Antisense oligonucleotides and crispr guide RNAS targeting KCTD20 for the treatment of neurodegeneration

Abstract

Antisense oligonucleotides are provided which target potassium channel tetramerization domain containing 20 (KCTD20) and can be used in treating, inhibiting, or reducing the severity of a neurodegenerative disease, especially those involving glutamate excitotoxicity.

Description

ANTISENSE OLIGONUCLEOTIDES AND CRISPR GUIDE RNAS TARGETING KCTD20 FOR THE TREATMENT OF NEURODEGENERATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 63 / 662,355, filed June 20, 2024, the entirety of which is hereby incorporated by reference. REFERENCE TO SEQUENCE LISTING

[0002] This application contains a Sequence Listing submitted as a computer readable form named “065715_000129WOPT_SequenceListing.xml”, having a size in bytes of 14,842,727 bytes, and created on June 11, 2025. The information contained in this computer readable form is hereby incorporated by reference in its entirety. FIELD OF INVENTION

[0003] This invention relates to oligonucleotide drugs for treating neurodegenerative diseases. BACKGROUND

[0004] Frontotemporal dementia (FTD) accounts for 5%–15% of all dementias, and is a leading cause of early-onset dementia. FTD with tau pathology (FTD-tau) is present in approximately 45% of all FTD cases. Over 50 disease-causing variants have been identified in the tau gene, MAPT, and they primarily affect the microtubule-binding domains and collectively account for 10%–20% of familial FTD patients. One commonly studied mutation, MAPT V337M, is located in exon 12 and is expressed in 3R and 4R tau isoforms. Similar to other mutations in the microtubule-binding domains, MAPT V337M decreases the affinity of tau to microtubules and enhances tau phosphorylation (p-tau), promoting neuronal deposits of hyperphosphorylated paired helical filaments in patients. Moreover, the tau-V337M mutation has been described to enhance formation of neurotoxic oligomeric tau, soluble multimeric intermediate structures preceding insoluble tau filaments. Oligomeric tau is indicated to be the most toxic species in disease, occurring before, and contributing to, neurodegeneration in the absence of filamentous tau species. Additionally, tau-V337M has been shown to promote hyperexcitability of induced pluripotent stem cell (iPSC)-derived neurons, and patient-derived tau-V337M cortical organoids are sensitized to glutamate excitotoxicity. Current FTD therapies primarily aim to manage neuropsychiatric symptoms, highlighting an urgent and unmet need to identify new treatments. 4934-0525-6010.2 Page 1 of 105065715-000129WOPT

[0005] Glutamate excitotoxicity is a pervasive mechanism across multiple neurodegenerative diseases, including frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Excitotoxicity refers to the toxic effects of excitatory neurotransmitters, primarily glutamate. In excitotoxicity, nerve cells suffer damage or death when the levels of otherwise necessary and safe neurotransmitters such as glutamate, α- amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), or N-methyl-D-aspartic acid (NMDA) become pathologically high resulting in excessive stimulation of receptors.

[0006] Pharmacological interventions to address this mechanism has thus far proven ineffective, highlighting a need to uncover novel modulators of glutamate-induced neuronal stress. For example, complete inhibition of glutamate receptor activity would likely alter brain function, possibly limiting the maximum efficacy of direct glutamate receptor blockade. In addition, tau can act downstream of glutamate receptors to increase neuronal network excitability, for example, by interacting with L-type voltage-gated calcium channels to increase calcium influx.

[0007] It is an object of the present disclosure to provide oligonucleotide compositions, especially antisense oligonucleotides, which may modulate key neuronal excitotoxicity regulators.

[0008] It is another object of the present disclosure to provide methods and oligonucleotide compositions for treating neurodegenerative diseases or reducing excitotoxic stress in neurons.

[0009] All publications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. The following description includes information that may be useful in understanding the present invention. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed invention, or that any publication specifically or implicitly referenced is prior art. SUMMARY OF THE DISCLOSURE

[0010] The following embodiments and aspects thereof are described and illustrated in conjunction with compositions and methods which are meant to be exemplary and illustrative, not limiting in scope.

[0011] Various embodiments provide single stranded antisense oligonucleotides (ASOs), especially ASOs that modulate expression of KCTD20. In various aspects, an ASO comprises 12-50 linked nucleosides, and the ASO has a nucleobase sequence having a4934-0525-6010.2Page 2 of 105 065715-000129WOPTguanine-cytosine content (GC content) of 30-70%, no cytidine (C) followed by a guanosine (G), and no more than three contiguous G’s.

[0012] In some embodiments, the ASO has a nucleobase sequence that comprises at least 12 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669 or of a variant of the any one of the SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669, wherein the variant has a substitution of one or more thymines if present by uracil or the variant has one or more nucleobases removed.

[0013] In some embodiments, the ASO has 18-20 linked nucleosides and comprises a nucleobase sequence set forth in any one of SEQ ID NOs:16718, 16719, 16750-16752, and 1- 16669 or of a variant of the any one of the SEQ ID NOs:16718, 16719, 16750-16752, and 1- 16669, wherein the variant substitutes a uracil for a thymine (i.e., U instead of T) and / or the variant has one or more nucleobases removed.

[0014] In some embodiments, the ASO has 20 linked nucleosides and comprises a nucleobase sequence of that of any one of SEQ ID NOs:648-1321 or a variant of the any one of SEQ ID NOs:648-1321.

[0015] In some embodiments at least one internucleoside linkage in an ASO is a modified internucleoside linkage. In some embodiments, at least one internucleoside linkage in an ASO is a modified internucleoside linkage, and at least another internucleoside linkage in the ASO is unmodified (that is, phosphodiester). In some embodiments, an ASO has alternating segments of nucleoside and deoxynucleoside, e.g., a gapmer comprising a 5’ wing of nucleoside, a gap segment of deoxynucleoside, and a 3’ wing of nucleoside; wherein the segment of deoxynucleoside has modified internucleoside linkages, such as a phosphorothioate linkage, and the segment of nucleoside has one or more internucleoside linkages being a modified internucleoside linkage.

[0016] In some embodiments, the modified internucleoside linkage is a phosphorothioate (PS) internucleoside linkage or a N3′-P5′ phosphoroamidate (NP) internucleoside linkage. In some embodiments, at least one internucleoside linkage is a phosphodiester internucleoside linkage, and at least another internucleoside linkage is a phosphorothioate internucleoside linkage.

[0017] In some embodiments, at least one nucleoside in an ASO comprises a modified nucleobase. In some embodiments, the modified nucleobase is an abasic site. In some embodiments, the modified nucleobase is 5mC.

[0018] In some embodiments, at least one nucleoside of the ASO comprises a modified sugar moiety, wherein the modified sugar moiety comprises a modification of 2’-O-4934-0525-6010.2Page 3 of 105 065715-000129WOPTmethoxyethyl group, 2’-O-methyl group, or constrained ethyl group, or the modified sugar moiety is a threose moiety.

[0019] In some embodiments, at least one nucleoside of the ASO comprises a locked nucleic acid (LNA), a phosphorodiamidate morpholino (PMO), a peptide nucleic acid (PNA), or a tricyclo DNA (tcDNA).

[0020] In some embodiments, the ASO is a gapmer comprising one or more segments of linked deoxynucleosides and one or more segments of linked nucleosides, wherein a nucleoside of the one or more segments of linked nucleosides comprises a modified sugar moiety. In further embodiments, the ASO comprises: a gap segment consisting of 10 to 12 linked deoxynucleosides, a 5’ wing segment consisting of 5 to 4 linked nucleosides, and a 3’ wing segment consisting of 5 to 4 linked nucleosides, wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment. In some embodiments, each nucleoside making up each wing segment comprises a modified sugar moiety comprising a 2’-O-methoxyethyl group or a 2’-O-methyl group, and wherein each internucleoside linkage between the deoxynucleosides making up the gap segment is a phosphorothioate internucleoside linkage. In further embodiments, the ASO has 20 linked nucleosides comprising: a gap segment consisting of 10 linked deoxynucleosides, a 5’ wing segment consisting of 5 linked nucleosides, and a 3’ wing segment consisting of 5 linked nucleosides, wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment, and wherein each nucleoside making up each wing segment comprises a modified sugar moiety comprising a 2’-O-methoxyethyl group, and the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage.

[0021] In some embodiments, the ASO has a nucleobase sequence having 100% or at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% complementarity to (segment(s) of) 3’UTR of human KCTD20 mRNA, or the ASO suppresses expression of potassium channel tetramerization domain containing 20 (KCTD20) by at least 15%.

[0022] In various embodiments, the GC content in an ASO is 40%-60%.

[0023] In some embodiments, the GC content is 40%-60%, having no more than two consecutive G’s, and the ASO has a nucleobase sequence having at least 6 cytidines (C’s), at least 6 thymines (T’s), 0-3 G’s, and a 3’ end not ending in 1-3 G’s; and optionally wherein the ASO has a change in Gibbs free energy (ΔG) associated with self-dimerization being ≥ -7 kcal / mole.4934-0525-6010.2Page 4 of 105 065715-000129WOPT

[0024] In other embodiments, the GC content is 40%-65%, and the ASO has a nucleobase sequence having at least 5 C’s and at least 5 T’s; and optionally wherein the ASO has a ΔG associated with self-dimerization being ≥ -7 kcal / mole, and if the nucleobase sequence of the ASO has 5 T’s and 5 C’s, the ASO has one G in the last three bases at the 3’ end.

[0025] In additional embodiments, the GC content is 40%-60%, and the ASO has a nucleobase sequence having no G at the 3’ end (e.g., the last base at the 3’ end is not G; or the 3’ wing segment has no G); and optionally wherein the ASO has a ΔG associated with self-dimerization being ≥ -7 kcal / mole.

[0026] Pharmaceutical compositions are also provided, which include an antisense oligonucleotide disclosed herein and a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the pharmaceutical composition is formulated for intracerebroventricular injection or intrathecal injection. In some embodiments, the pharmaceutical composition is formulated for parenteral delivery. In some embodiments, pharmaceutically acceptable carrier, diluent or excipient comprises artificial cerebrospinal fluid (CSF).

[0027] Various embodiments provide methods of reducing loss of neurons, which include contacting the neurons with an antisense oligonucleotide disclosed herein. In some embodiments, the neurons are in a subject with amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD). In some embodiments, the neurons are obtained from a subject with ALS or FTD. In some embodiments, the neurons are derived from induced pluripotent stem cells derived from a subject with ALS or FTD.

[0028] Methods of treating, inhibiting, and reducing the severity of a neurological or a neurodegenerative disease in a subject are also provided, which include administering to the subject a therapeutically effective amount of a pharmaceutical composition disclosed herein or an ASO that modulates expression of KCTD20 disclosed herein.

[0029] In some embodiments, the neurodegenerative disease comprises frontotemporal dementia (FTD), motor neuron disease / amyotrophic lateral sclerosis (MND / ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), progressive supranuclear palsy, tauopathies, chronic traumatic encephalopathy, Charcot Marie Tooth 2A and 4B, Huntington’s disease, dementia, transmissible spongiform encephalopathy, spinobulbar muscular atrophy, dentatorubro-pallidoluysian atrophy, spinocerebellar ataxias, Creutzfeldt- Jakob disease, or a combination thereof, or the subject exhibits symptoms of glutamate excitotoxicity. In some embodiments, the neurodegenerative disease comprises ALS. In some4934-0525-6010.2Page 5 of 105 065715-000129WOPTembodiments, the neurodegenerative disease comprises FTD. In some embodiments, the neurodegenerative disease comprises Alzheimer’s disease (AD). In some embodiments, the neurodegenerative disease comprises Parkinson’s disease (PD). In some embodiments, the neurodegenerative disease comprises tauopathies. In some embodiments, the neurological disease is associated with aberrant lysosomal storage. In further embodiments, the therapeutically effective amount is effective for reducing glutamate-induced tau oligomerization in the subject.

[0030] Some embodiments provide methods of screening for an oligonucleotide, which include: contacting an oligonucleotide of interest with an organoid derived from induced-pluripotent stem cells (iPSCs) from a patient with a neurodegenerative disease, and performing one or more of: assaying transcription or expression level of KCTD20, assaying expression levels of an AKT signaling, an mTOR signaling, or both, assaying level of transcription factor EB (TFEB) nuclear localization, assaying expression levels of p62, LC3, or both, assaying level of autolysosome formation, and assaying level of tau oligomerization.

[0031] In some embodiments, the organoid derived from iPSCs from a patient with a neurodegenerative disease is an organoid derived from iPSCs obtained from a patient with frontotemporal dementia (FTD) associated with the V337M microtubule-associated protein tau (MAPT) mutation. In some embodiments, the organoid derived from iPSCs from a patient with a neurodegenerative disease is an organoid derived from iPSCs obtained from a patient with amyotrophic lateral sclerosis (ALS) or FTD having an expansion of a hexanucleotide repeat in the gene C9orf72.

[0032] In some embodiments, performing the one or more of assaying comprises assaying the expression levels and / or the levels before contacting the oligonucleotide of interest and assaying the expression levels and / or the levels in the presence of the oligonucleotide of interest, wherein an decreased transcription or expression level of KCTD20, an increased expression level of the AKT signaling and / or the mTOR signaling, an increased level of TFEB nuclear localization, an increased expression level of p62 and / or LC3, an increased level of autolysosome formation, and / or a decreased level of tau oligomerization, in the presence of the oligonucleotide of interest relative to that before the contact thereof, indicates that the oligonucleotide of interest is a candidate drug for treating a neurodegenerative disease.

[0033] Various embodiments provide methods of modulating a neuronal excitotoxicity regulator in a subject in need thereof, which include administering an antisense oligonucleotide or a pharmaceutical composition disclosed herein to the subject in need4934-0525-6010.2Page 6 of 105 065715-000129WOPTthereof. In some embodiments, the neuronal excitotoxicity regulator is potassium channel tetramerization domain containing 20 (KCTD20) and the composition suppresses expression of KCTD20. In some embodiments, the neuronal excitotoxicity regulator is lysosomal exocytosis and the composition activates lysosomal exocytosis. In further embodiments, the subject in need thereof has a neurological disease.

[0034] Other features and advantages of the invention will become apparent from the following detailed description, taken in conjunction with the accompanying drawings, which illustrate, by way of example, various features of embodiments of the invention. BRIEF DESCRIPTION OF THE FIGURES

[0035] Exemplary embodiments are illustrated in referenced figures. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than restrictive.

[0036] Figures 1A-1G depict glutamate induces oligomeric tau formation and neurodegeneration. (1A) 2-month-old SYN1::eGFP-labeled organoids were immobilized using matrigel, treated with L-glutamic acid (glutamate) or vehicle control, and imaged for 7 days to longitudinally track neuron survival. (1B) Kaplan-Meier survival analysis of SYN1::eGFP organoids treated with vehicle control, 100 µM and 5mM glutamate. GFP+ neurons were tracked for 7 days to determine neuron survival (n = 100 neurons / condition; log- rank test; scale bars, 50 µm). Error bars represent mean ± SEM between conditions. (1C) Inter-organoid reproducibility of glutamate treatment on neurodegeneration in multiple wild- type iPSC lines (2 iPSC lines; 4 organoids / line / condition, 30 neurons / organoid; error bars omitted for clarity). (1D) Survival of SYN1::eGFP neurons in organoids treated with 5 mM glutamate and DMSO or ionotropic glutamate receptor inhibitors (3i; 2 µM nimodipine, 10 µM CNQX, 10 µM MK-801) (n = 200 neurons / group; log-rank test; scale bars, 50 µm). Error bars represent mean ± SEM between conditions. (1E) Quantification of high-molecular- weight species above 75 kDa. (1F) Quantification of high-molecular-weight species above 150 kDa. Quantification was based on images of oligomeric tau (T22) and total tau (tau-5) expression by western blot in organoids treated with glutamate for 48 h. Only the upper band of >150 kDa oligomers were used for T22 western blot quantifications throughout the manuscript (n = 6–7 organoids / group; one-way ANOVA with Dunnett’s correction). (1G) Quantification of high-molecular-weight (>150 kDa) oligomeric tau in organoids treated with 5 mM glutamate for 7 days (n = 6 organoids / group; unpaired t test, two-tailed). Error bars represent mean ± SEM between organoid averages unless otherwise stated.4934-0525-6010.2Page 7 of 105 065715-000129WOPT

[0037] Figures 1H-1K depict characterization of oligomeric and phosphorylated tau. (1H) Kaplan-Meier survival analysis of SYN1::Egfp organoids treated with vehicle control, 5mM glutamate, or 5mM GABA. GFP+ neurons were tracked for 7 days to determine neuron survival (n= 120 neurons / condition; Log-Rank test; scale bars, 50μm). (1I) Quantification of T22 signal in non-denaturing dot blot of organoids treated with vehicle or glutamate for 7 days (n=5-6 organoids / group, unpaired t test two-tailed). (1J) Quantification of pTau (S202 / T205) immunostaining in wild-type organoids treated with vehicle or 5mM glutamate for 7 days (n=6 organoids / group; unpaired t test two-tailed; scale bars, 10μm). (1K) Quantification of pTau (Thr231) immunostaining in organoids treated with vehicle or 5mM glutamate for 7 days (n = 3 organoids / condition, black circles; n = 20 neurons / organoid, light color; unpaired t test two-tailed [organoid average]; scale bars, 10μm). Error bars represent mean + / - s.e.m. between organoid averages unless otherwise stated.

[0038] Figures 2A-2C depict a CRISPRi screen in glutamate-treated neuronal spheroids. (2A) 70-day-old NGN2-spheroids (n = 300) stably expressing dCas9-BFP-KRAB were transduced with a lentiviral genome-wide sgRNA library (209,070 gRNAs [10 gRNAs / gene], 3,790 non-targeting gRNAs). Spheroids were treated with 5 mM glutamate (n = 150) and sgRNAs were enriched by PCR and sequenced to determine enrichment of sgRNAs compared with vehicle-treated organoids (n = 150) after 10 days. (2B) Volcano plot of protective and detrimental sgRNAs (p < 0.05) determined by MAGeCK-RRA, with most significant protective sgRNAs highlighted. (2C) Gene Ontology molecular function pathway enrichment of top protective and detrimental sgRNAs.

[0039] Figures 2D and 2E depict composition of neurons and tau isoforms in NGN2- spheroids. (2D) Schematic of NGN2-spheroid generation. (2E) Quantification of NMDAR1 (NR1) protein expression in age-matched NGN2-spheroids and patterned organoids, normalized to total protein (n=5 organoids / group; unpaired t test two-tailed). Error bars represent mean + / - s.e.m. between organoid averages unless otherwise stated.

[0040] Figures 3A-3C depict that a CRISPRi screen identifies KCTD20 as a modifier of glutamate excitotoxicity. (3A) Longitudinal survival of dCas9-BFP-KRAB-expressing NGN2-spheroids were transduced with SYN1::eGFP, KCTD20-targeting sgRNA or non- targeting sgRNA, and treated with glutamate. GFP+ / BFP+ neurons were longitudinally tracked for 7 days for survival. (n = 120–240 neurons / condition; log-rank test; scale bars, 50 µm). Error bars represent mean ± SEM between conditions. (3B) GFP+ neuron survival of organoids treated with 10 mM NT or KCTD20 ASO for 3 days followed by 7 days of glutamate treatment (n = 120 neurons / condition; log-rank test; scale bars, 50 µm). Error bars4934-0525-6010.2Page 8 of 105 065715-000129WOPTrepresent mean ± SEM between conditions. (3C) Quantification of the number of TUNEL- positive cells per total number of cells in organoids pre-treated with 10 µM NT or KCTD20 ASO, followed by 7 days of vehicle or 5 mM glutamate treatment (n = 6 organoids / group; one-way ANOVA with Tukey’s correction; scale bars, 50 µm). Error bars represent mean ± SEM between organoid averages unless otherwise stated.

[0041] Figures 3D-3G depict additional gene targets from CRISPRi screen. (3D) Hazard ratio (Mantel-Haenszel) graphs of survival analysis of SYN1::eGFP neurons in ASO- organoids treated with glutamate for 7 days for ASOs against KCTD20 (ASO-2), PPP4R1, BCAP29, TMEM63C, SSRP1, PECR, SLC39A8, TMEM37, CTBP2, and GNG13 relative to NT ASO (n=120 neurons / 4 organoids / group; One-way ANOVA with Dunnett’s correction). (3E) qPCR of KCTD20, PPP4R1, BCAP29, TMEM63C, SSRP1, PECR, SLC39A8, TMEM37, CTBP2, and GNG13 in organoids after 72 hour treatment with 10μM NT or target ASO, normalized to 18S (n=6 organoids / group; unpaired t test twotailed). (3F) 6-month wildtype organoids were treated with 10 μM NT or KCTD20 ASO for three days before glutamate treatment, and GFP+ neurons were tracked longitudinally over 7 days (n= 120 neurons / condition; Log-Rank test). (3G) Peak amplitude of calcium traces in organoids pre- treated with 10 μM KCTD20 or NT ASO followed by 48 hours of glutamate, then loaded with 1 μM Fluo-4-AM, then imaged for 2 min before and after glutamate stimulation (unpaired t test, two-tailed; n = 30 neurons / condition analyzed, error bars mean ± SEM between neurons). Error bars represent mean + / - s.e.m. between organoid averages unless otherwise stated.

[0042] Figure 4A depicts glutamate-induced neuron death depends on tau protein. Organoids grown from neurologically normal (wild type) and MAPT knockout (KO) iPSCs were transduced with SYN1::eGFP and treated with 10 µM NT or KCTD20 ASO for 3 days before 5mM glutamate treatment, and GFP+ neurons were tracked longitudinally over 7 days (n = 120 neurons / condition; log-rank test). Error bars represent mean ± SEM between conditions.

[0043] Figures 4B-4C depict tau knockout and neuron expression in MAPT KO organoids. Quantification of total tau (4B) and TUJ1 (4C) in 2-month MAPT KO and wildtype organoids (n=6 organoids / group; unpaired t test two-tailed). Error bars represent mean + / - s.e.m. between organoid averages unless otherwise stated.

[0044] Figures 5A-5E depict tau-V337M organoids display enhanced tau pathology and neuron death following glutamate. (5A) Quantification of untreated isogenic tau-V337V and tau-V337M organoids for oligomeric tau (n = 4 organoids / condition, black circles; n = 204934-0525-6010.2Page 9 of 105 065715-000129WOPTneurons / organoid, light color; unpaired t test two-tailed [organoid average]; scale bars, 10 µm). (5B) Isogenic tau-V337M and tau-V337V (patient pair 1) organoids were transduced with SYN1::eGFP and treated with 5 mM glutamate or vehicle. GFP+ neurons were tracked longitudinally for 7 days for survival. (n = 120 neurons / condition; log-rank test; scale bars, 50 µm). Error bars represent mean ± SEM between conditions. (5C) Survival of FTD organoids treated with 10 µM NT, KCTD20 ASO, or MAPT ASO for 3 days before 7 days of glutamate treatment. GFP+ neurons were tracked longitudinally over 7 days (n = 120 neurons / condition; log-rank test; scale bars, 50 µm). Error bars represent mean ± SEM between conditions. Quantification of (5D) high-molecular-weight (>150 kDa) oligomeric tau and (5E) total tau in tau-V337M organoids treated with 10 µM NT or KCTD20 ASO for 3 days and then 5mM glutamate for 7 days (n = 6 organoids / group; one-way ANOVA with Tukey’s correction). Error bars represent mean ± SEM between organoid averages unless otherwise stated.

[0045] Figures 5F-5K depict that survival of MAPT V337M FTD neurons is improved with KCTD20 ASO. (5F) Quantification of surface isolated NR1 in tau-V337V and tau-V337M organoids, normalized to transferrin receptor (n= 5 organoids / group; unpaired t test two-tailed). (5G and 5H) Kaplan-Meier survival analysis of SYN1::eGFP neurons in independent MAPT V337M FTD-patient organoids pre-treated with NT or KCTD20 ASO followed by 7 days glutamate treatment (n=120 neurons / group; Log-Rank test); wherein patient 2’s is shown in 5G, and patient 3’s is shown in 5H. (5I) Day 300 FTD organoids were treated with 10 μM NT, KCTD20 ASO, or MAPT ASO for three days before glutamate treatment, and GFP+ neurons were tracked longitudinally over 7 days (n= 120 neurons / condition; Log-Rank test). (5J) qPCR of MAPT in organoids after 72 hour treatment with 10μM NT or MAPT ASO, normalized to 18S (n=6 organoids / group; unpaired t test two tailed). (5K) Quantification of neuronal oligomeric tau in wild-type organoids treated with NT or KCTD20 ASO followed by glutamate for 7 days (n = 4 organoids / condition, black circles; n = 20 neurons / organoid, light color; One-way ANOVA with Tukey’s correction [organoid average]; scale bars 10 μm). Error bars represent mean + / - s.e.m. between organoid averages unless otherwise stated.

[0046] Figures 5L-5O depict survival of C9ORF72 ALS / FTD neurons is improved with KCTD20 ASO. (5L) Survival analysis of SYN1::eGFP neurons from NT ASO wild-type or C9ORF72 ALS / FTD organoids treated with vehicle or 5 mM glutamate (n=100 neurons / group; Log-Rank test). (5M) Survival analysis of SYN1::eGFP neurons of glutamate- treated wild-type or C9ORF72 ALS / FTD organoids pre-treated with NTor KCTD20 ASO4934-0525-6010.2Page 10 of 105 065715-000129WOPT(data from 5L and 5M are from the same experiment and contain shared datasets, separated across two graphs for clarity; n=100 neurons / group; Log-Rank test). (5N) qPCR for ratio of truncated STMN2 to full length STMN2 mRNA in WT organoids pre-treated with NT or KCTD20 ASO for 72 hours followed by 5 mM glutamate for 7 days, normalized to 18S (n=6 organoids / condition; One-way ANOVA with Tukey’s correction). (5O) Quantification of cytoplasmic TDP-43 in WT organoids pre-treated with NT or KCTD20 ASO for 72 hours followed by vehicle or 5 mM glutamate for 7 days (n = 5 organoids / condition, black circles; n = 15 neurons / organoid, light color; One-way ANOVA with Dunnett’s correction [organoid average]; scale bars 10 μm). Error bars represent mean + / - s.e.m. between organoid averages unless otherwise stated.

[0047] Figures 5P-5S depict proteostasis stressors induce tau oligomerization and neurodegeneration. (5P) Quantification of HMW oligomeric tau, normalized to tau5, in organoids treated with vehicle, 5 mM glutamate or 250 nM bafilomycin for 48 hours (n= 6-7 organoids / group; One-way ANOVA with Dunnett’s correction). (5Q) Neuron survival of organoids pre-treated with NT or KCTD20 ASO followed by vehicle or 250 nM bafilomycin (n=120 neurons / group; Log-Rank test). (5R-5S) Changes in secreted (5R) pTau (Thr231) and (5S) total tau in the supernatant of organoids pre-treated with 10μM NT or KCTD20 ASO for 72 hours followed by 5mM glutamate for 24hr measured by MSD electrochemical ELISA (n=6 organoids / group, unpaired t test two-tailed). Error bars represent mean + / - s.e.m. unless otherwise stated.

[0048] Figures 6A-6E depict improved neuron survival following KCTD20 knockdown depends on exocytosis. (6A) Experimental schematic.3-month-aged tau-V337M organoids were treated with 10 µM NT or KCTD20 ASO for 3 days followed by 5 mM glutamate for 3 and 6 days (n = 5 organoids / group). Samples were multiplexed with streptavidin antibody barcodes prior to single-cell reaction (10×) and sequencing. (6B and 6C) (6B) Elsevier and (6C) GO biological processes enrichment of significantly upregulated genes (average log2(fold change) > 0.25, adjusted p < 0.05) in KCTD20-low ExNs. (6D) Survival analysis of Syn1::eGFP neurons pre-treated with NT or KCTD20 ASO followed by DMSO, glutamate, or glutamate and 10 µM GW4869 (n = 120 neurons / group; log-rank test). Error bars represent mean ± SEM between conditions. (6E) Quantification of T22 in neurons following treatment with glutamate and 10 µM GW4869 (n = 3 organoids / condition, black circles; n = 20 neurons / organoid, light color; one-way ANOVA with Tukey’s correction [organoid average]; scale bars, 10 µm). Error bars represent mean ± SEM between organoid averages unless otherwise stated.4934-0525-6010.2Page 11 of 105 065715-000129WOPT

[0049] Figures 7A-7H depict that protective effects of KCTD20 knockdown are mediated by lysosomal exocytosis. (7A) Hazard ratio (Mantel-Haenszel) graphs of neuron survival in organoids pre-treated with ASO for 3 days followed by 7 days of glutamate or vehicle (n = 4 organoids / 30 neurons / group; one-way ANOVA with Dunnett’s correction). (7B) Quantification of Pearson’s R correlation for T22 and LAMP1 in SYN1::eGFP neurons pre-treated with ASO for 3 days then glutamate or vehicle for 7 days (n = 4 organoids / condition; one-way ANOVA with Tukey’s correction; scale bars, 10 µm). (7C-7D) Exosomes from conditioned medium of pooled organoids treated with 10 µM NT or KCTD20 ASO and 5 mM glutamate for 7 days were pulled down using the ExoView R100 and counterstained to quantify the number of (7C) LAMP1+ particles and (7D) T22+ particles (n = 3 samples / condition, each point representative of n = 5 pooled organoids, unpaired t test two-tailed). (7E) Quantification of nuclear TFEB levels in ASO-treated organoids after 7 days of vehicle or 5 mM glutamate (n = 3–4 organoids / condition, black circles; n = 10 neurons / organoid, light color; one-way ANOVA with Tukey’s correction [organoid average]). (7F) Quantification of cytoplasmic phosphorylated TFEB (Ser142) in ASO-treated organoids following 7 days of vehicle or 5 mM glutamate (n = 6 organoids / group; one-way ANOVA with Tukey’s correction). (7G) Schematic of proposed mechanism for KCTD20 suppression. (7H) Hazard ratio (Mantel-Haenszel) graphs of survival analysis of SYN1::eGFP neurons in organoids pre-treated with 10 µM NT or KCTD20 ASO followed by glutamate and 10 µM eltrombopag for 7 days (n = 120 neurons / 4 organoids / group; one-way ANOVA with Dunnett’s correction). Error bars represent mean ± SEM between organoid averages unless otherwise stated.

[0050] Figures 7I-7N depict the effect of exocytosis pathway inhibition on neuron survival. (7I) Hazard ratio (Mantel-Haenszel) graphs of neuron survival following 7 days of longitudinal imaging with glutamate, KCTD20 ASO, and ASOs against VAMP7, HSPA8, ATG7, RAB8A, ATG5, NSMAF, and GORASP (n=3 organoids / 30 neurons / group; One-way ANOVA with Dunnett’s correction). (7J) Neurologically normal organoids were treated with 10 μM NT, KCTD20, or MCOLN1 ASO for 3 days. Kaplan-Meier survival analysis of SYN1::eGFP neurons in ASO-organoids treated with glutamate for 7 days (n=120 neurons / group; Log-Rank test). (7K) qPCR of STXBP1 in organoids after 72 hour treatment with 10μM NT or STXBP1 ASO, normalized to 18S (n=6 organoids / group; unpaired t test two-tailed). (7L) Survival of wildtype organoids pre-treated with 10μM NT, STXBP1, and / or KCTD20 ASO for 72 hours followed by glutamate for 7 days (n=120 neurons / group; Log- Rank test). (7M) Neuron survival in tau-V337M organoids pre-treated with 10 μM NT,4934-0525-6010.2Page 12 of 105 065715-000129WOPTKCTD20, or MCOLN1 ASO for 3 days followed by 5 mM glutamate for 7 days (n=120 neurons / group; Log-Rank test). (7N) Kaplain-Meier survival of tau-V337V organoids with combinations of NT, KCTD20, and MCOLN1 ASO with vehicle or glutamate (n=120 neurons / group; Log-Rank test).

[0051] Figures 7O-7T depict upregulation of lysosomal biogenesis genes following KCTD20 knockdown. Organoids were infected with FIRE-pHLy lentivirus. (7O) Quantification of neuronal mTFP:mRFP ratio in FIRE-pHLY infected organoids pre-treated with NT or KCTD20 ASO followed by glutamate for 7 days (n = 5 organoids / condition, One- way ANOVA with Tukey’s correction). (7P-7R) Quantification based on immunostaining of neuronal phosphorylated AKT (Thr308) (7P) and total AKT (7Q) and relative p-Akt / Akt (7R) in organoids treated with NT or KCTD20 ASO and vehicle or 5 mM glutamate for 7 days (n = 6 organoids / condition; One-way ANOVA with Tukey’s correction). (7S) Quantification based on immunostaining of neuronal phosphorylated mTOR (Ser2448) and total mTOR in organoids treated with NT or KCTD20 ASO and vehicle or 5 mM glutamate for 7 days (n=5 organoids / group; One-way ANOVA with Tukey’s correction). (7T) Organoids were treated with 10 μM KCTD20 or NT ASO for 72 hours and RNA was isolated. qPCR for relative KCTD20, TFEB, LAMP1, and MCOLN1 mRNA expression, normalized to 18S (n=6 organoids / condition; unpaired t test, two-tailed). Error bars represent mean + / - s.e.m. between organoid averages unless otherwise stated.

[0052] Figures 8A-8I depict Kctd20 knockdown reduces tau pathology and improves neuron survival in vivo using MAPT transgenic mice. (8A) Quantification of oligomeric tau (T22) in the cortices of 6-month-old WT or MAPT P301S (MAPT) mice without ASO treatment (N = 13 mice / group; unpaired t test two-tailed. (8B) Quantification of upper-layer NeuN+ cells in the cortices of 6-month-old WT or MAPT P301S (MAPT) mice (N = 13 mice / group; each value representative of average counts across five 300 µm2regions; unpaired t test two-tailed. (8C) Quantification of nuclear TFEB in the cortices of 6-month-old WT or MAPT P301S (MAPT) mice (N = 7–8 mice / group; unpaired t test two-tailed. (8D) Schematic of experimental timeline for intracerebroventricular ASO injections in MAPT P301S mice. (8E and 8F) qPCR of Kctd20 in NT or Kctd20 ASO-injected MAPT P301S mice after (8E) 5 days and (8F) 4 weeks (N = 4–6 mice / group; unpaired t test two-tailed). (8G) Quantification of oligomeric tau in the cortices of 6-month-old MAPT mice 4 weeks after injection with NT or Kctd20 ASO (N = 7–8 mice / group; unpaired t test two-tailed). (8H) Quantification of nuclear TFEB in the cortices of 6-month-old MAPT mice 4 weeks after injection with NT or Kctd20 ASO (N = 7–8 mice / group; unpaired t test two-tailed). (8I)4934-0525-6010.2Page 13 of 105 065715-000129WOPTQuantification of the average number of NeuN+ cells per area in the cortices of 6-month-old MAPT mice 4 weeks after injection with NT or Kctd20 ASO (N = 7–8 mice / group; quantification representative of average counts across five 300 µm2regions; unpaired t test two-tailed). Error bars represent mean ± SEM between mouse averages unless otherwise stated.

[0053] Figures 8J-8N depict mouse pups injected ICV with glutamate recapitulate changes in tau and TFEB, which is mitigated by Kctd20 ASO. (8J) Experimental schematic. (8K) Representative western blot and (8L) quantification of Kctd20 from homogenized whole brain lysate of P5 mice, dissected into ipsilateral and contralateral hemispheres relative to ASO injection site, normalized to Gapdh (N=5 mice / group; One-way ANOVA with Tukey’s correction). (8M) Quantification of phosphorylated tau (Ser202 / Thr205) in cortex of P5 wildtype BL / 6J mice injected ICV with NT or Kctd20 ASO and vehicle or glutamate (N=5-8 mice / group; One-way ANOVA with Tukey’s correction). (8N) Quantification of nuclear TFEB in cortex of P5 WT mice injected ICV with NT or Kctd20 ASO and vehicle or glutamate (N=6-8 mice / group; One-way ANOVA with Tukey’s correction). Error bars represent mean + / - s.e.m. between mouse averages unless otherwise stated.

[0054] Figures 8O-8R depict tau pathology and neuron loss in adult transgenic MAPT P301S mice. (8O) Quantification of oligomeric tau (T22) in the hippocampus (CA1) of 6 month MAPT P301S mice injected with NT or Kctd20 ASO (N=5-8 mice / group; unpaired t test two-tailed). Quantification of phosphorylated tau (8P) Ser202 / Thr205 and (8Q) Ser396 in the cortex of 6 month MAPT mice injected with NT or Kctd20 ASO (N=7-8 mice / group; unpaired t test two-tailed). (8R) 4-month wildtype and MAPT P301S mice were injected ICV with NT or Kctd20 ASO. Rotarod test was performed prior to surgery and each subsequent week until 6-months of age. Data shown beginning from 1 week post-injection (17-week old) (N=3-8 mice / group; Two-way ANOVA with Sidak’s correction). Error bars represent mean + / - s.e.m. between mouse averages unless otherwise stated.

[0055] Figure 9 is a schematic summary of neuronal survival under glutamate excitotoxicity or under KCTD20 suppression. DESCRIPTION OF THE INVENTION

[0056] All references cited herein are incorporated by reference in their entirety as though fully set forth. Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton et al., Dictionary of Microbiology and Molecular Biology 3rded., Revised, J. Wiley & Sons (New York, NY 2006); March, Advanced Organic4934-0525-6010.2Page 14 of 105 065715-000129WOPTChemistry Reactions, Mechanisms and Structure 7thed., J. Wiley & Sons (New York, NY 2013); and Sambrook and Russel, Molecular Cloning: A Laboratory Manual 4thed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY 2012), provide one skilled in the art with a general guide to many of the terms used in the present application. For references on how to prepare antibodies, see D. Lane, Antibodies: A Laboratory Manual 2nded. (Cold Spring Harbor Press, Cold Spring Harbor NY, 2013); Kohler and Milstein, (1976) Eur. J. Immunol.6: 511; Queen et al. U. S. Patent No.5,585,089; and Riechmann et al., Nature 332: 323 (1988); U.S. Pat. No. 4,946,778; Bird, Science 242:423-42 (1988); Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988); Ward et al., Nature 334:544-54 (1989); Tomlinson I. and Holliger P. (2000) Methods Enzymol, 326, 461-479; Holliger P. (2005) Nat. Biotechnol. Sep;23(9):1126-36).

[0057] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. Indeed, the present invention is in no way limited to the methods and materials described. For purposes of the present invention, the following terms are defined below.

