6-(6-(((lr,2r,3s,5s)-2-fluoro-9-azabicyclo[3.3.1jnonan-3yl) (methyl)amino)pyridazine-3-yl)-2-methylbenzo[d]oxazol-5-ol to treat huntington's disease

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

6-(6-{[(1 R,2R,3S,5S)-2-FLUORO-8-AZABICYCLO[3.2.1 ]0CTAN-3-YL] (METHYL)AMINO }PYRIDAZIN-3-YL)-2-METHYL-L,3-BENZOXAZOL-5-OL TO TREAT HUNTINGTON'S DISEASERELATED APPLICATIONS

[0001] This application claims the benefit of U. S. Provisional Patent Application No. 63 / 716,282, filed on November 5, 2024; U. S. Provisional Patent Application No. 63 / 882,903, filed on September 16, 2025; and U. S. Provisional Patent Application No. 63 / 896,358, filed on October 9, 2025, 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 in XML format and is hereby incorporated by reference in its entirety. Said XML copy, created on October 9, 2025, is named 51503-775_602_SL.xml and is 28,486 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 with therapies 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-{[(lR,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-l,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-{[(lR,2R,3S,5S)-2-fluoro-8-azabicyclo [3.2.1 ]octan-3 -yl] (methyl)amino }pyridazin-3 -y 1) -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. 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 incirculation, etc.) can be affected by small changes between two similar compounds. Thus, compounds similar to 6-(6-{[(lR,2R,3S,5S)-2-fluoro-8-azabicyclo[3.2.1]octan-3-yl](methyl)amino}pyridazin-3-yl)-2-methyl-l,3-benzoxazol-5-ol, but with improved metabolic profdes 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 a human subject in need thereof, comprising administering to the human 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:wherein the human subject has been genetically confirmed by DNA sequencing to have cytosine, adenine, and guanine (CAG) repeat length of at least 40 in an HTT gene. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject in need thereof for a treatment period of at least 28 days.

[0009] In one aspect, described herein is a method of treating or preventing Huntington’s disease in a human subject in need thereof, comprising administering to the human 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:Structure B,wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject in need thereof for a treatment period of at least 28 days. In some embodiments, the human subject has a total motor score (TMS) of 6 or higher. In some embodiments, the human subject has an independence scale (IS) score of 70 or higher. In some embodiments, the human subject has a total functional capacity (TFC) score of 10 or higher.

[0010] In one aspect, described herein is a method of treating or preventing Huntington’s disease in a human subject in need thereof, comprising administering to the human 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:Structure B,wherein the human subject has one or more of: (i) a total motor score (TMS) of 6 or higher; (ii) has an independence scale (IS) score of 70 or higher; or (iii) a total functional capacity (TFC) score of 10 or higher.

[0011] In some embodiments, the amount of a mutant huntingtin (mHTT) is reduced in a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the amount of mHTT 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 a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

[0012] A method of treating or preventing Huntington’s disease in a human subject in need thereof, comprising administering to the human 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,wherein the amount of a mutant huntingtin (mHTT) 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 a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

[0013] In some aspects, the compound of structure B is 6-(6-(((lA,2A,3S,5S)-2-Fluoro-9-azabicyclo [3.3.1 ]nonan-3 -yl)(methyl)amino)pyridazine-3 -y 1) -2 -methylbenzo [o oxazol-5 -ol.

[0014] In some embodiments, the human subject has been genetically confirmed by DNA sequencing to have cytosine, adenine, and guanine (CAG) repeat length of at least 40 in an HTT gene. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject in need thereof for a treatment period of at least 28 days. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomerthereof is administered to the human subject in need thereof for a treatment period of at least 56 days or at least 84 days. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject in need thereof for a treatment period of about 12 months or more than 12 months. In some embodiments, the amount of the mHTT protein is reduced by at least 10% in the blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of at least 84 days. In some embodiments, the treatment period is followed by a drug holiday period. In some embodiments, the drug holiday period is about 14 days.

[0015] In some embodiments, the human subject is between the ages of 25 and 70. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human 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 human 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.

[0016] In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject at about 3 mg per dose. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject at about 4 mg per dose. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject at about 6 mg per dose. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject at about 9 mg per dose.

[0017] In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject daily. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject once a day. 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 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.

[0018] 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 human subject. In some embodiments, the human subject has Huntington’s Disease Integrated Staging System [HD-ISS] Stage 2 or 3 mild.

[0019] In some embodiments, the amount of a 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 a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof. In some embodiments, the amount of a 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 a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of at least 28 days, at least 56 days, or at least 84 days.

[0020] In some embodiments, the amount of a canonical isoform of an HTT mRNA, a canonical isoform of a 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 a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of at least 28 days, at least 56 days, or at least 84 days. In some embodiments, the amount of the canonical isoform of the HTT mRNA is reduced by at least 50% in the blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of at least 84 days. In some embodiments, the amount of the canonical isoform of the PMS1 mRNA is reduced by at least 20% in the blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of at least 84 days.

[0021] In some embodiments, the human subject’s genome encodes a wild-type PMS1. In some embodiments, the human subject’s genome comprises an allele comprising a genetic variation in a PMS1 gene. In some embodiments, the genetic variation is anon-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-fiinction 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 hgl9. 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.

[0022] In some embodiments, the human subject has been identified as having the genetic variation. In some embodiments, the genetic variation disrupts or modulates the PMS1 gene. In some embodiments, the human subject has been identified as not having the genetic variation.

[0023] In some embodiments, the genetic variation is a non-synonymous coding variant. In some embodiments, 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 hgl9. In some embodiments, the allele comprising a genetic variation in a PMS1 gene encodes a variant PMS1 comprising a mutation selectedfrom the group consisting ofaT75I, T110R, T112A, S264*, G501R, E537K, R569Q, Y793H, wherein * denotes a premature termination of protein translation.

[0024] In some embodiments, the human subject has been tested for a presence of the genetic variation with a genetic assay. In some embodiments, the human subject is heterozygous for the genetic variation. In some embodiments, the human subject is homozygous for the genetic variation.

[0025] In some embodiments, the method delays onset or slows progression of the Huntington’s disease. In some embodiments, the Huntington’s disease is associated with expression level of activity level of a protein encoded by an HTT gene or a PMS 1 gene.

[0026] In some embodiments, the Huntington’s disease is associated with a string of CAG repeats in the HTT 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 Huntington’s disease 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 Huntington’s disease 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 compound of structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof binds to an HTT pre-mRNA, a PMS 1 pre-mRNA, or both and modulates splicing of the HTT pre-mRNA, the PMS 1 pre-mRNA, or both at a splice site of the HTT pre-mRNA, the PMS 1 pre-mRNA, or both in a cell or cells of the human subject to produce a spliced product of the HTT pre-mRNA, the PMS 1 pre-mRNA or both. In some embodiments, the HTT pre-mRNA, the PMS1 pre-mRNA, or both comprise a splice site sequence.

[0027] In some embodiments, the expression level of a canonical isoform of a PMS 1 mRNA encoded by the PMS1 pre-mRNA is reduced by at least 20% 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 30% 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.

[0028] 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.

[0029] In some embodiments, the poison exon comprises a nucleic acid sequence located between exon 49 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 ofthe 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 PMS 1 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.

[0030] In some embodiments, the compound of structure B, or the pharmaceutically 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. 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).

[0031] In some embodiments, inclusion of a cryptic exon promotes destabilization or nonsense -mediated decay (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 PMS 1 pre-mRNA.

[0032] In some embodiments, the amount of an HTT mRNA, a PMS 1 mRNA, or both is reduced 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 HTT mRNA, the PMS 1 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.

[0033] In some embodiments, the amount of the mutant HTT protein is reduced 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 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.

[0034] In some embodiments, the amount of the PMS 1 protein is reduced 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 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.

[0035] In some embodiments, the cell or the cells comprise primary cells. In some embodiments, the cell or 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 cell or the cells.INCORPORATION BY REFERENCE

[0036] All publications, patents, and patent applications mentioned in this specification are herein incorporated 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

[0037] FIG. 1 depicts RNA structure analysis of a splice site, showing base-pairing between 5’ splice site sequence and U1 snRNA sequence that forms a bulge structure, ss: splice site,: pseudouridine. Figure discloses SEQ ID NOs: 18, 19, 18, 20, 18, 21, 18, and 22, respectively, in order of appearance.

[0038] FIG. 2 depicts RNA structure analysis of a splice site, showing base-pairing between 5’ splice site sequence and U1 snRNA sequence that forms a loop structure, ss: splice site, 'P: pseudouridine. Figure discloses SEQ ID NOs: 18, 23, 18, 24, 18, and 25, respectively, in order of appearance.

[0039] FIG. 3 shows that potential mechanisms by which SMSM can drive reduction of both HTT and PMS1 protein levels.

[0040] FIG. 4A and FIG. 4B depict a simplified schematic of an exemplary splicing event of Huntingtin (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.

[0041] FIG. 4C depicts RNA structure analysis of HTT target splice site, showing base-pairing between target 5’ splice site sequence and U1 snRNA sequence. Figure discloses SEQ ID NOs: 26 and 1, respectively, in order of appearance.

[0042] FIG. 4D shows SMSM binding to HTT exon 49b.

[0043] FIGs. 5A-5B and 5D-5E are graphs demonstrating a reduction in HIT mRNA due to a shift in the ratio between the canonical and non-canonical mRNA isoforms in the presence of SMSM (compound of Structure B) 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).

[0044] FIG. 5C and FIG. 5F are graphs showing mutant HTT (mHTT) protein levels after SMSM (compound of Structure B) treatment in mouse model brain (FIG. 5C) and in mouse model blood (FIG.5F).

