WO2024151770A2

WO2024151770A2 - Compositions and methods for the treatment, prevention, and/or amelioration of cognitive decline

Info

Publication number: WO2024151770A2
Application number: PCT/US2024/011093
Authority: WO
Inventors: Shelley L. Berger; Nancy BONINI; Gabor EGERVARI; Naemeh POURSHAFIE; Desiree ALEXANDER; Connor HOGAN
Original assignee: University of Pennsylvania Penn
Current assignee: University of Pennsylvania Penn
Priority date: 2023-01-11
Filing date: 2024-01-10
Publication date: 2024-07-18
Anticipated expiration: 2025-07-11
Also published as: WO2024151770A3

Abstract

The present disclosure relates, in part, to a recombinant viral vector comprising an expression cassette comprising a nucleic acid encoding acetyl-CoA synthetase 2 (ACSS2) and an expression control sequence operably linked to the nucleic acid, and pharmaceutical compositions thereof. In another aspect, the present disclosure relates to antisense oligonucleotide compositions. In certain embodiments, the antisense oligonucleotide compositions at least partially inhibit degradation of ACSS2 mRNA transcripts. In another aspect, the present disclosure relates to nucleic acid-lipid particles comprising a nucleic acid encoding ACSS2 and/or antisense oligonucleotide compositions. In another aspect, the present disclosure relates to methods of treating, preventing, and/or ameliorating cognitive decline in a subject in need thereof.

Description

TITLE OF THE INVENTION

Compositions and Methods for the Treatment, Prevention, and/or Amelioration of Cognitive

Decline

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Patent Application No. 63/479,468, filed January 11, 2023, which is incorporated herein by reference in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AA027202 and AA028577 awarded by the National Institutes of Health. The government has certain rights in the invention.

SEQUENCE LISTING

The XML filed named “046483-7417WO1 - Sequence Listing.xml” created on January 9, 2024, comprising 24.5 Kbytes, is hereby incorporated herein by reference in its entirety.

BACKGROUND

In a number of models of neurodegeneration, global histone acetylation is decreased in the affected neuronal tissue. Histone acetylation is controlled by the antagonistic actions of two protein families, the histone acetyltransferases (HATs) and the histone deacetylases (HDACs). Acetyl-CoA is the substrate used by HATs to generate histone acetylation by transferring the acetyl-group from acetyl-CoA to histone lysine residues.

Metabolic production of acetyl-CoA, catalyzed by acetyl-CoA synthetase 2 (ACSS2), is linked to histone acetylation and gene regulation. ACSS2 is recruited to specific promoters and maintains a local pool of acetyl-CoA that fuels histone acetylation and drives the expression of key neuronal genes that regulate learning and memory. ACSS2 is required for brain histone acetylation as well as learning and memory in vivo, and it has recently been shown that loss of protective histone acetylation potentially underlies Alzheimer’s disease (AD) and related dementia in humans.

Thus, there is a need in the art for compositions and/or methods for overexpressing and/or upregulating the expression of ACSS2 and/or promoting histone acetylation in a subject for the treatment, prevention, and/or amelioration of cognitive decline associated with neurodegenerative diseases or disorders ( .g., Alzheimer’s disease), aging, and trauma, inter alia. The present disclosure addresses this need.

SUMMARY OF THE INVENTION

In one aspect, the present disclosure provides a recombinant viral vector, the vector comprising:

(a) an expression cassette comprising a nucleic acid encoding acetyl-CoA synthetase 2 (ACSS2); and

(b) an expression control sequence operably linked to the nucleic acid.

In another aspect, the present disclosure provides an antisense oligonucleotide composition, wherein the antisense oligonucleotide comprises a nucleic acid sequence which is at least partially complementary to at least a portion of a 3 ’-untranslated region (3’-UTR) of an acetyl-CoA synthetase 2 (ACSS2) mRNA transcript. In certain embodiments, the nucleic acid sequence comprises at least one chemically modified nucleoside or intemucleoside linkage.

In another aspect, the present disclosure provides a composition comprising one or more nucleic acid-lipid particles, wherein each nucleic acid-lipid particle comprises:

(a) a cationic lipid;

(b) a non-cationic lipid;

(c) a conjugated lipid that inhibits aggregation of two or more nucleic acid lipid particles; and at least one selected from the group consisting of:

(d) a nucleic acid encapsulated within the nucleic acid-lipid particle, wherein the nucleic acid encodes acetyl-CoA synthetase 2 (ACSS2); and

(e) at least one antisense oligonucleotide of the present disclosure.

In another aspect, the present disclosure provides a pharmaceutical composition comprising the recombinant viral vector of the present disclosure, the antisense oligonucleotide composition of the present disclosure, or the nucleic acid-lipid particle composition of the present disclosure and a pharmaceutically acceptable carrier.

In another aspect, the present disclosure provides a method of treating, preventing, and/or ameliorating cognitive decline in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one recombinant viral vector of the present disclosure.

In another aspect, the present disclosure provides a method of treating, preventing, and/or ameliorating cognitive decline in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one antisense oligonucleotide composition of the present disclosure.

In another aspect, the present disclosure provides a method of treating, preventing, and/or ameliorating cognitive decline in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one nucleic acid-lipid particle composition of the present disclosure.

In another aspect, the present disclosure provides a method of treating, preventing, and/or ameliorating cognitive decline in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one pharmaceutical composition of the present disclosure.

In another aspect, the present disclosure provides a method of treating, preventing, and/or ameliorating cognitive decline in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of acetic acid, or a salt, solvate, or pharmaceutical composition thereof.

BRIEF DESCRIPTION OF THE FIGURES

The drawings illustrate generally, by way of example, but not by way of limitation, various embodiments of the present application.

FIGs. 1A-1F show that ACSS2 overexpression (OE) improves memory in aged mice. FIG. 1A: experimental design for ACSS2 OE in aged mice. FIGs. 1B-1C: contextual recall at 24-hour (FIG. IB) and 48-hour (FIG. 1C). FIG. ID: acquisition (n= 21 ACSS2 OE and n=17) mock from 3 independent batches, t-test, p**<0.01(three independent batches). FIG. IE: Y- maze, total arm entries. FIG, IF: alternation behavior were measured during an 8-min session (n^:=:9, two independent batches).

FIGs. 2A-2D show that ACSS2 overexpression increases H3K9ac levels in the aged hippocampus. FIG. 2A: untargeted histone post-translational mass-spectrometry showing a significant increase in H3K9ac 30 minutes after remote context recall, t-test, p**<0.01. FIG. 2B: Western blot analysis. FIG. 2C: quantification of ACSS2 levels. FIG. 2D: G3K9ac levels, normalized to actin levels, t-test. p*<0.05.

FIGs. 3A-3E show that ACSS2 OE reduces insoluble mouse tau pathology in neurons. FIG. 3A: ACSS2 OE in primary hippocampal neurons at different days in vitro (DIV). FIG. 3B: experimental design for ACSS2 OE in primary neurons. FIG. 3C: DIV 21 neuronal cultures stained with a mouse tau-specific antibody (R2295M). FIG. 3D: in vitro AD-tau seeding measured in a blinded manner. FIG. 3E: nuclear cell number. Average signal intensity was automatically quantified from 12 fields of views per replicate, unpaired t-test, ***p <0.001.

FIGs. 4A-4J show that ACSS2 OE ameliorates AD-tau related transcriptomic changes in neurons. FIG. 4A: Principal Component Analysis (PCA) plot. FIG. 4B: number of significantly differential genes (adjusted p-value <0.05). FIGs. 4C-4F: volcano plots from RNA-sequencing. FIGs. 4G-4J: Top gene ontology (GO) terms of significantly differentially expressed genes (DEGs).

FIGs. 5A-5E shows that the AD-Tau model recapitulates epigenetic changes observed in human AD. FIG. 5 A: schematic of stereotactic injections. FIG. 5B: immunohistochemistry showing hippocampal Tau phosphorylation in AD-Tau injected but not PBS-injected mice. FIG. 5C: H3K27ac enrichment at Bub3 gene in the hippocampus of PBS/AD-Tau injected mice (top) and in young/old/AD humans (bottom). FIG. 5D: H3K27ac peaks most specific to Tau are strongly enriched at genes previously shown to exhibit dysregulated H3K27ac in human brain. FIG. 5E: GO analysis of H3K27ac peaks induced by AD-Tau injection in mouse hippocampus.

FIGs. 6A-6D show that ACSS2 KO exacerbates learning and memory impairments in AD-Tau injected mice. FIG. 6A: schematic of contextual fear conditioning. FIG. 6B: freezing levels during 24 hour recall and remote recall. FIG. 6C: schematic of object location memory (OLM). FIG. 6D: discrimination index during OLM recall.

FIGs. 7A-7B show transcriptional changes in AD-Tau injected ACSS2 KO mice. FIG. 7A: volcano plots showing significantly increased and decreased genes in various comparisons of PBS/ AD-Tau injected WT/ACSS2 KO mice. FIG. 7B: GO analysis of differential genes in the WT PBS vs ACSS2 KO AD-Tau comparison.

FIGs. 8A-8E depict single nucleic RNAseq identified loss and transcriptional dysregulation of Cajal-Retzius neurons in AD-Tau injected ACSS2 KO mouse hippocampus. FIG. 8 A: Uniform Manifold Approximation and Projection (UMAP) clustering of hippocampal snRNAseq. FIG. 8B: cell ratios in each cell type per treatment. FIG. 8C: number of differential genes in each cell type in WT PBS vs ACSS2 KO AD-Tau comparison. FIG. 8D: heatmap showing top 20 upregulated and downregulated genes in WT PBS vs ACSS2 KO AD-Tau comparison in Cajal-Retzius neurons. FIG. 8E: GO analysis of differential genes in WT PBS vs ACSS2 KO AD-Tau comparison in Cajal -Retzius neurons.

FIGs. 9A-9J show that exogenous acetate promotes learning and memory. FIG. 9A: dose-dependent heavy labeling of mouse hippocampal histone acetylation following /i-acetate injection. FIG. 9B: temporal dynamics of heavy labeling of mouse hippocampal histone acetylation following 6/3-acetate injection. FIG. 9C: heavy labeling of histone acetylation in WT but not ACSS2 KO mice following rfe-acetate injection. FIG. 9D: H3K27ac peaks induced by acetate injection in WT and ACSS2 KD mouse hippocampus. FIG. 9E: GO analysis of H3K27ac peaks. FIG. 9F: H3K27ac enrichment s Pcdh genes in PBS or acetate- injected WT and ACSS2 KD mice. FIG. 9G: correlation between differential gene expression and differential H3K27 acetylation in WT (top) and ACSS2 KD (bottom) mouse hippocampus. FIG. 9H: GO analysis of acetate-induced H3K27ac peaks in WT (top) and ACSS2 KD (bottom) mouse hippocampus. FIG. 91: preference scores for cocaine and cocaine-acetate injected WT mice. FIG. 9J: preference score for cocaine-acetate injected WT and ACSS2 KD mice.

FIG. 10 shows that an acetate-enriched diet rescues memory impairments induced by AD-Tau mice. Freezing levels in PBS or AD-Tau injected mice maintained on control or acetate- enriched diet. FIGs. 11A-11R show that ACSS2 overexpression maintains histone acetylation and synaptic gene expression over time.

FIGs. 12A-12H provide supplemental data showing that ACSS2 overexpression maintains histone acetylation and synaptic gene expression over time.

FIGs. 13A-13H show that AD-tau induces global transcriptomic and epigenetic changes in neurons.

FIGs. 14A-14C provide supplemental data showing that AD-tau induces global transcriptomic and epigenetic changes in neurons.

FIGs. 15A-15E show that ACSS2 overexpression mitigates tau induced transcriptomics changes and enhances neuronal resilience to tau spread.

FIGs. 16A-16E provide supplemental data showing that ACSS2 overexpression mitigates tau induced transcriptomics changes and enhances neuronal resilience to tau spread.

FIGs. 17A-17Q show pre-symptomatic enhancement of ACSS2 maintains resilience to tau-induced memory decline, synaptic plasticity, and transcriptomic changes. FIGs. 17A-17C: 9- month-old PS19 mouse contextual fear conditioning. FIGs. 17D-17K: 3-month-old PS19 mouse in vivo recording. FIG. 17L-17Q: 9-month-old PS19 mouse immunohistochemistry.

FIGs. 18A-18N provide supplementary data showing pre-symptomatic enhancement of ACSS2 maintains resilience to tau-induced memory decline, synaptic plasticity, and transcriptomic changes. FIGs. 18A-18I: 9-month-old PS 19 mouse behavior. FIGs. 18J-18N: 3- month-old PS 19 mouse in vivo recording.

FIGs. 19A-19I show global changes to CpG DNA methylation in Alzheimer’s disease (AD) mouse are reversed with ACSS2 overexpression.

FIGs. 20A-20B provide supplementary data showing that global changes to CpG DNA methylation in Alzheimer’s disease (AD) mouse are reversed with ACSS2 overexpression.

FIGs. 21A-21L show that ACSS2 upregulation enhances cognitive longevity. FIGs. 21A- 21C: ACSS2 upregulation in adult mice. FIGs. 21D-21L: ACSS2 upregulation in aged mice.

FIGs. 22A-22D provide supplementary data showing that ACSS2 upregulation enhances cognitive longevity. FIGs. 22A-22C: aged wildtype mouse behavior. FIG. 22D: aged wildtype DNA methylation. FIG. 23: downregulation of dACSS2 in the nervous system shortens lifespan of Drosophila. Animals are expressing an RNAi line to dACSS2 or a control transgene (mCherry.RNAi) by a neural GAL4 driver, elav-GAL4.

FIG. 24: upregulation of dACSS2 in the nervous system extends lifespan in Drosophila. Animals are expressing a transgene that upregulates dACSS2 or a control protein GFP in the nervous system with the elav-GAL4 driver.

FIG. 25 provides a schematic depicting the use of antisense oligonucleotides (ASOs) to interfere with miRNA/RISC binding to the 3’-UTR of ACSS2 mRNA to prevent miRNA action and overall increasing ACSS2 protein levels.

FIG. 26 provides a bar graph depicting results of a 3’-UTR ACSS2 luciferase reporter assay showing significant reduction in luciferase activity in three independent clones (C1-C3) U2OS cells treated with 25 nM of mir-15a-5p and mir-15b-5p. n=3, *p<0.05, **p<0.01.

FIG. 27 provides a schematic depicting the design of antisense oligonucleotides (ASOs) for blocking miRNA/RISC binding to the 3’-UTR of ACSS2 mRNA.

FIG. 28 provides a bar graph depicting results of a 3’-UTR ACSS2 luciferase report assay showing significant reduction in luciferase activity in U2OS cells treated with 25 nM miRNAs and significant upregulation of luciferase activity upon ASO treatment. *p<0.05.

FIG. 29 provides a bar graph depicting a restoration assay using antisense oligonucleotides of the present disclosure (z.e., ASO1-ASO4), wherein significant restoration of luciferase activity with ASO2 is shown.

FIG. 30 provides a bar graph depicting dose response ACSS2-luciferase reporter assay using certain antisense oligonucleotides of the present disclosure.

FIG. 31 provides a bar graph depicting a luciferase recovery assay which shows that both ASO3 and ASO4 increase luciferase activity in the presence of miR-15b.

DETAILED DESCRIPTION OF THE INVENTION

Reference will now be made in detail to certain embodiments of the disclosed subject matter, examples of which are illustrated in part in the accompanying drawings. While the disclosed subject matter will be described in conjunction with the enumerated claims, it will be understood that the exemplified subject matter is not intended to limit the claims to the disclosed subject matter.

Throughout this document, values expressed in a range format should be interpreted in a flexible manner to include not only the numerical values explicitly recited as the limits of the range, but also to include all the individual numerical values or sub-ranges encompassed within that range as if each numerical value and sub-range is explicitly recited. For example, a range of "about 0.1% to about 5%" or "about 0.1% to 5%" should be interpreted to include not just about 0.1% to about 5%, but also the individual values (e.g., 1%, 2%, 3%, and 4%) and the sub-ranges (e.g., 0.1% to 0.5%, 1.1% to 2.2%, 3.3% to 4.4%) within the indicated range. The statement "about X to Y" has the same meaning as "about X to about Y," unless indicated otherwise. Likewise, the statement "about X, Y, or about Z" has the same meaning as "about X, about Y, or about Z," unless indicated otherwise.

In this document, the terms "a," "an," or "the" are used to include one or more than one unless the context clearly dictates otherwise. The term "or" is used to refer to a nonexclusive "or" unless otherwise indicated. The statement "at least one of A and B" or "at least one of A or B" has the same meaning as "A, B, or A and B." In addition, it is to be understood that the phraseology or terminology employed herein, and not otherwise defined, is for the purpose of description only and not of limitation. Any use of section headings is intended to aid reading of the document and is not to be interpreted as limiting; information that is relevant to a section heading may occur within or outside of that particular section. All publications, patents, and patent documents referred to in this document are incorporated by reference herein in their entirety, as though individually incorporated by reference.

In the methods described herein, the acts can be carried out in any order, except when a temporal or operational sequence is explicitly recited. Furthermore, specified acts can be carried out concurrently unless explicit claim language recites that they be carried out separately. For example, a claimed act of doing X and a claimed act of doing Y can be conducted simultaneously within a single operation, and the resulting process will fall within the literal scope of the claimed process.

Definitions The term "about" as used herein can allow for a degree of variability in a value or range, for example, within 10%, within 5%, or within 1% of a stated value or of a stated limit of a range, and includes the exact stated value or range.

As used herein, the term “AAV” is a standard abbreviation for adeno-associated virus. Adeno-associated virus is a single-stranded DNA parvovirus that grows only in cells in which certain functions are provided by a co-infecting helper virus. There are currently thirteen serotypes of AAV that have been characterized. General information and reviews of AAV can be found in, for example, Carter, 1989, Handbook of Parvoviruses, Vol. 1, pp. 169-228, and Berns, 1990, Virology, pp. 1743-1764, Raven Press, (New York). However, it is fully expected that these same principles will be applicable to additional AAV serotypes since it is well known that the various serotypes are quite closely related, both structurally and functionally, even at the genetic level. (See, for example, Blacklowe, 1988, pp. 165-174 of Parvoviruses and Human Disease, J. R. Pattison, ed.; and Rose, Comprehensive Virology 3:1-61 (1974)). For example, all AAV serotypes apparently exhibit very similar replication properties mediated by homologous rep genes; and all bear three related capsid proteins such as those expressed in AAV2. The degree of relatedness is further suggested by heteroduplex analysis which reveals extensive cross-hybridization between serotypes along the length of the genome; and the presence of analogous self-annealing segments at the termini that correspond to “inverted terminal repeat sequences” (ITRs). The similar infectivity patterns also suggest that the replication functions in each serotype are under similar regulatory control.

An “AAV vector” as used herein refers to a vector comprising one or more polynucleotides of interest (or transgenes) that are flanked by AAV terminal repeat sequences (ITRs). Such AAV vectors can be replicated and packaged into infectious viral particles when present in a host cell that has been transfected with a vector encoding and expressing rep and cap gene products.

An “AAV virion” or “AAV viral particle” or “AAV vector particle” refers to a viral particle composed of at least one AAV capsid protein and an encapsidated polynucleotide AAV vector. If the particle comprises a heterologous polynucleotide (z.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as an “AAV vector particle” or simply an “AAV vector”. Thus, production of AAV vector particle necessarily includes production of AAV vector, as such a vector is contained within an AAV vector particle.

As used herein, the term “active ingredient” refers to a therapeutic agent that is to be delivered to a subject to produce a therapeutic effect in the subject.

As referred to herein, the term "adenovirus" refers to a nonenveloped virus with an icosahedral nucleocapsid containing a double stranded DNA of the family Adenoviridae. Over 50 adenoviral subtypes have been isolated from humans and many additional subtypes have been isolated from other mammals and birds. See, e.g., Ishibashi et al., "Adenoviruses of animals," In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 497-562 (1984); Strauss, "Adenovirus infections in humans," In The Adenoviruses, Ginsberg, ed., Plenum Press, New York, N.Y., pp. 451-596 (1984). These subtypes belong to the family Adenoviridae, which is currently divided into two genera, namely Mastadenovirus and Aviadenovirus. All adenoviruses are morphologically and structurally similar. In humans, however, adenoviruses show diverging immunological properties and are, therefore, divided into serotypes. Two human serotypes of adenovirus, namely AV2 and AV5, have been studied intensively and have provided the majority of general information about adenoviruses.

The term “amphipathic lipid” refers, in part, to any suitable material wherein the hydrophobic portion of the lipid material orients into a hydrophobic phase, while the hydrophilic portion orients toward the aqueous phase. Hydrophilic characteristics derive from the presence of polar or charged groups such as carbohydrates, phosphate, carboxylic, sulfato, amino, sulfhydryl, nitro, hydroxyl, and other like groups. Hydrophobicity can be conferred by the inclusion of apolar groups that include, but are not limited to, long-chain saturated and unsaturated aliphatic hydrocarbon groups and such groups substituted by one or more aromatic, cycloaliphatic, or heterocyclic group(s). Examples of amphipathic compounds include, but are not limited to, phospholipids, aminolipids, and sphingolipids.

Representative examples of phospholipids include, but are not limited to, phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, phospha-tidic acid, palmitoyloleoyl phosphatidylcholine, lysophosphatidylcholine, lysophosphatidylethanolamine, dipalmitoylphosphatidylcholine, dioleoylphosphatidyl choline, distearoylphosphatidylcholine, and dilinoleoylphosphatidylcholine. Other compounds lacking in phosphorus, such as sphingolipid, glycosphingolipid families, diacylglycerols, and acyloxyacids, are also within the group desig-nated as amphipathic lipids. Additionally, the amphipathic lipids described above can be mixed with other lipids including triglycerides and sterols.

The term “anionic lipid” refers to any lipid that is negatively charged at physiological pH. These lipids include, but are not limited to, phosphatidylglycerols, cardiolipins, diacylphosphatidylserines, diacylphosphatidic acids, N-dodecanoyl phosphatidylethanolamines, N-succinyl phosphatidylethanolamines, N-glutarylphosphatidylethanolamines, lysylphosphatidylglycerols, palmitoyloleyolphosphatidylglycerol (POPG), and other anionic modifying groups joined to neutral lipids.

The term “cationic lipid” refers to any of a number of lipid species that carry a net positive charge at a selected pH, such as physiological pH (e.g., pH of about 7.0). It has been found that cationic lipids comprising alkyl chains with multiple sites of unsaturation, e.g., at least two or three sites of unsaturation, are particularly useful for forming lipid particles with increased membrane fluidity. A number of cationic lipids and related analogs, which are also useful in the present disclosure, have been described in U.S. Patent Publication Nos. 20060083780 and 20060240554; U.S. Pat. Nos. 5,208,036; 5,264,618; 5,279,833; 5,283,185; 5,753,613; and 5,785,992; and PCT Publication No. WO 96/10390, the disclosures of which are herein incorporated by reference in their entirety for all purposes. Non-limiting examples of cationic lipids are described in detail herein. In some cases, the cat-ionic lipids comprise a protonatable tertiary amine (e.g., pH titratable) head group, Cis alkyl chains, ether linkages between the head group and alkyl chains, and 0 to 3 double bonds. Such lipids include, e.g., DSDMA, DLinDMA, DLenDMA, and DODMA.

In one aspect, the terms “co-administered” and “co-administration” as relating to a subject refer to administering to the subject a compound/composition of the present disclosure or salt thereof along with a compound/composition that may also treat, ameliorate, and/or prevent any disease or disorder contemplated herein and/or with a compound that is useful in treating, ameliorating, and/or preventing other medical conditions but which in themselves may cause or facilitate any disease or disorder contemplated herein. In certain embodiments, the coadministered compounds are administered separately, or in any kind of combination as part of a single therapeutic approach. The co-administered compound may be formulated in any kind of combinations as mixtures of solids and liquids under a variety of solid, gel, and liquid formulations, and as a solution.

The term “cognitive decline” as used herein is defined as a deterioration of memory, attention, and cognitive function. Alternatively to the term cognitive decline, the term cognitive dysfunction, the term cognitive impairment or the term dementia may be used. The term preferably refers to a condition which can be characterized as a loss, usually progressive, of cognitive and intellectual functions, without impairment of perception or consciousness caused by a variety of disorders, but most commonly associated with structural brain disease. Cognitive testing may be done using the Montreal Cognitive Assessment (MoCA), as described in Conen et al. 2019, which evaluates visuospatial and executive functions, confrontation naming, memory, attention, language, and abstraction. Patients can obtain a maximum of 30 points, with higher scores indicating better cognitive function. The most common pathology underlying dementia and/or advanced cognitive decline is Alzheimer's disease, which makes up 50% to 70% of cases. Other common types include vascular dementia (25%), dementia with Lewy bodies, and frontotemporal dementia. The term “dementia” includes, but is not restricted to AIDS dementia, Alzheimer dementia, presenile dementia, senile dementia, catatonic dementia, Lewy body dementia (diffuse Lewy body disease), multi-infarct dementia (vascular dementia), paralytic dementia, posttraumatic dementia, dementia praecox, vascular dementia.

The term “dietary supplement” as used herein refers to a product intended to supplement or complement a subject's diet, the product comprising one or more substances with a nutritional and/or physiological effect on a subject. The dietary supplement can be a partial nutritional composition, which does not contain all the essential macro- and micronutrients and hence may not be used to replace one or more daily meals and/or may not be used as the sole source of nutrition of a subject. The dietary supplement may be a liquid, powder, gel, paste, solid, concentrate, suspension or ready-to-use formulation. The dietary supplement may further comprise one or more additional ingredients, including vitamins, minerals, herbs or other botanicals, amino acids, and substances such as enzymes, organ tissues, glandulars, and metabolites. As used herein, a “disease” is a state of health of a subject wherein the subject cannot maintain homeostasis, and wherein if the disease is not ameliorated then the subject's health continues to deteriorate.

