Methods for central nervous system nucleic acid delivery

Abstract

Provided herein are methods for treating a galactocerebrosidase (GALC) deficiency in a subject. Also provided herein are methods for delivering a polynucleotide to a oligodendrocytes of a subject.

Description

METHODS FOR CENTRAL NERVOUS SYSTEMNUCLEIC ACID DELIVERYCROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application Serial No. 63 / 590,321, filed on October 13, 2023, the disclosure of which is hereby incorporated by reference in its entirety.TECHNICAL FIELD

[0002] The present disclosure relates generally to methods for delivery of nucleic acids, such as RNA, to cells of the central nervous system (CNS).BACKGROUND

[0003] Many developments have recently been reported in the field of nucleic acid medicines. For instance, Nusinersen was approved by the U.S. Food and Drug Administration as the first nucleic acid therapeutic for spinal muscular atrophy (SMA) and other intractable neurological disorders. Nusinersen has been shown to significantly improve motor function and life span in SMA patients. Moreover, the development and rapidly advancing clinical application of COVID-19 vaccines have triggered great interest in mRNA-based therapy. Although mRNA and other nucleic acid therapies have a wide range of clinical applications, such as treatment of various cancers, major difficulties exist in achieving efficient nucleic acid delivery, especially into the brain, due to the obstacle presented by the blood-brain barrier (BBB). Many nucleic acid therapeutics have been reported to be capable of targeting neurons. However, nucleic acid therapeutics targeting other cell types, such as glial cells, have not been reported. In addition, cell specificity of nucleic acid delivery, including delivery of mRNA, siRNA, and antisense oligonucleotides, poses potential difficulties in clinical applications.

[0004] The inefficiency of gene transfer to oligodendrocytes is one of the primary reasons that fundamental treatments using nucleic acid therapeutics have not been established for leukodystrophies, despite several attempts. Highly efficient gene delivery into oligodendrocytes remains challenging, and methods for gene delivery into oligodendrocytes have not been applied clinically.SUMMARY

[0005] In view of the foregoing, there is exists a need for efficient methods for delivery of nucleic acid therapeutics to cells of the central nervous system, such as oligodendrocytes. The present disclosure provides methods to address this need, and provides other advantages as well.

[0006] In some aspects, provided herein are methods for treating a galactocerebrosidase (GALC) deficiency in a subject. In some embodiments, the method comprises delivering to oligodendrocytes of the subject a polynucleotide encoding a GALC, wherein the polynucleotide comprises one or more of: (a) chemically-modified nucleotides, (b) a non-naturally occurring nucleotide sequence encoding the GALC, or (c) a recombinant sequence; and further wherein the polynucleotide is effective to increase GALC activity in the oligodendrocytes.

[0007] In some aspects, provided herein are methods for delivering a polynucleotide to oligodendrocytes of a subject. In some embodiments, the method comprises administering to the subject a lipid nanoparticle (LNP) comprising the polynucleotide, wherein (i) the LNP comprises an ionizable cationic lipid; (ii) the administered polynucleotide is expressed in the oligodendrocytes of the subject; and optionally (iii) the polynucleotide encodes a GALC.

[0008] In some embodiments, the polynucleotide comprises a codon-optimized region encoding GALC as compared to SEQ ID NO: 24 or 25. In some embodiments, the polynucleotide comprises a sequence having at least 80% identity to the sequence of SEQ ID NO: 1. In some embodiments, the GALC comprises: (a) an amino acid sequence with a sequence identity of at least 80%, 85%, 90%, or 95% to SEQ ID NO:26; or (b) the amino acid sequence of SEQ ID NO: 26. In some embodiments, the GALC is a GALC polypeptide means for providing GALC activity to a cell. In some embodiments, the GALC deficiency comprises Krabbe disease.

[0009] In some embodiments, the polynucleotide comprises RNA. In some embodiments, (a) the polynucleotide comprises chemically-modified nucleotides; and (b) the chemically modified nucleotides comprise one or more of 5 -hydroxy cytidine, 5 -methylcytidine, 5- hydroxymethylcytidine, 5-carboxycytidine, 5-formylcytidine, 5 -methoxy cytidine, 5- propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5- methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'- amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5-hydroxymethyluridine, 5- carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5 -methoxyuridine, 5- propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl- pseudouridine, N^hydroxypseudouridine, N^methylpseudouridine, 2'-O-methyl-N1- methylpseudouridine, N^ethylpseudouridine, N^hydroxymethylpseudouridine, arauridine, N6- methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8-oxoadenosine,inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, or 6-O-methylguanosine. In some embodiments, the chemically modified nucleotides comprise ISf-methylpseudouridines. In some embodiments, the chemically modified nucleotides comprise 5-methoxyuridines. In some embodiments, at least 1% or at least 50% of the nucleotides in the polynucleotide are chemically- modified nucleotides.

[0010] In some embodiments, the polynucleotide further comprises a 5’ UTR. In some embodiments, the 5’ UTR comprises the sequence of any of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NOs:28-104. In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 13. In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 14.

[0011] In some embodiments, the polynucleotide further comprises a 3’ UTR. In some embodiments, the 3’ UTR comprises the sequence of any of SEQ ID NO: 17 or SEQ ID NOs: 105- 150. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 17.

[0012] In some embodiments, the polynucleotide further comprises a poly-A tail or a poly- C tail. In some embodiments, the polynucleotide comprises a poly-A tail having a length of about 60 nucleotides to about 120 nucleotides, about 90 nucleotides to about 110 nucleotides, about 95 nucleotides to about 100 nucleotides, or about 97 nucleotides.

[0013] In some embodiments, the polynucleotide further comprises a 5’ cap. In some embodiments, the 5’ cap has a Cap 1 structure, a Cap 1 (m6A) structure, a Cap 2 structure, or a Cap 0 structure.

[0014] In some embodiments, the polynucleotide is formulated with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises a lipid formulation. In some embodiments, the lipid formulation comprises a transfection reagent, a lipoplex, a liposome, a lipid nanoparticle (LNP), a polymer-based carrier, an exosome, a lamellar body, a micelle, or an emulsion. In some embodiments, the lipid formulation comprises a cationic liposome, a nanoliposome, a proteoliposome, a unilamellar liposome, a multilamellar liposome, a ceramide-containing nanoliposome, or a multivesicular liposome. In some embodiments, the lipid formulation comprises a lipid nanoparticle (LNP).

[0015] In some embodiments, the lipid formulation comprises one or more cationic lipids. In some embodiments, the one or more cationic lipids comprises one or more of 5- carboxyspermylglycinedioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), l,2-Dioleoyl-3- Dimethylammonium-Propane (DODAP), l,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1, 2-di oleyloxy -N,N- dimethyl-3 -aminopropane (DODMA), l,2-dilinoleyloxy-N,N-dimethyl-3 -aminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethyl-3 -aminopropane (DLenDMA), N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc- tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy l-l-(cis,cis-9',l-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- di oleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3 -dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'- Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (DLinCDAP), 2, 2-dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), or 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-K-XTC2- DMA).

[0016] In some embodiments, the one or more cationic lipids comprises an ionizable cationic lipid. In some embodiments, the ionizable cationic lipid has a structure of formula I:or a pharmaceutically acceptable salt or solvate thereof, wherein R5and R6are each independently selected from the group consisting of a linear or branched C1-C31 alkyl, C2-C31 alkenyl or C2-C31 alkynyl and cholesteryl; L5and L6are each independently selected from the group consisting of a linear C1-C20 alkyl and C2-C20 alkenyl; X5is -C(O)O-, whereby -C(O)O-R6is formed or -OC(O)- whereby -OC(O)-R6is formed; X6is -C(O)O- whereby -C(O)O-R5is formed or -OC(O)- whereby -OC(O)-R5is formed; X7is S or O; L7is absent or lower alkyl; R4is a linear or branched Ci-Ce alkyl; and R7and R8are each independently selected from the group consisting of a hydrogen and a linear or branched Ci-Ce alkyl. In some embodiments, the ionizable cationic lipid is selected from Table 1. In some embodiments, the ionizable cationic lipid is:

[0017] In some embodiments, the lipid formulation comprises an anionic lipid, a zwitterionic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, a phospholipid, a glycolipid, or a combination thereof. In some embodiments, the lipid formulation comprises a helper lipid. In some embodiments, the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidyl choline (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC). In some embodiments, the helper lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the lipid formulation comprises cholesterol. In some embodiments, the lipid formulation comprises a polyethylene glycol (PEG)- lipid conjugate. In some embodiments, the PEG-lipid conjugate is a PEG-DMG, optionally wherein the PEG-DMG is PEG2000-DMG. In some embodiments, the lipid formulation comprises about 40 mol% to about 60 mol% of the ionizable cationic lipid, about 4 mol% to about 16 mol% DSPC, about 30 mol% to about 47 mol% cholesterol, and about 0.5 mol% to about 3 mol% PEG2000-DMG.

[0018] In some embodiments, the lipids of the lipid formulation and the polynucleotides are in a total lipidmucleic acid molecule weight ratio of about 50: 1 to about 10: 1. In some embodiments, the polynucleotide is encapsulated within the lipid formulation or lipid nanoparticle (LNP). In some embodiments, the polynucleotide is complexed to the lipid formulation or lipid nanoparticle (LNP). In some embodiments, the lipid nanoparticle has a size of less than about 200 nm, less than about 150 nm, less than about 100 nm, or about 55 nm to about 90 nm.BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIGS. 1A-1D show efficient and specific lipid nanoparticle (LNP)-mediated transfer of EGFP mRNA into oligodendrocytes. Panels (a)-(c) of FIG. 1A show distribution patterns of EGFP, which was transfected using an illustrative LNP formulation (also referred to herein as “LUNAR ) in mice. Small round cells showing high EGFP expression (brighter areas) in the white matter of the striatum and corpus callosum in injected mice. Panels (d)-(h) of FIG. IB show EGFP-expressing cells colocalized with NeuN, Olig2, GSTK, Ibal, and GFAP. Panel (i) of FIG. ID shows percentage of EGFP-positive cells colocalized with Olig2, NeuN, Ibal, and GFAP. Data are represented as mean± SEM (n = 3). Panels (j)-(m) of FIG. 1C show the expression of EGFP (brighter areas) with LUNAR-delivery in rat primary culture, oligodendrocytes (OLGs), oligodendrocyte precursor cells (OPCs), neurons, and astrocytes after 0 h, 8 h, and 24 h. Panel (n) of FIG. ID shows the percentage of EGFP-positive cells in rat primary cultures after 24 h. Data are represented as mean ± SEM (n = 3). Tukey’s test. ***p < 0.001

[0020] FIGS. 2A-2C show LDLR-mediated delivery of LNP -EGFP mRNA into cells. Panels (a)-(l) show double immunofluorescence staining of (1) Olig2 and (2) LDLR, VLDLR, or ApoER2 in the mouse brain. Panel (m) of FIG. 2B shows representative images of differentiated MO3.13 cells expressing EGFP delivered with LUNAR showing EGFP expression suppressed by LDLR knockdown (KD). LUNAR: LNP -EGFP mRNA, NC: negative control siRNA. Panel (n) of FIG. 2C shows expression of LDLR, VLDLR, and ApoER2 mRNA significantly reduced by LDLR, VLDLR, and ApoER2 siRNA in MO3.13 cells. Data are represented as mean ± SEM (n = 6). t-test. **p < 0.01 and ***p < 0.001. Panel (o) of FIG. 2C shows LUNAR uptake significantly reduced by LDLR knockdown. Data are represented as mean ± SEM (n = 4). Tukey’s test. **p < 0.01, ***p < 0.001, and ****p < 0.0001.

[0021] FIGS. 3A-3D show the effect of GALC expression induced by LNP-mediated mRNA delivery on the phenotype of twitcher mice. Panels (a)-(e) of FIG. 3A show immunohistochemistry of GALC in mouse brain injected with LUNAR-GAZG mRNA. The arrow indicates the injection site. Panels (f) and (g) of FIG. 3B show representative images of Olig2-positive cells for control and injected hemispheres at P35. Panel (h) of FIG. 3B shows percent area of Olig2-positive cells for control and injected hemispheres. Data are represented as mean ± SEM (n = 4). Panel (i) of FIG. 3C shows the severity score and body weight of twitcher mice at P30 and P35. Control (non-injected) (n = 13) andtreated mice (n = 9). Data are represented as mean ± SEM. Panel (j) of FIG. 3C shows Kaplan-Meier survival curves in twitcher mice with or withoutinjections. Survival of untreated (noninjected) (n = 13) andThe overall difference in the survival among non-injected and mice was highly significant(p=0.0003 by the log-rank test). Panel (k) of FIG. 3D shows analysis of GalCer (dl8: l-C18:0), GalCer (dl8: 1-C24: 1), and GalSph of WT mice (n = 7), non-treated twitcher mice (n = 7), and treated twitcher mice (n = 6). Data are represented as mean ± SEM. Tukey’s test. *p < 0.05, ***p < 0.001, and ****p < 0.0001.

[0022] FIG. 4 shows a time course of EGFP expression by LUNAR-delivered EGFP mRNA in mice. Representative images of EGFP mRNA expression 8 hours to 14 days after LNP- mediated delivery are shown.

[0023] FIG. 5 shows LDLR, VLDLR, and ApoER2 mRNA expression in neurons, oligodendrocyte precursor cells (OPCs), oligodendrocytes, microglia, and astrocytes. Significantly increased LDLR and VLDLR mRNA levels were seen in oligodendrocytes compared to those in neurons, OPCs, microglia, and astrocytes. Data are represented as mean ± SEM (n = 4). Tukey’s test. *p < 0.05, **p < 0.01.

[0024] FIG. 6 shows LNP uptake in the presence of FBS or ApoE. LUNAR LNP uptake with and without FBS / ApoE is depicted. EGFP expression was significantly increased in the presence of FBS or ApoE. Data are represented as mean ± SEM (n = 4). Dunnett’s test. **p < 0.01, ****p < 0.0001.DETAILED DESCRIPTION

[0025] The present disclosure provides methods for delivery of polynucleotides such as RNA to cells of the central nervous system (CNS), such as oligodendrocytes. In some aspects, the present disclosure provides methods of treating a galactocerebrosidase (GALC) deficiency in a subject. In some aspects, the present disclosure provides methods of delivering a polynucleotide to oligodendrocytes of a subject..Definitions

[0026] As used herein, the term “nucleic acid” refers to any deoxyribonucleic acid (DNA) molecule, ribonucleic acid (RNA) molecule, or nucleic acid analogues. A DNA or RNA molecule can be double-stranded or single- stranded and can be of any size. Exemplary nucleic acids include, but are not limited to, chromosomal DNA, plasmid DNA, cDNA, cell-free DNA (cfDNA), mitochondrial DNA, chloroplast DNA, viral DNA, mRNA, tRNA, rRNA, long non-coding RNA, siRNA, micro RNA (miRNA or miR), hnRNA, and viral RNA. Exemplary nucleic analogues include peptide nucleic acid, morpholino- and locked nucleic acid, glycol nucleic acid, and threose nucleic acid. As used herein, the term “nucleic acid molecule” is meant to include fragments of nucleic acid molecules as well as any full-length or non-fragmented nucleic acid molecule, for example. As used herein, the terms “nucleic acid” and “nucleic acid molecule” can be used interchangeably, unless context clearly indicates otherwise.

[0027] As used herein, the term “polynucleotide” refers to a nucleic acid sequence that includes at least two nucleotide monomers. The term “polynucleotide” can refer to polymers of DNA, RNA, nucleic acid analogues, or combinations of these. A “polynucleotide” can be doublestranded or single-stranded and can be of any size. A polynucleotide can be a separate nucleic acid molecule or be a part of a nucleic acid molecule. Accordingly, the term “polynucleotide” can refer to a nucleic acid molecule or to a region of a nucleic acid molecule.

[0028] As used herein, the term “protein” refers to any polymeric chain of amino acids. The terms “peptide” and “polypeptide” can be used interchangeably with the term protein, unless context clearly indicates otherwise, and can also refer to a polymeric chain of amino acids. The term “protein” encompasses native or artificial proteins, protein fragments and polypeptide analogs of a protein sequence. A protein may be monomeric or polymeric. The term “protein” encompasses fragments and variants (including fragments of variants) thereof, unless otherwise contradicted by context.

[0029] In general, “sequence identity” or “sequence homology,” which can be used interchangeably, refer to an exact nucleotide-to-nucleotide or amino acid-to-amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Typically, techniques for determining sequence identity include determining the nucleotide sequence of a polynucleotide and / or determining the amino acid sequence encoded thereby or the amino acid sequence of a polypeptide, and comparing these sequences to a second nucleotide or amino acid sequence. As used herein, the term “percent (%) sequence identity” or “percent (%) identity,” also including “percent homology,” refers to the percentage of amino acid residues or nucleotides in a sequence that are identical with the amino acid residues or nucleotides in a reference sequenceafter aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity, and not considering any conservative substitutions as part of the sequence identity. Thus, two or more sequences (polynucleotide or amino acid) can be compared by determining their “percent identity,” also referred to as “percent homology.” The percent identity to a reference sequence (e.g., nucleic acid or amino acid sequences), which may be a sequence within a longer molecule (e.g., polynucleotide or polypeptide), may be calculated as the number of exact matches between two optimally aligned sequences divided by the length of the reference sequence and multiplied by 100. Percent identity may also be determined, for example, by comparing sequence information using the advanced BLAST computer program, including version 2.2.9, available from the National Institutes of Health. The BLAST program is based on the alignment method of Karlin and Altschul, Proc. Natl. Acad. Sci. USA 87:2264-2268 (1990) and as discussed in Altschul et al., J. Mol. Biol. 215:403-410 (1990); Karlin and Altschul, Proc. Natl. Acad. sci. USA 90:5873-5877 (1993); and Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997). Briefly, the BLAST program defines identity as the number of identical aligned symbols (i.e., nucleotides or amino acids), divided by the total number of symbols in the shorter of the two sequences. The program may be used to determine percent identity over the entire length of the sequences being compared. Default parameters are provided to optimize searches with short query sequences, for example, with the blastp program. The program also allows use of an SEG filter to mask-off segments of the query sequences as determined by the SEG program of Wootton and Federhen, Computers and Chemistry 17: 149-163 (1993). Ranges of desired degrees of sequence identity are approximately 80% to 100% and integer values in between. Percent identities between a reference sequence and a claimed sequence can be at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, or at least 99.9%. In general, an exact match indicates 100% identity over the length of the reference sequence. Additional programs and methods for comparing sequences and / or assessing sequence identity include the Needleman- Wunsch algorithm (see, e.g., the EMBOSS Needle aligner available at ebi.ac.uk / Tools / psa / emboss needle / , optionally with default settings), the Smith-Waterman algorithm (see, e.g., the EMBOSS Water aligner available at ebi.ac.uk / Tools / psa / emboss water / , optionally with default settings), the similarity search method of Pearson and Lipman, 1988, Proc. Natl. Acad. Sci. USA 85, 2444, or computer programs which use these algorithms (GAP, BESTFIT, FASTA, BLAST P, BLAST N and TFASTA in Wisconsin Genetics Software Package, Genetics Computer Group. 575 Science Drive, Madison, Wis.). In some embodiments, reference to percent sequence identity refers to sequence identity as measured using BLAST (Basic Local Alignment Search Tool). In some embodiments, ClustalW is used for multiple sequencealignment. Optimal alignment may be assessed using any suitable parameters of a chosen algorithm, including default parameters.

[0030] As used herein, the term “drug” or “medicament,” means a pharmaceutical formulation or composition as described herein.

[0031] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to “the method” includes one or more methods, and / or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0032] “About” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of +20%, or ±10%, or ±5%, or even ±1% from the specified value, as such variations are appropriate for the disclosed methods or to perform the disclosed methods.

[0033] The term “expression” refers to the process by which a nucleic acid sequence or a polynucleotide is transcribed from a DNA template (such as into mRNA or other RNA transcript) and / or the process by which a transcribed mRNA or other RNA is subsequently translated into peptides, polypeptides, or proteins. Transcripts and encoded polypeptides may be collectively referred to as “gene product.”

[0034] As used herein, “operably linked,” “operable linkage,” “operatively linked,” or grammatical equivalents thereof refer to juxtaposition of genetic elements, e.g., a promoter, an enhancer, a polyadenylation sequence, etc., wherein the elements are in a relationship permitting them to operate in the expected manner. For instance, a regulatory element, which can comprise promoter and / or enhancer sequences, is operatively linked to a coding region if the regulatory element helps initiate transcription of the coding sequence. There may be intervening residues between the regulatory element and coding region so long as this functional relationship is maintained.Methods for Delivery Polynucleotides

[0035] In some aspects, provided herein are methods for treating a galactocerebrosidase (GALC) deficiency in a subject. In some embodiments, the method comprises delivering to oligodendrocytes of the subject a polynucleotide encoding a GALC, wherein the polynucleotide comprises one or more of: (a) chemically-modified nucleotides, (b) a non-naturally occurring nucleotide sequence encoding the GALC, or (c) a recombinant sequence; and further wherein the polynucleotide is effective to increase GALC activity in the oligodendrocytes.