[0045] 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.

[0046] FIG. 6B depicts RNA structure analysis of PMS1 target splice site, showing base-pairing between target 5’ splice site sequence and U1 snRNA sequence. Figure discloses SEQ ID NOs: 26 and 2, respectively, in order of appearance.

[0047] FIG. 6C is a graph showing a reduction in PMS 1 mRNA due to a shift in the ratio between the canonical and non-canonical mRNA isoforms in the presence of SMSM (compound of Structure B).

[0048] FIG. 6D is a graph showing a reduction in PMS 1 mRNA due to a shift in the ratio between canonical and non-canonical (cryptic) mRNA isoforms in human lymphoblast cells treated with SMSM (compound of Structure B).

[0049] FIG. 6E shows a reduction in PMS 1 total protein level in human lymphoblast cells treated with SMSM (compound of Structure B).

[0050] 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.

[0051] FIG. 7B is a graph showing changes in HTT mRNA after a single dose SMSM (Compound of Structure B) treatment compared to pre-treatment.

[0052] FIG. 7C is a graph showing the mean concentration of SMSM (Compound of Structure B) by time profile following a single dose oral administration.

[0053] 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.

[0054] FIG. 8B is a graph showing the mean HTT mRNA in blood 24 hours after the last dose of 14 consecutive days. SMSM: Compound of Structure B.

[0055] FIG. 8C is a graph showing the mean SMSM (Compound of Structure B) concentrations after ascending oral dose administration in healthy volunteers.

[0056] FIG. 9 is a schematic of exemplary Cumulative Staging framework and landmarks illustrating graphical representation of temporal sequence of Stage progression and the associated landmark assessments that define Stage entry (note: time not to scale).

[0057] FIG. 10A is a Phase 1 - Part C clinical trial design to test SMSM in HD patients. HD-ISS:Huntington’s Disease Integrated Staging System, TMS: Total Motor Score, IS: Independence Scale, TFC: Total Functional Capacity, R: Randomized, SMSM: Compound of Structure B, QD: once daily, PK: Pharmacokinetics, CSF: Cerebrospinal fluid, cUHDRS: composite Unified Huntington’s Disease Rating Scale.

[0058] FIG. 10B is a graph showing the reduction in mutant huntingtin (mHTT) protein level in blood from pre-dose through 84 days of treatment (left) and mean % change from baseline HTT mRNA.Administration of SMSM (Compound of Structure B) achieves dose-dependent reductions of mHTT protein, with 62% lowering at Day 84 on the 9 mg daily oral dose. Error bars represent standard error of the mean. The graph excludes Day 84 data for a patient that discontinued 9 mg treatment at Day 73. Placebo (N=6), 3 mg (N=10), and 9 mg (N=10) patient treatment is ongoing.

[0059] FIG. 11 is a graph showing the mean pre-dose concentration of SMSM (Compound of Structure B) following a multiple ascending dose (MAD) oral administration.

[0060] FIG. 12 is a graph showing the plasma concentration of SMSM (Compound of Structure B) following oral administration.

[0061] FIGs. 13A-13B are graphs showing changes in HTT mRNA (FIG. 13A) and PMS1 mRNA (FIG. 13B) over 28 days with administration of SMSM (Compound of Structure B). Note: Error bars represent the standard error of the mean.

[0062] FIG. 14 is a graph showing cerebrospinal fluid (CSF) Neurofilament Light (NfL) levels at baseline and at 84 days of treatment with SMSM (Compound of Structure B). No group effects on CSF NfL were observed in patients treated with Compound of Structure B for three months. (One subject removed who had COVID just prior to 84-day sample).

[0063] FIG. 15 is a Phase 2 / 3 clinical trial design to test SMSM in HD patients. HD-ISS: Huntington’s Disease Integrated Staging System, TMS: Total Motor Score, IS: Independence Scale, TFC: Total Functional Capacity, R: Randomized, SMSM: Compound of Structure B, QD: once daily, AE: adverse effect, SAE: serious adverse effect, cUHDRS: composite Unified Huntington’s Disease Rating Scale.DETAILED DESCRIPTION

[0064] Certain specific details of this description are set forth in order to provide a thorough understanding 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.

[0065] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this 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

[0066] The terms “compound(s) of this disclosure”, “compound(s) of the present disclosure”, “small molecule 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.

[0067] Any open valency appearing on a carbon, oxygen, sulfur or nitrogen atom in the structures herein indicates the presence of hydrogen, unless indicated otherwise.

[0068] The terms “administer,” “administering”, “administration,” and the like, as used herein, refer to the 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.

[0069] The terms “co-administration” or the like, as used herein, are meant to encompass administration of 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.

[0070] The terms “effective amount” or “therapeutically effective amount,” as used herein, refer to a sufficient 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.

[0071] The terms “enhance” or “enhancing,” as used herein, means to increase or prolong either in amount, 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.

[0072] The term “subject” or “patient” encompasses mammals. Examples of mammals include, but are not 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.

[0073] 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.

[0074] The term “pharmaceutical combination” as used herein, means a product that results from mixing or 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.

[0075] The term “pharmaceutically acceptable” denotes an attribute of a material which is useful in preparing 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.

[0076] 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.

[0077] The term “pharmaceutically acceptable salts” denotes salts which are not biologically or otherwise 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.

[0078] 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.

[0079] As used herein, a “small molecular weight compound” can be used interchangeably with “small molecule” or “small organic molecule.” Small molecules refer to compounds other than peptides oroligonucleotides; and typically have molecular weights of less than about 2000 Daltons, e.g., less than about 900 Daltons.

[0080] The terms “effective amount” or “therapeutically effective amount,” as used herein, refer to a sufficient 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.

[0081] The terms “treat,” “treating” or “treatment,” as used herein, include alleviating, abating or ameliorating 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 the 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.

[0082] The term “preventing” or “prevention” of a disease state denotes causing the clinical symptoms of 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.

[0083] The term “pharmaceutical composition” denotes a mixture comprising an active pharmaceutical ingredient together with one or more pharmaceutically acceptable excipients that can be administered to a subject, e.g., a human in need thereof.

[0084] The term “nucleic acid” or “polynucleic acid” as used herein generally refers to one or more nucleobases, nucleosides, or nucleotides, and the term includes polynucleobases, polynucleosides, and polynucleotides.

[0085] The term “polynucleotide,” as used herein generally refers to a molecule comprising two or more linked 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 (POs) 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 ( / . 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 may comprise isolated and purified DNA / RNA molecules, synthetic DNA / RNA molecules, and / or synthetic DNA / RNA analogs.

[0086] As used herein, the terms “polypeptide,” “protein,” and “peptide” are used interchangeably and refer 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.

[0087] Methods for detection and / or measurement of polypeptides in biological material are well known in 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.

[0088] Methods for detection and / or measurement of RNA in biological material are well known in the art and include, but are not limited to, Northern-blotting, RNA protection assay, RT PCR. Suitablemethods 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.

[0089] A ribonucleoprotein (RNP) refers to a nucleoprotein that contains RNA. A RNP can be a complex 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).

[0090] 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 be components of hnRNPs, which can contain heterogeneous nuclear RNA (hnRNA) (e.g., pre-mRNA and nuclear RNA complexes) of various sizes.

[0091] Splicing complex components function in splicing and / or splicing regulation. Splicing complex components 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 / U2AFl-RS2, SF1 / BBP, CBP80, CBP 20, SF1 and PTB / hnRNPl. hnRNP proteins in humans include, but are not limited to, Al, A2 / B1, L, M, K, U, F, H, G, R, I and C1 / C2. Human genes encoding hnRNPs include HNRNP A0, HNRNP Al, HNRNP Al LI, HNRNPA1L2, HNRNP A3, HNRNP A2B1, HNRNP AB, HNRNPB1, HNRNPC, HNRNPCL1, HNRNPD, HNRPDL, HNRNPF, HNRNPH1, HNRNPH2, HNRNPH3, HNRNP K, HNRNPL, HNRPLL, HNRNPM, HNRNP R, HNRNP U, HNRNP ULI, HNRNP UL2, HNRNPUL3, and FMRI. Splicing complex components may be stably or transiently associated with a snRNP or with a transcript.

[0092] The term “intron” refers to both the DNA sequence within a gene and the corresponding sequence 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).

[0093] An “exon” can be any part of a gene that encodes a part of the final mature RNA produced by that 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.

[0094] A “spliceosome” can be assembled from snRNAs and protein complexes. The spliceosome can remove introns from a transcribed pre-mRNA.

[0095] The term “AUC” as used herein refers to an abbreviation for “area under the curve” in a graph of the 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.

[0096] The term “cryptic exon” can refer to an intronic sequence that may be flanked by apparent consensus 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.

[0097] A splicing event that promotes inclusion of a poison exon can further promote inclusion of an intron 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)

[0098] Described herein are compounds modifying splicing of gene products for use in the treatment, prevention and / or delay of progression of diseases or conditions.

[0099] In one aspect, described herein is a compound that has the structure ofpharmaceutically acceptable salt thereof.

[0100] In one aspect, described herein is a compound that has the structure of structure B, or a pharmaceutically acceptable salt or stereoisomer thereof:Structure B.

[0101] In another aspect, described herein is the compound 6-(6-((( l / ?.2 / ?.3. S'.5. S')-2-Fluoro-9-azabicyclo [3.3.1 ]nonan-3 -yl)(methyl)amino)pyridazine-3 -yl)-2 -methylbenzo [o oxazol-5 -ol.

[0102] In some embodiments, a stereoisomer of Structure B is a compound of Structure A

[0103] In one aspect, described herein is the compound 6-(6-(((lS,2S,3A,5A)-2-fluoro-9-azabicyclo [3.3.1 ]nonan-3 -yl)(methyl)amino)pyridazin-3 -y 1 )-2-methy 1 benzo[ d\ oxazol-5 -ol.

