Modifying neurons to treat or prevent parkinson's disease

Abstract

Provided herein are compositions and methods of treating and / or preventing Parkinson's Disease (PD) in a subject in need thereof, the methods including administering an agent that increases, enhances, and / or stimulates Kcnn1 expression or activity.

Description

MODIFYING NEURONS TO TREAT OR PREVENT PARKINSON’S DISEASEFIELD

[0001] The present disclosure relates to compositions for treating and / or preventing Parkinson’s Disease (PD). More specifically, the present disclosure relates to compositions for increasing levels and / or activity of the small conductance calcium activated potassium (SK) channel, Kcnnl.CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Nos. U.S. 63 / 554,025, filed February 15, 2024 and 63 / 706,279, filed October 11, 2024, the entire contents of which are hereby incorporated by reference in their entireties.DESCRIPTION OF THE XML FILE SUBMITTED ELECTRONICALLY

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. The XML file, created on February 12, 2025, is named“2681_155PC02_SequenceListing_ST26.xml” and is 20,287 bytes in size.BACKGROUND

[0004] Parkinson’s Disease (PD) is a common neurodegenerative disorder that becomes increasingly prevalent with age and affects about ten million people worldwide. PD presents as a multi-faceted disease characterized by both motor and non-motor symptoms, including tremor, bradykinesia / akinesia, muscular rigidity, postural instability and gait dysfunction, neuropsychiatric disorders (e.g., depression, psychotic symptoms, anxiety, apathy, mild cognitive impairment, and dementia), autonomic dysfunctions, and sleep disturbances.

[0005] The most effective therapeutic strategies available to patients suffering from PD, and aiming at controlling motor symptoms, are primarily indirect and direct dopamine agonists. The classic treatment regimen includes chronic oral intake of L-3, 4-dihydroxyphenylalanine (L-DOPA) which is decarboxylated in the brain to form dopamine, or administration of carbidopa, an inhibitor of Dopa decarboxylase, to correct the loss of dopaminergic functions. Other approaches include administration of dopamine receptor agonists such as apomorphine, which acts on both the DI and D2 receptors subtypes, or pramipexole, ropinirole, and others which are predominantly directed towards D2 receptors subtypes. However, after several years of treatment (ie., the so-called “honeymoon period”), complications often arise due the inherent progression of the disease (e.g., sustained loss of dopaminergic neurons) as well as poor pharmacokinetic (PK) profile of L-DOPA.

[0006] Clinical trials to develop disease-modifying agents for PD have seen numerous failures, such as the various NET-PD trials (Tilley and Galpem (2007), “Screening potential therapies: lessons learned from new paradigms used in Parkinson disease,” Stroke 38 800-803), the ADAGIO trial using the MAO inhibitor rasagiline (Rascol, et al. (2016), “Long-term effects of rasagiline and the natural history of treated Parkinson’s disease,” Mov. Disord. 31 1489-1496), the anti-Synuclein immunotherapy trials (Pagano et al. (2022), “Trial of Prasinezumab in Early-Stage Parkinson’s Disease,” N Engl J Med 2022; 387:421-432) and the trial using the glycosylceramide synthase inhibitors Venglustat (Giladi, et al. (2023), “Safety and efficacy of venglustat in GB Al -associated Parkinson’s disease: an international, multicenter, double-blind, randomized, placebo-controlled, phase 2 trial,” Lancet 22 (8) 661-671), each of which showed no benefit. Trials of intra- striatal injection (into putamen or substantia nigra) of recombinant AAV’s encoding AADC (L- amino acid decarboxylase), aimed to facilitate conversion of L-DOPA to dopamine, glial- derived neurotrophic factor (GDNF), or neurturin have also been carried out at early phase (reviewed and citations to specific studies in Daci and Flotte, “Delivery of adeno-associated virus vectors to the central nervous system for correction of single gene disorders” International Journal of Molecular Sciences 215, 1050-1069, 2024). Further trials are also underway with small molecule inhibitors of leucine rich repeat kinase, LRRK2, which is activated in the most common monogenic heritable form of PD, where it appears that LRRK2 phosphorylates a number of downstream Rab proteins, impairing their functions (Alessi and Pfeffer, “Leucine rich-repeat kinases” Annual Review of Biochemistry 93, 261- 287, 2024). Trials by Denali / Biogen (BI122) and by Brenig Therapeutics (BT-267) are ongoing in patients with dominant-inherited LRKK2 mutations, as well as other PD patients.

[0007] There is currently no cure for PD, and treatment is limited to improving symptoms and / or minimizing discomfort. Thus, there is an urgent need in the art to provide new and effective treatments for PD.SUMMARY

[0008] In aspects, the disclosure provides a method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising administering to the subject an effective amount of an agent that increases, enhances, and / or stimulates an expression (e.g., of protein), amount (e.g., of protein), and / or activity of an SK channel, e.g., without limitation, Kcnnl.

[0009] In aspects, the disclosure provides a method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a nucleic acid agent encoding for Kcnnl, or a fragment or derivative thereof.

[0010] In aspects, the disclosure provides a method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising administering to the subject an effective amount of a cell, the cell having been contacted (e.g., transduced, e.g., ex vivo) with an effective amount of a composition comprising a nucleic acid agent encoding for Kcnnl, or a fragment or variant thereof. In embodiments, the contacting is ex vivo. In embodiments, the cell is derived from the subject, embodiments, the cell is derived from an organism other than the subject, e.g, allogeneic.

[0011] In aspects, there is provided a method for treating or preventing Parkinson’s Disease(PD) in a subject in need thereof, comprising: (i) administering to the nigrostriatal region of the brain of the subject an effective amount of an AAV composition comprising a nucleic acid agent encoding for Kcnnl, or a fragment or variant thereof, or (ii) administering to the nigrostriatal region of the brain of the subject an effective amount of a cell, the cell having been contacted with (e.g., transduced, e.g., ex vivo) an effective amount of an AAV composition comprising a nucleic acid agent encoding for Kcnnl, or a fragment or variant thereof.

[0012] In embodiments, the administering is to the substantia nigra pars compacta of the brain of the subject. In embodiments, the administering is to the striatum (e.g., putamen) of the brain of the subject. In embodiments, the administering is by injection. In embodiments,the cell is derived from the subject (e.g., via iPSC production and differentiation) or the cell is derived from another organism (e.g., allogeneic). In embodiments, the cell is dopamine- producing cell derived from the subject, e.g., via iPSC production and differentiation. In embodiments, the cell is a fetal ventral mesencephalic cell.

[0013] In embodiments, the method reduces or prevents alpha-synuclein spread from endogenous affected cells, e.g., into a nigral graft. In embodiments, and without wishing to be bound by theory, Kcnnl transduction ex vivo causes protection of the grafted cells from spread of alpha synuclein from neighboring affected cells into the graft with damage / loss of the grafted neurons.

[0014] In embodiments, the agent encoding for Kcnnl, or a fragment or derivative thereof, is an expression cassette comprising: (a) a polynucleotide encoding Kcnnl, or a fragment or derivative thereof, operably linked to an expression control element; (b) an adeno- associated virus (AAV) inverted repeat (ITR) flanking the 5’ terminus of the polynucleotide; and (c) an AAV ITR flanking the 3’ terminus of the polynucleotide.

[0015] In embodiments, the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 1, 3, or 9, or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10, or a functional variant thereof having at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a codon-optimized form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10. In embodiments, the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a CpG-reduced form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10.

[0016] In embodiments, the expression control element is positioned 5’ of the polynucleotide encoding Kcnnl. In embodiments, the expression control element is CpG- reduced compared to the wild type expression control element. In embodiments, the expression control element comprises one or more of a cytomegalovirus (CMV) enhancerpromoter, a CMV enhancer fused to the chicken P-actin promoter (CAG), a chicken P-actinpromoter (CBA), a simian vacuolating virus 40 (SV40) enhancer-promoter, a polyubiquitin C gene promoter (UBC), an elongation-factor la subunit (EF-la) promoter, and a phosphoglycerate kinase promoter (PGK). In embodiments, the expression control element comprises a CMV enhancer-promoter and comprises a polynucleotide sequence of SEQ ID NO: 8 or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the expression control element comprises an enhancer-promoter combination or a promoter that is tissue-specific and / or inducible. In embodiments, the tissue-specific enhancer-promoter combination or promoter that is a neuron-specific enhancer-promoter combination or promoter. In embodiments, the neuron-specific enhancer-promoter or promoter is selected from a neuron-specific enolase (ENO2), a platelet-derived growth factor a-chain (PDGFA), a platelet-derived growth factor P-chain (PDGFB), a synapsin (SYN1), a methyl-CpG binding protein 2 (MECP2), a Ca2+ / calmodulin-dependent protein kinase II (CAMK2G), metabotropic glutamate receptor 2 (GRM2), a neurofilament light (NEFL) or heavy (NEFH) chain, a proenkephalin (PENK), an excitatory amino acid transporter 2 (SLC1A2) and a synthetic promoter (e.g., designed using artificial intelligence). In embodiments, the tissue-specific promoter or enhancer-promoter combination is a nigrostriatal-specific promoter or enhancer-promoter combination.

[0017] In embodiments, the 5’ ITR and the 3’ ITR are independently selected from a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74 and AAV3B ITR, or self-complementary versions thereof. In embodiments, the 5’ ITR and / or the 3’ ITR is modified to have reduced CpGs. In embodiments, the 5’ ITR and / or the 3’ ITR comprises a polynucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7 or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

[0018] In embodiments, the expression cassette further comprises a polyadenylation sequence positioned 3’ of the polynucleotide encoding Kcnnl, or a fragment or variant thereof. In embodiments, the expression cassette further comprises one or more of an intron, a transcriptional termination signal, an miRNA, and a post-transcriptional regulatory element (PRE). In embodiments, the expression cassette comprises, from 5’ to 3’ : a 5’ ITR, a CMV enhancer-promoter, an intron, a Kcnnl cDNA, a transcriptional terminationsequence, a poly A site, and a 3’ ITR. In embodiments, the expression cassette comprises, from 5’ to 3’: a 5’ ITR, a CMV enhancer-promoter, an intron, a human Kcnnl cDNA, a transcriptional termination sequence, a poly A site, and a 3’ ITR.

[0019] In embodiments, the agent encoding for Kcnnl, or a fragment or variant thereof, is a cDNA encoding Kcnnl, or a fragment or variant thereof. In embodiments, the cDNA encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 1, 3, or 9, or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the cDNA encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10, or a functional variant thereof having at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the cDNA encoding Kcnnl, or a fragment or variant thereof, comprises a codon optimized form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10. In embodiments, the cDNA encoding Kcnnl, or a fragment or variant thereof, comprises a CpG-reduced form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10.

[0020] In embodiments, the agent encoding for Kcnnl, or a fragment or variant thereof, is an mRNA encoding Kcnnl, or fragment thereof. In embodiments, the mRNA encoding Kcnnl, or a fragment or variant thereof, is transcribed from a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 1, 3, or 9, or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the mRNA encoding Kcnnl, or a fragment or variant thereof, is transcribed from a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10, or a functional variant thereof having at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the mRNA encoding Kcnnl, or a fragment or variant thereof, comprises a modified mRNA. In embodiments, the modified mRNA comprise at least one modified nucleoside optionally selected from a pseudouridine, 5-methylcytosine (m5C), 5-methyluridine (m5U), 2'-O- methyluridine (Um or m2'-0U), 2-thiouridine (s2U), and N6-methyladenosine (m6A).

[0021] In aspects, the disclosure provides a viral vector comprising an expression cassette disclosed herein. In embodiments, the viral vector comprises a recombinant adeno- associated virus (rAAV) vector. In embodiments, the AAV is selected from an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid serotype, a recombinant AAV (rAAV), or a functional variant of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9. In embodiments, the AAV is selected from an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, including self-complementary versions. In aspects, the AAV is an AAV9 or other AAV with a capsid altered to include a transferrin receptor binding element (e.g., the seven amino acid segment in Huang et al., “An AAV capsid reprogrammed to bind human transferrin receptor mediates brain-wide gene delivery”, Science 384, 1220-1227, 2024) or other element that facilitates blood-brain barrier passage of virus particles, allowing IV injection and selective / efficient transfer to the CNS. In embodiments, the viral vector comprises a recombinant anellovirus vector. In embodiments, the anellovirus vector is selected from TT virus (TTV), Torquetenomini virus (TTMV), and Torquetenomidivirus (TTMVD).

[0022] In aspects, the disclosure provides a method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising administering to the subject an effective amount of an agent that increases, enhances, and / or stimulates Kcnnl expression (e.g, of protein), amount (e.g., of protein), and / or activity. In aspects, the disclosure provides a method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising administering to the subject an effective amount of a cell, the cell having been contacted with an effective amount of an agent that increases, enhances, and / or stimulates Kcnnl expression (e.g., of protein), amount (e.g., of protein), and / or activity. In embodiments, the contacting is transduction. In embodiments, the contacting is ex vivo. In embodiments, the cell is derived from the subject (e.g, via iPSC production and differentiation) or the cell is derived from another organism, e.g., allogeneic. In embodiments, the cell is dopamine-producing cell derived from the subject, e.g., via iPSC production and differentiation. In embodiments, the cell is a fetal ventral mesencephalic cell. In embodiments, the agent that increases, enhances, and / or stimulates Kcnnl expression (e.g., of protein), amount (e.g., of protein), and / or activity is a small molecule optionally selected from (-)CM-TPMF, chlorzoxazone, DCEBIO, 1-EBIO, and riluzole, or a molecule derived therefrom.

[0023] In embodiments, the subject is a human subject. In embodiments, the human subject is at least 40 years old, at least 45 years old, at least 50 years old, at least 55 years old, at least 60 years old, at least about 65 years old, at least about 70 years old, at least about 75 years old, at least about 80 years old, or at least about 85 years old.

[0024] In embodiments, the subject demonstrates one or more of bradykinesia, akinesia, tremors in the hands, fingers, forearm, foot, mouth, or chin, muscular rigidity, poor balance, postural instability and gait dysfunction, Parkinsonian gait, neuropsychiatric disorders, depression, psychotic symptoms, anxiety, apathy, mild-cognitive impairment, dementia, autonomic dysfunctions, and sleep disturbances. In embodiments, the PD has responded poorly to a previous therapy, optionally conventional L-DOPA therapy.

[0025] In embodiments, the method prevents or delays progression from (i) a Stage 1 PD to a Stage 2 PD, a Stage 3 PD, a Stage 4 PD, or a Stage 5 PD; (ii) a Stage 2 PD to a Stage 3 PD, a Stage 4 PD, or a Stage 5 PD; (iii) a Stage 3 PD to a Stage 4 PD or a Stage 5 PD; or (iv) a Stage 4 PD to a Stage 5 PD. In embodiments, the method prevents or delays progression from (i) a mild / early PD to a moderate / mid-stage PD or a severe / advanced PD; or (ii) a moderate / mid-stage PD to a severe / advanced PD. In embodiments, the method prevents paralysis. In embodiments, the method reduces, ablates, or slows the progression of one or more symptoms of PD. In embodiments, the one or more symptoms are selected from bradykinesia, akinesia, tremors in the hands, fingers, forearm, foot, mouth, or chin, muscular rigidity, poor balance, postural instability and gait dysfunction, Parkinsonian gait, neuropsychiatric disorders, depression, psychotic symptoms, anxiety, apathy, mild- cognitive impairment, dementia, autonomic dysfunctions, and sleep disturbances.

[0026] In embodiments, the method improves and / or increases and / or enhances anti-PD efficacy, compared to treatment with conventional L-DOPA therapy. In embodiments, the method promotes or enhances clearance of a pathogenic cytosolic protein, compared to conventional L-DOPA therapy. In embodiments, the pathogenic cytosolic protein is wild type alpha-synuclein, serine- 129-phosphorylated alpha-synuclein, a A53T mutant form of alpha-synuclein, a E46K mutant form of alpha-synuclein, a H50Q mutant form of alpha- synuclein, a G51D mutant form of alpha-synuclein, a A30P mutant form of alpha- synuclein, tau, TAR DNA-binding protein 43 (TDP-43), and / or superoxide dismutase 1 (SOD1), or potentially abnormal secreted proteins. In embodiments, the method reduces, prevents, and / or delays accumulation of the pathogenic serine- 129-phosphorylated form of alpha-synuclein, compared to treatment with conventional L-DOPA therapy. Inembodiments, the method promotes or enhances neuronal protection from oxidative damage, compared to conventional L-DOPA therapy. In embodiments, the method promotes or enhances sparing of pre- or postsynaptic dopaminergic terminals, compared to conventional L-DOPA therapy.

[0027] In embodiments, the expression cassette, cDNA, mRNA, viral vector, agent, or pharmaceutical composition is administered by intrastriatal injection, substantia nigra pars compacta delivery, striatum (e.g., putamen) delivery and / or nigrostriatal region delivery.

[0028] In embodiments, the method further comprises evaluating analysis of a biological fluid. In embodiments, the biological fluid is cerebrospinal fluid (CSF) or blood. In embodiments, the method further comprises evaluating analysis of a brain image. In embodiments, the brain image is from one or more of computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI).

[0029] In aspects, the disclosure provides a pharmaceutical composition comprising an expression cassette, cDNA, mRNA, viral vector, or agent described herein, and a pharmaceutically accepted excipient, carrier or diluent. In embodiments, the composition is suitable for intrastriatal injection delivery, substantia nigra pars compacta delivery, striatum (e.g., putamen) delivery, and / or nigrostriatal region delivery.

[0030] In aspects, the disclosure provides a Parkinson’s Disease (PD) therapy comprising an effective amount of an expression cassette, cDNA, mRNA, viral vector, agent, or pharmaceutical composition described herein.

[0031] In aspects, the disclosure provides a method of making a PD therapy comprising (a) identifying the PD therapy by: (i) administering an effective amount of a test agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal; (ii) identifying the test agent as a PD therapy if the transgenic animal demonstrates one or more of increased expression of Kcnnl, increased activity of the gene product of Kcnnl, improved survival time, or reduced paralysis, as compared to a control; and (b) formulating the candidate agent for administration for the treatment of PD.

[0032] In aspects, the disclosure provides a method of making a PD therapy comprising (a) identifying a candidate agent that modulates Kcnnl, comprising: (i) providing a cell line expressing Kcnnl; (ii) treating the cell line with one or more test agents; (iii) measuring a voltage-independent potassium channel activity in the cell; and (iv) identifying the test agent as a candidate agent if voltage-independent potassium channel activity is increased in the presence of the test agent, compared to in the absence of the test agent and / or presenceof a control agent that is known not to be a candidate agent; and (b) administering an effective amount of the candidate agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal; (c) classifying the candidate agent as a PD therapy if the transgenic animal demonstrates one or more of increased expression of Kcnnl, increased activity of the gene product of Kcnnl, improved survival time, or reduced paralysis, as compared to a control; and (d) formulating the PD therapy for administration for the treatment of PD.

[0033] In aspects, the disclosure provides a method of making a PD therapy comprising (a) identifying a candidate agent that modulates Kcnnl, comprising: (i) providing a cell line expressing Kcnnl; (ii) treating the cell line with one or more test agents; (iii) measuring the effects on stress-response elements in the cell; and (iv) identifying the test agent as a candidate agent if one or more stress response is increased in the presence of the test agent, compared to in the absence of the test agent and / or presence of a control agent that is known not to be a candidate agent; and (b) administering an effective amount of the candidate agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal; (c) classifying the candidate agent as a PD therapy if the transgenic animal demonstrates one or more of increased expression of Kcnnl, increased activity of the gene product of Kcnnl, improved survival time, or reduced paralysis, as compared to a control; and (d) formulating the PD therapy for administration for the treatment of PD.

[0034] In aspects, there is provided a method of making a PD therapy, comprising: (a) identifying a candidate agent that modulates a cellular stress response, comprising: (i) providing a cell line overexpressing Kcnnl and having a cellular stress response substantially similar to a cellular stress response of spinal cord motor neurons overexpressing Kcnnl, (ii) treating the cell line with one or more test agents, (iii) measuring cellular stress responses in the cell, and (iv) identifying the test agent as a candidate agent if one or more of the cellular stress responses is similarly increased in the presence of the test agent, compared to in the absence of the test agent and / or presence of a control agent that is known not to be a candidate agent; and (b) administering an effective amount of the candidate agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal; (c) classifying the candidate agent as a PD therapy if the transgenic animal demonstrates one or more of increased expression of stress-related proteins in spinal cord neurons, improved survival time, or reduced paralysis, as compared to a control; and (d) formulating the PD therapy for administration for the treatment of PD, optionally whereinthe one or more of the cellular stress responses comprises one or more of ER, integrated, and mitochondrial cellular stress responses.

[0035] In aspects, the disclosure provides a method of identifying a PD therapy that is effective in treating or preventing PD comprising administering an effective amount of the agent to an A53T mutant alpha-synuclein transgenic animal and determining time to paralysis, wherein an increased time to paralysis compared to an A53T mutant alpha- synuclein transgenic animal that does not receive the agent indicates that the agent is a PD therapy.

[0036] In aspects, there is provided a method of making a PD therapy, comprising: (a) identifying a candidate agent that mimics the activity of Kcnnl, comprising: (i) providing a neuronal cell line or primary cultured neurons; (ii) treating the neuronal cell line or primary cultured neurons with one or more test agents, (iii) measuring the levels of selected stress-response elements in the neuronal cell lines or primary cultured neurons, and (iv) identifying the test agent as a candidate agent if stress response activity is increased in the presence of the test agent, compared to in the absence of the test agent and / or presence of a control agent that is known not to be a candidate agent; and (b) administering an effective amount of the candidate agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal; (c) classifying the candidate agent as a PD therapy if the transgenic animal demonstrates one or more of improved survival time and reduced motor compromise as compared to the wild type animal control; and (d) formulating the PD therapy for administration for the treatment of PD optionally wherein the test agent is a small molecule or peptide, and / or the candidate agent is a small molecule or peptide.

[0037] In embodiments, the PD therapy is a gene therapy, a biologic agent, a small molecule, or a polynucleotide agent. In embodiments, the gene therapy is a viral gene therapy. In embodiments, the viral gene therapy is or comprises a recombinant adeno- associated virus (rAAV). In embodiments, the rAAV comprises a Kcnnl nucleic acid. In embodiments, the biologic agent is an antibody or peptide. In embodiments, the polynucleotide agent is selected from mRNA, siRNA, shRNA, miRNA, and cDNA. In embodiments, the small molecule mimics action of overexpression of Kcnnl by preventing appearance of phospho-serine- 129 alpha-synuclein or by promoting phospho-serine- 129 alpha-synuclein clearance. In embodiments, the peptide mimics action of overexpression of Kcnnl.

[0038] In embodiments, the methods described herein further comprise evaluating a biological sample for levels of one or more ER stress response biomarkers, one or more integrated stress response (ISR) biomarkers, and / or one or more mitochondrial stress response biomarkers.

[0039] In embodiments, the biological sample is cerebrospinal fluid (CSF) or blood.

[0040] In embodiments, the methods described herein cause one or more of an ER stress response, an integrated stress response (ISR), and a mitochondrial stress response.

[0041] In embodiments, the methods described herein cause an ER stress response, wherein expression of one or more of Creb3Ll, GADD34, XBP1, ATF4, CHOP, Trib3, Sestrin2, ASNS, ATF5, ATF6, WFS1, Sec61 beta, Sec61 gamma, TRAM, Ssrl, Ssr4, SERP1, BiP, DnaJB9, GRP94, Cry AB, HYOU, Calnexin, Glucosidase 2, PdiA3, PdiA4, PdiA6, P4hb, Ostc, Magtl, TMEM258, Lman, GRASP55, Derl3, Sdf211, Herpudl, Edem2, and FBXO6 are increased or elevated relative to untreated or pre-treated.

[0042] In embodiments, the methods described herein cause an integrated stress response (ISR), wherein phosphorylation levels of Ser51 of eIF2alpha are increased or elevated relative to untreated or pre-treated, and / or induction of ATF4 transcription factor with attendant nuclear localization.

[0043] In embodiments, the methods described herein cause a mitochondrial stress response, wherein expression of one or more of FGF21, GDF15, Atf5, Aifm2, and MAVS are increased or elevated relative to untreated or pre-treated.

[0044] In aspects, the disclosure provides a method for evaluating the therapeutic benefit of a method for treating or preventing Parkinson’ s Disease (PD) in a subj ect in need thereof, comprising: (i) determining a baseline expression and / or activity level of one or more ER stress response biomarkers, one or more integrated stress response (ISR) biomarkers, and / or one or more mitochondrial stress response biomarkers at a first timepoint; (ii) administering to the subject the pharmaceutical composition described herein; (iii) determining the expression and / or activity level of the one or more ER stress response biomarkers, the one or more integrated stress response (ISR) biomarkers, and / or the one or more mitochondrial stress response biomarkers at a second timepoint; and (iv) determining whether the expression and / or activity level of the one or more ER stress response biomarkers, the one or more integrated stress response (ISR) biomarkers, and / or the one or more mitochondrial stress response biomarkers is increased between the first and second timepoints.