[0058] A “subject” or “patient” as used interchangeably refers to a human or a vertebrate animal. Usually the animal is a vertebrate such as a primate, rodent, domestic animal or game animal. Primates include chimpanzees, cynomologous monkeys, spider monkeys, and macaques, e.g., Rhesus. Rodents include mice, rats, woodchucks, ferrets, rabbits and hamsters. Domestic and game animals include cows, horses, pigs, deer, bison, buffalo, feline species, e.g., domestic cat, and canine species, e.g., dog, fox, wolf. The terms, “patient”, “individual” and “subject” are used interchangeably herein. In an embodiment, the subject is mammal. The mammal can be a human, non-human primate (for example, a monkey, such as a cynomolgous or rhesus monkey, chimpanzee, etc.), mouse, rat, dog, cat, horse, or cow, but are not limited to these examples. In an embodiment, the subject is human.

[0059] A “neurological disease” is any disease that causes electrical, biochemical, or structural abnormalities in the brain, spine, or neurons. For example, a neurological disease may be a neurodegenerative disease. The neurodegenerative disease may result in motor neuron degeneration, for example. The neurological disease may be amyloid lateral sclerosis, Huntington’s disease, Alzheimer’s disease, or frontotemporal dementia, for example. Further examples of neurological diseases include, but are not limited to Parkinson’s disease, multiple sclerosis, peripheral myopathy, Rasmussen’s encephalitis, attention deficit hyperactivity disorder, autism, central pain syndromes, anxiety, and / or depression, for example. In some embodiments, the neurological disease is amyloid lateral sclerosis. In some4934-0525-6010.2Page 15 of 105 065715-000129WOPTembodiments, the neurological disease is frontotemporal dementia. In some embodiments, the neurological disease is Alzheimer’s disease.

[0060] An “antisense oligonucleotides” or ASO is a short strand of deoxyribonucleotide analogue that hybridizes with the complementary mRNA in a sequence- specific manner via Watson–Crick base pairing. An ASO as conceived may include modifications (compared to natural phosphodiester oligonucleotides such as DNA) in the phosphate backbone, the sugar moiety, or the nucleobase itself, altering the biophysical properties of the oligonucleotide with the intent to enhance their utility as antisense drugs; and additionally, targeting ligands can be appended to an ASO to assist with tissue-specific delivery. Formation of the ASO–mRNA heteroduplex either triggers RNase H activity, leading to mRNA degradation, induces translational arrest by steric hindrance of ribosomal activity, interferes with mRNA maturation by inhibiting splicing or destabilizes pre-mRNA in the nucleus, resulting in downregulation of target protein expression. Since RNase H is present in both nucleus and cytoplasm, ASOs’ druggable targets could be extended to non- coding elements. Alternatively, binding of ASOs to RNA can also modulate splicing by preventing the binding of splicing factors through steric hindrance. Pre-mRNA is then prevented from undergoing appropriate splicing, downregulating its protein. Similarly, ribosomal binding could also be sterically inhibited, leading to translational arrest. The antisense strategy comprising of targeting pre-mRNA, mRNA, or non-coding RNAs (ncRNAs) can alter the production of disease-causing proteins for therapeutic interventions. In various embodiments, the ASOs disclosed herein target human KCTD20 gene and reduce or suppress expression by about 50% or more at the transcript and / or protein level of potassium channel tetramerization domain containing 20.

[0061] “Nucleobase” refers to an unmodified nucleobase or a modified nucleobase. As used herein an “unmodified nucleobase” is adenine (A), thymine (T), cytosine (C), uracil (U), and guanine (G). As used herein, a “modified nucleobase” is a group of atoms other than unmodified A, T, C, U, or G capable of pairing with at least one unmodified nucleobase. For example, a “5-methylcytosine” or “5mC” is a modified nucleobase. In certain embodiments, modified nucleobases are selected from: 5-substituted pyrimidines, 6-azapyrimidines, alkyl or alkynyl substituted pyrimidines, alkyl substituted purines, and N-2, N-6 and 0-6 substituted purines. In certain embodiments, modified nucleobases are selected from: 2- aminopropyladenine, 5-hydroxymethylcytosine, xanthine, hypoxanthine, 2-aminoadenine, 6- N-methyladenine, 2-propyladenine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5- propynyl (—C≡C—CH3) uracil, 5-propynylcytosine, 6-azouracil, 6-azocytosine, 6-4934-0525-6010.2Page 16 of 105 065715-000129WOPTazothymine, 5-ribosyluracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl, 8-aza and other 8-substituted purines, 5-halo, particularly 5-bromo, 5- trifluoromethyl, 5-halouracil, and 5-halocytosine, 7-methylguanine, 7-methyladenine, 2-F- adenine, 2-aminoadenine, 7-deazaguanine, 7-deazaadenine, 3-deazaguanine, 3-deazaadenine, 6-N-benzoyladenine, 2-N-isobutyrylguanine, 4-N-benzoylcytosine, 4-N-benzoyluracil, 5- methyl 4-N-benzoylcytosine, 5-methyl 4-N-benzoyluracil, universal bases, hydrophobic bases, promiscuous bases, size-expanded bases, and fluorinated bases. Further modified nucleobases include tricyclic pyrimidines, such as 1,3-diazaphenoxazine-2-one, 1,3- diazaphenothiazine-2-one and 9-(2-aminoethoxy)-1,3-diazaphenoxazine-2-one (G-clamp). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2- aminopyridine and 2-pyridone. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, Kroschwitz, J. I., Ed., John Wiley & Sons, 1990, 858-859; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, Crooke, S. T. andJY76WRF Lebleu, B., Eds., CRC Press, 1993, 273-288; and those disclosed in Chapters 6 and 15, Antisense Drug Technology, Crooke S. T., Ed., CRC Press, 2008, 163-166 and 442-443. A universal base is a modified nucleobase that can pair with any one of the five unmodified nucleobases.

[0062] As used herein, “nucleobase sequence” means the order of contiguous nucleobases in a nucleic acid or oligonucleotide independent of any sugar or internucleoside linkage modification.

[0063] “Nucleoside” means a compound comprising a nucleobase and a sugar moiety. The nucleobase and sugar moiety are each, independently, unmodified or modified. As used herein, “modified nucleoside” means a nucleoside comprising a modified nucleobase and / or a modified sugar moiety. Modified nucleosides include abasic nucleosides, which lack a nucleobase. “Linked nucleosides” are nucleosides that are connected in a continuous sequence (i.e., no additional nucleosides are presented between those that are linked).

[0064] “Oligonucleotide” refers to a strand of linked nucleosides connected via internucleoside linkages, wherein each nucleoside and internucleoside linkage may be modified or unmodified. Unless otherwise indicated, oligonucleotides have 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or a range that includes or is between of any two of the foregoing numbers, linked nucleosides. As used herein, “modified4934-0525-6010.2Page 17 of 105 065715-000129WOPToligonucleotide” means an oligonucleotide, wherein at least one nucleoside or internucleoside linkage is modified. In certain embodiments, modified oligonucleotides comprise one or more nucleoside comprising a modified nucleobase. As used herein, “unmodified oligonucleotide” means an oligonucleotide that does not comprise any nucleoside modifications or internucleoside modifications.

[0065] The term “internucleoside linkage” is the covalent linkage between adjacent nucleosides in an oligonucleotide. As used herein, “modified internucleoside linkage” means any internucleoside linkage other than a phosphodiester internucleoside linkage. For example, “phosphorothioate linkage” is a modified internucleoside linkage in which one of the non- bridging oxygen atoms of a phosphodiester internucleoside linkage is replaced with a sulfur atom. In various embodiments, internucleotide bond and internucleoside linkage are often used interchangeably in biological terms, to refer to the chemical bond that links nucleotides or nucleosides together.

[0066] Nucleosides of modified oligonucleotides may be linked together using any internucleoside linkage. The two main classes of internucleoside linking groups are defined by the presence or absence of a phosphorus atom. Representative phosphorus-containing internucleoside linkages include but are not limited to phosphates, which contain a phosphodiester bond ("P=O") (also referred to as unmodified or naturally occurring linkages), phosphotriesters, methylphosphonates, phosphoramidates, and phosphorothioates ("P=S"), and phosphorodithioates ("HS-P=S"). Representative non phosphorus containing internucleoside linking groups include but are not limited to methylenemethylimino (-CH2- N(CH3)-O-CH2-), thiodiester, thionocarbamate (-O-C(=O)(NH)-S-); siloxane (-O-SiH2-O-); and N,N'-dimethylhydrazine (-CH2-N(CH3)-N(CH3)-). Modified internucleoside linkages, compared to naturally occurring phosphate linkages, can be used to alter, typically increase, nuclease resistance of the oligonucleotide. Methods of preparation of phosphorous-containing and non-phosphorous-containing internucleoside linkages can be found in the art.

[0067] “Gapmer” refers to a modified oligonucleotide comprising an internal region having a plurality of nucleosides that support RNase H cleavage (thereby introducing RNase H susceptibility into the gapmer) positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.” Unless otherwise indicated, the sugar moieties of the nucleosides of the gap of a gapmer are unmodified 2′-deoxyfuranosyl. In some embodiments, when modified, the sugar moieties of a4934-0525-6010.2Page 18 of 105 065715-000129WOPTnucleoside has a 2’-O-methoxytheyl (2’-MOE, or 2MOE) modification, which typically displays nuclease resistance in concert with lower cell toxicity and increased binding affinity to the desired target. Thus, the term “MOE gapmer” indicates a gapmer having a sugar motif of 2’-MOE nucleosides in both wings and a gap of 2’-deoxynucleosides.

[0068] Other exemplary of 2’ substitutions (e.g., modification of 2’-hydroxyl of the ribose, also called 2’-ribose modification) include but are not limited to 2’-O-methyl (2’OMe), locked nucleic acid (LNA), (S)-constrained ethyl (S-cEt), and (R)-constrained ethyl (R-cEt). LNA has a bridge (chemical bond) between the 2’ oxygen and 4’ carbon of the ribose ring.

[0069] “Sugar moiety” or “sugar component” means an unmodified sugar moiety or a modified sugar moiety. As used herein, “unmodified sugar moiety” means a 2'-OH(H) furanosyl moiety, as found in RNA (an “unmodified RNA sugar moiety”), or a 2'-H(H) moiety, as found in DNA (an “unmodified DNA sugar moiety”). Unmodified sugar moieties have one hydrogen at each of the 1’, 3', and 4' positions, an oxygen at the 3' position, and two hydrogens at the 5' position. As used herein, “modified sugar moiety” or “modified sugar” means a modified furanosyl sugar moiety or a sugar surrogate. As used herein, modified furanosyl sugar moiety means a furanosyl sugar comprising a non-hydrogen substituent in place of at least one hydrogen of an unmodified sugar moiety. In certain embodiments, a modified furanosyl sugar moiety is a 2'-substituted sugar moiety. Such modified furanosyl sugar moieties include bicyclic sugars and non-bicyclic sugars. "Sugar surrogate" means a modified sugar moiety having other than a furanosyl moiety that can link a nucleobase to another group, such as an internucleoside linkage, conjugate group, or terminal group in an oligonucleotide. Modified nucleosides comprising sugar surrogates can be incorporated into one or more positions within an oligonucleotide and such oligonucleotides are capable of hybridizing to complementary oligomeric compounds or nucleic acids.

[0070] In certain embodiments, modified sugar moieties are non-bicyclic modified sugar moieties comprising a furanosyl ring with one or more substituent groups none of which bridges two atoms of the furanosyl ring to form a bicyclic structure. Such non bridging substituents may be at any position of the furanosyl, including but not limited to substituents at the 2', 4', and / or 5' positions. In certain embodiments one or more non-bridging substituent of non-bicyclic modified sugar moieties is branched. Examples of 2’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 2’-F, 2’- OCH3("OMe" or "O-methyl"), and 2’-O(CH2)2OCH3 ("MOE"). In certain embodiments, 2’- substituent groups are selected from among: halo, allyl, amino, azido, SH, CN, -OCN, -CF3, -4934-0525-6010.2Page 19 of 105 065715-000129WOPTOCF3, -O-C1-10 alkoxy, -O-C1-10 substituted alkoxy, -O-C1-10 alkyl, -O-C1-10 substituted alkyl, -S-alkyl, -N(Rm)-alkyl, -O-alkenyl, -S-alkenyl, -N(Rm)-alkenyl, -O-alkynyl, -S-alkynyl, - N(Rm)-alkynyl, -O-alkylenyl-O-alkyl, alkynyl, alkaryl, aralkyl, -O-alkaryl, -O-aralkyl, - O(CH2)2SCH3, -O(CH2)2ON(Rm)(Rn) or -OCH2C(=O)-N(Rm)(Rn), where each Rm and Rn is, independently, H, an amino protecting group, or substituted or unsubstituted C1-10 alkyl, and the 2’-substituent groups can be further substituted with one or more substituent groups independently selected from among: hydroxyl, amino, alkoxy, carboxy, benzyl, phenyl, nitro, thiol, thioalkoxy, thioalkyl, halogen, alkyl, aryl, alkenyl and alkynyl, or are as described in Cook et al., U.S. Pat. No. 6,531,584; Cook et al., U.S. Pat. No. 5,859,221; and Cook et al., U.S. Pat. No. 6,005,087. Examples of 4’- substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to alkoxy (e.g., methoxy), and alkyl. Examples of 5’-substituent groups suitable for non-bicyclic modified sugar moieties include but are not limited to: 5’-methyl (R or S), 5’- vinyl, and 5’-methoxy. In certain embodiments, non-bicyclic modified sugar moieties comprise more than one non-bridging sugar substituent, for example, 2’-F-5’-methyl sugar moieties and the like.

[0071] In certain embodiments, a 2’-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’-substituent group selected from: F, NH2, NS, -OCF3, -OCH3, -O(CH2)3NH2, -CH2CH=CH2, -OCH2CH=CH2, -OCH2CH2OCH3, - O(CH2)2SCH3, -O(CH2)2ON(Rm)(Rn), -O(CH2)2O(CH2)2N(CH3)2, and N-substituted acetamide (-OCH2C(=O)-N(Rm)(Rn)), where each Rmand Rnis, independently, H, an amino protecting group, or substituted or unsubstituted C1-10 alkyl.

[0072] In certain embodiments, a 2’-substituted nucleoside non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’-substituent group selected from: F, -OCF3, -OCH3, -OCH2CH2OCH3, -O(CH2)2SCH3, -O(CH2)2ON(CH3)2, - O(CH2)2O(CH2)2N(CH3)2, and -OCH2C(=O)-N(H)CH3 ("NMA").

[0073] In certain embodiments, a 2’-substituted non-bicyclic modified nucleoside comprises a sugar moiety comprising a non-bridging 2’-substituent group selected from: F, - OCH3, and -OCH2CH2OCH3.

[0074] Certain modified sugar moieties comprise a substituent that bridges two atoms of the furanosyl ring to form a second ring, resulting in a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety comprises a bridge between the 4’ and the 2’ furanose ring atoms. Examples of such 4’ to 2’ bridging sugar substituents include but are not limited to: 4’-CH2-2’, 4’-(CH2)2-2’, 4’-(CH2)3-2’, 4’-CH2-O-2’ ("LNA"), 4’-CH2-S-2’, 4’- (CH2)2-O-2’ ("ENA"), 4’-CH(CH3)-O-2’ (referred to as "constrained ethyl" or "cEt"), 4’-4934-0525-6010.2Page 20 of 105 065715-000129WOPTCH2-O-CH2- 2’, 4’-CH2-N(R)-2’, 4’-CH(CH2OCH3)-O-2’ ("constrained MOE" or "cMOE") and analogs thereof, 4’-C(CH3)(CH3)-O-2’ and analogs thereof, 4’-CH2-N(OCH3)-2’ and analogs thereof, 4’-CH2-O-N(CH3)-2’, 4’-CH2-C(H)(CH3)-2’, 4’-CH2-C(=CH2)-2’ and analogs thereof, 4’- C(RaRb)-N(R)-O-2’, 4’-C(RaRb)-O-N(R)-2’, 4’-CH2-O-N(R)-2’, and 4’- CH2-N(R)-O-2’, wherein each R, Ra, and Rb, is, independently, H, a protecting group, or C1-12alkyl.

[0075] In certain embodiments, such 4’ to 2’ bridges independently comprise from 1 to 4 linked groups independently selected from: -[C(Ra)(Rb)]n-, -[C(Ra)(Rb)]n-O-, - C(Ra)=C(Rb)-, -C(Ra)=N-, -C(=NRa)-, -C(=O)-, -C(=S)-, -O-, -Si(Ra)2-, -S(=O)x-, and -N(Ra); wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each Raand Rbis, independently, H, a protecting group, hydroxyl, C1-12 alkyl, substituted C1-12 alkyl, C1-12 alkenyl, substituted C2-12 alkenyl, C2-12 alkynyl, substituted C2-12 alkynyl, C5-20 aryl, substituted C5-20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-7alicyclic radical, substituted C5-7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(=O)-H), substituted acyl, CN, sulfonyl (S(=O)2-J1), or sulfoxyl (S(=O)-J1); and each J1 and J2 is, independently, H, C1-12alkyl, substituted C1-12alkyl, C2-12alkenyl, substituted C2-12alkenyl, C2-12alkynyl, substituted C2-12alkynyl, C5-20aryl, substituted C5-20aryl, acyl (C(=O)-H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-12 aminoalkyl, substituted C1-12aminoalkyl, or a protecting group.

[0076] Additional bi cyclic sugar moieties are known in the art.

[0077] The three regions of a gapmer motif include the “5’ wing”, the “gap” and the “3’ wing” which form a contiguous sequence of nucleosides wherein at least some of the sugar moieties of the nucleosides of each of the wings differ from at least some of the sugar moieties of the nucleosides of the gap. Specifically, at least the sugar moieties of the nucleosides of each wing that are closest to the gap (the 3’-most nucleoside of the 5’-wing and the 5’-most nucleoside of the 3’-wing) differ from the sugar moiety of the neighboring gap nucleosides, thus defining the boundary between the wings and the gap (i.e., the wing / gap junction). In certain embodiments, the sugar moieties within the gap are the same as one another. In certain embodiments, the gap includes one or more nucleoside having a sugar moiety that differs from the sugar moiety of one or more other nucleosides of the gap. In certain embodiments, the sugar motifs of the two wings are the same as one another (symmetric gapmer). In certain embodiments, the sugar motif of the 5’-wing differs from the sugar motif of the 3’-wing (asymmetric gapmer).4934-0525-6010.2Page 21 of 105 065715-000129WOPT

[0078] In certain embodiments, the wings of a gapmer comprise a number of nucleosides selected from 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or a range that includes or is between any two of the foregoing numbers (e.g., 1-5, 2-7, etc.). In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside. In certain embodiments, the gap of a gapmer comprises comprise a number of nucleosides selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or a range that includes or is between any two of the foregoing numbers (e.g., 7-15, 10-20, etc.). In certain embodiments, each nucleoside of the gap of a gapmer is an unmodified 2’-deoxy nucleoside.

[0079] In certain embodiments, the gapmer is a deoxy gapmer. In further embodiments, the nucleosides on the gap side of each wing / gap junction are unmodified 2’- deoxy nucleosides and the nucleosides on the wing sides of each wing / gap junction are modified nucleosides. In certain embodiments, each nucleoside of the gap is an unmodified 2’-deoxy nucleoside. In certain embodiments, each nucleoside of each wing of a gapmer is a modified nucleoside.

[0080] In another embodiments, modified oligonucleotides comprise, consist essentially of or consist of a region having a fully modified sugar motif. In such embodiments, each nucleoside of the fully modified region of the modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, each nucleoside of the entire modified oligonucleotide comprises a modified sugar moiety. In certain embodiments, modified oligonucleotides comprise or consist of a region having a fully modified sugar motif, wherein each nucleoside within the fully modified region comprises the same modified sugar moiety, referred to herein as a uniformly modified sugar motif. In certain embodiments, a fully modified oligonucleotide is a uniformly modified oligonucleotide. In certain embodiments, each nucleoside of a uniformly modified comprises the same 2’-modification.

[0081] In various embodiments, a standard gapmer ASO retains a central region of phosphorothioate-modified DNA nucleosides (also called DNA with PS linkages, or a PS DNA gap) sufficient to induce RNase H cleavage; and these nucleosides are flanked on both sides by blocks of 2’-ribose modifications (RNA wings) that will increase binding affinity to the target. In various embodiments, the DNA gap in an ASO gapmer is 8-10 residues, which is optimal for human RNase H1 enzyme activity. In various embodiments, an ASO gapmer has a 5-10-5 design, whereby a stretch of 5 contiguous high binding affinity modifications (such as modifications to 2’ ribose) surrounds a central stretch of 10 DNA with PS linkages (substrates for RNase H1) on both the 5’- and 3’-ends. Once delivered to cells, ASOs enter the nucleus and bind to their complementary, endogenous RNA target. Hybridization of the4934-0525-6010.2Page 22 of 105 065715-000129WOPTASO gapmers to target RNA forms a DNA:RNA heteroduplex in the central region, which becomes a substrate for cleavage by the enzyme RNase H1. Unless otherwise indicated, an MOE gapmer may comprise one or more modified internucleoside linkages and / or modified nucleobases and such modifications do not necessarily follow the gapmer pattern of the sugar modifications. Other gapmer configurations are also conceived, such as uneven lengths of the RNA wings in a gapmer, as well as a mixmer design which consists of an alternation of RNA and DNA nucleotides.

[0082] For RNase H1 degradative ASOs, the RNase H1 endonuclease specifically cleaves RNA only when it is hybridized as a heteroduplex with DNA (in this case, DNA residues which are a part of the ASO). Once cleavage of an RNA molecule occurs, the ASO can dissociate and be recycled multiple times to cleave new RNA molecules.

[0083] Gapmer antisense oligonucleotides are usually used for gene silencing by stimulating RNA cleavage through the recruitment of an endogenous endonuclease known as RNase H to cleave the RNA strand of a DNA-RNA duplex. In various embodiments, an ASO that suppresses the expression of a KCTD20 is designed to target an 18nt to 20-nt (e.g., 20- nt) region at the mRNA and / or pre-mRNA level of KCTD20.

[0084] Alternatively, a steric blocking ASO (SBO) is chemically modified so that it does not form a substrate for RNase H1 when hybridized to the RNA target, typically by using 2’-modified RNA residues throughout the ASO (excluding DNA). Instead, a steric blocking ASO molecule will bind tightly to a single RNA molecule with no turn-over, obstructing the ability of other biomolecules to functionally bind at that site. Steric blocking ASOs are also conceived.

[0085] Nucleobase modifications can be made to enhance ASO activity or allow variations. An exemplary nucleobase modification is at the C5 hydrogen of deoxycytidine (dC), such as substituting the hydrogen with a methyl group (5-methylcytosine or 5Me-dC). This modification does not interfere with RNase H activity and can be included into the PS DNA “gap” of a gapmer ASO, for increasing thermal stability and for mitigating immune response induced by toll-like receptor 9 recognition (when substituting dC with 5Me-dC in CpG motifs in an ASO). Another nucleobase modification is removal of a heterocyclic base to produce either an apurinic or an apyrimidinic site in a sequence, referred to as an abasic site. Abasic site may be produced by selective hydrolysis of an N-glycosidic bond; and the resulting position has no base pairing capability.

[0086] In various embodiments, “complementary” in reference to an oligonucleotide means that at least 70%, at 80%, at least 90%, at least 95%, at least 98%, or at least 99% of4934-0525-6010.2Page 23 of 105 065715-000129WOPTthe nucleobases of the oligonucleotide or one or more regions thereof and the nucleobases of another nucleic acid or one or more regions thereof are capable of hydrogen bonding with one another when the nucleobase sequence of the oligonucleotide and the other nucleic acid are aligned in opposing directions. Complementary nucleobases means nucleobases that are capable of forming hydrogen bonds with one another. Complementary nucleobase pairs include adenine (A) and thymine (T), adenine (A) and uracil (U), cytosine (C) and guanine (G), 5-methylcytosine (mC) and guanine (G). Complementary oligonucleotides and / or nucleic acids need not have nucleobase complementarity at each nucleoside. Rather, some mismatches are tolerated.

[0087] “Fully complementary” or “100% complementary” in reference to oligonucleotides means that oligonucleotides are complementary to another oligonucleotide or nucleic acid at each nucleoside of the oligonucleotide.

[0088] A “target nucleic acid” and “target RNA” mean a nucleic acid that an antisense compound is designed to affect.

[0089] In some embodiments, inhibitors of KCTD20 are inhibitory nucleic acids, which suppress (or reduce or inhibitor) expression of KCTD20. “Inhibitory nucleic acids” refers to an antisense compound that modulates a target nucleic acid and / or protein encoded by a target nucleic acid; and it includes, but is not limited to, double-stranded siRNA, single- stranded RNA (ssRNA), and microRNA, including microRNA mimics.

[0090] The phrase “guide RNA” is also called guide CRISPR RNA (crRNA). In various embodiments, the guide RNA is designed to detect protospacer adjacent motif (PAM) or the equivalent for a target RNA or DNA. The protospacer adjacent motif (PAM) or PAM- like motif directs binding of the CRISPR-associated nuclease / crRNA complex as disclosed herein to the target locus of interest. The term “PAM” may be used interchangeably with the term “PFS” or “protospacer flanking site” or “protospacer flanking sequence”. In other embodiments, the one or more guide RNAs are designed to detect a single nucleotide polymorphism, splice variant of a transcript, or a frameshift mutation in a target RNA or DNA.

[0091] “Proteostasis,” or “proteostatic function,” is the dynamic regulation of a balanced, functional proteome.

[0092] The “Akt signaling pathway” is a signal transduction pathway that promotes survival and growth in response to extracellular signals. Key proteins involved are phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt).4934-0525-6010.2Page 24 of 105 065715-000129WOPT

[0093] The “mammalian target of rapamycin (mTOR) signaling pathway” integrates both intracellular and extracellular signals and serves as a central regulator of cell metabolism, growth, proliferation and survival.

[0094] “Oligomeric compound” means an oligonucleotide and optionally one or more additional features, such as a conjugate group or terminal group. An oligomeric compound may be paired with a second oligomeric compound that is complementary to the first oligomeric compound or may be unpaired. A “singled stranded oligomeric compound” is an unpaired oligomeric compound. The term “oligomeric duplex” means a duplex formed by two oligomeric compounds having complementary nucleobase sequences. Each oligomeric compound of an oligomeric duplex may be referred to as a “duplexed oligomeric compound.”

[0095] The “3’UTR” or three prime untranslated region is part of messenger RNA (mRNA) that immediately follows the translation termination codon. The 3'UTR of mRNA is transcribed from DNA, but is not translated into protein. The 3′-UTR often contains regulatory regions that post-transcriptionally influence gene expression. The 3′-UTR contains binding sites for both regulatory proteins and microRNAs (miRNAs). By binding to specific sites within the 3′-UTR, miRNAs can decrease gene expression of various mRNAs by either inhibiting translation or directly causing degradation of the transcript. The 3′-UTR also has silencer regions which bind to repressor proteins and will inhibit the expression of the mRNA.

[0096] A human mRNA molecule typically has a structure from 5’ to 3’ as follows: the 5’ cap – 5’ UTR – coding sequence – 3’ UTR – poly-A tail. One class of human genes produces mRNAs with only one 3’UTR, also called single UTR genes; whereas the other class of human genes, called multi-UTR genes, generates alternative 3’UTR isoforms. C. Mayr in Trends Cell Biol.2016 Mar, 26(3):227-237 describes that the median 3′UTR length of single-UTR genes is about 600 nt, but is about 2,300 nt for multi-UTR genes. Sood et al. in PNAS 103(8):2746-2751 (2006) describe a human 3’UTR on average is about 950 nt long, and for highly expressed neuronal genes being about 1,300 nt or for genes specific for a nonneuronal tissue being about 700 nt. Since mRNA processing in terms of termination of transcription, its transport from the nucleus to the cytoplasm, its stability and translation efficiency are dependent on key 3’UTR elements, targeting these 3’ UTR elements with ASOs can induce gene silencing.

[0097] Tau is a cytosolic protein that functions in the assembly and stabilization of axonal microtubule networks. Its oligomerization or aggregation, forming tau oligomers, may mediate neurodegeneration.4934-0525-6010.2Page 25 of 105 065715-000129WOPT

[0098] To identify key regulators of neuronal excitotoxicity, we use cortical cerebral organoids to model excitotoxicity in vitro. We find that glutamate treatment induces tauopathy disease phenotypes in organoids, including increased oligomeric tau and accelerated neurodegeneration. Organoids generated from tau-V337M iPSCs, which harbor a tau mutation that drives tau pathology and neurodegeneration in humans, display enhanced tauopathy and neurodegeneration after glutamate treatment, indicating that tau pathology is a key mediator of excitotoxicity in this model. Using genome-wide CRISPR interference (CRISPRi) screening, we find that suppression of potassium channel tetramerization domain containing 20 (KCTD20) is one of the most potent targets for mitigating tau pathology and neuron death in both control and tau-V337Morganoids.The KCTD family of proteins are soluble non-channel proteins with a common BTB domain responsible for E3 ligase and transcriptional activity. However, KCTD20 is relatively isolated from other KCTD paralogs based on amino acid sequence identity and has been genetically associated with addiction, a pathway associated with glutamatergic signaling. Our analyses indicate that KCTD20 suppression ameliorates tau pathology and neurodegeneration by enhancing lysosomal exocytosis of oligomeric tau. Kctd20 suppression also reduces tau pathology and neurodegeneration in vivo in transgenic humanized tau P301S mice. Without wishing to be bound by a particular theory, these data indicate that glutamate excitotoxicity can drive neuron death through tau pathology and that suppressing KCTD20 or activating lysosomal exocytosis to clear toxic protein species may mitigate neurodegeneration in diseases that display excitotoxicity.

[0099] Various embodiments provide antisense oligonucleotides (ASOs), wherein the ASOs modulates expression of KCTD20. Various embodiments provide single stranded ASOs, which suppresses expression of KCTD20. Various embodiments provide single stranded ASOs, which comprise a nucleobase sequence that is complementary to a segment of KCTD20.

[0100] In some embodiments, the ASO comprises 12-50 linked nucleosides, and the ASO has a nucleobase sequence having a guanine-cytosine content (GC content) of 30-70%, no cytidine (C) followed by a guanosine (G), and no more than three contiguous G’s. In some embodiments, the ASO comprises 18-22 linked nucleosides, and the ASO has a nucleobase sequence having a GC content of 40-65%, no cytidine (C) followed by a guanosine (G), and no more than three contiguous G’s. In some embodiments, the ASO consists of 18-22 linked nucleosides, and the ASO has a nucleobase sequence having a GC4934-0525-6010.2Page 26 of 105 065715-000129WOPTcontent of 40-65%, no cytidine (C) followed by a guanosine (G), and no more than three contiguous G’s.

[0101] In some embodiments, the ASO has a nucleobase sequence that comprises at least 12, 15, or 18 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs: 1-647, 16718, 16719, and 16750-16752 or set forth in a variant thereof, wherein the variant has substitutions of one or more thymines (T’s) if present by uracil. In some embodiments, the ASO has a nucleobase sequence that comprises at least 12, 15, or 18 consecutive nucleobases of the nucleobase sequence SEQ ID NO: 103 or a variant thereof, wherein the variant has a substitution of thymine in the wing segments (e.g., first 5 nucleosides at the 5’ end and last 5 nucleosides closest to the 3’ end) by uracil. In some embodiments, the ASO has a nucleobase sequence that comprises at least 12, 15, or 18 consecutive nucleobases of the nucleobase sequence SEQ ID NO: 104 or a variant thereof, wherein the variant has a substitution of thymine in the wing segments (e.g., first 5 nucleosides at the 5’ end and last 5 nucleosides closest to the 3’ end) by uracil. In some embodiments, the ASO has a nucleobase sequence that comprises at least 12, 15, or 18 consecutive nucleobases of the nucleobase sequence SEQ ID NO: 105 or a variant thereof, wherein the variant has a substitution of thymine in the wing segments (e.g., first 5 nucleosides at the 5’ end and last 5 nucleosides closest to the 3’ end) by uracil. In some embodiments, the ASO has a nucleobase sequence that comprises at least 12, 15, or 18 consecutive nucleobases of the nucleobase sequence SEQ ID NO: 16750 or a variant thereof, wherein the variant has a substitution of thymine in the wing segments (e.g., first 5 nucleosides at the 5’ end and last 5 nucleosides closest to the 3’ end) by uracil. In some embodiments, the ASO has a nucleobase sequence that comprises at least 12, 15, or 18 consecutive nucleobases of the nucleobase sequence SEQ ID NO: 16751 or a variant thereof, wherein the variant has a substitution of thymine in the wing segments (e.g., first 5 nucleosides at the 5’ end and last 5 nucleosides closest to the 3’ end) by uracil. In some embodiments, the ASO has a nucleobase sequence that comprises at least 12, 15, or 18 consecutive nucleobases of the nucleobase sequence SEQ ID NO: 16752. In some embodiments, the ASO has a nucleobase sequence that comprises at least 18 consecutive nucleobases of the nucleobase sequence of any one of SEQ ID NOs: 1-647, 16718, 16719, and 16750-16752 or a variant thereof, wherein the variant has an otherwise same nucleobase sequence except that one or more thymines (T’s) if present is substituted by uracil. In some embodiments, the ASO has a nucleobase sequence of that in any one of SEQ ID NOs: 1-647, 16718, 16719, and 16750-16752 or a variant thereof, wherein the variant has an otherwise4934-0525-6010.2Page 27 of 105 065715-000129WOPTidentical nucleobase sequence as the sequence of respective SEQ ID except that one or more thymines (T’s) if present is substituted by uracil. In some embodiments, the ASO is a gapmer, comprising a gap segment consisting of 10 linked deoxynucleosides, a 5’ wing segment consisting of 5 linked nucleosides, and a 3’ wing segment consisting of 5 linked nucleosides; the ASO has a nucleobase sequence of a variant of any one of SEQ ID NOs: 1-647, 16718, 16719, and 16750-16752, wherein the variant has variances comprising or consisting of (1) unmodified 2′-deoxyfuranosyl, 2’-O-methoxytheyl modified deoxyfuranosyl, or 2’-O- methoxy modified deoxyfuranosyl as the sugar moiety in a gap segment consisting of 10 linked nucleosides, flanked by a 5’ wing segment and a 3’ wing segment, each wing segment consisting of 5 linked nucleosides whose sugar moiety is 2’-O-methoxytheyl modified furanosyl or 2’-O-methoxy modified furanosyl, and (2) substitution of all thymine’s by uracil, or substitution of thymine’s only in the 5’ wing segment and the 3’ wing segment by uracil; and the at least one internucleoside linkage is phosphorothioate internucleoside linkage.

[0102] In some embodiments, a variant of a nucleic acid sequence is one wherein at least one thymine (if present) is substituted by uracil. In some embodiments, a variant of a nucleic acid sequence is one otherwise identical except that at least one thymine (if present) is substituted by uracil. In some embodiments, a variant of a nucleic acid sequence is one otherwise identical except that all thymine’s (if present) are substituted by uracil. In some embodiments, a variant of a nucleic acid sequence is one otherwise identical except that thymine’s (if present) in the first 3-5 bases from the 5’ end and / or in the last 3-5 bases closest to the 3’ end are substituted by uracil. Generally, an ASO having a nucleobase sequence of that in such a variant may have a different sugar moiety in certain nucleosides and / or a different internucleoside linkage, although the nucleobase sequence is identical to such variant.

[0103] Further embodiments provide ASOs with a high stringency for improved safety and efficacy in suppressing KCTD20. In some embodiments, the ASO comprises 18-22 or 20 linked nucleosides, wherein the ASO has a nucleobase sequence having a G-C content of 40%-60%, having no C followed by a G, having no more than two consecutive G’s, and the ASO has a nucleobase sequence having at least 6 cytidines (C’s), at least 6 thymines (T’s), 0-3 G’s, and a 3’ end not ending in 1-3 G’s; and optionally wherein the ASO has a change in Gibbs free energy (ΔG) associated with self-dimerization being ≥ -7 kcal / mole. In some embodiments, the ASO comprises 20 linked nucleosides, wherein the ASO has a nucleobase sequence having a G-C content of 40%-60%, having no C followed by a G, having no more than two consecutive G’s, and the ASO has a nucleobase sequence having at4934-0525-6010.2Page 28 of 105 065715-000129WOPTleast 6 cytidines (C’s), at least 6 thymines (T’s), 0-3 G’s, and a 3’ end not ending in 1-3 G’s, and wherein the ASO has a change in Gibbs free energy (ΔG) associated with self- dimerization being ≥ -7 kcal / mole.

[0104] In some embodiments, an ASO has a nucleobase sequence that comprises at least 12, 15, or 18, or 20 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:648-1321. In some embodiments, an ASO has a nucleobase sequence that comprises at least 15 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:648-1321. In some embodiments, an ASO has a nucleobase sequence that comprises at least 18 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:648-1321. In some embodiments, an ASO has a nucleobase sequence that comprises the nucleobases of any one of SEQ ID NOs:648-1321. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:648-1321. In some aspects, the variant comprises substitutions of one, two, three, or more thymines (T’s) by uracil. In some aspects, the variant consists of substitutions of one, two, three, or more thymines (T’s) by uracil. In some aspects, the variant comprises one, two, three, or more nucleobases removed. In some aspects, the variant consists of one, two, three, or more nucleobases removed. In further aspects, the variant comprises both substitutions of a thymine by uracil and a removal of another nucleobase, thus resulting in the nucleobase sequence comprising uracil and abasic site. In further aspects, the variant consists of both substitutions of a thymine by uracil and a removal of another nucleobase. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:648-1321, wherein the variant has substitution of a thymine by a uracil. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:648-1321, wherein the variant has substitution of all thymine’s by uracil’s. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:648-1321, wherein the variant has one or more nucleobases removed, resulting in one or more abasic sites. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:648 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:649 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:650 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:651 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:652 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:653 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:6544934-0525-6010.2Page 29 of 105 065715-000129WOPTor a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:655 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:656 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:657 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:658 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:659 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:660 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:661 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:662 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:663 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:664 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:665 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:666 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of that of SEQ ID NO:667 or a variant thereof. In some embodiments, an ASO has a nucleobase sequence of the nucleobase sequence of any one of the first 20, 30, 40, 50, 75, 100, 200, 300, 400, 500 of SEQ ID NOs:648-1321 (in the order from small to large SEQ ID NOs) or a variant thereof.