[0104] The absolute stereochemistry for Structure A and Structure B is not identified, but relative stereochemistry is known and indicated.

[0105] In one aspect, disclosed herein is a method of modulating splicing comprising contacting a compound 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.

[0106] In one aspect, disclosed herein is a method of treating a disease or condition comprising administering a compound of the present disclosure.

[0107] The compound described herein may be formed as, and / or used as, a pharmaceutically acceptable salt. 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, benzene sulfonic acid, toluenesulfonic acid, 2-naphthalenesulfonic acid, 4-methylbicyclo-[2.2.2]oct-2-ene-l-carboxylic acid, glucoheptonic acid, 4,4’-methylenebis-(3-hydroxy-2-ene-l-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.

[0108] 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.

[0109] In some embodiments, the compound and methods of use described herein can modulate splicing, 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 isnot 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).

[0110] Described herein is a compound modifying splicing of gene products wherein the compound induces 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 stop 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

[0111] In some embodiments, the compounds described herein are formulated into pharmaceutical compositions. 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.

[0112] In some embodiments, disclosed herein is a pharmaceutical composition comprising a compound of 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

[0113] The present disclosure contemplates use of small molecules with favorable drug properties that modulate 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, theSMSMs 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-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.

[0114] Modulation of splicing by SMSMs described herein can include, but is not limited to, modulation of 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.

[0115] In some embodiments, the splice site sequence can comprise a native splice site sequence. In some 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 cancomprise 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).

[0116] Described herein are compounds for modifying splicing of gene products, such as a pre-mRNA of 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.

[0117] In some embodiments, 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 SMSM to a subject in need thereof, wherein the SMSM modulates splicing of an HTT pre-mRNA or a PMS1 pre-mRNA, or both. In some embodiments, 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.

[0118] 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 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 PMS 1 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.

[0119] 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 PMS 1 pre-mRNA with a splicesite sequence or cells comprising the HTT pre-mRNA or the PMS 1 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 PMS 1 pre-mRNA in a cell of a subject to produce a spliced product of the HTT pre-mRNA or the PMS1 pre-mRNA.

[0120] 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 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.

[0121] In some embodiments, the spliced product of the HTT pre-mRNA or the spliced product of the PMS1 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 PMS 1 pre-mRNA is increased compared to a spliced product of the PMS 1 pre-mRNA produced in the absence of the SMSM.

[0122] 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 PMS 1 pre-mRNA in a cell of a subject to produce a spliced product of the PMS 1 pre-mRNA.

[0123] 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 PMS 1 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).

[0124] 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 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 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).

[0125] 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 associated with PMS 1 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).

[0126] 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 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 toa 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).

[0127] The exemplary RNA structures of a splice site, showing base-pairing between 5’ splice site sequence 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 U 1 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.

[0128] In some embodiments, the modulating splicing can comprise modulating alternative splicing. In some embodiments, the modulating splicing can comprise promoting exon skipping. In some embodiments, the modulating splicing can comprise promoting exon inclusion. In some embodiments, 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.

[0129] In some embodiments, an SMSM can modulate cryptic exon inclusion. In some embodiments, an SMSM 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.

[0130] In some embodiments, the SMSM can induce splicing at the alternative splice site. In some embodiments, 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, blockingsplicing 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.

[0131] In some embodiments, promoting the splicing of the HTT pre-mRNA at an alternative splice site or 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.

[0132] In some embodiments, promoting the splicing of the PMS1 pre-mRNA at an alternative splice site or a cryptic splice site can promote NMD of the spliced product of the PMS 1 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.

[0133] In some embodiments, the splicing of a pre-mRNA at an alternative splice site or a cryptic splice site 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 immediatelydownstream 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.

[0134] In some embodiments, the cells can comprise primary cells. In some embodiments, the cells can comprise 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 can comprise a wildtype protein. In some embodiments, the protein encoded by the spiced product of the pre-mRNA can comprise a mutant protein.

[0135] In some embodiments, provided herein is a method of downregulating expression of a native protein 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).

[0136] 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.

[0137] In some aspects, a method can comprise contacting a SMSM to a pre-mRNA that modulates splicing 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. For example, a SMSM described herein can decrease expression one or more isoforms of a transcript that promotes cell proliferation.

[0138] In some embodiments, the invention provides compositions and methods for decreasing production 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-fimctional 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-fimctional 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.

[0139] In some embodiments, administering of the compounds described herein to a subject reduces HTT 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 byat 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 acceptable 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 someembodiments, 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. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered once a day. In some embodiments, the Compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof, is administered once a day for a minimum of 12 months.

[0140] In some embodiments, the total amount of an mRNA encoding a protein (e.g., a target protein) or functional 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 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.

[0141] In some embodiments, the total amount of an mRNA encoding a protein (e.g., a target protein) or functional 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.

[0142] In some embodiments, the amount of an HTT mRNA, PMS1 mRNA, or both HTT mRNA and PMS 1 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%, 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.

[0143] In some embodiments, the amount of an HTT protein, PMS 1 protein, or both HTT and PMS 1 protein 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.

[0144] 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.

[0145] In some embodiments, provided herein are methods of treating a disease or condition in a subject in 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.

[0146] In some embodiments, the splice modulating compounds and methods of use described herein can 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, 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.

[0147] In some embodiments, SMSMs and methods of use described herein can modulate splicing of HTT 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.

[0148] In some embodiments, a SMSM described herein can promote splicing of an HTT pre-mRNA at an alternative splice site sequence or a cryptic splice site sequence in intron 49, resulting in inclusion ofpoison 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 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.

[0149] In some embodiments, the splice modulating compounds and methods of use described herein can 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 PMS 1 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 PMS 1 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 PMS 1 spliced product is shown in FIG.6A.

[0150] In some embodiments, SMSMs and methods of use described herein can modulate splicing of PMS 1 pre-mRNA by promoting splicing of an PMS 1 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 ina spliced product of the PMS 1 pre-mRNA can lead to destabilization of the PMS 1 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 / hgl9: 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 / hgl9: chr2: 189818824 to GRCh38 / hgl9: chr2: 189818841.

[0151] In some embodiments, a PMS 1 may be a wild-type PMS 1. In some embodiments, a PMS 1 gene may comprise a genetic variation. Non-limiting examples of PMS 1 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, 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 hgl9. In some embodiments, an allele comprising a genetic variation in a PMS 1 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.

[0152] In some embodiments, a genetic variation may disrupt or modulate the PMS1 gene. In this embodiment, 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 hgl9. 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).

[0153] In some embodiments, a SMSM described herein can promote splicing of a PMS 1 pre-mRNA at an 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 PMS 1 mRNA lacking poison exon 5b produced or processed from PMS 1 pre-mRNA is reduced in the cell or in the cells contacted by a SMSM described herein.

[0154] In some embodiments, the amount of the spliced product of the PMS 1 pre-mRNA comprising exon 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%, 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.

[0155] In some embodiments, SMSMs and methods of use described herein can modulate splicing of both 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 PMS 1 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 PMS 1 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 least97%, 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.

[0156] In some embodiments, a SMSM can modulate splicing at a splice site sequence of a polynucleotide 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.

[0157] 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.Table 20A. Exemplary Targets for Exon InclusionTable 20B. PMS1 Variation* Premature stop codon loss-of-function variant

[0158] In some embodiments, a SMSM can modulate splicing at a splice site of a polynucleotide and does not exhibit significant toxicity. In some embodiments, an SMSM penetrates the blood brain barrier (BBB) when administered to a subject.

[0159] In some embodiments, following administration of a single dose of an SMSM to a subject under fasting conditions the Tmaxfor 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 fasting 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, fasting conditions may be characterized by the levels of nutrient in the blood of the subject.

[0160] In some embodiments, following administration of a single dose of an SMSM to a subject under fed conditions the Tmaxfor 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.

[0161] In some embodiments, following administration of a single dose of an SMSM to a subject 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 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.

[0162] In some embodiments, following administration of a single dose of an SMSM to a subject under fasting conditions the Cmaxfor 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 about1400 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.

[0163] In some embodiments, following administration of a single dose of an SMSM to a subject under fasting conditions the Cmaxfor 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, fasting conditions may be characterized by the levels of nutrient in the blood of the subject.

[0164] In some embodiments, following administration of a single dose of an SMSM to a subject under fed conditions the Cmaxfor 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, about600 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.

[0165] In some embodiments, following administration of a single dose of an SMSM to a subject under fed 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.

[0166] In some embodiments, following administration of a single dose of an SMSM to a subject 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, 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, about500 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.

[0167] In some embodiments, following administration of a single dose of an SMSM to a subject 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.

[0168] In some embodiments, a SMSM has a cell viability IC50 of 0.01-10 nM, 0.01-5 nM, 0.01-2.5 nM, 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.

[0169] In some embodiments, a SMSM has a cell viability IC50 of at most 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, 66nM, 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 pM, or 10 pM.

[0170] 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, 25-250 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.

[0171] 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 pM, or 10 pM 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.

[0172] In some embodiments, a SMSM reduces viability or increases apoptosis of diseased cells by more 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.

[0173] In some embodiments, a SMSM reduces viability or increases apoptosis of diseased cells by more 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, 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 pM, or 10 pM 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.

[0174] In some embodiments, a SMSM does not reduce viability or does not increase apoptosis of nondiseased 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.

[0175] In some embodiments, a SMSM does not reduce viability or does not increase apoptosis of nondiseased 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, 22nM, 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 pM, or 10 pM 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.