[0045] In embodiments, the ER stress response biomarkers are selected from Creb3Ll, GADD34, XBP1, ATF4, CHOP, Trib3, Sestrin2, ASNS, ATF5, ATF6, WFS1, Sec61 beta, Sec61 gamma, TRAM, Ssrl, Ssr4, SERP1, BiP, DnaJB9, GRP94, Cry AB, HYOU, Calnexin, Glucosidase 2, PdiA3, PdiA4, PdiA6, P4hb, Ostc, Magtl, TMEM258, Lman, GRASP55, Derl3, Sdf211, Herpudl, Edem2, and FBXO6.

[0046] In embodiments, the integrated stress response (ISR) biomarker is phosphorylation level of Ser51 of eIF2alpha, or induction of ATF protein.

[0047] In embodiments, the mitochondrial stress response biomarkers are selected from FGF21, GDF15, Atf5, Aifm2, and MAVS.BRIEF DESCRIPTION OF THE DRAWINGS

[0048] FIGs.lA-lB illustrate Kaplan-Meier survival plots for transgenic mice. FIG. 1A shows a Kaplan-Meier survival plot for transgenic mice carrying Thyl promoter-driven A53T alpha-synuclein alone in a B6 / SJL background (designated A53T line). FIG. IB shows a Kaplan-Meier survival plot for transgenic mice carrying A53T alone in the B6 / SJL background or such mice also hemizygously transgenic for either ~3 copies or ~6 copies of mouse Kcnnl (Kcnnl-3 or Kcnnl-6, respectively), produced by crossing A53T / B6SJL mice to transgenic Kcnnl / B6SJL mouse lines with the respective copy number (~3 copies or ~6 copies as measured by real time PCR of tail DNA).

[0049] FIG. 2 illustrates four end-stage A53T mice.

[0050] FIG. 3 depicts brain A53T human alpha-synuclein RNA levels of A53T / B6SJL mice, A53T / Kcnnl-3 / B6SJL mice, and A53T / Kcnnl-6 / B6SJL mice, measured as the ratio of RT-qPCR measurements of human A53T alpha-synuclein RNA relative to (control) GAPDH level (the latter a constant in these studies relative to amount of total RNA).

[0051] FIG. 4 illustrates clearance of A53T alpha-synuclein-mCherry by Kcnnl coexpression in a cultured cell system.

[0052] FIGs. 5A-5G illustrate histologic sagittal brain sections and a spinal cord cross section stained with antibody to the pathogenic serine 129-phosphorylated form of alpha- synuclein, from the brain of an A53T alpha synuclein end stage mouse (8 months; note that no immunostaining in any brain region was observed in 2-month-old mice that were similarly studied). FIG. 5A presents a reference sagittal view of an adult mouse brain at 1.2 mm (1200 microns) from the midline, capturing relevant structures, as labeled in theAllen Brain Atlas. FIGs. 5B-5E show immunostained images from that sagittal regiomcortex (CTX) and hippocampus (HP), including an expanded view of subfield CA3 (FIG. 5B); striatum (CP, caudate / putamen, in the reference diagram), thalamus (TH), subthalamic region including zona incerta (ZI) and subthalamic nucleus (STN), and substantia nigra (SN) (FIG. 5C); superior colliculus (SC), deep cerebellar nuclei (DCN), and vestibular nucleus (Ve, in medulla) (FIG. 5D); and a cross-section of cervical spinal cord, including an expanded view of motor neurons in the ventral horn (FIG. 5E). In additional sagittal section immunostaining analyses (FIG. 5F), sections from an endstage A53T transgenic brain were compared with those from a 1 year-old A53T / Kcnnl-6. Whereas noticeable punctate staining was observed in the deep cortical region in the A53T brain, no serine-129 phosphorylated alpha-synuclein staining was observed in the corresponding cortical region of the A53T / Kcnnl-6 mouse (lower left), where cortical neurons were observed to be strongly expressing Kcnnl (lower right). This supports, without wishing to be bound by theory, that Kcnnl-6 is affording protection from formation of the pathogenic (serine 129-phosphorylated) form of alpha-synuclein. FIG. 5G illustrates superior colliculus of an endstage A53T mouse and a 1 year-old asymptomatic A53T / Kcnnl-6 mouse, stained for both phospho-serine- 129 alpha-synuclein and Kcnnl.

[0053] FIG. 6 shows electron microscopy (EM) images of sections taken from the spinal cord of a mouse that is transgenic only for Kcnnl (the protecting gene; homozygous Thy 1- Kcnnl-3). Autophagosomes in the lower image are labeled “APG”.

[0054] FIG. 7 shows an EM image of sections taken from the spinal cord of a mouse that is transgenic only for Kcnnl (the protecting gene; homozygous Thy 1 -Kcnnl -3). Multivesicular bodies are labeled “MVB”.

[0055] FIG. 8 shows EM images of sections of spinal cord motor neurons from Kcnnl homozygous mice. Arrows point to invaginations in the nuclear envelope.

[0056] FIG. 9 shows EM images of cultured HeLa cells transiently transfected with a control plasmid (Bluescript) and with Kcnnl.

[0057] FIG. 10A shows an EM image of a spinal cord motor neuron from a 4 month old Kcnnl-6 mouse. Expanded whorls of ER are shown. The black objects lacking surrounding membrane are lipofuscin granules, and the dark objects surrounded by membrane are lysosomes.

[0058] FIG. 10B shows that overexpressed Kcnnl colocalizes with the ER. The immunostaining pattern of overexpressed Kcnnl in a spinal cord motor neuron of a Kcnnl-6 mouse (top left panel) matches closely to that of the endogenous ER-membrane- associated protein calnexin (top right panel).

[0059] FIG. 11 shows a heat map gene ontology analysis of RNAseq data comparing laser captured spinal cord motor neurons from Kcnnl-6 transgenic mice with those from B6SJL mice.

[0060] FIGs. 12A-12B show evaluation of an integrated stress response (ISR), and in particular, immunostaining with anti-phosphorylated-S51-eIF2alpha (FIG. 12A) and anti- ATF4 (FIG. 12B), comparing spinal cord sections of Kcnnl-6 / + mouse with nontransgenic (B6SJL). Anti-ChAT staining identifies motor neurons. Scale bar 20 microns in (FIG. 12A), 5 microns in (FIG. 12B).DETAILED DESCRIPTION

[0061] The following description and examples illustrate embodiments of the present disclosure in detail.

[0062] It is to be understood that the present disclosure is not limited to the particular embodiments described herein and as such can vary. Those of skill in the art will recognize that there are variations and modifications of the present disclosure, which are encompassed within its scope.

[0063] All terms are intended to be understood as they would be understood by a person skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosure pertains.

[0064] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0065] Although various features of the disclosure can be described in the context of a single embodiment, the features can also be provided separately or in any suitable combination. Conversely, although the present disclosure can be described herein in the context of separate embodiments for clarity, the present disclosure can also be implemented in a single embodiment.

[0066] The following definitions supplement those in the art and are directed to the current application and are not to be imputed to any related or unrelated case, e.g. , to any commonly owned patent or application. Although any methods and materials similar or equivalent tothose described herein can be used in the practice for testing of the present disclosure, the preferred materials and methods are described herein. Accordingly, the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.I. Overview

[0067] The present disclosure is based, inter alia, on the discovery that Kcnnl modulation is useful to treat Parkinson’s Disease (PD). The inventors of the present disclosure have surprisingly found that polynucleotides and expression constructs comprising polynucleotides encoding mouse Kcnnl can be used to effectively treat or prevent disease in a model of PD. An rAAV identical to one described here has also been used in vivo to improve survival of G93A SOD1 ALS mice, and is being used here to test therapy of the A53T mouse model of PD.

[0068] In embodiments, increasing an amount of protein and / or activity (e.g., without limitation, by overexpression) of Kcnnl, but not Kcnn2, can be used to effectively treat or prevent PD. In embodiments, increasing an amount of protein and / or activity (e.g., without limitation, by overexpression) of mouse or modified human Kcnnl, but not Kcnn2 or human KCNN1, can be used to effectively treat or prevent PD.

[0069] Without wishing to be bound by theory, increasing an amount of protein and / or activity (e.g., without limitation, by overexpression) of Kcnnl delays accumulation of the toxic alpha-synuclein protein, i.e., the pathogenic serine- 129-phosphorylated form of alpha-synuclein associated with PD. Without wishing to be bound by theory, increasing the amount of protein and / or activity (e.g., without limitation, by overexpression) of Kcnnl prevents formation of, causes, stimulates, or increases clearance of the pathogenic alpha- synuclein. In embodiments, clearance refers to preventing accumulation of or facilitating removal of pathogenic protein via the endolysosomal pathway or via an as yet unknown Kcnnl -dependent intracellular pathway. In embodiments, transgenic overexpression of Kcnnl delays accumulation of the pathogenic serine- 129-phosphorylated form of alpha- synuclein by way of an autophagosomal clearance mechanism that ingests cytosol, including pathogenic proteins such as wild type alpha-synuclein, serine- 129- phosphorylated alpha-synuclein, a A53T mutant form of alpha-synuclein, a E46K mutant form of alpha-synuclein, a H50Q mutant form of alpha-synuclein, a G51D mutant form of alpha-synuclein, a A30P mutant form of alpha-synuclein, tau, TAR DNA-binding protein43 (TDP-43), and / or superoxide dismutase 1 (SOD1), or potentially abnormal secreted proteins.

[0070] In embodiments, the Kcnnl is nonassembled. In embodiments, the Kcnnl is misfolded. In embodiments, the Kcnnl is not in the form of an active ion channel. In embodiments, the Kcnnl is a monomer. In embodiments, the Kcnnl is in the form of a superstructure.

[0071] In embodiments, the methods described herein cause one or more of an ER stress response, an integrated stress response (ISR), and a mitochondrial stress response. In embodiments, methods described herein cause an ER stress response, wherein expression of one or more of Creb3Ll, GADD34, XBP1, ATF4, CHOP, Trib3, Sestrin2, ASNS, ATF5, ATF6, WFS1, Sec61 beta, Sec61 gamma, TRAM, Ssrl, Ssr4, SERP1, BiP, DnaJB9, GRP94, Cry AB, HYOU, Calnexin, Glucosidase 2, PdiA3, PdiA4, PdiA6, P4hb, Ostc, Magtl, TMEM258, Lman, GRASP55, Derl3, Sdf21I, Herpudl, Edem2, and FBXO6 are increased or elevated relative to untreated or pre-treated. In embodiments, methods described herein cause an ER stress response, wherein expression of one or more of the genes of Table 1 are increased or elevated relative to untreated or pre-treated.

[0072] In embodiments, the methods described herein cause an integrated stress response (ISR), wherein phosphorylation levels of Ser51 of eIF2alpha are increased or elevated relative to untreated or pre-treated, and / or levels of ATF4 are increased and its nuclear localization is elevated.

[0073] In embodiments, the methods described herein cause a mitochondrial stress response, wherein expression of one or more of FGF21, GDF15, Atf5, Aifm2, and MAVS are increased or elevated relative to untreated or pre-treated. In embodiments, methods described herein cause an mitochondrial stress response, wherein expression of one or more of the genes of Table 2 are increased or elevated relative to untreated or pre-treated.II. Treatment of Parkinson’s Disease (PD)

[0074] In aspects, the disclosure provides a method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising administering to the subject an effective amount of a composition comprising a nucleic acid agent encoding for Kcnnl, or a fragment or variant thereof.

[0075] In aspects, the disclosure provides a method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising administering to the subject aneffective amount of a cell, the cell having been contacted (e.g., transduced, e.g., ex vivo) with an effective amount of a composition comprising a nucleic acid agent encoding for Kcnnl, or a fragment or variant thereof. In embodiments, the contacting is ex vivo. In embodiments, the cell is derived from the subject (e.g., via iPSC production and differentiation) or the cell is derived from another organism (e.g., allogeneic). In embodiments, the cell is dopamine-producing cell derived from the subject, e.g., via iPSC production and differentiation. In embodiments, the cell is a fetal ventral mesencephalic cell.

[0076] In aspects there is provided a method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising: (i) administering to the nigrostriatal region of the brain of the subject an effective amount of an AAV composition comprising a nucleic acid agent encoding for Kcnnl, or a fragment or variant thereof, or (ii) administering to the nigrostriatal region of the brain of the subject an effective amount of a cell, the cell having been contacted (e.g., transduced, e.g., ex vivo) with an effective amount of an AAV composition comprising a nucleic acid agent encoding for Kcnnl, or a fragment or variant thereof.

[0077] In embodiments, the administering is to the substantia nigra pars compacta of the brain of the subject. In embodiments, the administering is to the striatum (e.g., putamen) of the brain of the subject. In embodiments, the administering is by injection. In embodiments, the cell is derived from the subject (e.g., via iPSC production and differentiation) or the cell is derived from another organism (e.g., allogeneic). In embodiments, the cell is dopamine- producing cell derived from the subject, e.g., via iPSC production and differentiation. In embodiments, the cell is a fetal ventral mesencephalic cell.

[0078] In embodiments, the method reduces or prevents alpha-synuclein spread from endogenous affected cells, e.g., into a nigral graft. In embodiments, and without wishing to be bound by theory, Kcnnl transduction ex vivo causes protection of the grafted cells from spread of alpha synuclein from neighboring affected cells into the graft with damage / loss of the grafted neurons.

[0079] In embodiments, the methods and compositions described herein are used to treat one or more symptoms associated with PD. In embodiments, methods and compositions described herein are used to slow the progression of PD.

[0080] In embodiments, the methods described herein further comprise evaluating analysis of a biological fluid of the subject. In embodiments, the biological fluid is cerebrospinalfluid (CSF) or blood. In embodiments, methods described herein further comprise evaluating analysis of a brain image. In embodiments, the brain image is from one or more of computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI).

[0081] Human alpha-synuclein is an abundant neuronal protein of 140 amino acids, principally located in axonal terminals, that regulates presynaptic vesicle trafficking and subsequent neurotransmitter release. In a set of conditions known as synucleinopathies, including Parkinson’s Disease (PD), Parkinson’s Disease Dementia (PDD), and Dementia with Lewy bodies (DLB), alpha-synuclein forms filamentous inclusions known as Lewy bodies (in the cell soma) or Lewy neurites (in axons). In PD, such inclusions develop in the dopaminergic neurons of the substantia nigra, which are ultimately lost, with attendant loss of axonal nigrostriatal fibers. The consequent loss of dopamine in the striatum is associated with motor symptoms featuring resting tremor, muscle rigidity, bradykinesia, postural instability, and gait disturbance. More widespread alpha-synuclein pathology in neocortical areas leads to PDD or DLB, which are associated with cognitive decline and other neurologic features (e.g., apathy, depression, psychosis). In an additional synucleinopathy, multiple system atrophy (MSA), alpha-synuclein inclusions form in the cytosol of oligodendrocytes, and clinical features resemble those of PD but include prominent autonomic dysfunction and poor responsiveness to L-DOPA.

[0082] PD is the second most common neurodegenerative disease, and approximately 90% of cases are sporadic in character, with no family history and without an identifiable mutation, while -10% comprise heritable mutations in causal monogenic so-called PARK genes. Sporadic cases may involve both environment and complex genetics. Environmental exposure to pesticides / herbicides or to prior head trauma have been shown to increase disease risk. The collection of -20 known PARK genes includes alpha-synuclein itself. Kindreds with a dominant-inherited A53T mutation in alpha-synuclein were identified in 1997 (Polymeropoulos et cd.. “Mutation in the alpha-synuclein gene identified in families with Parkinson's disease,” Science. 1997 Jun 27;276(5321):2045-7), providing the first link between alpha-synuclein and PD. Immediately afterward, alpha-synuclein was identified by immunostaining to be a major component of Lewy bodies in patients with sporadic PD or with DLB (Spillantini et al. “Alpha-synuclein in Lewy bodies,” Nature. 1997 Aug 28;388(6645):839-40). In further studies of several kindreds with PD, multiplication of the alpha-synuclein gene, producing increased expression, was also shown to be associatedwith PD (Singleton et al. , “alpha-Synuclein locus triplication causes Parkinson's disease,” Science. 2003 Oct 31;302(5646):841; Chartier-Harlin et al., “Alpha-synuclein locus duplication as a cause of familial Parkinson's disease,” Lancet. 2004 Sep 25-Oct l;364(9440): 1167-9).

[0083] Another dominant-transmitting cause of PD involves mutation of LRRK2 (Paisan- Ruiz et al., “Cloning of the gene containing mutations that cause PARK8-linked Parkinson's disease,” Neuron. 2004 Nov 18;44(4):595-600; Zimprich et al., “Mutations in LRRK2 cause autosomal-dominant parkinsonism with pleomorphic pathology,” Neuron. 2004 Nov 18;44(4):601-7), a large protein whose dominantly-inherited G2019S kinase- activating mutation suppresses lysosomal degradative activity. Many of these patients accrete alpha-synuclein in Lewy bodies. Heterozygous mutation in the lysosomal enzyme glucocerebrosidase (GBA) increases risk of PD, and these patients uniformly accrete alpha- synuclein (Sidransky and Lopez, “The link between the GBA gene and parkinsonism,” Lancet Neurol. 2012 Nov; 11(11):986-98). Risk for PD is also conferred by >90 variants in other regions of the genome, identified by GWAS studies, that individually confer a small increase of lifetime risk for what would likely be synuclein-associated PD (Blauwendraat et al., “The genetic architecture of Parkinson's disease,” Lancet Neurol. 2020 Feb; 19(2): 170-178).

[0084] From early pathology studies, it appeared that alpha-synuclein-immunopositive Lewy neurites and Lewy bodies could spread progressively in a connected anatomic pattern in the course of PD (Braak etal., “Staging of brain pathology related to sporadic Parkinson's disease,” Neurobiol Aging. 2003 Mar-Apr;24(2): 197-211). Braak proposed commencement in the anterior olfactory system and dorsal motor nucleus of cranial nerves IX / X, ascending to substantia nigra and striatum to produce dopaminergic cell death, and ultimately to cortex. Thus, the topographic pattern could indicate the stage of PD. A more recent study in mice has extended a proposed vagus nerve origin to suggest a gut-to-brain transmission of alpha-synuclein: preformed alpha-synuclein fibrils injected into the muscular layer of the pylori s / duodenum reached the dorsal motor nucleus, then were found in caudal portions of hindbrain and eventually in substantia nigra. Truncal vagotomy prevented the spread of synuclein (Kim et al., “Transneuronal Propagation of Pathologic a-Synuclein from the Gut to the Brain Models Parkinson's Disease,” Neuron. 2019 Aug 21 ; 103(4):627-641). This raises the question of a gut origin of PD. Within the CNS, spread was further demonstrated when a graft of fetal nigral neurons into the striatum of a PDpatient was examined at postmortem 14 years later and found to contain Lewy -body inclusions that stained positively for alpha-synuclein (Kordower et al., “Lewy body-like pathology in long-term embryonic nigral transplants in Parkinson's disease,” Nat Med. 2008 May;14(5):504-6). Additional experiments in mice indicated that injection of preformed fibrils composed of recombinant alpha-synuclein into the striatum was followed by cell-to-cell transmission of alpha-synuclein fibrils and Lewy pathology in anatomically connected regions, with progressive loss of dopamine neurons in the substantia nigra and some loss of motor coordination (Luk et al., “Pathological a-synuclein transmission initiates Parkinson-like neurodegeneration in nontransgenic mice,” Science. 2012 Nov 16;338(6109):949-53).

[0085] In embodiments, the PD is sporadic PD. In embodiments, the PD is characterized by a mutation in a PARK gene. In embodiments, the PD is characterized by a mutation in an alpha-synuclein gene. In embodiments, the PD is characterized by a mutation in a LRRK2 gene. In embodiments, the PD is characterized by a mutation in a risk factor gene, e.g., a mutant form of the GBA gene.

[0086] In embodiments, Parkinson’s Disease or PD refers to a progressive, neurodegenerative disorder that affects the mobility and control of the skeletal muscular system. Clinically, PD is typically characterized by severe and progressing tremors, rigidity, bradykinetic movements, posture instability, and cognitive impairment. Neuropathologically, the hallmarks of PD can include the progressive degeneration of dopaminergic nigrostriatal neurons and the formation of aggregated a-synuclein, called Lewy bodies, in the brain. Treatments, such as levodopa therapies, may improve one or more symptoms of PD in a subject.

[0087] In embodiments, Parkinson’s Disease symptoms include the commonly observed symptoms of PD, such as those described in: Sulkava, Adv Neurol, 91 :411-413, 2003; Facca and Koller, Adv Neurol, 91 :383-396,2003; Maijama-Lyons and Koller, Geriatrics Aug;56(8):24-25, 29-30, and 33-35, 2001; Siderowf, Neurol Clin Aug; 19(3):565-578, 2001; and Poewe, Curr Opin Neurol Neurosurg Jun;6(3):333-338,1993. Non-limiting examples of symptoms of PD include bradykinesia, akinesia, tremor, including tremors in the hands, fingers, forearm, foot, mouth, or chin, muscular rigidity, poor balance, postural instability and gait dysfunction, Parkinsonian gait, neuropsychiatric disorders (e.g., depression, psychotic symptoms, anxiety, apathy, mild-cognitive impairment, anddementia), autonomic dysfunctions, and sleep disturbances. In embodiments, the present compositions and methods reduce or ablate one or more Parkinson’s Disease symptoms.

[0088] Bradykinesia, or slowness in voluntary movement, produces difficulty initiating movement as well as difficulty completing movement once it is in progress. The delayed transmission of signals from the brain to the skeletal muscles, due to diminished dopamine, produces bradykinesia. Tremors in the hands, fingers, forearm, or foot tend to occur when the limb is at rest but not when performing tasks. Tremor may occur in the mouth and chin as well. Rigidity, or stiff muscles, may produce muscle pain and an expressionless, masklike face. Rigidity tends to increase during movement. Poor balance, due to the impairment or loss of the reflexes that adjust posture in order to maintain balance, may occur. Falls are common in people with PD. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of bradykinesia. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of tremors. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of rigidity. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of poor balance. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of falls.

[0089] Parkinsonian gait is the distinctive unsteady walk associated with PD. There is a tendency to lean unnaturally backward or forward, and to develop a stooped, head-down, shoulders-drooped stance. Arm swing is diminished or absent and people with PD tend to take small shuffling steps (called festination). Someone with PD may have trouble starting to walk and / or appear to be falling forward as they walk, freeze in midstride, and have difficulty making a turn. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of Parkinsonian gait.

[0090] The progressive loss of voluntary and involuntary muscle control produces a number of secondary symptoms associated with PD. Non-limiting examples of secondary symptoms of PD include bradyphrenia; constipation; dementia late in the disease; dysphagia (including, e.g., saliva and food that collects in the mouth or back of the throat may cause choking, coughing, or drooling; hyperhidrosis (excessive sweating); hypersalivation (excessive salivation); hypophonia (soft, whispery voice); incontinence (loss of bladder and / or bowel control); micrographia (small, cramped handwriting); and psychosocial symptoms, such as: anxiety, depression, isolation; and seborrhea (scaling, dryskin on the face and scalp). Symptoms of PD are associated with changes in the substantia nigra of the brain. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of loss of voluntary and involuntary muscle control.

[0091] In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of bradyphrenia. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of constipation. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of dementia. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of dysphagia. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of hyperhidrosis. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of hypersalivation. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of hypophonia. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of incontinence. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of micrographia. In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of one or more psychosocial symptoms, such as: anxiety, depression, isolation; and seborrhea (scaling, dry skin on the face and scalp). In embodiments, the methods and compositions described herein reduce, ablate, or slow the progression of changes in the substantia nigra of the brain.

[0092] In embodiments, to evaluate whether a subject is benefiting from the PD treatment, one can examine the subject’s symptoms in a quantitative way. In embodiments, in a successful treatment, the subject status will have improved (z.e., the symptoms will have decreased), or the progression will have been retarded (e.g., the subject’s condition will have stabilized). In embodiments, the subject’s neurons are also evaluated, and a benefited subject will exhibit neuronal protection from oxidative damage e.g., by magnetic resonance imaging (MRI) in frequent, serial MRI studies, comparing the subject’s status measurement before and after treatment), SPECT or PET imaging techniques demonstrating sparing of pre- or postsynaptic dopaminergic terminals.

[0093] Various staging systems are used for classifying PD, based on patient symptoms.

[0094] The Hoehn-Yahr (H-Y) staging system is divided into five stages, based primarily on motor symptoms and ability to take care of oneself. The stages are:• Stage 1 - (Mild / Early PD) This is the earliest stage. This includes mild tremors (e.g., of one hand), rigidity, clumsy leg, and mild difficulty walking. It affects only 1 side of the body (unilateral involvement). Less facial expressions may be noticed. These symptoms do not interfere with daily life much.• Stage 2 - (Mild / Early PD) Symptoms worsen during this stage. Tremors and difficulty moving affect both sides of the body (bilateral involvement) or the midline, loss of facial expression on both sides, decreased blinking, speech abnormalities, and rigidity of the muscles in the trunk. Daily tasks are more difficult but still possible to complete independently.• Stage 3 - (Moderate / Mid- stage PD) Balance and coordination are affected, slowness of movement, and inability to make rapid, automatic, and involuntary adjustments. Falls are common. Assistance is needed for some tasks like dressing and feeding.• Stage 4 - (Severe / Advanced PD) Tasks become very challenging. Walking aids might be necessary. It may be difficult to live independently because most tasks require help.• Stage 5 - (Severe / Advanced PD) This is the most severe stage. It may be difficult to stand and walk even with assistance. Patients often fall when standing or turning and may freeze or stumble when walking. Patients most likely depend on a wheelchair or may be bed-bound. New symptoms may develop, such as hallucinations or delusions.