[0105] In some embodiments, the ASO having the nucleobase sequence of any one of SEQ ID NOs:648-1321 or a variant of SEQ ID NOs:648-1321 is 20 nucleotide long, being in a gapmer format comprising from 5’ to 3’: a 5’ wing segment consisting of 5 nucleosides, a gap segment consisting of 10 deoxynucleosides, and a 3’ wing segment consisting of 5 nucleosides. In various aspects, the ASO has phosphorothioate or phosphodiester linkages at any one of the internucleoside linkages. In some aspects, the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage. In further aspects, the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage; and the second, third, fourth, fifth, sixteenth, and seventeenth internucleoside linkage is a phosphodiester internucleoside linkage. In various aspects, an ASO in a gapmer format has a modified sugar moiety at any position on the wing segments, such as 2’MOE or 2’OMe or other sugar moieties like threose. In some aspects, the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is a modified sugar moiety. In some aspects, the sugar moiety in the first, second, third, fourth,4934-0525-6010.2Page 30 of 105 065715-000129WOPTfifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O-methoxyethyl (2’MOE) group. In some aspects, the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O-methyl (2’OMe) group. In some aspects, the sugar moiety in some of the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O-methoxyethyl group, and in other modified with a 2’-O-methyl group. In further aspects, the ASO is 20-nucleotide long, being in a gapmer format comprising from 5’ to 3’: a 5’ wing segment consisting of 5 nucleosides, a gap segment consisting of 10 deoxynucleosides, and a 3’ wing segment consisting of 5 nucleosides, wherein the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O- methoxyethyl or 2’O-methyl or threose or another group, and wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage.

[0106] Further embodiments provide ASOs with a medium stringency for improved safety and efficacy in suppressing KCTD20. In some embodiments, the ASO comprises 18-22 or 20 linked nucleosides, wherein the ASO has a nucleobase sequence with a GC content of 40%-65%, having no C followed by a G, having no more than 3 consecutive G’s, having at least 5 C’s and at least 5 T’s, and if the nucleobase sequence has 5 T’s and 5 C’s, the ASO has one G in the last three bases at the 3’ end; and optionally the ASO has a ΔG associated with self-dimerization being ≥ -7 kcal / mole. In some embodiments, the ASO comprises 20 linked nucleosides, wherein the ASO has a nucleobase sequence with a GC content of 40%- 65%, having no C followed by a G, having no more than 3 consecutive G’s, having at least 5 C’s and at least 5 T’s, and if the nucleobase sequence has 5 T’s and 5 C’s, the ASO has one G in the last three bases at the 3’ end; and the ASO has a ΔG associated with self-dimerization being ≥ -7 kcal / mole.

[0107] In some embodiments, an ASO has a nucleobase sequence that comprises at least 12, 15, or 18, or 20 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:1322-2584 or set forth in a variant thereof. In some embodiments, an ASO has a nucleobase sequence that comprises at least 15 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:1322-2584. In some embodiments, an ASO has a nucleobase sequence that comprises at least 18 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:1322-2584. In some embodiments, an ASO has a nucleobase sequence that comprises the nucleobases of any one of SEQ ID4934-0525-6010.2Page 31 of 105 065715-000129WOPTNOs:1322-2584. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:1322-2584. In some aspects, the variant comprises substitutions of one, two, three, or more thymines (T’s) by uracil. In some aspects, the variant consists of substitutions of one, two, three, or more thymines (T’s) by uracil. In some aspects, the variant comprises one, two, three, or more nucleobases removed. In some aspects, the variant consists of one, two, three, or more nucleobases removed. In further aspects, the variant comprises both substitutions of a thymine by uracil and a removal of another nucleobase, thus resulting in the nucleobase sequence comprising uracil and abasic site. In further aspects, the variant consists of both substitutions of a thymine by uracil and a removal of another nucleobase. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:1322-2584, wherein the variant has substitution of a thymine by a uracil. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:1322-2584, wherein the variant has substitution of all thymine’s by uracil’s. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:1322-2584, wherein the variant has one or more nucleobases removed, resulting in one or more abasic sites. In some embodiments, an ASO has a nucleobase sequence of the nucleobase sequence of any one of the first 20, 30, 40, 50, 75, 100, 200, 300, 400, 500 of SEQ ID NOs:1322-2584 (in the order from small to large SEQ ID NOs) or a variant thereof.

[0108] In some embodiments, the ASO having the nucleobase sequence of the variant of any one of SEQ ID NOs:1322-2584 is 20-nucleotide long, being in a gapmer format comprising from 5’ to 3’: a 5’ wing segment consisting of 5 nucleosides, a gap segment consisting of 10 deoxynucleosides, and a 3’ wing segment consisting of 5 nucleosides. In various aspects, the ASO has phosphorothioate or phosphodiester linkages at any one of the internucleoside linkages. In some aspects, the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage. In further aspects, the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage; and the second, third, fourth, fifth, sixteenth, and seventeenth internucleoside linkage is a phosphodiester internucleoside linkage. In various aspects, an ASO in a gapmer format has a modified sugar moiety at any position on the wing segments, such as 2’MOE or 2’OMe or other sugar moieties like threose. In some aspects, the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is a4934-0525-6010.2Page 32 of 105 065715-000129WOPTmodified sugar moiety. In some aspects, the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O-methoxyethyl (2’MOE) group. In some aspects, the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O-methyl (2’OMe) group. In some aspects, the sugar moiety in some of the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O-methoxyethyl group, and in other modified with a 2’-O-methyl group. In further aspects, the ASO is 20-nucleotide long, being in a gapmer format comprising from 5’ to 3’: a 5’ wing segment consisting of 5 nucleosides, a gap segment consisting of 10 deoxynucleosides, and a 3’ wing segment consisting of 5 nucleosides, wherein the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O- methoxyethyl or 2’O-methyl or threose or another group, and wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage.

[0109] Further embodiments provide ASOs with a low stringency for improved safety and efficacy in suppressing KCTD20. In some embodiments, the ASO comprises 18-22 or 20 linked nucleosides, wherein the ASO has a nucleobase sequence with a GC content of 40%- 60%, having no C followed by a G, having no more than 3 consecutive G’s, having no G on the 3’ end; and optionally wherein the ASO has a ΔG associated with self-dimerization being ≥ -7 kcal / mole. In some embodiments, the ASO comprises 20 linked nucleosides, wherein the ASO has a nucleobase sequence with a GC content of 40%-60%, having no C followed by a G, having no more than 3 consecutive G’s, having no G on the 3’ end; and wherein the ASO has a ΔG associated with self-dimerization being ≥ -7 kcal / mole.

[0110] In some embodiments, an ASO has a nucleobase sequence that comprises at least 12, 15, or 18, or 20 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:2585-5842 or set forth in a variant thereof. In some embodiments, an ASO has a nucleobase sequence that comprises at least 15 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:2585-5842. In some embodiments, an ASO has a nucleobase sequence that comprises at least 18 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:2585-5842. In some embodiments, an ASO has a nucleobase sequence that comprises the nucleobases of any one of SEQ ID NOs:2585-5842. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs: 2585-5842. In some aspects, the variant comprises4934-0525-6010.2Page 33 of 105 065715-000129WOPTsubstitutions of one, two, three, or more thymines (T’s) by uracil. In some aspects, the variant consists of substitutions of one, two, three, or more thymines (T’s) by uracil. In some aspects, the variant comprises one, two, three, or more nucleobases removed. In some aspects, the variant consists of one, two, three, or more nucleobases removed. In further aspects, the variant comprises both substitutions of a thymine by uracil and a removal of another nucleobase, thus resulting in the nucleobase sequence comprising uracil and abasic site. In further aspects, the variant consists of both substitutions of a thymine by uracil and a removal of another nucleobase. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:2585-5842, wherein the variant has substitution of a thymine by a uracil. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:2585-5842, wherein the variant has substitution of all thymine’s by uracil’s. In some embodiments, an ASO has a nucleobase sequence of that in a variant of any one of SEQ ID NOs:2585-5842, wherein the variant has one or more nucleobases removed, resulting in one or more abasic sites. In some embodiments, an ASO has a nucleobase sequence of the nucleobase sequence of any one of the first 20, 30, 40, 50, 75, 100, 200, 300, 400, 500 of SEQ ID NOs:2585-5842 (in the order from small to large SEQ ID NOs) or a variant thereof.

[0111] In some embodiments, the ASO having the nucleobase sequence of the variant of any one of SEQ ID NOs:2585-5842 is 20 nucleotide long, being in a gapmer format comprising from 5’ to 3’: a 5’ wing segment consisting of 5 nucleosides, a gap segment consisting of 10 deoxynucleosides, and a 3’ wing segment consisting of 5 nucleosides. In various aspects, the ASO has phosphorothioate or phosphodiester linkages at any one of the internucleoside linkages. In some aspects, the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage. In further aspects, the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage; and the second, third, fourth, fifth, sixteenth, and seventeenth internucleoside linkage is a phosphodiester internucleoside linkage. In various aspects, an ASO in a gapmer format has a modified sugar moiety at any position on the wing segments, such as 2’MOE or 2’OMe or other sugar moieties like threose. In some aspects, the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is a modified sugar moiety. In some aspects, the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a4934-0525-6010.2Page 34 of 105 065715-000129WOPT2’-O-methoxyethyl (2’MOE) group. In some aspects, the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O-methyl (2’OMe) group. In some aspects, the sugar moiety in some of the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O-methoxyethyl group, and in other modified with a 2’-O-methyl group. In further aspects, the ASO is 20-nucleotide long, being in a gapmer format comprising from 5’ to 3’: a 5’ wing segment consisting of 5 nucleosides, a gap segment consisting of 10 deoxynucleosides, and a 3’ wing segment consisting of 5 nucleosides, wherein the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’-O- methoxyethyl or 2’O-methyl or threose or another group, and wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage.

[0112] Additional embodiments provide ASOs with none stringency. In some embodiments, the ASO has a nucleobase sequence of any one of SEQ ID NOs:5843-16669 or a variant thereof, wherein the variant has substitution of one or more thymine’s by uracil’s and / or one or more nucleobases removed. In some embodiments, the ASO is 20-nucleotide long, being in a gapmer format comprising from 5’ to 3’: a 5’ wing segment consisting of 5 nucleosides, a gap segment consisting of 10 deoxynucleosides, and a 3’ wing segment consisting of 5 nucleosides, wherein the sugar moiety in the first, second, third, fourth, fifth, sixteenth, seventeenth, eighteenth, nineteen, and twentieth nucleoside is modified with a 2’- O-methylethyl group, and wherein the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage.

[0113] In a further embodiment, the disclosure provides modified oligonucleotides consisting of 12-30 linked nucleosides and having a nucleobase sequence comprising at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any of one of the nucleobase sequences of SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669. In some embodiments, the modified oligonucleotide is at least 75% to 100% (i.e., 75%, 80%, 82%, 84%, 86%, 88%, 90%, 92%, 94%, 96%, 98% or 100%; or any numerical range or value between any of the foregoing values) identical to any of the sequences of SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669.4934-0525-6010.2Page 35 of 105 065715-000129WOPT

[0114] Nucleobase sequences provided herein can be used to design antisense molecules. For example, gapmer oligonucleotides are designed using the nucleobase sequences and can comprise a 5′-wing of about 3-5 nucleotides, a 3′-wing of about 3-5 nucleotides and a gap region comprising 12-8 consecutive deoxyribonucleosides of any one of the nucleobase sequences of SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669. In one embodiment, an oligonucleotide of the disclosure comprises a gapmer having a gap segment of at least 8, at least 9, at least 10, at least 11 at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19 or at least 20 consecutive nucleotide bases of any of the nucleobase sequences of SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669; flanked by a 5′ and 3′ wing segments, wherein the gap segment is located between the 5′ and 3′ wing segments and wherein each of the wing segments comprises a modified sugar. In one embodiment, the gap segment is 8-10 nucleosides in length and each wing segment is 3-5 modified nucleosides in length. In one embodiment, the gap segment is 10 nucleosides in length and each wing segment is 5 modified nucleosides in length. In yet another embodiment, an oligonucleotide of the disclosure comprises a 5′ wing segment comprising modified sugars and having the nucleobase sequence of the first 3-5 nucleobases of any of SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669 and variants thereof containing substitution of thymine by uracil, followed by a gap of the next 8-12 unmodified nucleotides of the same sequence corresponding to SEQ ID NOs:16718, 16719, 16750- 16752, and 1-16669 or variants thereof, followed by a 3′ wing segment comprising modified sugars and having the nucleobase sequence of the last 3-5 nucleobases of the same sequence corresponding to SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669 and variants thereof. In various aspects,

[0115] The 5′ and / or 3′ wings can comprise the following chemistries: 2′-OMe, 2′- MOE, LNA or DNA, by themselves or used in combination with one another. The backbone linkage of the 5′ and / or 3′ wings can be phosphorothioate or a mixture of phosphodiester and phosphorothioate. Linkages in the gap region can be phosphorothioate.

[0116] In some embodiments, the oligonucleotide is single stranded. In some embodiments the oligonucleotide comprises or is complexed with a moiety that neutralizes charge on the oligonucleotide to promote uptake and transfer across a cell membrane.

[0117] In one embodiment, an ASO has the following 5-10-5 motif: 2MOE*2MOE- 2MOE-2MOE-2MOE-N*N*N*N*N*N*N*N*N*N*2MOE-2MOE-2MOE*2MOE*2MOE, where (i) 2MOE is a nucleobase with a 2′-OCH2CH2—OCH3group (i.e., 2′-MOE), (ii) N is a4934-0525-6010.2Page 36 of 105 065715-000129WOPTnucleobase, (iii) the asterisk (*) refers to a phosphorothioate linkage, and (iv) the dash (-) refers to a phosphodiester linkage.

[0118] In one embodiments, an ASO has the following 5-10-5 motif: 2OMe*2OMe*2OMe*2OMe*2OMe*N*N*N*N*N*N*N*N*N*N*2OMe*2OMe*2OMe*2 OMe*2OMe, where (i) 2OMe is a nucleobase with a 2’-O-CH3 group (i.e., 2’OMe), (ii) N is a nucleobase, and (iii) the asterisk (*) refers to a phosphorothioate linkage.

[0119] In some embodiments, an ASO having a gapmer configuration (e.g., a 5-10-5 motif) has a modified sugar moiety (e.g., 2’MOE, 2OMe, or threose) at any one or more or all positions on the wings.

[0120] In some embodiments, an ASO having a gapmer configuration (e.g., a 5-10-5 motif) has phosphorothioate linkages or phosphodiester linkages at any internucleoside position.

[0121] In some embodiments, an ASO has a nucleobase sequence that includes thymines (T’s) instead of uracils (U’s) throughout the nucleobase sequence. In other embodiments, an ASO has a nucleobase sequence wherein one or more T's are replaced by U’s. In some embodiments, an ASO has a nucleobase sequence wherein any one nucleotide position has its nucleobase replaced by an abasic (no nucleobase) site. Typically, an abasic site does not compromise the modulation function on target compared to the sequence without an abasic site.

[0122] In some embodiments, the KCTD20 ASOs can include any combination of the ASOs described above.

[0123] In some embodiments, a KCDT20 ASO suppresses KCDT20 expression by at least 15%. In some embodiments, a KCDT20 ASO suppresses KCDT20 expression by at least 20%. In some embodiments, a KCDT20 ASO suppresses KCDT20 expression by at least 30%. In some embodiments, a KCDT20 ASO suppresses KCDT20 expression by at least 40%. In some embodiments, a KCDT20 ASO suppresses KCDT20 expression by at least 50%. In some embodiments, a KCDT20 ASO suppresses KCDT20 expression by at least 60%. In some embodiments, a KCDT20 ASO suppresses KCDT20 expression by at least 70%. In some embodiments, a KCDT20 ASO suppresses KCDT20 expression by at least 80%. In some embodiments, a KCDT20 ASO suppresses KCDT20 expression by at least 90%.

[0124] Methods of treatment may include any number of modes of administering a disclosed composition. Modes of administration may include aqueous, lipid, oily or other solutions, solutions in simulated cerebrospinal fluid, emulsions such as oil-in-water4934-0525-6010.2Page 37 of 105 065715-000129WOPTemulsions, liposomes, aqueous or oily suspensions and the like. Typically, an ASO of the disclosure will be administered directly to the central nervous system of the subject. Accordingly, the formulation or composition will be sterile and more preferably be suitable for injection. The following formulations and methods are merely exemplary and are in no way limiting.

[0125] Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and may be stored as liquids or in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets. The formulation may be provided in a pre-filled syringe.

[0126] Additional therapeutic agent(s) may be administered simultaneously or sequentially with the disclosed one or more ASO compositions. Sequential administration includes administration before or after the disclosed one or more antisense or inhibitory nucleic acids or compositions. In some embodiments, the additional therapeutic agent or agents may be administered in the same composition as the disclosed one or more ASOs. In other embodiments, there may be an interval of time between administration of the additional therapeutic agent and the disclosed one or more antisense or inhibitory nucleic acids. In some embodiments, administration of an additional therapeutic agent with a disclosed ASO may allow lower doses of the other therapeutic agents and / or administration at less frequent intervals. When used in combination with one or more other active ingredients, the one or more ASOs of the disclosure and the other active ingredients may be used in lower doses than when each is used singly. Accordingly, the pharmaceutical compositions of the disclosure include those that contain one or more other active ingredients, in addition to one or more ASOs of the disclosure. The above combinations include combinations of one or more ASOs not only with one other active compound, but also with two or more other active compounds. For example, the compound of the disclosure may be combined with a variety of drugs to treat neurological diseases. The antisense oligonucleotide may be covalently linked to another oligonucleotide, such as one with a target other than CCDC146. The antisense oligonucleotide may be covalently linked to an antibody.4934-0525-6010.2Page 38 of 105 065715-000129WOPT

[0127] The disclosed one or more ASOs can be combined with the following, but are not limited, anticholinergic drugs, anticonvulsants, antidepressants, benzodiazepines, decongestants, muscle relaxants, pain medications, and / or stimulants. Additional types of therapy and treatment include, but are not limited to digital communication devices, feeding tubes, mechanical ventilation, nutritional support, deep brain stimulation, occupational therapy, physical therapy, and / or speech therapy. In some embodiments, the disclosed one or more ASOs are the sole active ingredient for administration.

[0128] The disclosed composition(s) may be incorporated into a pharmaceutical composition suitable for administration to a subject (such as a patient, which may be a human or non-human). The pharmaceutical compositions may comprise a carrier (e.g., a pharmaceutically acceptable carrier). Any suitable carrier can be used within the context of the disclosure, and such carriers are well known in the art. The choice of carrier will be determined, in part, by the particular use of the composition (e.g., administration to an animal) and the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of the composition of the present invention.

[0129] The pharmaceutical compositions may include a therapeutically effective amount or a prophylactically effective amount of the antisense oligonucleotide. A therapeutically effective amount of the composition may be determined by a person skilled in the art and may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the composition to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of one or more ASOs disclosed herein are outweighed by the therapeutically beneficial effects. A “prophylactically effective amount” refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount will be less than the therapeutically effective amount.

[0130] The pharmaceutical compositions may include one or more pharmaceutically acceptable carriers. The term “pharmaceutically acceptable carrier,” as used herein, means a non-toxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, corn starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not4934-0525-6010.2Page 39 of 105 065715-000129WOPTlimited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogenfree water; isotonic saline; Ringer’s solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as releasing agents, coating agents, preservatives and antioxidants may also be present in the composition, according to the judgment of the formulator.

[0131] The route by which the disclosed one or more antisense or inhibitory nucleic acids are administered, and the form of the composition will dictate the type of carrier to be used.

[0132] The pharmaceutical compositions of the disclosure can be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration can be parenteral including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal, intracerebroventricular, or intraventricular, administration. In one embodiment the antisense or inhibitory nucleic acid is administered by intravenous, intraperitoneal, or as a bolus injection or administered directly into the target organ. In another embodiment, the antisense or inhibitory nucleic acid is administered intrathecally or intra-cerebroventricular as a bolus injection.

[0133] Carriers for systemic administration typically include at least one of solvent, diluents, lubricants, binders, disintegrants, colorants, flavors, sweeteners, antioxidants, preservatives, glidants, solvents, suspending agents, wetting agents, surfactants, combinations thereof, and others. All carriers are optional in the compositions.

[0134] Compositions for parenteral administration typically include 0.1% to 10% of actives and 90% to 99.9% of a carrier including a diluent and a solvent. In some embodiments, a pharmaceutical composition is provided which include one or more ASOs disclosed herein and cerebrospinal fluid (CSF) or artificial CSF (ACSF). The composition of ACSF is similar to that of CSF, and may include sodium chloride (NaCl), potassium chloride (KCl), calcium chloride (CaCl2), sodium phosphate (NaH2PO4), magnesium sulfate (MgSO4), D-glucose, sodium bicarbonate (NaHCO3). In some embodiments, ACSF contains: 125 mM NaCl, 25 mM NaHCO3, 3 mM KCl, 1.25 mM NaH2PO4-H2O, 10 mM C6H12O6, 1 mM MgCl2, 2 mM CaCl2, pH adjusted to 7.4.4934-0525-6010.2Page 40 of 105 065715-000129WOPT

[0135] In various embodiments, inhibitors of KCTD20 include antisense oligonucleotides (ASOs) that target sense mRNA of KCTD20. Generally, ASOs are synthetic polymers in which some or all of the natural nucleotide monomers of the oligonucleotide are chemically-modified deoxynucleotides (in DNA) or ribonucleotides (in RNA). In various embodiments, ASOs contain 15 to 25 monomers, or 15 to 25 linked nucleosides. In antisense technology, single-stranded DNA or RNA molecules are used to target a specific sense mRNA (or pre-mRNA), ultimately facilitating the degradation of the RNA target. Without wishing to be bound by a particular theory, ASOs engage RNase H – an enzyme prevalent in the nucleus that binds and cleaves DNA / RNA heteroduplexes; and in other antisense mechanisms, ASOs engage RISC to mediate degradation of the target mRNA, or engage initiation complex to exert steric blocking and block the translation of the target mRNA. Cellular RNase H1 requires the heteroduplex to contain a segment of unmodified (with either native or phosphorothiate linkages) DNA that is ≥6 bases.

[0136] Exemplary ASOs that target human KCTD20 gene (e.g., targeting mRNA of KCTD20), specifically the 3’UTR of human KCTD20 gene, are shown in Table 1. Some embodiments provide that the ASO sequences are modified for increased stability, increased binding affinity, and / or lower toxicity, compared to unmodified oligonucleotides of an identical nucleotide monomer sequence with unmodified internucleoside bonds. In various embodiments, the ASOs that target KCTD20 are modified with (or to be) one or more of: (1) a phosphorothioate (PS) bond, which is added between bases to provide nuclease resistance and represented with an asterisk “*” between the bases, (2) an additional 2’-O-Methyl RNA (2’OMe, or OMe), creating a “gapmer” ASO and reducing toxicity, which is represented with a lower case “m” in front of each modified base, (3) an additional 2’-O-methoxyethyl RNA (MOE), which is represented with upper case “ / i2MOEr / ” in front of each internal modified base, and “ / 52MOEr / ” and “ / 32MOEr / ” at the 5’ and 3’ end respectively, (4) a locked nucleic acid (LNA), (5) phosphorodiamidate morpholino (PMO), (6) N3’^P5’ phosphoramidate (NP), (7) 2’-C-allyl uridine, (8) 2’-fluoro nucleotide, and (9) 2’-amino nucleotide. Without wishing to be bound by a particular theory, in ASOs with phosphorothioate as an internucleoside bond, one of the non-bridging oxygen atoms is replaced by sulfur, conferring resistance against nucleolytic degradation. RNA derivatives with methyl or a methoxyethyl group at the 2’-position of the ribose confer high nuclease resistance with reduced toxicity. LNAs combine the properties of nuclease resistance and a hybridization affinity towards complementary oligonucleotides. PMOs are resistant to nucleases and do not activate RNase H. NPs exhibit high affinity towards a complementary RNA strand and good nuclease4934-0525-6010.2Page 41 of 105 065715-000129WOPTresistance. In further embodiments, the ASOs of this disclosure contain a segment of at least 6 bases of unmodified (with either native or phosphorothiate linkages) DNA. Further modifications are described in US Patent Application Publication No. US20230098111, which is hereby incorporated by reference in its entirety.

[0137] The 3’ UTR of human KCTD20 mRNA has a polynucleotide sequence of: GGCCAGCTGTGGGTCTACTCCTTGTTGGAGCCCATCTCACCTGGGATGCCTGCAGCCAGCCC TCCCTCGTGATTTGTCTCACCTTGAGTAGGAGACATGCTTCTCCCCTAACCTTTTCCTTTCT GCCATAATTAACATATGTCCTTTTCAGTAAGTCCATGCCTCTGGCAGGGGATGAAGAAGTAC TCACTGGTAATTAGCTACCATCTTTGCAGCAGCCCTGGTAACTTGAAAAATTTGGGTCTGGT GCTGTTCATTGAGTCTTTGTGTAACTGCAAAAGCAGGAAAGGAAGTCAAGACTCCTGTTGCC TCGTGCTTAGCAAAGCAGTCCTTATCCTTTATACTCTGTTCTTGGGTTTTGTTTTTGTCTTG TTTTATACCAGGCAAATTGCTTAGTAGCAAAGGGACCAAACTGAAAAGGTGACAATCTCTAA CTTCTAAAAGCAGACACCAATCGGATGCTCATTAGAGGTTAATGAAGATGCCATTCTTGGTG GCCTCTGCACCCAAATTGCATCTGGAAAGAACTAGGGTCTCATTCAGAATGTCCAAAAGGAA ATTCTTAAGAGCTTAAATTCAGATTTGTGTCTCATTAATGCAGTGAACAATTCAAAACCACA CAGATTCCTTGGCAGGAAGGATAATGGAATAACAGTGTTGATGAGACCTTTTTAGCTTCAAG GTTTCGGAGTCTAAACAAATGGATGATTCATTTGGAATGAAACTCACAATGCAAGTAGAAGG ACCTCTCCAAATCAGGCCAGTTGGGTTATCCTGGCTTGGAATCTGGTGTGAAACCATAGGTC TTAACACTCTGGAGCAGCACATTGCTGTGGATATGTCCAGGAGACCTTAGATATGGCTTAAA GGCTTTCAAGATGAGGACAGAAATTGCTTACAATTGCTCAGTTTCTCAACAGAAAGACTCAT AAGAGTGCCAGCATGGGGTACATGGAGTGAAGCTGGGTGGGAAGCATCATCTGCACAGTCCC TGTCCTAGTGCAGGACTTTTCTCTGTATGTTTTCATACCATGGGATTTTTGGATATCAGTGT ATTTTGGTTCTTGAAATAGCCTAATAGCTGCTCACACATTGGGTAGGAATATTATACCAATG TCATCCCCAAAGGAAGGGTGAGCTGAATGGAAATTAAGCCCAGTCATTTTATTTGATCTATT AGCTCTGTTATCAGTGCATGATCACCCAGATCACCCTCCTCAGCCCACACAGTGCTGAACCA TCTTCCCTCCTGTTCTCCATGGCTATTAATAGTATAGCTAAATTTAGAGTGCAGAGCCAGAT ATAAGTATTTTGGAATTATCTCCCAGTTTGTGGTAGAAGCTGACTGGAATACAGGTTGAGTA TCTCTTATCCAAAATGCTAGGGACCAGAAAGGTTTCAGATTTTTTCAGATTTTGGAATACTT AACAGTTGAGCACCCCAAATCTGAAAGGCTTCTGAACGTCATGTCAGCACTCAAAAAAGTGG ATTTTGGAGCACTTCAAATTTCGGATTTTTGGATTTGGGATGCTCATCCTGTGTAGGAGAGG CTACTCGATTCCATTTAATGACTGTCCTAGTCATAATCATCCAAAGATAAAAGCCAGGTAGA TGTTGAAAGCTCTTTCCAGGGCTGAAAAAGTGTTCTTACGTTCTCTGCATGTGACTAGCATC ACTGTGGAAATTAATGCTCTGTTCTTCACTAGAATGTAGTAAGTGGTTAAACTGAGCTATCC CCCACCTGATGACTATTGGCATCCATTTGCAAGGCCAATGGCCTGGATTAAGGGTTAGGATT ATTTGTAGCTAGAAGGTAATTTTATTTCTGTGAAACTAATTGGCTCATATTTGAGGTTAGGT GTGGCCTTGACCTTACCAGTACATTTATACCCACTACCAGTTGACTAGCCCAGATAATTGTT AAATGGTGCTTCTTTTCTGCTTCTCAGTAGACTTCCATGCCATTACAAAGGAAATTTGAATT ACCTAGTGTTTGTATATTCCATGATAACTATGTATAACTTCTGTTACACAGCTTATGTATTG TTAACATTTAAGTGTAAACCATGCCACAGCTAACACTTAAAAATGAAAACTAATTAGTTCTT GCTTAGGGAAAATGCCAGGTATGAAGTATGGCATATACTTGACACTGTCCTGTGTAACCCTT TACTTTGCTCAGGCTTTCAAGATTGAGTCTTTTTTCCCCCAAATTAGGTTAACATGCATTTG ACCCCAACCTGTGGGGTTTGAGTAAGCTGGAAATCTGTGACGGTAGGCTTTCTAGTGTCACG AGGTGGTGGTGACTGAAGGAAAAGCTGGGATCACAGGTTCCTTCTGATGGAGAGGAAGGTTT ATTTCTATGCCCCTCCCACCACCCTCCACCTAGAGCTCACCCAAGCCTGCTCCAGTCCCAGG GGCAGGCCATTCTGCAAAAGCAGGACCTCACAGAAACAAGGGCTGGGTTGAGGTCACCCCCT TCAGAGTTGGTTCCTGGCCAGATGGGTAAGAGGCATTTGTAATTTTAAAAATGTGAAACTTG GGTTTGGTGTTTTCTTCTAAGTGCCTAAATAAGCAAGCCAGGCTGTTGATATTTTAGCCAGA GAAATCGGCAAGCCAAGATTAACCCGAATCTGAAGTTTAGAATCTTGAGTTTGCATCTGCAT4934-0525-6010.2Page 42 of 105 065715-000129WOPTCATATCATGCTGTTTTGATGAGGAAACATTTGCCACTGAGGAGTTGGAGGGAGGGCAAGACG ACAGTGTTAAGTCAGATCATTTAATGGTTTCCCCTAAGCCCTGGAAAAATATTTGAAAGAAT GGCAGCAAAAAGGTTAAGAAAGCAAGCCAGATTTACTGCACAATATGCAGTACCCAGTACTA CTTTAAATCCCAAGAGAACAGTGTGATGTCTAATATATACAGGTCTATGAAAATACTGTGGA ATAAGCCCAGGAAGGTTAGATGTGTTTGCAAATAAGTTGCCCAAAGGGTCCCCCTCTAAGTA AAACAAATATTCAGACCACAGGCTTTAATGTAAACTGTCAAAAAGTGGGATGTGGAGGATTT TTGTTAAGTGTCAATCGAAGTTAAAAAGCAAGGGTTTTTGGCCAGGCGTGGTGGCTCACGCC TGTAATCCCAGCACTTTGGGAGGCCGAGGCCGGCAAATCACCTAAGGTCAGGAGTTCGAGAC CAGCCTGGCCAACATGGTGAAACCCCGTCTCTACTAAAAATACAAAAAAATTAGCCCGGTGT GGTGGCAAGTGCCTGTAGTCCCAGCTACTTGGGAGGCTGAGGCAGGAGAACTGCTTGAACCC GGGAGGTAGAGGTTGCAGTGAGCCACGATCATGCCACTGCACTCCAGCCTGGGCAACAGAGC AAGACTCCATCTCAAAAAAGAAAAAAAAAAATTAAAGGTTTTGGCTGGGTGCAGTGGCTCAT GCCGGTAATCCCAGCACTTTGGGAGGCCAAGGTGGGAGGATCACTTGAGGTCAGGAGTTTCA GACTGGCCTGGGCAACGTAAAAAATTAAAAAAATAGCCAGGCATGGACATGGTAGTGCATAC CTGTGGTCCCAGCCACTCAAGAGGCTGAGACAGGATGATTGCTTGAGCCCAGGAGGATGAGG CTGCAGTGAGCCATGATGACGCTACTGCACTCCAGCCTGGATGACAGGGCAAGACCCTGTCT CAATATTTTAAGTCAAGGGTTTGTAGTAATGTATTCAGTGCCACTTCTTGCCATCACTTTGC AATTATTGAAATGGGAATACTGAGCTCAGAAAGCAAATATGATGCTTTCATGGGAGATGGAG CCACATTTGTGTTCTGGGTGGGATCACTAGTGCAGGAGAACATTACATTTTCTTCTGAAGGC AAAATGCTTGTAGGTTTTGCCTCTACTTTGTATTTACTTTTAAAATTGCACTTGTTCACCTA CCAGTGTTTACGAAATCCTGTATTTGGGATGCTTTTTCTATAATAAAATATTATAATTTG (SEQ ID NO:16757).