[0176] In some embodiments, a SMSM can reduce the amount of a canonical HTT mRNA (e.g., an 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 PMS 1 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 the amount of a canonical HTT mRNA (e.g., an 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.

[0177] In some embodiments, a SMSM can reduce the amount of a canonical HTT mRNA (e.g., an 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%, atleast 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., an HTT 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.

[0178] The compositions and methods described herein can be used for treating a human disease or disorder 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.

[0179] Provided herein are methods of treating a disease or condition in a subject in need thereof. The method 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.

[0180] In some embodiments, an effective amount in the context of the administration of a SMSM or a pharmaceutically 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) reduceshospitalization 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.

[0181] In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof 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 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 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.

[0182] In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof 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.

[0183] 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 (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 a 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.

[0184] In some embodiments, an effective amount of a SMSM or a pharmaceutically acceptable salt thereof 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.

[0185] Non-limiting examples of effective amounts of a SMSM or a pharmaceutically acceptable salt thereof 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.

[0186] A method of treating a disease or a condition in a subject in need thereof can comprise administering 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.

[0187] In some embodiments, a SMSM described herein can be used in the preparation of medicaments for 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.

[0188] In certain embodiments, a SMSM described herein can be administered for prophylactic and / or therapeutic 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 at 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.

[0189] In some embodiments, methods described herein can further comprise a genetic testing of a biological sample of a subject in need thereof. For example, methods can further comprise subjecting a biological 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 PMSlhlgh. 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 PMSlhlgh. 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

[0190] In some embodiments, the present disclosure relates to a pharmaceutical composition comprising a 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 neuronsand 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.

[0191] 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 motor 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.

[0192] In some embodiments, a subject with HD can benefit from lowering HTT protein level. In some embodiments, a subject with HD that has rs 13102260 single nucleotide polymorphism (SNP) may have lower HTT protein level. For example, a subject with HD that has rs 13102260 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 rsl3102260 SNP can have delayed disease onset by 5.6 years.

[0193] In some embodiments, SMSMs described herein can modulate splicing of a pre-mRNA comprising 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 an HTT protein encoded by a spliced product of the HTTpre-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.

[0194] In some embodiments, the aberrant expansion of CAG repeats in the HTT gene may be associated 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 MutSB 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, PMS 1 can 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 PMS 1 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 PMS 1 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 PMS lhlgh, or may express a variant PMS 1 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 PMSlhlgh.

[0195] In some embodiments, a subject may be suffering from one or more herein mentioned diseases or conditions, 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.

[0196] In some embodiments, SMSMs described herein can modulate splicing of a pre-mRNA that encodes 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 PMS 1 pre-mRNA can lead to a decrease in the expression level of a PMS 1 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 HDcells. 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.

[0197] In some embodiments, SMSMs described herein can modulate splicing of an HTT pre-mRNA and 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 mutant HTT (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. In some embodiments, the amount of the mHTT protein or 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 a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 15 days, at least 28 days, at least 56 days, or at least 84 days. In some embodiments, the amount of the mHTT protein is reduced by at least 17% in a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 15 days. In some embodiments, the amount of the mHTT protein is reduced by at least 39% in a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 28 days. In some embodiments, the amount of the mHTT protein is reduced by at least 51% in a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 56 days. In some embodiments, the amount of the mHTT protein is reduced by at least 50% in a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 84 days. In some embodiments, the amount of the mHTT protein is reduced by at least 62% in a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 84 days.

[0198] In some embodiments, the amount of the mHTT protein is reduced by at least 30% in a blood sample of a subject administered with an SMSM described herein (e.g., the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof) for a treatment period of at least 3 months. In some embodiments, the amount of the mHTT protein is reduced by at least 40% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 3 months. In some embodiments, the amount of the mHTT protein is reduced by at least 50% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 3 months. In some embodiments, the amount of the mHTT protein is reduced by at least 60% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 3 months. In some embodiments, the amount of the mHTT protein is reduced by at least 70% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 3months. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered daily. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 3 mg. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 4 mg. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 6 mg. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 9 mg.

[0199] In some embodiments, the amount of the mHTT protein is reduced by at least 5% in a blood sample of a subject administered with an SMSM described herein (e.g., the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof) for a treatment period of 1 month. In some embodiments, the amount of the mHTT protein is reduced by at least 10% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 1 month. In some embodiments, the amount of the mHTT protein is reduced by at least 20% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 1 month. In some embodiments, the amount of the mHTT protein is reduced by at least 30% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 1 month. In some embodiments, the amount of the mHTT protein is reduced by at least 40% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 1 month. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered daily. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 3 mg. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 9 mg.

[0200] In some embodiments, the amount of a canonical isoform of an HTT mRNA, a canonical isoform of a 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 a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 15 days, at least 28 days, at least 56 days, or at least 84 days. In some embodiments, the amount of the canonical isoform of the HTT mRNA is reduced by at least 23% in a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 15 days. In some embodiments the amount of the canonical isoform of the HTT mRNA is reduced by at least 50% in a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 28 days. In some embodiments, the amount of the canonical isoform of the HTT mRNA is reduced by at least 62% in a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 28 days. In some embodiments, the amount of the canonical isoform ofthe PMS1 mRNA is reduced by at least 20% in a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 28 days. In some embodiments, the amount of the canonical isoform of the PMS1 mRNA is reduced by at least 26% in a blood sample of a subject administered with an SMSM described herein for a treatment period of at least 28 days.

[0201] In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS1 mRNA, or both is reduced by at least 10% in a blood sample of a subject administered with an SMSM described herein (e.g., the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof) for a treatment period of at least 3 months. In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS 1 mRNA, or both is reduced by at least 20% in a blood sample of a subject administered with an SMSM described herein (e.g., the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof) for a treatment period of at least 3 months. In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS 1 mRNA, or both is reduced by at least 30% in a blood sample of a subject administered with an SMSM described herein (e.g., the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof) for a treatment period of at least 3 months. In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS1 mRNA, or both is reduced by at least 40% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 3 months. In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS 1 mRNA, or both is reduced by at least 50% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 3 months. In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS 1 mRNA, or both is reduced by at least 60% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 3 months. In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS1 mRNA, or both is reduced by at least 70% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 3 months. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered daily. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 3 mg. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 4 mg. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 6 mg. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 9 mg.

[0202] In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS 1 mRNA, or both is reduced by at least 5% in a blood sample of a subjectadministered with an SMSM described herein (e.g, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof) for a treatment period of 1 month. In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS 1 mRNA, or both is reduced by at least 10% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 1 month. In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS1 mRNA, or both is reduced by at least 20% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 1 month. In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS1 mRNA, or both is reduced by at least 30% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 1 month. In some embodiments, the amount of the canonical isoform of the HTT mRNA, the canonical isoform of the PMS 1 mRNA, or both is reduced by at least 40% in a blood sample of a subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of 1 month. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered daily. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 3 mg. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 4 mg. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 6 mg. In some embodiments, the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered at a daily dose of 9 mg.Dosing and Schedules

[0203] The SMSMs utilized in the methods of the disclosure can be, e.g., administered at dosages that may 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.

[0204] In some embodiments, the SMSM described herein can be administered to the subject at from about 1 mg to about 16 mg per dose. In some embodiments, the SMSM described herein can be administered to the subject at from about 3 mg to about 9 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. In some embodiments, the subject isadministered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof, in an amount 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 day.

[0205] 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. In some embodiments, the SMSM described herein can be administered to the subject at a dose range of from 1 mg to about 16 mg once a day. In some embodiments, the SMSM described herein can be administered to the subject at a dose range of from 3 mg to about 9 mg once a day. In some embodiments, the SMSM described herein can be administered to the subject at a dose range of from 1 mg to about 16 mg once a day for at least 1 week, at least 2 weeks, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 weeks, at least 10 weeks, at least 11 weeks, at least 12 weeks, at least 13 weeks, at least 14 weeks, at least 15 weeks, at least 16 weeks, at least 17 weeks, at least 18 weeks, at least 19 weeks, at least 20 weeks, at least 21 weeks, at least 22 weeks, at least 23 weeks, at least 24 weeks, at least 25 weeks, at least 26 weeks, at least 27 weeks, at least 28 weeks, at least 29 weeks, at least 30 weeks, at least 31 weeks, at least 32 weeks, at least 33 weeks, at least 34 weeks, at least 35 weeks, at least 36 weeks, at least 37 weeks, at least 38 weeks, at least 39 weeks, at least 40 weeks, or more than 40 weeks. In some embodiments, the SMSM described herein can be administered to the subject at a dose range of from 1 mg to about 16 mg once a day for at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, or more than 24 months. In some embodiments, the SMSM described herein can be administered to the subject at a dose range of from 1 mg to about 16 mg once a day for at least 12 months. In some embodiments, the SMSM described herein can be administered to the subject at a dose range of from 3 mg to about 9 mg once a day for at least 12 months. In some embodiments, the SMSM described herein can be administered to the subject at a dose range of from 1 mg to about 16 mg once a day for at least 28 days, 56 days, or 84 days. In some embodiments, the SMSM described herein can be administered to the subject at a dose range of from 1 mg to about 16 mg once a day for at least 12 months.

[0206] In some embodiments, a subject does not have a drug holiday.