[0095] In embodiments, the PD is classified using the H-Y staging system and is classified as Stage 1. In embodiments, the PD is classified using the H-Y staging system and is classified as Stage 2. In embodiments, the PD is classified using the H-Y staging system and is classified as Stage 3. In embodiments, the PD is classified using the H-Y staging system and is classified as Stage 4. In embodiments, the PD is classified using the H-Y staging system and is classified as Stage 5. In embodiments, the PD is classified using the H-Y staging system and is classified as mild / early PD. In embodiments, the PD is classified using the H-Y staging system and is classified as moderate / mid-stage PD. In embodiments, the PD is classified using the H-Y staging system and is classified as severe / advanced PD.

[0096] In embodiments, the method prevents or delays a Stage 1 PD from progressing to a Stage 2 PD, a Stage 3 PD, a Stage 4 PD, or a Stage 5 PD. In embodiments, the method prevents or delays a Stage 2 PD from progressing to a Stage 3 PD, a Stage 4 PD, or a Stage5 PD. In embodiments, the method prevents or delays a Stage 3 PD from progressing to a Stage 4 PD or a Stage 5 PD. In embodiments, the method prevents or delays a Stage 4 PD from progressing to a Stage 5 PD. In embodiments, the method prevents or delays a mild / early PD from progressing to a moderate / mid-stage PD or a severe / advanced PD. In embodiments, the method prevents or delays a moderate / mid-stage PD from progressing to a severe / advanced PD.

[0097] The Unified Parkinson’s Disease Rating Scale (UPDRS) is another scale that looks at more than the ability to move and live alone, as well as non-motor symptoms. It is used by physicians to monitor the response to agents used to improve the signs and symptoms of PD. The UPDRS contains 4 parts:• Part 1 - Mentation (thinking), behavior, mood• Part 2 - Activities of daily living• Part 3 - Motor function• Part 4 - Complications of therapy

[0098] Parts 1 to 3 are scored on a 0 to 4 rating scale. Part 4 is scored with yes and no ratings. Higher scores indicate increased severity of PD. Once parts 1 through 4 are completed, a doctor can then complete the H-Y staging system.

[0099] In embodiments, the PD is classified using the UPDRS system, and Part 1 is scored with a 0 rating. In embodiments, the PD is classified using the UPDRS system, and Part 1 is scored with a 2 rating. In embodiments, the PD is classified using the UPDRS system, and Part 1 is scored with a 3 rating. In embodiments, the PD is classified using the UPDRS system, and Part 1 is scored with a 4 rating. In embodiments, the PD is classified using the UPDRS system, and Part 2 is scored with a 0 rating. In embodiments, the PD is classified using the UPDRS system, and Part 2 is scored with a 2 rating. In embodiments, the PD is classified using the UPDRS system, and Part 2 is scored with a 3 rating. In embodiments, the PD is classified using the UPDRS system, and Part 2 is scored with a 4 rating. In embodiments, the PD is classified using the UPDRS system, and Part 3 is scored with a 0 rating. In embodiments, the PD is classified using the UPDRS system, and Part 3 is scored with a 2 rating. In embodiments, the PD is classified using the UPDRS system, and Part 3 is scored with a 3 rating. In embodiments, the PD is classified using the UPDRS system, and Part 3 is scored with a 4 rating. In embodiments, the PD is classified using the UPDRS system, and Part 4 is scored with a no rating. In embodiments, the PD is classified using the UPDRS system, and Part 4 is scored with a yes rating.

[0100] In embodiments, the method prevents or delays a Part 1 score from progressing from a 0 rating to a 1, 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 1 score from progressing from a 1 rating to a 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 1 score from progressing from a 2 rating to a 3 or 4 rating. In embodiments, the method prevents or delays a Part 1 score from progressing from a 3 rating to a 4 rating. In embodiments, the method prevents or delays a Part 2 score from progressing from a 0 rating to a 1, 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 2 score from progressing from a 1 rating to a 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 2 score from progressing from a 2 rating to a 3 or 4 rating. In embodiments, the method prevents or delays a Part 2 score from progressing from a 3 rating to a 4 rating. In embodiments, the method prevents or delays a Part 3 score from progressing from a 0 rating to a 1, 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 3 score from progressing from a 1 rating to a 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 3 score from progressing from a 2 rating to a 3 or 4 rating. In embodiments, the method prevents or delays a Part 3 score from progressing from a 3 rating to a 4 rating. In embodiments, the method prevents or delays a Part 4 score from progressing from a no rating to a yes rating.

[0101] The Movement Disorder Society revised this scale to create the MDS-UPDRS. This new scale has the same format as the original. However, it is reorganized and emphasizes the non-motor symptoms of Parkinson’s. It includes more symptoms such as depression, dementia, and nervous system function. These scales were created to be more thorough than H-Y staging, The 4 parts of the MDS-UPDRS include:• Part 1 - Non-motor experiences of daily living• Part 2 - Motor experiences of daily living• Part 3 - Motor examinations• Part 4 - Motor complications

[0102] Parts 1 through 4 of the MDS-UPDRS are scored on a 0 to 4 rating scale. Higher scores indicate increased severity of PD. The MDS-UPDRS also specifies whether a caretaker or person with PD is answering the questions.

[0103] In embodiments, the PD is classified using the MDS-UPDRS system, and Part 1 is scored with a 0 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 1 is scored with a 2 rating. In embodiments, the PD is classified using the MDS- UPDRS system, and Part 1 is scored with a 3 rating. In embodiments, the PD is classifiedusing the MDS-UPDRS system, and Part 1 is scored with a 4 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 2 is scored with a 0 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 2 is scored with a 2 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 2 is scored with a 3 rating. In embodiments, the PD is classified using the MDS- UPDRS system, and Part 2 is scored with a 4 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 3 is scored with a 0 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 3 is scored with a 2 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 3 is scored with a 3 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 3 is scored with a 4 rating. In embodiments, the PD is classified using the MDS- UPDRS system, and Part 4 is scored with a 0 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 4 is scored with a 2 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 4 is scored with a 3 rating. In embodiments, the PD is classified using the MDS-UPDRS system, and Part 4 is scored with a 4 rating.

[0104] In embodiments, the method prevents or delays a Part 1 score from progressing from a 0 rating to a 1, 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 1 score from progressing from a 1 rating to a 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 1 score from progressing from a 2 rating to a 3 or 4 rating. In embodiments, the method prevents or delays a Part 1 score from progressing from a 3 rating to a 4 rating. In embodiments, the method prevents or delays a Part 2 score from progressing from a 0 rating to a 1, 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 2 score from progressing from a 1 rating to a 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 2 score from progressing from a 2 rating to a 3 or 4 rating. In embodiments, the method prevents or delays a Part 2 score from progressing from a 3 rating to a 4 rating. In embodiments, the method prevents or delays a Part 3 score from progressing from a 0 rating to a 1, 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 3 score from progressing from a 1 rating to a 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 3 score from progressing from a 2 rating to a 3 or 4 rating. In embodiments, the method prevents or delays a Part 3 score from progressing from a 3 rating to a 4 rating. In embodiments, the method prevents or delays a Part 4 score from progressing from a 0 rating to a 1, 2, 3, or 4 rating. Inembodiments, the method prevents or delays a Part 4 score from progressing from a 1 rating to a 2, 3, or 4 rating. In embodiments, the method prevents or delays a Part 4 score from progressing from a 2 rating to a 3 or 4 rating. In embodiments, the method prevents or delays a Part 4 score from progressing from a 3 rating to a 4 rating.

[0105] In embodiments, the present compositions and methods are used to treat and / or prevent PD, to treat or prevent one or more symptoms associated with PD, and / or to slow the progression of PD. In embodiments, the present compositions and methods mimic action of, or cause mimicking of, overexpression of Kcnnl by preventing appearance of phospho-serine- 129 alpha-synuclein or by promoting phospho-serine- 129 alpha-synuclein clearance. In embodiments, without wishing to be bound by theory, the present compositions and methods mimic action of, or cause mimicking of, action of overexpression of Kcnnl in protect! on / survival derived from clogging membranes, itself misfolding / aggregating within membranes or outside of them and leading to activation of autophagic or endosomal clearance mechanisms (or reducing translation of A53T-alpha- synuclein or endosomal clearance mechanisms or reducing translation of A53T-alpha- synuclein e.g, by an integrated stress response, e.g., mediated by eIF2alpha phosphorylation).

[0106] In embodiments, the terms patient and subject are used interchangeably. In embodiments, the subject and / or animal is a mammal, e.g, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon. In embodiments, the subject and / or animal is a non-mammal, such as, for example, a zebrafish.

[0107] In embodiments, the compositions and methods described herein are useful in treatment of a human subject. In embodiments, the human is a pediatric human. In embodiments, the human is an adult human. In embodiments, the human is a geriatric human. In embodiments, the human may be referred to as a patient. In embodiments, the human is a female. In embodiments, the human is a male.

[0108] In embodiments, the human has an age in a range of from about 1 to about 18 months old, from about 18 to about 36 months old, from about 1 to about 5 years old, from about 5 to about 10 years old, from about 10 to about 15 years old, from about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, from about 40 to about 45 years old, from about 45 to about 50 years old, from about 50 to about 55 yearsold, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old.III. Polynucleotides, Expression Vectors, and Viral Vectors

[0109] In embodiments, provided herein are agents and polynucleotides encoding Kcnnl, or a fragment or variant thereof, expression cassettes comprising the polynucleotides encoding Kcnnl, or a fragment or variant thereof, and viral vectors comprising the polynucleotides encoding Kcnnl, or a fragment or variant thereof.

[0110] In embodiments, agents, polynucleotides, expression vectors, and rAAV vector genomes described herein can be prepared by using recombinant DNA technology methods known in the art.A. Polynucleotides[OHl] In embodiments, provided herein are agents encoding for Kcnnl, or a fragment or variant thereof, and polynucleotides encoding Kcnnl, or a fragment or variant thereof. In embodiments, an agent or polynucleotide encoding Kcnnl, or a fragment or variant thereof refers to a nucleic acid. In embodiments, the nucleic acid is a DNA or an RNA.

[0112] In embodiments, the agent encoding for Kcnnl, or a fragment or variant thereof comprises an expression cassette comprising (a) a polynucleotide encoding Kcnnl, or a fragment or variant thereof, operably linked to an expression control element; (b) an adeno- associated virus (AAV) inverted repeat (ITR) flanking the 5’ terminus of the polynucleotide; and (c) an AAV ITR flanking the 3’ terminus of the polynucleotide.

[0113] In embodiments, an agent or a polynucleotide encoding an amino acid sequence includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence that encodes a protein or an RNA may also include introns to the extent that the nucleotide sequence encoding the protein may in some versions contain an intron(s).

[0114] In embodiments, functional variant refers to a polypeptide or protein having substantial or significant sequence identity or similarity to a parent polypeptide or protein, wherein a functional variant retains at least a portion of the biological activity of the polypeptide or protein of which it is a variant. Functional variants and / or fragmentsencompass, for example, those variants and / or fragments of the polypeptide or protein described herein (the parent polypeptide or protein) that retain the ability to delay accumulation of the pathogenic serine- 129-phosphorylated form of alpha-synuclein to a similar extent, the same extent, or to a higher extent, as the parent polypeptide or protein. Functional variants and / or fragments encompass, for example, those variants and / or fragments of the polypeptide or protein described herein (the parent polypeptide or protein) that retain the ability to prolong survival of A53T transgenic mice to a similar extent, the same extent, or to a higher extent, as the parent polypeptide or protein.

[0115] In embodiments, in reference to the parent polypeptide or protein, the functional variant can, for instance, be at least about 30%, about 50%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99% or more identical in amino acid sequence to the parent polypeptide or protein. In embodiments, a functional variant can, for example, comprise the amino acid sequence of the parent polypeptide or protein with at least one conservative amino acid substitution. In embodiments, a functional variant can, for example, comprise the amino acid sequence of the parent polypeptide or protein with at least one nonconservative amino acid substitution. In embodiments, the non-conservative amino acid substitution does not interfere with or inhibit the biological activity of the functional variant. In embodiments, the non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent polypeptide or protein.

[0116] Conservative amino acid substitutions are known in the art and include amino acid substitutions in which one amino acid having certain physical and / or chemical properties is exchanged for another amino acid that has the same or similar chemical or physical properties. For instance, in embodiments, the conservative amino acid substitution can be an acidic / negatively charged polar amino acid substituted for another acidic / negatively charged polar amino acid (e.g., Asp or Glu), an amino acid with a nonpolar side chain substituted for another amino acid with a nonpolar side chain (e.g., Ala, Gly, Leu, Met, Phe, Pro, Trp, Cys, He and Vai), a basic / positively charged polar amino acid substituted for another basic / positively charged polar amino acid (e.g., Lys, His, and Arg), an uncharged amino acid with a polar side chain substituted for another uncharged amino acid with a polar side chain (e.g., Asn, Gin, Ser, Thr, and Tyr), an amino acid with a beta-branched side-chain substituted for another amino acid with a beta-branched side-chain (e.g., Leu,He, Thr, and Vai), an amino acid with an aromatic side-chain substituted for another amino acid with an aromatic side chain (e.g., His, Phe, Trp, and Tyr), etc.

[0117] In embodiments, nucleic acid and polynucleotide refer to all forms of nucleic acid, oligonucleotides, including deoxyribonucleic acid (DNA) and ribonucleic acid (RNA). In discussing polynucleotides, a sequence or structure of a particular polynucleotide can be described herein according to the convention of providing the sequence in the 5’ to 3’ direction.

[0118] In embodiments, polynucleotides include naturally occurring, synthetic, and intentionally modified or altered polynucleotides.

[0119] In embodiments, a transgene refers to a polynucleotide that is intended to be or has been introduced into a cell or organism. Transgenes include any nucleic acid, such as a heterologous polynucleotide sequence, such as a polynucleotide encoding Kcnnl, or a fragment or variant thereof. The term transgene and heterologous nucleic acid / polynucleotide sequences are used interchangeably herein.

[0120] In embodiments, “SK channels” refers to a family of four small conductance calcium activated potassium channels: KCa2.1 (SKI), KCa2.2 (SK2), KCa2.3 (SK3), KCa2.4 (SK4 / IK1), which are encoded by genes Kcnnl, Kcnn2, Kcnn3, and Kcnn4, respectively. Without wishing to be bound by theory, the individual protein subunits produced by these genes in many cases assemble into homotetramers that constitute the respective SK channels. Mixed tetramer heteromeric channels [e.g., (Kcnnl )2 / (Kcnn2)2] have been observed, however. Notably, Benton et al. (in “Small conductance Ca2+- activated K+ channels formed by the expression of rat SKI and SK2 genes in HEK 293 cells”. Journal of Physiology 533 Part 1, 13-19, 2003) observed that rat Kcnnl subunits overexpressed in HEK293 cells could not form an active channel that could conduct a calcium-dependent potassium current. In contrast, when rat Kcnnl was co-expressed with rat Kcnn2 (which by itself homotetramerizes and produces a calcium-dependent current), an increased current relative to that produced by Kcnn2 alone was observed, showing stepwise increases with increasing steps of voltage, suggesting that the subunits are able to form heteromeric assemblies. The same effect holds true for mouse Kcnnl. Thus, rodent Kcnnl’ s are channel-inactive by themselves (and probably not assembled) but are incorporated into active heterotetramers when Kcnn2 is co-expressed. Notably, Kcnnl and Kcnn2 are both expressed in many neuronal tissues. This behavior contrasts with that of human KCNN1, which appears to form active homotetramers and produces current insimilar transfection studies. SK channels are activated by an action potential-dependent increase in intracellular Ca2+ concentrations. Calcium binds to calmodulin molecules that are stably and constitutively associated with each of the intracellular C-termini of the four SK subunits of a channel, allosterically opening the potassium channel. This contributes to the afterhyperpolarization (AHP) phase of an action potential. In embodiments, Kcnnl refers to any polynucleotide or protein of Kcnnl which is or forms an inactive or substantially inactive channel.

[0121] In embodiments, Kcnnl refers to any polynucleotide or protein of Kcnnl . Examples of Kcnnl include any naturally occurring Kcnnl and functional variants thereof originating in any species including mouse, rat, and human. In embodiments, Kcnnl refers to any polynucleotide or protein of mouse Kcnnl. In embodiments, Kcnnl refers to any polynucleotide or protein of rat Kcnnl. In embodiments, Kcnnl refers to any polynucleotide or protein of human Kcnnl.

[0122] In embodiments, the Kcnnl is a Kcnnl variant. A Kcnnl variant has at least one or more amino acid modifications, including amino acid additions, deletions, and / or substitutions, as compared to the parental wild type sequence.

[0123] In embodiments, a polynucleotide encoding a Kcnnl refers to a recombinant polynucleotide molecule that encodes a protein having at least part of a function and / or activity of wild type Kcnnl protein.

[0124] In embodiments, modify means that a polynucleotide or protein deviates from a reference or parental sequence. A modified polynucleotide encoding Kcnnl, or a fragment or variant thereof, has been altered compared to reference (e.g., wild type) or parental polynucleotide. Modified polynucleotides can therefore have substantially the same, greater or less activity or function than a reference or parental polynucleotide, but at least retain partial activity, function and or sequence identity to the reference or parental polynucleotide. The modified polynucleotide can be genetically modified to encode a modified or variant Kcnnl .

[0125] In embodiments, a modified polynucleotide encoding Kcnnl, or a fragment or variant thereof means that the Kcnnl polynucleotide has alteration compared the parental unmodified polynucleotide encoding Kcnnl, or a fragment or variant thereof. A particular example of a modification is a nucleotide substitution. Nucleotide substitutions can be silent mutations that code for the same amino acid, or missense mutations that code for a different amino acid. Missense mutations can be conservative or non-conservativemutations. Other examples of modifications include, e.g., truncations and insertions. The modified polynucleotide can also include a codon optimized polynucleotide that encodes the same protein as that of the wild type protein or of the polynucleotide that has not been codon optimized. Codon optimization can be used in a broader sense, e.g., including removing the CpG dinucleotides. In embodiments, modified polynucleotides encoding Kcnnl, or a fragment or variant thereof, include polynucleotides with a reduced number of CpG dinucleotides compared to a reference polynucleotide encoding Kcnnl, or a fragment or variant thereof, referred to as CpG-reduced polynucleotides.

[0126] In embodiments, CpG-reduced or CpG-depleted refers to a polynucleotide sequence which is generated, either synthetically or by mutation of a polynucleotide sequence, such that one or more of the CpG dinucleotides (or motifs) are removed from the polynucleotide sequence. In embodiments, all CpG motifs are removed to provide what is termed herein as a modified CpG-free sequence. In embodiments, the CpG motifs are suitably reduced or eliminated not just in a coding sequence (e.g., a transgene), but also in the non-coding sequences, including, e.g., 5’ and 3’ untranslated regions (UTRs), promoter, enhancer, signal peptides, poly A, ITRs, introns, and any other sequences present in the polynucleotide molecule.

[0127] In embodiments, the polynucleotide encoding Kcnnl encodes Kcnnl, or a fragment or variant of Kcnnl . In embodiments, the Kcnnl is from mammals other than mouse, which protein functions in a substantially similar manner to the mouse protein. In embodiments, the Kcnnl is a variant of human KCNN1. In embodiments, the Kcnnl is from mouse. In embodiments, the Kcnnl comprises naturally occurring variants or recombinantly derived mutants of wild type Kcnnl, which variants or mutants render the Kcnnl encoded thereby either as therapeutically effective as full-length protein, or even more therapeutically effective than full-length protein in the gene therapy methods as disclosed herein. In embodiments, the Kcnnl comprises a variant Kcnnl that retains Kcnnl’ s salutary biological activity. In embodiments, such variants include proteins or polypeptides that have been or may be modified using recombinant DNA technology, such that the protein or polypeptide possesses additional properties which enhance its suitability for use in the methods described herein, for example, but not limited to, variants conferring enhanced intracellular stability on the protein and enhanced specific activity of the protein. Analogs can differ from naturally occurring proteins or peptides by conservative amino acid sequence differences or by modifications which do not affect sequence, or by both. Forexample, conservative amino acid changes may be made, which although they alter the primary sequence of the protein or peptide, do not normally alter its function.

[0128] In embodiments, the Kcnnl comprises a variant Kcnnl or a fragment or variant of Kcnnl which is capable of producing diminution of A53T alpha synuclein-mCherry in HeLa cell culture. In embodiments, the Kcnnl comprises a variant Kcnnl or fragment of Kcnnl which is capable of producing transgenic extension of survival of a mouse model, e.g., without limitation those disclosed herein. In embodiments, the Kcnnl comprises a variant Kcnnl or fragment of Kcnnl which is capable of producing prolongation of survival associated with reduction of serl29 phosphorylated pathogenic version of alpha-synuclein in a mouse model, e.g., without limitation those disclosed herein.

[0129] In embodiments, the Kcnnl comprises a variant Kcnnl or fragment of Kcnnl which is capable of producing clearance of A53T alpha-synuclein-mCherry associated with the same morphologic features at the EM level observed to be produced by Kcnnl transfection (e.g., MVB and autophagosome formation as well as nuclear envelope invagination).

[0130] In embodiments, the Kcnnl comprises a variant Kcnnl or fragment of Kcnnl which fails to form, or substantially form, channel activity when transiently overexpressed in cultured cells and colocalizes with the ER in such cells and neurons as does overexpressed mouse Kcnnl, optionally exhibiting a broad cytoplasmic immunostaining pattern and colocalizing with (by immunostaining) calnexin, an ER membrane protein.

[0131] In embodiments, human SKI (KCNN1) has the following amino acid sequence (SEQ ID NO: 1):>sp|Q92952|KCNNl_HUMAN Small conductance calcium-activated potassium channel protein 1 OS=Homo sapiens OX=9606 GN=KCNN1 PE=1 SV=2 MNSHSYNGSVGRPLGSGPGALGRDPPDPEAGHPPQPPHSPGLQVWAKSEPARPSPGSPR GQPQDQDDDEDDEEDEAGRQRASGKPSNVGHRLGHRRALFEKRKRLSDYALIFGMFGIW MVTETELSWGVYTKESLYSFALKCLISLSTAILLGLWLYHAREIQLFMVDNGADDWRIA MTCERVFLISLELAVCAIHPVPGHYRFTWTARLAFTYAPSVAEADVDVLLSIPMFLRLYL LGRVMLLHSKIFTDASSRSIGALNKITFNTRFVMKTLMTICPGTVLLVFSISSWIIAAWT VRVCERYHDKQEVTSNFLGAMWLISITFLSIGYGDMVPHTYCGKGVCLLTGIMGAGCTAL VVAVVARKLELTKAEKHVHNFMMDTQLTKRVKNAAANVLRETWLJIYKHTRLJVKKPDQARV RKHQRKFLQAIHQAQKLRSVKIEQGKLNDQANTLTDLAKTQTVMYDLVSELHAQHEELEA RLATLESRLDALGASLQALPGLIAQAIRPPPPPLPPRPGPGPQDQAARSSPCRWTPVAPS DCG

[0132] In embodiments, Kcnnl has an amino acid sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 1. In embodiments, Kcnnl has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 1 by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, or 105 or more amino acid modifications selected from amino acid additions, deletions, and substitutions.

