Compositions and methods for modulating splicing

Abstract

Described herein is a small molecule splicing modulator compound that modulates splicing of mRNA, such as pre-mRNA, encoded by genes, and methods of use of the small molecule splicing modulator compounds for modulating splicing and treating diseases and conditions.

Description

WSGR Docket No.51503-775.601 COMPOSITIONS AND METHODS FOR MODULATING SPLICING RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 656,229, filedon June 5, 2024; U.S. Provisional Patent Application No.63 / 656,174, filed on June 5, 2024; Provisional Patent Application No.63 / 666,363 filed on July 1, 2024; and Provisional Patent Application No. 63 / 693,450, filed on September 11, 2024, each of which is incorporated herein by reference in its entirety. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically inXML format and is hereby incorporated by reference in its entirety. Said XML copy, created on May 22, 2025, is named 51503-775_601_SL2.xml and is 23,172 bytes in size. BACKGROUND

[0003] The majority of protein-coding genes in the human genome are composed of multiple exons(coding regions) that are separated by introns (non-coding regions). Gene expression results in a single precursor messenger RNA (pre-mRNA). The intron sequences are subsequently removed from the pre- mRNA by a process called splicing, which results in the mature messenger RNA (mRNA). By including different combinations of exons, alternative splicing gives rise to multiple mRNAs encoding distinct protein isoforms. The spliceosome, an intracellular complex of multiple proteins and ribonucleoproteins, catalyzes splicing.

[0004] Small molecule splicing modulators (SMSMs) overcome many of the problems associated withtherapies such as oligonucleotide technologies (antisense, RNAi, etc.), including lack of oral bioavailability, and lack of blood-brain-barrier penetration, with the latter precluding delivery to the brain or spinal cord after parenteral drug administration for the treatment of diseases (e.g., neurological diseases, brain cancers).

[0005] SMSMs disclosed in WO2020 / 163541, such as, 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-1,3-benzoxazol-5-ol, are useful in treating and preventing a wide range of diseases and conditions through modulating splicing of pre- mRNA, including, but not limited to, neurodegenerative diseases, such as Huntington’s Disease. SMSMs, however, can also have challenges, such as metabolic profiles in patients, clearance rates, the amount of compound available to exert an effect (e.g., fraction unbound in plasma, half-life of the compound in circulation, etc.). SUMMARY

[0006] Provided herein are small molecule splicing modulators and uses thereof that fulfill this need.

[0007] As mentioned above, WO2020 / 163541 discloses 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-1,3-benzoxazol-5-ol, which is useful in treating and preventing a wide range of diseases and conditions through modulating splicing of pre-mRNA, including, but not limited to, neurodegenerative diseases, such as Huntington’s Disease.WSGR Docket No.51503-775.601 Certain parameters for SMSMs such as metabolic profiles in patients, clearance rates, the amount of compound available to exert an effect (e.g., fraction unbound in plasma, half-life of the compound in circulation, etc.) can be affected by small changes between two similar compounds. Thus, compounds similar to 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}pyridazin-3-yl)- 2-methyl-1,3-benzoxazol-5-ol, but with improved metabolic profiles are useful in developing therapies for neurodegenerative diseases, such as Huntington’s Disease.

[0008] In one aspect, described herein is a method of treating or preventing Huntington’s disease in asubject in need thereof, comprising administering to the subject in need thereof a therapeutically effective amount of a composition comprising a compound of structure B, or a pharmaceutically acceptable salt or stereoisomer thereof:

[0009] In some aspects, the compound of structure B is 6-(6-(((1R,2R,3S,5S)-2-Fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[d]oxazol-5-ol.

[0010] In some embodiments, the subject’s genome encodes a wild-type PMS1. In some embodiments,the subject’s genome comprises an allele comprising a genetic variation in a PMS1 gene. In some embodiments, the genetic variation is a non-synonymous coding variant. In some embodiments, the genetic variation does not disrupt or modulate the PMS1 gene, or wherein the genetic variation is not a loss-of-function genetic variation. In some embodiments, the genetic variation comprises chr2:190660537 G>A, chr2:190719296 A>G, chr2:190719569 T>C, or any combination thereof, wherein chromosome positions of the genetic variation are defined with respect to UCSC hg19. In some embodiments, the allele comprising a genetic variation in a PMS1 gene encodes a variant PMS1 comprising a mutation selected from the group consisting of a E59K mutation, a K433R mutation, a L524S mutation, and any combination thereof.

[0011] In some embodiments, the subject has been identified as having the genetic variation. In someembodiments, the genetic variation disrupts or modulates the PMS1 gene. In some embodiments, the subject has been identified as not having the genetic variation.

[0012] In some embodiments, the genetic variation is a non-synonymous coding variant. In someembodiments, the genetic variation comprises chr2:190660586 C>T, chr2:190670391 C>G, chr2:190670396 A>G, chr2:190717470 CA>C, chr2:190719499 G>A, chr2:190719607 G>A, chr2:190719704 G>A, chr2:190732559 T>C, or any combination thereof, wherein chromosome positions of the genetic variation are defined with respect to UCSC hg19. In some embodiments, the allele comprising a genetic variation in a PMS1 gene encodes a variant PMS1 comprising a mutation selectedWSGR Docket No.51503-775.601 from the group consisting of a T75I, T110R, T112A, S264*, G501R, E537K, R569Q, Y793H, wherein * denotes a premature termination of protein translation.

[0013] In some embodiments, the subject has been tested for a presence of the genetic variation with agenetic assay. In some embodiments, the subject is heterozygous for the genetic variation. In some embodiments, the subject is homozygous for the genetic variation.

[0014] In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt orstereoisomer thereof modulates splicing of an HTT pre-mRNA, a PMS1 pre-mRNA, or both. In some embodiments, the method delays onset or slows progression of the Huntington’s disease.

[0015] Also provided herein is a is a method of treating or preventing a disease or condition in a subjectin need thereof, comprising administering to the subject a therapeutically effective amount of a composition comprising a compound of structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof:Structure B, wherein the disease or condition is associated with expression level or activity level of a protein encoded by an HTT gene or a PMS1 gene.

[0016] In some embodiments, the disease or condition is associated with a string of CAG repeats in theHTT gene. In some embodiments, a pre-mRNA and / or an mRNA encoded by the HTT gene comprises the string of CAG repeats. In some embodiments, the disease or condition is associated with an aberrant expansion of a string of CAG repeats in the HTT gene. In some embodiments, a pre-mRNA encoded by the HTT gene comprises the aberrant expansion of the string of CAG repeats. In some embodiments, the protein encoded by the HTT gene comprises a mutant HTT protein. In some embodiments, the disease or the condition is associated with a string of CAG repeats or an aberrant expansion of a string of CAG repeats in the HTT gene caused by the protein encoded by the PMS1 gene. In some embodiments, the disease or condition is Huntington’s disease. In some embodiments, the compound of structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof binds to a pre-mRNA and modulates splicing of the pre-mRNA at a splice site of the pre-mRNA in a cell of a subject, wherein the pre-mRNA is encoded by the HTT gene or the PMS1 gene.

[0017] In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt orstereoisomer thereof is administered to the subject at about 1 mg to about 16 mg per dose. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the subject at about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg,WSGR Docket No.51503-775.601 about 14 mg, about 15 mg, or about 16 mg per dose. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the subject daily.

[0018] Also provided herein is a method of modulating splicing of an HTT pre-mRNA, a PMS1 pre-mRNA, or both, comprising contacting a compound of structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, to the HTT pre-mRNA, the PMS1 pre-mRNA, or both or cells comprising the HTT pre-mRNA, the PMS1 pre-mRNA, or bothStructure B, wherein the HTT pre-mRNA and the PMS1 pre-mRNA comprise a splice site sequence, and wherein the compound binds to the HTT pre-mRNA, the PMS1 pre-mRNA, or both and modulates splicing of the HTT pre-mRNA, the PMS1 pre-mRNA, or both in the cells or cells of a subject to produce a spliced product of the HTT pre-mRNA, the PMS1 pre-mRNA, or both.

[0019] Also provided herein is a method of modulating splicing of an HTT pre-mRNA, a PMS1 pre-mRNA, or both in a cell, comprising contacting the cell with a compound of structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof:Structure B.

[0020] In some embodiments, the expression level of a canonical isoform of a PMS1 mRNA encoded bythe PMS1 pre-mRNA is reduced by at least 50% in the cell contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the expression level of a canonical isoform of an HTT mRNA encoded by the HTT pre-mRNA is reduced by at least 10% in the cell contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is the compound of structure B.

[0021] In some embodiments, the method comprises administering to the subject a pharmaceuticalcomposition comprising the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable excipient or carrier. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof binds to the HTT pre-mRNA, the PMS1 pre-mRNA, or both. In some embodiments, the compound of structure B, orWSGR Docket No.51503-775.601 the pharmaceutically acceptable salt or stereoisomer thereof modulates the splicing of the HTT pre- mRNA, the PMS1 pre-mRNA, or both. In some embodiments, modulating splicing comprises promoting inclusion of an exon. In some embodiments, modulating splicing comprises promoting inclusion of a cryptic exon. In some embodiments, the cryptic exon comprises a poison exon. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof promotes inclusion of the poison exon in the spliced product of the HTT pre-mRNA, in the spliced product of the PMS1 pre-mRNA, or both.

[0022] In some embodiments, the poison exon comprises a nucleic acid sequence located between exon49 and exon 50 of the HTT pre-mRNA. In some embodiments, the poison exon is exon 49b. In some embodiments, the poison exon comprises a nucleic acid sequence located between exon 5 and exon 6 of the PMS1 pre-mRNA. In some embodiments, the poison exon is exon 5b. In some embodiments, the poison exon comprises an in-frame stop codon. In some embodiments, the in-frame stop codon is a premature termination codon. In some embodiments, the in-frame stop codon is at least 50 or 60 base pairs upstream of the 3’ end of the poison exon. In some embodiments, the in-frame stop codon is less than 60 base pairs upstream of the 3’ end of the poison exon, and wherein the exon immediately downstream of the poison exon is not the last exon in the HTT pre-mRNA or the PMS1 pre-mRNA. In some embodiments, the sum of (a) the number of base pairs in the exon immediately downstream of the poison exon and (b) the number of base pairs between the premature stop codon in the poison exon and the 3’ end of the poison exon is at least 50 or at least 60.

[0023] In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt orstereoisomer thereof binds to the nucleic acid sequence located between exon 49 and exon 50 of the HTT pre-mRNA, the nucleic acid sequence located between exon 5 and exon 6 of the PMS1 pre-mRNA, or both. In some embodiments, the nucleic acid sequence located between exon 49 and exon 50 of the HTT pre-mRNA comprises intron 49b comprising a 5’ splice site sequence. In some embodiments, the nucleic acid sequence located between exon 5 and exon 6 of the PMS1 pre-mRNA comprises intron 5b comprising a 5’ splice site sequence. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof promotes splicing at the 5’ splice site sequence. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof promotes inclusion of exon 49b in the spliced product of the HTT pre-mRNA, inclusion of exon 5b in the spliced product of the PMS1 pre-mRNA, or both. In some embodiments, the amount of the spliced product of the HTT pre-mRNA comprising exon 49b is increased in the cell or in the cells contacted by the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the amount of the spliced product of the PMS1 pre-mRNA comprising exon 5b is increased in the cell or in the cells contacted by the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the 5’ splice site sequence comprises AGCAGA / guaagggggcuu (SEQ ID NO: 1) or AAAUGA / guaagacugguu (SEQ ID NO: 2).WSGR Docket No.51503-775.601

[0024] In some embodiments, inclusion of a cryptic exon promotes destabilization or nonsense-mediateddecay (NMD) of the spliced product of the HTT pre-mRNA. In some embodiments, inclusion of a cryptic exon promotes destabilization or nonsense-mediated decay (NMD) of the spliced product of the PMS1 pre-mRNA.

[0025] In some embodiments, the amount of an HTT mRNA, a PMS1 mRNA, or both is reduced in thecell or in the cells contacted by the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the amount of the HTT mRNA, the PMS1 mRNA, or both is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% in the cell or in the cells contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

[0026] In some embodiments, the amount of the mutant HTT protein is reduced in the cell or in the cellscontacted by the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the amount of the mutant HTT protein is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% in the cell or in the cells contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

[0027] In some embodiments, the amount of the PMS1 protein is reduced in the cell or in the cellscontacted by the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the amount of the PMS1 protein is reduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% in the cell or in the cells contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

[0028] In some embodiments, the cell or the cells comprise primary cells. In some embodiments, the cellor the cells comprise disease cells. In some embodiments, the cell or the cells comprise Huntington’s disease patient-derived lymphoblastoid cell line, induced pluripotent stem cell (iPSC)-derived cortical neurons, blood cells, brain cells, or combinations thereof. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof modulates proliferation or survival of the cells.

[0029] In some embodiments, the composition or the pharmaceutical composition comprising thecompound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered orally. In some embodiments, the composition or the pharmaceutical composition comprising the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered as a solid dosage form. In some embodiments, the solid dosage form comprises a tablet or a capsule. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof penetrates a blood brain barrier (BBB) when administered to the subject.WSGR Docket No.51503-775.601 INCORPORATION BY REFERENCE

[0030] All publications, patents, and patent applications mentioned in this specification are hereinincorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] FIG. 1 depicts RNA structure analysis of a splice site, showing base-pairing between 5’ splicesite sequence and U1 snRNA sequence that forms a bulge structure. ss: splice site, Ψ: pseudouridine. Figure discloses SEQ ID NOs: 18, 19, 18, 20, 18, 21, 18, and 22, respectively, in order of appearance.

[0032] FIG. 2 depicts RNA structure analysis of a splice site, showing base-pairing between 5’ splicesite sequence and U1 snRNA sequence that forms a loop structure. ss: splice site, Ψ: pseudouridine. Figure discloses SEQ ID NOs: 18, 23, 18, 24, 18, and 25, respectively, in order of appearance.

[0033] FIG. 3 shows that potential mechanisms by which SMSM can drive reduction of both HTT andPMS1 protein levels.

[0034] FIG. 4A and FIG. 4B depict a simplified schematic of an exemplary splicing event ofHuntingtin (HTT) in the absence (left: canonical splicing) or in the presence (right: non-canonical splicing) of a small molecule splicing modulator (SMSM). CAGn: expansion of CAG repeats.

[0035] FIG. 4C depicts RNA structure analysis of HTT target splice site, showing base-pairing betweentarget 5’ splice site sequence and U1 snRNA sequence. Figure discloses SEQ ID NOs: 26 and 1, respectively, in order of appearance.

[0036] FIG. 4D shows SMSM binding to HTT exon 49b.

[0037] FIGs. 5A-5B and 5D-5E are graphs demonstrating a reduction in HTT mRNA due to a shift inthe ratio between the canonical and non-canonical mRNA isoforms in the presence of SMSM in Huntington’s disease (HD) cells (FIG.5A), in mouse model brain (FIG.5B), in induced pluripotent stem cell (iPSC)-derived cortical neurons (FIG.5D), and in mouse model blood (FIG.5E).

[0038] FIG. 5C and FIG. 5F are graphs showing mutant HTT (mHTT) protein levels after SMSMtreatment in mouse model brain (FIG.5C) and in mouse model blood (FIG.5F).

[0039] FIG. 6A depicts a simplified schematic of an exemplary splicing event of PMS1 homolog 1,mismatch repair system component (PMS1) in the absence (top) or in the presence (bottom) of a small molecule splicing modulator (SMSM). NMD: nonsense-medicated mRNA decay.

[0040] FIG. 6B depicts RNA structure analysis of PMS1 target splice site, showing base-pairingbetween target 5’ splice site sequence and U1 snRNA sequence. Figure discloses SEQ ID NOs: 26 and 2, respectively, in order of appearance.

[0041] FIG. 6C is a graph showing a reduction in PMS1 mRNA due to a shift in the ratio between thecanonical and non-canonical mRNA isoforms in the presence of SMSM.

[0042] FIG. 6D is a graph showing a reduction in PMS1 mRNA due to a shift in the ratio betweencanonical and non-canonical (cryptic) mRNA isoforms in human lymphoblast cells treated with SMSM.

[0043] FIG. 6E shows a reduction in PMS1 total protein level in human lymphoblast cells treated withSMSM.WSGR Docket No.51503-775.601

[0044] FIG. 7A is a Phase 1 clinical trial design to test SMSM in healthy volunteers and HD patients. R:Randomized, SAD: single ascending dose, MAD: multiple ascending dose, PK: Pharmacokinetics, PD: Pharmacodynamics, SMSM: Compound of Structure B, CSF: Cerebrospinal fluid, QD: once daily.

[0045] FIG. 7B is a graph showing changes in HTT mRNA after a single dose SMSM (Compound ofStructure B) treatment compared to pre-treatment.

[0046] FIG. 7C is a graph showing mean concentration of SMSM (Compound of Structure B) by timeprofile following a single dose oral administration.

[0047] FIG. 8A is a graph showing the mean ratio of HTT mRNA after multiple doses of SMSM(Compound of Structure B) from day 1 pre-dose by time.

[0048] FIG. 8B is a graph showing the mean HTT mRNA in blood 24 hours after the last dose of 14consecutive days. SMSM: Compound of Structure B.

[0049] FIG. 8C is a graph showing the mean SMSM (Compound of Structure B) concentrations afterascending oral dose administration in healthy volunteers. DETAILED DESCRIPTION

[0050] Certain specific details of this description are set forth in order to provide a thoroughunderstanding of various embodiments. However, one skilled in the art will understand that the present disclosure may be practiced without these details. In other instances, well-known structures have not been shown or described in detail to avoid unnecessarily obscuring descriptions of the embodiments.

[0051] Unless otherwise defined, all technical and scientific terms used herein have the same meaning ascommonly understood by one of ordinary skill in the art to which this disclosure belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. Definitions

[0052] The terms “compound(s) of this disclosure”, “compound(s) of the present disclosure”, “smallmolecule splicing modulator(s)”, “splicing modulator(s)”, “compound(s) that modify splicing” and “compound(s) modifying splicing”, or “SMSM” are interchangeably used herein and refer to compounds as disclosed herein and stereoisomers, tautomers, solvates, and salts (e.g., pharmaceutically acceptable salts) thereof.

[0053] Any open valency appearing on a carbon, oxygen, sulfur or nitrogen atom in the structures hereinindicates the presence of hydrogen, unless indicated otherwise.

[0054] The terms “administer,” “administering”, “administration,” and the like, as used herein, refer tothe methods that may be used to enable delivery of compounds or compositions to the desired site of biological action. These methods include, but are not limited to oral routes (p.o.), intraduodenal routes (i.d.), parenteral injection (including intravenous (i.v.), subcutaneous (s.c.), intraperitoneal (i.p.), intramuscular (i.m.), intravascular or infusion (inf.)), topical (top.) and rectal (p.r.) administration. Those of skill in the art are familiar with administration techniques that can be employed with the compounds and methods described herein. In some embodiments, the compounds and compositions described herein are administered orally.WSGR Docket No.51503-775.601

[0055] The terms “co-administration” or the like, as used herein, are meant to encompass administrationof the selected therapeutic agents to a single patient, and are intended to include treatment regimens in which the agents are administered by the same or different route of administration or at the same or different time.

[0056] The terms “effective amount” or “therapeutically effective amount,” as used herein, refer to asufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or condition being treated; for example a reduction and / or alleviation of one or more signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses can be an amount of an agent that provides a clinically significant decrease in one or more disease symptoms. An appropriate “effective” amount may be determined using techniques, such as a dose escalation study, in individual cases.

[0057] The terms “enhance” or “enhancing,” as used herein, means to increase or prolong either inamount, potency or duration a desired effect. For example, in regard to enhancing splicing of a target, the term “enhancing” can refer to the ability to increase or prolong splicing, either in amount, potency or duration, of a target.

[0058] The term “subject” or “patient” encompasses mammals. Examples of mammals include, but arenot limited to, any member of the mammalian class: humans, non-human primates such as chimpanzees, and other apes and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like. In one aspect, the mammal is a human. The term “animal” as used herein comprises human beings and non-human animals. In one embodiment, a “non-human animal” is a mammal, for example a rodent such as rat or a mouse. In one embodiment, a non-human animal is a mouse.

[0059] The terms “pharmaceutical composition” and “pharmaceutical formulation” (or “formulation”)are used interchangeably and denote a mixture or solution comprising a therapeutically effective amount of an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients to be administered to a subject, e.g., a human in need thereof.

[0060] The term “pharmaceutical combination” as used herein, means a product that results from mixingor combining more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination” means that the active ingredients, e.g., a compound described herein and a co-agent, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a compound described herein and a co-agent, are administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific intervening time limits, wherein such administration provides effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., administration of three or more active ingredients.WSGR Docket No.51503-775.601

[0061] The term “pharmaceutically acceptable” denotes an attribute of a material which is useful inpreparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable and is acceptable for veterinary as well as human pharmaceutical use. “Pharmaceutically acceptable” can refer to a material, such as a carrier or diluent, which does not abrogate the biological activity or properties of the compound, and is relatively nontoxic, i.e., the material may be administered to an individual without causing undesirable biological effects or interacting in a deleterious manner with any of the components of the composition in which it is contained.

[0062] The terms “pharmaceutically acceptable excipient”, “pharmaceutically acceptable carrier” and“therapeutically inert excipient” can be used interchangeably and denote any pharmaceutically acceptable ingredient in a pharmaceutical composition having no therapeutic activity and being non-toxic to the subject administered, such as disintegrators, binders, fillers, solvents, buffers, tonicity agents, stabilizers, antioxidants, surfactants, carriers, diluents, excipients, preservatives or lubricants used in formulating pharmaceutical products.

[0063] The term “pharmaceutically acceptable salts” denotes salts which are not biologically orotherwise undesirable. Pharmaceutically acceptable salts include both acid and base addition salts. A “pharmaceutically acceptable salt” can refer to a formulation of a compound that does not cause significant irritation to an organism to which it is administered and / or does not abrogate the biological activity and properties of the compound. In some embodiments, pharmaceutically acceptable salts are obtained by reacting an SMSM compound of Formula (I) with an acid. Pharmaceutically acceptable salts are also obtained by reacting a compound of Formula (I) with a base to form a salt.

[0064] The term “nucleic acid” as used herein generally refers to one or more nucleobases, nucleosides,or nucleotides, and the term includes polynucleobases, polynucleosides, and polynucleotides.

[0065] As used herein, a “small molecular weight compound” can be used interchangeably with “smallmolecule” or “small organic molecule.” Small molecules refer to compounds other than peptides or oligonucleotides; and typically have molecular weights of less than about 2000 Daltons, e.g., less than about 900 Daltons.

[0066] The terms “effective amount” or “therapeutically effective amount,” as used herein, refer to asufficient amount of an agent or a compound being administered which will relieve to some extent one or more of the symptoms of the disease or the condition being treated; for example a reduction and / or alleviation of one or more signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. For example, an “effective amount” for therapeutic uses can be an amount of an agent that provides a clinically significant decrease in one or more disease symptoms. An appropriate “effective” amount may be determined using techniques, such as a dose escalation study, in individual cases.

[0067] The terms “treat,” “treating” or “treatment,” as used herein, include alleviating, abating orameliorating at least one symptom of a disease or a condition, preventing additional symptoms, inhibiting the disease or the condition, e.g., arresting the development of the disease or the condition, relieving theWSGR Docket No.51503-775.601 disease or the condition, causing regression of the disease or the condition, relieving a condition caused by the disease or the condition, or stopping the symptoms of the disease or the condition.

[0068] The term “preventing” or “prevention” of a disease state denotes causing the clinical symptomsof the disease state not to develop in a subject that can be exposed to or predisposed to the disease state, but does not yet experience or display symptoms of the disease state.

[0069] The term “pharmaceutical composition” denotes a mixture comprising an active pharmaceuticalingredient together with one or more pharmaceutically acceptable excipients that can be administered to a subject, e.g., a human in need thereof.

[0070] The term “nucleic acid” or “polynucleic acid” as used herein generally refers to one or morenucleobases, nucleosides, or nucleotides, and the term includes polynucleobases, polynucleosides, and polynucleotides.

