Agents and / or compositions useful for modulating CIS-regulatory elements in synucleinopathies and methods for identifying agents and compositions thereof

Abstract

The present disclosure relates to methods of preventing, or delaying the progression of, death of neurons and / or microgliosis and / or astrogliosis that contributes to the death of neurons. The present disclosure also relates to methods of treating, preventing, or delaying the progression of, a synucleinopathy (e.g., Parkinson's disease). Also disclosed are related in vitro, ex vivo, and in vivo methods of identifying agents and / or compositions useful for preventing, or delaying the progression of, death of neurons and / or microgliosis and / or astrogliosis that contributes to the death of neurons and agents and / or compositions useful for treating, preventing, or delaying the progression of, a synucleinopathy. The agents and / or compositions of the present disclosure decrease the level and / or activity of a cis-regulatory element that propagates the misfolding and aggregation of proteins encoded by synucleinopathy-associated genes in neurons and / or glial cells.

Description

Atty. Docket: JHU-43272.601 AGENTS AND / OR COMPOSITIONS USEFUL FOR MODULATING CIS-REGULATORY ELEMENTS IN SYNUCLEINOPATHIES AND METHODS FOR IDENTIFYING AGENTS AND COMPOSITIONS THEREOF CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Application No.63 / 662,938, filed June 21, 2024, which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] The text of the computer readable sequence listing filed herewith, titled “JHU_43272_601_SequenceListing.xml”, created June 17, 2025, having a file size of 7,317 bytes, is hereby incorporated by reference in its entirety. FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0003] This invention was made with government support under grant NS128604 awarded by the National Institutes of Health. The government has certain rights in the invention. BACKGROUND

[0004] Parkinson's disease (PD) is a prevalent neurodegenerative disorder characterized by a combination of motor symptoms—such as tremors, rigidity, bradykinesia (slowness of movement), and postural instability—and non-motor symptoms, including cognitive impairment, mood disorders, sleep disturbances, and autonomic dysfunction. These symptoms progressively worsen over time, significantly impairing the quality of life for affected individuals.

[0005] PD is associated with a broader category of disorders known as synucleinopathies. Synucleinopathies are a group of chronic and progressive neurodegenerative conditions marked by the abnormal accumulation of alpha-synuclein (SNCA) proteins into insoluble aggregates within the brain. This aggregation disrupts normal cellular function and contributes to the neurodegenerative process. In addition to PD, synucleinopathies include neurodegenerative diseases such as, Gaucher's disease, Lewy body diffuse disease, dementia with Lewy bodies, a variant of Alzheimer's disease with Lewy bodies, sporadic Alzheimer's Disease, familial Alzheimer's Disease, true autonomic failure, dopa-responsive dystonia, multiple system atrophy (MSA) also Shy-Drager syndrome and the like. Collectively, these conditions affect millions of people worldwide, imposing a significant burden on individuals, families, and healthcare systems.

[0006] The etiology of PD and other synucleinopathies involves a complex interplay of genetic predispositions and environmental exposures. Notably, mutations and multiplications of the SNCA gene are directly linked to familial forms of these disorders, underscoring the crucial role of alpha-synuclein in theirAtty. Docket: JHU-43272.601 development. The aggregation of misfolded alpha-synuclein into Lewy bodies and Lewy neurites is a hallmark of these diseases and serves as a rate-limiting step in their pathological progression.

[0007] Current therapeutic strategies for synucleinopathies, and more specifically PD, primarily focus on managing symptoms rather than halting the disease's progression. For example, in treating PD, dopaminergic medications such as levodopa aim to alleviate motor symptoms by replenishing dopamine levels in the brain. However, these medications do not address the underlying disease mechanisms, and their effectiveness can diminish over time, leading to fluctuating symptoms. Consequently, non-motor symptoms often persist or worsen, highlighting the limitations of current therapies and the urgent need for treatments targeting the root causes of PD. Given these challenges, there is a critical need for novel interventions that target the molecular and cellular mechanisms driving synucleinopathies. SUMMARY

[0008] In an aspect, the present disclosure provides a method of preventing, or delaying the progression of, death of a neuron and / or microgliosis and / or astrogliosis that contributes to the death of the neuron, the method comprising contacting the neuron and / or a glial cell with an agent and / or composition that decreases the level and / or activity of a cis-regulatory element (CRE) that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in the neuron and / or glial cell, thereby reducing transcription of the synucleinopathy-associated gene encoding the protein in the neuron and / or glial cell and preventing, or delaying the progression of, death of the neuron and / or microgliosis and / or astrogliosis that contribute to the death of the neuron.

[0009] The agent and / or composition reduces transcription of the synucleinopathy-associated gene encoding the protein in the at least one neuron. The agent and / or composition reduces transcription of the synucleinopathy-associated gene encoding the protein in the at least one glial cell.

[0010] In an aspect, the present disclosure provides a method of treating, preventing, or delaying the progression of, a synucleinopathy in a subject in need thereof, comprising administering to the subject an effective amount of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in at least one neuron and / or glial cell in the subject, thereby reducing transcription of the synucleinopathy- associated gene encoding the protein in at least one neuron and / or glial cell of the subject and treating, preventing, or delaying the progression of, the synucleinopathy in the subject.

[0011] The agents and / or compositions of the present disclosure can be used to: (i) treat, prevent, or delay the progression of, microgliosis in the subject; (ii) treat, prevent, or delay the progression of, astrogliosis in the subject; (iii) treats prevent, or delay the progression of, the synucleinopathy in the subject; (iv) treat, prevent, or delay the progression of, a motor deficit in the subject; (v) treat, prevent, or delay the progressionAtty. Docket: JHU-43272.601 of, cognitive decline in the subject; (vi) treat, prevent, or delay the progression of, Lewy body aggregation in the cortex of the subject; (vii) delete the CRE that propagates the misfolding and aggregation of the protein encoded by the synucleinopathy-associated gene; and / or (viii) decrease the level of misfolded and aggregated alpha-synuclein protein in the subject.

[0012] In an aspect, the present disclosure provides a method of screening for an agent and / or composition which decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein in a neuron and / or glial cell, comprising: (a) contacting the neuron and / or glial cell with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of the CRE; and (b) identifying the candidate agent and / or composition as the agent and / or composition that decreases the level and / or activity of the CRE, if the candidate agent and / or composition decreases the transcription level and / or activity of a synucleinopathy-associated gene encoding the protein in the neuron and / or glial cell compared to the transcription level and / or activity of a synucleinopathy-associated gene encoding the protein in a neuron and / or glial cell that has not been contacted with the candidate agent and / or composition.

[0013] The method may include contacting the neuron with the agent and / or composition, contacting the glial cell with the agent and / or composition, or contacting both the neuron and glial cell with the agent and / or composition.

[0014] The neuron may be any neuron that is implicated in a synucleinopathy, for example, a dopaminergic (DA) neuron.

[0015] The CRE can be any CRE that propagates the misfolding and aggregation of a synucleinopathy- associated gene in a neuron and / or a glial cell, for example, an enhancer that otherwise by its role in modulating the transcription of its cognate synucleinopathy-associated gene propagates the misfolding and aggregation of protein in a neuron and / or a glial cell. Exemplary such enhancers include, without limitation, the following enhancers located at Human Chromosome 4 (Ch4): Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975-90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557- 90662154, Ch4: 90672325-90672709, Ch4: 90678450-90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4: 90735341-90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, Ch4: 90842771-90843114 and corresponding enhancers in non-human mammals.

[0016] The present disclosure contemplates decreasing the level and / or activity of any synucleinopathy- associated gene in a neuron and / or glial cell. Exemplary synucleinopathy-associated genes include, without limitation, SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2,Atty. Docket: JHU-43272.601 GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1.

[0017] The present disclosure contemplates any protein encoded by a synucleinopathy-associated gene that is misfolded and aggregated in neurons and / or glial cells. Exemplary proteins include, without limitation, alpha-synuclein and LRRK2.

[0018] The present disclosure contemplates treating, preventing, or delaying the progression of any synucleinopathy, for example, Parkinson's disease (“PD”).

[0019] The present disclosure contemplates the use of any agent and / or composition that decreases the level and / or activity of the CRE, for example, a gene editing system. Exemplary agents and / or compositions also include, without limitation, CRISPR-Cas system gRNA, a siRNA, a shRNA, a miRNA, an antisense DNA, an antisense RNA, a DNAzyme, a Ribozyme, an antagomir, a LMA, a MNA, and / or a chemical / small molecule.

[0020] The methods of the present disclosure may be performed in vitro or ex vivo.

[0021] The methods of the present disclosure may also be performed in vivo in a subject, for example, in a human, or non-human animal, such as a mouse.

[0022] The methods of the present disclosure may also include evaluating the ability of the agent and / or composition to: (i) decrease microgliosis that contributes to the death of the neuron; (ii) decrease astrogliosis that contributes to the death of the neuron; (iii) decrease the level of the CRE in the neuron and / or glial cell; (iv) decrease the activity of the CRE in the neuron and / or glial cell;(v) decrease the transcription level of the synucleinopathy associated gene in the neuron and / or glial cell; (vi) decrease the level of misfolded and aggregated protein encoded by the synucleinopathy-associated gene in the neuron and / or glial cell; (vii) treat, prevent, or delay the progression of, microgliosis in the subject; (viii) treat, prevent, or delay the progression of, astrogliosis in the subject; (ix) treat, prevent, or delay the progression of, a synucleinopathy in the subject; (x) treat, prevent, or delay the progression of, a motor deficit in the subject; (xi) treat, prevent, or delay the progression of, a cognitive deficit in the subject; (xii) treat, prevent, or delay the progression of, Lewy body aggregation in the cortex of the subject; (xiii) delete the CRE that propagates the misfolding and aggregation of the protein encoded by the synucleinopathy-associated gene; and / or (xiv) decrease the level of misfolded and aggregated protein encoded by the synucleinopathy- associated gene in the subject. BRIEF DESCRIPTION OF THE FIGURESAtty. Docket: JHU-43272.601

[0023] Having thus described the presently disclosed subject matter in general terms, reference will now be made to the accompanying Figures, which are not necessarily drawn to scale, and wherein:

[0024] FIG.1A-C. (A) shows relative position of the putative enhancer, SncaEnh+37, within mouse Snca intron 4. This element lies within a differentially accessible chromatin peak (pink) as identified by McClymont et al17. Mice lacking this element, spanning 2.76 kb (dark blue), were injected with α-syn PFF or PBS control prior to motor, molecular, and histological analyses. (B) smFISH / RNAscope reveals a significant reduction in Snca transcript among mice lacking SncaEnh+37. The bar chart shows the average number of Snca transcripts per Th+ cell in the SN of 3 mice / genotype / founder line. Error bars represent standard error of the mean (SEM). Significance was determined using a one-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test with a 95% confidence interval. (C) Representative smFISH / RNAscope images from SncaEnh+37 L1 founder mice. Scale bar = 100μm. See Table 2 for ANOVA summary statistics. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, ns = not significant.

[0025] FIG. 2A-D. Mice lacking SncaEnh+37 are protected against motor deficits as measured by: (A) Pole descent performance; (B) Grip strength; and (C) Rotarod performance. (D) Mice lacking SncaEnh+37 are also protected against anxiety-like behaviours in the open field test. Error bars represent standard error (SE). Significance was determined using a two-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test with a 95% confidence interval. See Tables 3-26 for ANOVA summary statistics. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.

[0026] FIG. 3A-M. Mice lacking SncaEnh+37 are protected against PD-like histopathological phenotypes: (A) Representative immunostaining for α-Synuclein phosphorylated serine 129 (pS129-α-Syn), indicative of LB inclusions, and Th+ dopaminergic neurons in the SN. Scale bar=20µm (B) Quantification of pS129-α-Syn + immunostained neurons in the SN. (C) Quantification of Th+ immunostained neurons in the SN. (D) Proportion of Th+ neurons that contain pS129-α-Syn / LB inclusions in the SN. (E) Representative photomicrographs of coronal sections showing Th+ and Nissl+ neurons in the SN. Scale bar=40µm. (F) Unbiased stereological quantification of Th+ DA neurons in the SN. (G) Unbiased stereological quantification of Nissl+ neurons in the SN. (H) Western blot of Th and DAT from TX-soluble fraction. (I) Relative quantification of Th, normalized to 13-actin. (J) Relative quantification of DAT, normalized to 13-actin. (K) Western blot from TX-insoluble fraction. (L) Relative quantification of insoluble α-Syn, normalized to 13- actin. (M) Relative quantification of insoluble pS129-α-Syn, normalized to 13-actin. Significance was determined using a two-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test with a 95% confidence interval. See Tables 27-31 (IHC); 32-S35 (WB) for ANOVA summary statistics. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.Atty. Docket: JHU-43272.601

[0027] FIG. 4A-F. Mice lacking SncaEnh+37 are protected against neuroinflammatory phenotypes: (A) Representative immunostaining of Iba1 and Gfap in the SN. Scale bar=40µm. (B) Quantification of Iba1+ immunostained cells. (C) Quantification of Gfap+ immunostained cells. (D) Western blot of neuroinflammatory markers, Iba1 (microglia) and Gfap (astrocytes) in the SN (E) Relative quantification of Iba1, normalized to β-actin. (F) Relative quantification of Gfap, normalized to β-actin. Significance was determined using a two-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test with a 95% confidence interval. See Tables 36-37 (IHC); 38-39 (WB) for ANOVA summary statistics * = p < 0.05, ** = p < 0.01, *** = p < 0.001, **** = p < 0.0001, ns = not significant.

[0028] FIG. 5A-B. Characterization of SncaEnh+37 mice. (A) A SnapGene map showing the location of genotyping primers, screening primers, guide RNA sequences, SncaEnh+37 deletion, and their relative position to the lifted-over open chromatin peak identified in McClymont et al

[0017] . (B) A 1% agarose gel run with 3 replicates of each SncaEnh+37 mouse genotype: WT(SncaEnh+37+ / +) = 686 bp amplicon between “Forward Genotyping Primer” and “Internal Genotyping Primer”; Hom(SncaEnh+37- / -) = 259 bp amplicon between “Forward Genotyping Primer” and “Reverse Screening / Genotyping Primer,” and Het(SncaEnh+37+ / -) = 686 bp and 259 bp amplicon.

[0029] FIG.6A-B. Representative images of (A) smFISH / RNAscope staining in the substantia nigra of a Het(SncaEnh+37+ / -) founder mouse (L1); blue = Hoechst, red = Th, green = Snca, yellow = colocalization of Th and Snca and (B) the HALO logic gate used to identify dopaminergic neurons from RNAscope images (purple = nucleus, light gray = cytoplasm of Th+ cells, dark gray = cytoplasm of Th- cells, red = cytoplasm of Th+ cells containing Snca transcripts).

[0030] FIG. 7. Representative smFISH / RNAscope images from SncaEnh+37 founder mice, showing WT(SncaEnh+37+ / +), Het(SncaEnh+37+ / -), and Hom(SncaEnh+37- / -) mice from founder lines L1, L2, Y1, and Y7. Scale bars = 100μm.

[0031] FIG.8A-C. Mice lacking SncaEnh+37 are protected against motor deficits as measured by: (A) pole descent performance; (B) Grip strength; and (C) Rotarod performance. (D) Mice lacking SncaEnh+37 are also protected against anxiety-like behaviours. Bar charts report both combined and sex-dependent analyses of 8-10 mice / sex / genotype / treatment group. Error bars represent standard error (SE). Significance was determined using a two-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test with a 95% confidence interval. See Tables 3-26 for ANOVA summary statistics. * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

[0032] FIG. 9A-B. Mice lacking both copies of SncaEnh+37 record more peripheral movement. (A) Bar chart showing center movement for 16-20 mice / genotype / treatment group. (B) Bar chart showing sex differences in peripheral movement of 8-10 mice / sex / genotype / treatment group. Error bars represent standard error (SE). Significance was determined using a two-way ANOVA and Tukey’s HonestlyAtty. Docket: JHU-43272.601 Significant Difference (HSD) test with a 95% confidence interval. See Tables 18-20 for ANOVA summary statistics. ▪ = p < 0.1, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

[0033] FIG.10A-B. α-Syn PFF injected animals recorded more total movement in the open field chamber compared to PBS mice. (A) Bar chart showing locomotion for 16-20 mice / genotype / treatment group. (B) Bar chart showing sex differences in locomotion of 810 mice / sex / genotype / treatment group. Error bars represent standard error (SE). Significance was determined using a two-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test with a 95% confidence interval. See Tables 15-17 for ANOVA summary statistics. ▪ = p < 0.1, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

[0034] FIG. 11A-B. Mice lacking both copies of SncaEnh+37 record more center movement. (A) Bar chart showing center movement for 16-20 mice / genotype / treatment group. (B) Bar chart showing sex differences in center movement of 8-10 mice / sex / genotype / treatment group. Error bars represent standard error (SE). Significance was determined using a two-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test with a 95% confidence interval. See Tables 21-23 for ANOVA summary statistics. ▪ = p < 0.1, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

[0035] FIG.12A-B. Mice lacking both copies of SncaEnh+37 show no differences in rearing behaviours. (A) Bar chart showing rearing behaviours for 16-20 mice / genotype / treatment group. (B) Bar chart showing sex differences in rearing behaviours of 8-10 mice / sex / genotype / treatment group. Error bars represent standard error (SE). Significance was determined using a two-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test with a 95% confidence interval. See Tables 24-26 for ANOVA summary statistics. ▪ = p < 0.1, * = p < 0.05, ** = p < 0.01, *** = p < 0.001.

[0036] FIG. 13A-B. Mice lacking SncaEnh+37 are protected against PD-like histopathological phenotypes in the cortex. A. Representative photomicrographs of coronal sections (Low Mag-scale bar=100μm and High Mag-scale bar=50μm) showing Th+ and Nissl+ neurons in the Cortex. WT-PBS, wild-type animals injected with PBS vehicle; WT-PFF, wild-type animals injected with α-syn PFF; Het- PFF, animals Heterozygous for deletion of SncaEnh+37 injected with α-syn PFF; Hom-PFF, animals Homozygous for deletion of SncaEnh+37 injected with α-syn PFF. B. Relative quantification of insoluble pS129-α-syn, normalized to β-actin. Significance was determined using a two-way ANOVA and Tukey’s Honestly Significant Difference (HSD) test with a 95% confidence interval. * = p < 0.05, ** = p < 0.01, ns = not significant. DEFINITIONS

[0037] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. However, inAtty. Docket: JHU-43272.601 case of conflict, the present specification, including definitions, will control. Accordingly, in the context of the embodiments described herein, the following definitions apply.

[0038] The terms “comprise(s),” “include(s),” “having,” “has,” “can,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that do not preclude the possibility of additional acts or structures. The singular forms “a,” “and” and “the” include plural references, i.e., “one or more,” unless the context clearly dictates otherwise.

[0039] The present disclosure also contemplates other embodiments “comprising,” “consisting of” and “consisting essentially of,” the embodiments or elements presented herein, whether explicitly set forth or not.

[0040] Likewise, the term “include”, and its grammatical variants are intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that can be substituted or added to the listed items.

[0041] As used herein, the term “about,” when used in connection with one or more numbers or numerical ranges, should be understood to refer to all such numbers, including all numbers in a range and modifies that range by extending the boundaries slightly above and slightly below the numerical values set forth by, for example, in some embodiments, + / -20%, + / -15%, + / -10%, + / -5%, + / -4%, + / -3%, + / -2%, and + / -1%. The recitation of numerical ranges by endpoints includes all numbers, e.g., whole integers, including fractions thereof, subsumed within that range (for example, the recitation of 1 to 5 includes 1, 2, 3, 4, and 5, as well as fractions thereof, e.g., 1.5, 2.25, 3.75, 4.1, and the like) and any range within that range.

[0042] As used herein, the term “administration” refers to the act of giving a drug, prodrug, or other agent, or therapeutic treatment (e.g., pharmaceutical compositions of the present disclosure) to a subject or in vivo, in vitro, or ex vivo cells, tissues, and organs. Exemplary routes of administration to the human body can be through the eyes (e.g., intraocularly, intravitreally, periocularly, ophthalmic, etc.), intrathecal (within or into the space around the membranes enveloping the spinal cord and brain), mouth (oral), skin (transdermal), nose (nasal), lungs (inhalant), oral mucosa (buccal), ear, rectal, by injection (e.g., intravenously, subcutaneously, intratumorally, intraperitoneally, etc.) and the like.

[0043] As used herein, the terms “alpha-synuclein nucleic acid” or “α-synuclein” or “Snca” or “SNCA” or “α-syn” means any nucleic acid encoding alpha-synuclein. For example, in certain embodiments, an alpha-synuclein nucleic acid includes a DNA sequence encoding alpha-synuclein, an RNA sequence transcribed from DNA encoding alpha-synuclein (including genomic DNA comprising introns and exons), and an mRNA sequence encoding alpha-synuclein. “alpha-synuclein mRNA” means an mRNA encoding an alpha-synuclein protein.

[0044] Alpha-synuclein (α-syn) is a protein associated with neurodegenerative synucleinopathies. It exists in both soluble and aggregated forms, with the latter contributing to disease pathology. ElevatedAtty. Docket: JHU-43272.601 levels of α-syn or mutations in the SNCA gene are implicated in the pathogenesis of various synucleinopathies that include but are not limited to, Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA) also Shy-Drager syndrome. The aggregation of α-syn into insoluble fibrils, known as Lewy bodies and Lewy neurites, is a hallmark of these diseases, marking a critical stage in their progression. In PD, α-syn pathology is linked to neuronal dysfunction and cell death, leading to motor symptoms such as tremors, rigidity, and bradykinesia, as well as non-motor symptoms like cognitive impairment and autonomic dysfunction. The role of α-syn in disrupting synaptic function and impairing neurotransmitter release, contributes to continued disease progression.

[0045] As used herein, the term “alteration” or “alteration of genetic information” refers to any change in the genome of a cell.

[0046] As used herein, the term “antisense” means an oligomeric compound that is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.

[0047] As used herein, the term “combination” is used in its broadest sense and means that a subject is administered at least two agents, more particularly an agent and / or composition disclosed herein and at least one other therapeutic agent. More particularly, the term “in combination” refers to the concomitant administration of two (or more) active agents for the treatment of a, e.g., single disease state. As used herein, the active agents may be combined and administered in a single dosage form, may be administered as separate dosage forms at the same time, or may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In one embodiment of the presently disclosed subject matter, the active agents are combined and administered in a single dosage form. In another embodiment, the active agents are administered in separate dosage forms (e.g., wherein it is desirable to vary the amount of one but not the other). The single dosage form may include additional active agents for the treatment of the disease state.

[0048] As used herein, the term "contacting" refers to the process of bringing a therapeutic agent, diagnostic tool, or any other treatment modality into direct or indirect interaction with a biological target or system in mammals. This interaction may occur through various means, including but not limited to administration, application, infusion, injection, or delivery of the agent or tool to the target tissue, cells, or organs. In some embodiments, contacting involves the administration of a pharmaceutical compound to a patient via oral, intravenous, intramuscular, subcutaneous, intrathecal, or topical routes. Other examples include the application of diagnostic probes, imaging agents, or therapeutic devices to the skin, mucous membranes, or internal tissues. Contacting can also encompass the exposure of cells or tissues in vitro to specific agents for research, diagnostic, or therapeutic purposes, ensuring that the agent or tool interacts with the biological system to elicit the desired effect.Atty. Docket: JHU-43272.601

[0049] As used herein, the term “correction” or “corrected” as used herein, refers to a change of one or more nucleotides of a genome in a cell, whether by insertion, deletion, or substitution. Such correction may result in a more favorable genotypic or phenotypic outcome, whether in structure or function, to the genomic site which was corrected. One non-limiting example of a “correction” includes the correction of a mutant or defective sequence to a wild-type sequence which restores structure or function to a gene or its gene product(s).

[0050] As used herein, the term “deletion” refers to a loss or removal of one or more nucleotides in a DNA sequence or a loss or removal of the function of a gene. In some cases, a deletion can include, for example, a loss of a few nucleotides, an exon, an intron, a gene segment, or the entire sequence of a gene. In some cases, deletion of a gene refers to the elimination or reduction of the function or expression of a gene or its gene product. This can result from not only a deletion of sequences within or near the gene, but also other events (e.g., insertion, nonsense mutation) that disrupt the expression of the gene.

[0051] As used herein, the terms “in one embodiment” or “in some embodiments” as used herein does not necessarily refer to the same embodiment, though it may. Furthermore, the phrase “in another embodiment” as used herein does not necessarily refer to a different embodiment, although it may. Thus, as described below, various embodiments of the invention may be readily combined, without departing from the scope or spirit of the invention.

[0052] As used herein, the term “individual” means a human or non-human animal selected for treatment or therapy.

[0053] As used herein, the term “insertion” refers to an addition of one or more nucleotides in a DNA sequence. Insertions can range from small insertions of a few nucleotides to insertions of large segments such as a cDNA or a gene.

[0054] As used herein, the term “in vitro ” refers to an artificial environment and to processes or reactions that occur within an artificial environment. In vitro environments can consist of, but are not limited to, test tubes and cell culture.

[0055] The term “ in vivo ” refers to the natural environment (e.g., an animal or a cell) and to processes or reaction that occur within a natural environment.