[0138] In various embodiments, ASOs that target 3’UTR of human KCTD20 mRNA are shown as follows: Table 1. Exemplary ASOs that target 3’UTR of human KCTD20 mRNA. AS Nucleobase ASO Format SEQ O Sequence ID ID NO: 428 GCTCCAACAAGGAG mG*mC*mU*mC*mC*A*A*C*A*A*G*G*A*G*T* TAGACCmA*mG*mA*mC*mC1429 CCAGGTGAGATGGG mC*mC*mA*mG*mG*T*G*A*G*A*T*G*G*G*C* CTCCAAmU*mC*mC*mA*mA2430 TACTCAAGGTGAGA mU*mA*mC*mU*mC*A*A*G*G*T*G*A*G*A*C* CAAATCmA*mA*mA*mU*mC3431 GGAGACATGCTTCT mG*mG*mA*mG*mA*C*A*T*G*C*T*T*C*T*C* CCCCTAmC*mC*mC*mU*mA4432 TATGGCAGAAAGGA mU*mA*mU*mG*mG*C*A*G*A*A*A*G*G*A*A* AAAGGTmA*mA*mG*mG*mU5433 CCTGCCAGAGGCAT mC*mC*mU*mG*mC*C*A*G*A*G*G*C*A*T*G* GGACTTmG*mA*mC*mU*mU6434 CAGTGAGTACTTCT mC*mA*mG*mU*mG*A*G*T*A*C*T*T*C*T*T* TCATCCmC*mA*mU*mC*mC7435 GCTGCTGCAAAGAT mG*mC*mU*mG*mC*T*G*C*A*A*A*G*A*T*G* GGTAGCmG*mU*mA*mG*mC8436 TCAATGAACAGCAC mU*mC*mA*mA*mU*G*A*A*C*A*G*C*A*C*C* CAGACCmA*mG*mA*mC*mC9437 GTCTTGACTTCCTT mG*mU*mC*mU*mU*G*A*C*T*T*C*C*T*T*T* 104934-0525-6010.2Page 43 of 105 065715-000129WOPTTCCTGC mC*mC*mU*mG*mC 438 TAAGGACTGCTTTG mU*mA*mA*mG*mG*A*C*T*G*C*T*T*T*G*C* CTAAGCmU*mA*mA*mG*mC11439 CCAAGAACAGAGTA mC*mC*mA*mA*mG*A*A*C*A*G*A*G*T*A*T* TAAAGGmA*mA*mA*mG*mG12440 TGCCTGGTATAAAA mU*mG*mC*mC*mU*G*G*T*A*T*A*A*A*A*C* CAAGACmA*mA*mG*mA*mC13441 TCACCTTTTCAGTT mU*mC*mA*mC*mC*T*T*T*T*C*A*G*T*T*T* TGGTCCmG*mG*mU*mC*mC14442 GCCATTCTTGGTGG mG*mC*mC*mA*mU*T*C*T*T*G*G*T*G*G*C* CCTCTGmC*mU*mC*mU*mG15443 CACCCAAATTGCAT mC*mA*mC*mC*mC*A*A*A*T*T*G*C*A*T*C* CTGGAAmU*mG*mG*mA*mA16444 TGGACATTCTGAAT mU*mG*mG*mA*mC*A*T*T*C*T*G*A*A*T*G* GAGACCmA*mG*mA*mC*mC17445 TCACTGCATTAATG mU*mC*mA*mC*mU*G*C*A*T*T*A*A*T*G*A* AGACACmG*mA*mC*mA*mC18446 CATTATCCTTCCTG mC*mA*mU*mU*mA*T*C*C*T*T*C*C*T*G*C* CCAAGGmC*mA*mA*mG*mG19447 AAGGTCTCATCAAC mA*mA*mG*mG*mU*C*T*C*A*T*C*A*A*C*A* ACTGTTmC*mU*mG*mU*mU20448 GTCCTTCTACTTGC mG*mU*mC*mC*mU*T*C*T*A*C*T*T*G*C*A* ATTGTGmU*mU*mG*mU*mG21449 AACTGGCCTGATTT mA*mA*mC*mU*mG*G*C*C*T*G*A*T*T*T*G* GGAGAGmG*mA*mG*mA*mG22450 GATTCCAAGCCAGG mG*mA*mU*mU*mC*C*A*A*G*C*C*A*G*G*A* ATAACCmU*mA*mA*mC*mC23451 AGACCTATGGTTTC mA*mG*mA*mC*mC*T*A*T*G*G*T*T*T*C*A* ACACCAmC*mA*mC*mC*mA24452 CAATGTGCTGCTCC mC*mA*mA*mU*mG*T*G*C*T*G*C*T*C*C*A* AGAGTGmG*mA*mG*mU*mG25453 TCTCCTGGACATAT mU*mC*mU*mC*mC*T*G*G*A*C*A*T*A*T*C* CCACAGmC*mA*mC*mA*mG26454 CCTTTAAGCCATAT mC*mC*mU*mU*mU*A*A*G*C*C*A*T*A*T*C* CTAAGGmU*mA*mA*mG*mG27455 TTCTGTCCTCATCT mU*mU*mC*mU*mG*T*C*C*T*C*A*T*C*T*T* TGAAAGmG*mA*mA*mA*mG28456 GAAACTGAGCAATT mG*mA*mA*mA*mC*T*G*A*G*C*A*A*T*T*G* GTAAGCmU*mA*mA*mG*mC29457 CATGCTGGCACTCT mC*mA*mU*mG*mC*T*G*G*C*A*C*T*C*T*T* TATGAGmA*mU*mG*mA*mG30458 CCAGCTTCACTCCA mC*mC*mA*mG*mC*T*T*C*A*C*T*C*C*A*T* TGTACCmG*mU*mA*mC*mC31459 ACTGTGCAGATGAT mA*mC*mU*mG*mU*G*C*A*G*A*T*G*A*T*G* GCTTCCmC*mU*mU*mC*mC32460 AGTCCTGCACTAGG mA*mG*mU*mC*mC*T*G*C*A*C*T*A*G*G*A* ACAGGGmC*mA*mG*mG*mG33461 TGTGTGAGCAGCTA mU*mG*mU*mG*mU*G*A*G*C*A*G*C*T*A*T* TTAGGCmU*mA*mG*mG*mC34462 TAATTTCCATTCAG mU*mA*mA*mU*mU*T*C*C*A*T*T*C*A*G*C* 354934-0525-6010.2Page 44 of 105 065715-000129WOPTCTCACC mU*mC*mA*mC*mC 463 ATCTGGGTGATCAT mA*mU*mC*mU*mG*G*G*T*G*A*T*C*A*T*G* GCACTGmC*mA*mC*mU*mG36464 GAAGATGGTTCAGC mG*mA*mA*mG*mA*T*G*G*T*T*C*A*G*C*A* ACTGTGmC*mU*mG*mU*mG37465 TAGCCATGGAGAAC mU*mA*mG*mC*mC*A*T*G*G*A*G*A*A*C*A* AGGAGGmG*mG*mA*mG*mG38466 TTATATCTGGCTCT mU*mU*mA*mU*mA*T*C*T*G*G*C*T*C*T*G* GCACTCmC*mA*mC*mU*mC39467 TCAGCTTCTACCAC mU*mC*mA*mG*mC*T*T*C*T*A*C*C*A*C*A* AAACTGmA*mA*mC*mU*mG40468 ATACTCAACCTGTA mA*mU*mA*mC*mU*C*A*A*C*C*T*G*T*A*T* TTCCAGmU*mC*mC*mA*mG41469 CCTAGCATTTTGGA mC*mC*mU*mA*mG*C*A*T*T*T*T*G*G*A*T* TAAGAGmA*mA*mG*mA*mG42470 AATCTGAAACCTTT mA*mA*mU*mC*mU*G*A*A*A*C*C*T*T*T*C* CTGGTCmU*mG*mG*mU*mC43471 AGATTTGGGGTGCT mA*mG*mA*mU*mU*T*G*G*G*G*T*G*C*T*C* CAACTGmA*mA*mC*mU*mG44472 CCTACACAGGATGA mC*mC*mU*mA*mC*A*C*A*G*G*A*T*G*A*G* GCATCCmC*mA*mU*mC*mC45473 CTAGGACAGTCATT mC*mU*mA*mG*mG*A*C*A*G*T*C*A*T*T*A* AAATGGmA*mA*mU*mG*mG46474 GCTTTCAACATCTA mG*mC*mU*mU*mU*C*A*A*C*A*T*C*T*A*C* CCTGGCmC*mU*mG*mG*mC47475 GTAAGAACACTTTT mG*mU*mA*mA*mG*A*A*C*A*C*T*T*T*T*T* TCAGCCmC*mA*mG*mC*mC48476 GCTAGTCACATGCA mG*mC*mU*mA*mG*T*C*A*C*A*T*G*C*A*G* GAGAACmA*mG*mA*mA*mC49477 ATTCTAGTGAAGAA mA*mU*mU*mC*mU*A*G*T*G*A*A*G*A*A*C* CAGAGCmA*mG*mA*mG*mC50478 GCCAATAGTCATCA mG*mC*mC*mA*mA*T*A*G*T*C*A*T*C*A*G* GGTGGGmG*mU*mG*mG*mG51479 GCCATTGGCCTTGC mG*mC*mC*mA*mU*T*G*G*C*C*T*T*G*C*A* AAATGGmA*mA*mU*mG*mG52480 TGAGCCAATTAGTT mU*mG*mA*mG*mC*C*A*A*T*T*A*G*T*T*T* TCACAGmC*mA*mC*mA*mG53481 TCAAGGCCACACCT mU*mC*mA*mA*mG*G*C*C*A*C*A*C*C*T*A* AACCTCmA*mC*mC*mU*mC54482 GCTAGTCAACTGGT mG*mC*mU*mA*mG*T*C*A*A*C*T*G*G*T*A* AGTGGGmG*mU*mG*mG*mG55483 AGAAGCAGAAAAGA mA*mG*mA*mA*mG*C*A*G*A*A*A*A*G*A*A* AGCACCmG*mC*mA*mC*mC56484 AATGGCATGGAAGT mA*mA*mU*mG*mG*C*A*T*G*G*A*A*G*T*C* CTACTGmU*mA*mC*mU*mG57485 AAGTGTTAGCTGTG mA*mA*mG*mU*mG*T*T*A*G*C*T*G*T*G*G* GCATGGmC*mA*mU*mG*mG58486 TGCCATACTTCATA mU*mG*mC*mC*mA*T*A*C*T*T*C*A*T*A*C* CCTGGCmC*mU*mG*mG*mC59487 TTACACAGGACAGT mU*mU*mA*mC*mA*C*A*G*G*A*C*A*G*T*G* 604934-0525-6010.2Page 45 of 105 065715-000129WOPTGTCAAG mU*mC*mA*mA*mG 488 TCAATCTTGAAAGC mU*mC*mA*mA*mU*C*T*T*G*A*A*A*G*C*C* CTGAGCmU*mG*mA*mG*mC61489 CACAGGTTGGGGTC mC*mA*mC*mA*mG*G*T*T*G*G*G*G*T*C*A* AAATGCmA*mA*mU*mG*mC62490 ATTTCCAGCTTACT mA*mU*mU*mU*mC*C*A*G*C*T*T*A*C*T*C* CAAACCmA*mA*mA*mC*mC63491 TTCCTTCAGTCACC mU*mU*mC*mC*mU*T*C*A*G*T*C*A*C*C*A* ACCACCmC*mC*mA*mC*mC64492 TCAGAAGGAACCTG mU*mC*mA*mG*mA*A*G*G*A*A*C*C*T*G*T* TGATCCmG*mA*mU*mC*mC65493 AGAAATAAACCTTC mA*mG*mA*mA*mA*T*A*A*A*C*C*T*T*C*C* CTCTCCmU*mC*mU*mC*mC66494 GTGAGCTCTAGGTG mG*mU*mG*mA*mG*C*T*C*T*A*G*G*T*G*G* GAGGGTmA*mG*mG*mG*mU67495 CCTGCTTTTGCAGA mC*mC*mU*mG*mC*T*T*T*T*G*C*A*G*A*A* ATGGCCmU*mG*mG*mC*mC68496 CCTTGTTTCTGTGA mC*mC*mU*mU*mG*T*T*T*C*T*G*T*G*A*G* GGTCCTmG*mU*mC*mC*mU69497 CCAGGAACCAACTC mC*mC*mA*mG*mG*A*A*C*C*A*A*C*T*C*T* TGAAGGmG*mA*mA*mG*mG70498 TGGCTTGCTTATTT mU*mG*mG*mC*mU*T*G*C*T*T*A*T*T*T*A* AGGCACmG*mG*mC*mA*mC71499 TGGCTAAAATATCA mU*mG*mG*mC*mU*A*A*A*A*T*A*T*C*A*A* ACAGCCmC*mA*mG*mC*mC72500 GATGCAGATGCAAA mG*mA*mU*mG*mC*A*G*A*T*G*C*A*A*A*C* CTCAAGmU*mC*mA*mA*mG73501 GTGGCAAATGTTTC mG*mU*mG*mG*mC*A*A*A*T*G*T*T*T*C*C* CTCATCmU*mC*mA*mU*mC74502 GTGCAGTAAATCTG mG*mU*mG*mC*mA*G*T*A*A*A*T*C*T*G*G* GCTTGCmC*mU*mU*mG*mC75503 AAAGTAGTACTGGG mA*mA*mA*mG*mU*A*G*T*A*C*T*G*G*G*T* TACTGCmA*mC*mU*mG*mC76504 ACATCACACTGTTC mA*mC*mA*mU*mC*A*C*A*C*T*G*T*T*C*T* TCTTGGmC*mU*mU*mG*mG77505 TTCCTGGGCTTATT mU*mU*mC*mC*mU*G*G*G*C*T*T*A*T*T*C* CCACAGmC*mA*mC*mA*mG78506 ACATTAAAGCCTGT mA*mC*mA*mU*mU*A*A*A*G*C*C*T*G*T*G* GGTCTGmG*mU*mC*mU*mG79507 CAAAGTGCTGGGAT mC*mA*mA*mA*mG*T*G*C*T*G*G*G*A*T*T* TACAGGmA*mC*mA*mG*mG80508 CCTGACCTTAGGTG mC*mC*mU*mG*mA*C*C*T*T*A*G*G*T*G*A* ATTTGCmU*mU*mU*mG*mC81509 AGACCAGCCTGGCC mA*mG*mA*mC*mC*A*G*C*C*T*G*G*C*C*A* AACATGmA*mC*mA*mU*mG82510 ACAGGCACTTGCCA mA*mC*mA*mG*mG*C*A*C*T*T*G*C*C*A*C* CCACACmC*mA*mC*mA*mC83511 CAGTTCTCCTGCCT mC*mA*mG*mU*mU*C*T*C*C*T*G*C*C*T*C* CAGCCTmA*mG*mC*mC*mU84512 TCACTGCAACCTCT mU*mC*mA*mC*mU*G*C*A*A*C*C*T*C*T*A* 854934-0525-6010.2Page 46 of 105 065715-000129WOPTACCTCC mC*mC*mU*mC*mC 513 TGGAGTCTTGCTCT mU*mG*mG*mA*mG*T*C*T*T*G*C*T*C*T*G* GTTGCCmU*mU*mG*mC*mC86514 CTGACCTCAAGTGA mC*mU*mG*mA*mC*C*T*C*A*A*G*T*G*A*T* TCCTCCmC*mC*mU*mC*mC87515 CCAGGCCAGTCTGA mC*mC*mA*mG*mG*C*C*A*G*T*C*T*G*A*A* AACTCCmA*mC*mU*mC*mC88516 ACCATGTCCATGCC mA*mC*mC*mA*mU*G*T*C*C*A*T*G*C*C*T* TGGCTAmG*mG*mC*mU*mA89517 TGGGACCACAGGTA mU*mG*mG*mG*mA*C*C*A*C*A*G*G*T*A*T* TGCACTmG*mC*mA*mC*mU90518 GTCTCAGCCTCTTG mG*mU*mC*mU*mC*A*G*C*C*T*C*T*T*G*A* AGTGGCmG*mU*mG*mG*mC91519 TGGGCTCAAGCAAT mU*mG*mG*mG*mC*T*C*A*A*G*C*A*A*T*C* CATCCTmA*mU*mC*mC*mU92520 TCACTGCAGCCTCA mU*mC*mA*mC*mU*G*C*A*G*C*C*T*C*A*T* TCCTCCmC*mC*mU*mC*mC93521 TCCAGGCTGGAGTG mU*mC*mC*mA*mG*G*C*T*G*G*A*G*T*G*C* CAGTAGmA*mG*mU*mA*mG94522 TATTGAGACAGGGT mU*mA*mU*mU*mG*A*G*A*C*A*G*G*G*T*C* CTTGCCmU*mU*mG*mC*mC95523 GATGGCAAGAAGTG mG*mA*mU*mG*mG*C*A*A*G*A*A*G*T*G*G* GCACTGmC*mA*mC*mU*mG96524 CTTTCTGAGCTCAG mC*mU*mU*mU*mC*T*G*A*G*C*T*C*A*G*T* TATTCCmA*mU*mU*mC*mC97525 ACAAATGTGGCTCC mA*mC*mA*mA*mA*T*G*T*G*G*C*T*C*C*A* ATCTCCmU*mC*mU*mC*mC98526 TTCTCCTGCACTAG mU*mU*mC*mU*mC*C*T*G*C*A*C*T*A*G*T* TGATCCmG*mA*mU*mC*mC99527 TACAAGCATTTTGC mU*mA*mC*mA*mA*G*C*A*T*T*T*T*G*C*C* CTTCAGmU*mU*mC*mA*mG100528 TACAAAGTAGAGGC mU*mA*mC*mA*mA*A*G*T*A*G*A*G*G*C*A* AAAACCmA*mA*mA*mC*mC101529 ACTGGTAGGTGAAC mA*mC*mU*mG*mG*T*A*G*G*T*G*A*A*C*A* AAGTGCmA*mG*mU*mG*mC1021 TCTCCTACTCAAGG mU*mC*mU*mC*mC*T*A*C*T*C*A*A*G*G*T* TGAGACmG*mA*mG*mA*mC1032 GCAGAAAGGAAAAG mG*mC*mA*mG*mA*A*A*G*G*A*A*A*A*G*G* GTTAGGmU*mU*mA*mG*mG1044 TGGACATTCTGAAT mU*mG*mG*mA*mC*A*T*T*C*T*G*A*A*T*G* GAGACCmA*mG*mA*mC*mC105* ASO ID 444 and ASO ID 4 share a same sequence.