[0071] The term “polynucleotide,” as used herein generally refers to a molecule comprising two or morelinked nucleic acid subunits, e.g., nucleotides, and can be used interchangeably with “oligonucleotide”. For example, a polynucleotide may include one or more nucleotides selected from adenosine (A), cytosine (C), guanine (G), thymine (T) and uracil (U), or variants thereof. A nucleotide generally includes a nucleoside and at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more phosphate (PO3) groups. A nucleotide can include a nucleobase, a five–carbon sugar (either ribose or deoxyribose), and one or more phosphate groups. Ribonucleotides include nucleotides in which the sugar is ribose. Deoxyribonucleotides include nucleotides in which the sugar is deoxyribose. A nucleotide can be a nucleoside monophosphate, nucleoside diphosphate, nucleoside triphosphate or a nucleoside polyphosphate. For example, a nucleotide can be a deoxyribonucleoside polyphosphate, such as a deoxyribonucleoside triphosphate (dNTP). Exemplary dNTPs include deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxyguanosine triphosphate (dGTP), uridine triphosphate (dUTP) and deoxythymidine triphosphate (dTTP). dNTPs can also include detectable tags, such as luminescent tags or markers (e.g., fluorophores). For example, a nucleotide can be a purine (i.e., A or G, or variant thereof) or a pyrimidine (i.e., C, T or U, or variant thereof). In some examples, a polynucleotide is deoxyribonucleic acid (DNA), ribonucleic acid (RNA), or derivatives or variants thereof. Exemplary polynucleotides include, but are not limited to, short interfering RNA (siRNA), a microRNA (miRNA), a plasmid DNA (pDNA), a short hairpin RNA (shRNA), small nuclear RNA (snRNA), messenger RNA (mRNA), precursor mRNA (pre–mRNA), antisense RNA (asRNA), and heteronuclear RNA (hnRNA), and encompasses both the nucleotide sequence and any structural embodiments thereof, such as single–stranded, double–stranded, triple–stranded, helical, hairpin, loop, stem loop, bulge, asymmetric loop, symmetric loop, etc. In some cases, a polynucleotide is circular. A polynucleotide can have various lengths. For example, a polynucleotide can have a length of at least about 7 bases, 8 bases, 9 bases, 10 bases, 20 bases, 30 bases, 40 bases, 50 bases, 100 bases, 200 bases, 300 bases, 400 bases, 500 bases, 1 kilobase (kb), 2 kb, 3, kb, 4 kb, 5 kb, 10 kb, 50 kb, or more. A polynucleotide can be isolated from a cell or a tissue. For example, polynucleotide sequences mayWSGR Docket No.51503-775.601 comprise isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and / or synthetic DNA / RNA analogs.

[0072] As used herein, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably andrefer to a polymer of amino acid residues linked via peptide bonds and which may be composed of two or more polypeptide chains. For example, a polypeptide can refer to a polymer of at least two amino acid monomers joined together through amide bonds. An amino acid may be the L–optical isomer or the D– optical isomer. More specifically, the terms “polypeptide,” “protein,” and “peptide” can refer to a molecule composed of two or more amino acids in a specific order; for example, the order as determined by the base sequence of nucleotides in the gene or RNA coding for the protein. Proteins are essential for the structure, function, and regulation of the body’s cells, tissues, and organs, and each protein has unique functions. Examples are hormones, enzymes, antibodies, and any fragments thereof. In some cases, a protein can be a portion of the protein, for example, a domain, a subdomain, or a motif of the protein. In some cases, a protein can be a variant (or mutation) of the protein, wherein one or more amino acid residues are inserted into, deleted from, and / or substituted into the naturally occurring (or at least a known) amino acid sequence of the protein. A protein or a variant thereof can be naturally occurring or recombinant.

[0073] Methods for detection and / or measurement of polypeptides in biological material are well knownin the art and include, but are not limited to, Western–blotting, flow cytometry, ELISAs, RIAs, and various proteomics techniques. An exemplary method to measure or detect a polypeptide is an immunoassay, such as an ELISA. This type of protein quantitation can be based on an antibody capable of capturing a specific antigen, and a second antibody capable of detecting the captured antigen. Exemplary assays for detection and / or measurement of polypeptides are described in Harlow, E. and Lane, D. Antibodies: A Laboratory Manual, (1988), Cold Spring Harbor Laboratory Press.

[0074] Methods for detection and / or measurement of RNA in biological material are well known in theart and include, but are not limited to, Northern–blotting, RNA protection assay, RT PCR. Suitable methods are described in Molecular Cloning: A Laboratory Manual (Fourth Edition) By Michael R. Green, Joseph Sambrook, Peter MacCallum 2012, 2,028 pp, ISBN 978–1–936113–42–2.

[0075] A ribonucleoprotein (RNP) refers to a nucleoprotein that contains RNA. A RNP can be acomplex of a ribonucleic acid and an RNA–binding protein. Such a combination can also be referred to as a protein–RNA complex. These complexes can function in a number of biological functions that include, but are not limited to, DNA replication, gene expression, metabolism of RNA, and pre–mRNA splicing. Examples of RNPs include the ribosome, the enzyme telomerase, vault ribonucleoproteins, RNase P, heterogeneous nuclear RNPs (hnRNPs) and small nuclear RNPs (snRNPs).

[0076] Nascent RNA transcripts from protein–coding genes and mRNA processing intermediates,collectively referred to as pre–mRNA, are generally bound by proteins in the nuclei of eukaryotic cells. From the time nascent transcripts first emerge from RNA polymerase (e.g., RNA polymerase II) until mature mRNAs are transported into the cytoplasm, the RNA molecules are associated with an abundant set of splicing complex components (e.g., nuclear proteins and snRNAs). These proteins can beWSGR Docket No.51503-775.601 components of hnRNPs, which can contain heterogeneous nuclear RNA (hnRNA) (e.g., pre–mRNA and nuclear RNA complexes) of various sizes.

[0077] Splicing complex components function in splicing and / or splicing regulation. Splicing complexcomponents can include, but are not limited to, ribonuclear proteins (RNPs), splicing proteins, small nuclear RNAs (snRNAs), small nuclear ribonucleoproteins (snRNPs), and heterogeneous nuclear ribonucleoproteins (hnRNPs). Splicing complex components include, but are not limited to, those that may be required for splicing, such as constitutive splicing, alternative splicing, regulated splicing, and splicing of specific messages or groups of messages. A group of related proteins, the serine arginine rich proteins (SR proteins), can function in constitutive pre–mRNA splicing and may also regulate alternative splice–site selection in a concentration–dependent manner. SR proteins typically have a modular structure that consists of one or two RNA–recognition motifs (RRMs) and a C–terminal rich in arginine and serine residues (RS domain). Their activity in alternative splicing may be antagonized by members of the hnRNP A / B family of proteins. Splicing complex components can also include proteins that are associated with one or more snRNAs. SR proteins in human include, but are not limited to, SC35, SRp55, SRp40, SRm300, SFRS10, TASR–1, TASR–2, SF2 / ASF, 9G8, SRp75, SRp30c, SRp20, and P54 / SFRS11. Other splicing complex components in human that can be involved in splice site selection include, but are not limited to, U2 snRNA auxiliary factors (e.g., U2AF65, U2AF35), Urp / U2AF1–RS2, SF1 / BBP, CBP80, CBP 20, SF1 and PTB / hnRNP1. hnRNP proteins in humans include, but are not limited to, A1, A2 / B1, L, M, K, U, F, H, G, R, I and C1 / C2. Human genes encoding hnRNPs include HNRNPA0, HNRNPA1, HNRNPA1L1, HNRNPA1L2, HNRNPA3, HNRNPA2B1, HNRNPAB, HNRNPB1, HNRNPC, HNRNPCL1, HNRNPD, HNRPDL, HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, HNRNPK, HNRNPL, HNRPLL, HNRNPM, HNRNPR, HNRNPU, HNRNPUL1, HNRNPUL2,HNRNPUL3, and FMR1. Splicing complex components may be stably or transiently associated with asnRNP or with a transcript.

[0078] The term “intron” refers to both the DNA sequence within a gene and the correspondingsequence in the unprocessed RNA transcript. As part of the RNA processing pathway, introns can be removed by RNA splicing either shortly after or concurrent with transcription. Introns are found in the genes of most organisms and many viruses. They can be located in a wide range of genes, including those that generate proteins, ribosomal RNA (rRNA), and transfer RNA (tRNA).

[0079] An “exon” can be any part of a gene that encodes a part of the final mature RNA produced bythat gene after introns have been removed by RNA splicing. The term “exon” refers to both the DNA sequence within a gene and to the corresponding sequence in RNA transcripts.

[0080] A “spliceosome” can be assembled from snRNAs and protein complexes. The spliceosome canremove introns from a transcribed pre–mRNA.

[0081] The term “AUC” as used herein refers to an abbreviation for “area under the curve” in a graph ofthe concentration of a therapeutic agent over time in a certain part or tissue, such as blood or plasma, of a subject to whom the therapeutic agent has been administered.WSGR Docket No.51503-775.601

[0082] The term “cryptic exon” can refer to an intronic sequence that may be flanked by apparentconsensus splice sites but are generally not spliced into the mature mRNA or the product of splicing. The term “poison exon” can refer to a cryptic exon that contains a premature termination codon (premTC) in the reading frame of the exon when included in an RNA transcript. “Poison exon” can also refer to a cryptic exon inclusion of which in an RNA transcript causes a reading frame shift in downstream exons resulting in a premature stop codon, which was not in frame prior to the frame-shift caused by inclusion of the cryptic exon. In some embodiments, the poison exon can be a variant of an existing exon. In some embodiments, the poison exon can be an extended form of an existing exon. In some embodiments, the poison exon can be a truncated form of an existing exon. The terms “poison exon” and “toxic exon” are used interchangeably in the present invention. The terms “stop codon” and “termination codon” are used interchangeably in the present invention.

[0083] A splicing event that promotes inclusion of a poison exon can further promote inclusion of anintron immediately following the poison exon in an RNA transcript. Inclusion of the poison exon and the intron immediately following the poison exon can result in “nuclear retention” of the RNA transcript, e.g., mRNA, wherein the RNA transcript is retained in the nucleus and not transported or exported to the cytoplasm and thus, not translated into a protein. Small Molecule Splicing Modulators (SMSMs)

[0084] Described herein are compounds modifying splicing of gene products for use in the treatment,prevention and / or delay of progression of diseases or conditions.

[0085] In one aspect, described herein is a compound that has the structure ofpharmaceutically acceptable salt thereof.

[0086] In one aspect, described herein is a compound that has the structure of structure B, or apharmaceutically acceptable salt or stereoisomer thereof:Structure B.

[0087] In another aspect, described herein is the compound 6-(6-(((1R,2R,3S,5S)-2-Fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[d]oxazol-5-ol.WSGR Docket No.51503-775.601

[0088] In some embodiments, a stereoisomer of Structure B is a compound of Structure A.

[0089] In one aspect, described herein is the compound 6-(6-(((1S,2S,3R,5R)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazol-5-ol.

[0090] The absolute stereochemistry for Structure A and Structure B is not identified, but relativestereochemistry is known and indicated.

[0091] In one aspect, disclosed herein is a method of modulating splicing comprising contacting acompound of the present disclosure to cells, wherein the compound modulates splicing at a splice site sequence of a pre-mRNA that encodes a mRNA, wherein the mRNA encodes a target protein or a functional RNA.

[0092] In one aspect, disclosed herein is a method of treating a disease or condition comprisingadministering a compound of the present disclosure.

[0093] The compound described herein may be formed as, and / or used as, a pharmaceutically acceptablesalt. The type of pharmaceutical acceptable salts, include, but are not limited to: (1) acid addition salts, formed by reacting the free base form of the compound with a pharmaceutically acceptable: inorganic acid, such as, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, metaphosphoric acid, and the like; or with an organic acid, such as, for example, acetic acid, propionic acid, hexanoic acid, cyclopentanepropionic acid, glycolic acid, pyruvic acid, lactic acid, malonic acid, succinic acid, malic acid, maleic acid, fumaric acid, trifluoroacetic acid, tartaric acid, citric acid, benzoic acid, 3-(4-hydroxybenzoyl)benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, 1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, toluenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-1-carboxylic acid, glucoheptonic acid, 4,4’-methylenebis-(3-hydroxy-2-ene-1-carboxylic acid), 3-phenylpropionic acid, trimethylacetic acid, tertiary butylacetic acid, lauryl sulfuric acid, gluconic acid, glutamic acid, hydroxynaphthoic acid, salicylic acid, stearic acid, muconic acid, butyric acid, phenylacetic acid, phenylbutyric acid, valproic acid, and the like; (2) salts formed when an acidic proton present in the parent compound is replaced by a metal ion, e.g., an alkali metal ion (e.g. lithium, sodium, potassium), an alkaline earth ion (e.g. magnesium, or calcium), or an aluminum ion. In some cases, compounds described herein may coordinate with an organic base, such as, but not limited to, ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, dicyclohexylamine, tris(hydroxymethyl)methylamine. In other cases, compounds described herein may form salts with amino acids such as, but not limited to, arginine, lysine, and the like. Acceptable inorganic bases used to form salts with compounds that include an acidic proton, include, but are not limited to, aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate, sodium hydroxide, and the like.WSGR Docket No.51503-775.601

[0094] In some embodiments, described herein is a compound modifying splicing of gene products,such as HTT pre-m RNA for use in the treatment, prevention, and / or delay of progression of diseases or conditions (e.g., Huntington’s disease). In some embodiments, the present disclosure relates to a pharmaceutical composition comprising a compound described herein for use in the treatment, prevention, and / or delay of progression of Huntington’s disease. In some embodiments, a compound described herein can be administered for treatment, prevention, and / or delay of progression of Huntington’s disease. In some embodiments, a subject is affected by Huntington’s disease associated with the HTT gene. In some embodiments, a subject is affected by Huntington’s disease associated with a splicing product of the HTT pre-mRNA. In some embodiments, the splicing product of the HTT pre- mRNA is an aberrant splicing product. In some embodiments, the splicing product of the HTT pre- mRNA encodes an aberrant polypeptide. In some embodiments, the splicing product of the HTT pre- mRNA is an aberrant splicing product resulted from a mutation in the HTT gene. In some embodiments, the splicing product of the HTT pre-mRNA may comprise a string of CAG repeats. In some embodiments, the splicing product of the HTT pre-mRNA may comprise an aberrant expansion of a string of CAG repeats. In some embodiments, the splicing product of the HTT pre-mRNA may comprise an aberrant expansion of a string of CAG repeats resulted from a mutation in the HTT gene.

[0095] In some embodiments, the compound and methods of use described herein can modulatesplicing, such as alternative splicing of a polynucleotide encoded by HTT gene. In some embodiments, alternative splicing of the HTT pre-mRNA may lead to the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 isoforms of the huntingtin protein. In some embodiments, the HTT gene may comprise a mutation. In some embodiments, the HTT gene may comprise a mutation associated with expansion of a CAG repeat. In some embodiments, the splice modulating compounds and methods of use described herein can modulate splicing of the HTT pre-mRNA that lead to inclusion of a cryptic exon (e.g., a poison exon) that is normally not included in the HTT spliced product, e.g., mRNA. In some embodiments, the cryptic exon (e.g., a poison exon) included in the HTT spliced product may lead to degradation of the HTT spliced product through nonsense-mediated decay (NMD) mediated RNA degradation. In a preferred embodiment, alternative splicing of the HTT pre-mRNA may lead to inclusion of a cryptic exon that is not normally included in between exon 49 and exon 50 of the HTT mRNA. In a preferred embodiment, alternative splicing of the HTT pre-mRNA may promote the inclusion of a poison exon 49b. In some embodiments, the HTT pre-mRNA comprises the sequence AGCAGA / guaagggggcuu (SEQ ID NO: 1). In a preferred embodiment, the compounds described herein bind to the 5’ss sequence AGCAGA / guaagggggcuu (SEQ ID NO: 1).

[0096] Described herein is a compound modifying splicing of gene products wherein the compoundinduces a post-transcriptionally unstable variant or transcript of a gene product. Described herein is a compound modifying splicing of gene products wherein the compound represses a transcript of a gene product. In some embodiments, an HTT transcript harbors a poison exon. In some embodiments, the poison exon results in a frame-shift in a downstream exon, for example in an exon immediately following the poison exon. In some embodiments, the frame-shift in a downstream exon contains an in-frame stopWSGR Docket No.51503-775.601 codon that would not be in frame in the absence of inclusion of the poison exon. In some embodiments, the poison exon comprises an in-frame premature termination codon (PTC). In some embodiments, the poison exon triggers NMD and degradation of the transcript. In some embodiments, the gene product is HTT. Pharmaceutical Compositions

[0097] In some embodiments, the compounds described herein are formulated into pharmaceuticalcompositions. Pharmaceutical compositions are formulated in a conventional manner using one or more pharmaceutically acceptable inactive ingredients that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Pharmaceutical compositions and methods of making the same can be found, for example, in Remington: The Science and Practice of Pharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995); Hoover, John E., Remington’s Pharmaceutical Sciences, Mack Publishing Co., Easton, Pennsylvania 1975; Liberman, H.A. and Lachman, L., Eds., Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; and Pharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed. (Lippincott Williams & Wilkins, 1999), herein incorporated by reference for such disclosure.

[0098] In some embodiments, disclosed herein is a pharmaceutical composition comprising a compoundof the disclosure or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable excipient or carrier. Splicing Modulation of Target Gene Products and Methods of Use

[0099] The present disclosure contemplates use of small molecules with favorable drug properties thatmodulate the activity of splicing of a target RNA. Provided herein are small molecule splicing modulators (SMSMs) that modulate splicing of a polynucleotide. In some embodiments, the SMSMs can bind and modulate target RNA. In some embodiments, the target RNA can be an mRNA. In some embodiments, the target RNA can be a noncoding RNA. In some embodiments, the target RNA can be a pre-mRNA. In some embodiments, the target RNA can be an hnRNA. In some embodiments, the SMSMs can modulate splicing of the target RNA. In some embodiments, a SMSM provided herein can modulate splicing at a sequence of the target RNA. In some embodiments, a SMSM provided herein can modulate splicing at a native splice site sequence of the target RNA. In some embodiments, a SMSM provided herein can modulate splicing at a cryptic splice site sequence of the target RNA. In some embodiments, a SMSM provided herein can modulate splicing at an alternative splice site sequence of the target RNA. In some embodiments, a SMSM provided herein can bind to a target RNA. In some embodiments, a SMSM provided herein can bind to a splicing complex or a component thereof. In some embodiments, a SMSM provided herein can bind to a target RNA and a splicing complex or a component thereof. In some embodiments, a SMSM provided herein can modulate binding affinity of a splicing complex component to a target RNA such as a pre-mRNA. In some embodiments, a SMSM provided herein can modulate binding affinity of a splicing complex component to a target RNA such as a pre-WSGR Docket No.51503-775.601 mRNA at a splice site sequence. In some embodiments, a SMSM provided herein can modulate binding affinity of a splicing complex component to a target RNA such as a pre-mRNA upstream of a splice site sequence or downstream of a splice site sequence.

[0100] Modulation of splicing by SMSMs described herein can include, but is not limited to, modulationof naturally occurring splicing, splicing of an RNA expressed in a diseased cell, splicing at cryptic splice site sequences of an RNA, or alternative splicing. Modulation of splicing by SMSMs described herein can restore or promote correct splicing or a desired splicing event. Modulation of splicing by SMSMs described herein can block a splicing event. Modulation of splicing by SMSMs described herein can include, but is not limited to, prevention of a naturally occurring splicing events or prevention of aberrant splicing events, e.g., splicing events caused by mutations or aberrant secondary or tertiary structures of RNA that are associated with conditions and diseases. In some embodiments, SMSMs described herein can prevent, block, or inhibit splicing at a splice site sequence. In some embodiments, SMSMs described herein can prevent, block, or inhibit a splicing event at a splice site that results in an inclusion or exclusion of an exon. In some embodiments, SMSMs described herein can promote or increase splicing at a splice site sequence. In some embodiments, SMSMs described herein can promote a splicing event at a splice site that results in an inclusion or exclusion of an exon. In some embodiments, SMSMs described herein can promote a splicing event at a splice site that results in an inclusion of an exon and an intron immediately following the exon. In some embodiments, the exon can be a naturally occurring or a canonical exon. In some embodiments, the exon can be a cryptic exon. In some embodiments, the exon can be a poison exon. In some embodiments, SMSMs described herein can modulate splicing at a specific splice site sequence. In some embodiments, a splice site sequence can comprise a 5’ splice site sequence, 3’ splice site sequence, an alternative splice site sequence, or a cryptic splice site sequence.

[0101] In some embodiments, the splice site sequence can comprise a native splice site sequence. Insome embodiments, the native splice site can comprise a canonical splice site. In some embodiments, the native splice site can comprise an alternative splice site. In some embodiments, the native splice site can comprise a cryptic splice site. In some embodiments, the alternative splice site can comprise a 5’ splice site sequence. In some embodiments, the alternative splice site or the cryptic splice site sequence can comprise AGCAGA / guaagggggcuu (SEQ ID NO: 1). In some embodiments, the alternative splice site or the cryptic splice site sequence can comprise AAAUGA / guaagacugguu (SEQ ID NO: 2).

[0102] Described herein are compounds for modifying splicing of gene products, such as a pre-mRNAof Huntingtin (HTT) gene or PMS1 homolog 1, mismatch repair system component (PMS1) gene, for use in the treatment, prevention, and / or delay of progression of diseases or conditions. In some embodiments, described herein is a method of stabilizing somatic expansion via PMS1, comprising administering a therapeutically effective amount of a SMSM (e.g., a compound of Structure B or a salt or stereoisomer thereof) to a subject in need thereof.

[0103] In some embodiments, described herein is a method of treating, preventing, delaying ofprogression of a disease or a condition, or ameliorating symptoms of the disease or the condition, comprising administering a therapeutically effective amount of a SMSM to a subject in need thereof,WSGR Docket No.51503-775.601wherein the SMSM modulates splicing of an HTT pre-mRNA or a PMS1 pre-mRNA, or both. In someembodiments, described herein is a method of treating, preventing, delaying of progression of a disease or a condition, or ameliorating symptoms of the disease or the condition, comprising administering a therapeutically effective amount of a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof.

[0104] In some embodiments, described herein is a method of treating, preventing, delaying of progressof a disease or condition, or ameliorating symptoms of a disease or condition associated with HTT, mutant HTT (mHTT), or PMS1 expression level or activity level in a subject in need thereof, comprising administering a therapeutically effective amount of a SMSM, wherein the SMSM binds to a pre-mRNA encoded by HTT or PMS1 and modulates splicing of the HTT pre-mRNA or the PMS1 pre-mRNA in a cell of the subject to produce a spliced product of the HTT pre-mRNA or the PMS1 pre-mRNA. In some embodiments, described herein is a method of treating, preventing, delaying of progress, or ameliorating symptoms of a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a SMSM, wherein the SMSM binds to a pre-mRNA encoded by HTT or PMS1 and modulates splicing of the HTT pre-mRNA or the PMS1 pre-mRNA in a cell of the subject to produce a spliced product of the HTT pre-mRNA or the PMS1 pre-mRNA, wherein the subject can benefit from modulating splicing of the HTT pre-mRNA or the PMS1 pre-mRNA. In some embodiments, described herein is a method of treating, preventing, delaying of progress of a disease or condition, or ameliorating symptoms of a disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof. In some embodiments, described herein is a method of treating, preventing, delaying of progress of a disease or condition, or ameliorating symptoms of a disease or condition, comprising administering a therapeutically effective amount of a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof.

[0105] In some embodiments, described herein is a method of modulating splicing of an HTT pre-mRNA or a PMS1 pre-mRNA, comprising contacting a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof to the HTT pre-mRNA or the PMS1 pre-mRNA with a splice site sequence or cells comprising the HTT pre-mRNA or the PMS1 pre-mRNA, wherein the compound binds to the HTT pre-mRNA or the PMS1 pre-mRNA and modulates splicing of the HTT pre-mRNA or the PMS1 pre-mRNA in a cell of a subject to produce a spliced product of the HTT pre-mRNA or the PMS1 pre-mRNA.

[0106] In some embodiments, described herein is use of a compound of Structure B, or apharmaceutically acceptable salt or a stereoisomer thereof, in the manufacture of a medicament for the treatment of a condition or disease. In some embodiments, described herein is use of a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, in the manufacture of a medicament for the treatment of a disease or condition, wherein the compound modulates splicing of an HTT pre-mRNA or a PMS1 pre-mRNA.WSGR Docket No.51503-775.601

[0107] In some embodiments, the spliced product of the HTT pre-mRNA or the spliced product of thePMS1 pre-mRNA undergoes non-sense mediated decay (NMD) or nuclear retention. In some embodiments, the nonsense-mediated decay (NMD) or nuclear retention of the spliced product of the HTT pre-mRNA or the spliced product of the PMS1 pre-mRNA is promoted. In some embodiments, the nonsense-mediated decay (NMD) or nuclear retention of the spliced product of the HTT pre-mRNA is increased compared to a spliced product of the HTT pre-mRNA produced in the absence of the SMSM. In some embodiments, the nonsense-mediated decay (NMD) or nuclear retention of the spliced product of the PMS1 pre-mRNA is increased compared to a spliced product of the PMS1 pre-mRNA produced in the absence of the SMSM.

[0108] In some embodiments, described herein is a method of modulating splicing of an HTT pre-mRNA, comprising contacting a SMSM to the HTT pre-mRNA with a splice site sequence or cells comprising the HTT pre-mRNA, wherein the SMSM binds to the HTT pre-mRNA and modulates splicing of the HTT pre-mRNA in a cell of a subject to produce a spliced product of the HTT pre-mRNA. In some embodiments, described herein is a method of modulating splicing of a PMS1 pre-mRNA, comprising contacting a SMSM to the PMS1 pre-mRNA with a splice site sequence or cells comprising the PMS1 pre-mRNA, wherein the SMSM binds to the PMS1 pre-mRNA and modulates splicing of the PMS1 pre-mRNA in a cell of a subject to produce a spliced product of the PMS1 pre-mRNA.