[0036] In some aspects, provided herein are methods for delivering a polynucleotide to oligodendrocytes of a subject. In some embodiments, the method comprises administering to the subject a lipid nanoparticle (LNP) comprising the polynucleotide, wherein (i) the LNP comprises an ionizable cationic lipid; (ii) the administered polynucleotide is expressed in the oligodendrocytes of the subject; and optionally (iii) the polynucleotide encodes a GALC.Polynucleotide Molecules

[0037] In some embodiments, polynucleotides according to methods provided herein encode a galactocerebrosidase (GALC). In some embodiments, the polynucleotide comprises one or more of: (a) chemically-modified nucleotides, (b) a non-naturally occurring nucleotide sequence encoding the GALC, or (c) a recombinant sequence. In some embodiments, the polynucleotide comprises a codon-optimized region encoding GALC as compared to SEQ ID NO: 24 or 25.Galactocerebrosidase (GALC)

[0038] The acid hydrolase Galactocerebrosidase (GALC) also known as beta- galactocerebrosidase, beta-Galactosidase, beta-Galactosylceramidase, Galactosylceramidase, Galactocerebroside Beta-Galactosidase, Galactosylceramide, and Beta-Galactosidase, is an enzyme that in humans is encoded by the GALC gene and is a lysosomal protein which hydrolyzes the galactose ester bonds of galactosylceramide, galactosylsphingosine, lactosylceramide, and monogalactosyldiglyceride. Functional deficiency of GALC is toxic to myelin-producing cells, which leads to progressive demyelination in both the central and peripheral nervous systems. It is hypothesized that accumulation of psychosine, which can only be degraded by GALC, is a primary initiator of pathologic cascades.

[0039] In some embodiments, the polynucleotide comprises or consists of RNA. In some embodiments, the polynucleotide comprises or consists of DNA. In some embodiments, the polynucleotide comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, or 100% identity to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a sequence having at least 80% identity to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a sequence having at least 90% identity to the nucleotide sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises a sequence having at least 95% identity to thenucleotide sequence of SEQ ID NO: 1. In some embodiments, the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 1.

[0040] In some embodiments, the GALC comprises an amino acid sequence with a sequence identity of at least 80%, 85%, 90%, or 95% to SEQ ID NO:26. In some embodiments, the GALC comprises an amino acid sequence with a sequence identity of at least 90% to SEQ ID NO:26. In some embodiments, the GALC comprises an amino acid sequence with a sequence identity of at least 95% to SEQ ID NO:26. In some embodiments, the GALC comprises the amino acid sequence of SEQ ID NO: 26. In some embodiments, the polynucleotide disclosed herein encodes GALC that is a GALC polypeptide means for providing GALC activity to a cell.Codon Optimization

[0041] In some embodiments, polynucleotides according to methods provided herein comprise codon-optimized sequences. For example, the polynucleotide may comprise a codon- optimized region encoding GALC, which is codon optimized as compared to SEQ ID NO: 24 or 25. As a result, protein-coding sequences provided herein may be substantially varied and still encode the same protein. As used herein, the term “codon-optimized” means a polynucleotide, nucleic acid sequence, or coding sequence has been redesigned as compared to a wild-type or reference polynucleotide, nucleic acid sequence, or coding sequence by choosing different codons without altering the amino acid sequence of the encoded protein. Accordingly, codon-optimization generally refers to replacement of codons with synonymous codons to optimize expression of a protein while keeping the amino acid sequence of the translated protein the same. Codon optimization of a sequence can increase protein expression levels (Gustafsson et al., Codon bias and heterologous protein expression. 2004, Trends Biotechnol 22: 346-53) of the encoded proteins, for example, and provide other advantages. Variables such as codon usage preference as measured by codon adaptation index (CAI), for example, the presence or frequency of U and other nucleotides, mRNA secondary structures, cis-regulatory sequences, GC content, and other variables may correlate with protein expression levels (Villalobos et al., Gene Designer: a synthetic biology tool for constructing artificial DNA segments. 2006, BMC Bioinformatics 7:285). Polynucleotides can be codon-optimized before modifying miRNA binding sites. miRNA binding sites can be modified to replace one or more codons with synonymous codons.

[0042] Any of a variety of methods of codon optimization can be used to codon optimize polynucleotides and nucleic acid molecules provided herein, and any of a variety of variables can be altered by codon optimization. Accordingly, a variety of codon optimization methods can be used. Exemplary methods include the high codon adaptation index (CAI) method, the Low U method, and others. The CAI method chooses a most frequently used synonymous codon for anentire protein coding sequence. As an example, the most frequently used codon for each amino acid can be deduced from 74,218 protein-coding genes from a human genome. The Low U method targets U-containing codons that can be replaced with a synonymous codon with fewer U moi eties, generally without changing other codons. If there is more than one choice for replacement, the more frequently used codon can be selected. Any polynucleotide, nucleic acid sequence, or codon sequence provided herein can be codon-optimized.

[0043] In some embodiments, the nucleotide sequence of any region of the RNA or DNA templates described herein may be codon optimized, including, for example, nucleotide sequences encoding GALC. Preferably, the primary cDNA template may include reducing the occurrence or frequency of appearance of certain nucleotides in the template strand. For example, the occurrence of a nucleotide in a template may be reduced to a level below 25% of said nucleotides in the template. In further examples, the occurrence of a nucleotide in a template may be reduced to a level below 20% of said nucleotides in the template. In some examples, the occurrence of a nucleotide in a template may be reduced to a level below 16% of said nucleotides in the template. Preferably, the occurrence of a nucleotide in a template may be reduced to a level below 15%, and preferably may be reduced to a level below 12% of said nucleotides in the template.

[0044] In some embodiments, the nucleotide reduced is uridine. For example, the present disclosure provides nucleic acids with altered uracil content wherein at least one codon in the wild-type sequence has been replaced with an alternative codon to generate a uracil-altered sequence. Altered uracil sequences can have at least one of the following properties:(i) an increase or decrease in global uracil content (i.e., the percentage of uracil of the total nucleotide content in the nucleic acid of a section of the nucleic acid, e.g., the open reading frame);(ii) an increase or decrease in local uracil content (i.e., changes in uracil content are limited to specific subsequences);(iii) a change in uracil distribution without a change in the global uracil content;(iv) a change in uracil clustering (e.g., number of clusters, location of clusters, or distance between clusters); or(v) combinations thereof.

[0045] In some embodiments, the percentage of uracil nucleobases in the nucleic acid sequence is reduced with respect to the percentage of uracil nucleobases in the wild-type nucleic acid sequence. For example, 30% of nucleobases may be uracil in the wild-type sequence but thenucleobases that are uracil are preferably lower than 15%, preferably lower than 12% and preferably lower than 10% of the nucleobases in the nucleic acid sequences of the disclosure. The percentage uracil content can be determined by dividing the number of uracil in a sequence by the total number of nucleotides and multiplying by 100.

[0046] In some embodiments, the percentage of uracil nucleobases in a subsequence of the nucleic acid sequence is reduced with respect to the percentage of uracil nucleobases in the corresponding subsequence of the wild-type sequence. For example, the wild-type sequence may have a 5'-end region (e.g., 30 codons) with a local uracil content of 30%, and the uracil content in that same region could be reduced to preferably 15% or lower, preferably 12% or lower and preferably 10% or lower in the nucleic acid sequences of the disclosure. These subsequences can also be part of the wild-type sequences of the heterologous 5’ and 3 ’ UTR sequences of the present disclosure.

[0047] In some embodiments, codons in the nucleic acid sequence of the disclosure reduce or modify, for example, the number, size, location, or distribution of uracil clusters that could have deleterious effects on protein translation. Although lower uracil content is desirable in certain embodiments, the uracil content, and in particular the local uracil content, of some subsequences of the wild-type sequence can be greater than the wild-type sequence and still maintain beneficial features (e.g., increased expression).

[0048] In some embodiments, the uracil content of polynucleotides disclosed herein is less than about 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% of the total nucleobases in the sequence in the reference sequence. In some embodiments, the uracil content of polynucleotides disclosed herein is between about 5% and about 25%. In some embodiments, the uracil content of polynucleotides disclosed herein is between about 15% and about 25%.

[0049] In some embodiments, the nucleotide that is increased or decreased is a nucleotide other than or in addition to uracil. Sequences with altered nucleotide content can have (i) an increase or decrease in local C content (i.e., changes in cytosine content are limited to specific subsequences); (ii) an increase or decrease in local G content (i.e., changes in guanosine content are limited to specific subsequences); or (iii) a combination thereof.Natural and Modified Nucleotides

[0050] A polynucleotide according to methods of the present disclosure can comprise one or more chemically modified nucleotides. Examples of nucleic acid monomers include nonnatural, modified, and chemically-modified nucleotides, including any such nucleotides known in the art. Nucleotides can be artificially modified at either the base portion or the sugar portion. In nature, most polynucleotides comprise nucleotides that are “unmodified” or “natural” nucleotides, which include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). These bases are typically fixed to a ribose or deoxy ribose at the 1’ position. The use of RNA polynucleotides comprising chemically modified nucleotides have been shown to improve RNA expression, expression rates, half-life and / or expressed protein concentrations. RNA polynucleotides comprising chemically modified nucleotides have also been useful in optimizing protein localization thereby avoiding deleterious bio-responses such as immune responses and / or degradation pathways.

[0051] Examples of modified or chemically-modified nucleotides include 5- hydroxycytidines, 5-alkylcytidines, 5-hydroxyalkylcytidines, 5-carboxycytidines, 5- formylcytidines, 5-alkoxycytidines, 5-alkynylcytidines, 5-halocytidines, 2-thiocytidines, N4- alkylcytidines, N4-aminocytidines, N4-acetylcytidines, and N4,N4-dialkylcytidines.

[0052] Examples of modified or chemically-modified nucleotides include 5- hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5- formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 5-bromocytidine, 5-iodocytidine, 2- thiocytidine; N4-methylcytidine, N4-aminocytidine, N4-acetylcytidine, and N4,N4- dimethylcytidine.

[0053] Examples of modified or chemically-modified nucleotides include 5- hydroxyuridines, 5-alkyluridines, 5-hydroxyalkyluridines, 5-carboxyuridines, 5- carboxyalkylesteruridines, 5-formyluridines, 5-alkoxyuridines, 5-alkynyluridines, 5-halouridines, 2-thiouridines, and 6-alkyluridines.

[0054] Examples of modified or chemically-modified nucleotides include 5- hydroxyuridine, 5-methyluridine, 5-hydroxymethyluridine, 5-carboxyuridine, 5- carboxymethylesteruridine, 5-formyluridine, 5 -methoxyuridine (also referred to herein as “5MeOU”), 5-propynyluridine, 5-bromouridine, 5-fluorouridine, 5-iodouridine, 2-thiouridine, and 6-methyluridine.

[0055] Examples of modified or chemically-modified nucleotides include 5- methoxycarbonylmethyl-2-thiouridine, 5-methylaminomethyl-2-thiouridine, 5-carbamoylmethyluridine, 5-carbamoylmethyl-2’-O-methyluridine, l-methyl-3-(3-amino-3- carboxypropy)pseudouridine, 5-methylaminomethyl-2-selenouridine, 5-carboxymethyluridine, 5- methyldihydrouridine, 5-taurinomethyluridine, 5-taurinomethyl-2-thiouridine, 5- (isopentenylaminomethyl)uridine, 2’-O-methylpseudouridine, 2-thio-2’O-methyluridine, and 3,2’-O-dimethyluridine.

[0056] Examples of modified or chemically-modified nucleotides include N6- methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 8-azaadenosine, 7-deazaadenosine, 8- oxoadenosine, 8-bromoadenosine, 2-methylthio-N6-methyladenosine, N6-isopentenyladenosine, 2-methylthio-N6-isopentenyladenosine, N6-(cis-hydroxyisopentenyl)adenosine, 2-methylthio- N6-(cis-hydroxyisopentenyl)adenosine, N6-glycinylcarbamoyladenosine, N6- threonylcarbamoyl-adenosine, N6-methyl-N6-threonylcarbamoyl-adenosine, 2-methylthio-N6- threonylcarbamoyl-adenosine, N6,N6-dimethyladenosine, N6- hydroxynorvalylcarbamoyladenosine, 2-methylthio-N6-hydroxynorvalylcarbamoyl-adenosine, N6-acetyl-adenosine, 7-methyl-adenine, 2-methylthio-adenine, 2-methoxy-adenine, alpha-thio- adenosine, 2'-O-methyl-adenosine, N6,2'-O-dimethyl-adenosine, N6,N6,2'-O-trimethyl- adenosine, l,2'-O-dimethyl-adenosine, 2'-O-ribosyladenosine, 2-amino-N6-methyl-purine, 1- thio-adenosine, 2'-F-ara-adenosine, 2'-F-adenosine, 2'-OH-ara-adenosine, and N6-(19-amino- pentaoxanonadecyl)-adenosine.

[0057] Examples of modified or chemically-modified nucleotides include Nl- alkylguanosines, N2-alkylguanosines, thienoguanosines, 7-deazaguanosines, 8-oxoguanosines, 8- bromoguanosines, O6-alkylguanosines, xanthosines, inosines, and Nl-alkylinosines.

[0058] Examples of modified or chemically-modified nucleotides include Nl- methylguanosine, N2-methylguanosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, 8- bromoguanosine, O6-methylguanosine, xanthosine, inosine, and Nl-methylinosine.

[0059] Examples of modified or chemically-modified nucleotides include pseudouridines. Examples of pseudouridines include Nl-alkylpseudouri dines, Nl-cycloalkylpseudouri dines, Nl- hydroxypseudouri dines, Nl-hydroxyalkylpseudouri dines, Nl-phenylpseudouri dines, Nl- phenylalkylpseudouri dines, Nl-aminoalkylpseudouri dines, N3 -alkylpseudouridines, N6- alkylpseudouridines, N6-alkoxypseudouridines, N6-hydroxypseudouridines, N6- hydroxyalkylpseudouridines, N6-morpholinopseudouridines, N6-phenylpseudouridines, and N6- halopseudouridines. Examples of pseudouridines include Nl-alkyl-N6-alkylpseudouridines, Nl- alkyl-N6-alkoxypseudouridines, Nl-alkyl-N6-hydroxypseudouridines, Nl-alkyl-N6- hydroxyalkylpseudouridines, Nl-alkyl-N6-morpholinopseudouridines, Nl-alkyl-N6- phenylpseudouridines, and Nl-alkyl-N6-halopseudouridines. In these examples, the alkyl,cycloalkyl, and phenyl substituents may be unsubstituted, or further substituted with alkyl, halo, haloalkyl, amino, or nitro substituents.

[0060] Examples of pseudouridines include Nl-methylpseudouridine (also referred to herein as “N1MPU”), Nl-ethylpseudouridine, Nl-propylpseudouridine, Nl- cyclopropylpseudouridine, Nl-phenylpseudouridine, Nl-aminom ethylpseudouridine, N3- m ethylpseudouridine, N1 -hydroxypseudouridine, and N1 -hydroxymethylpseudouridine.

[0061] Examples of nucleic acid monomers include modified and chemically-modified nucleotides, including any such nucleotides known in the art.

[0062] Examples of modified and chemically-modified nucleotide monomers include any such nucleotides known in the art, for example, 2'-O-methyl ribonucleotides, 2'-O-methyl purine nucleotides, 2'-deoxy-2'-fluoro ribonucleotides, 2'-deoxy-2'-fluoro pyrimidine nucleotides, 2'- deoxy ribonucleotides, 2'-deoxy purine nucleotides, universal base nucleotides, 5-C-methyl- nucleotides, and inverted deoxyabasic monomer residues.

[0063] Examples of modified and chemically-modified nucleotide monomers include 3'- end stabilized nucleotides, 3 '-glyceryl nucleotides, 3 '-inverted abasic nucleotides, and 3 '-inverted thymidine.

[0064] Examples of modified and chemically-modified nucleotide monomers include locked nucleic acid nucleotides (LNA), 2'-O,4'-C-methylene-(D-ribofuranosyl) nucleotides, 2'- methoxyethoxy (MOE) nucleotides, 2'-methyl-thio-ethyl, 2'-deoxy-2'-fluoro nucleotides, and 2'- O-methyl nucleotides. In an exemplary embodiment, the modified monomer is a locked nucleic acid nucleotide (LNA).

[0065] Examples of modified and chemically-modified nucleotide monomers include 2',4'-constrained 2'-O-methoxyethyl (cMOE) and 2'-O-Ethyl (cEt) modified DNAs.

[0066] Examples of modified and chemically-modified nucleotide monomers include 2'- amino nucleotides, 2'-O-amino nucleotides, 2'-C-allyl nucleotides, and 2'-O-allyl nucleotides.

[0067] Examples of modified and chemically-modified nucleotide monomers include N6- methyladenosine nucleotides.

[0068] Examples of modified and chemically-modified nucleotide monomers include nucleotide monomers with modified bases 5-(3-amino)propyluridine, 5-(2-mercapto)ethyluridine, 5-bromouridine; 8-bromoguanosine, or 7-deazaadenosine.

[0069] Examples of modified and chemically-modified nucleotide monomers include 2’- O-aminopropyl substituted nucleotides.

[0070] Examples of modified and chemically-modified nucleotide monomers include replacing the 2'-OH group of a nucleotide with a 2'-R, a 2'-OR, a 2'-halogen, a 2'-SR, or a 2'- amino, where R can be H, alkyl, alkenyl, or alkynyl.

[0071] Exemplary base modifications described above can be combined with additional modifications of nucleoside or nucleotide structure, including sugar modifications and linkage modifications. Certain modified or chemically-modified nucleotide monomers may be found in nature.

[0072] Preferred nucleotide modifications include Nkmethylpseudouridine and 5- methoxyuridine.

[0073] In some embodiments, the chemically modified nucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5- formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5 -hydroxyuridine, 5- methyluridine, 5,6-dihydro-5-methyluridine, 2'-O-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5- hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5- methoxyuridine, 5-propynyluridine, 5-bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, Nkhydroxypseudouridine, Nkmethylpseudouridine, 2'-O-methyl-N1-methylpseudouridine, Nkethylpseudouridine, Nkhydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8- oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, or 6-O- methylguanosine. In some embodiments, the chemically modified nucleotides comprise N1- methylpseudouridines. In some embodiments, the chemically modified nucleotides comprise 5- methoxyuridines.

[0074] In some embodiments, at least 1% of the nucleotides (e.g., at least 5%, 10%, 20%, 30%, 40%, 50%, 75% or more of the nucleotides) are chemically-modified nucleotides. In some embodiments, at least 25% of the nucleotides are chemically modified nucleotides. In some embodiments, at least 50% of the nucleotides are chemically modified nucleotides.5’ Untranslated Region (5’-UTR)

[0075] Polynucleotides according to methods provided herein can further comprise untranslated regions (UTRs). Untranslated regions, including 5’ UTRs and 3’ UTRs, for example, can affect RNA stability and / or efficiency of RNA translation, such as translation of cellular mRNAs, for example.

[0076] In some embodiments, polynucleotides according to methods provided herein further include a 5’ untranslated region (5’ UTR). Any 5’ UTR sequence can be included in nucleic acid molecules provided herein. In some embodiments, nucleic acid molecules provided herein include a viral 5’ UTR. In some embodiments, nucleic acid molecules provided herein include a non-viral 5’ UTR. Any non-viral 5’ UTR can be included in nucleic acid molecules provided herein, such as 5’ UTRs of transcripts expressed in any cell or organ, including muscle, skin, subcutaneous tissue, liver, spleen, lymph nodes, antigen-presenting cells, and others. In some embodiments, nucleic acid molecules provided herein include a 5’ UTR comprising viral and non- viral sequences. Accordingly, a 5’ UTR included in nucleic acid molecules provided herein can comprise a combination of viral and non-viral 5’ UTR sequences. In some embodiments, the 5’ UTR included in nucleic acid molecules provided herein is located upstream of or 5’ of a polynucleotide that encodes a GALC.

[0077] In some embodiments, the 5’ UTR of nucleic acid molecules provided herein comprises an alphavirus 5’ UTR. A 5’ UTR from any alphavirus can be included in nucleic acid molecules provided herein, including 5’ UTR sequences from Venezuelan Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Everglades Virus (EVEV), Mucambo Virus (MUCV), Semliki Forest Virus (SFV), Pixuna Virus (PIXV), Middleburg Virus (MIDV), Chikungunya Virus (CHIKV), O'Nyong-Nyong Virus (ONNV), Ross River Virus (RRV), Barmah Forest Virus (BFV), Getah Virus (GETV), Sagiyama Virus (SAGV), Bebaru Virus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), Sindbis Virus (SINV), Aura Virus (AURAV), Whataroa Virus (WHAV), Babanki Virus (BABV), Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus (WEEV), Highland J Virus (HJV), Fort Morgan Virus (FMV), Ndumu Virus (NDUV), Salmonid Alphavirus (SAV), or Buggy Creek Virus (BCRV). In some embodiments, the 5’ UTR comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and any number or range in between, identity to the sequence of any of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NOs:28-104, for example. In some embodiments, the 5’ UTR comprises a sequence having at least 90% identity to the sequence of any of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NOs:28-104. In some embodiments, the 5’ UTR comprises a sequence having at least 95% identity to the sequence of any of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NOs:28-104. In some embodiments, the 5’ UTR comprises the sequence of any of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NOs:28-104. In some embodiments, the 5’ UTRcomprises the sequence of SEQ ID NO: 13. In some embodiments, the 5’ UTR comprises the sequence of SEQ ID NO: 14.