[0207] In some embodiments, a subject may take a drug holiday or dosing holiday (e.g., a break from taking any SMSM described herein). In some embodiments, a subject can take a drug holiday or dosing holiday of at least 1 day, at least 2 days, at least 3 days, 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 days, at least 9 days, at least 10 days, at least 11 days, at least 12 days, at least 13 days, at least 14 days, at least 15 days, at least 16 days, at least 17 days, at least 18 days, at least 19 days, at least 20 days, at least 21 days, at least 22 days, at least 23 days, at least 24 days, at least 25 days, at least 26 days, at least 27 days, at least 28 days, or more than 28 days. In some embodiments, a subject can take adrug holiday or dosing holiday of 1 to 4 weeks. In some embodiments, a subject can take a drug holiday or dosing holiday of 2 to 3 weeks. In some embodiments, a subject can take a drug holiday or dosing holiday of 4 to 8 weeks. For example, the SMSM described herein can be administered to the subject at a dose range of 1 mg to 16 mg for a period of time and the subject can take a drug holiday or dosing holiday for another period of time. In some embodiments, the subject can be administered with an SMSM described at a dose of 9 mg for 28 days and take a drug holiday or dosing holiday for 14 days. In some embodiments, the drug holiday is taken by the subject after 1-8 weeks of continuous treatment. In some embodiments, the drug holiday is taken by the subject after 3-5 weeks of continuous treatment. In some embodiments, the drug holiday is taken by the subject after 8-20 weeks of continuous treatment.

[0208] In some embodiments, a method described herein comprises administering a SMSM (e.g., compound of Structure B) to a subject in one or more cycles. In some embodiments, the one or more cycles can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 cycles. In some embodiments, the one or more cycles can comprise more than 20 cycles. In some embodiments, the one or more cycles can comprise about 2-100 cycles. In some embodiments, each of the one or more cycles comprises a treatment period and a drug holiday period. In some embodiments, the treatment period is about 1 to 52 weeks. In some embodiments, the treatment period is about 1 to 16 weeks. In some embodiments, the treatment period is 1 to 12 weeks. In some embodiments, the treatment period is 2 to 8 weeks. In some embodiments, the treatment period is about 2 to 6 weeks. In some embodiments, the treatment period is about 4 weeks. In some embodiments, the treatment period is about 8 weeks. In some embodiments, the treatment period is about 12 weeks. In some embodiments, the treatment period is about 16 weeks. In some embodiments, the treatment period is about 20 weeks. In some embodiments, the treatment period is about 52 weeks. In some embodiments, the treatment period is about 28 days. In some embodiments, the treatment period is about 56 days. In some embodiments, the treatment period is about 84 days. In some embodiments, the treatment period is about 12 months or more than 12 months. In some embodiments, the one or more cycles can comprise at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or at least 20 cycles, wherein each cycle comprises a treatment period of about 28 days.

[0209] In some embodiments, the drug holiday period is 1 to 6 weeks. In some embodiments, the drug holiday period is 1 to 2 weeks. In some embodiments, the drug holiday period is about 14 days. In some embodiments, each of the cycles comprises 6 weeks. In some embodiments, each of the cycles comprises 5-7 weeks. In some embodiments, each of the cycles comprises 2-10 weeks. In some embodiments, the method comprises administering the SMSM for about 8-10 cycles. In some embodiments, the method comprises administering the SMSM for about 9 cycles.

[0210] The compositions of the SMSM described herein can be administered as a solid dosage form. In some 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 stereoisomerthereof, 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.

[0211] In some embodiments, a compound of Structure B, or a pharmaceutically acceptable salt or a stereoisomer 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

[0212] The subjects that can be treated with the SMSMs and methods described herein can be any subject 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 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.

[0213] In some embodiments, a subject may have HD. In some embodiments, a subject has early-stage HD. 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 mHTThlgh. 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 mHTThlgh. 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 mHTThlgh. In some embodiments, a subject can comprise a subject that has been genetically confirmed to have HD. For example, a subject can comprise a subject that has been genetically confirmed to have CAG repeat length of at least 40, or greater than or equal to 40 in HTT gene by DNA sequencing. In some embodiments, a subject disclosed herein is a male or female aged 25-70 (inclusive) with genetically confirmed HD (cytosine, adenine, and guanine [CAG]) repeat length of at least 40, or greater than or equal to 40 in HTT gene by direct DNA testing and Huntington’s Disease Integrated Staging System [HD-ISS] Stage 2 or 3 mild). 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 PMSlhlgh. 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 PMSlhlgh. In some embodiments, asubject 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 PMSlhlgh. 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.

[0214] In some embodiments, a subject’s genome can encode a PMS1. In some embodiments, a subject’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.

[0215] In some embodiments, a genetic variation can comprise a non-synonymous coding variant. In some 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 hgl9. 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.

[0216] In some embodiments, a genetic variation may disrupt or modulate the PMS1 gene. In this embodiment, 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 hgl9. 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).

[0217] In some embodiments, a subject disclosed herein is between the ages of 30 and 50. In some embodiments, a subject disclosed herein is 30 years of age or older. In some embodiments, a subject disclosed herein is between the ages of 25 and 70.

[0218] In some embodiments, a subject disclosed herein has symptoms that are characterized by the progressive 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 someembodiments, a subject disclosed herein exhibits complications such as pneumonia and heart failure. In some embodiments, a subject can have Total Motor Score (TMS) score of 6 or higher. In some embodiments, a subject can have independence scale (IS) score of 70 or higher. In some embodiments, a subject can have Total Functional Capacity (TFC) score of 10 or higher.

[0219] In some embodiments, a subject disclosed herein has HD-ISS Stage 0. In some embodiments, a subject 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

[0220] The compound of structure B, 6-(6-(((lR,2R,3S,55)-2-Fluoro-9-azabicyclo[3.3. l]nonan-3-yl)(methyl)amino)pyridazine-3-yl)-2-methylbenzo[<7]oxazol-5-ol, can be made by the techniques and processes known in the art, for example as described in Example 1.EXAMPLES

[0221] These examples are provided for illustrative purposes only and not to limit the scope of the claims 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, Acres 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.

[0222] Suitable reference books and treatise that detail the synthesis of reactants useful in the preparation 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, “Modem 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 Chemistrv, An Intermediate Text” (1996) Oxford UniversityPress, 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) “Modem 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.

[0223] In the reactions described, it may be necessary to protect reactive functional groups, for example hydroxy, 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).

[0224] Examples can be made using known techniques and further chemically modified, in some embodiments, 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:

[0225] (±) 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 is based on known stereochemistry of similar compounds and or reactions and the product is a racemic mixture of enantiomers of both (1S,2S,3A,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.

[0226] Example 1: Synthesis of 6-(6-(((15, 25,37?, 57?)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[< / ]oxazol-5-ol (Structure A) and 6-(6-(((17?,27?,35,55)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo [< Z]oxazol-5-ol (Structure B).

[0227] Synthesis of 2-amino-4-methoxyphenol.

[0228] 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 20 °C 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 [M+H]+; tR= 0.93 min.

[0229] Synthesis of 5-methoxy-2-methylbenzo[r / | oxazole.

[0230] A mixture of 2-amino-4-methoxyphenol (20.0 g, 0.14 mol) in trimethyl orthoacetate (50 mL) was heated to 100 °C 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-mcthoxy-2-mcthylbcnzo|t / |oxazolc as orange oil (17.5 g, yield 74.6 %). LCMS: m / z 164.1 [M+H]+; tR= 1.41 min.

[0231] Synthesis of 6-bromo-5-methoxy-2-methylbenzo[< / ]oxazole.

[0232] NBS (19.6 g, 0.11 mol) was added to a mixture of 5-mcthoxy-2-mcthylbcnzo|t / |oxazolc (17.5 g, 0.11 mol) in AcOH (150 mL). This resulting mixture was stirred at 20 °C for 18 h. The mixture was quenched with ice water, neutralized with Na2COs aqueous, 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 [<7] oxazole as pink solid (20.5 g, yield 77.0 %). LCMS: m / z 242.1; 243.9 [M+H]+; tR= 1.70 min. ’HNMR(500 MHz, CDCWs) 57.68 (s, 1H), 7.17 (s, 1H), 3.93 (s, 3H), 2.61 (s, 3H).

[0233] Synthesis of 6-bromo-2-methylbenzo[< / ]oxazol-5-ol.

[0234] BBr, (210 mL, 1 mol / 1, 0.21 mol) was added to a mixture of 6-bromo-5-methoxy-2-mcthylbcnzo|t / | oxazole (20.5 g, 0.085 mol) in DCM (30 mL) at 0 °C. This resulting mixture was stirred at 0 °C for 10 min and then warmed to 20 °C with stirring and held for 3 d. The mixture was quenched with ice water, neutralized with NaHCOs aqueous, 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[<7]oxazol-5-ol as grey solid (19.0 g, yield 98.6 %). LCMS: m / z 228.0; 230.0 [M+H]+; A = 1.50 min.

[0235] Synthesis of 6-bromo-5-(methoxymethoxy)-2-methylbenzo[< / | oxazole.

[0236] MOMBr (15.6 g, 0.12 mol) was added drop wise to a mixture of 6-bromo-2-methylbenzo[<7|oxazol-5-ol (19.0 g, 0.08 mol) and DIPEA (37.7 g, 0.19 mol) in ACN (300 mL) at 5 °C. This resulting mixture was stirred at 5 °C 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 product 6-bromo-5-(mcthoxymcthoxy)-2-mcthylbcnzo|t / |oxazolc as pink solid (18.0 g, yield 79.5 %). LCMS: m / z 272.0; 274.0 [M+H]+; tR= 1.77 min.

[0237] Synthesis of 5-(methoxymethoxy)-2-methyl-6-(4, 4, 5, 5-tetramethyl-l, 3, 2-dioxaborolan-2-yl)benzo | z / | oxazole.

[0238] A mixture of 6-bromo-5-(methoxymethoxy)-2-methylbenzo[aQoxazole (50 g, 0.02 mol), pinaolboron (28.0 g, 0.11 mol), PdCLdppf (1.1 g, 1.5 mmol)and KOAc (10.8 g, 0.11 mol) in 1, 4-dioxane (300 mL) was heated to 100 °C 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-l, 3, 2-dioxaborolan-2-yl)bcnzo|t / |oxazolc as pink solid (4.0 g, yield 68.2 %). LCMS: m / z 320.2 [M+H]+; tR= 1.86 min.