[0139] In various embodiments, KCTD20 inhibitors comprise ASOs that target any one or more of 5’UTR, 3’UTR, exons, introns, and / or exon-spanning regions of human KCTD20 mRNA as follows: Table 2. Exemplary ASOs that target human KCTD20 mRNA. Nucleobase ASO Format SEQ ID4934-0525-6010.2Page 47 of 105 065715-000129WOPTSequence NO: GCCTATTTCTCCTGA mG*mC*mC*mU*mA*T*T*T*C*T*C*C*T*G*A GAACA*mG*mA*mA*mC*mA106GGAATGCTAGCTGTC mG*mG*mA*mA*mU*G*C*T*A*G*C*T*G*T*C AGTTT*mA*mG*mU*mU*mU107CATGCATATTCCTGT mC*mA*mU*mG*mC*A*T*A*T*T*C*C*T*G*T GTTCC*mG*mU*mU*mC*mC108GGATTTCTCTCTGAT mG*mG*mA*mU*mU*T*C*T*C*T*C*T*G*A*T CAAAC*mC*mA*mA*mA*mC109GGACAGTTCAGGACT mG*mG*mA*mC*mA*G*T*T*C*A*G*G*A*C*T CAGAA*mC*mA*mG*mA*mA110TCTAAGGATGAATGT mU*mC*mU*mA*mA*G*G*A*T*G*A*A*T*G*T TCACC*mU*mC*mA*mC*mC111GTGGCAGTGACAGTG mG*mU*mG*mG*mC*A*G*T*G*A*C*A*G*T*G ACAGG*mA*mC*mA*mG*mG112GGAGGCCAGCTGCTT mG*mG*mA*mG*mG*C*C*A*G*C*T*G*C*T*T AGTGG*mA*mG*mU*mG*mG113GATACTTTAGCTGTA mG*mA*mU*mA*mC*T*T*T*A*G*C*T*G*T*A GCCCA*mG*mC*mC*mC*mA114AGAAAAAGAAGCAAA mA*mG*mA*mA*mA*A*A*G*A*A*G*C*A*A*A CAGCC*mC*mA*mG*mC*mC115GGCTTCATCTGGTCC mG*mG*mC*mU*mU*C*A*T*C*T*G*G*T*C*C TCATA*mU*mC*mA*mU*mA116CTTACTTATCCTCTA mC*mU*mU*mA*mC*T*T*A*T*C*C*T*C*T*A GGTCC*mG*mG*mU*mC*mC117CAGGAATGAAGGTAC mC*mA*mG*mG*mA*A*T*G*A*A*G*G*T*A*C AGTGA*mA*mG*mU*mG*mA118GGCTTTCCAATACTA mG*mG*mC*mU*mU*T*C*C*A*A*T*A*C*T*A GAGCA*mG*mA*mG*mC*mA119CTACTCTACCCAGAA mC*mU*mA*mC*mU*C*T*A*C*C*C*A*G*A*A GGCAT*mG*mG*mC*mA*mU120GCTGACCTTCACAGA mG*mC*mU*mG*mA*C*C*T*T*C*A*C*A*G*A AACTC*mA*mA*mC*mU*mC121CAGCCACAGGTTTGG mC*mA*mG*mC*mC*A*C*A*G*G*T*T*T*G*G AGTCA*mA*mG*mU*mC*mA122CTAGCTCAGCTGCTT mC*mU*mA*mG*mC*T*C*A*G*C*T*G*C*T*T ATTAG*mA*mU*mU*mA*mG123GTAACCATGGATGAG mG*mU*mA*mA*mC*C*A*T*G*G*A*T*G*A*G ATCCT*mA*mU*mC*mC*mU124CAGTCTTTCTGAGCC mC*mA*mG*mU*mC*T*T*T*C*T*G*A*G*C*C CTGCT*mC*mU*mG*mC*mU125GGTCTCACTCTTCAC mG*mG*mU*mC*mU*C*A*C*T*C*T*T*C*A*C CCAGG*mC*mC*mA*mG*mG126GATCTTGGCTCACTG mG*mA*mU*mC*mU*T*G*G*C*T*C*A*C*T*G CAGCT*mC*mA*mG*mC*mU127GGTACCACCACACCC mG*mG*mU*mA*mC*C*A*C*C*A*C*A*C*C*C AGCTA*mA*mG*mC*mU*mA128GGTTTCACTATTTTG mG*mG*mU*mU*mU*C*A*C*T*A*T*T*T*T*G CCAAG*mC*mC*mA*mA*mG129GCTGGCAAGCCTCAC mG*mC*mU*mG*mG*C*A*A*G*C*C*T*C*A*C 1304934-0525-6010.2Page 48 of 105 065715-000129WOPTTTTCT *mU*mU*mU*mC*mU GCCTATAATCCCAGC mG*mC*mC*mU*mA*T*A*A*T*C*C*C*A*G*C ACTTT*mA*mC*mU*mU*mU131GGCCAAAGCAGGTGG mG*mG*mC*mC*mA*A*A*G*C*A*G*G*T*G*G ATCAC*mA*mU*mC*mA*mC132GAGGTCAGGACTTCA mG*mA*mG*mG*mU*C*A*G*G*A*C*T*T*C*A AGACC*mA*mG*mA*mC*mC133GAAACCCCCATCTCT mG*mA*mA*mA*mC*C*C*C*C*A*T*C*T*C*T ACTAA*mA*mC*mU*mA*mA134GTGGTGGCAGATGCC mG*mU*mG*mG*mU*G*G*C*A*G*A*T*G*C*C TGTAA*mU*mG*mU*mA*mA135GAGTATGAGGCACAA mG*mA*mG*mU*mA*T*G*A*G*G*C*A*C*A*A GAATC*mG*mA*mA*mU*mC136GGAGGTTGCAGTGAG mG*mG*mA*mG*mG*T*T*G*C*A*G*T*G*A*G CCAAG*mC*mC*mA*mA*mG137GGAGACTGTGTCTCA mG*mG*mA*mG*mA*C*T*G*T*G*T*C*T*C*A AAAAA*mA*mA*mA*mA*mA138GCACCAATAATAGCA mG*mC*mA*mC*mC*A*A*T*A*A*T*A*G*C*A ACTAC*mA*mC*mU*mA*mC139GAACATTCACTGTAC mG*mA*mA*mC*mA*T*T*C*A*C*T*G*T*A*C TAGGA*mU*mA*mG*mG*mA140GGTGGCAAACTGGTC mG*mG*mU*mG*mG*C*A*A*A*C*T*G*G*T*C AAATC*mA*mA*mA*mU*mC141GCCATCATGTCCATA mG*mC*mC*mA*mU*C*A*T*G*T*C*C*A*T*A TTGTC*mU*mU*mG*mU*mC142GGCTGCCTTCCTGCT mG*mG*mC*mU*mG*C*C*T*T*C*C*T*G*C*T ACAGT*mA*mC*mA*mG*mU143GTGGCAGAGACTGAC mG*mU*mG*mG*mC*A*G*A*G*A*C*T*G*A*C CACAA*mC*mA*mC*mA*mA144GACTCTACTCCAGAT mG*mA*mC*mU*mC*T*A*C*T*C*C*A*G*A*T TAGTC*mU*mA*mG*mU*mC145CAGATGAGGAAACTG mC*mA*mG*mA*mU*G*A*G*G*A*A*A*C*T*G AGGCT*mA*mG*mG*mC*mU146GAAAGCTTAGGTAAC mG*mA*mA*mA*mG*C*T*T*A*G*G*T*A*A*C TTGCT*mU*mU*mG*mC*mU147CAGGTTCACATTGCT mC*mA*mG*mG*mU*T*C*A*C*A*T*T*G*C*T AGGAG*mA*mG*mG*mA*mG148GTGAGCTGAGATGAT mG*mU*mG*mA*mG*C*T*G*A*G*A*T*G*A*T TTCTA*mU*mU*mC*mU*mA149GCTGAAGAGTACACC mG*mC*mU*mG*mA*A*G*A*G*T*A*C*A*C*C ATTAG*mA*mU*mU*mA*mG150GGAGAACTTGTGCAA mG*mG*mA*mG*mA*A*C*T*T*G*T*G*C*A*A GGAGT*mG*mG*mA*mG*mU151GCCCATTTAAGCAGG mG*mC*mC*mC*mA*T*T*T*A*A*G*C*A*G*G ATAGA*mA*mU*mA*mG*mA152ACCTGAGGTGTCTTC mA*mC*mC*mU*mG*A*G*G*T*G*T*C*T*T*C TTTGG*mU*mU*mU*mG*mG153GCTATTCTCCTGCCT mG*mC*mU*mA*mU*T*C*T*C*C*T*G*C*C*T CAGCC*mC*mA*mG*mC*mC154ACTACAGGCACCTGC mA*mC*mU*mA*mC*A*G*G*C*A*C*C*T*G*C 1554934-0525-6010.2Page 49 of 105 065715-000129WOPTCACCA *mC*mA*mC*mC*mA GGTTTCACTGTGTTA mG*mG*mU*mU*mU*C*A*C*T*G*T*G*T*T*A GCCAG*mG*mC*mC*mA*mG156GATCTCCTGACCTCA mG*mA*mU*mC*mU*C*C*T*G*A*C*C*T*C*A TGATC*mU*mG*mA*mU*mC157GTGTGAAAGCCTATC mG*mU*mG*mU*mG*A*A*A*G*C*C*T*A*T*C CTGGT*mC*mU*mG*mG*mU158GTTACCTGGTGATTG mG*mU*mU*mA*mC*C*T*G*G*T*G*A*T*T*G CCTGT*mC*mC*mU*mG*mU159GTAGCTTTTGGTGTG mG*mU*mA*mG*mC*T*T*T*T*G*G*T*G*T*G CAAAC*mC*mA*mA*mA*mC160CTAAGTGTCCTCTAC mC*mU*mA*mA*mG*T*G*T*C*C*T*C*T*A*C CCAAC*mC*mC*mA*mA*mC161GGTTGTCTGAGTGTA mG*mG*mU*mU*mG*T*C*T*G*A*G*T*G*T*A GGAAA*mG*mG*mA*mA*mA162GGCATTAATCATGTT mG*mG*mC*mA*mU*T*A*A*T*C*A*T*G*T*T CCTGA*mC*mC*mU*mG*mA163CTTGCCAAATGTATG mC*mU*mU*mG*mC*C*A*A*A*T*G*T*A*T*G TAGGT*mU*mA*mG*mG*mU164GTGCTTCCCTATGTT mG*mU*mG*mC*mU*T*C*C*C*T*A*T*G*T*T TGGAA*mU*mG*mG*mA*mA165TGTGGTGGCTCACAC mU*mG*mU*mG*mG*T*G*G*C*T*C*A*C*A*C CTGTA*mC*mU*mG*mU*mA166GGATCACTTGAGGAG mG*mG*mA*mU*mC*A*C*T*T*G*A*G*G*A*G ACTGC*mA*mC*mU*mG*mC167AAGAACCCTTTGGTA mA*mA*mG*mA*mA*C*C*C*T*T*T*G*G*T*A ATCCT*mA*mU*mC*mC*mU168GAGACAGAGTCTTGC mG*mA*mG*mA*mC*A*G*A*G*T*C*T*T*G*C TCTGT*mU*mC*mU*mG*mU169TAGGCTGGAGTGCAG mU*mA*mG*mG*mC*T*G*G*A*G*T*G*C*A*G TGGTA*mU*mG*mG*mU*mA170CAATCTTGGCTTGCT mC*mA*mA*mU*mC*T*T*G*G*C*T*T*G*C*T GCAAC*mG*mC*mA*mA*mC171TCCACCTCCCAGGTT mU*mC*mC*mA*mC*C*T*C*C*C*A*G*G*T*T CCAGA*mC*mC*mA*mG*mA172GATTCTCCTGCCTCA mG*mA*mU*mU*mC*T*C*C*T*G*C*C*T*C*A GCCTC*mG*mC*mC*mU*mC173CTGATAGCTGGAATT mC*mU*mG*mA*mU*A*G*C*T*G*G*A*A*T*T ACAGG*mA*mC*mA*mG*mG174ACCACCACTTCTGGC mA*mC*mC*mA*mC*C*A*C*T*T*C*T*G*G*C TAATT*mU*mA*mA*mU*mU175GACAGAGTCTTGCTC mG*mA*mC*mA*mG*A*G*T*C*T*T*G*C*T*C TGTCA*mU*mG*mU*mC*mA176GGTTCACACCATTCA mG*mG*mU*mU*mC*A*C*A*C*C*A*T*T*C*A CCTGC*mC*mC*mU*mG*mC177CTCAGCCTCCTGAGT mC*mU*mC*mA*mG*C*C*T*C*C*T*G*A*G*T AGCTG*mA*mG*mC*mU*mG178GCCACCACACTCAGC mG*mC*mC*mA*mC*C*A*C*A*C*T*C*A*G*C TAATT*mU*mA*mA*mU*mU179GGCTTCACCATGTTA mG*mG*mC*mU*mU*C*A*C*C*A*T*G*T*T*A 1804934-0525-6010.2Page 50 of 105 065715-000129WOPTGCCAG *mG*mC*mC*mA*mG CCTCAGATGATCTGC mC*mC*mU*mC*mA*G*A*T*G*A*T*C*T*G*C CCACC*mC*mC*mA*mC*mC181GTGAAACAGCCCTGT mG*mU*mG*mA*mA*A*C*A*G*C*C*C*T*G*T GAGGT*mG*mA*mG*mG*mU182CCTCAATATAGATGA mC*mC*mU*mC*mA*A*T*A*T*A*G*A*T*G*A TGGAC*mU*mG*mG*mA*mC183GGTGGCTCATGCCTG mG*mG*mU*mG*mG*C*T*C*A*T*G*C*C*T*G TAATC*mU*mA*mA*mU*mC184GAGATCAGGAGATAG mG*mA*mG*mA*mU*C*A*G*G*A*G*A*T*A*G AGACC*mA*mG*mA*mC*mC185GATGAAACCTTGTCT mG*mA*mU*mG*mA*A*A*C*C*T*T*G*T*C*T CTACT*mC*mU*mA*mC*mU186CTCCTGTAGTCCCAG mC*mU*mC*mC*mU*G*T*A*G*T*C*C*C*A*G CTGCT*mC*mU*mG*mC*mU187GGAGGCTGAGGCAGG mG*mG*mA*mG*mG*C*T*G*A*G*G*C*A*G*G AGAAT*mA*mG*mA*mA*mU188GGAGCTTGCAGTGAG mG*mG*mA*mG*mC*T*T*G*C*A*G*T*G*A*G CTGAG*mC*mU*mG*mA*mG189GACAGTGTGAGACTC mG*mA*mC*mA*mG*T*G*T*G*A*G*A*C*T*C TATCT*mU*mA*mU*mC*mU190CATGAATGAATCCTA mC*mA*mU*mG*mA*A*T*G*A*A*T*C*C*T*A ATGCC*mA*mU*mG*mC*mC191GCACTGGAACTTTTC mG*mC*mA*mC*mU*G*G*A*A*C*T*T*T*T*C TGCTG*mU*mG*mC*mU*mG192GGTGATGTTAGGATT mG*mG*mU*mG*mA*T*G*T*T*A*G*G*A*T*T CTCCT*mC*mU*mC*mC*mU193GGTACATGTGCACAT mG*mG*mU*mA*mC*A*T*G*T*G*C*A*C*A*T TGTGC*mU*mG*mU*mG*mC194GCTGCACCCACTAAC mG*mC*mU*mG*mC*A*C*C*C*A*C*T*A*A*C TCATC*mU*mC*mA*mU*mC195CTAGCATTAGCTATA mC*mU*mA*mG*mC*A*T*T*A*G*C*T*A*T*A TCTCC*mU*mC*mU*mC*mC196ACAACAGTCACCAGA mA*mC*mA*mA*mC*A*G*T*C*A*C*C*A*G*A GTGTA*mG*mU*mG*mU*mA197GGCACATATTAGTAG mG*mG*mC*mA*mC*A*T*A*T*T*A*G*T*A*G CAGGT*mC*mA*mG*mG*mU198GTGATTGCCTCTGAA mG*mU*mG*mA*mU*T*G*C*C*T*C*T*G*A*A CCAAC*mC*mC*mA*mA*mC199GGCCTCCACATAAAT mG*mG*mC*mC*mU*C*C*A*C*A*T*A*A*A*T TCTTC*mU*mC*mU*mU*mC200GCATCCTGCCTTCAT mG*mC*mA*mU*mC*C*T*G*C*C*T*T*C*A*T CCTCT*mC*mC*mU*mC*mU201CACATGTACATGGAA mC*mA*mC*mA*mU*G*T*A*C*A*T*G*G*A*A GAGAT*mG*mA*mG*mA*mU202GCTTGCTTTTTACTT mG*mC*mU*mU*mG*C*T*T*T*T*T*A*C*T*T GCTGT*mG*mC*mU*mG*mU203GGCTTTTGCTCTCAA mG*mG*mC*mU*mU*T*T*G*C*T*C*T*C*A*A GTTGT*mG*mU*mU*mG*mU204GACCTCTCACTTGAC mG*mA*mC*mC*mU*C*T*C*A*C*T*T*G*A*C 2054934-0525-6010.2Page 51 of 105 065715-000129WOPTTATGC *mU*mA*mU*mG*mC CTCTCAGCCAGCAAA mC*mU*mC*mU*mC*A*G*C*C*A*G*C*A*A*A TCTTC*mU*mC*mU*mU*mC206GTTCCCTCACATAAT mG*mU*mU*mC*mC*C*T*C*A*C*A*T*A*A*T GCCCC*mG*mC*mC*mC*mC207GCTGAAGACATCAAA mG*mC*mU*mG*mA*A*G*A*C*A*T*C*A*A*A GGTTC*mG*mG*mU*mU*mC208GCTCCAGAAAGATTT mG*mC*mU*mC*mC*A*G*A*A*A*G*A*T*T*T GGAAA*mG*mG*mA*mA*mA209GGCTTTGGCAGTAAT mG*mG*mC*mU*mU*T*G*G*C*A*G*T*A*A*T TCCCA*mU*mC*mC*mC*mA210CCCAAGCACCAGAGA mC*mC*mC*mA*mA*G*C*A*C*C*A*G*A*G*A AAGTG*mA*mA*mG*mU*mG211GCTTCTTGTAGATGG mG*mC*mU*mU*mC*T*T*G*T*A*G*A*T*G*G CACAC*mC*mA*mC*mA*mC212GTTTTGTTGTGAATC mG*mU*mU*mU*mU*G*T*T*G*T*G*A*A*T*C CACAG*mC*mA*mC*mA*mG213CCTGCTGTTTATCTT mC*mC*mU*mG*mC*T*G*T*T*T*A*T*C*T*T GCATT*mG*mC*mA*mU*mU214GTCTGTAATCCCAGC mG*mU*mC*mU*mG*T*A*A*T*C*C*C*A*G*C ACTTT*mA*mC*mU*mU*mU215GAGGCAGGCAGATCA mG*mA*mG*mG*mC*A*G*G*C*A*G*A*T*C*A CCTGA*mC*mC*mU*mG*mA216GGTCAGGAGTTTGAG mG*mG*mU*mC*mA*G*G*A*G*T*T*T*G*A*G ACCAG*mA*mC*mC*mA*mG217CCTGGCCAACATGGT mC*mC*mU*mG*mG*C*C*A*A*C*A*T*G*G*T GAAAC*mG*mA*mA*mA*mC218GCACCTATAATCCCA mG*mC*mA*mC*mC*T*A*T*A*A*T*C*C*C*A GCTGC*mG*mC*mU*mG*mC219GGAGGCTGAGGCAGG mG*mG*mA*mG*mG*C*T*G*A*G*G*C*A*G*G AGAAT*mA*mG*mA*mA*mU220GCCTGAACCTGGAAG mG*mC*mC*mU*mG*A*A*C*C*T*G*G*A*A*G GTGGA*mG*mU*mG*mG*mA221GAGACTGCACCATTG mG*mA*mG*mA*mC*T*G*C*A*C*C*A*T*T*G CACTC*mC*mA*mC*mU*mC222CTGCCCTCATACATT mC*mU*mG*mC*mC*C*T*C*A*T*A*C*A*T*T TGTGA*mU*mG*mU*mG*mA223GGCAAGCTGGTAAAA mG*mG*mC*mA*mA*G*C*T*G*G*T*A*A*A*A GCCTA*mG*mC*mC*mU*mA224GCCAGTATAGTAAAA mG*mC*mC*mA*mG*T*A*T*A*G*T*A*A*A*A TGAGC*mU*mG*mA*mG*mC225CCTGCTAAATCTCAT mC*mC*mU*mG*mC*T*A*A*A*T*C*T*C*A*T TCTCT*mU*mC*mU*mC*mU226GCTCCTCTACAAATT mG*mC*mU*mC*mC*T*C*T*A*C*A*A*A*T*T CACTC*mC*mA*mC*mU*mC227GGTAAGGTGAGACCT mG*mG*mU*mA*mA*G*G*T*G*A*G*A*C*C*T AAATC*mA*mA*mA*mU*mC228CTGCATGGCACAAGA mC*mU*mG*mC*mA*T*G*G*C*A*C*A*A*G*A ACAAA*mA*mC*mA*mA*mA229CCCTGCCATCTATTG mC*mC*mC*mU*mG*C*C*A*T*C*T*A*T*T*G 2304934-0525-6010.2Page 52 of 105 065715-000129WOPTCAAGT *mC*mA*mA*mG*mU GCTTGGCAACAGATG mG*mC*mU*mU*mG*G*C*A*A*C*A*G*A*T*G CTATA*mC*mU*mA*mU*mA231GCCTTCCATTACTAT mG*mC*mC*mU*mU*C*C*A*T*T*A*C*T*A*T TCAAG*mU*mC*mA*mA*mG232GGTCCATTCTCTGTG mG*mG*mU*mC*mC*A*T*T*C*T*C*T*G*T*G CTGCT*mC*mU*mG*mC*mU233GTCTCTGGCCCCCAG mG*mU*mC*mU*mC*T*G*G*C*C*C*C*C*A*G AGTTT*mA*mG*mU*mU*mU234GGTTAGAGTTTAGGA mG*mG*mU*mU*mA*G*A*G*T*T*T*A*G*G*A AATGG*mA*mA*mU*mG*mG235GCCTACTAAGGAGGA mG*mC*mC*mU*mA*C*T*A*A*G*G*A*G*G*A ACACT*mA*mC*mA*mC*mU236GTCCAGGAAGGAGTC mG*mU*mC*mC*mA*G*G*A*A*G*G*A*G*T*C TCAGA*mU*mC*mA*mG*mA237GGCTAGGAGTAAAGA mG*mG*mC*mU*mA*G*G*A*G*T*A*A*A*G*A GATTG*mG*mA*mU*mU*mG238CTTTCTGGCCAACCA mC*mU*mU*mU*mC*T*G*G*C*C*A*A*C*C*A TAGAC*mU*mA*mG*mA*mC239CTTAACCTAAGGAAG mC*mU*mU*mA*mA*C*C*T*A*A*G*G*A*A*G AGCAA*mA*mG*mC*mA*mA240GAGATCTAACTCAGG mG*mA*mG*mA*mU*C*T*A*A*C*T*C*A*G*G ACTTT*mA*mC*mU*mU*mU241CTAGGCTAAATAGAA mC*mU*mA*mG*mG*C*T*A*A*A*T*A*G*A*A TCCAG*mU*mC*mC*mA*mG242GACAGAGTCTCACTC mG*mA*mC*mA*mG*A*G*T*C*T*C*A*C*T*C TGTCA*mU*mG*mU*mC*mA243GTGCCTCAGCCTCCC mG*mU*mG*mC*mC*T*C*A*G*C*C*T*C*C*C AAGTA*mA*mA*mG*mU*mA244GGATCACAGGTGTGT mG*mG*mA*mU*mC*A*C*A*G*G*T*G*T*G*T GCCAC*mG*mC*mC*mA*mC245GTTGGTCAGGCTGGT mG*mU*mU*mG*mG*T*C*A*G*G*C*T*G*G*T CTTGA*mC*mU*mU*mG*mA246GCCCACCTTAGCCTC mG*mC*mC*mC*mA*C*C*T*T*A*G*C*C*T*C CCAAA*mC*mC*mA*mA*mA247GGATTACAGGCATGA mG*mG*mA*mU*mU*A*C*A*G*G*C*A*T*G*A GCCAC*mG*mC*mC*mA*mC248CCAGACTGGCCCTCT mC*mC*mA*mG*mA*C*T*G*G*C*C*C*T*C*T ATTTT*mA*mU*mU*mU*mU249TCAGGTTTGACTTCC mU*mC*mA*mG*mG*T*T*T*G*A*C*T*T*C*C TGTTC*mU*mG*mU*mU*mC250GGTATTACCATCATT mG*mG*mU*mA*mU*T*A*C*C*A*T*C*A*T*T CCCAC*mC*mC*mC*mA*mC251CCCTGAAAAAAGTGT mC*mC*mC*mU*mG*A*A*A*A*A*A*G*T*G*T TTCTC*mU*mU*mC*mU*mC252GTGCAGGCTGGCTGT mG*mU*mG*mC*mA*G*G*C*T*G*G*C*T*G*T TTTAG*mU*mU*mU*mA*mG253GTATGTTGCCTCTGA mG*mU*mA*mU*mG*T*T*G*C*C*T*C*T*G*A AAGTA*mA*mA*mG*mU*mA254GTGATCAAGAGAAGA mG*mU*mG*mA*mU*C*A*A*G*A*G*A*A*G*A 2554934-0525-6010.2Page 53 of 105 065715-000129WOPTAAGGC *mA*mA*mG*mG*mC GGTGACACTGGTTTT mG*mG*mU*mG*mA*C*A*C*T*G*G*T*T*T*T TTGAA*mU*mU*mG*mA*mA256GGTTGTCCCATGAAG mG*mG*mU*mU*mG*T*C*C*C*A*T*G*A*A*G AGGAG*mA*mG*mG*mA*mG257CTGGTTCTTTATCTG mC*mU*mG*mG*mU*T*C*T*T*T*A*T*C*T*G TATGG*mU*mA*mU*mG*mG258CATTCAGCTGAATTG mC*mA*mU*mU*mC*A*G*C*T*G*A*A*T*T*G GCAAG*mG*mC*mA*mA*mG259CTGTTCTGTGGCTAC mC*mU*mG*mU*mU*C*T*G*T*G*G*C*T*A*C CTGTC*mC*mU*mG*mU*mC260GTAAGCCTAGCCAGT mG*mU*mA*mA*mG*C*C*T*A*G*C*C*A*G*T GAGCT*mG*mA*mG*mC*mU261GGAGAAGCTGAGAGA mG*mG*mA*mG*mA*A*G*C*T*G*A*G*A*G*A AAGAG*mA*mA*mG*mA*mG262GGATTAATGCAGATC mG*mG*mA*mU*mU*A*A*T*G*C*A*G*A*T*C CATGC*mC*mA*mU*mG*mC263CTCTGAGCTGAAGTC mC*mU*mC*mU*mG*A*G*C*T*G*A*A*G*T*C CAGCT*mC*mA*mG*mC*mU264CAGAGCTCTCTGAGC mC*mA*mG*mA*mG*C*T*C*T*C*T*G*A*G*C TAGAC*mU*mA*mG*mA*mC265CCAGAGCATTCCAGA mC*mC*mA*mG*mA*G*C*A*T*T*C*C*A*G*A AAAAC*mA*mA*mA*mA*mC266CTGCATTCTTTGGAG mC*mU*mG*mC*mA*T*T*C*T*T*T*G*G*A*G GCTTT*mG*mC*mU*mU*mU267TGTGTGGTACAGTTA mU*mG*mU*mG*mU*G*G*T*A*C*A*G*T*T*A TTTGG*mU*mU*mU*mG*mG268GATCTCAGCTCACTG mG*mA*mU*mC*mU*C*A*G*C*T*C*A*C*T*G CAAGC*mC*mA*mA*mG*mC269GGTTCAGCCATTCTC mG*mG*mU*mU*mC*A*G*C*C*A*T*T*C*T*C CTGCC*mC*mU*mG*mC*mC270GGTTTCACCATTCTA mG*mG*mU*mU*mU*C*A*C*C*A*T*T*C*T*A GCCAG*mG*mC*mC*mA*mG271GGTCTCTATCTCCTG mG*mG*mU*mC*mU*C*T*A*T*C*T*C*C*T*G ACCTC*mA*mC*mC*mU*mC272CCTTTGAAAAGCAGC mC*mC*mU*mU*mU*G*A*A*A*A*G*C*A*G*C CAGTT*mC*mA*mG*mU*mU273GCCTGTAATCCCAGC mG*mC*mC*mU*mG*T*A*A*T*C*C*C*A*G*C ACTTT*mA*mC*mU*mU*mU274GAAACCCTGTCTCTA mG*mA*mA*mA*mC*C*C*T*G*T*C*T*C*T*A CTAAA*mC*mU*mA*mA*mA275GCCTGTAGTCCCAAC mG*mC*mC*mU*mG*T*A*G*T*C*C*C*A*A*C TGCTG*mU*mG*mC*mU*mG276GGAGGCTAAGGCAGG mG*mG*mA*mG*mG*C*T*A*A*G*G*C*A*G*G AGAAT*mA*mG*mA*mA*mU277GGAGGACAACAGTTG mG*mG*mA*mG*mG*A*C*A*A*C*A*G*T*T*G TGAAC*mU*mG*mA*mA*mC278GCTGAACAGGCAACA mG*mC*mU*mG*mA*A*C*A*G*G*C*A*A*C*A CTTCC*mC*mU*mU*mC*mC279CAGCAGTTTAAGGAA mC*mA*mG*mC*mA*G*T*T*T*A*A*G*G*A*A 2804934-0525-6010.2Page 54 of 105 065715-000129WOPTGGAAA *mG*mG*mA*mA*mA GGCAGTTAACCCCCA mG*mG*mC*mA*mG*T*T*A*A*C*C*C*C*C*A TTTGA*mU*mU*mU*mG*mA281GGAAGTCAAATCTTA mG*mG*mA*mA*mG*T*C*A*A*A*T*C*T*T*A GGCTG*mG*mG*mC*mU*mG282GTTGAAGATAGCAGC mG*mU*mU*mG*mA*A*G*A*T*A*G*C*A*G*C TGCCT*mU*mG*mC*mC*mU283GCCCTATCCTTTGTG mG*mC*mC*mC*mU*A*T*C*C*T*T*T*G*T*G TTATC*mU*mU*mA*mU*mC284CCATTCCACAGCTCA mC*mC*mA*mU*mU*C*C*A*C*A*G*C*T*C*A TACAC*mU*mA*mC*mA*mC285GTGCAGCAGGAGTGG mG*mU*mG*mC*mA*G*C*A*G*G*A*G*T*G*G AAGTC*mA*mA*mG*mU*mC286GGAGAGGCAAATAAG mG*mG*mA*mG*mA*G*G*C*A*A*A*T*A*A*G AAAAG*mA*mA*mA*mA*mG287CTCTTCTCCCTGTAC mC*mU*mC*mU*mU*C*T*C*C*C*T*G*T*A*C CCTCA*mC*mC*mU*mC*mA288GACTTCTGAGTTCAT mG*mA*mC*mU*mU*C*T*G*A*G*T*T*C*A*T GCTGA*mG*mC*mU*mG*mA289CAGCAAGCCTGTCTC mC*mA*mG*mC*mA*A*G*C*C*T*G*T*C*T*C TCTGA*mU*mC*mU*mG*mA290GCAGTAAACTGCTGA mG*mC*mA*mG*mU*A*A*A*C*T*G*C*T*G*A TAAGC*mU*mA*mA*mG*mC291CTAATACCTACTACC mC*mU*mA*mA*mU*A*C*C*T*A*C*T*A*C*C CTCCT*mC*mU*mC*mC*mU292CTACTTGATCTGAAA mC*mU*mA*mC*mU*T*G*A*T*C*T*G*A*A*A ATCCC*mA*mU*mC*mC*mC293GAGTAAGGCCATTTT mG*mA*mG*mU*mA*A*G*G*C*C*A*T*T*T*T TCCCC*mU*mC*mC*mC*mC294CATAGCTGCATCTAA mC*mA*mU*mA*mG*C*T*G*C*A*T*C*T*A*A CAGAA*mC*mA*mG*mA*mA295GGCATATTAGATAGG mG*mG*mC*mA*mU*A*T*T*A*G*A*T*A*G*G TCAGT*mU*mC*mA*mG*mU296GGACAGGAATAAAAA mG*mG*mA*mC*mA*G*G*A*A*T*A*A*A*A*A GGAGC*mG*mG*mA*mG*mC297CTAGATGCTGTCTTT mC*mU*mA*mG*mA*T*G*C*T*G*T*C*T*T*T CTGAG*mC*mU*mG*mA*mG298CTGTCAGTATAGAAA mC*mU*mG*mU*mC*A*G*T*A*T*A*G*A*A*A AGCAG*mA*mG*mC*mA*mG299GGCATGAAAGGCCAC mG*mG*mC*mA*mU*G*A*A*A*G*G*C*C*A*C CATCT*mC*mA*mU*mC*mU300GGCCACCTCTGCAGT mG*mG*mC*mC*mA*C*C*T*C*T*G*C*A*G*T TGCTA*mU*mG*mC*mU*mA301CTGCATTTACTGCCA mC*mU*mG*mC*mA*T*T*T*A*C*T*G*C*C*A AAGAA*mA*mA*mG*mA*mA302CTCACTACAGATGAA mC*mU*mC*mA*mC*T*A*C*A*G*A*T*G*A*A CTTTG*mC*mU*mU*mU*mG303GGACTTGATCCTTCA mG*mG*mA*mC*mU*T*G*A*T*C*C*T*T*C*A TTCTT*mU*mU*mC*mU*mU304GCCTTTTTTTCCTGT mG*mC*mC*mU*mU*T*T*T*T*T*C*C*T*G*T 3054934-0525-6010.2Page 55 of 105 065715-000129WOPTCTGAA *mC*mU*mG*mA*mA GGAGCTTCCTAAGAC mG*mG*mA*mG*mC*T*T*C*C*T*A*A*G*A*C TTGAG*mU*mU*mG*mA*mG306GGTTCCAGTGCTGTG mG*mG*mU*mU*mC*C*A*G*T*G*C*T*G*T*G TTTTT*mU*mU*mU*mU*mU307GGATGGAGTCTTCCA mG*mG*mA*mU*mG*G*A*G*T*C*T*T*C*C*A GAATG*mG*mA*mA*mU*mG308GGAGAAATCTCATCT mG*mG*mA*mG*mA*A*A*T*C*T*C*A*T*C*T GTGAA*mG*mU*mG*mA*mA309GCCTGGATATCTTTC mG*mC*mC*mU*mG*G*A*T*A*T*C*T*T*T*C AGGAT*mA*mG*mG*mA*mU310GGACCAGGAAGAGAG mG*mG*mA*mC*mC*A*G*G*A*A*G*A*G*A*G TACAA*mU*mA*mC*mA*mA311GAGAGTATGAGATTG mG*mA*mG*mA*mG*T*A*T*G*A*G*A*T*T*G CTGAA*mC*mU*mG*mA*mA312GGCATCAGTGCAACT mG*mG*mC*mA*mU*C*A*G*T*G*C*A*A*C*T GTATT*mG*mU*mA*mU*mU313GCACAGTGCTGGTGT mG*mC*mA*mC*mA*G*T*G*C*T*G*G*T*G*T GTGGT*mG*mU*mG*mG*mU314GGCATGTGTGATCAA mG*mG*mC*mA*mU*G*T*G*T*G*A*T*C*A*A TAGCC*mU*mA*mG*mC*mC315GAGAGTTCCATCAAC mG*mA*mG*mA*mG*T*T*C*C*A*T*C*A*A*C TATGT*mU*mA*mU*mG*mU316GGTCTCTGGTTTCCA mG*mG*mU*mC*mU*C*T*G*G*T*T*T*C*C*A GTTGT*mG*mU*mU*mG*mU317CCTTTCTCCCTTTGT mC*mC*mU*mU*mU*C*T*C*C*C*T*T*T*G*T TTTCT*mU*mU*mU*mC*mU318GTCCTTGCTTGTTTG mG*mU*mC*mC*mU*T*G*C*T*T*G*T*T*T*G TATGT*mU*mA*mU*mG*mU319GAGCTCTATCAGTTA mG*mA*mG*mC*mU*C*T*A*T*C*A*G*T*T*A CCAAG*mC*mC*mA*mA*mG320GCCAGGAGCTGTTTT mG*mC*mC*mA*mG*G*A*G*C*T*G*T*T*T*T GCCAT*mG*mC*mC*mA*mU321GGTGATAATTCCAGT mG*mG*mU*mG*mA*T*A*A*T*T*C*C*A*G*T AGCTC*mA*mG*mC*mU*mC322CTTGTTCTCTGCCTA mC*mU*mU*mG*mU*T*C*T*C*T*G*C*C*T*A CATTT*mC*mA*mU*mU*mU323GGTATCATCAATTGT mG*mG*mU*mA*mU*C*A*T*C*A*A*T*T*G*T CCTGA*mC*mC*mU*mG*mA324GGCATCTCTATCCCA mG*mG*mC*mA*mU*C*T*C*T*A*T*C*C*C*A GATCT*mG*mA*mU*mC*mU325GATGTCAAGATCTGA mG*mA*mU*mG*mU*C*A*A*G*A*T*C*T*G*A GTAAG*mG*mU*mA*mA*mG326AGCAGGTGCCAGCTG mA*mG*mC*mA*mG*G*T*G*C*C*A*G*C*T*G CACTT*mC*mA*mC*mU*mU327GCAGCTGAACTTTCA mG*mC*mA*mG*mC*T*G*A*A*C*T*T*T*C*A GTTTT*mG*mU*mU*mU*mU328CAGTTTTCTTGGAAG mC*mA*mG*mU*mU*T*T*C*T*T*G*G*A*A*G TCACC*mU*mC*mA*mC*mC329GAGGCTGGAGTGCAG mG*mA*mG*mG*mC*T*G*G*A*G*T*G*C*A*G 3304934-0525-6010.2Page 56 of 105 065715-000129WOPTTGGCA *mU*mG*mG*mC*mA GGATTTTCCCTGCCT mG*mG*mA*mU*mU*T*T*C*C*C*T*G*C*C*T CAGCC*mC*mA*mG*mC*mC331GGATTACACATACCT mG*mG*mA*mU*mU*A*C*A*C*A*T*A*C*C*T GCCAC*mG*mC*mC*mA*mC332CACACCTGGCTAATT mC*mA*mC*mA*mC*C*T*G*G*C*T*A*A*T*T TTTGT*mU*mU*mU*mG*mU333GAGCTGGTTTTGAAC mG*mA*mG*mC*mU*G*G*T*T*T*T*G*A*A*C TCCTG*mU*mC*mC*mU*mG334CACCAAGCCCAGCTG mC*mA*mC*mC*mA*A*G*C*C*C*A*G*C*T*G ATATT*mA*mU*mA*mU*mU335GGTTGAAAATCTTCT mG*mG*mU*mU*mG*A*A*A*A*T*C*T*T*C*T GCAGA*mG*mC*mA*mG*mA336GGCAGAGATGTGATA mG*mG*mC*mA*mG*A*G*A*T*G*T*G*A*T*A CTAAC*mC*mU*mA*mA*mC337CCCTTTTCCTCTCTT mC*mC*mC*mU*mU*T*T*C*C*T*C*T*C*T*T AGTTC*mA*mG*mU*mU*mC338CTGTGAGGAACTGGA mC*mU*mG*mU*mG*A*G*G*A*A*C*T*G*G*A GTAGA*mG*mU*mA*mG*mA339AAATGGAGCCTGACC mA*mA*mA*mU*mG*G*A*G*C*C*T*G*A*C*C AGTGG*mA*mG*mU*mG*mG340GAGACCAAGCTGACT mG*mA*mG*mA*mC*C*A*A*G*C*T*G*A*C*T GCTTC*mG*mC*mU*mU*mC341CTGATTGTCCAACAT mC*mU*mG*mA*mU*T*G*T*C*C*A*A*C*A*T GAGGA*mG*mA*mG*mG*mA342CAGCACAAATTGGCC mC*mA*mG*mC*mA*C*A*A*A*T*T*G*G*C*C AGGTT*mA*mG*mG*mU*mU343CTCCTGATCATGATA mC*mU*mC*mC*mU*G*A*T*C*A*T*G*A*T*A TTGCC*mU*mU*mG*mC*mC344GGTCTTGCTACTATG mG*mG*mU*mC*mU*T*G*C*T*A*C*T*A*T*G TTGCT*mU*mU*mG*mC*mU345CAGGCTGGTCTCAAA mC*mA*mG*mG*mC*T*G*G*T*C*T*C*A*A*A CTTCT*mC*mU*mU*mC*mU346GGACTACAGGTACAT mG*mG*mA*mC*mU*A*C*A*G*G*T*A*C*A*T GCCAC*mG*mC*mC*mA*mC347GTTTTGGAGACAAGA mG*mU*mU*mU*mU*G*G*A*G*A*C*A*A*G*A GTCTC*mG*mU*mC*mU*mC348GGAGTGCAGTGGCAC mG*mG*mA*mG*mU*G*C*A*G*T*G*G*C*A*C AATCT*mA*mA*mU*mC*mU349GCCTCCTGGATTCAA mG*mC*mC*mU*mC*C*T*G*G*A*T*T*C*A*A GCAAT*mG*mC*mA*mA*mU350GGTTTCACCATGTTG mG*mG*mU*mU*mU*C*A*C*C*A*T*G*T*T*G GCCAG*mG*mC*mC*mA*mG351GAACTCCTGACCTCA mG*mA*mA*mC*mU*C*C*T*G*A*C*C*T*C*A AGTGA*mA*mG*mU*mG*mA352GGTTACAGGCATGAG mG*mG*mU*mU*mA*C*A*G*G*C*A*T*G*A*G CCACT*mC*mC*mA*mC*mU353GCTTATTCAGAGCTC mG*mC*mU*mU*mA*T*T*C*A*G*A*G*C*T*C TAGGC*mU*mA*mG*mG*mC354GGCCAGGATGAGAAC mG*mG*mC*mC*mA*G*G*A*T*G*A*G*A*A*C 3554934-0525-6010.2Page 57 of 105 065715-000129WOPTTCAGA *mU*mC*mA*mG*mA GTCTGACTGCAAACT mG*mU*mC*mU*mG*A*C*T*G*C*A*A*A*C*T CCATG*mC*mC*mA*mU*mG356CCCTGCATGTTGCAT mC*mC*mC*mU*mG*C*A*T*G*T*T*G*C*A*T GAAAG*mG*mA*mA*mA*mG357CCCTGTACATGTGAT mC*mC*mC*mU*mG*T*A*C*A*T*G*T*G*A*T TTCTT*mU*mU*mC*mU*mU358GAGTAAGTCCTTTGG mG*mA*mG*mU*mA*A*G*T*C*C*T*T*T*G*G TGTCA*mU*mG*mU*mC*mA359CTCCATGAAATCAGG mC*mU*mC*mC*mA*T*G*A*A*A*T*C*A*G*G ATCCA*mA*mU*mC*mC*mA360CCATCTTCAGAAGAG mC*mC*mA*mU*mC*T*T*C*A*G*A*A*G*A*G GTACT*mG*mU*mA*mC*mU361GAATCATCCCCAAAT mG*mA*mA*mU*mC*A*T*C*C*C*C*A*A*A*T GATGA*mG*mA*mU*mG*mA362CATGGTCTGACCTTG mC*mA*mU*mG*mG*T*C*T*G*A*C*C*T*T*G TAAGT*mU*mA*mA*mG*mU363GTGGTAGAATAGGAG mG*mU*mG*mG*mU*A*G*A*A*T*A*G*G*A*G ACATA*mA*mC*mA*mU*mA364CACCCTGGAGCAGGA mC*mA*mC*mC*mC*T*G*G*A*G*C*A*G*G*A AACAA*mA*mA*mC*mA*mA365CTCTTTGCCTTTAAA mC*mU*mC*mU*mU*T*G*C*C*T*T*T*A*A*A TGTGC*mU*mG*mU*mG*mC366CACTTCTCTGAACCT mC*mA*mC*mU*mU*C*T*C*T*G*A*A*C*C*T CATCA*mC*mA*mU*mC*mA367GATAACAGCCATTCC mG*mA*mU*mA*mA*C*A*G*C*C*A*T*T*C*C CTCAT*mC*mU*mC*mA*mU368CACTCCAGTAATATG mC*mA*mC*mU*mC*C*A*G*T*A*A*T*A*T*G TGCAT*mU*mG*mC*mA*mU369GGCAGAAAGCATGTT mG*mG*mC*mA*mG*A*A*A*G*C*A*T*G*T*T CACCT*mC*mA*mC*mC*mU370CTCTCTGCAGGTGCT mC*mU*mC*mU*mC*T*G*C*A*G*G*T*G*C*T TTACT*mU*mU*mA*mC*mU371GTGCTCATAAGCAGT mG*mU*mG*mC*mU*C*A*T*A*A*G*C*A*G*T TTGAT*mU*mU*mG*mA*mU372GAGCTCATCTTGCCC mG*mA*mG*mC*mU*C*A*T*C*T*T*G*C*C*C ATCAT*mA*mU*mC*mA*mU373GAGTGCCACATTGTT mG*mA*mG*mU*mG*C*C*A*C*A*T*T*G*T*T GTGCT*mG*mU*mG*mC*mU374GATGAGGATTCTGTG mG*mA*mU*mG*mA*G*G*A*T*T*C*T*G*T*G GACTG*mG*mA*mC*mU*mG375GGATGAAGACCACCC mG*mG*mA*mU*mG*A*A*G*A*C*C*A*C*C*C TCCAC*mU*mC*mC*mA*mC376GGAGGAATATTCCCA mG*mG*mA*mG*mG*A*A*T*A*T*T*C*C*C*A AAGTA*mA*mA*mG*mU*mA377CTTGCTACCTGGAGT mC*mU*mU*mG*mC*T*A*C*C*T*G*G*A*G*T AGCAG*mA*mG*mC*mA*mG378CTTGATCCTAGAACC mC*mU*mU*mG*mA*T*C*C*T*A*G*A*A*C*C AATGT*mA*mA*mU*mG*mU379GTGAATATCAATGTT mG*mU*mG*mA*mA*T*A*T*C*A*A*T*G*T*T 3804934-0525-6010.2Page 58 of 105 065715-000129WOPTGAGCC *mG*mA*mG*mC*mC GTGATGAGCACACTC mG*mU*mG*mA*mU*G*A*G*C*A*C*A*C*T*C ACCTG*mA*mC*mC*mU*mG381GGATGACAAATCCCT mG*mG*mA*mU*mG*A*C*A*A*A*T*C*C*C*T AGTGA*mA*mG*mU*mG*mA382CAGGTCTAGCCTTTA mC*mA*mG*mG*mU*C*T*A*G*C*C*T*T*T*A TGCTG*mU*mG*mC*mU*mG383GGAACACAGTGAATG mG*mG*mA*mA*mC*A*C*A*G*T*G*A*A*T*G TTGTT*mU*mU*mG*mU*mU384CACCATTCACTCTTT mC*mA*mC*mC*mA*T*T*C*A*C*T*C*T*T*T CTCTG*mC*mU*mC*mU*mG385CTGTGTAAGATACTG mC*mU*mG*mU*mG*T*A*A*G*A*T*A*C*T*G AGGCA*mA*mG*mG*mC*mA386GATGGTGTCATTCTT mG*mA*mU*mG*mG*T*G*T*C*A*T*T*C*T*T CTCTT*mC*mU*mC*mU*mU387CTCACTCCTGCCCCA mC*mU*mC*mA*mC*T*C*C*T*G*C*C*C*C*A TCTAT*mU*mC*mU*mA*mU388CAGGCTTCCAGGTAT mC*mA*mG*mG*mC*T*T*C*C*A*G*G*T*A*T ATTCA*mA*mU*mU*mC*mA389GTAGCTCATGCCTGT mG*mU*mA*mG*mC*T*C*A*T*G*C*C*T*G*T AATCC*mA*mA*mU*mC*mC390CATGAGGTCAGGAGT mC*mA*mU*mG*mA*G*G*T*C*A*G*G*A*G*T TCAAG*mU*mC*mA*mA*mG391CCAGTCTGGCCAACA mC*mC*mA*mG*mU*C*T*G*G*C*C*A*A*C*A TAGTG*mU*mA*mG*mU*mG392CCTGTAGTCCCAGCT mC*mC*mU*mG*mU*A*G*T*C*C*C*A*G*C*T ACTCA*mA*mC*mU*mC*mA393GGAGGCTGAGGCAGG mG*mG*mA*mG*mG*C*T*G*A*G*G*C*A*G*G AGAAT*mA*mG*mA*mA*mU394CAGTGTGAGACTCCA mC*mA*mG*mU*mG*T*G*A*G*A*C*T*C*C*A TCTCA*mU*mC*mU*mC*mA395CCTTTGCACAAATGT mC*mC*mU*mU*mU*G*C*A*C*A*A*A*T*G*T GCCAT*mG*mC*mC*mA*mU396GACTTAACCAGCCAT mG*mA*mC*mU*mU*A*A*C*C*A*G*C*C*A*T TATTG*mU*mA*mU*mU*mG397GTGGCTCATGCCTGT mG*mU*mG*mG*mC*T*C*A*T*G*C*C*T*G*T AATCC*mA*mA*mU*mC*mC398GGCTGATTGCTTGAG mG*mG*mC*mU*mG*A*T*T*G*C*T*T*G*A*G GTCAA*mG*mU*mC*mA*mA399GACCAGCCTTGCCAA mG*mA*mC*mC*mA*G*C*C*T*T*G*C*C*A*A TATAG*mU*mA*mU*mA*mG400GGCTCATGCCTGTAA mG*mG*mC*mU*mC*A*T*G*C*C*T*G*T*A*A TCCTA*mU*mC*mC*mU*mA401GCTACTCTGGAGGCT mG*mC*mU*mA*mC*T*C*T*G*G*A*G*G*C*T GAAAC*mG*mA*mA*mA*mC402GGAGAATCACTTGAA mG*mG*mA*mG*mA*A*T*C*A*C*T*T*G*A*A CCCAG*mC*mC*mC*mA*mG403GAGGTTGCAGTGAGA mG*mA*mG*mG*mU*T*G*C*A*G*T*G*A*G*A CAAGA*mC*mA*mA*mG*mA404CACCACTGCACTTCA mC*mA*mC*mC*mA*C*T*G*C*A*C*T*T*C*A 4054934-0525-6010.2Page 59 of 105 065715-000129WOPTGCCTG *mG*mC*mC*mU*mG GCCTGTAATCCCAGC mG*mC*mC*mU*mG*T*A*A*T*C*C*C*A*G*C TACTC*mU*mA*mC*mU*mC406GGCTGAGAGGCAGGA mG*mG*mC*mU*mG*A*G*A*G*G*C*A*G*G*A GAATC*mG*mA*mA*mU*mC407GATCATGCCATTGCA mG*mA*mU*mC*mA*T*G*C*C*A*T*T*G*C*A CTACA*mC*mU*mA*mC*mA408CATGACAGGTTTGTT mC*mA*mU*mG*mA*C*A*G*G*T*T*T*G*T*T TCCTA*mU*mC*mC*mU*mA409CCTCACCAAAACAGT mC*mC*mU*mC*mA*C*C*A*A*A*A*C*A*G*T GGCTT*mG*mG*mC*mU*mU410GAGACAGAGTCCCAC mG*mA*mG*mA*mC*A*G*A*G*T*C*C*C*A*C CTCCT*mC*mU*mC*mC*mU411GGTTCAAGTGATTCT mG*mG*mU*mU*mC*A*A*G*T*G*A*T*T*C*T CATGC*mC*mA*mU*mG*mC412CTCAACCTCCCAAGA mC*mU*mC*mA*mA*C*C*T*C*C*C*A*A*G*A AGCTG*mA*mG*mC*mU*mG413GGTTTCACCATGTTG mG*mG*mU*mU*mU*C*A*C*C*A*T*G*T*T*G GCCAG*mG*mC*mC*mA*mG414GCTGGTCTCAAATTC mG*mC*mU*mG*mG*T*C*T*C*A*A*A*T*T*C CTGAC*mC*mU*mG*mA*mC415GGATTGCAGGCACCA mG*mG*mA*mU*mU*G*C*A*G*G*C*A*C*C*A TGGCT*mU*mG*mG*mC*mU416GGCCAACAGTGGCTT mG*mG*mC*mC*mA*A*C*A*G*T*G*G*C*T*T CTTAA*mC*mU*mU*mA*mA417GCAGTGGTGCAATCA mG*mC*mA*mG*mU*G*G*T*G*C*A*A*T*C*A TGGCT*mU*mG*mG*mC*mU418CACTGCAGCCTCAAA mC*mA*mC*mU*mG*C*A*G*C*C*T*C*A*A*A CTCCT*mC*mU*mC*mC*mU419GTCTTAAGTGATTCT mG*mU*mC*mU*mU*A*A*G*T*G*A*T*T*C*T CCCAT*mC*mC*mC*mA*mU420GCAGGTGTGTGCCAC mG*mC*mA*mG*mG*T*G*T*G*T*G*C*C*A*C CATGA*mC*mA*mU*mG*mA421GGTTCTATGTTGCCT mG*mG*mU*mU*mC*T*A*T*G*T*T*G*C*C*T TGCCT*mU*mG*mC*mC*mU422GGCTAGTCTCAAACT mG*mG*mC*mU*mA*G*T*C*T*C*A*A*A*C*T CCAGG*mC*mC*mA*mG*mG423CCTCAAGCCATCTTC mC*mC*mU*mC*mA*A*G*C*C*A*T*C*T*T*C CTGCA*mC*mU*mG*mC*mA424GCAGAATCTCATGAT mG*mC*mA*mG*mA*A*T*C*T*C*A*T*G*A*T TGGCA*mU*mG*mG*mC*mA425CATGAGTGAGGCTGA mC*mA*mU*mG*mA*G*T*G*A*G*G*C*T*G*A ATTTT*mA*mU*mU*mU*mU426GAGATGGTGTCTCCC mG*mA*mG*mA*mU*G*G*T*G*T*C*T*C*C*C TTCTC*mU*mU*mC*mU*mC427GTGATCTTGGCTCAC mG*mU*mG*mA*mU*C*T*T*G*G*C*T*C*A*C TGCAA*mU*mG*mC*mA*mA428GGTTCAAGTGCTTCT mG*mG*mU*mU*mC*A*A*G*T*G*C*T*T*C*T CCTGC*mC*mC*mU*mG*mC429GGATTACAGGCACCC mG*mG*mA*mU*mU*A*C*A*G*G*C*A*C*C*C 4304934-0525-6010.2Page 60 of 105 065715-000129WOPTACCAC *mA*mC*mC*mA*mC GGTTTCACCATGTTA mG*mG*mU*mU*mU*C*A*C*C*A*T*G*T*T*A GTCAG*mG*mU*mC*mA*mG431GCTGGTCTCCAACTC mG*mC*mU*mG*mG*T*C*T*C*C*A*A*C*T*C CCAAC*mC*mC*mA*mA*mC432CTCAACTGATCTGCC mC*mU*mC*mA*mA*C*T*G*A*T*C*T*G*C*C TGCCT*mU*mG*mC*mC*mU433GGTTACAGGTGTGAG mG*mG*mU*mU*mA*C*A*G*G*T*G*T*G*A*G CCACC*mC*mC*mA*mC*mC434GCTCTATTTGTGGCA mG*mC*mU*mC*mU*A*T*T*T*G*T*G*G*C*A GCTCT*mG*mC*mU*mC*mU435CAGTGTCTTTTTAGC mC*mA*mG*mU*mG*T*C*T*T*T*T*T*A*G*C TTTGG*mU*mU*mU*mG*mG436GTTTAGGCTATGCAG mG*mU*mU*mU*mA*G*G*C*T*A*T*G*C*A*G AGATG*mA*mG*mA*mU*mG437GGCTTCTAGGTTTAA mG*mG*mC*mU*mU*C*T*A*G*G*T*T*T*A*A TGTTG*mU*mG*mU*mU*mG438GATCCATCTGGATTA mG*mA*mU*mC*mC*A*T*C*T*G*G*A*T*T*A TTTGG*mU*mU*mU*mG*mG439GCTTCTCTCTTTCCT mG*mC*mU*mU*mC*T*C*T*C*T*T*T*C*C*T CTTAG*mC*mU*mU*mA*mG440GTCTTTACTCCATGG mG*mU*mC*mU*mU*T*A*C*T*C*C*A*T*G*G CTAAC*mC*mU*mA*mA*mC441GCTCCAAGCTCTACA mG*mC*mU*mC*mC*A*A*G*C*T*C*T*A*C*A GATTC*mG*mA*mU*mU*mC442GGATGTTGCAAAAAC mG*mG*mA*mU*mG*T*T*G*C*A*A*A*A*A*C AGTGT*mA*mG*mU*mG*mU443CCCAGTCAACATTCA mC*mC*mC*mA*mG*T*C*A*A*C*A*T*T*C*A GGTTG*mG*mG*mU*mU*mG444GGTCTATTGCCACAG mG*mG*mU*mC*mU*A*T*T*G*C*C*A*C*A*G CTTAC*mC*mU*mU*mA*mC445CCTCAAAATTTAGCC mC*mC*mU*mC*mA*A*A*A*T*T*T*A*G*C*C TCCAG*mU*mC*mC*mA*mG446CCAGAGGCCTAGAAA mC*mC*mA*mG*mA*G*G*C*C*T*A*G*A*A*A ATAGG*mA*mU*mA*mG*mG447CTCCAGTTGAAAATG mC*mU*mC*mC*mA*G*T*T*G*A*A*A*A*T*G GTTCA*mG*mU*mU*mC*mA448AGCAGCCACAGAAAC mA*mG*mC*mA*mG*C*C*A*C*A*G*A*A*A*C AAGTT*mA*mA*mG*mU*mU449CAGGATAAAGAGCAG mC*mA*mG*mG*mA*T*A*A*A*G*A*G*C*A*G CAAGG*mC*mA*mA*mG*mG450CTGCCACCAGTCTTG mC*mU*mG*mC*mC*A*C*C*A*G*T*C*T*T*G GTGAA*mG*mU*mG*mA*mA451GATGTTTCTGGTATC mG*mA*mU*mG*mU*T*T*C*T*G*G*T*A*T*C TGGAA*mU*mG*mG*mA*mA452GGCACAGTTTGTTGT mG*mG*mC*mA*mC*A*G*T*T*T*G*T*T*G*T GCCTA*mG*mC*mC*mU*mA453GGATACCAAGGTAAT mG*mG*mA*mU*mA*C*C*A*A*G*G*T*A*A*T TGCAC*mU*mG*mC*mA*mC454CCATACTTTTGAGGA mC*mC*mA*mU*mA*C*T*T*T*T*G*A*G*G*A 4554934-0525-6010.2Page 61 of 105 065715-000129WOPTATGTC *mA*mU*mG*mU*mC ATGATCTTGAGCAGT mA*mU*mG*mA*mU*C*T*T*G*A*G*C*A*G*T GGCTC*mG*mG*mC*mU*mC456GGAAGGCTCTCAAGA mG*mG*mA*mA*mG*G*C*T*C*T*C*A*A*G*A GACTG*mG*mA*mC*mU*mG457CCTGGAGAGTGCTAA mC*mC*mU*mG*mG*A*G*A*G*T*G*C*T*A*A AGTCA*mA*mG*mU*mC*mA458GAGATGGAGTCTCAC mG*mA*mG*mA*mU*G*G*A*G*T*C*T*C*A*C TCTGT*mU*mC*mU*mG*mU459TCTCAGCTCACTGCC mU*mC*mU*mC*mA*G*C*T*C*A*C*T*G*C*C AGCTC*mA*mG*mC*mU*mC460GCCAGGATGGTCTCA mG*mC*mC*mA*mG*G*A*T*G*G*T*C*T*C*A ATCTC*mA*mU*mC*mU*mC461GGATTACAGATGTGA mG*mG*mA*mU*mU*A*C*A*G*A*T*G*T*G*A GCCAC*mG*mC*mC*mA*mC462GTGGATCTTTGTTTG mG*mU*mG*mG*mA*T*C*T*T*T*G*T*T*T*G ACTCT*mA*mC*mU*mC*mU463GTGCTTTGCTACTCT mG*mU*mG*mC*mU*T*T*G*C*T*A*C*T*C*T TGTTG*mU*mG*mU*mU*mG464CCTCATTTCTGCCCA mC*mC*mU*mC*mA*T*T*T*C*T*G*C*C*C*A CAGAA*mC*mA*mG*mA*mA465CCATCAAAGCTTCAC mC*mC*mA*mU*mC*A*A*A*G*C*T*T*C*A*C TGTGT*mU*mG*mU*mG*mU466GGTCTCACTCTTCTG mG*mG*mU*mC*mU*C*A*C*T*C*T*T*C*T*G CTTAC*mC*mU*mU*mA*mC467CTGGAGTACAGTGAC mC*mU*mG*mG*mA*G*T*A*C*A*G*T*G*A*C ACAAT*mA*mC*mA*mA*mU468CATGGCTCACTGCAG mC*mA*mU*mG*mG*C*T*C*A*C*T*G*C*A*G CCTTA*mC*mC*mU*mU*mA469CTCAAGTGGTCCTCC mC*mU*mC*mA*mA*G*T*G*G*T*C*C*T*C*C TGCCT*mU*mG*mC*mC*mU470CAGCCTTCCAAGTAG mC*mA*mG*mC*mC*T*T*C*C*A*A*G*T*A*G CAGAG*mC*mA*mG*mA*mG471CCACAGGCATGCACC mC*mC*mA*mC*mA*G*G*C*A*T*G*C*A*C*C ACTAT*mA*mC*mU*mA*mU472GAGATGAGGTCTCCC mG*mA*mG*mA*mU*G*A*G*G*T*C*T*C*C*C TCTGG*mU*mC*mU*mG*mG473CCAGGCTGGTTTTGA mC*mC*mA*mG*mG*C*T*G*G*T*T*T*T*G*A ACTCC*mA*mC*mU*mC*mC474CCAAAGTGCTAGGAT mC*mC*mA*mA*mA*G*T*G*C*T*A*G*G*A*T TACAG*mU*mA*mC*mA*mG475CAGTGTCCAGCCTTG mC*mA*mG*mU*mG*T*C*C*A*G*C*C*T*T*G GAAGA*mG*mA*mA*mG*mA476GTCTTTCCCTTGTCT mG*mU*mC*mU*mU*T*C*C*C*T*T*G*T*C*T GTTAG*mG*mU*mU*mA*mG477GTGCATTTCCCAGAA mG*mU*mG*mC*mA*T*T*T*C*C*C*A*G*A*A CAACC*mC*mA*mA*mC*mC478CCATTAGAACATTCT mC*mC*mA*mU*mU*A*G*A*A*C*A*T*T*C*T GCCAT*mG*mC*mC*mA*mU479GCCTTCCAGTTAATA mG*mC*mC*mU*mU*C*C*A*G*T*T*A*A*T*A 4804934-0525-6010.2Page 62 of 105 065715-000129WOPTGCCTC *mG*mC*mC*mU*mC CCCATTTCCATGTGA mC*mC*mC*mA*mU*T*T*C*C*A*T*G*T*G*A CAGCC*mC*mA*mG*mC*mC481CACATATTTTAGGCA mC*mA*mC*mA*mU*A*T*T*T*T*A*G*G*C*A GCATC*mG*mC*mA*mU*mC482GGCTAAACATTCCAC mG*mG*mC*mU*mA*A*A*C*A*T*T*C*C*A*C ATTTC*mA*mU*mU*mU*mC483GTATGCCCTCTAAGC mG*mU*mA*mU*mG*C*C*C*T*C*T*A*A*G*C AATAG*mA*mA*mU*mA*mG484GGTAGCCATGCTTTG mG*mG*mU*mA*mG*C*C*A*T*G*C*T*T*T*G GCAGG*mG*mC*mA*mG*mG485GGTATTGTTGCCAGC mG*mG*mU*mA*mU*T*G*T*T*G*C*C*A*G*C AACAA*mA*mA*mC*mA*mA486CAGGTTCCTGAACTG mC*mA*mG*mG*mU*T*C*C*T*G*A*A*C*T*G GTAAG*mG*mU*mA*mA*mG487GGCTTCTCTCCTCTT mG*mG*mC*mU*mU*C*T*C*T*C*C*T*C*T*T TCCAT*mU*mC*mC*mA*mU488CTCAGATTAGTCCCT mC*mU*mC*mA*mG*A*T*T*A*G*T*C*C*C*T TCTAG*mU*mC*mU*mA*mG489GGACAAGACCTACTA mG*mG*mA*mC*mA*A*G*A*C*C*T*A*C*T*A ATCTG*mA*mU*mC*mU*mG490GGAGAAAACTGATGT mG*mG*mA*mG*mA*A*A*A*C*T*G*A*T*G*T GGTTA*mG*mG*mU*mU*mA491GGAAGGAGAAAGTAT mG*mG*mA*mA*mG*G*A*G*A*A*A*G*T*A*T GGACA*mG*mG*mA*mC*mA492CCAGAGTGAGCACAA mC*mC*mA*mG*mA*G*T*G*A*G*C*A*C*A*A TTTGT*mU*mU*mU*mG*mU493TAGTCATGAGAATGG mU*mA*mG*mU*mC*A*T*G*A*G*A*A*T*G*G CCTTT*mC*mC*mU*mU*mU494GTTACCCTACCTGTA mG*mU*mU*mA*mC*C*C*T*A*C*C*T*G*T*A AAGAA*mA*mA*mG*mA*mA495CCCTTCATCCAGATG mC*mC*mC*mU*mU*C*A*T*C*C*A*G*A*T*G TCATG*mU*mC*mA*mU*mG496CCATGTGGATTTCCA mC*mC*mA*mU*mG*T*G*G*A*T*T*T*C*C*A GTGTG*mG*mU*mG*mU*mG497CTGGTAGCTGCTGGA mC*mU*mG*mG*mU*A*G*C*T*G*C*T*G*G*A GATGA*mG*mA*mU*mG*mA498GTCTTGGAGGACCAG mG*mU*mC*mU*mU*G*G*A*G*G*A*C*C*A*G GAGAT*mG*mA*mG*mA*mU499AATGCATCACCCACC mA*mA*mU*mG*mC*A*T*C*A*C*C*C*A*C*C CCAAG*mC*mC*mA*mA*mG500CCCAAGTGGATGAAC mC*mC*mC*mA*mA*G*T*G*G*A*T*G*A*A*C TTGAC*mU*mU*mG*mA*mC501GCTAAATGCCCCACT mG*mC*mU*mA*mA*A*T*G*C*C*C*C*A*C*T TTCTC*mU*mU*mC*mU*mC502GGTCTACTCCTTGTT mG*mG*mU*mC*mU*A*C*T*C*C*T*T*G*T*T GGAGC*mG*mG*mA*mG*mC503TTGGAGCCCATCTCA mU*mU*mG*mG*mA*G*C*C*C*A*T*C*T*C*A CCTGG*mC*mC*mU*mG*mG504GATTTGTCTCACCTT mG*mA*mU*mU*mU*G*T*C*T*C*A*C*C*T*T 5054934-0525-6010.2Page 63 of 105 065715-000129WOPTGAGTA *mG*mA*mG*mU*mA GGAGACATGCTTCTC mG*mG*mA*mG*mA*C*A*T*G*C*T*T*C*T*C CCCTA*mC*mC*mC*mU*mA506ACCTTTTCCTTTCTG mA*mC*mC*mU*mU*T*T*C*C*T*T*T*C*T*G CCATA*mC*mC*mA*mU*mA507AAGTCCATGCCTCTG mA*mA*mG*mU*mC*C*A*T*G*C*C*T*C*T*G GCAGG*mG*mC*mA*mG*mG508GGATGAAGAAGTACT mG*mG*mA*mU*mG*A*A*G*A*A*G*T*A*C*T CACTG*mC*mA*mC*mU*mG509GCTACCATCTTTGCA mG*mC*mU*mA*mC*C*A*T*C*T*T*T*G*C*A GCAGC*mG*mC*mA*mG*mC510GGTCTGGTGCTGTTC mG*mG*mU*mC*mU*G*G*T*G*C*T*G*T*T*C ATTGA*mA*mU*mU*mG*mA511GCAGGAAAGGAAGTC mG*mC*mA*mG*mG*A*A*A*G*G*A*A*G*T*C AAGAC*mA*mA*mG*mA*mC512GCTTAGCAAAGCAGT mG*mC*mU*mU*mA*G*C*A*A*A*G*C*A*G*T CCTTA*mC*mC*mU*mU*mA513CCTTTATACTCTGTT mC*mC*mU*mU*mU*A*T*A*C*T*C*T*G*T*T CTTGG*mC*mU*mU*mG*mG514GTCTTGTTTTATACC mG*mU*mC*mU*mU*G*T*T*T*T*A*T*A*C*C AGGCA*mA*mG*mG*mC*mA515GGACCAAACTGAAAA mG*mG*mA*mC*mC*A*A*A*C*T*G*A*A*A*A GGTGA*mG*mG*mU*mG*mA516GCCATTCTTGGTGGC mG*mC*mC*mA*mU*T*C*T*T*G*G*T*G*G*C CTCTG*mC*mU*mC*mU*mG517CACCCAAATTGCATC mC*mA*mC*mC*mC*A*A*A*T*T*G*C*A*T*C TGGAA*mU*mG*mG*mA*mA518GGTCTCATTCAGAAT mG*mG*mU*mC*mU*C*A*T*T*C*A*G*A*A*T GTCCA*mG*mU*mC*mC*mA519GTGTCTCATTAATGC mG*mU*mG*mU*mC*T*C*A*T*T*A*A*T*G*C AGTGA*mA*mG*mU*mG*mA520CCTTGGCAGGAAGGA mC*mC*mU*mU*mG*G*C*A*G*G*A*A*G*G*A TAATG*mU*mA*mA*mU*mG521AACAGTGTTGATGAG mA*mA*mC*mA*mG*T*G*T*T*G*A*T*G*A*G ACCTT*mA*mC*mC*mU*mU522CACAATGCAAGTAGA mC*mA*mC*mA*mA*T*G*C*A*A*G*T*A*G*A AGGAC*mA*mG*mG*mA*mC523CTCTCCAAATCAGGC mC*mU*mC*mU*mC*C*A*A*A*T*C*A*G*G*C CAGTT*mC*mA*mG*mU*mU524GGTTATCCTGGCTTG mG*mG*mU*mU*mA*T*C*C*T*G*G*C*T*T*G GAATC*mG*mA*mA*mU*mC525TGGTGTGAAACCATA mU*mG*mG*mU*mG*T*G*A*A*A*C*C*A*T*A GGTCT*mG*mG*mU*mC*mU526CACTCTGGAGCAGCA mC*mA*mC*mU*mC*T*G*G*A*G*C*A*G*C*A CATTG*mC*mA*mU*mU*mG527CTGTGGATATGTCCA mC*mU*mG*mU*mG*G*A*T*A*T*G*T*C*C*A GGAGA*mG*mG*mA*mG*mA528CCTTAGATATGGCTT mC*mC*mU*mU*mA*G*A*T*A*T*G*G*C*T*T AAAGG*mA*mA*mA*mG*mG529CTTTCAAGATGAGGA mC*mU*mU*mU*mC*A*A*G*A*T*G*A*G*G*A 5304934-0525-6010.2Page 64 of 105 065715-000129WOPTCAGAA *mC*mA*mG*mA*mA GCTTACAATTGCTCA mG*mC*mU*mU*mA*C*A*A*T*T*G*C*T*C*A GTTTC*mG*mU*mU*mU*mC531CTCATAAGAGTGCCA mC*mU*mC*mA*mU*A*A*G*A*G*T*G*C*C*A GCATG*mG*mC*mA*mU*mG532GGTACATGGAGTGAA mG*mG*mU*mA*mC*A*T*G*G*A*G*T*G*A*A GCTGG*mG*mC*mU*mG*mG533GGAAGCATCATCTGC mG*mG*mA*mA*mG*C*A*T*C*A*T*C*T*G*C ACAGT*mA*mC*mA*mG*mU534CCCTGTCCTAGTGCA mC*mC*mC*mU*mG*T*C*C*T*A*G*T*G*C*A GGACT*mG*mG*mA*mC*mU535GCCTAATAGCTGCTC mG*mC*mC*mU*mA*A*T*A*G*C*T*G*C*T*C ACACA*mA*mC*mA*mC*mA536GGTGAGCTGAATGGA mG*mG*mU*mG*mA*G*C*T*G*A*A*T*G*G*A AATTA*mA*mA*mU*mU*mA537CAGTGCATGATCACC mC*mA*mG*mU*mG*C*A*T*G*A*T*C*A*C*C CAGAT*mC*mA*mG*mA*mU538CACAGTGCTGAACCA mC*mA*mC*mA*mG*T*G*C*T*G*A*A*C*C*A TCTTC*mU*mC*mU*mU*mC539CCTCCTGTTCTCCAT mC*mC*mU*mC*mC*T*G*T*T*C*T*C*C*A*T GGCTA*mG*mG*mC*mU*mA540GAGTGCAGAGCCAGA mG*mA*mG*mU*mG*C*A*G*A*G*C*C*A*G*A TATAA*mU*mA*mU*mA*mA541CAGTTTGTGGTAGAA mC*mA*mG*mU*mU*T*G*T*G*G*T*A*G*A*A GCTGA*mG*mC*mU*mG*mA542CTGGAATACAGGTTG mC*mU*mG*mG*mA*A*T*A*C*A*G*G*T*T*G AGTAT*mA*mG*mU*mA*mU543CTCTTATCCAAAATG mC*mU*mC*mU*mU*A*T*C*C*A*A*A*A*T*G CTAGG*mC*mU*mA*mG*mG544GACCAGAAAGGTTTC mG*mA*mC*mC*mA*G*A*A*A*G*G*T*T*T*C AGATT*mA*mG*mA*mU*mU545CAGTTGAGCACCCCA mC*mA*mG*mU*mU*G*A*G*C*A*C*C*C*C*A AATCT*mA*mA*mU*mC*mU546GGATGCTCATCCTGT mG*mG*mA*mU*mG*C*T*C*A*T*C*C*T*G*T GTAGG*mG*mU*mA*mG*mG547CCATTTAATGACTGT mC*mC*mA*mU*mU*T*A*A*T*G*A*C*T*G*T CCTAG*mC*mC*mU*mA*mG548GCCAGGTAGATGTTG mG*mC*mC*mA*mG*G*T*A*G*A*T*G*T*T*G AAAGC*mA*mA*mA*mG*mC549GGCTGAAAAAGTGTT mG*mG*mC*mU*mG*A*A*A*A*A*G*T*G*T*T CTTAC*mC*mU*mU*mA*mC550GTTCTCTGCATGTGA mG*mU*mU*mC*mU*C*T*G*C*A*T*G*T*G*A CTAGC*mC*mU*mA*mG*mC551GCTCTGTTCTTCACT mG*mC*mU*mC*mU*G*T*T*C*T*T*C*A*C*T AGAAT*mA*mG*mA*mA*mU552CCCACCTGATGACTA mC*mC*mC*mA*mC*C*T*G*A*T*G*A*C*T*A TTGGC*mU*mU*mG*mG*mC553CCATTTGCAAGGCCA mC*mC*mA*mU*mU*T*G*C*A*A*G*G*C*C*A ATGGC*mA*mU*mG*mG*mC554CTGTGAAACTAATTG mC*mU*mG*mU*mG*A*A*A*C*T*A*A*T*T*G 5554934-0525-6010.2Page 65 of 105 065715-000129WOPTGCTCA *mG*mC*mU*mC*mA GAGGTTAGGTGTGGC mG*mA*mG*mG*mU*T*A*G*G*T*G*T*G*G*C CTTGA*mC*mU*mU*mG*mA556CCCACTACCAGTTGA mC*mC*mC*mA*mC*T*A*C*C*A*G*T*T*G*A CTAGC*mC*mU*mA*mG*mC557GGTGCTTCTTTTCTG mG*mG*mU*mG*mC*T*T*C*T*T*T*T*C*T*G CTTCT*mC*mU*mU*mC*mU558CAGTAGACTTCCATG mC*mA*mG*mU*mA*G*A*C*T*T*C*C*A*T*G CCATT*mC*mC*mA*mU*mU559CCATGCCACAGCTAA mC*mC*mA*mU*mG*C*C*A*C*A*G*C*T*A*A CACTT*mC*mA*mC*mU*mU560GCCAGGTATGAAGTA mG*mC*mC*mA*mG*G*T*A*T*G*A*A*G*T*A TGGCA*mU*mG*mG*mC*mA561CTTGACACTGTCCTG mC*mU*mU*mG*mA*C*A*C*T*G*T*C*C*T*G TGTAA*mU*mG*mU*mA*mA562GCTCAGGCTTTCAAG mG*mC*mU*mC*mA*G*G*C*T*T*T*C*A*A*G ATTGA*mA*mU*mU*mG*mA563GCATTTGACCCCAAC mG*mC*mA*mU*mU*T*G*A*C*C*C*C*A*A*C CTGTG*mC*mU*mG*mU*mG564GGTTTGAGTAAGCTG mG*mG*mU*mU*mU*G*A*G*T*A*A*G*C*T*G GAAAT*mG*mA*mA*mA*mU565GGTGGTGGTGACTGA mG*mG*mU*mG*mG*T*G*G*T*G*A*C*T*G*A AGGAA*mA*mG*mG*mA*mA566GGATCACAGGTTCCT mG*mG*mA*mU*mC*A*C*A*G*G*T*T*C*C*T TCTGA*mU*mC*mU*mG*mA567GGAGAGGAAGGTTTA mG*mG*mA*mG*mA*G*G*A*A*G*G*T*T*T*A TTTCT*mU*mU*mU*mC*mU568ACCCTCCACCTAGAG mA*mC*mC*mC*mU*C*C*A*C*C*T*A*G*A*G CTCAC*mC*mU*mC*mA*mC569GGCCATTCTGCAAAA mG*mG*mC*mC*mA*T*T*C*T*G*C*A*A*A*A GCAGG*mG*mC*mA*mG*mG570AGGACCTCACAGAAA mA*mG*mG*mA*mC*C*T*C*A*C*A*G*A*A*A CAAGG*mC*mA*mA*mG*mG571CCTTCAGAGTTGGTT mC*mC*mU*mU*mC*A*G*A*G*T*T*G*G*T*T CCTGG*mC*mC*mU*mG*mG572GTGCCTAAATAAGCA mG*mU*mG*mC*mC*T*A*A*A*T*A*A*G*C*A AGCCA*mA*mG*mC*mC*mA573GGCTGTTGATATTTT mG*mG*mC*mU*mG*T*T*G*A*T*A*T*T*T*T AGCCA*mA*mG*mC*mC*mA574CTTGAGTTTGCATCT mC*mU*mU*mG*mA*G*T*T*T*G*C*A*T*C*T GCATC*mG*mC*mA*mU*mC575GATGAGGAAACATTT mG*mA*mU*mG*mA*G*G*A*A*A*C*A*T*T*T GCCAC*mG*mC*mC*mA*mC576GCAAGCCAGATTTAC mG*mC*mA*mA*mG*C*C*A*G*A*T*T*T*A*C TGCAC*mU*mG*mC*mA*mC577GCAGTACCCAGTACT mG*mC*mA*mG*mU*A*C*C*C*A*G*T*A*C*T ACTTT*mA*mC*mU*mU*mU578CTGTGGAATAAGCCC mC*mU*mG*mU*mG*G*A*A*T*A*A*G*C*C*C AGGAA*mA*mG*mG*mA*mA579CAGACCACAGGCTTT mC*mA*mG*mA*mC*C*A*C*A*G*G*C*T*T*T 5804934-0525-6010.2Page 66 of 105 065715-000129WOPTAATGT *mA*mA*mU*mG*mU CCTGTAATCCCAGCA mC*mC*mU*mG*mU*A*A*T*C*C*C*A*G*C*A CTTTG*mC*mU*mU*mU*mG581GCAAATCACCTAAGG mG*mC*mA*mA*mA*T*C*A*C*C*T*A*A*G*G TCAGG*mU*mC*mA*mG*mG582AGACCAGCCTGGCCA mA*mG*mA*mC*mC*A*G*C*C*T*G*G*C*C*A ACATG*mA*mC*mA*mU*mG583GTGTGGTGGCAAGTG mG*mU*mG*mU*mG*G*T*G*G*C*A*A*G*T*G CCTGT*mC*mC*mU*mG*mU584AGGCTGAGGCAGGAG mA*mG*mG*mC*mU*G*A*G*G*C*A*G*G*A*G AACTG*mA*mA*mC*mU*mG585GGAGGTAGAGGTTGC mG*mG*mA*mG*mG*T*A*G*A*G*G*T*T*G*C AGTGA*mA*mG*mU*mG*mA586GGCAACAGAGCAAGA mG*mG*mC*mA*mA*C*A*G*A*G*C*A*A*G*A CTCCA*mC*mU*mC*mC*mA587GGAGGATCACTTGAG mG*mG*mA*mG*mG*A*T*C*A*C*T*T*G*A*G GTCAG*mG*mU*mC*mA*mG588GGAGTTTCAGACTGG mG*mG*mA*mG*mU*T*T*C*A*G*A*C*T*G*G CCTGG*mC*mC*mU*mG*mG589TAGCCAGGCATGGAC mU*mA*mG*mC*mC*A*G*G*C*A*T*G*G*A*C ATGGT*mA*mU*mG*mG*mU590AGTGCATACCTGTGG mA*mG*mU*mG*mC*A*T*A*C*C*T*G*T*G*G TCCCA*mU*mC*mC*mC*mA591GCCACTCAAGAGGCT mG*mC*mC*mA*mC*T*C*A*A*G*A*G*G*C*T GAGAC*mG*mA*mG*mA*mC592AGGATGATTGCTTGA mA*mG*mG*mA*mU*G*A*T*T*G*C*T*T*G*A GCCCA*mG*mC*mC*mC*mA593GGAGGATGAGGCTGC mG*mG*mA*mG*mG*A*T*G*A*G*G*C*T*G*C AGTGA*mA*mG*mU*mG*mA594CTACTGCACTCCAGC mC*mU*mA*mC*mU*G*C*A*C*T*C*C*A*G*C CTGGA*mC*mU*mG*mG*mA595GGCAAGACCCTGTCT mG*mG*mC*mA*mA*G*A*C*C*C*T*G*T*C*T CAATA*mC*mA*mA*mU*mA596CAGTGCCACTTCTTG mC*mA*mG*mU*mG*C*C*A*C*T*T*C*T*T*G CCATC*mC*mC*mA*mU*mC597GGAATACTGAGCTCA mG*mG*mA*mA*mU*A*C*T*G*A*G*C*T*C*A GAAAG*mG*mA*mA*mA*mG598GGAGATGGAGCCACA mG*mG*mA*mG*mA*T*G*G*A*G*C*C*A*C*A TTTGT*mU*mU*mU*mG*mU599GGATCACTAGTGCAG mG*mG*mA*mU*mC*A*C*T*A*G*T*G*C*A*G GAGAA*mG*mA*mG*mA*mA600CTGAAGGCAAAATGC mC*mU*mG*mA*mA*G*G*C*A*A*A*A*T*G*C TTGTA*mU*mU*mG*mU*mA601GGTTTTGCCTCTACT mG*mG*mU*mU*mU*T*G*C*C*T*C*T*A*C*T TTGTA*mU*mU*mG*mU*mA602GCACTTGTTCACCTA mG*mC*mA*mC*mU*T*G*T*T*C*A*C*C*T*A CCAGT*mC*mC*mA*mG*mU603GGATTTCACCATGTT mG*mG*mA*mU*mU*T*C*A*C*C*A*T*G*T*T GGTCA*mG*mG*mU*mC*mA604ACTCCTGACCTCAGG mA*mC*mU*mC*mC*T*G*A*C*C*T*C*A*G*G 6054934-0525-6010.2Page 67 of 105 065715-000129WOPTTGATC *mU*mG*mA*mU*mC GCAGTTGTATCTGTG mG*mC*mA*mG*mU*T*G*T*A*T*C*T*G*T*G TATTG*mU*mA*mU*mU*mG606GCTAAGGCTGCCAAA mG*mC*mU*mA*mA*G*G*C*T*G*C*C*A*A*A GCAAA*mG*mC*mA*mA*mA607GAGGCCTCTGTTTGA mG*mA*mG*mG*mC*C*T*C*T*G*T*T*T*G*A CTTGT*mC*mU*mU*mG*mU608GGTCTTGCTCTGTCA mG*mG*mU*mC*mU*T*G*C*T*C*T*G*T*C*A CCCAG*mC*mC*mC*mA*mG609TGAGGTGTAGTGGCA mU*mG*mA*mG*mG*T*G*T*A*G*T*G*G*C*A AGATT*mA*mG*mA*mU*mU610CAGCTCACTGCAGCT mC*mA*mG*mC*mU*C*A*C*T*G*C*A*G*C*T TTGAC*mU*mU*mG*mA*mC611GGATTACAGGCATGA mG*mG*mA*mU*mU*A*C*A*G*G*C*A*T*G*A GCCAC*mG*mC*mC*mA*mC612GCCTACTTTATGGCT mG*mC*mC*mU*mA*C*T*T*T*A*T*G*G*C*T CTTTA*mC*mU*mU*mU*mA613GCCACCCATTCTACA mG*mC*mC*mA*mC*C*C*A*T*T*C*T*A*C*A GTGTT*mG*mU*mG*mU*mU614AAGTGATCCTCCTGC mA*mA*mG*mU*mG*A*T*C*C*T*C*C*T*G*C CTTGG*mC*mU*mU*mG*mG615GAGCCACTGTGTCTG mG*mA*mG*mC*mC*A*C*T*G*T*G*T*C*T*G GATTA*mG*mA*mU*mU*mA616GGATTTGCTTGGTAG mG*mG*mA*mU*mU*T*G*C*T*T*G*G*T*A*G GCATT*mG*mC*mA*mU*mU617GCTAACACAGGAAAA mG*mC*mU*mA*mA*C*A*C*A*G*G*A*A*A*A GAGCA*mG*mA*mG*mC*mA618GTAGTGAAGATGTTT mG*mU*mA*mG*mU*G*A*A*G*A*T*G*T*T*T GATGG*mG*mA*mU*mG*mG619GGTGTCACCAGAGAG mG*mG*mU*mG*mU*C*A*C*C*A*G*A*G*A*G CATTA*mC*mA*mU*mU*mA620GGTGGTGAACCTCTG mG*mG*mU*mG*mG*T*G*A*A*C*C*T*C*T*G GTTCT*mG*mU*mU*mC*mU621GCACATTGCTGTGAC mG*mC*mA*mC*mA*T*T*G*C*T*G*T*G*A*C CTTCA*mC*mU*mU*mC*mA622CTGCTAAACTCCAAG mC*mU*mG*mC*mU*A*A*A*C*T*C*C*A*A*G TACAT*mU*mA*mC*mA*mU623GGAGCCCTGGTTTTA mG*mG*mA*mG*mC*C*C*T*G*G*T*T*T*T*A TAAAC*mU*mA*mA*mA*mC624GGCTTATACAGTCTA mG*mG*mC*mU*mU*A*T*A*C*A*G*T*C*T*A GTCCA*mG*mU*mC*mC*mA625GCACAGCTGAAGTGT mG*mC*mA*mC*mA*G*C*T*G*A*A*G*T*G*T AACTT*mA*mA*mC*mU*mU626CCTGGAGTGTTGACT mC*mC*mU*mG*mG*A*G*T*G*T*T*G*A*C*T GGCAA*mG*mG*mC*mA*mA627GGCAATTAATTGGCT mG*mG*mC*mA*mA*T*T*A*A*T*T*G*G*C*T CCATT*mC*mC*mA*mU*mU628GGACTTCCTCCTGAA mG*mG*mA*mC*mU*T*C*C*T*C*C*T*G*A*A CTCTG*mC*mU*mC*mU*mG629CTAGGTCCCAGGAAT mC*mU*mA*mG*mG*T*C*C*C*A*G*G*A*A*T 6304934-0525-6010.2Page 68 of 105 065715-000129WOPTGAAGA *mG*mA*mA*mG*mA GGTCCCAGGAATGAA mG*mG*mU*mC*mC*C*A*G*G*A*A*T*G*A*A GACCT*mG*mA*mC*mC*mU631GGAATGAAGACCTCT mG*mG*mA*mA*mU*G*A*A*G*A*C*C*T*C*T CACTT*mC*mA*mC*mU*mU632CCTCTCACTTGACTA mC*mC*mU*mC*mU*C*A*C*T*T*G*A*C*T*A TGCCC*mU*mG*mC*mC*mC633CTCTCACTTGACTAT mC*mU*mC*mU*mC*A*C*T*T*G*A*C*T*A*T GCCCC*mG*mC*mC*mC*mC634GGAAGGATGTTTGGA mG*mG*mA*mA*mG*G*A*T*G*T*T*T*G*G*A CCAGG*mC*mC*mA*mG*mG635GAAGGATGTTTGGAC mG*mA*mA*mG*mG*A*T*G*T*T*T*G*G*A*C CAGGA*mC*mA*mG*mG*mA636AAGGATGTTTGGACC mA*mA*mG*mG*mA*T*G*T*T*T*G*G*A*C*C AGGAA*mA*mG*mG*mA*mA637ATGTTTGGACCAGGA mA*mU*mG*mU*mU*T*G*G*A*C*C*A*G*G*A AGAGC*mA*mG*mA*mG*mC638GGACCAGGAAGAGCT mG*mG*mA*mC*mC*A*G*G*A*A*G*A*G*C*T GTATT*mG*mU*mA*mU*mU639CACAGTGCTGGATTA mC*mA*mC*mA*mG*T*G*C*T*G*G*A*T*T*A TTACA*mU*mU*mA*mC*mA640GTCAAGATCTGAGTG mG*mU*mC*mA*mA*G*A*T*C*T*G*A*G*T*G CTTTA*mC*mU*mU*mU*mA641GATCTGAGTGCTTTA mG*mA*mU*mC*mU*G*A*G*T*G*C*T*T*T*A CTCCA*mC*mU*mC*mC*mA642CCCAAATTCTTTATA mC*mC*mC*mA*mA*A*T*T*C*T*T*T*A*T*A GCTCC*mG*mC*mU*mC*mC643TTATAGCTCCAAGCT mU*mU*mA*mU*mA*G*C*T*C*C*A*A*G*C*T CCATT*mC*mC*mA*mU*mU644CATTGGAATTGAAGG mC*mA*mU*mU*mG*G*A*A*T*T*G*A*A*G*G TTACC*mU*mU*mA*mC*mC645GGAATTGAAGGTTAC mG*mG*mA*mA*mU*T*G*A*A*G*G*T*T*A*C CCTAC*mC*mC*mU*mA*mC646GAAGGTTACCCTACC mG*mA*mA*mG*mG*T*T*A*C*C*C*T*A*C*C TGTAA*mU*mG*mU*mA*mA647