As used herein, a “disorder” in a subject is a state of health in which the subject is able to maintain homeostasis, but in which the subject's state of health is less favorable than it would be in the absence of the disorder. Left untreated, a disorder does not necessarily cause a further decrease in the subject's state of health.

The term “edible base” as used herein refers to any edible material, hard or soft, including vary degrees of hardness or softness. Examples of suitable bases and/or substrates include, but are not limited to, inulin, starch, modified starches, xanthan gum, carboxymethyl cellulose, methyl cellulose, hydroxypropylmethyl cellulose, konjac, chitosan, tragacanth, karaya, ghatti, larch, carageenan, alginate, chemically modified alginate, agar, guar, locust bean, psyllium, tara, gellan, curdlan, pullan, gum arabic, gelatin, pectin, and combinations thereof. In certain embodiments, the edible base is intended to include any edible food, such as fish, chicken, vegetables, legumes, fruits, meats, and the like.

In particular, in the case of a therapeutic nucleic acid (e.g., DNA) an “effective amount” or “therapeutically effective amount” of a therapeutic nucleic acid is an amount sufficient to produce the desired effect, e.g., DNA-directed expression of an amount of a protein that causes a desirable biological effect in the organism within which the protein is expressed. For example, in some embodiments, the expressed protein is an active form of a protein that is normally expressed in a cell type within the body, and the therapeutically effective amount of the DNA is an amount that produces an amount of the encoded protein that is at least 50% (e.g., at least 60%, or at least 70%, or at least 80%, or at least 90%) of the amount of the protein that is normally expressed in the cell type of a healthy individual. For example, in some embodiments, the expressed protein is a protein that is normally expressed in a cell type within the body, and the therapeutically effective amount of the DNA is an amount that produces a similar level of expression as observed in a healthy individual in an individual with aberrant expression of the protein (i.e., protein deficient individual). Suitable assays for measuring the expression of a nucleic acid or protein of interest include, but are not limited to dot blots, Northern blots, in situ hybridization, ELISA, immunoprecipitation, enzyme function, as well as phenotypic assays known to those of skill in the art.

The term “encapsulated” indicates that the active agent or therapeutic agent in the lipid particle is not significantly degraded after exposure to serum or a nuclease or protease assay that would significantly degrade free DNA, RNA, or protein. In a fully encapsulated system, preferably less than about 25% of the active agent or therapeutic agent in the particle is degraded in a treatment that would normally degrade 100% of free active agent or therapeutic agent, more preferably less than about 10%, and most preferably less than about 5% of the active agent or therapeutic agent in the particle is degraded. In the context of nucleic acid therapeutic agents, full encapsulation may be determined by an Oligreen® assay. Oligreen® is an ultra-sensitive fluorescent nucleic acid stain for quantitating oligonucleotides and single-stranded DNA or RNA in solution (available from Invitrogen Corporation; Carlsbad, Calif.). “Fully encapsulated” also indicates that the lipid particles are serum stable, that is, that they do not rapidly decompose into their component parts upon in vivo administration.

The term “encode” as used herein refers to the product specified (e. , protein and RNA) by a given sequence of nucleotides in a nucleic acid (i.e., DNA and/or RNA), upon transcription or translation of the DNA or RNA, respectively. In certain embodiments, the term “encode” refers to the RNA sequence specified by transcription of a DNA sequence. In certain embodiments, the term “encode” refers to the amino acid sequence (e.g., polypeptide or protein) specified by translation of mRNA. In certain embodiments, the term “encode” refers to the amino acid sequence specified by transcription of DNA to mRNA and subsequent translation of the mRNA encoded by the DNA sequence. In certain embodiments, the encoded product may comprise a direct transcription or translation product. In certain embodiments, the encoded product may comprise post-translational modifications understood or reasonably expected by one skilled in the art.

As used herein, “expression cassette” refers to a nucleic acid molecule encoding a gene product of interest, a promoter, and other regulatory sequences for it, wherein the cassette is a viral vector (e.g., a viral particle). In certain embodiments, the expression cassette is packaged within a capsid (/.< ., viral vector). Usually, such expression cassettes for making viral vectors are adjacent to the packaging signals of the viral genome and other expression control sequences. For example, in the case of AAV viral vectors, the packaging signals are 5-'inverted terminal repeats (ITR) and 3'-ITR.

The term “gene” refers to a nucleic acid (e.g., DNA or RNA) sequence that comprises partial length or entire length coding sequences necessary for the production of a polypeptide or precursor polypeptide.

The term “gene product,” as used herein, refers to a product of a gene such as a RNA transcript or a polypeptide.

In general, when referring to “identity,” “homology,” or “similarity” between two different sequences, “identity,” “homology,” or “similarity” is that of an “aligned” sequence. Determined in relation to. An “aligned” sequence or “alignment” refers to a plurality of nucleic acid or protein (amino acid) sequences that often contain corrections for missing or additional bases or amino acids compared to the reference sequence.

The term "independently selected from" as used herein refers to referenced groups being the same, different, or a mixture thereof, unless the context clearly indicates otherwise. Thus, under this definition, the phrase "X¹, X², and X³ are independently selected from noble gases" would include the scenario where, for example, X¹, X², and X³ are all the same, where X¹, X², and X³ are all different, where X¹ and X² are the same but X³ is different, and other analogous permutations.

The term "lentivirus vector" as used herein refers to an enveloped virus with a small spherical shape containing two single stranded RNA molecules belonging to the family Retroviridae. Lentiviruses contain gag, pol, and env genes and are further distinguished from other retrovirus family members by having two regulatory genes, tat and rev. Lentivirus vectors are widely known in the art as useful tools in molecular biology to induce expression of genes of interest in cultured cells and animal tissues.

The term “lipid” refers to a group of organic compounds that include, but are not limited to, esters of fatty acids and are characterized by being insoluble in water, but soluble in many organic solvents. They are usually divided into at least three classes: (1) “simple lipids,” which include fats and oils as well as waxes; (2) “compound lipids,” which include phospholipids and glycolipids; and (3) “derived lipids” such as steroids. The term “lipid conjugate” refers to a conjugated lipid that inhibits aggregation of lipid particles. Such lipid conjugates include, but are not limited to, polyamide oligomers (e.g., ATTA-lipid conjugates), PEG-lipid conjugates, such as PEG coupled to dialkyloxypropyls, PEG coupled to di acylglycerols, PEG coupled to cholesterol, PEG coupled to phosphatidylethanolamines, PEG conjugated to ceramides (see, e.g., U.S. Pat. No. 5,885,613, the disclosure of which is herein incorporated by reference in its entirety for all purposes), cationic PEG lipids, and mixtures thereof. PEG can be conjugated directly to the lipid or may be linked to the lipid via a linker moiety. Any linker moiety suitable for coupling the PEG to a lipid can be used including, e.g., non-ester containing linker moieties and ester-containing linker moieties. In preferred embodiments, non-ester containing linker moieties are used.

As used herein, “lipid encapsulated” can refer to a lipid particle that provides an active agent or therapeutic agent, such as a nucleic acid (e.g., a messenger RNA), with full encapsulation, partial encapsulation, or both. In a preferred embodiment, the nucleic acid is fully encapsulated in the lipid particle (e.g., to form an SPLP, pSPLP, SNALP, or other nucleic acid- lipid particle).

The term “lipid particle” is used herein to refer to a lipid formulation that can be used to deliver an active agent or therapeutic agent, such as a nucleic acid, to a target site of interest. In the lipid particle of the disclosure, which is typically formed from a cationic lipid, a non-cationic lipid, and a conjugated lipid that prevents aggregation of the particle, the active agent or therapeutic agent may be encapsulated in the lipid, thereby protecting the agent from enzymatic degradation.

The term “mammal” refers to any mammalian species such as a human, mouse, rat, dog, cat, hamster, guinea pig, rabbit, livestock, and the like.

The term “nucleic acid” as used herein refers to a polymer containing at least two deoxyribonucleotides or ribonucleotides in either single- or double-stranded form and includes DNA and RNA. DNA may be in the form of, e.g., antisense molecules, plasmid DNA, precondensed DNA, a PCR product, vectors (Pl, PAC, BAC, YAC, artificial chromosomes), expression cassettes, chimeric sequences, chromosomal DNA, or derivatives and combinations of these groups. RNA may be in the form of siRNA, asymmetrical interfering RNA (aiRNA), microRNA (miRNA), mRNA, tRNA, rRNA, tRNA, viral RNA (vRNA), and combinations thereof. Nucleic acids include nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, and which have similar binding properties as the reference nucleic acid. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2'-O-methyl ribonucleotides, and peptide-nucleic acids (PNAs). Unless specifically limited, the term encompasses nucleic acids containing known analogues of natural nucleotides that have similar binding properties as the reference nucleic acid. Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka et al., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mai. Cell. Probes, 8:91-98 (1994)).

The term “neutral lipid” refers to any of a number of lipid species that exist either in an uncharged or neutral zwitterionic form at a selected pH. At physiological pH, such lipids include, for example, diacylphosphatidylcholine, diacylphosphatidylethanolamine, ceramide, sphingomyelin, cephalin, cholesterol, cerebrosides, and diacylglycerols.

The term “non-cationic lipid” refers to any amphipathic lipid as well as any other neutral lipid or anionic lipid.

As used herein, the term “nucleic acid” includes any oligonucleotide or polynucleotide, with fragments containing up to 60 nucleotides generally termed oligonucleotides, and longer fragments termed polynucleotides. In particular embodiments, oligonucleotides of the disclosure are from about 15 to about 60 nucleotides in length. Nucleic acid may be administered alone in the lipid particles of the disclosure, or in combination (e.g., co-administered) with lipid particles of the disclosure comprising peptides, polypeptides, or small molecules such as conventional drugs. In other embodiments, the nucleic acid may be administered in a viral vector.

“Nucleotides” contain a sugar deoxyribose (DNA) or ribose (RNA), a base, and a phosphate group. Nucleotides are linked together through the phosphate groups. “Bases” include purines and pyrimidines, which further include natural compounds adenine, thymine, guanine, cytosine, uracil, inosine, and natural analogs, and synthetic derivatives of purines and pyrimidines, which include, but are not limited to, modifications which place new reactive groups such as, but not limited to, amines, alcohols, thiols, carboxylates, and alkyl halides.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer et al., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini etal., Mol. Cell. Probes, 8:91-98 (1994)).

These control sequences are “operably linked” coding sequence. As used herein, the term “operably linked” refers to an expression control sequence that is close to a gene of interest and an expression control that acts trans or distantly to control the gene of interest. Refers to both with an array.

As used herein, the term “pharmaceutical composition” or “composition” refers to a mixture of at least one composition or recombinant viral vector useful within the present disclosure with a pharmaceutically acceptable carrier. The pharmaceutical composition facilitates administration of the compound to a subject.

As used herein, the term “pharmaceutically acceptable” refers to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound useful within the present disclosure, and is relatively non-toxic, i.e., the material may be administered to a subject without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

As used herein, the term “pharmaceutically acceptable carrier” means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the present disclosure within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the present disclosure, and not injurious to the subject. Some examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as com starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. As used herein, “pharmaceutically acceptable carrier” also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents, and the like that are compatible with the activity of the compound useful within the present disclosure, and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. The “pharmaceutically acceptable carrier” may further include a pharmaceutically acceptable salt of the compound useful within the present disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the present disclosure are known in the art and described, for example in Remington's Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

As used herein, the language “pharmaceutically acceptable salt” refers to a salt of the administered compound prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic acids, inorganic bases, organic acids, inorganic bases, solvates, hydrates, and clathrates thereof.

The terms “polynucleotide” and “oli onucleotide” as used herein, refer to a polymer or oligomer of nucleotide or nucleoside monomers comprising naturally occurring bases, sugars and intersugar (backbone) linkages. The terms “polynucleotide” and “oligonucleotide” also include polymers or oligomers comprising non-naturally occurring monomers, or portions thereof, which function similarly. Such modified or substituted oligonucleotides are often preferred over native forms because of properties such as, for example, enhanced cellular uptake, reduced immunogenicity, and increased stability in the presence of nucleases. Oligonucleotides are generally classified as deoxyribooligonucleotides or ribooligonucleotides. A deoxyribooligonucleotide consists of a 5-carbon sugar called deoxyribose joined covalently to phosphate at the 5' and 3' carbons of this sugar to form an alternating, unbranched polymer. A ribooligonucleotide consists of a similar repeating structure where the 5-carbon sugar is ribose.

The term “prevent,” “preventing” or “prevention,” as used herein, means avoiding or delaying the onset of symptoms associated with a disease or condition in a subject that has not developed such symptoms at the time the administering of an agent or compound commences. Disease, condition and disorder are used interchangeably herein.

The term “salt” includes any anionic and cationic complex, such as the complex formed between a cationic lipid and one or more anions. Non-limiting examples of anions include inorganic and organic anions, e.g., hydride, fluoride, chloride, bromide, iodide, oxalate (e.g, hemioxalate), phosphate, phosphonate, hydrogen phosphate, dihydrogen phosphate, oxide, carbonate, bicarbonate, nitrate, nitrite, nitride, bisulfite, sulfide, sulfite, bisulfate, sulfate, thiosulfate, hydrogen sulfate, borate, formate, acetate, benzoate, citrate, tartrate, lactate, acrylate, polyacrylate, fumarate, maleate, itaconate, glycolate, gluconate, malate, mandelate, tiglate, ascorbate, salicylate, polymethacrylate, perchlorate, chlorate, chlorite, hypochlorite, bromate, hypobromite, iodate, an alkyl sulfonate, an aryl sulfonate, arsenate, arsenite, chromate, dichromate, cyanide, cyanate, thiocyanate, hydroxide, peroxide, permanganate, and mixtures thereof. In particular embodiments, the salts of the cationic lipids disclosed herein are crystalline salts.

The terms “sequence homology,” “percent identity (%),” “sequence identity,” “sequence identity percent,” or “percent identity” in the context of nucleic acid and/or amino acid sequences refers to a quantitative measurement of the similarity between two nucleic acid or amino acid sequences (e.g., DNA, amino acid or otherwise).

The term “SNALP” as used herein, refers to a stable nucleic acid-lipid particle, which term may be used interchangeably with nucleic acid-lipid particle. A SNALP represents a particle made from lipids (e.g, a cationic lipid, a non-cationic lipid, and a conjugated lipid that prevents aggregation of the particle), wherein the nucleic acid (e.g., mRNA, siRNA, aiRNA, miRNA, ssDNA, dsDNA, ssRNA, short hairpin RNA (shRNA), dsRNA, or a plasmid, including plasmids from which an interfering RNA is transcribed) is fully encapsulated within the lipid. As used herein, the term “SNALP” includes an SPLP, which is the term used to refer to a nucleic acid-lipid particle comprising a nucleic acid (e.g., a plasmid) encapsulated within the lipid. SNALP and SPLP typically contain a cationic lipid, a non-cationic lipid, and a lipid conjugate (e.g., a PEG-lipid conjugate). SNALP and SPLP are useful for systemic applications, as they can exhibit extended circulation lifetimes following intravenous (i.v.) injection, they can accumulate at distal sites (e.g., sites physically separated from the administration site), and they can mediate expression of the transfected gene or silencing of target gene expression at these distal sites.

SPLP include “pSPLP,” which comprise an encapsulated condensing agent-nucleic acid complex as set forth in PCT Publication No. WO 2000/03683, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

The nucleic acid-lipid particles of the present disclosure typically have a mean diameter of 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, or from about 70 to about 90 nm, and are substantially non-toxic. In addition, nucleic acids, when present in the lipid particles of the disclosure, are resistant in aqueous solution to degradation with a nuclease. Nucleic acid-lipid particles and their method of preparation are disclosed in, e.g., U.S. Patent Publication Nos. 20040142025 and 20070042031, the disclosures of which are herein incorporated by reference in their entirety for all purposes.

As used herein, a “subject” may be a human or non-human mammal or a bird. Nonhuman mammals include, for example, livestock and pets, such as ovine, bovine, porcine, canine, feline and murine mammals. In certain embodiments, the subject is human.

The terms “substantially identical” or “substantial identity,” in the context of two or more nucleic acids, refer to two or more sequences or subsequences that are the same or have a specified percentage of nucleotides that are the same (i.e., at least about 60%, in certain embodiments at least about 65%, 70%, 75%, 80%, 85%, 90%, or 95% identity over a specified region), when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. This definition, when the context indicates, also refers analogously to the complement of a sequence. In certain embodiments, the substantial identity exists over a region that is at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, or 60 nucleotides in length.

The term "substantially" as used herein refers to a majority of, or mostly, as in at least about 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, 99.99%, or at least about 99.999% or more, or 100%. The term "substantially free of as used herein can mean having none or having a trivial amount of, such that the amount of material present does not affect the material properties of the composition including the material, such that the composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less. The term "substantially free of can mean having a trivial amount of, such that a composition is about 0 wt% to about 5 wt% of the material, or about 0 wt% to about 1 wt%, or about 5 wt% or less, or less than, equal to, or greater than about 4.5 wt%, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.01, or about 0.001 wt% or less, or about 0 wt%.

The term “treat,” “treating” or “treatment,” as used herein, means reducing the frequency or severity with which symptoms of a disease or condition are experienced by a subject by virtue of administering an agent or compound to the subject.

The term “vehicle” as used herein refer to a carrier and/or inert medium in which an active agent (e. , nucleic acid) is formulated and/or administered.

Unless otherwise indicated, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (e.g., degenerate codon substitutions), alleles, orthologs, SNPs, and complementary sequences as well as the sequence explicitly indicated. Specifically, degenerate codon substitutions may be achieved by generating sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (Batzer etal., Nucleic Acid Res., 19:5081 (1991); Ohtsuka etal., J. Biol. Chem., 260:2605-2608 (1985); Rossolini et al., Mol. Cell. Probes, 8:91-98 (1994)).

Recombinant Viral Vectors In one aspect, the present disclosure provides a recombinant viral vector, the vector comprising:

(b) an expression control sequence operably linked to the nucleic acid.

In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 85% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 90% sequence homology with SEQ ID NO:1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 95% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 96% sequence homology with SEQ ID NO:1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 97% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 98% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 99% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares 100% sequence homology with SEQ ID NO: 1.

In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 85% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 90% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 95% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 96% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 97% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 98% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 99% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares 100% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 85% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 90% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 95% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 96% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 97% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 98% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 99% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares 100% sequence homology with SEQ ID NO:3.

In certain embodiments, the vector is an Adeno-associated virus (AAV) vector.

In certain embodiments, the vector is AAV-PHP.eB.

Recombinant AAV (rAAV) genomes of the invention may comprise nucleic acid molecule of the invention and one or more AAV ITRs flanking a nucleic acid molecule. AAV DNA in the rAAV genomes may be from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692. Other types of rAAV variants, for example rAAV with capsid mutations, are also contemplated. See, for example, Marsic etal., Molecular Therapy, 22(11): 1900-1909 (2014). As indicated elsewhere herein, the nucleotide sequences of the genomes of various AAV serotypes are known in the art. To promote neuron specific expression AAV2, inter alia, may be used.

DNA plasmids of the invention comprise rAAV genomes of the invention. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, El -deleted adenovirus or herpes vims) for assembly of the rAAV genome into infectious viral particles. Techniques to produce rAAV particles, in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell, are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions. The AAV rep and cap genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV- 1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAVrh.74, AAV-8, AAV-9, AAV-10, AAV-11, AAV-12 and AAV-13. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.

A method of generating a packaging cell is to create a cell line that stably expresses all the necessary components for AAV particle production. For example, a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell. AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA, 79:2077-2081), addition of synthetic linkers containing restriction endonuclease cleavage sites (Laughlin etal., 1983, Gene, 23:65-73) or by direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem., 259:4661-4666). The packaging cell line is then infected with a helper virus such as adenovirus. The advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV. Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells.

General principles of rAAV production are reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbial, and Immunol., 158:97-129). Various approaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81 :6466 (1984); Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J. Virol., 62: 1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol., 7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat. No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO 97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243 (PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine 13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark e/ al. (1996) Gene Therapy 3: 1124-1132; U.S. Pat. Nos. 5,786,211; 5,871,982; and 6,258,595. The foregoing documents are hereby incorporated by reference in their entirety herein, with particular emphasis on those sections of the documents relating to rAAV production.

The invention thus provides packaging cells that produce infectious rAAV. In one embodiment packaging cells may be stably transformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line). In another embodiment, packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with El of adenovirus), MRC-5 cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).

Recombinant AAV (i.e., infectious encapsidated rAAV particles) of the invention comprise a rAAV genome. In exemplary embodiments, the genomes of both rAAV lack AAV rep and cap DNA, that is, there is no AAV rep or cap DNA between the ITRs of the genomes. Examples of rAAV that may be constructed to comprise the nucleic acid molecules of the invention are set out in International Patent Application No. PCT/US2012/047999 (WO 2013/016352) incorporated by reference herein in its entirety.

The rAAV may be purified by methods standard in the art such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in, for example, Clark etal., Hum. Gene Then, 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69 427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98/09657.

In another embodiment, the invention contemplates compositions comprising rAAV of the present invention. Compositions of the invention comprise rAAV and a pharmaceutically acceptable carrier. The compositions may also comprise other ingredients such as diluents and adjuvants. Acceptable carriers, diluents and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, di saccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counter ions such as sodium; and/or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG).

Titers of rAAV to be administered in methods of the invention will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of rAAV may range from about l *10⁶, about l * 10⁷, about l^x 10⁸, about l ><10⁹, about I x lO¹⁰, about 1 x 10¹¹, about 1 x 10¹², about 1 x 10¹³ to about 1 x 10¹⁴ or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg).

Methods of transducing a target cell with rAAV, in vivo or in vitro, are contemplated by the invention. The in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a rAAV of the invention to an animal (including a human being) in need thereof. If the dose is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose is administered after the development of a disorder/disease, the administration is therapeutic. In embodiments of the invention, an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease state being treated, that slows or prevents progression to a disorder/disease state, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival. An example of a disease contemplated for treatment, prevention, and/or amelioration with methods of the invention is cognitive decline and/or cognitive decline associated with neurodegeneration (e.g., Alzheimer’s disease).

Combination therapies are also contemplated by the invention. Combination as used herein includes both simultaneous treatment and sequential treatments. Combinations of methods of the invention with standard medical treatments are specifically contemplated, as are combinations with novel therapies.

Administration of an effective dose of the compositions may be by routes standard in the art including, but not limited to, intrathecal, intramuscular, parenteral, intravenous (e.g., retro- orbital injection), oral, buccal, nasal, pulmonary, intracranial, intraosseous, intraocular (e.g., retro-orbital injection), rectal, or vaginal. Route(s) of administration and serotype(s) of AAV components of the rAAV (in particular, the AAV ITRs and capsid protein) of the invention may be chosen and/or matched by those skilled in the art taking into account the disease and/or disorder state being treated and the target cells/tissue(s) that are to express the ACSS2 (e.g., neuronal cells).

The invention provides for local administration and systemic administration of an effective dose of rAAV and compositions of the invention including combination therapy of the invention. For example, systemic administration is administration into the circulatory system so that the entire body is affected. Systemic administration includes enteral administration such as absorption through the gastrointestinal tract and parental administration through injection, infusion or implantation.

In particular, actual administration of rAAV of the present invention may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal. Administration according to the invention includes, but is not limited to, intrathecal, retro-orbital injection, and/or alternative routes of administration suitable for delivery to the brain and/or cerebrospinal fluid of a subject.

Capsid proteins of a rAAV may be modified such that the rAAV is targeted to a particular target of interest, e.g., brain. See, for example, PCT/US2019/052969, the disclosure of which is incorporated herein by reference. Numerous formulations for selective delivery of the rAAV to the brain have been previously developed and can be used in the practice of the present invention. The rAAV can be used with any of a number of pharmaceutically acceptable carriers for ease of administration and handling.

The dose of rAAV to be administered in methods disclosed herein will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of each rAAV administered may range from about I x lO⁶, about 1 x 10⁷, about I x lO⁸, about l * 10⁹, about l ^z I O¹⁰, about IxlO¹¹, about l ^x 10¹², about l ^x 10¹³, about l ^x 10¹⁴, or to about I x lO¹⁵ or more DNase resistant particles (DRP) per ml. Dosages may also be expressed in units of viral genomes (vg) (i.e., l x l0⁷ vg, l ^x 10⁸ vg, l x l0⁹ vg, l x lO^lovg, lxlOⁿ vg, l ^x 10¹² vg, I x lO¹³ vg, 1 x 10¹⁴ vg, I x lO¹⁵ respectively). Dosages may also be expressed in units of viral genomes (vg) per kilogram (kg) of bodyweight (i.e., 1 x IO¹⁰ vg/kg, 1 x 10¹¹ vg/kg, 1 * 10¹² vg/kg, 1 x io¹³ vg/kg, 1 x 10¹⁴ vg/kg, 1 x 10¹⁵ vg/kg respectively). Methods for titering AAV are described in Clark et al., Hum. Gene Then, 10: 1031-1039 (1999).

The pharmaceutical carriers, diluents or excipients suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization. Generally, dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.

In certain embodiments, the vector is a lentivirus or lentiviral vector. In certain embodiments,

As used herein a “lentiviral vector producer cell” refers to a cell that contains, integrated into its genome, the elements required to produce a lentiviral vector. In certain embodiments, the method utilizes a lentiviral vector producer cell including integrated into its genome a lentiviral regulator of expression of virion proteins (REV) gene under control of a first promoter, a lentiviral envelope gene (an envelope glycoprotein gene) under control of a second promoter, and a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene both under control of a third promoter. In certain embodiments, the nucleic acid sequence is flanked on both the 5' and 3' ends by sequences resulting from the recombination of transposon-specific inverted terminal repeats (ITRs).