[0133] Human KCNN1 cDNA has the following nucleotide sequence (SEQ ID NO: 2): >NM_002248.5:323-1954 Homo sapiens potassium calcium-activated channel subfamily N member 1 (KCNN1), transcript variant 1, mRNA ATGAACAGCCACAGCTACAATGGCAGCGTGGGGCGGCCGCTGGGCAGCGGGCCGGGCGCC CTGGGACGAGACCCTCCGGACCCTGAGGCCGGCCACCCCCCACAACCCCCGCACAGCCCG GGCCTCCAGGTGGTAGTGGCCAAGAGTGAGCCAGCCCGGCCCTCACCCGGCAGCCCCCGG GGGCAGCCCCAGGACCAGGACGATGACGAGGATGATGAGGZtAGATGAGGCCGGCAGGCAG AGAGCCTCGGGGAAACCCTCAAATGTGGGCCACCGCCTGGGCCACCGGCGGGCGCTCTTC GAGAAGCGGAAGCGCCTCAGCGACTATGCCCTCATTTTCGGCATGTTTGGCATCGTCGTC ATGGTGACGGAGACCGAGCTGTCCTGGGGGGTGTACACCAAGGAGTCTCTGTACTCATTC GCACTCAAATGCCTCATCAGCCTCTCCACGGCCATCCTGCTGGGTCTCGTTGTCCTCTAC CATGCCCGGGAGATCCAGCTGTTCATGGTGGACAACGGGGCTGATGACTGGCGCATCGCC ATGACCTGCGAGCGCGTGTTCCTCATCTCGCTAGAGCTGGCAGTGTGCGCCATTCACCCG GTGCCCGGCCACTACCGCTTCACGTGGACGGCGCGGCTGGCCTTCACGTACGCGCCCTCG GTGGCCGAGGCCGACGTGGACGTGCTGCTGTCCATCCCCATGTTCCTGCGCCTCTACCTG CTGGGCCGGGTGATGCTACTGCACAGCAAAATCTTCACGGACGCCTCGAGCCGCAGCATC GGGGCCCTCAACAAGATCACCTTCAACACGCGCTTCGTCATGAAGACACTCATGACCATC TGCCCCGGCACCGTGCTGCTGGTCTTCAGCATCTCCTCCTGGATCATCGCAGCCTGGACC GTGCGCGTCTGCGAGAGGTACCACGACAAGCAGGAAGTGACCAGCAACTTCCTGGGGGCC ATGTGGCTGATTTCCATCACCTTCCTCTCCATTGGCTACGGCGACATGGTGCCCCACACC TACTGCGGGAAGGGTGTGTGCCTGCTCACTGGCATCATGGGAGCTGGCTGTACCGCGCTCGTGGTGGCTGTGGTGGCTCGGAAGCTGGAGCTCACCAAGGCTGAGAAGCACGTGCACAAC TTCATGATGGACACTCAGCTCACCAAGCGGGTAAAAAACGCCGCTGCTAACGTTCTCAGG GAGACGTGGCTCATCTACAAACATACCAGGCTGGTGAAGAAGCCAGACCAAGCCCGGGTT CGGAAACACCAGCGTAAGTTCCTCCAAGCCATCCATCAGGCTCAGAAGCTCCGGAGTGTG AAGATCGAGCAAGGGAAGCTGAACGACCAGGCTAACACGCTTACCGACCTAGCCAAGACC CAGACCGTCATGTACGACCTTGTATCGGAGCTGCACGCTCAGCACGAGGAGCTGGAGGCC CGCCTGGCCACCCTGGAAAGCCGCTTGGATGCGCTGGGTGCCTCTCTACAGGCCCTGCCT GGCCTCATCGCCCAAGCCATACGCCCACCCCCGCCTCCCCTGCCTCCCAGGCCCGGCCCC GGCCCCCAAGACCAGGCAGCCCGGAGCTCCCCCTGCCGGTGGACGCCCGTGGCCCCCTCG GACTGCGGGTGA

[0134] In embodiments, Kcnnl is encoded by an mRNA sequence at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 2. In embodiments, Kcnnl is encoded by an mRNA sequence that is a codon-optimized variation of SEQ ID NO: 2.

[0135] Mouse SKI (Kcnnl) has the following amino acid sequence (SEQ ID NO: 3) >tr|A0A140T8Q8|A0A140T8Q8_MOUSE Small conductance calcium-activated potassium channel protein 1 OS=Mus musculus OX=10090 GN=Kcnnl PE=4 SV=1 MSSHSHNGSVGQPLGSGPGFLGWEPVDPEAGRPLQPTQGPGLQMVAKGQPVRLSPGGSRG HPQEQEEEEEEEEEEEDKTGSGKPPTVSHRLGHRRALFEKRKRLSDYALIFGMFGIWMV TETELSWGVYTKESLCSFALKCLISLSTVILLGLVILYHAREIQLFLVDNGADDWRIAMT WERVSLISLELWCAIHPVPGHYRFTWTARLAFSLVPSAAEADLDVLLSIPMFLRLYLLA RVMLLHSRIFTDASSRSIGALNRVTFNTRFVTKTLMTICPGTVLLVFSVSSWIVAAWTVR VCERYHDKQEVTSNFLGAMWLISITFLSIGYGDMVPHTYCGKGVCLLTGIMGAGCTALW AVVARKLELTKAEKHVHNFMMDTQLTKRVKNAAANVLRETWLJIYKHTRLJVKKPDQGRVRK HQRKFLQAIHQAQKLRSVKIEQGKVNDQANTLAELAKAQSIAYEWSELQAQQEELEARL AALESRLDVLGASLQALJPGLIAQAICPLPPPWPGPGHLJATATQSPQSHWLJPTMGSDCG

[0136] In embodiments, Kcnnl has an amino acid sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, atleast about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 3. In embodiments, Kcnnl has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 3 by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, or 105 or more amino acid modifications selected from amino acid additions, deletions, and substitutions.

[0137] Mouse SKI (Kcnnl) cDNA has the following nucleotide sequence (SEQ ID NO: 4)>NM_001363407.2: Mus musculus potassium intermediate / small conductance calcium- activated channel, subfamily N, member 1 (Kcnnl), transcript variant 1, mRNA ATGAGTAGCCACAGCCACAATGGCAGCGTGGGGCAGCCTCTGGGCAGCGGGCCTGGATTC CTGGGCTGGGAGCCTGTGGACCCTGAGGCAGGCCGCCCCCTGCAGCCCACCCAAGGCCCA GGCCTGCAGATGGTGGCCAAGGGTCAGCCTGTCAGGCTGTCACCCGGTGGTTCCAGGGGC CACCCCCAGGAGCAGGAGGAGGAAGAGGAAGAGGAGGAGGAGGAGGAGGACAAGACAGGC TCAGGGAAGCCCCCAACAGTCAGCCACCGCCTGGGACACCGCAGGGCCCTCTTTGAAAAG CGTAAACGGCTCAGTGACTATGCGCTCATCTTTGGCATGTTTGGGATTGTCGTCATGGTG ACAGAAACAGAGCTGTCCTGGGGTGTATACACCAAGGAGTCACTCTGCTCTTTTGCTCTG AAATGCCTCATCAGCCTGTCCACTGTCATCTTGCTTGGCCTTGTCATCCTGTACCACGCC CGAGAGATCCAGCTGTTCTTGGTGGACAATGGTGCCGACGACTGGCGTATCGCCATGACG TGGGAGCGCGTGTCCCTGATCTCGCTGGAGTTGGTCGTGTGTGCCATCCACCCGGTGCCC GGCCACTATCGCTTCACGTGGACGGCACGACTGGCCTTCTCTCTGGTGCCGTCGGCAGCC GAGGCAGACCTGGATGTGCTGCTGTCCATCCCCATGTTCCTGCGCCTCTACCTGCTGGCT CGGGTCATGCTCCTGCACAGCCGCATCTTCACCGATGCATCCAGCCGCAGCATCGGGGCC CTTAACCGCGTCACCTTCAACACACGCTTCGTCACCAAGACGCTCATGACCATCTGCCCC GGCACTGTGTTGTTGGTCTTCAGCGTCTCCTCCTGGATCGTTGCTGCGTGGACAGTGCGC GTGTGTGAGAGGTACCACGATAAGCAGGAAGTGACCAGCAACTTCCTGGGAGCCATGTGG CTCATCTCCATCACCTTCTTGTCCATTGGCTATGGAGACATGGTGCCGCATACCTACTGT GGGAAGGGTGTGTGTCTGCTCACTGGCATCATGGGAGCAGGCTGCACTGCACTCGTGGTG GCCGTCGTGGCTCGGAAGTTGGAACTCACCAAGGCTGAGAAACACGTGCACAACTTCATGATGGACACACAGCTCACCAAGCGGGTCAAAAATGCTGCTGCAAACGTTCTCAGGGAGACA TGGCTCATCTACAAACACACCAGGCTGGTGAAGAAGCCAGACCAAGGCCGGGTTCGGAAA CACCAGCGTAAGTTCCTTCAGGCCATCCATCAGGCTCAGAAGCTCCGAAGTGTGAAGATT GAACAAGGGAAGGTGAACGATCAGGCCAACACGCTGGCTGAGCTGGCCAAGGCACAGAGC ATCGCATATGAGGTGGTGTCAGAGCTGCAGGCCCAGCAGGAGGAGTTGGAGGCACGCCTA GCCGCCTTGGAGAGCCGACTGGATGTCCTGGGTGCCTCCCTGCAGGCTCTACCAGGCCTT ATAGCCCAAGCCATATGCCCTCTACCACCACCCTGGCCTGGGCCTGGTCACCTGGCCACA GCCACCCAGAGCCCACAAAGCCACTGGCTGCCCACCATGGGATCAGACTGTGGGTGA

[0138] In embodiments, Kcnnl is encoded by an mRNA sequence at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 4. In embodiments, Kcnnl is encoded by an mRNA sequence that is a codon-optimized variation of SEQ ID NO: 4.

[0139] Truncated mouse Kcnnl cDNA has the following nucleotide sequence (SEQ ID NO: 5) ATGAGTAGCCACAGCCACAATGGCAGCGTGGGGCAGCCTCTGGGCAGCGGGCCTGGATTC CTGGGCTGGGAGCCTGTGGACCCTGAGGCAGGCCGCCCCCTGCAGCCCACCCAAGGCCCA GGCCTGCAGATGGTGGCCAAGGGTCAGCCTGTCAGGCTGTCACCCGGTGGTTCCAGGGGC CACCCCCAGGAGCAGGAGGAGGAAGAGGAAGAGGAGGAGGAGGAGGAGGACAAGACAGGC TCAGGGAAGCCCCCAACAGTCAGCCACCGCCTGGGACACCGCAGGGCCCTCTTTGAAAAG CGTAAACGGCTCAGTGACTATGCGCTCATCTTTGGCATGTTTGGGATTGTCGTCATGGTG ACAGAAACAGAGCTGTCCTGGGGTGTATACACCAAGGAGTCACTCTGCTCTTTTGCTCTG AAATGCCTCATCAGCCTGTCCACTGTCATCTTGCTTGGCCTTGTCATCCTGTACCACGCC CGAGAGATCCAGCTGTTCTTGGTGGACAATGGTGCCGACGACTGGCGTATCGCCATGACG TGGGAGCGCGTGTCCCTGATCTCGCTGGAGTTGGTCGTGTGTGCCATCCACCCGGTGCCC GGCCACTATCGCTTCACGTGGACGGCACGACTGGCCTTCTCTCTGGTGCCGTCGGCAGCC GAGGCAGACCTGGATGTGCTGCTGTCCATCCCCATGTTCCTGCGCCTCTACCTGCTGGCT CGGGTCATGCTCCTGCACAGCCGCATCTTCACCGATGCATCCAGCCGCAGCATCGGGGCC CTTAACCGCGTCACCTTCAACACACGCTTCGTCACCAAGACGCTCATGACCATCTGCCCC GGCACTGTGTTGTTGGTCTTCAGCGTCTCCTCCTGGATCGTTGCTGCGTGGACAGTGCGCGTGTGTGAGAGGTACCACGATAAGCAGGAAGTGACCAGCAACTTCCTGGGAGCCATGTGG CTCATCTCCATCACCTTCTTGTCCATTGGCTATGGAGACATGGTGCCGCATACCTACTGT GGGAAGGGTGTGTGTCTGCTCACTGGCATCATGGGAGCAGGCTGCACTGCACTCGTGGTG GCCGTCGTGGCTCGGAAGTTGGAACTCACCAAGGCTGAGAAACACGTGCACAACTTCATG ATGGACACACAGCTCACCAAGCGGGTCAAAAATGCTGCTGCAAACGTTCTCAGGGAGACA TGGCTCATCTACAAACACACCAGGCTGGTGAAGAAGCCAGACCAAGGCCGGGTTCGGAAA CACCAGCGTAAGTTCCTTCAGGCCATCCATCAGGCTCAGAAGCTCCGAAGTGTGAAGATT GAACAAGGGAAGGTGAACGATCAGGCCAACACGCTGGCTGAGCTGGCCAAGGCACAGAGC ATCGCATATGAGGTGGTGTCAGAGCTGCAGGCCTAG

[0140] In embodiments, Kcnnl is encoded by an mRNA sequence at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 5. In embodiments, Kcnnl is encoded by an mRNA sequence that is a codon-optimized variation of SEQ ID NO: 5.

[0141] Rattus norvegicus (Rat) SKI (Kcnnl) has the following amino acid sequence (SEQID NO: 9) >sp|P70606|KCNNl_RAT Small conductance calcium-activated potassium channel protein 1 OS=Rattus norvegicus OX=10116 GN=Kcnnl PE=1 SV=2 MSSRSHNGSVGRPLGSGPGFLGWEPVDPEAGRPRQPTQGPGLQMMAKGQPAGLSPSGPRG HSQAQEEEEEEEDEDRPGSGKPPTVSHRLGHRRALFEKRKRLSDYALIFGMFGIWMVTE TELSWGVYTKESLCSFALKCLISLSTVILLGLVILYHAREIQLFLVDNGADDWRIAMTWE RVSLISLELAVCAIHPVPGHYRFTWTARLAFSLVPSAAEADVDVLLSIPMFLRLYLLARV MLLHSRIFTDASSRSIGALNRVTFNTRFVTKTLMTICPGTVLLVFSISSWIVAAWTVRVC ERYHDKQEVTSNFLGAMWLISITFLSIGYGDMVPHTYCGKGVCLLTGIMGAGCTALWAV VARKLELTKAEKHVHNFMMDTQLTKRVKNAAANVLRETWLIYKHTRLVKKPDQSRVRKHQ RKFLQAIHQAQKLRTVKIEQGKVNDQANTLADLAKAQS IAYEWSELQAQQEELJEARLJAA LESRLDVLGASLQALPSLIAQAICPLPPPWPGPSHLTTAAQSPQSHWLJPTTASDCG .

[0142] In embodiments, Kcnnl has an amino acid sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at leastabout 99% identical, or 100% identical to SEQ ID NO: 9. In embodiments, Kcnnl has an amino acid sequence that differs from the amino acid sequence of SEQ ID NO: 9 by 1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 11 or more, 12 or more, 13 or more, 14 or more, 15 or more, 16 or more, 17 or more, 18 or more, 19 or more, 20 or more, 21 or more, 22 or more, 23 or more, 24 or more, 25 or more, 26 or more, 27 or more, 28 or more, 29 or more, 30 or more, 35 or more, 40 or more, 45 or more, 50 or more, 55 or more, 60 or more, 65 or more, 70 or more, 75 or more, 80 or more, 85 or more, 90 or more, 95 or more, 100 or more, or 105 or more amino acid modifications selected from amino acid additions, deletions, and substitutions.

[0143] Rattus norvegicus (Rat) SKI (Kcnnl) cDNA has the following nucleotide sequence (SEQ ID NO: 10)>NM_019313.2 Rattus norvegicus potassium calcium-activated channel subfamily N member 1 (Kcnnl), mRNA ATGAGTAGCCGCAGCCACAATGGCAGTGTGGGGCGGCCTCTGGGCAGTGGGCCTGGATTC CTGGGCTGGGAACCTGTGGACCCCGAGGCAGGCCGCCCCCGACAGCCCACCCAAGGCCCC GGCCTGCAGATGATGGCCAAGGGTCAGCCTGCTGGGCTGTCACCCAGTGGCCCCAGGGGC CACTCCCAGGCGCAGGAGGAGGAAGAGGAAGAGGAGGATGAGGACAGGCCAGGCTCAGGG AAGCCCCCGACCGTCAGTCACCGCCTGGGCCACCGTAGGGCCCTCTTCGAGAAGCGTAAA CGACTCAGTGACTATGCACTCATCTTTGGCATGTTCGGGATTGTCGTCATGGTGACAGAA ACAGAGCTGTCCTGGGGTGTGTACACCAAGGAGTCTCTGTGCTCATTCGCCCTGAAATGC CTAATCAGCCTCTCCACTGTCATCCTGCTTGGCCTTGTCATCCTCTACCACGCCCGAGAG ATCCAGCTGTTCCTGGTGGACAATGGTGCCGATGACTGGCGCATTGCCATGACGTGGGAG CGAGTGTCCCTGATCTCGCTGGAGTTGGCTGTGTGTGCCATCCACCCAGTGCCTGGCCAC TACCGCTTCACATGGACGGCGCGGCTGGCCTTCTCCCTGGTGCCGTCAGCAGCCGAGGCG GATGTGGATGTGCTTCTGTCCATCCCCATGTTTCTGCGCCTCTATCTGCTGGCTCGGGTC ATGCTCCTGCACAGCCGCATCTTCACGGACGCATCCAGTCGCAGCATCGGAGCCCTGAAC CGTGTCACCTTCAACACACGCTTTGTCACCAAGACACTCATGACCATCTGCCCTGGCACT GTGCTGTTGGTCTTCAGCATCTCCTCCTGGATCGTCGCTGCATGGACAGTGCGCGTGTGT GAGAGGTACCATGATAAACAGGAAGTGACCAGCAACTTCCTGGGGGCCATGTGGCTCATC TCCATTACCTTCCTGTCCATCGGCTACGGGGACATGGTGCCGCACACCTACTGTGGGAAG GGCGTGTGTCTGCTCACCGGCATCATGGGAGCAGGCTGCACTGCACTCGTGGTGGCCGTC GTGGCCCGGAAGTTGGAACTCACCAAGGCTGAGAAACACGTGCACAACTTCATGATGGAC ACACAGCTCACCAAGCGGGTTAAAAACGCCGCTGCAAACGTTCTCAGGGAGACATGGCTCATCTACAAACACACCAGGCTAGTGAAGAAGCCAGACCAAAGCCGGGTTCGGAAACACCAG CGTAAGTTCCTTCAGGCCATCCATCAGGCGCAGAAGCTCCGGACTGTGAAGATTGAACAA GGGAAGGTGAATGATCAGGCCAACACGCTGGCTGACCTGGCCAAGGCACAGAGCATCGCA TATGAGGTGGTGTCGGAGCTGCAGGCCCAGCAGGAGGAGTTGGAGGCCCGTCTGGCTGCC CTGGAGAGCCGCCTGGATGTCCTAGGCGCCTCCCTGCAGGCCCTACCAAGTCTCATAGCC CAAGCCATATGCCCTCTACCACCACCCTGGCCCGGGCCCAGTCACCTGACCACAGCCGCC CAGAGCCCACAAAGCCACTGGCTGCCCACCACGGCATCAGACTGTGGGTGA

[0144] In embodiments, Kcnnl is encoded by an mRNA sequence at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 10. In embodiments, Kcnnl is encoded by an mRNA sequence that is a codon-optimized variation of SEQ ID NO: 10.

[0145] In embodiments, the agent encoding for Kcnnl, or a fragment or variant thereof comprises a cDNA encoding Kcnnl, or a fragment or variant thereof. In embodiments, the cDNA encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 1, 3, or 9, or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the cDNA encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10, or a functional variant thereof having at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the cDNA encoding Kcnnl, or a fragment or variant thereof, comprises a codon optimized form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10. In embodiments, the cDNA encoding Kcnnl, or a fragment or variant thereof, comprises a CpG-reduced form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10.

[0146] In embodiments, the agent encoding for Kcnnl, or a fragment or variant thereof comprises an mRNA encoding Kcnnl, or a fragment or variant thereof. In embodiments, the mRNA encoding Kcnnl, or a fragment or variant thereof, is transcribed from apolynucleotide sequence encoding the polypeptide of SEQ ID NO: 1, 3, or 9, or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the mRNA encoding Kcnnl, or a fragment or variant thereof, is transcribed from a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10, or a functional variant thereof having at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto. In embodiments, the mRNA encoding Kcnnl, or a fragment or variant thereof, comprises a modified mRNA. In embodiments, the modified mRNA comprise at least one modified nucleoside optionally selected from a pseudouridine, 5-methylcytosine (m5C), 5- methyluridine (m5U), 2'-O-methyluridine (Um or m2'-0U), 2-thiouridine (s2U), and N6- methyladenosine (m6A).B. Expression Cassettes

[0147] Also provided herein are expression cassettes comprising the polynucleotides comprising the nucleic acids encoding Kcnnl, or a fragment or variant thereof, as described herein, operably linked to an expression control element. In embodiments, the expression cassette comprises a nucleic acid encoding a Kcnnl, wherein the nucleic acid is selected from: (1) a polynucleotide that encodes an amino acid sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 1; (2) a polynucleotide that encodes an amino acid sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 3; (3) a polynucleotide that is at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 2; (4) a polynucleotide that is at least about 50% identical, at least about 55% identical, at leastabout 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 4; and (5) a polynucleotide that is at least about 50% identical, at least about 55% identical, at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 5.

[0148] In embodiments, an expression control element is positioned 5’ of a nucleic acid encoding a Kcnnl.

[0149] In embodiments, expression cassette refers to a nucleic acid construct comprising nucleic acid elements sufficient for the expression of a polynucleotide molecule of the present disclosure. In embodiments, an expression cassette comprises a polynucleotide molecule of the present disclosure operably linked to a promoter sequence.

[0150] In embodiments, an expression control element refers to nucleic acid sequence(s) that influence expression of an operably linked nucleic acid. In embodiments, expression control elements comprise promoters and enhancers. In embodiments, vector sequences including rAAV vectors can include one or more expression control elements. In embodiments, such elements are included to facilitate proper heterologous polynucleotide transcription and as appropriate translation (e.g., a promoter, enhancer, splicing signal for introns, maintenance of the correct reading frame of the gene to permit in-frame translation of mRNA, stop codons, and termination / polyadenylation sequence). In embodiments, such elements act in cis. In embodiments, such elements act in trans.

[0151] In embodiments, expression control can be effected at the level of transcription, translation, splicing, message stability, etc. In embodiments, an expression control element that modulates transcription is juxtaposed near the 5’ end (ie., upstream) of a transcribed nucleic acid. In embodiments, expression control elements can also be located at the 3’ end (z.e., downstream) of the transcribed sequence or within the transcript (e.g., in an intron). In embodiments, expression control elements can be located adjacent to or at a distance away from the transcribed sequence (e.g., about 1-10, about 10-25, about 25- 50, about 50- 100, about 100-500, or more nucleotides from the polynucleotide), even at considerabledistances. Nevertheless, owing to the length limitations of AAV vectors, in embodiments, expression control elements in rAAV vectors are within 1 to 1000 nucleotides from the transcription start site of the heterologous nucleic acid.

[0152] In embodiments, expression of an operably linked nucleic acid is at least in part controllable by the element (e.g. , a promoter) such that the element modulates transcription of the nucleic acid and, as appropriate, translation of the transcript. In embodiments, a specific example of an expression control element is a promoter, which is usually located 5’ of the transcribed nucleic acid sequence. In embodiments, a promoter increases an amount expressed from operably linked nucleic acid as compared to an amount expressed when no promoter exists.

[0153] In embodiments, operably linked means that the regulatory sequences necessary for expression of a nucleic acid sequence are placed in the appropriate positions relative to the sequence so as to mediate expression of the nucleic acid sequence. This same definition is sometimes applied to the arrangement of nucleic acid sequences and transcription control elements (e.g, promoters, enhancers, and termination elements) in an expression vector, e.g, rAAV vector or non- viral vector. Encoding sequences can be operably linked to regulatory sequences in sense or antisense orientation. In embodiments, the promoter is a heterologous promoter.

[0154] In embodiments, heterologous promoter refers to a promoter that is not found to be operably linked to a given encoding sequence in nature. In embodiments, an expression cassette can comprise additional elements, for example, an intron, an enhancer, a polyadenylation site, a woodchuck response element (WRE), and / or other elements known to affect expression levels of the encoding sequence.

[0155] In embodiments, promoter refers to a nucleotide sequence capable of controlling the expression of a coding sequence or functional RNA. In embodiments, nucleic acid molecules of the present disclosure are located 3’ of a promoter sequence. In embodiments, a promoter sequence consists of proximal and more distal upstream elements and can comprise an enhancer element.

[0156] In embodiments, enhancer refers to a sequence that is located adjacent to the heterologous nucleic acid. Enhancer elements are typically located upstream of a promoter element but also function and can be located downstream of or within a sequence. In embodiments, an enhancer element can be located about 10-50 base pairs, about 50-100 base pairs, about 100-200 base pairs, or about 200-300 base pairs, or more base pairsupstream or downstream of a heterologous nucleic acid sequence. In embodiments, enhancer elements increase expression of an operably linked nucleic acid afforded by a promoter element.

[0157] In embodiments, expression control elements comprise ubiquitous or promiscuous promoter s / enhancers which are capable of driving expression of a polynucleotide in many different cell types. Non-limiting examples of such elements include the cytomegalovirus (CMV) immediate early promoter / enhancer sequences, the Rous sarcoma virus (RSV) promoter / enhancer sequences and the other viral promoters / enhancers active in a variety of mammalian cell types, or synthetic elements that are not present in nature (see, e.g., Boshart et al., Cell, 41 :521-530 (1985)), the SV40 promoter, the dihydrofolate reductase promoter, the cytoplasmic b-actin promoter and the phosphoglycerate kinase (PGK) promoter. Other examples of promoters include, but are not limited to, the phosphoglycerate kinase (PKG) promoter, CAG (composite of the CMV enhancer, the chicken beta actin promoter (CBA) and the rabbit beta globin intron), and other constitutive promoters; NSE (neuronal specific enolase), synapsin, andNeuN promoters; the SV40 early promoter, mouse mammary tumor virus LTR promoter; adenovirus major late promoter (Ad MLP); a herpes simplex virus (HSV) promoter, SFFV promoter, rous sarcoma virus (RSV) promoter; rat insulin promoter, TBG promoter and other liver-specific promoters; the desmin promoter and similar muscle-specific promoters; the EFl -alpha promoter, hybrid promoters, promoters with multi-tissue specificity, and the like, all of which are promoters well known and readily available to those of skill in the art. Promoters can be of human origin or from other species, including from mice. Other enhancers / promoters can be synthetic in origin, derived by machine learning and tested in various cultured cell systems in high-throughput reporter assays, as in Gosai et al., “Machine-guided design of cell-type-targeting cis-regulatory elements”, Nature 634, 1211-1220, 2024.