[0140] In various embodiments, inhibitors of KCTD20 are ASOs of 20-nucleotide long, having 40%-60% GC content, no C followed by a G, and no more than three consecutive G’s. In some embodiments, the ASO sequences are fully complimentary to the target gene sequence, i.e., no degenerative or alternate sequences. In some embodiments, 2’OMe modification is added to reduce toxicity of the oligonucleotide. In preferably embodiments, inhibitors of KCTD20 include any one, or two or more combinations, of ASOs selected from a group that collectively target the full spread of human KCTD20 gene.

[0141] In further embodiments, inhibitors of KCTD20 for use in methods disclosed herein are oligonucleotides having at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%,4934-0525-6010.2Page 69 of 105 065715-000129WOPT91%, 90%, 85%, 80%, 75%, 70%, 65%, or 60% sequence identity to a sequence shown in Table 1 or Table 2.

[0142] Some embodiments provide a method of treating a subject in need thereof, or treating, inhibiting, and reducing the severity of a neurodegenerative disease in a subject, which includes administering to the subject a composition comprising, or consisting essentially of, one or more ASOs targeting 3’UTR of KCTD20 as shown in Table 1.

[0143] Some embodiments provide a method of treating a subject in need thereof, or treating, inhibiting, and reducing the severity of a neurodegenerative disease in a subject, which includes administering to the subject a composition comprising, or consisting essentially of, one or more ASOs targeting KCTD20 as shown in Table 2.

[0144] Some embodiments provide a method of treating a subject in need thereof, or treating, inhibiting, and reducing the severity of a neurodegenerative disease in a subject, which includes administering to the subject a composition comprising, or consisting essentially of, one or more ASOs as shown in Table 1 and / or Table 2.

[0145] Exemplary neurodegenerative diseases or conditions include, but are not limited to, frontotemporal dementia (FTD), motor neuron disease / amyotrophic lateral sclerosis (MND / ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), progressive supranuclear palsy, and tauopathies.

[0146] In various embodiments, the present invention provides a pharmaceutical composition. The pharmaceutical composition includes an inhibitor of KCTD20 (for example, one or more oligonucleotides that bind to at least a portion of the DNA or mRNA of human gene KCTD20 and lead to cleavage, degradation or blocking of translation thereof). The pharmaceutical compositions according to the invention can contain any pharmaceutically acceptable excipient. “Pharmaceutically acceptable excipient” means an excipient that is useful in preparing a pharmaceutical composition that is generally safe, non- toxic, and desirable, and includes excipients that are acceptable for veterinary use as well as for human pharmaceutical use. Such excipients may be solid, liquid, semisolid, or, in the case of an aerosol composition, gaseous. Examples of excipients include but are not limited to starches, sugars, microcrystalline cellulose, diluents, granulating agents, lubricants, binders, disintegrating agents, wetting agents, emulsifiers, coloring agents, release agents, coating agents, sweetening agents, flavoring agents, perfuming agents, preservatives, antioxidants, plasticizers, gelling agents, thickeners, hardeners, setting agents, suspending agents, surfactants, humectants, carriers, stabilizers, and combinations thereof.4934-0525-6010.2Page 70 of 105 065715-000129WOPT

[0147] In various embodiments, the pharmaceutical compositions according to the invention may be formulated for delivery via any route of administration. “Route of administration” may refer to any administration pathway known in the art, including but not limited to intracerebroventricular, intrathecal, parenteral, aerosol, nasal, oral, transmucosal, transdermal, or enteral. “Parenteral” refers to a route of administration that is generally associated with injection, including intraorbital, infusion, intraarterial, intracapsular, intracardiac, intradermal, intramuscular, intraperitoneal, intrapulmonary, intraspinal, intrasternal, intrathecal, intrauterine, intravenous, subarachnoid, subcapsular, subcutaneous, transmucosal, or transtracheal. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection, or as lyophilized powders. Via the parenteral route, the compositions may be in the form of solutions or suspensions for infusion or for injection. Via the enteral route, the pharmaceutical compositions can be in the form of tablets, gel capsules, sugar-coated tablets, syrups, suspensions, solutions, powders, granules, emulsions, microspheres or nanospheres or lipid vesicles or polymer vesicles allowing controlled release. Typically, the compositions are administered by injection. Methods for these administrations are known to one skilled in the art.

[0148] Formulations suitable for parenteral administration include aqueous and non- aqueous, isotonic sterile injection solutions, which may contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient, and aqueous and non-aqueous sterile suspensions that may include suspending agents, solubilizers, thickening agents, stabilizers, and preservatives. The formulations may be presented in unit-dose or multi-dose sealed containers, such as ampules and vials, and may be stored in a freeze-dried (lyophilized) condition requiring only the addition of the sterile liquid excipient, for example, water, for injections, immediately prior to use. Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules, and tablets.

[0149] The pharmaceutical compositions according to the invention can contain any pharmaceutically acceptable carrier. “Pharmaceutically acceptable carrier” as used herein refers to a pharmaceutically acceptable material, composition, or vehicle that is involved in carrying or transporting a compound of interest from one tissue, organ, or portion of the body to another tissue, organ, or portion of the body. For example, the carrier may be a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, or a combination thereof. Each component of the carrier must be “pharmaceutically acceptable” in that it must be compatible with the other ingredients of the formulation. It must also be suitable for use in4934-0525-6010.2Page 71 of 105 065715-000129WOPTcontact with any tissues or organs with which it may come in contact, meaning that it must not carry a risk of toxicity, irritation, allergic response, immunogenicity, or any other complication that excessively outweighs its therapeutic benefits.

[0150] Additional therapeutic agent(s) may be administered simultaneously or sequentially with the disclosed one or more antisense or inhibitory nucleic acids and compositions.

[0151] Various embodiments provide a method of treating a subject with a neurodegenerative disease or condition, comprising administering to the subject an effective amount of an inhibitor of potassium channel tetramerization domain containing 20 (KCTD20), such as one or more ASOs disclosed herein. Other embodiments provide a method of treating a subject in need thereof, comprising administering to the subject an effective amount of an inhibitor of KCTD20, such as one or more disclosed ASOs herein. Further embodiments provide a method for treating or reducing the severity or likelihood of a neurodegenerative disease or condition, comprising administering to the subject an effective amount of an KCTD20 ASO or a pharmaceutical composition thereof. In some embodiments, the method is for treating one or more of sporadic ALS, C9orf72-FTD / ALS, and TARDBP- related ALS-FTD in a subject in need thereof.

[0152] In some embodiments, a method of treating a subject in need thereof comprises administering to the subject a therapeutically effective amount of a pharmaceutical composition comprising an ASO disclosed herein and a pharmaceutically acceptable carrier, diluent or excipient. In some embodiments, the method does not include administering to the subject an inhibitor of mTOR, rapamycin, or LY-294002. In some embodiments, the method does not include administering to the subject a sphingomyelinase 2 inhibitor or a neutral sphingomyelinase 2 (nSMase2) inhibitor, wherein the nSMase2 inhibitor optionally comprises GW4869. In some embodiments, an ASO that targets human KCTD20 gene is not used with an nSMase2 inhibitor. In some embodiments, the method does not include administering to the subject an inhibitor of SMPD3 or an inhibitor of MCOLN1; for example, the method does not include administering to the subject an ASO that targets SMPD3 or an ASO that targets MCOLN1. In some embodiments, an ASO that targets human KCTD20 gene is not used with an ASO that targets SMPD3 or an ASO that targets MCOLN1. In some embodiments, an ASO disclosed herein is not used with eltrombopag. In some embodiments, an ASO disclosed herein (e.g., modulating KCTD20) is not used in combination with eltrombopag, neither concurrently nor sequentially,4934-0525-6010.2Page 72 of 105 065715-000129WOPT

[0153] In some implementations, a method of treating a subject in need thereof consists of administering to the subject a pharmaceutical composition that has a therapeutically effective amount of a KCTD20 ASO and one or more pharmaceutically acceptable excipients. In further implementations, a method of treating a subject in need thereof includes administering an effective amount of A KCTD20 ASO in one dose, which may have one exposure or a number of exposures within a short amount of time (e.g., 2 days, 3 days, 4 days, or a week), and the subject experiences beneficial results and so the method does not include or require a second dose.

[0154] In some embodiments, a subject in need of the treatment is a human diagnosed with a neurodegenerative disease or condition, or a sign of a neurodegenerative disease or condition. In some embodiments, a subject in need of the treatment is a human diagnosed as at risk of developing a neurodegenerative disease or condition. In some embodiments, a subject in need of the treatment is a human suffering from a neurodegenerative disease or condition. In some embodiments, a subject in need of the treatment is a human being at an age of over 50 years old, over 55 years old, over 60 years old, over 65 years old, over 70 years old, over 80 years old, or over 90 years old.

[0155] Methods of screening for an oligonucleotide are also provided, which include contacting an oligonucleotide of interest with an organoid derived from induced-pluripotent stem cells (iPSCs) from a patient with a neurodegenerative disease, and performing one or more of assaying transcription or expression level of KCTD20, assaying expression levels of an AKT signaling, an mTOR signaling, or both, assaying level of transcription factor EB (TFEB) nuclear localization, assaying expression levels of p62, LC3, or both, assaying level of autolysosome formation, and assaying level of tau oligomerization.