[0056] As used herein, the term "gliosis" refers to a reactive process involving the proliferation and hypertrophy of glial cells in the central nervous system (CNS) in response to injury, disease, or other pathological conditions. In some embodiments, the terms "astrogliosis" and "microgliosis," which describe the specific activation and response of astrocytes and microglia, respectively, refer to the specific type of glial cell in a reactive process. As used herein, the term "astrogliosis" refers to a reactive process involving the proliferation and hypertrophy of astrocytes, a type of glial cell in the CNS, in response to injury, disease, or other pathological conditions in mammals. This process is characterized by the increased expression ofAtty. Docket: JHU-43272.601 glial fibrillary acidic protein (GFAP) and other markers of astrocyte activation. In some embodiments, astrogliosis can lead to the formation of a glial scar, which can inhibit axonal regeneration and contribute to the chronicity of CNS injuries and diseases. As used herein, the term "microgliosis" refers to the activation and proliferation of microglia, the resident immune cells of the CNS, in response to injury, infection, or neurodegenerative diseases in mammals. In some embodiments, the process is characterized by changes in microglial morphology, increased expression of immune-related markers, and the release of pro-inflammatory cytokines and chemokines. Microgliosis plays a critical role in the CNS immune response, but chronic activation can contribute to neuroinflammation and neuronal damage.

[0057] As used herein “level”, refers to a measure of the amount of, or a concentration of a transcription product, for instance an mRNA, or a translation product, for instance a protein or polypeptide. As used herein “activity” refers to a measure for the ability of a transcription product or a translation product to produce a biological effect or a measure for a level of biologically active molecules. As used herein “level and / or activity” further refer to gene expression levels or gene activity. Gene expression can be defined as the utilization of the information contained in a gene by transcription and translation leading to the production of a gene product.

[0058] As used herein, the term “neurodegenerative disease” means a disease characterized by progressive loss of structure or function of neurons, including death of neurons.

[0059] As used herein, the term “nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).

[0060] As used herein, the terms “preventing”, “delaying”, “amelioration” and the like refers to prophylactic steps taken to reduce the likelihood of a subject (e.g., an at-risk subject) from contracting or suffering from a particular disease, disorder, or condition. The likelihood of the disease, disorder, or condition occurring in the subject need not be reduced to zero for the preventing to occur; rather, if the steps reduce the risk of a disease, disorder, or condition across a population, then the steps prevent the disease, disorder, or condition for an individual subject within the scope and meaning herein. In some embodiments, preventing refers to causing a disease, disorder, condition, or symptom or manifestation of such, or worsening of the severity of such, not to occur. The severity of indicators may be determined by subjective or objective measures, which are known to those skilled in the art.

[0061] As used herein, the term “siRNA” refers to a double stranded stretch of RNA or modified RNA monomers. In a typical siRNA compound, the two strands usually have 19 nucleotides complementary to each other thereby creating a double strand that is 19 nucleotides long and each strand having a 3′-end of two overhanging nucleotides. This is not a strict definition of siRNA, which may be slightly longer orAtty. Docket: JHU-43272.601 shorter, and with or without overhangs. In siRNA one strand is guiding and complementary to the target RNA (antisense strand), and the other strand (sense strand) has the same sequence as the target RNA and hence is complementary to the guiding / antisense strand. Herein, regulatory RNAs such as “micro RNA” (“miRNA”) and “short RNA” (“shRNA”) and a variety of structural RNAs such as tRNA, snRNA, scRNA, rRNA are used interchangeably with the term “siRNA”.

[0062] As used herein, the term “subject” refers to the presently disclosed methods in their many embodiments is desirably a human subject, although it is to be understood that the methods described herein are effective with respect to all vertebrate species, which are intended to be included in the term “subject.” Accordingly, a “subject” can include a human subject for medical purposes, such as for the treatment of an existing condition or disease or the prophylactic treatment for preventing the onset of a condition or disease, or an animal subject for medical, veterinary purposes, or developmental purposes. Suitable animal subjects include mammals including, but not limited to, primates, e.g., humans, monkeys, apes, and the like; bovines, e.g., cattle, oxen, and the like; ovines, e.g., sheep and the like; caprines, e.g., goats and the like; porcines, e.g., pigs, hogs, and the like; equines, e.g., horses, donkeys, zebras, and the like; felines, including wild and domestic cats; canines, including dogs; lagomorphs, including rabbits, hares, and the like; and rodents, including mice, rats, and the like. An animal may be a transgenic animal. In some embodiments, the subject is a human including, but not limited to, fetal, neonatal, infant, juvenile, and adult subjects. Further, a “subject” can include a patient afflicted with or suspected of being afflicted with a condition or disease. Thus, the terms “subject” and “patient” are used interchangeably herein. The term “subject” also refers to an organism, tissue, cell, or collection of cells from a subject.

[0063] As used herein, the term “synucleinopathy” refers to a group of neurodegenerative disorders characterized by the abnormal accumulation of alpha-synuclein protein aggregates within the central nervous system. In some embodiments, the conditions involve the progressive dysfunction and loss of neurons, contributing to a decline in neurological function over time. Synucleinopathies encompass diseases such as PD, multiple system atrophy (MSA) also Shy-Drager syndrome, and dementia with Lewy bodies, each of which manifests with varying degrees of motor, cognitive, and autonomic impairments. The hallmark pathology of synucleinopathies involves the formation of Lewy bodies and Lewy neurites, disrupting normal neuronal processes and ultimately leading to neuronal death in affected regions of the brain and spinal cord.

[0064] The terms “test compound” and “candidate compound” refer to any chemical entity, pharmaceutical, drug, and the like that is a candidate for use to treat or prevent a disease, illness, sickness, or disorder of bodily function (e.g., synucleinopathy, PD). Test compounds comprise both known and potential therapeutic compounds. A test compound can be determined to be therapeutic by screening using the screening methods of the present disclosure.Atty. Docket: JHU-43272.601

[0065] As used herein, the terms “therapeutically effective amount”, “effective amount” and the like of a therapeutic agent refers to the amount of the agent necessary to elicit the desired biological response. As will be appreciated by those of ordinary skill in the art, the effective amount of an agent may vary depending on such factors as the desired biological endpoint, the agent to be delivered, the composition of the pharmaceutical composition, the target tissue or cell, and the like. In some embodiments, the term “therapeutically effective amount” refers to an amount sufficient to reduce or ameliorate the severity, duration, progression, or onset of a disease, disorder, or condition, or one or more symptoms thereof; prevent the advancement of a disease, disorder, or condition, cause the regression of a disease, disorder, or condition; prevent the recurrence, development, onset or progression of a symptom associated with a disease, disorder, or condition, or enhance or improve the prophylactic or therapeutic effect(s) of another therapy.

[0066] As used herein, the terms “treatment,” “treating,” and the like refer to obtaining a desired pharmacologic and / or physiologic effect against a particular disease, disorder, or condition. Preferably, the effect is therapeutic, i.e., the effect partially or completely cures the disease and / or adverse symptom attributable to the disease. The terms “treatment” and “treating” refers to reversing, alleviating, slowing down, inhibiting the progression of, preventing, or reducing the likelihood of the disease, disorder, or condition to which such term applies, or one or more symptoms or manifestations of such disease, disorder, or condition. In some embodiments, for example, the terms “treatment” and “treating” of a disease also includes providing relief from the symptoms or side-effects of the disease (including palliative treatment).

[0067] As used herein, the terms “treatment with” or “treatment of ” or “administration of” and variants thereof refers to the administration of at least two agent(s) or therapies to a subject. In some embodiments, the co-administration of two or more agents or therapies is concurrent (e.g., in the same or separate formulations). As used herein, the active agents may be administered as separate dosage forms that are administered alternately or sequentially on the same or separate days. In other embodiments, a first agent / therapy is administered prior to a second agent / therapy. Those of skill in the art understand that the formulations and / or routes of administration of the various agents or therapies used may vary. The appropriate dosage for co-administration can be readily determined by one skilled in the art. In some embodiments, when agents or therapies are co-administered, the respective agents or therapies are administered at lower dosages than appropriate for their administration alone. The single dosage form may include additional active agents for the treatment of the disease state. Thus, co-administration is especially desirable in embodiments where the co-administration of the agents or therapies lowers the requisite dosage of a potentially harmful (e.g., toxic) agent(s). DETAILED DESCRIPTIONAtty. Docket: JHU-43272.601

[0068] The presently disclosed subject matter now will be described more fully hereinafter with reference to the accompanying Figures, in which some, but not all embodiments of the inventions are shown. Like numbers refer to like elements throughout. The presently disclosed subject matter may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Therefore, it is to be understood that the presently disclosed subject matter is not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims.

[0069] The presently discloses subject matter relates to methods of preventing, or delaying the progression of, death of neurons, and / or microgliosis and / or astrogliosis that contribute to the death of neurons. The presently disclosed subject matter also relates to methods of treating, preventing, and / or delaying the progression of a synucleinopathy in subjects. The inventive methods of the present disclosure relate to decreasing the level and / or activity of a cis-regulatory element (CRE) that otherwise by its role in modulating the transcription of its cognate synucleinopathy-associated gene propagates the misfolding and aggregation of protein in neurons and / or glial cells. Also disclosed are methods of identifying agents and / or compositions useful for decreasing the level and / or activity of CREs in neurons and / or glial cells in vitro, ex vivo, and / or in vivo. Methods of identifying agents and / or compositions useful for decreasing microgliosis and / or astrogliosis that contribute to the death of neurons are also contemplated.

[0070] Surprisingly and unexpectedly, the work described herein demonstrates that decreasing the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons by significantly decreasing transcription of the synucleinopathy-associated gene in neurons and decreasing the propagation of the misfolding and aggregation of the protein in the neurons, thereby promoting survival of the neurons.

[0071] The data provided herein demonstrarte that deletion (via CRISPR) of an intronic cis-regulatory sequence (CRE) of Snca (SncaCRE+37) significantly mitigates PD-relevant pathology in mice, initiated by intra-striatal delivery of α-syn pre-formed fibrils (PFF). This verifies that the reduction in the acquisition of Lewy bodies; the reduction in neurodegeneration; the abrogation of microglial and astrocytic neuroinflammatory response are all mediated by the cell dependent modulation of alpha synuclein – all seen in the context of the ventral midbrain region associated with motor function. Further, the examples provide robust evidence of the mitigation of motor deficits in the engineered mice, demonstrating clearly that cell-dependent reduction in alpha-synuclein can protect against the onset PD and related progressive decline in motor function. PD is often accompanied by distressing non-motor symptoms, including cognitive decline, anxiety / depression, constipation and anosmia. The data provide herein further demonstrates that regulatory modulation of alpha synuclein via SncaCRE+37 deletion protects from corticalAtty. Docket: JHU-43272.601 Lewy body aggregates (See Fig.13) and reduces anxiety scores, which can lead to protection from cognitive deficits. Thus, the present invention provides compositions and methods useful for mitigating risk of cognitive decline in PD, diffuse lewy body dementia, other synucleinopathies and Alzheimer’s Disease.

[0072] Accordingly, in an aspect, the present disclosure provides a method of preventing, or delaying the progression of, death of a neuron and / or microgliosis and / or astrogliosis that contributes to the death of the neuron comprising contacting a neuron and / or a glial cell with an agent and / or composition that decreases the level and / or activity of a cis-regulatory element (CRE) that propagates the misfolding and aggregation of a protein in the neuron and / or glial cell, thereby reducing transcription of a synucleinopathy-associated gene encoding the protein in the neuron and / or glial cell and preventing, or delaying the progression of, death of the neuron and / or microgliosis and / or astrogliosis that contribute to the death of the neuron.

[0073] In an aspect, the present disclosure provides a method of preventing, or delaying the progression of, death of a neuron comprising contacting the neuron with an agent and / or composition that decreases the level and / or activity of a cis-regulatory element (CRE) that propagates the misfolding and aggregation of a protein in the neuron, thereby reducing transcription of a synucleinopathy-associated gene encoding the protein in the neuron and preventing, or delaying the progression of, death of the neuron.

[0074] In an aspect, the present disclosure provides a method of preventing, or delaying the progression of, death of a neuron comprising contacting a glial cell in proximity to, or in cellular communication with, the neuron with an agent and / or composition that decreases the level and / or activity of a cis-regulatory element (CRE) that propagates the misfolding and aggregation of a protein in the glial cells, thereby reducing transcription of a synucleinopathy-associated gene encoding the protein in the glial cell and preventing, or delaying the progression of, death of the neuron.

[0075] In an aspect, the present disclosure provides a method of preventing, or delaying the progression of, microgliosis and / or astrogliosis that contributes to the death of a neuron comprising contacting a neuron and / or a glial cell with an agent and / or composition that decreases the level and / or activity of a cis- regulatory element (CRE) that propagates the misfolding and aggregation of a protein in the neuron and / or glial cell, thereby reducing transcription of a synucleinopathy-associated gene encoding the protein in the neuron and / or glial cell and preventing, or delaying the progression of, microgliosis and / or astrogliosis that contribute to the death of the neuron.

[0076] Surprisingly and unexpectedly, the work described herein demonstrates in a mouse model of a synucleinopathy (e.g., PD), that the deletion of a CRE (e.g., enhancer) showed a significant reduction in SNCA alpha-synuclein misfolding and aggregation and neuronal loss compared to controls, as well as improved motor function, thereby supporting the efficacy of targeting CREs in synucleinopathies. Decreasing the level and / or activity of the CRE using an agent and / or composition of the present disclosure significantly decreased SNCA transcription, and not only decreased the death of dopaminergic neurons butAtty. Docket: JHU-43272.601 also reduced markers of microglia and astrocytes, as confirmed by immunohistochemical analysis and quantification of neuronal survival markers and glial cells. This coincides with a reduction in glial cell activation and overall deleterious effects, leading to decreased progression of neurodegenerative symptoms. This offers a promising therapeutic strategy for synucleinopathies such as PD, multiple system atrophy, and dementia with Lewy bodies.

[0077] Accordingly, in an aspect, the present disclosure provides a method of treating, preventing, or delaying the progression of, a synucleinopathy in a subject in need thereof, comprising administering to the subject an effective amount of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein in at least one neuron and / or glial cell of the subject, thereby reducing transcription of a synucleinopathy-associated gene encoding the protein in the at least one neuron and / or glial cell of the subject and treating, preventing, or delaying the progression of, the synucleinopathy in the subject.

[0078] Accordingly, in an aspect, the present disclosure provides a method of preventing, or delaying the progression of, microgliosis and / or astrogliosis in a subject in need thereof (e.g., a subject suffering from a synucleinopathy, such as PD), comprising administering to the subject an effective amount of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein in at least one neuron and / or glial cell of the subject, thereby reducing transcription of a synucleinopathy-associated gene encoding the protein in the at least one neuron and / or glial cell of the subject and preventing, or delaying the progression of, microgliosis and / or astrogliosis in the subject.

[0079] It is well known that a critical aspect of synucleinopathies is the vicious cycle of protein misfolding and aggregation, leading to microgliosis and astrogliosis. Microgliosis refers to the sustained activation and proliferation of microglia, the brain's resident immune cells, in response to injury or disease, whereas astrogliosis involves the continuous activation and proliferation of astrocytes, which support and protect neurons. This persistent activation results in a harmful feedback loop, causing increased inflammation and further neuronal death. These processes exacerbate neuroinflammation and contribute to neuronal cell death, ultimately leading to neurodegenerative diseases and synucleinopathies like PD. Thus, the use of the agents and / or compositions disclosed herein can potentially prevent the initial propagation of neuronal stress by reducing transcription levels of genes associated with synucleinopathies (e.g., PD), thereby preventing the development of neuroinflammation and the subsequent proliferation of microgliosis and astrogliosis. Additionally, by decreasing the activation of these glial cells, such agents and / or compositions not only disrupt the pathological aggregation of proteins but also reduce neuroinflammatory responses that are detrimental to neuronal survival. Therefore there is a need in identifying agents and / or compositions for effective modulation that leads to the reduction in glial cell activation and preservation of neuronal cells, ultimately providing a therapeutic strategy to combat the progression of synucleinopathies.Atty. Docket: JHU-43272.601

[0080] Accordingly, an aspect of the present disclosure provides a method of screening for an agent and / or composition which decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein in a neuron and / or glial cell, comprising: (a) contacting the neuron and / or glial cell with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of the CRE; and (b) identifying the candidate agent and / or composition as the agent and / or composition that decreases the level and / or activity of the CRE, if the candidate agent and / or composition decreases the transcription level and / or activity of a synucleinopathy-associated gene encoding the protein in the neuron and / or glial cell compared to the transcription level and / or activity of a synucleinopathy-associated gene encoding the protein in a neuron and / or glial cell that has not been contacted with the candidate agent and / or composition.

[0081] In an aspect, the present disclosure provides a method of identifying an agent and / or composition which decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein in at least one neuron and / or glial cell of a subject comprising: (a) administering to the subject a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of the CRE in the at least one neuron and / or glial cell; and (b) identifying the candidate agent and / or composition as the agent and / or composition that decreases the level and / or activity of the CRE, if the candidate agent and / or composition decreases the transcription level and / or activity of a synucleinopathy-associated gene encoding the protein in the neuron and / or glial cell compared to the transcription level and / or activity of the synucleinopathy-associated gene in at least one neuron and / or glial cell of a subject who has not been administered the candidate agent and / or composition. Decreasing the level and / or activity of CREs

[0082] Aspects of the present disclosure relate to decreasing the level and / or activity of a CRE that otherwise by its role in modulating the transcription of its cognate synucleinopathy-associated gene propagates the misfolding and aggregation of protein in neurons and / or glial cells. The present disclosure is not limited in the way in which the level and / or activity of a CRE can be decreased. The present disclosure contemplates methods comprising contacting neurons and / or glial cells in vitro, ex vivo, and / or in vivo with agents and / or compositions that decrease the level and / or activity of the CRE.

[0083] The level and / or activity of the CRE can be decreased by any amount in the neuron and / or glial cell, preferably by an amount that sufficient to reduce the transcription level of a synucleinopathy associated gene in the neurons and / or glial cells, reduce the propagation of misfolding and aggregation of proteins encoded by the synucleinopathy-associated genes in the neurons and / or glial cells, and / or reduce microgliosis and / or astrogliosis that contribute to death of the neurons.Atty. Docket: JHU-43272.601

[0084] In some embodiments, the level and / or activity of the CRE is decreased by an absolute amount compared to a baseline level and / or activity of the CRE in the neuron and / or glial cell in vitro, ex vivo and / or in vivo. In some embodiments, the level and / or activity of the CRE is decreased in a neuron and / or glial cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to the baseline level and / or activity of the CRE in the neuron and / or glial cell before being contacted with an agent and / or composition of the present disclosure.

[0085] In some embodiments, the level and / or activity of the CRE is decreased by a relative amount in the neuron and / or glial cell in vitro, ex vivo and / or in vivo compared to a neuron and / or glial cell that is not contacted with an agent and / or composition of the present disclosure. In some embodiments, the level and / or activity of the CRE is decreased in a neuron and / or glial cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to level and / or activity of the CRE in a control neuron and / or glial cell which has not been contacted with an agent and / or composition of the present disclosure. Decreasing the transcription levels of synucleinopathy-associated genes

[0086] Aspects of the present disclosure relate to decreasing the transcription level of a synucleinopathy- associated gene in a neuron and / or glial cell in vitro, ex vivo and / or in vivo. The present disclosure is not limited in the way in which the transcription level of a synucleinopathy-associated gene is decreased. The present disclosure contemplates methods comprising contacting neurons and / or glial cells in vitro, ex vivo, and / or in vivo with agents and / or compositions that decrease the transcription level of the synucleinopathy- associated gene, for example, by decreasing the level and / or activity of the CRE.

[0087] The transcription level can be decreased by any amount, preferably by an amount that sufficient to reduce the propagation of misfolding and aggregation of proteins encoded by the synucleinopathy- associated gene in the neurons and / or glial cells, and / or reduce microgliosis and / or astrogliosis that contribute to death of the neurons.

[0088] In some embodiments, the transcription level of the synucleinopathy-associated gene is decreased by an absolute amount compared to a baseline transcription level of the synucleinopathy-associated geneAtty. Docket: JHU-43272.601 in the neuron and / or glial cell in vitro, ex vivo and / or in vivo. In some embodiments, the transcription level of the synucleinopathy associated gene is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline transcription level of the synucleinopathy-associated gene prior to contacting the neuron and / or glial cell with an agent and / or composition of the present disclosure.

[0089] In some embodiments, the transcription level of the synucleinopathy-associated gene is decreased by a relative amount in a neuron and / or glial cell in vitro, ex vivo and / or in vivo compared to a transcription level of the synucleinopathy-associated gene in a neuron and / or glial cell not contacted with an agent and / or composition of the present disclosure. In some embodiments, the transcription level of the synucleinopathy associated gene is decreased in a neuron and / or glial cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to the transcription level of the synucleinopathy-associated gene in a neuron and / or glial cell not contacted with an agent and / or composition of the present disclosure. Decreasing the propagation of misfolding and aggregation of protein in neurons and / or glial cells

[0090] Aspects of the present disclosure relate to decreasing the propagation of misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in a neuron and / or glial cell in vitro, ex vivo and / or in vivo. The present disclosure is not limited in the way in which the propagation of misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene is decreased. The present disclosure contemplates methods comprising contacting neurons and / or glial cells in vitro, ex vivo, and / or in vivo with agents and / or compositions that decrease the propagation of misfolding and aggregation of proteins encoded by synucleinopathy-associated genes in neurons and / or glial cells, for example, by decreasing the transcription level of the synucleinopathy-associated gene, and / or by decreasing the level and / or activity of the CRE.

[0091] The propagation of misfolding and aggregation of protein in neurons and / or can be decreased by any amount, preferably by an amount that sufficient to reduce microgliosis and / or astrogliosis that contribute to death of the neurons.Atty. Docket: JHU-43272.601

[0092] In some embodiments, the propagation of misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells is decreased by an absolute amount compared to a baseline level of misfolded and / or aggregated protein in vitro, ex vivo and / or in vivo. In some embodiments, the propagation of misfolding and aggregation of a protein encoded by a synucleinopathy- associated gene in neurons and / or glial cells is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline level of propagation of misfolding and aggregation of the protein prior to contacting the neuron and / or glial cell with an agent and / or composition of the present disclosure.

[0093] In some embodiments, the propagation of misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells is decreased by a relative amount in a neuron and / or glial cell in vitro, ex vivo and / or in vivo compared to the level of propagation of misfolding and aggregation of a protein in neurons and / or glial cells not contacted with an agent and / or composition of the present disclosure. In some embodiments, the propagation of misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in decreased neurons and / or glial cells by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to the level propagation of misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells not contacted with an agent and / or composition of the present disclosure. Decreasing the level of microgliosis and / or astrogliosis that contributes to death of neurons

[0094] Aspects of the present disclosure relate to decreasing the level microgliosis and / or astrogliosis that contributes to death of a neuron in vitro, ex vivo and / or in vivo. The present disclosure is not limited in the way in which the microgliosis and / or astrogliosis that contributes to death of a neuron is decreased. The present disclosure contemplates methods comprising contacting neurons and / or glial cells in vitro, ex vivo, and / or in vivo with agents and / or compositions that decrease the level of microgliosis and / or astrogliosis that contribute to death of neurons, for example, by decreasing the propagation of misfolding and aggregated proteins encoded by synucleinopathy-associated genes, decreasing the transcription level of theAtty. Docket: JHU-43272.601 synucleinopathy-associated gene, and / or by decreasing the level and / or activity of the CRE, in neurons and / or glial cells.

[0095] The levels of microgliosis and / or astrogliosis that contribute to death of a neuron can be decreased by any amount, preferably by an amount that sufficient to prevent, or delay the progression of, death of neurons in vitro, ex vivo, and / or in vivo.

[0096] In some embodiments, the level of microgliosis and / or astrogliosis that contributes to death of a neuron is decreased by an absolute amount compared to a baseline level of microgliosis and / or astrogliosis that contributes to death of a neuron in vitro, ex vivo and / or in vivo. In some embodiments, the level of microgliosis and / or astrogliosis that contributes to death of a neuron is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% in glial cells compared to a baseline level microgliosis and / or astrogliosis in the glial cells that contributes to death of the neuron prior to contacting the neuron and / or glial cell with an agent and / or composition of the present disclosure.

[0097] In some embodiments, the level of microgliosis and / or astrogliosis that contributes to death of a neuron is decreased by a relative amount in a glial cell in vitro, ex vivo and / or in vivo compared to the level of microgliosis and / or astrogliosis that contributes to death of a neuron in glial cells not contacted with an agent and / or composition of the present disclosure. In some embodiments, the level of microgliosis and / or astrogliosis that contributes to death of a neuron is decreased by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% in glial cells contacted with an agent and / or composition of the present disclosure compared to the level of microgliosis and / or astrogliosis that contributes to death of a neuron in glial cells not contacted with an agent and / or composition of the present disclosure.