As disclosed herein, the lentiviral regulator of expression of virion proteins (REV) is an RNA-binding protein that promotes late phase gene expression. It is also important for the transport of the unspliced or singly-spliced mRNAs, which encode viral structural proteins, from the nucleus to the cytoplasm.

The envelope glycoprotein gene, suitably a Vesicular Somatitis Virus Glycoprotein (VSV-G) gene, is expressed and displayed on the surface of lentiviral vectors and mediates the transduction of lentiviral vector into the target cells.

GAG encodes a polyprotein that is translated from an unspliced mRNA which is then cleaved by the viral protease (PR) into the matrix protein, capsid, and nucleocapsid proteins. The lentiviral polymerase (POL) is expressed as a GAG-POL polyprotein as a result of ribosomal frameshifting during GAG mRNA translation, and encodes the enzymatic proteins reverse transcriptase, protease, and integrase. These three proteins are associated with the viral genome within the virion. Suitably the GAG gene is an HIV GAG gene and the POL gene is an HIV POL gene.

In certain embodiments, the expression cassette is flanked on both the 5' and 3' ends by transposon-specific inverted terminal repeats (ITR). Exemplary promoters for use in the lentiviral vector-producing cells are known in the art and include derepressible promoters, and suitably the expression cassette further encodes a repressor element of the first, second and third derepressible promoters. In embodiments, the derepressible promoters comprises a functional promoter and a tetracycline operator sequence (TetO), and the repressor element is a tetracycline repressor protein, as described herein. In certain embodiments, the method to produce a lentiviral vector includes transducing the mammalian lentiviral vector producer cell with a vector encoding a gene of interest. In embodiments, the gene of interest is a gene of therapeutic interest.

In certain embodiments, the method includes activation of the first, second, and third promoters within the lentiviral vector producer cell and expanding the transduced viral producer cell. In certain In embodiments, the method includes suitably isolating the produced lentiviral vector. Methods for isolated produced viral vectors are described herein. In certain embodiments, embodiments, the method is performed in a closed an automated process.

In certain embodiments, methods are provided for automated production of a lentiviral vector, comprising: introducing a mammalian cell into a fully enclosed cell engineering system; transducing a mammalian cell with: a first nucleic acid encoding a lentiviral regulator of expression of virion proteins (REV) gene under control of a first promoter and an envelope glycoprotein gene under control of a second promoter; a second nucleic acid encoding a gene of interest under control of a third promoter; and a third nucleic acid encoding a lentiviral group specific antigen (GAG) gene and a lentiviral polymerase (POL) gene both under control of a fourth promoter, expanding the transduced cell and producing the lentiviral vector within the transduced cell; and isolating the viral vector, wherein (a) through (d) are performed in a closed and automated process. Methods for production of transient production of lentiviral vectors can be found in U.S. Provisional Patent Application No. 62/949,848, filed December 18, 2019, the disclosure of which is incorporated by reference herein in its entirety.

In certain embodiments, the method of automated production of a lentiviral viral vector produces at least about IO¹⁰ viral vectors. For example, the amount of lentiviral vectors produced by the methods described herein is at least about IO¹⁰ lentiviral vectors, or at least about 10¹¹ lentiviral vectors, or at least about 10¹² lentiviral vectors, or at least about 10¹³ lentiviral vectors, or at least about 10¹⁴ lentiviral vectors, or about 10¹⁰-l 0¹⁴ lentiviral vectors, or about IO¹⁰- 10¹³ lentiviral vectors, or about 10¹⁰-l 0¹² lentiviral vectors, or about IO¹⁰, about 10¹¹, about 10¹², or about 10¹³ lentiviral vectors.

Thus, the invention provides methods of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of rAAV or lentiviral vectors that encode ACSS2 to a subject in need thereof. Antisense Oligonucleotide Compositions

In one aspect, the present disclosure provides an antisense oligonucleotide composition. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid sequence which is at least partially complementary to at least a portion of a 3 ’-untranslated region (3’-UTR) of an acetyl-CoA synthetase 2 (ACSS2) mRNA transcript. In certain embodiments, the nucleic acid is complementary to a substantial fraction of the 3’-UTR of ACSS2 mRNA transcript. In certain embodiments, the nucleic acid is complementary to substantially all of the 3’-UTR of ACSS2 mRNA transcript. In certain embodiments, the nucleic acid sequence comprises at least one chemically modified nucleoside or internucleoside linkage. In certain embodiments, the antisense oligonucleotide binds to at least a portion of the 3’-UTR of an ACSS2 mRNA transcript. In certain embodiments, binding of the antisense oligonucleotide binds to at least a portion of the 3’-UTR of an ACSS2 mRNA transcript at least partially inhibits degradation of the ACSS2 mRNA transcript. In certain embodiments, the at least partial inhibition of degradation of the ACSS2 mRNA transcript results in increased expression and/or translation of ACSS2.

In certain embodiments, the 3’-UTR of ACSS2 mRNA transcript comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO:4. In certain embodiments, the 3’-UTR of ACSS2 mRNA transcript comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO:4. In certain embodiments, the 3’-UTR of ACSS2 mRNA transcript comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO:4. In certain embodiments, the 3’-UTR of ACSS2 mRNA transcript comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO:4. In certain embodiments, the 3’-UTR of ACSS2 mRNA transcript comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO:4. In certain embodiments, the 3’-UTR of ACSS2 mRNA transcript comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO:4. In certain embodiments, the 3’-UTR of ACSS2 mRNA transcript comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO:4. In certain embodiments, the 3’-UTR of ACSS2 mRNA transcript comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO:4. In certain embodiments, the at least a portion of the 3’-UTR of the ACSS2 mRNA transcript ranges from about nucleic acid 435 to about nucleic acid 465 of SEQ ID NO:4.

In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO:5. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO:5. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO: 5. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO:5. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO: 5. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO:5. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO: 5. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO:5.

In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO:6. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO:6. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO:6. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO:6. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO:6. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO:6. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO:6. In certain embodiments, the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO:6.

In certain embodiments, the nucleic acid comprises at least one chemically modified nucleoside. In certain embodiments, the chemical modification comprises 2’ -hydroxy substitution. In certain embodiments, the 2’ -hydroxy substitution comprises 2 ’-methoxy ethyl substitution. In certain embodiments, each nucleoside of the nucleic acid is chemically modified.

In certain embodiments, the nucleic acid comprises at least one chemically modified internucleoside linkage. In certain embodiments, the chemically modified internucleoside linkage comprises a phosphorothioate linkage. In certain embodiments, each internucleoside linkage comprises a phosphorothioate linkage.

In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO: 7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO:7.

In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO: 8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO: 8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO: 8.

In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO: 10.

Nucleic Acid-Lipid Particles

In one aspect, the present disclosure provides a composition comprising one or more nucleic acid-lipid particles, wherein each nucleic acid-lipid particle comprises:

(a) a cationic lipid;

(b) a non-cationic lipid;

(c) a conjugated lipid that inhibits aggregation of two or more nucleic acid lipid particles; and

(d) a nucleic acid at least partially encapsulated within the nucleic acid-lipid particle, wherein the nucleic acid encodes acetyl-CoA synthetase 2 (ACSS2).

(a) a cationic lipid;

(b) a non-cationic lipid;

(c) a conjugated lipid that inhibits aggregation of two or more nucleic acid lipid particles; and (e) at least one antisense oligonucleotide of the present disclosure at least partially encapsulated within the nucleic acid-lipid particle.

(a) a cationic lipid;

(b) a non-cationic lipid;

(c) a conjugated lipid that inhibits aggregation of two or more nucleic acid lipid particles; and at least one selected from (d) and (e):

(d) a nucleic acid at least partially encapsulated within the nucleic acid-lipid particle, wherein the nucleic acid encodes acetyl-CoA synthetase 2 (ACSS2); and

(e) at least one antisense oligonucleotide of the present disclosure at least partially encapsulated within the nucleic acid-lipid particle.

In certain embodiments, the cationic lipid comprises about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90 mol% of the total lipid present in the nucleic acid-lipid particle.

In certain embodiments, the cationic lipid comprises less than about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90 mol% of the total lipid present in the nucleic acid- lipid particle.

In certain embodiments, the cationic lipid comprises more than about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, or about 90 mol% of the total lipid present in the nucleic acid- lipid particle.

In certain embodiments, the non-cationic lipid is cholesterol. In certain embodiments, the non-cationic lipid is a phospholipid.

In certain embodiments, the non-cationic lipid comprises 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mol% of the total lipid present in the nucleic acid-lipid particle. In certain embodiments, the non-cationic lipid comprises less than 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mol% of the total lipid present in the nucleic acid-lipid particle.

In certain embodiments, the non-cationic lipid comprises more than 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, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or about 50 mol% of the total lipid present in the nucleic acid-lipid particle.

In certain embodiments, the conjugated lipid that inhibits aggregation of two or more nucleic acid-lipid particles comprises a polyethyleneglycol (PEG)-lipid conjugate.

In certain embodiments, the conjugated lipid comprises about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 mol% of the total lipid present in the nucleic acid-lipid particle.

In certain embodiments, the conjugated lipid comprises less than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 mol% of the total lipid present in the nucleic acid-lipid particle.

In certain embodiments, the conjugated lipid comprises more than about 0.1, 0.2, 0.3, 0.4, 0.5, 0 6, 0 7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, or about 2.0 mol% of the total lipid present in the nucleic acid-lipid particle.

In certain embodiments, the nucleic acid at least partially encapsulated within the nucleic acid-lipid particle is a nucleic acid which encodes acetyl-CoA synthetase 2 (ACSS2). In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 85% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 90% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 95% sequence homology with SEQ ID NO: 1 . In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 96% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 97% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 98% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 99% sequence homology with SEQ ID NO: 1. In certain embodiments, the nucleic acid comprises a DNA sequence which shares 100% sequence homology with SEQ ID NO: 1.

In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 85% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 90% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 95% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 96% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 97% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 98% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares at least 99% sequence homology with SEQ ID NO:2. In certain embodiments, the nucleic acid comprises a DNA sequence which shares 100% sequence homology with SEQ ID NO:2.

In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 85% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 90% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 95% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 96% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 97% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 98% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 99% sequence homology with SEQ ID NO:3. In certain embodiments, the nucleic acid comprises a DNA sequence which encodes a protein that shares 100% sequence homology with SEQ ID NO:3. In certain embodiments, the particle at least partially encapsulates a antisense oligonucleotide of the present disclosure. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO:7. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO:8. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO:9. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 85% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 90% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 95% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 96% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 97% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 98% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares at least 99% sequence homology with SEQ ID NO: 10. In certain embodiments, the antisense oligonucleotide comprises a nucleic acid which shares 100% sequence homology with SEQ ID NO: 10.

The lipid particles of the present disclosure typically comprise an active agent or therapeutic agent, a cationic lipid, a non-cationic lipid, and a conjugated lipid that inhibits aggregation of particles. In some embodiments, the active agent or therapeutic agent is fully encapsulated within the lipid portion of the lipid particle such that the active agent or therapeutic agent in the lipid particle is resistant in aqueous solution to enzymatic degradation, e.g., by a nuclease or protease. In other embodiments, the lipid particles described herein are substantially non-toxic to mammals such as humans. The lipid particles of the disclosure typically have a mean diameter of from about 40 nm to about 150 nm, from about 50 nm to 10 about 150 nm, from about 60 nm to about 130 nm, from about 70 nm to about 110 nm, or from about 70 to about 90 nm.

In certain embodiments, the nucleic acid-lipid particles of the present disclosure are serum-stable nucleic acid-lipid particles (SNALP) which comprise RNA (e.g., mRNA), a cationic lipid, a non-cationic lipid (e.g., cholesterol alone or mixtures of one or more phospholipids and cholesterol), and a conjugated lipid that inhibits aggregation of the particles (e.g., one or more PEG-lipid conjugates). The SNALP may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more unmodified and/or modified mRNA molecules. Nucleic acid-lipid particles and their method of preparation are described in, e.g., U.S. Pat. Nos. 5,753,613; 5,785, 992; 5,705,385; 5,976,567; 5,981,501; 6,110,745; 6,320, 25 017; 8,058,069; 8,492,359; 8,822,668; 9,364,435; 9,504,651; and 11,141,378; and PCT Publication No. WO 96/40964, the disclosures of which are each herein incorporated by reference in their entirety for all purposes.

Cationic Lipids

In the nucleic acid-lipid particles of the disclosure, the cationic lipid may comprise, e.g., one or more of the following: l,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2- dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA), 2,2-dilinoleyl-4-(2- dimethylaminoethyl)-[l,3]-dioxolane (DLin-K-C2-DMA; “XTC2”), 2,2-dilinoleyl-4-(3- 45 dimethy laminopropyl )-[ 1,3 ]-dioxolane (D Lin-K-C3-D MA), 2,2-dilinoleyl-4-( 4- dimethylaminobutyl)-[l,3]-dioxolane (DLin-K-C4-DMA), 2,2-dilinoleyl-5-dimethylaminom- ethyl-[l,3]-dioxane (DLin-K6-DMA), 2, 2-dilinoleyl-4-Nmethylpepiazino-[l,3]-di oxolane (DLin-K-MPZ), 2,2-dili-noleyl-4-dimethylaminomethyl-[l,3]-dioxolane (DLin-KDMA), 1,2- dilinoleylcarbamoyloxy-3-dimethy laminopropane (D Lin-C-DAP), 1,2-dilinoley oxy-3 - (dimethylaminoacetoxypropane (DLin-DAC), l-2dilinoley oxy-3 -morpholinopropane (DLin- MA), l,2-dilinoleoyl-3 -dimethylaminopropane (DLinDAP), l,2-dilinoleylthio-3- dimethylaminopropane (DLin-2-DMAP), l,2-dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), l,2-dilinoleoyl-3 -trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2- dilinoleyloxy-3-(N-methy Ipiperazino )propane (D Lin-MPZ), 3-(N,Ndilinoley lamino )- 1 ,2- propanediol (D LinAP), 3-(N ,Ndioley lamino )-l,2-propanedio (DOAP), l,2-dilinoleyloxo-3-(2- N,N-dimethy lamino )ethoxypropane (D Lin-EG-D MA), N,N-dioleyl-N,N-dimethylanrmonium chloride (DODAC), l,2-dioleyloxy-N,N-dimethylaminopropane (DODMA), 1,2-disteary loxy- N,N-dimethy laminopropane (DSD MA), N-(l-(2,3-dioleyloxy)propyl)-N,N,N- trimethylammonium chloride (DOTMA), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(l -(2,3 -di oleoyloxy )propyl)-N,N, N-trimethylammonium chloride (DOTAP), 3-(N- (N',N'dimethylaminoethane)-carbamoyl)cholesterol (DC-Chol), N-(l,2-dimyristyloxyprop-3-yl)- N,N-dimethyl-N-hydroxyethyl ammonium bromide (DMRIE), 2,3-dioleyloxy-N-[2 ( spermine- carboxamidoethyl]-N,N-dimethy 1-1 -propanaminiumtrifluoroacetate (DOSPA), dioctadecylamidoglycyl spermine (DOGS), 3-dimethylamino-2-( cholest-5-en-3-beta-oxybutan- 4-oxy )-l-(cis,cis-9,12-octadecadienoxy )propane (CLinDMA), 2-[5'-(cholest-5-en-3-beta-oxy)- 3'-oxapentoxy )-3-dimethyl-l-(cis,cis-9',l-2'-octadecadienoxy) propane (CpLinDMA), N,N- dimethyl-3,4-dioleyloxybenzylamine (DMOBA), l,2-N,N'dioleylcarbamyl-3- dimethylaminopropane (DOcarbDAP), l,2-N,N'-dilinoleylcarbamyl-3 -dimethylaminopropane (DLincarbDAP), or mixtures thereof. In certain embodiments, the cationic lipid is DLinDMA, DLin-K-C2-DMA (“XTC2”), or mixtures thereof.

The synthesis of cationic lipids such as DLin-K-C2-DMA (“XTC2”), DLin-K-C3-DMA, DLin-K-C4-DMA, DLin-K6-DMA, and DLin-K-MPZ, as well as additional cationic lipids, is described in U.S. Provisional Application No. 61/104, 212, filed Oct. 9, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as DLin-K-DMA, DLin-CDAP, DLin-DAC, DLin-MA, DLinDAP, DLin-S- DMA, DLin-2-DMAP, DLin-TMA.Cl, DLin-TAP.Cl, DLin-MPZ, DLinAP, DOAP, and DLin- EG-DMA, as well as additional cationic lipids, is described in PCT Application No. PCT/ US08/88676, filed Dec. 31, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. The synthesis of cationic lipids such as CLinDMA, as well as additional cationic lipids, is described in U.S. Patent Publication No. 20060240554, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

Non-cationic Lipid

In the nucleic acid-lipid particles of the present disclosure, the non-cationic lipid may comprise, e.g., one or more anionic lipids and/or neutral lipids. In preferred embodiments, the non-cationic lipid comprises one of the following neutral lipid components: (1) cholesterol or a derivative thereof (2) a phospholipid; or (3) a mixture of a phospholipid and cholesterol or a derivative thereof.

Examples of cholesterol derivatives include, but are not limited to, cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'- hydroxybutyl ether, and mixtures thereof. The synthesis of cholesteryl-2'-hydroxyethyl ether is known to one skilled in the art and described in U.S. Patent Nos. 8,058,069, 8,492,359, 8,822,668, 9,364,435, 9,504,651, and 11,141,378, all of which are hereby incorporated herein in their entireties for all purposes.

Non-limiting examples of non-cationic lipids include phospholipids such as lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanolamine, phosphatidylserine, phosphatidylinositol, sphingomyelin, egg sphingomyelin (ESM), cephalin, cardiolipin, phosphatidic acid, cerebrosides, dicetylphosphate, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), ioleoylphosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), palmitoyl oleyolphosphatidylglycerol (POPG), dioleoylphosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-l-carboxylate DOPE-mal), dipalmitoylphosphatidylethanolamine (DPPE), dimyristoylphosphatidylethanolamine (DMPE), distearoylphosphatidylethanolamine (DSPE), monomethylphosphatidy lethanolamine, dimethylphosphatidylethanolamine, dielaidoylphosphatidylethanolamine (DEPE), stearoyloleoylphosphatidylethanolamine (SOPE), lysophosphatidylcholine, dilinoleoylphosphatidylcholine, and mixtures thereof.

Other diacylphosphatidylcholine and diacylphosphatidylethanolamine phospholipids can also be used. The acyl groups in these lipids are preferably acyl groups derived from fatty acids having C10-C24 carbon chains, e. , lauroyl, myristoyl, palmitoyl, stearoyl, or oleoyl. Additional examples of non-cationic lipids include sterols such as cholesterol and derivatives thereof such as cholestanol, cholestanone, cholestenone, coprostanol, cholesteryl-2'-hydroxyethyl ether, cholesteryl-4'-hydroxybutyl ether, and mixtures thereof. In certain embodiments, the phospholipid is DPPC, DSPC, or mixtures thereof. Conjugated Lipid

In the nucleic acid-lipid particles of the present disclosure, the conjugated lipid that inhibits aggregation of particles may comprise, e.g., one or more of the following: a polyethyleneglycol (PEGjlipid conjugate, a polyamide (ATTA)-lipid conjugate, a cationic- polymer-lipid conjugates (CPLs), or mixtures thereof. In one preferred embodiment, the nucleic acid-lipid particles comprise either a PEG-lipid conjugate or an ATTA-lipid conjugate.

PEG is a linear, water-soluble polymer of ethylene PEG repeating units with two terminal hydroxyl groups. PEGs are classified by their molecular weights; for example, PEG 2000 has an average molecular weight of about 2,000 daltons, and PEG 5000 has an average molecular weight of about 5,000 daltons. PEGs are commercially available from Sigma Chemical Co. and other companies and include, for example, the following: monomethoxypolyethylene glycol (MePEGOH), monomethoxypolyethylene glycolsuccinate (MePEGS), monomethoxypolyethylene glycolsuccinimidyl succinate (MePEG-S-NHS), monomethoxypolyethylene glycolamine (MePEG-NH2), monomethoxypolyethylene glycoltresylate (MePEG-TRES), and monomethoxypolyethylene glycolimidazolylcarbonyl (MePEG-IM). Other PEGs such as those described in U.S. Pat. Nos. 6,774,180 and 7,053,150 (e.g., mPEG (20 KDa) amine) are also useful for preparing the PEG-lipid conjugates of the present disclosure. The disclosures of these patents are herein incorporated by reference in their entirety for all purposes. In addition, monomethoxypolyethyleneglycolacetic acid (MePEG- CH2COOH) is particularly useful for preparing PEG-lipid conjugates including, e.g., PEG-DAA conjugates.

In certain embodiments, the PEG-lipid conjugate or ATTA-lipid conjugate is used together with a CPL. The conjugated lipid that inhibits aggregation of particles may comprise a PEG-lipid including, e.g., a PEG-diacylglycerol (DAG), a PEG dialkyloxypropyl (DAA), a PEG- phospholipid, a PEG-ceramide (Cer), or mixtures thereof. The PEGDAA conjugate may be PEG- di lauryl oxypropyl (C12), a PEG-di myristyl oxy propyl (C14), a PEG-dipalmityloxypropyl (Cis), a PEG-distearyloxypropyl (Cis), or mixtures thereof.

Additional PEG-lipid conjugates suitable for use in the disclosure include, but are not limited to, mPEG2000-l,2-diO-alkyl-sn3-carbomoylglyceride (PEG-C-DOMG). The synthesis of PEG-C-DOMG is described in PCT Application No. PCT/US08/88676, filed Dec. 31, 2008, the disclosure of which is herein incorporated by reference in its entirety for all purposes. Yet additional PEG-lipid conjugates suitable for use in the disclosure include, without limitation, 1- [8'-(l,2-dimyristoyl-3-propanoxy)-carboxamido-3',6'-dioxaoctanyl] carbamoyl-methyl- poly(ethylene glycol) (2 KPEG-DMG). The synthesis of 2 KPEG-DMG is described in U.S. Pat. No. 7,404,969, the disclosure of which is herein incorporated by reference in its entirety for all purposes.

The PEG moiety of the PEG-lipid conjugates described herein may comprise an average molecular weight ranging from about 550 daltons to about 10,000 daltons. In certain instances, the PEG moiety has an average molecular weight of from about 750 daltons to about 5,000 daltons (e.g., from about 1,000 daltons to about 5,000 daltons, from about 1,500 daltons to about 3,000 daltons, from about 750 daltons to about 3,000 daltons, from about 750 daltons to about 2,000 daltons, etc.). In preferred embodiments, the PEG moiety has an average molecular weight of about 2,000 daltons or about 750 daltons.

In addition to the foregoing, it will be readily apparent to those of skill in the art that other hydrophilic polymers can be used in place of PEG. Examples of suitable polymers that can be used in place of PEG include, but are not limited to, polyvinylpyrrolidone, polymethyloxazoline, polyethyloxazoline, polyhydroxypropyl methacrylamide, polymethacrylamide and polydimethylacrylamide, polylactic acid, polyglycolic acid, and derivatized celluloses such as hydroxymethylcellulose or hydroxyethylcellulose.

In addition to the foregoing components, the particles (e.g., SNALP or SPLP) of the present disclosure can further comprise cationic poly(ethylene glycol) (PEG) lipids or CPLs (see, e.g., Chen et al., Bioconj. Chem., 11 :433-437 (2000)). Suitable SPLPs and SPLP-CPLs for use in the present disclosure, and methods of making and using SPLPs and SPLPCPLs, are disclosed, e.g., in U.S. Pat. No. 6,852,334 and PCT Publication No. WO 00/62813, the disclosures of which are herein incorporated by reference in their entirety for all purposes.

In the nucleic acid-lipid particles of the present disclosure, the active agent or therapeutic agent may be fully encapsulated within the lipid portion of the particle, thereby protecting the active agent or therapeutic agent from enzymatic degradation. In preferred embodiments, a nucleic acid-lipid particle comprising a nucleic acid such as DNA or mRNA is fully encapsulated within the lipid portion of the particle, thereby protecting the nucleic acid from nuclease degradation. In certain instances, the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after exposure of the particle to a nuclease at 37° C. for at least about 20, 30, 45, or 60 minutes. In certain other instances, the nucleic acid in the nucleic acid-lipid particle is not substantially degraded after incubation of the particle in serum at 37° C. for at least about 30, 45, or 60 minutes or at least about 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 other embodiments, the active agent or therapeutic agent (e.g., nucleic acid such as DNA) is complexed with the lipid portion of the particle. One of the benefits of the formulations of the present disclosure is that the lipid particle compositions are substantially non-toxic to mammals such as humans.

Lipic Active Agent Ratios

Typically, the nucleic acid-lipid particles of the present disclosure have a lipid:active agent (e.g., lipidmucleic acid) ratio (mass/mass ratio) of from about 1 to about 100. In some instances, the lipid:active agent (e.g., lipidmucleic acid) ratio (mass/mass ratio) ranges from about 1 to about 50, from about 2 to 40 about 25, from about 3 to about 20, from about 4 to about 15, or from about 5 to about 10. In certain embodiments, the lipid particles of the disclosure have a lipid:active agent (e.g., lipidmucleic acid) ratio (mass/mass ratio) of from about 5 to about 15, e.g., about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 (or any fraction thereof or range therein).

Typically, the nucleic acid-lipid particles of the present disclosure have a mean diameter of from about 40 nm to about 150 nm. In certain embodiments, the lipid particles (e.g., SNALP) of the disclosure have a mean diameter of from about 40 nm to 50 about 130 nm, from about 40 nm to about 120 nm, from about 40 nm to about 100 nm, from about 50 nm to about 120 nm, from about 50 nm to about 100 nm, from about 60 nm to about 120 nm, from about 60 nm to about 110 nm, from about 60 nm to about 100 nm, from about 60 nm to about 90 nm, from 55 about 60 nm to about 80 nm, from about 70 nm to about 120 nm, from about 70 nm to about 110 nm, from about 70 nm to about 100 nm, from about 70 nm to about 90 nm, from about 70 nm to about 80 nm, or less than about 120 nm, 110 nm, 100 nm, 90 nm, or 80 nm (or any fraction thereof or range therein).

