer, Oropharyngeal Cancer, Osteosarcoma and Undifferentiated Pleomorphic Sarcoma of Bone, Ovarian Cancer, Pancreatic Cancer, Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer, Parathyroid 25 Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm (including Multiple Myeloma), Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Pulmonary Inflammatory Myofibroblastic Tumors, Rectal Cancer, Recurrent Cancer, Renal Cell Cancer, Rhabdomyosarcoma, Salivary Gland Cancer, Various Sarcoma (including Uterine Sarcoma, Soft 30 Tissue Sarcoma, and more), Sezary Syndrome, Skin Cancer, Small Intestine Cancer, Squamous Cell Carcinoma of the Skin, Squamous Neck Cancer with Occult Primary, Stomach (Gastric) GRH-00361 Cancer, T-Cell Lymphoma, Throat Cancer (including Nasopharyngeal Cancer, Oropharyngeal Cancer, and Hypopharyngeal Cancer), Thymoma and Thymic Carcinoma, Thyroid Cancer, Tracheobronchial Tumors, Transitional Cell Cancer of the Renal Pelvis and Ureter, Urethral Cancer, Vaginal Cancer, Vascular Tumors, Vulvar Cancer, Wilms Tumor and Other Childhood 5 Kidney Tumors, and other diseases and disorders resulting in tumors or cancer formations. In cells, the purpose of the TP53 gene is to respond to genotoxic and cellular stresses and induce cell cycle arrest and apoptosis when a cell's DNA has unrepairable DNA damage and mutations. p53 also plays a role in cellular metabolism and antioxidant response. Due to these roles, p53 plays the largest role in blocking tumor progression and the progression of cancer 10 growth in general. GRH-00361 An exemplary payload for the LNPs contemplated herein is an mRNA strand derived from the TP53 gene. Provided herein is mRNA sequence which codes for the protein p53 which 5 is located on the gene TP53. Utilizing specifically designed primers, the following sequence was amplified and applied to the lipid nanoparticles disclosed herein. Table 9 GRH-00361 Intended payloads of the LNPS provided herein may be derived from the TP53 gene utilizing specifically designed primers as follows: 5 Table 10 GRH-00361 GRH-00361 Thus, p53 encoded by mRNAs having a sequence as disclosed above, or encoding a p53 polypeptide, or functional fragment thereof, as provided above, can be utilized in native mRNA 5 compositions and methods disclosed herein. However in certain preferred embodiments, the intended mRNA payload of the LNPs (e.g., the solid lipid nanoparticles (SLNs)) of the invention disclosed herein comprises an open reading frame encoding tumor protein p53 polypeptide, or a functional fragment thereof. In some embodiments, the tumor protein p53 polypeptide, or functional fragment thereof is selected from cellular tumor antigen p53 isoform a transcript 10 variant 1, cellular tumor antigen p53 isoform a transcript variant 2, cellular tumor antigen p53 isoform a transcript variant 9, cellular tumor antigen p53 isoform a transcript variant 10, cellular tumor antigen p53 isoform a transcript variant 11, cellular tumor antigen p53 isoform b transcript variant 3, cellular tumor antigen p53 isoform b transcript variant 12, cellular tumor antigen p53 isoform b transcript variant 13, cellular tumor antigen p53 isoform c, cellular tumor antigen p53 15 isoform d, cellular tumor antigen p53 isoform e, cellular tumor antigen p53 isoform f, cellular tumor antigen p53 isoform g transcript variant 8, cellular tumor antigen p53 isoform g transcript variant 1, cellular tumor antigen p53 isoform g transcript variant 2, cellular tumor antigen p53 isoform g transcript variant 9, cellular tumor antigen p53 isoform g transcript variant 10, cellular tumor antigen p53 isoform g transcript variant 11, cellular tumor antigen p53 isoform h, cellular 20 tumor antigen p53 isoform i transcript variant 3, cellular tumor antigen p53 isoform i transcript variant 12, cellular tumor antigen p53 isoform i transcript variant 13, cellular tumor antigen p53 GRH-00361 isoform, cellular tumor antigen p53 isoform k, or cellular tumor antigen p53 isoform 1. Preferably the tumor protein p53 polypeptide, or functional fragment thereof is cellular tumor antigen p53 isoform a transcript variant 1. Definitions 5 For convenience, certain terms employed in the specification, examples, and appended claims are collected here. The 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 10 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 15 ingredients or conditions such as temperature, these values include the stated value with a variation of 0-10%R around the value (X ± 10%). Ranges are stated in the 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 of the range. For example, a range of 0-.1-1.0 represents the terminal values of 0.1 and 1.0, 20 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 25 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 30 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. GRH-00361 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 5 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 10 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 15 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 20 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 disease or disorder disclosed herein) 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 25 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- 30 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 GRH-00361 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 5 may have suppressive, disruptive, or enhancing properties. Nucleic acids and vectors Nucleic acids and vectors disclosed herein include polynucleotides and polynucleotide vectors encoding for native human proteins that allow expression in the disclosed cells, tissues, 10 organs, and / or systems. Nucleic acid sequences contemplated herein can be obtained using methods known in the art, e.g., molecular biology and recombinant methodologies / techniques. Alternatively, the sequence of interest can be produced synthetically, rather than cloned, e.g., in vitro, enzymatically or chemically. 15 In addition to the polypeptide-encoding sequences, other structural properties as disclosed herein for mRNA constructs (e.g., modified nucleobases, 5' cap, 5' UTR, 3' UTR, miR-30 binding site(s), poly-A 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 20 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, poly-A 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. 25 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, WO 2014093924, WO2014164253, WO2014159813, WO2018144775, WO2018081459, each of which are incorporated herein their entirety. 30 In some embodiments, the mRNAs of the disclosure can include a combination of modifications to the sugar, the nucleobase, and / or the inter-nucleoside linkage. These combinations can include any one or more modifications described herein. As a non-limiting GRH-00361 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%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 99.9% of the natural uridines) with at least 5 one of the modified nucleosides disclosed herein, e.g., pseudouridine. Thus, the native human mRNA sequences disclosed herein include the following: • Nucleoside Bases: o Adenine / Adenosine o Uracil / Uridine 10 o Cytosine / Cytidine o Guanine / Guanosine • Coding Sequence o Native human mRNA with the exact coding sequence as found in humans, i.e., no deviations from the coding sequence of the wild type native human mRNA. 15 • 5' Cap: o 7-methylguanosine (m7G) Cap-0 (m7Gppp-), or o Cap-1 (m7GpppNm-) added by 2' -O-methyltransferase to the 2' -0 position of the initiating nucleotide of the native human mRNA sequence. • Poly-A Tail: 20 o Unmodified sequence of adenines at the 3' end of the native human mRNA. In such embodiments, the native human mRNA sequence does not comprise synthetic or chemically modified adenine (adenosine). o In some variations, a Poly-A Tail is absent. • 5' UTR 25 o The native human mRNA will include most of the 5' UTR of the native human mRNA being synthesized. o The only modification which can occur is removal of some of the front end (upstream) 5' UTR sequence. In some embodiments, 2-90% from the upstream portion of the 5' UTR has been deleted. 30 • 3' UTR GRH-00361 o The native human mRNA will include most of the 3' UTR of the native human mRNA being synthesized. o The only modification which can occur is removal of some of the back end (downstream)3' UTR sequence. In some embodiments, 2-90% from the 5 downstream end of the 3' UTR has been deleted. Expression of nucleic acids encoding native human mRNA is typically achieved by operably linking a nucleic acid encoding native human 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 10 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. 15 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), 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 20 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 25 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) 30 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. GRH-00361 Another example of a suitable promoter is Elongation Growth Factor-l(EF-la). However, other constitutive promoter sequences may also be used, including, but not limited to the simian virus 40 (SV4O) early promoter, MND (myeloproliferative sarcoma virus) promoter, mouse mammary tumor virus (MMTV), human immunodeficiency virus (HIV) long terminal repeat (LTR) 5 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 10 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 15 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 native human 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 expression 20 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 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. 25 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 30 introduced into the recipient cells. Suitable reporter genes may include genes encoding luciferase, beta-galactosidase, chloramphenicol acetyl transferase, secreted alkaline phosphatase, GRH-00361 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 5 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. 10 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). 15 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 20 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 25 associated with a lipid may be encapsulated in the aqueous interior of a liposome, interspersed 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 30 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 GRH-00361 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 5 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 ("Chol") can be obtained from Calbiochem-Behring; 10 dimyristyl phosphatidylglycerol ("DMPG") and other lipids may be obtained from Avanti Polar Lipids, me, (Birmingham, Ala.). In 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. & 15 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 20 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, 25 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 30 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 GRH-00361 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 5 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., 10 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 15 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, 20 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 25 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 30 mRNA contemplated herein). Such contemplated methods comprise using any of the compositions disclosed herein and producing lipid nanoparticles in accordance with methods of GRH-00361 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 Biopharmaceuticals. Part I: Nanoparticles and Microparticles" Curr. Pharm. Biotechnol. 