[0109] In some embodiments, described herein, is a method of modulating splicing of HTT pre-mRNA,comprising contacting a SMSM to the HTT pre-mRNA with a splice site sequence or cells comprising the HTT pre-mRNA, wherein the SMSM binds to the HTT pre-mRNA and modulates splicing of the HTT pre-mRNA in a cell of a subject to produce a spliced product of the HTT pre-mRNA, wherein the splice site sequence comprises AGCAGA / guaagggggcuu (SEQ ID NO: 1). In some embodiments, described herein, is a method of modulating splicing of PMS1 pre-mRNA, comprising contacting a SMSM to the PMS1 pre-mRNA with a splice site sequence or cells comprising the PMS1 pre-mRNA, wherein the SMSM binds to the PMS1 pre-mRNA and modulates splicing of the PMS1 pre-mRNA in a cell of a subject to produce a spliced product of the PMS1 pre-mRNA, wherein the splice site sequence comprises AAAUGA / guaagacugguu (SEQ ID NO: 2).

[0110] In some embodiments, described herein is a method of treating, preventing, delaying of progressof a disease or condition, or ameliorating symptoms of a disease or condition associated with HTT protein or mutant mHTT (HTT) protein expression level or activity level in a subject in need thereof, comprising administering a therapeutically effective amount of a SMSM to the subject, wherein the SMSM binds to an HTT pre-mRNA with a splice site sequence and modulates splicing of the HTT pre- mRNA in a cell of the subject, wherein a spliced product of the HTT pre-mRNA undergoes nonsense- mediated decay (NMD), and wherein the splice site sequence comprises AGCAGA / guaagggggcuu (SEQ ID NO: 1). In some embodiments, described herein is a method of treating, preventing, delaying of progress of a disease or condition, or ameliorating symptoms of a disease or condition in a subject in need thereof (e.g., a subject that can benefit from modulating splicing of the HTT pre-mRNA), comprising administering a therapeutically effective amount of a SMSM to the subject, wherein theWSGR Docket No.51503-775.601 SMSM binds to an HTT pre-mRNA with a splice site sequence and modulates splicing of the HTT pre- mRNA in a cell of the subject, wherein a spliced product of the HTT pre-mRNA undergoes nonsense- mediated decay (NMD), and wherein the splice site sequence comprises AGCAGA / guaagggggcuu (SEQ ID NO: 1).

[0111] In some embodiments, described herein is a method of treating, preventing, delaying of progressof a disease or condition, or ameliorating symptoms of a disease or condition associated with PMS1 protein expression level or activity level in a subject in need thereof, comprising administering a therapeutically effective amount of a SMSM to the subject, wherein the SMSM binds to a PMS1 pre- mRNA with a splice site sequence and modulates splicing of the PMS1 pre-mRNA in a cell of the subject, wherein a spliced product of the PMS1 pre-mRNA undergoes nonsense-mediated decay (NMD), and wherein the splice site sequence comprises AAAUGA / guaagacugguu (SEQ ID NO: 2). In some embodiments, described herein is a method of treating, preventing, delaying of progress of a disease or condition, or ameliorating symptoms of a disease or condition in a subject in need thereof (e.g., a subject that can benefit from modulating splicing of the PMS1 pre-mRNA), comprising administering a therapeutically effective amount of a SMSM to the subject, wherein the SMSM binds to a PMS1 pre- mRNA with a splice site sequence and modulates splicing of the PMS1 pre-mRNA in a cell of the subject, wherein a spliced product of the PMS1 pre-mRNA undergoes nonsense-mediated decay (NMD), and wherein the splice site sequence comprises AAAUGA / guaagacugguu (SEQ ID NO: 2).

[0112] In some embodiments, described herein is a method of treating, preventing, delaying of progressof Huntington’s disease (HD), or ameliorating symptoms of HD in a subject in need thereof, comprising administering a therapeutically effective amount of a SMSM to the subject, wherein the SMSM binds to an HTT pre-mRNA with a splice site sequence and modulates splicing of the HTT pre-mRNA in a cell of the subject, wherein a spliced product of the HTT pre-mRNA undergoes nonsense-mediated decay (NMD), and wherein the splice site sequence comprises AGCAGA / guaagggggcuu (SEQ ID NO: 1). In some embodiments, described herein is a method of treating, preventing, delaying of progress of Huntington’s disease (HD), or ameliorating symptoms of HD in a subject in need thereof, comprising administering a therapeutically effective amount of a SMSM to the subject, wherein the SMSM binds to a PMS1 pre-mRNA with a splice site sequence and modulates splicing of the PMS1 pre-mRNA in a cell of the subject, wherein a spliced product of the PMS1 pre-mRNA undergoes nonsense-mediated decay (NMD), and wherein the splice site sequence comprises AAAUGA / guaagacugguu (SEQ ID NO: 2).

[0113] The exemplary RNA structures of a splice site, showing base-pairing between 5’ splice sitesequence and U1 snRNP sequence that forms a bulge or a loop structure are shown in FIG.1 and FIG. 2. The exemplary RNA structure analysis of target splice site of HTT and PMS1, showing base-pairing between target 5’ splice site sequence and U1 snRNA sequence is shown in FIG.4C and FIG.6B, respectively. The exemplary binding of SMSM to HTT pre-mRNA exon 49b is shown in FIG.4D.

[0114] In some embodiments, the modulating splicing can comprise modulating alternative splicing. Insome embodiments, the modulating splicing can comprise promoting exon skipping. In some embodiments, the modulating splicing can comprise promoting exon inclusion. In some embodiments,WSGR Docket No.51503-775.601 the modulating splicing can comprise modulating nonsense-mediated mRNA decay or nonsense- mediated degradation (NMD). In some embodiments, the modulating NMD can comprise promoting NMD. In some embodiments, the modulating splicing can comprise modulating nuclear retention of the spliced product of the pre-mRNA. In some embodiments, the modulating splicing can comprise modulating intron intention. In some embodiments, the modulating intron retention can comprise promoting nuclear retention of the spliced product of the pre-mRNA.

[0115] In some embodiments, an SMSM can modulate cryptic exon inclusion. In some embodiments, anSMSM can modulate inclusion of a poison exon. In some embodiments, an SMSM can promote NMD. In some embodiments, an SMSM can promote inclusion of an exon that causes a reading frame shift in a downstream exon which introduces a premature stop codon to occur in the adjacent or in a downstream exon where the premature stop codon is at least ~50 to 55 nucleotides upstream of the final exon-exon junction. In some embodiments, an SMSM can promote inclusion of a poison exon that harbors an early termination codon within the reading frame, thereby triggering NMD. In some embodiments, an SMSM can promote inclusion of an upstream open reading frame (uORF), thereby triggering NMD. In some embodiments, an SMSM can promote inclusion of an intron after a termination codon, thereby triggering NMD. In some embodiments, an SMSM can modulate splicing at a cryptic splice site within an exon, causing truncation or extension of the exon, which can result in a reading frame shift in the exon or in a downstream exon that introduces a premature stop codon that is at least ~50 to 55 nucleotides upstream of the final exon-exon junction, triggering NMD. In some embodiments, an SMSM can modulate inclusion or exclusion of a native exon which is alternatively spliced. In some embodiments, the native exon harbors a premature stop codon, triggering NMD when included in an RNA transcript such as an mRNA.

[0116] In some embodiments, the SMSM can induce splicing at the alternative splice site. In someembodiments, the splicing at the alternative splice site can result in a frameshift in a downstream exon in the spliced product. In some embodiments, the downstream exon can comprise an in-frame stop codon that is not in frame in the absence of splicing at the alternative splice site. In some embodiments, the SMSM can block splicing at the native splice site or the 5’ splice site. In some embodiments, blocking splicing at the native splice site or the 5’ splice site can result in a frameshift in a downstream exon in the spliced product. In some embodiments, the downstream exon can comprise an in-frame stop codon that is not in frame in the presence of splicing at the native splice site or the 5’ splice site. In some embodiments, blocking splicing at the native splice site or the 5’ splice site can result in a frameshift in one or more downstream exons in the spliced product. In some embodiments, one of the one or more downstream exon can comprise an in-frame stop codon that is not in frame in the presence of splicing at the native splice site or the 5’ splice site. In some embodiments, the in-frame stop codon in the downstream exon may be at least 50 or at least 60 base pairs upstream of the 3’ end of the downstream exon. In some embodiments, the in-frame stop codon in the downstream exon may be at least 50 or at least 60 base pairs upstream of a final exon-exon junction.WSGR Docket No.51503-775.601

[0117] In some embodiments, promoting the splicing of the HTT pre-mRNA at an alternative splice siteor a cryptic splice site can promote NMD of the spliced product of the HTT pre-mRNA. In some embodiments, promoting the splicing of the HTT pre-mRNA at the alternative splice site or the cryptic splice site can result in destabilization of the spliced product of the HTT pre-mRNA. In some embodiments, promoting the splicing of the HTT pre-mRNA at the alternative splice site or the cryptic splice site can result in a decreased expression of a protein expressed by the spliced product of the HTT pre-mRNA. In some embodiments, promoting the splicing of the HTT pre-mRNA at the alternative splice site or the cryptic splice site can result in a decreased expression of a full-length HTT protein.

[0118] In some embodiments, promoting the splicing of the PMS1 pre-mRNA at an alternative splicesite or a cryptic splice site can promote NMD of the spliced product of the PMS1 pre-mRNA. In some embodiments, promoting the splicing of the PMS1 pre-mRNA at the alternative splice site or the cryptic splice site can result in destabilization of the spliced product of the PMS1 pre-mRNA. In some embodiments, promoting the splicing of the PMS1 pre-mRNA at the alternative splice site or the cryptic splice site can result in a decreased expression of a protein expressed by the spliced product of the PMS1 pre-mRNA. In some embodiments, promoting the splicing of the PMS1 pre-mRNA at the alternative splice site or the cryptic splice site can result in a decreased expression of a full-length PMS1 protein.

[0119] In some embodiments, the splicing of a pre-mRNA at an alternative splice site or a cryptic splicesite can promote NMD of a spliced product of the pre-mRNA. In some embodiments, the spliced product can comprise an alternative exon. In some embodiments, the SMSM can promote inclusion of the alternative exon in the spliced product. In some embodiments, the alternative exon can comprise a poison exon. In some embodiments, the SMSM can promote inclusion of the poison exon in the spliced product. In some embodiments, the poison exon can comprise an in-frame stop codon. In some embodiments, the in-frame stop codon can comprise a premature termination codon. In some embodiments, the in-frame stop codon may be at least 50 or 60 base pairs upstream of the 3’ end of the poison exon. In some embodiments, the in-frame stop codon may be less than 60 base pairs upstream of the 3’ end of the poison exon and the exon immediately downstream of the poison exon may not be the last exon in the pre-mRNA. In some embodiments, the sum of (a) the number of base pairs in the exon immediately downstream of the poison exon and (b) the number of base pairs between the premature termination codon in the poison exon and the 3’ end of the poison exon may be at least 50 or at least 60.

[0120] In some embodiments, the cells can comprise primary cells. In some embodiments, the cells cancomprise disease cells. In some embodiments, the cells can comprise Huntington’s disease cells. In some embodiments, the SMSM can modulate proliferation or survival of the cells. For example, the SMSM can modulate proliferation or survival of disease cells. In some embodiments, the SMSM can modulate cell death or apoptosis. For example, the SMSM can modulate cell death or apoptosis of disease cells. In some embodiments, modulating splicing with SMSMs can modulate DNA damage repair pathway (e.g., DNA mismatch repair pathway) in a cell, for example, in a disease cell. In some embodiments, the SMSM can modulate the expression level of a protein encoded by the spliced product of the pre-mRNA in the cells. In some embodiments, the protein encoded by the spiced product of the pre-mRNA canWSGR Docket No.51503-775.601 comprise a wildtype protein. In some embodiments, the protein encoded by the spiced product of the pre- mRNA can comprise a mutant protein.

[0121] In some embodiments, provided herein is a method of downregulating expression of a nativeprotein in a cell containing a DNA sequence encoding the native protein. In some embodiments, provided herein is a method of downregulating expression of a mutant protein in a cell containing a DNA sequence encoding the mutant protein. The method can comprise introducing into the cell a SMSM provided herein that can promote a splicing event at an alternative splice site or a cryptic splice site, promoting inclusion of a cryptic exon or a poison exon, and the level of the native protein or the mutant protein produced by the cell is reduced. In some embodiments, provided herein is a method of downregulating expression of a canonical isoform of an mRNA or a canonical isoform of a spliced product of a pre-mRNA. In some embodiments, provided herein is a method of upregulating expression of a cryptic (or non-canonical) isoform of an mRNA or a cryptic (or non-canonical) isoform of a spliced product of a pre-mRNA. For example, a SMSM provided herein can promote a splicing event at an alternative splice site or a cryptic splice site, promoting inclusion of a cryptic (or non-canonical) exon or a poison exon, and the level of the canonical isoform of an mRNA or a canonical isoform of a spliced product of a pre-mRNA is reduced and the level of a cryptic (or non-canonical) isoform of an mRNA or a cryptic (or non-canonical) isoform of a spliced product of a pre-mRNA is increased (e.g., an mRNA or a spliced product of a pre-mRNA with a cryptic exon or a poison exon).

[0122] Also provided herein is a method of altering the ratio of splice variants produced from a gene.The method can comprise contacting a pre-mRNA molecule and / or other elements and / or factors of the splicing machinery with a SMSM described herein to modulate alternative splicing events. The SMSMs described herein can be used to act upon 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 alternative splicing events that can occur within a pre-mRNA. In some embodiments, a first splice variant may be downregulated or inhibited, resulting in an altered ratio of splice variants of the RNA. In some embodiments, a first splice variant may be upregulated while a second splice variant may be unaffected, thereby altering the ratio of the RNA. In some embodiments, a first splice variant may be upregulated while a second splice variant may be downregulated, thereby altering the ratio of the RNA. In some embodiments, a first splice variant may be downregulated while a second splicing variant may be unaffected thereby altering the ratio of the RNA. In some embodiments, a first splice variant may be downregulated while a second splicing variant may be upregulated thereby altering the ratio of the RNA. In some embodiments, a first splice variant can comprise a canonical isoform or a cryptic (or non- canonical) isoform. In some embodiments, a second variant can comprise a canonical isoform or a cryptic (or non-canonical) isoform.

[0123] In some aspects, a method can comprise contacting a SMSM to a pre-mRNA that modulatessplicing of the pre-mRNA to favor expression of a transcript that promotes cell proliferation. For example, a SMSM described herein can increase one or more isoforms of a transcript that promotes cell proliferation. In some aspects, a method comprises contacting a SMSM to a pre–mRNA that modulates splicing of the pre-mRNA to favor expression of a transcript that prevents or inhibits cell proliferation.WSGR Docket No.51503-775.601 For example, a SMSM described herein can decrease expression one or more isoforms of a transcript that promotes cell proliferation.

[0124] In some embodiments, the invention provides compositions and methods for decreasingproduction of mature mRNA and, in turn, protein, in cells of a subject in need thereof, for example, a subject that can benefit from decreased production of a protein. In one embodiment, the described methods may be used to treat subjects having a disease or condition caused by a mutation in a gene, including missense, splicing, frameshift and nonsense mutations, as well as whole gene deletions, which can result in increased protein production. In some embodiments, the described methods may be used to treat subjects having a disease or condition caused by an aberrant expansion of a nucleotide sequence repeat in a gene (e.g., aberrant expansion of a string of CAG repeat in a gene), which can result in expression and / or accumulation of a mutant protein or a non-functional protein. In some embodiments, the described methods may be used to treat subjects having a disease or condition caused by accumulation of a mutant protein or a non-functional protein. In another embodiment, the described methods may be used to treat subjects having a disease or condition not caused by gene mutation. In some embodiments, the compositions and methods of the present invention can be used to treat subjects having a disease or condition, who can benefit from decreased production of a protein. In some embodiments, the compositions and methods of the present invention can be used to treat subjects having a disease or condition, who can benefit from decreased production of decreased activity of a protein resulting from the decreased production of the protein.

[0125] In some embodiments, administering of the compounds described herein to a subject reducesHTT mRNA in the subject. In some embodiments, an average HTT mRNA in a subject is reduced by at least 25% by the oral administering of the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof. In some embodiments, an average HTT mRNA in a subject is reduced by at least 50% by the oral administering of the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof. In some embodiments, an average HTT mRNA in a subject is reduced by at least 60% by the oral administering of the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof. In some embodiments, an average HTT mRNA in a subject is reduced by at least 70% by the oral administering of the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof. In some embodiments, an average HTT mRNA in a subject is reduced by at least 80% by the oral administering of the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof. In some embodiments, an average HTT mRNA in a subject is reduced by at least 90% by the oral administering of the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof. In some embodiments, an average HTT mRNA in a subject is reduced by at most 99% by the oral administering of the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof. In some embodiments, an average HTT mRNA in a subject is reduced by at most 90% by the oral administering of the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof. In some embodiments, an average HTT mRNA in a subject is reduced by at most 80% by the oral administering of the Compound of Structure B, or a pharmaceutically acceptableWSGR Docket No.51503-775.601 salt or a stereoisomer thereof. In some embodiments, an average HTT mRNA in a subject is reduced by at most 99%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, or 50% by the oral administering of the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered daily. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 1 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 2 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 3 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 4 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 5 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 6 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 7 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 8 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 9 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 10 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 11 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 12 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 13 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 14 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 15 mg. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered at a daily oral dose of 16 mg.

[0126] In some embodiments, the total amount of an mRNA encoding a protein (e.g., a target protein) orfunctional RNA produced in the cell contacted by a SMSM or a pharmaceutically acceptable salt thereof is decreased about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100% about 90% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, aboutWSGR Docket No.51503-775.601 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, 70% to about 80%, about 70% to about 90%, about 80% to about 90%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 99%, compared to the total amount of the mRNA encoding the target protein or functional RNA produced in a control cell, e.g., a cell not contacted by a SMSM.

[0127] In some embodiments, the total amount of an mRNA encoding a protein (e.g., a target protein) orfunctional RNA produced in the cell contacted by a SMSM or a pharmaceutically acceptable salt thereof is decreased about 10% to about 100%, about 20% to about 100%, about 30% to about 100%, about 40% to about 100%, about 50% to about 100%, about 60% to about 100%, about 70% to about 100%, about 80% to about 100% about 90% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 40% to about 50%, about 40% to about 60%, about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, 70% to about 80%, about 70% to about 90%, about 80% to about 90%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, or at least about 100%, compared to the total amount of target protein produced by a control cell, e.g., a cell not contacted by a SMSM.

[0128] In some embodiments, the amount of a HTT mRNA, PMS1 mRNA, or both HTT mRNA andPMS1 mRNA is reduced in the cell or in the cells contacted by the SMSM compared to a cell or cells not contacted by the SMSM. In some embodiments, the amount of the HTT mRNA, PMS1 mRNA, or both HTT mRNA and PMS1 mRNA is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, by at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, by at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, by at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%,WSGR Docket No.51503-775.601 at least 90%, by at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the cell or in the cells contacted by the SMSM compared to a cell or cells not contacted by the SMSM.

[0129] In some embodiments, the amount of a HTT protein, PMS1 protein, or both HTT and PMS1protein is reduced in the cell or in the cells contacted by the SMSM compared to a cell or cells not contacted by the SMSM. In some embodiments, the amount of the HTT protein, PMS1 protein, or both HTT and PMS1 protein is reduced by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, by at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, by at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, by at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, by at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the cell or in the cells contacted by the SMSM compared to a cell or cells not contacted by the SMSM.

[0130] In some embodiments, the ratio of a first isoform (e.g., a canonical isoform) to a second isoform(e.g., a cryptic isoform or a non-canonical isoform) may be 1:1, 1:1.2, 1:1.4, 1:1.6, 1:1.8, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, or 1:5. In some embodiments, the ratio of a first isoform (e.g., a canonical isoform) to a second isoform (e.g., a cryptic isoform or a non-canonical isoform) may be from about 1:1 to about 1:1.1, from about 1:1 to about 1:1.2, from about 1:1 to about 1:1.3, from about 1:1 to about 1:1.4, from about 1:1 to about 1:1.5, from about 1:1 to about 1:1.6, from about 1:1 to about 1:1.8, from about 1:1 to about 1:2, from about 1:1 to about 1:3, from about 1:1 to about 1:3.5, from about 1:1 to about 1:4, from about 1:1 to about 1:4.5, from about 1:1 to about 1:5, 1:2 to about 1:3, from about 1:2 to about 1:4, from about 1:2 to about 1:5, from about 1:3 to about 1:4, from about 1:3 to about 1:5, or from about 1:4 to about 1:5.

[0131] In some embodiments, provided herein are methods of treating a disease or condition in a subjectin need thereof by decreasing the amount of a target protein or mRNA in the cells of the subject, wherein the target protein is encoded by a gene listed in Table 20A.

[0132] In some embodiments, the splice modulating compounds and methods of use described hereincan modulate splicing of a polynucleotide encoded by HTT gene. In some embodiments, modulating splicing of the HTT pre-mRNA may lead to the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 isoforms of the HTT spliced product. In some embodiments, modulating splicing of the HTT pre-mRNA can lead to the expression of a cryptic isoform (or a non-canonical isoform) of the HTT mRNA. In some embodiments,WSGR Docket No.51503-775.601 modulating splicing of the HTT pre-mRNA can lead to an increase in the expression of a cryptic isoform (or a non-canonical isoform) of the HTT mRNA. In some embodiments, modulating splicing of the HTT pre-mRNA can lead to a decrease in the expression of a canonical isoform of the HTT mRNA (e.g., a full-length mRNA). An exemplary splicing event that can lead to a canonical isoform or a cryptic isoform (or a non-canonical isoform) of the HTT spliced product is shown in FIG.4A and FIG.4B.

[0133] In some embodiments, SMSMs and methods of use described herein can modulate splicing ofHTT pre-mRNA by promoting splicing of an HTT pre-mRNA at an alternative splice site or a cryptic splice site sequence in intron 49 that can result in inclusion of a poison exon 49b in a spliced product of the HTT pre-mRNA. In some embodiments, inclusion of the poison exon 49b in a spliced product of the HTT pre-mRNA can lead to degradation of the HTT spliced product or the HTT mRNA through NMD mediated RNA degradation. In some embodiments, inclusion of poison exon 49b in a spliced product of the HTT pre-mRNA can lead to destabilization of the HTT spliced product or the HTT mRNA. For example, poison exon 49b comprise an in-frame stop codon or a premature termination codon, leading to NMD. In some embodiments, the HTT pre-mRNA can comprise a cryptic splice site sequence comprising AGCAGA / guaagggggcuu (SEQ ID NO: 1). In some embodiments, the alternative splice site sequence or the cryptic splice site sequence may be located within intron 49. In some embodiments, the alternative splice site sequence or the cryptic splice site sequence in intron 49 may be located between the nucleotide encoded by genomic site GRCh38 / hg38: chr4: 3213731 and the nucleotide encoded genomic site GRCh38 / hg38: chr4: 3213748. In some embodiments, the alternative splice site sequence or the cryptic splice site sequence in intron 49 can comprise the nucleotide sequence encoded by genomic coordinates GRCh38 / hg38: chr4: 3213731 to GRCh38 / hg38: 3213748.

[0134] In some embodiments, a SMSM described herein can promote splicing of a HTT pre-mRNA atan alternative splice site sequence or a cryptic splice site sequence in intron 49, resulting in inclusion of poison exon 49b in a spliced product of the HTT pre-mRNA. In some embodiments, the amount of HTT mRNA produced or processed from HTT pre-mRNA is reduced in the cell or in the cells contacted by a SMSM described herein. In some embodiments, the amount of HTT mRNA lacking exon 49b produced or processed from HTT pre-mRNA is reduced in the cell or in the cells contacted by a SMSM described herein. In some embodiments, the amount of the spliced product of the HTT pre-mRNA comprising exon 49b is increased in the cell or in the cells contacted by the SMSM. In some embodiments, the amount of the spliced product of the HTT pre-mRNA comprising exon 49b is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, by at least 61%, at least 62%, at least 63%, at leastWSGR Docket No.51503-775.601 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, by at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, by at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, by at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the cell or in the cells contacted by the SMSM compared to in the cell or in the cells not contacted by the SMSM.

[0135] In some embodiments, the splice modulating compounds and methods of use described hereincan modulate splicing of a polynucleotide encoded by PMS1 gene. In some embodiments, modulating splicing of the PMS1 pre-mRNA may lead to the expression of 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 isoforms of the PMS1 spliced product. In some embodiments, modulating splicing of the PMS1 pre-mRNA can lead to the expression of a cryptic isoform (or a non-canonical isoform) of the PMS1 mRNA. In some embodiments, modulating splicing of the PMS1 pre-mRNA can lead to an increase in the expression of a cryptic isoform (or a non-canonical isoform) of the PMS1 mRNA. In some embodiments, modulating splicing of the PMS1 pre-mRNA can lead to a decrease in the expression of a canonical isoform of the PMS1 mRNA (e.g., a full-length mRNA). An exemplary splicing event that can lead to a canonical isoform or a cryptic isoform (or a non-canonical isoform) of the PMS1 spliced product is shown in FIG. 6A.