[0098] The methods of the present disclosure can be performed in vitro, ex vivo, or in vivo and contemplate agents and / or compositions capable of (i) decreasing the level and / or activity of a CRE in neurons; (ii) decreasing the transcription levels of synucleinopathy-associated genes in neurons; (iii) decreasing the propagation of misfolding and aggregation of proteins encoded by synucleinopathy-associated genes in neurons; (iv) decreasing microgliosis and / or astrogliosis that contribute to death of neurons. NeuronsAtty. Docket: JHU-43272.601

[0099] The present disclosure contemplates methods comprising contacting a neuron with an agent / or composition. The methods of the present disclosure can be performed in vitro, ex vivo, or in vivo and contemplate agents and / or compositions capable of (i) decreasing the level and / or activity of a CRE in neurons; (ii) decreasing the transcription levels of synucleinopathy-associated genes in neurons; (iii) decreasing the propagation of misfolding and aggregation of proteins encoded by synucleinopathy- associated genes in neurons; (iv) decreasing microgliosis and / or astrogliosis that contribute to death of neurons. Neurons are specialized cells in the nervous system that play a fundamental role in transmitting information throughout the body. They are responsible for processing and transmitting electrical and chemical signals, enabling communication within the brain, spinal cord, and the rest of the body. Neurons consist of several components, including dendrites (which receive signals), a cell body (which integrates incoming signals), and an axon (which transmits signals to other neurons or effector cells). In synucleinopathies such as PD, the misfolding and aggregation of proteins in neurons causes neuroinflammation ultimately leading to progressive dysfunction and death of neurons. This process leads to the deterioration of cognitive abilities, motor functions, or both, depending on the specific areas of the nervous system affected. Therefore, there is a need for agent and / or composition that slows and / or halts the progression of synucleinopathies aiming to preserve neuronal function and alleviate symptoms for affected individuals. The present disclosure is not limited to the type of neuron in which the level and / or activity of a CRE, the transcription levels of synucleinopathy-associated genes, the propagation of misfolding and aggregation of proteins encoded by synucleinopathy-associated genes, and / or microgliosis and / or astrogliosis that contribute to death of neurons can be decreased. The neuron may be a catecholaminergic neuron. The neuron is a dopaminergic neuron, a noradrenergic neuron, adrenergic neuron, a midbrain dopaminergic (DA) neuron, substantia nigra (SN) neuron, a ventral tegmental area (VTA) neuron, a periaqueductal gray area (PAG) neuron, a brain stem neuron, a hypothalamic neuron, and / or a pons neuron. In some embodiments, the neuron is a dopaminergic neuron. In some embodiments, the neuron is a noradrenergic neuron. In some embodiments, the neuron is an adrenergic neuron. In some embodiments, the neuron is a midbrain dopaminergic neuron. In some embodiments, the neuron is present in the substantia nigra (SN). In some embodiments, the neuron is present in the ventral tegmental area (VTA). In some embodiments, the neuron is present in the brain stem. In some embodiments, the neuron is present in the hypothalamus. In some embodiments, the neuron is present in the midbrain. In some embodiments, the neuron is present in the pons. Techniques for evaluating neurons are known to those skilled in the art. These techniques include, but are not limited to, immunohistochemistry, electrophysiology, neuroimaging such as MRI and PET, genetic and molecular analysis, behavioral assessments linking behavior to neurological health, and biomarker analysis. Glial CellsAtty. Docket: JHU-43272.601

[0100] The present disclosure contemplates methods comprising contacting a glial cell with an agent / or composition. The methods of the present disclosure can be performed in vitro, ex vivo, or in vivo and contemplate agents and / or compositions capable of (i) decreasing the level and / or activity of a CRE in glial cells; (ii) decreasing the transcription levels of synucleinopathy-associated genes in glial cells; (iii) decreasing the propagation of misfolding and aggregation of proteins encoded by synucleinopathy- associated genes in glial cells; and / or (iv) decreasing microgliosis and / or astrogliosis that contribute to death of neurons in close proximity to or in cellular communication with glial cells. Glial cells are essential components of the central nervous system, play pivotal roles in maintaining neuronal health and function. In the context of synucleinopathies, such as PD, their involvement extends beyond mere support to active participation in disease progression. Microglia, the resident immune cells of the brain, respond to pathological stimuli by becoming activated, contributing to neuroinflammation and the clearance of aggregated proteins like alpha-synuclein, characteristic of PD. However, prolonged activation can lead to neurotoxicity, exacerbating neuronal damage. Astrocytes, another type of glial cell, regulate the brain's chemical environment, provide metabolic support to neurons, and influence the blood-brain barrier's integrity. Dysfunction in astrocytes can contribute to neuroinflammation and impair the clearance of toxins, further compromising neuronal health in PD. Evaluating glial cells in PD involves techniques such as immunohistochemistry, gene expression analysis, advanced imaging, cell culture models, animal studies, and clinical biomarkers. These methods collectively shed light on how glial cells interact with neurons and contribute to disease pathology, offering potential targets for therapeutic interventions aimed at mitigating neurodegeneration in PD and related synucleinopathies. The present disclosure is not limited to the type of glial cell in which the level and / or activity of a CRE, the transcription levels of synucleinopathy-associated genes, the propagation of misfolding and aggregation of proteins encoded by synucleinopathy-associated genes, and / or microgliosis and / or astrogliosis that contribute to death of neurons can be decreased. The glial cell may be an astrocyte, oligodendrocyte, microglia, Schwann cell and / or ependymal cells. In some embodiments, the glial cell is an astrocyte. In some embodiments, the glial cell is an oligodendrocyte. In some embodiments, the glial cell comprises microglia. In some embodiments, the glial cell comprises a Schwann cell. In some embodiments, the glial cell is an ependymal cell. Cis-Regulatory Elements (CREs)

[0101] Aspects of the present disclosure contemplate reducing the level and / or activity of any CRE that otherwise by its role in modulating the transcription of its cognate synucleinopathy-associated gene propagates the misfolding and aggregation of protein in neurons and / or glial cells and / or increases microgliosis and / or astrogliosis that contributes to the death of neurons in vitro, ex vivo, and / or in vivo.Atty. Docket: JHU-43272.601

[0102] A cis-regulatory element (CRE) is a non-coding DNA sequence that regulates the expression of nearby genes. Unlike coding sequences that directly specify the amino acid sequence of proteins, CREs control when, where, and to what extent genes are expressed. These elements can be located proximal to the transcriptional start site (promoters), upstream / downstream, intragenic, or intergenic (enhancers / silencers) of the gene they regulate, or delineating boundaries between genes sharing aspects of regulatory control (insulators). CREs achieve their regulatory function by binding specific transcription factors or other regulatory proteins, which in turn influence the recruitment of RNA polymerase and other components of the transcriptional machinery to the gene's promoter region. CREs implicated in PD can be evaluated or quantitated using a variety of techniques well known in the art. These methods include chromatin immunoprecipitation followed by sequencing (ChIP-seq), which identifies DNA-protein interactions, and reporter assays, where CRE activity is measured by linking the regulatory sequence to a reporter gene. Other techniques include electrophoretic mobility shift assays (EMSA) to assess DNA- protein binding, DNase I hypersensitivity assays to determine accessible chromatin regions, and quantitative PCR (qPCR) to measure changes in gene expression associated with specific CREs. Additionally, high-throughput methods such as ATAC-seq (Assay for Transposase-Accessible Chromatin with high-throughput sequencing) can be employed to map open chromatin regions indicative of active regulatory elements. Overall, by modulating gene expression, CREs play critical roles in various biological processes, including development, differentiation, response to environmental cues, and disease pathways.

[0103] The present disclosure contemplates modulating the level and / or activity of any CRE that propagates the misfolding and aggregation of proteins encoded by synucleinopathy-associated genes in neurons and / or glial cells. The CRE may be an enhancer, a promoter, a silencer, an insulator, a repressor or it may represent a combinations of functional outcomes in a cell / state-dependent manner. In some embodiments, the CRE is a promoter. In some embodiments, the CRE is a silencer. In some embodiments, the CRE is an insulator. In some embodiments, the CRE is a repressor. In some embodiments, the CRE is an enhancer. In some embodiments, the CRE represents a combination of functional outcomes in a cell / state-dependent manner. Promoters

[0104] An agent and / or composition of the present disclosure can be selected for its ability to decrease the level and / or activity of a promoter that propagates the misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo. In some embodiments, the agent and / or composition of the present disclosure decreases the level and / or activity of a promoter that propagates the misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo by at least 5%, atAtty. Docket: JHU-43272.601 least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to the level and / or activity of the promoter in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. Silencers

[0105] An agent and / or composition of the present disclosure can be selected for its ability to increase the level and / or activity of a silencer that represses the level and / or activity of a CRE that propagates the misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo. In some embodiments, the agent and / or composition increases the level and / or activity of the silencer in neurons and / or glial cells contacted in vitro, ex vivo or in vivo by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% compared to the level and / or activity of the silencer in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition.

[0106] In some embodiments, the agent and / or composition increases the level and / or activity of the silencer in neurons and / or glial cells contacted in vitro, ex vivo or in vivo by at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.2 fold, at least 2.4 fold, at least 2.6 fold, at least 2.8 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold compared to the level and / or activity of the silencer in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. An agent and / or composition of the present disclosure can be selected for its ability to decrease the level and / or activity of a silencer that represses the level and / or activity of a CRE that inhibits the propagation of the misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo. In some embodiments, the agent and / or composition of the present disclosure decreases the level and / or activity of the silencer that represses the level and / or activity of a CRE that inhibits the propagation of the misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,Atty. Docket: JHU-43272.601 at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to the level and / or activity of the silencer in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. Repressors

[0107] An agent and / or composition of the present disclosure can be selected for its ability to increase the level and / or activity of a repressor that represses the level and / or activity of a CRE that propagates the misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo. In some embodiments, the agent and / or composition increases the level and / or activity of the repressor in neurons and / or glial cells contacted in vitro, ex vivo or in vivo by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% compared to the level and / or activity of the repressor in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. In some embodiments, the agent and / or composition increases the level and / or activity of the repressor in neurons and / or glial cells contacted in vitro, ex vivo or in vivo by at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.2 fold, at least 2.4 fold, at least 2.6 fold, at least 2.8 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold compared to the level and / or activity of the repressor in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. An agent and / or composition of the present disclosure can be selected for its ability to decrease the level and / or activity of a repressor that represses the level and / or activity of a CRE that inhibits the propagation of the misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo. In some embodiments, the agent and / or composition of the present disclosure decreases the level and / or activity of the repressor that represses the level and / or activity of a CRE that inhibits the propagation of the misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%,Atty. Docket: JHU-43272.601 at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to the level and / or activity of the repressor in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. Insulators

[0108] An agents and / or composition of the present disclosure can be selected for its ability to increase the level and / or activity of an insulator that blocks a CRE (e.g., an enhancer) from propagating the misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo. In some embodiments, the agent and / or composition increases the level and / or activity of the insulator in neurons and / or glial cells contacted in vitro, ex vivo or in vivo by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100% compared to the level and / or activity of the insulator in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. In some embodiments, the agent and / or composition increases the level and / or activity of the insulator in neurons and / or glial cells contacted in vitro, ex vivo or in vivo by at least 1.1 fold, at least 1.2 fold, at least 1.3 fold, at least 1.4 fold, at least 1.5 fold, at least 1.6 fold, at least 1.7 fold, at least 1.8 fold, at least 1.9 fold, at least 2 fold, at least 2.2 fold, at least 2.4 fold, at least 2.6 fold, at least 2.8 fold, at least 3 fold, at least 3.5 fold, at least 4 fold, at least 4.5 fold, at least 5 fold, at least 7 fold, at least 10 fold, at least 15 fold, at least 20 fold, at least 25 fold, at least 30 fold, at least 40 fold, at least 50 fold, or at least 100 fold compared to the level and / or activity of the insulator in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. Enhancers

[0109] An agents and / or composition of the present disclosure can be selected for its ability to decrease the level and / or activity of an enhancer that propagates the misfolding and aggregation of proteins encoded by a synucleinopathy-associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo. In some embodiments, an agent and / or composition of the present disclosure decreases the level and / or activity of an enhancer that propagates the misfolding and aggregation of proteins encoded by a synucleinopathy- associated gene in neurons and / or glial cells in vitro, ex vivo or in vivo by at least 5%, at least 10%, at leastAtty. Docket: JHU-43272.601 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to the level and / or activity of the enhancer in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. In some embodiments, the CRE is an enhancer located in Human Chromosome 4 (Ch4) selected from the group consisting of the following nucleotide start and stop position in Ch4: Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975-90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557-90662154, Ch4: 90672325- 90672709, Ch4: 90678450-90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4: 90735341-90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, and / or Ch4: 90842771-90843114. It should be noted that the above Human Ch4 genomic coordinates are referenced from the Human CRCh37 / hg19 build. The present disclosure contemplates decreasing the level and / or activity of CREs that contain single nucleotide polymorphisms (“SNP”) and / or single nucleotide variants (“SNV”), for example, that are associated with the risk of development and / or progression of a synucleinopathy. The methods contemplate contacting neurons and / or glial cells in vitro, ex vivo, and / or in vivo with agents and / or compositions of the present disclosure that decrease the level and / or activity of a CRE that contains a SNP and / or SNV, associated with the disease or present in the diseased individual but absent from those not predisposed to the disease. The agent and / or composition may be designed to decrease the level and / or activity of the CRE by modulation of the CRE itself and / or manipulation of the sequence of the CRE (e.g., correction of the SNP and / or SNV, e.g., base and / or genomic editing). In some embodiments, the least one SNP is in an enhancer. In some embodiments, the CRE (e.g., enhancer) has a SNP selected from the group consisting of rs2583959, rs2737024, rs2583959, rs2737024, rs356220, rs356182, rs737029, rs356225, rs356168 and combinations thereof. In some embodiments, a subject has at least one SNP in the CRE (e.g., enhancer) that propagates the misfolding and aggregation of alpha- synuclein in at least one neuron. In some embodiments, the SNP in the enhancer of the subject is selected from the group consisting of rs2583959, rs2737024, rs2583959, rs2737024 and combinations thereof. Synucleinopathy-associated genes

[0110] Aspects of the present disclosure relate to decreasing the transcription level of a synucleinopathy-associated gene in a neuron and / or glial cell in vitro, ex vivo, or in vivo, for example, by decreasing the level and / or activity of a CRE that propagates the misfolding and aggregation of the synucleinopathy-associated gene in the neuron and / or glial cell. The present disclosure contemplatesAtty. Docket: JHU-43272.601 decreasing the transcription level and / or activity of any synucleinopathy-associated gene that encodes a protein that is misfolded and aggregated in neurons and / or glial cells. In some embodiments, the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and CPLX1. Synucleinopathy- associated genes can be evaluated or quantitated in a variety of ways well known in the art. These methods include quantitative PCR (qPCR) for measuring gene expression levels, RNA sequencing (RNA-seq) for a comprehensive analysis of gene expression profiles, and microarray analysis. An agent and / or composition of the present disclosure can be selected for its ability to decrease the transcription level and / or activity of PRKN, UCHL1, PARK7, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1 and / or an enhancer that increases the transcription level of PRKN, UCHL1, PARK7, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1 in neurons and / or glial cells in vitro, ex vivo or in vivo, thereby propagating the misfolding and aggregation of proteins encoded by PRKN, UCHL1, PARK7, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1 in the neurons and / or glial cells. In some embodiments, an agent and / or composition of the present disclosure decreases the transcription level and / or activity of PRKN, UCHL1, PARK7, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1 and / or the enhancer that increases the transcription level of PRKN, UCHL1, PARK7, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1 in the neurons and / or glial cells in vitro, ex vivo or in vivo by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to the transcription level and / or activity of SNCA and / or the enhancer in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. In some embodiments, the synucleinopathy associated gene is SNCA. An agent and / or composition of the present disclosure can be selected for its ability to decrease the transcription level and / or activity of SNCA and / or an enhancer that increases the transcription level of SNCA in neurons and / or glial cells in vitro, ex vivo or in vivo, thereby propagating the misfolding and aggregation of alpha-synuclein protein in the neurons and / or glial cells. In a preferred embodiment, the present disclosure provides aAtty. Docket: JHU-43272.601 method of preventing, or delaying the progression of, death of a neuron (e.g., a substantia nigra neuron, e.g., dopaminergic) and / or microgliosis and / or astrogliosis that contributes to the death of the neuron comprising contacting a neuron (e.g., a SN neuron, e.g., dopaminergic) and / or a glial cell with an agent and / or composition that decreases the level and / or activity of an enhancer that otherwise by its role in modulating the transcription of SNCA propagates the misfolding and aggregation of alpha-synuclein protein in the neuron, thereby reducing the transcription levels of SNCA and concomitant propagation of misfolded and aggregated alpha-synuclein protein in the neuron and / or glial cells, thereby preventing, or delaying the progression of, death of the neuron and / or microgliosis and / or astrogliosis that contribute to the death of the neuron. In a preferred embodiment, the present disclosure provides a method of screening for an agent and / or composition which prevents, or delays the progression of, death of a neuron (e.g., a SN neuron, e.g., dopaminergic) and / or microgliosis and / or astrogliosis that contributes to the death of the neuron, comprising (a) contacting a neuron (e.g., a SN neuron, e.g., dopaminergic) and / or glial cell with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of an enhancer that otherwise by its role in modulating the transcription of SNCA propagates the misfolding and aggregation of alpha-synuclein protein in the neuron and / or glial cell; and (b) identifying the candidate agent and / or composition as an agent and / or composition which prevents, or delays the progression of, death of the neuron (e.g., a SN neuron, e.g., dopaminergic) and / or microgliosis and / or astrogliosis that contributes to the death of the neuron, if the candidate agent and / or composition decreases the transcription level and / or activity of the SNCA in the neuron and / or glial cell compared to the transcription level and / or activity of SNCA in a neuron and / or glial cell that has not been contacted with the candidate agent and / or composition. In a preferred embodiment, the present disclosure provides a method of screening for an agent and / or composition which prevents, or delays the progression of, death of a neuron (e.g., a SN neuron, e.g., dopaminergic), comprising (a) contacting a neuron (e e.g., a SN neuron, e.g., dopaminergic) with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of an enhancer that otherwise by its role in modulating the transcription of SNCA propagates the misfolding and aggregation of alpha-synuclein protein in the neuron; and (b) identifying the candidate agent and / or composition as an agent and / or composition which prevents, or delays the progression of, death of the neuron (e.g., a SN neuron, e.g., dopaminergic), if the candidate agent and / or composition decreases the transcription level of SNCA in the neuron compared to the transcription level of SNCA in a neuron that has not been contacted with the candidate agent and / or composition. In a preferred embodiment, the present disclosure provides a method of screening for an agent and / or composition which prevents, or delays the progression of, microgliosis and / or astrogliosis that contributes to the death of a neuron (e.g., dopaminergic neuron), comprising (a) contacting a neuron (e.g., a SN neuron, e.g., dopaminergic) and / or glial cell with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of an enhancerAtty. Docket: JHU-43272.601 that otherwise by its role in modulating the transcription of SNCA propagates the misfolding and aggregation of alpha-synuclein protein in the neuron and / or glial cell; and (b) identifying the candidate agent and / or composition as an agent and / or composition which prevents, or delays the progression of, microgliosis and / or astrogliosis that contributes to death of the neuron (e.g., a SN neuron, e.g., dopaminergic), if the candidate agent and / or composition decreases the transcription level of SNCA in the neuron and / or glial cell compared to the transcription level of SNCA in a neuron and / or glial cell that has not been contacted with the candidate agent and / or composition. In a preferred embodiment, the present disclosure provides a method of screening for an agent and / or composition which decreases the level and / or activity of an enhancer that otherwise by its role in modulating the transcription of SNCA propagates the misfolding and aggregation of alpha-synuclein protein in a neuron and / or glial cell, comprising: (a) contacting a neuron and / or glial cell with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of the enhancer; and (b) identifying the candidate agent and / or composition as the agent and / or composition that decreases the level and / or activity of the enhancer, if the candidate agent and / or composition decreases the transcription level of SNCA in the neuron and / or glial cell compared to the transcription level of SNCA in a neuron and / or glial cell that has not been contacted with the candidate agent and / or composition. In some embodiments, the agent and / or composition of the present disclosure decreases the transcription level and / or activity of SNCA and / or the enhancer that increases the transcription level of SNCA in the neurons and / or glial cells in vitro, ex vivo or in vivo by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to the transcription level and / or activity of SNCA and / or the enhancer in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition.

[0111] In some embodiments, the synucleinopathy associated gene is LRRK2. An agent and / or composition of the present disclosure can be selected for its ability to decrease the transcription level and / or activity of LRRK2 and / or an enhancer that increases the transcription level of LRRK2 in neurons and / or glial cells in vitro, ex vivo or in vivo, thereby propagating the misfolding and aggregation of LRRK2 protein in the neurons and / or glial cells. In a preferred embodiment, the present disclosure provides a method of preventing, or delaying the progression of, death of a neuron (e.g., a SN neuron, e.g., dopaminergic) and / or microgliosis and / or astrogliosis that contributes to the death of the neuron comprising contacting a neuron (e e.g., a SN neuron, e.g., dopaminergic) and / or a glial cell with an agent and / or composition that decreases the level and / or activity of an enhancer that otherwise by its role in modulating the transcription of LRRK2Atty. Docket: JHU-43272.601 propagates the misfolding and aggregation of LRRK2 protein in the neuron, thereby reducing the transcription levels of LRRK2 and concomitant propagation of misfolded and aggregated LRRK2 protein in the neuron and / or glial cells, thereby preventing, or delaying the progression of, death of the neuron and / or microgliosis and / or astrogliosis that contribute to the death of the neuron. In a preferred embodiment, the present disclosure provides a method of screening for an agent and / or composition which prevents, or delays the progression of, death of a neuron (e.g., a SN neuron, e.g., dopaminergic) and / or microgliosis and / or astrogliosis that contributes to the death of the neuron, comprising (a) contacting a neuron (e.g., a SN neuron, e.g., dopaminergic) and / or glial cell with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of an enhancer that otherwise by its role in modulating the transcription of LRRK2 propagates the misfolding and aggregation of LRRK2 protein in the neuron and / or glial cell; and (b) identifying the candidate agent and / or composition as an agent and / or composition which prevents, or delays the progression of, death of the neuron (e.g., a SN neuron, e.g., dopaminergic) and / or microgliosis and / or astrogliosis that contributes to the death of the neuron, if the candidate agent and / or composition decreases the transcription level and / or activity of LRRK2 in the neuron and / or glial cell compared to the transcription level and / or activity of LRRK2 in a neuron and / or glial cell that has not been contacted with the candidate agent and / or composition. In a preferred embodiment, the present disclosure provides a method of screening for an agent and / or composition which prevents, or delays the progression of, death of a neuron (e.g., a SN neuron, e.g., dopaminergic), comprising (a) contacting a neuron (e.g., a SN neuron, e.g., dopaminergic) with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of an enhancer that otherwise by its role in modulating the transcription of LRRK2 propagates the misfolding and aggregation of LRRK2 protein in the neuron; and (b) identifying the candidate agent and / or composition as an agent and / or composition which prevents, or delays the progression of, death of the neuron (e.g., a SN neuron, e.g., dopaminergic), if the candidate agent and / or composition decreases the transcription level of LRRK2 in the neuron compared to the transcription level of LRRK2 in a neuron that has not been contacted with the candidate agent and / or composition. In a preferred embodiment, the present disclosure provides a method of screening for an agent and / or composition which prevents, or delays the progression of, microgliosis and / or astrogliosis that contributes to the death of a neuron (e e.g., a SN neuron, e.g., dopaminergic), comprising (a) contacting a neuron (e.g., a SN neuron, e.g., dopaminergic) and / or glial cell with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of an enhancer that otherwise by its role in modulating the transcription of LRRK2 propagates the misfolding and aggregation of LRRK2 protein in the neuron and / or glial cell; and (b) identifying the candidate agent and / or composition as an agent and / or composition which prevents, or delays the progression of, microgliosis and / or astrogliosis that contributes to death of the neuron (e.g., a SN neuron, e.g., dopaminergic), if the candidate agent and / or composition decreases theAtty. Docket: JHU-43272.601 transcription level of LRRK2 in the neuron and / or glial cell compared to the transcription level of LRRK2 in a neuron and / or glial cell that has not been contacted with the candidate agent and / or composition. In a preferred embodiment, the present disclosure provides a method of screening for an agent and / or composition which decreases the level and / or activity of an enhancer that otherwise by its role in modulating the transcription of LRRK2 propagates the misfolding and aggregation of LRRK2 protein in a neuron and / or glial cell, comprising: (a) contacting a neuron and / or glial cell with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of the enhancer; and (b) identifying the candidate agent and / or composition as the agent and / or composition that decreases the level and / or activity of the enhancer, if the candidate agent and / or composition decreases the transcription level of LRRK2 in the neuron and / or glial cell compared to the transcription level of LRRK2 in a neuron and / or glial cell that has not been contacted with the candidate agent and / or composition. In some embodiments, the agent and / or composition of the present disclosure decreases the transcription level and / or activity of LRRK2 and / or the enhancer that increases the transcription level of LRRK2 in the neurons and / or glial cells in vitro, ex vivo or in vivo by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to the transcription level and / or activity of LRRK2 and / or the enhancer in the neurons and / or glial cells prior to contact with, or not contacted with, the agent and / or composition. The methods of identifying and screening for agents and / or compositions that prevent, or delay the progression of, death of a neuron, decrease the level and / or activity of a CRE (e.g., enhancer), decrease the level of misfolded and aggregated protein in neurons and / or glial cells, or decrease microgliosis and / or astrogliosis that contribute to death of neurons can be performed in vitro, ex vivo, or in vivo. In some embodiments, the screening / identifying method is performed in vitro or ex vivo and the neuron and / or glial cell is obtained from a subject that has been diagnosed with and / or is at risk of developing a synucleinopathy. In some embodiments, the screening / identifying method is performed in vitro or ex vivo and the neuron and / or glial cell is obtained from a subject that is displaying a cognitive deficit. In some embodiments, the screening / identifying method is performed in vitro or ex vivo and the neuron and / or glial cell is obtained from a subject presenting with a cognitive deficit selected from the group consisting of memory loss, memory decline, attention deficits, executive function impairment, language and communication deficits, visuospatial deficits, processing speed deficits, learning difficulties, reasoning and judgement deficits, and / or social cognition deficits. In some embodiments, the screening / identifying method is performed in vitro or ex vivo and the neuron and / or glial cell is obtained from a subject that is displaying a motor deficit. In some embodiments, the screening / identifying methodAtty. Docket: JHU-43272.601 is performed in vitro or ex vivo and the neuron and / or glial cell is obtained from a subject having a motor deficit selected from the group consisting of tremors, bradykinesia, rigidity, and / or postural instability. In some embodiments, the screening / identifying method is performed in vitro or ex vivo and the neuron and / or glial cell is obtained from a subject that has a synucleinopathy-associated gene selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1. In some embodiments, the screening / identifying method is performed in vitro or ex vivo and the neuron and / or glial cell is obtained from a subject that has at least one SNP or SNV in the regulatory element that propagates the misfolding and aggregation of the protein in the neuron. In some embodiments, the screening / identifying method is performed in vitro or ex vivo and the neuron and / or glial cell is obtained from a subject who has at least one SNP in an enhancer selected from the group consisting of rs2583959, rs2737024, rs2583959, rs2737024, rs356220, rs356182, rs737029, rs356225, rs356168 and combinations thereof. Proteins encoded by synucleinopathy-associated genes that are misfolded and aggregated