[0158] In embodiments, expression control elements comprise tissue-specific promoters and / or enhancers. In embodiments, expression control elements comprise inducible promoters and / or enhancers.

[0159] In embodiments, expression control elements comprise a native control element (e.g., promoter). In embodiments, other native expression control elements, such as introns, polyadenylation sites or Kozak consensus sequences can also be used.

[0160] In embodiments, expression constructs include one or more of an expression control (e.g., promoter / enhancer) element, a transcription termination signal, 5’ or 3’ untranslatedregions (e.g., polyadenylation (poly A) sequences) which flank a sequence, such as one or more copies of an AAV ITR sequence, an intron, filler or stuffer polynucleotide sequences. In embodiments, polynucleotides encoding protein(s) useful within the present disclosure, for example, SKI channel proteins, are used in gene therapy protocols for the treatment of diseases or disorders contemplated herein. In embodiments, the disease or disorder comprises Parkinson’s Disease (PD). In embodiments, constructs encoding the protein(s) are inserted into the appropriate gene therapy vector and administered to a subject to treat or prevent the disease or disorder of interest.C. Viral vectors

[0161] Also provided herein are viral vectors comprising polynucleotides encoding Kcnnl, or a fragment or variant thereof, as set forth herein. Any known vector known to those skilled in the art in view of the present disclosure can be used in the compositions disclosed herein. A variety of vectors, both viral vectors and plasmid vectors are known in the art (see, e.g., U.S. Patent No. 5,252,479 and WO 93 / 07282, each of which is incorporated by reference herein in its entirety). In particular, a number of viruses have been used as gene transfer vectors, including papovaviruses such as SV40, vaccinia virus, herpes viruses including HS V and EB V, retroviruses, and parvoviruses such as AAV. Many gene therapy protocols in the art have employed disabled murine retroviruses. Several issued patents are directed to methods and compositions for performing gene therapy (see, e.g., U.S. Patent Nos. 6,168,916; 6,135,976; 5,965,541 and 6,129,705, each of which is incorporated by reference herein in its entirety).

[0162] In embodiments, provided herein are viral vectors such as adeno-associated virus (AAV) vectors comprising polynucleotides encoding Kcnnl, or a fragment or variant thereof, as set forth herein.

[0163] In embodiments, vector or gene transfer vector refers to a nucleic acid molecule comprising a gene of interest. Examples of vectors include, but are not limited to, viral vectors delivered by viral particles such as retroviral, adenoviral, adeno-associated viral, and lenti viral particles, or virus-like particles (VLPs) that resemble viral particles but are non-infectious; and non-viral vectors delivered by non-viral gene transfer systems, such as microinjection, electroporation, liposomes, large natural polymers, large synthetic polymers, and polymers comprised of both natural and synthetic components.

[0164] In embodiments, a vector nucleic acid sequence generally contains at least an origin of replication for propagation in a cell and optionally additional elements, such as a heterologous polynucleotide sequence, expression control element (e.g., a promoter, enhancer), intron, an inverted terminal repeat (ITR), selectable marker (e.g., antibiotic resistance), polyadenylation signal.

[0165] In embodiments, gene transfer system refers to any means of delivering a composition comprising a nucleic acid sequence to a cell or tissue. In embodiments, a gene transfer system can be a viral gene transfer system, e.g., intact viruses, modified viruses and VLPs, used to facilitate delivery of a viral vector to a desired cell or tissue.

[0166] A viral vector is derived from or based upon one or more nucleic acid elements that comprise a viral genome. In embodiments, viral vectors include lentiviral and adeno- associated virus (AAV) vectors.

[0167] In embodiments, recombinant, as a modifier of vector, such as recombinant AAV (rAAV) vector, as well as a modifier of sequences such as recombinant polynucleotides and polypeptides, means that the compositions have been manipulated (z.e., engineered) in a fashion that generally does not occur in nature. Although the term recombinant is not always used herein in reference to AAV vectors, as well as sequences such as polynucleotides, recombinant forms including polynucleotides, are expressly included in spite of any such omission.

[0168] In embodiments, a recombinant AAV vector or rAAV is derived from the wild type genome of AAV by using molecular methods to remove the wild type coding sequence from an AAV genome and replacing it with a non-native nucleic acid sequence, referred to as a heterologous nucleic acid. Typically, for AAV one or both inverted terminal repeat (ITR) sequences of AAV genome are retained in the AAV vector. rAAV is distinguished from an AAV genome, since all or a part of the native AAV genome has been replaced with a non-native sequence with respect to the AAV genomic nucleic acid. In embodiments, incorporation of a non-native sequence therefore defines the AAV vector as a recombinant vector, which can be referred to as a rAAV vector.

[0169] In embodiments, an rAAV sequence can be packaged, referred to herein in embodiments as a particle, for subsequent infection (transduction) of a cell, ex vivo, in vitro or in vivo. Where a recombinant AAV vector sequence is encapsidated or packaged into an AAV particle, the particle can also be referred to as an rAAV vector or rAAV particle. SuchrAAV particles include proteins that encapsidate or package the vector genome and in the case of AAV, they are referred to as capsid proteins.

[0170] In embodiments, a vector genome refers to the portion of the recombinant plasmid sequence that is ultimately packaged or encapsidated to form a viral (e.g., rAAV) particle. In cases where recombinant plasmids are used to construct or manufacture recombinant vectors, the vector genome does not include the portion of the plasmid that does not correspond to the vector genome sequence of the recombinant plasmid. In embodiments, this non vector genome portion of the recombinant plasmid can be referred to as the plasmid backbone, which is used for cloning and amplification of the plasmid, a process that is used for propagation and recombinant virus production. Except for possible 3’ ITR and / or 5’ ITR cloning remnants, the plasmid backbone is not itself packaged or encapsidated into virus (e.g., AAV) particles. Thus, in embodiments, a vector genome refers to the polynucleotide that is packaged or encapsidated by virus (e.g., AAV).

[0171] Host cells for producing rAAV particles include but are not limited to microorganisms, yeast cells, insect cells, and mammalian cells that can be, or have been, used as recipients of heterologous rAAV vectors. Cells from the stable human cell line, HEK293 (readily available through, e.g., the American Type Culture Collection under Accession Number ATCC CRL1573) can be used, as well as derivatives of HEK293, e.g., with modifications to enhance viral production. In embodiments, a modified human embryonic kidney cell line (e.g., HEK293), which is transformed with adenovirus type-5 DNA fragments and expresses the adenoviral Ela and Elb genes, is used to generate rAAV particles. The modified HEK293 cell line is readily transfected and provides a particularly convenient platform in which to produce rAAV particles. Other host cell lines appropriate for rAAV production are described in International Application PCT / 2017 / 024951, the disclosure of which is herein incorporated in its entirety.

[0172] In embodiments, AAV helper functions are introduced into the host cell by transfecting the host cell with an AAV helper construct either prior to, or concurrently with, the transfection of an AAV expression vector. In embodiments, a host cell having AAV helper functions can be referred to as a helper cell or packaging helper cell. AAV helper constructs are thus sometimes used to provide at least transient expression of AAV rep and / or cap genes to complement missing AAV functions necessary for productive AAV transduction. AAV helper constructs often lack AAV ITRs and can neither replicate nor package themselves. These constructs can be in the form of a plasmid, phage, transposon,cosmid, virus, or virion. A number of AAV helper constructs have been described, such as the commonly used plasmids pAAV / Ad and pIM29+45 which encode both Rep and Cap expression products. A number of other vectors are known which encode Rep and / or Cap expression products.

[0173] Methods of generating rAAV particles capable of transducing mammalian cells are known in the art. For example, rAAV particles can be produced as described in US Patent 9,408,904; and International Applications PCT / US2017 / 025396 and PCT / US2016 / 064414, the disclosures of which are herein incorporated in their entirety.

[0174] The present disclosure provides cells comprising polynucleotides encoding Kcnnl, or a fragment or variant thereof, cells comprising expression cassettes comprising the polynucleotides encoding Kcnnl, or a fragment or variant thereof, and cells comprising viral vectors such as rAAV vectors comprising polynucleotides encoding Kcnnl, or a fragment or variant thereof. In embodiments, the cell produces a viral vector. In embodiments, the cell produces an rAAV vector as set forth herein.

[0175] Also provided are methods of producing viral vectors such as rAAV vectors as set forth herein. In embodiments, a method of producing rAAV vectors includes introducing an rAAV vector genome comprising a polynucleotide encoding Kcnnl, or a fragment or variant thereof, or expression cassette comprising a polynucleotide encoding Kcnnl, or a fragment or variant thereof, as set forth herein into a packaging helper cell; and culturing the helper cell under conditions to produce the rAAV vectors. In embodiments, a method of producing rAAV vectors includes introducing a polynucleotide encoding Kcnnl, or a fragment or variant thereof, or expression cassette comprising a polynucleotide encoding Kcnnl, or a fragment or variant thereof, as set forth herein into a packaging helper cell; and culturing the helper cells under conditions to produce the rAAV vector.

[0176] In embodiments, the cells are mammalian cells.

[0177] In embodiments, cells for vector production provide helper functions, such as AAV helper functions, that package the vector into a viral particle. In embodiments, the helper functions are Rep and / or Cap proteins for AAV vector packaging. In embodiments, cells for vector production can be stably or transiently transfected with polynucleotide(s) encoding Rep and / or Cap protein sequence(s). In embodiments, cells for vector production provide Rep78 and / or Rep68 proteins. In such cells, the cells can be stably or transiently transfected with Rep78 and / or Rep68 proteins polynucleotide encoding sequence(s).

[0178] In embodiments, cells for vector production are human embryonic kidney cells. In embodiments, cells for vector production are HEK-293 cells.

[0179] In embodiments, transduce refers to introduction of a molecule such as an rAAV vector into a cell or host organism. In embodiments, the heterologous polynucleotide / transgene may or may not be integrated into genomic polynucleotide of the recipient cell. In embodiments, the introduced heterologous polynucleotide can also exist in the recipient cell or host organism extrachromosomally, or only transiently.

[0180] In embodiments, a transduced cell is a cell into which the transgene has been introduced. Accordingly, in embodiments, a transduced cell (e.g., in a mammal, such as a cell or tissue or organ cell), means a genetic change in a cell following incorporation, for example, of a polynucleotide (e.g., a transgene) into the cell. Thus, in embodiments, a transduced cell is a cell or a progeny thereof into which an exogenous polynucleotide has been introduced. The cell(s) can be propagated, and the introduced protein expressed. For gene therapy uses and methods, a transduced cell can be in a subject.

[0181] In embodiments, isolated, when used as a modifier of a composition, means that the compositions are made by the hand of man or are separated, completely or at least in part, from their naturally occurring in vivo environment. Generally, isolated compositions are substantially free of one or more materials with which they normally associate with in nature, for example, one or more protein, nucleic acid, lipid, carbohydrate, or cell membrane. In embodiments, isolated does not exclude combinations produced by the hand of man, for example, a rAAV sequence, or rAAV particle that packages or encapsidates an AAV vector genome and a pharmaceutical formulation. In embodiments, isolated also does not exclude alternative physical forms of the composition, such as hybrids / chimeras, multimers / oligomers, modifications (e.g., phosphorylation, glycosylation, lipidation) or derivatized forms, or forms expressed in host cells produced by the hand of man.

[0182] In embodiments, recombinant AAV vectors, as well as methods and uses thereof, include any viral strain or serotype. As a non-limiting example, a recombinant AAV vector can be based upon any AAV genome, such as LK03, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV 12, RhlO, Rh74, AAV3B or AAV- 2i8. In embodiments, such vectors can be based on the same strain or serotype (or subgroup or variant) or be different from each other. As a non-limiting example, a recombinant AAV vector based upon a particular serotype genome can be identical to the serotype of the capsid proteins that package the vector. In addition, a recombinant AAV vector genomecan be based upon an AAV serotype genome distinct from the serotype of the AAV capsid proteins that package the vector. For example, the AAV vector genome can be based upon AAV2, whereas at least one of the three capsid proteins could be an LK03, AAV1, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74, AAV3B or AAV-2i8, or variant thereof or a self-complementary version thereof.

[0183] In embodiments, AAV vectors include LK03, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74, AAV3B and AAV-2i8, as well as variants (e.g., capsid variants, such as amino acid insertions, additions, substitutions, and deletions, e.g., that may change the tissue tropism or ability to cross the blood-brain barrier) thereof, for example, as set forth in WO 2013 / 158879, WO 2015 / 013313, US 2013 / 0059732, and WO 2016 / 210170, the disclosures of which are herein incorporated in their entirety.

[0184] In embodiments, the present methods employ the composition comprising a nucleic acid agent encoding Kcnnl, which is a viral vector comprising the nucleic acid agent encoding Kcnnl and / or the expression cassette. In embodiments, the viral vector comprises a recombinant adeno-associated virus (rAAV) vector. In embodiments, the AAV is selected from an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid serotype, a recombinant AAV (rAAV), or a functional variant of AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9, or a self-complementary version thereof. In embodiments, the viral vector comprises a recombinant anellovirus vector. In embodiments, the anellovirus vector is selected from TT virus (TTV), Torquetenomini virus (TTMV), and Torquetenomidivirus (TTMVD).

[0185] In embodiments, serotype is a distinction used to refer to an AAV having a capsid that is serologically distinct from other AAV serotypes. Serologic distinctiveness is determined on the basis of the lack of cross-reactivity between antibodies to one AAV as compared to another AAV. Such cross-reactivity differences are usually due to differences in capsid protein sequences / antigenic determinants (e.g., due to VP1, VP2, and / or VP3 sequence differences of AAV serotypes). Despite the possibility that AAV variants including capsid variants might not be serologically distinct from a reference AAV or other AAV serotype, they differ by at least one nucleotide or amino acid residue compared to the reference or other AAV serotype.

[0186] In embodiments, AAV capsid proteins can exhibit less than 100% sequence identity to a reference or parental AAV serotype such as AAV1, AAV2, AAV3, AAV4, AAV5,AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74, AAV3B, LK03, and AAV-2i8. In embodiments, a modified / variant AAV capsid protein comprises or consists of a sequence at least about 80%, about 85%, about 85%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.9%, or about 100% identical to a reference or parental AAV capsid protein, such as AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74, AAV3B, LK03, or AAV-2i8.

[0187] In embodiments, a viral vector such as an AAV vector or an rAAV vector comprises any of the polynucleotides comprising the polynucleotides encoding Kcnnl, or a fragment or variant thereof, as set forth herein operably linked to an expression control element.

[0188] In embodiments, a viral vector such as an AAV vector or an rAAV vector comprises any of the expression cassettes comprising the polynucleotides comprising the polynucleotides encoding Kcnnl, or a fragment or variant thereof, as set forth herein.

[0189] In embodiments, an rAAV vector comprises: one or more of an AAV capsid; and one or more AAV inverted terminal repeats (ITRs), wherein the AAV ITR(s) flanks the 5’ or 3’ terminus of the polynucleotide or the expression cassette. In embodiments, an AAV vector further comprises an intron positioned 5’ or 3’ of one or more ITRs.

[0190] In embodiments, delivery of a protein of interest to the cells of a mammal is accomplished by first generating an rAAV vector comprising a polynucleotide encoding the protein of interest and then administering the rAAV vector to the mammal. Thus, the present disclosure should be construed to include rAAV vectors comprising a polynucleotide encoding the protein of interest.

[0191] In embodiments, an rAAV vector comprises at least two copies of an AAV ITR sequence (SEQ ID NO: 6 and / or SEQ ID NO: 7), a promoter / enhancer element, a transcription termination / polyadenylation signal, any necessary 5’ or 3’ untranslated regions which flank a polynucleotide encoding the protein of interest, or a biologically active fragment or variant thereof. In embodiments, rAAV vectors of the present disclosure also include an intron known to produce splicing. In embodiments, rAAV vectors of the present disclosure comprise a polynucleotide encoding a mutated protein of interest.

[0192] AAV2 ITR has the following DNA sequence (SEQ ID NO: 6):AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGG CCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGC GAGCGCGCAG

[0193] In embodiments, one or more of the ITRs comprises a sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 6.

[0194] A modified AAV2 ITR has the following DNA sequence (SEQ ID NO: 7): CTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGC CTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0195] In embodiments, one or more of the ITRs comprises a sequence at least about 80% identical, at least about 85% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, or 100% identical to SEQ ID NO: 7.

[0196] In embodiments, an rAAV vector comprises a promoter / regulatory sequence that comprises a promiscuous promoter, which is capable of driving expression of a heterologous polynucleotide to high levels in many different cell types. In embodiments, the promoter is selected from a cytomegalovirus (CMV) immediate early promoter / enhancer sequence (e.g., SEQ ID NO: 8), Rous sarcoma virus promoter / enhancer sequences, and the like. In embodiments, the promoter sequence used to drive expression of the heterologous polynucleotide may be a tissue-specific promoter, such as, but not limited to, the transthyretin promoter, which is liver-specific, or the muscle creatine kinase promoter / enhancer, and the like, or may be an inducible promoter, for example, but not limited to, a steroid inducible promoter.

[0197] CMV enhancer / promoter sequence (SEQ ID NO: 8): GACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCC CATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCA ACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGA CTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATC AAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCT GGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTAT TAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGC GGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACC7 V\ATC7\ACGGGACTTTCC7VV\ATGTCGT7\AC7\ACTCCGCCCCATTGACGC7V\A TGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCT

[0198] In embodiments, the rAAV vector comprises a signal for transcription termination and polyadenylation. While any transcription termination signal may be included in the vector of the present disclosure, in embodiments, the transcription termination signal is the SV40 transcription termination / polyA signal.

[0199] The present disclosure should be construed to include any suitable AAV vector, including, but not limited to, vectors based on AAV1, AAV2, AAV3, AAV4 and AAV6, and the like, including self-complementary versions. Also disclosed is a method of treating a mammal having a disease or disorder in an amount effective to provide a therapeutic effect. The method comprises administering to the mammal a rAAV vector comprising the protein of interest. Preferably, the mammal is a human.IV. Agents That Increase Kcnnl Activity or Modulate Kcnnl or Small Molecules and Peptides That Mimic Protective Action of Kcnnl

[0200] In embodiments, provided herein are agents that increase Kcnnl activity, and methods of using the agents to treat or prevent PD, to treat or prevent one or more symptoms associated with PD, and / or to slow the progression of PD.

[0201] In embodiments, small molecule Kcnnl activators and / or positive modulators with varying specificity for Kcnnl may be used to treat and / or prevent PD, to treat or prevent one or more symptoms associated with PD, and / or to slow the progression of PD. In embodiments, the small molecule mimics action of overexpression of Kcnnl by preventing appearance of phospho-serine- 129 alpha-synuclein or by promoting phospho-serine- 129 alpha-synuclein clearance. In embodiments, without wishing to be bound by theory, the small molecule mimics overexpression of Kcnnl in protect! on / survival by binding to the ER membrane or by induction of an ER stress response, an integrated stress response, and / or mitochondrial stress response which mimics overexpression of Kcnnl in protect! on / survival by binding to the ER membrane or by induction of an ER stress response, an integrated stress response, and / or mitochondrial stress response. In embodiments, without wishing to be bound by theory, the small molecule mimics action of overexpression of Kcnnl via autophagic or endosomal clearance mechanisms.

[0202] In embodiments, the small molecule increases Kcnnl channel activity. Nonlimiting examples of agents that increase Kcnnl activity include a triazolo pyrimidinecalled N-{7-[l-(4-chloro-2-methylphenoxy)ethyl]-[l,2,4]triazolo[l,5-a]pyrimidin-2-yl}- N'-methoxy-formamidine (“(-)CM-TPMF”), and derivatives; chlorzoxazone; 5,6- Dichloro- 1 -ethyl- 1 ,3 -dihydro-2H-benzimidazol-2-one (DCEBIO); 1 -ethyl-2- benzimidazolinone (1-EBIO) and / or riluzole; or a pharmaceutically acceptable salt thereof or a small molecule derived from such compounds. (-)CM-TPMF, a potent KCNN1- specific activator, was reported by Hougaard et al. (“Evidence for a common pharmacological interaction site on K(Ca)2 channels providing both selective activation and selective inhibition of the human K(Ca)2.1 subtype,” Mol Pharmacol. 2012 Feb;81(2):210-9), see also (U.S. PatentNo. 8,765,770 and U.S. Patent No. 8,685,987), each of which is incorporated herein by reference in its entirety. (-)CM-TPMF was shown to have specificity for human KCNN1.

[0203] The activity of (-)CM-TPMF has been confirmed using HeLa cells transiently transfected with Kcnnl, measuring outward current following compound addition in patch recordings. However, the level of activity that could be attained by activating the number of channels present endogenously may not parallel the activity obtained from the increased number of channels in the setting of transgenesis. In embodiments, the methods described herein comprise administration of (-)CM-TPMF, chlorzoxazone, DCEBIO, 1-EBIO, and / or riluzole to treat one or more symptoms associated with PD and / or to slow the progression ofPD.

[0204] In embodiments, peptides may be used to treat and / or prevent PD, to treat or prevent one or more symptoms associated with PD, and / or to slow the progression of PD. In embodiments, the peptide mimics action of overexpression of Kcnnl.V. Pharmaceutical Compositions and Administration

[0205] Also provided herein are pharmaceutical compositions comprising the agents, rAAV particles, viral vectors, expression cassettes, agents encoding for Kcnnl, or a fragment or variant thereof, and polynucleotides encoding human KCNN1, or a fragment or variant thereof, methods of treating or preventing PD comprising administration of the pharmaceutical compositions, and the various constructs provided herein for use in treating PD.

[0206] In embodiments, the expression cassette, cDNA, mRNA, viral vector, agent, or pharmaceutical composition is administered by intrastriatal injection, substantia nigra pars compacta delivery, striatum (e.g., putamen) delivery, and / or nigrostriatal region delivery.

[0207] In embodiments, the delivery is to the substantia nigra of the brain of the subject. In embodiments, the delivery is to one of or both left and right substantiae nigra of the brain of the subject. In embodiments, the delivery is to one or both of the pars reticulata (SNpr) and / or the delivery is to one or both of the pars compacta (SNpc).

[0208] In embodiments, the delivery is MRI-guided. In embodiments, the delivery is intraoperatively MRI-guided.

[0209] In embodiments, pharmaceutical composition refers to a mixture of at least one agent useful within the present disclosure with other chemical components, such as carriers, stabilizers, diluents, dispersing agents, suspending agents, controlled release agents, thickening agents, and / or excipients. The pharmaceutical composition facilitates administration of the agent to an organism. Multiple techniques of administering an agent or pharmaceutical composition comprising the agent exist in the art including, but not limited to: intrastriatal, nasal, inhalational, topical, oral, buccal, rectal, pleural, peritoneal, vaginal, intramuscular, subcutaneous, transdermal, epidural, intratracheal, optic, intraocular, intracranial, intrathecal, and intravenous routes. In embodiments, parenteral administration of an agent or pharmaceutical composition comprising the agent includes, e.g., subcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), intrastemal injection, and / or infusion techniques.

[0210] In embodiments, pharmaceutically acceptable carrier means a pharmaceutically acceptable material, composition or carrier, such as a liquid or solid filler, stabilizer, dispersing agent, suspending agent, diluent, excipient, thickening agent, solvent or encapsulating material, involved in carrying or transporting a compound useful within the present disclosure within or to the subject such that it may perform its intended function. Typically, such constructs are carried or transported from one organ, or portion of the body, to another organ, or portion of the body. In embodiments, each carrier is acceptable in the sense of being compatible with the other ingredients of the formulation, including the compound useful within the present disclosure, and not injurious to the subject. Some nonlimiting examples of materials that may serve as pharmaceutically acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols, such as propylene glycol; polyols,such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; surface active agents; alginic acid; pyrogen-free water; isotonic saline; Ringer’s solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations.

[0211] In embodiments, pharmaceutically acceptable carrier also includes any and all coatings, antibacterial and antifungal agents, and absorption delaying agents including controlled release materials (see, e.g., Uhrich, Cannizzaro, Langer, and Shakesheff, Chem. Rev. 99, 3181-3198, 1999 “Polymeric Systems for Controlled Drug Release”), and the like that are compatible with the activity of the compound useful within the present disclosure and are physiologically acceptable to the subject. Supplementary active compounds may also be incorporated into the compositions. In embodiments, pharmaceutically acceptable carrier may further include a pharmaceutically acceptable salt of the compound useful within the present disclosure. Other additional ingredients that may be included in the pharmaceutical compositions used in the practice of the present disclosure are known in the art and described, for example in Remington’s Pharmaceutical Sciences (Genaro, Ed., Mack Publishing Co., 1985, Easton, PA), which is incorporated herein by reference.