In certain embodiments, the nucleic acid-lipid particle comprises: (a) unmodified and/or modified DNA that encodes a functional protein (i.e., gene product); (b) a cationic lipid comprising from about 56.5 mol% to about 66.5 mol% of the total lipid present in the 65 particle; (c) a non-cationic lipid comprising from about 31.5 mol% to about 42.5 mol% of the total lipid present in the particle; and (d) a conjugated lipid that inhibits aggregation of particles comprising from about 1 mol% to about 2 mol% of the total lipid present in the particle. In certain embodiments, this nucleic acid-lipid particle is referred to herein as the “1 :62” formulation. In certain embodiments, the cationic lipid is DLinDMA or DLin-K-C2- DMA(“XTC2”), the non-cationic lipid is cholesterol, and the conjugated lipid is a PEG-DAA conjugate. Although these are preferred embodiments of the 1 :62 formulation, those of skill in the art will appreciate that other cationic lipids, non-cationic lipids (including other cholesterol derivatives), and conjugated lipids can be used in the 1 :62 formulation as described herein.

In another embodiment of the disclosure, the nucleic acid-lipid particle comprises: (a) unmodified and/or modified DNAthat encodes a functional protein (i.e., gene product); (b) a cationic lipid comprising from about 52 mol% to about 62 mol% of the total lipid present in the particle; (c) a non-cationic lipid comprising from about 36 mol% to about 47 mol% of the total lipid present in the particle; and (d) a conjugated lipid that inhibits aggregation of particles comprising from about 1 mol% to about 2 mol% of the total lipid present in the particle. This specific embodiment of nucleic acid-lipid particle is generally referred to herein as the “1 :57” formulation. In this embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA (“XTC2”), the non-cationic lipid is a mixture of a phospholipid (such as DPPC) and cholesterol, wherein the phospholipid comprises from about 5 mol% to about 9 mol% of the total lipid present in the particle (e.g., about 7.1 mol%) and the cholesterol (or cholesterol derivative) comprises from about 32 mol% to about 37 mol% of the total lipid present in the particle e.g., about 34.3 mol%), and the PEG-lipid is a PEG-DAA (e.g., PEG-cDMA). In another preferred embodiment, the cationic lipid is DLinDMA or DLin-K-C2-DMA (“XTC2”), the non-cationic lipid is a mixture of a phospholipid (such as DPPC) and cholesterol, wherein the phospholipid comprises from about 15 mol% to about 25 mol% of the total lipid present in the particle (e.g., about 20 mol%) and the cholesterol (or cholesterol derivative) comprises from about 15 mol% to about 25 mol% of the total lipid present in the particle (e.g., about 20 mol%), and the PEG-lipid is a PEGDAA (e.g., PEG-cDMA). Those of skill in the art will appreciate that other cationic lipids, non-cationic lipids (including other phospholipids and other cholesterol derivatives), and conjugated lipids can be used in the 1 :57 formulation as described herein.

In certain embodiments, the 1 :62 nucleic acid-lipid particle formulation is a three- component system which is phospholipid-free and comprises about 1.5 mol% PEG-cDMA (or PEG-IDSA), about 61.5 mol% DLinDMA (or XTC2), and about 36.9 mol% cholesterol (or derivative thereof). In other embodiments, the 1:57 nucleic acid-lipid particle formulation is a four-component system which comprises about 1.4 mol% PEG-cDMA (or PEG-cDSA), about 57.1 mol% DLinDMA (or XTC2), about 7.1 mol% DPPC, and about 34.3 mol% cholesterol (or derivative thereof). In yet other preferred embodiments, the 1 :57 nucleic acid-lipid particle formulation is a four-component system which comprises about 1.4 mol% PEG-cDMA (or PEG- cDSA), about 57.1 mol% DLinDMA (or XTC2), about 20 mol% DPPC, and about 20 mol% cholesterol (or derivative thereof). It should be understood that these nucleic acid-lipid particle formulations are target formulations, and that the amount of lipid (both cationic and noncationic) present and the amount of lipid conjugate present in the nucleic acid-lipid particle formulations may vary.

Methods

In another aspect, the present disclosure provides a method of treating, preventing, and/or ameliorating cognitive decline in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one nucleic acid-lipid particle composition of the present disclosure. In another aspect, the present disclosure provides a method of treating, preventing, and/or ameliorating cognitive decline in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one pharmaceutical composition of the present disclosure.

In certain embodiments, the cognitive decline is associated with a neurodegenerative disease or disorder. In certain embodiments, the cognitive decline is associated with age. In certain embodiments, the cognitive decline is associated with trauma.

In certain embodiments, the neurodegenerative disease or disorder is Alzheimer’s disease.

In certain embodiments, the neurodegenerative disease or disorder is vascular dementia. In certain embodiments, the neurodegenerative disease or disorder is Lewy bodies. In certain embodiments, the neurodegenerative disease or disorder is Parkinson’s disease. In certain embodiments, the neurodegenerative disease or disorder is frontotemporal dementia. In certain embodiments, the neurodegenerative disease or disorder is Huntington’s disease. In certain embodiments, the neurodegenerative disease or disorder is HIV-associated neurocognitive disorder. In certain embodiments, the neurodegenerative disease or disorder is Creutzfeldt-Jakob disease. In certain embodiments, the neurodegenerative disease or disorder is alcohol-related dementia. In certain embodiments, the neurodegenerative disease or disorder is inflammation- derived dementia (e.g, Behcet’s disease, multiple sclerosis, sarcoidosis, Sjogren’s syndrome, lupus, celiac disease, and non-celiac gluten sensitivity).

In certain embodiments, the subject is administered the recombinant viral vector of the present disclosure, or a pharmaceutical composition thereof.

In certain embodiments, the recombinant viral vector and/or pharmaceutical composition is administered by at least one route selected from the group consisting of intravenous, intrathecal, intraocular, intranasal, and/or intraparenchymal.

In certain embodiments, the expression of ACSS2 is promoted in the subject. In certain embodiments, the subject is administered acetate.

In certain embodiments, the acetate is sodium acetate.

In certain embodiments, the acetate is administered to the subject by at least one route selected from the group consisting of intravenous and oral.

In certain embodiments, the oral administration comprises dietary supplementation.

In certain embodiments, histone acetylation is promoted in the subject.

In certain embodiments, the subject is a mammal. In certain embodiments, the mammal is a human.

Pharmaceutical Compositions

The present disclosure provides pharmaceutical compositions comprising a nucleic acid- lipid particle and a pharmaceutically acceptable carrier. The present disclosure further provides a pharmaceutical composition comprising a recombinant viral vector and a pharmaceutically acceptable carrier.

Such a pharmaceutical composition may consist of at least one composition or vector of the invention, in a form suitable for administration to a subject, or the pharmaceutical composition may comprise at least one composition or vector, and one or more pharmaceutically acceptable carriers, one or more additional ingredients, or any combinations of these. At least one composition or vector of the invention may be present in the pharmaceutical composition in the form of a physiologically acceptable salt, such as in combination with a physiologically acceptable cation or anion, as is well known in the art.

In certain embodiments, the pharmaceutical compositions useful for practicing the method of the invention may be administered to deliver a dose of between 1 ng/kg/day and 100 mg/kg/day. In other embodiments, the pharmaceutical compositions useful for practicing the invention may be administered to deliver a dose of between 1 ng/kg/day and 1,000 mg/kg/day.

The relative amounts of the active ingredient, the pharmaceutically acceptable carrier, and any additional ingredients in a pharmaceutical composition of the invention will vary, depending upon the identity, size, and condition of the subject treated and further depending upon the route by which the composition is to be administered. By way of example, the composition may comprise between 0.1% and 100% (w/w) active ingredient. Pharmaceutical compositions that are useful in the methods of the invention may be suitably developed for nasal, inhalational, oral, rectal, vaginal, pleural, peritoneal, parenteral, topical, transdermal, pulmonary, intranasal, buccal, ophthalmic, epidural, intrathecal, intravenous, or another route of administration. A composition useful within the methods of the invention may be directly administered to the brain, the brainstem, or any other part of the central nervous system of a mammal or bird. Other contemplated formulations include projected nanoparticles, microspheres, liposomal preparations, coated particles, polymer conjugates, resealed erythrocytes containing the active ingredient, and immunologically-based formulations.

In certain embodiments, the compositions of the invention are part of a pharmaceutical matrix, which allows for manipulation of insoluble materials and improvement of the bioavailability thereof, development of controlled or sustained release products, and generation of homogeneous compositions. By way of example, a pharmaceutical matrix may be prepared using hot melt extrusion, solid solutions, solid dispersions, size reduction technologies, molecular complexes (e.g., cyclodextrins, and others), microparticulate, and particle and formulation coating processes. Amorphous or crystalline phases may be used in such processes.

The route(s) of administration will be readily apparent to the skilled artisan and will depend upon any number of factors including the type and severity of the disease being treated, the type and age of the veterinary or human patient being treated, and the like.

The formulations of the pharmaceutical compositions described herein may be prepared by any method known or hereafter developed in the art of pharmacology and pharmaceutics. In general, such preparatory methods include the step of bringing the active ingredient into association with a carrier or one or more other accessory ingredients, and then, if necessary or desirable, shaping or packaging the product into a desired single-dose or multi-dose unit.

As used herein, a "unit dose" is a discrete amount of the pharmaceutical composition comprising a predetermined amount of the active ingredient. The amount of the active ingredient is generally equal to the dosage of the active ingredient that would be administered to a subject or a convenient fraction of such a dosage such as, for example, one-half or one-third of such a dosage. The unit dosage form may be for a single daily dose or one of multiple daily doses (e.g, about 1 to 4 or more times per day). When multiple daily doses are used, the unit dosage form may be the same or different for each dose. Although the descriptions of pharmaceutical compositions provided herein are principally directed to pharmaceutical compositions suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats, and dogs.

In certain embodiments, the compositions of the invention are formulated using one or more pharmaceutically acceptable excipients or carriers. In certain embodiments, the pharmaceutical compositions of the invention comprise a therapeutically effective amount of at least one compound of the invention and a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers, which are useful, include, but are not limited to, glycerol, water, saline, ethanol, recombinant human albumin (e.g., RECOMBUMIN®), solubilized gelatins (e.g., GELOFUSINE®), and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

The carrier may be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), recombinant human albumin, solubilized gelatins, suitable mixtures thereof, and vegetable oils. The proper fluidity may be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms may be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, isotonic agents, for example, sugars, sodium chloride, or polyalcohols such as mannitol and sorbitol, are included in the composition. Prolonged absorption of the injectable compositions may be brought about by including in the composition an agent that delays absorption, for example, aluminum monostearate or gelatin. Formulations may be employed in admixtures with conventional excipients, i.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for oral, parenteral, nasal, inhalational, intravenous, subcutaneous, transdermal enteral, or any other suitable mode of administration, known to the art. The pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring, and/or fragranceconferring substances and the like. They may also be combined where desired with other active agents, e.g., other analgesic, anxiolytics or hypnotic agents. As used herein, "additional ingredients" include, but are not limited to, one or more ingredients that may be used as a pharmaceutical carrier.

The composition of the invention may comprise a preservative from about 0.005% to 2.0% by total weight of the composition. The preservative is used to prevent spoilage in the case of exposure to contaminants in the environment. Examples of preservatives useful in accordance with the invention include but are not limited to those selected from the group consisting of benzyl alcohol, sorbic acid, parabens, imidurea and any combinations thereof. One such preservative is a combination of about 0.5% to 2.0% benzyl alcohol and 0.05-0.5% sorbic acid.

The composition may include an antioxidant and a chelating agent that inhibit the degradation of the compound. Antioxidants for some compounds are BHT, BHA, alphatocopherol and ascorbic acid in the exemplary range of about 0.01% to 0.3%, or BHT in the range of 0.03% to 0.1% by weight by total weight of the composition. The chelating agent may be present in an amount of from 0.01% to 0.5% by weight by total weight of the composition. Exemplary chelating agents include edetate salts (e.g. disodium edetate) and citric acid in the weight range of about 0.01% to 0.20%, or in the range of 0.02% to 0.10% by weight by total weight of the composition. The chelating agent is useful for chelating metal ions in the composition that may be detrimental to the shelf life of the formulation. While BHT and disodium edetate are exemplary antioxidant and chelating agent, respectively, for some compounds, other suitable and equivalent antioxidants and chelating agents may be substituted therefore as would be known to those skilled in the art.

Liquid suspensions may be prepared using conventional methods to achieve suspension of the active ingredient in an aqueous or oily vehicle. Aqueous vehicles include, for example, water, and isotonic saline. Oily vehicles include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin. Liquid suspensions may further comprise one or more additional ingredients including, but not limited to, suspending agents, dispersing or wetting agents, emulsifying agents, demulcents, preservatives, buffers, salts, flavorings, coloring agents, and sweetening agents. Oily suspensions may further comprise a thickening agent. Known suspending agents include, but are not limited to, sorbitol syrup, hydrogenated edible fats, sodium alginate, polyvinylpyrrolidone, gum tragacanth, gum acacia, and cellulose derivatives such as sodium carboxymethylcellulose, methylcellulose, hydroxypropylmethyl cellulose. Known dispersing or wetting agents include, but are not limited to, naturally-occurring phosphatides such as lecithin, condensation products of an alkylene oxide with a fatty acid, with a long chain aliphatic alcohol, with a partial ester derived from a fatty acid and a hexitol, or with a partial ester derived from a fatty acid and a hexitol anhydride (e.g., polyoxyethylene stearate, heptadecaethyleneoxycetanol, polyoxyethylene sorbitol monooleate, and polyoxyethylene sorbitan monooleate, respectively). Known emulsifying agents include, but are not limited to, lecithin, acacia, and ionic or non-ionic surfactants. Known preservatives include, but are not limited to, methyl, ethyl, or w-propyl para-hydroxybenzoates, ascorbic acid, and sorbic acid. Known sweetening agents include, for example, glycerol, propylene glycol, sorbitol, sucrose, and saccharin.

Liquid solutions of the active ingredient in aqueous or oily solvents may be prepared in substantially the same manner as liquid suspensions, the primary difference being that the active ingredient is dissolved, rather than suspended in the solvent. As used herein, an "oily" liquid is one which comprises a carbon-containing liquid molecule and which exhibits a less polar character than water. Liquid solutions of the pharmaceutical composition of the invention may comprise each of the components described with regard to liquid suspensions, it being understood that suspending agents will not necessarily aid dissolution of the active ingredient in the solvent. Aqueous solvents include, for example, water, and isotonic saline. Oily solvents include, for example, almond oil, oily esters, ethyl alcohol, vegetable oils such as arachis, olive, sesame, or coconut oil, fractionated vegetable oils, and mineral oils such as liquid paraffin.

A pharmaceutical composition of the invention may also be prepared, packaged, or sold in the form of oil-in-water emulsion or a water-in-oil emulsion. The oily phase may be a vegetable oil such as olive or arachis oil, a mineral oil such as liquid paraffin, or a combination of these. Such compositions may further comprise one or more emulsifying agents such as naturally occurring gums such as gum acacia or gum tragacanth, naturally-occurring phosphatides such as soybean or lecithin phosphatide, esters or partial esters derived from combinations of fatty acids and hexitol anhydrides such as sorbitan monooleate, and condensation products of such partial esters with ethylene oxide such as polyoxyethylene sorbitan monooleate. These emulsions may also contain additional ingredients including, for example, sweetening or flavoring agents.

Methods for impregnating or coating a material with a chemical composition are known in the art, and include, but are not limited to methods of depositing or binding a chemical composition onto a surface, methods of incorporating a chemical composition into the structure of a material during the synthesis of the material (z.e., such as with a physiologically degradable material), and methods of absorbing an aqueous or oily solution or suspension into an absorbent material, with or without subsequent drying. Methods for mixing components include physical milling, the use of pellets in solid and suspension formulations and mixing in a transdermal patch, as known to those skilled in the art.

Administration/Dosing

The regimen of administration may affect what constitutes an effective amount. The therapeutic formulations may be administered to the patient either prior to or after the onset of a disease or disorder. Further, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. Further, the dosages of the therapeutic formulations may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

Administration of the compositions of the present disclosure to a patient, such as a mammal, such as a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated herein. An effective amount of therapeutic (z.e., composition and/or recombinant viral vector) necessary to achieve a therapeutic effect may vary according to factors such as the activity of the particular therapeutic employed; the time of administration; the rate of excretion of the composition and/or recombinant viral vector; the duration of the treatment; other drugs, compounds or materials used in combination with the composition and/or recombinant viral vector; the state of the disease or disorder, age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well-known in the medical arts. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, several divided doses may be administered daily or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. A non-limiting example of an effective dose range for a therapeutic composition and/or recombinant viral vector of the disclosure is from about 0.01 mg/kg to 100 mg/kg of body weight/per day of active agent (i.e., nucleic acid). One of ordinary skill in the art would be able to study the relevant factors and make the determination regarding the effective amount of the therapeutic composition and/or recombinant viral vector without undue experimentation.

The composition and/or recombinant viral vector may be administered to an animal as frequently as several times daily, or it may be administered less frequently, such as once a day, once a week, once every two weeks, once a month, or even less frequently, such as once every several months or even once a year or less. It is understood that the amount of composition and/or recombinant viral vector dosed per day may be administered, in non-limiting examples, every day, every other day, every 2 days, every 3 days, every 4 days, or every 5 days. For example, with every other day administration, a 5 mg per day dose may be initiated on Monday with a first subsequent 5 mg per day dose administered on Wednesday, a second subsequent 5 mg per day dose administered on Friday, and so on. The frequency of the dose is readily apparent to the skilled artisan and depends upon a number of factors, such as, but not limited to, type and severity of the disease being treated, and type and age of the animal.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of this disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

A medical doctor, e.g., physician or veterinarian, having ordinary skill in the art may readily determine and prescribe the effective amount of the pharmaceutical composition required. For example, the physician or veterinarian could start doses of the compounds of the disclosure employed in the pharmaceutical composition at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

In particular embodiments, it is especially advantageous to formulate the compound in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the patients to be treated; each unit containing a predetermined quantity of therapeutic composition and/or recombinant viral vector calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. The dosage unit forms of the disclosure are dictated by and directly dependent on (a) the unique characteristics of the therapeutic composition and/or recombinant viral vector and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding/formulating such a therapeutic composition and/or recombinant viral vector for the treatment of a disease or disorder in a patient.

In certain embodiments, the compositions of the disclosure are administered to the patient in dosages that range from one to five times per day or more. In other embodiments, the compositions of the disclosure are administered to the patient in range of dosages that include, but are not limited to, once every day, every two days, every three days to once a week, and once every two weeks. It will be readily apparent to one skilled in the art that the frequency of administration of the various combination compositions of the disclosure will vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, the disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any patient will be determined by the attending physician taking all other factors about the patient into account.

The amount of active agent of the composition(s) and/or recombinant viral vector(s) of the disclosure for administration may be in the range of from about 1 pg to about 7,500 mg, about 20 pg to about 7,000 mg, about 40 pg to about 6,500 mg, about 80 p g to about 6,000 mg, about 100 p g to about 5,500 mg, about 200 p g to about 5,000 mg, about 400 p g to about 4,000 mg, about 800 p g to about 3,000 mg, about 1 mg to about 2,500 mg, about 2 mg to about 2,000 mg, about 5 mg to about 1,000 mg, about 10 mg to about 750 mg, about 20 mg to about 600 mg, about 30 mg to about 500 mg, about 40 mg to about 400 mg, about 50 mg to about 300 mg, about 60 mg to about 250 mg, about 70 mg to about 200 mg, about 80 mg to about 150 mg, and any and all whole or partial increments there-in-between.

In some embodiments, the dose of active agent (i.e., nucleic acid) present in the composition and/or recombinant viral vector of the disclosure is from about 0.5 pg and about 5,000 mg. In some embodiments, a dose of active agent present in the composition and/or recombinant viral vector of the disclosure used in compositions described herein is less than about 5,000 mg, or less than about 4,000 mg, or less than about 3,000 mg, or less than about 2,000 mg, or less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 200 mg, or less than about 50 mg. Similarly, in some embodiments, a dose of a second compound as described herein is less than about 1,000 mg, or less than about 800 mg, or less than about 600 mg, or less than about 500 mg, or less than about 400 mg, or less than about 300 mg, or less than about 200 mg, or less than about 100 mg, or less than about 50 mg, or less than about 40 mg, or less than about 30 mg, or less than about 25 mg, or less than about 20 mg, or less than about 15 mg, or less than about 10 mg, or less than about 5 mg, or less than about 2 mg, or less than about 1 mg, or less than about 0.5 mg, and any and all whole or partial increments thereof.

In certain embodiments, the present disclosure is directed to a packaged pharmaceutical composition comprising a container holding a therapeutically effective amount of the composition and/or recombinant viral vector of the disclosure, alone or in combination with a second pharmaceutical agent; and instructions for using the compound to treat, prevent, or reduce one or more symptoms of a disease or disorder in a patient.

The term "container" includes any receptacle for holding the pharmaceutical composition or for managing stability or water uptake. For example, in certain embodiments, the container is the packaging that contains the pharmaceutical composition, such as liquid (solution and suspension), semisolid, lyophilized solid, solution and powder or lyophilized formulation present in dual chambers. In other embodiments, the container is not the packaging that contains the pharmaceutical composition, i.e., the container is a receptacle, such as a box or vial that contains the packaged pharmaceutical composition or unpackaged pharmaceutical composition and the instructions for use of the pharmaceutical composition. Moreover, packaging techniques are well known in the art. It should be understood that the instructions for use of the pharmaceutical composition may be contained on the packaging containing the pharmaceutical composition, and as such the instructions form an increased functional relationship to the packaged product.

However, it should be understood that the instructions may contain information pertaining to the compound’s ability to perform its intended function, e.g., treating, preventing, or reducing a disease or disorder in a patient.

Administration

Routes of administration of any of the compositions of the disclosure include inhalational, oral, nasal, rectal, parenteral, sublingual, transdermal, transmucosal (e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal e.g., trans- and perivaginally), (intra)nasal, and (trans)rectal), intravesical, intrapulmonary, intraduodenal, intragastrical, intrathecal, epidural, intrapleural, intraperitoneal, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

Suitable compositions and dosage forms include, for example, tablets, capsules, caplets, pills, gel caps, troches, emulsions, dispersions, suspensions, solutions, syrups, granules, beads, transdermal patches, gels, powders, pellets, magmas, lozenges, creams, pastes, plasters, lotions, discs, suppositories, liquid sprays for nasal or oral administration, dry powder or aerosolized formulations for inhalation, compositions and formulations for intravesical administration and the like. It should be understood that the formulations and compositions that would be useful in the present disclosure are not limited to the particular formulations and compositions that are described herein.

Parenteral Administration

As used herein, "parenteral administration" of a pharmaceutical composition includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to, subcutaneous, intrahepatic, intravenous, intraperitoneal, intramuscular, intrasternal injection, loco-regional delivery, and kidney dialytic infusion techniques.

Formulations of a pharmaceutical composition suitable for parenteral administration comprise the active ingredient combined with a pharmaceutically acceptable carrier, such as sterile water or sterile isotonic saline. Such formulations may be prepared, packaged, or sold in a form suitable for bolus administration or for continuous administration. Injectable formulations may be prepared, packaged, or sold in unit dosage form, such as in ampules or in multidose containers containing a preservative. Injectable formulations may also be prepared, packaged, or sold in devices such as patient-controlled analgesia (PCA) devices. Formulations for parenteral administration include, but are not limited to, suspensions, solutions, emulsions in oily or aqueous vehicles, pastes, and implantable sustained-release or biodegradable formulations. Such formulations may further comprise one or more additional ingredients including, but not limited to, suspending, stabilizing, or dispersing agents. In one embodiment of a formulation for parenteral administration, the active ingredient is provided in dry (i.e., powder or granular) form for reconstitution with a suitable vehicle (e.g., sterile pyrogen-free water) prior to parenteral administration of the reconstituted composition.

The pharmaceutical compositions may be prepared, packaged, or sold in the form of a sterile injectable aqueous or oily suspension or solution. This suspension or solution may be formulated according to the known art, and may comprise, in addition to the active ingredient, additional ingredients such as the dispersing agents, wetting agents, or suspending agents described herein. Such sterile injectable formulations may be prepared using a non-toxic parenterally acceptable diluent or solvent, such as water or 1,3 -butanediol, for example. Other acceptable diluents and solvents include, but are not limited to, Ringer’s solution, isotonic sodium chloride solution, and fixed oils such as synthetic mono- or di-glycerides. Other parentally-administrable formulations which are useful include those which comprise the active ingredient in microcrystalline form in a recombinant human albumin, a fluidized gelatin, in a liposomal preparation, or as a component of a biodegradable polymer system. Compositions for sustained release or implantation may comprise pharmaceutically acceptable polymeric or hydrophobic materials such as an emulsion, an ion exchange resin, a sparingly soluble polymer, or a sparingly soluble salt.

EXAMPLES

Various embodiments of the present application can be better understood by reference to the following Examples which are offered by way of illustration. The scope of the present application is not limited to the Examples given herein.

Materials and Methods

Mouse lines

C57BL6/J wild-type mice were obtained from the NIA aged Rodent Colonies. ACSS2 KO mice were created on a C57BL6/J background. Animals were housed under 12-hour light/dark cycles (7 am to 7 pm, and 70-72 °F ambient temperature and 40-50% humidity. For all aging experiments, mice ages 21-22 months old were used. All behavioral experiments were conducted between 7-11 am to reduce time-of-day effects.