[0078] In some embodiments, the 5’ UTR comprises a sequence selected from the 5’ UTRs of human IL-6, alanine aminotransferase 1, human apolipoprotein E, human fibrinogen alpha chain, human transthyretin, human haptoglobin, human alpha- 1 -anti chymotrypsin, human antithrombin, human alpha- 1 -antitrypsin, human albumin, human beta globin, human complement C3, human complement C5, SynK (thylakoid potassium channel protein derived from the cyanobacteria, Synechocystis sp.), mouse beta globin, mouse albumin, and a tobacco etch virus, or fragments of any of the foregoing. In some preferred embodiments, the 5’ UTR is derived from a tobacco etch virus (TEV).

[0079] An RNA, mRNA or any other RNA described herein can comprise any 5’ UTR sequence provided herein. For example, an RNA described herein can comprise a 5’ UTR sequence that is derived from a gene expressed by Arabidopsis thaliana. In some embodiments, the 5’ UTR sequence of a gene expressed by Arabidopsis thaliana is AT1G58420. Examples of 5’ UTRs and 3’ UTRs are described in US20190002906A1, the contents of which are herein incorporated by reference.3’ Untranslated Region (3’ UTR)

[0080] In some embodiments, polynucleotides according to methods provided herein further include a 3 ’ untranslated region (3 ’ UTR). Any 3 ’ UTR sequence can be included in nucleic acid molecules provided herein. In some embodiments, polynucleotides provided herein include a viral 3’ UTR. In some embodiments, nucleic acid molecules provided herein include a non-viral 3’ UTR. Any non-viral 3’ UTR can be included in nucleic acid molecules provided herein, such as 3’ UTRs of transcripts expressed in any cell or organ, including muscle, skin, subcutaneous tissue, liver, spleen, lymph nodes, antigen-presenting cells, and others. In some embodiments, nucleic acid molecules provided herein include a 3’ UTR comprising viral and non-viral sequences. Accordingly, a 3’ UTR included in nucleic acid molecules provided herein can comprise a combination of viral and non-viral 3’ UTR sequences. In some embodiments, the 3’ UTR is located 3’ of or downstream of a polynucleotide encoding a GALC.

[0081] In some embodiments, the 3’ UTR of polynucleotides provided herein comprises an alphavirus 3’ UTR. A 3’ UTR from any alphavirus can be included in nucleic acid molecules provided herein, including 3’ UTR sequences from Venezuelan Equine Encephalitis Virus (VEEV), Eastern Equine Encephalitis Virus (EEEV), Everglades Virus (EVEV), Mucambo Virus (MUCV), Semliki Forest Virus (SFV), Pixuna Virus (PIXV), Middleburg Virus (MIDV),Chikungunya Virus (CHIKV), O'Nyong-Nyong Virus (ONNV), Ross River Virus (RRV), Barmah Forest Virus (BFV), Getah Virus (GETV), Sagiyama Virus (SAGV), Bebaru Virus (BEBV), Mayaro Virus (MAYV), Una Virus (UNAV), Sindbis Virus (SINV), Aura Virus (AURAV), Whataroa Virus (WHAV), Babanki Virus (BABV), Kyzylagach Virus (KYZV), Western Equine Encephalitis Virus (WEEV), Highland J Virus (HJV), Fort Morgan Virus (FMV), Ndumu Virus (NDUV), Salmonid Alphavirus (SAV), or Buggy Creek Virus (BCRV).

[0082] In some embodiments, the 3’ UTR comprises a sequence having at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 97.5%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, at least 99.9%, and any number or range in between, identity to the sequence of any of SEQ ID NO: 17 or SEQ ID N0s:105-150, for example. In some embodiments, the 3’ UTR comprises a sequence having at least 90% identity to the sequence of any of SEQ ID NO: 17 or SEQ ID NOs: 105-150. In some embodiments, the 3’ UTR comprises a sequence having at least 95% identity to the sequence of any of SEQ ID NO: 17 or SEQ ID NOs: 105-150. In some embodiments, the 3’ UTR comprises the sequence of any of SEQ ID NO: 17 or SEQ ID NO: 105-150. In some embodiments, the 3’ UTR comprises the sequence of SEQ ID NO: 17. In some embodiments, the 3’ UTR comprises an XbG 3’ UTR sequence.

[0083] In some embodiments, the 3’ UTR comprises a sequence selected from the 3’ UTRs of alanine aminotransferase 1, human apolipoprotein E, human fibrinogen alpha chain, human haptoglobin, human antithrombin, human alpha globin, human beta globin, human complement C3, human growth factor, human hepcidin, MALAT-1, mouse beta globin, mouse albumin, and Xenopus beta globin, or fragments of any of the foregoing. In some embodiments, the 3’ UTR is derived from Xenopus beta globin.

[0084] In some embodiments, the 3’ UTR further comprises a poly-A tail or a poly-C tail.Polynucleotide Tail (e.g., Poly-A Tail)

[0085] Polyadenylation is the addition of a poly(A) tail, a chain of adenine nucleotides usually about 100-120 monomers in length, to a mRNA or an RNA that can function as an mRNA. In eukaryotes, polyadenylation is part of the process that produces mature mRNA for translation and begins as the transcription of a gene terminates. The 3 '-most segment of a newly made pre- mRNA is first cleaved off by a set of proteins; these proteins then synthesize the poly(A) tail at the 3' end. The poly(A) tail is important for the nuclear export, translation, and stability of natural mRNA. The tail is shortened over time, and, when it is short enough, the mRNA is enzymatically degraded. However, in a few cell types, mRNAs with short poly(A) tails are stored for lateractivation by re-polyadenylation in the cytosol. In some embodiments, the poly-A tail is transcribed from a template polynucleotide (e.g, the template encoding the RNA), or is added post-transcriptionally.

[0086] In some embodiments, a polynucleotide according to methods of the disclosure comprises a 3’ tail region, which can serve to protect the RNA from exonuclease degradation. The tail region may be a 3’poly(A) and / or 3 ’poly (C) region. Preferably, the tail region is a 3’ poly(A) tail. However, it is to be understood that embodiments referring to a poly(A) tail may be applied to poly(C) tails as well. As used herein a “3’ poly(A) tail” is a polymer of sequential adenine nucleotides that can range in size from, for example: 10 to 250 sequential adenine nucleotides; 60-125 sequential adenine nucleotides, 90-125 sequential adenine nucleotides, 95-125 sequential adenine nucleotides, 95-121 sequential adenine nucleotides, 100 to 121 sequential adenine nucleotides, 110-121 sequential adenine nucleotides; 112-121 sequential adenine nucleotides; 114-121 adenine sequential nucleotides; or 115 to 121 sequential adenine nucleotides. In some embodiments, a 3’ poly(a) tail as described herein includes about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, 240, 250, 260, 270, 280, 290, 300, and any number or range in between, sequential adenine nucleotides. Preferably, a 3’ poly(A) tail as described herein comprises 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106, 107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149, or 150 sequential adenine nucleotides. In some embodiments, the 3’ poly(A) tail as described herein comprises about 97 sequential adenine nucleotides. In some embodiments, the 3’ poly(A) tail as described herein comprises about 106 sequential adenine nucleotides. In some embodiments, the 3’ poly(A) tail as described herein comprises about 117 sequential adenine nucleotides. In some embodiments, the 3’ poly(A) tail as described herein comprises about 141 sequential adenine nucleotides. 3’ Poly(A) tails can be added using a variety of methods known in the art, e.g., using poly(A) polymerase to add tails to synthetic or in vitro transcribed RNA. Other methods include the use of a transcription vector to encode poly(A) tails or the use of a ligase (e.g., via splint ligation using a T4 RNA ligase and / or T4 DNA ligase), wherein poly(A) may be ligated to the 3' end of a sense RNA. In some embodiments, a combination of any of the above methods is utilized.

[0087] In some embodiments, the 3’ UTR comprises a poly-A tail of about 20-300 nucleotides. In some embodiments, the poly-A tail comprises about 20-300 consecutive A nucleotides. In some embodiments, the poly-A tail comprises about 10, about 20, about 30, about40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, about 160, about 170, about 180, about 190, about 200, about 210, about 220, about 230, about 240, about 250, about 260, about 270, about 280, about 290, about 300, and any number or range in between, nucleotides, for example, about 60 to 120 nucleotides, about 90 nucleotides to about 110 nucleotides, about 95 nucleotides to about 100 nucleotides, or about 97 nucleotides in length. In some embodiments, the nucleotides of the poly-A tail are A nucleotides. In some embodiments, the 3’ UTR includes a poly-C tail. A 3’ UTR comprising any sequence can have a tail region, such as a poly-A tail or a poly-C tail.Triple Stop Codon

[0088] In some embodiments, polynucleotides according to methods described herein comprise a sequence immediately downstream of a coding region (i.e., ORF) that creates a triple stop codon. A triple stop codon is a sequence of three consecutive stop codons. The triple stop codon can ensure total insulation of an expression cassette and may be incorporated to enhance the efficiency of translation. In some embodiments, RNA molecules of the disclosure may comprise a triple combination of any of the sequences UAG, UGA, or UAA immediately downstream of an ORF described herein. The triple combination can be three of the same codons, three different codons, or any other permutation of the three stop codons.Translation Enhancers and Kozak Sequences

[0089] Fortranslation initiation, proper interactions between ribosomes and mRNAs must be established to determine the exact position of the translation initiation region. However, ribosomes also must dissociate from the translation initiation region to slide toward the downstream sequence during mRNA translation. Translation enhancers upstream from initiation sequences of mRNAs enhance the yields of protein biosynthesis. Several studies have investigated the effects of translation enhancers. In some embodiments, a polynucleotide according to methods described herein, such as a polynucleotide encoding a GALC, comprises a translation enhancer sequence. These translation enhancer sequences enhance the translation efficiency of the polynucleotide and thereby provide increased production of the protein encoded by the polynucleotide. The translation enhancer region may be located in the 5’ or 3’ UTR of a protein coding sequence. Examples of translation enhancer regions include naturally-occurring enhancer regions from the TEV 5’ UTR and the Xenopus beta-globin 3’ UTR. Exemplary 5’ UTR enhancer sequences include but are not limited to those derived from mRNAs encoding human heat shock proteins (HSP) including HSP70-P2, HSP70-M1 HSP72-M2, HSP17.9 and HSP70-P1. Exemplary translation enhancer sequences used in accordance with the embodiments of the present disclosure are represented by SEQ ID NOs: 151-155.

[0090] In some embodiments, a polynucleotide according to methods of the disclosure comprises a Kozak sequence. As is understood in the art, a Kozak sequence is a short consensus sequence centered around the translational initiation site of eukaryotic mRNAs that allows for efficient initiation of translation of the self-replicating RNA or mRNA. See, for example, Kozak, Marilyn (1988) Mol. and Cell Biol, 8:2737-2744; Kozak, Marilyn (1991) J. Biol. Chem, 266: 19867-19870; Kozak, Marilyn (1990) Proc Natl. Acad. Sci. USA, 87:8301-8305; and Kozak, Marilyn (1989) J. Cell Biol, 108:229-241. It ensures that a protein is correctly translated from the genetic message, mediating ribosome assembly and translation initiation. The ribosomal translation machinery recognizes the AUG initiation codon in the context of the Kozak sequence. A Kozak sequence may be inserted upstream of the coding sequence for the protein of interest, downstream of a 5’ UTR or inserted upstream of the coding sequence for the protein of interest and downstream of a 5’ UTR. In some embodiments, the Kozak sequence has the sequence GCCACC. A polynucleotide described herein can comprise a partial Kozak sequence “p” having the nucleotide sequence GCCA.RNA Molecules - Exemplary Features

[0091] Polynucleotides according to methods provided herein can be DNA molecules or RNA molecules. It will be appreciated that T present in DNA is substituted with U in RNA, and vice versa. In some embodiments, nucleic acid molecules provided herein are RNA molecules, wherein a first polynucleotide is located 5’ of a second polynucleotide (which second polynucleotide may optionally be located 5’ of a third polynucleotide, and so on). In some embodiments, first, second, third, or more polynucleotides of DNA or RNA molecules provided herein each encode a transgene. Where more than one transgene is included in DNA or RNA molecules provided herein, transgenes may be the same or different. Transgenes included in DNA or RNA molecules provided herein can be expressed from separate promoters, internal ribosomal entry sites (IRES), sequences encoding 2A self-cleaving peptides, or a combination thereof. Any suitable promoter, IRES, or sequence encoding a 2A self-cleaving peptide can be included in polynucleotides provided herein. Exemplary 2A self-cleaving peptide sequences include P2A, T2A, E2A, and F2A peptides. Sequences presented herein as RNA sequences may be encoded by a corresponding DNA sequence, in which U is replaced with T. Similarly, sequences presented herein as DNA sequences may be converted to the corresponding RNA sequence encoded thereby by replacing T with U. In general, the RNA sequence “encoded” by a DNA sequence refers herein to an RNA with the same 5’ to 3’ orientation and order of bases (except for U replacing T), and not the reverse complement. Both the DNA and RNA versions of a given sequence are contemplated herein, unless context clearly indicates otherwise. In some cases, a DNA sequencemay include elements not found in an RNA encoded thereby (e.g., a promoter sequence). In some cases, an RNA sequence may include elements not found in a DNA construct encoding the RNA (e.g., a poly-A tail when added post-transcriptionally).

[0092] An RNA molecule according to methods provided herein can be generated by in vitro transcription (IVT) of DNA molecules provided herein. In some embodiments, RNA molecules according to methods provided herein are self-replicating RNA molecules. In some embodiments, RNA molecules according to methods provided herein are mRNA molecules. In some embodiments, RNA molecules according to methods provided herein further comprise a 5’ cap. Any 5’ cap can be included in RNA molecules provided herein, including 5’ caps having a Cap 1 structure, a Cap 1 (m6A) structure, a Cap 2 structure, or a Cap 0 structure. A population or plurality of RNA molecules provided herein can have the same 5’ cap or can have different 5’ caps. For example, a population or plurality of RNA molecules can have 5’ caps having a Cap 1 structure, a Cap 1 (m6A) structure, a Cap 2 structure, a Cap 0 structure, or any combination thereof.

[0093] In some embodiments, RNA molecules according to methods provided herein include a 5’ cap having Cap 1 structure. In some embodiments, the RNA molecules are self- replicating RNA molecules comprising a 5’ cap having a Cap 1 structure. In some embodiments, the RNA molecules comprise a cap having a Cap 1 structure, wherein a m7G is linked via a 5’ -5’ triphosphate to the 5’ end of the 5’ UTR. Any method of capping can be used, including, but not limited to using a Vaccinia Capping enzyme (New England Biolabs, Ipswich, Mass.) and co- transcriptional capping or capping at or shortly after initiation of in vitro transcription (IVT), by for example, including a capping agent as part of an in vitro transcription (IVT) reaction. (Nuc. Acids Symp. (2009) 53: 129).

[0094] Only those RNA molecules, such mRNAs and RNAs that can function as mRNAs, that carry the Cap structure are active in Cap dependent translation; “decapitation” of mRNA results in an almost complete loss of their template activity for protein synthesis (Nature, 255:33- 37, (1975); J. Biol. Chem., vol. 253:5228-5231, (1978); and Proc. Natl. Acad. Sci. USA, 72: 1189- 1193, (1975)).

[0095] Another element of eukaryotic mRNA is the presence of 2'-O-methyl nucleoside residues at transcript position 1 (Cap 1), and in some cases, at transcript positions 1 and 2 (Cap 2). The 2'-O-methylation of mRNA provides higher efficacy of mRNA translation in vivo (Proc. Natl. Acad. Sci. USA, 77:3952-3956 (1980)) and further improves nuclease stability of the 5'- capped mRNA. The mRNA with Cap 1 (and Cap 2) is a distinctive mark that allows cells torecognize the bona fide mRNA 5' end, and in some instances, to discriminate against transcripts emanating from infectious genetic elements (Nucleic Acid Research 43: 482-492 (2015)).

[0096] Some examples of 5' cap structures and methods for preparing mRNAs comprising the same are given in WO2015 / 051169A2, WO / 2015 / 061491, US 2018 / 0273576, and US Patent Nos. 8,093,367, 8,304,529, and U.S. 10,487,105. In some embodiments, the 5’ cap is m7GpppAmpG, which is known in the art. In some embodiments, the 5’ cap is m7GpppG or m7GpppGm, which are known in the art. Structural formulas for embodiments of 5’ cap structures are provided below.

[0097] In some embodiments, an RNA molecule according to methods of the disclosure comprises a 5’ cap having the structure of Formula (Cap I).wherein B1is a natural or modified nucleobase; R1and R2are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phophorothioate, and boranophosphate wherein each L is linked by diester bonds; n is 0 or 1. and mRNA represents an mRNA of the present disclosure linked at its 5’ end. In some embodiments B1is G, m7G, or A. In some embodiments, n is 0. In some embodiments n is 1. In some embodiments, B1is A or m6A and R1is OCH3; wherein G is guanine, m7G is 7-methylguanine, A is adenine, and m6A is N6-methyladenine.

[0098] In some embodiments, an RNA molecule according to methods of the disclosure comprises a 5’ cap having the structure of Formula (Cap II).wherein B1and B2are each independently a natural or modified nucleobase; R1, R2, and R3are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phophorothioate, and boranophosphate wherein each L islinked by diester bonds; mRNA represents an mRNA of the present disclosure linked at its 5’ end; and n is 0 or 1. In some embodiments B1is G, m7G, or A. In some embodiments, n is 0. In some embodiments, n is 1. In some embodiments, B1is A or m6A and R1is OCH3; wherein G is guanine, m7G is 7-methylguanine, A is adenine, and m6A is N6-methyladenine.

[0099] In some embodiments, an RNA molecule according to methods of the disclosure comprises a 5’ cap having the structure of Formula (Cap III).wherein Bl, B2, and B3 are each independently a natural or modified nucleobase; Rl, R2, R3, and R4 are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of the present disclosure linked at its 5’ end; and n is 0 or 1. In some embodiments, at least one of Rl, R2, R3, and R4 is OH. In some embodiments B 1 is G, m7G, or A. In some embodiments, Bl is A or m6A and Rl is OCH3; wherein G is guanine, m7G is 7-methylguanine, A is adenine, and m6A is N6-methyladenine. In some embodiments, n is 1.

[0100] In some embodiments, an RNA molecule according to methods of the disclosure comprises a m7GpppG 5’ cap analog having the structure of Formula (Cap IV).wherein, Rl, R2, andR3are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of thepresent disclosure linked at its 5’ end; n is 0 or 1. In some embodiments, at least one of R1, R2, and R3is OH. In some embodiments, the 5’ cap is m7GpppG wherein R1, R2, andR3are each OH, n is 1, and each L is a phosphate. In some embodiments, n is 1. In some embodiments, the 5’ cap is m7GpppGm, wherein R1and R2are each OH, R3is OCH3, each L is a phosphate, and n is 1.

[0101] In some embodiments, an RNA molecule according to methods of the disclosure comprises a m7Gpppm7G 5’ cap analog having the structure of Formula (Cap V).wherein, R1, R2, and R3are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of the present disclosure linked at its 5’ end; and n is 0 or 1. In some embodiments, at least one of R1, R2, and R3is OH. In some embodiments, n is 1.

[0102] In some embodiments, an RNA molecule according to methods of the disclosure comprises a m7Gpppm7GpN, 5’ cap analog, wherein N is a natural or modified nucleotide, the 5’ cap analog having the structure of Formula (Cap VI).wherein B3is a natural or modified nucleobase; R1, R2, R3, and R4are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of the present disclosure linked at its 5’ end; and n is 0 or 3. In someembodiments, at least one of R1, R2, R3, and R4is OH. In some embodiments B1is G, m7G, or A. In some embodiments, B1is A or m6A and R1is OCH3; wherein G is guanine, m7G is 7- methylguanine, A is adenine, and m6A is N6-methyladenine. In some embodiments, n is 1.

[0103] In some embodiments, an RNA molecule according to methods of the disclosure comprises a m7Gpppm7GpG 5’ cap analog having the structure of Formula (Cap VII).wherein, R1, R2, R3, and R4are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of the present disclosure linked at its 5’ end; and n is 0 or 1. In some embodiments, at least one of R1, R2, R3, andR4is OH. In some embodiments, n is 1.

[0104] In some embodiments, an RNA molecule according to methods of the disclosure comprises a m7Gpppm7Gpm7G 5’ cap analog having the structure of Formula (Cap VIII).wherein, R1, R2, R3, andR4are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of thepresent disclosure linked at its 5’ end; n is 0 or 1. In some embodiments, at least one of R1, R2, R3, andR4is OH. In some embodiments, n is 1.

[0105] In some embodiments, an RNA molecule according to methods of the disclosure comprises a m7GpppA 5’ cap analog having the structure of Formula (Cap IX).wherein, R1, R2, andR3are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of the present disclosure linked at its 5’ end; and n is 0 or 1. In some embodiments, at least one of R1, R2, and R3is OH. In some embodiments, n is 1.

[0106] In some embodiments, an RNA molecule according to methods of the disclosure comprises a m7GpppApN 5’ cap analog, wherein N is a natural or modified nucleotide, and the 5’ cap has the structure of Formula (Cap X).wherein B3is a natural or modified nucleobase; R1, R2, R3, and R4are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of the present disclosure linked at its 5’ end; and n is 0 or 1. In some embodiments, at least one of R1, R2, R3, andR4is OH. In some embodiments B3is G, m7G, A or m6A; wherein G is guanine, m7G is 7-methylguanine, A is adenine, and m6A is N6-methyladenine. In some embodiments, n is 1.