[0212] In embodiments, pharmaceutically acceptable salt refers to a salt of the administered compounds prepared from pharmaceutically acceptable non-toxic acids, including inorganic acids or bases, organic acids or bases, solvates, hydrates, or clathrates thereof.

[0213] In embodiments, the number of viral vector genomes / mammal that are administered in a single injection ranges from about IxlO8to about 5xl016. In embodiments, the number of viral vector genomes / mammal that are administered in a single injection is from about IxlO10to about IxlO15. In embodiments, the number of viral vector genomes / mammal that are administered in a single injection is from about 5xlO10to about 5xl015. In embodiments, the number of viral vector genomes / mammal that are administered in a single injection is from about 5xl0nto about 5xlO14

[0214] In embodiments, when the method of the present disclosure comprises multiple site simultaneous injections, or several multiple site injections comprising injections into different sites over a period of several hours (for example, from about less than one hour to about two or three hours), the total number of viral vector genomes administered may beidentical, or a fraction thereof or a multiple thereof, to that recited in the single site injection method.

[0215] In embodiments, for administration of an rAAV vector in a single site injection, a composition comprising the virus is injected directly into the CNS of the subject.

[0216] In embodiments, for administration to a mammal, an rAAV vector may be suspended in a pharmaceutically acceptable carrier, for example, HEPES buffered saline at a pH of about 7.8. In embodiments, other useful pharmaceutically acceptable carriers include, but are not limited to, glycerol, water, saline, ethanol and other pharmaceutically acceptable salt solutions such as phosphates and salts of organic acids. Examples of these and other pharmaceutically acceptable carriers are described in Remington’s Pharmaceutical Sciences (1991, Mack Publication Co., New Jersey).

[0217] In embodiments, pharmaceutical compositions of the present disclosure are useful in the methods described herein, when used in combination with at least one additional agent useful for treating or preventing PD. In embodiments, this additional agent may comprise compounds identified herein or compounds, e.g., commercially available compounds, known to treat, prevent or reduce the symptoms of PD in a subject. Nonlimiting examples of such agents include L-DOPA, carbidopa, apomorphine, pramipexole, and ropinirole.

[0218] In embodiments, the regimen of administration may affect what constitutes an effective amount. In embodiments, pharmaceutical compositions of the disclosure may be administered to the subject either prior to or after the onset of a disease or disorder contemplated in the present disclosure. In embodiments, several divided dosages, as well as staggered dosages may be administered daily or sequentially, or the dose may be continuously infused, or may be a bolus injection. In embodiments, the dosages of the pharmaceutical compositions may be proportionally increased or decreased as indicated by the exigencies of the therapeutic or prophylactic situation.

[0219] In embodiments, administration of the pharmaceutical compositions of the present disclosure to a subject, optionally a mammal, optionally a human, may be carried out using known procedures, at dosages and for periods of time effective to treat a disease or disorder contemplated in the present disclosure. An effective amount of the agent necessary to achieve a therapeutic effect may vary according to factors such as the state of the disease or disorder in the subject; the age, sex, and weight of the subject; and the ability of the agentto treat a disease or disorder contemplated in the present disclosure. In embodiments, dosage regimens may be adjusted to provide the optimum therapeutic response.

[0220] In embodiments, actual dosage levels of the pharmaceutical compositions of the present disclosure may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being unduly toxic to the subject.

[0221] In embodiments, the selected dosage level depends upon a variety of factors including the activity of the particular agent employed, the time of administration, the rate of excretion of the agent, the duration of the treatment, other drugs, compounds or materials used in combination with the agent, the age, sex, weight, condition, general health and prior medical history of the subject being treated, and like factors well-known in the medical arts.

[0222] In embodiments, it is advantageous to formulate the agent in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit containing a predetermined quantity of agent calculated to produce the desired therapeutic effect in association with the required pharmaceutical vehicle. In embodiments, dosage unit forms of the present disclosure are dictated by and directly dependent on (a) the unique characteristics of the agent and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding / formulating such an agent for the treatment of a disease or disorder contemplated in the present disclosure.

[0223] The frequency of administration of the pharmaceutical compositions of the present disclosure can vary from subject to subject depending on many factors including, but not limited to, age, disease or disorder to be treated, gender, overall health, and other factors. Thus, embodiments of the present disclosure should not be construed to be limited to any particular dosage regime and the precise dosage and composition to be administered to any subject is determined by the attending physician taking all other factors about the subject into account.

[0224] In embodiments, formulations can be employed in admixtures with conventional excipients, z.e., pharmaceutically acceptable organic or inorganic carrier substances suitable for intrastriatal, oral, parenteral, nasal, intravenous, subcutaneous, enteral, or any other suitable mode of administration, known to the art. In embodiments, the pharmaceutical preparations may be sterilized and if desired mixed with auxiliary agents,e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure buffers, coloring, flavoring and / or aromatic substances and the like. In embodiments, they may also be combined where desired with other active agents.

[0225] In embodiments, routes of administration of any of the pharmaceutical compositions of the present disclosure include, but are not limited to, intrastriatal, oral, nasal, rectal, intravaginal, parenteral, buccal, sublingual, subcutaneous, intravenous, intradermal, intramuscular, intraperitoneal, or topical. In embodiments, the pharmaceutical compositions for use of the present disclosure may be formulated for administration by any suitable route, such as intrastriatal, oral, or parenteral: for example, transdermal, transmucosal [e.g., sublingual, lingual, (trans)buccal, (trans)urethral, vaginal (e.g., trans- and perivaginally), (intra)nasal and (trans)rectal)], intravesicular, intrapulmonary, intraduodenal, intragastrical, intrathecal, intracerebral, intracerebellar, intracerebroventricular, subcutaneous, intramuscular, intradermal, intra-arterial, intravenous, intrabronchial, inhalation, and topical administration.

[0226] In embodiments, agent refers to biologically active substances including viruses, compounds, and / or means to alter a biological mechanism. Agents can exhibit biological effects on one or more cells, tissues, circuits, or organs.VI. Methods of Making PD Therapies

[0227] Also provided herein are methods of making a PD therapy.

[0228] In embodiments, provided herein is a method of making a PD therapy comprising(a) identifying the PD therapy by: (i) administering an effective amount of a test agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal; (ii) identifying the test agent as a PD therapy if the transgenic animal demonstrates one or more of increased expression of Kcnnl, increased activity of the gene product of Kcnnl, improved survival time, or reduced paralysis, as compared to a control; and (b) formulating the candidate agent for administration for the treatment of PD.

[0229] In embodiments, provided herein is a method of making a PD therapy comprising (a) identifying a candidate agent that modulates Kcnnl, comprising: (i) providing a cell line expressing Kcnnl; (ii) treating the cell line with one or more test agents; (iii) measuring a voltage-independent potassium channel activity in the cell; and (iv) identifying the test agent as a candidate agent if voltage-independent potassium channel activity is increased in the presence of the test agent, compared to in the absence of the test agent and / or presenceof a control agent that is known not to be a candidate agent; and (b) administering an effective amount of the candidate agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal; (c) classifying the candidate agent as a PD therapy if the transgenic animal demonstrates one or more of increased expression of Kcnnl, increased activity of the gene product of Kcnnl, improved survival time, or reduced paralysis, as compared to a control; and (d) formulating the PD therapy for administration for the treatment of PD.

[0230] In embodiments, provided herein is a method of making a PD therapy comprising (a) identifying a candidate agent that modulates Kcnnl, comprising: (i) providing a cell line expressing Kcnnl; (ii) treating the cell line with one or more test agents; (iii) measuring the levels of selected stress-response elements in the cell; and (iv) identifying the test agent as a candidate agent if stress response activity is increased in the presence of the test agent, compared to in the absence of the test agent and / or presence of a control agent that is known not to be a candidate agent; and (b) administering an effective amount of the candidate agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal; (c) classifying the candidate agent as a PD therapy if the transgenic animal demonstrates one or more of increased expression of Kcnnl, increased activity of the gene product of Kcnnl, improved survival time, or reduced paralysis, as compared to a control; and (d) formulating the PD therapy for administration for the treatment of PD.

[0231] In embodiments, provided herein is a method of making a PD therapy comprising (a) identifying a candidate agent that that mimics the activity of Kcnnl, comprising: (i) providing a neuronal cell line or primary cultured neurons; (ii) treating the neuronal cell line or primary cultured neurons with one or more test agents; (iii) measuring the levels of selected stress-response elements in the neuronal cell line or primary cultured neurons; and (iv) identifying the test agent as a candidate agent if stress response activity is increased in the presence of the test agent, compared to in the absence of the test agent and / or presence of a control agent that is known not to be a candidate agent; and (b) administering an effective amount of the candidate agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal; (c) classifying the candidate agent as a PD therapy if the transgenic animal demonstrates one or more of improved survival time and reduced motor compromise as compared to a control; and (d) formulating the PD therapy for administration for the treatment of PD.

[0232] In embodiments, the test agent is a small molecule or peptide. In embodiments, the candidate agent is a small molecule or peptide.

[0233] In embodiments, provided herein is a method of identifying a PD therapy that is effective in treating or preventing PD comprising administering an effective amount of the agent to an A53T mutant alpha-synuclein transgenic animal and determining time to paralysis, wherein an increased time to paralysis compared to an A53T mutant alpha- synuclein transgenic animal that does not receive the agent indicates that the agent is a PD therapy.

[0234] In embodiments, the PD therapy is a gene therapy, a biologic agent, a small molecule, or a polynucleotide agent. In embodiments, the gene therapy is a viral gene therapy. In embodiments, the viral gene therapy is or comprises a recombinant adeno- associated virus (rAAV). In embodiments, the rAAV comprises a Kcnnl nucleic acid. In embodiments, the biologic agent is an antibody or peptide. In embodiments, the polynucleotide agent is selected from mRNA, siRNA, shRNA, miRNA, and cDNA. In embodiments, the small molecule mimics action of overexpression of Kcnnl by preventing appearance of phospho-serine- 129 alpha-synuclein or by promoting phospho-serine- 129 alpha-synuclein clearance. In embodiments, the peptide mimics action of overexpression of Kcnnl.

[0235] In embodiments, there is provided a method of mimicking the effect of Kcnnl using cultured HeLa cells to identify small molecule compound(s) that promote the clearance of A53T alpha-synuclein-mCherry associated with the same morphologic features at the EM level observed to be produced by Kcnnl transfection (e.g., MVB and autophagosome formation as well as nuclear envelope invagination). In embodiments, such candidate small molecules are administered to A53T alpha synuclein transgenic mice, and prolongation of survival associated with reduction of serl29 phosphorylated pathogenic version of alpha- synuclein is observed.VII. Longevity

[0236] In embodiments, there is provided methods for increasing a subject’s longevity or lifespan. For instance, in embodiments, the present disclosure relates to the administration of the described Kcnnl compositions to a subject to increase longevity or lifespan. For example, the present disclosure may increase a subject’s longevity or lifespan by at least about 5, at least about 10, at least about 15, at least about 20, or at least about 25 years, ascompared to a subject that is not administered the described Kcnnl compositions and / or as compared to a life expectancy calculation, as described herein. Further, embodiments of the present disclosure contemplates methods that reduce or decrease cellular senescence and / or immunosenescence in a subject.

[0237] In embodiments, an increase in longevity or lifespan is assessed relative to a comparable population. For example, an increase in longevity or lifespan is assessed relative to a cohort - e.g., cohort LEB, the mean length of life of an actual birth cohort (all individuals bom a given year) or a period - e.g., period LEB, the mean length of life of a hypothetical cohort assumed to be exposed, from birth through death, to the mortality rates observed at a given year. Such assessments can be made relative to various reports on lifespan and / or longevity in the art (e.g., World Health Organization (WHO)’s Health Status Statistics: Mortality). In embodiments, the present methods provide for increased longevity or lifespan than what is expected relative to comparable populations. In embodiments, the present methods provide for increased longevity or lifespan than what is expected relative to various reports on lifespan and / or longevity in the art (e.g., World Health Organization (WHO)’s Health Status Statistics: Mortality).

[0238] In embodiments, an increase in longevity or lifespan is assessed with reference to one or more actuarial life tables, e.g., Life Tables For The United States Social Security Area 1900-2100 (Actuarial Study No. 120, Bell and Miller). In embodiments, the present methods provide for increased longevity or lifespan than what is expected relative to one or more actuarial life tables.

[0239] In embodiments of the present disclosure, the subject is a young human, a middle- aged human, or an elderly human. For example, in embodiments, the subject is between about 18 and about 35 years, or between about 18 and about 30 years, or between about 18 and about 25 years, or between about 18 and about 20 years. In embodiments, the subject is between about 36 and about 55 years, or between about 40 and about 55 years, or between about 45 and about 55 years, or between about 36 and about 50 years, or between about 36 and about 45 years, or between about 36 and about 40 years, or between about 40 and about 50 years old, or between about 45 and about 55 years old. In embodiments, the subject is between about 56 and about 85 years, or between about 60 and about 85 years, or about 65 and about 85 years, or between about 70 and about 85 years, or between about 75 and about 85 years, or between 80 and about 85 years, or between 56 and about 80 years, or between 56 and about 75 years, or between 56 and about 70 years, or between 56 and about 65 years,or between 56 and about 60 years, or between about 60 years and about 80 years, or about 65 years and about 75 years.

[0240] In embodiments, the subject is about 1, or about 2, or about 3, or about 4, or about5, or about 6, or about 7, or about 8, or about 9, or about 10, or about 11, or about 12, or about 13, or about 14, or about 15, or about 16, or about 17, or about 18, or about 19, or about 20, or about 21, or about 22, or about 23, or about 24, or about 25, or about 26, or about 27, or about 28, or about 29, or about 30, or about 31, or about 32, or about 33, or about 34, or about 35, or about 36, or about 37, or about 38, or about 39, or about 40, or about 41, or about 42, or about 43, or about 44, or about 45, or about 46, or about 47, or about 48, or about 49, or about 50, or about 51, or about 52, or about 53, or about 54, or about 55, or about 56, or about 57, or about 58, or about 59, or about 60, or about 61, or about 62, or about 63, or about 64, or about 65, or about 66, or about 67, or about 68, or about 69, or about 70, or about 71, or about 72, or about 73, or about 74, or about 75, or about 76, or about 77, or about 78, or about 79, or about 80, or about 81, or about 82, or about 83, or about 84, or about 85, or about 86, or about 87, or about 88, or about 89, or about 90, or about 91, or about 92, or about 93, or about 94, or about 95, or about 96, or about 97, or about 98, or about 99, or about 100 years old. In embodiments, the subject is at least 55 years old.

[0241] A person of skill in the art will contemplate that age ranges with respect to “young,” “middle-aged,” and “elderly” definitions can vary based on geographic region, among other factors. Petry, Gerontologist 2002 Feb;42(l):92-9 describes age-related definitions and is hereby incorporated by reference in its entirety.

[0242] In embodiments, there is provided methods of improving or reducing and / or treating or preventing frailty in a subject, where the method includes: identifying a subject desiring or in need of frailty treatment or prevention, and administering to the subject the described Kcnnl composition(s).

[0243] In embodiments, the frailty is age-related. In embodiments, frailty comprises an accumulation of deficiencies in major physiological functions, reduction of regeneration capabilities, impaired wound healing and increased risk of age-related diseases. For example, in embodiments, frailty is associated with natural aging or accelerated aging. Frailty can be measured according to any number of indices or tests known to one of skill in the art. For example, one such index, the Physiological Frailty Index (PFI), includes measurement of one or more parameters selected from grip strength, systolic bloodpressure, diastolic blood pressure, blood flow volume, number of blood neutrophils, percentage of blood neutrophils, number of blood monocytes, percentage of blood monocytes, number of lymphocytes, number of red blood cells, hemoglobin levels, hematocrit levels, mean corpuscular volume, mean corpuscular hemoglobin levels, mean corpuscular hemoglobin concentration and keratinocyte-derived cytokine levels. Deviation from a reference standard in any one individual is known as a deficit, and the overall average PFI score of the individual is a ratio of deficits to the total number of parameters measured.

[0244] In embodiments, the present disclosure provides methods of improving or reducing and / or treating or preventing frailty in a subject, as measured by a reduction in the PFI score of the subject. In embodiments, methods and compositions of the present disclosure for improving or reducing and / or treating or preventing frailty in a subject include maintaining a PFI score over time so that the score increases at a rate slower than if the subject were not being administered the described Kcnnl composition(s). In embodiments of the present disclosure, the PFI score of the subject remains nearly the same over time. In embodiments, methods of the present disclosure provide for a reduction in cellular senescence and immunosenescence associated with natural aging and / or accelerated aging (e.g., accelerated aging induced by, e.g., cancer or a cancer treatment).

[0245] In embodiments, the present disclosure provides for methods of treating or preventing an age-related disease or disorder in a subject, where the method includes: identifying a subject desiring or in need of treatment or prevention of an age-related disease or disorder, and administering to the subject the described Kcnnl composition(s). In embodiments, the age-related disease or disorder is characterized by increased cellular senescence or immunosenescence.

[0246] In embodiments, an age-related disease or disorder is selected from accelerated aging, cardiovascular disease, cerebrovascular disease, peripheral vascular disease, cardiac diastolic dysfunction, benign prostatic hypertrophy, aortic aneurysm, emphysema, atherosclerosis, diabetes, pulmonary fibrosis, blindness, dementia, Alzheimer’s disease, kidney dysfunction, osteoarthritis, low grade chronic sterile inflammation, herniated intervertebral disc, frailty, hair loss, hearing loss, vision loss, muscle fatigue, skin conditions, skin nevi, wrinkly skin, hyperpigmentation, scarring, keloid, rosacea, vitiligo, ichthyosis vulgaris, dermatomyositis, actinic keratosis, and sarcopenia.

[0247] In embodiments, methods of the present disclosure include treating or preventing accelerated aging. In embodiments, accelerated aging is a Progeroid syndrome or symptom thereof, including, but not limited to, Hutchinson-Gilford progeria syndrome (HGPS), Werner syndrome (WS), Bloom syndrome (BS), Rothmund-Thomson syndrome (RTS), Cockayne syndrome (CS), xeroderma pigmentosum (XP), trichothiodystrophy (TTD), combined xeroderma pigmentosum-Cockayne syndrome (XP-CS), or restrictive dermopathy (RD). Subjects having one of these diseases or disorders typically have reduced longevity (i.e., lifespan).VIII. Definitions

[0248] As used herein, “a,” “an,” or “the” can mean one or more than one.

[0249] The use of “or” means “and / or” unless stated otherwise. The terms “and / or” and“any combination thereof’ and their grammatical equivalents as used herein, can be used interchangeably. These terms can convey that any combination is specifically contemplated. Solely for illustrative purposes, the following phrases “A, B, and / or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.” The term “or” can be used conjunctively or disjunctively, unless the context specifically refers to a disjunctive use.

[0250] The use of “some embodiments,” “an embodiment,” “one embodiment” or “other embodiments” means that a particular feature, structure, or characteristic described in connection with the embodiments is included in at least some embodiments, but not necessarily all embodiments, of the present disclosures.

[0251] Further, the term “about” when used in connection with a referenced numeric indication means the referenced numeric indication plus or minus up to 10% of that referenced numeric indication. For example, the language “about 50%” covers the range of 45% to 55%.

[0252] Ranges: throughout this disclosure, it should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the present disclosure. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well asindividual numbers within that range, for example, 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0253] An “effective amount,” when used in connection with medical uses is an amount that is effective for providing a measurable treatment, prevention, or reduction in the rate of pathogenesis of a disorder of interest.

[0254] As used herein, the term “modulate” refers to any change in biological state, e.g., increasing, decreasing, and the like.

[0255] As used herein, something is “decreased” if a read-out of activity and / or effect is reduced by a significant amount, such as by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100%, in the presence of an agent or stimulus relative to the absence of such modulation. As will be understood by one of ordinary skill in the art, in embodiments, activity is decreased, and some downstream readouts will decrease but others can increase.

[0256] Conversely, activity is “increased” if a read-out of activity and / or effect is increased by a significant amount, for example by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 97%, at least about 98%, or more, up to and including at least about 100% or more, at least about 2-fold, at least about 3-fold, at least about 4-fold, at least about 5-fold, at least about 6-fold, at least about 7-fold, at least about 8-fold, at least about 9-fold, at least about 10-fold, at least about 50-fold, at least about 100-fold, in the presence of an agent or stimulus, relative to the absence of such agent or stimulus.

[0257] As referred to herein, all compositional percentages are by weight of the total composition, unless otherwise specified. As used herein, the word “include,” and its variants, is intended to be non-limiting, such that recitation of items in a list is not to the exclusion of other like items that may also be useful in the compositions and methods of this technology. Similarly, the terms “can” and “may” and their variants are intended to be non-limiting, such that recitation that an embodiment can or may comprise certain elements or features does not exclude other embodiments of the present technology that do not contain those elements or features.

[0258] Although the open-ended term “comprising,” as a synonym of terms such as including, containing, or having, is used herein to describe and claim the disclosure, the present disclosure, or embodiments thereof, may alternatively be described using alternative terms such as “consisting of’ or “consisting essentially of.”

[0259] As used herein, the words “preferred” and “preferably” refer to embodiments of the technology that afford certain benefits, under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the technology.

[0260] “Naturally occurring” as applied to an object refers to the fact that the object can be found in nature. For example, a polypeptide or polynucleotide sequence that is present in an organism (including viruses) that can be isolated from a source in nature, and which has not been intentionally modified by man is a naturally occurring sequence.

[0261] As used herein, the term “wild type” or “normal” refers to the genotype and phenotype that is characteristic of most of the members of a species occurring naturally and contrasting with the genotype and phenotype of a mutant.

[0262] As used herein, “methods of treatment” are equally applicable to use of a composition for treating the diseases or disorders described herein and / or compositions for use and / or uses in the manufacture of a medicaments for treating the diseases or disorders described herein.

[0263] As used herein, the term “prevent” or “prevention” means no disorder or disease development if none had occurred, or no further disorder or disease development if there had already been development of the disorder or disease. Also considered is the ability to prevent some or all of the symptoms associated with the disorder or disease.

[0264] As used herein, “treating” or “treatment” of a state, disorder or condition includes inhibiting the state, disorder or condition, z.e., arresting or reducing the development of the disease or at least one clinical or subclinical symptom thereof, or relieving the disease, z.e., causing regression of the state, disorder or condition or at least one of its clinical or subclinical symptoms.EXAMPLESExample 1. Evaluation of Kcnnl as a Modifier of PD

[0265] A mouse study was carried out to examine effect of Kcnnl expression in a PD mouse model.

[0266] Mouse models offer an approach to investigate mechanisms and potential treatments of alpha-synuclein-associated PD. To date, at least four transgenic alpha- synuclein mouse models have been investigated in detail (Aniszewska et al., “Modeling Parkinson's disease-related symptoms in alpha-synuclein overexpressing mice,” Brain Behavior 2022, Jul;12(7):e2628). Using either a Thyl promoter or a PDGFbeta promoter, all of the alpha-synuclein-expressing mouse lines present aggregation of alpha-synuclein in the CNS as well as dopaminergic alterations, but each exhibits a somewhat different distribution of alpha-synuclein expression and aggregation (see Fig. 2 in Aniszewska). Phenotypic features include hindlimb clasping, spasticity, decreased locomotor activity (bradykinesia), and muscular rigidity, the latter two features cardinal to human PD. Lifespan is shortened in these mice by the progression of the motor symptoms, leaving them ultimately immobilized and unable to acquire food and water.

[0267] The Thyl-A53T human alpha-synuclein transgenic line described by Martin et al (“Mitochondrial permeability transition pore regulates Parkinson's disease development in mutant a-synuclein transgenic mice,” Neurobiol Aging . 2014 May;35(5): 1132-52) provides an example of the phenotypic and histologic progression in a C57BL / 6 mouse model of synuclein disease. Mice developed bradykinesia, tremors, and ataxia by 6 months of age and were completely immobile by 10-15 months of age. Immunostaining with anti-human alpha-synuclein antibodies showed robust expression in brain at 6 months of age - in cortex (L5), olfactory bulb, striatum, diencephalon, brainstem, and cerebellar deep nuclei. Neurons exhibited immunostaining, but glia did not. Cortical neurons showed cytosolic and nuclear aggregates of alpha-synuclein. Neurons in substantia nigra were strongly immunoreactive as was neuropil of the striatum, the latter consistent with alpha-synuclein in nigrostriatal projection neurons. By six months of age, there was dropout of larger nigral neurons as compared with counts from control nontransgenic littermates, with some remaining neurons showing cytoplasmic Lewy-like inclusions. Thus, this A53T mouse strain shares major clinical and pathologic features with human PD.