Systemic delivery ofAAV.PHP.eB

Mice were anesthetized with isoflurane using a nose cone, and 1.50E+11 VG of AAV- PHP.eB- ACSS2 or AAV-PHP.eB-mPLUM (100 uL) was delivered through retro-orbital injection.

Plasmid

EFla-ACSS2-Flag-P2A-mPLUM was synthesized by Genewiz and packaged into a PHP.eB (titer 1.52 x 10¹³ VG/ml) by the PENN Vector core. The vector backbone expressing the mPLUM fluorophore was obtained from PENN Vector (5.82 x 10¹³ VG/ml).

Hippocampal AD-Tau injection

1 ug of human pathological AD-Tau was injected into the right hippocampus of C57BL6/J mice. Mice were anaesthetized with isoflurane gas (1-5% to maintain surgical plane) and placed in a sterile field within a stereotaxic device. Artificial tears were applied to eyes to ensure sufficient lubrication. Mice received an injection of bupivacaine (2.5 mg/kg) for local anesthesia before the skin was disinfected with betadine solution and the skull exposed with a short incision using sterile surgical equipment. A small hole (about 0.5 mm) was drilled in the skull over the target area using a stereotax and a stereotactic drill. A microsyringe filled with AD-Tau was inserted into the hippocampus and slowly removed following injection (AP, -2.5 mm; DV, -2.4 mm; ML, ± 2 mm from bregma). All mice received a single dose of subcutaneous meloxicam (5 mg kg— 1) as analgesia at induction and one dose per day for two days postoperatively as needed. Behavioral testing and molecular characterization were performed 6 months post-injection.

Hippocampal ACSS2 knock-down

Stereotactic surgeries were performed as described for AD-Tau injection using coordinates AP, -2.0 mm; DV, -1.4 mm; ML, ± 1.5 mm from bregma. The vector for ACSS2 knockdown was AAV2/9.U6.shACSS2.CMV.EGFP.

Mouse behavior - cued and contextual fear conditioning

Mice will be handled for three consecutive days before the start of the experiment. On the day of fear acquisition, mice will be individually placed in conditioning chambers (Med Associates) and habituated to the novel environment. An auditory cue (an un-modulating tone: 80 dB and 5 kHz) was presented for 30 seconds, co-terminating with a mild 2s, 1 mA foot shock. Chambers will be wiped down with 70% ethanol between each round.

After an inter-tone interval of 1 minute, the tone-shock pairing will be presented twice more. Mice will be promptly removed 30 s after shock onset and returned to their home cages. To assess the retention of long-term memory related to the auditory cue, chambers will be modified to remove spatial and olfactory cues. The shape of the chamber will be modified with cardboard inserts, and the barred flooring replaced with a solid layer. The scent of ethanol will be masked with vanilla extract. The same tone (minus the shock) will be repeated in the same pattern as the previous day. Freezing behavior will be monitored automatically using FreezeScanTM software (CleverSys, Inc) for the entire recall period. For the contextual fear conditioning paradigm, mice will be placed in the same chamber as the day of fear acquisition (minus the shock and auditory cue), and the freezing response will be tested for

6.5 consecutive minutes.

Mouse behavior - Y-maze

Mice will be placed at the end of an arm in a Y-shaped arena and will be allowed to explore freely for 6 minutes. Arm entries will be scored by eye. Arm entries will be called when the mouse’s hindquarters passed fully into the new arm. Percent spontaneous alternations will be quantified according to the formula [SAR = [Number of alternations/(total arm entries - 2)] x 100.

Mouse behavior - object location memory

The object location memory procedure is used to test spatial memory. The procedure consists of a training phase and a testing phase. Prior to training, each mouse was handled for 3 min a day for 3 days. On the training day, mice are placed in an arena (approx. 1 square foot) containing three different objects. The objects used were a glass bottle, a metal tower (h x _w ^x 1, 5 x 2 x 2 inches), and a plastic cylinder. Mice were habituated to an empty arena, followed by object exposure in three 6-min trials with an interval of 3 min. The arena and objects were cleaned with 70% EtOH between trials. After 24 h, the individual mice were placed back in the arena used in the testing phase. For testing, one of the objects was moved to different location in the arena. Mice were allowed to explore freely for 5 min. Each session was recorded using a video camera and time spent exploring (approaches and sniffing) each object was assessed using AnyMaze software. All animals were randomized and preassigned to arena and object the day before testing to ensure that every treatment group explored every object configuration.

Mouse behavior - conditioned place preference

Mouse CPP boxes (Ugo Basile; 42553) with external dimensions 35 x 18 x 29 cm were used. The apparatus was divided into two chambers (16 x 15 x 25 cm) that differed in wall and floor pattern. Striped walls were paired with circle cutouts (1 cm) and solid grey walls were paired with square cutouts (0.5 cm). Sessions were run in a dark room at ambient temperature. Boxes were cleaned with 70% ethanol between mice and allowed to dry between rounds. The paradigm consisted of 1 habituation day (5 min exploration in neutral environment), 1 pre-training session (20 min with access to both chambers), a training day (biased subject assignment, intraperitoneal injection of saline, 1.5 g/kg acetate and/or 10 mg/kg cocaine immediately before the 30-min session) and 1 post-training test session (20 min with access to both chambers). The percentage of time spent in the conditioned chamber was measured by blinded investigators. Preference scores were calculated as the difference between the time spent in the conditioned chamber and the unconditioned chamber.

Stable isotope labeling of brain histone acetylation

Mice were injected intraperitoneally with sodium acetate-cfe or control saline, and deuterium incorporation into acetylated histones was assessed. Using quantitative liquid chromatography-mass spectrometry (LC-MS), the relative abundance of isotopically labelled histone acetylation was quantified in the brain by measuring increases of the [M+3] species. Histones were extracted by lysing tissue in nuclear isolation buffer (15 mM Tris-HCl, 15 mM NaCl, 60 mM KC1, 5 mM MgCH, 1 mM CaCh and 250 mM sucrose at pH 7.5; 0.5 mM AEBSF, 10 mM sodium butyrate, 5 nM microcy stein and 1 mM DTT added fresh) with 0.2% NP-40 on ice for 5 min. The nuclei were collected by centrifuging at 700 g at 4 °C for 5 min. The resulting nuclear pellet was washed twice with the same volume of nuclear isolation buffer without NP-40. Histones were then acid- extracted with 0.2 M H2SO4 for 3 h at 4 °C with rotation. The insoluble nuclear debris was pelleted at 3,400 g at 4 °C for 5 min, and the supernatant was retained. Next, histone proteins were precipitated by adding 100% trichloroacetic acid in a 1 :3 ratio (v/v) for 1 h at 4 °C. The pellet was washed with acetone to remove residual acid. Histones were resuspended in 30 pL of 50 mM NH4HCO3 (pH 8.0). The sample was mixed with 15 pL derivatization mix, consisting of propionic anhydride and acetonitrile in a 1 :3 ratio (v/v), and this was immediately followed by the addition of 7.5 pL ammonium hydroxide to maintain pH 8.0. The sample was incubated for 15 min at RT and the derivatization procedure was repeated one more time. Samples were then resuspended in 50 mM NH4HCO3 and incubated with trypsin (enzyme:sample ratio of 1 :20) overnight at room temperature. After digestion, the derivatization reaction was performed again twice to derivatize the N termini of the peptides. Samples were desalted using Cl 8 stage tips before LC-MS analysis.

Untargeted histone mass spectrometry

Histones were extracted from the mouse hippocampus by using a Nuclei Isolation Buffer (NIB) as previously described in the literature. The tissue was incubated in NIB (15 mM Tris-HCl, 15 mM NaCl, 60 mM KC1, 5 mM MgCh, 1 mM CaCk, and 250 mM sucrose at pH 7.5; 0.5 mM AEBSF, 10 mM sodium butyrate, 5 pM microcystin and 1 mM DTT added fresh) with 0.2% NP-40 on ice for 5 min. Two rounds of NIB incubation were performed at a volume buffer: cell pellet of 10: 1; the first round 0.2% NP-40 was added to lyse the cell membrane, and the second without NP-40 to remove the detergent from the nuclear pellet. Each step included centrifugation at 700 x g for 5 min to pellet the intact nuclei. Next, the pellet was incubated in 0.2 M H2SO4 for 2 hours, and the supernatant was collected after centrifugation for 5 min at 3,400 x g. Finally, histones were precipitated with 33% trichloroacetic acid (TCA) overnight. The histone pellet was then washed with ice-cold acetone to remove residual TCA. Histones were derivatized and digested as previously described (7). Histone pellets were resuspended in 20 pL of 50 mM ammonium bicarbonate (pH 8.0), and 10 pL derivatization mix was added to the samples, which consist of propionic anhydride and acetonitrile in a 1 :3 ratio (v/v), and this was immediately followed by the addition of 5 pL ammonium hydroxide to maintain pH 8.0. The sample was incubated for 15 min at 37 °C, dried and the derivatization procedure was repeated one more time to ensure complete derivatization of unmodified and monomethylated lysine residues. Samples were then resuspended in 50 pL of 50 mM ammonium bicarbonate and incubated with trypsin (enzyme: sample ratio of 1 :20) overnight at room temperature. After digestion, the derivatization reaction was performed again twice to derivatize the N-termini of the peptides. Samples were desalted using Cl 8 stage tips before LC-MS analysis and dried. Finally, the peptides were resuspended in 0.1% formic acid (FA) before nLC-MS/MS.

Primary hippocampal neuron culture

CD1 wild-type (WT) mice were purchased from Charles River. CD1 mouse cortices and hippocampi were dissected at embryo day 16-18 and dissociated with papain (Worthington Biochemical Corporation). Neurons were resuspended in neural basal medium (Gibco, 21,103) with 2% B27 (Gibco), 1 x Glutamax (Gibco), and 1 x penicillin/streptomycin (Gibco). Plates were coated with poly-d-lysine (0.1 mg/ml, Sigma-Aldrich) in borate buffer (0.05 M boric acid, pH 8.5) overnight at room temperature. Cells were plated at a density of 50,000 cells/cm² for all types of plates. When plating, 5% FBS was added to the cell suspension. The plating medium was replaced with a neural basal medium without FBS on day 1 in vitro (DIV1). At DIV7, the plating medium was replaced with a conditioned medium containing a 1 : 1 ratio of old and fresh medium. Tau fibrils were diluted in PBS to the desired concentrations, added on top of the cells, and incubated for 14 days until DIV 21. The dose of tau fibrils was scaled based on cell densities (pg tau/10⁶ cells) per different plate.

Total RNA extraction and sequencing

Total RNA was extracted and purified from primary hippocampal neurons and mouse brain using Trizol-chloroform and RNeasy kit (Qiagen) following the manufacturer's instructions. Total RNA quality was assessed on the Bioanalyzer platform using the RNA 6000 Nano assay (Agilent). mRNA was isolated from 130 ng total RNA using the NEBNext® Poly(A) mRNA Magnetic Isolation Module (E7490L), and libraries were prepared using the NEBNext® Ultra™ II RNA Library Prep Kit for Illumina® (E7770). All RNA-seq data were prepared for analysis as follows: NextSeq sequencing data were demultiplexed using native applications on BaseSpace. Demultiplexed FASTQs were aligned by RNA-STAR 2.5.2 to assembly mmlO (GRCm38). Aligned reads were mapped to genomic features using HTSeq 0.9.1 The significance of gene alterations was determined using the Wald test with multiple test correction according to the Benjamini Hochberg method with FDR < 0.05. Gene ontology (GO) analysis was performed using the DAVID bioinformatics suite, and top terms associated with biological processes were reported.

Single nuclei RNAseq

Hippocampi were dissected from PBS or AD-Tau injected WT and ACSS2 KO mice. Nuclei isolation was performed following lOxGenomics manufacturer’s recommendation (Protocol CG000375-RevB). Libraries were prepared using lOxGenomics Multiome kit following manufacturer’s recommendation (Protocol CG000338-RevC) and sequenced on the NextSeq 550 platform (Illumina) in accordance with the manufacturer’s protocol.

H3K27ac ChlP-seq in mouse hippocampus

Approximately 20 mg of hippocampal tissue from each mouse was minced on ice, cross-linked with 1% formaldehyde for 10 min and quenched with 125 mM glycine for 5 min. Nuclei were prepared by dounce homogenization of cross-linked tissue in nuclei isolation buffer (50 mM Tris- HC1 at pH 7.5, 25 mM KC1, 5 mM MgCh, 0.25 M sucrose) with freshly added protease inhibitors and sodium butyrate. Nuclei were lysed in nuclei lysis buffer (10 mM Tris-HCl at pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% sodium deoxycholate, 0.5% N-lauroylsarcosine) with freshly added protease inhibitors and sodium butyrate, and chromatin was sheared using a Covaris S220 sonicator to approximately 250 bp in size. Equal aliquots of sonicated chromatin were used per immunoprecipitation reaction with 4 pL H3K27ac antibody (Abeam; 4729, GR323132-1) preconjugated to Protein G Dynabeads (Life Technologies). Ten percent of the chromatin was saved as input DNA. ChIP reactions were incubated overnight at 4 °C with rotation and washed three times in wash buffer. Immuno- precipitated DNA was eluted from the beads, reversed cross-linked and purified together with the input DNA. Exactly 10 ng DNA (either ChIP or input) was used to construct sequencing libraries using the NEBNext Ultra II DNA library preparation kit for Illumina (New England Biolabs; NEB). Libraries were multiplexed using NEBNext Multiplex Oligos for Illumina (dual index primers) and sequenced on the NextSeq 550 platform (Illumina) in accordance with the manufacturer’s protocol.

Western blot

Cells and tissue were lysed in RTPA buffer containing 50 mM Tris pH 8.0, 0.5 mM EDTA, 150 mM NaCl, 1% NP40, 1% SDS, supplemented with HALT protease and phosphatase inhibitor cocktail (Life Technologies, number 78446). Protein concentration was determined by Bradford assay and equal amounts of protein were loaded onto 4-12% Bis-Tris polyacrylamide gels (NuPAGE). Proteins were transferred to PVDF membrane and subsequently blocked with 5% milk in TBS-T (blocking buffer). Membranes were incubated with primary antibodies diluted in blocking buffer for 4 °C overnight. Membranes were washed three times in TBS-T for 10 minutes each before incubation with HRP-conjugated secondary antibodies in blocking buffer. Membranes were washed again as before, developed with SuperSignal west pico PLUS chemiluminescent substrate (Thermo Fisher) then imaged with an Amersham Imager 600. Antibodies used: Rabbit anti-ACSS2 (1 : 1000, CST #3658 ), Mouse anti-mPLUM (1: 1000, Origene TA180072), Rabbit anti-Actin (1 :2000 CST #4970), Rabbit anti-H3K9ac (1 : 1,000, Active motif 39137), Mouse anti-flag (1 : 1,000, Sigma- Aldrich Fl 804).

Example 1: ACSS2 upregulation/overexpression improves cognitive function in aged mice

It has been previously demonstrated that depletion of ACSS2 leads to a reduction in histone acetylation associated with the transcription of neuronal genes. However, the extent to which ACSS2 upregulation may enhance neuronal function and increases resilience to age- associated cognitive decline remains unknown. Because ACSS2 functions together with other proteins and enzymes to modify gene expression, it was not clear whether upregulation of ACSS2 alone would affect age-associated cognitive dysfunction and/or neurodegenerative disease. Thus, to determine whether upregulating ACSS2-dependent chromatin processes improves age-associated cognitive decline, ACSS2 was overexpressed in the brain of aged (i.e. 21-22 months old) wildtype C57BL6 mice for two months.

A mouse ACSS2 OE plasmid (SEQ ID NO. 1) was developed, which was tagged with a C-terminal flag and a mPlum fluorophore reporter (ACSS2- flag-mPLUM). The plasmid was packaged into an AAV -PHP. eB (FIG. 1A). Given the translational potential of broad overexpression, AAV-PHP.eB carrying the ACSS2 gene was delivered to the hippocampus via retro-orbital injection. The vector backbone expressing only the mPLUM fluorophore was used for mock treatment (control). The PHP.eB stereotype is known to predominantly transduce NeuN+ neurons and therefore minimizes potential heterogeneous effects of AAV injection.

Previous studies show that retro-orbital injection of PHP.eB produces effective transduction of the central nervous system with minimal off-target transduction of the liver. A blinded behavioral study on three independent cohorts of aged mice detected a significant enhancement in cued memory recall (FIGs. 1B-1C), without differences in baseline learning two months post-injection (FIG. ID). Cued and contextual fear memory formation is dependent on the hippocampus. Mice with ACSS2 OE did not show any signs of hyperactivity (Y-maze: assessed by the number of arm entries) and had no changes in short-term memory recall (Y-maze: assessed by the number of spontaneous alterations) (FIGs. 1E-1F), suggesting that ACSS2 OE impacts long-term memory. Furthermore, after 2 months of overexpression, ACSS2 did not induce tumor growth in the brain or the liver.

Example 2: ACSS2 overexpression increases H3K9ac levels in the aged hippocampus

To capture histone modification changes in response to remote memory recall, mice were sacrificed 30 minutes after remote contextual recall (FIG. 2A). Remote memories are more stable than recent ones, therefore these histone modification changes represent stable changes for the retrieval of long-term memory. To unbiasedly assess the changes in histone modification that occur in the hippocampus (crucial for memory-related neuronal plasticity) of mice with ACSS2 overexpression, untargeted histone post-translational mass spectrometry was performed.

Proteomic screening detected significant upregulation of histone acetyltransferase for H3K9ac and modest upregulation in H3K14ac, H4K12ac, and H4K16ac levels with ACSS2 OE. Furthermore, western blot analysis validated H3K9ac overexpression in mice hippocampus treated with ACSS2 OE (FIGs. 2B-2D). Among perturbations linked to AD are changes in H3K9ac and H4K16ac levels. H3K9ac is associated with neuronal plasticity and broadly associated with tau pathology. This suggests that ACSS2 overexpression may protect the brain from transcriptional and epigenetic changes related to tau.

Example 3: ACSS2 overexpression reduces AD-tau pathology in primary hippocampal neurons

AD is the most common tauopathy and tau pathology correlates closely to neurodegeneration and cognitive decline. Reduced histone acetylation and dysregulation of critical neuronal genes are extensively reported in aging and AD brains. To determine whether ACSS2 upregulation can prevent AD-tau-related transcriptomic and epigenetic changes, primary hippocampal neurons isolated from CD1 WT mice were treated with human AD-tau as a model for the spread of AD-tau and AD-tau associated toxicity. Primary hippocampal neurons showed a significant reduction in ACSS2 protein level with AD-tau treatment (FIG. 3A).

Using immunohistochemistry with mouse tau antibody (T49) and high-content imaging, it was assessed whether the upregulation of ACSS2 impacts AD-tau pathology. ACSS2 was overexpressed in primary hippocampal neurons 2 days before AD-tau treatment (FIG. 3B). In a blinded analysis, DIV 21 WT neurons showed a significant reduction in mouse-tau pathology with ACSS2 OE (FIGs. 3C-3D), with minimal toxicity (FIG. 3E). A decrease in tau-pathology was unexpected, and indicates that epigenetic priming of the neurons with ACSS2 before AD-tau treatment improves neuronal resilience to pathological AD-tau insult.

Example 4: ACSS2 overexpression ameliorates AD-tau-induced transcriptomic changes in primary hippocampal neurons

The transcriptomic changes by which ACSS2 OE operates were investigated, with the operating hypothesis that ACSS2 OE would increase histone-acetylation associated with key neuronal genes. Following the same experimental paradigm as in FIG. 3B, ACSS2 was overexpressed before AD-tau treatment and RNA-sequencing was performed. The quality of data was first assessed using Principal component analysis (PCA). PCA showed that biological replicates cluster within the same treatment group (FIG. 4A). Next, AD-tau- induced transcriptomic changes were defined. Comparing AD-tau and PBS in mock-treated neurons shows that AD-tau reduces overall gene expression (533 significantly downregulated and 420 significantly upregulated, adjusted p-value <0.05) (FIGs. 4B-4C). Gene Ontology (GO) analysis of significantly dysregulated genes reveals changes in neuronal and synapse- related genes (FIG. 4E). This observation indicates that AD-Tau burden has a broad effect on the transcriptome.

Among the significantly dysregulated genes were the family of solute carrier (SLC) genes that have been linked to AD and other neurodegenerative diseases. Comparing transcriptomic changes between the ACSS2 OE and mock in PBS-treated samples, 607 differentially expressed genes (DEGs, adjusted p-value <0.05) were identified. It was found that ACSS2 OE alone increases overall transcription (403 significantly upregulated and 204 significantly downregulated, adjusted p-value <0.05) (FIG. 4B and FIG. 4D). Like AD-tau, GO analysis of significantly dysregulated genes was mostly related to neuronal and synapse- related genes. (FIG. 4F).

Interestingly, in conjugation with AD-tau, ACSS2 OE globally induced gene expression related to neurons (553 significantly upregulated and 251 significantly downregulated, adjusted p-value <0.05) (FIG. 4B, FIG. 4D, and FIG. 4G). These data indicates that ACSS2 OE operates to ameliorate gene dysregulation induced by AD-tau. Comparing ACSS2 OE in AD-tau treated neurons with PBS, suggests a transcriptional “tug-of- war”, related to neuronal genes, between ACSS2 OE and AD-tau (FIG. 4F and FIG. 4H).

Example 5: Injection of AD-Tau into mouse hippocampus recapitulates human AD-like pathology as well as gene expression and histone acetylation changes

Human pathological AD-Tau protein, obtained from post-mortem brains of Alzheimer’s disease (AD) patients, was injected into the hippocampus of 6 months old wildtype C57B16/J mice (FIG. 5A). This procedure induces AD-like pathology in mice characterized by hyperphosphorylation of endogenous Tau protein. Consistent with previous data, Tau phosphorylation was observed in the hippocampus of AD-Tau mice but not PBS- injected littermates (FIG. 5B), in a pattern mostly localized to the dentate gyrus with limited spreading to the entorhinal cortex. Next, molecular characterization of the hippocampus was performed in this model, assessing gene expression and the enrichment of specific types of histone acetylation implicated in the human AD brain. Brain tissue was collected 6 months post-injection, focusing on a time point where mice exhibit peak AD-like pathological changes.

While transcriptional changes were overall limited, differential expression of several genes previously linked to AD and neurodegeneration was nevertheless observed. These included e.g. Hpcall (Hippocalcin Like 1) anA Itga4 (Integrin Subunit Alpha 4), genes with polymorphisms associated with increased AD risk. Downregulation of Crmpl (Collapsin Response Mediator Protein 1) and Madd (MAP Kinase Activating Death Domain) was also observed, which comprise changes that have been linked to neuronal death and specifically correlate with neuron loss in AD, respectively. Of note, altered expression of enzymes that regulate histone acetylation, such as Anp32a (Acidic Nuclear Phosphoprotein 32 Family Member A), was also observed, which has been linked to synapse and memory loss in mouse models of AD and Hdac2 (Histone Deacetylase 2), which has been implicated in neurodegeneration and cognitive dysfunction.

On the epigenetic level, it was found that AD-Tau injection recapitulated changes observed in human AD. Increased acetylation of histone H3 lysine 27 (H3K27ac) was observed in the mouse hippocampus at loci analogous to those of the human brain. For example, both mouse and human showed induced H3K27ac at the transcription start site of Bnb3 (FIG. 5C), a gene encoding the Bub3 mitotic checkpoint protein implicated in neurodegeneration and other aging-associated phenotypes. Genome-wide, it was found that the H3K27ac peaks that were most specific to AD-Tau (top 10%) were strongly enriched at genes previously identified as showing H3K27ac dysregulation in human AD (FIG. 5D). Gene ontology analysis of genes associated with AD-Tau-specific H3K27ac peaks revealed enrichment of genes related to the regulation of axonogenesis, neurological system processes, long-term synaptic depression and the regulation of synaptic plasticity (FIG. 5E), which might contribute to AD-like learning and memory impairments in this model (FIGs. 6A-6D).

Example 6: ACSS2 knock-out exacerbates learning and memory impairments induced by AD-Tau injection in the mouse hippocampus

To characterize learning and memory impairments associated with hippocampal AD- Tau injection, a battery of behavioral assays were performed in both WT and ACSS2 KO mice 6 months post- injection. As reported previously for AD-Tau injection, no evidence was found for anxiety-like behavior in Open Field or elevated zero maze, and no impairments of short-term memory in Y maze. Further, locomotor behavior was not affected in any of the behavioral assays performed, emphasizing the lack of gross behavioral abnormalities in these mice.

Strikingly, however, marked impairments of long-term memory were observed using contextual fear conditioning (FC, FIG. 6A) and object location memory (OLM, FIG. 6C). Impaired performance of both ACSS2 KO mice and mice with dorsal hippocampal knock- down of ACSS2, respectively, has been previously reported. To eliminate potential investigator bias, experiments were performed in a blinded manner and analyzed using built- in software (FC) and AnyMaze from EthoVision (OLM). In the FC assay, mice were trained to associate a specific environment with an aversive stimulus (mild foot-shock). Upon reexposure to the context 24 hours later, impairments of contextual recall in were observed AD- Tau -injected and ACSS2 KO mice (one-way ANOVA, F3, 80=4.001, p=0.0104; FIG. 6B). Post-hoc multiple comparisons revealed that this was mostly driven by a significant decrease of freezing in AD-Tau-injected ACSS2 KO mice compared to PBS-injected WT mice (Tukey’s test, p=0.0049), suggesting that the “double hit” of hippocampal AD-Tau injection and loss ofACSS2 significantly exacerbated the long-term memory impairments in these mice. Even more pronounced differences were observe during remote recall that was performed two weeks following FC training (one-way ANOVA F3, 80-5.592, p=0.0016, driven by Tukey’s test p=0.0008 in the AD-Tau-KO vs PBS-Wt comparison; FIG. 6B). The observed deficits of long-term memory were further emphasized by similar impairments in a cohort of mice that underwent OLM.