[0112] Aspects of the present disclosure relate to preventing, or delaying the progression of, death of a neuron and treatment, prevention, and / or delaying the progression of, a synucleinopathy (e.g., PD) in subjects, for example, by decreasing the level of propagation of misfolded and aggregated proteins encoded by a synucleinopathy-associated gene, for example, using an agent and / or composition of the present disclosure. The present disclosure contemplates decreasing the levels of any misfolded and aggregated proteins in neurons and / or glial cells in vitro, ex vivo or in vivo. In some embodiments, the misfolded and aggregated protein in neurons and / or glial cells is selected from the group consisting of PRKN, UCHL1, PARK7, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, CPLX1, and combinations thereof. In some embodiments, the misfolded and aggregated protein is alpha-synuclein. An agent and / or composition of the present disclosure can be selected for its ability to reduce the misfolding and aggregation of alpha-synuclein protein in neurons and / or glial cells in vitro, ex vivo or in vivo. In some embodiments, the agent and / or composition decreases the misfolding and aggregation of alpha-synuclein protein in a neuron and / or glial cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a neuron and / or glial cell prior to contact with, or not contacted with, the agent and / or composition.Atty. Docket: JHU-43272.601

[0113] In some embodiments, the misfolded and aggregated protein is LRRK2. An agent and / or composition of the present disclosure can be selected for its ability to reduce the misfolding and aggregation of LRRK2 protein in neurons and / or glial cells in vitro, ex vivo or in vivo. In some embodiments, the agent and / or composition decreases the misfolding and aggregation of LRRK2 protein in a neuron and / or glial cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a neuron and / or glial cell prior to contact with, or not contacted with, the agent and / or composition. Protein levels of synucleinopathy-associated gene products can be assessed using immunoprecipitation, Western blot analysis (immunoblotting), enzyme-linked immunosorbent assay (ELISA), and quantitative protein assays. Additionally, immunohistochemistry and immunocytochemistry can be employed to visualize protein localization and distribution within tissues or cells. Fluorescence-activated cell sorting (FACS) can be used for cell-specific protein quantitation. Antibodies directed to these gene products can be identified and obtained from various sources, such as the MSRS catalog of antibodies (Aerie Corporation, Birmingham, Mich.), or can be prepared via conventional monoclonal or polyclonal antibody generation methods well known in the art. Treating, Preventing, or Delaying Progression of, a Synucleinopathy

[0114] The present disclosure contemplates methods of treating, preventing, or delaying the progression of, a synucleinopathy in a subject in need thereof. An agent and / or composition of the present disclosure can be selected for treating, preventing, or delaying the progression of, any synucleinopathy in a subject. In some embodiments, the synucleinopathy is PD, Gaucher's disease, Lewy body diffuse disease, dementia with Lewy bodies, a variant of Alzheimer's disease with Lewy bodies, sporadic Alzheimer's Disease, familial Alzheimer's Disease, true autonomic failure, dopa-responsive dystonia, or multiple system atrophy (MSA) also Shy-Drager syndrome. These synucleinopathies are associated with the abnormal accumulation of alpha-synuclein in neurons, disrupting cellular function and leading to neuronal damage, gliosis, and a progressive decline in motor and cognitive abilities. The treatment approach as described herein contemplates reducing the transcription of synucleinopathy-associated genes encoding proteins in the neuron and / or glial cells through the use of small molecules, immunotherapies, and / or gene therapies. By targeting the root cause of protein aggregation, these therapies aim to slow disease progression and protect neuronal integrity. In some embodiments, the synucleinopathy or PD is associated with activation of CNS resident innate immune cells. This activation is often mediated by abnormal proteins, such as aggregated alpha-synuclein. These abnormal proteins trigger an immune response in microglia andAtty. Docket: JHU-43272.601 astrocytes, the primary innate immune cells in the central nervous system. Activated microglia and astrocytes can contribute to neuroinflammation and neuronal damage, exacerbating the progression of the synucleinopathy. Understanding the role of these immune cells in response to protein aggregation is crucial for developing targeted therapies aimed at mitigating their harmful effects and slowing disease progression. In a preferred embodiment, the present disclosure provides a method of treating, preventing, or delaying the progression of, PD in a subject in need thereof, comprising administering to the subject an effective amount of an agent and / or composition that decreases the level and / or activity of an enhancer that propagates the misfolding and aggregation of alpha-synuclein protein in at least one neuron of the subject, thereby reducing transcription of SNCA in the at least one neuron of the subject and treating, preventing, or delaying the progression of, PD in the subject.

[0115] In a preferred embodiment, the present disclosure provides a method of preventing, or delaying the progression of, microgliosis and / or astrogliosis in a subject diagnosed with or suspected of having PD, comprising administering to the subject an effective amount of an agent and / or composition that decreases the level and / or activity of an enhancer that propagates the misfolding and aggregation of alpha-synuclein protein in at least one neuron and / or glial cell of the subject, thereby reducing transcription of SNCA in the at least one neuron and / or glial cell of the subject and preventing, or delaying the progression of, microgliosis and / or astrogliosis in the subject. In some embodiments, the agent and / or composition reduces the level and / or activity of the enhancer at least one neuron of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline level and / or activity of the enhancer in at least one neuron of the subject prior to administration of the agent and / or composition. In some embodiments, the agent and / or composition reduces the transcription level of SNCA in at least one neuron of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline transcription level of SNCA in the subject prior to administration of the agent and / or composition. In some embodiments, the agent and / or composition reduces the level of misfolded and aggregated alpha-synuclein protein in at least one neuron of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at leastAtty. Docket: JHU-43272.601 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline level of misfolded and aggregated alpha-synuclein protein in at least one neuron of the subject prior to administration of the agent and / or composition. In some embodiments, the agent and / or composition reduces the transcription level of SNCA in at least one glial cell of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline transcription level of SNCA in at least one glial cell of the subject prior to administration of the agent and / or composition. In some embodiments, the agent and / or composition reduces the level of misfolded and aggregated alpha-synuclein protein in at least one glial cell of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline level of misfolded and aggregated alpha-synuclein protein in at least one glial cell of the subject prior to administration of the agent and / or composition. In some embodiments, the agent and / or composition reduces the level of microgliosis and / or astrogliosis in at least one glial cell the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline level of microgliosis and / or astrogliosis in at least one glial cell of the subject prior to administration of the agent and / or composition.

[0116] In a preferred embodiment, the present disclosure provides a method of treating, preventing, or delaying the progression of, PD in a subject in need thereof, comprising administering to the subject an effective amount of an agent and / or composition that decreases the level and / or activity of an enhancer that propagates the misfolding and aggregation of LRRK2 protein in at least one neuron of the subject, thereby reducing transcription of LRRK2 in the at least one neuron of the subject and treating, preventing, or delaying the progression of, PD in the subject. In a preferred embodiment, the present disclosure provides a method of preventing, or delaying the progression of, microgliosis and / or astrogliosisAtty. Docket: JHU-43272.601 in a subject diagnosed with or suspected of having PD, comprising administering to the subject an effective amount of an agent and / or composition that decreases the level and / or activity of an enhancer that propagates the misfolding and aggregation of LRRK2 protein in at least one neuron and / or glial cell of the subject, thereby reducing transcription of LRRK2 in the at least one neuron and / or glial cell of the subject and preventing, or delaying the progression of, microgliosis and / or astrogliosis in the subject. In some embodiments, the agent and / or composition reduces the level and / or activity of the enhancer at least one neuron of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline level and / or activity of the enhancer in at least one neuron of the subject prior to administration of the agent and / or composition. In some embodiments, the agent and / or composition reduces the transcription level of LRRK2 in at least one neuron of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline transcription level of LRRK2 in the subject prior to administration of the agent and / or composition. In some embodiments, the agent and / or composition reduces the level of misfolded and aggregated LRRK2 protein in at least one neuron of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline level of misfolded and aggregated LRRK2 protein in at least one neuron of the subject prior to administration of the agent and / or composition. In some embodiments, the agent and / or composition reduces the transcription level of LRRK2 in at least one glial cell of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline transcription level of LRRK2 in at least one glial cell of the subject prior to administration of the agent and / or composition. In some embodiments, the agent and / orAtty. Docket: JHU-43272.601 composition reduces the level of misfolded and aggregated LRRK2 protein in at least one glial cell of the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline level of misfolded and aggregated LRRK2 protein in at least one glial cell of the subject prior to administration of the agent and / or composition. In some embodiments, the agent and / or composition reduces the level of microgliosis and / or astrogliosis in at least one glial cell the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, and / or at least 100% compared to a baseline level of microgliosis and / or astrogliosis in at least one glial cell of the subject prior to administration of the agent and / or composition.

[0117] In some embodiments, the PD is familial PD. In some embodiments, the PD presents with dementia. In some embodiments, the PD is combined with Alzheimer's disease. In some embodiments, the PD is sporadic PD. In some embodiments, the PD is idiopathic PD. In some embodiments, the clinical presentation may be defined as Parkinsonian. In some embodiments, the method comprises contacting a microglia and / or astrocyte with the agent and / or composition. In some embodiments, the method comprises contacting a neuron with the agent and / or composition.

[0118] In some embodiments, the agent and / or composition is administered to a subject who presents with a Parkinsonian disorder or symptom. In some embodiments, the agent and / or composition is administered to a subject who has underwent, or is currently undergoing, treatment with a conventional therapeutic agent for treating the synucleinopathy. In some embodiments, the method comprises administering a conventional therapeutic agent for treating PD. Exemplary PD therapeutics include, without limitation, any treatment used to directly treat or reduce the symptoms of Parkinson’s disease. Treatments used to directly treat or reduce the symptoms of PD include, without limitation, MAO-B inhibitors, dopamine agonists, catechol-O- methyltransferase (COMT) inhibitors, adenosine A2A antagonists such as istradefylline; aromatic L-amino acid decarboxylase inhibitor (AAADI or DDCI), anticholinergic drugs, Amantadine, Levodopa, a gene therapy directed to AADC, tyrosine decarboxylase inhibitors, or any combination thereof. In some embodiments, the PD therapeutic is levodopa and carbidopa. In some embodiments, the PD therapeutic is levodopa, carbidopa, and an additional intervention disclosed herein. When MAO-B inhibitors are used as a treatment, any MAO-B inhibitor deemed appropriate may be used.Atty. Docket: JHU-43272.601 MAO-B inhibitors that find use in the present disclosure include, without limitation, selegiline, selegiline HCL, rasagiline, safinamide, etc. When dopamine agonists are used as a treatment, any dopamine agonist deemed necessary may be used. Dopamine agonists that find use in the present disclosure include, without limitation, pramipexole, pramipexole dihydrochloride, ropinirole, apomorphine hydrochloride, rotigotine, etc. Non-limiting examples of COMT inhibitors that may be used in the treatment of PD include entacapone, nebicapone, nitecapone, opicapone, and tolcapone. When a AAADI or DDCI is used in the treatment of PD, any AAADI or DDCI may be administered. AAADI or DDCI that find use in the present disclosure include without limitation, benserazide, carbidopa, methyldopa, alpha-Difluoromethyl-DOPA, 3’4’,5,7- Tetrahydroxy-8- methoxyisoflavone, epigallocatechin gallate, epigallocatechin, etc. Non-limiting examples of anticholinergic drugs that find use in the present disclosure include benztropine and trihexyphenidyl HCL. When a gene therapy directed to AADC is used as a treatment, any gene therapy deemed useful may be used. Gene therapies directed to AADC have been described, for example, in Pearson et al. (Nat Commun. 2021 Jul 12;12(1):4251) , Nutt et al. (Mov Disord. 2020 May;35(5):851-858), Hwu et al. (Sci Transl Med.2012 May 16;4(134):134ra61), Kojima et al. (Brain.2019 Feb 1;142(2):322-333), Hwu et al. (Eladocagene Exuparvovec Gene Therapy Improves Motor Development in Patients With Aromatic L- Amino Acid Decarboxylase Deficiency (S39.006), Neurology May 2022, 98 (18 Supplement) 2034), Christine, C.W. et al. Neurology, 2022 Jan 4;98(1):e40-e50, and each specifically incorporated by reference herein. In some embodiments, the gene therapy directed to AADC is VY-AADC01. When tyrosine decarboxylase inhibitors as a treatment, any tyrosine decarboxylase inhibitor may be used that reduces or inhibits the conversion of levodopa to dopamine by a tyrosine decarboxylase enzyme. Non-limiting tyrosine decarboxylase inhibitors include a tyrosine decarboxylase from Enterococcus faecalis and alpha- fluoromethyltyrosine (AFMT). Tyrosine decarboxylase inhibitors are known in the art and have been described in, for example, U.S. Patent publication number US20230181493A1 and International Patent application number PCT / US2019 / 064896 each of which are specifically incorporated by reference herein. Identifying agents and / or compositions useful for treating, preventing, or delaying the progression of a synucleinopathy

[0119] The present disclosure contemplates methods of identifying agents and / or compositions useful for treating, preventing, or delaying the progression of, a synucleinopathy in a subject in need thereof. In a preferred embodiment, the present disclosure provides a method of identifying an agent and / or composition that treats, prevents, or delays the progression of, a synucleinopathy in a subject, comprising (a) administering to the subject a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of an enhancer that propagates the misfolding and aggregation of alpha-synuclein protein in at least one neuron of the subject, and (b) identifying the candidate agent and / or composition asAtty. Docket: JHU-43272.601 the agent and / or composition that treats, prevents, or delays the progression of, the synucleinopathy, if the candidate agent and / or composition decreases the transcription level of SNCA in at least one neuron of the subject compared to the transcription level of SNCA in at least one neuron in a subject who has not been administered the candidate agent and / or composition. In a preferred embodiment, the present disclosure provides a method of identifying an agent and / or composition that decreases the level and / or activity of an enhancer that propagates the misfolding and aggregation of alpha-synuclein protein in at least one neuron and / or glial cell of a subject comprising (a) administering to the subject a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of the enhancer in the at least one neuron of the subject, and (b) identifying the candidate agent and / or composition as the agent and / or composition that decreases the level and / or activity of the enhancer, if the candidate agent and / or composition decreases the transcription level of SNCA in at least one neuron of the subject compared to the transcription level of SNCA in at least one neuron in a subject who has not been administered the candidate agent and / or composition. In a preferred embodiment, the present disclosure provides a method of identifying an agent and / or composition that decreases the misfolding and aggregation of alpha-synuclein protein in at least one neuron and / or glial cell of a subject comprising (a) administering to the subject a candidate agent and / or composition to evaluate its ability to decrease misfolding and aggregation of alpha- synuclein protein in at least one neuron of the subject, and (b) identifying the candidate agent and / or composition as the agent and / or composition that decreases the misfolding and aggregation of alpha- synuclein protein in the at least one neuron, if the candidate agent and / or composition decreases the transcription level of SNCA in at least one neuron of the subject compared to the transcription level of SNCA in at least one neuron in a subject who has not been administered the candidate agent and / or composition. In some embodiments, the candidate agent and / or composition decreases the transcription level of SNCA in at least one neuron of the subject compared to the transcription level of SNCA in at least one neuron in a subject who has not been administered the candidate agent and / or composition by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the candidate agent and / or composition decreases the level of misfolding and aggregation of alpha-synuclein protein at least one neuron of the subject compared to the level of misfolding and aggregation of alpha-synuclein protein in at least one neuron in a subject who has not been administered the candidate agent and / or composition by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, atAtty. Docket: JHU-43272.601 least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. An agent and / or composition of the present disclosure can also be selected for its ability to reduces the risk of the development and / or progression of the synucleinopathy. In some embodiments, the candidate agent and / or composition reduces the risk of the development and / or progression of the synucleinopathy in a subject compared to risk in a subject who has not been administered the candidate agent and / or composition by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. An agent and / or composition of the present disclosure can be selected for its ability to reduce the risk of the development and / or progression of microgliosis in the subject. In some embodiments, the candidate agent and / or composition reduces the risk of the development and / or progression of microgliosis in the subject compared to the risk in a subject who has not been administered the candidate agent and / or composition by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. An agent and / or composition of the present disclosure can be selected for its ability to reduce the risk of the development and / or progression of astrogliosis in the subject. In some embodiments, the candidate agent and / or composition reduces the risk of the development and / or progression of astrogliosis in the subject compared to the risk in a subject who has not been administered the candidate agent and / or composition by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. An agent and / or composition of the present disclosure can be selected for its ability to reduce the risk of the development and / or progression of Parkinson’s disease. The Parkinson’s disease may be familial Parkinson’s disease, Parkinson’s disease with dementia, Parkinson’s disease combined with Alzheimer's disease, sporadic Parkinson’s disease, idiopathic Parkinson’s disease. In some embodiments, the subject has a Parkinsonian disorder. In some embodiments, the candidate agent and / or composition reduces the risk of the development and / or progression of Parkinson’s disease in the subject compared to the risk in a subject who has not been administered the candidate agent and / orAtty. Docket: JHU-43272.601 composition by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. An agent and / or composition of the present disclosure can be selected for its ability to reduce the risk of the development and / or progression of a motor deficit in the subject. The motor deficit can be selected from the group consisting of tremors, bradykinesia, rigidity, and / or postural instability. In some embodiments, the candidate agent and / or composition reduces the risk of the development and / or progression of a motor deficit in the subject compared to the risk in a subject who has not been administered the candidate agent and / or composition by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. An agent and / or composition of the present disclosure can be selected for its ability to reduce the risk of the development and / or progression of cognitive decline in the subject. The cognitive decline can be selected from the group consisting of memory loss, memory decline, attention deficits, executive function impairment, language and communication deficits, visuospatial deficits, processing speed deficits, learning difficulties, reasoning and judgement deficits, and / or social cognition deficits. In some embodiments, the candidate agent and / or composition reduces the risk of the development and / or progression of cognitive decline in the subject compared to the risk in a subject who has not been administered the candidate agent and / or composition by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. An agent and / or composition of the present disclosure can be selected for its ability to protect against Lewy body aggregation in the cortex of the subject. In some embodiments, the candidate agent and / or composition reduces the risk of Lewy body aggregation in the cortex of the subject compared to the risk in a subject who has not been administered the candidate agent and / or composition by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%,Atty. Docket: JHU-43272.601 at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. Subjects

[0120] The present disclosure contemplates methods of treating, preventing, or delaying the progression of, a synucleinopathy in a subject in need thereof. In some embodiments, the subject has been diagnosed with and / or is at risk of developing a synucleinopathy. In some embodiments, the subject has been diagnosed with and / or is at risk of developing Parkinson’s disease. In some embodiments the methods of the invention comprise the step of identifying individuals “at-risk” for development of, or in the “early- stages” of, PD. “At risk” for development of PD includes: (1) individuals who are at increased risk for development of PD, and (2) individuals exhibiting a “pre-clinical” disease state, but do not meet the diagnostic criteria for PD and thus are not formally considered to have PD, i.e. overt PD. Individuals “at increased risk” for development (also termed “at-risk” for development) of PD are individuals with a higher likelihood of developing PD or condition compared to the general population. Such individuals can be identified based on their exhibiting or possessing one or more of the following: the presence of certain physical findings, laboratory test results, imaging findings, or biomarker test results associated with development of PD; the presence of clinical signs related to PD; the presence of certain symptoms related to PD (although the individual is frequently asymptomatic); the presence of markers (also termed “biomarkers” or “PD-related polypeptides”) of PD; and other findings that indicate an individual has an increased likelihood over the course of their lifetime to develop PD. Individuals at increased risk for development of PD can be asymptomatic and are not experiencing any symptoms related to the disease that they are at an increased risk for developing. Included, without limitation, in the group of individuals at increased risk of developing PD, are individuals exhibiting “a pre-clinical disease state”. The pre-disease state may be diagnosed based on developing symptoms, physical findings, laboratory test results, imaging results, and other findings that result in the individual meeting the diagnostic criteria for the PD, and thus being formally diagnosed. Individuals with “pre-clinical disease” exhibit findings that suggest that the individual is in the process of developing PD, but do not exhibit findings, including the symptoms, clinical findings, laboratory findings, and / or imaging findings, etc. that are necessary to meet the diagnostic criteria for a formal diagnosis of overt PD. In some embodiments, these individuals have laboratory results, or physical findings, or symptoms, or imaging findings that place them at increased risk for development of PD. In some embodiments, individuals with preclinical disease states are asymptomatic. In some embodiments, individuals with pre-clinical disease states exhibit increased or decreased levels of the expression of certain genes, expression of certain proteins, metabolic markers, and other markers. In some embodiments the methods of the invention comprise the step of identifying and treating individuals atAtty. Docket: JHU-43272.601 increased risk for development of PD. These individuals at increased risk for development of PD can have risk factors for disease and / or be in a “pre-clinical” state as described herein and are sometimes asymptomatic. Prior to the individual being determined to have PD or at risk of developing PD, the individual may be screened for one or more PD related polypeptides to determine the level of the PD related polypeptide. Individuals who are screened may be suspected of having or developing PD. Individuals who are suspected of having or developing PD may have a familial history of PD. In some embodiments, the individual who is suspected to have or develop PD has a mutation in a gene associated with PD. Mutations in genes associated with PD include mutations in leucine- rich repeat kinase 2 (LRRK2), PRKN, alpha- synuclein (SNCA), PTEN-induced kinase 1 (PINK1), Vacuolar protein sorter-35 (VPS35), coiled-coil-helix- coiled-coil-helix domain containing 2 (CHCHD2) and prosaposin (PSAP), DJ-1, ATP13A2, GIGYF2, HTRA2, PLA2G6, FBXO7, EIF4G1, DNAJC6, SYNJ1, DNAJC13, glucocerebrosidase (GBA1), UCHL1, NURR1 nuclear receptor superfamily protein (NR4A2), synphilin-1 (SNCAIP), apolipoprotein E (APOE), and microtubule-associated protein tau (MAPT). Individuals who have a familial history of PD or have a mutation in a gene associated with PD may be particularly at risk for PD and may develop PD at an earlier age, e.g., juvenile parkinsonism or early-onset PD. Individuals who are at risk may be screened at a variety of ages including, without limitation, 10 years old, 11, 12, 13, 14, 15, 16, 17, 18, 1920, 25, 30, 35, 40, 45, 50, 55, 60 or greater than 60 years. In some embodiments, the screening involves quantifying one or more PD related polypeptides and determining if the individual has PD or has an increased likelihood of PD. In some embodiments, individuals that are at risk for developing PD are screened at multiple timepoints such that if at a first timepoint the individual is not determined to have PD or be at increased likelihood of developing PD then the individual may be screened at additional timepoints to determine if the individual has PD or is at increased likelihood of developing PD at the additional timepoints. Following screening, the individual may be provided with a timely intervention. As used herein “timely intervention” is intended to mean an intervention, e.g., a treatment such as those described above, that occurs during the early stages of disease, e.g., when there are minimal symptoms present. In some embodiments, the Parkinson’s disease is familial Parkinson’s disease. In some embodiments, the Parkinson’s disease presents with dementia. In some embodiments, the Parkinson’s disease is combined with Alzheimer’s disease. In some embodiments, the Parkinson’s disease is sporadic Parkinson’s disease. In some embodiments, the Parkinson’s disease is idiopathic Parkinson’s disease. In some embodiments, the subject presents with Parkinsonian symptom or disorder. In some embodiments, the subject is displaying a cognitive deficit. In some embodiments, the subject presenting with a cognitive deficit selected from the group consisting of memory loss, memory decline, attention deficits, executive function impairment, language and communication deficits, visuospatial deficits, processing speed deficits, learning difficulties, reasoning and judgement deficits, and / or social cognition deficits. In some embodiments, the subject that is displaying a motor deficit. InAtty. Docket: JHU-43272.601 some embodiments, the subject has a motor deficit selected from the group consisting of tremors, bradykinesia, rigidity, and / or postural instability. In some embodiments, the subject has a synucleinopathy- associated gene selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1. In some embodiments, the subject has at least one SNP or SNV in the CRE that propagates the misfolding and aggregation of the protein encoded by the synucleinopathy associated gene in a neuron and / or glial cell. In some embodiments, the subject has at least one SNP in an enhancer (e.g., SNCA enhancer) selected from the group consisting of rs2583959, rs2737024, rs2583959, rs2737024, rs356220, rs356182, rs737029, rs356225, rs356168 and combinations thereof. In some embodiments, the synucleinopathy-associated gene In some embodiments, the subject is a mammal. In some embodiments, the subject is a non-human mammal. In some embodiments, the subject is a mouse. In a preferred embodiment, the present disclosure provides a method of assessing and / or evaluating the subject’s clinical condition prior to, during and after the administering the candidate agent and / or composition comprising: (a) assessing the subject’s clinical condition prior to administering the candidate agent and / or composition; and (b) evaluating the subject’s clinical condition after administering the candidate agent and / or composition; wherein the assessment comprises measuring one or more clinical parameters. In some embodiments, the clinical condition prior to treatment is evaluated using the MDS- sponsored Revision of the Unified Parkinsion’s Disease Rating Scale (MDS-UPDRS). The MDS-UPDRS serves as a robust and widely adopted tool for evaluating both the severity and progression of Parkinson's disease and related synucleinopathies. This scale comprises multiple comprehensive parts that meticulously assess various facets of the condition, including motor function, non-motor experiences of daily living, and specific motor complications. It thoroughly evaluates symptoms such as tremors, bradykinesia, rigidity, and postural instability, offering a standardized framework for clinicians to gauge the extent of these manifestations. Moreover, the MDS-UPDRS includes assessments for cognitive impairments and mood disturbances, ensuring a comprehensive evaluation of the patient's overall health status. By establishing a baseline measurement of the subject's condition, healthcare providers can effectively track disease progression, monitor the response to treatment interventions, and adjust therapeutic strategies as needed to optimize patient care and outcomes. Additionally, the subject’s clinical condition is assessed at multiple time points after the administration of the candidate agent to monitor the agent’s treatment progress. The subjects and treatment approach described herein highlights the role of the MDS-UPDRS in evaluating the efficacy of the agent and / or composition on a subject in need thereof, by facilitating informed decision- making and personalized management plans tailored to the individual needs of patients with PD and synucleinopathies. Agents and / or CompositionsAtty. Docket: JHU-43272.601