[0268] A strain of A53T mutant alpha-synuclein transgenic mice, a mouse model for Parkinson’s disease, was obtained (A53 / BL6). The model develops hypokinesis, lower extremity clenching, and paralysis due to the accretion of alpha-synuclein aggregates from the transgene. In this manner it provides a mouse model of the symptoms of PD. It was observed that the symptoms were slow to develop in the A53 / BL6 model, and therefore the model was changed to B6SJL status (by crossing the original A53T / BL6 to SJL / J to yield A53T / B6SJL). The resultant A53T / B6SJL mice become motor-deficient, unable achieve upright position or walk by just over 300 days old, with a median of 265 days.

[0269] Thyl-Kcnnl mice were crossed to the A53T / B6SJL disease strain to create a genetic transfer of the Kcnnl gene into the mice, and the resultant transferred mice showed considerable improvement in motor function and survival.

[0270] FIGs. 1A-1B illustrate Kaplan-Meier survival plots for transgenic mice carrying either (a) Thyl promoter-driven A53T alpha-synuclein alone in a B6 / SJL background (designated A53T line), or (b) further sets of mice with A53T alone in the B6 / SJL background or such mice also hemizygously transgenic for either -3 copies or -6 copies of mouse Kcnnl (Kcnnl-3 or Kcnnl-6, respectively), produced by crossing A53T / B6SJL mice to transgenic Kcnnl / B6SJL mouse lines with the respective copy number (-3 copies or -6 copies as measured by real time PCR of tail DNA).

[0271] As illustrated in FIGs. 1A and IB, the A53T / B6SJL line had a median survival time to end-stage immobility of -260 days (257 or 265, respectively for the two experiments in FIG. 1A and FIG. IB), and a range of survival time to end-stage from -180 to 320 days. Penetrance was complete in the experiment, as all animals reached end-stage. This contrasts with A53T carried in a C57BL / 6 background, where penetrance is not complete and where the mice that reach end-stage do so at -15 months.

[0272] In contrast with the A53T / B6SJL strain, the Kcnnl-3 / A53T line exhibited extended survival, with a median of 392 days and a range of -285 to 450 days. The median survival of 392 days amounts to an -50% extension of survival of the Kcnnl-3 / A53T line as compared with the A53T line alone. When the strain Kcnnl-6 was mated to the A53T / B6SJL strain, the median survival of the resultant strain was 539 days, amounting to an -100% extension of survival, with a sixth animal alive and well at 654 days (Fig. IB). Mice of the Kcnnl-3 cohort exhibited the same clinical features as exhibited by the mice transgenic for A53T alone, but with onset occurring later. That is, mice were unable to maintain upright posture (Fig. 2), rolling and writhing. Early symptoms include pulling inor clenching of the lower extremities when picked up by the tail, reported previously in A53T transgenic mice (e.g., Martin et al.. “The mitochondrial permeability transition pore regulates Parkinson’s disease development in mutant alpha-synuclein transgenic mice.” Neurobiology of Aging 35, 1132-1152, 2014), as well as hypokinesis, manifested most prominently as a delay to initiate walking when touched or gently prodded. These symptoms were progressive. For example, unilateral clenching of one lower extremity progressed over time to clenching together of both lower extremities when picked up by the tail. Hypokinesis became more severe, with the mouse unable to initiate movement when pushed upon. Eventually mice became, in effect, paralyzed, but the behavior differed from that of ALS mouse strains. That is, mice were unable to maintain upright posture, rolling and writhing via their extremities but unable to balance upright. Lower extremities were found to assume positions of either flexion or extension, often with one extremity held in flexion and the other extended (as compared to usual full extension of lower extremities in ALS mice). Upper paws were often held in a clenched state. One mouse exhibited absence seizures from an early time, but the others did not exhibit seizures.

[0273] The Kcnnl-6 / A53T line, with the higher copy number of Kcnnl, exhibited no visible symptoms, with no pulling in or clenching when picked up by the tail, no evidence of hypokinesis, and normal walking at a year of age. It appeared that the higher copy number of Kcnnl in the Kcnnl-6 / A53T mice conferred additional protection from clinical disease. At ~14 months of age, however, the mice demonstrated mild hypokinesis and a mild degree of unilateral lower extremity clenching when picked up by the tail but remained fully mobile. This progressed over the following 2-4 months to more significant hypokinesis and bilateral clenching, with an inability to get to an upright posture occurring at endstage, resembling the behavior of mice with A53T alone. One mouse progressed more slowly, reaching endstage at 650 days, and another one remains alive but hypokinetic at 680 days.

[0274] FIG. 2 illustrates four end-stage A53T mice. All are on-side, their lower extremities exhibit various states of flexion / extension, and their upper paws appear clenched. They were unable to maintain an upright posture.

[0275] FIG. 3 depicts brain A53T human alpha-synuclein RNA levels of A53T / B6SJL mice, A53T / Kcnnl-3 / B6SJL mice, and A53T / Kcnnl-6 / B6SJL mice, measured as the ratio of RT-qPCR measurements of human A53T alpha-synuclein RNA relative to (control) GAPDH level, which was a constant in these studies relative to amount of total RNA. Threeindependent mice of each strain were measured (shown as dots at extremities of a bar and one dot each within the bar). The bars overlapped, but the variance across them was large, with the largest and smallest values of each bar differing by nearly a factor of two. While the values were roughly similar, the large variance precluded assignment of a statistical measure. However, it appeared that RNA expression of Kcnnl from a Thy 1.2 promoter was not substantially affecting (e.g., depressing) the level of RNA expressed from the transgene encoding A53T alpha-synuclein, also directed by a Thy 1.2 promoter. Because both the promoter driving A53T alpha-synuclein and that driving Kcnnl were the same Thy 1.2 promoter, the common promoter afforded the advantage that the very same cell types expressing the pathogenic protein would also be expressing the protein species responsible for rescue.

[0276] FIG. 4 illustrates clearance of A53T alpha-synuclein-mCherry by Kcnnl coexpression in a cultured cell system. Cultured HeLa cells were transiently transfected with a plasmid, 0.3 microgram, driving expression of A53T alpha-synuclein fused at its C- terminus with mCherry from a CAG promoter and co-transfected in two cases with a second plasmid encoding Kcnnl, driven from a CAG promoter. Kcnnl was either not added (A53T alone) or added at 0.3 microgram or 1.0 micrograms. To apply equal amounts of total plasmid DNA, an unrelated third plasmid (BlueScript) was added to bring amounts of DNA to equality. At 40 hr, the transfected cultures were examined for mCherry fluorescence.

[0277] FIGs. 5A-5G illustrate histologic sagittal brain sections and a spinal cord cross section from an A53T alpha synuclein end stage mouse (8 months) stained with antibody to the pathogenic serine 129-phosphorylated form of alpha-synuclein,. No immunostaining in any brain region was observed in 2-month-old mice that were similarly studied, whereas at end-stage there was florid staining in a deep cortical layer and in many other parts of the brain. FIG. 5A presents a reference sagittal view of a mouse brain at 1.2 mm (1200 microns) from the midline, capturing relevant structures, as labeled [taken from the Allen Brain Atlas (atlas.brain-map.org)]. FIGs. 5B-5E show immunostained images from a sagittal section of that region: cortex (CTX) and hippocampus (HP), including an expanded view of subfield CA3 (FIG. 5B); striatum (CP, caudate / putamen, in the reference diagram), thalamus (TH), subthalamic region including zona incerta (ZI) and subthalamic nucleus (STN), and substantia nigra (SN) (FIG. 5C); superior colliculus (SC), deep cerebellar nuclei (DCN), and vestibular nucleus (Ve, in medulla) (FIG. 5D); and cross-section of cervical spinal cord, including an expanded view of motor neurons in the ventral horn (FIG.5E). Without wishing to be bound by theory, it is thought that in most cases immunostaining of neurons is likely to result from ongoing primary expression driven by the Thy 1.2 promoter, but some component of fluorescent pathogenic synuclein may comprise “spread”, particularly where there is extracellular manifestation of pathogenic synuclein (e.g., in striatum).

[0278] FIG. 5F illustrates cortical regions of an endstage A53T mouse and a 1 year-old asymptomatic A53T / Kcnnl-6 mouse, stained for both phospho-serine- 129 alpha-synuclein and Kcnnl . Adjacent sagittal sections were stained with the respective antibodies. Whereas noticeable punctate staining was observed in the deep cortical region in the A53T brain (upper left), no staining was observed in the corresponding cortical region of the A53T / Kcnnl-6 mouse (lower left), associated with strong anti-Kcnnl immunostaining (lower right). This supports, without wishing to be bound by theory, that Kcnnl-6 is affording protection from formation of the pathogenic (serine 129-phosphorylated) form of alpha-synuclein.

[0279] FIG. 5G illustrates superior colliculus of an endstage A53T mouse and a 1 year- old asymptomatic A53T / Kcnnl-6 mouse, stained for both phospho-serine- 129 alpha- synuclein and Kcnnl. Adjacent sagittal sections were stained with the respective antibodies. Whereas noticeable punctate phospho-serine- 129 alpha-synuclein staining was observed in the superior colliculus of the A53T brain (upper left), no staining was observed in the corresponding superior colliculus region of the A53T / Kcnnl-6 mouse (lower left), associated with strong anti-Kcnnl immunostaining (lower right). This supports, without wishing to be bound by theory, that Kcnnl-6 is affording protection from formation of the pathogenic (serine 129-phosphorylated) form of alpha-synuclein.

[0280] FIG. 6 and FIG. 7 illustrate EM sections taken from a mouse that is transgenic only for Kcnnl (the protecting gene; in this case homozygous Thy 1 -Kcnnl -3). The sections shown are from spinal cord ventral horn, focusing on motor neurons. Structures in the Kcnnl motor neurons include phagophores (an early-stage structure in formation of an autophagosome), where engulfment of an apparent organelle can be noted; autophagosomes (APG) with a double membrane surrounding captured cytosol; and multivesicular bodies (MVB), which are derived from in-budding of late endosomes, containing multiple vesicles. These three structures appeared with significant frequency in cells examined and were not readily observed in motor neurons from non-transgenic mice. The first two structures, in particular, are consistent with the activation by Kcnnloverexpression of an autophagosomal “clearance” mechanism, which would continuously be “ingesting” cytosol, including pathogenic proteins such as A53T alpha-synuclein, and delivering them to lysosomes for degradation. Such clearance may also include segments of expanded ER (see FIG. 10 A) by a mechanism of “ER-phagy”.

[0281] FIG. 8 shows an additional feature observed in EM analysis of spinal cord motor neurons from Kcnnl homozygous mice, namely the presence of “fingers” of nuclear envelope pointing into the nuclear matrix. At least 50% of motor neurons exhibited this feature of unusual protrusions of nuclear envelope into nuclear matrix in spinal cord motor neurons, whereas it was not observed in similar analysis of motor neurons from control B6SJL mice. This may be consistent with ER expansion (Fig. 10A-B) - note that the outer nuclear envelope is continuous with the ER membrane - and with ER stress (Fig. 12 and Table 1)

[0282] FIG. 9 shows that similar morphologic disturbances involving membranes and vesicles as observed in vivo in Kcnnl overexpressing motor neurons can be seen in EM of cultured HeLa cells transiently transfected with and overexpressing Kcnnl (40 hrs posttransfection). The two Kcnnl -transfected cells at bottom exhibit a distorted nuclear envelope (with finger protrusions not observed in these two cells, but seen rarely in a few other cells), and presence of cytosolic MVB-like single membrane-bounded structures as well as a double-membrane-bounded structure that appears to be an autophagosome (APG).

[0283] Many spinal cord motor neurons of homozygous Kcnnl -3 mice exhibited expanded ER, in some cells manifesting as tight stacks of parallel ER cisternae, but most strikingly in some cells presenting as a whorl pattern of such parallel cisternae, as depicted in FIG. 10A. In some cases, ribosomes studded the cisternae, but in others the ER appeared smooth. This seemed indicative of an ER stress response.

[0284] FIG.10B shows that overexpressed Kcnnl colocalizes with the ER. The immunostaining pattern of overexpressed Kcnnl in a spinal cord motor neuron of a Kcnnl - 6 mouse (top left panel) matches closely to that of the endogenous ER-membrane- associated protein calnexin (top right panel). This matchup was observed for every motor neuron examined. By contrast, in B6SJL control mouse motor neurons, the same calnexin immunostaining pattern is observed (lower right panel) but there is no obvious relationship of the very weakly staining endogenous Kcnnl with the calnexin (lower left panel). This supports that overexpressed mouse Kcnnl is targeted to the ER, as is normal for all potassium channels, and associates with it. Its increased presence at the ER in the settingof overexpression is likely to account for ER expansion (see FIG.10A) and for the induction of an ER stress response observed at the RNA level (FIG. 11, FIG. 12A-B and Table 1)

[0285] To assess for a Kcnnl -mediated stress response, RNAseq was carried out on RNA from pools of 1000 laser capture-microdissected spinal cord motor neuron somata from each of four B6SJL (wt) and four Kcnnl -6 transgenic mice. An average of 67 million paired reads were obtained from each sample. Reads were aligned to the mouse genome assembly (mm 10), identifying -21,000 genes. Differential gene expression (DGE) analysis was performed using DESeq2, and p-values for the differences were adjusted for multiple comparisons to give p-adjust (padj) values. Significantly differentially expressed genes (padj <0.05; n=1788: 968 down, 820 up) were assessed for functional enrichments using Metascape. Of the 20 most significantly enriched categories in the gene ontology analysis, only two showed strongly upregulated transcription in Kcnnl-6 transgenics: G0:0034976 “Response to ER stress” and KEGG pathway mmu04141, “Protein processing in the ER,” as shown in FIG. 11.

[0286] Table 1 lists the two sets of upregulated ER genes, including all of those exhibiting 30% or greater increases, along with significance values (as well as several relevant genes with a 20% increase). The genes could be assigned to functional categories of ER stress, reflecting the presence of effects at multiple levels of ER physiology.

[0287] Under “Stress Signaling”, XBP1 mRNA was modestly induced, but more revealing, the activating splice of XBP1 RNA was observed in 36% of the Kcnnl-6 reads and in none of the B6SJL reads. This result implies that the IRE1 limb of the UPR is activated in Kcnnl- 6 motor neurons, contributing, e.g., to induction of RNAs encoding ER chaperones (BiP / Hspa5 and cooperating component DnaJB9 as examples). RNAs for components of the translocon were also induced, as well as for (ER-localized) protein disulfide isomerases and glycosylation components. Significantly, RNAs encoding ERAD components such as Derlin 3, a component of the HRD1 retrotranslocation complex known to be induced under ER stress, were also induced. Without wishing to be bound by theory, it is thought that there is a chronic degree of ER stress response in the spinal cord motor neurons of the Kcnnl-6 expressing mice, and that this may contribute to neuroprotection.Table 1. ER Stress Response Genes Upregulated By Kcnnl TransgenesisFold change P adjSTRESS SIGNALINGCreb3L1 4.0 X 2x 10E-5GADD34 (Ppp1r15a) 1.4X 4.5 x 10E-3XBP1 1.3X 6.2 x 10E-3ATF4 1.3 X 2.4 x 10E-2CHOP (Ddit3) 1.6X 2.9 x 10E-7Trib3 3.0 X 1.4 x 10E-2Sestrin2 1.3X 5.4 x 10E-5ASNS 1.2X 2.5 x 10E-2ATF5 2.7 X 4.5x 10E-13ATF6 1.2 X 1.2 x 10E-3WFS1 1.5X 1.3 x 10E-6TRANSLOCATIONSec61 beta 1.6 X 3x 10E-8Sec61 gamma 1.6 X 2.2 x 10E-9TRAM 1.5X 1.2 x 10E-7Ssr1 (signal seq R) 1.3 X 4x 10E-7 Ssr4 (signal seq R) 1.3X 5 x 10E-4 SERP1 1.4 X 2.4 x 10E-8PROTEIN FOLDINGChaperonesBiP (HspA5) 1.5X 9.7x10E-13DnaJB9 1.5X 6.8x10E-12GRP94 1.3 X 5.2x10E-5CryAB 1.6X 9.2x10E-11HYOU (ER Hsp110) 1.5X 3x10E-15Calnexin 1.2 X 2.2x10E-4Glucosidase 2 (Prkcsh) 1.3 X 5x10E-4Protein disulfide isomerasesPdiA3 1.2X 1.6x10E-3PdiA4 1.3 X 1 x 10E-2PdiA6 1.6X 4x10E-9P4hb (disulfide isom / prolyl4OHase) 1.5X 1.6x10E-17GLYCOSYLATION / MODIFICATIONOstc (oligosacch.xferase subunit) 1.6 X 1.6x 10E-10 Magtl (oligosacch xferase subunit) 1.9 X 3.8 x 10E-10 TMEM258 (oligosacch xf subunit) 1.4 X 3.3 x 10E-4 Lman (ERGIC 53) 1.3 X 3x 10E-3GRASP55 1.3 X 2.7 x 10E-5ER-ASSOCIATED DEGRADATIONDerl3 3.4 X 1.0 x 10E-2Sdf2l1 2.0 X 6.0 x 10E-12Herpudl 1.3 X 3.9 x 10E-5Edem2 1.3 X 3 x 10E-4FBX06 (FBS2) 1.4 X 2.4 x 10E-2

[0288] Without wishing to be bound by theory, it is thought that the trigger of the ER stress response is the Kcnnl protein itself, which, like other potassium channels, is normally targeted to the ER for biogenesis. Here, in an overproduced state, the Kcnnl subunit may at least mildly stress the ER, possibly as the result of misfolding or misassembling of some fraction of the overexpressed Kcnnl-encoded subunits.

[0289] A low level of induction of ATF4 RNA was observed, as well as induction of RNAs of several of ATF4’s downstream transcriptional targets, CHOP (Ddit3), Trib3, Sestrin2 (Sesn2), and Asns (see Table 1), raising a question of whether there might be activation of the integrated stress response (ISR). Hallmark features of an ISR include phosphorylation of Ser51 of eIF2alpha to activate the ISR (by any one of four different kinase sensors, including PERK (EIF2AK3), which recognizes unfolded / misfolded ER proteins). Such phosphorylation of eIF2alpha reduces translation initiation at a global scale and is accompanied by translation of a select group of uORF-containing mRNAs, including that for ATF4 (Costa-Mattioli, M. and Walter, P. “The integrated stress response: From mechanism to disease.” Science 368, eaat5314, 2020). When immunostained spinal cord cross-sections of B6SJL were compared with those of Kcnnl-6, increased levels of both phosphorylated eIF2alpha and ATF4 in ventral horn motor neurons were observed (FIGS.12A-12B)

[0290] FIG. 12A shows an at least several-fold increase of anti-phospho-S51-eIF2alpha signal in Kcnnl-6 motor neurons as compared with those of the nontransgenic mouse, reflecting activation of the ISR.

[0291] FIG. 12B shows three representative ventral horn sections from a Kcnnl-6 mouse and three from a B6SJL mouse, stained with anti-ATF4 and anti-ChAT. Motor neurons are identified by anti-ChAT antibody staining. In the case of ATF4, many but not all motor neurons exhibited an increase of ATF4 signal strength and also a change of localization - whereas the protein was cytosolic and barely detectable in B6SJL, it was prominent and nuclear-localized in many Kcnnl-6 motor neurons. This reflects the selective translation that occurs as a feature of the ISR.

[0292] Thus, an ISR appears to be present. Without wishing to be bound by theory, it is thought that the effect of reduced translational initiation could extend to mutant A53T alpha-synuclein, lowering its overall levels, preventing aggregation / toxicity.

[0293] Strong induction of several RNAs implicated in a mitochondrial stress response was also observed (Table 2). This included the regulatory transcription factor ATF5 (which can be induced by ATF4 from the ISR pathway), as well as downstream factors induced by it, such as FGF21 and GDF15 (the latter two factors noted to be induced, for example, in children with primary mitochondrial diseases). Without wishing to be bound by theory, the partitioning between nucleus and mitochondria of the regulatory factor, ATF5, may be affected here to favor ATF5 nuclear localization and induction of a mitochondrial stress response (also referred to as UPRmt).Table 2. Mitochondrial Stress Response Genes Upregulated by KcnnlFold change P adjFGF21 36 X 1.2 x 10E-12GDF15 11.6 X 8.3 x 10E-5Atf5 2.7 X 4.5 x 10E-13Aifm2 1.8 X 6 x 10E-8MAVS 1.7 X 2 x 10E-2

[0294] RNA encoding the Hsp70 protein, Hsplal, was also upregulated in the Kcnnl transgenic motor neurons (1.56 X; p=0.035). RNA encoding the autophagy component, ATG9b, implicated in pre-autophagosome formation was also induced (1.4X, padj = 1 X 10E-4). A preliminary immunostaining with anti-phospho S757 ULK1 antibodies of spinal cord sections observed reduced signal in Kcnnl-6 relative to B6SJL, also supporting upregulation of autophagy (data not shown). There was also evidence for an oxidative stress response, with RNA for aldehyde dehydrogenases Aldhla2 and Aldhla7 induced 4.3-fold and 5-fold, respectively (padj = 0.01 and 0.03; see Singh et al, 2013).Example 2, Clinical effects on mice of Thyl,2-Kcnnl expression alone

[0295] The beneficial effects of transgenic Kcnnl-3 / + were without any visible additional effects. That is, Kcnnl-3 mice were normal in size, mobility, and behavior, including breeding. In fact, the Kcnnl-3 mice may be especially long-lived. Three mice that were set aside during early experiments have survived to near or beyond 3 years of age. In particular, one mouse died at 1111 days (3 yr = 1095 days); a second mouse died of abdominal tumor at 958 days, 4 months short of 3 years; and a third mouse died of urinary obstruction at1043 days of age, 2 months short of 3 years. Additional Kcnnl-3 / + mice as well as control B6SJL mice are being monitored (we note that several of the control mice have died by 15 months of age). This said, the higher copy Thy 1 ,2-Kcnnl-6 transgenic mice developed mild lower extremity spasticity at 4-6 months of age (not present in any of the Kcnnl-3 / + mice), but it did not progress, and these mice are in many cases now 2 years old.Example 3, Efficacy of rAAV9 bearing CMV-mouse Kcnnl cDNA to prevent synuclein aggregation indicated by P-Serl29 alpha-synuclein following injection of virus into regions of A53T / B6SJL mouse brain that become affected.

[0296] The ability of self-complementary rAAV9 containing a CMV promoter driving mouse Kcnnl cDNA to prevent alpha-synuclein aggregation following injection into regions of the brain of the A53T / B6SJL mice that become affected is tested. The first target for injection is the superior colliculus (see FIGs. 5D and 5G), which strongly accumulates P-Serl29 alpha-synuclein in endstage A53T / B6SJL mice (about 8-10 months of age). How early this form of synuclein appears in superior colliculus is assessed, in order to gauge injections such that they precede onset of such aggregation, which could be about 5-6 months of age, illustrating a preventive action of virus. Alternatively, by injecting at about 7-9 months, a time when mice are usually exhibiting early lower extremity symptoms of motor disease, testing for whether there is prevention of further aggregation once it has commenced may be undertaken. The superior colliculis, while affected with aggregation in parallel to motor areas like motor cortex and basal ganglia, does not exhibit direct action in the motor system (it is involved, rather, in processing sensory information to guide eye / head positioning). Nonetheless, it is a possible target because it becomes heavily populated with phosphorylated synuclein aggregates, is a relatively large region of brain, and lies sufficiently dorsal that needle injection through a small appropriately positioned hole in the skull is straightforward. In further tests, the same type of study on motor cortex, which is possibly the earliest region to display P-129Ser alpha-synuclein, at about 6-7 months, may be undertaken. For both regions, virus is injected into the relevant structure on one side of the brain, while mock injected into the opposite side, a varying passage of time is allowed, and then the mouse is harvested for tissue analysis as in FIGs. 5A-G, comparing the injected with uninjected side. Given the results with the presence of transgenic Kcnnl, as shown in FIGs. 5F and 5G, it is expected that beneficial effects of the virus upon P-Serl29 alpha-synuclein accumulation will be observed. Note that this same virus has been effective, when injected ICV into P0 SOD1 G93A ALS mouse pups, in prolonging theirsurvival, associated with presence of overexpressed Kcnnl in spinal cord motor neurons as detected by immunostaining (see doi.org / 10.1101 / 2024.10.11.617887, “Neuronal overexpression of potassium channel subunit Kcnnl prolongs survival of SOD 1 -linked ALS and A53T alpha-synuclein mouse models”, e.g., at Fig. 6 and Suppl Fig. 9).EQUIVALENTS

[0297] While the disclosure has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the disclosure following, in general, the principles of the disclosure and including such departures from the present disclosure as come within known or customary practice within the art to which the disclosure pertains and as may be applied to the essential features hereinbefore set forth and as follows in the scope of the appended claims.

[0298] Those skilled in the art will recognize, or be able to ascertain, using no more than routine experimentation, numerous equivalents to the specific embodiments described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.INCORPORATION BY REFERENCE

[0299] All patents and publications referenced herein are hereby incorporated by reference in their entireties.

[0300] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present disclosure is not entitled to antedate such publication by virtue of prior disclosure.