[0107] In some embodiments, an RNA molecule according to methods of the disclosure comprises a m7GpppAmpG 5’ cap analog having the structure of Formula (Cap XI).wherein, R1, R2, andR4are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of the present disclosure linked at its 5’ end; and n is 0 or 1. In some embodiments, at least one of R1, R2, and R4is OH. In some embodiments, the compound of Formula Cap XI is m7GpppAmpG, wherein R1, R2, and R4are each OH, n is 1, and each L is a phosphate linkage. In some embodiments, n is 1.

[0108] In some embodiments, an RNA molecule according to methods of the disclosure comprises a m7GpppApm7G 5’ cap analog having the structure of Formula (Cap XII).wherein, R1, R2, R3, andR4are each independently selected from a halogen, OH, and OCH3; eachL is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of the present disclosure linked at its 5’ end; and n is 0 orl. In some embodiments, at least one of R1, R2, R3, andR4is OH. In some embodiments, n is 1.

[0109] In some embodiments, an RNA molecule according to methods of the disclosure comprises a m7GpppApm7G 5’ cap analog having the structure of Formula (Cap XIII).wherein, R1, R2, and R4are each independently selected from a halogen, OH, and OCH3; each L is independently selected from the group consisting of phosphate, phosphorothioate, and boranophosphate wherein each L is linked by diester bonds; mRNA represents an mRNA of the present disclosure linked at its 5’ end; and n is 0 or 1. In some embodiments, at least one of R1, R2, andR4is OH. In some embodiments, n is 1.DNA Molecules

[0110] In some embodiments, polynucleotides according to methods provided herein are DNA molecules, such as DNA encoding an RNA molecule according to methods disclosed herein. In some embodiments, DNA molecules according to methods provided herein further comprise a promoter. As used herein, the term “promoter” refers to a regulatory sequence that initiates transcription. A promoter can be operably linked to one or more polynucleotides of DNA molecules provided herein, with the one or more polynucleotides of DNA molecules encoding one or more polynucleotides of RNA molecules provided herein. Generally, promoters included in DNA molecules provided herein include promoters for in vitro transcription (IVT). Any suitable promoter for in vitro transcription can be included in DNA molecules provided herein, such as a T7 promoter, a T3 promoter, an SP6 promoter, and others. In some embodiments, DNA molecules provided herein comprise a T7 promoter. In some embodiments, the promoter is located 5’ of the 5’ UTR included in DNA molecules provided herein. In some embodiments, the promoter is a T7 promoter located 5’ of the 5’ UTR included in DNA molecules provided herein. In some embodiments, the promoter overlaps with the 5’ UTR. A promoter and a 5’ UTR can overlap by about one nucleotide, about two nucleotides, about three nucleotides, about four nucleotides, about five nucleotides, about six nucleotides, about seven nucleotides, about eight nucleotides, about nine nucleotides, about ten nucleotides, about 11 nucleotides, about 12 nucleotides, about 13nucleotides, about 14 nucleotides, about 15 nucleotides, about 16 nucleotides, about 17 nucleotides, about 18 nucleotides, about 19 nucleotides, about 20 nucleotides, about 21 nucleotides, about 22 nucleotides, about 23 nucleotides, about 24 nucleotides, about 25 nucleotides, about 26 nucleotides, about 27 nucleotides, about 28 nucleotides, about 29 nucleotides, about 30 nucleotides, about 31 nucleotides, about 32 nucleotides, about 33 nucleotides, about 34 nucleotides, about 35 nucleotides, about 36 nucleotides, about 37 nucleotides, about 38 nucleotides, about 39 nucleotides, about 40 nucleotides, about 41 nucleotides, about 42 nucleotides, about 43 nucleotides, about 44 nucleotides, about 45 nucleotides, about 46 nucleotides, about 47 nucleotides, about 48 nucleotides, about 49 nucleotides, about 50 nucleotides, or more nucleotides.

[0111] In some embodiments, DNA molecules according to methods provided herein include a promoter for in vivo transcription. Typically, the promoter for in vivo transcription is an RNA polymerase II (RNA pol II) promoter. Any RNA pol II promoter can be included in DNA molecules provided herein, including constitutive promoters, inducible promoters, and tissuespecific promoters. Exemplary constitutive promoters include a cytomegalovirus (CMV) promoter, an EFla promoter, an SV40 promoter, a PGK1 promoter, a Ubc promoter, a human beta actin promoter, a CAG promoter, and others. Any tissue-specific promoter can be included in DNA molecules provided herein. In some embodiments, the RNA pol II promoter is a musclespecific promoter, skin-specific promoter, subcutaneous tissue-specific promoter, liver-specific promoter, spleen-specific promoter, lymph node-specific promoter, or a promoter with any other tissue specificity. DNA molecules provided herein can also include an enhancer. Any enhancer that increases transcription can be included in DNA molecules provided herein.Design and Synthesis of RNA and DNA Molecules

[0112] RNA molecules according to methods provided herein can include any combination of the RNA sequences provided herein, including, for example, any 5’ UTR sequences, any sequences encoding any transgene disclosed herein (e.g., GALC), and any 3’ UTR sequences provided herein.

[0113] In some embodiments, RNA molecules according to methods provided herein include modified nucleotides. For example, 0% to 100%, 1% to 100%, 25% to 100%, 50% to 100% and 75% to 100% of the uracil nucleotides of the RNA molecules can be modified. In some embodiments, 1% to 100% of the uracil nucleotides are N1 -methylpseudouridine or 5- methoxyuridine. In some embodiments, 100% of the uracil nucleotides are Nl- methylpseudouridine. In some embodiments, 100% of the uracil nucleotides are 5- methoxyuridine.

[0114] An RNA molecule according to methods of the disclosure may be obtained by any suitable means. A variety of methods for the manufacture of RNA molecules are available. An RNA molecule of the disclosure may be prepared according to any available technique, including, but not limited to chemical synthesis, in vitro transcription (IVT) or enzymatic or chemical cleavage of a longer precursor, etc.

[0115] In some embodiments, an RNA molecule according to methods of the disclosure is produced from a primary complementary DNA (cDNA) construct. The cDNA constructs can be produced on an RNA template by the action of a reverse transcriptase (e.g., RNA-dependent DNA-polymerase). The process of design and synthesis of the primary cDNA constructs described herein generally includes the steps of gene construction, RNA production (either with or without modifications) and purification. In an illustrative IVT method, a target polynucleotide sequence encoding an RNA molecule of the disclosure is first selected for incorporation into a vector which will be amplified to produce a cDNA template. Optionally, the target polynucleotide sequence and / or any flanking sequences may be codon optimized. The cDNA template is then used to produce an RNA molecule of the disclosure through in vitro transcription (IVT). After production, the RNA molecule of the disclosure may undergo purification and clean-up processes. The steps of which are provided in more detail below.

[0116] The step of gene construction may include, but is not limited to gene synthesis, vector amplification, plasmid purification, plasmid linearization and clean-up, and cDNA template synthesis and clean-up. Once a protein of interest is selected for production, a primary construct is designed. Within the primary construct, a first region of linked nucleosides encoding the polypeptide of interest may be constructed using an open reading frame (ORF) of a selected nucleic acid (DNA or RNA) transcript. The ORF may comprise the wild type ORF, an isoform, variant or a fragment thereof. As used herein, an “open reading frame” or “ORF” is meant to refer to a nucleic acid sequence (DNA or RNA) which can encode a polypeptide of interest. ORF s often begin with the start codon, ATG and end with a nonsense or termination codon or signal.

[0117] The cDNA templates may be transcribed to produce an RNA molecule of the disclosure using an in vitro transcription (IVT) system. The system typically comprises a transcription buffer, nucleotide triphosphates (NTPs), an RNase inhibitor and a polymerase. The NTPs may be selected from, but are not limited to, those described herein including natural and unnatural (modified) NTPs. The polymerase may be selected from, but is not limited to, T7 RNA polymerase, T3 RNA polymerase and mutant polymerases such as, but not limited to, polymerases able to incorporate modified nucleic acids.

[0118] The primary cDNA template or transcribed RNA sequence may also undergo capping and / or tailing reactions. A capping reaction may be performed by methods known in the art to add a 5' cap to the 5' end of the primary construct. Methods for capping include, but are not limited to, using a Vaccinia Capping enzyme (New England Biolabs, Ipswich, Mass.) or capping at initiation of in vitro transcription, by for example, including a capping agent as part of the IVT reaction. (Nuc. Acids Symp. (2009) 53: 129). A poly(A) tailing reaction may be performed by methods known in the art, such as, but not limited to, 2' O-m ethyltransferase and by methods as described herein. If the primary construct generated from cDNA does not include a poly-T, it may be beneficial to perform the poly(A)-tailing reaction before the primary construct is cleaned.

[0119] In some embodiments, the present methods provide expression vectors comprising a polynucleotide sequence encoding an RNA that is preferably operably linked to at least one regulatory sequence. Regulatory sequences are art-recognized and are selected to direct expression of the encoded polypeptide.

[0120] Accordingly, the term regulatory sequence includes promoters, enhancers, and other expression control elements. The design of the expression vector may depend on such factors as the choice of the host cell to be transformed and / or the type of protein desired to be expressed.

[0121] In some embodiments, polynucleotides (e.g. DNA, RNA, cDNA, mRNA, etc.) directed to RNA molecules according to methods of the disclosure may be operably linked to one or more regulatory nucleotide sequences in an expression construct, such as a vector or plasmid. In certain embodiments, such constructs are DNA constructs. Regulatory nucleotide sequences will generally be appropriate for a host cell used for expression. Numerous types of appropriate expression vectors and suitable regulatory sequences are known in the art for a variety of host cells.

[0122] Typically, the one or more regulatory nucleotide sequences may include, but are not limited to, promoter sequences, leader or signal sequences, ribosomal binding sites, transcriptional start and termination sequences, translational start and termination sequences, and enhancer or activator sequences. Constitutive or inducible promoters as known in the art are contemplated by the embodiments of the present disclosure. The promoters may be either naturally occurring promoters, or hybrid promoters that combine elements of more than one promoter.

[0123] An expression construct may be present in a cell on an episome, such as a plasmid, or the expression construct may be inserted in a chromosome. In some embodiments, the expression vector contains a selectable marker gene to allow the selection of transformed host cells. Selectable marker genes are well known in the art and will vary with the host cell used.

[0124] In some embodiments, a polynucleotide according to methods disclosed herein is in a host cell transfected with the polynucleotide. The polynucleotide can encode any protein of interest, for example a GALC. The host cell may be any prokaryotic or eukaryotic cell.Formulations

[0125] In some embodiments, a polynucleotide according to methods disclosed herein is formulated with a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier comprises a lipid formulation. In some embodiments, the lipid formulation comprises a transfection reagent, a lipoplex, a liposome, a lipid nanoparticle, a polymer-based carrier, an exosome, a lamellar body, a micelle, or an emulsion. In some embodiments, the lipid formulation comprises a liposome (e.g., a cationic liposome). In some embodiments, the lipid formulation comprises a lipid nanoparticle. In some embodiments, the lipid formulation comprises one or more cationic lipids (e.g., an ionizable cationic lipid). In some embodiments, the lipid formulation comprises an anionic lipid, a zwitterionic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, a phospholipid, a glycolipid, or a combination thereof. In some embodiments, the lipid formulation comprises a helper lipid, cholesterol, a polyethylene glycol (PEG)-lipid conjugate, or any combination of one or more of these.Liposomes

[0126] Conventional liposomes are vesicles that consist of at least one bilayer and an internal aqueous compartment. Bilayer membranes of liposomes are typically formed by amphiphilic molecules, such as lipids of synthetic or natural origin that comprise spatially separated hydrophilic and hydrophobic domains (Lasic, Trends Biotechnol., 16: 307-321, 1998). Bilayer membranes of the liposomes can also be formed by amphiphilic polymers and surfactants (e.g., polymerosomes, niosomes, etc.). They generally present as spherical vesicles and can range in size from 20 nm to a few microns. Liposomal formulations can be prepared as a colloidal dispersion or they can be lyophilized to reduce stability risks and to improve the shelf-life for liposome-based drugs. Methods of preparing liposomal compositions are known in the art and would be within the skill of an ordinary artisan.

[0127] Liposomes that have only one bilayer are referred to as being unilamellar, and those having more than one bilayer are referred to as multilamellar. The most common types of liposomes are small unilamellar vesicles (SUV), large unilamellar vesicle (LUV), and multilamellar vesicles (MLV). In contrast to liposomes, lysosomes, micelles, and reversed micelles are composed of monolayers of lipids. Generally, a liposome is thought of as having a single interior compartment, however some formulations can be multivesicular liposomes (MVL),which consist of numerous discontinuous internal aqueous compartments separated by several nonconcentric lipid bilayers.

[0128] Liposomes have long been perceived as drug delivery vehicles because of their superior biocompatibility, given that liposomes are basically analogs of biological membranes, and can be prepared from both natural and synthetic phospholipids (Int J Nanomedicine. 2014; 9: 1833-1843). In their use as drug delivery vehicles, because a liposome has an aqueous solution core surrounded by a hydrophobic membrane, hydrophilic solutes dissolved in the core cannot readily pass through the bilayer, and hydrophobic compounds will associate with the bilayer. Thus, a liposome can be loaded with hydrophobic and / or hydrophilic molecules. When a liposome is used to carry a nucleic acid such as RNA, the nucleic acid will be contained within the liposomal compartment in an aqueous phase.

[0129] In some embodiments, the lipid formulation comprises a cationic liposome, a nanoliposome, a proteoliposome, a unilamellar liposome, a multilamellar liposome, a ceramide- containing nanoliposome, or a multivesicular liposome.Cationic Liposomes

[0130] Liposomes can be composed of cationic, anionic, and / or neutral lipids. As an important subclass of liposomes, cationic liposomes are liposomes that are made in whole or part from positively charged lipids, or more specifically a lipid that comprises both a cationic group and a lipophilic portion. In addition to the general characteristics profiled above for liposomes, the positively charged moieties of cationic lipids used in cationic liposomes provide several advantages and some unique structural features. For example, the lipophilic portion of the cationic lipid is hydrophobic and thus will direct itself away from the aqueous interior of the liposome and associate with other nonpolar and hydrophobic species. Conversely, the cationic moiety will associate with aqueous media and more importantly with polar molecules and species with which it can complex in the aqueous interior of the cationic liposome. For these reasons, cationic liposomes are increasingly being researched for use in gene therapy due to their favorability towards negatively charged nucleic acids via electrostatic interactions, resulting in complexes that offer biocompatibility, low toxicity, and the possibility of the large-scale production required for in vivo clinical applications. Cationic lipids suitable for use in cationic liposomes are listed herein below.Lipid Nanoparticles

[0131] In contrast to liposomes and cationic liposomes, lipid nanoparticles (LNP) have a structure that can include a single monolayer or bilayer of lipids that encapsulates a compound ina solid phase. Thus, unlike liposomes, lipid nanoparticles do not have an aqueous phase or other liquid phase in its interior, but rather the lipids from the bilayer or monolayer shell are directly complexed to the internal compound thereby encapsulating it in a solid core. Lipid nanoparticles are typically spherical vesicles having a relatively uniform dispersion of shape and size. While sources vary on what size qualifies a lipid particle as being a nanoparticle, there is some overlap in agreement that a lipid nanoparticle can have a diameter in the range of from 10 nm to 1000 nm. However, more commonly they are considered to be smaller than 120 nm or even 100 nm.

[0132] For lipid nanoparticle nucleic acid delivery systems in accordance with some embodiments, the lipid shell is formulated to include an ionizable cationic lipid which can complex to and associate with the negatively charged backbone of the nucleic acid core. Ionizable cationic lipids with apparent pKa values below or about 7 have the benefit of providing a cationic lipid for complexing with the nucleic acid’s negatively charged backbone and loading into the lipid nanoparticle at pH values below the pKa of the ionizable lipid where it is positively charged. Then, at physiological pH values, the lipid nanoparticle can adopt a relatively neutral exterior allowing for a significant increase in the circulation half-lives of the particles following i.v. administration. In the context of nucleic acid delivery, lipid nanoparticles offer many advantages over other lipid-based nucleic acid delivery systems including high nucleic acid encapsulation efficiency, potent transfection, improved penetration into tissues to deliver therapeutics, and low levels of cytotoxicity and immunogenicity.

[0133] Prior to the development of lipid nanoparticle delivery systems for nucleic acids, cationic lipids were widely studied as synthetic materials for delivery of nucleic acid medicines. In these early efforts, after mixing together at physiological pH, nucleic acids were condensed by cationic lipids to form lipid-nucleic acid complexes known as lipoplexes. However, lipoplexes proved to be unstable and characterized by broad size distributions ranging from the submicron scale to a few microns. Lipoplexes, such as the Lipofectamine® reagent, have found considerable utility for in vitro transfection. However, these first-generation lipoplexes have not proven useful in vivo. The large particle size and positive charge (Imparted by the cationic lipid) result in rapid plasma clearance, hemolytic and other toxicities, as well as immune system activation. In some embodiments, nucleic acid molecules provided herein and lipids or lipid formulations provided herein form a lipid nanoparticle (LNP).

[0134] In some embodiments, polynucleotides according to methods provided herein are incorporated into a lipid formulation (e.g., a lipid-based delivery vehicle). In some embodiments, the polynucleotide is encapsulated within the lipid formulation or lipid nanoparticle. In some embodiments, the polynucleotide is complexed to the lipid formulation or lipid nanoparticle.

[0135] In the context of the present disclosure, a lipid-based delivery vehicle typically serves to transport a desired RNA to a target cell or tissue. The lipid-based delivery vehicle can be any suitable lipid-based delivery vehicle known in the art. In some embodiments, the lipid- based delivery vehicle is a liposome, a cationic liposome, or a lipid nanoparticle containing an RNA or mRNA of the disclosure. In some embodiments, the lipid-based delivery vehicle comprises a nanoparticle or a bilayer of lipid molecules and a polynucleotide of the disclosure. In some embodiments, the lipid-based delivery vehicle (e.g., lipid nanoparticle, lipid monolayer or bilayer, solid lipid nanoparticle, nanostructured lipid formulation, or other lipid formulation) further comprises a neutral lipid or a polymer. In some embodiments, the lipid formulation comprises a liquid medium. In some embodiments, the formulation further encapsulates a nucleic acid. In some embodiments, the lipid formulation further comprises a nucleic acid and a neutral lipid or a polymer. In some embodiments, the lipid formulation encapsulates the nucleic acid.

[0136] In the nucleic acid-lipid formulations according to some embodiments of the present methods, the polynucleotide may be fully encapsulated within the lipid portion of the formulation, thereby protecting the nucleic acid from nuclease degradation. In some embodiments, a lipid formulation comprising a polynucleotide is fully encapsulated within the lipid portion of the lipid formulation, thereby protecting the nucleic acid from nuclease degradation. In some embodiments, the polynucleotide in the lipid formulation is not substantially degraded after exposure of the particle to a nuclease at 37°C for at least 20, 30, 45, or 60 minutes. In some embodiments, the polynucleotide in the lipid formulation is not substantially degraded after incubation of the formulation in serum at 37°C for at least 30, 45, or 60 minutes or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, or 36 hours. In some embodiments, the polynucleotide is complexed with the lipid portion of the formulation. One of the benefits of the formulations of the present disclosure is that the nucleic acid-lipid compositions are substantially non-toxic to animals such as humans and other mammals.

[0137] In the context of nucleic acids, full encapsulation may be determined by performing a membrane-impermeable fluorescent dye exclusion assay, which uses a dye that has enhanced fluorescence when associated with nucleic acid. Encapsulation is determined by adding the dye to a lipid formulation, measuring the resulting fluorescence, and comparing it to the fluorescence observed upon addition of a small amount of nonionic detergent. Detergent-mediated disruption of the lipid layer releases the encapsulated nucleic acid, allowing it to interact with the membrane-impermeable dye. Nucleic acid encapsulation may be calculated as E = (10 - I) / I0, where / and 10 refers to the fluorescence intensities before and after the addition of detergent.

[0138] In some embodiments, the present disclosure provides methods of delivering a nucleic acid-lipid composition comprising a plurality of nucleic acid-liposomes, nucleic acidcationic liposomes, or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid- lipid composition comprises a plurality of RNA-liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-lipid nanoparticles.

[0139] In some embodiments, the lipid formulations according to methods disclosed herein comprise RNA that is fully encapsulated within the lipid portion of the formulation, such that from about 30% to about 100%, from about 40% to about 100%, from about 50% to about 100%, from about 60% to about 100%, from about 70% to about 100%, from about 80% to about 100%, from about 90% to about 100%, from about 30% to about 95%, from about 40% to about 95%, from about 50% to about 95%, from about 60% to about 95%, from about 70% to about95%, from about 80% to about 95%, from about 85% to about 95%, from about 90% to about95%, from about 30% to about 90%, from about 40% to about 90%, from about 50% to about90%, from about 60% to about 90%, from about 70% to about 90%, from about 80% to about90%, or at least about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (or any fraction thereof or range therein) of the particles have the RNA encapsulated therein. The amount may be any value or subvalue within the recited ranges, including endpoints.

[0140] Depending on the intended use of the lipid formulation, the proportions of the components can be varied, and the delivery efficiency of a particular formulation can be measured using assays known in the art.

[0141] In some embodiments, a nucleic acid-lipid composition according to methods disclosed herein comprises a plurality of nucleic acid-liposomes, nucleic acid-cationic liposomes, or nucleic acid-lipid nanoparticles. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-cationic liposomes. In some embodiments, the nucleic acid-lipid composition comprises a plurality of RNA-lipid nanoparticles.