[0239] Synthesis of tert-butyl (liS,2iS,57?)-2-fluoro-3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylate.

[0240] LHMDS (94 mL, 94 mmol, 1 N solution in THF) was added to a stirred solution of / c77-biityl 3-oxo-9-azabicyclo[3.3.1]nonane-9-carboxylate (15 g, 62.8 mmol) in 150 mL of anhydrous THF at -78 °C 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 -78 °C for 4 h, quenchend with saturated NH4CI aqueous solution (30 mL), estracted with EtOAc (80 mL X 3). The combined organic phases were dried over anhydrous Na2SC>4, conentrated and purified by silica gel chromatography (0-5% EtOAc / petroleum ether) to give 6.5 g of tert-butyl ( lS,2S,5A)-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.

[0241] Synthesis of tert-butyl (liS,2iS,57?)-2-fluoro-3-(methylimino)-9-azabicyclo[3.3.1]nonane-9-carboxylate.

[0242] Methylamine (58.5 mL, 117 mmol, 2N solution in THF) and Ti(zPrO)4 (32.8 g, 117 mmol) were added to a stirred solution of tert-butyl ( I. S'.2. S'.5 / ?)-2-fluoro-3-oxo-9-azabicyclo|3.3.1 ]nonane-9-carboxylate (20 g, 77 mmol) in THF (I L) under N2 protection. The reaction mixture was stirred at room temperature for 2 h. Water (I L) was added to quench the reaction. The mixture was extracted with EtOAc (IL X 3). The combined organic phases were washed with brine, dried over anhydrous Na2SO4 and concentrated to give the crude product ( I. S'.2. S'.5 / ?)-2-fluoro-3-(mcthylimino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (20 g, 95% yield), which was directly used in next step. LCMS: m / z Tl\2 [M+H]+; tR= 1.58, 1.80 min.

[0243] Synthesis of tert-butyl (liS,27?,37?,57?)-2-fluoro-3-(methylamino)-9-azabicyclo[3.3.1]nonane-9-carboxylate.

[0244] NaBH4 (4 g, 104 mmol) was added to a stirred solution of ( I. S'.2. S'.5 / ?)-2-fluoro-3 -(meth l imino)-9-azabicyclo[3.3.1]nonane-9-carboxylate (7 g, 26 mmol) and MgCL (2.46 g, 26 mmol) in 30 mb 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 ISfeSCL, concentrated and purified by silica gel chromatography (0-5% McOH / CTLCL) give 2 g of tert-butyl (I. S'.2 / ?.3 / ?.5 / ?)-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.

[0245] Synthesis of tert-butyl (liS,27?,37?,57?)-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate.

[0246] A mixture of tert-butyl (lS,2A,3A,5A)-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 120 °C 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 ( I. S'.2 / ?.3 / ?.5 / ?)-3-((6-chloropyridazin-3-yl)(mcthyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate white solid (54% yield). LCMS: m / z 385.2 [M+H]+; R = 1.93 min.

[0247] Chiral separation of tert-butyl (liS,27?,37?,57?)-3-((6-chloropyridazin-3-yl)(methyl)amino)-2-fluoro-9-azabicyclo[3.3.1]nonane-9-carboxylate.

[0248] 1500 mg of racemic intermediate was separated by below chial condition to give 630 mg of Pl isomer (1.596 min) and 630 mg of P2 isomer (4.811 min).

[0249] Instrument: SFC-150 (Waters)Column: AD 20*250mm, lOum (Daicel)Column temperature: 35 °CMobile phase: CO2 / MEOH(0.2%Methanol Ammonia) = 65 / 35Flow rate: lOOg / minBackpressure: 100 barDetection wavelength: 214 nmCycle time: 3.5minSample solution: 1500mg dissolved in 100ml MethanolInjection volume: 3ml

[0250] Synthesis of tert-butyl (LS',2 / ?,3 / ?,5 / ?)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-m ethylbenzo [< / ] oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9- carboxylate.

[0251] A mixture of tert-butyl ( I. S'.2 / ?.3 / ?.5 / ?)-3-((6-chloropyridazin-3-yl)(mcthyl)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[aQoxazole (560 mg, 1.76 mmol), Pd(dppf)C12 (86 mg, 0.117 mmol) and K2CO3 (324 mg, 2.34 mmol) in 1,4-Dioxane (15 mb), water (5 ml) was stirred at 110°C for 2 h under N2 atmosphere. After cooling to room temperature, the mixture was concentrated and purified by silica gel chromatography (0-50% EtOAc / petroleum ether) to give 550 mg of tert-butyl ( \S.2R. R,5 / ?)-2-fl uoro-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]+; = 1.98 min.

[0252] Synthesis of 6-(6-(((15, 25,37?, 57?)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[< / ]oxazol-5-ol.

[0253] To a solution of tert-butyl ( I. S'.2 / ?.3 / ?.5 / ?)-2-fluoro-3-((6-(5-(mcthoxymcthoxy)-2-methylbenzo [d] oxazol-6-yl)pyridazin-3 -yl)(methyl)amino)-9-azabicyclo [3.3.1 ]nonane-9-carboxylate(550 mg, 1.02 mmol) in CH2Q2 (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-(((lS,2S,37?,57?)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[<7]oxazol-5-ol (41% yield). 'H NMR (400 MHz, MeOD -< A) 58.17 (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]+; fe = 1.40 min.

[0254] Synthesis of tert-butyl (17?, 2S,3S, 5, S’)-2-fluoro-3-((6-(5-(methoxymethoxy)-2-m ethylbenzo [< / ] oxazol-6-yl)pyridazin-3-yl)(methyl)amino)-9-azabicyclo[3.3.1]nonane-9-carboxylate.

[0255] A mixture of tert-butyl ( l / ?.2. S'.3. S'.5. S')-3-((6-chloropyridazin-3-yl)(mcthyl)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[aQoxazole (560 mg, 1.76 mmol), Pd(dppf)C12 (86 mg, 0.117 mmol) and K2CO3 (324 mg, 2.34 mmol) in 1,4-Dioxane (15 mL), water (5 ml) was stirred at 110 °C for 2 h under N2 atmosphere. After cooling to room temperature, the mixture was concentrated and purified by silica gel chromatography (0-50% EtOAc / petroleum ether) to give 500 mg of tert-butyl ( l / ?.2. S'.3. S'.5. S')-2-fluoro-3-((6-(5-(mcthoxymcthoxy)-2-mcthylbcnzo|t / |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]+; = 1.98 min.

[0256] Synthesis of 6-(6-(((17?,27?,3iS,5A)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[< / ]oxazol-5-ol.Structure B

[0257] To a solution of tert-butyl (l / .2. S'.3. S'.5. S)-2-fluoro-3-((6-(5-(mcthoxymcthoxy)-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 CH2Q2 (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) togive 133 mg of 6-(6-(((lR,2R,3S,5S)-2-fluoro-9-azabicyclo[3.3.1]nonan-3-yl)(methyl)amino)pyridazin-3-yl)-2-methylbenzo[<7]oxazol-5-ol (36% yield). 'H NMR (400 MHz, MeOD -d ) 58.17 (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]+; fe = 1.40 min.

[0258] Example 2: Metabolite ID.

[0259] Metabolite identification was performed for both 6-(6-{[(lR,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 (liverPool™ 10-Doner) from BioIVT (cat. No. X008001) for Compound 117 and (liverPool™ 20-Doner) from BioIVT (cat. No. X008000) for Structure B at 1.0 X 106cells / ml were incubated with test compound (50pM Compound 117, lOpM Structure B) for 240 minutes at 37°C. 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 pL acetonitrile (0.1% FA) fraction was dried by centrifugal vacuum evaporator and reconstituted with 50 pL water and 50 pL methanol. The following equipment and conditions were used for the analysis:

[0260] Instrumentation: Vanquish UHPLC system (Thermo Fisher Scientific, USA); Vanquish Variable Wavelength (Thermo Fisher Scientific, USA); Thermo Scientific Q Exactive (Thermo Fisher Scientific, USA).

[0261] EC conditions: Column: Waters XSelect HSS T3, 100 x 2.1 mm, 2.5 pm; Solvents: A, water (0.1% formic acid); B, acetonitrile (0.1% formic acid); Flow rate: 500 pL / 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.

[0262] MS conditions: Ionisation mode: Positive mode; Spray Voltage: 3.5 kV; Aux gas flow rate: 15; Aux gas heater temp: 350°C; Scan type: Full MS / ddMS2; Resolution: 70,000; AGC Target: 3 x e6; NCE / stepped NCE (Full Mass) for Compound 117: 25, 35, 45; NCE / stepped NCE (Full Mass) for Structure B: 30, 35, 40.

[0263] Three metabolites were detected for Compound 117 (Table 1), seven metabolites were detected for Structure B (Table 2).Table 1.Table 2

[0264] Example 3: Protein Binding Assay.

[0265] Protein binding of Structure B was determined in human, rat and mouse plasma using an equilibrium dialysis method (using 96-well Equilibrium Dialysis Plate (HTDialysis LLC, Gales Perry, 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-I strain, mixed gender plasma (pH 7.23) was obtained from IPHASE (batch no. M21005657.

[0266] A working solution of test compound and control compound (ketoconazole) was prepared in DMSO 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 dialysisbuffer 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 pL of blank plasma solution was added into each vial of a new plastic plate or separate plastic tube by addition of 3 pL 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 pM. 50 pL 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.