[0301] As used herein, all headings are simply for organization and are not intended to limit the disclosure in any manner. The content of any individual section may be equally applicable to all sections.

Claims

WHAT IS CLAIMED IS:

1. A method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising:(i) administering to the subject an effective amount of a composition comprising a nucleic acid agent encoding for Kcnnl, or a fragment or variant thereof, or(ii) administering to the subject an effective amount of a cell, the cell having been contacted with an effective amount of a composition comprising a nucleic acid agent encoding for Kcnnl, or a fragment or variant thereof.

2. The method of claim 1, wherein the agent encoding for Kcnnl, or a fragment or variant thereof is an expression cassette comprising:(a) a polynucleotide encoding Kcnnl, or a fragment or variant thereof, operably linked to an expression control element;(b) an adeno-associated virus (AAV) inverted repeat (ITR) flanking the 5’ terminus of the polynucleotide; and(c) an AAV ITR flanking the 3’ terminus of the polynucleotide.

3. The method of claim 2, wherein the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 1, 3, or 9, or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

4. The method of claim 2 or 3, wherein the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10, or a functional variant thereof having at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

5. The method of any one of claims 2-4, wherein the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a codon-optimized form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 105.

6. The method of any one of claims 2-5, wherein the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a CpG-reduced form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10.

7. The method of any one of claims 2-6, wherein the expression control element is positioned 5’ of the polynucleotide encoding Kcnnl, or a fragment or variant thereof.

8. The method of any one of claims 2-7, wherein the expression control element is CpG- reduced compared to the wild type expression control element.

9. The method of any one of claims 2-8, wherein the expression control element comprises one or more of a cytomegalovirus (CMV) enhancer-promoter, a CMV enhancer fused to the chicken P-actin promoter (CAG), a chicken P-actin promoter (CBA), a simian vacuolating virus 40 (SV40) enhancer-promoter, a polyubiquitin C gene promoter (UBC), an elongation-factor la subunit (EF-la)promoter, a phosphoglycerate kinase promoter (PGK), and a synthetic enhancer / promoter.

10. The method of any one of claims 2-9, wherein the expression control element comprises a CMV enhancer-promoter and comprises a polynucleotide sequence of SEQ ID NO: 8 or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

11. The method of any one of claims 2-10, wherein the expression control element comprises an enhancer-promoter combination or a promoter that is tissue-specific and / or inducible.

12. The method of claim 11, wherein the tissue-specific enhancer-promoter combination or promoter is a neuron-specific enhancer-promoter combination or promoter.

13. The method of claim 12, wherein the neuron-specific enhancer-promoter or promoter is selected from a neuron-specific enolase (EN02), a platelet-derived growth factor a-chain (PDGFA), a platelet-derived growth factor P-chain (PDGFB), a synapsin (SYN1), a methyl-CpG binding protein 2 (MECP2), a Ca2+ / calmodulin-dependent protein kinase II (CAMK2G), metabotropic glutamate receptor 2 (GRM2), a neurofilament light (NEFL) or heavy (NEFH) chain, a proenkephalin (PENK), an excitatory amino acid transporter 2 (SLC1A2), or a synthetic promoter.

14. The method of claim 11, wherein the tissue-specific promoter or enhancer-promoter combination is a nigrostriatal-specific promoter or enhancer-promoter combination.

15. The method of any one of claims 2-14, wherein the 5’ ITR and the 3’ ITR are independently selected from a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74, and AAV3B ITR, or self-complementary versions thereof.

16. The method of any one of claims 2-15, wherein the 5’ ITR and / or the 3’ ITR is modified to have reduced CpGs.

17. The method of any one of claims 2-16, wherein the 5’ ITR and / or the 3’ ITR comprises a polynucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7 or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto, or self-complementary versions thereof.

18. The method of any one of claims 2-17, wherein the expression cassette further comprises a polyadenylation sequence positioned 3’ of the polynucleotide encoding Kcnnl, or a fragment or variant thereof.

19. The method of any one of claims 2-18, wherein the expression cassette further comprises one or more of an intron, a transcriptional termination signal, an miRNA, and a post- transcriptional regulatory element (PRE).

20. The method of any one of claims 2-19, wherein the expression cassette comprises, from 5’ to 3’ : a 5’ ITR, a CMV enhancer-promoter, an intron, a Kcnnl cDNA, a transcriptional termination sequence, a polyadenylation site, and a 3’ ITR.

21. The method of claim 1, wherein the agent encoding Kcnnl, or a fragment or a variant thereof, is a cDNA encoding Kcnnl, or a fragment or a variant thereof.

22. The method of claim 21, wherein the cDNA encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 1, 3, or 9, or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

23. The method of claim 21 or 22, wherein the cDNA encoding Kcnnl, or fragment thereof, comprises a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10, or a functional variant thereof having at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

24. The method of any one of claims 21-23, wherein the cDNA encoding Kcnnl, or a fragment or variant thereof, comprises a codon optimized form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10.

25. The method of any one of claims 21-24, wherein the cDNA encoding Kcnnl, or fragment or variant thereof, comprises a CpG-reduced form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10.

26. The method of claim 1, wherein the agent encoding Kcnnl, or a fragment or variant thereof, is an mRNA encoding Kcnnl, or a fragment or variant thereof.

27. The method of claim 26, wherein the mRNA encoding Kcnnl, or a fragment or variant thereof, is transcribed from a polynucleotide sequence encoding the polypeptide of SEQ IDNO: 1, 3, or 9, or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

28. The method of claim 26 or 27, wherein the mRNA encoding Kcnnl, or a fragment or variant thereof, is transcribed from a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10, or a functional variant thereof having at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

29. The method of any one of claims 26-28, wherein the mRNA encoding Kcnnl, or a fragment or variant thereof, comprises a modified mRNA, optionally wherein the modified mRNA comprises at least one modified nucleoside optionally selected from a pseudouridine, 5- methylcytosine (m5C), 5-methyluridine (m5U), 2'-O-methyluridine (Um or m2'-0U), 2- thiouridine (s2U), and N6-methyladenosine (m6A).

30. The method of any one of claims 1-29, wherein the cell is derived from the subject, or the cell is derived from another organism, optionally wherein the cell is derived from the subject via iPSC production and differentiation, optionally wherein the cell is a dopamine- producing cell or a fetal ventral mesencephalic cell.

31. The method of any one of claims 1-30, wherein the composition comprising a nucleic acid agent encoding Kcnnl is a viral vector comprising the nucleic acid agent encoding Kcnnl and / or the expression cassette.

32. The method of claim 31, wherein the viral vector comprises a recombinant adeno- associated virus (rAAV) vector.

33. The method of claim 32, wherein the AAV is selected from an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9 capsid serotype, a recombinant AAV (rAAV) capsid serotype, or a functional variant of an AAV1, AAV2, AAV4, AAV5, AAV6, AAV7,AAV8, or AAV9 capsid serotype, optionally comprising a modification permitting transfer across the blood-brain barrier.

34. The method of claim 31, wherein the viral vector comprises a recombinant anellovirus vector, optionally wherein the anellovirus vector is selected from TT virus (TTV), Torquetenomini virus (TTMV), and Torquetenomi divirus (TTMVD).

35. The method of any one of claims 1-34, wherein the contacting is transduction.

36. A method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising:(i) administering to the subject an effective amount of an agent that increases, enhances, and / or stimulates Kcnnl expression (e.g., of protein), amount (e.g., of protein), and / or activity; or(ii) administering to the subject an effective amount of a cell, the cell having been contacted with an effective amount of an agent that increases, enhances, and / or stimulates Kcnnl expression (e.g., of protein), amount (e.g., of protein), or activity.

37. The method of claim 36, wherein the agent that increases, enhances, and / or stimulates Kcnnl expression (e.g., of protein), amount (e.g., of protein), or activity is a small molecule optionally selected from (-)CM-TPMF, chlorzoxazone, DCEBIO, 1-EBIO, and riluzole.

38. The method of any one of claims 1-37, wherein the subject is a human subject.

39. The method of claim 38, wherein the human subject is at least 40 years old, at least 45 years old, at least 50 years old, at least 55 years old, at least 60 years old, at least about 65 years old, at least about 70 years old, at least about 75 years old, at least about 80 years old, or at least about 85 years old.

40. The method of any one of claims 1-39, wherein the subject demonstrates one or more of bradykinesia, akinesia, tremors in the hands, fingers, forearm, foot, mouth, or chin, muscular rigidity, poor balance, postural instability and gait dysfunction, Parkinsonian gait,neuropsychiatric disorders, depression, psychotic symptoms, anxiety, apathy, mild- cognitive impairment, dementia, autonomic dysfunctions, and sleep disturbances.

41. The method of any one of claims 1-40, wherein the PD has responded poorly to a previous therapy, optionally conventional L-DOPA therapy.

42. The method of any one of claims 1-41, wherein the method prevents or delays progression from (i) a Stage 1 PD to a Stage 2 PD, a Stage 3 PD, a Stage 4 PD, or a Stage 5 PD; (ii) a Stage 2 PD to a Stage 3 PD, a Stage 4 PD, or a Stage 5 PD; (iii) a Stage 3 PD to a Stage 4 PD or a Stage 5 PD; or (iv) a Stage 4 PD to a Stage 5 PD.

43. The method of any one of claims 1-42, wherein the method prevents or delays progression from (i) a mild / early PD to a moderate / mid- stage PD or a severe / advanced PD; or (ii) a moderate / mid-stage PD to a severe / advanced PD.

44. The method of any one of claims 1-43, wherein the method prevents paralysis.

45. The method of any one of claims 1-44, wherein the method reduces, ablates, or slows the progression of one or more symptoms of PD.

46. The method of any one of claims 1-45, wherein the one or more symptoms are selected from bradykinesia, akinesia, tremors in the hands, fingers, forearm, foot, mouth, or chin, muscular rigidity, poor balance, postural instability and gait dysfunction, Parkinsonian gait, neuropsychiatric disorders, depression, psychotic symptoms, anxiety, apathy, mild- cognitive impairment, dementia, autonomic dysfunctions, and sleep disturbances.

47. The method of any one of claims 1-46, wherein the method improves and / or increases and / or enhances anti-PD efficacy, compared to treatment with conventional L-DOPA therapy.

48. The method of any one of claims 1-47, wherein the method promotes or enhances clearance of a pathogenic cytosolic protein, compared to conventional L-DOPA therapy.

49. The method of claim 48, wherein the pathogenic cytosolic protein is wild type alpha- synuclein, serine- 129-phosphorylated alpha-synuclein, a A53T mutant form of alpha- synuclein, a E46K mutant form of alpha-synuclein, a H50Q mutant form of alpha- synuclein, a G51D mutant form of alpha-synuclein, a A30P mutant form of alpha- synuclein, tau, TAR DNA-binding protein 43 (TDP-43), and / or superoxide dismutase 1(SOD1), or potentially abnormal secreted proteins.

50. The method of any one of claims 1-49, wherein the method reduces, prevents, and / or delays accumulation of the pathogenic serine- 129-phosphorylated form of alpha-synuclein, compared to treatment with conventional L-DOPA therapy.

51. The method of any one of claims 1-50, wherein the method promotes or enhances neuronal protection from oxidative damage, compared to conventional L-DOPA therapy.

52. The method of any one of claims 1-51, wherein the method promotes or enhances sparing of pre- or postsynaptic dopaminergic terminals, compared to conventional L-DOPA therapy.

53. The method of any one of claims 1-52, wherein the expression cassette, cDNA, mRNA, viral vector, agent, or pharmaceutical composition is administered by intrastriatal injection, substantia nigra pars compacta delivery, striatum (e.g., putamen) delivery, and / or nigrostriatal region delivery.

54. The method of any one of claims 1-53, wherein the method further comprises evaluating analysis of a biological fluid.

55. The method of claim 54, wherein the biological fluid is cerebrospinal fluid (CSF) or blood.

56. The method of any one of claims 1-55, wherein the method further comprises evaluating analysis of a brain image, optionally wherein the brain image is from one or more of computed tomography (CT), positron emission tomography (PET), and magnetic resonance imaging (MRI).

57. The method of any one of claims 1-56, wherein the contacting is transduction.

58. A pharmaceutical composition comprising the expression cassette or cell of any one of claims 1-20, the cDNA of any one of claims 21-25, the mRNA of any one of claims 26-30, the viral vector of any one of claims 31-35, or the agent of claim 36 or 37, and a pharmaceutically accepted excipient, carrier or diluent.

59. The pharmaceutical composition of claim 58, wherein the composition is suitable for intrastriatal injection, substantia nigra pars compacta delivery, striatum (e.g., putamen) delivery, and / or nigrostriatal region delivery.

60. A Parkinson’s Disease (PD) therapy comprising an effective amount of the expression cassette of any one of claims 2-20, the cDNA of any one of claims 21-25, the mRNA of any one of claims 26-30, the viral vector of any one of claims 31-35, or the agent of claim 36 or 37, or the pharmaceutical composition of claim 58 or 59.

61. A method of making a PD therapy, comprising:(a) identifying the PD therapy by:(i) administering an effective amount of a test agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal;(ii) identifying the test agent as a PD therapy if the transgenic animal demonstrates one or more of increased expression of Kcnnl, increased activity of the gene product of Kcnnl, improved survival time, or reduced paralysis, as compared to a control; and(b) formulating the candidate agent for administration for the treatment of PD.

62. A method of making a PD therapy, comprising:(a) identifying a candidate agent that modulates a cellular stress response, comprising:(i) providing a cell line overexpressing Kcnnl and having a cellular stress response substantially similar to a cellular stress response of spinal cord motor neurons overexpressing Kcnnl,(ii) treating the cell line with one or more test agents,(iii) measuring cellular stress responses in the cell, and(iv) identifying the test agent as a candidate agent if one or more of the cellular stress responses is similarly increased in the presence of the test agent, compared to in the absence of the test agent and / or presence of a control agent that is known not to be a candidate agent; and(b) administering an effective amount of the candidate agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal;(c) classifying the candidate agent as a PD therapy if the transgenic animal demonstrates one or more of increased expression of stress-related proteins in spinal cord neurons, improved survival time, or reduced paralysis, as compared to a control; and(d) formulating the PD therapy for administration for the treatment of PD, optionally wherein the one or more of the cellular stress responses comprises one or more of ER, integrated, and mitochondrial cellular stress responses.

63. A method of identifying a PD therapy that is effective in treating or preventing PD comprising:(a) administering an effective amount of the agent to an A53T mutant alpha- synuclein transgenic animal and(b) determining time to paralysis, wherein an increased time to paralysis compared to an A53T mutant alpha-synuclein transgenic animal that does not receive the agent indicates that the agent is a PD therapy.

64. A method of making a PD therapy, comprising:(a) identifying a candidate agent that mimics the activity of Kcnnl, comprising:(i) providing a neuronal cell line or primary cultured neurons;(ii) treating the neuronal cell line or primary cultured neurons with one or more test agents;(iii) measuring the levels of selected stress-response elements in the neuronal cell line or primary cultured neurons; and(iv) identifying the test agent as a candidate agent if stress response activity is increased in the presence of the test agent, compared to in the absence of the test agent and / or presence of a control agent that is known not to be a candidate agent;(b) administering an effective amount of the candidate agent to an A53T mutant alpha-synuclein transgenic animal or a wild type animal;(c) classifying the candidate agent as a PD therapy if the transgenic animal demonstrates one or more of improved survival time and reduced motor compromise as compared to the wild type animal control; and(d) formulating the PD therapy for administration for the treatment of PD, optionally wherein the test agent is a small molecule or peptide, and / or the candidate agent is a small molecule or peptide.

65. The method of any one of claims 61-63, wherein the PD therapy is a gene therapy, a biologic agent, a small molecule, or a polynucleotide agent, optionally wherein the gene therapy is a viral gene therapy.

66. The method of claim 65, wherein the viral gene therapy is or comprises a recombinant adeno-associated virus (rAAV).

67. The method of claim 66, wherein the rAAV comprises a Kcnnl nucleic acid.

68. The method of claim 64, wherein the biologic agent is an antibody or peptide.

69. The method of claim 64, wherein the polynucleotide agent is selected from mRNA, siRNA, shRNA, miRNA, and cDNA.

70. A method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising:(i) administering to the nigrostriatal region of the brain of the subject an effective amount of an AAV composition comprising a nucleic acid agent encoding Kcnnl, or a fragment or variant thereof, or(ii) administering to the nigrostriatal region of the brain of the subject an effective amount of a cell, the cell having been contacted with an effective amount of an AAV composition comprising a nucleic acid agent encoding Kcnnl, or a fragment or variant thereof.

71. The method of claim 70, wherein the administering is to the substantia nigra pars compacta or striatum (e.g., putamen) of the brain of the subject.

72. The method of claim 70 or 71, wherein the administering is by injection.

73. The method of any one of claims 70-72, wherein the cell is derived from the subject or the cell is derived from another organism, optionally wherein the cell is derived from the subject via iPSC production and differentiation.

74. The method of any one of claims 70-73, wherein the cell is a dopamine-producing cell.

75. The method of any one of claims 70-74, wherein the cell is a fetal ventral mesencephalic cell.

76. The method of any one of claims 70-75, wherein the AAV composition is an AAV9, or self-complementary version thereof.

77. The method of any one of claims 70-76, wherein the agent encoding Kcnnl, or a fragment or variant thereof is an expression cassette comprising: a. a polynucleotide encoding Kcnnl, or a fragment or variant thereof, operably linked to an expression control element; b. an adeno-associated virus (AAV) inverted repeat (ITR) flanking the 5’ terminus of the polynucleotide; and c. an AAV ITR flanking the 3’ terminus of the polynucleotide.

78. The method of any one of claims 70-77, wherein the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence encoding the polypeptide of SEQ ID NO: 1, 3, or 9, or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

79. The method of any one of claims 70-77, wherein the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10, or a functional variant thereof having at least about 50%, or at least about 55%, or at least about 60%, or at least about 65%, or at least about 70%, or at least about 75%, or at least about 80%, or at least about 85%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

80. The method of claim 79, wherein the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a codon-optimized form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10.

81. The method of claim 79 or 80, wherein the polynucleotide encoding Kcnnl, or a fragment or variant thereof, comprises a CpG-reduced form of a polynucleotide sequence of SEQ ID NO: 2, 4, 5, or 10.

82. The method of any one of claims 77-81, wherein the expression control element is positioned 5’ of the polynucleotide encoding Kcnnl, or a fragment or variant thereof.

83. The method of any one of claims 77-82, wherein the expression control element is CpG- reduced compared to the wild type expression control element.

84. The method of any one of claims 77-83, wherein the expression control element comprises one or more of a cytomegalovirus (CMV) enhancer-promoter, a CMV enhancer fused to the chicken P-actin promoter (CAG), a chicken P-actin promoter (CBA), a simian vacuolating virus 40 (SV40) enhancer-promoter, a polyubiquitin C gene promoter (UBC), an elongation-factor la subunit (EF-la)promoter, a phosphoglycerate kinase promoter (PGK) or a synthetic promoter.

85. The method of claim 84, wherein the expression control element comprises a CMV enhancer-promoter and comprises a polynucleotide sequence of SEQ ID NO: 8 or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto.

86. The method of any one of claims 77-85, wherein the expression control element comprises an enhancer-promoter combination or a promoter that is tissue-specific and / or inducible.

87. The method of claim 86, wherein the tissue-specific enhancer-promoter combination or promoter that is a neuron-specific enhancer-promoter combination or promoter.

88. The method of claim 86, wherein the neuron-specific enhancer-promoter or promoter is selected from a neuron-specific enolase (EN02), a platelet-derived growth factor a-chain (PDGFA), a platelet-derived growth factor P-chain (PDGFB), a synapsin (SYN1), a methyl-CpG binding protein 2 (MECP2), a Ca2+ / calmodulin-dependent protein kinase II (CAMK2G), metabotropic glutamate receptor 2 (GRM2), a neurofilament light (NEFL) or heavy (NEFH) chain, a proenkephalin (PENK), or an excitatory amino acid transporter 2 (SLC1A2) or a synthetic promoter.

89. The method of any one of claims 77-88, wherein the 5’ ITR and the 3’ ITR are independently selected from a AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74 and AAV3B ITR, or self- complementary versions thereof.

90. The method of any one of claims 77-89, wherein the 5’ ITR and / or the 3’ ITR is modified to have reduced CpGs.

91. The method of any one of claims 77-90, wherein the 5’ ITR and / or the 3’ ITR comprises a polynucleotide sequence of SEQ ID NO: 6 or SEQ ID NO: 7 or a functional variant thereof having at least about 80%, or at least about 90%, or at least about 95%, or at least about 97%, or at least about 99% identity thereto, or self-complementary versions thereof.

92. The method of any one of claims 77-91, wherein the expression cassette further comprises a polyadenylation sequence positioned 3’ of the polynucleotide encoding Kcnnl, or a fragment or variant thereof.

93. The method of any one of claims 77-92, wherein the expression cassette further comprises one or more of an intron, a transcriptional termination signal, an miRNA, and a post- transcriptional regulatory element (PRE).

94. The method of any one of claims 77-93, wherein the expression cassette comprises, from 5’ to 3’: a 5’ ITR, a CMV enhancer-promoter, an intron, a Kcnnl cDNA, a transcriptional termination sequence, a poly-adenylation site, and a 3’ ITR.

95. The method of any one of claims 77-94, wherein the AAV is selected from an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, RhlO, Rh74 and AAV3B ITR, or self-complementary versions thereof, or an AAV with a capsid altered to optionally comprise an element that facilitates blood-brain barrier passage of virus particles.

96. The method of any one of claims 77-95, wherein the method reduces or prevents alpha- synuclein spread from endogenous affected cells, e.g., into a nigral graft.

97. The method of any one of claims 70-95, wherein the contacting is transduction.

98. The method of any one of claims 1-57 and 70-97, wherein the method further comprises evaluating a biological sample for levels of one or more ER stress response biomarkers, one or more integrated stress response (ISR) biomarkers, and / or one or more mitochondrial stress response biomarkers.

99. The method of claim 98, wherein the biological sample is cerebrospinal fluid (CSF) or blood.

100. The method of any one of claims 1-57 and 70-99, wherein the method causes one or more of an ER stress response, an integrated stress response (ISR), and a mitochondrial stress response.

101. The method of any one of claims 1-57 and 70-100, wherein the method causes an ER stress response, wherein expression of one or more of Creb3Ll, GADD34, XBP1, ATF4, CHOP, Trib3, Sestrin2, ASNS, ATF5, ATF6, WFS1, Sec61 beta, Sec61 gamma, TRAM, Ssrl, Ssr4, SERP1, BiP, DnaJB9, GRP94, CryAB, HYOU, Calnexin, Glucosidase 2, PdiA3, PdiA4, PdiA6, P4hb, Ostc, Magtl, TMEM258, Lman, GRASP55, Derl3, Sdf211, Herpudl, Edem2, and FBXO6 are increased or elevated relative to untreated or pre-treated.

102. The method of any one of claims 1-57 and 70-101, wherein the method causes an integrated stress response (ISR), wherein phosphorylation levels of Ser51 of eIF2alpha are increased or elevated relative to untreated or pre-treated.

103. The method of any one of claims 1-57 and 70-102, wherein the method causes a mitochondrial stress response, wherein expression of one or more of FGF21, GDF15, Atf5, Aifm2, and MAVS are increased or elevated relative to untreated or pre-treated.

104. A method for evaluating the therapeutic benefit of a method for treating or preventing Parkinson’s Disease (PD) in a subject in need thereof, comprising:(i) determining a baseline expression and / or activity level of one or more ER stress response biomarkers, one or more integrated stress response (ISR) biomarkers, and / or one or more mitochondrial stress response biomarkers at a first timepoint;(ii) administering to the subject the pharmaceutical composition of claim 58 or 59;(iii) determining the expression and / or activity level of the one or more ER stress response biomarkers, the one or more integrated stress response (ISR) biomarkers, and / or the one or more mitochondrial stress response biomarkers at a second timepoint; and(iv) determining whether the expression and / or activity level of the one or more ER stress response biomarkers, the one or more integrated stress response (ISR) biomarkers, and / or the one or more mitochondrial stress response biomarkers is increased between the first and second timepoints.

105. The method of claim 104, wherein the ER stress response biomarkers are selected from Creb3Ll, GADD34, XBP1, ATF4, CHOP, Trib3, Sestrin2, ASNS, ATF5, ATF6, WFS1,Sec61 beta, Sec61 gamma, TRAM, Ssrl, Ssr4, SERP1, BiP, DnaJB9, GRP94, Cry AB, HYOU, Calnexin, Glucosidase 2, PdiA3, PdiA4, PdiA6, P4hb, Ostc, Magtl, TMEM258, Lman, GRASP55, Derl3, Sdf211, Herpudl, Edem2, and FBXO6.

106. The method of claim 104, wherein the integrated stress response (ISR) biomarker is phosphorylation level of Ser51 of eIF2alpha, or induction of ATF protein.

107. The method of claim 104, wherein the mitochondrial stress response biomarkers are selected from FGF21, GDF15, Atf5, Aifm2, and MAVS.