[0142] In some embodiments, the lipid formulations according to methods of the disclosure have a total lipidmucleic acid molecule weight ratio (mass / mass ratio) of from about 1 : 1 to about 100: 1, from about 1 : 1 to about 50: 1, from about 2: 1 to about 45: 1, from about 3: 1 to about 40: 1, from about 5: 1 to about 45: 1, or from about 10: 1 to about 40: 1, or from about 15: 1 to about 40: 1, or from about 20: 1 to about 40: 1; or from about 25: 1 to about 45: 1; or from about 30: 1to about 45: 1; or from about 32: 1 to about 42: 1; or from about 34: 1 to about 42: 1. In some embodiments, the total lipid: nucleic acid molecule weight ratio (mass / mass ratio) is from about 50: 1 to about 10: 1. The ratio may be any value or subvalue within the recited ranges, including endpoints.

[0143] In some embodiments, the lipid formulation according to methods disclosed herein has a mean diameter of from about 30 nm to about 150 nm, from about 40 nm to about 150 nm, from about 50 nm to about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 80 nm to about 100 nm, from about 90 nm to about 100 nm, from about 70 to about 90 nm, from about 80 nm to about 90 nm, from about 70 nm to about 80 nm, or about 30 nm, about 35 nm, about 40 nm, about 45 nm, about 50 nm, about 55 nm, about 60 nm, about 65 nm, about 70 nm, about 75 nm, about 80 nm, about 85 nm, about 90 nm, about 95 nm, about 100 nm, about 105 nm, about 110 nm, about 115 nm, about 120 nm, about 125 nm, about 130 nm, about 135 nm, about 140 nm, about 145 nm, or about 150 nm, and are substantially non-toxic. The diameter may be any value or sub-value within the recited ranges, including endpoints. In some embodiments, the lipid nanoparticle has a size of less than about 200 nm, less than about 150 nm, less than about 100 nm, or about 55 nm to about 90 nm. In some embodiments, the lipid nanoparticle has a size of about 55 nm to about 90 nm. In addition, nucleic acids, when present in the lipid nanoparticles of the present disclosure, generally are resistant in aqueous solution to degradation with a nuclease.

[0144] In some embodiments, a lipid formulation is a cationic liposome or a lipid nanoparticle (LNP) comprising: (a) an RNA of the present disclosure, (b) a cationic lipid, (c) an aggregation reducing agent (such as polyethylene glycol (PEG) lipid or PEG-modified lipid), (d) optionally a non-cationic lipid (such as a neutral lipid), and optionally (e) a sterol. In some embodiments, the cationic liposome or lipid nanoparticle (LNP) comprises an ionizable lipid. In some embodiments, the cationic liposome or lipid nanoparticle (LNP) comprises an ionizable cationic lipid

[0145] In some embodiments, pharmaceutically acceptable carriers according to methods of the present disclosure comprise substances to approximate physiological conditions, such as pH adjusting and buffering agents, tonicity adjusting agents, and wetting agents, for example, sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate, triethanolamine oleate, and mixtures thereof. For solid compositions, conventional nontoxic pharmaceutically acceptable carriers can be used which include, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like.Cationic Lipids

[0146] In some embodiments, the pharmaceutically acceptable carrier according to methods disclosed herein comprises a lipid formulation comprising one or more cationic lipids. In some embodiments, the one of more cationic lipids comprises one or more of 5- carboxyspermylglycinedioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), l,2-Dioleoyl-3- Dimethylammonium-Propane (DODAP), l,2-Dioleoyl-3-Trimethylammonium-Propane (DOTAP), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1, 2-di oleyloxy -N,N- dimethyl-3 -aminopropane (DODMA), l,2-dilinoleyloxy-N,N-dimethyl-3 -aminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethyl-3 -aminopropane (DLenDMA), N-dioleyl-N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc- tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy l-l-(cis,cis-9',l-2'-octadecadienoxy)propane (CpLinDMA), N,N-dimethyl-3,4- di oleyloxybenzylamine (DMOBA), l,2-N,N'-dioleylcarbamyl-3 -dimethylaminopropane(DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'- Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (DLinCDAP), 2, 2-dilinoleyl-4-dimethylaminomethyl-[l,3]-di oxolane (DLin-K-DMA), or 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-K-XTC2- DMA). In some embodiments, the one or more cationic lipids comprises an ionizable cationic lipid.

[0147] In some embodiments, the ionizable cationic lipid has a structure of formula I:or a pharmaceutically acceptable salt or solvate thereof, wherein R5 and R6 are each independently selected from the group consisting of a linear or branched C1-C31 alkyl, C2-C31 alkenyl or C2-C31 alkynyl and cholesteryl; L5 and L6 are each independently selected from the group consisting of a linear C1-C20 alkyl and C2-C20 alkenyl; X5 is -C(O)O-, whereby -C(O)O-R6 is formed or -OC(O)- whereby -0C(0)-R6 is formed; X6 is -C(O)O- whereby -C(O)O-R5 is formed or -OC(O)- whereby -0C(0)-R5 is formed; X7 is S or O; L7 is absent or lower alkyl; R4 is a linear or branched C1-C6 alkyl; and R7 and R8 are each independently selected from the group consisting of a hydrogen and a linear or branched C1-C6 alkyl.

[0148] In some embodiments, X7 is S.

[0149] In some embodiments, X5 is -C(O)O-, whereby -C(O)O-R6 is formed and X6 is -C(O)O- whereby -C(O)O-R5 is formed.

[0150] In some embodiments, R7 and R8 are each independently selected from the group consisting of methyl, ethyl and isopropyl.

[0151] In some embodiments, L5 and L6 are each independently a Cl -CIO alkyl. In some embodiments, L5 is C1-C3 alkyl, and L6 is C1-C5 alkyl. In some embodiments, L6 is C1-C2 alkyl. In some embodiments, L5 and L6 are each a linear C7 alkyl. In some embodiments, L5 and L6 are each a linear C9 alkyl.

[0152] In some embodiments, R5 and R6 are each independently an alkenyl. In some embodiments, R6 is alkenyl. In some embodiments, R6 is C2-C9 alkenyl. In some embodiments, the alkenyl comprises a single double bond. In some embodiments, R5 and R6 are each alkyl. In some embodiments, R5 is a branched alkyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of a C9 alkyl, C9 alkenyl and C9 alkynyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of a Cl l alkyl, Cl l alkenyl and Cl l alkynyl. In some embodiments, R5 and R6 are each independently selected from the group consisting of a C7 alkyl, C7 alkenyl and C7 alkynyl. In some embodiments, R5 is -CH((CH2)pCH3)2 or -CH((CH2)pCH3)((CH2)p-lCH3), wherein p is 4-8. In some embodiments, p is 5 and L5 is a C1-C3 alkyl. In some embodiments, p is 6 and L5 is a C3 alkyl. In some embodiments, p is 7. In some embodiments, p is 8 and L5 is a C1-C3 alkyl. In some embodiments, R5 consists of-CH((CH2)pCH3)((CH2)p-lCH3), wherein p is 7 or 8.

[0153] In some embodiments, R4 is ethylene or propylene. In some embodiments, R4 is n-propylene or isobutylene.

[0154] In some embodiments, L7 is absent, R4 is ethylene, X7 is S and R7 and R8 are each methyl. In some embodiments, L7 is absent, R4 is n-propylene, X7 is S and R7 and R8 are each methyl. In some embodiments, L7 is absent, R4 is ethylene, X7 is S and R7 and R8 are each ethyl.

[0155] In some embodiments, X7 is S, X5 is -C(O)O-, whereby -C(O)O-R6 is formed, X6 is -C(O)O- whereby -C(O)O-R5 is formed, L5 and L6 are each independently a linear C3-C7 alkyl, L7 is absent, R5 is -CH((CH2)pCH3)2, and R6 is C7-C12 alkenyl. In some further embodiments, p is 6 and R6 is C9 alkenyl.

[0156] In some embodiments, the ionizable cationic lipid is selected from Table 1.

[0157] In some embodiments, the ionizable cationic lipid is:(Compound-B).

[0158] In some embodiments, the ionizable cationic lipid is:(Compound-PP).

[0159] In some embodiments, the ionizable cationic lipid is:(Compound-WWW).

[0160] In some embodiments, the ionizable cationic lipid isp .

[0161] Therapies based on the intracellular delivery of nucleic acids to target cells face both extracellular and intracellular barriers. Indeed, naked nucleic acid materials cannot be easily systemically administered due to their toxicity, low stability in serum, rapid renal clearance, reduced uptake by target cells, phagocyte uptake and their ability in activating the immune response, all features that preclude their clinical development. When exogenous nucleic acid material (e.g., mRNA) enters the human biological system, it is recognized by the reticuloendothelial system (RES) as foreign pathogens and cleared from blood circulation before having the chance to encounter target cells within or outside the vascular system. It has been reported that the half-life of naked nucleic acid in the blood stream is around several minutes (Kawabata K, Takakura Y, Hashida MPharm Res. 1995 Jun; 12(6):825-30). Chemical modification and a proper delivery method can reduce uptake by the RES and protect nucleic acids from degradation by ubiquitous nucleases, which increase stability and efficacy of nucleic acidbased therapies. In addition, RNAs or DNAs are anionic hydrophilic polymers that are not favorable for uptake by cells, which are also anionic at the surface. The success of nucleic acidbased therapies thus depends largely on the development of vehicles or vectors that can efficientlyand effectively deliver genetic material to target cells and obtain sufficient levels of expression in vivo with minimal toxicity.

[0162] Moreover, upon internalization into a target cell, nucleic acid delivery vectors are challenged by intracellular barriers, including endosome entrapment, lysosomal degradation, nucleic acid unpacking from vectors, translocation across the nuclear membrane (for DNA), release at the cytoplasm (for RNA), and so on. Successful nucleic acid-based therapy thus depends upon the ability of the vector to deliver the nucleic acids to the target sites inside of the cells in order to obtain sufficient levels of a desired activity such as expression of a gene.

[0163] Some art-recognized lipid-formulated delivery vehicles for nucleic acid therapeutics include, according to various embodiments, polymer based carriers, such as polyethyleneimine (PEI), lipid nanoparticles and liposomes, nanoliposomes, ceramide-containing nanoliposomes, multivesicular liposomes, proteoliposomes, both natural and synthetically- derived exosomes, natural, synthetic and semi-synthetic lamellar bodies, nanoparticulates, micelles, and emulsions. These lipid formulations can vary in their structure and composition, and as can be expected in a rapidly evolving field, several different terms have been used in the art to describe a single type of delivery vehicle. At the same time, the terms for lipid formulations have varied as to their intended meaning throughout the scientific literature, and this inconsistent use has caused confusion as to the exact meaning of several terms for lipid formulations. Among the several potential lipid formulations, liposomes, cationic liposomes, and lipid nanoparticles are specifically described in detail and defined herein for the purposes of the present disclosure.

[0164] In some embodiments, the lipid formulation according to methods disclosed herein comprises an anionic lipid, a zwitterionic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, a phospholipid, a glycolipid, or a combination thereof.

[0165] In some embodiments, the lipid formulation according to methods disclosed herein comprises a helper lipid. In some embodiments, the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidyl choline (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoylphosphatidyl glycerol (DPPG), dipalmitoyl phosphatidylcholine (DPPC), dioleoylphosphatidyl phosphatidylcholine (DOPC), and phosphatidylcholine (PC). In some embodiments, the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidyl choline (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC). In some embodiments, the helper lipid is distearoylphosphatidylcholine (DSPC). In some embodiments, the lipid formulation comprises cholesterol.

[0166] In some embodiments, the lipid formulation comprises a polyethylene glycol (PEG)-lipid conjugate. In some embodiments, the PEG-lipid conjugate is PEG-DMG (e.g., PEG2000-DMG). In some embodiments, the lipid formulation comprises about 40 mol% to about 60 mol% of the ionizable cationic lipid, about 4 mol% to about 16 mol% DSPC, about 30 mol% to about 47 mol% cholesterol, and about 0.5 mol% to about 3 mol% PEG2000-DMG. In some embodiments, the composition has a total lipidmucleic acid molecule weight ratio of about 50: 1 to about 10: 1.

[0167] In some embodiments, the helper lipid comprises from about 2 mol% to about 20 mol%, from about 3 mol% to about 18 mol%, from about 4 mol% to about 16 mol%, about 5 mol% to about 14 mol%, from about 6 mol% to about 12 mol%, from about 5 mol% to about 10 mol%, from about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation.

[0168] The lipid portion, or the cholesterol or cholesterol derivative in the lipid formulation may comprise up to about 40 mol%, about 45 mol%, about 50 mol%, about 55 mol%, or about 60 mol% of the total lipid present in the lipid formulation. In some embodiments, the cholesterol or cholesterol derivative comprises about 15 mol% to about 45 mol%, about 20 mol% to about 40 mol%, about 25 mol% to about 35 mol%, or about 28 mol% to about 35 mol%; or about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, or about 37 mol% of the total lipid present in the lipid formulation.

[0169] In some embodiments, the lipid portion of the lipid formulation is about 35 mol% to about 42 mol% cholesterol.

[0170] In some embodiments, the phospholipid component in the mixture may comprise from about 2 mol% to about 20 mol%, from about 3 mol% to about 18 mol%, from about 4 mol % to about 16 mol %, about 5 mol % to about 14 mol %, from about 6 mol % to about 12 mol%, from about 5 mol% to about 10 mol%, from about 5 mol% to about 9 mol%, or about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, or about 12 mol% (or any fraction thereof or the range therein) of the total lipid present in the lipid formulation.

[0171] In some embodiments, the lipid portion of the lipid formulation comprises about, but is not necessarily limited to, 40 mol% to about 60 mol% of the ionizable cationic lipid, about4 mol% to about 16 mol% DSPC, about 30 mol% to about 47 mol% cholesterol, and about 0.5 mol% to about 3 mol% PEG2000-DMG.

[0172] In some embodiments, the lipid portion of the lipid formulation may comprise, but is not necessarily limited to, about 42 mol% to about 58 mol% of the ionizable cationic lipid, about 6 mol% to about 14 mol% DSPC, about 32 mol% to about 44 mol% cholesterol, and about 1 mol% to about 2 mol% PEG2000-DMG.

[0173] In some embodiments, the lipid portion of the lipid formulation may comprise, but is not necessarily limited to, about 45 mol% to about 55 mol% of the ionizable cationic lipid, about 8 mol% to about 12 mol% DSPC, about 35 mol% to about 42 mol% cholesterol, and about 1.25 mol% to about 1.75 mol% PEG2000-DMG.

[0174] The percentage of helper lipid present in the lipid formulation is a target amount, and the actual amount of helper lipid present in the formulation may vary, for example, by ± 5 mol%.

[0175] A lipid formulation that includes a cationic lipid compound or ionizable cationic lipid compound may be on a molar basis about 30-70% cationic lipid compound, about 25-40 % cholesterol, about 2-15% helper lipid, and about 0.5-5% of a polyethylene glycol (PEG) lipid, wherein the percent is of the total lipid present in the formulation. In some embodiments, the composition is about 40-65% cationic lipid compound, about 25- 35% cholesterol, about 3-9% helper lipid, and about 0.5-3% of a PEG-lipid, wherein the percent is of the total lipid present in the formulation.

[0176] The formulation may be a lipid particle formulation, for example containing 8-30% nucleic acid compound, 5-30% helper lipid, and 0-20% cholesterol; 4-25% cationic lipid, 4-25% helper lipid, 2- 25% cholesterol, 10- 35% cholesterol -PEG, and 5% cholesterol-amine; or 2-30% cationic lipid, 2-30% helper lipid, 1-15% cholesterol, 2-35% cholesterol -PEG, and 1-20% cholesterol-amine; or up to 90% cationic lipid and 2-10% helper lipids, or even 100% cationic lipid.Methods for treating GALC deficiency

[0177] In some embodiments, the present disclosure provides methods for treating a GALC deficiency. In some embodiments, the methods utilize a composition and / or polynucleotide according to any of the various aspects or embodiments herein. In some embodiments, the method comprises delivery of an RNA molecule and a lipid provided herein. In some embodiments, the method comprises delivery of an RNA molecule and a lipid formulation provided herein.

[0178] In some embodiments, the present disclosure provides methods for delivering a polynucleotide to cells of a central nervous system of a subject to treat a GALC deficiency, wherein the cells of the central nervous system comprise oligodendrocytes, neurons, astrocytes, ependymal cells, microglial cells, satellite cells, Schwann cells, choroid plexus cells, endothelial cells, or a combination thereof. In some embodiments, the cells of the central nervous system comprise oligodendrocytes.

[0179] As used herein, the term “subject” refers to any individual or patient on which the methods disclosed herein are performed. The term “subject” can be used interchangeably with the term “individual” or “patient.” The subject can be a human, although the subject may be an animal, as will be appreciated by those in the art. Thus, other animals, including mammals such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject. As used herein, the term “effective amount” or “therapeutically effective amount” refers to that amount of an RNA molecule, composition, or pharmaceutical composition described herein that is sufficient to effect the intended application, including but not limited to inducing an immune response and / or disease treatment, as defined herein. The therapeutically effective amount may vary depending upon the intended application (e.g., inducing an immune response, treatment, application in vivo), or the subject or patient and disease condition being treated, e.g., the weight and age of the subject, the species, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The term also applies to a dose that will induce a particular response in a target cell. The specific dose will vary depending on the particular RNA molecule, composition, or pharmaceutical composition chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds, timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried.

[0180] Exemplary doses of nucleic molecules that can be administered include about 0.01 pg, about 0.02 pg, about 0.03 pg, about 0.04 pg, about 0.05 pg, about 0.06 pg, about 0.07 pg, about 0.08 pg, about 0.09 pg, about 0.1 pg, about 0.2 pg, about 0.3 pg, about 0.4 pg, about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1.0 pg, about 1.5 pg, about 2.0 pg, about 2.5 pg, about 3.0 pg, about 3.5 pg, about 4.0 pg, about 4.5 pg, about 5.0 pg, about 5.5 pg, about 6.0 pg, about 6.5 pg, about 7.0 pg, about 7.5 pg, about 8.0 pg, about 8.5 pg, about 9.0 pg, about 9.5 pg, about 10 pg, about 11 pg, about 12 pg, about 13 p, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, about 100 pg, about 125 pg, about 150 pg, about 175 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, about 1,000 pg, or more, and any number or range in between. In some embodiments, the nucleic acid molecules are RNA molecules. In some embodiments, the nucleic acid molecules are DNA molecules. In some embodiments, nucleic acid molecules include one RNA molecule or one DNA molecule. In some embodiments, nucleic acid molecules include two, three, four, five, six, seven, eight, or more different RNA molecules or DNA molecules. Nucleic acid molecules can have a unit dosage comprising about 0.01 pg to about 1,000 pg or more nucleic acid in a single dose.

[0181] In some embodiments, the method includes administering about 0.01 pg, about 0.02 pg, about 0.03 pg, about 0.04 pg, about 0.05 pg, about 0.06 pg, about 0.07 pg, about 0.08 pg, about 0.09 pg, about 0.1 pg, about 0.2 pg, about 0.3 pg, about 0.4 pg, about 0.5 pg, about 0.6 pg, about 0.7 pg, about 0.8 pg, about 0.9 pg, about 1.0 pg, about 1.5 pg, about 2.0 pg, about 2.5 pg, about 3.0 pg, about 3.5 pg, about 4.0 pg, about 4.5 pg, about 5.0 pg, about 5.5 pg, about 6.0 pg, about 6.5 pg, about 7.0 pg, about 7.5 pg, about 8.0 pg, about 8.5 pg, about 9.0 pg, about 9.5 pg, about 10 pg, about 11 pg, about 12 pg, about 13 pg, about 14 pg, about 15 pg, about 16 pg, about 17 pg, about 18 pg, about 19 pg, about 20 pg, about 21 pg, about 22 pg, about 23 pg, about 24 pg, about 25 pg, about 26 pg, about 27 pg, about 28 pg, about 29 pg, about 30 pg, about 35 pg, about 40 pg, about 45 pg, about 50 pg, about 55 pg, about 60 pg, about 65 pg, about 70 pg, about 75 pg, about 80 pg, about 85 pg, about 90 pg, about 95 pg, about 100 pg, about 125 pg, about 150 pg, about 175 pg, about 200 pg, about 250 pg, about 300 pg, about 350 pg, about 400 pg, about 450 pg, about 500 pg, about 600 pg, about 700 pg, about 800 pg, about 900 pg, about 1,000 pg, or more, and any number or range in between, nucleic acid and lipid (e.g., in a lipid formulation). In some embodiments, methods include administering one RNA molecule or one DNA molecule. In some embodiments, methods include delivering two, three, four, five, six, seven, eight, or more different RNA molecules or DNA molecules.

[0182] In some embodiments, methods comprise administering a unit dosage comprising about 0.01 pg to about 1,000 pg or more nucleic acid and lipid in a single dose. In some embodiments, methods comprise administering a unit dosage comprising about 0.01 pg to about 1,000 pg or more nucleic acid and lipid formulation in a single dose. A unit dosage can correspond to the unit dosage of nucleic acid molecules, compositions, or pharmaceutical compositions provided herein and that can be administered to a subject. In some embodiments, the unit dosagecomprises about 0.01 pg to about 1,000 pg or more nucleic acid and lipid formulation in a single dose. In some embodiments, the unit dosage comprises about 0.01 pg to about 50 pg nucleic acid and lipid formulation in a single dose. In some embodiments, the unit dosage comprises about 0.2 pg to about 20 pg nucleic acid and lipid formulation in a single dose.