[0136] In some embodiments, SMSMs and methods of use described herein can modulate splicing ofPMS1 pre-mRNA by promoting splicing of an PMS1 pre-mRNA at an alternative splice site sequence or a cryptic splice site sequence in intron 5 that can result in inclusion of a poison exon 5b in a spliced product of the PMS1 pre-mRNA. In some embodiments, inclusion of the poison exon 5b in a spliced product of the PMS1 pre-mRNA can lead to degradation of the PMS1 spliced product or the PMS1 mRNA through NMD mediated RNA degradation. In some embodiments, inclusion of poison exon 5b in a spliced product of the PMS1 pre-mRNA can lead to destabilization of the PMS1 spliced product or the PMS1 mRNA. For example, poison exon 5b can comprise an in-frame stop codon or a premature termination codon, leading to NMD. In some embodiments, the PMS1 pre-mRNA can comprise an alternative splice site sequence or a cryptic splice site sequence comprising AAAUGA / guaagacugguu (SEQ ID NO: 2). In some embodiments, the alternative splice site sequence or the cryptic splice site sequence may be located within intron 5. In some embodiments, the alternative splice site sequence or the cryptic splice site sequence in intron 5 may be between the nucleotide encoded by genomic site GRCh38 / hg38: chr2: 189818824 and the nucleotide encoded by genomic site GRCh38 / hg19: chr2: 189818841. In some embodiments, the alternative splice site sequence or the cryptic splice site sequence of intron 5 can comprise the nucleotide sequence encoded by genomic coordinates GRCh38 / hg19: chr2: 189818824 to GRCh38 / hg19: chr2: 189818841.

[0137] In some embodiments, a PMS1 may be a wild-type PMS1. In some embodiments, a PMS1 genemay comprise a genetic variation. Non-limiting examples of PMS1 genetic variations are listed in Table 20B. In some embodiments, a genetic variation is a non-synonymous coding variant. In some embodiments, a genetic variation can comprise a non-synonymous coding variant. In some embodiments,WSGR Docket No.51503-775.601 a genetic variation may not disrupt or modulate the PMS1 gene. In some embodiments, a genetic variation may not be a loss-of-function genetic variation. In these embodiments, a genetic variation can comprise chr2:190660537 G>A, chr2:190719296 A>G, chr2:190719569 T>C, or any combination thereof, wherein chromosome positions of the genetic variation are defined with respect to UCSC hg19. In some embodiments, an allele comprising a genetic variation in a PMS1 gene can encode a variant PMS1 comprising a mutation. In some embodiments, the mutation can comprise E59K mutation, a K433R mutation, a L524S mutation, or any combination thereof.

[0138] In some embodiments, a genetic variation may disrupt or modulate the PMS1 gene. In thisembodiment, a genetic variation can comprise chr2:190660586 C>T, chr2:190670391 C>G, chr2:190670396 A>G, chr2:190717470 CA>C, chr2:190719499 G>A, chr2:190719607 G>A, chr2:190719704 G>A, chr2:190732559 T>C, or any combination thereof, wherein chromosome positions of the genetic variation are defined with respect to UCSC hg19. In some embodiments, an allele comprising a genetic variation in a PMS1 gene can encode a variant PMS1 comprising a mutation. In some embodiments, a variant PMS1 can comprise a mutation selected from the group consisting of T75I, T110R, T112A, S264*, G501R, E537K, R569Q, Y793H, wherein * denotes a premature termination of protein translation (e.g., loss-of-function variant due to premature stop codon).

[0139] In some embodiments, a SMSM described herein can promote splicing of a PMS1 pre-mRNA atan alternative splice site sequence or a cryptic splice site sequence in intron 5, resulting in inclusion of poison exon 5b in a spliced product of the PMS1 pre-mRNA. In some embodiments, the amount of PMS1 mRNA produced or processed from PMS1 pre-mRNA is reduced in the cell or in the cells contacted by a SMSM described herein. In some embodiments, the amount of PMS1 mRNA lacking poison exon 5b produced or processed from PMS1 pre-mRNA is reduced in the cell or in the cells contacted by a SMSM described herein.

[0140] In some embodiments, the amount of the spliced product of the PMS1 pre-mRNA comprisingexon 5b is increased in the cell or in the cells contacted by the SMSM. In some embodiments, the amount of the spliced product of the PMS1 pre-mRNA comprising exon 5b is increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, by at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, by at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, by at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, by at least 91%, at least 92%, at least 93%, at least 94%, atWSGR Docket No.51503-775.601 least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the cell or in the cells contacted by the SMSM compared to in the cell or in the cells not contacted by the SMSM.

[0141] In some embodiments, SMSMs and methods of use described herein can modulate splicing ofboth HTT1 pre-mRNA and PMS1 pre-mRNA by promoting splicing of an HTT pre-mRNA and an PMS1 pre-mRNA at an alternative splice site sequence or a cryptic splice site sequence in a cell or cells. In some embodiments, both the amount of the spliced product of the HTT pre-mRNA comprising exon 49b and the amount of the spliced product of the PMS1 pre-mRNA comprising exon 5b are increased in the cell or in the cells contacted by the SMSM. In some embodiments, both the amount of the spliced product of the HTT pre-mRNA comprising exon 49b and the amount of the spliced product of the PMS1 pre-mRNA comprising exon 5b are increased by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, by at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, by at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, by at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, by at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the cell or in the cells contacted by the SMSM compared to in the cell or in the cells not contacted by the SMSM.

[0142] In some embodiments, a SMSM can modulate splicing at a splice site sequence of apolynucleotide in primary cells or disease cells. In some embodiments, a SMSM can modulate proliferation or survival of primary cells or disease cells. In some embodiments, primary cells can comprise primary diseased cells.

[0143] In some embodiments, at least about 5%, 10%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%,85%, 90%, 95%, 97%, 98%, 99%, or 100% of disease cells may be killed. In some embodiments, at least about 5%, 10%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, or 100% of disease cells may undergo apoptosis. In some embodiments, at least about 5%, 10%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, or at least about 99%, or 100% of disease cells may undergo necrosis. In some embodiments, cell proliferation is reduced or inhibited in at least about 5%, 10%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99% or 100% of disease cells.WSGR Docket No.51503-775.601 Table 20A. Exemplary Targets for Exon InclusionTable 20B. PMS1 Variation* Premature stop codon loss-of-function variant

[0144] In some embodiments, a SMSM can modulate splicing at a splice site of a polynucleotide anddoes not exhibit significant toxicity. In some embodiments, an SMSM penetrates the blood brain barrier (BBB) when administered to a subject.

[0145] In some embodiments, following administration of a single dose of an SMSM to a subject underfasting conditions the Tmax for an SMSM is from about 0.25 hours to about 12 hours, about 0.25 hours to about 10 hours, 0.25 about hours to about 8 hours, about 0.25 hours to about 6 hours, about 0.25 hours to about 4 hours, about 0.25 hours to about 2 hours, about 0.5 hours to about 12 hours, about 0.5 hours to about 10 hours, about 0.5 hours to about 8 hours, about 0.5 hours to about 6 hours, about 0.5 hours to about 4 hours, about 0.5 hours to about 2 hours, about 0.75 hours to about 12 hours, about 0.75 hours to about 10 hours, about 0.75 hours to about 8 hours, about 0.75 hours to about 6 hours, about 0.75 hours to about 4 hours, about 0.75 hours to about 2 hours, about 1 hour to about 12 hours, about 1 hour to about 10 hour, about 1 hour to about 8 hours, about 1 hour to about 6 hours, about 1 hour to about 4 hours, about 1 hour to about 2 hours, about 2 hours to about 12 hours, about 2 hours to about 10 hours, about 2 hours to about 8 hours, about 2 hours to about 6 hours, about 2 hours to about 4 hours, about 4 hours to about 12 hours, about 4 hours to about 10 hours, about 4 hours to about 8 hours, about 4 hours to about 6 hours, about 6 hours to about 12 hours, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 12 hours, about 8 hours to about 10 hours, or about 10 hours to about 12 hours. InWSGR Docket No.51503-775.601 some embodiments, following a single dose of an SMSM under fasting conditions the Tmax for an SMSM is about 0.25 hours, about 0.5 hours, about 0.75 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In some embodiments, fasting conditions may be characterized by the levels of nutrient in the blood of the subject.

[0146] In some embodiments, following administration of a single dose of an SMSM to a subject underfed conditions the Tmax for an SMSM is from about 0.25 hours to about 12 hours, about 0.25 hours to about 10 hours, 0.25 about hours to about 8 hours, about 0.25 hours to about 6 hours, about 0.25 hours to about 4 hours, about 0.25 hours to about 2 hours, about 0.5 hours to about 12 hours, about 0.5 hours to about 10 hours, about 0.5 hours to about 8 hours, about 0.5 hours to about 6 hours, about 0.5 hours to about 4 hours, about 0.5 hours to about 2 hours, about 0.75 hours to about 12 hours, about 0.75 hours to about 10 hours, about 0.75 hours to about 8 hours, about 0.75 hours to about 6 hours, about 0.75 hours to about 4 hours, about 0.75 hours to about 2 hours, about 1 hour to about 12 hours, about 1 hour to about 10 hour, about 1 hour to about 8 hours, about 1 hour to about 6 hours, about 1 hour to about 4 hours, about 1 hour to about 2 hours, about 2 hours to about 12 hours, about 2 hours to about 10 hours, about 2 hours to about 8 hours, about 2 hours to about 6 hours, about 2 hours to about 4 hours, about 4 hours to about 12 hours, about 4 hours to about 10 hours, about 4 hours to about 8 hours, about 4 hours to about 6 hours, about 6 hours to about 12 hours, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 12 hours, about 8 hours to about 10 hours, or about 10 hours to about 12 hours. In some embodiments, following a single dose of an SMSM under fed conditions the Tmaxfor an SMSM is about 0.25 hours, about 0.5 hours, about 0.75 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours. In some embodiments, a fed condition may be characterized by the levels of nutrient in the blood of the subject.

[0147] In some embodiments, following administration of a single dose of an SMSM to a subject theTmaxfor an SMSM is from about 0.25 hours to about 12 hours, about 0.25 hours to about 10 hours, 0.25 about hours to about 8 hours, about 0.25 hours to about 6 hours, about 0.25 hours to about 4 hours, about 0.25 hours to about 2 hours, about 0.5 hours to about 12 hours, about 0.5 hours to about 10 hours, about 0.5 hours to about 8 hours, about 0.5 hours to about 6 hours, about 0.5 hours to about 4 hours, about 0.5 hours to about 2 hours, about 0.75 hours to about 12 hours, about 0.75 hours to about 10 hours, about 0.75 hours to about 8 hours, about 0.75 hours to about 6 hours, about 0.75 hours to about 4 hours, about 0.75 hours to about 2 hours, about 1 hour to about 12 hours, about 1 hour to about 10 hour, about 1 hour to about 8 hours, about 1 hour to about 6 hours, about 1 hour to about 4 hours, about 1 hour to about 2 hours, about 2 hours to about 12 hours, about 2 hours to about 10 hours, about 2 hours to about 8 hours, about 2 hours to about 6 hours, about 2 hours to about 4 hours, about 4 hours to about 12 hours, about 4 hours to about 10 hours, about 4 hours to about 8 hours, about 4 hours to about 6 hours, about 6 hours to about 12 hours, about 6 hours to about 10 hours, about 6 hours to about 8 hours, about 8 hours to about 12 hours, about 8 hours to about 10 hours, or about 10 hours to about 12 hours. In some embodiments,WSGR Docket No.51503-775.601 following a single dose of an SMSM the Tmax for an SMSM is about 0.25 hours, about 0.5 hours, about 0.75 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 7 hours, about 8 hours, about 9 hours, about 10 hours, about 11 hours, or about 12 hours.

[0148] In some embodiments, following administration of a single dose of an SMSM to a subject underfasting conditions the Cmax for an SMSM is from about 1 ng / mL to about 1000 ng / mL, from about 1 ng / mL to about 900 ng / mL, from about 1 ng / mL to about 800 ng / mL, from about 1 ng / mL to about 700 ng / mL, from about 1 ng / mL to about 600 ng / mL, from about 1 ng / mL to about 500 ng / mL, from about 1 ng / mL to about 400 ng / mL, from about 1 ng / mL to about 300 ng / mL, from about 1 ng / mL to about 200 ng / mL, from about 1 ng / mL to about 100 ng / mL, from about 1 ng / mL to about 50 ng / mL, from about 50 ng / mL to about 500 ng / mL, from about 50 ng / mL to about 400 ng / mL, from about 50 ng / mL to about 300 ng / mL, from about 50 ng / mL to about 200 ng / mL, from about 100 ng / mL to about 1000 ng / mL, from about 100 ng / mL to about 900 ng / mL, from about 100 ng / mL to about 800 ng / mL, from about 100 ng / mL to about 700 ng / mL, from about 100 ng / mL to about 600 ng / mL, from about 100 ng / mL to about 500 ng / mL, from about 500 ng / mL to about 1500 ng / mL, about 500 ng / mL to about 1400 ng / mL, about 500 ng / mL to about 1300 ng / mL, about 500 ng / mL to about 1200 ng / mL, about 500 ng / mL to about 1100 ng / mL, about 500 ng / mL to about 1000 ng / mL, about 500 ng / mL to about 900 ng / mL, about 500 ng / mL to about 800 ng / mL, about 500 ng / mL to about 700 ng / mL, about 500 ng / mL to about 600 ng / mL, about 600 ng / mL to about 1500 ng / mL, about 600 ng / mL to about 1400 ng / mL, about 600 ng / mL to about 1300 ng / mL, about 600 ng / mL to about 1200 ng / mL, about 600 ng / mL to about 1100 ng / mL, about 600 ng / mL to about 1000 ng / mL, about 600 ng / mL to about 900 ng / mL, about 600 ng / mL to about 800 ng / mL, about 600 ng / mL to about 700 ng / mL, about 700 ng / mL to about 1500 ng / mL, about 700 ng / mL to about 1400 ng / mL, about 700 ng / mL to about 1300 ng / mL, about 700 ng / mL to about 1200 ng / mL, about 700 ng / mL to about 1100 ng / mL, about 700 ng / mL to about 1000 ng / mL, about 700 ng / mL to about 900 ng / mL, about 700 ng / mL to about 800 ng / mL, about 800 ng / mL to about 1500 ng / mL, about 800 ng / mL to about 1400 ng / mL, about 800 ng / mL to about 1300 ng / mL, about 800 ng / mL to about 1200 ng / mL, about 800 ng / mL to about 1100 ng / mL, about 800 ng / mL to about 1000 ng / mL, about 800 ng / mL to about 900 ng / mL, about 900 ng / mL to about 1500 ng / mL, about 900 ng / mL to about 1400 ng / mL, about 900 ng / mL to about 1300 ng / mL, about 900 ng / mL to about 1200 ng / mL, about 900 ng / mL to about 1100 ng / mL, about 900 ng / mL to about 1000 ng / mL, about 1000 ng / mL to about 1500 ng / mL, about 1000 ng / mL to about 1400 ng / mL, about 1000 ng / mL to about 1300 ng / mL, about 1000 ng / mL to about 1200 ng / mL, about 1000 ng / mL to about 1100 ng / mL, about 1100 ng / mL to about 1500 ng / mL, about 1100 ng / mL to about 1400 ng / mL, about 1100 ng / mL to about 1300 ng / mL, about 1100 ng / mL to about 1200 ng / mL, about 1200 ng / mL to about 1500 ng / mL, about 1200 ng / mL to about 1400 ng / mL, about 1200 ng / mL to about 1300 ng / mL, about 1300 ng / mL to about 1500 ng / mL, about 1300 ng / mL to about 1400 ng / mL, or about 1400 ng / mL to about 1500 ng / mL.

[0149] In some embodiments, following administration of a single dose of an SMSM to a subject underfasting conditions the Cmax for an SMSM is about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45WSGR Docket No.51503-775.601 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 80 ng / mL, about 95 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 600 ng / mL, about 700 ng / mL, about 800 ng / mL, about 900 ng / mL, about 1000 ng / mL, about 1100 ng / mL, about 1200 ng / mL, about 1300 ng / mL, about 1400 ng / mL, or about 1500 ng / mL. In some embodiments, fasting conditions may be characterized by the levels of nutrient in the blood of the subject.

[0150] In some embodiments, following administration of a single dose of an SMSM to a subject underfed conditions the Cmax for an SMSM is from about 1 ng / mL to about 1000 ng / mL, from about 1 ng / mL to about 900 ng / mL, from about 1 ng / mL to about 800 ng / mL, from about 1 ng / mL to about 700 ng / mL, from about 1 ng / mL to about 600 ng / mL, from about 1 ng / mL to about 500 ng / mL, from about 1 ng / mL to about 400 ng / mL, from about 1 ng / mL to about 300 ng / mL, from about 1 ng / mL to about 200 ng / mL, from about 1 ng / mL to about 100 ng / mL, from about 1 ng / mL to about 50 ng / mL, from about 50 ng / mL to about 500 ng / mL, from about 50 ng / mL to about 400 ng / mL, from about 50 ng / mL to about 300 ng / mL, from about 50 ng / mL to about 200 ng / mL, from about 100 ng / mL to about 1000 ng / mL, from about 100 ng / mL to about 900 ng / mL, from about 100 ng / mL to about 800 ng / mL, from about 100 ng / mL to about 700 ng / mL, from about 100 ng / mL to about 600 ng / mL, from about 100 ng / mL to about 500 ng / mL, from about 500 ng / mL to about 1500 ng / mL, about 500 ng / mL to about 1400 ng / mL, about 500 ng / mL to about 1300 ng / mL, about 500 ng / mL to about 1200 ng / mL, about 500 ng / mL to about 1100 ng / mL, about 500 ng / mL to about 1000 ng / mL, about 500 ng / mL to about 900 ng / mL, about 500 ng / mL to about 800 ng / mL, about 500 ng / mL to about 700 ng / mL, about 500 ng / mL to about 600 ng / mL, about 600 ng / mL to about 1500 ng / mL, about 600 ng / mL to about 1400 ng / mL, about 600 ng / mL to about 1300 ng / mL, about 600 ng / mL to about 1200 ng / mL, about 600 ng / mL to about 1100 ng / mL, about 600 ng / mL to about 1000 ng / mL, about 600 ng / mL to about 900 ng / mL, about 600 ng / mL to about 800 ng / mL, about 600 ng / mL to about 700 ng / mL, about 700 ng / mL to about 1500 ng / mL, about 700 ng / mL to about 1400 ng / mL, about 700 ng / mL to about 1300 ng / mL, about 700 ng / mL to about 1200 ng / mL, about 700 ng / mL to about 1100 ng / mL, about 700 ng / mL to about 1000 ng / mL, about 700 ng / mL to about 900 ng / mL, about 700 ng / mL to about 800 ng / mL, about 800 ng / mL to about 1500 ng / mL, about 800 ng / mL to about 1400 ng / mL, about 800 ng / mL to about 1300 ng / mL, about 800 ng / mL to about 1200 ng / mL, about 800 ng / mL to about 1100 ng / mL, about 800 ng / mL to about 1000 ng / mL, about 800 ng / mL to about 900 ng / mL, about 900 ng / mL to about 1500 ng / mL, about 900 ng / mL to about 1400 ng / mL, about 900 ng / mL to about 1300 ng / mL, about 900 ng / mL to about 1200 ng / mL, about 900 ng / mL to about 1100 ng / mL, about 900 ng / mL to about 1000 ng / mL, about 1000 ng / mL to about 1500 ng / mL, about 1000 ng / mL to about 1400 ng / mL, about 1000 ng / mL to about 1300 ng / mL, about 1000 ng / mL to about 1200 ng / mL, about 1000 ng / mL to about 1100 ng / mL, about 1100 ng / mL to about 1500 ng / mL, about 1100 ng / mL to about 1400 ng / mL, about 1100 ng / mL to about 1300 ng / mL, about 1100 ng / mL to about 1200 ng / mL, about 1200 ng / mL to about 1500 ng / mL, about 1200 ng / mL to about 1400 ng / mL, about 1200 ng / mL toWSGR Docket No.51503-775.601 about 1300 ng / mL, about 1300 ng / mL to about 1500 ng / mL, about 1300 ng / mL to about 1400 ng / mL, or about 1400 ng / mL to about 1500 ng / mL.

[0151] In some embodiments, following administration of a single dose of an SMSM to a subject underfed conditions the Cmax for an SMSM is about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 80 ng / mL, about 95 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 600 ng / mL, about 700 ng / mL, about 800 ng / mL, about 900 ng / mL, about 1000 ng / mL, about 1100 ng / mL, about 1200 ng / mL, about 1300 ng / mL, about 1400 ng / mL, or about 1500 ng / mL. In some embodiments, a fed condition may be characterized by the levels of nutrient in the blood of the subject.

[0152] In some embodiments, following administration of a single dose of an SMSM to a subject theCmaxfor an SMSM is from about 1 ng / mL to about 1000 ng / mL, from about 1 ng / mL to about 900 ng / mL, from about 1 ng / mL to about 800 ng / mL, from about 1 ng / mL to about 700 ng / mL, from about 1 ng / mL to about 600 ng / mL, from about 1 ng / mL to about 500 ng / mL, from about 1 ng / mL to about 400 ng / mL, from about 1 ng / mL to about 300 ng / mL, from about 1 ng / mL to about 200 ng / mL, from about 1 ng / mL to about 100 ng / mL, from about 1 ng / mL to about 50 ng / mL, from about 50 ng / mL to about 500 ng / mL, from about 50 ng / mL to about 400 ng / mL, from about 50 ng / mL to about 300 ng / mL, from about 50 ng / mL to about 200 ng / mL, from about 100 ng / mL to about 1000 ng / mL, from about 100 ng / mL to about 900 ng / mL, from about 100 ng / mL to about 800 ng / mL, from about 100 ng / mL to about 700 ng / mL, from about 100 ng / mL to about 600 ng / mL, from about 100 ng / mL to about 500 ng / mL, about 500 ng / mL to about 1500 ng / mL, about 500 ng / mL to about 1400 ng / mL, about 500 ng / mL to about 1300 ng / mL, about 500 ng / mL to about 1200 ng / mL, about 500 ng / mL to about 1100 ng / mL, about 500 ng / mL to about 1000 ng / mL, about 500 ng / mL to about 900 ng / mL, about 500 ng / mL to about 800 ng / mL, about 500 ng / mL to about 700 ng / mL, about 500 ng / mL to about 600 ng / mL, about 600 ng / mL to about 1500 ng / mL, about 600 ng / mL to about 1400 ng / mL, about 600 ng / mL to about 1300 ng / mL, about 600 ng / mL to about 1200 ng / mL, about 600 ng / mL to about 1100 ng / mL, about 600 ng / mL to about 1000 ng / mL, about 600 ng / mL to about 900 ng / mL, about 600 ng / mL to about 800 ng / mL, about 600 ng / mL to about 700 ng / mL, about 700 ng / mL to about 1500 ng / mL, about 700 ng / mL to about 1400 ng / mL, about 700 ng / mL to about 1300 ng / mL, about 700 ng / mL to about 1200 ng / mL, about 700 ng / mL to about 1100 ng / mL, about 700 ng / mL to about 1000 ng / mL, about 700 ng / mL to about 900 ng / mL, about 700 ng / mL to about 800 ng / mL, about 800 ng / mL to about 1500 ng / mL, about 800 ng / mL to about 1400 ng / mL, about 800 ng / mL to about 1300 ng / mL, about 800 ng / mL to about 1200 ng / mL, about 800 ng / mL to about 1100 ng / mL, about 800 ng / mL to about 1000 ng / mL, about 800 ng / mL to about 900 ng / mL, about 900 ng / mL to about 1500 ng / mL, about 900 ng / mL to about 1400 ng / mL, about 900 ng / mL to about 1300 ng / mL, about 900 ng / mL to about 1200 ng / mL, about 900 ng / mL to about 1100 ng / mL, about 900 ng / mL to about 1000 ng / mL, about 1000 ng / mL to about 1500 ng / mL, about 1000 ng / mL to about 1400 ng / mL,WSGR Docket No.51503-775.601 about 1000 ng / mL to about 1300 ng / mL, about 1000 ng / mL to about 1200 ng / mL, about 1000 ng / mL to about 1100 ng / mL, about 1100 ng / mL to about 1500 ng / mL, about 1100 ng / mL to about 1400 ng / mL, about 1100 ng / mL to about 1300 ng / mL, about 1100 ng / mL to about 1200 ng / mL, about 1200 ng / mL to about 1500 ng / mL, about 1200 ng / mL to about 1400 ng / mL, about 1200 ng / mL to about 1300 ng / mL, about 1300 ng / mL to about 1500 ng / mL, about 1300 ng / mL to about 1400 ng / mL, or about 1400 ng / mL to about 1500 ng / mL.