[0121] The present disclosure contemplates methods comprising contacting a neuron with an agent / or composition. The present disclosure also contemplates administering an agent and / or composition of the present disclosure to subjects. The methods of the present disclosure can be performed in vitro, ex vivo, or in vivo and contemplate agents and / or compositions capable of (i) decreasing the level and / or activity of a CRE in neurons; (ii) decreasing the transcription levels of synucleinopathy-associated genes in neurons; (iii) decreasing the propagation of misfolding and aggregation of proteins encoded by synucleinopathy-associated genes in neurons; (iv) decreasing microgliosis and / or astrogliosis that contribute to death of neurons. In some embodiments, the agent and / or composition decreases the level and / or activity of a CRE in a neuron by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the agent and / or composition decreases the level and / or activity of a CRE in a glial cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the agent and / or composition decreases the transcription levels of a synucleinopathy-associated gene in a neuron by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the agent and / or composition decreases the transcription levels of synucleinopathy- associated gene in a glial cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the agent and / or composition decreases the transcription levels of synucleinopathy-associated genes in neurons in the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at leastAtty. Docket: JHU-43272.601 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the agent and / or composition decreases the level of misfolding and aggregation of alpha-synuclein protein in a neuron by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the agent and / or composition decreases the level of misfolding and aggregation of alpha-synuclein protein in a glial cell by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the agent and / or composition decreases the level and / or activity of a CRE in neurons in the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the agent and / or composition decreases the transcription levels of synucleinopathy- associated genes in neurons in the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%. In some embodiments, the agent and / or composition decreases the level of misfolding and aggregation of alpha- synuclein protein in at least one neuron in the subject by at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 33%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 66%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 100%.

[0122] Examples of agents and / or compositions that can be used for decreasing the level and / or activity of the CRE (e.g., enhancer, insulator, repressor, promotor, silencer, etc.) that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in a neuron and / orAtty. Docket: JHU-43272.601 glial cell, for example, in a subject, include, without limitation, CRISPR-Cas system guide RNA (gRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), microRNA (miRNA), an antisense DNA, an antisense RNA, a DNAzyme, a Ribozyme, an antagomir, a locked nucleid acid (LMA), a morpholino nucleic acid (MNA), and / or a chemical / small molecule.

[0123] Aspects of the present disclosure contemplate an siRNA agent targeted to a CRE that propagates the misfolding and aggregation of protein encoded by a synucleinopathy gene in a neuron and / or glial cell in vitro, ex vivo, or in vivo. In some embodiments, the siRNA agent is targeted to a enhancer that propagates the misfolding and aggregation of the protein. In some embodiments, the siRNA is targeted to a SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in neurons (e.g., SN dopaminergic neurons). In an embodiment, the agent and / or composition comprises a siRNA against an SNCA enhancer located at Human Ch4 (“HCh4”): 90578786-90579367. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90578838-90579505. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90629975-90630782. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90629981-90630880. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90636762- 90637480. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90642092-90642763. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90658878-90659419. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90661417-90661898. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90661557-90662154. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90672325-90672709. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90678450-90679064. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90720580- 90722663. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90720648-90722351. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90735341-90735761. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90756615-90759904. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90757134-90759904. In an embodiment, the agent and / or composition comprises siRNA against a SNCA enhancer located at HCh4: 90761441-90762196. In an embodiment, the agent and / or composition comprises a siRNA against a SNCA enhancer located at HCh4: 90842771-90843114. It should be noted that the above Human Ch4 genomic coordinates are referenced from the Human CRCh37 / hg19 build.InAtty. Docket: JHU-43272.601 some embodiments, the agent and / or composition comprises a siRNA against a SNCA promoter. In some embodiments, the agent and / or composition comprises a siRNA against a SNCA silencer. In some embodiments, the agent and / or composition comprises a siRNA against a SNCA insulator. In some embodiments, the agent and / or composition comprises a siRNA against a SNCA repressor.

[0124] Aspects of the present disclosure contemplate an miRNA agent targeted to a CRE that propagates the misfolding and aggregation of protein encoded by a synucleinopathy gene in a neuron and / or glial cell in vitro, ex vivo, or in vivo. In some embodiments, the miRNA agent is targeted to a enhancer that propagates the misfolding and aggregation of the protein. In some embodiments, the miRNA is targeted to a SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in neurons (e.g., dopaminergic neurons). In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at Human Ch4 (“HCh4”): 90578786-90579367. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90578838-90579505. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90629975-90630782. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90629981-90630880. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90636762-90637480. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90642092-90642763. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90658878-90659419. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90661417-90661898. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90661557-90662154. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90672325-90672709. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90678450-90679064. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90720580-90722663. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90720648-90722351. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90735341-90735761. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90756615-90759904. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90757134-90759904. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90761441-90762196. In an embodiment, the agent and / or composition comprises a miRNA targeted to a SNCA enhancer located at HCh4: 90842771-90843114. It should be noted that the above Human Ch4Atty. Docket: JHU-43272.601 genomic coordinates are referenced from the Human CRCh37 / hg19 build. In some embodiments, the agent and / or composition comprises a miRNA against a SNCA promoter. In some embodiments, the agent and / or composition comprises a miRNA against a SNCA silencer. In some embodiments, the agent and / or composition comprises a miRNA against a SNCA insulator. In some embodiments, the agent and / or composition comprises a miRNA against a SNCA repressor.

[0125] Aspects of the present disclosure contemplate an shRNA agent targeted to a CRE that propagates the misfolding and aggregation of protein encoded by a synucleinopathy gene in a neuron and / or glial cell in vitro, ex vivo, or in vivo. In some embodiments, the shRNA agent is targeted to a enhancer that propagates the misfolding and aggregation of the protein. In some embodiments, the shRNA is targeted to a SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in neurons (e.g., dopaminergic neurons). In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at Human Ch4 (“HCh4”): 90578786-90579367. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90578838-90579505. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90629975-90630782. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90629981-90630880. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90636762-90637480. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90642092-90642763. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90658878-90659419. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90661417-90661898. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90661557-90662154. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90672325-90672709. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90678450-90679064. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90720580-90722663. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90720648-90722351. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90735341-90735761. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90756615-90759904. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90757134-90759904. In an embodiment, the agent and / or composition comprises a shRNA targeted to a SNCA enhancer located at HCh4: 90761441-90762196. In an embodiment, the agent and / or composition comprises a shRNA targetedAtty. Docket: JHU-43272.601 to a SNCA enhancer located at HCh4: 90842771-90843114. It should be noted that the above Human Ch4 genomic coordinates are referenced from the Human CRCh37 / hg19 build. In some embodiments, the agent and / or composition comprises a shRNA against a SNCA promoter. In some embodiments, the agent and / or composition comprises a shRNA against a SNCA silencer. In some embodiments, the agent and / or composition comprises a shRNA against a SNCA insulator. In some embodiments, the agent and / or composition comprises a shRNA against a SNCA repressor.

[0126] Aspects of the present disclosure contemplate an antisense oligonucleotide (e.g., antisense DNA and / or antisense RNA) targeted to a CRE that propagates the misfolding and aggregation of protein encoded by a synucleinopathy gene in a neuron and / or glial cell in vitro, ex vivo, or in vivo. In some embodiments, the antisense oligonucleotide is targeted to a enhancer that propagates the misfolding and aggregation of the protein. In some embodiments, the antisense oligonucleotide is targeted to a SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in neurons (e.g., dopaminergic neurons). In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at Human Ch4 (“HCh4”): 90578786- 90579367. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90578838-90579505. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90629975-90630782. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90629981-90630880. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90636762-90637480. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90642092-90642763. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90658878-90659419. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90661417-90661898. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90661557-90662154. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90672325-90672709. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90678450-90679064. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90720580-90722663. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90720648-90722351. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90735341-90735761. In an embodiment,Atty. Docket: JHU-43272.601 the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90756615-90759904. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90757134-90759904. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90761441-90762196. In an embodiment, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA enhancer located at HCh4: 90842771-90843114. It should be noted that the above Human Ch4 genomic coordinates are referenced from the Human CRCh37 / hg19 build. In some embodiments, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA promoter. In some embodiments, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA silencer. In some embodiments, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA insulator. In some embodiments, the agent and / or composition comprises an antisense oligonucleotide targeted to a SNCA repressor.

[0127] Aspects of the present disclosure contemplate a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a CRE that propagates the misfolding and aggregation of protein encoded by a synucleinopathy gene in a neuron and / or glial cell in vitro, ex vivo, or in vivo. In some embodiments, the ribozyme, DNAzyme, antagomir, LMA, and / or MNA is targeted to a enhancer that propagates the misfolding and aggregation of the protein. In some embodiments, the ribozyme, DNAzyme, antagomir, LMA, and / or MNA is targeted to a SNCA enhancer that propagates the misfolding and aggregation of alpha- synuclein protein encoded by SNCA in neurons (e.g., dopaminergic neurons). In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at Human Ch4 (“HCh4”): 90578786-90579367. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90578838-90579505. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90629975-90630782. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90629981- 90630880. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90636762-90637480. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90642092-90642763. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90658878-90659419. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90661417-90661898. In an embodiment, the agent and / or composition comprises a ribozyme, aAtty. Docket: JHU-43272.601 DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90661557- 90662154. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90672325-90672709. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90678450-90679064. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90720580-90722663. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90720648-90722351. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90735341- 90735761. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90756615-90759904. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90757134-90759904. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90761441-90762196. It should be noted that the above Human Ch4 genomic coordinates are referenced from the Human CRCh37 / hg19 build. In an embodiment, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA enhancer located at HCh4: 90842771-90843114. In some embodiments, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA promoter. In some embodiments, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA silencer. In some embodiments, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA insulator. In some embodiments, the agent and / or composition comprises a ribozyme, a DNAzyme, antagomir, a LMA, and / or a MNA targeted to a SNCA repressor.

[0128] Aspects of the present disclosure contemplate an antibody or functional antibody fragment targeted to a CRE that propagates the misfolding and aggregation of protein encoded by a synucleinopathy gene in a neuron and / or glial cell in vitro, ex vivo, or in vivo. In some embodiments, the antibody or functional antibody fragment is targeted to a enhancer that propagates the misfolding and aggregation of the protein. In some embodiments, the antibody or functional antibody fragment is targeted to a SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in neurons (e.g., dopaminergic neurons). In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at Human Ch4 (“HCh4”): 90578786-90579367. In an embodiment, the agent and / or composition comprises an antibody or functionalAtty. Docket: JHU-43272.601 antibody fragment targeted to a SNCA enhancer located at HCh4: 90578838-90579505. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90629975-90630782. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90629981-90630880. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90636762-90637480. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90642092-90642763. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90658878-90659419. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90661417-90661898. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90661557-90662154. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90672325-90672709. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90678450-90679064. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment e targeted to a SNCA enhancer located at HCh4: 90720580-90722663. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90720648-90722351. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90735341-90735761. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90756615-90759904. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90757134-90759904. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90761441-90762196. In an embodiment, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA enhancer located at HCh4: 90842771-90843114. It should be noted that the above Human Ch4 genomic coordinates are referenced from the Human CRCh37 / hg19 build. In some embodiments, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA promoter. In some embodiments, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA silencer. In some embodiments, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA insulator. In some embodiments, the agent and / or composition comprises an antibody or functional antibody fragment targeted to a SNCA repressor.Atty. Docket: JHU-43272.601

[0129] In some embodiments, the method utilizes a gene editing system. The present disclosure provides strategies and techniques for the targeted, specific alteration of the genetic information (genome) in neurons and / or glial cells, for example, of a subject (e.g., a human, mouse, rodent, or other model of human disease). In the context of treating genetic disorders, alterations may include, but are not limited to, insertion, deletion, and correction. Depending on the nature of the mutation, correction may be achieved via various strategies disclosed herein. In one non-limiting example, a missense mutation may be corrected by replacing the region containing the mutation with its wild-type counterpart. As another example, duplication mutations (e.g., repeat expansions) in a gene may be corrected by removing the extra sequences. In an embodiment, the disclosure provides a method of preventing, or delaying the progression of, death of a neuron and / or microgliosis and / or astrogliosis that contributes to the death of the neuron comprising contacting a neuron and / or a glial cell with a gene editing system that decreases the level and / or activity of a cis-regulatory element (CRE) (e.g., enhancer, promoter, etc.) that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in the neuron and / or glial cell, thereby reducing transcription of the synucleinopathy-associated gene encoding the protein in the neuron and / or glial cell and preventing, or delaying the progression of, death of the neuron and / or microgliosis and / or astrogliosis that contribute to the death of the neuron. In an embodiment, the disclosure provides a method of preventing, or delaying the progression of, death of a neuron and / or microgliosis and / or astrogliosis that contributes to the death of the neuron comprising contacting a neuron and / or a glial cell with a gene editing system that decreases the level and / or activity of an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in the neuron and / or glial cell, thereby reducing the transcription level of SNCA in the neuron and / or glial cell and preventing, or delaying the progression of, death of the neuron and / or microgliosis and / or astrogliosis that contribute to the death of the neuron. In an embodiment, the disclosure provides a method of treating, preventing, or delaying the progression of, a synucleinopathy in a subject in need thereof, comprising administering to the subject an effective amount of a gene editing system that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in at least one neuron and / or glial cell of the subject, thereby reducing the transcription level of the synucleinopathy- associated gene encoding the protein in the least one neuron and / or glial cell of the subject and treating, preventing, or delaying the progression of, the synucleinopathy (e.g., PD) in the subject. In an embodiment, the disclosure provides a method of treating, preventing, or delaying the progression of, a synucleinopathy in a subject in need thereof, comprising administering to the subject an effective amount of a gene editing system that decreases the level and / or activity of an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in at least one neuron of the subject, therebyAtty. Docket: JHU-43272.601 reducing the transcription level of SNCA in the least one neuron of the subject and treating, preventing, or delaying the progression of, the synucleinopathy (e.g., PD) in the subject.

[0130] In some embodiments, the gene editing system is a CRISPR / Cas system (Haft et al. PLoS Comput Biol. 2005; l(6)e60), a transposon-based gene editing system, a zinc finger nuclease (ZFN), and a transcription activator-like effector nuclease (TALEN) system. In some embodiments, the CRISPR / Cas system is a CRISPR type II system. In some embodiments, the CRISPR / Cas system is a CRISPR type V system. In some embodiments, the gene editing agent is a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease / Cas (CRISPR / Cas). The CRISPR / Cas-like protein can be a wild type CRISPR / Cas protein, a modified CRISPR / Cas protein, or a fragment of a wild type or modified CRISPR / Cas protein. The CRISPR / Cas-like protein can be modified to increase nucleic acid binding affinity and / or specificity, alter an enzymatic activity, and / or change another property of the protein. For example, nuclease (i.e., DNase, RNase) domains of the CRISPR / Cas-like protein can be modified, deleted, or inactivated. Alternatively, the CRISPR / Cas-like protein can be truncated to remove domains that are not essential for the function of the protein. The CRISPR / Cas-like protein can also be truncated or modified to optimize the activity of the effector domain of the protein. In general, CRISPR / Cas proteins comprise at least one RNA recognition and / or RNA binding domain. RNA recognition and / or RNA binding domains interact with guide RNAs. CRISPR / Cas proteins can also comprise nuclease domains (i.e., DNase or RNase domains), DNA binding domains, helicase domains, RNAse domains, protein-protein interaction domains, dimerization domains, as well as other domains. In some embodiments, the CRISPR / Cas system can be a type I, a type II, or a type III system. Non-limiting examples of suitable CRISPR / Cas proteins include Cas3, Cas4, Cas5, Cas5e (or CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1, Cas8a2, Cas8b, Cas8c, Cas9, Cas10, CaslOd, CasF, CasG, CasH, Csy 1, Csy2, Csy3, Csel (or CasA), Cse2 (or CasB), Cse3 (or CasE), Cse4 (or CasC), Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Cszl, Csx15, Csfl, Csf2, Csf3, Csf4, and Cu1966.

[0131] In some embodiments, the RNA-guided endonuclease is derived from a type II CRISPR / Cas system. In other embodiments, the RNA-guided endonuclease is derived from a Cas9 protein. The CRISPR / Cas system may include one or more single guide RNAs (sgRNA) designed to flank a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy associated gene in neurons and / or glial cells.

[0132] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90578786-90579367.

[0133] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90578838-90579505.Atty. Docket: JHU-43272.601

[0134] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90629975-90630782.

[0135] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90629981-90630880.

[0136] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90636762-90637480.

[0137] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90642092-90642763.

[0138] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90658878-90659419.

[0139] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90661417-90661898.

[0140] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90661557-90662154.

[0141] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90672325-90672709.

[0142] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90678450-90679064.

[0143] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90720580-90722663.

[0144] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90720648-90722351.

[0145] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90735341-90735761.

[0146] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90756615-90759904.

[0147] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90757134-90759904.

[0148] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90761441-90762196.

[0149] In an embodiment, the CRISPR / Cas system includes a pair of sgRNAs designed to flank an enhancer located at Human Ch4: 90842771-90843114.Atty. Docket: JHU-43272.601

[0150] In some embodiments, the gene editing system comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0151] In some embodiments, the gene editing system comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in a dopaminergic neuron; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0152] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at Human Ch4 (“HCh4”): 90578786-90579367; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs. It should be noted that the above Human Ch4 genomic coordinates are referenced from the Human CRCh37 / hg19 build.

[0153] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90578838-90579505; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs. In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90629975- 90630782; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0154] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90629981-90630880; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0155] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90636762-90637480; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0156] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90642092-90642763; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0157] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4:Atty. Docket: JHU-43272.601 90658878-90659419; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0158] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90661417-90661898; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0159] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90661557-90662154; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0160] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90672325-90672709; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0161] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90678450-90679064; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0162] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90720580-90722663; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0163] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90720648-90722351; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0164] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90735341-90735761; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0165] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90756615-90759904; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.Atty. Docket: JHU-43272.601

[0166] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90757134-90759904; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0167] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90761441-90762196; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0168] In an embodiment, the agent and / or composition comprises a Cas12a / AsCpfl system comprising: (i) a pair of single guide RNA (sgRNA) designed to flank a SNCA enhancer located at HCh4: 90842771-90843114; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs.

[0169] In the preceding embodiments, the AsCpf1 scaffold has a nucleotide sequence of SEQ ID NO:7. It should be noted that the above Human Ch4 genomic coordinates are referenced from the Human CRCh37 / hg19 build. In the preceding embodiments, the AsCpf1 scaffold has a nucleotide sequence having at least one, two, three, four, our five different nucleotides relative to SEQ ID NO:7. It should be noted that the above Human Ch4 genomic coordinates are referenced from the Human CRCh37 / hg19 build. In some embodiments, the gene editing system is modified to facilitate activation (CRISPR activation, CRISPRa) or interference (CRISPR interference, CRISPRi) of an endogenous output of a CRE target. CRISPR technology for editing the genes of eukaryotes is disclosed in US Patent Application Publications 2016 / 0138008A1 and US2015 / 0344912A1, and in US Patents 8,697,359, 8,771,945, 8,945,839, 8,999,641, 8,993,233, 8,895,308, 8,865,406, 8,889,418, 8,871,445, 8,889,356, 8,932,814, 8,795,965, and 8,906,616. In an embodiment, the disclosure provides a method of preventing, or delaying the progression of, death of a neuron and / or microgliosis and / or astrogliosis that contributes to the death of the neuron comprising contacting a dopaminergic neuron with CRISPR Cas12a / AsCpfl system with single guide RNA designed to flank an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in the dopaminergic neuron, thereby reducing the transcription level of SNCA in the neuron and preventing, or delaying the progression of, death of the neuron and / or microgliosis and / or astrogliosis that contribute to the death of the neuron. In an embodiment, the disclosure provides a method of preventing, or delaying the progression of, death of a neuron comprising contacting a dopaminergic neuron with a CRISPR Cas12a / AsCpfl system with single guide RNA designed to flank an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in the dopaminergic neuron, thereby reducing the transcription level of SNCA in the neuron and preventing, or delaying the progression of, death of the neuron.Atty. Docket: JHU-43272.601

[0170] In an embodiment, the disclosure provides a method of preventing, or delaying the progression of, death of a neuron comprising contacting a dopaminergic neuron with a CRISPR Cas12a / AsCpfl system comprising: (i) single guide RNA (sgRNA) designed to flank an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in the dopaminergic neuron; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs, thereby reducing the transcription level of SNCA in the neuron and preventing, or delaying the progression of, death of the neuron.

[0171] In an embodiment, the disclosure provides a method of preventing, or delaying the progression of, death of a neuron comprising contacting a dopaminergic neuron with a CRISPR Cas12a / AsCpfl system comprising: (i) single guide RNA (sgRNA) designed to flank an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in the dopaminergic neuron; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs, wherein the CRISPR Cas12a / AsCpfl system induces a deletion between mm 9 60,724,326- 60,745,103 (mm39 chr6: 60,769,316-60,772,093), 37.49 kb downstream from the Snca transcription start site (MGI: Tssr61200 – mm39 chr6:60,806,810-60,806,833), thereby reducing the transcription level of SNCA in the neuron and preventing, or delaying the progression of, death of the neuron.

[0172] In an embodiment, the disclosure provides a method of treating, preventing, or delaying the progression of, a synucleinopathy (e.g., PD) in a subject in need thereof, comprising administering to the subject an effective amount of a gene editing system that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in at least one neuron and / or glial cell of the subject, thereby reducing the transcription level of the synucleinopathy-associated gene encoding the protein in the least one neuron and / or glial cell of the subject and treating, preventing, or delaying the progression of, the synucleinopathy (e.g., PD) in the subject.

[0173] In an embodiment, the disclosure provides a method of treating, preventing, or delaying the progression of, a synucleinopathy (e.g., PD) in a subject in need thereof, comprising administering to the subject an effective amount of a CRISPR Cas12a / AsCpfl system with single guide RNA designed to flank an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in at least one dopaminergic neuron of the subject, thereby reducing the transcription level of SNCA in the at least one dopaminergic neuron of the subject and treating, preventing, or delaying the progression of, the synucleinopathy (e.g., PD) in the subject.