[0156] In various embodiments of the screening methods, an decreased transcription or expression level of KCTD20, an increased expression level of the AKT signaling and / or the mTOR signaling, an increased level of TFEB nuclear localization, an increased expression level of p62 and / or LC3, an increased level of autolysosome formation, and / or a decreased level of tau oligomerization, in the presence of the oligonucleotide of interest relative to that before the contact thereof, indicates that the oligonucleotide of interest is a candidate drug for treating a neurodegenerative disease. Table 3. Additional exemplary antisense oligonucleotide sequences. ID Gene Nucleobase Sequence ASO format4934-0525-6010.2Page 73 of 105 065715-000129WOPTMs NC NC CCTATAGGACTATCCAG / 52MOErC / * / i2MOErC / / i2MOErT / / i2 GAA (SEQ ID NO:16746) MOErA / / i2MOErT / A*G*G*A*C*T* A*T*C*C* / i2MOErA / / i2MOErG / / i2 MOErG / * / i2MOErA / * / 32MOErA / msKct Kctd20 GCACAGACTGTTTCTCT / 52MOErG / * / i2MOErC / / i2MOErA / / i2 d20- GAC (SEQ ID NO:16747) MOErC / / i2MOErA / G*A*C*T*G*T* ASO-1 T*T*C*T* / i2MOErC / / i2MOErT / / i2M OErG / * / i2MOErA / * / 32MOErC / msKct Kctd20 TTACCAGAGAGCTTCTT / 52MOErT / * / i2MOErT / / i2MOErA / / i2 d20- CAC (SEQ ID NO:16748) MOErC / / i2MOErC / A*G*A*G*A*G* ASO-2 C*T*T*C* / i2MOErT / / i2MOErT / / i2M OErC / * / i2MOErA / * / 32MOErC / msKct Kctd20 AACAGTTCACTTCCTCT / 52MOErA / * / i2MOErA / / i2MOErC / / i2 d20- CCT (SEQ ID NO:16749) MOErA / / i2MOErG / T*T*C*A*C*T*T ASO-3 *C*C*T* / i2MOErC / / i2MOErT / / i2M OErC / * / i2MOErC / * / 32MOErT / ASO1 KCTD20 TCTCCTACTCAAGGTGA mU*mC*mU*mC*mC*T*A*C*T*C* GAC (SEQ ID NO:16750) A*A*G*G*T*mG*mA*mG*mA*mC ASO2 KCTD20 GCAGAAAGGAAAAGGT mG*mC*mA*mG*mA*A*A*G*G*A TAGG (SEQ ID NO:16751) *A*A*A*G*G*mU*mU*mA*mG*m G ASO4 KCTD20 UGGACATTCTGAATGA mU*mG*mG*mA*mC*A*T*T*C*T* GACC (SEQ ID NO:16752) G*A*A*T*G*mA*mG*mA*mC*mC Table 4. Exemplary guide RNA sequences. ID Gene Sequence 0855 KCTD20 GCGCCGCGCTGTCCCGCTCG (SEQ ID NO:16753) 1064 KCTD20 GCTGGGCGCGAGGAACCTGT (SEQ ID NO:16754) 2482 FIGF GGAAAGCTGGAACCCAACTC (SEQ ID NO:16755) 5010 SMC6 GTGACCGCCGCTGGCCCCCT (SEQ ID NO:16756) EXAMPLES

[0157] The following examples are provided to better illustrate the claimed invention and are not to be interpreted as limiting the scope of the invention. To the extent that specific materials are mentioned, it is merely for purposes of illustration and is not intended to limit4934-0525-6010.2Page 74 of 105 065715-000129WOPTthe invention. One skilled in the art may develop equivalent means or reactants without the exercise of inventive capacity and without departing from the scope of the invention. Example 1. KCTD20 suppression mitigates excitotoxicity in tauopathy patient organoids

[0158] Glutamate excitotoxicity promotes tau oligomerization and neurodegeneration

[0159] To identify key regulators of neuronal excitotoxicity, we used cortical cerebral organoids to model excitotoxicity in vitro. We generated iPSC-derived human cortical organoids as described by Pasca et al. Nat. Methods 12, 671–678 (2015). Cortical organoids contain a mix of deep- and superficial-layer neurons, radial glia, and astrocytes with extended culture and have been shown to be electrically active. To model excitotoxicity, we treated 2- month-old organoids derived from neurologically normal individuals with glutamate and tracked the survival of Synapsin1::eGFP (SYN1::eGFP) lentivirus-infected neurons over 7 days (Figure 1A). We observed a dose-dependent detrimental effect on neuron survival, with approximately 50% of tracked neurons degenerating following 5 mM glutamate treatment, a concentration similar to ranges observed in the synaptic cleft during action potentials (Figure 1B). This effect was consistent between organoid replicates from the same healthy iPSC line and also between different healthy donors (Figure 1C). Co-treatment with ionotropic glutamate receptor inhibitors nimodipine, cyanquixaline (CNQX), and MK-801 reduced glutamate-induced neurodegeneration, demonstrating the selectivity of this phenotype (Figure 1D). This effect was specific to glutamate, as an equal dose of 5 mM gamma-aminobutyric acid (GABA) did not affect survival of excitatory neurons (ExNs) (Figure 1H). We also observed that glutamate induced a dose-dependent increase in high-molecular-weight (HMW, >150 kDa band) oligomeric tau (T22) as early as in 48 h (Figures 1E–1F), with pronounced increases in HMW T22 species within 7 days (Figure 1G). Consistent with prior reports, we observed multiple bands for T22 and restricted all subsequent western blot quantification of T22 to the >150 kDa oligomer band. Because our western blot contained denaturing conditions, we confirmed the specificity of the T22 antibody via native dot blot under non- denaturing conditions, which shows a significant increase in antibody signal in glutamate- treated organoids (Figure 1I). This increase in oligomeric tau was in contrast to an observed decrease in intracellular phosphorylated tau in glutamate-treated organoids (Figures 1J-1K). Thus, glutamate treatment of cortical organoids induces tau pathology and causes neurodegeneration. This is similar to phenotypes observed in FTD, in which the tau-V337M mutation has been demonstrated to promote neuron death and tau oligomerization.

[0160] KCTD20 suppression mitigates glutamate-induced neurodegeneration4934-0525-6010.2Page 75 of 105 065715-000129WOPT

[0161] Although excitotoxicity has been linked to neurodegenerative diseases such as ALS / FTD and Alzheimer’s disease, the most effective genetic modifiers of this response remain unclear. To identify potential genetic modifiers of glutamate-induced neurodegeneration in neurons, we conducted a genome-wide CRISPRi screen on an enriched neuronal population (Figure 2D). In order to decrease the number of non-neuronal cells and increase the number of neurons receiving guide RNAs, we used the neurogenin 2 (NGN2)- over-expression system, which yields mature, diverse neuron types in the absence of glia (Figure 2D). NGN2-spheroids contain broad expression of neuronal markers, including MAP2 and NeuN, with an enrichment of BRN2-positive upper-layer neurons. Similar to patterned cortical organoids, NGN2-spheroids express 3R and 4R tau isoforms and do not have significantly different expression of ionotropic glutamate receptors (Figure 2E). We used a previously reported iPSC line stably expressing dCas9-BFP-KRAB and dox-inducible NGN2 constructs to generate NGN2-spheroids (n = 300) for the screen. Day 70 spheroids were transduced with a genome-wide lentiviral single-guide RNA (sgRNA) library, then divided into glutamate-treated and vehicle (PBS) control groups (Figure 2A). We isolated total DNA after 10 days of treatment with 5 mM glutamate or an equal volume of PBS, PCR- amplified the guides, and performed next-generation sequencing. We analyzed sgRNA reads using the standard MAGeCK-RRA pipeline to determine enriched and de-enriched gene targets from the glutamate-treated organoids relative to PBS controls (Figure 2B). In this analysis, enriched gene targets indicate that knockdown of a given gene improved survival of glutamate-treated neurons. Significantly enriched genes (-log10(p) > 1.3) were assessed using Gene Ontology (GO) molecular function, which identified multiple ion channel etiologies (Figure 2C). Multiple ionotropic glutamate receptor subunits were among the significant hits but were ranked below the top 50 genes, indicating that genetic inhibition of individual subunits was not the most effective approach.

[0162] We designed antisense oligonucleotides (ASOs) against the top enriched gene targets based on significance and fold-change enrichment for secondary validation to test an alternate modality of knockdown (Figures 3D-3E). We tested the top 10 genes from our screen and found that KCTD20 suppression most significantly improved the survival of glutamate-treated neurons using individually cloned sgRNAs relative to non-targeting (NT) sgRNA (Figure 3A) and using ASOs relative to NT control ASO, respectively (Figure 3B and 3E). Knock- down of KCTD20 in vehicle-treated neurologically normal organoids did not alter neuron survival (Figure 3B). We next performed TUNEL staining in NT and KCTD20 ASO glutamate-treated organoids as an orthogonal measure of neuron death. Consistent with4934-0525-6010.2Page 76 of 105 065715-000129WOPTour longitudinal survival assay, glutamate induced a significant increase in TUNEL-positive cells, which was reduced by KCTD20 suppression (Figure 3C). Because the 2-month-aged organoids used in our study are not fully electrically mature, we tested this phenotype in 6- month-aged organoids and found KCTD20 suppression also mitigated neuron death in older glutamate-treated organoids (Figure 3F). To determine whether KCTD20 suppression affects neuronal activity, we performed calcium imaging using Fluo-4 AM in ASO-treated organoids following glutamate treatment. We found that surviving neurons were electrically active and that KCTD20 ASO did not alter the peak amplitude (Figure 3G).

[0163] KCTD20 suppression reduces tau oligomerization and neurodegeneration in tau-V337M organoids

[0164] Our previous data showed that glutamate induced tau oligomerization and neurodegeneration in organoids derived from neurologically normal individuals. To determine whether tau is a driver of excitotoxic neuron death, we measured neuron survival in a MAPT knockout (MAPT KO) iPSC line. Organoids grown from this line indeed do not express tau protein but maintain expression of typical neuron markers relative to wild-type organoids (Figure 4B-4C). We found that loss of tau in the MAPT KO line prevented the majority of neuron death associated with glutamate treatment, indicating that tau pathology is responsible for much of the observed excitotoxic neuron death (Figure 4A). However, it is likely that other factors beyond tau pathology are also involved.

[0165] Next, to determine whether excitotoxicity is exacerbated in organoids harboring a tau mutation known to drive tauopathy, we generated organoids from iPSCs harboring the FTD-causing MAPT V337M mutation or a CRISPR-edited isogenic control line. We chose this mutation because iPSC neurons typically express relatively low levels of 4R tau, and the V337 residue is included in 3R tau, thus allowing for the assessment of the mutation phenotype. In addition, as opposed to many Alzheimer’s disease-associated genetic variants, which are highly expressed in non-neuronal cell types, including microglia, tau- V337M is more directly linked to neuronal biology and is capable of driving tau pathology in neurons. In the absence of stressors, tau-V337M organoids displayed elevated phosphorylated and oligomeric tau compared with CRISPR-corrected tau-V337V controls (Figure 5A). Compared with isogenic tau-V337V organoids, tau-V337M organoids showed enhanced neurodegeneration following 5 mM glutamate treatment (Figure 5B). This corresponded to previous reports, where we found enhanced neurodegeneration in three independent pairs of tau-V337M and tau-V337V lines. This effect was not due to expression levels of the NMDA subunit NR1, which was unchanged between mutant and isogenic organoids (Figure 5F).4934-0525-6010.2Page 77 of 105 065715-000129WOPTImportantly, KCTD20 ASO significantly improved neuron survival in glutamate-treated tau- V337M organoids derived from three independent donors (Figures 5C, 5G, and 5H). The effect on neuron survival was significantly greater than a MAPT ASO, which was found to be protective in tau transgenic mice and in non-human primate FTD models in both 2-month- and 10-month-aged organoids (Figures 5C, 5I, and 5J). We found a corresponding reduction in oligomeric tau in glutamate-treated organoids following KCTD20 knockdown in both tau- V337M and in neurologically normal organoids (Figures 5D and 5K). Interestingly, KCTD20 suppression did not confer a protective effect on survival following glutamate treatment in MAPT KO organoids, indicating that a reduction in tau may be part of its therapeutic mechanism (Figure 4A).

[0166] Because glutamate excitotoxicity has been described in neurodegenerative diseases beyond FTD, we tested whether KCTD20 suppression was neuroprotective in other genetic models. We found that, similar to tau-V337M, organoids grown from C9ORF72 ALS / FTD patients displayed a significant survival deficit compared with neurologically normal organoids after glutamate treatment (Figure 5L). Co-treatment with KCTD20 ASO significantly improved survival of C9ORF72 neurons (Figure 5M). Decreased survival in glutamate-treated C9ORF72 organoids was associated with dysregulated splicing of the TDP- 43 target gene STMN2, indicating that excess glutamate may induce loss of TDP-43 function in addition to tau pathology as has been observed early in AD progression (Figure 5N). Indeed, we found that glutamate induced the accumulation of mis-localized cytoplasmic TDP-43 and that KCTD20 suppression partially prevented this (Figure 5O).

[0167] Taken together, tau is a driver of excitotoxic neuron death and we found that KCTD20 suppression mitigated tau oligomerization and neurodegeneration following excitotoxic stress in multiple genetic backgrounds, including organoids harboring a tau variant known to drive tau pathology and disease, and in the context of TDP-43 proteinopathy.

[0168] Improved neuron survival following KCTD20 knockdown depends on exocytosis

[0169] Because KCTD20 suppression reduced oligomeric tau in neurons, we asked whether ASO treatment could mitigate neurodegeneration caused by another stressor capable of disrupting proteostasis, the autophagy inhibitor bafilomycin. Treatment with bafilomycin increased HMW oligomeric tau and induced a corresponding decrease in neuron survival, whereas co-treatment with KCTD20 ASO reduced neurodegeneration (Figure 5P-5Q). We observed a decrease in intracellular total tau signal as oligomeric tau increases in glutamate-4934-0525-6010.2Page 78 of 105 065715-000129WOPTtreated organoids by western blot (Figure 5E). Simultaneously, we observed increased tau in conditioned media, measured by electrochemical ELISA (Figures 5R-5S). These results indicate that KCTD20 suppression can mitigate neurodegeneration driven by mis-folded protein accumulation even if the pathology is not driven by excess glutamate.

[0170] To determine how KCTD20 suppression might alleviate misfolded protein accumulation, we performed single-cell RNA sequencing (scRNA-seq) on 3-month-aged tau- V337M organoids following glutamate treatment in the absence or addition of the KCTD20 ASO (Figure 6A). We multiplexed samples using oligonucleotide-tagged streptavidin antibodies to barcode distinct experimental conditions, and we recovered ~39,000 high- quality cells. Following sample demultiplexing, scRNA-seq normalization, reduction, and clustering, we annotated cells using reference expression data from gestational week 17 human neocortex. Similar to our longitudinal imaging results, we found that 5 mM glutamate resulted in a significant decrease in the proportion of ExNs over time in the scRNA-seq data, which was mitigated by KCTD20 ASO treatment. We next derived unsupervised cell pseudotime trajectories using Monocle 2 in ExNs. We calculated cell proportions per treatment and state and used significant marker genes (adjusted p < 0.05) to determine defining enriched biological processes in Enrichr. To identify pathways affected by KCTD20 knockdown, and taking advantage of our single-cell dataset to base our analysis on KCTD20 expression levels, we bisected all ExN neurons based on their relative expression of KCTD20 into KCTD20-low and KCTD20-high groups (relative expression<0.9 and >1.3). Genes that were upregulated in KCTD20-low neurons (average log2(fold change) > 0.25, adjusted p < 0.05), which corresponded with the neuroprotective state in our previous experiments, were used to determine enriched biological processes. GO biological processes and Elsevier pathway analysis identified pathways involving exocytosis and secretion in KCTD20-low neurons among the most significantly enriched pathways (Figures 6B and 6C). Genes involved in these pathways, including SYT1, VAMP2, SNAP25, and AP2B1, encode proteins reported to play general roles in secretion, whereas others, including RAB3A and RAB11A, are associated with lysosomal exocytosis and are collectively implicated in dementia. We hypothesized that KCTD20 suppression may therefore decrease oligomeric tau species and improve neuron survival by promoting exocytosis. This is similar to our previous findings in ALS / FTD demonstrating that upregulating unconventional protein clearance mechanisms involving exocytosis is neuro-protective, in part by clearing aggregation-prone proteins. We tested this by treating organoids with glutamate, KCTD20 ASO, and the neutral sphingomyelinase 2 (nSMase2) inhibitor GW4869, which broadly blocks exocytosis.4934-0525-6010.2Page 79 of 105 065715-000129WOPTLongitudinal tracking of neuron survival showed that inhibiting exocytosis with GW4869 abolished the neuroprotective effect of KCTD20 knockdown on neuron survival following glutamate treatment (Figure 6D). GW4869 also exacerbated oligomeric tau levels in glutamate-treated organoids (Figure 6E). Together, these results indicated that the proteostatic and neuroprotective effects of KCTD20 suppression may be dependent on exocytosis.

[0171] Protective effects of KCTD20 knockdown are mediated by lysosomal exocytosis

[0172] Inhibition of nSMase2 via GW4869 treatment broadly blocks cellular exocytosis pathways by inhibiting ceramide production. To identify which specific exocytosis mechanisms mediate the effects of KCTD20 knockdown, we employed previously validated ASOs against key targets involved in membrane fusion of multivesicular bodies (SMPD3 and VAMP7), macroautophagy and amphisome exocytosis (ATG7 and RAB8A), chaperone-mediated autophagy (HSPA8), LC3-dependent extracellular vesicle loading and secretion (NSMAF), lysosomal exocytosis (MCOLN1), and secretory autophagy (GORASP1). ASOs were treated in combination with KCTD20 ASO and glutamate and compared with the NT ASO vehicle control condition. Of the genes tested, ASOs against SMPD3 and MCOLN1 significantly reduced neuronal survival, increasing the hazard ratio (Figures 7A, 7I, and 7J). The SMPD3 gene encodes nSMase2 protein, and therefore its knockdown mimics the broad effect of GW4869 treatment. However, MCOLN1 is specifically required for lysosomal exocytosis and we therefore chose it for further validation. To test the involvement of synaptic secretion, we assayed survival by adding STXBP1 ASO. STXBP1 haploinsufficiency affects synaptic transmission and causes neurodevelopmental disease. Compared with glutamate treatment alone, neurons given a combination treatment of STXBP1 and KCTD20 ASOs and glutamate had a trend of increased survival but were not fully rescued to the level of KCTD20 ASO only, indicating that synaptic secretion may also be involved in the protective mechanism of KCTD20 suppression (Figures 7K and 7L). However, given that MCOLN1 suppression more strongly abolished the rescue conferred by KCTD20 ASO, we chose to pursue this pathway.

[0173] We confirmed that the MCOLN1 ASO abolished the protective effect of KCTD20 ASO treatment on neuron survival in the presence of excess glutamate in tau-V337M and isogenic tau-V337V organoids (Figures 7M and 7N). Interestingly, previous proximity labeling experiments identified direct intracellular interactions between tau and autophagosome-lysosome fusion sites among multiple vesicle proteins, including an4934-0525-6010.2Page 80 of 105 065715-000129WOPTenhanced interaction between mutant tau-V337M protein and the lysosomal protein LAMP1. Therefore, we hypothesized that excess glutamate treatment might lead to oligomeric tau accumulation in lysosomes, which could be secreted upon KCTD20 suppression. Indeed, we observed a statistically significant glutamate-induced increase in the co-localization of oligomeric tau and LAMP1 in neurons, as measured by Pearson’s correlation coefficient (Figure 7B). We next isolated exosomes using the ExoView R100, which uses an affinity- based chip to selectively bind exosomes and quantify biomarkers using single-particle imaging. We found that exosomes from tau-V337M organoids treated with glutamate contained both oligomeric tau and LAMP1. KCTD20 suppression significantly increased levels of secreted oligomeric tau and LAMP1 and decreased co-localization of these proteins within neurons (Figures 7B–7D). In addition to inducing oligomeric tau and lysosome secretion, an mTFP / mCherry reporter assay indicated that KCTD20 suppression helped to maintain lysosomes at an acidic pH after glutamate treatment, which alone induced a trend of increased lysosomal pH (Figure 7O). Together, these data indicate that excess glutamate leads to accumulation of oligomeric tau in lysosomes and KCTD20 suppression causes secretion of oligomeric tau-containing lysosomes from neurons.

[0174] Although little is known about KCTD20 function, it is reported to positively regulate the activity of Akt. We confirmed that KCTD20 suppression reduced activated (phosphorylated Thr308) Akt levels in glutamate-treated neurons (Figures 7P-7R). Because Akt promotes activation of mTOR via phosphorylation, and mTOR inhibition is believed to be neuroprotective, we next assessed mTOR activity following KCTD20 suppression. We found that KCTD20 suppression lowered activated mTOR (phosphorylated Ser2448) (Figure 7S).

[0175] The transcription factor EB (TFEB) is a master regulator of autophagy and lysosomal biogenesis-related genes, including MCOLN1 and LAMP1, and is regulated by Akt through mTOR- dependent and mTOR-independent mechanisms. Active TFEB increases the number of lysosomes near the plasma membrane and promotes their exocytosis by activating MCOLN1, which elevates intracellular calcium levels. We therefore aimed to determine whether KCTD20 suppression affects TFEB expression or activity. We measured the nuclear localization of TFEB as a proxy for its function and found that glutamate induced a reduction in nuclear TFEB localization, indicating decreased transcriptional activity, which was reversed with KCTD20 ASO (Figure 7E). Similarly, glutamate alone significantly increased TFEB phosphorylation (p-TFEB Ser142), which inhibits its transcriptional activity, and we found that co-treatment with KCTD20 ASO decreased p-TFEB levels (Figure 7F). KCTD204934-0525-6010.2Page 81 of 105 065715-000129WOPTsuppression elevated but did not significantly increase TFEB mRNA levels, although it did increase LAMP1 and MCOLN1 expression (Figure 7T). These data indicate that KCTD20 suppression helps to maintain TFEB activity during excitotoxic stress and supports neuron survival by increasing lysosomal exocytosis to clear oligomeric tau (Figure 7G). We tested whether TFEB was required for this mechanism by combining KCTD20 suppression with glutamate and eltrombopag, a chemical TFEB inhibitor. We found that TFEB inhibition alone did not affect neuron survival but that eltrombopag indeed abolished the protective effect of KCTD20 knockdown (Figure 7H).

[0176] Kctd20 knockdown reduces tau pathology and improves neuron survival in vivo using MAPT transgenic mice

[0177] Given that KCTD20 suppression in vitro reduced tau pathology and neurodegeneration, we sought to determine whether Kctd20 suppression could alleviate tau pathology and neurodegeneration in wild-type C57BL / 6J mice and in a tauopathy transgenic mouse model. To achieve widespread ASO delivery to the central nervous system, we performed intracerebroventricular (i.c.v.) injection of Kctd20 ASO in wild-type mice at postnatal day 1 (P1), resulting in a significant reduction of Kctd20 protein (Figures 8J–8L). We found that NT ASO-injected wild-type mice given a subsequent i.c.v. injection of 40 nmol glutamate showed increased p-tau (Ser202 / Thr205) in the cortex at P5, whereas Kctd20 ASO administration prevented this p-tau increase (Figures 8M). Similar to our in vitro experiments, glutamate treatment decreased nuclear TFEB, which was prevented by the Kctd20 ASO (Figure 8N). Thus, in glutamate-treated wild-type mice, we observed results similar to those in our in vitro experiments with respect to tau pathology and TFEB localization. We therefore explored the effects of Kctd20 suppression in mice with tau pathology driven by MAPT P301S overexpression.

[0178] Previous studies indicate that transgenic MAPT P301S (tau-P301S) mice, a tauopathy model overexpressing humanized mutant tau associated with FTD, accumulate significant tau pathology by 5 months, with a corresponding reduction in upper-layer cortical neurons. Indeed, in the absence of glutamate treatment we observed increased oligomeric tau staining in the cortices of 6-month-old tau-P301S mice relative to wild-type animals (Figure 8A). We confirmed that MAPT transgenic mice displayed a neurodegeneration phenotype, as evidenced by decreased numbers of NeuN-positive cells in the cortex relative to wild-type mice (Figure 8B). We also performed immunostaining for TFEB and found that nuclear TFEB was significantly reduced in MAPT P301S mice compared with wild type (Figure 8).4934-0525-6010.2Page 82 of 105 065715-000129WOPT

[0179] To test whether Kctd20 suppression could alleviate these changes, we performed i.c.v. injection of 500ug Kctd20 or NT control ASO in 5-month-old tau-P301S mice (Figure 8D). A single Kctd20 ASO injection significantly lowered Kctd20 within 5 days, and this change was sustained over 4 weeks until tissue was collected (Figures 8E and 8F). We found that Kctd20 ASO-injected transgenic mice displayed significantly reduced levels of oligomeric tau in the cortex and hippocampus compared with mice treated with NT ASO (Figures 8G and 8O). We also found a reduction in p-tau following Kctd20 suppression (Figures 8P and 8Q). Similar to our in vitro data, tau-P301S mice displayed increased nuclear TFEB localization when treated with Kctd20 ASO, indicative of greater TFEB activity (Figure 8H). We also used rotarod to assess motor function in mice injected with ASO at 4 months. MAPT mice injected with the NT ASO showed a significant motor deficit relative to wild-type mice by 6 months, whereas treatment with Kctd20 ASO prevented a significant decline, appearing to slow progression of this phenotype (Figure 8R). However, Kctd20 ASO did not significantly improve motor performance in MAPT mice relative to NT ASO-treated mice. Nevertheless, we found that Kctd20 knockdown significantly increased the number of NeuN-positive neurons in the cortices of tau-P301S mice (Figure 8I). Collectively, these data indicate that Kctd20 suppression can reduce tau pathology and improve neuron survival in vivo.

[0180] Overall, Glutamate-induced excitotoxicity has long been appreciated to contribute to the pathogenesis of multiple tauopathies, including Alzheimer’s disease and FTD, in which mutations such as tau-V337M have been shown to increase neuron excitability. However, how glutamate signaling drives neuron degeneration and how to most effectively mitigate these mechanisms are less clear. Many previous excitotoxicity studies have focused on neurotoxic effects of mitochondrial damage. Here, we show that glutamate is a driver for key aspects of tauopathies, promoting oligomeric tau formation and neurodegeneration in a dose-dependent manner. In addition, these effects are enhanced in neurons harboring a tau-V337M mutation. Importantly, our data using MAPT KO organoids demonstrate that tau is essential for glutamate-induced neuron death, as neurons lacking tau are dramatically less susceptible to glutamate compared with tau-expressing neurons. However, given our observation of mis-localized TDP-43, in addition to the existing knowledge of mechanisms underlying excitotoxicity, it is likely that tau is one driver of excitotoxic neuron death and that additional mechanisms are also involved in concert with genetic pre-disposition that may influence the impact of distinct protein pathologies. Although neurofibrillary tangles (NFTs) are pathologic hallmarks of neurodegenerative4934-0525-6010.2Page 83 of 105 065715-000129WOPTdiseases, including FTD, evidence indicates that memory loss and neurodegeneration can be dissociated from NFTs. Instead, tau oligomers are hypothesized to be the more toxic species and have been shown to accumulate in tauopathy models (including tau-V337M) and cause synaptic dysfunction, memory loss, and neurotoxicity in vitro and in vivo. In our system, intracellular oligomeric tau correlates with neurodegeneration following glutamate stress. Moreover, we observe reduced levels of multiple epitopes of phosphorylated tau and total tau in glutamate-treated neurons, which may relate to conflicting findings of increased and decreased phosphorylated tau in a mouse model of glutamate receptor inhibition. Our model highlights the role of excess glutamate in promoting tau protein oligomerization in addition to its effects on mitochondria function.

[0181] Our organoid excitotoxicity model is scalable, allowing for genome-wide screening to help address an urgent need for novel therapeutic targets. Although previous drug targets have aimed to mitigate glutamate signaling, they either have limited efficacy or have failed in clinical trials, possibly because directly limiting the activity of a key neurotransmission has negative consequences. One such strategy to directly inhibit NMDA receptors caused severe side effects, including cataplexy, motor and memory deficits, and reduced consciousness. Using a CRISPRi platform, we performed a phenotypic screen to identify gene targets that could mitigate excitotoxic neuron death, with secondary assays to assess associated tau pathology downstream of glutamate receptor activation. Multiple ionotropic glutamate receptor subunits were represented in the enriched genes but were not among the top significant hits, indicating that genetic inhibition of individual subunits is not the most effective approach. Given the prominent role of glutamate across multiple neurodegenerative diseases, we hypothesized that the screen may identify targets that may be widely applicable. Suppression of one of our top screen candidates, KCTD20, significantly improved survival of neurologically normal, tau-V337M FTD, and C9ORF72 ALS / FTD neurons following glutamate treatment, in part by reducing oligomeric tau levels. Although FTD and ALS fall within clinical disease spectra with overlapping phenotypes, the primary pathology and affected regions differ, with TDP-43 as the predominant pathologic protein in up to 97% of ALS cases whereas tau and TDP-43 cases are roughly equal in FTD. We find that excess glutamate also promotes TDP-43 loss of function, evidenced by its cytoplasmic mis-localization and increased levels of mis- spliced STMN2 cryptic transcripts, similar to clinical data of early molecular changes associated with tauopathies. Our STMN2 cryptic transcript data indicates that KCTD20 ASO did not rescue TDP-43 loss of function. Our mechanism of action indicates that pathologic TDP-43 may be secreted, consistent with our4934-0525-6010.2Page 84 of 105 065715-000129WOPTfinding of reduced cyto-plasmic TDP-43, which would confer a protective effect on neuron survival without restoring endogenous TDP-43 splicing function similar to previous data antagonizing TDP-43 phase separation. It is notable that KCTD20 suppression was also neuroprotective in the C9ORF72 genetic background associated with significant TDP-43 pathology, indicating that its therapeutic effects may be applicable to a range of proteinopathies associated with excitotoxicity. Similarly, in transgenic tau-P301S mice, which display elevated oligomeric tau staining and neurodegeneration compared with wild- type animals, we found that Kctd20 suppression improved neuron survival, with a corresponding reduction in oligomeric tau. Although Kctd20 ASO did not significantly improve motor function relative to NT ASO in MAPT mice, it did prevent a significant decline in rotarod performance compared with wild-type mice, indicating that disease progression may be slowed.

[0182] The KCTD family of proteins are primarily soluble non-channel proteins with a common BTB domain responsible for E3 ligase and transcriptional activity. However, KCTD20 is relatively isolated from other KCTD paralogs based on amino acid sequence identity, with its only reported function as an allosteric regulator of Akt activity, along with predicted involvement in Akt / mTOR signaling. We confirmed that KCTD20 suppression reduced Akt and mTOR activation in glutamate-treated organoids and hypothesized that, because Akt / mTOR inhibition is strongly linked with protein clearance pathways in neurodegenerative diseases, in part through TFEB activation, this may provide a link to explain the reduction in oligomeric tau species observed with KCTD20 knockdown. We found that an alternate proteostasis stressor, bafilomycin, increased oligomeric tau and neurodegeneration, which could be reversed by KCTD20 ASO. This indicates that the effects of KCTD20 knockdown may not be strictly limited to excitotoxicity but may generally serve as a strategy to reduce toxic protein inclusions, alluding to its efficacy in patient lines dominated by either tau or TDP-43 pathology. Interestingly, a genome-wide association study (GWAS) linked KCTD20 to cocaine addiction, a pathway implicated with excitatory glutamatergic signaling, providing additional evidence of its relationship to this pathway. Kctd20 was also found to be elevated in synaptoneurosomes of alcohol-treated mice, and further study of its role in addiction would be of interest. Additionally, KCTD family proteins, including KCTD7, contain structural domains of a potassium channel that could affect neuronal activity. However, KCTD20 has not been reported to impact potassium conductance, and our data indicates that neuronal activity is not changed in glutamate-treated organoids, with or without KCTD20 ASO.4934-0525-6010.2Page 85 of 105 065715-000129WOPT

[0183] Using scRNA-seq, we found that neurons with low KCTD20 expression had differential gene expression enriched for exocytosis and secretion pathways. We found that co-treatment of KCTD20 ASO with SMPD3 and MCOLN1 ASOs negated the neuroprotective effects of KCDT20 knockdown alone. SMPD3 encodes nSMase2 protein and, therefore, serves as an orthogonal validation to GW4869 treatment. However, MCOLN1 plays an important role in lysosomal exocytosis by regulating local Ca2+levels to promote membrane fusion between lysosomes and the plasma membrane. We also found that co-treatment of STXBP1 and KCTD20 ASO in the presence of glutamate induced a trend of increased survival but did not rescue neurons to the level of KCTD20 ASO alone, indicating a potential additional involvement of synaptic secretion in the clearance of toxic protein species. Based on the following data that (1) STXBP1 may also participate in other secretion pathways via SNAP25-SNARE complex interactions, (2) KCTD20 suppression did not affect neuronal calcium flux, and (3) MCOLN1 suppression fully blocked the effect of KCTD20 ASO, we pursued lysosomal exocytosis as the most promising mechanism of action.

[0184] TFEB is a primary regulator of lysosomal exocytosis, upregulating key genes including MCOLN1 and the lysosomal protein LAMP1, to promote clearance of proteins, including tau. Indeed, activation of lysosomal exocytosis via TFEB has been shown to reduce tau pathology in vivo without cross-seeding tau aggregates, potentially via glymphatic clearance. Tau protein is also reported to localize to autophagosome-lysosome fusion sites with an enriched interaction between LAMP1 and tau-V337M relative to wild-type tau, providing explanation for how tau may accumulate in lysosomal vesicles. Similar strategies that activate lysosomal exocytosis using an MCOLN1 agonist have been shown to mitigate a- synuclein toxicity in Parkinson’s disease neurons, alluding to the broad therapeutic potential of targeting this pathway. We found that glutamate induced a reduction in TFEB activity based on its nuclear localization and phosphorylation state and that KCTD20 suppression reversed this in iPSC cerebral organoids as well as in glutamate-injected mice. Additionally, unstressed MAPT P301S mice exhibit reduced nuclear TFEB localization relative to wild-type animals, which was increased following injection with Kctd20 ASO. It is possible that KCTD20 affects TFEB activity via its effect on Akt through an mTOR-dependent or - independent mechanism. Regardless, we found that TFEB activity was required for improved neuron survival following KCTD20 suppression, given that chemical inhibition of TFEB together with KCTD20 ASO caused neurons to degenerate more rapidly and to the level of glutamate-only-treated neurons, which indicates that TFEB mediates the therapeutic effects of KCTD20 suppression. Importantly, we found that secreted LAMP1 and oligomeric tau in4934-0525-6010.2Page 86 of 105 065715-000129WOPTpurified exosome fractions were increased following KCTD20 suppression, providing evidence that lysosomal secretion is upregulated and that tau oligomers are removed from the cell in part by this mechanism. Based on our data showing upregulation of LAMP1 and MCOLN1 following KCTD20 ASO treatment, it is possible that KCTD20 knockdown primes lysosomal exocytosis, which, in the context of an excitotoxic calcium influx, provides the biochemical means to promote fusion of lysosomes with the plasma membrane and remove protein cargo from the cell. This may relate to previous findings that lysosomal exocytosis is triggered by calcium influx during neuron firing. Further experiments may facilitate demonstrating that KCTD20-mediated TFEB activity directly enhances lysosomal exocytosis. Together, these data point toward enhancing pathways of protein clearance as an important therapeutic strategy following excitotoxic stress, rather than aiming to limit glutamate channel activity.

[0185] Collectively, we describe a human organoid model of excitotoxicity to test genetic modifiers of injury in healthy and diseased genetic backgrounds. This model recapitulates several key tauopathy phenotypes in neurologically normal organoids, while enhancing them in FTD-patient-derived organoids. We show that KCTD20 suppression is neuroprotective in non-diseased, tau-V337M, and C9ORF72 organoids and in vivo using an established tauopathy mouse model, in part by clearing tau oligomers via lysosomal exocytosis. We conceive that KCTD20 suppression is useful in treating related neurodegenerative diseases and lysosomal storage disorders. Our in vivo data show that a single ASO injection is effective at reducing gene expression for up to 1 month. It is also conceived that continued treatment can maintain knockdown for long-term therapeutic effect. Our data show that Kctd20 ASO administration mitigates the decline in motor performance in P301S mice, although this phenotype is not fully rescued.

[0186] Materials and Techniques

[0187] Cell Lines – Human iPSCs

[0188] Lymphocytes from healthy donors used for neurologically normal control lines (WT iPSC line 1: ND03231; WT iPSC line 2: ND05280) were obtained from the NINDS Biorepository at the Coriell Institute and reprogrammed into iPSCs. Cell lines derived from FTLD-Tau patients harbouring the V337M point mutation, and their corresponding CRISPR-corrected isogenic control, were acquired and generated by the Tau Consortium Stem Cell Group: GIH6-E11 (WT / WT), GIH6-A02 (V337M / WT), GIH7-B12 (WT / WT), GIH7-A01 (V337M / WT), ND-B06 (WT / WT), ND-B09 (V337M / WT). iPSC’s were maintained under feeder-free conditions in six-well plates (Corning) coated with4934-0525-6010.2Page 87 of 105 065715-000129WOPTMatrigel (BD) in mTeSR1 medium (Stem Cell Technologies) at 37⁰C and 5% CO2. Cells were fed daily with 2 mL mTeSR1 per well. Cultures were not grown in excess of 80% confluency, and passaging of iPSC colonies was performed using 10 µM EDTA.

[0189] The CRISPRi screen was conducted on an iPSC line expressing a Tet-ON hNGN2 and a constitutive dCas9-KRAB each stably integrated into the CLYBL locus (Velasco et al., Nature, 570, 523-527 (2019)). Gene editing of cell lines and insertion of hNGN2 in control lines was performed by transfection of iPSCs using Lipofectamine Stem Transfection Reagent together with the following plasmids: CLYBL-TO-hNGN2-BSD- mApple (Addgene #124229). Transfected cells were allowed to recover and subsequently selected with Geneticin (Thermo) and blasticidin (Thermo).

[0190] Mouse Model

[0191] All animal care and use were in accordance with local institution guidelines of the University of Southern California and approved by the Institutional Animal Care and Use Committee (IACUC) board of the University of Southern California under protocol numbers 11938 and 21174. Wildtype C57Bl / 6J mice were purchased from Jackson Laboratory (Stock No: 000664). Transgenic humanized-tau mice was strain: Tg(Thy1- MAPT*P301S)2541Godt). All mice were bred and housed at University of Southern California under standard conditions with food and water ad libitum. The animals were housed in cages under a temperature and humidity-controlled environment and subjected to a standard 12-hour light / dark cycle. Male and female littermate neonatal mice (Figure 8J-8N) or 6-month-old adult mice (Figures 8A-8I and 8O-8R) were used for all experiments.

[0192] Generation of Cortical Organoids and induced Spheroids

[0193] All organoids and spheroids used in this study were two-months old, unless otherwise stated. The generation of human cortical organoids was carried out as follows. In brief, iPSCs at ~70% confluence were dissociated into single cells with Accutase (Stem Cell Technologies) and 10,000 cells / well were seeded into a 96-well U-Bottom Low-Attachment plate (Corning) in mTeSR1 + 10 µM ROCK inhibitor (Y-27632; Tocris) to generate iPSC spheroids. Fresh mTeSR1 without ROCK inhibitor was replenished after 24 hours. For the following 5 days, neural induction was initiated through daily feeding with DMEM / F12 containing 20% KnockOut Serum (Gibco), 1 mM non-essential amino acids (Gibco), 1X GlutaMAX and 0.1 mM β-mercaptoethanol, supplemented with 10 µM dorsomorphin (Cayman) and 10 µM SB-431542 (Cayman). From day 6-15, media was refreshed daily with neural medium (Neurobasal-A (Invitrogen), B-27 Supplement without vitamin A (Gibco), 1X GlutaMAX, and 2% Pen / Strep (Corning)) supplemented with 20 ng / mL bFGF (Peprotech)4934-0525-6010.2Page 88 of 105 065715-000129WOPTand 20 ng / mL EGF (Peprotech), and every other day from day 16-25. On day 20, organoids were transferred to 6-well low attachment plates (Corning) and placed on an orbital shaker rotating at 60 rpm. To promote differentiation of neural progenitors into neurons, media was refreshed every other day with neural medium containing 20 ng / mL NT-3 (Peprotech) and 20 ng / mL BDNF (R&D) from day 25-43. Organoids were maintained from day 43 onwards in neural medium and refreshed every 3-4 days.

[0194] NGN2-induced cortical spheroids (iCS) were produced using iPSC lines with an integrated Tet-ON hNGN2 in the CLYBL locus. iPSC’s were grown to 80% confluency, treated with 2 µg / mL doxycycline for 60 minutes in mTeSR1, then dissociated into single cells with Accutase and seeded into 96-well U-Bottom Low-Attachment plates at 30,000 cells / well in neural medium supplemented with 2 µg / mL doxycycline, 10 ng / mL NT-3, 10 ng / mL BDNF, 0.33% Matrigel, and 10 µM ROCK inhibitor. Fresh medium with 2 µg / mL doxycycline, 10 ng / mL NT-3, 10 ng / mL BDNF, and 0.33% Matrigel was added daily on day 1-5, and every other day from day 6-10. Spheroids were maintained from day 10 onwards in neural medium with 10 ng / mL NT-3 and 10 ng / mL BDNF refreshed every 3 days.