[0153] In some embodiments, following administration of a single dose of an SMSM to a subject theCmax for an SMSM is about 1 ng / mL, about 5 ng / mL, about 10 ng / mL, about 15 ng / mL, about 20 ng / mL, about 25 ng / mL, about 30 ng / mL, about 35 ng / mL, about 40 ng / mL, about 45 ng / mL, about 50 ng / mL, about 55 ng / mL, about 60 ng / mL, about 65 ng / mL, about 70 ng / mL, about 75 ng / mL, about 80 ng / mL, about 85 ng / mL, about 80 ng / mL, about 95 ng / mL, about 100 ng / mL, about 150 ng / mL, about 200 ng / mL, about 250 ng / mL, about 300 ng / mL, about 350 ng / mL, about 400 ng / mL, about 450 ng / mL, about 500 ng / mL, about 600 ng / mL, about 700 ng / mL, about 800 ng / mL, about 900 ng / mL, about 1000 ng / mL, about 1100 ng / mL, about 1200 ng / mL, about 1300 ng / mL, about 1400 ng / mL, or about 1500 ng / mL.

[0154] In some embodiments, a SMSM has a cell viability IC50 of 0.01–10 nM, 0.01–5 nM, 0.01–2.5nM, 0.01–1 nM, 0.01–0.75 nM, 0.01–0.5 nM, 0.01–0.25 nM, 0.01–0.1 nM, 0.1–100 nM, 0.1–50 nM, 0.1–25 nM, 0.1–10 nM, 0.1–7.5 nM, 0.1–5 nM, 0.1–2.5 nM, 2–1000 nM, 2–500 nM, 2–250 nM, 2–100 nM, 2–75 nM, 2–50 nM, 2–25 nM, 2–10 nM, 10–1000 nM, 10–500 nM, 10–250 nM, 10–100 nM, 10–75 nM, 10–50 nM, 10–25 nM, 25–1000 nM, 25–500 nM, 25–250 nM, 25–100 nM, 25–75 nM, 25–50 nM, 50–1000 nM, 50–500 nM, 50–250 nM, 50–100 nM, 50–75 nM, 60–70 nM, 100–1000 nM, 100–500 nM, 100–250 nM, 250–1000 nM, 250–500 nM, or 500–1000 nM.

[0155] In some embodiments, a SMSM has a cell viability IC50 of at most 2 nM, 3 nM, 4 nM, 5 nM, 6nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 73 nM, 74 nM, 75 nM, 76 nM, 77 nM, 78 nM, 79 nM, 80 nM, 81 nM, 82 nM, 83 nM, 84 nM, 85 nM, 90 nM, 95 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM, 400 nM, 425 nM, 450 nM, 475 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 µM, or 10 µM.

[0156] In some embodiments, a SMSM reduces cell proliferation of diseased cells by more than 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% when the cells are treated with the SMSM at a concentration of 2– 1000 nM, 2–500 nM, 2–250 nM, 2–100 nM, 2–75 nM, 2–50 nM, 2–25 nM, 2–10 nM, 10–1000 nM, 10– 500 nM, 10–250 nM, 10–100 nM, 10–75 nM, 10–50 nM, 10–25 nM, 25–1000 nM, 25–500 nM, 25–250WSGR Docket No.51503-775.601 nM, 25–100 nM, 25–75 nM, 25–50 nM, 50–1000 nM, 50–500 nM, 50–250 nM, 50–100 nM, 50–75 nM, 60–70 nM, 100–1000 nM, 100–500 nM, 100–250 nM, 250–1000 nM, 250–500 nM, or 500–1000 nM for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0157] In some embodiments, a SMSM reduces cell proliferation of diseased cells by more than 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% when the cells are treated with the SMSM at a concentration of at least 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 73 nM, 74 nM, 75 nM, 76 nM, 77 nM, 78 nM, 79 nM, 80 nM, 81 nM, 82 nM, 83 nM, 84 nM, 85 nM, 90 nM, 95 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM, 400 nM, 425 nM, 450 nM, 475 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 µM, or 10 µM for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0158] In some embodiments, a SMSM reduces viability or increases apoptosis of diseased cells bymore than 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% when the cells are treated with the SMSM at a concentration of 2–1000 nM, 2–500 nM, 2–250 nM, 2–100 nM, 2–75 nM, 2–50 nM, 2–25 nM, 2–10 nM, 10–1000 nM, 10–500 nM, 10–250 nM, 10–100 nM, 10–75 nM, 10–50 nM, 10–25 nM, 25–1000 nM, 25–500 nM, 25–250 nM, 25–100 nM, 25–75 nM, 25–50 nM, 50–1000 nM, 50–500 nM, 50–250 nM, 50–100 nM, 50–75 nM, 60–70 nM, 100–1000 nM, 100–500 nM, 100–250 nM, 250–1000 nM, 250–500 nM, or 500–1000 nM for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0159] In some embodiments, a SMSM reduces viability or increases apoptosis of diseased cells bymore than 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% when the cells are treated with the SMSM at a concentration of at least 2 nM, 3 nM, 4 nM, 5WSGR Docket No.51503-775.601 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 73 nM, 74 nM, 75 nM, 76 nM, 77 nM, 78 nM, 79 nM, 80 nM, 81 nM, 82 nM, 83 nM, 84 nM, 85 nM, 90 nM, 95 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM, 400 nM, 425 nM, 450 nM, 475 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 µM, or 10 µM for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0160] In some embodiments, a SMSM does not reduce viability or does not increase apoptosis of non-diseased cells by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, or 50% when the cells are treated with the SMSM at a concentration of 2–1000 nM, 2–500 nM, 2–250 nM, 2–100 nM, 2–75 nM, 2–50 nM, 2–25 nM, 2–10 nM, 10–1000 nM, 10–500 nM, 10–250 nM, 10–100 nM, 10–75 nM, 10– 50 nM, 10–25 nM, 25–1000 nM, 25–500 nM, 25–250 nM, 25–100 nM, 25–75 nM, 25–50 nM, 50–1000 nM, 50–500 nM, 50–250 nM, 50–100 nM, 50–75 nM, 60–70 nM, 100–1000 nM, 100–500 nM, 100–250 nM, 250–1000 nM, 250–500 nM, or 500–1000 nM for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0161] In some embodiments, a SMSM does not reduce viability or does not increase apoptosis of non-diseased cells by more than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 30%, 35%, 40%, 45%, or 50% when the cells are treated with the SMSM at a concentration of at least 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 11 nM, 12 nM, 13 nM, 14 nM, 15 nM, 16 nM, 17 nM, 18 nM, 19 nM, 20 nM, 21 nM, 22 nM, 23 nM, 24 nM, 25 nM, 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 51 nM, 52 nM, 53 nM, 54 nM, 55 nM, 56 nM, 57 nM, 58 nM, 59 nM, 60 nM, 61 nM, 62 nM, 63 nM, 64 nM, 65 nM, 66 nM, 67 nM, 68 nM, 69 nM, 70 nM, 71 nM, 72 nM, 73 nM, 74 nM, 75 nM, 76 nM, 77 nM, 78 nM, 79 nM, 80 nM, 81 nM, 82 nM, 83 nM, 84 nM, 85 nM, 90 nM, 95 nM, 100 nM, 110 nM, 120 nM, 130 nM, 140 nM, 150 nM, 160 nM, 170 nM, 180 nM, 190 nM, 200 nM, 210 nM, 220 nM, 230 nM, 240 nM, 250 nM, 275 nM, 300 nM, 325 nM, 350 nM, 375 nM, 400 nM, 425 nM, 450 nM, 475 nM, 500 nM, 550 nM, 600 nM, 650 nM, 700 nM, 750 nM, 800 nM, 850 nM, 900 nM, 950 nM, 1 µM, or 10 µM for at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 21, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, or 48 hours.

[0162] In some embodiments, a SMSM can reduce the amount of a canonical HTT mRNA (e.g., a HTTmRNA not comprising exon 49b), a canonical PMS1 mRNA (e.g., a PMS1 mRNA not comprising exon 5b), or both canonical HTT mRNA and canonical PMS1 mRNA in a cell or in cells contacted by the SMSM or when administered to a subject in need thereof. In some embodiments, a SMSM can reduce theWSGR Docket No.51503-775.601 amount of a canonical HTT mRNA (e.g., a HTT mRNA not comprising exon 49b), a canonical PMS1 mRNA (e.g., a PMS1 mRNA not comprising exon 5b), or both canonical HTT mRNA and canonical PMS1 mRNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, by at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, by at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, by at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, by at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the cell or in the cells contacted by the SMSM compared to in the cell or in the cells not contacted by the SMSM; or in the subject administered with the SMSM compared to the subject not administered with the SMSM.

[0163] In some embodiments, a SMSM can reduce the amount of a canonical HTT mRNA (e.g., a HTTmRNA not comprising exon 49b), a canonical PMS1 mRNA (e.g., a PMS1 mRNA not comprising exon 5b), or both canonical HTT mRNA and canonical PMS1 mRNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 6%, at least 7%, 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%, at least 20%, at least 21%, at least 22%, at least 23%, at least 24%, at least 25%, at least 26%, at least 27%, at least 28%, at least 29%, at least 30%, at least 31%, at least 32%, at least 33%, at least 34%, at least 35%, at least 36%, at least 37%, at least 38%, at least 39%, at least 40%, at least 41%, at least 42%, at least 43%, at least 44%, at least 45%, at least 46%, at least 47%, at least 48%, at least 49%, at least 50%, at least 51%, at least 52%, at least 53%, at least 54%, at least 55%, at least 56%, at least 57%, at least 58%, at least 59%, at least 60%, by at least 61%, at least 62%, at least 63%, at least 64%, at least 65%, at least 66%, at least 67%, at least 68%, at least 69%, at least 70%, by at least 71%, at least 72%, at least 73%, at least 74%, at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, by at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, by at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% in the cell or in the cells when the cell or the cells are treated with the SMSM at a concentration of about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, or about 16 mg per dose compared to in the cell or in the cells not treated with the SMSM. In some embodiments, the amount of the canonical HTT mRNA (e.g., a HTTWSGR Docket No.51503-775.601 pre-mRNA not comprising exon 49b) are decreased by at least 30% in the cell or in the cells when the cell or the cells are treated with the SMSM at a concentration of about 8 mg per dose compared to in the cell or in the cells not treated with the SMSM. In some embodiments, the cell or the cells comprise whole blood cell or whole blood cells. In some embodiments, a subject can have a dose-dependent reduction in HTT mRNA in whole blood cells when administered with a single administration of a SMSM at a concentration of 8 mg per dose.

[0164] The compositions and methods described herein can be used for treating a human disease ordisorder associated with aberrant splicing, such as aberrant pre-mRNA splicing. The compositions and methods described herein can be used for treating a disease or condition. The compositions and methods described herein can be used for treating a human disease or disorder by modulating expression level of an mRNA, such as a pre-mRNA. In some embodiments, the compositions and methods described herein can be used for treating a human disease or disorder by modulating splicing of a nucleic acid even when that nucleic acid is not aberrantly spliced in the pathogenesis of the disease or disorder being treated.

[0165] Provided herein are methods of treating a disease or condition in a subject in need thereof. Themethod can comprise administering a therapeutically effective amount of a SMSM described herein or a pharmaceutically acceptable salt thereof, to a subject with a disease or condition. In some embodiments, the present disclosure relates to the use of a SMSM as described herein for the preparation of a medicament for the treatment, prevention and / or delay of progression of disease or condition.

[0166] In some embodiments, an effective amount in the context of the administration of a SMSM or apharmaceutically acceptable salt thereof, or composition or medicament thereof refers to an amount of a SMSM or a pharmaceutically acceptable salt thereof to a patient which has a therapeutic effect and / or beneficial effect. In certain specific embodiments, an effective amount in the context of the administration of a SMSM or a pharmaceutically acceptable salt thereof, or composition or medicament thereof to a patient results in one, two or more of the following effects: (i) reduces or ameliorates the severity of a disease; (ii) delays onset of a disease; (iii) inhibits the progression of a disease; (iv) reduces hospitalization of a subject; (v) reduces hospitalization length for a subject; (vi) increases the survival of a subject; (vii) improves the quality of life of a subject; (viii) reduces the number of symptoms associated with a disease; (ix) reduces or ameliorates the severity of a symptom associated with a disease; (x) reduces the duration of a symptom associated with a disease; (xi) prevents the recurrence of a symptom associated with a disease; (xii) inhibits the development or onset of a symptom of a disease; and / or (xiii) inhibits of the progression of a symptom associated with a disease.

[0167] In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable saltthereof is an amount effective to reduce the amount of disease cells. In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to reduce the cell viability or increase apoptosis of disease cells. In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to reduce the amount of an RNA transcript of a gene in patients with a disease or cells from patients with a disease compared to the amount of the RNA transcript detectable in healthy patients or cells from healthy patients. In someWSGR Docket No.51503-775.601 embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to reduce the amount of an RNA transcript of a gene in disease cells compared to the amount of the RNA transcript detectable in non-disease cells. In other embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to restore the amount an RNA isoform and / or protein isoform of a gene in disease cells to the amount of the RNA isoform and / or protein isoform detectable in non-disease cells. In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to inhibit a DNA damage repair pathway (e.g., mismatch repair pathway) in disease cells. In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to induce or promote apoptosis in disease cells. In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to restore the amount of an RNA transcript of a gene to the amount of the RNA transcript detectable in healthy patients or cells from healthy patients. In other embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to restore the amount an RNA isoform and / or protein isoform of a gene to the amount of the RNA isoform and / or protein isoform detectable in healthy patients or cells from healthy patients. In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to increase the amount of an RNA transcript of a gene in patients with a disease or cells from patients with a disease compared to the amount of the RNA transcript detectable in healthy patients or cells from healthy patients. In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to increase the amount of an RNA transcript of a gene in disease cells compared to the amount of the RNA transcript detectable in non-disease cells. In other embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to increase the amount an RNA isoform and / or protein isoform of a gene in patients with a disease or cells from patients with a disease compared to the amount of the RNA isoform and / or protein isoform detectable in healthy patients or cells from healthy patients.

[0168] In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable saltthereof is an amount effective to decrease the aberrant amount of an RNA transcript of a gene which associated with a disease. In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to decrease the amount of the aberrant expression of an isoform of a gene. In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to result in a substantial change in the amount of an RNA transcript (e.g., an mRNA transcript), alternative splice variant, or isoform.

[0169] In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable saltthereof is an amount effective to increase the amount of an RNA transcript (e.g., an mRNA transcript) of a gene that is beneficial for the prevention and / or treatment of a disease. In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof is an amount effective to increase the amount of an alternative splice variant of an RNA transcript of a gene that is beneficial for the prevention and / or treatment of a disease. In some embodiments, an effective amount of a SMSM or aWSGR Docket No.51503-775.601 pharmaceutically acceptable salt thereof is an amount effective to increase the amount of an isoform of a gene that is beneficial for the prevention and / or treatment of a disease.

[0170] In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable saltthereof is an amount effective to decrease the amount of an RNA transcript (e.g., an mRNA transcript) which causes or is related to the symptoms of the condition or disease. In particular embodiments, the SMSM can decrease the amount of an RNA transcript that causes or relates to the symptoms of the condition or disease by modulating one or more splicing elements of the RNA transcript. In some embodiments, the SMSM promotes skipping of one or more exons. In some embodiments, the SMSM promotes inclusion of one or more exons. In some embodiments, the SMSM promotes inclusion of a cryptic exon. In some embodiments, the SMSM promotes inclusion of a poison exon. In some embodiments, the SMSM promotes inclusion of one or more exons and / or introns that relate to nonsense- mediated mRNA decay (NMD). In some embodiments, the one or more exons harbor a premature termination codon. In particular embodiments, the premature stop codon is an in-frame codon that does not cause frameshift of the downstream exon(s). In some embodiments, inclusion of the one or more exons causes a reading frameshift in a downstream exon, for example, in the immediately downstream exon, introducing a premature termination codon. In some embodiments, skipping of one or more exons causes a reading frameshift in an exon downstream of the one or more of skipped exons, for example, in the exon immediately downstream of a skipped exon(s), introducing a premature termination codon. In some embodiments, a poison exon can comprise a premature termination codon or a premature stop codon that can cause NMD.

[0171] Non-limiting examples of effective amounts of a SMSM or a pharmaceutically acceptable saltthereof are described herein. In general, the effective amount will be in a range of from about 0.001 mg / kg / day to about 500 mg / kg / day for a patient having a weight in a range of between about 1 kg to about 200 kg. The typical adult subject is expected to have a median weight in a range of between about 70 and about 100 kg.

[0172] A method of treating a disease or a condition in a subject in need thereof can compriseadministering to the subject a therapeutically effective amount of a compound described herein or a pharmaceutically acceptable salt thereof. In some embodiments, the present disclosure relates to a method for the treatment, prevention and / or delay of progression of a disease or a condition associated with a gene listed in Table 20A.

[0173] In some embodiments, a SMSM described herein can be used in the preparation of medicamentsfor the treatment of diseases or conditions described herein. In addition, a method for treating any of the diseases or conditions described herein in a subject in need of such treatment, can involve administration of pharmaceutical compositions that include at least one SMSM described herein or a pharmaceutically acceptable salt, thereof, in a therapeutically effective amount to a subject.

[0174] In certain embodiments, a SMSM described herein can be administered for prophylactic and / ortherapeutic treatments. In certain therapeutic applications, the compositions are administered to a patient already suffering from a disease or condition, in an amount sufficient to cure or at least partially arrest atWSGR Docket No.51503-775.601 least one of the symptoms of the disease or condition. Amounts effective for this use depend on the severity and course of the disease or condition, previous therapy, the patient’s health status, weight, and response to the drugs, and the judgment of the treating physician. Therapeutically effective amounts are optionally determined by methods including, but not limited to, a dose escalation clinical trial. In prophylactic applications, compositions containing a SMSM described herein can be administered to a patient susceptible to or otherwise at risk of a particular disease, disorder, or condition.

[0175] In some embodiments, methods described herein can further comprise a genetic testing of abiological sample of a subject in need thereof. For example, methods can further comprise subjecting abiological sample of a subject to a genetic testing. In some embodiments, methods described herein can further comprise identifying a subject as a subject that expresses PMS1, as a subject that is PMS1+, or as a subject that is PMS1high. In some embodiments, a subject may have been previously identified as a subject that expresses PMS1, as a subject that is PMS1+, or as a subject that is PMS1high. Any suitable methods can be used for a genetic testing to determine expression of a gene or a protein (e.g., PMS1), or expression level of a gene or a protein. Conditions and Diseases

[0176] In some embodiments, the present disclosure relates to a pharmaceutical composition comprisinga SMSM described herein for use in the treatment, prevention and / or delay of progression of a disease or condition. In some embodiments, the disease or condition can comprise Huntington’s disease (HD). HD is a hereditary neurodegenerative disease, characterized by motor, psychiatric, and cognitive dysfunction. HD is caused by a mutation in the huntingtin (HTT) gene that encodes HTT protein. Huntingtin is a ubiquitously expressed nuclear protein that binds to a number of transcription factors to regulate transcription. Abnormal expansion of a polyglutamine tract (e.g., CAG repeats) in the N terminus of HTT gene (exon 1) can encode a mutant HTT protein thereby causing HD. In some embodiments, HTT gene can have about 20 CAG repeats. In some embodiments, mutant HTT gene or HTT gene of HD patients can have about 40 or more CAG repeats. In some embodiments, the CAG repeats can expand in neurons and other cells (CAG somatic expansion) in HD patients. The extra CAG repeats in HD patients can result in the production of mutant HTT protein containing an extended polyglutamine tract near the N- terminus. In some embodiments, mutant HTT proteins can accumulate in neurons, causing neuronal cell damage and cell death. In some embodiments, mutant HTT proteins can be regarded as the main pathogenic factor contributing to the genesis of HD symptoms. In other embodiments, alternative toxic species, including but not limited to HTT mRNAs with expanded CAG repeats, can potentially contribute to the HD disease pathology. In some embodiments, interfering with the activity of the expanded CAG repeats in the HTT RNA transcript can reverse motor symptoms in an HD mouse model, without modifying HTT RNA or protein levels. In some embodiments, regulating mutant HTT production at the RNA level can confer additional benefits compared to interfering at the mutant HTT protein level alone.

[0177] Approximately, 160,000 people globally are known to be chronically affected with HD.Symptoms typically begin to manifest between the ages of 30 to 50 and progress as a devastating neurodegenerative disorder. Symptoms can include, but are not limited to, progressive decline of motorWSGR Docket No.51503-775.601 and cognitive functions, abnormal involuntary movements (e.g., chorea) spreading to all muscles, progressive dementia, and a range of behavioral and psychiatric disturbances, including depression. One part of the brain affected by HD includes striatum, which controls movement, mood, and memory. HD patients also exhibit a variety of symptoms outside the central nervous system such as muscle atrophy and metabolic dysfunctions. Affected individuals may succumb to pneumonia, heart failure, or other fatal complications. Life expectancy after symptom onset is approximately 20 years, and median life expectancy after symptom onset is approximately 15-18 years. In some embodiments, HD patients can have an average of 10-20 year survival following diagnosis. According to the Huntington’s Disease Society of America, there are approximately 41,000 symptomatic Americans with HD and estimated 200,000 individuals in the United States who have a 50% risk of developing HD because of their family relationship to HD patients. Globally, it is estimated that approximately 160,000 people are chronically affected by HD.

[0178] In some embodiments, a subject with HD can benefit from lowering HTT protein level. In someembodiments, a subject with HD that has rs13102260 single nucleotide polymorphism (SNP) may have lower HTT protein level. For example, a subject with HD that has rs13102260 SNP may have a 50 percent reduction in wild-type HTT protein and / or a 25 percent reduction of mutant Huntingtin (mHTT) protein. In some embodiments, a subject with HD that has rs13102260 SNP can have delayed disease onset by 5.6 years.

[0179] In some embodiments, SMSMs described herein can modulate splicing of a pre-mRNAcomprising a string of CAG repeats. In some embodiments, SMSMs described herein can modulate splicing of a pre-mRNA that encodes a wild-type HTT protein. In some embodiments, SMSMs described herein can modulate splicing of a pre-mRNA that encodes a mutant Huntingtin (mHTT) protein. In some embodiments, SMSMs described herein can modulate splicing of an HTT pre-mRNA that may contain an aberrant expansion of CAG repeats. In some embodiments, modulating splicing of an HTT pre-mRNA can lead to a decrease in the expression level of a HTT protein encoded by a spliced product of the HTT pre-mRNA. In some embodiments, modulating splicing of an HTT pre-mRNA can lead to a decrease in the expression level of a mutant HTT protein encoded by a spliced product of the HTT pre-mRNA comprising an aberrant expansion of CAG repeats. In some embodiments, a decrease in the expression level of a mutant HTT protein can be beneficial to treat HD. In some embodiments, SMSMs described herein can penetrate the blood brain barrier and modulate splicing of an HTT pre-mRNA in brain tissues or peripheral tissues to reduce the expression level of mHTT protein in a subject with HD. In some embodiments, SMSMs described herein can penetrate the blood brain barrier and achieve systemic distribution within a subject or a patient with HD.

[0180] In some embodiments, the aberrant expansion of CAG repeats in the HTT gene may beassociated with another protein, for example, PMS1. In some embodiments, CAG repeat expansion can be accelerated in striatal neurons in a transcription-dependent manner and can be driven by the mismatch repair (MMR) complex comprising MutSß and MutLs. PMS1 is a member of the DNA mismatch repair mutL / hexB family, is thought to be involved in the repair of DNA mismatches. For example, PMS1 canWSGR Docket No.51503-775.601 form a heterodimer with MLH1 to form a MutL complex, which can facilitate CAG repeat expansion. Genome-wide associated studies indicate that altered expression and / or activity of many MMR genes can modify HD onset and progression. In some embodiments, depletion of PMS1 can delay onset and slow the progression of HD. In some embodiments, PMS1 may be involved in repeat expansion diseases. In some embodiments, PMS1 may be involved in CAG repeat expansion. In some embodiments, depletion of PMS1 can delay onset of HD and / or slow progression of HD. In some embodiments, HD cells may express PMS1 or a variant PMS1 described herein. In some embodiments, HD cells may be PMS1+, may be PMS1high, or may express a variant PMS1 described herein. In some embodiments, target cells for SMSM treatment can comprise cells that express PMS1 or a variant PMS1 described herein, cells that are PMS1+, or cells that are PMS1high.

[0181] In some embodiments, a subject may be suffering from one or more herein mentioned diseases orconditions, wherein the subject can benefit from a decreased expression level or activity level of a protein involved in aberrant expansion of CAG repeats in the HTT gene. In some embodiments, a subject may have a condition or disease associated with the expression level or activity level of a protein in DNA damage repair pathway. In some embodiments, a subject with a condition or disease may benefit from a decreased expression level or a decreased activity level of a protein associated with a DNA damage repair pathway. In some embodiments, SMSMs described herein can modulate splicing of a pre-mRNA that encodes a protein associated with a DNA damage repair pathway or expansion of CAG repeats. In some embodiments, the DNA damage repair pathway can comprise DNA mismatch repair.

[0182] In some embodiments, SMSMs described herein can modulate splicing of a pre-mRNA thatencodes a protein associated with DNA mismatch repair. In some embodiments, SMSMs described herein can modulate splicing of a PMS1 pre-mRNA. In some embodiments, modulating splicing of a PMS1 pre-mRNA can lead to a decrease in the expression level of a PMS1 protein encoded by a spliced product of the PMS1 pre-mRNA. In some embodiments, a decrease in the expression level of a PMS1 protein in HD cells can reduce the amount of aberrant expansion of CAG repeats in the HTT gene in HD cells. In some embodiments, SMSMs described herein can penetrate the blood brain barrier and modulate splicing of a PMS1 pre-mRNA in brain tissues or peripheral tissues to reduce the amount of aberrant expansion of CAG repeats in the HTT gene and / or to reduce the expression level of mHTT protein in a subject with HD.