16: 940-954; Naseri et al. (2015) "Solid Lipid Nanoparticles and 5 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 (LNP5) comprise lipids including an ionizable lipid, a structural lipid, a phospholipid, a stabilizing lipid, and one or more 10 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, a sterol and a phospholipid. In some embodiments, the LNP has a molar ratio of about 20- 15 60% ionizable lipid:about 5-25% phospholipid:about 25-55% sterol: and about 0.5-15% PEG- modified lipid, about 38.5% cholesterol and about 10% phospholipid. In some embodiments, the LNP comprises a 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 20 cholesterol. The ionizable lipids contemplated herein include cationic and / or ionizable lipids. Such cationic and / or ionizable lipids include, but are not limited to, SM-102, 9-Heptadecanyl 8-{(2- hydroxyethyl)[6-oxo-6-(undecyloxy)hexyl]amino }octanoate, 3-(didodecylamino)-Nl ,N4,N4- tridodecyk-1-piperazineethanamine (KLlO), Nl-[2-(didodecylamino)ethyl]-Nl,N4,N4- 25 tridodecyl-1,4-piperazinediethanamine (KL22), 14,25-ditridecyl-15,18,21,24-tetraaza- octatriacontane (KL25), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLin-DMA), 2,2- dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), heptatriaconta-6,9,28,31- tetraen-19-yl 4-(dimethylamino)butanoate (DLin-MC3-DMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-KC2-DMA), 2-({8-[(3^)-cholest-5-en-3- 30 yloxy]octyl}oxy)-N,N-dimethyl-3-[(9Z,12Z)-octadeca-9,12-dien-1-yloxy]propan-1-amine (Octyl-CLinDMA), (2R)-2-({8-[(3^)-cholest-5-en-3-yloxy]octyl }oxy)-N,N-dimethyl-3-[(9Z, GRH-00361 12Z)-octadeca-9, 12-dien-1-yloxy]propan-(Octyl-CLinDMA (2R)), (2S)-2-({8-[(3^)-cholest-5- en-3-yloxyl]octyl} oxy)-N,N-dimethyl-3 -[(9Z,12Z)-octadeca-9,12-dien-1-yloxylpropan-1-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- 5 dimethylammonium bromide ("DDAB"); N-(2,3-dioleoyloxy)propyl)-N,N,N- trimethy!ammonium chloride ("DOTAP"); 1,2-Dioleyloxy-3-trirnethylarninopropane chloride salt ("DOTAP.0 1"); 3-^-(N^(N',N'-dimethylaminoethane)-carbamoyl)cholesterol ("DC-Chol"), N- (1-(2,3-dioleyloxy)propyl)-N-2-(sperminecarboxamido)ethyl)-N,N-dimethyl-ammonium trifluoroacetate ("DOSPA"), dioctadecylamidoglycyl carboxyspermine ("DOGS"), 1,2-dioleoyl- 10 3-35 dimethylammonium propane ("DODAP"), N,N-dimethyl-2,3-dioleyloxy)propylamine ("DODMA"), and N-(l,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, 15 available from GIBCO / BRL). KLlO, KL22, and KL25 are described, for example, in U.S. Pat. No.8,691,750, which is incorporated herein by reference in 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 20 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 25 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 30 (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 GRH-00361 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 branches, oxidation, cyclization, and alkynes are also contemplated. For 5 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 lipid monolayer or bilayer) of a nanoparticle composition to a useful component such as a 10 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 15 sphingomyelin. The lipid composition of a pharmaceutical composition disclosed herein can compromise 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 20 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 25 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- 30 modified dialkylamines and PEG-modified 1,2-diacyloxpropan-3-amines. Such lipids are also GRH-00361 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 is not limited to 1,2-dimyristoyl-sn- glycerol methoxypolyethylene glycol (PEG-DMG), 1,2-distearoyl-sn-glycero-3- 5 phosphoethanolamine-N-[amino(polyethylene glycol)] (PEG-DSPE), PEG-disteryl glycerol (PEG-DSG), PEG-dipalmitoyl phosphatidylethanolamine (PEG-DPPE), or PEG-1,2- dimyristyloxypropyl-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- 10 modified phosphatidylethanolamine, a PEG-modified phosphatidic acid, a PEG-modified ceramide, a PEG-modified dialkylamine, a PEG-modified diacylglycerol, a PEG-modified dialkylglycerol, and mixture 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 15 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. 20 PEG-lipids are known in the art, such as those described in U.S. Pat. No, 8,158,601 and International Publ. No. WO2015130584 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. 25 PCT / US2016 / 000129, filed Dec.10, 2016, entitled "Compositions and methods for Delivery of Therapeutic Agents," which is now 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 alternatively referred to as PEGylated lipids. A PEG lipid is a lipid modified with 30 polyethylene glycol. A PEG lipid may be selected from the non-limiting group including PEG- modified phosphatidylethanolamines, PEG-modified phosphatidic acids, PEG-modified GRH-00361 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, 5 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 of ALC-0315, a synthetic ionizable cationic 10 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 polyethylene glycol (PEG)-conjugated lipid. In other embodiments, the polymer 15 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 PEGF on the outer 20 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 25 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 (Pl). 30 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 GRH-00361 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). Compositions 5 In some aspects, provided herein is a composition (e.g., a pharmaceutical composition, such as a therapeutic or vaccine composition), containing the nucleic acid disclosed herein, formulated together with a pharmaceutically acceptable carrier, (e.g., a composition of the nanoparticles disclosed herein) as well as methods of administering such pharmaceutical compositions. 10 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 15 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. 20 Compositions contemplated herein may be administered intrapleurally, intravenously, subcutaneously, intranodally, intratumorally, intrathecally, intraperitoneally, intracranially, or by direct administration to an organ. Said compositions may comprise one or more pharmaceutically acceptable sterile isotonic aqueous or nonaqueous solution, dispersions, suspensions, or emulsions, or sterile powders which may be reconstituted into sterile injectable 25 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 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, 30 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 GRH-00361 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 50 mL (50,000 μL) or any intermediate value encompassed therein, particularly, about: 100 μL, 200 μL, 5 300 μL, 400 μL, 500 μL, 600 μL, 700 μL, 800 μL, 900 μL, 1000 μL (1 mL), 2 mL, 3 mL, 4 mL, 5 mL, 6 mL, 7 mL, 8 mL, 9 mL, 10 mL, 11 mL, 12 mL, 13 mL, 14 mL, 15 mL, 16 mL, 17 mL, 18 mL, 19 mL, 20 mL, 21 mL, 22 mL, 23 mL, 24 mL, 25 mL, 26 mL, 27 mL, 28 mL, 29 mL, 30 mL, 31 mL, 32 mL, 33 mL, 34 mL, 35 mL, 36 mL, 37 mL, 38 mL, 39 mL, 40 mL, 41 mL, 42 mL, 43 mL, 44 mL, 45 mL, 46 mL, 47 mL, 48 mL, 49 mL, or 50 mL. In some preferred 10 embodiments, the dose size is 10 mL. In some such embodiments, the dose comprises an mRNA amount of about 50 μg to about 2,500 μg or any intermediate value encompassed therein, particularly, about: 100 μg, 150 μg, 200 μg, 250 μg, 300 μg, 350 μg, 400 μg, 450 μg, 500 μg, 550 μg, 600 μg, 650 μg, 700 μg, 750 μg, 800 μg, 850 μg, 900 μg, 950 μg, 1,000 μg, 1,050 μg, 1,100 μg, 1,150 μg, 1,200 μg, 1,250 μg, 1,300 μg, 1,350 μg, 1,400 μg, 1,450 μg, 1,500 μg, 1,550 μg, 15 1,600 μg, 1,650 μg, 1,700 μg, 1,750 μg, 1,800 μg, 1,850 μg, 1,900 μg, 1,950 μg, 2,000 μg, 2,050 μg, 2,100 μg, 2,150 μg, 2,200 μg, 2,250 μg, 2,300 μg, 2,350 μg, 2,400 μg, or 2,450 μg. In some such embodiments, the dose administered may be at a concentration of about 0.002 mg / kg to about 0.03 mg / kg mRNA or any intermediate value encompassed therein such as about: 0.003 mg / kg, 0.008mg / kg, 0.01 mg / kg, 0.012 mg / kg, 0.015 mg / kg, 0.016mg / kg, 0.02 mg / kg, 0.021 20 mg / kg, 0.022mg / kg, 0.023 mg / kg, or 0.025 mg / kg. By way of example, the dose may comprise an mRNA concentration of 0.33 μg / μL. Accordingly, without being bound by any particular theory or methodology, the dose size may be 1 mL, comprising 660 μL of mRNA (concentration of 500 μg / mL, i.e., 330 μg) and 340 μL of ethanol lipid nanoparticle solution. The ethanol may be removed via dialysis after particle formation, leaving the lipid nanoparticles containing 25 mRNA in saline buffer alone. This may then be stored at 4°C until use. Thus, in some embodiments, the administered dose may comprise an mRNA concentration of 0.025 μg / μL - 0.33 μg / μL for the suspension of LNPs in saline buffer (preferably phosphate buffered saline) or any intermediate value encompassed therein, particularly, about: 0.03 μg / μL, 0.033 μg / μL, 0.035 μg / μL, 0.04 μg / μL, 0.045 μg / μL, 0.05 μg / μL, 0.055 μg / μL, 0.06 μg / μL, 0.065 μg / μL 0.07 30 μg / μL, 0.075 μg / μL, 0.08 μg / μL, 0.085 μg / μL, 0.09 μg / μL, 0.095, μg / μL, 0.1 μg / μL, 0.15 μg / μL, 0.2 μg / μL, 0.25 μg / μL, or 0.3 μg / μL. GRH-00361 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 native human proteins 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, 5 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 10 In certain embodiments, provided herein are methods of treating a subject, comprising administering to the subject a therapeutic composition provided herein. The subject may be a pediatric subject (e.g., the subject is under 18 years of age). The subject may be an adult (e.g., 18 years old or older). The subject may be a fetus (e.g., a developing fetus within a pregnant person). In some embodiments, the subject is no more than 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 15 14, 15, 16, 17, 18, 19,20,21,22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39, 40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,or 65 years of age. In some embodiments, the condition disclosed herein may be a condition in children. Thus, the condition may be a pediatric condition. The child may be less than about 1 week old. The child may be less than about 1 month old. The child may be less than about 6 20 months old. The child may be less than about 12 months old. The child may be less than about 2 years old. The child may be less than about 3 years old. The child may be less than about 4 years old. The child may be less than about 5 years old. The child may be less than about 6 years old. The child may be less than about 7 years old. The child may be less than about 8 years old. The child may be less than about 9 years old. The child may be less than about 10 years old. The 25 child may be less than about 12 years old. In some embodiments, the methods provided herein are used to treat or prevent any diseases caused by a native human protein which has been mutated or downregulated. As a non- limiting example, and without being bound by theory or mechanism, the methods provided herein replace an absent or non-functioning protein, increase the level of active protein, and / or 30 outcompete a mutant protein via translation of the mRNAs delivered. Such disease include but are not limited to Alzheimer's Disease, Dementia, Parkinson's Disease, Acute Lymphoblastic GRH-00361 Leukemia, Acute Myeloid Leukemia, Adenocarcinoma, Adrenocortical Carcinoma, Bladder Cancer, Brain Cancer, Bone Cancer, Breast Cancer, Cervical Cancer, Cystic Fibrosis, Ductal Carcinoma, Esophageal Cancer, Fallopian Tube Cancer, Gallbladder Cancer, KCNTl Related Epilepsies, Angelman Syndrome, GABA-A Variants, Rett Syndrome, SLC6Al-NDD, Non- 5 Alcoholic Fatty Liver Disease, Nonalcoholic Steatohepatitis, Liver Cancer, SYNGAPl Syndrome, Lafora Disease, Niemann Pick Type C, Li-Fraumeni Syndrome (LFS), Ehlers-Danlos Syndrome, Neurofibromatosis Type 1, Tay-Sachs Disease, Friedreich Ataxia, Genetic Amyotrophic Lateral Sclerosis, Huntington's Disease, Sickle Cell Disease, Beta Thalassemia, Spinal Muscular Atrophy, Ichthyosis, FOXGl Disease, Creatine Deficiency Disorder, Ovarian 10 Cancer, Colorectal Cancer, and Lung Cancer. 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. 15 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 20 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 25 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 30 intravenous (i.v.) injection. The compositions may also be injected directly into a tumor, lymph node, organ, or site of disease or disorder (e.g., inflammation). In certain embodiments, the GRH-00361 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 additional therapy 5 as disclosed herein. As non-limiting examples, such therapies may comprise an immune checkpoint inhibitor, an immunosuppressive agent, a disease-modifying antirheumatic drug (DMARD), a pain-control drug, a steroid, a non-steroidal anti-inflammatory drug (NSAID), or a cytokine antagonist, and combinations thereof. Thus, without limitation, the compositions and methods contemplated herein may be used 10 in combination with additional therapeutic procedures and agents selected from the following representative list: cyclosporin, retinoids, corticosteroids, propionic acid derivative, acetic acid derivative, enolic acid derivatives, fenamic acid derivatives, Cox-2 inhibitors, lumiracoxib, ibuprophen, choline magnesium salicylate, fenoprofen, salsalate, difunisal, tolmetin, ketoprofen, flurbiprofen, oxaprozin, indomethacin, sulindac, etodolac, ketorolac, nabumetone, naproxen, 15 valdecoxib, etoricoxib, MK0966; rofecoxib, acetominophen, Celecoxib, Diclofenac, tramadol, piroxicam, meloxicam, tenoxicam, droxicam, lornoxicam, isoxicam, mefanamic acid, meclofenamic acid, flufenamic acid, tolfenamic, valdecoxib, parecoxib, etodolac, indomethacin, aspirin, ibuprophen, firocoxib, methotrexate (MTX), antimalarial drugs (e.g., hydroxychloroquine and chloroquine), sulfasalazine, Leflunomide, azathioprine, cyclosporin, 20 gold salts, minocycline, cyclophosphamide, D-penicillamine, minocycline, auranofin, tacrolimus, myocrisin, chlorambucil, TNF alpha antagonists (e.g., TNF alpha antagonists or TNF alpha receptor antagonists), e.g., ADALIMUMAB (Humira®), ETANERCEPT (Enbrel®), INFLIXIMAB (Remicade®; TA-650), CERTOLIZUMAB PEGOL (Cimzia®; CDP870), GOLIMUMAB (Simponi®; CNTO 148), ANAKINRA (Kineret®), RITUXIMAB (Rituxan®; 25 MabThera®), ABATACEPT (Orencia®), TOCILIZUMAB (RoActemra / Actemra®), integrin antagonists (TYSABRI® (natalizumab)), IL-1 antagonists (ACZ885 (Haris)), Anakinra (Kineret®)), CD4 antagonists, IL-23 antagonists, IL-20 antagonists, IL-6 antagonists, BLyS antagonists (e.g., Atacicept, Benlysta®, LymphoStat-B® (belimumab)), p38 Inhibitors, CD20 antagonists (Ocrelizumab, Ofatumumab (Arzerra®)), interferon gamma antagonists 30 (Fontolizumab), prednisolone, Prednisone, dexamethasone, Cortisol, cortisone, hydrocortisone, GRH-00361 methylprednisolone, betamethasone, triamcinolone, beclometasome, fludrocortisone, deoxycorticosterone, aldosterone, Doxycycline, vancomycin, pioglitazone, SBI-087, SCIO-469, Cura-100, Oncoxin + Viusid, TwHF, Methoxsalen, Vitamin D - ergocalciferol, Milnacipran, Paclitaxel, rosig tazone, Tacrolimus (Prograf®), RAD00l, rapamune, rapamycin, fostamatinib, 5 Fentanyl, XOMA 052, Fostamatinib disodium, rosiglitazone, Curcumin (Longvida™), Rosuvastatin, Maraviroc, ramipril, Milnacipran, Cobiprostone, somatropin, tgAAC94 gene therapy vector, MK0359, GW856553, esomeprazole, everolimus, trastuzumab, JAKl and JAK2 inhibitors, pan JAK inhibitors, e.g., tetracyclic pyridone 6 (P6), 325, PF-956980, denosumab, IL- 6 antagonists, CD2O antagonists, CTLA4 antagonists, IL-8 antagonists, IL-21 antagonists, IL-22 10 antagonist, integrin antagonists (Tysabri® (natalizumab)), VGEF antagonists, CXCL antagonists, MMP antagonists, defensin antagonists, IL-1 antagonists (including IL-1 beta antagonists), and IL-23 antagonists (e.g., receptor decoys, antagonistic antibodies, etc.). EXAMPLES 15 The 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. 