[0174] In an embodiment, the disclosure provides a method of treating, preventing, or delaying the progression of, a synucleinopathy (e.g., PD) in a subject in need thereof, comprising administering to the subject an effective amount of a CRISPR Cas12a / AsCpfl system with pair of single guide RNAs designed to flank an SNCA enhancer that propagates the misfolding and aggregation of alpha-synucleinAtty. Docket: JHU-43272.601 protein encoded by SNCA in the subject’s dopaminergic neurons, thereby reducing the transcription level of SNCA in the subject’s dopaminergic neurons and treating, preventing, or delaying the progression of, the synucleinopathy (e.g., PD) in the subject.

[0175] In an embodiment, the disclosure provides a method of treating, preventing, or delaying the progression of, a synucleinopathy (e.g., PD) in a subject in need thereof, comprising administering to the subject an effective amount of a CRISPR Cas12a / AsCpfl system comprising: (i) a pair of single guide RNAs (sgRNAs) designed to flank an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in the subject’s dopaminergic neurons; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs, thereby reducing the transcription level of SNCA in the subject’s dopaminergic neurons and treating, preventing, or delaying the progression of, the synucleinopathy (e.g., PD) in the subject.

[0176] In an embodiment, the disclosure provides a method of treating, preventing, or delaying the progression of, a synucleinopathy (e.g., PD) in a subject in need thereof, comprising administering to the subject an effective amount of a CRISPR Cas12a / AsCpfl system comprising: (i) a pair of single guide RNAs (sgRNAs) designed to flank an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in the dopaminergic neuron; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs, wherein the CRISPR Cas12a / AsCpfl system induces a deletion between mm 9 60,724,326-60,745,103 (mm39 chr6: 60,769,316-60,772,093), 37.49 kb downstream from the Snca transcription start site (MGI: Tssr61200 – mm39 chr6:60,806,810-60,806,833), thereby reducing the transcription level of SNCA in the subject’s dopaminergic neurons and treating, preventing, or delaying the progression of, the synucleinopathy (e.g., PD) in the subject. In some embodiments, the subject is a non-human mammal.

[0177] In an embodiment, the disclosure provides a method of treating, preventing, or delaying the progression of, a synucleinopathy (e.g., PD) in a non-human animal, comprising administering to the subject an effective amount of a CRISPR Cas12a / AsCpfl system, the CRISPR Cas12a / AsCpf1 system comprising: (i) a pair of single guide RNAs (sgRNAs) designed to flank an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in a dopaminergic neuron of the subject, wherein the pair of sgRNAs comprises a first sgRNA having a nucleotide sequence of SEQ ID NO: 1 and a second sgRNA having a nucleotide sequence of SEQ ID NO: 2; and (ii) a AsCpf1 scaffold ligated to the 5’ end of each of the flanking sgRNAs, the scaffold comprising a nucleotide sequence of SEQ ID NO: 7, wherein the CRISPR Cas12a / AsCpf1 system deletes the SNCA enhancer, thereby reducing the transcription level of SNCA in the subject’s dopaminergic neurons and treating, preventing, or delaying the progression of, the synucleinopathy (e.g., PD) in the subject.Atty. Docket: JHU-43272.601

[0178] In an embodiment, the disclosure provides a method of identifying agents and / or compositions that prevent, or delay the progression of the progression of, a synucleinopathy (e.g., PD) in a non-human animal, comprising administering to the subject an effective amount of a CRISPR Cas12a / AsCpfl system comprising: (i) a pair of single guide RNAs (sgRNAs) designed to flank an SNCA enhancer that propagates the misfolding and aggregation of alpha-synuclein protein encoded by SNCA in dopaminergic neurons of the animal, wherein the pair of sgRNAs comprises a first sgRNA comprising a nucleotide sequence of SEQ ID NO: 1 and a second sgRNA comprising a nucleotide sequence of SEQ ID NO: 2; and (ii) a AsCpf1 scaffold sequence ligated to the 5’ end of each of the flanking sgRNAs, the scaffold comprising a nucleotide sequence of SEQ ID NO: 7, wherein the CRISPR Cas12a / AsCpf1 system deletes the SNCA enhancer in the dopaminergic neurons of the animal, thereby decreasing the level of misfolded and aggregated alpha-synuclein protein in neurons and / or glial cells, or decrease microgliosis and / or astrogliosis that contribute to death of neurons in the subject. In the preceding embodiments, the first sgRNA comprises a nucleotide sequence having at least one, two, three, four, or five different nucleotides relative to SEQ ID NO:1. In the preceding embodiments, the second sgRNA comprises a nucleotide sequence having at least one, at least two, at least three, at least four, or at least five different nucleotides relative to SEQ ID NO: 2. In the preceding embodiments, the scaffold comprises a nucleotide sequence having at least one, two, three, four, or five different nucleotides relative to SEQ ID NO:7.

[0179] In some embodiments, the subject is a mouse. In some embodiments, the non-human animal is a mouse. In some embodiments, deletion of the SNCA enhancer reduces transcription of SNCA and the misfolding and aggregation of alpha-synuclein protein in the subject (e.g., human, mouse, etc.). In some embodiments, deletion of the SNCA enhancer ameliorations a motor deficits in the subject. In some embodiments, deletion of the SNCA enhancer ameliorates a cognitive deficit in the subject. In some embodiments, the method further comprises assessing the subject (e.g., human, non-human animal, mouse, etc.) after administration with the agent and / or composition for one or more clinical conditions or symptoms associated with the synucleinopathy (e.g., PD). In some embodiments, the method further comprises assessing the subject after administration with the agent and / or composition for improvement of a motor deficit. In some embodiments, the method further comprises assessing the subject after administration with the agent and / or composition for improvement of a cognitive deficit. In some embodiments, the method further comprises assessing the subject after administration with the agent and / or composition for improvement of a motor deficit. In some embodiments, the method further comprises administering to the subject a conventional therapeutic agent for treating a synucleinopathy (e.g., PD) in combination with the agent and / or composition and assessing the subject’s clinical condition (e.g., for improvement of a motor deficit and / or cognitive deficit). In some embodiments, the combination treatment is evaluated for synergy in ameliorating a motor deficit and / or cognitive deficit in the subject.Atty. Docket: JHU-43272.601 Administration of Agents and / or Compositions of the Present Disclosure

[0180] Aspects of the present disclosure involve administering an agent and / or composition of the present disclosure to a neuron, glial cell, or a subject. The present disclosure is not limited to any particular method of administering the presently disclosed agents and / or compositions. In some embodiments, the agents and / or compositions are delivered to the CNS by methods and compositions that promote transfer across the blood brain barrier (BBB). In some embodiments, the methods and compositions comprise a bi- specific immunoglobulin to a highly expressed protein including basigin, Glutl, and CD98hc. Immunoglobulins to these targets are significantly enriched in the brain after administration in vivo. Immunoglobulins against CD98hc exhibit robust accumulation in brain after systemic dosing. In some embodiments, transfer across the BBB is enhanced by transient disruption, for example, osmotic or pharmacologic disruption, and / or by other membrane protein pathways using receptor-mediate transcytosis comprising, for example, antibodies against the transferrin receptor.

[0181] In some embodiments, the agent and / or composition is delivered using stereotaxic injection, wherein the agent and / or composition is injected and / or infused into the brain parenchyma directly or into a ventricle. In some embodiments, the stereotaxic injection is a plasmid-free DNA, a plasmid free -RNA and / or a plasmid vector. In some embodiments, the stereotaxic injection is a viral vector selected from the group consisting of adeno-associated virus (AAV), lentivirus, and adenovirus.

[0182] In some embodiments, the agent and / or composition is delivered using an injection method selected from the group consisting of intracerebral injection, intra-thecal, intracerebroventricular (ICV) injection, intrastriatal injection, intranigral injection, intra-arterial injection, and subcutaneous injection.

[0183] In some embodiments, the agent and / or composition is delivered using cannulation and an external pump delivery.

[0184] Further, the agent and / or composition disclosed herein can be administered alone or in combination with adjuvants that enhance stability of the agents and / or compositions, alone or in combination with one or more therapeutic agents, facilitate administration of pharmaceutical compositions containing them in certain embodiments, provide increased dissolution or dispersion, increase inhibitory activity, provide adjunct therapy, and the like, including other active ingredients. Advantageously, such combination therapies utilize lower dosages of the conventional therapeutics, thus avoiding possible toxicity and adverse side effects incurred when those agents are used as monotherapies.

[0185] The timing of administration of an agent and / or composition disclosed herein and at least one additional therapeutic agent can be varied so long as the beneficial effects of the combination of these agents are achieved. When administered sequentially, the agents and / or compositions can be administered within 1, 5, 10, 30, 60, 120, 180, 240 minutes or longer of one another. In other embodiments, agents and / orAtty. Docket: JHU-43272.601 compositions administered sequentially, can be administered within 1, 5, 10, 15, 20 or more days of one another. Where the agent and / or composition described herein and at least one additional therapeutic agent are administered simultaneously, they can be administered to the subject as separate pharmaceutical compositions, each comprising either an agent and / or composition or at least one additional therapeutic agent, or they can be administered to a subject as a single pharmaceutical composition comprising both agents. When administered in combination, the effective concentration of each of the agents and / or compositions to elicit a particular biological response may be less than the effective concentration of each agent when administered alone, thereby allowing a reduction in the dose of one or more of the agents and / or compositions relative to the dose that would be needed if the agent was administered as a single agent. The effects of multiple agents and / or compositions may, but need not be, additive or synergistic. The agents and / or compositions may be administered multiple times. In some embodiments, when administered in combination, the two or more agents can have a synergistic effect. As used herein, the terms “synergy,” “synergistic,” “synergistically” and derivations thereof, such as in a “synergistic effect” or a “synergistic combination” or a “synergistic composition” refer to circumstances under which the biological activity of a combination of a compound described herein and at least one additional therapeutic agent is greater than the sum of the biological activities of the respective agents when administered individually.

[0186] Synergy can be expressed in terms of a “Synergy Index (SI),” which generally can be determined by the method described by F. C. Kull et al., Applied Microbiology 9, 538 (1961), from the ratio determined by: Qa / QA + Qb / QB = Synergy Index (SI) wherein: QA is the concentration of a component A, acting alone, which produced an end point in relation to component A; Qais the concentration of component A, in a mixture, which produced an end point; QBis the concentration of a component B, acting alone, which produced an end point in relation to component B; and Qbis the concentration of component B, in a mixture, which produced an end point.

[0187] Generally, when the sum of Qa / QAand Qb / QBis greater than one, antagonism is indicated. When the sum is equal to one, additivity is indicated. When the sum is less than one, synergism is demonstrated. The lower the SI, the greater the synergy shown by that particular mixture. Thus, a “synergistic combination” has an activity higher that what can be expected based on the observed activities of the individual components when used alone. Further, a “synergistically effective amount” of a component refers to the amount of the component necessary to elicit a synergistic effect in, for example, another therapeutic agent present in the composition.Atty. Docket: JHU-43272.601

[0188] Depending on the specific conditions being treated, the “therapeutic agent(s)”, “agent(s)”,“composition(s)” and the like may be formulated into liquid or solid dosage forms and administered systemically or locally. The agents may be delivered, for example, in a timed- or sustained- slow release form as is known to those skilled in the art. Techniques for formulation and administration may be found in Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000). Suitable routes may include oral, buccal, by inhalation spray, sublingual, rectal, transdermal, vaginal, transmucosal, nasal or intestinal administration; parenteral delivery, including intra- muscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intra-articular, intra-sternal, intra-synovial, intra-hepatic, intralesional, intracranial, intraperitoneal, intranasal, or intraocular injections or other modes of delivery.

[0189] For injection, the agents of the disclosure may be formulated and diluted in aqueous solutions, such as in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological saline buffer. For such transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0190] Use of pharmaceutically acceptable inert carriers to formulate the agents and / or compositions herein disclosed for the practice of the disclosure into dosages suitable for systemic administration is within the scope of the disclosure. With proper choice of carrier and suitable manufacturing practice, the compositions of the present disclosure, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The agents and / or compositions of the present disclosure can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the agents and / or compositions of the disclosure to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a subject (e.g., patient) to be treated.

[0191] For nasal or inhalation delivery, the agents of the disclosure also may be formulated by methods known to those of skill in the art, and may include, for example, but not limited to, examples of solubilizing, diluting, or dispersing substances, such as saline; preservatives, such as benzyl alcohol; absorption promoters; and fluorocarbons.

[0192] In particular embodiments, the agents and / or compositions disclosed herein are administered intranasally in a form selected from the group consisting of a nasal spray, a nasal drop, a powder, a granule, a cachet, a tablet, an aerosol, a paste, a cream, a gel, an ointment, a salve, a foam, a paste, a lotion, a cream, an oil suspension, an emulsion, a solution, a patch, and a stick. As used herein, the term administrating via an "intranasal route" refers to administering by way of the nasal structures.

[0193] Pharmaceutical compositions suitable for use in the present disclosure include compositions wherein the active ingredients are contained in an effective amount to achieve its intendedAtty. Docket: JHU-43272.601 purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. Generally, the agents and / or compositions according to the disclosure are effective over a wide dosage range. For example, in the treatment of adult humans, dosages from 0.01 to 1000 mg, from 0.5 to 100 mg, from 1 to 50 mg per day, and from 5 to 40 mg per day are examples of dosages that may be used. A non-limiting dosage is 10 to 30 mg per day. The exact dosage will depend upon the route of administration, the form in which the agent and / or composition is administered, the subject to be treated, the body weight of the subject to be treated, the bioavailability of the agent(s) and / or composition(s), the adsorption, distribution, metabolism, and excretion (ADME) toxicity of the agent(s) and / or composition(s), and the preference and experience of the attending physician.

[0194] In addition to the active ingredients, these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active agents and / or compositions into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the form of tablets, dragees, capsules, or solutions.

[0195] Pharmaceutical preparations for oral use can be obtained by combining the active agents and / or compositions with solid excipients, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethyl-cellulose (CMC), and / or polyvinylpyrrolidone (PVP: povidone). If desired, disintegrating agents may be added, such as the cross- linked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0196] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinylpyrrolidone, carbopol gel, polyethylene glycol (PEG), and / or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dye-stuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active agent and / or composition doses.

[0197] Pharmaceutical preparations that can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin, and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and / or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active agents and / or compositions may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols (PEGs). In addition, stabilizers may be added.Atty. Docket: JHU-43272.601

[0198] Further, one of ordinary skill in the art will recognize that the presently disclosed agents and / or compositions, and pharmaceutical compositions thereof, include pharmaceutically acceptable salts. Pharmaceutically acceptable salts are generally well known to those of ordinary skill in the art and include salts of active agents and / or compositions that can be prepared with relatively nontoxic acids or bases, depending on the particular substituent moieties found on the agents and / or compositions described herein. The parent form of the agents and / or compositions can differ from the various salt forms in certain physical properties, such as solubility, and the like.

[0199] When agents and / or compositions of the present disclosure contain relatively acidic functionalities, base addition salts can be obtained by contacting the neutral form of such agents and / or compositions with a sufficient amount of the desired base, either neat or in a suitable inert solvent or by ion exchange, whereby one basic counterion (base) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable base addition salts include sodium, potassium, calcium, ammonium, organic amino, magnesium, and the like.

[0200] When agents and / or compositions of the present disclosure contain relatively basic functionalities, acid addition salts can be obtained by contacting the neutral form of such agents and / or compositions with a sufficient amount of the desired acid, either neat or in a suitable inert solvent or by ion exchange, whereby one acidic counterion (acid) in an ionic complex is substituted for another. Examples of pharmaceutically acceptable acid addition salts include those derived from inorganic acids, organic acids, and amino acids. See, for example, Berge et al, “Pharmaceutical Salts”, Journal of Pharmaceutical Science, 1977, 66, 1-19. Agents and / or compositions containing both basic and acidic functionalities allow such agents and / or compositions to be converted into either base or acid addition salts.

[0201] Accordingly, pharmaceutically acceptable salts suitable for use with the presently disclosed subject matter include, by way of example but not limitation, acetate, arginate, benzenesulfonate, benzoate, bicarbonate, bitartrate, bromide, calcium edetate, camsylate, carbonate, monohy- drogencarbonate, citrate, edetate, edisylate, estolate, esylate, fumarate, galactonate, gluceptate, gluconate, glutamate, glycollylarsanilate, hexylresorcinate, hydrabamine, hydriodic, hydrobromide, hydrochloride, hydroxynaphthoate, iodide, isethionate, isobutyrate, lactate, lactobionate, malate, maleate, malonate, mandelate, mesylate, methanesulfonate, mucate, napsylate, nitrate, pamoate (embonate), pantothenate, phosphate, phthalate, diphosphate, monohydrogen phosphate, dihydrogen phosphate, polygalacturonate, propionate, salicylate, stearate, subacetate, suberate, succinate, sulfate, monohydrogensulfate, tannate, tartrate, including (+)-tartrates, (-)-tartrates, and mixtures thereof including racemic mixtures, teoclate, p- toluenesulfonate and tri-fluoroacetate. Other pharmaceutically acceptable salts may be found in, for example, Remington: The Science and Practice of Pharmacy (20th ed.) Lippincott, Williams & Wilkins (2000).Atty. Docket: JHU-43272.601

[0202] Unless otherwise noted, the chemical definitions provided immediately herein below are intended to comply with IUPAC. Compendium of Chemical Terminology, 2nd ed. (the "Gold Book"). Compiled by A. D. McNaught and A. Wilkinson. Blackwell Scientific Publications, Oxford (1997).

[0203] In certain embodiments, pharmaceutical compositions comprising agent and / or composition of the present disclosure that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a synucleinopathy-associated gene in neurons are used for the preparation of a medicament for treating a patient suffering or susceptible to a synucleinopathy.

[0204] In certain embodiments, pharmaceutical compositions comprising agent and / or composition of the present disclosure that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a synucleinopathy-associated gene in glial cells are used for the preparation of a medicament for treating a patient suffering or susceptible to a synucleinopathy. Kits and Articles of Manufacture

[0205] Kits and articles of manufacture of the present disclosure include an agent and / or composition of the present disclosure identified according to the methods discussed herein, e.g., combined with a pharmaceutically acceptable carrier, in a pharmaceutical formulation, e.g., in a pharmaceutical dosage form such as a pill, a powder, an injectable liquid, a tablet, dispersible granules, a capsule, a cachet or a suppository; optionally in association with a further therapeutic agent, e.g., as discussed herein. See for example, Gilman et al. (eds.) (1990), The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences, supra, Easton, Pa.; Avis et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications Dekker, New York; Lieberman et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets Dekker, New York; and Lieberman et al. (eds.) (1990), Pharmaceutical Dosage Forms: Disperse Systems Dekker, New York.

[0206] The kits and articles of manufacture of the present disclosure may also include information, for example, in the form of a package insert or label indicating that the agent and / or composition that decreases the level or activity of a CRE (e.g., an enhancer) that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells is intended to be administered to patients having, suspected of having, or at risk of developing, a synucleinopathy. The insert or label may take any form, such as paper or on electronic media such as a magnetically recorded or digital medium.

[0207] The label or insert may also include other information concerning the pharmaceutical compositions and dosage forms in the kit or article of manufacture. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectivelyAtty. Docket: JHU-43272.601 and safely. For example, the following information regarding the agent and / or composition that decreases the level and / or activity of the CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references and patent information.

[0208] In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having Parkinson’s disease. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having familial Parkinson’s disease. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having Parkinson’s disease with dementia. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having Parkinson’s disease and Alzheimer's disease. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having sporadic Parkinson’s disease. In some embodiments, the disclosure provides an article of manufactureAtty. Docket: JHU-43272.601 comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having idiopathic Parkinson’s disease. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having Gaucher's disease. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having Lewy body diffuse disease. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having dementia with Lewy bodies. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having Alzheimer's disease with Lewy bodies. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having sporadic Alzheimer's Disease. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a proteinAtty. Docket: JHU-43272.601 encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having familial Alzheimer's Disease. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having multiple system atrophy (MSA) also Shy-Drager syndrome. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having true autonomic failure. In some embodiments, the disclosure provides an article of manufacture comprising, packaged together, an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells or a pharmaceutical composition thereof comprising a pharmaceutically acceptable carrier; and a label stating that the agent and / or composition or pharmaceutical composition is indicated for treating patients having dopa-responsive dystonia. Uses

[0209] Aspects of the disclosure relate to the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of a synucleinopathy in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of Parkinson’s disease in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of familial Parkinson’s disease in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases theAtty. Docket: JHU-43272.601 level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of Parkinson’s disease with dementia in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of Parkinson’s disease and Alzheimer's disease in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of sporadic Parkinson’s disease in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of idiopathic Parkinson’s disease in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy- associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of a Parkinsonian disorder or symptom in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of Gaucher's disease in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of Lewy body diffuse disease in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of dementia with Lewy bodies in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of Alzheimer's disease with Lewy bodies in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding andAtty. Docket: JHU-43272.601 aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of sporadic Alzheimer's Disease in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of familial Alzheimer's Disease in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy- associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of multiple system atrophy (MSA) also Shy-Drager syndrome in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of true autonomic failure in a subject in need thereof. In an embodiment, the disclosure contemplates the use of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in neurons and / or glial cells for treating, preventing, or delaying the progression of dopa-responsive dystonia in a subject in need thereof.

[0210] The present disclosure is further summarized in the following paragraphs:

[0211] 1. A method of preventing, or delaying the progression of, death of a neuron and / or microgliosis and / or astrogliosis that contributes to the death of the neuron comprising contacting the neuron and / or a glial cell with an agent and / or composition that decreases the level and / or activity of a cis- regulatory element (CRE) that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in the neuron and / or glial cell, thereby reducing transcription of the synucleinopathy-associated gene encoding the protein in the neuron and / or glial cell and preventing, or delaying the progression of, death of the neuron and / or microgliosis and / or astrogliosis that contribute to the death of the neuron.

[0212] 2. The method of paragraph 1, wherein the method comprises contacting the neuron with the agent and / or composition.

[0213] 3. The method of paragraphs 1 or 2, wherein the neuron is a catecholaminergic neuron.

[0214] 4. The method of any one of paragraphs 1-3, wherein the neuron is a dopaminergic (DA) neuron.

[0215] 5. The method of any one of paragraphs 1-4, wherein the neuron is a midbrain DA neuron.

[0216] 6. The method of any one of paragraphs 1-5, wherein the CRE is an enhancer.Atty. Docket: JHU-43272.601

[0217] 7. The method of any one of paragraphs 1-6, wherein the CRE is selected from the group consisting of Human Chromosome 4 (Ch4): Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975-90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557-90662154, Ch4: 90672325- 90672709, Ch4: 90678450-90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4: 90735341-90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, and / or Ch4: 90842771-90843114.

[0218] 8. The method of any one of paragraphs 1-7, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1.

[0219] 9. The method of any one of paragraphs 1-8, wherein the protein is alpha-synuclein.

[0220] 10. The method of any one of claims 1-8, wherein the protein is LRRK2.

[0221] 11. The method of claim 1, wherein the method comprises contacting a glial cell with an agent / or composition.

[0222] 12. The method of paragraphs 1 or 11, wherein the glial cell is selected from the group consisting of astrocytes, oligodendrocytes, microglia, Schwann cells and / or ependymal cells.

[0223] 13. The method of any one of paragraphs 1,11 or 12, wherein the CRE is an enhancer.

[0224] 14. The method of any one of paragraphs 1, 11-13, wherein the CRE is selected from the group consisting of Human Ch4: Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975- 90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557-90662154, Ch4: 90672325-90672709, Ch4: 90678450-90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4: 90735341- 90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, and / or Ch4: 90842771-90843114.

[0225] 15. The method of any one of paragraphs 1, 11-14, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, and / or LRP10.

[0226] 16. The method of any one of paragraphs 1, 11-15, wherein the protein is alpha-synuclein.

[0227] 17. The method of any one of paragraphs 1, 11-15, wherein the protein is LRRK2.

[0228] 18. The method of any one of paragraphs 1-17, wherein the agent and / or composition comprises a gene editing system.Atty. Docket: JHU-43272.601

[0229] 19. The method of paragraph 18, wherein the gene editing system is a CRISPR / Cas system, a transposon-based gene editing system, a zinc finger nuclease (ZFN), and / or a transcription activator-like effector nuclease (TALEN) system.

[0230] 20. The method of any one of paragraphs 1-19, wherein said agent and / or composition is selected from the group consisting of a CRISPR-Cas system guide RNA (gRNA), a small interfering RNA (siRNA), a short hairpin RNA (shRNA), a microRNA (miRNA), an antisense DNA, an antisense RNA, a DNAzyme, a Ribozyme, an antagomir, a locked nucleid acid (LMA), a morpholino nucleic acid (MNA), and / or a chemical / small molecule.

[0231] 21. The method of any one of paragraphs 1-20, wherein the gene editing system is modified to facilitate activation or interference of the endogenous output of a CRE target via a CRISPR activation (CRISPRa) or a CRISPR interference (CRISPRi), respectively.

[0232] 22. A method of treating, preventing, or delaying the progression of, a synucleinopathy in a subject in need thereof, comprising administering to the subject an effective amount of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in at least one neuron and / or glial cell, thereby reducing transcription of the synucleinopathy-associated gene encoding the protein in at least one neuron and / or glial cell of the subject and treating, preventing, or delaying the progression of, the synucleinopathy in the subject.