[0183] In some embodiments, SMSMs described herein can modulate splicing of an HTT pre-mRNAand a PMS1 pre-mRNA. In some embodiments, modulating splicing of an HTT pre-mRNA and a PMS1 pre-mRNA can lead to a decrease in the expression level of a mHTT protein encoded by a spliced product of the HTT pre-mRNA and a PMS1 protein encoded by a spliced product of the PMS1 pre- mRNA. In some embodiments, a decrease in the expression level of a PMS1 protein in HD cells can reduce the amount of aberrant expansion of CAG repeats in the HTT gene, leading to a decrease in mHTT protein level in HD cells.WSGR Docket No.51503-775.601 Dosing and Schedules

[0184] The SMSMs utilized in the methods of the disclosure can be, e.g., administered at dosages thatmay be varied depending upon the requirements of the subject, the severity of the condition being treated and / or imaged, and / or the SMSM being employed. For example, dosages can be empirically determined considering the type and stage of disease diagnosed in a particular subject and / or the type of imaging modality being used in conjunction with the SMSMs. The dose administered to a subject, in the context of the present disclosure should be sufficient to affect a beneficial diagnostic or therapeutic response in the subject. The size of the dose also can be determined by the existence, nature, and extent of any adverse side-effects that accompany the administration of a SMSM in a particular subject.

[0185] In some embodiments, the SMSM described herein can be administered to the subject at fromabout 1 mg to about 16 mg per dose. In some embodiments, the SMSM described herein can be administered to the subject at about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, or about 16 mg per dose.

[0186] The compositions of the SMSM described herein can be administered as frequently as necessary.In some embodiments, the SMSM described herein can be administered to the subject daily.

[0187] The compositions of the SMSM described herein can be administered as a solid dosage form. Insome embodiments, compositions of the SMSM described herein are administered as a tablet. In some embodiments, compositions of the SMSM described herein are administered as a capsule. In some embodiments, compositions of the SMSM described herein are administered orally. In some embodiments, a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered as a tablet. In some embodiments, a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered as a capsule. In some embodiments, a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered orally.

[0188] In some embodiments, a compound of Structure B, or a pharmaceutically acceptable salt or astereoisomer thereof, is administered with food. In some embodiments, a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered without food. In some embodiments, a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered to a subject under fasting condition. In some embodiments, a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered to a subject under fed condition. Subjects

[0189] The subjects that can be treated with the SMSMs and methods described herein can be anysubject that produces mRNA that is subject to splicing or alternative splicing, e.g., the subject may be a eukaryotic subject, such as a plant or an animal. In some embodiments, the subject is a mammal, e.g., a human. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. In some embodiments, the subject is a non-human primate such as chimpanzee, and other apesWSGR Docket No.51503-775.601 and monkey species; farm animals such as cattle, horses, sheep, goats, swine; domestic animals such as rabbits, dogs, and cats; laboratory animals including rodents, such as rats, mice and guinea pigs, and the like.

[0190] In some embodiments, a subject may have HD. In some embodiments, a subject has early-stageHD. In some embodiments, a subject can have HD that may be refractory to a prior treatment. In some embodiments, a subject can have HD that may have relapsed from a prior treatment. In some embodiments, a subject can have a cell expressing mutant HTT (mHTT) protein. In some embodiments, a subject can have cells that are mHTT+ or mHTThigh. In some embodiments, a subject can comprise a subject identified from a genetic testing as a subject expressing mHTT, as a subject that is mHTT+, or as a subject that is mHTThigh. In some embodiments, a subject can comprise a subject that had been identified from a genetic testing as a subject expressing mHTT, as a subject that is mHTT+, or as a subject that is mHTThigh. In some embodiments, a subject can have a cell expressing a PMS1 protein. In some embodiments, a subject can have cells that are PMS1+ or PMS1high. In some embodiments, a subject can comprise a subject identified from a genetic testing as a subject expressing PMS1, as a subject that is PMS1+, or as a subject that is PMS1high. In some embodiments, a subject can comprise a subject that had been identified from a genetic testing as a subject expressing PMS1, as a subject that is PMS1+, or as a subject that is PMS1high. In some embodiments, a subject can comprise a subject or a patient at high-risk for developing HD. In some embodiments, a subject at high-risk for developing HD can have a family member diagnosed with HD.

[0191] In some embodiments, a subject’s genome can encode a PMS1. In some embodiments, asubject’s genome can encode a wild-type PMS1. In some embodiments, a subject’s genome can comprise an allele that comprises a genetic variation in a PMS1 gene. In some embodiments, a subject may be tested or may have been tested for a presence of a genetic variation. In some embodiments, a genetic assay, e.g., any suitable genetic assay, can be used to test the presence of a genetic variation. In some embodiments, a subject may have been identified as not having a genetic variation. In some embodiments, a subject may have been identified as having a genetic variation. In some embodiments, a subject may be heterozygous for a genetic variation. In some embodiments, a subject may be homozygous for a genetic variation.

[0192] In some embodiments, a genetic variation can comprise a non-synonymous coding variant. Insome embodiments, a genetic variation may not disrupt or modulate the PMS1 gene. In some embodiments, a genetic variation may not be a loss-of-function genetic variation. In these embodiments, a genetic variation can comprise chr2:190660537 G>A, chr2:190719296 A>G, chr2:190719569 T>C, or any combination thereof, wherein chromosome positions of the genetic variation are defined with respect to UCSC hg19. In some embodiments, an allele comprising a genetic variation in a PMS1 gene can encode a variant PMS1 comprising a mutation. In some embodiments, the mutation can comprise E59K mutation, a K433R mutation, a L524S mutation, or any combination thereof.

[0193] In some embodiments, a genetic variation may disrupt or modulate the PMS1 gene. In thisembodiment, a genetic variation can comprise chr2:190660586 C>T, chr2:190670391 C>G,WSGR Docket No.51503-775.601 chr2:190670396 A>G, chr2:190717470 CA>C, chr2:190719499 G>A, chr2:190719607 G>A, chr2:190719704 G>A, chr2:190732559 T>C, or any combination thereof, wherein chromosome positions of the genetic variation are defined with respect to UCSC hg19. In some embodiments, an allele comprising a genetic variation in a PMS1 gene can encode a variant PMS1 comprising a mutation. In some embodiments, a variant PMS1 can comprise a mutation selected from the group consisting of T75I, T110R, T112A, S264*, G501R, E537K, R569Q, Y793H, wherein * denotes a premature termination of protein translation (e.g., loss-of-function variant due to premature stop codon).

[0194] In some embodiments, a subject disclosed herein is between the ages of 30 and 50. In someembodiments, a subject disclosed herein is 30 years of age or older.

[0195] In some embodiments, a subject disclosed herein has symptoms that are characterized by theprogressive decline of motor and cognitive functions, abnormal involuntary movements (known as chorea), which eventually spread to all muscles, progressive dementia, and / or a range of behavioral and psychiatric disturbances, including depression. In some embodiments, a subject disclosed herein exhibits a variety of symptoms outside the CNS such as muscle atrophy and metabolic dysfunctions. In some embodiments, a subject disclosed herein exhibits complications such as pneumonia and heart failure.

[0196] In some embodiments, a subject disclosed herein has HD-ISS Stage 0. In some embodiments, asubject disclosed herein has HD-ISS Stage 1. In some embodiments, a subject disclosed herein has HD- ISS Stage 2. In some embodiments, a subject disclosed herein has HD-ISS Stage 3. Methods of Making Compounds

[0197] The compound of structure B, 6-(6-(((1R,2R,3S,5S)-2-Fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[d]oxazol-5-ol, can be made by the techniques and processes known in the art, for example as described in Example 1. EXAMPLES

[0198] These examples are provided for illustrative purposes only and not to limit the scope of theclaims provided herein. The compound described herein can be synthesized using standard synthetic techniques or using methods known in the art in combination with methods described herein. Unless otherwise indicated, conventional methods of mass spectroscopy, NMR, HPLC, protein chemistry, biochemistry, recombinant DNA techniques and pharmacology can be employed. The compound can be prepared using standard organic chemistry techniques such as those described in, for example, March’s Advanced Organic Chemistry, 6th Edition, John Wiley and Sons, Inc. Alternative reaction conditions for the synthetic transformations described herein may be employed such as variation of solvent, reaction temperature, reaction time, as well as different chemical reagents and other reaction conditions. The starting materials and reagents used for the synthesis of the compound described herein may be synthesized or can be obtained from commercial sources, such as, but not limited to, Sigma-Aldrich, Acros Organics, Fluka, and Fischer Scientific. The starting materials can be available from commercial sources or can be readily prepared. By way of example only, provided are schemes for preparing the Examples described herein.WSGR Docket No.51503-775.601

[0199] Suitable reference books and treatise that detail the synthesis of reactants useful in thepreparation of the compound described herein, or provide references to articles that describe the preparation, include for example, “Synthetic Organic Chemistry”, John Wiley & Sons, Inc., New York; S. R. Sandler et al., “Organic Functional Group Preparations,” 2nd Ed., Academic Press, New York, 1983; H. O. House, “Modern Synthetic Reactions”, 2nd Ed., W. A. Benjamin, Inc. Menlo Park, Calif. 1972; T. L. Gilchrist, “Heterocyclic Chemistry”, 2nd Ed., John Wiley & Sons, New York, 1992; J. March, “Advanced Organic Chemistry: Reactions, Mechanisms and Structure”, 4th Ed., Wiley Interscience, New York, 1992. Additional suitable reference books and treatise that detail the synthesis of reactants useful in the preparation of compounds described herein, or provide references to articles that describe the preparation, include for example, Fuhrhop, J. and Penzlin G. “Organic Synthesis: Concepts, Methods, Starting Materials”, Second, Revised and Enlarged Edition (1994) John Wiley & Sons ISBN: 3 527–29074–5; Hoffman, R.V. “Organic Chemistry, An Intermediate Text” (1996) Oxford University Press, ISBN 0–19–509618–5; Larock, R. C. “Comprehensive Organic Transformations: A Guide to Functional Group Preparations” 2nd Edition (1999) Wiley–VCH, ISBN: 0–471–19031–4; March, J. “Advanced Organic Chemistry: Reactions, Mechanisms, and Structure” 4th Edition (1992) John Wiley & Sons, ISBN: 0–471–60180–2; Otera, J. (editor) “Modern Carbonyl Chemistry” (2000) Wiley–VCH, ISBN: 3–527–29871–1; Patai, S. “Patai’s 1992 Guide to the Chemistry of Functional Groups” (1992) Interscience ISBN: 0–471–93022–9; Solomons, T. W. G. “Organic Chemistry” 7th Edition (2000) John Wiley & Sons, ISBN: 0–471–19095–0; Stowell, J.C., “Intermediate Organic Chemistry” 2nd Edition (1993) Wiley–Interscience, ISBN: 0–471–57456–2; “Industrial Organic Chemicals: Starting Materials and Intermediates: An Ullmann’s Encyclopedia” (1999) John Wiley & Sons, ISBN: 3–527–29645–X, in 8 volumes; “Organic Reactions” (1942–2000) John Wiley & Sons, in over 55 volumes; and “Chemistry of Functional Groups” John Wiley & Sons, in 73 volumes.

[0200] In the reactions described, it may be necessary to protect reactive functional groups, for examplehydroxy, amino, imino, thio or carboxy groups, where these are desired in the final product, in order to avoid their unwanted participation in reactions. A detailed description of techniques applicable to the creation of protecting groups and their removal are described in Greene and Wuts, Protective Groups in Organic Synthesis, 3rd Ed., John Wiley & Sons, New York, NY, 1999, and Kocienski, Protective Groups, Thieme Verlag, New York, NY, 1994, which are incorporated herein by reference for such disclosure).

[0201] Examples can be made using known techniques and further chemically modified, in someembodiments, to facilitate intranuclear transfer to, e.g., a splicing complex component, a spliceosome or a pre–mRNA molecule. One of ordinary skill in the art will appreciate the standard medicinal chemistry approaches for chemical modifications for intranuclear transfer (e.g., reducing charge, optimizing size, and / or modifying lipophilicity). Stereochemistry:

[0202] (±) or racemic indicates that the product is a racemic mixture of enantiomers. For example (±)(1S,2S,3R,5R) or racemic (1S,2S,3R,5R) indicates that the relative product stereochemistry shown isWSGR Docket No.51503-775.601 based on known stereochemistry of similar compounds and or reactions and the product is a racemic mixture of enantiomers of both (1S,2S,3R,5R) and (1R,2R,3S,5S) stereoisomers. A compound in which the absolute stereochemistry of separated enantiomers is undetermined is represented as being either of the single enantiomers, for example (1S,2S,3R,5R) or (1R,2R,3S,5S) or drawn as being either possible single enantiomer. In such cases, the product is pure and a single enantiomer, but absolute stereochemistry is not identified, but relative stereochemistry is known and indicated.

[0203] Example 1: Synthesis of 6-(6-(((1S,2S,3R,5R)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazol-5-ol (Structure A) and 6-(6- (((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2- methylbenzo[d]oxazol-5-ol (Structure B).

[0204] Synthesis of 2-amino-4-methoxyphenol.

[0205] A mixture of 4-methoxy-2-nitrophenol (25.0 g, 0.15 mol) and Pd / C (2.5 g) in MeOH (500 mL)was stirred, degassed with hydrogen 3 times and then held with stirring at 20oC under hydrogen for 2 d. The reaction mixture was filtered and the cake was washed with MeOH (350 mL*3). The filtrated was combined and concentrated in vacuum to afford the product 2-amino-4-methoxyphenol as brown solid (20.0 g, yield 97.2 %). LCMS: m / z 140.1 = 0.93 min.

[0206] Synthesis of 5-methoxy-2-methylbenzo[d]oxazole.WSGR Docket No.51503-775.601

[0207] A mixture of 2-amino-4-methoxyphenol (20.0 g, 0.14 mol) in trimethyl orthoacetate (50 mL) washeated to 100oC with stirring and held for 1 h. The mixture was concentrated and the residue was purified by combi-flash (Biotage, Silica gel column, 330 g, 60 mL / min, EA in PE 0 % ~ 35 %, 30 min, 35 %, 12 min, UV 254280) to give the desired product 5-methoxy-2-methylbenzo[d]oxazole as orange oil (17.5 g, yield 74.6 %). LCMS: m / z 164.1 [M+H]+; tR = 1.41 min.

[0208] Synthesis of 6-bromo-5-methoxy-2-methylbenzo[d]oxazole.

[0209] NBS (19.6 g, 0.11 mol) was added to a mixture of 5-methoxy-2-methylbenzo[d]oxazole (17.5 g,0.11 mol) in AcOH (150 mL). This resulting mixture was stirred at 20oC for 18 h. The mixture was quenched with ice water, neutralized with Na2CO3aqueous, extracted with EtOAc (200 mL*3). The extracts were concentrated and the residue was purified by combi-flash (Biotage, Silica gel column, 330 g, 60 mL / min, EA in PE 0 % ~ 10 %, 30 min, 20 %, 15 min, UV 254280) to give the desired product 6- bromo-5-methoxy-2-methylbenzo[d]oxazole as pink solid (20.5 g, yield 77.0 %). LCMS: m / z 242.1; 243.9 [M+H]+; tR= 1.70 min.1H NMR (500 MHz, CDCl3-d3) δ 7.68 (s, 1H), 7.17 (s, 1H), 3.93 (s, 3H), 2.61 (s, 3H).

[0210] Synthesis of 6-bromo-2-methylbenzo[d]oxazol-5-ol.

[0211] BBr3 (210 mL, 1 mol / l, 0.21 mol) was added to a mixture of 6-bromo-5-methoxy-2-methylbenzo[d]oxazole (20.5 g, 0.085 mol) in DCM (30 mL) at 0oC. This resulting mixture was stirred at 0oC for 10 min and then warmed to 20oC with stirring and held for 3 d. The mixture was quenched with ice water, neutralized with NaHCO3aqueous, extracted with EtOAc (360 mL*3). The extracts were concentrated and the residue was purified by combi-flash (Biotage, Silica gel column, 330 g, 80 mL / min, EA in PE 0 % ~ 50 %, 30 min, 40 %, 10 min, then MeOH in DCM 20% UV 254280) to give the desired product 6-bromo-2-methylbenzo[d]oxazol-5-ol as grey solid (19.0 g, yield 98.6 %). LCMS: m / z 228.0; 230.0 [M+H]+; tR= 1.50 min.

[0212] Synthesis of 6-bromo-5-(methoxymethoxy)-2-methylbenzo[d]oxazole.

[0213] MOMBr (15.6 g, 0.12 mol) was added drop wise to a mixture of 6-bromo-2-methylbenzo[d]oxazol-5-ol (19.0 g, 0.08 mol) and DIPEA (37.7 g, 0.19 mol) in ACN (300 mL) at 5oC. This resulting mixture was stirred at 5oC and held for 30 min. The mixture was quenched with ice water, extracted with EtOAc (200 mL*3). The extracts were washed with brine (300 mL) and concentrated. The residue was purified by combi-flash (Biotage, Silica gel column, 330 g, 60 mL / min, EA in PE 0 % ~ 15 %, 20 min, 15 %, 5 min, 15 % ~ 25 %, 10 min, 25 %, 15 min, UV 254280) to give the desired productWSGR Docket No.51503-775.601 6-bromo-5-(methoxymethoxy)-2-methylbenzo[d]oxazole as pink solid (18.0 g, yield 79.5 %). LCMS: m / z 272.0; 274.0 [M+H]+; tR = 1.77 min.

[0214] Synthesis of 5-(methoxymethoxy)-2-methyl-6-(4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl)benzo[d]oxazole.

[0215] A mixture of 6-bromo-5-(methoxymethoxy)-2-methylbenzo[d]oxazole (50 g, 0.02 mol),pinaolboron (28.0 g, 0.11 mol), PdCl2dppf (1.1 g, 1.5 mmol)and KOAc (10.8 g, 0.11 mol) in 1, 4-dioxane (300 mL) was heated to 100oC with stirring and held for 50 h. The mixture was quenched with ice water, extracted with EtOAc (200 mL*3). The extracts were washed with brine (200 mL) and concentrated. The residue was purified by combi-flash (Biotage, Silica gel column, 20 g, 30 mL / min, EA in PE 0 % ~ 15 %, 20 min, 15 %, 6 min, 15 % ~ 25 %, 15 min, 25 %, 10 min , UV 254280) to give the desired product 5- (methoxymethoxy)-2-methyl-6-(4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl)benzo[d]oxazole as pink solid (4.0 g, yield 68.2 %). LCMS: m / z 320.2 [M+H]+; tR= 1.86 min.

[0216] Synthesis of tert-butyl (1S,2S,5R)-2-fluoro-3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylate.

[0217] LHMDS (94 mL, 94 mmol, 1 N solution in THF) was added to a stirred solution of tert-butyl 3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylate (15 g, 62.8 mmol) in 150 mL of anhydrous THF at -78oC under nitrogen atmosphere. After stirring for 30 min, NFSI (26.6 g, 75 mmol) in 100 mL of anhydrous THF was added dropwise. The mixture was then stirred at -78oC for 4 h, quenchend with saturated NH4Cl aqueous solution (30 mL), estracted with EtOAc (80 mL X 3). The combined organic phases were dried over anhydrous Na2SO4, conentrated and purified by silica gel chromatography (0-5% EtOAc / petroleum ether) to give 6.5 g of tert-butyl (1S,2S,5R)-2-fluoro-3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylate as a white solid (40% yield). LCMS: m / z 202.1 [M-55]+; tR = 1.75 min.

[0218] Synthesis of tert-butyl (1S,2S,5R)-2-fluoro-3-(methylimino)-9-azabicyclo[3.3.1]nonane-9-carboxylate.

[0219] Methylamine (58.5 mL, 117 mmol, 2N solution in THF) and Ti(iPrO)4 (32.8 g, 117 mmol) wereadded to a stirred solution of tert-butyl (1S,2S,5R)-2-fluoro-3-oxo-9-azabicyclo[3.3.1]nonane-9- carboxylate (20 g, 77 mmol) in THF (1 L) under N2 protection. The reaction mixture was stirred at room temperature for 2 h. Water (1 L) was added to quench the reaction. The mixture was extracted with EtOAc (1L X 3). The combined organic phases were washed with brine, dried over anhydrous Na2SO4WSGR Docket No.51503-775.601 and concentrated to give the crude product (1S,2S,5R)-2-fluoro-3-(methylimino)-9- azabicyclo[3.3.1]nonane-9-carboxylate (20 g, 95% yield), which was directly used in next step. LCMS: m / z 271.2 [M+H]+; tR = 1.58, 1.80 min.

[0220] Synthesis of tert-butyl (1S,2R,3R,5R)-2-fluoro-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate.

[0221] NaBH4 (4 g, 104 mmol) was added to a stirred solution of (1S,2S,5R)-2-fluoro-3-(methylimino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (7 g, 26 mmol) and MgCl2 (2.46 g, 26 mmol) in 30 mL of MeOH. After the addition, the mixture was stirred at room temperature for 2 h. Additional NaBH4 may be needed till LCMS indicated the imine was consumed completely.100 mL of water was added to quench the reaction. The resulting mixture was extracted with EtOAc (180 mL X 3). The combined organic phases were washed with brine, dried over anhydrous Na2SO4, concentrated and purified by silica gel chromatography (0-5% MeOH / CH2Cl2) give 2 g of tert-butyl (1S,2R,3R,5R)-2-fluoro-3- (methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate as colorless oil (40% yield), (high polar isomer). LCMS: m / z 273.2 [M+H]+; tR = 1.42 min.

[0222] Synthesis of tert-butyl (1S,2R,3R,5R)-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate.

[0223] A mixture of tert-butyl (1S,2R,3R,5R)-2-fluoro-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (2 g, 7.35 mmol), 3,6-dichloropyridazine (2.19 g, 14.7 mmol) and DIPEA (3.8 g, 29.4 mmol) in DMSO (10 ml) was stirred at 1200C for 12 h. After cooling to room temperature, the mixture was quenched with H2O (100 mL) and extracted with EtOAc (150 mL X 3). The combined organic layers were concentrated and purified with silica gel chromatography (0-50% EtOAc / petroleum ether) to give 1.5 g of tert-butyl (1S,2R,3R,5R)-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9- azabicyclo[3.3.1]nonane-9-carboxylate white solid (54% yield). LCMS: m / z 385.2 [M+H]+; tR= 1.93 min.

[0224] Chiral separation of tert-butyl (1S,2R,3R,5R)-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate.WSGR Docket No.51503-775.601

[0225] 1500 mg of racemic intermediate was separated by below chial condition to give 630 mg of P1isomer (1.596 min) and 630 mg of P2 isomer (4.811 min).

[0226] Instrument: SFC-150 (Waters)Column: AD 20*250mm, 10um (Daicel) Column temperature: 35 ºC Mobile phase: CO2 / MEOH(0.2%Methanol Ammonia) = 65 / 35 Flow rate: 100 g / min Back pressure: 100 bar Detection wavelength: 214 nm Cycle time: 3.5min Sample solution: 1500mg dissolved in 100ml Methanol Injection volume: 3ml

[0227] Synthesis of tert-butyl (1S,2R,3R,5R)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9- carboxylate.

[0228] A mixture of tert-butyl (1S,2R,3R,5R)-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate (450 mg, 1.17 mmol), 5-(methoxymethoxy)-2-methyl-6-(4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl)benzo[d]oxazole (560 mg, 1.76 mmol), Pd(dppf)Cl2 (86 mg， 0.117 mmol) and K2CO3 (324 mg, 2.34 mmol) in 1,4-Dioxane (15 mL), water (5 ml) was stirred at 1100C for 2 h under N2atmosphere. After cooling to room temperature, the mixture was concentrated andpurified by silica gel chromatography (0-50% EtOAc / petroleum ether) to give 550 mg of tert-butyl(1S,2R,3R,5R)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (86% yield). LCMS: m / z 541.9 [M+H]+; tR= 1.98 min.

[0229] Synthesis of 6-(6-(((1S,2S,3R,5R)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazol-5-ol.

[0230] To a solution of tert-butyl (1S,2R,3R,5R)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylateWSGR Docket No.51503-775.601 (550 mg, 1.02 mmol) in CH2Cl2 (7 mL) was added TFA (3 mL) and the mixture was stirred at room temperature for 2 h, monitored by LCMS. Then the mixture was concentrated and water (10 mL) was added. pH value was adjusted to 8-9 with saturated K2CO3 aqueous solution. The product was collected, concentrated and purified by C18 reversed phase column (0-70% 0.01% NH4HCO3 in H2O / CH3OH) to give 166 mg of 6-(6-(((1S,2S,3R,5R)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazol-5-ol (41% yield). 1H NMR (400 MHz, MeOD –d4) δ 8.17 (d, J = 9.9 Hz,1H), 7.98 (s, 1H), 7.32 (d, J = 9.9 Hz, 1H), 7.10 (s, 1H), 6.01 – 5.86 (m, 1H), 5.07 – 4.90 (m, 1H), 3.57 – 3.47 (m, 2H), 3.11 (s, 3H), 2.71 – 2.64 (m, 1H), 2.62 (s, 3H), 2.15 – 2.02 (m, 3H), 1.97 – 1.78 (m, 4H). LCMS: m / z 398.1 [M+H]+; tR = 1.40 min.