20 For Example, the composition comprises, 1. the ionizable lipid SM-102, 2. the phospholipid DSPC (distearoylphosphatidylcholine), 3. cholesterol, and 4. DMG-PEG 2000. 25 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 ug / mL was diluted to achieve a desired mass ratio. In some embodiments, the mRNA to lipid mass-to-mass 30 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 GRH-00361 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 5 The four primary components comprise a molar ratio of about: 50 (SM-102) :10 (DSPC) :38.5 (Cholesterol) :1.5 (PEG-Lipid) The molar ratio may be adjusted to optimally deliver the payload based on this starting ratio. For example, • the ionizable lipid rage can be adjusted± 5, 10 • 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 15 mixture prepared as described in Table 11, 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 of DSPC was mixed with 400 μL ethanol, and so on, in accordance with Table 11 below. The appropriate volume of each lipid mixture component, as 20 listed in Table 11, was then transferred to a single tube to prepare the ethanolic lipid mixture and pipetted several times to ensure mixing and avoid precipitation and cloudiness. Table 11 GRH-00361 Aqueous mRNA Solution: A lipid:mRNA (w:w) ratio of 10:1 and an ethanol:aqueous ratio of 1:3 was used. 5 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). The ethanolic lipid mixture was mixed with mRNA in a microfluidic mixer. The output, comprising the LNPs, was collected and injected into a dialysis cartridge. The cartridge was 10 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: Standard Operating Procedure for Creation of native human mRNA encapsulated in SLNs: Plasmid carrying the target DNA sequence of interest was reconstituted in nuclease free 15 water to a concentration of 0.2 μg / μL. This stock was stored at -20°C and made into a working concentration of 0.25 ng / μL as needed. Predetermined primers were reconstituted in nuclease free water to achieve a concentration of 100 mM. PCR (DNA amplification) Primers were diluted to a concentration of 10 mM by adding 1 μL of reconstituted primer 20 to 9 μL of nuclease-free H2O. PCR tubes were loaded with reaction components in the following order. Table 12 GRH-00361 *final volume of each tube should be 50 μL After adding the last component, reaction tubes were briefly centrifuged and placed in a thermal cycler programmed with the following parameters: 5 Table 13 *anneal temperature depends on the TM of the primers used. Following completion of the thermal cycled reaction, the completed reaction tubes can be stored at -20°C until needed. 10 DNA Agarose Gel: DNA agarose gels were used to verify size and purity of the PCR product by visualization with Ethidium Bromide (EtBr). DNA samples (PCR products) were prepared for electrophoresis by addition of 3 μL of 15 lX DNA loading dye to 1 μL of PCR product. As outlined below, 6 μL of 1 kb DNA ladder was loaded into the first well of a 1% agarose gel. Into each of the remaining wells, 4 μL of each dyed DNA sample was loaded. An example set up is as follows: GRH-00361 Table 14 5 The samples were electrophoresed for 1 hour at a constant 100V. The expected length of the target sequence was confirmed relative to the DNA ladder. Optionally, or in addition, the sequence was confirmed by sequencing analysis, e.g., Sanger sequencing (i.e., chain termination method) analysis. DNA agarose gels can be seen in Figures 6, 58, and 71. 10 mRNA synthesis: Each mRNA synthesis reaction was conducted in a thin-walled, nuclease-free PCR tube. Reaction components were added to each tube in the following order. 15 Table 15 *Total volume of each tube should be 20 μL. If needed, the volume of Template DNA and nuclease free water can be adjusted to allow for more or less Template DNA. After adding all components, PCR reaction tubes were briefly centrifuged and the tubes 20 placed in an incubator set to 37°C for 2 hours (e.g., a thermal cycler set to 37°C for 2 hours followed by an indefinite 4°C hold) before proceeding to the capping reaction. A DNase treatment may optionally be done before proceeding to the capping reaction. GRH-00361 RNA Capping: 5 Each mRNA synthesis reaction (synthesized RNA in 20 μL) received 24.2 μL of nuclease free water (44.2 μL total). Capping reaction components were added to each synthesized RNA tube in the following order: Table 16 10 *total volume in each tube should be 65 μL After a brief centrifugation, reaction tubes were incubated at 37°C for 1 hour (e.g., a thermal cycler set to 37°C for 1 hour followed by an indefinite 4°C hold). RNA Cleaning: 15 RNA purification and concentration following enzymatic reaction was performed using commercially available kits, such as the Monarch® RNA Cleanup Kit. Briefly, 100 μL of RNA Cleanup Binding buffer was added to each synthesized RNA tube and pipetted carefully until the solution was a uniform consistency (165 μL total). This solution was transferred to a provided 20 filter tube / spin column and 165 μL of 100% Ethanol was further added and mixed by pipetting. Filter tube / spin column, assembled with a corresponding collection tube, were centrifuged at 13,000 rpm for 1 minute. The liquid flow through in the collection tube was discarded and the filter and collection tubes reassembled. RNA Cleanup Wash buffer (500 μL) was added to the filter tube, and the GRH-00361 Concentration, Size, and Purity Analysis: RNA concentration was measured using a Nanodrop™ spectrophotometer according to 5 manufacturer protocols. Agarose gels were used to verify size and purity of the synthesized RNA. Briefly, a 1% agarose (e.g., comprising Ethidium Bromide (EtBr)) was prepared and the RNA sample prepared for electrophoresis. Cleaned and capped RNA was diluted in nuclease-free water to a concentration between 300 and 400 μg / mL. In a PCR reaction tube 1 μL of the diluted RNA was 10 combined with 3 μL of RNA loading dye and mixed by pipetting. Commercially available single-stranded RNA (ssRNA) ladder was prepared per manufacturer protocols. Samples were loaded into the agarose gel as exemplified below. Table 17 15 The samples were electrophoresed for 1 hour at a constant 100V. The expected size and purity of the RNA was confirmed relative to the RNA ladder. Optionally, or in addition, the sequence was further confirmed by sequence analysis. RNA agarose gels can be seen in Figures 7, 59, and 72. 20 Lipid Nanoparticle Stock Solution & Mix Creation: Lipid nanoparticle stock solutions were prepared as indicated in the table below, i.e., second column. Lipid solutions were vortexed to mix until they were a uniform color / consistency. Stock solutions were then stored at -20°C until needed. 25 Table 18 GRH-00361 "LNP Mix" was prepared from lipid stock solutions, e , as described in the third column of the table above. All components were added to a 15 mL Falcon™ tube and vortexed to mix 5 prior to storage at -20°C until use. Aqueous RNA solution was prepared as described herein and using the following equations: • mRNA Volume: (75 / Concentration of mRNA) * 1,000 = _ μL • Sodium acetate Volume: 1,500 μL- mRNA Volume=_ μL 10 LNP Mix and aqueous RNA solution were each loaded into a microfluidic mixer in their respective receptacles (tubes), with the mixer flow rate set to 3.0 mL / min and all the tubes screwed in tightly. The output, comprising the mRNA-LNPs, was collected in a "Collection" tube and set aside. Dialysis: 15 The collected mRNA-LNPs were injected into a dialysis cartridge of appropriate volume, e.g., depending on the volume of the dose made, a 0.5-3 mL or 3-12 mL dialysis cassette was selected and placed in a 1 or 2 L beaker filled with PBS (200-300x the volume of dose made) for 5 minutes to moisten the dialysis tubing in the cassette prior to injection. A newly cleaned syringe was used to collect the mRNA-LNP dose and inject into the 20 dialysis cassette. The cassette was placed in the PBS and kept at 4°C for 1 hour. The PBS was exchanged with fresh PBS solution and returned to 4°C overnight. The next day, take out the dose from the dialysis cassette from the opposite side from where the syringe was initially inserted. The dialyzed RNA-LNPs were collected via syringe and GRH-00361 dispensed through a 0.22 μm filter into an appropriate container, e.g., a conical tube. A 1-1.5 mL sample of the newly dialyzed mRNA-LNPs was checked for size and purity via dynamic light scattering (DLS). The mRNA-LNPs were then stored at 4°C until use. Example 3: Dynamic Light Scattering (DLS) 5 In order for the LNP formulation disclosed herein to be used as a treatment for neurological diseases, disorders, and / or cancers, 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 effectively pass through the blood brain barrier (Cena & Javita, 2018). Furthermore, 10 particles over 120 nm have a high likelihood of contamination and / or error and will therefore be discarded. 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 15 solvent, which is related in tum 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 20 consistently comprised particles with a diameter of 110 nm or smaller. In a typical DLS analysis the Z-Average is the estimated average size of the particles being measured. The D50 showed 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 89.7 nm or below in size (see Histogram Operations: % Cumulative (6), the aforementioned D50). The Count Rate (measured in kilo counts per second 25 (kCPS)) correlates with the concentration of the sample being measured. A Count Rate above 400 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 native human mRNA. This without being bound by theory or methodology the higher the concentration, the more effective that dose can be. 30 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 GRH-00361 illustrated in the overlay of the Z-Average (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. (88.1 nm to 89.7 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 5 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 measure in intensity, one larger particle can block out many more of the smaller particles and skew the data 10 to show a larger Z-Average and D50 than is actually true. The test sample was prepared and stored at 4°C (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 time point a portion of sample was applied to a cuvette and read on the DLS machine and retired, e.g., to a 15 mL conical tube for storage at 4°C again. Results showed that 15 even after 145 days in storage, there was no aggregation of particles. Thus, the LNPs of the invention are stable at 4°C for long periods of time without changing size. In summary, the DLS data showed that particles at 110 nm or smaller can be made consistently. This will allow LNPs to cross the blood-brain barrier to deliver native human mRNA to the cells of the brain. The size of the mRNA-LNPs is also consistent within each 20 triplicate reading. Said particles do not aggregate together or change in size over time and can be stored at 4°C with no degradation or aggregation over 145 days of repeated testing. Example 4: HSP70 Cellular ELISA Data Enzyme-linked immunoassays (ELISAs) are currently considered the preferred 25 measurement of protein concentration within a solution. The ELISA employed herein was a sandwich ELISA, meaning that the analyte to be measured (HSP70) 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, HSP70, was initially bound to capture antibody adsorbed / linked to the bottom of wells of a microplate. After this initial binding a biotin 30 conjugated anti-HSP70 was attached to the bound HSP70. 