In this assay, mice were familiarized with three inanimate objects and allowed to interact with these 24 hours later with one object’s location changed (FIG. 6C). While PBS- injected WT mice tended to explore the moved object more, demonstrating intact long-term spatial memory, the discrimination index was markedly decreased in AD-Tau-injected WT and PBS-injected KO mice (FIG. 6D). As seen in FC, decreased memory was further impacted by the “double hit” of AD-Tau injection and ACSS2 loss (one-way ANOVA F3, 67=3.281, p=0.0261; Tukey’s test p=0.0310 in the PBS-WT vs AD-Tau-KO comparison).

Taken together, it was found that while AD-Tau injection and ACSS2 knock-out both impaired long-term memory formation, these deficits were strongly exacerbated with the “double hit” of Tau seeding in transgenic mice that do not express ACSS2. As short-term memory, anxiety- like behavior and general locomotion were not affected, these results suggest that loss of ACSS2 specifically contributes to a more severe impairment of long-term memory in a mouse model of Alzheimer’s disease.

Example 7: Transcriptional characterization of AD-Tau injected WT and ACSS2 KO mice Next, transcriptional profiling in AD-Tau injected ACSS2 KO mice and WT littermates was performed. Intriguingly, and in line with the behavioral results, the combination of AD-Tau injection and loss of ACSS2 led to a striking increase of transcriptional dysregulation. While only a handful of differentially expressed genes (DEGs) were observed with AD-Tau in both WT and ACSS2 KO mice, the comparison of WT PBS mice to ACSS2 KO AD-Tau littermates (z.e., “double hit” of Tau seeding and gene loss) revealed over 1,000 DEGs (FIG. 7A). Gene ontology analysis showed that dysregulated genes were related to nervous system development, axon guidance, neuronal apoptosis and ion channel function (FIG. 7B), suggesting a broad impairment of neuronal function. In line with previous findings linking ACSS2 to the regulation of immediate early genes, this family of genes was strongly represented among the DEGs with Npas4 being the most significantly affected transcript other than Acss2.

Example 8: Cell-type specific profiling of gene expression highlights impairments of Cajal- Retzius cells in AD-Tau-injected ACSS2 KO mice

To delineate the specific cell populations affected by AD-Tau injection and loss of ACSS2, transcriptional characterization was performed on the single cell level. The lOx Genomics multiomics platform was used to assess gene expression (RNAseq) from single nuclei. A total of 21,161 nuclei from the hippocampi of PBS or hpADT-injected ACSS2 KO mice and WT littermates were sequenced. Using this strategy, 9 neuronal and non-neuronal cell types were identified in the hippocampus (FIG. 8A). The majority of these were remarkably stable among the four conditions. No obvious differences in the number of excitatory neurons, inhibitory neurons, oligodendrocytes/OPCs, astrocytes or microglia were observed in ACSS2 KO mice with or without AD-Tau injection (FIG. 8B).

Strikingly, however, the number of Cajal -Retzius cells (CR) was markedly reduced in AD-Tau injected mice, especially in the transgenic background (FIG. 8B). CR cells are glutamatergic neurons in the cortex and hippocampus that play an important role developmentally and have been linked to learning and memory in adult animals. Loss of CR cells at different developmental stages has been previously reported in transgenic mouse models of AD. In addition to the decreased number of cells, CR neurons also showed the strongest transcriptional impairments when WT mice were compared to AD-Tau-injected ACSS2 KO animals (FIG. 8C).

821 differentially expressed genes were identified in CR neurons in the WT PBS vs. KO Tau comparison. As with the bulk RNAseq (FIGs. 7A-7B), transcriptional changes in CR cells were less pronounced with AD-Tau injection only (333 DEGs) or ACSS2 KO only (145 DEGs). Remarkably, the pattern of gene expression changes matched the severity of behavioral impairments in the fear conditioning assay (FIGs. 6A-6D): The top upregulated and top downregulated genes were increased or decreased both by hippocampal AD-Tau injection and loss of ACSS2, with significantly more pronounced changes in the “double hit” mice (FIG. 8D). GO analysis indicated the enrichment of genes related to axonogenesis, synapse organization, as well as metabolic functions (FIG. 8E), suggesting that both the number and activity of CR cells are severely compromised in AD-Tau-injected ACSS2 KO mice.

Example 9: Exogenous acetate promotes hippocampal histone acetylation and memory formation in vivo

It was reasoned that, since inhibiting metabolic-epigenetic interactions by knocking out the nuclear metabolic enzyme ACSS2 exacerbates molecular and behavioral phenotypes observed in the AD model described herein, stimulating this pathway by supplying the substrate of ACSS2 (z.e., acetate) might have beneficial effects. To test this hypothesis, it was first demonstrated that exogenous acetate is incorporated into hippocampal histone acetylation. Mice were injected with <A-acetate i.p., and heavy labeling of acetylated histones at different timepoints was measured using mass spectrometry. A transient and dosedependent deposition of acetate on hippocampal histones (j.e., peak labeling at 30 min) was observed (FIGs. 9A-9B). Heavy label incorporation was dependent on ACSS2 and was not observed in KO mice (FIG. 9C). Further, H3K27ac ChlPseq and RNAseq were performed in the hippocampus of saline or acetate-injected mice. It was found that 262 peaks were induced by acetate, the vast majority of which were not observed in ACSS2 knock-down (KD) mice (FIG. 9D). Peaks were assigned to the nearest transcription start site to perform GO analysis, and found enrichment of genes related to membrane function, axon extension, and intracellular signaling (FIG. 9E). Example genes included Pcdhbl5 and Pcdhbl6 encoding for protocadherins implicated in dendrite development, neuronal circuit formation and neuropsychiatric disorders (FIG. 9F).

Notably, while there was a strong positive correlation between differential acetylation and differential gene expression in WT mice (Pearson’s r=0.31, p=3.7xl0'⁸), this was significantly blunted in ACSS2 KD animals (Pearson’s r=0.1, p=3.4xl0'⁴; FIG. 9G), emphasizing the role of ACSS2-mediated acetate incorporation at sites that directly regulate transcription. Further, GO analysis revealed that genes induced by acetate in an ACSS2- dependent manner were related to cell junction, synapse and postsynaptic membrane functions, while those that were still elevated in ACSS2 KD mice were less specific to neuronal activity, such as protein binding and kinase function (FIG. 9H). These results suggest that acetate incorporated by ACSS2 could enhance neuronal activity in the hippocampus, potentially driving learning and memory.

To test this more directly in behaving animals, a sub-threshold cocaine conditioned place paradigm (CPP) was utilized. This model comprises only one pairing between a specific spatial environment and a low dose (1 mg/kg) of cocaine administered i.p. Strikingly, while WT mice did not readily learn this association (paired Student’s t test, t6=1.425, p=0.2039), strong place preference was found when cocaine was co-injected with 1.5 g/kg acetate (paired Student’s t test, t6=6.445, p=0.0007, FIG. 91). Importantly, this dose of acetate did not induce place preference or aversion when administered alone, suggesting that it acts by facilitating the encoding of association between cocaine reward and spatial clues. This effect of acetate was dependent on ACSS2, as mice with hippocampal knock-down of ACSS2 expression performed poorly in this assay (paired Student’s t test tl0=6.554, p=0.0001 in WT mice and pair Student’s t test, tl 1 = 1.22, p=0.248 in ACSS2 KD mice; FIG. 9J).

Taken together, it was unexpectedly found that exogenous acetate is readily incorporated into hippocampal histone acetylation and drives gene expression, learning and memory in an ACSS2-dependent manner.

Example 10: Acetate-enriched diet ameliorates AD-Tau-induced learning and memory deficits Next, it was evaluated whether stimulating ACSS2-driven histone acetylation by supplying exogenous acetate could reverse learning and memory impairments in the context of AD. WT mice were injected with 1 ug AD-Tau in the hippocampus and underwent fear conditioning training 6 months post-seeding. Since acute exposure to acetate (1.5 g/kg i.p. prior to acquisition) did not rescue decreased freezing in this model, it was hypothesized that chronic administration of acetate might be required to reverse the long-lasting effects of AD-Tau seeding. To test this, mice were maintained on a special diet enriched in acetate (5% w/w) throughout the 6 months incubation period post AD-Tau -injection. As previously reported, mice administered the acetate diet had slightly reduced body weights compared to controls maintained on a caloric equivalent diet (Student’s t test, t46=2.236, p=0.0302), but showed no signs of sickness, anxiety or any impairments of general locomotion in Open Field (OF). Further, chronic acetate had no effect on freezing behavior in mice that were not exposed to AD-Tau (Student’s t test, t22=1.276, p=0.2153, FIG. 10). In the AD model, however, the acetate diet completely reversed learning impairments and resulting in freezing levels similar to that observed in mice injected with vehicle (Student’s t test, t22=2.397, p=0.0254, FIG. 10). These data are remarkable in hat they indicate that long-term dietary supplementation of acetate protects from AD-tau associated cognitive deficits in mice. Importantly, this procedure may be translatable to humans with no expected adverse side effects.

Example 11: ACSS2 overexpression (OE) Maintains Histone Acetylation and Preserves Expression of Synaptic Genes in Hippocampal Neurons Over Time

Primary mouse hippocampal neurons were transduced with adeno-associated virus (AAV)-PHP.eB to upregulate ACSS2 tagged with a C-terminal flag and a mPlum fluorophore (ACSS2 OE) under the control of the constitutive elongation factor 1 (EFla) promoter (FIG. HA). The viral vector expressing mPlum (vector) only was used as a control throughout the experiments. Upregulation of ACSS2 protein was confirmed in neuronal culture (FIGs. 1 IB-11C and FIG. 12A). In accordance with the canonical role of ACSS2 in promoting histone acetylation, there was increased expression of transcriptional-linked H3K9ac and H3K27ac in neurons transduced with the ACSS2 OE plasmid compared to vector control (FIGs. 1 IB-1 IE). Notably, neurons exhibited an intrinsic reduction in the expression of these two histone acetylation markers over time, a trend that was counteracted by the continuing acetylation promoted by ACSS2 upregulation (FIGs. 1 IB-1 IE). Consistent with previous finding that established ACSS2 as a nuclear-metabolic enzyme in neurons, the nuclear localization of exogenous ACSS2 within the mouse hippocampal neurons was validated (FIG. 1 IF). Moreover, to evaluate potential toxicity linked to ACSS2 upregulation, neuronal survival was quantified following viral transduction. Interestingly, rather than loss, a modest but non-significant enhancement in neuronal survival was detected with ACSS2 OE at a higher dose (FIG. 1 IB).

To comprehensively characterize the impact of ACSS2 upregulation on the transcriptional dynamics within primary hippocampal neurons, RNA-sequencing (RNA-seq) was conducted at two distinct time points. 9 days and 21 days in vitro (DIV) neurons were used to capture gene expression changes with ACSS2 upregulation during different phases of neuronal development. Between the two-time points, there were 7,587 differentially expressed genes (DEGs) (P-adj 0.05) (FIG. 11G). This captured a snapshot of gene regulation during the maturation of these neurons. There was a notable downregulation of synaptic genes in 21 DIV neurons, enriched in pathways related to axon guidance, glutamatergic and cholinergic synapses (FIGs. 11G-11H and FIG. 12C). These neurotransmitter systems are critical in hippocampal memory function and undergo degeneration and dysfunction in aging and disease. A decrease in the expression of genes related to long-term potentiation (LTP) and longevity was also observed, including insulin singling pathways and FoxO signaling pathways (FIGs. 11G-11H and FIG. 12C). In parallel, there was an upregulation in mitochondrial, lysosomal, and senescence-related genes (FIG. 11G, FIG. 1 II, and FIG. 12D). These results indicate a dynamic shift in the molecular landscape of neurons with time, predominantly affecting the expression of genes associated with synaptic integrity and function. These changes are consistent with the intrinsic reduction observed in transcription-linked H3K9ac and H3K27ac over time (FIGs. 1 IB-1 IE).

To gain insight into the immediate and long-term impact of ACSS2 upregulation on gene expression, differentially expressed genes (DEGs) were assessed between ACSS2 OE and control at 9 DIV (4 days post-transduction) and 21 DIV (16 days post-transduction). 1,076 and 627 DEGs (P-adj<0.05), were observed, respectively (FIGs. 11J-1 IK). A notable overlap in the upregulated pathways between the two time points, related to neuronal system and synapses

(FIG. 12E). The downregulated pathways were more distinct between the two time points, with a reduction in RNA metabolism and stress response enriched at 9 DIV and lipid metabolism at 21 DIV (FIG. 12F).

By examining DEGs that overlap between these two time points (FIG. 1 IL and FIG. 121), it was found that ACSS2 OE upregulates the expression of consensus signature genes associated with the regulation of synaptic transmission and AMPA receptor activity (Mef2c, Sytl, Nlgn2, and others), learning and memory (Grinl, Shankl, Netol, Shisa7, and others), (hypergeometric test p < 1 669e-21) (FIGs. 1 IL-1 IM). Myocyte enhancer factor 2c (Mef2c) is a gene implicated in cognitive resilience and its upregulation confers protection against neurodegeneration. In parallel, a small number of DEGs commonly downregulated between the two time points were detected (FIG. 121). Although the number of commonly downregulated DEGs was not sufficient for Gene Ontology analysis (GO), among these genes, the high-temperature requirement serine peptidase Al (HtrAl) was found, with important proteolysis activity, required for the degradation of pathological deposits. HtrAl inactivity has been implicated in age-related macular degeneration and is associated with tau and amyloid aggregation, two major hallmarks of AD (FIG. 121). Genes associated with ACSS2 OE were further examined in the context of synaptic location using the SynGO database and the results suggested that ACSS2 OE regulates the expression of genes associated with both the pre-and postsynaptic compartments (FIG. 1 IN). The upregulation of pre-synaptic marker synapsin in hippocampal neurons was further confirmed with ACSS2 OE by immunofluorescence (FIGs. 110-1 IP). Synapsin controls the release of neurotransmitter, including critical molecules like glutamate, which has a crucial role in synaptic plasticity, a process fundamental to learning and memory.

Hierarchical clustering of z-score normalized DEGs revealed distinct patterns of gene regulation over time and with ACSS2 OE (FIGs. 1 IQ-11R). Neurons exhibited an intrinsic reduction in the expression of genes associated with nervous system development and axon guidance from 9 to 21 DIV, which was countered by ACSS2 OE (clusters 2 and 3). An upregulation in the expression profile of mitochondrial genes (clusters 4 and 5), and proteasomal genes (cluster 6) became evident over time, a pattern that was once again countered by ACSS2 OE (clusters 4 and 5) (FIGs. 1 IQ- 11R). The observed reduction in mitochondrial and proteasomal genes by ACSS2 upregulation may be attributed to a cellular state in which metabolic modifications are unnecessary for the maintenance of neuronal health and function, indicating that the neurons may be operating at a higher level of homeostasis and overall health with ACSS2 upregulation. A decrease in the expression of histone-modifying genes was also observed, including histone acetyltransferase 2A, 6A, and 6B (Kat2a, Kat6a, Kat6b), and histone deacetylases (HD AC 2, 4, and 6), in neurons at 21 DIV. Reduction in epigenetic and histone- modifying enzymes has been observed during aging and disease. Notably, this altered gene expression profile was sustained with ACSS2 OE (FIG. 12J). Collectively, these data indicate that ACSS2 upregulation enhances and maintains the expression of neuronal and synaptic genes over time in hippocampal neurons in vitro.

Example 12: AD-tau pathology Diminishes Synaptic Gene Expression and Reduces H3K27ac Hippocampal Neurons

Tau plays a prominent role in the pathology of ADRD with pathological tau thought to spreads across the brain, converting normal tau proteins into the pathological hyperphosphorylated form. Given the insights gained from the transcriptomic analysis highlighting the potential of ACSS2 to maintain and enhance synaptic gene expression, it was asked whether upregulating ACSS2 could promote resilience against tau-associated pathology. To do this, an in vitro model of primary hippocampal neurons was adapted and treated with human AD-tau (hAD-tau), as a model for the spread and toxicity of AD-tau. First, gene expression changes with tau treatment alone were characterized, performing RNA-seq of hippocampal neurons treated with tau for 14 days. Comparing transcriptomic changes between the tau and PBS treated (control) neurons identified 299 differentially expressed genes (DEGs, P-adj<Q.Q5) (FIG. 13A). Among the significantly upregulated genes were the apolipoprotein E gene (APOIP), Adrenomedullin (Adm), and serpin peptidase inhibitor (Serpina3n),' importantly, these genes are associated with AD. Further, the activator protein 1 (AP-1) superfamily members (JUN and FOS) were among the significantly upregulated genes (FIGs. 13A-13B) and are associated with aging. GO analysis of significantly downregulated genes revealed decreased expression of genes of synapse biology and synaptic membrane potential (FIG. 13C). Moreover, Mef2c was among the significantly downregulated genes with hAD-tau (FIG. 13 A). Comparison of these 299 significant DEG with the AD brain gene expression data sets from Mayo Clinic and the Religious Orders Study and Rush Memory and Aging Project (ROSMAP) showed that these same genes are significantly (p<0.0001) dysregulated in human AD patient brains (FIG. 13D). These data underscore that tau burden has a broad effect on the epigenome. To assess the extent to which the observed transcriptional changes are associated with changes in H3K27ac levels, Cut&Run was performed. hAD-tau treated neurons showed a significant reduction in global H3K27ac levels (FIGs. 13E-13F), which is highly correlated with gene expression dynamics from the RNA-seq data (R=) (FIG. 13G).

Motif enrichment analysis of significant H3K27ac peaks identified the neuronal transcription factor NeuroDl associated with neurogenesis, NFATC2 known for its role in efficient axonal growth, and NF 1 highly associated with learning and cognitive function (FIG. 13H). Together, these data identify a decrease in learning-related genes and upregulation of age- associated genes associated with reduced histone acetylation (H3K27ac) in primary hippocampal neurons upon hAD-tau treatment. Further, hippocampal neurons showed a significant reduction (P<0.05) in ACSS2 protein level with hAD-tau treatment, and a modest reduction in H3K9ac levels (FIGs. 14A-14C), indicating that changes induced by tau impact ACSS2 expression.

Example 13: ACSS2 OE Counters AD-Tau Induced Transcriptomic Changes and Enhances Neuronal Resilience to Tau Pathology

To determine whether ACSS2 upregulation could counter transcriptomic changes induced by hAD-tau, the hippocampal neurons were primed with ACSS2 OE before hAD-tau treatment (FIG. 15 A). hAD-tau-induced transcriptomic changes with ACSS2 OE were assessed at 9 and 21 DIV, corresponding to early and late stages of hAD-tau pathology, respectively (FIGs. 16A-16E). Upregulation of ACSS2 globally enhanced the transcriptional response in the presence of hAD-tau (FIG. 16 A), with hierarchical clustering of z-score normalized DEGs identifying 7 clusters (FIG. 15B). At 9 DIV there was a similar pattern of transcriptional changes induced by hAD-tau in hippocampal neurons treated with ACSS2 OE compared to control (FIG. 15B); given the absence of tau pathology at 9 DIV, this early time point may represent compensatory transcriptional changes against tau insult. Strikingly, at 21 DIV, when tau pathology is fully developed, hippocampal neurons primed with ACSS2 OE maintained a similar pattern of gene expression changes from 9 DIV (FIG. 15B). GO analysis of genes from cluster 2 represented biological processes related to synaptic function, which remained upregulated with ACSS2 OE and with the progression of pathology, whereas their expression was diminished with hAD-tau alone (vector) (FIG. 15C). Cluster 6 represented proteasomal genes that were upregulated only at 21 DIV with ACSS2 OE (FIG. 15C).

It was also examined whether ACSS2 OE could confer resilience against tau pathology. Using high-content imaging of 21 DIV neurons stained with a mouse specific antibody to pathological tau (AT8), a significant reduction in endogenous tau pathology was observed in primary hippocampal neurons with ACSS2 OE (FIGs. 15D-15E). This was unexpected, and collectively these data suggest that ACSS2 OE can counter AD-tau induced transcriptomic changes and confer resilience against tau pathology and its impact on synaptic dysfunction, as well as mitigate pathological hallmarks associated with pathogenic tau.

Example 14: Pre-symptomatic enhancement of ACSS2 Rescues Tan-Induced Memory Decline, Plasticity, and Pathology in Vivo

Tau pathology is a major driver of cognitive dysfunction in ADRD and exerts its most detrimental effects within the hippocampus, which is crucial for memory. As tau pathology advances, synaptic function required for cognitive function and memory is disrupted. This observation underscores the crucial role of preserving synaptic integrity in AD and other tau- related dementias. The data indicated that ACSS2 OE was marked effective in a spreading model in vitro, to mitigate transcriptional changes and tau pathology. To extend these data from primary neurons in vitro to the brain in vivo, it was assessed whether ACSS2 OE could increase neuronal resilience to tau-induced memory decline of the P301S (PS19) mouse, a transgenic mouse model of tauopathy known for its age-associated spatial memory deficits. Animals were treated via bilateral stereotaxic injection into the dHPC (FIG. 17A) with PHP.eB ACSS2 OE plasmid or Vector control. PHP.eB serotype is known to predominantly transduce NeuN+ neurons and therefore minimizes potential heterogeneous effects of AAV injection. Mice were injected at 2.5 months of age to assess if early intervention can enhance resilience to disease-associated perturbations, which emerge ~8-9 month.

In blinded behavioral studies, anxiety levels and spatial memory were examined in the mice at 9 months of age using the open field test and contextual fear conditioning (FC), respectively. There were no differences in anxiety-related behavior between the ACSS2 OE and control PS 19 mice (FIGs. 18A-18D) and, importantly, at 9 months the PS 19 mice displayed full mobility with no signs of motor deficits during the open field test (FIGs. 18E-18H). Moreover, there were no noticeable distinctions in the acquisition of fear memory between the ACSS2 OE and control PS19 mice (FIG. 181). However, the ACSS2 OE PS19 mice exhibited a significant increase in memory for freezing behavior in 24-hour contextual memory recall compared to the control group (FIGs. 17B-17C). These findings indicate that ACSS2 OE enhanced the consolidation and long-term retention of contextual fear memory of PS 19 mice. Given the known synaptic dysfunction and LTP deficits extensively characterized in the PS 19 mouse, it was hypothesized that the observed memory improvement with ACSS2 OE may be due to enhanced LTP. LTP is a primary synaptic plasticity change required for memory storage and retrieval and its dysfunction underlies memory loss. Neuronal firing patterns induce changes in synaptic plasticity that can selectively strengthen or weaken neuronal networks. In neurodegenerative diseases involving tau pathology, a subpopulation of neurons demonstrates a reduction in intrinsic excitability. To examine LTP, PS19 mice were injected with ACSS2 OE or empty viral vector as before (FIG. 17A) and assessed the background excitability of CAI pyramidal neurons, which are central in the consolidation and retrieval of hippocampal-dependent memories, 1- month post injection (mpi). Spontaneous action potential firing was recorded while the neurons were maintained in tight-seal, cell-attached recording mode for about 5-10 minutes. Spontaneous firing in CAI pyramidal neurons from ACSS2 OE mice (11.0 ± 7.7 Hz, n=13) was statistically faster (FIG. 17E, p < 0.04) than those from vector-control mice (5.0 ± 2.9 Hz, n=9) (FIG. 17D). Subsequently, the whole-cell recording mode was established to examine the electrophysiological properties of the neurons further. The response to a hyperpolarizing current (injected -40 pA) and to a depolarizing current (injected 80 pA) under current clamp mode (FIG. 17F) were examined. CAI pyramidal neurons from ACSS2 OE mice fired more action potentials (8.8 ± 1.9 Hz, n=14) than those neurons from the control mice (1.8 ± 1.0 Hz, n= 6) (FIG. 17G, p=0.04). Furthermore, the amplitudes of the evoked action potentials were also significantly greater in ACSS2 OE mice (101.2 ± 4.1 mV, n= 14) than in vector controls (79.6 ± 4.7 mV, n= 6) (FIG. 17H, p=0.01). Importantly, no differences were found in either resting membrane potential, action potential threshold, or input resistance (FIGs. 17J-17L). These findings indicate that the excitability of CAI pyramidal neurons in ACSS2 OE mice was significantly higher than in vector-control mice.

Next, the induction of LTP was examined. First, the input-output relationship of the basal synaptic strength in each CAI pyramidal neuron (FIGs. 17M-17N) was determined, and no significant differences were detected between the control and ACSS2 OE mice. The stimulus intensity was then adjusted to obtain a small stable baseline with comparably sized EPSPs (p = 0.8) in neurons from ACSS2 OE (1.5 ± 0.3 mV, n=5) and vector-control (1.6 ± 0.2 mV, n=6) mice. After LTP induction by high-frequency stimulation (HFS), the postsynaptic currents were assessed: the postsynaptic currents were larger in the ACSS2 OE mice than in the vector-control mice, indicating significantly greater LTP was induced by HFS in neurons from ACSS2 OE mice (p=0.01, 231.2 ± 33.4% vs baseline, n=5, paired t-test) compared to vector-control mice (p=0.3, 125.0 ± 20.2% vs baseline, n=6, paired t-test) (FIGs. 17I-17K; p=0.02). The difference in LTP formation indicates improved CAI pyramidal neuron function in ACSS2 OE mice.

Neuropathological protein accumulation in AD disrupts the balance of inhibitory and excitatory synaptic transmission, propagating neuronal dysfunction. To determine whether the improved electrophysiological features and memory of PS 19 mice with ACCS2 OE were correlated with a change in tau pathology, control vs ACSS2 OE mice at 9 months were immunostained for pathological tau (AT8). Immunohistochemical analysis revealed a significant reduction in tau pathology in PS19 mouse hippocampus (FIGs. 17L-17M) and improved NeuN+ immunostaining (FIGs. 17N-17O), indicative of reduced neurodegeneration, with ACSS2 OE. These observations extend the earlier findings in the in vitro model showing that ACSS2 OE protects neurons against pathological tau accumulation, to now show that ACSS2 upregulation can protect from pathological tau accumulation in the brain in vivo. Together these data indicate a protective role of ACSS2 upregulation against tau-induced detrimental effects on gene expression, histone acetylation, memory Isos, neural electrophysiological decline, and tau pathology in vivo.