[0183] A dosage form according to methods of this disclosure can be solid, which can be reconstituted in a liquid prior to administration. The solid can be administered as a powder. The solid can be in the form of a capsule, tablet, or gel. In some embodiments, the pharmaceutical composition comprises a nucleic acid lipid formulation that has been lyophilized. In some embodiments, the lyophilized composition may comprise one or more lyoprotectants, such as, including but not necessarily limited to, glucose, trehalose, sucrose, maltose, lactose, mannitol, inositol, hydroxypropyl-P-cyclodextrin, and / or polyethylene glycol. In some embodiments, the lyophilized composition comprises a poloxamer, potassium sorbate, sucrose, or any combination thereof. In specific embodiments, the poloxamer is poloxamer 188. In some embodiments, the lyophilized compositions described herein may comprise about 0.01 to about 1.0% w / w of a poloxamer. In some embodiments, the lyophilized compositions described herein may comprise about 1.0 to about 5.0% w / w of potassium sorbate. The percentages may be any value or subvalue within the recited ranges, including endpoints.

[0184] In some embodiments, the lyophilized composition may comprise about 0.01 to about 1.0 % w / w of the nucleic acid molecule. In some embodiments, the composition may comprise about 1.0 to about 5.0 % w / w lipids. In some embodiments, the composition may comprise about 0.5 to about 2.5 % w / w of TRIS buffer. In some embodiments, the composition may comprise about 0.75 to about 2.75 % w / w of NaCl. In some embodiments, the composition may comprise about 5 to about 95 % w / w of a sugar, about 10 to about 95 % w / w of a sugar, about 15 to about 95 % w / w of a sugar, about 20 to about 95 % w / w of a sugar, about 25 to about 95 % w / w of a sugar, about 30 to about 95 % w / w of a sugar, about 35 to about 95 % w / w of a sugar, about 40 to about 95 % w / w of a sugar, about 45 to about 95 % w / w of a sugar, about 50 to about 95 % w / w of a sugar, about 55 to about 95 % w / w of a sugar, about 60 to about 95 % w / w of a sugar, about 65 to about 95 % w / w of a sugar, about 70 to about 95 % w / w of a sugar, about 75 to about 95 % w / w of a sugar, about 80 to about 95 % w / w of a sugar, or about 85 to about 95 % w / w of a sugar. In some embodiments, the composition may comprise about 1 to about 50 % w / w of a sugar, about 5 to about 50 % w / w of a sugar, about 10 to about 50 % w / w of a sugar, about 15 to about 50 % w / w of a sugar, about 20 to about 50 % w / w of a sugar, about 25 to about 50 % w / w of a sugar, about 30 to about 50 % w / w of a sugar, about 35 to about 50 % w / w of a sugar, about 40 to about 50 % w / w of a sugar, or about 45 to about 50 % w / w of a sugar. In someembodiments, the composition may comprise about 1 to about 20 % w / w of a sugar, about 2 to about 20 % w / w of a sugar, about 3 to about 20 % w / w of a sugar, about 4 to about 20 % w / w of a sugar, about 5 to about 20 % w / w of a sugar, about 6 to about 20 % w / w of a sugar, about 7 to about 20 % w / w of a sugar, about 8 to about 20 % w / w of a sugar, about 9 to about 20 % w / w of a sugar, about 10 to about 20 % w / w of a sugar, about 11 to about 20 % w / w of a sugar, about 12 to about 20 % w / w of a sugar, about 13 to about 20 % w / w of a sugar, about 14 to about 20 % w / w of a sugar, about 15 to about 20 % w / w of a sugar, about 16 to about 20 % w / w of a sugar, about 17 to about 20 % w / w of a sugar, about 18 to about 20 % w / w of a sugar, or about 19 to about 20 % w / w of a sugar. In some embodiments, the composition may comprise about 1 to about 18 % w / w of a sugar, about 2 to about 18 % w / w of a sugar, about 3 to about 18 % w / w of a sugar, about 4 to about 18 % w / w of a sugar, about 5 to about 18 % w / w of a sugar, about 6 to about 18 % w / w of a sugar, about 7 to about 18 % w / w of a sugar, about 8 to about 18 % w / w of a sugar, about 9 to about 18 % w / w of a sugar, about 10 to about 18 % w / w of a sugar, about 11 to about 18 % w / w of a sugar, about 12 to about 18 % w / w of a sugar, about 13 to about 18 % w / w of a sugar, about 14 to about 18 % w / w of a sugar, about 15 to about 18 % w / w of a sugar, about 16 to about 18 % w / w of a sugar, or about 17 to about 18 % w / w of a sugar. In some embodiments, the composition may comprise about 1 to about 16 % w / w of a sugar, about 2 to about 16 % w / w of a sugar, about 3 to about 16 % w / w of a sugar, about 4 to about 16 % w / w of a sugar, about 5 to about 16 % w / w of a sugar, about 6 to about 16 % w / w of a sugar, about 7 to about 16 % w / w of a sugar, about 8 to about 16 % w / w of a sugar, about 9 to about 16 % w / w of a sugar, about 10 to about 16 % w / w of a sugar, about 11 to about 16 % w / w of a sugar, about 12 to about 16 % w / w of a sugar, about 13 to about 16% w / w of a sugar, about 14 to about 16 % w / w of a sugar, or about 15 to about 16 % w / w of a sugar. In some embodiments, the composition may comprise about 1 to about 12 % w / w of a sugar, about 2 to about 12 % w / w of a sugar, about 3 to about 12 % w / w of a sugar, about 4 to about 12 % w / w of a sugar, about 5 to about 12 % w / w of a sugar, about 6 to about 12 % w / w of a sugar, about 7 to about 12 % w / w of a sugar, about 8 to about 12 % w / w of a sugar, about 9 to about 12 % w / w of a sugar, about 10 to about 12 % w / w of a sugar, or about 11 to about 12 % w / w of a sugar. The percentages may be any value or subvalue within the recited ranges, including endpoints. Compositions provided herein can be lyophilized, a liquid, a frozen liquid, or a liquid suspension.

[0185] In some embodiments, the dosage form according to methods described herein can be a liquid suspension of RNA lipid nanoparticles described herein. In some embodiments, the RNA of RNA lipid nanoparticles is an mRNA. In some embodiments, the liquid suspension is in a buffered solution. In some embodiments, the buffered solution comprises a buffer selected fromthe group consisting of HEPES, MOPS, TES, and TRIS. In some embodiments, the buffer has a pH of about 7.4. In some preferred embodiments, the buffer is HEPES. In some further embodiments, the buffered solution further comprises a cryoprotectant. In some embodiments, the cryoprotectant is selected from a sugar and glycerol or a combination of a sugar and glycerol. In some embodiments, the sugar is a dimeric sugar. In some embodiments, the sugar is sucrose. In some preferred embodiments, the buffer comprises HEPES, sucrose, and glycerol at a pH of 7.4. In certain embodiments, the composition comprises a HEPES, MOPS, TES, or TRIS buffer at a pH of about 7.0 to about 8.5. In some embodiments, the HEPES, MOPS, TES, or TRIS buffer may at a concentration ranging from 7 mg / ml to about 15 mg / ml. The pH or concentration may be any value or subvalue within the recited ranges, including endpoints.

[0186] In some embodiments, the suspension is frozen during storage and thawed prior to administration. In some embodiments, the suspension is frozen at a temperature below about 70 °C. In some embodiments, the suspension is diluted with sterile water during intravenous administration. In some embodiments, intravenous administration comprises diluting the suspension with about 2 volumes to about 6 volumes of sterile water. In some embodiments, the suspension comprises about 0.1 mg to about 3.0 mg RNA / mL, about 15 mg / mL to about 25 mg / mL of an ionizable cationic lipid, about 0.5 mg / mL to about 2.5 mg / mL of a PEG-lipid, about 1.8 mg / mL to about 3.5 mg / mL of a helper lipid, about 4.5 mg / mL to about 7.5 mg / mL of a cholesterol, about 7 mg / mL to about 15 mg / mL of a buffer, about 2.0 mg / mL to about 4.0 mg / mL of NaCl, about 70 mg / mL to about 110 mg / mL of sucrose, and about 50 mg / mL to about 70 mg / mL of glycerol. In some embodiments, a lyophilized RNA-lipid nanoparticle formulation can be resuspended in a buffer as described herein.

[0187] In some embodiments, the polynucleotide according to methods of the disclosure is formulated to be administered to a subject such that a RNA concentration of at least about 0.05 mg / kg, at least about 0.1 mg / kg, at least about 0.5 mg / kg, at least about 1.0 mg / kg, at least about 2.0 mg / kg, at least about 3.0 mg / kg, at least about 4.0 mg / kg, at least about 5.0 mg / kg of body weight is administered in a single dose or as part of single treatment cycle. In some embodiments, the method comprises administering to a subject a total amount of at least about 0.1 mg, at least about 0.5 mg, at least about 1.0 mg, at least about 2.0 mg, at least about 3.0 mg, at least about 4.0 mg, at least about 5.0 mg, at least about 6.0 mg, at least about 7.0 mg, at least about 8.0 mg, at least about 9.0 mg, at least about 10 mg, at least about 15 mg, at least about 20 mg, at least about 25 mg, at least about 30 mg, at least about 35 mg, at least about 40 mg, at least about 45 mg, at least about 50 mg, at least about 55 mg, at least about 60 mg, at least about 65 mg, at least about 70 mg, at least about 75 mg, at least about 80 mg, at least about 85 mg, at least about 90 mg, atleast about 95 mg, at least about 100 mg, at least about 105 mg, at least about 110 mg, at least about 115 mg, at least about 120 mg, or at least about 125 mg RNA in one or more doses up to a maximum dose of about 300 mg, about 350 mg, about 400 mg, about 450 mg, or about 500 mg RNA.

[0188] Polynucleotide delivery according to methods of the present disclosure may be by any suitable route of administration. In some embodiments, administration according to methods provided herein comprises intravenous, epidural, intrathecal, intraparenchyma, intracisterna magna, or direct intraventricular administration. In some embodiments, administration is intravenous. In some embodiments, the administration is local rather than in a systemic manner, for example, via injection directly into a targeted tissue.

[0189] In some embodiments, the RNA delivered is expressed in a tissue different from the tissue in which the lipid formulation or pharmaceutical composition was administered. In preferred embodiments, RNA is formulated for delivery to and expressed in the central nervous system (e.g., in oligodendrocytes).

[0190] As used herein, the terms “treat,” “treatment,” “therapy,” “therapeutic,” and the like refer to obtaining a desired pharmacologic and / or physiologic effect, including, but not limited to, alleviating, delaying or slowing the progression, reducing the effects or symptoms, preventing onset, inhibiting, ameliorating the onset of a diseases or disorder, obtaining a beneficial or desired result with respect to a disease, disorder, or medical condition, such as a therapeutic benefit and / or a prophylactic benefit. “Treatment,” as used herein, includes any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject, including a subject which is predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease (e.g., arresting its development); and (c) relieving the disease (e.g., causing regression of the disease). A therapeutic benefit includes eradication or amelioration of the underlying disorder being treated. Also, a therapeutic benefit is achieved with the eradication or amelioration of one or more of the physiological symptoms associated with the underlying disorder such that an improvement is observed in the subject, notwithstanding that the subject may still be afflicted with the underlying disorder. In some embodiments, for prophylactic benefit, treatment or compositions for treatment, including pharmaceutical compositions, are administered to a subject at risk of developing a particular disease, or to a subject reporting one or more of the physiological symptoms of a disease, even though a diagnosis of this disease may not have been made. The methods of the present disclosure may be used with any mammal or other animal. In some embodiments, treatment results in a decrease or cessation of symptoms. A prophylactic effect includes delayingor eliminating the appearance of a disease or condition, delaying or eliminating the onset of symptoms of a disease or condition, slowing, halting, or reversing the progression of a disease or condition, or any combination thereof.

[0191] In some embodiments, polynucleotides (e.g., RNA or DNA molecules) according to methods provided herein are administered once or multiple times. In some embodiments, polynucleotides are administered one, two, three, four, five, six, seven, eight, nine, ten, or more times. Timing between two or more administrations can be one week, two weeks, three weeks, four weeks, five weeks, six weeks, seven weeks, eight weeks, nine weeks, weeks, ten weeks, 11 weeks, 12 weeks, 13 weeks, 14 weeks, 15 weeks, 16 weeks, 17 weeks, 18 weeks, 19 weeks, 20 weeks, 21 weeks, 22 weeks, 23 weeks, 24 weeks, 25 weeks, 26 weeks, 27 weeks, 28 weeks, 29 weeks, 30 weeks, 31 weeks, 32 weeks, 33 weeks, 34 weeks, 35 weeks, 36 weeks, 37 weeks, 38 weeks, 39 weeks, 40 weeks, 41 weeks, 42 weeks, 43 weeks, 44 weeks, 45 weeks, 46 weeks, 47 weeks, 48 weeks, 49 weeks, 50 weeks, 51 weeks, 52 weeks, or more weeks, and any number or range in between. In some embodiments, timing between two or more administrations is one month, two months, three months, four months, five months, six months, seven months, eight months, nine months, ten months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, or more months, and any number or range in between. In some embodiments, timing between two or more administrations can be one year, two years, three years, four years, five years, six years, seven years, eight years, nine years, ten years, or more years, and any number or range in between. Timing between the first and any subsequent administration can be the same or different. In some embodiments, nucleic acid molecules, compositions, or pharmaceutical compositions provided herein are administered once.

[0192] More than one nucleic acid molecule, composition, or pharmaceutical composition can be administered in the methods provided herein. In some embodiments, two or more nucleic acid molecules, compositions, or pharmaceutical compositions provided herein are administered simultaneously. In some embodiments, two or more nucleic acid molecules, compositions, or pharmaceutical compositions provided herein are administered sequentially. Simultaneous and sequential administrations can include any number and any combination of nucleic acid molecules, compositions, or pharmaceutical compositions provided herein. Multiple nucleic acid molecules, compositions, or pharmaceutical compositions that are administered together or sequentially can include transgenes encoding different proteins or fragments thereof. In this manner, expression of different genes can be achieved. In some embodiments, transgene expression results in restoration of wild-type protein expression. Two, three, four, five, six, seven, eight, nine, ten, or more nucleic acid molecules, compositions, or pharmaceutical compositionsincluding transgenes encoding different proteins or fragments thereof can be administered simultaneously or sequentially. Any combination of nucleic acid molecules, compositions, and pharmaceutical compositions including any combination of transgenes can be administered simultaneously or sequentially. In some embodiments, administration is simultaneous.

[0193] In some embodiments, administration is once per month. In some embodiments, administration is twice per month. In some embodiments, administration is three times per month. In some embodiments, administration is four times per month.

[0194] In some embodiments, the present disclosure provides methods for the treatment of leukodystrophies, a group of hereditary neurological disorders. Leukodystrophies include diseases such as Krabbe disease, heterozygous leukodystrophy, adrenoleukodystrophy, Alexander disease, and Pelizaeus-Merzbacher disease (PMD). Krabbe’s Leukodystrophy is a lipid storage disorder caused by a deficiency of GALC, which is necessary for the breakdown (metabolism) of the sphingolipids galactosylceremide and psychosine. Failure to break down these sphingolipids results in degeneration of the myelin sheath surrounding nerves in the brain (demyelination).

[0195] Following administration of the composition to the subject, the protein product encoded by an RNA according to the methods of the disclosure (e.g., a transgene) is detectable in the target tissues for at least about one to seven days or longer. For example, the protein product may be detectable in the target tissues at a concentration (e.g., a therapeutic concentration) of at least about 0.025-1.5 pg / ml (e.g., at least about 0.050 pg / ml, at least about 0.075 pg / ml, at least about 0.1 pg / ml, at least about 0.2 pg / ml, at least about 0.3 pg / ml, at least about 0.4 pg / ml, at least about 0.5 pg / ml, at least about 0.6 pg / ml, at least about 0.7 pg / ml, at least about 0.8 pg / ml, at least about 0.9 pg / ml, at least about 1.0 pg / ml, at least about 1.1 pg / ml, at least about 1.2 pg / ml, at least about 1.3 pg / ml, at least about 1.4 pg / ml, or at least about 1.5 pg / ml), for at least about 1, 2, 3, 4,5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 days or longer, and any number or range in between, following administration of the composition to the subject.Ranges

[0196] Throughout this disclosure, various aspects can be presented in range format. It should be understood that any description in range format is merely for convenience and brevity and not meant to be limiting. Accordingly, the description of a range should be considered to have specifically disclosed all possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2to 6, from 3 to 6, etc., as well as individual numbers within that range, for example 1, 2, 2.1, 2.2, 2.5, 3, 4, 4.75, 4.8, 4.85, 4.95, 5, 5.5, 5.75, 5.9, 5.00, and 6. This applies to a range of any breadth.MATERIALS AND METHODS FOR EXAMPLES 1-4 mRNA synthesis

[0197] Codon optimization of human GALC (hGALC, NCBI Reference Sequence: NP 000144.2), and selection of 5’- and 3’-UTR sequences was performed. The human codon- optimized hGALC was cloned between the 5’-UTR and 3’-UTR in a plasmid that was engineered for in vitro transcription (IVT). The sequence of the cloned portion in the plasmid was verified by DNA sequencing. The plasmid was linearized immediately after the poly(A) stretch located downstream of the 3-UTR and used as a template for IVT reaction with T7 RNA polymerase.

[0198] The IVT reactions were performed as previously described10, but with 100% substitution of UTP with Nl-methyl-pseudouri dine (Nlm'P). The RNA quality and integrity were verified by 0.8%-1.2% non-denaturing agarose gel electrophoresis as well as Fragment Analyzer (Advanced Analytical). The purified RNAs were stored in RNase-free water at -80°C until further use.Preparation of lipid nanoparticles (LNPs) and mRNA

[0199] Lipid nanoparticles (LNPs) were prepared for delivery of RNA. The illustrative LNP of the present examples were complexes containing Compound-B, and are referred to in the present examples and corresponding figures as “LUNAR”. LNPs were prepared by using a microfluidic device to mix appropriate volumes of lipids in ethanol with an aqueous phase containing EGFP or hGALC mRNA and then subjecting the mixture to downstream processing. For the encapsulation of mRNA by the lipid nanoparticles, the desired amount of RNA was dissolved in 5 mmol / L citric acid buffer (pH 3.5). Lipids having the desired molar concentrations were dissolved in ethanol. The mole concentrations for the constituent lipids were 0.16 mM Compound-B, 0.02 mM l,2-dioctadecanoyl-sn-glycerol-3 -phosphocholine (Avanti Polar Lipids, Alabaster, AL), 0.12 mM cholesterol (Avanti Polar Lipids, Alabaster, AL), and 0.05 mMl,2- dimyristoyl-sn-glycerol, methoxypolyethylene glycol, PEG chain molecular weight: 2000 (NOF America Corporation, White Plains, NY). At a 1 :3 flow ratio of ethanol: aqueous phases, the solutions were combined in a microfluidic device (Precision NanoSystems, Vancouver, Canada). The total combined flow rate was 12 mL / min per microfluidic chip. Lipid nanoparticles thus formed were purified by dialysis against phosphate buffer overnight using 100 kDa Spectra / Por Float-A-Lyzer ready to use dialysis device (Repligen, MA, US) followed by concentration using Amicon Ultra- 15 centrifugal filters (Merck Millipore). The particle size was determined bydynamic light scattering (ZEN3600, Malvern Instruments, UK). The encapsulation efficiency was calculated by determining the unencapsulated mRNA content by measuring the fluorescence upon the addition of RiboGreen (Molecular Probes, Eugene, OR) to the LNP slurry (Fi) and comparing this value to the total mRNA content obtained upon lysis of the LNPs with 1 % Triton X-100 (Ft), where percent encapsulation = (Ft - Fi) I Ft x 100.Animals

[0200] C57BL / 6J male mice at 2 months of age (purchased from Japan SLC, Shizuoka, Japan) (n = 60) and Sprague-Dawley rats (n = 15) were used for the experiments. The mice were maintained at 25°C with 55% humidity on a 12-h light-dark cycle and given free access to food and drinking water. All experimental procedures used in this study followed Japanese national guidelines on animal experimentation. Ethical approval and permission were obtained from The Animal Experimentation Committee of Kyoto University (MedKyo, 19219). Twitcher mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The genotypes were determined by analyzing DNA from the mouse tail that was extracted using Hypercool Primer & Probe (Nihon Gene Laboratory, Miyagi, Japan). Primers and probes used were as follows: forward primer, reverse primer, probe, FAM-aaTACCAGccTGGTTGaGTA-BHQ (SEQ ID NO: 166); and mutant probe, HEX- aaTaTcAGccTGGTTGaGTaA-BHQ (SEQ ID NO: 167). FAM, HEX, and BHQ are labels used in the FRET probes to detect the presence and quantifying the amount of specific target sequences. The signal was detected using Luna Universal Probe qPCR Master Mix (M3004, New England Biolabs, MA, USA) with LightCycler® 480 (Roche, Basel, Switzerland).Surgery

[0201] Adult mice were anesthetized with isoflurane for injections of the LNPs into the dorsal striatum. Unilateral stereotaxic injections (2 pl) of LUNAR carrying EGFP or hGALC mRNA were administered into the dorsal striatum (coordinates: 2.0 mm relative to bregma; 0.2 mm from the midline; 2.5 mm beneath the skull surface) using a 33-gauge syringe. For neonatal mice, injections of 0.5 pl of LUNAR-AGHZC mRNA were administered into the bilateral striatum using a 30-gauge syringe.Histological analysis of mouse brain tissues