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 GRH-00361 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 HSP70 protein was being overexpressed with application of the LNP-encapsulated mRNA. Briefly, SH-SY5Y Neuroblastoma cells were grown in DMEM media containing 9.1% 5 PBS 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, HSP70 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 HSP70 mRNA-LNPs and were returned to the incubator for 12 hours (overnight). After the 12- 10 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 HSP70 concentration. Microplates were loaded as described in Tables 19 and 20 and ELISAs were run using a Crocodile ELISA MiniWorkstation (Berthold Technologies, Germany). Following Biotin- 15 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 was read on a spectrophotometer 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 HSP70 mRNA-LNP5) and "Control" 20 (collected from cells that did not receive HSP70 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 19 and 20. Table 19 25 1st ELISA run Layout: GRH-00361 Table 20 2nd and 3rd ELISA run layout: 5 Overall, the fluorescence readings were shown to be higher in the sample columns than the control columns, which indicates that HSP70 was overexpressed following application of the LNP particles. GRH-00361 Using this data, and the data from the standard wells, the average concentration of HSP70 per well and the total amount of HSP70 present in the original 850 μL sample was calculated. All three of the trials showed higher average fluorescence, average HSP70 in ng / mL per well, and total HSP-70 concentration in the original 850 μL sample for samples dosed with HSP70 5 mRNA-LNPs. See Table 21 Table 21 The ELISA data confirms that the HSP70 mRNA-LNPs can induce an overproduction of 10 the HPS70 protein in human cells. Repeated tests showed that cells that have been dosed with HSP70 mRNA-LNPs produce significantly more HSP70 protein than cells that have not been dosed. Example 5: HSP70 Cell line Data 15 In the human body, HSP70 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 GRH-00361 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 HSP70 mRNA-LNPs. 5 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. Experiments 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 10 group, two 12-well plates were used, one plate as a control and the other as experimental (lead- poisoned). Cells treated with HSP70 mRNA-LNPs had between a 15%-40% higher viability than the cells without HSP70 mRNA-LNPs. The control plate also showed that HSP70 mRNA-LNPs was not damaging or killing the SH-SY5Y cell lines, thus demonstrating that HSP70 mRNA- 15 LNPs is not toxic to human cells. This data suggests that HSP70 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. 24 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 HSP70 mRNA- 20 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 HSP70 mRNA-LNPs. The control well plate consisted of 3 wells treated with lead acetate, 3 wells treated with HSP70 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 25 when treated with just lead acetate. Another 3 wells were treated with just HSP70 mRNA-LNPs 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 HSP70 mRNA-LNPs is in, or PBS, is not harming the cells and causing a decrease in viability. The test well plate showed that HSP70 mRNA-LNPs were able to slow down / stop 30 the effects of lead acetate on the cells. GRH-00361 Briefly, SH-SY5Y Neuroblastoma cells were grown in DMEM media containing 9.1% PBS 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 5 of HSP70 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 22: Test well plate 10 In the second plate (control well plate), after the 12-hour incubation, cells in wells 1, 5, and 9 received 50 μL of 80 mM lead acetate; cells in wells 2, 6, and 10 received 17 μL of HSP70 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. 15 Table 23: Control well plate Both 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 of the test well plate received 17 μL of HSP70 mRNA-LNPs GRH-00361 and cells in wells 7-12 received 17 μL of PBS. In the control well plate, cells in wells 1, 5, and 9 were dosed with 50 μL lead acetate, cells in wells 2, 6, and 10 were dosed with 17 μL of HSP70 mRNA-LNPs, cells in wells 3, 7, and 11 received 17 μL PBS, and cells in wells 4, 8, and 12 did not receive anything. 5 Both plates were returned to the incubator for an additional 4 hours at 37°C / 5.0% CO2. After the second 4-hour incubation, cell viability in each well was assessed by microscopy on a hemocytometer using trypan blue staining. Data showed that there was an average of 15% increase in cell viability (HSP70 mRNA- LNP + Lead) compared to the cells with the addition of lead acetate, i.e., Lead Only and PBS + 10 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 HSP70 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. 15 48 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. This test well plate consisted of 6 wells of cells that were treated with lead acetate and HSP70 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 with or without HSP70 mRNA-LNPs. The control well plate consisted of 3 wells 20 treated with lead acetate, 3 wells treated with HSP70 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 HSP70 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 25 last 3 wells treated with just PBS confirm that the solution comprising the HSP70 mRNA-LNPs is not harming the cells and causing a decrease in viability. Briefly, after overnight incubation (12 hours) at 37°C / 5.0% CO2 to allow cells to adhere and grow, the first 6 wells of the first plate (test well plate) received 17 μL of HSP70 mRNA- LNPs and was returned to the incubator for 3 hours, and then 50 μL of 80 mM lead acetate was 30 added to all 12 wells, and thee plates returned to the incubator for 8 hours. The control well plate, after the initial 12 hour incubation, received 50 μL of 80 mM lead acetate and 17 μL of GRH-00361 PBS in wells 1-6 and 17 μL of HSP70 mRNA LNPs in wells 7-12, before returning to the incubator for 8 hours. After the 8 hour incubation period, wells 1-6 of the test well plate received 17 μL of HSP70 mRNA-LNPs. In the control well plate, wells 1-6 received 17 μL of PBS and wells 7-12 5 received 17 μL of HSP70 mRNA-LNPs. Plates were then returned to the incubator for an additional 8 hours before cell viability was assessed as described above in the 24 hour cell count. Data from the test well plate showed that HSP70 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. 10 Data from the wells containing cells dosed with only HSP70 mRNA-LNPs (Dose Only) had an average viability of 87%, which displays that the HSP70 mRNA-LNPs do not negatively affect human neuroblastoma cells. The wells containing cells dosed with HSP70 mRNA-LNPS and lead acetate (Dose+ Lead) showed a 28% cell viability while wells containing cells dosed with lead acetate only (Lead Only) showed a 7% cell viability. The wells containing cells dosed 15 with PBS and lead acetate (PBS+ Lead) showed a 2% cell viability. This data shows that the HSP70 mRNA-LNPs increased the viability of human neuroblastoma cells by about 21% when both were dosed with 80 mM lead acetate (Figure 4). Overall, the data from the 24- and 48-hour cell counts show that HSP70 mRNA-LNPs increase the viability of lead poisoned cells, i.e., exposure to a 50 μL dose of 80 mM lead acetate 20 solution. The SH-SY5Y cells dosed with HSP70 mRNA-LNPs and lead acetate had a significantly higher viability than cells dosed 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 HSP70 mRNA-LNPs or PBS was compared to cells that were treated 25 with just lead acetate, just HSP70 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 HSP70 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 HSP70 30 mRNA-LNPs, the HSP70 has the capability to save cells from high levels of toxicity from lead GRH-00361 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. Example 6: HSP70 mRNA-LNP Encapsulation Data 5 These experiments were performed to ensure that mRNA coding for HSP70 was being successfully insured via hydro-fluidic (microfluidic) mixing into the solid lipid nanoparticles to successfully form the HSP70 mRNA-LNPs of the invention. Using ethidium bromide (EtBr), which is a DNA and RNA intercalator, particles were confirmed to contain fully formed mRNA strands coding for HSP70 as the fluorescence in said particles was more than double the 10 fluorescence of a control solution and control solid lipid nanoparticles. EtBr fluorescence 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 EtBr binds to intact strands more readily than it does to single nucleotides. Essentially EtBr 15 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 HSP70 mRNA-LNPs was prepared. A 5% Triton X-100 was made using nuclease-free H2O. A 15 mL conical tube of EtBr solution using 15 mL of DI water and 5 μg of EtBr was made. Test solution was prepared from 200 μL of 20 HSP70 mRNA-LNPs, 800 μL of 5% Triton X-100, and 50 μL of EtBr solution. Control solution 1, not containing HSP70 mRNA-LNPs, comprised 200 μL PBS, 800 μL of 5% Triton X-100, and 50 μL of EtBr solution. Control solution 2, containing empty solid lipid nanoparticles with no mRNA, comprised 200 μL Empty LNPs, 800 μL of 5% Triton X-100, and 50 μL of EtBr solution. Samples were transferred one at a time to a 1 mL cuvette and inserted into a 25 fluorometer where each sample was read at an emission wavelength of 540 nm to 750 nm. Figure 5A displays the fluorescence from EtBr when solid lipid nanoparticles containing mRNA coding for HSP70 are broken apart using Triton X. The final solution read for this test sample was composed of HSP70 mRNA-LNPs, EtBr solution, and Triton X. The Sl c curve (the corrected Sl curve) has a peak fluorescence of 4.34 x 106CPS for this test sample. The first 30 control curve depicted in Figure 5B displays the fluorescence of EtBr without any LNPs (empty or HSP70 mRNA-LNPs). The solution being measured was EtBr, Triton X, and PBS. The fluorescence peak of this control sample was 1.75 x 106CPS. The second control sample GRH-00361 depicted in Figure 5C consisted of Empty LNPs. These particles were lysed using Triton X and then exposed to EtBr. The final solution was composed of lysed Empty LNPs, EtBr solution, and Triton X. This solution had a measured fluorescence of 2.14 x 106CPS. This data displays that HSP70 mRNA was successfully encapsulated into LNPs as the fluorescence in the test sample 5 was over 2 million CPS higher than either control samples. Figure 5A also displayed a different peak wavelength than the controls with the peak being read at 607 nm compared to the 614 nm peak wavelength of Figure 5B and 613 nm peak wavelength of Figure 5C. This shift in 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 10 test sample peak wavelength was observed (607 nm). Using the inbred mouse line MS-NASH, an experimental group of mice were dosed with 75 μL of HSP70 mRNA-LNPs and a control group were dosed with 75 μL of Empty LNPs. The 15 experimental group consisted of 3 MS-NASH mice that were dosed via lateral tail vein injection every 24 hours. The control group consisted of 3 MS-NASH mice that were dosed via lateral tail vein injection every 24 hours. The experimental and control mice were kept in separate cages and assigned a number within each cage. The mice were weighed every day (see Table 24). 20 Table 24 GRH-00361 GRH-00361 GRH-00361 GRH-00361 After 2 weeks (14 days) of injection, all 6 mice were euthanized via CO2 inhalation. This was followed by a secondary method of cervical dislocation to ensure the death. Following the 5 euthanasia, a complete craniotomy was performed on each mouse. Following surgery, the brains and livers of the MS-NASH mice were placed in separate 15 mL conical tubes with 7 mL of 10% Neutral Buffered Formalin. These samples were subsequently processed for paraffin-embedded sectioning as well as hematoxylin and eosin (H&E) staining. Example 8: MS-NASH Mouse Weight Data (HSP70 Cohort 2) 10 The same MS-NASH procedure from example 8 was repeated, but with 2 control MS- NASH mice instead of 3, i.e., 5 total MS-NASH mice were used for the second trial of efficacy testing. All 5 mice were kept in the same cage, and each mouse was assigned a number as indicated below. The mice were weighed every day (see Table 25). 15 Table 25 GRH-00361 GRH-00361 GRH-00361 After 2 weeks (15 days) of injection, all 5 mice were euthanized, and brain and liver samples were processed for paraffin-embedded section and H&E staining as described above. 5 Example 9: MS-NASH Mouse Weight Data (HSP70 Cohort 3) The same MS-NASH procedure from example 8 was repeated. All 5 mice were kept in the same cage, and each mouse was assigned a mouse number. The mice were weighed every day (see Table 26). 10 Table 26 GRH-00361 GRH-00361 GRH-00361 After 2 weeks (14 days) of injection, all 5 mice were euthanized, and brain and liver samples were processed for paraffin-embedded section and H&E staining as described above. 5 Example 10: MS-NASH Mouse Weight Data (HSP70 Cohort 4) 4 MS-NASH mice were dosed every 24 hours with various amounts of HSP70 mRNA- LNPs for 2 weeks (14 days). All 4 mice were kept in the same cage, and each mouse was assigned a mouse number. Mouse #1 received a dose of 30 μL. Mouse #2 received a dose of 150 10 μL. Mouse #3 received a dose of 250 μL. Mouse #4 received a dose equal to 1% of its body GRH-00361 weight in volume (i.e., a 39.5g mouse would receive a 395 μL dose). The mice were weighed every day (see Table 27). Table 27 5 GRH-00361 GRH-00361 After 2 weeks (14 days) of injection, all 4 mice were euthanized, and brain and liver samples were processed for paraffin-embedded section and H&E staining as described above. 5 Example 11: HSP70 mRNA-LNP Dosed BALB / c.J Mouse Weight Data (HSP70 Cohort A) Toxicology mouse testing began with BALB / cJ mice. An experimental group dosed with 75 μL of HSP70 mRNA-LNPs and a control group dosed with 75 μL of Empty LNPs. The experimental group consisted of 3 BALB / cJ mice that were dosed via lateral tail vein injection 10 every 48 hours. The control group consisted of 2 BALB / cJ mice that were dosed via lateral tail vein injection every 48 hours. All 5 mice were kept in the same cage, and each mouse was assigned a mouse number. The mice were weighed every day, regardless of injections (see Table 28).