[0267] At T=6 hours, 50 pL 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 pL 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% CO2 on an orbital shaker in the CO2 incubator. At the end of incubation, the lid was removed and 50 pL of post-dialysis samples from both buffer and plasma solution chambers were transferred into separated 96-well plate for analysis, respectively. 50 pL 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 pL 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 pL of the supernatant was transferred to new 96-well plates and centrifuged again (3,220 g, 30 minutes).100 pL of the supernatant was transferred to new 96-well plates for analysis. 100 pL 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) x 100; % Bound = 100 - % Unbound; % Recovery = (Area ratio buffer chamber + Area ratio plasma solution chamber) / (Area ratio Total sample) x 100; % Remaining = Area ratio 6hr / Area ratio Ohr x 100. The following chromatography conditions were used:

[0268] UC system: Shimadzu; MS analysis: Triple Quad 5500+ instrument from AB Inc with an ESI interface; Column temperature: 40°C; Injection volume: 1 pE; Column: XSelectHSS T3 2.5pm2.1 x 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.

[0269] The following MS parameters were used: Ion source: Turbo spray; Ionization model: ESI; Scan type: MRM; Collision gas: 9 L / min; Curtain gas: 40 L / min; Nebulize gas: 55 L / min; Auxiliary gas: 55 L / min; Temperature: 500°C; lonspray voltage: +5500 V. Results for Structure B are in Table 4.Table 4.

[0270] Compound 117 was subjected to a similar protocol. With some variations. The test concentration was IpM, control compounds were warfarin and quinidine. Incubation time was 5 hours. Results for Compound 117 are in Table 5.Table 5.

[0271] Example 4: PK Study in nonhuman primates of Structure B.

[0272] Cynomolgus monkeys were administered Structure B at 2 different dose levels. POA was a dose level of 2mg / kg (dosing solution of 0.4mg / ml) and POB was a dose level of lOmg / 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. l%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.

[0273] POB was prepared by dissolving 109.96 mg of Structure B in 54.980 mL of 0.5%MC,0. l%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.

[0274] 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-C18 4 pm (50 x 2.1 mm). Mobile Phase: Solution A: 5% Acetonitrile in Water (0. l%Formic acid); Solution B: 95% Acetonitrile in Water (0. l%Formic acid). Flow rate: 0.6 mL / min, with the following gradient in Table 6.Table 6.

[0275] Inj ection volume: 3.1.

[0276] The desired serial concentrations of working solutions were achieved by diluting stock solution of analyte with DMSO. 5 pL of working solutions (5, 10, 20, 50, 100, 500, 1000, 5000, 10000 ng / mL) were added to 50 pL 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 pL. 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 pL of standards, 55 pL of QC samples and 55 pL of unknown samples (50 pL of male and female plasma with 5 pL of blank solution) were added to 200 pL 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 pL of diluted supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0277] 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.

[0278] Results are shown in Table 7 for the male cynomolgus monkeys and in Table 8 for the female cynomolgus monkeys.Table 7.Table 8.

[0279] BLOQ = Below quantifiable limit of 1 ng / mL for Male; BLOQ = Below quantifiable limit of 0.5 ng / mL for Female. PK Parameters were estimated by non-compartmental model using WinNonlin 6.1. The bioavailability (F%) was calculated asfollows: AUClast-PO / AUCINF-PO > 80%: F=(AUCINF-PO* DoseIV) / (mean AUCINF-IV* DosePO)

[0280] 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 cynomolgus monkeys and Table 10 for the female cynomolgus monkeys.Table 9.Table 10.

[0281] Example 5: PK Study in nonhuman primates of Compound 117.

[0282] Female cynomolgus monkeys were administered Compound 117 at 2 different dose levels. POA was a dose level of Img / 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. l%Tween80), followed by vortexing and sonication to obtain a solution with concentration at 0.2 mg / mL of Compound 117.

[0283] 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.

[0284] 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 C18 90A 2.7pm (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

[0285] Injection volume: Ipl.

[0286] The desired serial concentrations of working solutions were achieved by diluting stock solution of analyte with 50% acetonitrile in water solution. 5 pL of working solutions (5, 10, 20, 100, 500, 1000, 5000, 10000 ng / mL) were added to 50 pL 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 pL. Four qualitycontrol 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 pL of standards, 55 pL of QC samples and 55 pL of unknown samples (50 pL of monkey plasma with 5 pL of blank solution) were added to 200 pL 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 pL of diluted supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0287] 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.

[0288] Results are shown in Table 12.Table 12.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.

[0289] The PK parameters are set forth in Table 13 as a mean for all the animals given a particular dose.Table 13.

[0290] Example 6: RAT PK following IV administration of Structure B

[0291] Female Sprague Dawley rats were administered Structure B by IV at a dose level of Img / kg (dosing solution of 0.2mg / ml). The IV dose was prepared by dissolving 1.06 mg of Structure B in 5.3 mb of 30%HP-[3-CD followed by vortexing and sonication to obtain a solution with concentration at 0.2 mg / mL of Structure B.

[0292] 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-C8 4 pm (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.

[0293] Injection volume: 5 pl.

[0294] The desired serial concentrations of working solutions were achieved by diluting stock solution of analyte with 50% acetonitrile in water solution. 5 pL of working solutions (1, 2, 4, 10, 20, 100, 200, 1000, 2000 ng / mL) were added to 50 pL 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 pL. 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 pL standards, 50 pL QC samples and 50 pL unknown samples (50 pL plasma with 5 pL blank solution) were added to 200 pL 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 pL of diluted supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0295] 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.

[0296] Results are shown in Table 15.Table 15.BLOQ = Below quantifiable limit of 0.5 ng / mL; PK parameters were estimated by non-compartmenta 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.

[0297] The PK parameters are set forth in Table 16.Table 16.

[0298] Example 7: Rat PK for Compound 117.

[0299] Male Sprauge Dawley rats were administered Compound 117 by IV at a dose level of Img / kg (dosing solution of 0.5mg / ml). The IV dose was prepared by dissolving 1.17 mg of Compound 117 in 0.117 mb of DMSO followed by vortexing for 2 minutes and sonication for 3 minutes, then 0.117 mb 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.

[0300] 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 (2.1x50 mm, 1.7 pm) column at 50°C, using solution A: H20-0.025% FA-lmM NEfiOAc; solution B: ACN-0.025% FA-lmM NEfiOAc, a flow rate of 0.6 mL / min, with the following gradient set forth in Table 17:Table 17.

[0301] 1 pL of supernatant was injected into the LC / MS / MS system for quantitative analysis.

[0302] 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.

[0303] Results are shown in Table 18.Table 18.

[0304] PK parameters were estimated by non-compartmental model using WinNonlin 8.2; NA = Not available. The PK parameters are set forth in Table 19.Table 19.

[0305] Example 8: HTT Quantitative Splicing Assay.

[0306] Human HD patient-derived lymphoblastoid cells (GM04724) were plated in 12-well plates at 1 x 106cells / 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 culture incubator (37°C, 5% carbon dioxide (CO2)). Following treatment for 24 hours, cells were collected bycentrifugation 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 pg 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 21 A. Reagents for Reverse Transcription ReactionTable 21B. Reverse Transcription Reaction

[0307] Each plasmid standard was diluted to 0.5 x107copies per pL in nuclease free water containing 100 ng / pl yeast tRNA. Next, 10-fold dilutions were prepared to have 8 concentrations in total, from 1 x 107to 1 x100per 2 pL. 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 pL of each standard or cDNA (25 ng) and 8 pL 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 PCRTable 21D. qPCRTable 21E. Primers and Probes

[0308] The primary target tissue of Compound of Structure B is the brain. The effects of Compound of Structure B on HTT pre-mRNA splicing were therefore assessed in iPSC-derived cortical neurons using qRT-PCR.

[0309] 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 SuperScript™ IV VILO (Thermo Cat # 11756500) according to the manufacturer’s instructions, as described in Table 21F and Table 21G.Table 21F. Reagents for Reverse Transcription ReactionTable 21G. Reverse Transcription Reaction

[0310] Each plasmid standard was diluted to 0.5 x107copies per uL in nuclease free water containing 100 ng / pl yeast tRNA. Next, 10-fold dilutions were prepared to have 8 concentrations in total, from 1 x107to 1 x100per 2 pL. 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 pL of each standard or cDNA and 16 pL 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 211. The data is shown in FIG. 5D.Table 21H. Reagents for PCRTable 211. qPCR settingsTable 21 J. Primers and Probes

[0311] Example 9: SMSM Treatment in a Humanized Mouse Models.

[0312] Treatment with Compound of Structure B decreased HTT mRNA level in brain (FIG. 5B) and blood (FIG. 5E) in a humanized HTT mouse model. In addition, treatment with Compound of Structure B demonstrated a significant decrease in mutant HTT protein level in brain (FIG. 5C) and blood (FIG.5F) in a humanized HTT mouse disease model.

[0313] Hul8 / 18 mice carry two normal 18-CAG-repeat alleles of the human HTT gene in the absence of the 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).

[0314] In a single dose study, 2-month-old male and female Hul8 / 18 mice (n=2 to 3 per sex / 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.

[0315] 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.

[0316] To measure total HTT and mHTT protein, blood draws were performed at 4 hours after the last dose 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.

[0317] Example 10: PMS1 Quantitative Splicing Assay.

[0318] Human lymphoblastoid cells (GM07491) were plated in 96-well plates at 50,000 cells / well and treated 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°C, 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 pL of lysis solution containing DNAse. For cDNA synthesis, 10 pL of lysate was added to 40 pL of RT reaction mix and reverse transcribed, as described in Table 22A and Table 22BTable 22A. Reagents for Reverse Transcription ReactionTable 22B. Reverse Transcription Reaction

[0319] Each gBlock was diluted to 0.5x107copies / pl in nuclease free water containing 100 ng / pl yeast tRNA. Next, 10-fold dilutions were prepared to have 8 concentrations in total, from 1 x107to 1 x100per 2 pL. 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 pL of each gBlock or cDNA and 8 pL 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.