Example 15: Global changes to CpG DNA methylation in AD mice are reversed with ACSS2 upregulation To capture gene expression changes associated with improved memory in PS19 mice, RNA-seq was conducted on the dorsal hippocampus (dHPC) of mice euthanized 30 min post context recall (FIG. 19A). It is well-established that contextual recall is dependent on the dHPC. RNA-seq was performed on 1 mpi and 6 mpi treated mice with confirmed improved memory at both time points (FIG. 20A). Analysis of expression changes showed upregulation of synaptic genes with ACSS2 OE at 1 mpi (FIG. 19B and FIG. 19D), which points to an early and robust effect of ACSS2 on synaptic function and indicative of molecular mechanisms that may underlie the improved LTP and memory observed in PS 19 mice. The analysis also revealed changes in fundamental cellular processes, including metabolic and ribosomal genes, at 6 mpi (FIGs. 19C- 19D). These data highlight a dynamic temporal profile of gene expression changes, with early synaptic changes followed by alteration in broader cellular processes that likely support longterm memory enhancement. Taken together, these findings indicate that the impact of ACSS2 upregulation on synapses is most pronounced early after injection (by 1 month); this upregulation leads to strengthening of synapses and LTP and, even as synaptic gene expression returns to baseline levels, the memory-enhancing effects persist over time.

The significant impact of a single dose injection of ACSS2 OE in reversing disease phenotype and rescuing memory deficits 6 months post-injection in the PS 19 mice, signifies the presence of long-term and persistent epigenetic effects. While histone acetylation is an integral part of epigenetic modification regulated in part by ACSS2, the extent and sustainability of the changes observed with ACSS2 upregulation were unexpected and suggested the involvement of additional layers of epigenetic modification. There is a strong association between increased DNA methylation, elevation of pathology and decline in global cognitive function in AD patients. This suggests the role of these epigenetic mechanisms in driving AD. Therefore, it was considered that ACSS2 OE may counteract the impact of tau on DNA methylation. In 9-month- old PS 19 mice with ACSS2 upregulation, CpG DNA methylation levels closely resembled those observed in young (3-month) PS 19 mice (FIG. 19E), suggesting a reversal of CpG DNA methylation signatures induced by tau and age-related factors.

Examining both hypo- and hypermethylated CpG regions indicated enrichment in promoter and intronic regions (FIGs. 19F-19G). GO analysis of the hypom ethylated CpG regions (5482 single CpGs) revealed a reduction in histone methylation at the promoters of genes associated with NMDA glutamate receptor signaling, astrocyte-dopaminergic neuron signaling, and neurogenesis (FIG. 19H). GO of the hypermethylated regions (1759 single CpGs) showed enrichment of immune system-related pathways (FIG. 191). Together, these data unexpectedly suggest an indirect role of ACSS2 in regulating DNA methylation, and a dynamic interplay between ACSS2 and the broader cellular machinery governing gene expression.

Example 16: ACSS2 upregulation enhances age-associated cognitive decline

Aging is the predominant risk factor for ADRD. The remarkable outcomes of ACSS2 upregulation in preventing tau pathology and tau-associated memory decline, coupled with reversing the DNA methylation pattern in adult AD mice hold profound implications for its potential to rejuvenate the aging brain and mitigate the primary risk factor for this disease. Given these findings, it was investigated whether ACSS2 upregulation could function to protect against normal age-associated memory decline, in the absence of underlying pathology or disease.

To assess the impact of ACSS2 on the normal aging brain, ACSS2 was upregulated in adult (10-11 months) mice through retro-orbital injection. Retro-orbital injection of PHP.eB produces more effective transduction of the CNS and with minimal off-target transduction of the liver. To capture early gene expression changes, wild-type mice were euthanized two weeks post ACSS2 OE and the dHPC harvested 30 min after the acquisition of FC (FIG. 21A). Consistent with the role of ACSS2 in enhancing activity-dependent transcriptional response, RNA-seq analysis showed a significant increase in upregulated genes (FIG. 21A), with 328 upregulated and 40 downregulated genes with aP-adj <0.1, or 199 upregulated and 11 downregulated genes with aP-adj <0.05, in the dHPC of WT mice, following the acquisition of fear memory (FIG. 21B). Consistently, ACSS2 OE upregulated the expression of consensus signature genes associated with neuron projection and axons (FIG. 21C). ACSS2 OE also upregulated the expression of key genes involved in longevity-regulating pathways (Foxo3, Irs2, Mtor), circadian clock (Perl, Kcnj6, Cacnalh), and chromatin -modifying enzymes (Ep300, Ep400, Kat6a, Kmt2a, Jade2).

It was then asked whether ACSS2 upregulation could improve age-associated cognitive decline. Aged mice (21-22 months old) were injected via retro-orbital injection (FIG. 21D) with ACSS2 upregulation and H3K9ac levels in the hippocampus of these mice confirmed at two months post-injection (FIG. 2 IE). Long-term and short-term memory were examined. In a blinded behavioral study, there was a notable increase in the percentage of freezing behavior upon ACSS2 upregulation in aged mice with cued FC (FIGs. 21F-21G); contextual FC showed a trend toward upregulation, but did not reach statistical significance (FIGs. 21F-21H). No difference in baseline freezing behavior between the two groups was found during the acquisition phase (FIG. 22A). ACSS2 OE did not show an impact on short-term memory through the Y-maze paradigm (FIGs. 22B-22C), underlining the role of ACSS2 in enhancing long-term memory.

Following the behavioral assays, activity-dependent global histone modifications and gene expression changes in aged mice hippocampus were examined. Mice were euthanized 30 min post context recall to assess ACSS2-dependent activity-induced changes. Consistent with immunoblotting results and in vitro data, unbiased profiling of histone post-translational modification changes within the hippocampus of aged mice by mass spectrometry detected a significant upregulation in H3K9ac levels (FIG. 211) a mark associated with active transcription and open chromatin configuration. H3K27ac, a low-abundant modification, was not strongly detectable. Although not significant, a marked upregulation was detected in H4K16ac and H3K14ac, which are lost in AD, and H4K12ac, known to be lost in normal aging. Notably, restoring H4K12ac has previously been shown to ameliorate age-associated memory decline. Concurrently, gene expression changes were analyzed in aged mice with ACSS2 upregulation. Transcription analysis of genes correlated with cued memory recall signified upregulation of consensus signature genes associated with neuronal system (Gabbr2, Abcc8, Cam2g, Comt, Cacng3, and others), potassium channels (Kcncl, Kcnj2, Kcnj 10, Kcnj 11, and others), CREB1 phosphorylation (Camkk2 and Camk2g), and downregulation of proteasomal genes (Psmal, Psma2, Psma6, Psma7, and Psmd6).

Given the remarkable memory enhancement observed in aged mice (FIG. 21G), and the upregulation of longevity-related pathways in the dHPC of adult wildtype mice (FIGs. 21B-21C) with ACSS2 OE, the potential efficacy of ACSS2 OE in countering age-associated DNA methylation was assessed. The DNA methylation profiles of aged mice (21-22 months) treated with ACSS2 OE were compared to young mice and those of aged-matched control mice treated with an empty vector (FIG. 22D). There were no significant changes, suggesting that ACSS2 exerts its effect on memory decline in a manner independent of DNA methylation changes in the brain, at least at this level of detection.

Example 17: Manipulation of the Drosophila homolog of ACSS2 modulates lifespan

To extend the findings described herein, studies of the Drosophila counterpart of ACSS2 (i.e., AcCoAS, hereinafter “dACSS2”) have been performed. dACSS2. RNAi (to knock down dACSS2) and \JA -dACSS2 (to upregulate dACSS2)' have been expressed selectively in neurons, to determine if manipulation of the gene in the brain has effects on lifespan and/or healthspan. Lifespan is a robust assay used to define and identify genes required for age/aging of the animal, where some of the most impactful pathways that modulate health and lifespan have been discovered from model organisms like Drosophila and C. elegans. Hence, an effect on lifespan upon manipulation of the gene in the brain is an indicator that the gene has the potential to also modulate age-associated events of the nervous system in mammals, including humans.

It has been found that knockdown of dACSS2 selectively in neurons shortens lifespan, and the animals are also less healthy. It has been found that upregulation of dACSS2 in the nervous system extends lifespan, and the animals are more robust. These data support that manipulation of ACSS2 can modulate events associated with aging of the brain and nervous system, including general health of brain cells and cognitive abilities.

Example 18: ASO-mediated upregulation of ACSS2 by blocking miRNA function

MicroRNAs (miRNA) regulate RNA levels by binding to the 3' UTR of mRNA, leading to degradation or translational silencing via the RNA-induced silencing complex (RISC). In one aspect, the present disclosure relates to the design of an antisense oligonucleotide (ASO) approach to block miRNA/RISC binding to ACSS2 3’ UTR, in order to prevent ACSS2 mRNA regulation by miRNAs, thereby increasing translation of ACSS2 (FIG. 25).

Two miRNAs (miR-15a-5p and miR-15b-5p) known to regulate ACSS2 levels were validated using a luciferase reporter assay in U2O2 human cells. Three independent clones of U2OS cells with integrated ACSS2-luciferase reporter (Origene, SC209651) were transfected with 25 nM of miR-15a-5p or miR-15b-5p. A significant reduction in luciferase activity upon miRNA treatment confirmed binding of these miRNAs to the seed region on the ACSS2 3 'UTR and downregulation of luciferase reporter expression (FIG. 26). Therefore, miR-15a-5p or miR- 15b-5p were validated as positive controls for subsequent experiments.

Based on a comprehensive analysis utilizing the miRWalk database and cross-referencing with a miRNA profile specific to human neurons, a list of miRNAs suitable to target the human ACSS2 3'UTR was identified and/or generated. The top candidates were tested (/.<?., hsa-miR-16- 5p, hsa-miR-15b-5p, hsa-miR-15a-5p, hsa-miR-424-5p, hsa-miR-195-5p, has-miR-497-5p) for their potential to downregulate the ACSS2 reporter (FIG. 28). The selected miRNAs share a common seed location in the 3'UTR and are predicted to bind within the same 7-nucleotide region (FIG. 27). To attempt to block their impact, four exemplary ASOs were prepared, each comprising a 2’-O-Methoxyethyl (2’-M0E) modification on the sugar backbone of each nucleotide that encompassed the common seed motif in the 3’UTR (FIG. 27 and Table 1). The 2’ -MOE modification was chosen to inhibit Ribonuclease H activity, preventing degradation. ASOs 1 and 2 were additionally designed with phosphorothioate bonds due to their potential to enhance ASO half-life by inhibiting endo- and exonuclease activation. ASOs 3 and 4 were designed without phosphorothioate bonds, given the uncertainty of the need for this additional modification.

Table 1. Design of antisense oligonucleotides (ASOs) for blocking miRNA/RISC binding to the 3’UTR of ACSS2 mRNA

/*/ indicates phosphiorothioate linkage; “52MOEr” indicates a methoxy ethyl-modified 5’- terminal nucleic acid residue; “i2M0Er” indicates an internal methoxy ethyl-modified nucleic acid residue; and “32MOEr” indicates a methoxymethyl-modified 3 ’terminal nucleic acid residue; whereby the letter after “MOEr” provides the identity of the modified nucleic acid base.

The results showed a statistically significant decrease in luciferase activity following treatment with miRNAs hsa-miR-15b-5p, hsa-miR-15a-5p, hsa-miR-16-5p, hsa-miR-424-5p, has-miR-497-5p, and hsa-miR-195-5p (FIG. 28). Intriguingly, a remarkable reversal was observed when cells were treated with the ASOs, with ASO3 and ASO4 showing significant upregulation of the reporter (FIG. 28). The significant increase in luciferase activity suggests the successful blockade of miRNA-mediated downregulation of the ACSS2 reporter levels by the ASOs in U2O2 cells. Further, these findings indicate this region of the ACSS2 3’UTR (i.e., the 5’ 435-465) is a promising region to target with site blocking oligonucleotides. That said, the present invention is not limited to ASOs which are complementary to the 435-465 region of the ACSS2 3’UTR. In view of the teachings of the present application, one of ordinary skill in the art appreciates that alternative regions of the ACSS2 3’UTR may be utilized to identify and/or prepare ASOs suitable to promote expression and/or translation of ACSS2, by preventing and/or reducing degradation of ACSS2 mRNA transcripts.

To evaluate the effectiveness of each ASO in restoring the expression of ACSS2 in the presence of miRNAs, U2O2 cells were treated with 25 nM has-miR-15b-5p along with 25 nM of ASOs that were previously tested. Cells treated with miR-15b and ASO2 exhibited a significant (p<0.05) restoration (FIG. 29).

To explore the dosage effect of ASOs, U2O2 cells were treated with 5, 10, 25, and 50 nM of ASO3 or ASO4 (FIG. 30). Significantly higher luciferase activity was detected in the presence of ASO3 and ASO4, suggesting that both ASOs were able to block miRNAs binding to ACSS2 and therefore prevent its degradation.

The restoration assay was repeated using 5 and 25 nM of miRNA15b to further assess the efficacy of AS03 and AS04. At a miRNA concentration of 25 nM, an increase in luciferase activity was detected with both AS03 and ASO4, indicating that these ASOs are effective in blocking the binding of miRNAs to ACSS2 3’UTR (FIG. 31). All experiments were conducted in three biological replicates. Cells were treated with miRNA, ASO, ASO/miRNA at the time of plating. The oligonucleotide was seeded first in OMEM/Lipofectamine and then the cells were plated on top. The luciferase assay was performed 72 hours after treatment.

The observed increase in luciferase activity underscores the efficacy of this ASO strategy, demonstrating its capability to disrupt the binding of miRNAs that target ACSS2 for downregulated expression. This encouraging outcome provides compelling evidence for the potential therapeutic application of these ASOs in modulating ACSS2 expression, paving the way for further exploration and refinement of this approach.

Sequence Listing

SEQ ID NO : 1 (human ACSS2)

ATGGGGCTTCCTGAGGAGCGGGTCCGGAGCGGCAGCGGGAGCCGGGGCCAGGAGGAAGCTGGAG CCGGAGGCCGGGCGCGGAGTTGGTCTCCGCCGCCCGAGGTCAGCCGCTCCGCGCACGTCCCCTC GCTGCAGCGCTACCGCGAGCTGCACCGGCGCTCCGTGGAGGAGCCGCGGGAATTCTGGGGAGAC ATTGCCAAGGAATTTTACTGGAAGACTCCATGCCCTGGCCCATTCCTTCGGTACAACTTTGATG T GAG T AAAG GGAAAAT 0 T T T AT T GAG T G GAT GAAAG GAG C AAC TAG C AAC AT C T GC T AC AAT G T ACTGGATCGAAATGTCCATGAGAAAAAGCTTGGAGATAAAGTTGCTTTTTACTGGGAGGGCAAT GAGCCAGGGGAGACCACTCAGATCACATACCATCAGCTTCTGGTCCAAGTGTGTCAGTTCAGCA ATGTTCTCCGAAAACAGGGCATTCAGAAGGGGGACCGAGTGGCCATCTACATGCCTATGATCCC AGAGCTTGTGGTGGCCATGCTGGCATGTGCCCGCATTGGGGCTTTGCACTCCATTGTGTTTGCA GGCTTCTCTTCAGAGTCTCTATGTGAACGGATCTTGGATTCCAGCTGCAGTCTTCTCATCACTA CAGATGCCTTCTACAGGGGGGAAAAGCTTGTGAACCTGAAGGAGCTGGCTGACGAGGCCCTGCA GAAGTGTCAGGAGAAGGGTTTCCCAGTAAGATGCTGCATTGTGGTCAAGCACCTGGGGCGGGCA GAGCTCGGCATGGGTGACTCCACCAGCCAGTCCCCCCCAATTAAGAGGTCATGCCCAGATGTGC AGATCTCATGGAACCAAGGGATTGACTTGTGGTGGCATGAGCTCATGCAAGAGGCAGGGGATGA GTGTGAGCCCGAGTGGTGTGATGCCGAGGACCCACTCTTCATCCTGTACACCAGTGGCTCCACA GGCAAACCCAAGGGTGTGGTTCACACAGTTGGGGGCTACATGCTCTATGTAGCCACAACCTTCA AGTATGTGTTTGACTTCCATGCAGAGGATGTGTTCTGGTGCACGGCAGACATTGGTTGGATCAC TGGTCATTCCTACGTCACCTATGGGCCACTGGCCAATGGTGCCACCAGTGTTTTGTTTGAGGGG ATTCCCACATATCCGGACGTGAACCGCCTGTGGAGCATTGTGGACAAATACAAGGTGACCAAGT TCTACACAGCACCCACAGCCATCCGTCTGCTCATGAAGTTTGGAGATGAGCCTGTCACCAAGCA TAGCCGGGCATCCTTGCAGGTGTTAGGCACAGTGGGTGAACCCATCAACCCTGAGGCCTGGCTA TGGTACCACCGGGTGGTAGGTGCCCAGCGCTGCCCCATCGTGGACACCTTCTGGCAAACAGAGA CAGGTGGCCACATGTTGACTCCCCTTCCTGGTGCCACACCCATGAAACCCGGTTCTGCTACTTT CCCATTCTTTGGTGTAGCTCCTGCAATCCTGAATGAGTCCGGGGAAGAGTTGGAAGGTGAAGCT

GAAGGTTATCTGGTGTTCAAGCAGCCCTGGCCAGGGATCATGCGCACAGTCTATGGGAACCACG AAC GC T T T GAGAC AAC C T AC T T T AAGAAG TTTCCTGGATACTATGT T ACAG GAGAT G G C T GC C A GCGGGACCAGGATGGCTATTACTGGATCACTGGCAGGATTGATGACATGCTCAATGTATCTGGA CACCTGCTGAGTACAGCAGAGGTGGAGTCAGCACTTGTGGAACATGAGGCTGTTGCAGAGGCAG CTGTGGTGGGCCACCCTCATCCTGTGAAGGGTGAATGCCTCTACTGCTTTTTCACCTTGTGTGA TGGCCACACCTTCAGCCCCAAGCTCACCGAGGAGCTCAAGAAGCAGATTAGAGAAAAGATTGGC CCCATTGCCACACCAGACTACATCCAGAATGCACCTGGCTTGCCTAAAACCCGCTCAGGGAAAA TCATGAGGCGAGTGCTTCGGAAGATTGCTCAGAATGACCATGACCTCGGGGACATGTCTACTGT GGCTGACCCATCTGTCATCAGTCACCTCTTCAGCCACCGCTGCCTGACCATCCAG

SEQ ID NO : 2 (ACSS2 overexpression plasmid)

CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCAAAGCCCGGGCGTCGGGCGACCTTTGGTC GCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTC CTTGTAGTTAATGATTAACCCGCCATGCTACTTATCTACGTAGCCATGCTCTAGGAAGATCCGA TGTACGGGCCAGATATACGCGTTGACATTGATTATTGACTAGGCTTTTGCAAAAAGCTTTGCAA AGATGGATAAAGT T T TAAACAGAG GGAAT C T T T GCAGC TAAT GGACC T T C TAGGT C T T GAAAG GAGTGGGAATTGGCTCCGGTGCCCGTCAGTGGGCAGAGCGCACATCGCCCACAGTCCCCGAGAA GTTGGGGGGAGGGGTCGGCAATTGAACCGGTGCCTAGAGAAGGTGGCGCGGGGTAAACTGGGAA AGTGATGTCGTGTACTGGCTCCGCCTTTTTCCCGAGGGTGGGGGAGAACCGTATATAAGTGCAG TAGTCGCCGTGAACGTTCTTTTTCGCAACGGGTTTGCCGCCAGAACACAGGTAAGTGCCGTGTG TGGTTCCCGCGGGCCTGGCCTCTTTACGGGTTATGGCCCTTGCGTGCCTTGAATTACTTCCACC TGGCTGCAGTACGTGATTCTTGATCCCGAGCTTCGGGTTGGAAGTGGGTGGGAGAGTTCGAGGC CTTGCGCTTAAGGAGCCCCTTCGCCTCGTGCTTGAGTTGAGGCCTGGCCTGGGCGCTGGGGCCG CCGCGTGCGAATCTGGTGGCACCTTCGCGCCTGTCTCGCTGCTTTCGATAAGTCTCTAGCCATT TAAAATTTTTGATGACCTGCTGCGACGCTTTTTTTCTGGCAAGATAGTCTTGTAAATGCGGGCC AAGATCTGCACACTGGTATTTCGGTTTTTGGGGCCGCGGGCGGCGACGGGGCCCGTGCGTCCCA GCGCACATGTTCGGCGAGGCGGGGCCTGCGAGCGCGGCCACCGAGAATCGGACGGGGGTAGTCT CAAGCTGGCCGGCCTGCTCTGGTGCCTGGCCTCGCGCCGCCGTGTATCGCCCCGCCCTGGGCGG CAAGGCTGGCCCGGTCGGCACCAGTTGCGTGAGCGGAAAGATGGCCGCTTCCCGGCCCTGCTGC AGGGAGCTCAAAATGGAGGACGCGGCGCTCGGGAGAGCGGGCGGGTGAGTCACCCACACAAAGG AAAAGGGCCTTTCCGTCCTCAGCCGTCGCTTCATGTGACTCCACGGAGTACCGGGCGCCGTCCA GGCACCTCGATTAGTTCTCGAGCTTTTGGAGTACGTCGTCTTTAGGTTGGGGGGAGGGGTTTTA TGCGATGGAGTTTCCCCACACTGAGTGGGTGGAGACTGAAGTTAGGCCAGCTTGGCACTTGATG

TAATTCTCCTTGGAATTTGCCCTTTTTGAGTTTGGATCTTGGTTCATTCTCAAGCCTCAGACAG TGGTTCAAAGTTTTTTTCTTCCATTTCAGGTGTCGTGAGGAATTAGCTTGGTACTAATACGACT CACTATAGGGAGACCCAAGCTGGCTAGGTAAGCTTGGTACCGAGCTCGGAATTCGCCGCCACCA TGGGGCTTCCCGAGGAGCGGCGCAAGAGCGGCAGTGGAAGCCGGGCTCGTGAGGAGACCGGCGC CGAGGGCCGGGTGCGGGGTTGGTCCCCGCCGCCGGAGGTCAGACGCTCGGCGCACGTCCCCTCT CTGCAGCGCTACCGCGAGCTGCACCGGCGTTCTGTGGAGGAGCCACGGGAGTTTTGGGGAAACA TTGCCAAGGAATTTTACTGGAAAACTGCATGCCCTGGCCCATTCCTCCAGTACAACTTTGATGT GAG TAAAGGGAAAATAT T CACT GAGT GGAT GAAAGGAGCAAC TACAAACAT C T GCTACAACGT G CTGGATCGAAATGTCCATGAGAAAAAACTTGGCGACAAAGTTGCTTTTTACTGGGAGGGCAATG AGCCAGGGGAGACCACCAAGATCACATACCGTGAACTCCTGGTCCAGGTGTGTCAGTTCAGCAA TGTTCTCCGTAAACAGGGCATTCAGAAGGGTGACCGAGTGGCCATCTACATGCCTATGATCTTG

GAACTTGTGGTGGCTATGCTGGCATGTGCTCGCCTTGGAGCTTTGCACTCCATTGTGTTTGCAG GCTTCTCTGCAGAGTCTCTCTGTGAAAGGATCTTGGATTCCAGTTGCTGCCTGCTCATCACTAC AGATGCCTTCTACAGGGGGGAAAAACTTGTGAACCTGAAGGAGCTGGCTGATGAGTCCTTGGAG AAGTGCCGAGAGAAGGGCTTCCCAGTGAGATGCTGCATTGTGGTCAAACATCTGGGGCGGGCAG AGCTGGGCATGAATGACTCCCCCAGCCAGTCCCCACCAGTTAAGAGGCCATGTCCAGATGTCCA GATCTGCTGGAACGAAGGGGTTGACTTATGGTGGCATGAACTCATGCAGCAGGCAGGAGACGAG TGTGAGCCTGAGTGGTGTGATGCTGAGGACCCACTCTTCATCTTGTACACCAGTGGCTCCACAG

GCAAACCTAAGGGTGTGGTGCACACAATTGGAGGCTATATGCTCTATGTGGCTACAACTTTCAA

GTATGTGTTTGATTTCCACCCGGAAGATGTGTTCTGGTGCACAGCAGACATCGGCTGGATCACT

GGTCATTCCTATGTCACCTATGGGCCACTGGCTAATGGTGCCACTAGTGTTTTGTTTGAGGGGA

TCCCCACATACCCAGATGAAGGGCGCTTGTGGAGCATTGTGGACAAATACAAGGTGACCAAGTT

CTACACGGCACCAACAGCCATCCGGATGCTCATGAAGTTTGGAGATGATCCTGTCACCAAGCAT

AGCCGGGCATCCCTGCAGGTGCTGGGCACAGTAGGTGAACCCATCAACCCTGAAGCCTGGCTAT

GGTACCACCGGGTAGTAGGTTCCCAGCGCTGCCCCATTGTAGACACCTTCTGGCAAACAGAAAC

AGGTGGCCATATGCTGACCCCTCTCCCTGGCGCCACACCCATGAAACCTGGTTCTGCTTCTTTC

CCATTCTTCGGTGTAGCGCCTGCAATCCTGAATGAGTCCGGGGAGGAGCTGGAAGGGGAAGCTG

AAGGTTATCTGGTGTTCAAGCAGCCCTGGCCAGGGATCATGCGCACAGTCTATGGGAACCACAC

ACGGTTTGAGACCACCTACTTTAAGAAGTTCCCTGGCTACTATGTGACCGGAGATGGCTGCCGA

CGGGATCAGGATGGCTATTACTGGATCACGGGCAGGATTGATGACATGCTCAATGTGTCTGGAC

ATCTCCTGAGTACAGCAGAGGTGGAATCGGCACTTGTGGAACACGAGGCTGTCGCAGAGGCAGC

TGTGGTGGGCCACCCTCATCCTGTGAAGGGCGAATGCCTCTACTGCTTTGTTACCTTGTGTGAT

GGCCATACCTTCAGCCCCACACTCACAGAGGAACTCAAGAAGCAGATTAGAGAAAAGATTGGCC

CCATTGCCACACCAGACTACATCCAGAATGCACCTGGCTTGCCTAAAACACGCTCAGGGAAAAT

CATGAGGCGAGTTCTCCGGAAGATTGCTCAGAATGACCATGACCTGGGGGATACATCTACGGTG

GCTGACCCATCTGTCATCAACCATCTCTTCAGTCACCGCTGCCTGACCACCCAGGACTACAAAG

ACGATGACGACAAGGGATCCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCAGGAGACGTGGA

AGAAAACCCCGGTCCTATGGTGAGCAAGGGCGAGGAGGTCATCAAGGAGTTCATGCGCTTCAAG

GAGCACATGGAGGGCTCCGTGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCC

CCTACGAGGGCACCCAGACCGCCAGGCTGAAGGTGACCAAGGGTGGCCCCCTGCCCTTCGCCTG

GGACATCCTGTCCCCTCAGATCATGTACGGCTCCAAGGCCTACGTGAAGCACCCCGCCGACATC

CCCGACTACTTGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGG

ACGGCGGCGTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCGAGTTCATCTACAAGGT

GAAGGTGCGCGGCACCAACTTCCCCTCCGACGGCCCCGTAATGCAGAAGAAGACCATGGGCTGG

GAGGCCTCCTCCGAGCGGATGTACCCCGAGGACGGCGCCCTGAAGGGCGAGATGAAGATGAGGC

TGAGGCTGAAGGACGGCGGCCACTACGACGCCGAGGTCAAGACCACCTACATGGCCAAGAAGCC

CGTGCAGCTGCCCGGCGCCTACAAGACCGACATCAAGCTGGACATCACCTCCCACAACGAGGAC TACACCATCGTGGAACAGTACGAGCGCGCCGAGGGCCGCCACTCCACCGGCGCCTAAGTCGACC CGGGCGGCCTCGACCGCTTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACA AC TAGAAT GCAGT GAAAAAAAT GC T T TAT T T GT GAAAT T T GT GAT GC TAT T GC T T TAT T T GTAA C C AT T AT AAGC T G C AAT AAAC AAG T T AAC AACAAC AAT TGCATTCATTTTATGTTT C AG G T T C A GGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCGATAAG GATCTTCCTAGAGCATGGCTACGTAGATAAGTAGCATGGCGGGTTAATCATTAACTACAAGGAA CCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGAC CAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAGCCT TAATTAACCTAATTCACTGGCCGTCGTTTTACAACGTCGTGACTGGGAAAACCCTGGCGTTACC CAACTTAATCGCCTTGCAGCACATCCCCCTTTCGCCAGCTGGCGTAATAGCGAAGAGGCCCGCA CCGATCGCCCTTCCCAACAGTTGCGCAGCCTGAATGGCGAATGGGACGCGCCCTGTAGCGGCGC ATTAAGCGCGGCGGGTGTGGTGGTTACGCGCAGCGTGACCGCTACACTTGCCAGCGCCCTAGCG CCCGCTCCTTTCGCTTTCTTCCCTTCCTTTCTCGCCACGTTCGCCGGCTTTCCCCGTCAAGCTC TAAATCGGGGGCTCCCTTTAGGGTTCCGATTTAGTGCTTTACGGCACCTCGACCCCAAAAAACT TGATTAGGGTGATGGTTCACGTAGTGGGCCATCGCCCTGATAGACGGTTTTTCGCCCTTTGACG TTGGAGTCCACGTTCTTTAATAGTGGACTCTTGTTCCAAACTGGAACAACACTCAACCCTATCT CGGTCTATTCTTTTGATTTATAAGGGATTTTGCCGATTTCGGCCTATTGGTTAAAAAATGAGCT GAT T TAACAAAAAT T TAACGCGAAT T T TAACAAAATAT TAACGCT TAGAAT T TAGGT GGCAC T T TTCGGGGAAATGTGCGCGGAACCCCTATTTGTTTATTTTTCTAAATACATTCAAATATGTATCC G C T C AT GAGAC AT AC C C T GAT AAAT G C T T CAAT AAT AT T GAAAAAG G AGAG TAT GAG T AT T CAACATTTCCGTGTCGCCCTTATTCCCTTTTTTGCGGCATTTTGCCTTCCTGTTTTTGCTCACC CAGAAACGCTGGTGAAAGTAAAAGATGCTGAAGATCAGTTGGGTGCACGAGTGGGTTACATCGA ACTGGATCTCAACAGCGGTAAGATCCTTGAGAGTTTTCGCCCCGAAGAACGTTTTCCAATGATG AGCACTTTTAAAGTTCTGCTATGTGGCGCGGTATTATCCCGTATTGACGCCGGGCAAGAGCAAC TCGGTCGCCGCATACACTATTCTCAGAATGACTTGGTTGAGTACTCACCAGTCACAGAAAAGCA TCTTACGGATGGCATGACAGTAAGAGAATTATGCAGTGCTGCCATAACCATGAGTGATAACACT GCGGCCAACTTACTTCTGACAACGATCGGAGGACCGAAGGAGCTAACCGCTTTTTTGCACAACA TGGGGGATCATGTAACTCGCCTTGATCGTTGGGAACCGGAGCTGAATGAAGCCATACCAAACGA CGAGCGTGACACCACGATGCCTGTAGCAATGGCAACAACGTTGCGCAAACTATTAACTGGCGAA CTACTTACTCTAGCTTCCCGGCAACAATTAATAGACTGGATGGAGGCGGATAAAGTTGCAGGAC CACTTCTGCGCTCGGCCCTTCCGGCTGGCTGGTTTATTGCTGATAAATCTGGAGCCGGTGAGCG TGGGTCTCGCGGTATCATTGCAGCACTGGGGCCAGATGGTAAGCCCTCCCGTATCGTAGTTATC TACACGACGGGGAGTCAGGCAACTATGGATGAACGAAATAGACAGATCGCTGAGATAGGTGCCT GAG T GAT T AAG C AT T G G T AAC T G T C AGAC C AAG TTTACTCATATATACTT T AGAT T GAT T T AAA ACTTCATTTTTAATT T AAAAGG T C T AG G T G AGAT CCTTTTTGATAATCTCAT GAG C AAAAT C CCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTT GAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGT GGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCG CAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAG CACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTC GTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACG GGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGC GTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGG CAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGT CCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGA GCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGC TCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTATTACCGCCTTTGAGTGA GCTGATACCGCTCGCCGCAGCCGAACGACCGAGCGCAGCGAGTCAGTGAGCGAGGAAGCGGAAG AGCGCCCAATACGCAAACCGCCTCTCCCCGCGCGTTGGCCGATTCATTAATGCAGCTGGCACGA CAGGTTTCCCGACTGGAAAGCGGGCAGTGAGCGCAACGCAATTAATGTGAGTTAGCTCACTCAT TAGGCACCCCAGGCTTTACACTTTATGCTTCCGGCTCGTATGTTGTGTGGAATTGTGAGCGGAT AACAAT T T CACACAGGAAACAGC TAT GACCATGAT TACGCCAGAT T TAAT TAAGGCC T TAAT TA GG

SEQ ID NO : 3 (ACSS2 protein)

MGLPEERVRSGSGSRGQEEAGAGGRARSWSPPPEVSRSAHVPSLQRYRELHRRSVEEPREFWGD IAKEFYWKTPCPGPFLRYNFDVTKGKI FIEWMKGATTNICYNVLDRNVHEKKLGDKVAFYWEGN EPGETTQI TYHQLLVQVCQFSNVLRKQGIQKGDRVAIYMPMI PELWAMLACARIGALHS IVFA GFSSESLCERILDSSCSLLI TTDAFYRGEKLVNLKELADEALQKCQEKGFPVRCCIWKHLGRA ELGMGDSTSQSPPIKRSCPDVQI SWNQGIDLWWHELMQEAGDECEPEWCDAEDPLFILYTSGST GKPKGWHTVGGYMLYVATTFKYVFDFHAEDVFWCTADIGWI TGHSYVTYGPLANGATSVLFEG I PTYPDVNRLWS IVDKYKVTKFYTAPTAIRLLMKFGDEPVTKHSRASLQVLGTVGEPINPEAWL WYHRWGAQRCPIVDTFWQTETGGHMLTPLPGATPMKPGSATFPFFGVAPAILNESGEELEGEA EGYLVFKQPWPGIMRTVYGNHERFETTYFKKFPGYYVTGDGCQRDQDGYYWI TGRIDDMLNVSG HLLSTAEVESALVEHEAVAEAAWGHPHPVKGECLYCFVTLCDGHTFSPKLTEELKKQIREKIG PIATPDYIQNAPGLPKTRSGKIMRRVLRKIAQNDHDLGDMSTVADPSVISHLFSHRCLT IQ

SEQ ID NO : 4 (ACSS2 3 ' -UTR)

ACATGATCCTGACCTTTACCTAGGATTCCTCCTGCTCCAAACTTTGCCCATCCTCTTTGCCCCC TCAGGAGTGCTGAGGGCCAGTGTTGACCCACACTACCCTCCCTTGACCAGCTGTCTGGGACCGG AAACCAGCTTTGTCTCCAGGTAGAGACAACATCCTGTGACTGCCAGGCAGAAAGGACAGGGCCC AGGTCAGCCTCAGTCTGCTGTGCCTCCAGACTGCAGAGCTCTCAGAACCCAGAACAGAGACGAA AAGGCTACCTCTCCTACCCAAGTTAAGTGTTCAAAGGGGATGTGAGGGCCTCCACTGAAGCAGG GAGGCAGCTGTGTAATCCTATGTCAGCTCTCTTAGGAAGCCCCAGTACTTATATTGGGCATGCA C T T GC C C T T AAAAACAAT GAT T T G T GAG T C C AG GAAC AAT T T AC T AT T T T TAAAAT AT T T T G C T GCTTCTGTTCTGGGTCTGAATTCCCTTTTGTGCCAGATGCCAGTACTGTCTGCCCATTGGCTCC

AGGGGCTGTATGGGCAGATTCAGTCTCCAGAGGGTATTCAGATCATCTGCTTCTTTGAAGGAGT AAATGTGTTTTGTTCCTAGGGCCAGAGGAGCTTGTCTTCCTTGTCCTCTGTTCCCACCCTCCCC TGAACAGAACCCAGCCCATAAGAGACATTCTCAGATGAAACTCTGTTTTCTTGCCCCAGTCAGG CTCAAGCCCTGTGGTTGTAGGAATAAAGCCTGTGATCTCAA

SEQ ID NO : 5 (Core 1 )

AGAACAGAAGCAGCAAAAT T T

SEQ ID NO : 6 (Core 2 )

GACCCAGAACAGAAGCAGC

SEQ ID NO : 7 (ASO1 )

AGAACAGAAGCAGCAAAATAT T , wherein each 2 ' -hydroxy of each base is methoxyethyl substituted and each phosphodiester linkage is substituted for a phosphorothioate linkage .

SEQ ID NO : 8 (AS02 )

GACCCAGAACAGAAGCAGC , wherein each 2 ' -hydroxy of each base is methoxyethyl substituted and each phosphodiester linkage is substituted for a phosphorothioate linkage .

SEQ ID NO : 9 (AS03)

AGAACAGAAGCAGCAAAATATT , wherein each 2 ' -hydroxy of each base is methoxyethyl substituted .

SEQ ID NO : 10 (ASO4 )

GACCCAGAACAGAAGCAGC , wherein each 2 ' -hydroxy of each base is methoxyethyl substituted .

Enumerated Embodiments

The following exemplary embodiments are provided, the numbering of which is not to be construed as designating levels of importance:

Embodiment 1 provides a recombinant viral vector, the vector comprising:

(b) an expression control sequence operably linked to the nucleic acid. Embodiment 2 provides the recombinant viral vector of Embodiment 1, wherein the nucleic acid comprises a DNA sequence which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO: 1 or SEQ ID NO:2.

Embodiment 3 provides the recombinant viral vector of Embodiment 1 or 2, wherein the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO:3.

Embodiment 4 provides the recombinant viral vector of any one of Embodiments 1-3, wherein the vector is an Adeno-associated virus (AAV) vector.

Embodiment 5 provides the recombinant viral vector of any one of Embodiments 1-4, wherein the vector is AAV-PHP.eB.

Embodiment 6 provides the recombinant viral vector of any one of Embodiments 1-3, wherein the vector is a lentivirus vector.

Embodiment 7 provides an antisense oligonucleotide composition, wherein the antisense oligonucleotide comprises a nucleic acid sequence which is at least partially complementary to at least a portion of a 3 ’-untranslated region (3’-UTR) of an acetyl-CoA synthetase 2 (ACSS2) mRNA transcript, and wherein the nucleic acid sequence comprises at least one chemically modified nucleoside or internucleoside linkage.

Embodiment 8 provides the antisense oligonucleotide composition of Embodiment 7, wherein the 3’-UTR of ACSS2 mRNA transcript comprises a nucleic acid which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO:4.

Embodiment 9 provides the antisense oligonucleotide composition of Embodiment 7 or 8, wherein the at least a portion of the 3’-UTR of the ACSS2 mRNA transcript ranges from about nucleic acid 435 to about nucleic acid 465 of SEQ ID NO:4.

Embodiment 10 provides the antisense oligonucleotide composition of any one of Embodiments 7-9, wherein the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO: 5 or SEQ ID NO:6.

Embodiment 11 provides the antisense oligonucleotide composition of any one of Embodiments 7-10, wherein the nucleic acid comprises at least one chemically modified nucleoside, optionally wherein chemical modification comprises 2’-hydroxy substitution, optionally wherein the 2’-hydroxy substitution comprises 2 ’-meth oxy ethyl substitution, and optionally wherein each nucleoside of the nucleic acid is chemically modified.

Embodiment 12 provides the antisense oligonucleotide composition of any one of Embodiments 7-11, wherein the nucleic acid comprises at least one chemically modified internucleoside linkage, optionally wherein the chemically modified internucleoside linkage comprises a phosphorothioate linkage, and optionally wherein each intemucleoside linkage comprises a phosphorothioate linkage.

Embodiment 13 provides the antisense oligonucleotide composition of any one of Embodiments 7-12, wherein the nucleic acid comprises a nucleic acid which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with at least one selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10.

Embodiment 14 provides a composition comprising one or more nucleic acid-lipid particles, wherein each nucleic acid-lipid particle comprises:

(a) a cationic lipid;

(b) a non-cationic lipid;

(e) the antisense oligonucleotide of any one of Embodiments 7-13.

Embodiment 15 provides the composition of Embodiment 14, wherein the cationic lipid comprises about 50 mol% to about 90 mol% of the total lipid present in the nucleic acid-lipid particle.

Embodiment 16 provides the composition of Embodiment 14 or 15, wherein the noncationic lipid is at least one selected from the group consisting of cholesterol and a phospholipid.

Embodiment 17 provides the composition of any one of Embodiments 14-16, wherein the non-cationic lipid comprises about 9.9 mol% to about 49.9 mol% of the total lipid present in the nucleic acid-lipid particle. Embodiment 18 provides the composition of any one of Embodiments 14-17, wherein the conjugated lipid that inhibits aggregation of two or more nucleic acid-lipid particles comprises a polyethyleneglycol (PEG)-lipid conjugate.

Embodiment 19 provides the composition of any one of Embodiments 14-18, wherein the conjugated lipid comprises about 0.1 mol% to about 2 mol% of the total lipid present in the nucleic acid-lipid particle

Embodiment 20 provides the composition of any one of Embodiments 14-19, wherein the nucleic acid comprises a DNA sequence which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO: 1 or SEQ ID NO:2.

Embodiment 21 provides the composition of any one of Embodiments 14-20, wherein the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO:3.

Embodiment 22 provides the composition of any one of Embodiments 14-19, wherein the antisense oligonucleotide comprises a nucleic acid which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with at least one selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10.

Embodiment 23 provides a pharmaceutical composition comprising the recombinant viral vector of any one of Embodiments 1-6, the antisense oligonucleotide composition of any one of Embodiments 7-13, or the composition of any one of Embodiments 14-22 and a pharmaceutically acceptable carrier.

Embodiment 24 provides a method of treating, preventing, and/or ameliorating cognitive decline in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one selected from the group consisting of:

(a) the recombinant viral vector of any one of Embodiments 1-6;

(b) the antisense oligonucleotide of any one of Embodiments 7-13;

(c) the composition of any one of Embodiments 14-22;

(d) the pharmaceutical composition of Embodiment 23; and/or

(e) acetic acid, or a salt, solvate, or pharmaceutical composition thereof.

Embodiment 25 provides the method of Embodiment 24, wherein the cognitive decline is associated with a neurodegenerative disease or disorder, age, and/or trauma. Embodiment 26 provides the method of Embodiment 25, wherein the neurodegenerative disease or disorder is selected from the group consisting of Alzheimer’s disease, vascular dementia, Lewy bodies, Parkinson’s disease, frontotemporal dementia, Huntington’s disease, HIV-associated neurocognitive disorder, Creutzfeldt-Jakob disease, alcohol-related dementia, and inflammation-derived dementia (e.g, Behcet’s disease, multiple sclerosis, sarcoidosis, Sjogren’s syndrome, lupus, celiac disease, and non-celiac gluten sensitivity), or any combination thereof.

Embodiment 27 provides the method of any one of Embodiments 24-26, wherein the subject is administered the recombinant viral vector of any one of Embodiments 1-6, the antisense oligonucleotide composition of any one of Embodiments 7-13, the composition of any one of Embodiments 14-22, and/or the pharmaceutical composition of Embodiment 13.

Embodiment 28 provides the method of any one of Embodiments 24-27, wherein the recombinant viral vector and/or pharmaceutical composition is administered by at least one route selected from the group consisting of intravenous, intrathecal, intraocular, intranasal, and/or intraparenchymal.

Embodiment 29 provides the method of any one of Embodiments 24-28, wherein the translation and/or expression of ACSS2 is promoted in the subject.

Embodiment 30 provides the method of any one of Embodiments 24-29, wherein the subject is administered acetate.

Embodiment 31 provides the method of Embodiment 30, wherein the acetate is sodium acetate.

Embodiment 32 provides the method of Embodiment 30 or 31, wherein the acetate is administered to the subject by at least one route selected from the group consisting of intravenous and oral.

Embodiment 33 provides the method of Embodiment 32, wherein the oral administration comprises dietary supplementation.

Embodiment 34 provides the method of any one of Embodiments 24-33, wherein histone acetylation is promoted in the subject.

Embodiment 35 provides the method of any one of Embodiments 24-34, wherein the subject is a mammal. Embodiment 36 provides the method of Embodiment 35, wherein the mammal is a human.

The terms and expressions employed herein are used as terms of description and not of limitation, and there is no intention in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the embodiments of the present application. Thus, it should be understood that although the present application describes specific embodiments and optional features, modification and variation of the compositions, methods, and concepts herein disclosed may be resorted to by those of ordinary skill in the art, and that such modifications and variations are considered to be within the scope of embodiments of the present application.

Claims

CLAIMS What is claimed is:

1. A recombinant viral vector, the vector comprising:

(b) an expression control sequence operably linked to the nucleic acid.

2. The recombinant viral vector of claim 1, wherein the nucleic acid comprises a DNA sequence which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO:1 or SEQ ID NO: 2.

3. The recombinant viral vector of claim 1 or 2, wherein the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO:3.

4. The recombinant viral vector of any one of claims 1-3, wherein the vector is an Adeno- associated virus (AAV) vector.

5. The recombinant viral vector of any one of claims 1 -4, wherein the vector is AAV- PHP.eB.

6. The recombinant viral vector of any one of claims 1-3, wherein the vector is a lentivirus vector.

7. An antisense oligonucleotide composition, wherein the antisense oligonucleotide comprises a nucleic acid sequence which is at least partially complementary to at least a portion of a 3 ’-untranslated region (3’-UTR) of an acetyl-CoA synthetase 2 (ACSS2) mRNA transcript, and wherein the nucleic acid sequence comprises at least one chemically modified nucleoside or internucleoside linkage.

8. The antisense oligonucleotide composition of claim 7, wherein the 3’-UTR of ACSS2 mRNA transcript comprises a nucleic acid which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO:4.

9. The antisense oligonucleotide composition of claim 7 or 8, wherein the at least a portion of the 3’-UTR of the ACSS2 mRNA transcript ranges from about nucleic acid 435 to about nucleic acid 465 of SEQ ID NO:4.

10. The antisense oligonucleotide composition of any one of claims 7-9, wherein the nucleic acid sequence which is at least partially complementary to the 3’-UTR of the ACSS2 mRNA transcript comprises a nucleic acid which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO:5 or SEQ ID NO:6.

11. The antisense oligonucleotide composition of any one of claims 7-10, wherein the nucleic acid comprises at least one chemically modified nucleoside, optionally wherein chemical modification comprises 2’-hydroxy substitution, optionally wherein the 2’-hydroxy substitution comprises 2 ’-methoxy ethyl substitution, and optionally wherein each nucleoside of the nucleic acid is chemically modified.

12. The antisense oligonucleotide composition of any one of claims 7-11, wherein the nucleic acid comprises at least one chemically modified internucleoside linkage, optionally wherein the chemically modified intemucleoside linkage comprises a phosphorothioate linkage, and optionally wherein each internucleoside linkage comprises a phosphorothioate linkage.

13. The antisense oligonucleotide composition of any one of claims 7-12, wherein the nucleic acid comprises a nucleic acid which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with at least one selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NOTO.

14. A composition comprising one or more nucleic acid-lipid particles, wherein each nucleic acid-lipid particle comprises:

(a) a cationic lipid;

(b) a non-cationic lipid;

(e) the antisense oligonucleotide of any one of claims 7-13.

15. The composition of claim 14, wherein the cationic lipid comprises about 50 mol% to about 90 mol% of the total lipid present in the nucleic acid-lipid particle.

16. The composition of claim 14 or 15, wherein the non-cationic lipid is at least one selected from the group consisting of cholesterol and a phospholipid.

17. The composition of any one of claims 14-16, wherein the non-cationic lipid comprises about 9.9 mol% to about 49.9 mol% of the total lipid present in the nucleic acid-lipid particle.

18. The composition of any one of claims 14-17, wherein the conjugated lipid that inhibits aggregation of two or more nucleic acid-lipid particles comprises a polyethyleneglycol (PEG)- lipid conjugate.

19. The composition of any one of claims 14-18, wherein the conjugated lipid comprises about 0.1 mol% to about 2 mol% of the total lipid present in the nucleic acid-lipid particle

20. The composition of any one of claims 14-19, wherein the nucleic acid comprises a DNA sequence which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO:1 or SEQ ID NO: 2.

21. The composition of any one of claims 14-20, wherein the nucleic acid comprises a DNA sequence which encodes a protein that shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with SEQ ID NO:3.

22. The composition of any one of claims 14-19, wherein the antisense oligonucleotide comprises a nucleic acid which shares at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence homology with at least one selected from the group consisting of SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, and SEQ ID NO: 10.

23. A pharmaceutical composition comprising the recombinant viral vector of any one of claims 1-6, the antisense oligonucleotide composition of any one of claims 7-13, or the composition of any one of claims 14-22 and a pharmaceutically acceptable carrier.

24. A method of treating, preventing, and/or ameliorating cognitive decline in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of at least one selected from the group consisting of:

(a) the recombinant viral vector of any one of claims 1-6;

(b) the antisense oligonucleotide of any one of claims 7-13;

(c) the composition of any one of claims 14-22;

(d) the pharmaceutical composition of claim 23; and/or

(e) acetic acid, or a salt, solvate, or pharmaceutical composition thereof.

25. The method of claim 24, wherein the cognitive decline is associated with a neurodegenerative disease or disorder, age, and/or trauma.

26. The method of claim 25, wherein the neurodegenerative disease or disorder is selected from the group consisting of Alzheimer’s disease, vascular dementia, Lewy bodies, Parkinson’s disease, frontotemporal dementia, Huntington’s disease, HIV-associated neurocognitive disorder, Creutzfeldt-Jakob disease, alcohol-related dementia, and inflammation-derived dementia (e. , Behcet’s disease, multiple sclerosis, sarcoidosis, Sjogren’s syndrome, lupus, celiac disease, and non-celiac gluten sensitivity), or any combination thereof.

27. The method of any one of claims 24-26, wherein the subject is administered the recombinant viral vector of any one of claims 1-6, the antisense oligonucleotide composition of any one of claims 7-13, the composition of any one of claims 14-22, and/or the pharmaceutical composition of claim 13.

28. The method of any one of claims 24-27, wherein the recombinant viral vector and/or pharmaceutical composition is administered by at least one route selected from the group consisting of intravenous, intrathecal, intraocular, intranasal, and/or intraparenchymal.

29. The method of any one of claims 24-28, wherein the translation and/or expression of ACSS2 is promoted in the subject.

30. The method of any one of claims 24-29, wherein the subject is administered acetate.

31. The method of claim 30, wherein the acetate is sodium acetate.

32. The method of claim 30 or 31, wherein the acetate is administered to the subject by at least one route selected from the group consisting of intravenous and oral.

33. The method of claim 32, wherein the oral administration comprises dietary supplementation.

34. The method of any one of claims 24-33, wherein histone acetylation is promoted in the subject.

35. The method of any one of claims 24-34, wherein the subject is a mammal.

36. The method of claim 35, wherein the mammal is a human.