[0003] GRH-00361 Table 28 GRH-00361 GRH-00361 *On 03-30-24 and 03-31-24 (Days 4 and 5), the roads to the vivarium were flooded, preventing the dosing and weighing of the mice. 5 After 2 weeks (14 days) of injection, all 5 mice were euthanized, and brain and liver GRH-00361 samples were processed for paraffin-embedded section and H&E staining as described above. Example 12: H&E Stained Imaging (MS-NASH Mice) 5 H&E-stained slides of sectioned liver from MS-NASH mice dosed with HSP70 mRNA- LNPs were compared to MS-NASH mice dosed with Empty LNPs. H&E staining of sectioned liver can reveal lipid accumulation as clear vacuoles in hepatocytes and these images illustrated major differences between the two groups of mice. The HSP70 mRNA-LNP dosed mice had significantly less steatosis (fat buildup) of the liver, as indicated by the significantly lower 10 amount of white space (see Figures 14, 16, & 18) relative to the Empty-LNP-dosed mice (control group). (See Figures 13, 15, & 17). The area of the white space was also calculated using image J software. On average the HSP70 mRNA-LNP dosed MS-NASH mice had a fat percentage of 4.51%. The Empty LNP dosed MS-NASH mice had a fat percentage of 13.04%. The brain sections were also compared but showed no noticeable or significant differences between the two 15 groups. Before the livers were placed into formalin and processed for H&E staining or paraffin embedding, images of the livers were taken. The livers of MS-NASH mice dosed with HSP70 mRNA-LNPs (Figures 23, 24, and 25) were noticeably darker in color than those of MS-NASH mice dosed with Empty LNPs (Figures 26, 27, and 28). The mice given Empty LNPs had large 20 clumps of visual fat in their livers. The livers of the Empty LNP dosed mice were also noticeably more pale when compared to the HSP70 mRNA-LNP dosed livers. Overall, mice dosed with HSP70 mRNA-LNPs had livers that visually appeared healthy with no visual clumps of fat. The Empty LNP dosed mice had livers that were pale in color with large clumps of fat visible to the naked eye. These results were confirmed with the H&E staining mentioned above. 25 Example 13: H&E Stained Imaging (HSP70 mRNA-LNP dosed BALB / c.J Mice) Whole brains and livers were sectioned at 5-micron thick slices and stained with H&E. Slides were imaged on a fluorescent microscope and BALB / cJ mice dosed with HSP70 mRNA- LNPs (Figure 20 & 22) were compared to BALB / cJ mice dosed with Empty LNPs (Figures 19 & 30 21). No significant or notable differences between the two groups of mice. Example 14: IHC Imaging (HSP70 mRNA-LNP dosed MS-NASH and BALB / c.J Mice) GRH-00361 To see how much the HSP70 mRNA-LNP is increasing HSP70 expression in the liver of the BALB / cJ and MS-NASH mice, immunohistochemistry (IHC) was performed on paraffin embedded tissue sections. Slides of the embedded tissue sections were heated in a dry oven for 20 minutes at about 55°C. After this, the slides were placed in a tray and covered with a nonpolar 5 solvent, such as Risto-Clear II, and allowed to sit for 5 minutes. After this 5-minute wash, the slides were removed and washed again in the same manner for a total of three washes. Slides were then subjected to the following wash cycle: 100% Ethanol (2 washes for 5 minutes each), 90% Ethanol (1 wash for 5 minutes), 70% Ethanol (1 wash for 5 minutes), and finally DI H20 (1 wash for 5 minutes). After the wash cycle, each slide was placed in a 500 mL beaker with 10 enough Tris-EDTA antigen Retrieval solution to comfortably cover each slide (about 40-80 mL). The slides in the Tris- EDTA solution were then microwaved until boiling (about 30-45 seconds) and then placed on a preheated hotplate set to 95°C- 100°C and allowed to sit for 15 minutes. After the 15-minute heating period, the beakers were removed from the hot plate and allowed to come down to room temperature (about 15-25 minutes). After cooling, the slides were placed in 15 PBS and on a shaker set to 40 rpm at room temperature for 5 minutes. Following this PBS wash, the edges of the slides were dried and nail polish, or a hydrophobic barrier pen, was used along the edges of the slide to create a barrier. The slides were then placed on a wet paper towel in a tray and 600-900 μL of 2% PBS in PBS solution) was then carefully added to each slide. The slides were then covered and protected from light and allowed to incubate at room temperature 20 for 1 hour. After the incubation period, the 2% PBS solution was removed, and the slides were placed in PBS and put on a shaker set to 40 rpm for 5 minutes. The PBS was replaced, and the slides were returned to the shaker for another 5 minutes. During these washes, a primary antibody solution was made with an anti-human HSP70 monoclonal rabbit antibody at a dilution of 1:2,000 in 0.1% PBS. The slides were removed from the PBS wash and placed on a wet paper 25 towel in a tray and 600-900 μL of the primary antibody solution was carefully pipetted onto each slide. The slides were covered and protected from light and were incubated at 4°C overnight. After the overnight incubation the slides underwent the same PBS washing procedure for a total of 3 washes. During the PBS washes, a secondary antibody solution was made with an anti- rabbit secondary antibody tagged with Alexa FluorTM 568 and Hoechst 33342. Both 30 components were diluted in the same 0.1% PBS / PBS solution at a concentration of 1:2,000. After the 3 PBS washes, the slides were placed on a wet paper towel-lined tray and moved to a GRH-00361 dark area and 600-900 μL of the secondary antibody solution was carefully pipetted onto each slide. The slides were covered and protected from light while they incubated at room temperature for 1 hour. The secondary antibody solution was removed, and the slides were stored in PBS, protected from light, at 4°C until ready for analysis on a fluorescent microscope. Prior to 5 imaging, the slides were removed from PBS and allowed to dry in a light protected space. Said imaging (Figures 82 - 85) confirmed increased HSP70 expression in the liver, which was further confirmed using enzyme-linked immunosorbent assay (ELISA). Example 15: HSP70 mRNA-LNP Dosed MS-NASH ELISA Data 10 In order to determine how much HSP70 mRNA-LNPs were increasing HSP70 expression in the livers of mice, an ELISA was run on the livers of MS-NASH mice. This assay was run with a commercially available HSP70 sandwich ELISA kit from Thermo Fisher Scientific. The kit recommended procedure was used for this assay. Tissue samples were prepped by first removing the livers of MS-NASH mice (both mice dosed with HSP70 mRNA-LNPs and empty 15 LNPs) and storing them at -80°C until the ELISA was run. The livers were then placed in a flat bottom conical tube and 1 mL of PBS was added. Then a tissue homogenizer was used to blend the organs and PBS for at least 30 seconds, or until no visible tissue chunks were left (the organ was uniformly blended and mixed with the 1 mL of added PBS). The tubes were then spun in a centrifuge set to 2,000-2,500xg for 5-7 minutes. After the centrifugation, the tubes were 20 inspected to see a visual separation of organ solids and supernatant.50 μL of the supernatant was added to 50 μL of antibody diluent in the correct wells of a 96 well plate coated with an anti- human HSP70 antibody. As the tissue samples are added to the 96 well plate, the HSP70 standards are also added to the 96 well plate. After all samples and standards were added, the ELISA was run according to kit specifications. See Table 29 for results. 25 The ELISA data above shows that HSP70 mRNA-LNPs can almost double the concentration of HSP70 found in the livers of MS-NASH mice. The concentration paired with GRH-00361 the whole organ images and H&E stained images suggests that the cause of the liver's healing and returning to normal functioning is the increased expression of HSP70 in the livers caused by the HSP70 mRNA-LNPs. Example 16: HSP70 mRNA-LNP Dosed MS-NASH Bloodwork Data Overview 5 In order to determine the altered levels of relevant components within the blood from the MS-NASH mice, blood samples were collected and analyzed using IDEXX's Hematology for Veterinary Diagnostics service. A post-euthanasia cardiac punch was performed on the MS- NASH mice for blood collection before being sent off for analysis. The two types of hematology 10 diagnostics that were used were complete blood count (CBC) and expanded toxicology. The CBC test is a method of analysis that evaluates the quantity of a variety of cells and components in the whole blood. It assists in the diagnosis of certain medical conditions as it provides data on red blood cell count, platelets, hemoglobin, and the relative concentration of the different types of white blood cells. The expanded toxicology test provides insight on the varying levels of 15 relevant enzymes present in the serum component of the blood. It also assists in the diagnosis of medical conditions due to the insight it provides on liver enzymes, glucose, and cholesterol levels. Table 30 20 LOR: Low (Outside of Range) LWR: Low (Within Range) Average:(Near Mean) HWR: High (Within Range) HOR: High (Outside of Range) GRH-00361 The above CBC blood work data displays several key points. Primarily, MS-NASH mice treated with HSP70 mRNA-LNPs display healthy levels of red blood cells as well as average hemoglobin concentrations. Control MS-NASH mice, receiving empty LNP doses, had higher than average red blood cell counts but average hemoglobin concentrations. Normal MS-NASH 5 mice have oxygen transport issues due to their difficulty breathing and high blood pressure from the symptoms induced by obesity (Kosmalski et al, 2018). This shows the broad impact and improvement of the experimental group of MS-NASH mice treated with HSP70 mRNA-LNPs as their RBC and HGB levels were significantly lowered in comparison to the control group. The NASH & NAFLD diseases have been linked to compromised immune systems (Kosmalski et al, 10 2018). Low WBC, lymphocytes, and platelet counts are all factors associated with hepatitis and cirrhosis of the liver. Therefore both experimental and control groups had decreased levels of lymphocytes and other immune system related blood components. Table 31 15 LOR: Low (Outside of Range) LWR: Low (Within Range) Average:(Near Mean) HWR: High (Within Range) HOR: High (Outside of Range) The above blood work data displays the ability of HSP70 mRNA-LNPs to rescue liver function inside of NASH affected mice. HSP70 mRNA-LNPs successfully brought AST and 20 ALT levels back to normal ranges as well as massively lowering glucose and cholesterol in these GRH-00361 mice. This suggests that HSP70 mRNA-LNPs anti-inflammatory abilities are able to revitalize the liver and return it to its normal functioning levels. Example 17: Immunofluorescence (IF) Imaging on Cells Treated with MECP2 mRNA- LNPs 5 SK-N-FI human neuroblastoma cells were grown in recommended DMEM media at 5% CO2 at 37°C in a 75 cm2tissue culture flask until cells were about 75% confluent. These cellswere then split and seeded onto a 4-chamber slide with 30,000 cells being added to each well. 500 μL of DMEM media was added to each well before the addition of 30,000 cells. The cells were allowed to adhere overnight (at least 12 hours) in a cell incubator set to 5% CO2 and 37°C. 10 After 12 hours, 2 wells were dosed with 5 μL of MECP2 mRNA-LNPs, and the other 2 were dosed with Empty LNPs. After 48 hours of incubation in the cell incubator, the media was removed, and each well was washed with 500 μL of PBS three times. Following the PBS washes, 400 μL of 4% paraformaldehyde was added to each well. The cells were then incubated at 37°C for 10 minutes. Following the 10-minute incubation, the 4% paraformaldehyde was 15 removed, and each well was washed with 500 μL of PBS three times. Following this wash, 400 μL of ice cold 100% methanol was added to each well. The cells were then incubated at -20°C for 5 minutes. After the 5-minute incubation, the 400 μL of methanol was removed. Each well was then washed 3 times with 500 μL of PBS. After the 3 washes, 400 μL of 0.1% Triton X-100 in PBS was added to each of the wells. The cells were then incubated at room temperature for 15 20 minutes. After the 15-minute incubation period, the Triton X-100 solution was removed, and the wells were washed 3 times with 500 μL of PBS. After this PBS wash, 500 μL of 2% PBS (fetal bovine serum) in PBS was added to each well. These cells were then incubated for 1 hour at room temperature. After this incubation period, the 500 μL of 2% PBS in PBS was removed. MECP2 monoclonal antibody (8H4A5B9) from Thermo Fisher Scientific was used as the 25 primary antibody for the immunostaining. This antibody was diluted in 0.1% PBS in PBS at a concentration of 1:500 (1 μL of MECP2 monoclonal antibody in 499 μL of 0.1% PBS in PBS). After removing the 2% PBS in PBS from each well, 500 μL of the MECP2 monoclonal antibody solution was added to each well. The slide was then incubated at room temperature for 3 hours (or optionally at 4°C overnight). Following the incubation with MECP2 monoclonal antibody, 30 the 500 μL of the antibody solution was removed and each well was washed with PBS 3 times. GRH-00361 A secondary antibody and nuclear dye solution was used for the fluorescence. The secondary antibody used was Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 from Thermo Fisher Scientific at a concentration of 1:2,000. The nuclear dye used was Hoechst 33342, Trihydrochloride, Trihydrate - 10 mg / mL Solution in Water from 5 Thermo Fisher Scientific at a concentration of 1:2,000. Both the secondary antibody and Hoechst 