[0202] LUNAR-injected mice were sacrificed at the indicated time points. Following perfusion with 4 % (w / v) paraformaldehyde in PBS, the brains were removed and subjected to overnight immersion in 4 % (w / v) paraformaldehyde in PBS at 4 °C. Tissues were sectioned usinga vibratome (Neo-LinearSlicer, DOSAKA, Kyoto, Japan) or embedded in paraffin for sectioning. Paraffin sections of 8 pm thickness were prepared using an HM 325 rotary microtome (Microm, Thermo Fisher Scientific, MA, USA).Immunohi stochemi stry

[0203] The following primary antibodies were used for immunohistochemical analysis: anti-NeuN (Millipore, ABN78, 1 :400), anti-Ibal (Wako, #019-19741, 1 :200), anti-GFAP (Invitrogen, #13-0300, 1 :200), anti-GST7t (MBL, #312, 1 :500), anti-01ig2 (Millipore, ab9610, 1 :300), anti-GFP (Abeam, abl3970, 1 :300), and anti-GALC (Abeam, ab232972, 1 : 1000). The sections were incubated overnight at 4 °C with primary antibodies and then processed for visualization. Histofine® Simple Stain (Nichirei Bioscience) was used as a secondary antibody for diaminobenzidine staining, and Alexa Fluor 488-or 594-conjugated antibodies (Thermo Fisher Scientific, MA, USA) were used for immunofluorescence. Sections were examined using a BZ- X710 fluorescence microscope (KEYENCE, Osaka, Japan) and an FV-1000 confocal laser scanning microscope (Olympus, Tokyo, Japan).Cultures of primary oligodendrocyte lineage cells and other glial cells

[0204] Primary rat OPCs were prepared according to methods described in previous publications25'27. Cerebral cortices from 1- to 2- day-old Sprague-Dawley rats (Shimizu Laboratory Supplies, Kyoto, Japan) were dissected, minced, and digested. Dissociated cells were plated in 75 cm2poly D-lysine-coated flasks and maintained in Dulbecco’s modified Eagle’s medium (DMEM) (Thermo Fisher, Waltham, MA, USA) containing 20 % heat-inactivated FBS and 1 % penicillin / streptomycin. When the cells became confluent (~ 10 days), the flasks were shaken for 1 h on an orbital shaker (220 rpm) at 37 °C to remove microglia. The flasks were then replaced with fresh medium and shaken overnight (~ 20 h). After incubation for 1 h at 37 °C on non-coated cell culture dishes, the non-adherent cells (OPCs) were replated at a density of 20,000 cells / cm2in Neurobasal™ Medium (Thermo Fisher, Waltham, MA, USA) containing 2 mM glutamine, 1 % penicillin / streptomycin, lO ng / mL PDGF-AA, lO ng / mL FGF-2, and 2 % B27 supplement onto poly DL-ornithine-coated plates. The culture medium was replaced with DMEM containing 1 % penicillin / streptomycin, 10 ng / mL CNTF, 15 nM T3, and 2 % B27 supplement for the OPCs to differentiate into mature oligodendrocytes. The OPCs and oligodendrocytes were treated with LUNAR-EGFP mRNA (0.2 pl / well), and the EGFP expression of these cells was observed using a BZ-X710 fluorescence microscope after 0, 8, and 24 h.Primary neuronal cell cultures

[0205] Cortical neuronal cultures were prepared from 17-day-old Sprague-Dawley rat embryos according to a method described in a previous publication25'27. Cortices were first dissected and dissociated. The cells were then plated into dishes coated with poly-D-lysine in DMEM containing 5 % FBS and 1 % penicillin / streptomycin at a density of 200,000 cells / cm2. At 24 h after seeding, the medium was changed to Neurobasal™ Medium (Thermo Fisher, Waltham, MA, USA) containing 0.5 mM L-glutamine, 1 % penicillin / streptomycin, and 2 % B- 27 supplement. Cultured neurons were used for the experiments 14 days after seeding.Cell line culture

[0206] Cells from the human oligodendrocyte cell line MO3.13 (RRID: CVCL D357) were cultured according to a method described previously28. MO3.13 cells were cultured for 48- 72 h in high glucose DMEM (Wako, Osaka, Japan, cat# 043-30085) with 10 % FBS (Gibco, Grand Island, NY, USA) and a mixture of penicillin 100 U / ml and streptomycin 100 pg / ml (Nacalai Tesque, Kyoto, Japan, cat# 26253-84) in a humidified incubator with 5 % CO2 at 37 °C. For cell maturation studies, 16-24 h after seeding cells, the culture medium was replaced with DMEM containing FBS 0 %, penicillin 100 U / ml, streptomycin 100 pg / mL, and 100 nM phorbol 12- myristate 13-acetate (Sigma-Aldrich, St. Louis, MO, USA, cat# P1585). Cells were allowed to mature for 3-7 days with a change in medium on alternate days. siRNA treatments

[0207] Seeded cells were incubated for 24 h and transfected with siRNA using Lipofectamine RNAiMAX reagent (Thermo Fisher Scientific, MA, USA), according to the manufacturer’s instructions. After 24 h of incubation, the medium was replaced with fresh medium, and the cells were treated with LUNAR EGFP mRNA for another 24 h. ON- TARGETp / z / .s siRNAs were used to knockdown LDLR, VLDLR, and ApoER2 (Horizon Discoveries, Cambridge, UK, cat#L-011073, L-003721, and L-011802, respectively). Silencer™ Select Negative Control #2 siRNA (Thermo Fisher Scientific, MA, USA, cat#4390846) was used as the control siRNA (negative control: NC).Quantitative Real-Time PCR (qPCR)

[0208] Total RNA was isolated using a QIAGEN RNeasy Mini Kit (Qiagen, Venlo, Nederland, #74106), reverse-transcribed into cDNA (PrimeScript™ RT Master Mix, Takara, Shiga, Japan, RR036), amplified, and quantified by SYBR Green (Thermofisher, MA, USA, cat#K0222) detection. All qPCRs were run with LightCycler® 480 (Roche). Relative mRNAexpression was normalized with the glyceraldehyde 3 -phosphate dehydrogenase (GAPDH) gene. All primers are listed in Table 2.Neuropathological analysis

[0209] To assess the distribution and efficiency of LUNAR-FGFF mRNA in vivo, two locations in each brain (n = 3) region injected with LUNAR were screened at 20* magnification using a BZ-X710 fluorescence microscope (KEYENCE, Osaka, Japan). Cells positive for cellspecific markers (Olig2, NeuN, Ibal, and GFAP) and EGFP-cell marker double-positive cells were counted using ImageJ software. The EGFP-positive ratio in vivo was calculated as (number of double-positive cells) / (number of cell marker-positive cells). For in vitro experiments, four areas in each well (n = 3) of neurons were screened, oligodendrocytes, OPCs, and GFAP at 0, 8, and 24 h after treatment with LUNAR-FGFF mRNA. Total and EGFP-positive cells were counted. The in vitro EGFP-positive ratio was calculated as (number of LUNAR-EGFP-positive cells) / (total number of cells). Tukey’s test was used to compare the statistical significance.Assessment of clinical symptoms

[0210] Body weight and neurological symptoms of mice at P30 and P35 were assessed using the following system for scoring the severity of the twitching, as was reported in a previous publication29: frequency: 1, rare; 2, intermittent; 3, constant; and severity: 1, asymptomatic; 2, mild; 3, mild moderate; 4, moderate; 5, severe. The final score is the sum of the two parameters.Materials for glycolipid analysis

[0211] P-D-Glucopyranosyl-l l ^ l j-N-lauroyl-D-c / yV / i / vi-sphingosine (GlcCer [dl8: l- C12:0]) and P-D-glucopyranosyl-(l^l)-D-erj7Aro-sphingosine-t / 5 (GlcSph-t / 5) were purchased from Avanti Polar Lipids (Alabaster, AL, USA). Liquid chromatography (LC)-electrospray ionization tandem mass spectrometry (ESLMS / MS) was performed using high-performance LC- grade acetonitrile, methanol, and distilled water purchased from Kanto Chemical Co., Inc. (Tokyo, Japan), chloroform and formic acid purchased from FUJIFILM Wako (Osaka, Japan), and ammonium formate purchased from Sigma-Aldrich, Japan.Lipid extraction for analysis of the glycolipid profile

[0212] Frozen tissue (half of the brain, approximately 1 g) was lyophilized. The lyophilized tissues comprising half of the brain (approximately 25 mg) and the frozen striatum (approximately 40 mg) were homogenized, and total lipids were extracted with a chlorofomrmethanol (C:M) (2: 1 [v / v], 3-5 ml) mixture added to 5 pmol / mg lyophilized tissue or to 1 pmol / mg frozen tissue of GlcCer (d!8: l-C12:0), and GlcSph-t / 5 served as an internalstandard. The extracts were dried under a flow of N2 gas and hydrolyzed for 2 h at room temperature in C:M (2: 1 [v / v], 2 ml) containing 0.1 M KOH. The reaction mixture was neutralized with 7.5 pL of glacial acetic acid. The neutralized reaction mixture was subjected to Folch’s partition, and the lower phase was dried under a flow of N2 gas. The resulting lipid films were suspended in C:M (2: 1, v / v) at a concentration of 20 pg of lyophilized tissue / pL or 100 pg of frozen tissue / pL, and aliquots were subjected to LC-ESI-MS / MS.LC-ESI-MS / MS for glycolipid analysis

[0213] LC-ESI-MS / MS was performed on an LC system (Nexera X2, Shimadzu, Kyoto, Japan) attached to a triple-quadrupole linear ion trap mass spectrometer (QTRAP4500; SCIEX, Tokyo, Japan). The LC-ESI-MS / MS datasets were analyzed using MultiQuant™ (ver. 2.1), and Analyst® (SCIEX) software programs. Target lipids were monitored in multiple reaction monitoring (MRM) mode using specific precursor-product ion pairs, as described in Table 2.

[0214] GalCer and GalSph were analyzed by hydrophilic interaction chromatography (HILIC)-ESI-MS / MS. HILIC enables the separation of glucosylceramide (GlcCer) and GalCer, and glucosylsphingosine (GlcSph) and GalSph30. The lipid extracts dissolved in C:M (2: 1, v / v) were diluted 10-fold with mobile phase A (acetonitrile:methanol:formic acid, 97:2: 1 [v / v / v], with 5 mM ammonium formate), and aliquots (10 pL) were applied to an Atlantis silica HILIC column (2.1 mm i.d.x150 mm, particle size, 3 pm; Waters, Milford, MA, USA) maintained at 40 °C. HILIC-ESI-MS / MS analysis for GalCer was performed according to a previously established method31,32with minor modifications. The mass spectrometer was set to positive ion mode (ion spray voltage, 5500 V; curtain gas pressure, 30 psi; nebulizer gas pressure, 50 psi; heating gaspressure, 30 psi; temperature, 100 °C) using MRM detection for targeted analysis. HILIC-ESI- MS / MS analysis for GalSph was performed according to a previously established method32,33with minor modifications. For this process, the mass spectrometer was set to positive ion mode (ion spray voltage, 5500 V; curtain gas pressure, 30 psi; nebulizer gas pressure, 70 psi; heating gas pressure, 80 psi; temperature, 700 °C) using MRM detection for targeted analysis. The ionization efficiencies of GlcCer and GalCer, and GlcSph and GalSph were similar under the implemented conditions. Peak areas were integrated and quantified relative to the associated internal standard.Statistical analysis

[0215] The experiments were performed independently 3-4 times. All quantitative data were analyzed using GraphPad Prism software version 7. Multiple comparisons were evaluated by a one-way ANOVA followed by the Tukey-Kramer test or Dunnett's test. All values are represented as mean±standard error of the mean (SEM). Statistical significance was defined as P<0.05.Primer Sequences for qPCR

[0216] Primer sequences for qPCR are shown in Table3.Table 3. Primer sequences for qPCRGene Forward primer Reverse primerEXAMPLE 1

[0217] This example describes EGFP mRNA expression patterns in the brain following lipid-mediated delivery.

[0218] To assess the distribution of mRNA transduced by LUNAR LNPs in the brain, LUNAR-FGFF mRNA was injected into the unilateral striatum of the brains of C57BL / 6J mice that were sacrificed 24 h later for the brain tissue to be removed for histological analysis. Results of the histological analysis showed EGFP proteins to be widely expressed mainly in the striatum (FIG. 1A, panels a-c). For example, EGFP was strongly expressed in small round cells in the corpus callosum (FIG. 1A, panel b) and the white matter of the striatum (FIG. 1A, panel c), suggesting EGFP expression in oligodendrocytes. EGFP expression in mice was observed after 8 h, peaked at 24 h to 3 days, and decreased after 7 days (FIG. 4). To further analyze which cells specifically express EGFP proteins, the percentage of EGFP-expressing cells that colocalized with cell-specific markers was evaluated: neurons (NeuN), astrocytes (GFAP), microglia (Ibal), and oligodendrocytes (Olig2 or GSTK). The expression of EGFP was seen to colocalize with the oligodendrocyte markers Olig2 or GSTK, but not with NeuN, GFAP, or Ibal (FIG. IB, panels d- h). Moreover, more than 65 % of Olig2 was seen to be colocalized with EGFP, which was significantly higher than the colocalization of NeuN, GFAP, or Ibal expression (FIG. ID, panel i). Next, the oligodendrocyte-selectivity of the LNP-mediated delivery was confirmed using primary cell cultures from brain tissues. Primary cultures of neurons, astrocytes, oligodendrocyte precursor cells (OPCs), and oligodendrocytes were prepared from the cortex of Sprague-Dawley rats and treated with LUNAR-FGFF mRNA. Time lapse imaging revealed that EGFP transferred with LUNAR-FGFF mRNA was gradually expressed only in oligodendrocytes, but not in neurons (data not shown). More than 30% of oligodendrocytes were seen to strongly express EGFP 24 h after treatment with LUNAR-FGFF mRNA (FIG. 1C, panel j). Other cells showed negligible expression of EGFP, thus indicating a significant difference from the EGFP expression in oligodendrocytes (FIGS. 1C-1D, panels k-n).

[0219] These results show efficient lipid nanoparticle (LNP)-mediated mRNA delivery to the brain.EXAMPLE 2

[0220] This example describes uptake of lipid-formulated RNA in the brain.

[0221] ApoE protein and LDLR have been reported to be associated with liposome uptake.First, we examined the association of LDLR, very low-density lipoprotein receptor (VLDLR), and apoprotein E receptor 2 (ApoER2) with LUNAR LNP uptake in the mouse brain. EGFP and LDLR were seen to colocalize with Olig2 -positive cells, while ApoER2 or VLDLR did not show colocalization (FIG. 2A, panels a— 1). In vitro protein expression analysis of the primary culture demonstrated significant expression of LDLR and VLDLR mRNA in oligodendrocytes comparedto that in other cell types (FIG. 5). Next, a key molecule involved in the uptake of LUNAR was analyzed using differentiated MO3.13 cells, a human oligodendrocytic cell line. LDLR knockdown significantly suppressed LUNAR uptake in differentiated MO3.13, while knockdown of VLDLR or ApoER2 did not have an effect on uptake (FIGS. 2B-2C, panels m-o). Moreover, the uptake of LUNAR did not occur in the absence of ApoE or fetal bovine serum (FBS) containing ApoE (Fig. 6). These data indicate that the uptake of LUNAR occurs via LDLR in the presence of ApoE.

[0222] In summary, these data show that the uptake of mRNA formulated with LUNAR LNPs into oligodendrocytes occurs via LDLR and ApoE receptor 2 (ApoER2).EXAMPLE 3

[0223] This example describes rescue of mutant mice by LNP-mediated delivery of humanGALC (hGALC) mRNA.

[0224] As mRNA can be delivered into oligodendrocytes with high efficiency and specificity by means of LUNAR LNPs, the therapeutic effect of the LUNAR-ZG^ZC mRNA in the treatment of Krabbe disease, which is associated with abnormalities in oligodendrocytes, was evaluated. The injection of LUNAR-AGAZC mRNA into the unilateral striatum of adult mice resulted in GALC proteins being expressed in oligodendrocytes in the injected hemisphere (FIG. 3 A, panels a-e). The injection of LUNAR-ZG^ZC mRNA into the unilateral striatum of the Krabbe disease model mice (a GALC-deficient mouse, GalcC', twitcher mice) at Pl resulted in a significant increase in the Olig2-positive area in the injected hemisphere compared to that on the control side at P35 (FIG. 3B, panels f-h). LUN AR. -AGAZE mRNA was also injected into the bilateral striatum of twitcher mice at PL The injected twitcher mice presented a significant decrease in the severity score compared to that of the control at P30 and P35, while exhibiting no significant difference in body weight (FIG. 3C, panel i; and data not shown). LUNAR-AGAZC mRNA injection extended the lifespan of twitcher mice up to 48 days, whereas that of the control mice was up to 42 days, thus showing a significant difference among the two groups (FIG. 3C, panel j).

[0225] These results show that LNP-mediated delivery of human GALC (hGALC) mRNA rescued or ameliorated the phenotype of twitcher mice, a mouse model of Krabbe disease.EXAMPLE 4

[0226] This example describes glycolipid analysis of twitcher and wild-type (WT) mice.

[0227] GALC hydrolyzes galactosylceramide (GalCer) to galactose and ceramide and galactosylsphingosine (GalSph, psychosine) to galactose and sphingosine. Glycolipid profile analyses of WT, non-treated twitcher mice, and treated twitcher mice were performed at P14. Levels of GalCer were lower in non-treated twitcher mice and significantly increased in treated twitcher mice compared to those in WT mice (FIG. 3D, panel k). However, GalSph levels were elevated in both treated and non-treated twitcher mice compared to those in WT mice and did not differ significantly between the two groups (FIG. 3D, panel k).

[0228] These results show that treatment of twitcher mice with hGALC mRNA delivered via LNPs increased levels GalCer levels as compared to untreated twitcher mice, with GalCer levels restored to levels comparable to GalCer levels seen in WT mice.DISCUSSION OF EXAMPLES 1-4

[0229] The field of drug delivery systems (DDSs) involving the use of LNPs has made remarkable progress. However, technologies having high specificity in delivering drugs to particular cell types have not been developed. Although previous studies investigated the efficiency of luciferase expression on an organ-by-organ basis, there are few reports that present detailed histological analyses, especially of the brain. In this study, a histological analysis of cell types transduced by EGFP mRNA complexed with lipid nanoparticles (LNPs) for delivery to the brain was conducted. LUNAR was found to be a cell type-specific nanocarrier having high efficiency of transduction and specificity to oligodendrocytes.

[0230] In general, LNPs containing cholesterol tend to be taken up by the liver. Previous studies have reported that the uptake of LNPs by the liver occurs via LDLR by means of their binding to ApoE. Considering the importance of LDLR in the process of LNP uptake, apoprotein receptors in the brain were examined and oligodendrocytes were found to have a high expression of LDLR. Furthermore, in vitro cell line studies showed the uptake of LUNAR to occur via LDLR in the presence of ApoE. Without being limited by theory, it was hypothesized that oligodendrocytes, which specifically express LDLR in the brain, may efficiently take up LUNAR bound to ApoE. In the present study, cell-specific nanocarriers were combined with therapeutic mRNA, resulting in successful introduction of mRNA into target cells.

[0231] Moreover, it was demonstrated herein that the characteristic of oligodendrocytespecific uptake of LUNAR could be utilized to make it a useful DDS for mRNA-based treatment of Krabbe disease. LUNAR was used in the studies described herein to deliver GN / .C-mRNA specifically into oligodendrocytes in twitcher mice and improvement in motor function and survival was observed in the treated mice. GalCer is enriched in myelin and is important forstabilizing myelin. GalCer is one of the precursors of cytotoxic GalSph production. The accumulation of cytotoxic GalSph in the brain has been proposed to play a central role in the pathological mechanism of Krabbe disease. It has been reported that GalSph levels increased whereas GalCer levels decreased in the brains from patients with Krabbe disease. Without being limited by theory, it was hypothesized that the reduction in GalCer levels might be due to demyelination. Results of the present study do not show a decrease in GalSph levels, but show an increase in GalCer levels in treated twitcher mice. In the central nervous system, GalCer is synthesized in oligodendrocytes; hence, without being limited by theory, the observed increase in GalCer levels in treated twitcher mice may be interpreted as being due to the recovery of oligodendrocytes. In addition, the observed increase in GalSph levels may be considered to be due to conversion of excessive GalCer into GalSph.

[0232] Results provided herein thus show cationic lipid formulations, such as LUNAR, to be capable of delivering mRNAs to oligodendrocytes with high efficiency and specificity, where efficient gene transfer was previously difficult. Lipid formulation disclosed herein can therefore be considered novel mRNA therapeutic tools for diseases affecting oligodendrocytes, such as multiple system atrophy, Alexander disease, PMD, adrenoleukodystrophy, and other leukodystrophies. In addition, the present lipid formulations have the advantage of being capable of mRNA transduction without involving the use of a viral vector, and hence does not require consideration of virus-induced inflammation or infectivity. The development of mRNA-based vaccines against COVID-19 has led to increased attention towards mRNA therapy because of concerns regarding a higher risk of thrombosis associated with viral vector-based vaccines compared to the risk posed by mRNA-based vaccines.