[0233] 23. The method of paragraph 22, wherein the agent and / or composition reduces transcription of the synucleinopathy-associated gene encoding the protein in the at least one neuron.

[0234] 24. The method of paragraphs 22 or 23, wherein the agent and / or composition reduces the risk of the development and / or progression of microgliosis.

[0235] 25. The method of any one of paragraphs 22-24, wherein the agent and / or composition reduces the risk of the development and / or progression of astrogliosis.

[0236] 26. The method of any one of paragraphs 22-25, wherein the agent and / or composition reduces the risk of the development and / or progression of the synucleinopathy.

[0237] 27. The method of any one of paragraphs 22-26 wherein the agent and / or composition ameliorates a motor deficit in the subject.

[0238] 28. The method of paragraph 27, wherein the motor deficit is selected from the group consisting of tremors, bradykinesia, rigidity, and / or postural instability.

[0239] 29. The method of any one of paragraph 22-26, wherein the agent and / or composition reduces the risk of cognitive decline in the subject.Atty. Docket: JHU-43272.601

[0240] 30. The method of paragraph 29, wherein the cognitive deficit is selected from the group consisting of memory loss, memory decline, attention deficits, executive function impairment, language and communication deficits, visuospatial deficits, processing speed deficits, learning difficulties, reasoning and judgement deficits, and / or social cognition deficits.

[0241] 31. The method of any one of paragraphs 22-30, wherein the agent and / or composition protects against Lewy body aggregation in the cortex of the subject.

[0242] 32. The method of any one of paragraphs 22-31, wherein the agent and / or composition reduces the propagation of misfolded and aggregated alpha-synuclein protein in at least one neuron.

[0243] 33. The method of any one of paragraphs 22-32, wherein the synucleinopathy is Parkinson's disease (“PD”), Gaucher's disease, Lewy body diffuse disease, dementia with Lewy bodies, a variant of Alzheimer's disease with Lewy bodies, sporadic Alzheimer's Disease, familial Alzheimer's Disease, true autonomic failure, dopa-responsive dystonia, or multiple system atrophy (MSA) also Shy-Drager syndrome.

[0244] 34. The method of paragraph 33, wherein the PD is familial PD, PD with dementia, combined with Alzheimer’s and PD, sporadic PD, or idiopathic PD.

[0245] 35. The method of paragraph 22, wherein the agent and / or composition is delivered using stereotaxic injection, wherein the agent and / or composition is injected and / or infused into the brain parenchyma directly or into a ventricle.

[0246] 36. The method of paragraph 35, wherein the stereotaxic injection is a plasmid-free DNA, a plasmid free -RNA and / or a plasmid vector.

[0247] 37. The method of paragraphs 35 or 36, wherein the stereotaxic injection is a viral vector selected from the group consisting of adeno-associated virus (AAV), lentivirus, and / or adenovirus.

[0248] 38. The method of paragraph 22, wherein the agent and / or composition is delivered using an injection method selected from the group consisting of intracerebral injection, intra-thecal injection, intracerebroventricular (ICV) injection, intrastriatal injection, intranigral injection, intra-arterial injection, and / or subcutaneous injection.

[0249] 39. The method of paragraph 22, wherein the agent and / or composition is delivered using cannulation and an external pump delivery.

[0250] 40. The method of paragraph 22, wherein the adminstering comprises delivering an agent and / or composition in a nanoparticle encapsulation.

[0251] 41. The method of any one of paragraphs 22-40, further comprising administering a conventional therapeutic agent for treating a synucleinopathy.Atty. Docket: JHU-43272.601

[0252] 42. The method of any one of paragraphs 22-41, further comprising administering a conventional therapeutic agent for treating PD.

[0253] 43. The method of any one of paragraphs 22-42, wherein the agent and / or composition is administered to a subject who has underwent, or is currently undergoing, treatment with a conventional therapeutic agent for treating the synucleinopathy.

[0254] 44. The method of any one of paragraphs 22-43, wherein the subject has been diagnosed with and / or is at risk of developing a synucleinopathy.

[0255] 45. The method of any one of paragraphs 22-44, wherein the subject is displaying a cognitive deficit.

[0256] 46. The method of paragraph 45, wherein the cognitive deficit is selected from the group consisting of memory loss, memory decline, attention deficits, executive function impairment, language and communication deficits, visuospatial deficits, processing speed deficits, learning difficulties, reasoning and judgement deficits, and / or social cognition deficits.

[0257] 47. The method of any one of paragraphs 22-44, wherein the subject is displaying a motor deficit.

[0258] 48. The method of paragraph 47, wherein the motor deficit is selected from the group consisting of tremors, bradykinesia, rigidity, and / or postural instability.

[0259] 49. The method of any one of paragraphs 22-48, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1.

[0260] 50. The method of any one of paragraphs 22-49, wherein the subject has at least one SNP or SNV in the CRE that propagates the misfolding and aggregation of the protein in the neuron.

[0261] 51. The method of paragraph 50, wherein the at least one SNP in the CRE is selected from the group consisting of rs2583959, rs2737024, rs2583959, rs2737024, rs356220, rs356182, rs737029, rs356225, rs356168 and combinations thereof.

[0262] 52. The method of any one of paragraph 22-51, wherein the subject has at least one SNP in the CRE that propagates the misfolding and aggregation of alpha-synuclein in the at least one neuron.

[0263] 53. The method of paragraph 52, wherein the SNP is selected from the group consisting of rs2583959, rs2737024, rs2583959, rs2737024 and combinations thereof.

[0264] 54. A method of screening for an agent and / or composition which decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein in a neuron and / or glial cell, comprising: (a) contacting the neuron and / or glial cell with a candidate agent and / or composition to evaluateAtty. Docket: JHU-43272.601 its ability to decrease the level and / or activity of the CRE; and (b) identifying the candidate agent and / or composition as the agent and / or composition that decreases the level and / or activity of the CRE, if the candidate agent and / or composition decreases the transcription level and / or activity of a synucleinopathy- associated gene encoding the protein in the neuron and / or glial cell compared to the transcription level and / or activity of a synucleinopathy-associated gene encoding the protein in a neuron and / or glial cell that has not been contacted with the candidate agent and / or composition.

[0265] 55. The method of paragraph 54, wherein the neuron is a catecholaminergic neuron.

[0266] 56. The method of paragraphs 54 or 55, wherein the neuron is a DA neuron.

[0267] 57. The method of any one of paragraphs 54-56, wherein the neuron is a midbrain DA neuron.

[0268] 58. The method of any one of paragraphs 54-57, wherein the CRE is an enhancer.

[0269] 59. The method of any one of paragraphs 54-58, wherein the CRE is selected from the group consisting of Human Ch4: Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975- 90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557-90662154, Ch4: 90672325-90672709, Ch4: 90678450-90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4: 90735341- 90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, and Ch4: 90842771-90843114.

[0270] 60. The method of any one of paragraphs 54-59, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1.

[0271] 61. The method of any one of paragraphs 54-60, wherein the protein is alpha-synuclein.

[0272] 62. The method of any one of paragraphs 54-60, wherein the protein is LRRK2.

[0273] 63. The method of paragraph 62, wherein the glial cell is selected from the group consisting of astrocytes, oligodendrocytes, microglia, Schwann cells and / or ependymal cells.

[0274] 64. The method of any one of paragraphs 54 or 63, wherein the CRE is an enhancer.

[0275] 65. The method of any one of paragraphs 54, 63 or 64, wherein the CRE is selected from the group consisting of Human Ch4: Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975-90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557-90662154, Ch4: 90672325- 90672709, Ch4: 90678450-90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4:Atty. Docket: JHU-43272.601 90735341-90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, and / or Ch4: 90842771-90843114.

[0276] 66. The method of any one of paragraphs 54, 63-65, wherein the synucleinopathy- associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, and / or LRP10.

[0277] 67. The method of any one of paragraphs 54, 63-67, wherein the protein is alpha-synuclein.

[0278] 68. The method of any one of paragraphs 54, 63-67, wherein the protein is LRRK2.

[0279] 69. The method of any one of paragraphs 54-68, wherein the agent and / or composition comprises a gene editing system.

[0280] 70. The method of paragraph 69, wherein the gene editing system is a CRISPR / Cas system, a transposon-based gene editing system, a ZFN, and / or a TALEN system.

[0281] 71. The method of any one of paragraphs 54-70, wherein said agent and / or composition is selected from the group consisting of CRISPR-Cas system gRNA, a siRNA, a shRNA, a miRNA, an antisense DNA, an antisense RNA, a DNAzyme, a Ribozyme, an antagomir, a LMA, a MNA, and / or a chemical / small molecule.

[0282] 72. The method of any one of paragraphs 54-71, wherein the gene editing system is modified to facilitate activation or interference of the endogenous output of a CRE target via a CRISPRa or a CRISPRi, respectively.

[0283] 73. The method of any one of paragraphs 54-72, wherein the neuron was obtained from a subject that has been diagnosed with and / or is at risk of developing a synucleinopathy.

[0284] 74. The method of any one of paragraphs 54-73, wherein the neuron was obtained from a subject that is displaying a cognitive deficit.

[0285] 75. The method of paragraph 74, wherein the cognitive deficit is selected from the group consisting of memory loss, memory decline, attention deficits, executive function impairment, language and communication deficits, visuospatial deficits, processing speed deficits, learning difficulties, reasoning and judgement deficits, and / or social cognition deficits.

[0286] 76. The method of any one of paragraphs 54-73, wherein the neuron was obtained from a subject that is displaying a motor deficit.

[0287] 77. The method of paragraph 76, wherein the motor deficit is selected from the group consisting of tremors, bradykinesia, rigidity, and / or postural instability.

[0288] 78. The method of any one of paragraphs 54-77, wherein the neuron was obtained from a subject that has a synucleinopathy-associated gene.Atty. Docket: JHU-43272.601

[0289] 79. The method of any one of paragraphs 54-78, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1.

[0290] 80. The method of any one of paragraphs 54-79, wherein the neuron was obtained from a subject that has at least one SNP or SNV in the regulatory element that propagates the misfolding and aggregation of the protein in the neuron.

[0291] 81. The method of paragraph 80, wherein the at least one SNP in the CRE is selected from the group consisting of rs2583959, rs2737024, rs2583959, rs2737024, rs356220, rs356182, rs737029, rs356225, rs356168 and combinations thereof.

[0292] 82. The method of any one of paragraphs 54-81, wherein the neuron was obtained from a subject that has at least one SNP in the CRE that propagates the misfolding and aggregation of alpha- synuclein in at least one neuron.

[0293] 83. The method of paragraph 82, wherein the SNP is selected from the group consisting of rs2583959, rs2737024, rs2583959, rs2737024 and combinations thereof.

[0294] 84. The method of any one of paragraphs 54-83, wherein the method is in vitro or ex vivo.

[0295] 85. A method of identifying an agent and / or composition which decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein in at least one neuron and / or glial cell of a subject comprising: (a) administering to the subject a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of the CRE in the at least one neuron and / or glial cell; and (b) identifying the candidate agent and / or composition as the agent and / or composition that decreases the level and / or activity of the CRE, if the candidate agent and / or composition decreases the transcription level and / or activity of a synucleinopathy-associated gene encoding the protein in the neuron and / or glial cell compared to the transcription level and / or activity of the synucleinopathy-associated gene in at least one neuron and / or glial cell of a subject who has not been administered the candidate agent and / or composition.

[0296] 86. The method of paragraph 85, wherein the neuron is a catecholaminergic neuron.

[0297] 87. The method of paragraphs 85 or 86, wherein the neuron is a DA neuron.

[0298] 88. The method of any one of paragraphs 85-87, wherein the neuron is a midbrain DA neuron.

[0299] 89. The method of any one of paragraphs 85-88, wherein the CRE is an enhancer.

[0300] 90. The method of any one of paragraphs 85-89, wherein the CRE is selected from the group consisting of Human Ch4: Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975-Atty. Docket: JHU-43272.601 90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557-90662154, Ch4: 90672325-90672709, Ch4: 90678450-90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4: 90735341- 90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, and / or Ch4: 90842771-90843114.

[0301] 91. The method of any one of paragraphs 85-90, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1.

[0302] 92. The method of any one of paragraphs 85-91, wherein the protein is alpha-synuclein.

[0303] 93. The method of any one of paragraphs 85-92, wherein the protein is LRRK2.

[0304] 94. The method of paragraph 85, wherein the glial cell is selected from the group consisting of astrocytes, oligodendrocytes, microglia, Schwann cells and / or ependymal cells.

[0305] 95. The method of any one of paragraphs 85 or 94, wherein the CRE is an enhancer or repressor.

[0306] 96. The method of any one of paragraphs 85, 94 or 95, wherein the CRE is selected from the group consisting of Human Ch4: Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975-90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557-90662154, Ch4: 90672325- 90672709, Ch4: 90678450-90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4: 90735341-90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, and / or Ch4: 90842771-90843114.

[0307] 97. The method of any one of paragraphs 85 -96, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, and / or LRP10.

[0308] 98. The method of any one of paragraphs 85-97, wherein the protein is alpha-synuclein.

[0309] 99. The method of any one of paragraphs 85-98, wherein the protein is LRRK2.

[0310] 100. The method of any one of paragraphs 85-99, wherein the agent and / or composition comprises a gene editing system.

[0311] 101. The method of paragraph 100, wherein the gene editing system is a CRISPR / Cas system, a transposon-based gene editing system, a ZFN, and / or a TALEN system.Atty. Docket: JHU-43272.601

[0312] 102. The method of any one of paragraphs 85-101, wherein said agent and / or composition is selected from the group consisting of CRISPR-Cas system gRNA, a siRNA, a shRNA, a miRNA, an antisense DNA, an antisense RNA, a DNAzyme, a Ribozyme, an antagomir, a LMA, a MNA, and / or a chemical / small molecule.

[0313] 103. The method of any one of paragraphs 85-102, wherein the gene editing system is modified to facilitate activation or interference of the endogenous output of a CRE target via a CRISPRa or a CRISPRi, respectively.

[0314] 104. The method of any one of paragraphs 85-103, wherein the neuron was obtained from a subject that has been diagnosed with and / or is at risk of developing a synucleinopathy.

[0315] 105. The method of any one of paragraphs 85-104, wherein the neuron was obtained from a subject that is displaying a cognitive deficit.

[0316] 106. The method of paragraph 105, wherein the cognitive deficit is selected from the group consisting of memory loss, memory decline, attention deficits, executive function impairment, language and communication deficits, visuospatial deficits, processing speed deficits, learning difficulties, reasoning and judgement deficits, and / or social cognition deficits.

[0317] 107. The method of any one of paragraphs 85-104, wherein the neuron was obtained from a subject that is displaying a motor deficit.

[0318] 108. The method of paragraph 107, wherein the motor deficit is selected from the group consisting of tremors, bradykinesia, rigidity, and / or postural instability.

[0319] 109. The method of any one of paragraphs 85-108, wherein the neuron was obtained from a subject that has a synucleinopathy-associated gene.

[0320] 110. The method of any one of paragraphs 85-109, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1.

[0321] 111. The method of any one of paragraphs 85-110, wherein the neuron was obtained from a subject that has at least one SNP or SNV in the regulatory element that propagates the misfolding and aggregation of the protein in the neuron.

[0322] 112. The method of paragraph 111, wherein the at least one SNP in the CRE is selected from the group consisting of rs2583959, rs2737024, rs2583959, rs2737024, rs356220, rs356182, rs737029, rs356225, rs356168 and combinations thereof.Atty. Docket: JHU-43272.601

[0323] 113. The method of any one of paragraphs 85-112, wherein the neuron was obtained from a subject that has at least one SNP in the CRE that propagates the misfolding and aggregation of alpha- synuclein in at least one neuron.

[0324] 114. The method of paragraph 113, wherein the SNP is selected from the group consisting of rs2583959, rs2737024, rs2583959, rs2737024 and combinations thereof.

[0325] 115. The method of any one of paragraphs 85-114, wherein the subject is a mammal.

[0326] 116. The method of any one of paragraphs 85-115, wherein the subject is a non-human mammal.

[0327] 117. The method of any one of paragraphs 85-116, further comprising: (a) assessing the subject’s clinical condition prior to administering the candidate agent and / or composition; and (b) evaluating the subject’s clinical condition after administering the candidate agent and / or composition; wherein the assessment comprises measuring one or more clinical parameters.

[0328] 118. The method of paragraph 117, wherein the clinical condition prior to treatment is evaluated using the MDS-sponsored Revision of the Unified Parkinsion’s Disease Rating Scale (MDS- UPDRS).

[0329] 119. The method of paragraph 117, wherein the clinical parameter is a cognitive deficit.

[0330] 120. The method of paragraph 119, wherein the cognitive deficit is selected from the group consisting of memory loss, memory decline, attention deficits, executive function impairment, language and communication deficits, visuospatial deficits, processing speed deficits, learning difficulties, reasoning and judgement deficits, and / or social cognition deficits.

[0331] 121. The method of paragraph 117, wherein the clinical parameter is a motor deficit.

[0332] 122. The method of paragraph 121, wherein the motor deficit is selected from the group consisting of tremors, bradykinesia, rigidity, and / or postural instability.

[0333] 123. The method of paragraph 117, wherein the subject’s clinical condition is assessed at multiple time points after the administration of the candidate agent to monitor the candidate agent’s treatment progress.

[0334] EXAMPLES

[0335] The following Examples have been included to provide guidance to one of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill can appreciate that the following Examples are intended to be exemplary only and that numerous changes, modifications, and alterations canAtty. Docket: JHU-43272.601 be employed without departing from the scope of the presently disclosed subject matter. The synthetic descriptions and specific examples that follow are only intended for the purposes of illustration and are not to be construed as limiting in any manner to make agents and / or compositions of the disclosure by other methods. MATERIALS / METHODS Generation of Snca Enhancer Deletion Mouse Model

[0336] All mice were maintained on a 12-hour light-dark cycle in a temperature and humidity-controlled facility with ad libitum access to food and water. All experiments were performed in strict accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) at the Johns Hopkins University School of Medicine (MO18M427; MO23M44; MO24M315), which were developed in accordance with National Research Council guidelines.

[0337] Snca enhancer deletion founder mice (C57BL / 6J-Sncaem1Asm / J, or SncaEnh+37-) were created using a CRISPR Cas12a / AsCpf1 system, as we have done previously1. Single guide RNAs (sgRNAs) were designed to flank the Snca enhancer interval and induce a 2.76 kb deletion between mm9 chr6: 60,742,326-60,745,103 (mm39 chr6: 60,769,316-60,772,093), 37.49 kb downstream from the Snca transcription start site (MGI: Tssr61200 – mm39 chr6:60,806,810- 60,806,833). AsCpf1 scaffold sequences (5′-TAATTTCTACTCTTGTAGAT-3′ (SEQ ID NO.: 7)) were ligated to the 5’ end of each sgRNA (Integrated DNA Technologies; IDT). Guides were diluted in Opti-MEM media (Thermo-Fisher Scientific # 31985070) to a final concentration of 2 uM, and CPF1 protein (IDT) was diluted in Opti-MEM media (Thermo-Fisher Scientific # 31985070) to a final concentration of 5 ng / μl. A total of 50 μl guide-CPF1 solution was electroporated into approximately 150 C57BL / 6J × FVB / N F1hybrid zygotes using a Nepa21 electroporator (NepaGene, Co. Ltd., Japan) with set pulse conditions as recommended by the manufacturer. Hybrid zygotes were allowed to rest for 30-60 minutes at 5% CO2, washed in M2 media (Sigma Aldrich # M7167), and implanted using standard embryo transfer surgery protocols1. The resulting founder mice were screened for deletion alleles using the flanking PCR primers (Table 1; Figure 6). Amplicons were Sanger sequenced to confirm the presence of the intended deletion. At sexual maturity, five founder animals and their offspring were backcrossed on a C57BL / 6J (The Jackson Laboratory, Bar Harbor, Maine) background for 10 generations (N10) toAtty. Docket: JHU-43272.601 generate congenic strains. One of the five founder lines (V9) was slower to reproduce, reaching N8 when the other lines (L1, L2, Y1, and Y7) reached N10, and was excluded from subsequent analysis in the interest of time. Mouse genotyping

[0338] Mouse genomic DNA was isolated from ≤ 20 mg of ear tissue, obtained from 1- month-old mice. Tissue was incubated in lysis buffer (50 uM KCl, 10 uM Tris HCl pH9.0, 0.1% Triton X- 100) and 5 tl of proteinase K (18.7 mg / ml; Sigma Aldrich #3115879001) at 60°C for 3 hours, followed by a 95°C incubation for 10 minutes. Extracted DNA was purified using a DNA Clean & ConcentratorTM-5 kit (Zymo Research #D4067), eluted in 6 tl of elution buffer, and quantified using a Nanodrop. All genomic DNA samples were diluted to a concentration of 20 ng / tl using nuclease free water. Mouse genotype was assessed by polymerase chain reaction (PCR) and agarose gel electrophoresis using custom primers designed to flank and bridge the enhancer locus (Table 1; Figure 5). A 25 tl reaction volume – consisting of 12.5 tl GoTaq® Green Master Mix (Promega #M7123); 1 tl each of forward, reverse, and internal primers at a concentration of 10 tM; 8.5 tl nuclease free water; and 1 tl of template DNA at a concentration of 20 ng / tl – was amplified using an initial denaturation step of 95°C for 5 minutes; followed by 30 cycles of 95°C for 30 seconds, 56°C for 30 seconds, and 72°C for 60 seconds; and a final extension step of 72°C for 5 minutes. Amplicon size was assessed on a 1% (w / v) agarose gel. Snca Quantification via Single Molecule Fluorescent in situ Hybridization

[0339] Single molecule fluorescent in situ hybridization (smFISH) was performed using the RNAscope® Multiplex Fluorescent Reagent Kit v2 Assay (ACDBio, user manual document 323100-USM / Rev Date: 02272019), following protocols for fixed-frozen tissue samples, with some modifications. All buffers, probes, and OpalTM dyes were prepared according to the ACD user manual.

[0340] First, N10 mice from each of the 4 founder lines (3 mice / genotype / line) were given 10 μg / g body weight of a 77.5% (v / v) saline, 15% (v / v) ketamine (100 mg / mL), 7.5% (v / v) xylazine (100 mg / mL) anesthesia cocktail via intraperitoneal (i.p.) injection. Next, they underwent transcardial perfusion with 4% (w / v) paraformaldehyde (PFA) solution composed of 40 g PFA (Sigma-Aldrich #P6148) in 100 uL of 10x PBS (pH 7.4) and 900 uL water treated with 0.1% (v / v) DEPC (Diethyl pyrocarbonate; VWR #E174-100G). Brains were dissected and post-fixed in 4% PFA solution overnight at 4°C. After 24 hours, brains were moved to a 30% (w / v) sucrose solution,Atty. Docket: JHU-43272.601 where they remained for another 24 h or until the brain had sunk to the bottom of a 50 mL falcon tube. Brains were frozen using CytocoolTM II, Aerosol Freezing Spray (EprediaTM #8323), mounted on a microtome disk using O.C.T. Compound (Tissue-Tek® #4583), and placed in a microtome at - 20°C. Midbrain coronal sections were cut to 12 μm thickness, and 10 sections at ~120 μm intervals were transferred to Superfrost® Plus microscope slides (FisherbrandTM #12-550-15). Slides were stored at -80°C until slide pre-treatment.

[0341] Prior to pre-treatment, slides were moved from -80°C to -20°C for 1 hour. Since brains were not mounted in O.C.T. Compound, the slides were next incubated for 1 hour in a HybEZTM oven set to 37°C. Next, RNAscope® hydrogen peroxide treatment and target retrieval steps were performed as recommended by the manufacturer. Slides were dried at room temperature for 5 minutes before the hydrophobic barrier was applied and left to dry overnight at room temperature. The following day, RNAscope® Protease III reagent was applied to each slide, incubated for 30 minutes at 40°C, and then treated with the appropriate positive control probe, negative control probe, or probe mix (50 volumes of RNAscopeTM Probe-Mm-Snca #313281: 1 volume RNAscopeTM Probe- Mm-Th-C2 #317621-C2). All hybridization steps were performed as written. Meanwhile, OpalTM dyes (Akoya Biosciences; FP1487001KT and FP1488001KT), reconstituted in 75 tL of DMSO, were diluted according to our fluorophore optimization efforts. OpalTM 520 (Akoya Biosciences #FP1487001KT) was diluted 1:100 in TSA buffer (ACDBio) and OpalTM 570 (Akoya Biosciences #FP1488001KT) was diluted 1:1750 in TSA buffer (ACDBio). HRP-C1 and HRP-C2 signals were developed, as written, except that 300 tL of each OpalTM dye was added at the appropriate time. Finally, slides were counterstained using 250 tL of Hoechst 34580 (Thermo- Fisher Scientific #H21486) solution made from 3 tL Hoechst in 1.5 mL DEPC-PBS. Slides were incubated at room temperature for 10 minutes before the Hoechst stain was removed and 1-2 drops of ProLong Gold Antifade Mountant (InvitrogenTM #P36930) was added to each slide. A 24x50 mm glass coverslip (FisherbrandTM #12-545-F) was carefully placed on each slide and slides were left to dry overnight in a cool, dark place.