[0231] Synthesis of tert-butyl (1R,2S,3S,5S)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9- carboxylate.

[0232] A mixture of tert-butyl (1R,2S,3S,5S)-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate (450 mg, 1.17 mmol), 5-(methoxymethoxy)-2-methyl-6-(4, 4, 5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl)benzo[d]oxazole (560 mg, 1.76 mmol), Pd(dppf)Cl2 (86 mg， 0.117 mmol) and K2CO3(324 mg, 2.34 mmol) in 1,4-Dioxane (15 mL), water (5 ml) was stirred at 1100C for 2 h under N2atmosphere. After cooling to room temperature, the mixture was concentrated andpurified by silica gel chromatography (0-50% EtOAc / petroleum ether) to give 500 mg of tert-butyl(1R,2S,3S,5S)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (79% yield). LCMS: m / z 541.9 [M+H]+; tR= 1.98 min.

[0233] Synthesis of 6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazol-5-ol.

[0234] To a solution of tert-butyl (1R,2S,3S,5S)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-methylbenzo[d]oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (500 mg, 0.92 mmol) in CH2Cl2(7 mL) was added TFA (3 mL) and the mixture was stirred at room temperature for 2 h, monitored by LCMS. Then the mixture was concentrated and water (10 mL) was added. pH value was adjusted to 8-9 with saturated K2CO3aqueous solution. The product was collected， concentrated and purified by C18 reversed phase column (0-70% 0.01% NH4HCO3 in H2O / CH3OH) toWSGR Docket No.51503-775.601 give 133 mg of 6-(6-(((1R,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[d]oxazol-5-ol (36% yield). 1H NMR (400 MHz, MeOD –d4) δ 8.17 (d, J = 9.9 Hz,1H), 7.98 (s, 1H), 7.32 (d, J = 9.9 Hz, 1H), 7.10 (s, 1H), 6.01 – 5.86 (m, 1H), 5.07 – 4.90 (m, 1H), 3.57 – 3.47 (m, 2H), 3.11 (s, 3H), 2.71 – 2.64 (m, 1H), 2.62 (s, 3H), 2.15 – 2.02 (m, 3H), 1.97 – 1.78 (m, 4H). LCMS: m / z 398.1 [M+H]+; tR = 1.40 min.

[0235] Example 2: Metabolite ID.

[0236] Metabolite identification was performed for both 6-(6-{[(1R,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-1,3-benzoxazol-5-ol (Compound 117) and Structure B. Human hepatocytes (liverPoolTM10-Doner) from BioIVT (cat. No. X008001) for Compound 117 and (liverPoolTM20-Doner) from BioIVT (cat. No. X008000) for Structure B at 1.0 X 106cells / ml were incubated with test compound (50^M Compound 117, 10^M Structure B) for 240 minutes at 37oC. Incubations were quenched with 2 volumes of acetonitrile (0.1% FA) followed by centrifugation for 15 min at 16,000 g; Supernatant was then analyzed by LC-MS / MS. For UV analysis, 500 ^L acetonitrile (0.1% FA) fraction was dried by centrifugal vacuum evaporator and reconstituted with 50 ^L water and 50 ^L methanol. The following equipment and conditions were used for the analysis:

[0237] Instrumentation: Vanquish UHPLC system (Thermo Fisher Scientific, USA); Vanquish VariableWavelength (Thermo Fisher Scientific, USA); Thermo Scientific Q Exactive (Thermo Fisher Scientific , USA).

[0238] LC conditions: Column: Waters XSelect HSS T3, 100 x 2.1 mm, 2.5 ^m; Solvents: A, water(0.1% formic acid); B, acetonitrile (0.1% formic acid); Flow rate: 500 ^L / min; Program for Compound 117: 0-1.5 min, 5%B, 1.5-9 min, 5%-25%B, 9-12 min, 25%-100%B, 12-14 min, 100%B, 14-14.3 min, 100%-5%B, 14.3-15 min, 5%B. Program for Structure B: 0-1.5 min, 5%B, 1.5-9 min, 5%-30%B, 9-12 min, 30%-100%B, 12-14 min, 100%B, 14-14.3 min, 100%-5%B, 14.3-15 min, 5%B.

[0239] MS conditions: Ionisation mode: Positive mode; Spray Voltage: 3.5 kV; Aux gas flow rate: 15;Aux gas heater temp: 350oC; Scan type: Full MS / ddMS2; Resolution: 70,000; AGC Target: 3 × e6; NCE / stepped NCE (Full Mass) for Compound 117: 25, 35, 45; NCE / stepped NCE (Full Mass) for Structure B: 30, 35, 40.

[0240] Three metabolites were detected for Compound 117 (Table 1), seven metabolites were detectedfor Structure B (Table 2).WSGR Docket No.51503-775.601 Table 1.Table 2.WSGR Docket No.51503-775.601

[0241] Example 3: Protein Binding Assay.

[0242] Protein binding of Structure B was determined in human, rat and mouse plasma using anequilibrium dialysis method (using 96-well Equilibrium Dialysis Plate (HTDialysis LLC, Gales Ferry, CT) and HTD 96a / b Dialysis Membrane Strips, MWCO 12-14K). Human mixed gender plasma (pH 7.46) was obtained from BioIVT (batch no. HMN575149), Rat SD strain, mixed gender plasma (pH 7.49) was obtained from BioIVT (batch no. RAT463303) and Mouse CD-1 strain, mixed gender plasma (pH 7.23) was obtained from IPHASE (batch no. M21005657.

[0243] A working solution of test compound and control compound (ketoconazole) was prepared inDMSO at a concentration of 1 mM. A basic solution was prepared by dissolving 14.2 g / L Na2HPO4 and 8.77 g / L NaCl in deionized water and the solution could be stored at 4°C for up to 7 days. An acidic solution was prepared by dissolving 12.0 g / L NaH2PO4 and 8.77 g / L NaCl in deionized water and the solution could be stored at 4°C for up to 7 days. The basic solution was titrated with the acidic solution to pH 7.4 and stored at 4°C for up to 7 days. pH was checked on the day of experiment and was adjusted if outside specification of 7.4 ± 0.1. The temperature of a water bath was set to 37°C. Frozen Plasma (stored at -80°C) was thawed immediately in a 37°C water bath. The dialysis membranes were soaked in ultrapure water for 60 minutes to separate strips, then in 20% ethanol for 20 minutes, finally in dialysisWSGR Docket No.51503-775.601 buffer for 20 minutes. Prepared membranes were loaded into the dialysis device and the device was installed following the manufacturers guidelines. The air bath was turned on and allow to pre-heat to 37°C. 597 μL of blank plasma solution was added into each vial of a new plastic plate or separate plastic tube by addition of 3 μL of the working solution of test compound, vortex at 1000 rpm for 2 minutes. The final percent volume of organic solvent was 0.5% and the final concentration for test compound was 5 μM.50 μL of the spiked plasma solution suspension was transferred to a 96-well plate to act as T=0 control sample. All remaining spiked plasma solution is placed in the incubator for the duration of the study. At the same time, the remaining spiked plasma solution sample in the plastic plate or separate plastic tube was incubated for 6 hours at 37°C with 5% CO2 in the CO2 incubator.

[0244] At T=6 hours, 50 μL of the original spiked plasma solution suspension was transferred to the 96-well plate for analysis. The dialysis set up was assembled following the manufacturer’s instructions. Cells were loaded with 120 μL of plasma sample and dialyzed against equal volume of dialysis buffer (PBS). The assay was performed in duplicate. The unit was covered with a gas permeable lid and incubated for 6 hours at 37°C at 100 rpm with 5% CO2on an orbital shaker in the CO2incubator. At the end of incubation, the lid was removed and 50 μL of post-dialysis samples from both buffer and plasma solution chambers were transferred into separated 96-well plate for analysis, respectively. 50 μL of plasma solution was added to the buffer samples, and an equal volume of PBS to the collected plasma solution samples. The plate at was shaken at 1000 rpm for 2 minutes and 400 μL of acetonitrile was added containing an appropriate internal standard (IS) to precipitate protein and release compound. Samples were vortexed at 1000 rpm for 10 minutes and then centrifuged for 30 minutes at 3,220 g.250 μL of the supernatant was transferred to new 96-well plates and centrifuged again (3,220 g, 30 minutes). 100 μL of the supernatant was transferred to new 96-well plates for analysis.100 μL of distilled water was added to each sample and mixed for analysis by LC-MS / MS. Concentrations of test compound and control compound in the buffer and plasma solution chambers was determined. Percentages of test compound(s) and control compound bound were calculated as follows: % Unbound = (Area ratio buffer chamber / Area ratio plasma solution chamber) × 100; % Bound = 100 - % Unbound; % Recovery = (Area ratio buffer chamber + Area ratio plasma solution chamber) / (Area ratio Total sample) × 100; % Remaining = Area ratio 6hr / Area ratio 0hr × 100. The following chromatography conditions were used:

[0245] LC system: Shimadzu; MS analysis: Triple Quad 5500+ instrument from AB Inc with an ESIinterface; Column temperature: 40°C; Injection volume: 1 µL; Column: XSelect HSS T32.5μm 2.1×50mm Column; Mobile phase: 0.1% formic acid in water (A) and 0.1% formic acid in acetonitrile (B); Elution rate: 0.8 mL / min; with the following program in Table 3: Table 3.WSGR Docket No.51503-775.601

[0246] The following MS parameters were used: Ion source: Turbo spray; Ionization model: ESI; Scantype: MRM; Collision gas: 9 L / min; Curtain gas: 40 L / min; Nebulize gas: 55 L / min; Auxiliary gas: 55 L / min; Temperature: 500°C; Ionspray voltage: +5500 V. Results for Structure B are in Table 4. Table 4.

[0247] Compound 117 was subjected to a similar protocol. With some variations. The testconcentration was 1^M, control compounds were warfarin and quinidine. Incubation time was 5 hours. Results for Compound 117 are in Table 5. Table 5.

[0248] Example 4: PK Study in nonhuman primates of Structure B.

[0249] Cynomologous monkeys were administered Structure B at 2 different dose levels. POA was adose level of 2mg / kg (dosing solution of 0.4mg / ml) and POB was a dose level of 10mg / kg (dosing solution of 2mg / ml). POA was prepared by dissolving 28.8 mg of Structure B in 72.00 mL of 0.5%MC, 0.1%Tween80, 30 mM citrate pH3.5-4 followed by vortexing and sonication to obtain a solution with concentration at 0.4 mg / mL of Structure B.

[0250] POB was prepared by dissolving 109.96 mg of Structure B in 54.980 mL of 0.5%MC,0.1%Tween80, 30 mM citrate pH3.5-4 followed by vortexing and sonication to obtain a solution with concentration at 2 mg / mL of Structure B.

[0251] HPLC was performed on the samples using the following equipment and parameters. HPLC:Instrument: Shimadzu (DGU-20A5R, Serial No: L20705826727 IX; LC-30AD Serial No: L20555913986 AE and L20555913987 AE; SIL-30AC, Serial No: L20565906455 AE; Rack Changer II Serial No. L20585901289 SS; CTO-30A: Serial No.L20575801653 CD; CBM-20A: Serial No.L20235941035 CD). MS: AB API 5500+ LC / MS / MS instrument (Serial No. EX227122104). Column: Agilent Poroshell 120 EC-C184 µm (50 × 2.1 mm). Mobile Phase: Solution A: 5% Acetonitrile in Water (0.1%Formic acid); Solution B: 95% Acetonitrile in Water (0.1%Formic acid). Flow rate: 0.6 mL / min, with the following gradient in Table 6. Table 6.WSGR Docket No.51503-775.601

[0252] Injection volume: 3^l.

[0253] The desired serial concentrations of working solutions were achieved by diluting stock solutionof analyte with DMSO.5 µL of working solutions (5, 10, 20, 50, 100, 500, 1000, 5000, 10000 ng / mL) were added to 50 μL of the blank male or female cynomolgus monkeys plasma to achieve calibration standards of 0.5~1000 ng / mL (0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng / mL) in a total volume of 55 μL. Five quality control samples at 1 ng / mL, 2 ng / mL, 5 ng / mL, 50 ng / mL and 800 ng / mL for plasma were prepared independently of those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards.55 μL of standards, 55 μL of QC samples and 55 μL of unknown samples (50 µL of male and female plasma with 5 µL of blank solution) were added to 200 μL of acetonitrile containing IS mixture for precipitating protein respectively. Then the samples were vortexed for 30 s. After centrifugation at 4 degree Celsius, 3900 rpm for 15 min. The supernatant was diluted 3 times with water.3 µL of diluted supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0254] Blood samples were taken at the following time points post-does administration: 0.083, 0.17,0.33, 0.5, 1, 2, 4, 7, 11, 24, and 48 hours.

[0255] Results are shown in Table 7 for the male cynomologous monkeys and in Table 8 for the femalecynomologous monkeys. Table 7.WSGR Docket No.51503-775.601Table 8.

[0256] BLOQ = Below quantifiable limit of 1 ng / mL for Male; BLOQ = Below quantifiable limit of 0.5ng / mL for Female. PK Parameters were estimated by non-compartmental model using WinNonlin 6.1.WSGR Docket No.51503-775.601 The bioavailability (F%) was calculated asfollows: AUClast-PO / AUCINF-PO > 80%: F=(AUCINF- PO*DoseIV) / (mean AUCINF-IV*DosePO)

[0257] AUClast-PO / AUCINF-PO ≤ 80% or AUCINF was not available: F=(AUClast-PO*DoseIV) / (mean AUClast-IV*DosePO). The PK parameters are set forth in Table 9 for the male cynomologous monkeys and Table 10 for the female cynomologous monkeys. Table 9.Table 10.

[0258] Example 5: PK Study in nonhuman primates of Compound 117.

[0259] Female cynomologous monkeys were administered Compound 117 at 2 different dose levels.POA was a dose level of 1mg / kg (dosing solution of 0.2mg / ml) and POB was a dose level of 3mg / kg (dosing solution of 0.6mg / ml). POA was prepared by dissolving 15.79 mg of Compound 117 in 78.95 mL of deionized water (0.5%MC, 0.1%Tween80), followed by vortexing and sonication to obtain a solution with concentration at 0.2 mg / mL of Compound 117.WSGR Docket No.51503-775.601

[0260] POB was prepared by dissolving 48.85 mg of Compound 117 in 81.417 mL of deionized water(0.5%MC, 0.1%Tween80), followed by vortexing and sonication to obtain a solution with concentration at 3 mg / mL of Compound 117.

[0261] HPLC was performed on the samples using the following equipment and parameters. HPLC:Instrument: Shimadzu (DGU-20A5R, Serial No: L20705518888 IX; LC-30AD Serial No: L20555510784 AE and L20555510780AE; SIL-30AC, Serial No: L20565504983AE; Rack Changer II Serial No. L20585501070 SS; CTO-30A: Serial No. L20575501292 CD; CBM-20A: Serial No. L20235533956 CD). MS: AB API 5500 LC / MS / MS instrument (Serial No. EF20381804). Column: HALO C1890A 2.7µm (50*2.1 mm). Mobile Phase: Solution A: 5% Acetonitrile in Water (0.1%Formic acid); Solution B: 95% Acetonitrile in Water (0.1%Formic acid). Flow rate: 0.6 mL / min, with the following gradient in Table 11: Table 11

[0262] Injection volume: 1^l.

[0263] The desired serial concentrations of working solutions were achieved by diluting stock solutionof analyte with 50% acetonitrile in water solution.5 µL of working solutions (5, 10, 20, 100, 500, 1000, 5000, 10000 ng / mL) were added to 50 μL of the blank monkey plasma to achieve calibration standards of 0.5~1000 ng / mL (0.5, 1, 2, 10, 50, 100, 500, 1000 ng / mL) in a total volume of 55 μL. Four quality control samples at 1 ng / mL, 2 ng / mL, 50 ng / mL and 800 ng / mL for plasma were prepared independently of those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards.55 μL of standards, 55 μL of QC samples and 55 μL of unknown samples (50 µL of monkey plasma with 5 µL of blank solution) were added to 200 μL of acetonitrile containing IS mixture for precipitating protein respectively. Then the samples were vortexed for 30 s. After centrifugation at 4 degree Celsius, 3900 rpm for 15 min. The supernatant was diluted 3 times with water.1 µL of diluted supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0264] Blood samples were taken at the following time points post-does administration: 0.083, 0.17,0.33, 0.5, 1, 2, 4, 7, 11, 24, and 48 hours.

[0265] Results are shown in Table 12.WSGR Docket No.51503-775.601 Table 12.WSGR Docket No.51503-775.601model using WinNonlin 6.1; The bioavailability (F%) was calculated as following: AUClast- PO / AUCINF-PO > 80%: F=(AUCINF-PO*DoseIV) / (mean AUCINF-IV*DosePO) AUClast- PO / AUCINF-PO ≤ 80% or AUCINF was not available: F=(AUClast-PO*DoseIV) / (mean AUClast- IV*DosePO); NA = Not available.WSGR Docket No.51503-775.601

[0266] The PK parameters are set forth in Table 13 as a mean for all the animals given a particular dose.Table 13.

[0267] Example 6: RAT PK following IV administration of Structure B

[0268] Female Sprague Dawley rats were administered Structure B by IV at a dose level of 1mg / kg(dosing solution of 0.2mg / ml). The IV dose was prepared by dissolving 1.06 mg of Structure B in 5.3 mL of 30%HP-β-CD followed by vortexing and sonication to obtain a solution with concentration at 0.2 mg / mL of Structure B.

[0269] HPLC was performed on the samples using the following equipment and parameters. HPLC:Instrument: Shimadzu (DGU-20A5R, Serial No: L20705826739 IX; LC-30AD Serial No: L20555913985 AE and L20555913969 AE; SIL-30AC, Serial No: L20565806434 AE; Rack Changer II Serial No. L20585801286 SS; CTO-30A: Serial No.L20575801652 CD; CBM-20A: Serial No.L20235941033 CD). MS: AB API 5500 LC / MS / MS instrument (Serial No. EX227152104). Column: Agilent Poroshell 120 EC-C84 µm (50 × 2.1 mm). Mobile Phase: Solution A: 5% Acetonitrile in Water (0.1%Formic acid); Solution B: 95% Acetonitrile in Water (0.1%Formic acid). Flow rate: 0.6 mL / min, with the following gradient in Table 14: Table 14.

[0270] Injection volume: 5^l.WSGR Docket No.51503-775.601

[0271] The desired serial concentrations of working solutions were achieved by diluting stock solutionof analyte with 50% acetonitrile in water solution.5 µL of working solutions (1, 2, 4, 10, 20, 100, 200, 1000, 2000 ng / mL) were added to 50 μL of the blank SD Rat plasma to achieve calibration standards of 0.5~1000 ng / mL ( 0.5, 1, 2, 5, 10, 50, 100, 500, 1000 ng / mL) in a total volume of 55 μL. Five quality control samples at 1 ng / mL, 2 ng / mL, 5 ng / mL, 50 ng / mL and 800 ng / mL for plasma were prepared independently of those used for the calibration curves. These QC samples were prepared on the day of analysis in the same way as calibration standards. 50 μL standards, 50 μL QC samples and 50 μL unknown samples（50 µL plasma with 5 µL blank solution）were added to 200 μL of acetonitrile containing IS mixture for precipitating protein respectively. Then the samples were vortexed for 30 s. After centrifugation at 4 degree Celsius, 3900 rpm for 15 min, the supernatant was diluted 3 times with water.5 µL of diluted supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0272] Blood samples were taken at the following time points post-dose administration: 0.083, 0.17,0.33, 0.5, 1, 2, 4, 7, 11 and 24 hours.

[0273] Results are shown in Table 15.Table 15.WSGR Docket No.51503-775.601BLOQ = Below quantifiable limit of 0.5 ng / mL; PK parameters were estimated by non-compartmental model using WinNonlin 6.1; The bioavailability (F%) was calculated as following: AUClast- PO / AUCINF-PO > 80%: F=(AUCINF-PO*DoseIV) / (mean AUCINF-IV*DosePO) AUClast- PO / AUCINF-PO ≤ 80% or AUCINF was not available: F=(AUClast-PO*DoseIV) / (mean AUClast- IV*DosePO); NA = Not available.WSGR Docket No.51503-775.601

[0274] The PK parameters are set forth in Table 16.Table 16.

[0275] Example 7: Rat PK for Compound 117.

[0276] Male Sprauge Dawley rats were administered Compound 117 by IV at a dose level of 1mg / kg(dosing solution of 0.5mg / ml). The IV dose was prepared by dissolving 1.17 mg of Compound 117 in 0.117 mL of DMSO followed by vortexing for 2 minutes and sonication for 3 minutes, then 0.117 mL of Solutol HS15 was added and vortexed for 3 minutes and finally 2.106mL of saline was added and vortexed for 3 minutes to obtain a solution with concentration at 0.5 mg / mL of Compound 117.

[0277] Analysis was carried out on samples using the following equipment and parameters: LCMSMS-39 (Triple Quad 6500+); Positive ion, ESI; MRM detection. HPLC was performed with a Waters X- Bridge BEH C18 (2.1×50 mm, 1.7 µm) column at 50oC, using solution A: H2O-0.025% FA-1mM NH4OAc; solution B: ACN-0.025% FA-1mM NH4OAc, a flow rate of 0.6 mL / min, with the following gradient set forth in Table 17: Table 17.

[0278] 1 µL of supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0279] Blood samples were taken at the following time points post-dose administration: 0.083, 0.25, 0.5,1, 2, 4, 8 and 24 hours after administration.WSGR Docket No.51503-775.601

[0280] Results are shown in Table 18.Table 18.

[0281] PK parameters were estimated by non-compartmental model using WinNonlin 8.2; NA = Notavailable. The PK parameters are set forth in Table 19. Table 19.

[0282] Example 8: HTT Quantitative Splicing Assay.

[0283] Human HD patient-derived lymphoblastoid cells (GM04724) were plated in 12-well plates at 1 x106cells / well and treated with 0.1% DMSO (vehicle) or Compound of Structure B at final concentrations of 2500, 625, 156, 39, 9.7, 2.4, or 0.6 nM and incubated for 24 hours in a humidified cell cultureWSGR Docket No.51503-775.601 incubator (37℃, 5% carbon dioxide (CO2)). Following treatment for 24 hours, cells were collected by centrifugation for 5 minutes at 100 x g. Homogenization and total RNA extraction were performed using QIAshredder columns (Qiagen Cat# 79656) and RNeasy Mini kit (Qiagen Cat# 74104) on an automated QIAcube Connect instrument, according to the manufacturer's instructions. An on-column DNase digestion step (DNase I, Qiagen Cat# 79254) was included to remove genomic deoxyribonucleic acid (DNA).. For complementary DNA (cDNA) synthesis, 1 µg of purified total cellular RNA per reaction was reverse-transcribed using SuperScript™ III First-Strand Synthesis kit (Invitrogen Cat. # 11752) according to the manufacturer’s instructions, as described in Table 21A and Table 21B. Table 21A. Reagents for Reverse Transcription ReactionTable 21B. Reverse Transcription Reaction

[0284] Each plasmid standard was diluted to 0.5 x107 copies per µL in nuclease free water containing100 ng / µl yeast tRNA. Next, 10-fold dilutions were prepared to have 8 concentrations in total, from 1 x107to 1 x100per 2 µL. The qPCR reaction mixture was prepared using TaqMan™ Fast Advanced Master Mix (Applied Biosystems Cat # 4444965) with primers and probes shown in Table 21E. Two µL of each standard or cDNA (25 ng) and 8 µL reaction mixture were added to a qPCR 384-well plate (Table 21C).. The plate was sealed with optical adhesive film and centrifuged briefly to bring the qPCR reaction mixture to the bottom of the plate. HTT isoform-specific primers and 5’nuclease probes, in duplex with endogenous control (TBP) primers and probes, were used to measure the levels of annotated (E49E50) and non-canonical (E49bE50) HTT mRNAs. Reactions were run in a Quant Studio 7 qPCR instrument with settings as described in Table 21D. The data is shown in FIG.5A. Table 21C. Reagents for PCRWSGR Docket No.51503-775.601 Table 21D. qPCRTable 21E. Primers and Probes

[0285] The primary target tissue of Compound of Structure B is the brain. The effects of Compound ofStructure B on HTT pre-mRNA splicing were therefore assessed in iPSC-derived cortical neurons using qRT-PCR.