33342 were added to 0.1% PBS in PBS (1 μL of secondary antibody, 1 μL of Hoechst dye, 1,998 μL of 0.1% PBS in PBS). Following the washes after the primary antibody incubation, 500 μL of the secondary antibody and Hoechst solution was added to each well. The slide was then placed in the dark and incubated at room temperature for 45 minutes. After this 45-minute incubation 10 period, the secondary antibody and Hoechst solution was removed, and each well was washed 3 times with PBS. After the PBS washes, the slides were left to air dry for 5 minutes. (Optionally the slides can be mounted by air drying the slide, removing the well chambers and adding mounting medium. Following the mounting medium, a cover slip can be added). The slides were then imaged on a fluorescent microscope with wavelengths set to DAPI 15 and GFP as the secondary antibody and Hoechst were fluorescing green and blue respectively. The images were then processed using image J software to determine the overlap between the blue fluorescence in the nucleus due to the Hoechst and the green fluorescence from the secondary antibody. The cells dosed with MECP2 mRNA-LNPs saw on average less area from the blue wavelength when compared to cells dosed with Empty LNPs. The cells dosed with 20 MECP2 mRNA-LNPs also had more area from the green fluorescence when compared to the cells dosed with Empty LNPs. The green fluorescence area is divided by the blue fluorescence area to calculate the mean staining intensity. The mean staining intensity of the cells dosed with MECP2 mRNA-LNPs was higher than the mean staining intensity of the cells dosed with Empty LNPs. Table 32 lists the mean staining intensity of the samples. With experimental being dosed 25 with MECP2 mRNA-LNPs and control being dosed with Empty LNPs. See Figures 29 and 30 for examples of the area of blue and green fluorescence of experimental and control cells. Table 32 96 GRH-00361 5 The cellular immunofluorescence data disclosed herein acts as a means of quantification of MECP2 protein expression within SK-N-FI Neuroblastoma cells. A 12-well cell culture plate is treated with 250,000 SK-N-FI Neuroblastoma cells and DMEM media with 9.1% PBS per well. After being grown out for 24-48 hours, 6 of the wells are treated with 40μL of MECP2 mRNA-LNPs per well and the other 6 wells are treated with 40μL of empty LNPs per well. Once 10 the cells have been treated, they are left over a certain period of time. The time periods disclosed herein are 1, 2, 3, 4 and 12 hours. After the determined time period has passed, the plate is subjected to removal of the DMEM media with 9.1% PBS wherein each well is subsequently washed with 700 μL PBS with MgCh and CaCh 3 times. Following is the addition of 500 μL of methanol that has been chilled 15 to -20°C. The plate containing the methanol is quickly placed into the -20°C freezer for 5 minutes. After the allotted 5-minute period, the methanol is quickly removed from each well which is followed by subsequent washing of each well with 700 μL PBS with MgCh and CaCh 3 times. Following is the addition of 500 μL of 0.1% Triton X in PBS with MgCh and CaCh to 20 each well, followed by a 15-minute hold at room temperature. After the allotted 15-minute time period, the 0.1% Triton X solution is removed and each well is washed with 700 μL PBS GRH-00361 comprising MgCh and CaCh, 3 times. After the washes, 1 mL of 2% BSA in PBS with MgCh and CaCh is added to each well and is left at room temperature for 1 hour. When there is approximately 5-10 minutes left within the 1-hour hold, 1 μL of a primary antibody solution (containing an Invitrogen MECP2 Polyclonal Antibody diluted 1:6000 with 5 0.1% BSA in PBS with MgCh and CaCh) is made and stored at 4°C until ready to be used. Once the 1-hour period is over, the 2% BSA solution is removed from each well and each well is then washed 3x times with 700 μL of PBS with MgCh and CaCh. After the washes, 500 μL of the primary antibody solution is added to each well and left to incubate at room temperature for 3 hours. When there is approximately 5-10 minutes left within the 3 hour hold, 1 μL of a secondary 10 antibody solution (containing an Invitrogen Qdot 655 goat F(ab')2 anti-rabbit IgG conjugate (H+L) diluted 1:50 with 0.1% BSA in PBS with MgCh and CaCh) is made and stored at 4°C until ready to be used. Following the 3-hour period, the primary antibody solution is removed from each well, and each well is then washed 3x times with 700 μL of PBS with MgCh and CaCh. After the 15 wash, 400 μL of the secondary antibody solution is added to each well and left to incubate at room temperature for 45 minutes. Once the 45-minute incubation period has completed, the secondary antibody solution is removed from each well, and each well is then washed 3x times with 700 μL of PBS with MgCh and CaCh. Following the washing, 220 μL of trypsin and 220 μL of 1% Triton-X in PBS (with no 20 MgCh or CaCh) is added to each well. The plate is placed in a cell incubator (set to 37°C I 5% CO2) for approximately 5 minutes. During the 5-minute hold, a blank solution is added to 1 well in a black Nunc 96-well plate composed of 110 μL of trypsin and 110 μL of 1% Triton-X. Once the 5-minute hold is completed, each well in the 12-well plate is added to 2 new wells of the black Nunc 96-well plate. Hence, each well with liquid in the 96-well plate should contain 220 25 μL of volume. Once the control and experimental wells are transferred to the 96-well plate, the plate is taken to a fluorometer where the fluorescence is read. Table 33 GRH-00361 Table 34 5 The fluorescence measurements were observed in the Control wells (Empty LNP) and the Experimental wells (MECP2 mRNA-LNPs). Table 33 and Figure 31 both depict the average percent changes in fluorescence over time periods of 1,2,3,4 and 12 hours of the cells treated with MECP2 mRNA-LNP when compared to cells treated with Empty LNPs. Table 34 depicts the average fluorescence of the Control and Experimental at each time period. Protein production 10 created by MECP2 mRNA LNPs can be most seen between hours 1-3 when the particles are exposed directly on to SK-N-FI cells. After 4 hours, the particles cease to cause an overexpression of MECP2. This is ideal to control MECP2 expression in patients as the overexpression is not long lasting. 15 Al Toxicology mouse testing began with BALB / cJ mice. An experimental group dosed with 75 μL of MECP2 mRNA-LNPs and a control group dosed with 75 μL of Empty LNPs. The experimental group consisted of 3 BALB / cJ mice that were dosed via lateral tail vein injection every 48 hours. The control group consisted of 2 BALB / cJ mice that were dosed via lateral tail 20 vein injection every 48 hours. All 5 mice were kept in the same cage, and each mouse was GRH-00361 assigned a mouse number. The mice were weighed every day, regardless of injections (see Table 35). Table 35 5 GRH-00361 GRH-00361 GRH-00361 GRH-00361 After 3 weeks (21 days) of injection, all 5 mice were euthanized, and brain and liver samples were processed for paraffin-embedded section and H&E staining as described above in 5 Example 7. Example 20: MECP2 mRNA-LNP Dosed Weight Data (MECP2 Cohort ID. The same BALB / cJ procedure from example 19 was repeated. All 5 mice were kept in the same cage, and each mouse was assigned a mouse number. The mice were weighed every 10 day (see Table 36). Table 36 GRH-00361 GRH-00361 GRH-00361 GRH-00361 *On Day 6, Experimental (#1) was not weighed After 3 weeks (21 days) of injection, all 5 mice were euthanized, and brain and liver samples were processed for paraffin-embedded section and H&E staining as described above in 5 Example 7. Example 21: Male B6.129 dl.1Weight Data (MECP2 Cohort 1) GRH-00361 Using the inbred mouse line B6.l29P2(C)-Mecp2tml.IBird / J, an experimental group of mice were dosed with 75 μL of MECP2 mRNA-LNPs and a control group were dosed with 75 μL of Empty LNPs. The experimental group consisted of 3 B6.l29P2(C)-Mecp2tml.IBird / J mice that were dosed via lateral tail vein injection every 24 hours. The control group consisted of 2 5 B6. l29P2(C)-Mecp2tml.IBird / J mice that were dosed via lateral tail vein injection every 24 hours. All 5 mice were kept in the same cage, and each mouse was assigned a number as indicated below. The mice were weighed every day (see Table 37and Figure 32). Prior to injection every day, each mouse was subjected to a hindlimb clasping test that lasted 60 seconds. 10 Table 37 GRH-00361 GRH-00361 111 GRH-00361 GRH-00361 After 22 days of injections, all 5 mice were euthanized, and brain and liver samples were processed for paraffin-embedded section and H&E staining as described above in Example 7. 5Native MECP2 mRNA-LNP treated B6.l29P2(C)-Mecp2tml.IBird / J mice saw morecontrolled weight gain than empty LNP treated B6. l29P2(C)-Mecp2tml.IBird / J. This is due to RettSyndrome's impact on the liver and the inflammation and NASH it creates. Native MECP2 mRNA-LNPs treat this damage and allow the mice to not put on excess fat and instead grow more naturally. 10 Example 22: Male B ml.1Birdl.1Weight Data (MECP2 Cohort 2) The same B6. l29P2(C)-Mecp2tml.IBird / J procedure from example 21 was repeated, exceptwith 4 experimental and 4 control mice. The control and experimental mice were kept in separate GRH-00361 cages, with each mouse assigned a number for its cage. The mice were weighed every day (see Table 38and Figure 33). The mice were all subjected to a hindlimb clasping test for 60 seconds before injection each day. 5 Table 38 GRH-00361 GRH-00361 GRH-00361 GRH-00361 GRH-00361 GRH-00361 *On day 12, Experimental (#4) was euthanized due to severe weight loss. On day 14, Control (#1) was euthanized due to a prolapsed penis that did not heal. 5 After 3 weeks (22 days) of injection, all remaining of the 8 mice were euthanized, and brain and liver samples were processed for paraffin-embedded section and H&E staining as described above in Example 7. Discussion of the controlled versus uncontrolled weight gain is the same as written in Example 21 GRH-00361 A hindlimb clasping test was performed daily on all B6.l29P2(C)-Mecp2tml.IBird / J mice. The hindlimb clasping test has been applied to this study for its non-invasive nature as well as its ease of reproducibility. This test involves suspending the designated mouse by its tail for one minute about 6 inches to a foot above the lab bench surface. Observations on the positioning of 5 the mouse's hindlimbs upon whole body suspension provide insight related to early indications of neurological deterioration. A healthy mouse being held up by its tail will splay its limbs outwards to reach for a surface or to brace for impact from a fall. Mice with neurological deterioration from the MECP2 gene deletion were observed with their hindlimbs clasping inwards towards their midline while they are held up from their tail. This hindlimb positioning is 10 regarded as a sign that their survival instinct is impaired and is scored accordingly for data collection and analysis. Under the established hindlimb clasping scoring system, as shown below, a lower score is more indicative of improved neurological function while a higher score would indicate more neurological deterioration. The Hindlimb clasping scoring system has been determined as follows: 15 Score of 0: Both limbs splayed out for more than 90% of the time with little to no retraction towards abdomen (0-9 seconds of retraction). Score of 1: One or both hindlimbs partially retracted for 10-20 seconds. Score of 2: One or both hindlimbs partially retracted for 21- 30 seconds. Score of 3: One or both hindlimbs partially retracted for 31-40 seconds. Score of 4: One or both hindlimbs partially retracted for 41-50 seconds. Score of 5: One or both hindlimbs 20 partially retracted for 51-60 seconds. Table 39 GRH-00361 *Lower score indicates a healthier mouse As shown in Figure 34 and Table 39, the B6 mice dosed with MECP2 mRNA-LNPs experienced, on average, improved hindlimb clasping scoring in comparison to the B6 mice dosed with Empty LNPs. Over the course of the 20 days after the B6 neurological falloff point 5 until the end of the dosing regimen, the B6 mice dosed with MECP2 mRNA-LNPs had an average score improvement by a factor of two or more, in comparison to the control B6 mice dosed with Empty LNPs. Example 24: H&E Stained Imaging (MECp2 mRNA-LNP and Empty LNP Dosed BALB / c.J) 10 Whole brains and livers were sectioned at 5-micron thick slices and stained with H&E. Slides were imaged on a fluorescent microscope and BALB / cJ mice dosed with MECP2 mRNA- LNPs (Figures 39-42) were compared to BALB / cJ mice dosed with Empty LNPs (Figures 43- 46). No significant or notable differences between the two groups of mice. 15 H&E-stained slides of sectioned livers and brains from B6.l29P2(C)-Mecp2tml.IBirdlJ Mice dosed with MECP2 mRNA-LNPs were compared to B6.l29P2(C)-Mecp2tml.IBird / J mice dosed with Empty LNPs. The brains were not seen to have notable differences on the H&E stained slides as represented in Figures 47-49 for MECP2 mRNA LNP treated brains and Figures 53-55 for Empty LNP treated brains. However, MECP2 mRNA LNP treated B6.l29P2(C)- 20 Mecp2tml.IBird / J livers showed far less hepatocyte ballooning and steatosis (Figures 50-52) when compared to empty LNP treated B6.l29P2(C)-Mecp2tml.IBird / J livers (Figures 56-58). This shows MECP2 mRNA LNP' s ability to reduce inflammation and repair liver and hepatocyte damage as seen in these images. Whole liver images of MECP2 mRNA-LNP dosed male B6.l29P2(C)-Mecp2tml.IBird / J 25 mice were compared to whole liver images of male B6. l29P2(C)-Mecp2tml.IBird / J mice dosed with empty LNPs. The MECP2 mRNA-LNP dosed mice had livers that were far healthier than mice dosed with empty LNPs (see Figure 37). The empty LNP dosed mice livers were pale and had signs of necrosis (see Figure 38). MECP2 mRNA-LNP dosed mice had livers that were dark read and healthy. GRH-00361 Example 26: IHC Imaging (MECP2 mRNA-LNP dosed B6.129P2(C)-Mecp2tml.IBirdl.1andBALB / c.J Mice) To see how much the MECP2 mRNA-LNP is increasing MECP2 expression in the liver and brain of the BALB / cJ and B6.l29P2(C)-Mecp2tml.IBird!J mice, immunohistochemistry (IHC)5 was performed on paraffin embedded tissue sections. The same protocol was used as in Example 14. The IHC imaging displayed higher expression levels of MECP2 in both the brain and liver of both BALB / cJ and B6.l29P2(C)-Mecp2tml.IBird!J mice, which was further confirmed usingenzyme-linked immunosorbent assay (ELISA). 