[0233] LNP -based nanocarriers targeting specific cells has potential applications in the treatment of many neurological diseases owing to its ability to specifically control various cell types in the brain.REFERENCES1. Finkel, R. S. et al. Nusinersen versus Sham Control in Infantile-Onset Spinal Muscular Atrophy. N. Engl. J. Med. 377, 1723-1732, (2017).2. Mercuri, E. et al. Nusinersen versus Sham Control in Later-Onset Spinal Muscular Atrophy. N. Engl. J. Med. 378, 625-635, (2018).3. Moser, H. W. Peripheral nerve involvement in Krabbe disease: a guide to therapy selection and evaluation. Neurology. 61, 201-202, (2006).4. Engelen, M. et al. X-linked adrenoleukodystrophy (X-ALD): clinical presentation and guidelines for diagnosis, follow-up and management. Orphanet. J. Rare. Dis. 7, 51, (2012).5. von Jonquieres, G. et al. Recombinant Human Myelin-Associated Glycoprotein Promoter Drives Selective AAV-Mediated Transgene Expression in Oligodendrocytes. Front. Mol. Neurosci. 9, 13, (2016).Powell, S. K. et al. Characterization of a novel adeno-associated viral vector with preferential oligodendrocyte tropism. Gene. Ther. 23, 807-814, (2016). Li, H. et al. Gene suppressing therapy for Pelizaeus-Merzbacher disease using artificial microRNA. JCI. Insight. 4, (2019). Georgiou, E. et al. Gene therapy targeting oligodendrocytes provides therapeutic benefit in a leukodystrophy model. Brain. 140, 599-616, (2017). Tamaru, M. et al. Application of apolipoprotein E-modified liposomal nanoparticles as a carrier for delivering DNA and nucleic acid in the brain. Int. J. Nanomedicine . 9, 4267- 4276, (2014). Ramaswamy, S. et al. Systemic delivery of factor IX messenger RNA for protein replacement therapy. Proc. Natl. Acad. Sci. U.S.A. 114, E1941-el950, (2017). Yanagi, T. et al. Lipid Nanoparticle-mediated siRNA Transfer Against PCTAIRE1 / PCTK1 / Cdkl6 Inhibits In Vivo Cancer Growth. Mol. Ther. Nucleic. Acids. 5, e327, (2016). Suzuki, K. & Suzuki, Y. Globoid cell leucodystrophy (Krabbe's disease): deficiency of galactocerebroside beta-galactosidase. Proc Natl Acad Sci U SA. 66, 302-309, (1970). Nagara, H., Ogawa, H., Sato, Y, Kobayashi, T. & Suzuki, K. The twitcher mouse: degeneration of oligodendrocytes in vitro. Brain. Res. 391, 79-84, (1986). Tanaka, K. & Webster, H. D. Effects of psychosine (galactosylsphingosine) on the survival and the fine structure of cultured Schwann cells. J. Neuropathol. Exp. Neurol. 52, 490-498, (1993). Taniike, M. et al. An apoptotic depletion of oligodendrocytes in the twitcher, a murine model of globoid cell leukodystrophy. J Neuropathol Exp Neurol. 58, 644-653, (1999). Akita, H. et al. Molecular Tuning of a Vitamin E-Scaffold pH-Sensitive and Reductive Cleavable Lipid-like Material for Accelerated in Vivo Hepatic siRNA Delivery. ACS. Biomater. Sci. Eng. 1, 834-844, (2015). Baumann, N. A., Harpin, M. L. & Bourre, J. M. Long chain fatty acid formation: key step in myelination studied in mutant mice. Nature. 227, 960-961, (1970). Li, Y. et al. Genetic ablation of acid ceramidase in Krabbe disease confirms the psychosine hypothesis and identifies a new therapeutic target. Proc. Natl. Acad. Sci. U.S.A. 116, 20097-20103, (2019). Yaghootfam, C., Gehrig, B., Sylvester, M., Gieselmann, V. & Matzner, U. Deletion of fatty acid amide hydrolase reduces lyso-sulfatide levels but exacerbates metachromatic leukodystrophy in mice. J. Biol. Chem. 297, 101064, (2021). Igisu, H. & Suzuki, K. Progressive accumulation of toxic metabolite in a genetic leukodystrophy. Science. 224, 753-755, (1984). Svennerholm, L., Vanier, M. T. & Mansson, J. E. Krabbe disease: a galactosylsphingosine (psychosne) lipidosis. J. Lipid. Res. 21, 53-64, (1980). Schulte, S. & Stoffel, W. Ceramide UDPgalactosyltransferase from myelinating rat brain: purification, cloning, and expression. Proc. Natl. Acad. Sci. U.S.A. 90, 10265-10269, (1993). Rzymski, P, Perek, B. & Flisiak, R. Thrombotic Thrombocytopenia after COVID-19 Vaccination: In Search of the Underlying Mechanism. Vaccines (Basel). 9, (2021). Rafi, M. A., Luzi, P. & Wenger, D. A. Conditions for combining gene therapy with bone marrow transplantation in murine Krabbe disease. Bioimpacts. 10, 105-115, (2020). Kaji, S. et al. Pathological Endogenous a-Synuclein Accumulation in Oligodendrocyte Precursor Cells Potentially Induces Inclusions in Multiple System Atrophy. Stem. Cell. Reports. 10, 356-365, (2018). Kaji, S. et al. B CAS 1 -positive immature oligodendrocytes are affected by the a-synuclein- induced pathology of multiple system atrophy. Acta. Neuropathol. Commun. 8, 120, (2020). Maki, T. et al. Potential interactions between pericytes and oligodendrocyte precursor cells in perivascular regions of cerebral white matter. Neurosci Lett. 597, 164-169, (2015).28. Hoshino, T., Yamakado, H., Takahashi, R. & Matsuzawa, S. I. Susceptibility to erastin- induced ferroptosis decreases during maturation in a human oligodendrocyte cell line. FEBS. Open. Bio. 10, 1758-1764, (2020).29. Shen, J. S., Watabe, K., Ohashi, T. & Eto, Y. Intraventricular administration of recombinant adenovirus to neonatal twitcher mouse leads to clinicopathological improvements. Gene. Ther. 8, 1081-1087, (2001).30. von Gerichten, J. et al. Diastereomer-specific quantification of bioactive hexosylceramides from bacteria and mammals. J. Lipid. Res. 58, 1247-1258, (2017).31. Akiyama, H. et al. Glucocerebrosidases catalyze a transgalactosylation reaction that yields a newly-identified brain sterol metabolite, galactosylated cholesterol. J. Biol. Chem. 295, 5257-5277, (2020).32. Ikuno, M. et al. GBA haploinsufficiency accelerates alpha-synuclein pathology with altered lipid metabolism in a prodromal model of Parkinson's disease. Hum. Mol. Genet. 28, 1894-1904, (2019).33. Nakanishi, E. et al. Impact of Gba2 on neuronopathic Gaucher's disease and a-synuclein accumulation in medaka (Oryzias latipes). Mol. Brain. 14, 80, (2021).SEQUENCESSEQ ID NO:1 - Human GALC ORF - RNAAUGGCUGAGUGGCUGCUGAGCGCCAGCUGGCAGAGGAGGGCCAAGGCCAUGACCGCCGCCGCCGGCAGCGCCGGCAGGGCCGCCGUGCCCCUGCUGCUGUGCGCCCUGCUCGCCCCUGGCGGCGCCUACGUGCUGGACGACAGCGACGGCCUGGGCCGGGAGUUCGACGGCAUCGGCGCCGUGAGCGGCGGCGGCGCCACCAGCAGGCUGCUGGUGAACUACCCCGAGCCCUACAGGAGCCAGAUCCUGGACUACCUGUUCAAGCCCAACUUCGGCGCCAGCCUGCACAUCCUGAAGGUGGAGAUCGGCGGCGACGGCCAGACCACCGACGGCACCGAGCCCAGCCACAUGCACUACGCCCUGGACGAGAACUACUUCAGGGGCUACGAGUGGUGGCUGAUGAAGGAGGCCAAGAAGAGGAACCCCAACAUCACCCUGAUCGGCCUGCCCUGGAGCUUCCCCGGCUGGCUGGGCAAGGGCUUCGACUGGCCCUACGUGAACCUGCAGCUGACCGCCUACUACGUGGUGACCUGGAUCGUGGGCGCCAAGAGGUACCACGACCUGGACAUCGACUACAUCGGCAUCUGGAACGAGAGGAGCUACAACGCCAACUACAUCAAGAUCCUGAGGAAGAUGCUGAACUACCAGGGCCUGCAGAGGGUGAAGAUCAUCGCCAGCGACAACCUGUGGGAGAGCAUCAGCGCCAGCAUGCUGCUGGACGCCGAGCUGUUCAAGGUGGUGGACGUGAUCGGCGCCCACUACCCCGGCACCCACAGCGCCAAGGACGCCAAGCUGACCGGCAAGAAGCUGUGGAGCAGCGAGGACUUCAGCACCCUGAACAGCGACAUGGGCGCCGGCUGCUGGGGCAGGAUCCUGAACCAGAACUACAUCAACGGCUACAUGACCAGCACCAUCGCCUGGAACCUGGUGGCCAGCUACUACGAGCAGCUGCCCUACGGCAGGUGCGGCCUGAUGACCGCCCAGGAGCCCUGGAGCGGCCACUACGUGGUGGAGAGCCCCGUGUGGGUGAGCGCCCACACCACCCAGUUCACCCAGCCCGGCUGGUACUACCUGAAGACCGUGGGCCACCUGGAGAAGGGCGGCAGCUACGUGGCCCUGACCGACGGCCUGGGCAACCUGACCAUCAUCAUCGAGACAAUGAGCCACAAGCACAGCAAGUGCAUCCGGCCCUUCCUGCCCUACUUCAACGUGAGCCAGCAGUUCGCCACCUUCGUGCUGAAGGGCAGCUUCAGCGAGAUCCCCGAGCUGCAGGUGUGGUACACCAAGCUGGGCAAGACCAGCGAGAGGUUCCUGUUCAAGCAGCUGGACAGCCUGUGGCUGCUGGACAGCGACGGCAGCUUCACCCUGAGCCUGCACGAGGACGAGCUGUUCACCCUGACCACCCUGACCACCGGCAGGAAGGGCAGCUACCCUCUGCCUCCCAAGAGCCAGCCCUUCCCCAGCACCUACAAGGACGACUUCAACGUGGACUACCCCUUCUUCAGCGAGGCCCCUAACUUCGCCGACCAGACCGGCGUGUUCGAGUACUUCACCAACAUCGAGGACCCCGGCGAGCACCACUUCACCCUGAGGCAGGUGCUGAACCAGAGGCCCAUCACCUGGGCCGCCGACGCCAGCAACACCAUCAGCAUCAUCGGCGACUACAACUGGACCAACCUGACCAUCAAGUGCGACGUGUAUAUCGAGACACCUGACACCGGCGGCGUGUUCA

[0234] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which this invention belongs.

[0235] Any and all references and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, that have been made throughout this disclosure are hereby incorporated herein in their entirety for all purposes. Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.

Claims

CLAIMSWhat is claimed is:

1. A method for treating a galactocerebrosidase (GALC) deficiency in a subject, the method comprising delivering to oligodendrocytes of the subject a polynucleotide encoding a GALC, wherein the polynucleotide comprises one or more of: (a) chemically-modified nucleotides, (b) a non-naturally occurring nucleotide sequence encoding the GALC, or (c) a recombinant sequence; and further wherein the polynucleotide is effective to increase GALC activity in the oligodendrocytes.

2. A method for delivering a polynucleotide to oligodendrocytes of a subject, the method comprising administering to the subject a lipid nanoparticle (LNP) comprising the polynucleotide, wherein (i) the LNP comprises an ionizable cationic lipid; (ii) the administered polynucleotide is expressed in the oligodendrocytes of the subject; and optionally (iii) the polynucleotide encodes a GALC.

3. The method of claim 1 or 2, wherein the polynucleotide comprises a codon-optimized region encoding GALC as compared to SEQ ID NO: 24 or 25.

4. The method of any one of claims 1-3, wherein the polynucleotide comprises a sequence having at least 80% identity to the sequence of SEQ ID NO: 1.

5. The method of any one of claims 1-4, wherein the GALC comprises: (a) an amino acid sequence with a sequence identity of at least 80%, 85%, 90%, or 95% to SEQ ID NO:26; or (b) the amino acid sequence of SEQ ID NO: 26.

6. The method of any one of claims 1-4, wherein the GALC is a GALC polypeptide means for providing GALC activity to a cell.

7. The method of any one of claims 1-6, wherein the GALC deficiency comprises Krabbe disease.

8. The method of any one of claims 1-7, wherein the polynucleotide comprises RNA.

9. The method of any one of claims 1-8, wherein: (a) the polynucleotide comprises chemically-modified nucleotides; and (b) the chemically modified nucleotides comprise one or more of 5-hydroxycytidine, 5-methylcytidine, 5-hydroxymethylcytidine, 5-carboxycytidine, 5- formylcytidine, 5-methoxycytidine, 5-propynylcytidine, 2-thiocytidine, 5-hydroxyuridine, 5-methyluridine, 5,6-dihydro-5-methyluridine, 2'-0-methyluridine, 2'-O-methyl-5-methyluridine, 2'-fluoro-2'-deoxyuridine, 2'-amino-2'-deoxyuridine, 2'-azido-2'-deoxyuridine, 4-thiouridine, 5- hydroxymethyluridine, 5-carboxyuridine, 5-carboxymethylesteruridine, 5-formyluridine, 5- methoxyuridine, 5-propynyluridine, 5 -bromouridine, 5-iodouridine, 5-fluorouridine, pseudouridine, 2'-O-methyl-pseudouridine, N^hydroxypseudouridine, N^methylpseudouridine, 2'-O-methyl-N1-methylpseudouridine, N^ethylpseudouridine, N^hydroxymethylpseudouridine, arauridine, N6-methyladenosine, 2-aminoadenosine, 3 -methyladenosine, 7-deazaadenosine, 8- oxoadenosine, inosine, thienoguanosine, 7-deazaguanosine, 8-oxoguanosine, or 6-0- methylguanosine.

10. The method of claim 9, wherein the chemically modified nucleotides comprise N1- methylpseudouridines.

11. The method of claim 9, wherein the chemically modified nucleotides comprise 5- methoxyuridines.

12. The method of any one of claims 1-11, wherein at least 1% or at least 50% of the nucleotides in the polynucleotide are chemically-modified nucleotides.

13. The method of any one of claims 1-12, wherein the polynucleotide further comprises a 5’ UTR.

14. The method of claim 13, wherein the 5’ UTR comprises the sequence of any of SEQ ID NO: 13, SEQ ID NO: 14, or SEQ ID NOs:28-104.

15. The method of claim 14, wherein the 5’ UTR comprises the sequence of SEQ ID NO: 13.

16. The method of claim 14, wherein the 5’ UTR comprises the sequence of SEQ ID NO: 14.

17. The method of any one of claims 1-16, wherein the polynucleotide further comprises a 3’ UTR.

18. The method of claim 17, wherein the 3’ UTR comprises the sequence of any of SEQ ID NO: 17 or SEQ ID NOs: 105-150.

19. The method of claim 18, wherein the 3’ UTR comprises the sequence of SEQ ID NO: 17.

20. The method of any one of claims 1-19, wherein the polynucleotide further comprises a poly- A tail or a poly-C tail.

21. The method of claim 20, wherein the polynucleotide comprises a poly-A tail having a length of about 60 nucleotides to about 120 nucleotides, about 90 nucleotides to about 110 nucleotides, about 95 nucleotides to about 100 nucleotides, or about 97 nucleotides.

22. The method of any one of claims 1-21, wherein the polynucleotide further comprises a 5’ cap.

23. The method of claim 22, wherein the 5’ cap has a Cap 1 structure, a Cap 1 (m6A) structure, a Cap 2 structure, or a Cap 0 structure.

24. The method of any one of claims 1-23, wherein the polynucleotide is formulated with a pharmaceutically acceptable carrier.

25. The method of claim 24, wherein the pharmaceutically acceptable carrier comprises a lipid formulation.

26. The method of claim 25, wherein the lipid formulation comprises a transfection reagent, a lipoplex, a liposome, a lipid nanoparticle (LNP), a polymer-based carrier, an exosome, a lamellar body, a micelle, or an emulsion.

27. The method of claim 25 or 26, wherein the lipid formulation comprises a cationic liposome, a nanoliposome, a proteoliposome, a unilamellar liposome, a multilamellar liposome, a ceramide-containing nanoliposome, or a multivesicular liposome.

28. The method of claim 25 or 26, wherein the lipid formulation comprises a lipid nanoparticle (LNP).

29. The method of any one of claims 25-28, wherein the lipid formulation comprises one or more cationic lipids.

30. The method of claim 29, wherein the one or more cationic lipids comprises one or more of 5-carboxyspermylglycinedioctadecylamide (DOGS), 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-l-propanaminium (DOSPA), l,2-Dioleoyl-3- Dimethylammonium -Propane (DODAP), l,2-Dioleoyl-3 -Trimethylammonium -Propane (DOTAP), l,2-distearyloxy-N,N-dimethyl-3-aminopropane (DSDMA), 1, 2-di oleyloxy -N,N- dimethyl-3 -aminopropane (DODMA), l,2-dilinoleyloxy-N,N-dimethyl-3 -aminopropane (DLinDMA), l,2-dilinolenyloxy-N,N-dimethyl-3 -aminopropane (DLenDMA), N-di oleyl -N,N- dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide(DDAB), N-(l,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 3-dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-l-(cis,cis-9,12-oc- tadecadienoxy)propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethy 1-1 -(ci s, ci s-9 1 -2 '-octadecadi enoxy)propane (CpLinDMA), N,N-dimethyl-3 ,4- dioleyloxybenzyl amine (DMOBA), l,2-N,N'-dioleylcarbamyl-3 -dimethylaminopropane (DOcarbDAP), 2,3-Dilinoleoyloxy-N,N-dimethylpropylamine (DLinDAP), 1,2-N,N'- Dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), l,2-Dilinoleoylcarbamyl-3- dimethylaminopropane (DLinCDAP), 2,2-dilinoleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-K-DMA), or 2,2-dilinoleyl-4-dimethylaminoethyl-[l,3]-dioxolane (DLin-K-XTC2- DMA).

31. The method of claim 29, wherein the one or more cationic lipids comprises an ionizable cationic lipid.

32. The method of claim 31, wherein the ionizable cationic lipid has a structure of formula I:or a pharmaceutically acceptable salt or solvate thereof, wherein R5and R6are each independently selected from the group consisting of a linear or branched C1-C31 alkyl, C2-C31 alkenyl or C2-C31 alkynyl and cholesteryl; L5and L6are each independently selected from the group consisting of a linear C1-C20 alkyl and C2-C20 alkenyl; X5is -C(O)O-, whereby -C(O)O-R6is formed or -OC(O)- whereby -OC(O)-R6is formed; X6is -C(O)O- whereby -C(O)O-R5is formed or -OC(O)- whereby -OC(O)-R5is formed; X7is S or O; L7is absent or lower alkyl; R4is a linear or branched Ci-Ce alkyl; and R7and R8are each independently selected from the group consisting of a hydrogen and a linear or branched Ci-Ce alkyl.

33. The method of claim 31, wherein the ionizable cationic lipid is selected from Table 1.

34. The method of claim 33, wherein the ionizable cationic lipid is:

35. The method of any one of claims 25-34, wherein the lipid formulation comprises an anionic lipid, a zwitterionic lipid, a neutral lipid, a steroid, a polymer conjugated lipid, a phospholipid, a glycolipid, or a combination thereof.

36. The method of any one of claims 25-34, wherein the lipid formulation comprises a helper lipid.

37. The method of claim 36, wherein the helper lipid is selected from dioleoylphosphatidyl ethanolamine (DOPE), dimyristoylphosphatidyl choline (DMPC), distearoylphosphatidyl choline (DSPC), dimyristoylphosphatidyl glycerol (DMPG), dipalmitoyl phosphatidylcholine (DPPC), and phosphatidylcholine (PC).

38. The method of claim 37, wherein the helper lipid is distearoylphosphatidylcholine (DSPC).

39. The method of any one of claims 25-38, wherein the lipid formulation comprises cholesterol.

40. The method of any one of claims 25-39, wherein the lipid formulation comprises a polyethylene glycol (PEG)-lipid conjugate.

41. The method of claim 40, wherein the PEG-lipid conjugate is a PEG-DMG, optionally wherein the PEG-DMG is PEG2000-DMG.

42. The method of any one of claims 25-41, wherein the lipid formulation comprises about 40 mol% to about 60 mol% of the ionizable cationic lipid, about 4 mol% to about 16 mol% DSPC, about 30 mol% to about 47 mol% cholesterol, and about 0.5 mol% to about 3 mol% PEG2000-DMG.

43. The method of any one of claims 25-41, wherein the lipids of the lipid formulation and the polynucleotides are in a total lipidmucleic acid molecule weight ratio of about 50: 1 to about 10: 1.

44. The method of any one of claims 25-43, wherein the polynucleotide is encapsulated within the lipid formulation or lipid nanoparticle (LNP).

45. The method of any one of claims 25-43, wherein the polynucleotide is complexed to the lipid formulation or lipid nanoparticle (LNP).

46. The method of any one of claims 28-45, wherein the lipid nanoparticle has a size of less than about 200 nm, less than about 150 nm, less than about 100 nm, or about 55 nm to about 90 nm.

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