[0286] Cortical neurons were generated from XCL-1 cells, a commercially available control iPSC line,via a tetracycline-inducible neurogenin 2 (Ngn2) lentiviral system. Neuronal differentiation and maturation were carried out for 8 days. At Day 8 of maturation, replicate wells of cortical neurons were treated with 0.1% DMSO (vehicle control) or Compound of Structure B at final concentrations of 2500, 625, 156, 39, 10, 2.4, or 0.6 nM for 24 hours. Following treatment with Compound of Structure B or vehicle for 24 hours, cells were lysed using lysis buffer from a TaqMan Fast Advanced Cells-to-CT kit (Invitrogen Cat # A35377) supplemented with DNase I (Qiagen Cat # 79254) and complementary deoxyribonucleic acid (cDNA) was reverse-transcribed using SuperScriptTMIV VILO (Thermo Cat # 11756500) according to the manufacturer’s instructions, as described in Table 21F and Table 21G.WSGR Docket No.51503-775.601 Table 21F. Reagents for Reverse Transcription ReactionTable 21G. Reverse Transcription Reaction

[0287] Each plasmid standard was diluted to 0.5 x107 copies per uL in nuclease free water containing100 ng / µl yeast tRNA. Next, 10-fold dilutions were prepared to have 8 concentrations in total, from 1 x107to 1 x100per 2 µL. The qPCR reaction mixture was prepared using TaqMan™ Fast Advanced Master Mix (Applied Biosystems Cat # 4444965) with primers and probes shown in Table 21J. Four µL of each standard or cDNA and 16 µL reaction mixture were added to a qPCR 384-well plate (Table 21H). The plate was sealed with optical adhesive film and centrifuged briefly to bring the qPCR reaction mixture to the bottom of the plate. HTT isoform-specific primers and 5’ nuclease probes, in duplex with endogenous control (TBP) primers and probes, were used to measure the levels of annotated (E49E50) and non-canonical (E49bE50) HTT mRNAs. Reactions were run on a QuantStudio FLEX 7 instrument, with settings as described in Table 21I. The data is shown in FIG.5D. Table 21H. Reagents for PCRTable 21I. qPCR settingsWSGR Docket No.51503-775.601Table 21J. Primers and Probes

[0288] Example 9: SMSM Treatment in a Humanized Mouse Models.

[0289] Treatment with Compound of Structure B decreased HTT mRNA level in brain (FIG. 5B) andblood (FIG. 5E) in a humanized HTT mouse model. In addition, treatment with Compound of StructureB demonstrated a significant decrease in mutant HTT protein level in brain (FIG. 5C) and blood (FIG.5F) in a humanized HTT mouse disease model.

[0290] Hu18 / 18 mice carry two normal 18-CAG-repeat alleles of the human HTT gene in the absence ofthe endogenous mouse Htt gene and Hu97 / 18 mice carry disease-causing 97-CAG-repeat alleles of the human HTT gene in the absence of the endogenous mouse Htt gene (Southwell et al, 2013).

[0291] In a single dose study, 2-month-old male and female Hu18 / 18 mice (n=2 to 3 persex / timepoint / group) were given oral doses of 0, 1, 3 and 10 mg / kg of Compound of Structure B. RNA was extracted from the forebrain and whole blood samples; the RNA was reverse transcribed and used to perform multiplex qRT-PCR for the amplification of HTT RNA (annotated [E49E50] or non-canonical [E49bE50]) and the mouse Tbp housekeeping gene simultaneously.

[0292] A single administration of Compound of Structure B resulted in a gradual decrease of annotated(E49E50) HTT mRNA that was mirrored by a corresponding increase of non-canonical (E49bE50)mRNA level in brain (FIG. 5B) and blood (FIG. 5E), consistent with the proposed mechanism of action.A second study characterized the dose response of total HTT and mHTT protein and mRNA in Hu97 / 18 mice following daily oral gavage administration of Compound of Structure B. This study was conducted in 2-month-old male and female Hu97 / 18 mice (N= 10 per group) at doses of 0, 1, 3, 10 and 30 mg / kg of Compound of Structure B. For all animals, the vehicle and the test compound were orally administered once daily for 30 days.WSGR Docket No.51503-775.601

[0293] To measure total HTT and mHTT protein, blood draws were performed at 4 hours after the lastdose on Day 30; forebrains were also collected 4 hours after the last dose on Day 30, immediately after the blood collection. Protein was extracted from brain tissues using Native Protein Purification Kit (ThermoFisher Scientific). Meso Scale Discovery (MSD) 2B7-D7F7 and 2B7-MW1 assays were used to measure the total and mutant HTT proteins, respectively, in whole blood (FIG.5F) and brain (FIG.5C) samples.

[0294] Example 10: PMS1 Quantitative Splicing Assay.

[0295] Human lymphoblastoid cells (GM07491) were plated in 96-well plates at 50,000 cells / well andtreated with 0.1% DMSO (vehicle) or Compound of Structure B at final concentrations of 10000, 2500, 625, 156, 39, 9.7, or 2.4 nM and incubated for 24 hours in a humidified cell culture incubator (37℃, 5% carbon dioxide (CO2)). Following treatment for 24 hours, cells were collected by centrifugation for 5 minutes at 123 x g, and processed using TaqMan fast advanced Cells-to-Ct kit (Invitrogen, Cat. # 35377). Cells were lysed with 50 µL of lysis solution containing DNAse. For cDNA synthesis, 10 µL of lysate was added to 40 µL of RT reaction mix and reverse transcribed, as described in Table 22A and Table 22B. Table 22A. Reagents for Reverse Transcription ReactionTable 22B. Reverse Transcription Reaction

[0296] Each gBlock was diluted to 0.5x107 copies / µl in nuclease free water containing 100 ng / µl yeasttRNA. Next, 10-fold dilutions were prepared to have 8 concentrations in total. from 1 x107to 1 x100per 2 µL. The qPCR reaction mixture was prepared using TaqMan Mastermix from TaqMan fast advanced Cells-to-Ct kit (Invitrogen, Cat. # 35377) with primers and probes shown in Table 22E. Two µL of each gBlock or cDNA and 8 µL reaction mixture were added to a qPCR 384-well plate (Table 22C). The plate was sealed with optical adhesive film and centrifuged briefly to bring the qPCR reaction mixture to the bottom of the plate. PMS1 isoform-specific primers and 5’nuclease probes, in duplex with endogenous control (TBP) primers and probes, were used to measure the levels of annotated (E5E6) and non- canonical (E5E5b) PMS1 mRNAs. Reactions were run on a QuantStudio FLEX 6 or FLEX 7 instrument, with settings as described in Table 22D. The data is shown in FIG.6C and FIG.6D.

[0297] In addition, PMS1 protein expression level was measured in human lymphoblastoid cells treatedwith Compound of Structure B for 48 hours and the data is shown in FIG.6E. Briefly, human lymphoblastoid cells were seeded at 5E5 cells / well in 12 well plates and treated with Compound ofWSGR Docket No.51503-775.601 Structure B or DMSO. The concentrations of compounds were tested at appropriate doses ranging from 10 µM to 2.4 nM. After incubation for 48 hours, the cells were lysed with 100 µL of lysis buffer containing EDTA and protease inhibitors, and total PMS1 protein levels were assessed by a Jess Automated Western Blot System assay developed with one anti-PMS1 antibody and one β-Actin antibody, raised in rabbit and mouse, respectively.0.2 µg / µL protein was loaded and run on the Separation Module, and Anti-Mouse / Anti-Rabbit Detection Module was used to detect chemiluminescence via Jess Automated Western Blot System. PMS1 protein expression was subsequently normalized to β-Actin. Table 22C. Reagents for PCRTable 22D. qPCRTable 22E. Primers and ProbesWSGR Docket No.51503-775.601

[0298] Example 11: Phase 1 Clinical Trial

[0299] Phase 1 clinical trial was designed to evaluate the safety, tolerability, pharmacokinetics,pharmacodynamics, and blood biomarker modulation activity of Compound of Structure B in healthy volunteers and early diagnosed or early-stage HD patients. The clinical trial design is shown in FIG.7A. The clinical trial comprises Part A, Part B, and Part C.

[0300] Part A is a double-blind placebo-controlled single ascending dose (SAD) design in healthy adultvolunteers. Part A has five cohorts, up to eight subjects per cohort, receiving ascending doses of Compound of Structure B (ranging from 1 mg to 16 mg) or placebo (3:1 ratio of active:placebo). Doses were administered daily from Day 1 to Day 14 (inclusive). The influence of food on the pharmacokinetics (PK) of Compound of Structure B was also examined in a dedicated cohort. The food intake had a minor effect on exposure of Compound of Structure B with a difference below 50% compared to the fasting condition. An additional cohort can be added to the study depending on the drug exposure in Cohort 5.

[0301] Part B is a double-blind placebo-controlled multiple ascending dose (MAD) design in healthyadult volunteers. Part B includes three to four cohorts, up to eight subjects per cohort, randomized to receive multiple ascending doses of Compound of Structure B (ranging from 1 mg to 9 mg) or placebo (3:1 ratio of active:placebo). Doses were administered daily from Day 1 to Day 14 (inclusive).

[0302] Parts C is a double-blind placebo-controlled multiple ascending dose (MAD) design in early-stage HD patients (HD-Integrated Staging System or HD-ISS Stage 1, 2, or mild Stage 3), preceded by an observational period lasting a minimum of 28 days, which aims to establish a stable baseline of pharmacodynamic parameters such as HTT protein and mRNA. HD-ISS, is a recently developed evidence-based staging centered on biological, clinical, and functional assessments to standardize evaluation in clinical trials. Stage 0 includes individuals with the HD genetic mutation without any detectable pathological changes or symptoms by using a genetic definition of HD. Disease progression is marked by measurable indicators of underlying pathophysiology (Stage 1), a detectable clinical phenotype (Stage 2), and a decline in function (Stage 3). Individuals can be classified into stages based on thresholds of stage-specific landmark assessments. Two dose levels of Compound of Structure B, identified in Parts A and B, will be evaluated in Part C. Part C comprises two active dose groups and one placebo group with a maximum enrollment of 24 patients, randomized to receive low or high doses of Compound of Structure B or placebo. The open-label extension is designed to generate long-term safety and efficacy data in patients early in clinical development. Doses will be administered daily from Day 1 to Day 28. The clinical pharmacodynamics (PD) / efficacy biomarkers for HTT decreasing therapies have been established. In Phase 1 studies, blood biomarkers (HTT mRNA and protein) can be used to establish pharmacodynamic correlations. Also, PMS1, a unique biomarker, can be used to identify potential patients with a high likelihood of response to the therapeutics.

[0303] A regional randomized, controlled Phase 2 trial can also be initiated to allow participants in PartC to continue receiving Compound of Structure B, while also enrolling additional HD patients, toWSGR Docket No.51503-775.601 ultimately generate longer-term data from an independent study which could support potential global pivotal clinical trials. Table 23. Phase 1 Clinical Trial Summary

[0304] 54 healthy participants were exposed to at least one dose of Compound of Structure B duringPart A and Part B of the study. Compound of Structure B was generally well-tolerated in healthy participants at doses up to 16 mg (single dose) and up to 9 mg (multiple dose, QD over 14 days). No safety signs have been identified (see summary table in Table 24 below). Table 24. Overall Summary of TEAEs - Part B (Safety Analysis Set)TEAE = Treatment emergent adverse event. Related TEAEs were defined as TEAEs that are possibly, probably, or definitely related to the study drug.

[0305] As shown in FIG. 7B, administration of Compound of Structure B demonstrated dose-dependent decrease of HTT mRNA in SAD study. In particular, 16 mg single dose administration demonstrated about 67% reduction in HTT mRNA (FIG.7B). In addition, administration of 1 mg / day ofWSGR Docket No.51503-775.601 Compound B for 14 days demonstrated about 19% reduction in HTT mRNA in MAD study (FIG.8B). Linear PK was demonstrated across Phase 1 cohorts after a single oral administration of Compound of Structure B (FIG.7C).

[0306] As shown in FIG 8B, administration of Compound of Structure B demonstrated dose-dependentdecrease of HTT mRNA in MAD study. In particular, 9 mg multiple dose administration demonstrated about 72% reduction in HTT mRNA. A concomitant increase in the NMD-targeted cryptic HTT mRNA isoform was observed following single or multiple daily administration.

[0307] Exposure to Compound of Structure B increased in a dose proportional manner following singleor multiple ascending doses (FIG.7C and FIG.8C). Following daily dose administration, exposure increased about 3-fold, indicating accumulation with multiple doses. Mean exposure values were similar between the fasted and fed states, indicating that food has a minimal impact on exposure to Compound of Structure B. The median Tmaxvalues shifted about 4 hours in the presence of food, with no impact on mean Cmaxvalues.

[0308] The half-life following single or multiple dose administration is about 30 hours. Following dailyCompound of Structure B oral dose administration as single (1-16 mg) or multiple dose (1-9 mg) for 14 days, peak pharmacodynamic effects on canonical HTT mRNA in blood were highly correlated with Compound of Structure B exposures, both Cmax(r2= 0.813 and 0.801 for SD and MD, respectively) and AUClast(r2= 0.836 and 0.845 for SD and MD, respectively.

[0309] The examples and embodiments described herein are for illustrative purposes only and variousmodifications or changes suggested to persons skilled in the art are to be included within the spirit and purview of this application and scope of the appended claims.

Claims

WSGR Docket No.51503-775.601 CLAIMS What is claimed is:

1. A method of treating or preventing Huntington’s disease in a subject in need thereof, comprisingadministering to the subject in need thereof a therapeutically effective amount of a composition comprising a compound of structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof:Structure B.

2. The method of claim 1, wherein the subject wherein the subject’s genome encodes a wild-typePMS1.

3. The method of claim 1, wherein the subject’s genome comprises an allele comprising a geneticvariation in a PMS1 gene.

4. The method of claim 3, wherein the genetic variation is a non-synonymous coding variant.

5. The method of claim 3 or 4, wherein the genetic variation does not disrupt or modulate the PMS1gene, or wherein the genetic variation is not a loss-of-function genetic variation.

6. The method of any one of claims 3 to 5, wherein the genetic variation comprises chr2:190660537G>A, chr2:190719296 A>G, chr2:190719569 T>C, or any combination thereof, wherein chromosome positions of the genetic variation are defined with respect to UCSC hg19.

7. The method of any one of claims 3 to 6, wherein the allele comprising a genetic variation in a PMS1gene encodes a variant PMS1 comprising a mutation selected from the group consisting of a E59K mutation, a K433R mutation, a L524S mutation, and any combination thereof.

8. The method of any one of claims 3 to 7, wherein the subject has been identified as having the geneticvariation.

9. The method of claim 3, wherein the genetic variation disrupts or modulates the PMS1 gene.

10. The method of claim 3, wherein the subject has been identified as not having the genetic variation.

11. The method of claim 9 or 10, wherein the genetic variation is a non-synonymous coding variant.

12. The method of any one of claims 9 to 11, wherein the genetic variation comprises chr2:190660586C>T, chr2:190670391 C>G, chr2:190670396 A>G, chr2:190717470 CA>C, chr2:190719499 G>A, chr2:190719607 G>A, chr2:190719704 G>A, chr2:190732559 T>C, or any combination thereof, wherein chromosome positions of the genetic variation are defined with respect to UCSC hg19.

13. The method of any one of claims 9 to 12, wherein the allele comprising a genetic variation in aPMS1 gene encodes a variant PMS1 comprising a mutation selected from the group consisting of a T75I, T110R, T112A, S264*, G501R, E537K, R569Q, Y793H, wherein * denotes a premature termination of protein translation.WSGR Docket No.51503-775.60114. The method of any one of claims 3 to 13, wherein the subject has been tested for a presence of thegenetic variation with a genetic assay.

15. The method of any one of claims 3 to 14, wherein the subject is heterozygous for the geneticvariation.

16. The method of any one of claims 3 to 14, wherein the subject is homozygous for the geneticvariation.

17. The method of any one of claims 1 to 16, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof modulates splicing of an HTT pre-mRNA, a PMS1 pre-mRNA, or both.

18. The method of any one of claims 1 to 17, wherein the method delays onset or slows progression ofthe Huntington’s disease.

19. A method of treating or preventing a disease or condition in a subject in need thereof, comprisingadministering to the subject a therapeutically effective amount of a composition comprising a compound of structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof:Structure B, wherein the disease or condition is associated with expression level or activity level of a protein encoded by an HTT gene or a PMS1 gene.

20. The method of claim 19, wherein the disease or condition is associated with a string of CAG repeatsin the HTT gene.

21. The method of claim 19 or 20, wherein a pre-mRNA and / or an mRNA encoded by the HTT genecomprises the string of CAG repeats.

22. The method of any one of claims 19 to 21, wherein the disease or condition is associated with anaberrant expansion of a string of CAG repeats in the HTT gene.

23. The method of any one of claims 19 to 22, wherein a pre-mRNA encoded by the HTT genecomprises the aberrant expansion of the string of CAG repeats.

24. The method of any one of claims 19 to 23, wherein the protein encoded by the HTT gene comprises amutant HTT protein.

25. The method of any one of claims 19 to 24, wherein the disease or the condition is associated with astring of CAG repeats or an aberrant expansion of a string of CAG repeats in the HTT gene caused by the protein encoded by the PMS1 gene.

26. The method of any one of claims 19 to 25, wherein the disease or condition is Huntington’s disease.

27. The method of any one of claims 19 to 26, wherein the compound of structure B, or apharmaceutically acceptable salt or a stereoisomer thereof binds to a pre-mRNA and modulatesWSGR Docket No.51503-775.601 splicing of the pre-mRNA at a splice site of the pre-mRNA in a cell of a subject, wherein the pre- mRNA is encoded by the HTT gene or the PMS1 gene.

28. The method of any one of claims 1 to 27, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof is administered to the subject at about 1 mg to about 16 mg per dose.

29. The method of any one of claims 1 to 27, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof is administered to the subject at about 1 mg, about 2 mg, about 3 mg, about 4 mg, about 5 mg, about 6 mg, about 7 mg, about 8 mg, about 9 mg, about 10 mg, about 11 mg, about 12 mg, about 13 mg, about 14 mg, about 15 mg, or about 16 mg per dose.

30. The method of any one of claims 1 to 29, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof is administered to the subject daily.

31. A method of modulating splicing of an HTT pre-mRNA, a PMS1 pre-mRNA, or both, comprisingcontacting a compound of structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, to the HTT pre-mRNA, the PMS1 pre-mRNA, or both or cells comprising the HTT pre- mRNA, the PMS1 pre-mRNA, or bothStructure B, wherein the HTT pre-mRNA and the PMS1 pre-mRNA comprise a splice site sequence, and wherein the compound binds to the HTT pre-mRNA, the PMS1 pre-mRNA, or both and modulates splicing of the HTT pre-mRNA, the PMS1 pre-mRNA, or both in the cells or cells of a subject to produce a spliced product of the HTT pre-mRNA, the PMS1 pre-mRNA, or both.

32. A method of modulating splicing of an HTT pre-mRNA, a PMS1 pre-mRNA, or both in a cell,comprising contacting the cell with a compound of structure B, or a pharmaceutically acceptable saltor a stereoisomer thereof:Structure B.WSGR Docket No.51503-775.60133. The method of claim 31 or 32, wherein the expression level of a canonical isoform of a PMS1 mRNAencoded by the PMS1 pre-mRNA is reduced by at least 50% in the cell contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

34. The method of any one of claims 31 to 33, wherein the expression level of a canonical isoform of anHTT mRNA encoded by the HTT pre-mRNA is reduced by at least 10% in the cell contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

35. The method of any one of claims 1 to 34, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof is the compound of structure B.

36. The method of any one of claims 1 to 24 or 35, comprising administering to the subject apharmaceutical composition comprising the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof, and a pharmaceutically acceptable excipient or carrier.

37. The method of any one of claims 1 to 36, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof binds to the HTT pre-mRNA, the PMS1 pre-mRNA, or both.

38. The method of any one of claims 1 to 37, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof modulates the splicing of the HTT pre- mRNA, the PMS1 pre-mRNA, or both.

39. The method of any one of claims 1 to 38, wherein modulating splicing comprises promotinginclusion of an exon.

40. The method of claim 39, wherein modulating splicing comprises promoting inclusion of a crypticexon.

41. The method of claim 40, wherein the cryptic exon comprises a poison exon.

42. The method of claim 41, wherein the compound of structure B, or the pharmaceutically acceptablesalt or stereoisomer thereof promotes inclusion of the poison exon in the spliced product of the HTT pre-mRNA, in the spliced product of the PMS1 pre-mRNA, or both.

43. The method of claim 41 or 42, wherein the poison exon comprises a nucleic acid sequence locatedbetween exon 49 and exon 50 of the HTT pre-mRNA.

44. The method of claim 43, wherein the poison exon is exon 49b.

45. The method of claim 41 or 42, wherein the poison exon comprises a nucleic acid sequence locatedbetween exon 5 and exon 6 of the PMS1 pre-mRNA.

46. The method of claim 43, wherein the poison exon is exon 5b.

47. The method of any one of claims 41 to 46, wherein the poison exon comprises an in-frame stopcodon.

48. The method of claim 47, wherein the in-frame stop codon is a premature termination codon.

49. The method of claim 47 or 48, wherein the in-frame stop codon is at least 50 or 60 base pairsupstream of the 3’ end of the poison exon.WSGR Docket No.51503-775.60150. The method of claim 47 or 48, wherein the in-frame stop codon is less than 60 base pairs upstream ofthe 3’ end of the poison exon, and wherein the exon immediately downstream of the poison exon is not the last exon in the HTT pre-mRNA or the PMS1 pre-mRNA.

51. The method of claim 50, wherein the sum of(a) the number of base pairs in the exon immediately downstream of the poison exon and (b) the number of base pairs between the premature stop codon in the poison exon and the 3’ end of the poison exon is at least 50 or at least 60.

52. The method of any one of claims 1 to 51, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof binds to the nucleic acid sequence located between exon 49 and exon 50 of the HTT pre-mRNA, the nucleic acid sequence located between exon 5 and exon 6 of the PMS1 pre-mRNA, or both.

53. The method of claim 52, wherein the nucleic acid sequence located between exon 49 and exon 50 ofthe HTT pre-mRNA comprises intron 49b comprising a 5’ splice site sequence.

54. The method of claim 52, wherein the nucleic acid sequence located between exon 5 and exon 6 of thePMS1 pre-mRNA comprises intron 5b comprising a 5’ splice site sequence.

55. The method of claim 53 or 54, wherein the compound of structure B, or the pharmaceuticallyacceptable salt or stereoisomer thereof promotes splicing at the 5’ splice site sequence.

56. The method of any one of claims 52 to 55, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof promotes inclusion of exon 49b in the spliced product of the HTT pre-mRNA, inclusion of exon 5b in the spliced product of the PMS1 pre- mRNA, or both.

57. The method of any one of claims 1 to 56, wherein the amount of the spliced product of the HTT pre-mRNA comprising exon 49b is increased in the cell or in the cells contacted by the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

58. The method of any one of claims 1 to 56, wherein the amount of the spliced product of the PMS1pre-mRNA comprising exon 5b is increased in the cell or in the cells contacted by the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

59. The method of any one of claims 53 to 58, wherein the 5’ splice site sequence comprisesAGCAGA / guaagggggcuu (SEQ ID NO: 1) or AAAUGA / guaagacugguu (SEQ ID NO: 2).

60. The method of any one of claims 1 to 59, wherein inclusion of a cryptic exon promotesdestabilization or nonsense-mediated decay (NMD) of the spliced product of the HTT pre-mRNA.

61. The method of any one of claims 1 to 59, wherein inclusion of a cryptic exon promotesdestabilization or nonsense-mediated decay (NMD) of the spliced product of the PMS1 pre-mRNA.

62. The method of claim 60 or 61, wherein the amount of an HTT mRNA, a PMS1 mRNA, or both isreduced in the cell or in the cells contacted by the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

63. The method of claim 62, wherein the amount of the HTT mRNA, the PMS1 mRNA, or both isreduced by at least 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%,WSGR Docket No.51503-775.601 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% in the cell or in the cells contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

64. The method of any one of claims 60 to 63, wherein the amount of the mutant HTT protein is reducedin the cell or in the cells contacted by the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

65. The method of claim 64, wherein the amount of the mutant HTT protein is reduced by at least 1%,2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% in the cell or in the cells contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

66. The method of any one of claims 60 to 63, wherein the amount of the PMS1 protein is reduced in thecell or in the cells contacted by the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

67. The method of claim 66, wherein the amount of the PMS1 protein is reduced by at least 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% in the cell or in the cells contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

68. The method of any one of claims 1 to 67, wherein the cell or the cells comprise primary cells.

69. The method of any one of claims 1 to 68, wherein the cell or the cells comprise disease cells.

70. The method of any one of claims 1 to 69, wherein the cell or the cells comprise Huntington’s diseasepatient-derived lymphoblastoid cell line, induced pluripotent stem cell (iPSC)-derived cortical neurons, blood cells, brain cells, or combinations thereof.

71. The method of any one of claims 1 to 70, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof modulates proliferation or survival of the cells.

72. The method of any one of claims 1 to 31 or 36 to 70, wherein the composition or the pharmaceuticalcomposition comprising the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered orally.

73. The method of any one of claims 1 to 31 or 36 to 70, wherein the composition or the pharmaceuticalcomposition comprising the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered as a solid dosage form.

74. The method of claim 73, wherein the solid dosage form comprises a tablet or a capsule.

75. The method of any one of claims 1 to 74, wherein the compound of structure B, or thepharmaceutically acceptable salt or stereoisomer thereof penetrates a blood brain barrier (BBB) when administered to the subject.

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