[0342] Slides were imaged using a Nikon Eclipse Ti confocal microscope, with all imaging settings (offset, HV / gain, and laser percentage) normalized to Hoescht / DAPI signal. Images were exported in .tiff format at 16-bits / px. Snca expression was quantified using HALO software (indica labs; v3.3.2541.383), at a resolution of 0.41tm / px, in which DA neurons, defined as Th- and Hoechst / DAPI-expressing cells within the SN, were selected for inclusionAtty. Docket: JHU-43272.601 in FISH scoring analysis. The area for analysis was manually defined, such that only DA neurons of the SN, and not adjacent brain regions (i.e., the ventral tegmental area), were included in analysis. Snca copy intensity was calculated for each slide image and was averaged over the right and left hemispheres for each animal (Data not shown). The geometric mean, standard deviation (SD), and standard error or the mean (SEM) of Snca copy intensity was calculated to include all three animals in each genotype group. Again, to determine if enhancer genotype significantly affects Snca expression, we employed one-way ANOVA and post-hoc Tukey HSD tests to calculate pair-wise significance for each founder line. Preparation and Stereotaxic Injection of α-synuclein Preformed Fibrils

[0343] Mouse recombinant full-length α-Synuclein protein was purified as previously described, using the IPTG independent inducible pRK172 vector system3. α-Syn PFF were also prepared as we have previously described4–7, by diluting endotoxin using the ToxinEraserTM Endotoxin Removal Kit (GenScript #L00338), then using a magnetic stirrer (1,000 r.p.m.) to mix 5 mg / mL α-Syn PFF in PBS at 37°C. The α-Synuclein protein was incubated for a week before aggregates were diluted to 0.1 mg / mL in PBS and sonicated for 30 seconds (0.5 second pulse on / off) at 10% amplitude (Branson Digital sonifier, Danbury, CT, USA). α-Syn PFF was validated using atomic force microscopy and transmission electron microscopy and stored at −80 °C until stereotaxic injection.

[0344] At 3 months of age (postnatal day 90; P90), 10 mice / sex / genotype were randomly assigned to experimental (α-Syn PFF) or control (PBS) groups, and stereotaxic injections were performed as previously described5–9. All mice were anesthetized with 250 mg / kg of a 1.25% working solution of Tribromoethanol / Avertin made from 25g 2,2,2-Tribromoethanol powder (Sigma- Aldrich T48402) with 15.5ml 2-Methyl-2 butanol (Sigma-Aldrich 240486) via i.p. injection. A26.5-gauge injection cannula was unilaterally inserted into the right hemisphere of the striatum guided by stereotaxic coordinates (mediolateral, 2.0 mm from bregma; anteroposterior, 0.2 mm; dorsoventral, 2.6 mm). Each mouse within the experimental and control groups was administered a 2 μL injection of α-Syn PFF (2.5 μg / μL in PBS) or PBS, respectively, at an infusion rate of 0.2 μL / minute. α-Syn PFF-induced PD pathology was allowed to develop for 6- months (180 days) following injection surgeries5–10, and by 9-months of age, all mice in the α-Syn PFF and PBS groups progressed to experimental endpoints consisting of motor, behavioural, and histopathological assays.Atty. Docket: JHU-43272.601 Rotarod Test of Motor Control

[0345] All behaviour tests were performed in the Behavioral Core Facility at the Johns Hopkins University School of Medicine between 09:00–16:00 during the lights-on cycle. Rotarod tests were performed on 9-month-old mice, 6-months post α-Syn PFF / PBS injection in accordance with published methods4,5,11. Briefly, mice were acclimatized to the procedure room for 30 minutes before being placed on a rotamex V instrument equipped with photobeams and a sensor to automatically detect mice that fall from the rotarod. This rotarod cylinder slowly accelerated from 4 r.p.m. to 40 r.p.m. over 5 minutes. Rotamex settings remained constant throughout all trials: start speed, 4.0 r.p.m.; maximum speed, 40 r.p.m.; acceleration interval, 15 seconds; acceleration step, 2 r.p.m. The duration over which each animal remained on the rotarod was recorded (in seconds), and a trial ended when the animal fell off the rungs. All animals underwent three consecutive training days, each consisting of three trials. Next, over five test days, each mouse was given one warm-up run and one evaluated test run. The mean duration over which each mouse remained on the rotarod during their test run was calculated.

[0346] To determine if enhancer genotype, α-Syn PFF / PBS injection, or pairwise interactions of these variables significantly impacted motor function, we employed a two-way ANOVA, using R (v.4.3.3) function “aov”, and post-hoc Tukey HSD tests, using R function “TukeyHSD” specifying “conf.level=.95” to calculate pair-wise significance for each genotype and α-Syn PFF / PBS injection group. Outliers were removed such that data from n = 8-9 mice / sex / genotype / injection group (N = 106) were included in analysis. Open Field Test

[0347] Open field testing was performed on 9-month-old mice, 6-months post α-Syn PFF / PBS injection, as we have done previously4,11. In a quiet room, each mouse was placed against the wall of the open field arena – a rectangular plastic box (40 cm × 40 cm × 40 cm) divided into 36 (6 × 6) identical squares (6.6 cm × 6.6 cm), where the “central” sector was defined as the 4 central squares (2 × 2) and the “peripheral” sector was defined as the remaining squares. Each mouse was allowed to explore the arena for 15 minutes (900 seconds). Photobeam activity system (PAS) software connected to the open field equipment recorded the total number of beam breaks, which was used to determine gross locomotor activity of each mouse. Photobeam Activity SystemTM (PAS) software was used to measure ambulatory (A) and fine (F) movements at the center (cen) and periphery (per). Total center beam breaks were calculated by cenA+cenF; total peripheralAtty. Docket: JHU-43272.601 beam breaks were calculated by perA+perF; locomotion was defined as total beam breaks (cenA+cenF+perA+perF); rears were defined as total number of vertical beam breaks; and anxiety was defined as the percentage of peripheral movements [perF+perA) / (cenA+cenF+perA+perF)*100].

[0348] To determine if enhancer genotype, α-Syn PFF / PBS injection, or pairwise interactions of these variables significantly impacted anxiety, locomotor, and exploratory behaviours, we employed two-way ANOVA and post-hoc Tukey HSD tests to calculate pair-wise significance for each genotype and α-Syn PFF / PBS injection group. Outliers were removed such that data from n= 9-10 mice / sex / genotype / injection group (N = 112) were included in analysis. Pole Descent Test of Motor Control

[0349] Pole descent tests were performed on 9-month-old mice, 6-months post α-Syn PFF / PBS injection as previously described4,5. Mice were placed on a gauze-wrapped metal rod 75 cm in height and 9 mm in diameter. Mice were placed on the top of the pole, facing down, and the total time (in seconds; 60 seconds maximum) taken for each mouse to initiate downward movement and reach the base of the pole with their front paws was recorded. All animals underwent three consecutive training days, each consisting of three trials, prior to four test days, during which mice were evaluated over two trials.

[0350] To determine if enhancer genotype, α-Syn PFF / PBS injection, or pairwise interactions of these variables significantly impacted motor control, we employed two-way ANOVA and post hoc Tukey HSD tests to calculate pair-wise significance for each genotype and α-Syn PFF / PBS injection group. Outliers were removed such that data from n = 8-10 mice / sex / genotype / injection group (N = 114) were included in analysis. Grip Strength Test

[0351] Grip strength testing was performed on ≥9-month-old mice, ≥6-months post α- Syn PFF / PBS injection. Mice were acclimatized to the procedure room for 30 minutes before their forelimbs were placed on a specially designed grid connected to a Bioseb grip strength meter (BIO- GS4; v 3.47). Neuromuscular function was determined by the maximal peak force (g) required for an experimenter to pull the mouse off the grid by pulling each animal’s tail down at a constant speed. To reduce variability, one experimenter performed this assay for all mice. The grip strength meter was reset to 0.0 g before and after each test. All mice were naively evaluated over three trials.Atty. Docket: JHU-43272.601

[0352] To determine if enhancer genotype, α-Syn PFF / PBS injection, or pairwise interactions of these variables significantly impacted neuromuscular function and grip strength, we employed two-way ANOVA and post-hoc Tukey HSD tests to calculate pair-wise significance for each genotype and α-Syn PFF / PBS injection group. Outliers were removed such that data from n = 9- mice / sex / genotype / injection group (N = 118) were included in analysis. Immunohistochemistry and Immunofluorescence

[0353] Following the acquisition of all motor and behavioural data, 5 mice / sex / treatment / genotype were anesthetized with 250 mg / kg Tribromoethanol / Avertin by i.p. injection and underwent transcardial perfusion with 4% PFA (pH7.4; Boster #AR1068). Brains were dissected from each animal and post-fixed in 4% PFA overnight. The following day, brains were moved to a cryoprotectant 30% (w / v) sucrose solution, where they remained for 2- 3 days or until the brain had sunk to the bottom of a 50 mL falcon tube. Next, brains were frozen using O.C.T. Compound, coronal sections were cut to 30 μm thickness using a microtome, and sections were placed, free- floating, in 9 wells of a 12-well tissue culture plate in a storage solution of 0.01 M PBS. Sections were stored at -20°C overnight prior to IHC staining.

[0354] Immunohistochemistry (IHC) was performed with 30µm thick brain sections. The sections were washed in PBS 3 times for 10 minutes before blocking in PBS containing 5% (v / v) goat serum (Jackson ImmunoResearch #005-000-121) and 0.3% (v / v) TritonTM X-100 (Millipore Sigma #T9284). After 1 h in blocking buffer, sections were washed in PBS 3 times for 10 minutes. Next, sections were incubated in 1:500 dilution of an anti-TH primary antibody (Novus Biologicals #NB300-109) and were left to shake gently overnight at 4°C. The next day, sections were washed in PBS 3 times for 10 minutes and then incubated with 1:250 dilution of biotin-conjugated anti- rabbit IgG secondary antibody (Vector laboratories #BA-1000) in PBS containing 0.3% TritonTM X-100 for 2h at RT. After washing with PBS again, brain sections were placed in ABC solution (Vector Laboratories #PK-6100) for 2 hours at room temperature (RT). After a final wash in PBS 3 times for 10 minutes, brain sections were developed using SigmaFast DAB peroxidase substrate (Millipore Sigma #D4293). The sections were mounted on a gelatin-coated slide, followed by counterstaining with Nissl (0.09% v / v thionin; Millipore Sigma, 861340-25G). Image analysis was performed using Stereo Investigator software (MicroBright-Field, VT, USA), including an Axiophot photomicroscope (Carl Zeiss) and Hitachi HV C20 camera. TH- and Nissl-positive dopaminergic neurons in SN region were counted by a blinded investigator.Atty. Docket: JHU-43272.601

[0355] For immunofluorescence, sections were incubated in 1:500 dilution of anti-pS129- α-syn antibodies (BioLegend #825701), anti-GFAP antibodies (Invitrogen #14-9892-82) and anti- Iba-1 antibodies (Wako #019-19741) overnight at 4°C. The next day, sections were incubated in 1:250 dilution of Alexa-Fluor 488- and 594- conjugated secondary antibodies (Invitrogen). The images were obtained by confocal microscopy (LSM 880, Carl Zeiss), using Zen software. Signal intensity and counting were conducted using ImageJ software (v1.48). GraphPad Prism (v10.3.0) was used to plot the data and perform a Two-way ANOVA (or Mixed-Model) with the following parameters: “Multiple comparisons test” = Tukey, “Multiple comparisons options” = Report multiplicity adjusted P value for each comparison, “Family-wise alpha threshold and confidence” = 0.05 (95% confidence interval). Tissue Lysate and Western Blot Analysis

[0356] After perfusion with cold 1x-PBS, nonionic detergent-soluble and -insoluble fractions were made by homogenization of tissue in soluble lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl;1% Triton x100; phosphatase inhibitor cocktail II and III (Sigma-Aldrich); and complete protease inhibitor; DW). The homogenate was centrifuged at 22,000 x g for 20 minutes at 4°C, and the resulting pellet (P1) and supernatant (S1, soluble part) fractions were collected. The P1 was washed 2 times in soluble lysis buffer, re-suspended using insoluble lysis buffer (50 mM Tris-HCl, pH 7.4; 150 mM NaCl; 1% Triton x100; 2% SDS; phosphatase inhibitor cocktail II and III (Sigma- Aldrich); and complete protease inhibitor; DW) and then sonicated (20% amplitude, 1 s pulse on / off, total 5s). The P1 was centrifuged at 22,000 x g for 20 minutes at room temperature, and the resulting pellet (P2) and supernatant (S2, insoluble part) fractions were collected. For western blot analysis, protein concentrations were measured using a Bicinchoninic Acid (BCA) assay (Pierce, Rockford, IL, USA). The protein samples were separated on 8 - 16% gradient SDS- PAGE gels and transferred to nitrocellulose (NC) membrane (0.45µm, Bio-Rad). After blocking with 5% non-fat milk or 3% BSA in TES-T (Tris-buffered saline with 0.1% tween-20) for 1h at room temperature, and incubated overnight at 4°C with anti-TH (1:1000, Novus Biologicals #NB300- 109), anti-DAT (1:1000, Millipore Sigma #D6944), anti-pS129-a-syn (1:1000, Cell Signaling #23706), anti-a-syn (1:1000, Cell Signaling #2642S), anti-IBA1 (1:1000, Wako #019- 19741), anti-GFAP (1:1000, Invitrogen #14-9892-82). Membranes were then washed and incubated with

[0357] HRP-conjugated secondary antibodies (1:10000, Millipore #32230, #32260) for 1hAtty. Docket: JHU-43272.601 at RT. Protein were detected using ECL solution and an Amersham Imager 800 (GE healthcare Life Sciences,). Results of western blot was quantified using ImageJ (v1.48). Plotting and statistical analysis was performed using GraphPad Prism (v10.3.0), as stated above. REFERENCES FOR MATERIALS AND METHODS

[0358] 1. Watkins-Chow, D. E. et al. Highly Efficient Cpf1-Mediated Gene Targeting in Mice Following High Concentration Pronuclear Injection. G3 (Bethesda, Md.) 7, 719–722 (2017).

[0359] 2. Taylor, S. C. et al. The ultimate qPCR experiment: Producing publication quality, reproducible data the first time. Trends in Biotechnology 37, 761–774 (2019).

[0360] 3. Volpicelli-Daley, L. A., Luk, K. C. & Lee, V. M. Y. Addition of exogenous α- Synuclein Pre- formed fibrils to Primary Neuronal Cultures to seed recruitment of endogenous α- Synuclein to Lewy body and Lewy Neurite-like aggregates. Nature protocols 9, 2135 (2014).

[0361] 4. Kim, S. et al. Transneuronal Propagation of Pathologic α-Synuclein from the Gut to the Brain Models Parkinson’s disease. Neuron 103, 627 (2019).

[0362] 5. Yun, S. P. et al. Block of A1 astrocyte conversion by microglia is neuroprotective in models of Parkinson’s disease. Nature medicine 24, 931 (2018).

[0363] 6. Verma, D. K. et al. Alpha-synuclein preformed fibrils induce cellular senescence in Parkinson’s disease models. Cells 10, (2021).

[0364] 7. Mao, X. et al. Pathological α-Synuclein transmission initiated by binding lymphocyte-activation gene 3. Science 353, (2016).

[0365] 8. Lee, S. et al. The c-Abl inhibitor, Radotinib HCl, is neuroprotective in a preclinical Parkinson’s disease mouse model. Human Molecular Genetics 27, 2344 (2018).

[0366] 9. Kim, D. et al. Graphene quantum dots prevent α-synucleinopathy in Parkinson’s disease. Nature nanotechnology 13, 812 (2018).

[0367] 10. Kam, T. I. et al. Poly (ADP-ribose) Drives Pathologic α-Synuclein Neurodegeneration in Parkinson’s Disease. Science 362, (2018).

[0368] 11. Lee, Y. et al. Parthanatos Mediates AIMP2 Activated Age Dependent Dopaminergic 277 Neuronal Loss. Nature neuroscience 16, 1392 (2013). EXAMPLE 1Atty. Docket: JHU-43272.601 Founder mice lacking SncaEnh+37 demonstrate reduced Snca expression.

[0369] To test whether this neuronal enhancer is required for transcriptional control of Snca, mice lacking this sequence were engineered. A single guide RNAs (sgRNAs) was designed to flank the Snca enhancer interval and induce a 2.76 kb deletion, 37.49 kb downstream from the Snca transcription start site within intron 4 (SncaEnh+37; Figure 5A-B; Table 1). Five independent founder mice were identified that were heterozygous for this element, which were progressively backcrossed to C57BL / 6J to ensure an isogenic background.

[0370] The degree to which deletion of SncaEnh+37 impacts Snca expression in mbDA neurons was evaluated via single molecule fluorescent in situ hybridization (smFISH) / RNAscope in four of the founder lines displaying the highest fecundity. Mice were assayed across all three genotypes (n = 36; 3 / genotype / founder), wild-type / WT(SncaEnh+37+ / +), heterozygous / Het(SncaEnh+37+ / -), and homozygous / Hom(SncaEnh+37- / -), at N8 generation. These genotypes are designated as WT(SncaEnh+ / +); Het(SncaEnh+ / -), and Hom(SncaEnh- / -), respectively.

[0371] To determine whether the engineered mice displayed a dose-dependent reduction in Snca within mbDA neurons, the smFISH / RNAscope assay was designed with probes against Snca and tyrosine hydroxylase (Th...

Claims

Atty. Docket: JHU-43272.601 THAT WHICH IS CLAIMED:

1. A method of preventing, or delaying the progression of, death of a neuron and / or microgliosis and / or astrogliosis that contributes to the death of the neuron comprising contacting the neuron and / or a glial cell with an agent and / or composition that decreases the level and / or activity of a cis-regulatory element (CRE) that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy- associated gene in the neuron and / or glial cell, thereby reducing transcription of the synucleinopathy- associated gene encoding the protein in the neuron and / or glial cell and preventing, or delaying the progression of, death of the neuron and / or microgliosis and / or astrogliosis that contribute to the death of the neuron.

2. The method of claim 1, comprising contacting the neuron with the agent and / or composition.

3. The method of claim 2, wherein the neuron is a dopaminergic (DA) neuron.

4. The method of claim 1, comprising contacting the glial cell with the agent and / or composition.

5. The method of any one of claims 1-4, wherein the CRE is an enhancer.

6. The method of any one of claims 1-5, wherein the enhancer is selected from the group consisting of Human Chromosome 4 (Ch4): Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975- 90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557-90662154, Ch4: 90672325-90672709, Ch4: 90678450-90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4: 90735341- 90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, Ch4: 90842771-90843114 and corresponding enhancers in non-human mammals.

7. The method of any one of claims 1-6, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1.

8. The method of any one of claims 1-7, wherein the protein is alpha-synuclein.Atty. Docket: JHU-43272.601 9. The method of any one of claims 1-8, wherein the protein is LRRK2.

10. A method of treating, preventing, or delaying the progression of, a synucleinopathy in a subject in need thereof, comprising administering to the subject an effective amount of an agent and / or composition that decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein encoded by a synucleinopathy-associated gene in at least one neuron and / or glial cell in the subject, thereby reducing transcription of the synucleinopathy-associated gene encoding the protein in at least one neuron and / or glial cell of the subject and treating, preventing, or delaying the progression of, the synucleinopathy in the subject.

11. The method of claim 10, wherein the agent and / or composition reduces transcription of the synucleinopathy-associated gene encoding the protein in the at least one neuron.

12. The method of claim 11, wherein the agent and / or composition reduces transcription of the synucleinopathy-associated gene encoding the protein in the at least one glial cell.

13. The method of any one of claims 10-12, wherein the CRE is an enhancer.

14. The method of any one of claims 10-13, wherein the enhancer is selected from the group consisting of Human Ch4: Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975-90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557-90662154, Ch4: 90672325-90672709, Ch4: 90678450- 90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4: 90735341-90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, Ch4: 90842771-90843114 and corresponding enhancers in non-human mammals.

15. The method of any one of claims 10-14, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1.

16. The method of any one of claims 10-15, wherein the protein is alpha-synuclein.

17. The method of any one of claims 10-16, wherein the protein is LRRK2.Atty. Docket: JHU-43272.601 18. The method of any one of claim 10-17, wherein the agent and / or composition: (i) treats, prevents, or delays the progression of, microgliosis in the subject; (ii) treats, prevents, or delays the progression of, astrogliosis in the subject; (iii) treats, prevents, or delays the progression of, the synucleinopathy in the subject; (iv) treats, prevents, or delays the progression of, a motor deficit in the subject; (v) treats, prevents, or delays the progression of, cognitive decline in the subject; (vi) treats, prevents, or delays the progression of, Lewy body aggregation in the cortex of the subject; (vii) deletes the CRE that propagates the misfolding and aggregation of the protein encoded by the synucleinopathy-associated gene; and / or (viii) decreases the level of misfolded and aggregated alpha-synuclein protein in the subject.

19. The method of any one of claims 10-18, wherein the synucleinopathy is Parkinson's disease (“PD”).

20. A method of screening for an agent and / or composition which decreases the level and / or activity of a CRE that propagates the misfolding and aggregation of a protein in a neuron and / or glial cell, comprising: (a) contacting the neuron and / or glial cell with a candidate agent and / or composition to evaluate its ability to decrease the level and / or activity of the CRE; and (b) identifying the candidate agent and / or composition as the agent and / or composition that decreases the level and / or activity of the CRE, if the candidate agent and / or composition decreases the transcription level and / or activity of a synucleinopathy-associated gene encoding the protein in the neuron and / or glial cell compared to the transcription level and / or activity of a synucleinopathy- associated gene encoding the protein in a neuron and / or glial cell that has not been contacted with the candidate agent and / or composition.

21. The method of claim 20, comprising contacting the neuron with the agent and / or composition.

22. The method of claims 20 or 21, wherein the neuron is a DA neuron.

23. The method of claim 20, comprising contacting the glial cell with the agent and / or composition.

24. The method of any one of claims 20-23, wherein the CRE is an enhancer.Atty. Docket: JHU-43272.601 25. The method of any one of claims 20-24, wherein the enhancer is selected from the group consisting of Human Ch4: Ch4: 90578786-90579367, Ch4: 90578838-90579505, Ch4: 90629975-90630782, Ch4: 90629981-90630880, Ch4: 90636762-90637480, Ch4: 90642092-90642763, Ch4: 90658878-90659419, Ch4: 90661417-90661898, Ch4: 90661557-90662154, Ch4: 90672325-90672709, Ch4: 90678450- 90679064, Ch4: 90720580-90722663, Ch4: 90720648-90722351, Ch4: 90735341-90735761, Ch4: 90756615-90759904, Ch4: 90757134-90759904, Ch4: 90761441-90762196, Ch4: 90842771-90843114 and corresponding enhancers in a non-human mammal.

26. The method of any one of claims 20-25, wherein the synucleinopathy-associated gene is selected from the group consisting of SNCA, PRKN, UCHL1, PARK7, LRRK2, PINK1, POLG, HTRA2, ATP13A2, FBX07, GIGYF2, GBA2, PLA2G6, EIF4G1, VPS35, DNAJC61, DNAJC13, TMEM230, VPS13C, LRP10, GCH1, MAPT, and / or CPLX1.

27. The method of any one of claims 20-26, wherein the protein is alpha-synuclein.

28. The method of any one of claims 20-27, wherein the protein is LRRK2.

29. The method of any one of claims 21-28, wherein the agent and / or composition comprises a gene editing system.

30. The method of any one of claims 1-29, wherein the agent and / or composition is selected from the group consisting of CRISPR-Cas system gRNA, a siRNA, a shRNA, a miRNA, an antisense DNA, an antisense RNA, a DNAzyme, a Ribozyme, an antagomir, a LMA, a MNA, and / or a chemical / small molecule.

31. The method of any one of claims 20-30, wherein the method is performed in vitro or ex vivo.

32. The method of any one of claims 20-31, wherein the method is performed in vivo in a subject.

33. The method of claim 32, wherein the subject is a mouse.

34. The method of any one of claims 10-33, further comprising evaluating the ability of the agent and / or composition to:Atty. Docket: JHU-43272.601 (i) decrease microgliosis that contributes to the death of the neuron; (ii) decrease astrogliosis that contributes to the death of the neuron; (iii) decrease the level of the CRE in the neuron and / or glial cell; (iv) decrease the activity of the CRE in the neuron and / or glial cell; (v) decrease the transcription level of the synucleinopathy associated gene in the neuron and / or glial cell; (vi) decrease the level of misfolded and aggregated protein encoded by the synucleinopathy- associated gene in the neuron and / or glial cell; (vii) treat, prevent, or delay the progression of, microgliosis in the subject; (viii) treat, prevent, or delay the progression of, astrogliosis in the subject; (ix) treat, prevent, or delay the progression of, a synucleinopathy in the subject; (x) treat, prevent, or delay the progression of, a motor deficit in the subject; (xi) treat, prevent, or delay the progression of, a cognitive deficit in the subject; (xii) treat, prevent, or delay the progression of, Lewy body aggregation in the cortex of the subject; (xiii) delete the CRE that propagates the misfolding and aggregation of the protein encoded by the synucleinopathy-associated gene; and / or (xiv) decrease the level of misfolded and aggregated protein encoded by the synucleinopathy- associated gene in the subject.

Images