10 Example 27: Biodistribution of MECP2 mRNA-LNPs in female B6.129P2(C)- Mecp2tm1.1nird / .J mice The biodistribution of MECP2 mRNA-LNPs was observed through 5 days (120 hours). 35 female B6.l29P2(C)-Mecp2tml.IBird!J mice were randomly split into 7 cages of 5 mice. These15 cages were then randomly assigned a number 1-7. The mice in each cage were randomly assigned a number 1-5. Mice 4 and 5 in cage 7 were assigned to be control mice that were left undosed so a baseline MECP2 reading could be done in the liver, gonads, brain, heart, lungs, spleen, pancreas, and kidneys of female B6.l29P2(C)-Mecp2tml.IBird!J. In addition to this, 4BALB / cJ mice were undosed to provide a healthy baseline for the organs listed above. The other20 33 female B6.l29P2(C)-Mecp2tml.IBird!J mice were dosed with 75 μL of MECP2 mRNA-LNPsonce and then groups of 1-5 mice were euthanized at timepoints of 2 hours, 4 hours, 12 hours, 24 hours, 36 hours, 48 hours, 60 hours, 72 hours, 84 hours, 96 hours, 108 hours, and 120 hours. After the mice were euthanized, the livers, gonads, brains, hearts, lungs, spleens, pancreas, and kidneys were removed. A tissue MECP2 ELISA was then performed on the organs to measure 25 the concentration of MECP2 protein in the organs listed above. See Table 40 for results of B6. l29P2(C)-Mecp2tml.IBird / J mice. Table 41 lists the results of BALB / cJ tissue ELISAs.30 GRH-00361 GRH-00361 GRH-00361 GRH-00361 Table 41 GRH-00361 This biodistribution study of MECP2 mRNA-LNPs displays MECP2 expression induced in several organs by the MECP2 mRNA-LNPs. The data indicates that MECP2 mRNA-LNP's 5 MECP2 expression occurs primarily between the 0-24 hour mark post administration. The organs which MECP2 mRNA-LNPs cause expression in appears to be the liver, lungs, heart, gonads, pancreas, and most importantly, the brain. All these organs experienced MECP2 expression above the control Female B6. l29P2(C)-Mecp2tml.IBird!J mice at varying timesbetween the 0-24 hour mark post administration. MECP2 mRNA-LNPs do not appear to cause 10 expression of MECP2 in the spleen or kidneys. MECP2 mRNA-LNPs were seen to express a large amount of MECP2 between the 0-12 hour mark before returning to normal levels following 108 hours (see figure 61). MECP2 mRNA-LNPs were seen to express a large amount of MECP2 between the 0-12 hour mark before returning to normal levels for the following 108 hours (see figure 62). MECP2 mRNA-LNPs were seen to express MECP2 in the heart between the 0-2 hour 15 mark before returning to normal levels for the following 118 hours (see figure 63). The expression in the heart only lasted for 2 hours and falls sharply after expression. This is expected as the product was administered intravenously and particles will be passed through the heart. MECP2 mRNA-LNPs were seen to not increase the expression of MECP2 in the kidneys (see figure 64). It should be noted that the kidneys already had incredibly high expression of MECP2 20 in all mouse models studied. MECP2 mRNA-LNPs were seen to increase the expression of MECP2 in the pancreas, but not to the level of the healthy BALB / cJ mice (see figure 65). GRH-00361 MECP2 expression was seen to be higher than undosed B6. l29P2(C)-Mecp2tml.IBird / J mice at all timepoints, but the healthy level of expression was never reached. MECP2 mRNA-LNPs were seen to elevate MECP2 expression in B6. l29P2(C)-Mecp2tml.IBird / J mice at the 0-2 hour timepoint and the 60-108 hour timepoint (see figure 66). The expression of MECP2 fell below 5 the average of undosed B6.l29P2(C)-Mecp2tml.IBird / J mice at all other timepoints. MECP2 mRNA-LNPs were seen to increase the expression of MECP2 in the brain at the 0-12 hour timepoint (see figure 67). This increase in expression displays the ability of MECP mRNA-LNPs to cross the blood brain barrier and express the desired protein. MECP2 mRNA-LNPs were seen to increase the expression of MECP2 in the gonads of female B6.l29P2(C)-Mecp2tml.IBird / J mice 10 at the points of 0-12 hour mark and the 48-108 hour mark (see figure 68). The expression of MECP2 returned to normal after 108 hours. Figures 61, 62, 63, 64, 65, 66, 67, and 68 show the induced expression of MECP2 compared to undosed female B6. l29P2(C)-Mecp2tml.IBird / J and female BALB / cJ mice. Example 28: Pharmacokinetic Testing MECP2 mRNA-LNPs 15 Female BALB / cJ mice were used to test the lifespan of MECP2 mRNA-LNPs in the blood stream. Female BALB / cJ mice were dosed once with 75 μL of MECP2 mRNA-LNPs. The mice were then euthanized at different timepoints ranging from 4 hours after administration to 28 days after administration. After the mice were euthanized, a cardiac puncture blood draw was 20 performed on the mice and the whole blood was separated from blood serum. The serum was then analyzed for PEG lipid by running an ELISA assay specific for PEG. After 48 hours, PEG levels were almost zero, and by 120 hours after MECP2 mRNA-LNP administration, there was no detectable PEG. See Figure 69 for results of the PEG ELISA test. Example 29: Inflammatory Testing with MECP2 mRNA-LNPs 25 Understanding the inflammatory response to MECP2 mRNA-LNPs is important when addressing any safety concerns. Inflammatory responses were measured in female B6. l29P2(C)- Mecp2tmI.IBird / J mice. These mice were split into 5 groups.1 group was left undosed to serve as a control for cytokine levels. The other 4 groups were dosed a single time with MECP2 mRNA- 30 LNPs. These groups were euthanized at 24 hours, 48 hours, and 72 hours after MECP2 mRNA- LNP administration. The groups were dosed with 2 μL, 15 μL, 30 μL, or 47 μL of MECP2 mRNA-LNPs. GRH-00361 After euthanasia, a cardiac puncture blood draw was performed, and blood serum was separated from whole blood. The serum was then sent out for cytokine testing. All results are shown in tables 42, 43, 44, and 45 (cells in bold indicate elevated cytokine levels). 5 Table 42 GRH-00361 Table 43 GRH-00361 Table 44 GRH-00361 Table 45 GRH-00361 Cytokine levels for the three different dosage volumes at each timepoint (24 hours, 48 hours, and 72 hours after MECP2 mRNA-LNP administration) were compared to the average 5immune responses from undosed female B6. l29P2(C)-Mecp2tml.IBird / J mice. At a dosage level of2 μL of MECP2 mRNA-LNP the only change in cytokine levels observed was a slight elevation of IL-17 and IP-10 seen 24 hours after administration of MECP2 mRNA-LNP. All other cytokines were within the normal range for female B6.l29P2(C)-Mecp2tml.IBird / J mice at 24hours. At 48 and 72 hours after MECP2 mRNA-LNP administration, all cytokines, including IL-10 17 and IP-10, were within the normal limits of female B6. l29P2(C)-Mecp2tml.IBird / J mice.At a dosage level of 15 μL, IL-la, IL-5, IL-17, IP-10, MCP-1, MIP-lB, and RANTES were observed at slightly elevated levels 24 hours after RTT-1 administration. At both 48 and 72 hours after MECP2 mRNA-LNP administration, IL-la, IL-5, IL-17, IP-10, MCP-1, MIP-lB, and RANTES all fell within the average range for female B6. l29P2(C)-Mecp2tml.IBird / J mice. IL-615 was observed at elevated levels at both 24 and 48 hours after MECP2 mRNA-LNP administration but decreased to within the expected range by 72 hours after MECP2 mRNA-LNP administration. At a dosage level of 30 μL, IL-la, IL-5, IL-17, MCP-1, MIP-lB, and RANTES were observed at slightly elevated levels 24 hours after MECP2 mRNA-LNP administration. At both GRH-00361 48 and 72 hours after MECP2 mRNA-LNP administration, IL-la, IL-5, IL-17, MCP-1, MIP-lB, and RANTES all dropped back to the expected levels in female B6. l29P2(C)-Mecp2tml.IBird / J mice. IP-10 was observed at elevated levels at all time points after MECP2 mRNA-LNP administration. It should be again noted that an increase in IP-10 has been seen as a positive sign 5 in Rett Syndrome as it is normally lowered in Rett Syndrome patients. At the highest dosage level tested with 47 μL of MECP2 mRNA-LNP, IL-la, IL-12 (p70), IL-17, MCP-1, and MIP- lB were observed at elevated levels 24 hours after administration of RTT-1. These cytokines decreased to within the normal range at 48 and 72 hours after administration of MECP2 mRNA-LNP. IL-6 and RANTES were observed at elevated levels at 10 both 24 and 48 hours after dose administration. At 72 hours, both IL-6 and RANTES were observed at regular levels for female B6. l29P2(C)-Mecp2tml.IBird / J mice. Both IL-5 and IP-10 were seen to be elevated at all time points studied when compared to undosed female B6. l29P2(C)-Mecp2tml.JBirdlJ mice. It should be noted that IP-10 is known to be lowered in Rett Syndrome mouse models and 15 patients and so an increase in IP-10 could be seen as positive (Gonc;alez et al.2024). It should also be noted that IL-17 is elevated during wound repair, which explains the increased levels 24 after dosing, as the wound from lateral tail vein injection is still healing (Mu et al.2024). RANTES levels has been seen to be decreased in patients with Rett Syndrome, so an increase when compared to undosed female B6. l29P2(C)-Mecp2tml.IBird / J mice could be a positive sign 20 (Gonc;alez et al.2024). The slightly elevated cytokine levels posed no threat or significant impact on quality of life as on-site veterinarians did not report any side effects or signs of immune responses. The increase in cytokines is unlikely to be due to the inclusion of SM-102. A study done by Modema found that there were no genotoxic effects of their COVID-19 vaccine, which 25 contains SM-102 (European Medicines Agency, 2021). Example 30: Cellular Viability Testing with p53 mRNA-LNPs p53 is one of the main proteins involved with apoptosis in cells. It is also often mutated to become ineffective or is inhibited in many forms of cancer. To test the effectiveness of p53 30 mRNA-LNPs on the treatment of cancers, cellular viability testing was run on both SW-1417 and SK-N-FI human neuroblastoma cells. Experiments were performed 24 and 48 hours after the initial dosing of cells with p53 mRNA-LNPs or Empty LNPs. The p53 mRNA-LNP dosed SW- GRH-00361 1417 cells saw a decrease in viability between 50% and 70% when compared to SW-1417 cells dosed with Empty LNPs. The p53 mRNA-LNP dosed SK-N-FI cells saw a decrease in viability between 11% and 21% when compared to SK-N-FI cells dosed with Empty LNPs. Both cell types also show a significant decrease in the total number of cells in each well when treated with 5 p53 mRNA-LNPs instead of Empty LNPs. All experiments were performed in 12 well plates where each well was seeded with 250,000 cells and 1 mL of complete DMEM media. They were then allowed to grow and adhere overnight in an incubator set to 37°C I 5% CO2. 24-hour cell count: After being allowed to grow and adhere overnight, 6 wells of a 12 well plate were dosed with 40 μL of p53 mRNA-LNPs. The other 6 wells were dosed with 40 μL 10 of Empty LNPs. The 12 well plate was then returned to a cell incubator (set to 37°C / 5% CO2) for 24 hours. After the 24-hour incubation, the cell viability in each well was assessed by microscopy on a hemocytometer using trypan blue staining. See Figures 70-73 for results. 48-hour (single dosed) cell count: After being allowed to grow and adhere overnight, 6 wells of a 12 well plate (seeded with SW-1417 cells) were dosed with 40 μL of p53 mRNA- 15 LNPs. The other 6 wells were dosed with 40 μL of Empty LNPs. The 12 well plate was then returned to a cell incubator (set to 37°C I 5% CO2) for 48 hours. After the 48-hour incubation, the cell viability in each well was assessed by microscopy on a hemocytometer using trypan blue staining. See Figures 74 and 75 for results. 48-hour (dosing every 24 hours) cell count: After being allowed to grow and adhere 20 overnight, 6 wells of a 12 well plate were dosed with 40 μL of p53 mRNA-LNPs. The other 6 wells were dosed with 40 μL of Empty LNPs. The 12 well plate was then returned to a cell incubator (set to 37°C / 5% CO2) for 24 hours. After this 24-hour incubation, the cells were dosed again following the same procedure as above. The 12 well plate was then returned to the incubator for another 24-hour incubation period. After the second 24-hour incubation, the cell 25 viability in each well was assessed by microscopy on a hemocytometer using trypan blue staining. See Figures 76-79 for results. INCORPORATION BY REFERENCE All publications and patents mentioned herein are hereby incorporated by reference in 30 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 GRH-00361 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 5 equivalents, and the specification, along with such variations. 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 10 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 modification and alternative arrangements within the spirit and scope as set forth in the appended claims.