[0195] Virus Production and Transduction

[0196] Lentivirus was produced through transfection of 80-90% confluent HEK293T cells on 0.1% gelatin-coated 10 cm plates grown in DMEM / F12 with 10% FBS. Prior to transfection, polyethylenimine (PEI, Sigma) was mixed with OptiMEM and incubated for five minutes at room temperature. The packaging plasmids pPAX2 and VSVG, and viral vector plasmid were subsequently added followed by a 15-minute incubation at room temperature. The transfection mix was then added to cells dropwise. Fresh DMEM / F12 with 10% FBS was replenished after 24 hours, and supernatant was collected after 48 and 72 hours. Supernatant was filtered using 0.45 µM filters and concentrated with 1 / 3 volume of Lenti-X (Clontech) overnight at 4⁰C. Concentrated virus was centrifuged at 1500g, 4⁰C, for 45 minutes, then resuspended in DMEM / F12 and stored at -80⁰C. Viral vectors used: pHR- hSyn-EGFP (Addgene #114215); pLJM1-FIRE-pHLy (Addgene #170775). Organoids were transduced with virus by combining concentrated virus with 1:1000 polybrene in neural medium. Medium was refreshed after 24 hours to remove virus.

[0197] Cryopreservation and Cryosectioning

[0198] Organoids were fixed in 4% paraformaldehyde for 1 hour at 4°C, washed three times with phosphate buffered saline (PBS), and dehydrated in 30% sucrose overnight. The following day, organoids were embedded in Tissue-Tek O.C.T. Compound (Sakura) and snap-frozen in a dry ice-ethanol bath. Frozen tissue blocks were then processed into 14-µm4934-0525-6010.2Page 89 of 105 065715-000129WOPTsections using a Leica cryostat (CM3050S) adhered to Superfrost Plus Microscope slides (Fisher) and stored at -80°C until use.

[0199] Immunofluorescent Staining

[0200] Tissue sections were thawed to room temperature and allowed to dry, and sections were partitioned using an ImmEdge pen (Vector Labs). Sections were rehydrated in 1X Tris Buffered Saline (TBS; Sigma) containing 0.1% tween-20 (TBS-T), permeabilized for 15 minutes at room temperature in TBS + 0.1% triton X-100, blocked with TBS-T + 5% fetal bovine serum for 30 minutes at room temperature, and stained overnight with primary antibodies diluted in blocking buffer. The following day, slides were washed three times with 1X TBS-T and Alexa Fluor conjugated secondary antibodies were added (1:500) for 1 hour in blocking buffer. Slides were washed thoroughly with 1X TBS-T and coverslips were mounted using Vectashield (Vector Laboratories).

[0201] Microscopy and Image Analysis

[0202] A Zeiss AxioZoom.v16 wide-field fluorescent microscope was used for longitudinal imaging of SYN1::eGFP-labelled neurons. Confocal images were captured using a Zeiss LSM 800: AxioObserver.M2 upright confocal microscope with a 20X, 40X, or 63X objective. Images were analyzed using NIH ImageJ software. Single-neuron measurements were calculated by thresholding neuronal cell bodies based on SYN1::eGFP intensity to create individual cell regions of interest (ROIs) then measuring target signal. Nuclear:cytoplasmic measurements were calculated by thresholding DAPI and SYN1::eGFP then measuring target signal. Target intensity was normalized to DAPI intensity.

[0203] Longitudinal Tracking of Neuron Survival

[0204] Survival tracking of neurons in organoids was performed. Briefly, organoids were transduced with Synapsin1::eGFP (SYN::eGFP) lentivirus (with 1:1000 polybrene) for five days, then fixed in Matrigel prior to experimental use. For the survival time course, glutamate (L-Glutamic acid monosodium salt hydrate in PBS) was added to culture medium at day zero, and was replenished after one, two, four, and six days. Images spanning 150 µM from the organoid surface were captured daily on a Zeiss Axiozoom.v16 wide-field fluorescent microscope. Post-processing and alignment were performed using ImageJ software (NIH). Staining with 1:500 DRAQ5 (Biolegend 424101) for 15 minutes at room temperature was used to assess live cells remaining at experimental endpoint. Detection of live neurons was determined by co-localization of SYN::eGFP and DRAQ5.

[0205] Single-cell RNA Sequencing

[0206] Organoid Dissociation and Cell Hashing4934-0525-6010.2Page 90 of 105 065715-000129WOPT

[0207] Organoids were dissociated for single-cell RNA sequencing. In brief, organoids were incubated in Papain (Worthington LK003176) with DNase1 (Worthington LK003170) reconstituted per manufacturer instructions for 60 minutes at 37⁰C with gentle shaking at 27 rpm with regular pipetting. Single-cell suspension was added to Earle’s Balanced Salt Solution (EBSS) with Ovomucoid Inhibitor (Worthington LK003182) and DNase1, pelleted, resuspended in ice-cold Neurobasal-A (Invitrogen) with 0.2% bovine serum albumin (BSA), and filtered through 40 µM Flowmi tips (Sigma BAH136800040- 50EA). Cell count and viability was determined by Trypan Blue staining using a hemocytometer. Cells were labeled with hashtag-oligonucleotide (HTO) antibodies (Biolegend TotalSeq-B, 405287, 405289, 405291) and libraries were prepared following manufacturer protocols (10X Genomics, CG000206 Rev D).

[0208] Single-Cell RNA Seq Alignment and QC

[0209] Data alignment and QC was conducted through the University of Southern California Center for Advanced Research Computing (CARC) using the Cell Ranger count Feature Barcode Analysis pipeline (10× Genomics) for concurrent UMI identification, reference genome (human hg38 genome) alignment, and HTO assignment. Samples were demultiplexed using the HTODemux pipeline in R version 4.0.1. Data was further processed using Seurat v4.0 to remove cells with < 200 detectable genes and > 50% mitochondrial rate. We retrieved roughly 39,000 high quality cells with a depth of 20,000-25,000 reads / cell.

[0210] Data Analysis

[0211] Data from different lanes and cell lines were integrated using SCTransform to regress out percentage of mitochondrial genes and anchored using the top 5,000 variable features. Principle Component Analysis (PCA) was run using the top 5,000 variable genes, and Uniform Manifold Approximation and Projection (UMAP) was implemented for data reduction. Data was then log-normalized and re-scaled, and clusters were identified using default parameters. Differentially expressed genes (DEGs) were calculated based on raw gene count data using MAST with default covariates. Significant DEGs between conditions were submitted to Enrichr and Ingenuity Pathway Analysis (IPA) for pathway analysis.

[0212] Calculation of Pseudotime Trajectories

[0213] Excitatory neuron clusters in V337M, V337V, and WT (5280) organoids were separately ordered in pseudotime using Monocle2. Genes were ordered based on significance as defined by p < 0.05 and clusters were generated by unsupervised clustering. FindAllMarkers function was implemented to identify marker genes of pseudotime clusters,4934-0525-6010.2Page 91 of 105 065715-000129WOPTand genes with adjusted p < 0.05 were submitted for ontology enrichment through Enrichr. Proportions of cells in each state across conditions were calculated using Fisher’s Exact test.

[0214] CRISPRi screen

[0215] Library and Experimental Design

[0216] NGN2-spheroids (n=150 spheroids / group) were transduced with the lentiviral CRISPRi-v2 sgRNA library (Addgene # 1000000090), containing 209,070 sgRNAs and 3,790 non-targeting sgRNAs, 7 days prior to glutamate treatment. Glutamate (5 mM) was added to neural medium containing 10 ng / mL NT3 and BDNF and refreshed daily from day 1-3. From day 4-10, medium containing glutamate was refreshed every other day. Degeneration was monitored throughout the experiment to validate neuronal death.

[0217] Library preparation

[0218] Total genomic DNA (gDNA) was isolated using the Monarch Genomic DNA Purification Kit (NEB #T3010S). To amplify the sgRNA cassette, the total gDNA was split and amplified using custom mirrored Illumina indexing primers using Q5 High-Fidelity DNA polymerase (NEB #M0491). The desired amplicon (~150 bp) was further enriched using SPRIselect (Beckman Coulter) double size selection beads and quantified by Bioanalyzer and Qubit.

[0219] Next-generation Sequencing & Bioinformatics

[0220] Amplicon libraries were sequenced with the NextSeq 500 / 550 High Output Kit v2.5 (75 cycles) with a 20% Phi-X spike-in. Trimming and alignments were performed in Python 3.8.3 and analysis was conducted using the MAGeCKFlute pipeline in R version 4.0.1.

[0221] Western Blot analysis

[0222] Organoids collected for western blot analysis were lysed in RIPA buffer (Santa Cruz) supplemented with 1X cOmplete EDTA-free protease inhibitor cocktail (Roche), 1% (v / v) phosphatase inhibitor cocktail 3 (Sigma) and 5 nM Trichostatin A deacetylase inhibitor (Cayman). Lysed samples were incubated on ice for 20 minutes followed by centrifugation at 12000g for 15 minutes at 4⁰C. Supernatant was transferred to fresh microcentrifuge tubes and stored at -80⁰C. Protein quantification was measured by bicinchoninic acid assay (BCA, Thermo) following manufacturer protocols.

[0223] Samples were run on 10% or 15% SDS-PAGE gels. Odyssey One-Color protein molecular weight marker (LI-COR) was used as a size marker. Gels were transferred onto Immobilon-FL PVDF membranes (Millipore). Total protein was then measured using the Revert 700 total protein stain kit (LI-COR) using standard protocols. After washing,4934-0525-6010.2Page 92 of 105 065715-000129WOPTmembranes were blocked in Intercept TBS blocking buffer (LI-COR) overnight at 4⁰C. Primary antibodies were added in Intercept buffer + 0.2% tween-20 and incubated overnight at 4⁰C. Membranes were washed 3 times with 1X TBS + 0.1% tween-20 (TBS-T), and IRDye secondary antibodies (LI-COR) were added in Intercept buffer containing 0.2% tween-20 and 0.1% SDS for 1 hour at room temperature followed by 3 washes in 1X TBS-T and 2 washes in 1X TBS. Membranes were visualized on the Odyssey CLx imaging system (LI-COR). Target bands were normalized to total protein for quantification per manufacturer protocols.

[0224] qRT-PCR

[0225] Total RNA was extracted from organoid or mouse tissue with RNeasy Mini Kit (Qiagen 74104) and reverse transcribed with random primer using Protoscript II First Strand Synthesis Kit (NEB) using manufacturer protocols. RNA concentration was measured by Nanodrop (Thermo). Real-time PCR was conducted using iTaq SYBR Green Supermix (Bio-Rad) with the following primers: (Table 6). Relative expression was calculated using the ΔΔCt method and normalized to housekeeping controls.

[0226] In vitro Antisense Oligonucleotide Treatment

[0227] Organoids were treated with 10 µM antisense oligonucleotide (ASO) for 72 hours prior to the start of the experiment. ASO sequences were custom designed and synthesized (IDT). The following sequences were used: (Table 5).

[0228] Exosome Purification and Analysis

[0229] Conditioned medium from organoids was collected and spun down at 4⁰C for 300g, 10 minutes followed by 2000g, 10 minutes to clear cell debris. Conditioned medium was sent to the Extracellular Vesicle Core at Children’s Hospital Los Angeles to analyze with the ExoView R100 per standard protocols. A chip containing the tetraspanins CD63, CD81, and CD9 was used to pull down purified exosomes and particles were counterstained with oligomeric tau (T22) and LAMP1 (CD107a) for quantification.

[0230] Calcium Imaging

[0231] Organoids were incubated with 1 µM of Fluo-4 AM (Thermo) for 15 minutes and washed twice in complete neural medium. Organoids were then immobilized in Matrigel and imaged on a Zeiss Axiozoom.v16 before and immediately after injury for 2 minutes at 24 frames per second. Change in fluorescence was calculated by measuring peak fluorescent intensity per frame normalized to background fluorescence.

[0232] Antisense Oligonucleotide and Glutamate Intracranial Ventricular Administration4934-0525-6010.2Page 93 of 105 065715-000129WOPT

[0233] Neonatal mice were cryo-anesthetized on ice prior to injection at postnatal day 1 and 3 (P1, P3). Anesthetic depth was assessed by toe-pinch. Antisense oligonucleotides (ASOs) were administered at P1 via intracerebroventricular (ICV) injection using a model 1701RN, 33-gauge 10 µL Neuros Syringe (Hamilton). 22.5 µg ASO in 1.5 µL phosphate- buffered saline (PBS, pH=7.4) was given per animal. 40 nmol glutamate in 3 µL PBS (or 3 µL PBS control) was injected ICV at P3 contralateral to ASO injection site. Mice were placed in a clean, pre-warmed container to recover for 10 minutes or until responsiveness returned. Animals were euthanized 48 hours later. Brain tissue was snap frozen, stored at - 80⁰C, and processed into 25 µM sections using a Leica CM3050S cryostat (Leica).

[0234] Adult mice (age 8 weeks and up) were injected with ASO and glutamate ICV. Briefly, mice were anesthetized with 1%-5% isoflurane and given 0.5 mg / kg Buprenephorine via subcutaneous injection for analgesia. Mice were placed in a stereotactic apparatus (RWD) to immobilize the head. Craniotomy was performed using a cordless microdrill (RWD) at bregma lateral 1.00 mm, anterior +0.30 mm. A model 1701RN small removable 32-gauge needle (Hamilton) was aligned and lowered to a ventral depth of z= -3.00 mm from the pial surface. ASO (22.5 µg in 1.5 µL PBS), glutamate (40 nmol in 3 µL PBS), or PBS (3 µL) was infused at a rate of 1 µL / min. The needle was left in place for an additional 7 minutes to prevent backflow before removal. Animals were placed in a clean, pre-warmed recovery area until responsiveness returned, and were monitored daily for 3 days post-op. Mice were transcardially perfused at experimental endpoints with PBS followed by 4% formaldehyde and cryoprotection in 30% sucrose. Brain tissue was snap frozen and stored at -80⁰C, and later processed into 25 µM sections.

[0235] ASO sequences were custom-designed and synthesized at IDT with modifications. The following sequences were used: (Table 5).

[0236] Rotarod Motor Assay

[0237] Mouse motor function, coordination, and equilibrium was tested using the Rotor Rod (SD Instruments) rotarod assay as previously described (Behavioral and Functional Neuroscience Laboratory, Stanford University). In brief, animals were placed into the device and run under the following conditions: 0 seconds 5 rpm; 300 seconds 5 to 50 rpm ramp. Experiment was halted after completion of the time course, and latency to fall was recorded. Animals were tested in triplicate for each timepoint.

[0238] Genotyping of Transgenic Mice

[0239] Crude DNA was isolated from tail snips using Tail Lysis Buffer with 1:20 ProteinaseK at 55⁰C overnight, followed by 85⁰C for 45 minutes. DNA was purified using the4934-0525-6010.2Page 94 of 105 065715-000129WOPTMonarch Genomic DNA Purification Kit (NEB #T3010S) and quantification was determined by nanodrop. Purified DNA was diluted to 7.5 ng / µL in UltraPure Distilled Water. Zygosity of Tg(Thy1-MAPT*P301S)2541Godt mice was determined by qPCR. Primers were designed against human MAPT and mouse Actb (β-actin) with the following sequences: MAPT-FWD- 5'-GATTGGGTCCCTGGACAATA-3' (SEQ ID NO:16714); MAPT-REV- 5'- GTGGTCTGTCTTGGCTTTGG-3' (SEQ ID NO:16715); bActin-FWD-5'- CGAGGCCCAGAGCAAGAGAG -3' (SEQ ID NO:16716); bActin-REV- 5'- CGGTTGGCCTTAGGGTTCAG-3' (SEQ ID NO:16717). DNA was mixed with 10 µL SYBRGreen Master mix, 0.40 µM FWD primer, 0.40 µM REV primer, and UltraPure water for a final reaction volume of 20 µL / well. Samples were tested in triplicate for MAPT and Actb and run on a Roche LightCycler qPCR machine under the following cycle conditions: 95⁰C for 3:30 minutes; (95⁰C for 0:10 minutes, 65⁰C for 0:30 minutes) x 45 cycles. Expression was calculated using ΔΔCt. Mice homozygous for the transgene were identified as having a Ct value of 1 lower than the heterozygous mice.

[0240] Statistical Analysis

[0241] Analysis was performed using the statistical package Prism (GraphPad Prism Version 9.3.1). Statistical analysis of neuron survival experiments was conducted using a two-sided log-rank test. For each condition, survival data from 100 Syn1::eGFP neurons were randomly selected and used to generate a survival curve. Hazard ratios (Mantel-Haenszel) were plotted relative to controls, and hazard ratio of all other conditions were divided by the control. For all other experiments, differences between two groups were calculated using a two-tailed Student’s t-test (when data was normally distributed) or two-sided Mann-Whitney test to compare ranks if data was not normally distributed. Differences between three or more groups were analyzed by One-way ANOVA with Tukey correction for multiple testing. Significance was assessed by P < 0.05. Error bars represent mean + / - s.e.m. unless otherwise stated. Table 5: Exemplary antisense oligonucleotide (ASO) sequences. Gene Species Sequence (5’->3’) Nucleobase SEQ ID NO: KCTD20 ASO-4 Human mU*mG*mG*mA*mC*A*T*T*C*T*G*A* 16718 (ASO ID 4) A*T*G*mA*mG*mA*mC*mC KCTD20 ASO-2 Human mG*mC*mA*mG*mA*A*A*G*G*A*A*A 16719 *A*G*G*mU*mU*mA*mG*mG TMEM63C ASO- Human mG*mA*mC*mA*mU*G*T*G*C*T*T*G* 16720 1 C*C*T*mG*mU*mA*mU*mC TMEM63C ASO- Human mG*mC*mA*mG*mC*A*G*C*A*G*A*A 16721 2 *G*C*T*mC*mC*mU*mU*mG4934-0525-6010.2Page 95 of 105 065715-000129WOPTSSRP1 ASO-1 Human mG*mG*mA*mC*mU*G*C*A*T*T*T*C* 16722 A*T*G*mA*mG*mG*mA*mG SSRP1 ASO-2 Human mC*mC*mU*mA*mG*T*G*A*C*A*T*A* 16723 C*A*C*mA*mC*mC*mA*mA SLC39A8 ASO-1 Human mG*mG*mC*mA*mU*C*T*C*A*G*A*C* 16724 T*G*A*mC*mA*mC*mU*mG PECR ASO Human mU*mC*mA*mA*mG*A*T*G*T*G*A*A 16738 *G*G*C*mA*mC*mU*mG*mG TMEM37 ASO Human mG*mC*mU*mG*mA*C*C*A*C*C*C*A* 16739 C*A*A*mG*mU*mU*mA*mU PPP4R1 ASO Human mA*mA*mG*mC*mU*A*T*C*C*C*A*G* 16740 G*T*C*mA*mC*mA*mU*mG BCAP29 ASO Human mA*mG*mU*mU*mA*C*T*C*T*C*A*C* 16741 A*A*C*mC*mA*mA*mG*mG CTBP2 ASO Human mC*mA*mC*mU*mG*C*A*A*C*A*T*C* 16742 A*A*T*mG*mA*mU*mG*mC GNG13 ASO Human mG*mG*mU*mC*mU*C*A*C*A*G*G*A 16743 *T*G*G*mA*mG*mU*mG*mA MAPT ASO Human mC*mC*mG*mU*mU*T*T*C*T*T*A*C* 16725 C*A*C*mA*mC*mC*mC*mU Non-targeting Human mG*mC*mG*mA*mC*T*A*T*A*C*G*C* 16726 (NC) ASO G*C*A*mA*mU*mA*mU*mG MCOLN1 ASO Human mG*mG*mU*mU*mA*G*A*T*G*T*A*C 16727 *C*T*T*mC*mA*mC*mA*mU SMPD3 ASO Human mG*mG*mA*mU*mU*G*T*C*A*A*A*A 16728 *A*C*A*mG*mU*mC*mC*mC VAMP7 ASO Human mC*mU*mA*mG*mG*C*T*A*A*A*C*A 16729 *G*G*T*mG*mG*mC*mU*mA HSPA8 ASO Human mC*mA*mA*mG*mG*A*A*G*G*T*A*G 16730 *T*T*G*mC*mC*mA*mA*mC ATG7 ASO Human mG*mU*mU*mG*mA*G*T*G*C*C*A*T* 16731 A*C*C*mA*mG*mU*mA*mG RAB8A ASO Human mU*mG*mG*mU*mU*G*A*C*C*T*G*G 16732 *T*C*C*mC*mA*mG*mU*mC ATG5 ASO Human mG*mU*mG*mG*mU*A*A*T*A*G*C*A 16733 *T*A*G*mU*mC*mC*mA*mA NSMAF ASO Human mA*mU*mC*mU*mG*C*C*C*T*A*A*G* 16734 A*G*A*mA*mU*mA*mG*mC GORASP1 ASO Human mG*mG*mA*mU*mA*G*A*C*C*T*A*G 16735 *T*C*A*mG*mG*mU*mA*mGSTXBP1 ASO HumanmC*mU*mG*mG*mA*G*A*A*T*G*A*G16745 *A*G*C*mU*mG*mC*mU*mG Non-targeting Mouse / 52MOErC / * / i2MOErC / / i2MOErT / / i2MOEr 16736 ASO A / / i2MOErT / A*G*G*A*C*T*A*T*C*C* / i 2MOErA / / i2MOErG / / i2MOErG / * / i2MOErA / * / 32MOErA / Kctd20 ASO Mouse / 52MOErG / * / i2MOErC / / i2MOErA / / i2MOEr 16737 C / / i2MOErA / G*A*C*T*G*T*T*T*C*T* / i2 MOErC / / i2MOErT / / i2MOErG / * / i2MOErA / *4934-0525-6010.2Page 96 of 105 065715-000129WOPT / 32MOErC / Table 6: RT-qPCR primers used in this study. Gene Species Primer KCTD20 Human Forward CGGGGAGCGACGAGATT (SEQ ID NO:16670) KCTD20 Human Reverse CTCCTGCCGCAATAACCTGT (SEQ ID NO:16671) MCOLN1 Human Forward GCGACAAGTTTCGAGCCAAG (SEQ ID NO:16672) MCOLN1 Human Reverse AAACAGGATGAGCTGCACCG (SEQ ID NO:16673) LAMP1 Human Forward ATGTGTTAGTGGCACCCAGG (SEQ ID NO:16674) LAMP1 Human Reverse TGTTCACAGCGTGTCTCTCC (SEQ ID NO:16675) TFEB Human Forward GCAAGCTCAGGCTGGGAG (SEQ ID NO:16676) TFEB Human Reverse TATTGATGGCCGGGGTGGG (SEQ ID NO:16677) 18S Human Forward CTCAACACGGGAAACCTCAC (SEQ ID NO:16678) 18S Human Reverse CGCTCCACCAACTAAGAACG (SEQ ID NO:16679) MAP2 Human Forward GGAACCAACTCTCTCTGGATTT (SEQ ID NO:16680) MAP2 Human Reverse GCATTCTCTCTTCAGCCTTCT (SEQ ID NO:16681) MAPT Human Forward GGTGGCCAGGTGGAAGTAAA (SEQ ID NO:16682) MAPT Human Reverse GACACCACTGGCGACTTGTA (SEQ ID NO:16683) STXBP1 Human Forward CATGACCGAGGGCATAACGA (SEQ ID NO:16684) STXBP1 Human Reverse AGGGCATCTGGACAAGAGTC (SEQ ID NO:16685) PPP4R1 Human Forward GACGCAGACGGATTTGGTGT (SEQ ID NO:16686) PPP4R1 Human Reverse ACAAAGTCCAGGGCTGAAGG (SEQ ID NO:16687) BCAP29 Human Forward GCCGGCGGGTGTGAA (SEQ ID NO:16688) BCAP29 Human Reverse TGCCATCTCTGAGGAGGAAT (SEQ ID NO:16689) TMEM63C Human Forward GCTAGAGGAGCAGCTAACGG (SEQ ID NO:16690) TMEM63C Human Reverse ATCCTTACGGACACGCTTGG (SEQ ID NO:16691) SSRP1 Human Forward CCGCAGCCTGAGGAGATTC (SEQ ID NO:16692)4934-0525-6010.2Page 97 of 105 065715-000129WOPTSSRP1 Human Reverse AAGTAAACTGGGAGCTGGGC (SEQ ID NO:16693) PECR Human Forward TCGAATGTCCCTGAGACCCA (SEQ ID NO:16694) PECR Human Reverse CCACATTACTCCCCAGCTCC (SEQ ID NO:16695) SLC39A8 Human Forward TCCCCACGAGTTAGGAGACT (SEQ ID NO:16696) SLC39A8 Human Reverse GTTGCCCACCAAAATGCCAA (SEQ ID NO:16697) TMEM37 Human Forward GGCGTGCAGGCCCAG (SEQ ID NO:16698) TMEM37 Human Reverse TGAGGGTCCGGATGAAGGAT (SEQ ID NO:16699) CTBP2 Human Forward AAGAGCAAATAGCCGGGAGG (SEQ ID NO:16700) CTBP2 Human Reverse GACGCCACTATGAACCCAAT (SEQ ID NO:16701) GNG13 Human Forward GCTGTCACCTTTTCAAGCCCC (SEQ ID NO:16702) GNG13 Human Reverse GTCCTCGATCCACTTCAGCA (SEQ ID NO:16703) STMN2 Human Forward AGCTGTCCATGCTGTCACTG (SEQ ID NO:16704) STMN2 Human Reverse GGTGGCTTCAAGATCAGCTC (SEQ ID NO:16705) trncSTMN2 Human Forward GGACTCGGCAGAAGACCTTC (SEQ ID NO:16706) trncSTMN2 Human Reverse GCAGGCTGTCTGTCTCTCTC (SEQ ID NO:16707) Gapdh Mouse Forward TGTCAAGCTCATTTCCTGGTATG (SEQ ID NO:16708) Gapdh Mouse Reverse TTATGGGGGTCTGGGATGGA (SEQ ID NO:16709) Hprt Mouse Forward GCAGCGTTTCTGAGCCATTG (SEQ ID NO:16710) Hprt Mouse Reverse CATCATCGCTAATCACGACGC (SEQ ID NO:16711) Kctd20 Mouse Forward GGAGTATGTGATCGCGGAGG (SEQ ID NO:16712) Kctd20 Mouse Reverse TCGTGCAGTAGAGCACTCAG (SEQ ID NO:16713)

[0242] Various embodiments of the invention are described above in the Detailed Description. While these descriptions directly describe the above embodiments, it is understood that those skilled in the art may conceive modifications and / or variations to the specific embodiments shown and described herein. Any such modifications or variations that fall within the purview of this description are intended to be included therein as well. Unless specifically noted, it is the intention of the inventors that the words and phrases in the4934-0525-6010.2Page 98 of 105 065715-000129WOPTspecification and claims be given the ordinary and accustomed meanings to those of ordinary skill in the applicable art(s).

[0243] The foregoing description of various embodiments of the invention known to the applicant at this time of filing the application has been presented and is intended for the purposes of illustration and description. The present description is not intended to be exhaustive nor limit the invention to the precise form disclosed and many modifications and variations are possible in the light of the above teachings. The embodiments described serve to explain the principles of the invention and its practical application and to enable others skilled in the art to utilize the invention in various embodiments and with various modifications as are suited to the particular use contemplated. Therefore, it is intended that the invention not be limited to the particular embodiments disclosed for carrying out the invention.

[0244] While particular embodiments of the present invention have been shown and described, it will be obvious to those skilled in the art that, based upon the teachings herein, changes and modifications may be made without departing from this invention and its broader aspects and, therefore, the appended claims are to encompass within their scope all such changes and modifications as are within the true spirit and scope of this invention. It will be understood by those within the art that, in general, terms used herein are generally intended as “open” terms (e.g., the term “including” should be interpreted as “including but not limited to,” the term “having” should be interpreted as “having at least,” the term “includes” should be interpreted as “includes but is not limited to,” etc.).4934-0525-6010.2Page 99 of 105 065715-000129WOPT

Claims

WHAT IS CLAIMED IS:

1. A single stranded antisense oligonucleotide (ASO), wherein the ASO comprises 12- 50 linked nucleosides, and the ASO has a nucleobase sequence having a guanine- cytosine content (GC content) of 30-70%, no cytidine (C) followed by a guanosine (G), and no more than three contiguous G’s.

2. The ASO of claim 1, wherein the ASO has a nucleobase sequence that comprises at least 12 consecutive nucleobases of the nucleobase sequence set forth in any one of SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669 or of a variant of the any one of the SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669, wherein the variant has a substitution of one or more thymines if present by uracil or the variant has one or more nucleobases removed.

3. The ASO of claim 1, wherein the ASO has 18-20 linked nucleosides and comprises a nucleobase sequence set forth in any one of SEQ ID NOs:16718, 16719, 16750- 16752, and 1-16669 or of a variant of the any one of the SEQ ID NOs:16718, 16719, 16750-16752, and 1-16669, wherein the variant has a substitution of one or more thymines if present by uracil or the variant has one or more nucleobases removed.

4. The ASO of claim 1, wherein the ASO has 20 linked nucleosides and comprises a nucleobase sequence of that of any one of SEQ ID NOs:648-1321 or a variant of the any one of SEQ ID NOs:648-1321.

5. The ASO of claim 1, wherein at least one internucleoside linkage is a modified internucleoside linkage.

6. The ASO of claim 5, wherein the modified internucleoside linkage is a phosphorothioate (PS) internucleoside linkage or a N3′-P5′ phosphoroamidate (NP) internucleoside linkage.

7. The ASO of claim 1, wherein at least one internucleoside linkage is a phosphodiester internucleoside linkage, and at least another internucleoside linkage is a phosphorothioate internucleoside linkage.

8. The ASO of claim 1, wherein at least one nucleoside comprises a modified nucleobase.

9. The ASO of claim 8, wherein the modified nucleobase is an abasic site.

10. The ASO of claim 1, wherein at least one nucleoside of the ASO comprises a modified sugar moiety, wherein the modified sugar moiety comprises a modification4934-0525-6010.2Page 100 of 105 065715-000129WOPTof 2’-O-methoxyethyl group, 2’-O-methyl group, or constrained ethyl group, or the modified sugar moiety is a threose moiety.

11. The ASO of claim 1, wherein at least one nucleoside of the ASO comprises a locked nucleic acid (LNA), a phosphorodiamidate morpholino (PMO), a peptide nucleic acid (PNA), or a tricyclo DNA (tcDNA).

12. The ASO of claim 1, wherein the ASO is a gapmer comprising one or more segments of linked deoxynucleosides and one or more segments of linked nucleosides, wherein a nucleoside of the one or more segments of linked nucleosides comprises a modified sugar moiety.

13. The ASO of claim 12, wherein the ASO comprises: a gap segment consisting of 10 to 12 linked deoxynucleosides, a 5’ wing segment consisting of 5 to 4 linked nucleosides, and a 3’ wing segment consisting of 5 to 4 linked nucleosides, wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment.

14. The ASO of claim 13, wherein each nucleoside making up each wing segment comprises a modified sugar moiety comprising a 2’-O-methoxyethyl group or a 2’-O- methyl group, and wherein each internucleoside linkage between the deoxynucleosides making up the gap segment is a phosphorothioate internucleoside linkage.

15. The ASO of claim 12, wherein the ASO has 20 linked nucleosides comprising: a gap segment consisting of 10 linked deoxynucleosides, a 5’ wing segment consisting of 5 linked nucleosides, and a 3’ wing segment consisting of 5 linked nucleosides, wherein the gap segment is positioned between the 5’ wing segment and the 3’ wing segment, and wherein each nucleoside making up each wing segment comprises a modified sugar moiety comprising a 2’-O-methoxyethyl group, and the first, sixth, seventh, eighth, ninth, tenth, eleventh, twelfth, thirteenth, fourteenth, fifteenth, eighteenth, and nineteenth internucleoside linkage is a phosphorothioate internucleoside linkage.

16. The ASO of claim 12, wherein the ASO has a nucleobase sequence having 100% or at least 99%, 98%, 97%, 96%, 95%, 90%, 85%, 80%, or 75% complementarity to 3’UTR of human KCTD20 mRNA, or wherein the ASO suppresses expression of potassium channel tetramerization domain containing 20 (KCTD20) by at least 15%.4934-0525-6010.2Page 101 of 105 065715-000129WOPT17. The ASO of any one of claims 1-16, wherein the GC content is 40%-60%.

18. The ASO of any one of claims 1-16, wherein the GC content is 40%-60%, having no more than two consecutive G’s, and the ASO has a nucleobase sequence having at least 6 cytidines (C’s), at least 6 thymines (T’s), 0-3 G’s, and a 3’ end not ending in 1- 3 G’s; and optionally wherein the ASO has a change in Gibbs free energy (ΔG) associated with self-dimerization being ≥ -7 kcal / mole.

19. The ASO of any one of claims 1-3 and 5-16, wherein the GC content is 40%-65%, and the ASO has a nucleobase sequence having at least 5 C’s and at least 5 T’s; and optionally wherein the ASO has a ΔG associated with self-dimerization being ≥ -7 kcal / mole, and if the nucleobase sequence of the ASO has 5 T’s and 5 C’s, the ASO has one G in the last three bases at the 3’ end.

20. The ASO of any one of claims 1-3 and 5-16, wherein the GC content is 40%-60%, and the ASO has a nucleobase sequence having no G at the 3’ end; and optionally wherein the ASO has a ΔG associated with self-dimerization being ≥ -7 kcal / mole.

21. A pharmaceutical composition, comprising the antisense oligonucleotide of any one of claims 1-20 and a pharmaceutically acceptable carrier, diluent or excipient.

22. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is formulated for intracerebroventricular injection or intrathecal injection.

23. The pharmaceutical composition of claim 21, wherein the pharmaceutical composition is formulated for parenteral delivery.

24. The pharmaceutical composition of claim 21, wherein pharmaceutically acceptable carrier, diluent or excipient comprises artificial cerebrospinal fluid (CSF).

25. A method of reducing loss of neurons, comprising contacting the neurons with an antisense oligonucleotide of any one of claims 1-20.

26. The method of claim 25, wherein the neurons are in a subject with amyotrophic lateral sclerosis (ALS) or frontotemporal dementia (FTD), or derived from induced pluripotent stem cells derived from the subject with the ALS or FTD.

27. A method of treating, inhibiting, and reducing the severity of a neurological or a neurodegenerative disease in a subject, comprising administering to the subject a therapeutically effective amount of a pharmaceutical composition of claim 21.

28. The method of claim 27, wherein the neurodegenerative disease comprises frontotemporal dementia (FTD), motor neuron disease / amyotrophic lateral sclerosis (MND / ALS), Alzheimer’s disease (AD), Parkinson’s disease (PD), progressive4934-0525-6010.2Page 102 of 105 065715-000129WOPTsupranuclear palsy, tauopathies, chronic traumatic encephalopathy, Charcot Marie Tooth 2A and 4B, Huntington’s disease, dementia, transmissible spongiform encephalopathy, spinobulbar muscular atrophy, dentatorubro-pallidoluysian atrophy, spinocerebellar ataxias, Creutzfeldt- Jakob disease, or a combination thereof, or the subject exhibits symptoms of glutamate excitotoxicity.

29. The method of claim 27, wherein the neurological disease is associated with aberrant lysosomal storage.

30. The method of claim 27, wherein the therapeutically effective amount is effective for reducing glutamate-induced tau oligomerization in the subject.

31. A method of screening for an oligonucleotide, comprising: contacting an oligonucleotide of interest with an organoid derived from induced- pluripotent stem cells (iPSCs) from a patient with a neurodegenerative disease, and performing one or more of: assaying transcription or expression level of KCTD20, assaying expression levels of an AKT signaling, an mTOR signaling, or both, assaying level of transcription factor EB (TFEB) nuclear localization, assaying expression levels of p62, LC3, or both, assaying level of autolysosome formation, and assaying level of tau oligomerization.

32. The method of claim 31, wherein the organoid derived from iPSCs from a patient with a neurodegenerative disease is an organoid derived from iPSCs obtained from a patient with frontotemporal dementia (FTD) associated with the V337M microtubule- associated protein tau (MAPT) mutation.

33. The method of claim 14, wherein the organoid derived from iPSCs from a patient with a neurodegenerative disease is an organoid derived from iPSCs obtained from a patient with amyotrophic lateral sclerosis (ALS) or FTD having an expansion of a hexanucleotide repeat in the gene C9orf72.

34. The method of claim 14, wherein performing the one or more of assaying comprises assaying the expression levels and / or the levels before contacting the oligonucleotide of interest and assaying the expression levels and / or the levels in the presence of the oligonucleotide of interest, wherein an decreased transcription or expression level of KCTD20, an increased expression level of the AKT signaling and / or the mTOR signaling, an increased level of TFEB nuclear localization, an increased expression level of p62 and / or LC3, an increased level of autolysosome formation, and / or a4934-0525-6010.2Page 103 of 105 065715-000129WOPTdecreased level of tau oligomerization, in the presence of the oligonucleotide of interest relative to that before the contact thereof, indicates that the oligonucleotide of interest is a candidate drug for treating a neurodegenerative disease.

35. A method of modulating a neuronal excitotoxicity regulator in a subject in need thereof, comprising: administering an antisense oligonucleotide of any one of claims 1-20 or a pharmaceutical composition of any one of claims 21-24 to the subject in need thereof.

36. The method of claim 35, wherein the neuronal excitotoxicity regulator is potassium channel tetramerization domain containing 20 (KCTD20) and the composition suppresses KCTD20.

37. The method of claim 35, wherein the neuronal excitotoxicity regulator is lysosomal exocytosis and the composition activates lysosomal exocytosis.

38. The method of any one of claims 35-36, wherein the subject in need thereof has a neurological disease.

39. The method of claim 38, wherein the neurological disease is selected from the group consisting of amyloid lateral sclerosis, Huntington’s disease, Alzheimer’s disease, frontotemporal dementia, Parkinson’s disease, multiple sclerosis, peripheral myopathy, Rasmussen’s encephalitis, attention deficit hyperactivity disorder, autism, central pain syndromes, anxiety, and combinations thereof.

40. The method of claim 38, wherein the neurological disease is amyloid lateral sclerosis, frontotemporal dementia, Alzheimer’s disease, or combinations thereof.4934-0525-6010.2Page 104 of 105 065715-000129WOPT

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