Claims

GRH-00361 What is claimed is:

1. A therapeutic composition comprising a native nucleic acid formulated in a lipid nanoparticle (LNP), wherein the nucleic acid comprises an open reading frame encoding a human protein, e.g., wherein the human protein is not a human HSP70 protein, a human heat 5 shock protein, a human MECP2 protein, or a human MBD family protein.

2. The therapeutic composition of claim 1, wherein the native nucleic acid is a native human mRNA. 10 3. The therapeutic composition of claim 2, wherein the coding sequence of the native human mRNA does not comprise a synthetic and / or chemically modified nucleotide.

4. The therapeutic composition of claim 2 or 3, wherein the coding sequence of the native human mRNA has 100% sequence identity to a wildtype human mRNA sequence. 15 5. The therapeutic composition of any one of claims 2 to 4, wherein the native human mRNA does not comprise a 5' Cap analog, such as an Anti-Reverse Cap Analog (ARCA), a two headed cap, an S Cap, or a 2S Cap. 20 6. The therapeutic composition of any one of claims 2 to 5, wherein the native human mRNA comprises a Cap-0 or Cap-15' Cap structure.

7. The therapeutic composition of any one of claims 2 to 6, wherein the native human mRNA does not comprise synthetic or chemically modified adenine. 25 8. The therapeutic composition of any one of claims 2 to 7, wherein the 5' untranslated region (UTR) of the native human mRNA has 100% sequence identity to the 5' UTR of the wildtype mRNA, without addition or deletion of nucleotides.GRH-00361 9. The therapeutic composition of any one of claims 2 to 8, wherein the 3' UTR of the native human mRNA has 100% sequence identity to the 3' UTR of the wildtype mRNA, without addition or deletion of nucleotides. 5 10. The therapeutic composition of any one of claims 1 to 9, wherein the LNP comprises an ionizable lipid, a phospholipid, a sterol, a PEG-modified lipid, or any combination thereof.

11. The therapeutic composition of any one of claims 1 to 9, wherein the LNP consists essentially of an ionizable lipid, a phospholipid, a sterol, a PEG-modified lipid, or any 10 combination thereof.

12. The therapeutic composition of claims 10 or 11, wherein the ionizable lipid is SM-102. 15 13. The therapeutic composition of any one of claims 10 to 12, wherein the phospholipid is distearoylphosphatidy!choline (DSPC).

14. The therapeutic composition of any one of claims 10 to 13, wherein the sterol is cholesterol, 20 15. The therapeutic composition of any one of claims 10 to 14, wherein the PEG-modified lipid is DMG-PEG 2000.

16. A cell comprising any one of claims 1 to 15. 25 17. A cell of claim 16, expressing the human polypeptide encoded by a native human mRNA. 30 18. The cell of claim 16 or 17, wherein the cell is an endothelial cell, epithelial cell, neuronal cell, non-neuronal cell, or haematopoietic cell.

19. The cell of claim 18, wherein the haematopoietic cell is an immune cell selected from a lymphocyte, a monocyte, a dendritic cell, a mast cell, a neutrophil, a basophil, or an eosinophil.GRH-00361 20. The cell of claim 19, wherein the immune cell is lymphocyte selected from a a^T cell, y8T cell, a Natural Killer (NK) cell, a Natural Killer T (NKT) cell, a B cell, an innate lymphoid 5 cell (ILC) a cytokine induced killer (CIK) cell, a cytotoxic T lymphocyte (CTL), a lymphokine activated killer (LAK) cell, or a regulatory T cell.

21. The cell of any one of claims 16 to 20, wherein the cell is a cell derived from bone marrow. 10 22 The cell of any one of claims 16 to 20, wherein the cell is a cell of the central nervous system (CNS) or peripheral nervous system (PNS).

23. The cell of any one of claims 16 to 20, wherein the cell is a cell of the central nervous 15 system (CNS) or peripheral nervous system (PNS).

24. The cell of claim 22 or 23, wherein the cell is a neuronal cell. 20 25. The cell of claim 24, wherein the neuronal cell is a sensory neuron, a motor neuron, or an intemeuron.

26. The cell of claim 22 or 23, wherein the cell is a non-neuronal cell. 25 27. The cell of claim 26, wherein the non-neuronal cell is a glial cell.

28. The cell of claim 27, wherein the glial cell is an astrocyte cell, an oligodendrocyte cell, an 30 ependymal cell, a radial glial cell, a Schwann cell, a satellite cell, an enteric glial cell, or a microglial cell.

29. A method of preparing the therapeutic composition of anyone of claims 1 to 15, comprising encapsulating a native human mRNA in an LNP, wherein the LNP comp[rises an 35 ionizable lipid, a phospholipid, a sterol, a PEG-modified lipid, or any combination thereof.GRH-00361 30. The method of claim 29, wherein the coding sequence of the native human mRNA does not comprise a synthetic and / or chemically modified nucleotide. 5 31. The method of claim 29 or 30, wherein the coding sequence of the native human mRNA has 100% sequence identity toa wildtype human mRNA sequence.

32. The method of any one of claims 29 to 31, wherein the native human mRNA does not 10 comprise a 5' Cap analog, such as an ARCA, a two-headed cap, an S Cap, or a 2S Cap.

33. The method of any one of claims 29 to 32, wherein the native human mRNA comprises a Cap-0 or Cap-15' Cap structure. 15 34. The method of any one of claims 29 to 33, wherein the native human mRNA does not comprise synthetic or chemically modified adenine.

35. The method of any one of claims 29 to 34, wherein the 5' UTR of the native human mRNA has 100% sequence identity to the 5' UTR of the wildtype mRNA, without addition or 20 deletion of nucleotides.

36. The method of any one of claims 29 to 35, wherein the 3' UTR of the native human mRNA has 100% sequence identity to the 3' UTR of the wildtype mRNA, without addition or deletion of nucleotides. 25 37. The method of any one of claims 29 to 36, wherein the LNP consists essentially of an ionizable lipid, a phospholipid, a sterol, a PEG-modified lipid, or any combination thereof.

38. The method of any one of claims 29 to 37, wherein the ionizable lipid is SM-102. 30 39. The method of any one of claims 29 to 38, wherein the phospholipid is distearoylphosphatidy!choline (DSPC).GRH-00361 40. The method of any one of claims 29 to 39, wherein the sterol is cholesterol. 5 41. The method of any one of claims 29 to 40, wherein the PEG-modified lipid is DMG-PEG 2000.

42. The method of any one of claims 29 to 41, wherein the LNP has a molar ratio of 10 50:38.5:10:1.5 of SM-102: cholesterol: DSPC (Distearoylphosphatidylcholine): DMG-PEG 2000.