[0320] In addition, PMS 1 protein expression level was measured in human lymphoblastoid cells treated with 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 of Structure B or DMSO. The concentrations of compounds were tested at appropriate doses ranging from 10 pM to 2.4 nM. After incubation for 48 hours, the cells were lysed with 100 pL of lysis buffer containing EDTA and protease inhibitors, and total PMS 1 protein levels were assessed by a Jess Automated Western Blot System assay developed with one anti -PMS 1 antibody and one [3-Actin antibody, raised in rabbit and mouse, respectively. 0.2 pg / pL 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 [3-Actin.Table 22C. Reagents for PCRTable 22D. qPCRTable 22E. Primers and Probes

[0321] Example 11: Phase 1 Clinical Trial

[0322] 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.

[0323] Part A is a double-blind placebo-controlled single ascending dose (SAD) design in healthy adult volunteers. 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.

[0324] Part B is a double-blind placebo-controlled multiple ascending dose (MAD) design in healthy adult volunteers. Part B includes three to four cohorts, up to eight subjects per cohort, randomized toreceive 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).

[0325] 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.

[0326] A regional randomized, controlled Phase 2 trial can also be initiated to allow participants in Part C to continue receiving Compound of Structure B, while also enrolling additional HD patients, to ultimately generate longer-term data from an independent study which could support potential global pivotal clinical trials.Table 23. Phase 1 Clinical Trial Summary

[0327] 54 healthy participants were exposed to at least one dose of Compound of Structure B during Part 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.

[0328] 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 of 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 (FIG. 7C) or multiple (FIG. 11) oral administration of Compound of Structure B.

[0329] As shown in FIG. 8B, administration of Compound of Structure B demonstrated dose-dependent decrease 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.

[0330] Exposure to Compound of Structure B increased in a dose proportional manner following single or 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 Tmax values shifted about 4 hours in the presence of food, with no impact on mean C max values.

[0331] The half-life following single or multiple dose administration is about 30 hours. Following daily Compound of Structure B oral dose administration as single (1-16 mg) or multiple dose (1-9 mg) for 14days, 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 AUCiast (r2= 0.836 and 0.845 for SD and MD, respectively.

[0332] Example 12: Clinical Trial

[0333] This example describes a randomized, double blind, placebo-controlled, dose ranging study to evaluate the efficacy, safety, and pharmacodynamics of Compound of Structure B in participants with Huntington’s Disease (HD).

[0334] Investigational Product: Compound of Structure B (a small molecule designed to target expression of the HTT gene for the treatment of HD)

[0335] Comparator: Placebo (microcrystalline cellulose) capsules

[0336] Mode of Administration: Oral

[0337] Study Population

[0338] Male or female participants aged 25-70 (inclusive) with genetically confirmed HD (cytosine, adenine, and guanine [CAG]) repeat length greater than or equal to 40 in HTT gene by direct DNA testing and Huntington’s Disease Integrated Staging System [HD-ISS] Stage 2 or 3 mild). Additional selection criteria apply as described in the study inclusion and exclusion criteria.

[0339] Study Rationale

[0340] HD is caused by expansion of a CAG repeat in the first exon of the HTT gene. CAG repeat expansion results in a mutant HTT (mHTT) protein containing an extended polyglutamine tract near the N-terminus. Although the precise molecular mechanism of disease pathogenesis remains elusive, overwhelming evidence implicates novel toxic fiinction(s) of the mHTT protein as the primary cause of HD.

[0341] Compound of Structure B is an orally bioavailable small molecule splicing modulator that acts to shift the ratio between the canonical HTT mRNA and a non-canonical mRNA isoform containing a cryptic exon 49b located between annotated exons 49 and 50. This results in an overall lowering of HTT mRNA and protein for both wild type and mutant alleles with the therapeutic benefit associated with the latter.Table 25. Study Objectives and Endpoints

[0342] Overall Study Design

[0343] This is a double blind randomized placebo-controlled study. Participants will be enrolled and randomized to 5 parallel treatment arms and receive placebo or Compound of Structure B at a dose range of 3-9 mg once a day (QD) for a minimum of 12 months. Doses are to be administered orally once a day in the morning for the duration of the study.

[0344] Dose Range and Dosing justification

[0345] Planned dose levels of Compound of Structure B to be evaluated are shown in Table 26.Table 26. Compound of Structure B dose levels

[0346] In phase 1 studies a dose range of 1-16 mg as a single dose and 1-9 mg daily have been administered for up to 14 days in healthy volunteers. Compound of Structure B has been generally well tolerated with no significant safety findings. No SAE or grade 3 AE was reported; the majority of AEs were mild and unlikely or unrelated to the study drug and resolved with no intervention. The exposure after a single administration in the range of 1-16 mg increased in a linear fashion with a Tmax of 6-12 hours and half-life of approximately 30 hours. After multiple dose administration the steady state was achieved within 5 days with an accumulation of 3-fold compared with single administration.Administration of Compound of Structure B in this range have resulted in an average reduction in bloodHTT mRNA of 67% after a single 16 mg and 72% after multiple once daily 9 mg dose administration in healthy volunteers (see Example 11).

[0347] Number of Participants

[0348] Initially 150 participants (randomized 1: 1: 1: 1: 1) are planned to be enrolled. The sample size may increase during the pre-defined interim analysis based on observed conditional power to achieve the objectives of the study. Enrollment will be stratified based on TFC (normal = 13 (stage 2) or < 13 (stage 3 mild)). The details of sample size adjustment will be outlined in the statistical analysis plan; however, the maximum sample size will not be greater than a total ofN=300 for the study.

[0349] Study Duration

[0350] All randomized participants will receive blinded treatment until the last participant enrolled reaches 12 months of treatment. Therefore, the duration of participation may vary for each participant. The last participant will be in the study up to 15 months, inclusive of screening (2 months), treatment (12 months), and follow up (1 month) period.

[0351] Study Procedures:• Medical history• Prior and concomitant medication assessments• Height, weight, and BMI• Demographics (including age, race, sex, and ethnicity)• Physical examination• Adverse events (AEs) and treatment-emergent AEs• Vital signs (systolic and diastolic blood pressure, pulse rate, temperature, respiration rate) • 12-lead ECG• Clinical laboratory safety assessments (hematology, serum chemistry, and coagulation)• Blood samples will be collected for analysis of Compound of Structure B PK.• Blood samples will be collected for analyses of HTT mRNA and mHTT / total HTT protein, neurofilament light (NfL), and other HD exploratory biomarkers.• Cerebrospinal fluid will be collected for analysis of Compound of Structure B concentration, mHTT protein, NfL, and / or other HD exploratory biomarkers.• Volumetric MRI (vMRI)• Unified Huntington’s Disease Rating Scale (UHDRS)• Symbol Digit Modalities Test (SDMT)• Montreal Cognitive Assessment (MoCA)• Patient Global Impression of Change• Clinician Global Impression -Improvement and Clinician Global Impression - Severity Scale • Stroop Word Reading Test (SWRT)

[0352] Pharmacodynamic Primary Endpoint: Blood mHTT protein

[0353] Pharmacodynamic and Exploratory Efficacy Measures• Blood / Plasma PD markerso Blood mHTT / Total HTT proteino Blood HTT mRNAo Plasma NfLo Other HD markers• CSF PD markerso mHTT / Total HTT proteino NfLo Other HD markers• Composite UHDRS (cUHDRS)• SDMT• TMS• TFC• SWRT• MoCA• Volumetric MRI (vMRI) parameters

[0354] Participant population'. Participant population criteria is described in Table 27.Table 27. Participant Criteria

[0355] Pharmacodynamic (PD) Markers

[0356] HD is caused by an expansion of a CAG repeat in the first exon of the HTT gene, resulting in the translation of a mutant HTT protein isoform (mHTT). Compound of Structure B has a direct effect on HTT mRNA lowering, and subsequently HTT / mHTT protein lowering. As HTT mRNA and protein are ubiquitously expressed, pharmacodynamic (PD) effects of Compound of Structure B on HTT mRNA and HTT / mHTT protein can be measured in blood, and results can be used to model the predicted PD effects in the brain. Mutant HTT protein in CSF is considered to be a correlate of brain mHTT protein concentration. The combination of mHTT lowering in blood and CSF can provide additional insight into the efficacy of Compound of Structure B in the CNS.

[0357] The concentrations of neurofilament light chain (NfL) in cerebrospinal fluid (CSF) and plasma have become key biomarkers of many neurodegenerative diseases, including HD. NfL protein in CSF and plasma can be a prognostic marker of neurodegeneration and longitudinal increases in NfL in HD patients are associated with neuronal damage and disease progression. A stable or reduced NfL level may provide early signs of treatment efficacy and, at a minimum, indicated that the treatment is not exacerbating the disease.

[0358] The following validated assays will be used to measure HTT mRNA and protein, and NfL.• HTT mRNA canonical and cryptic isoforms from whole blood (ddPCR)• mHTT and / or total HTT protein from whole blood (MSD)• mHTT from CSF (SMCxPro)• NfL from plasma and / or CSF (Quanterix)

[0359] Additional Information

[0360] UHDRS

[0361] UHDRS is a comprehensive clinical assessment tool specifically designed for individuals with Huntington's disease (HD). Developed by the Huntington Study Group, the UHDRS is widely used in both clinical practice and research settings to evaluate various aspects of HD, including motor function,cognitive performance, and psychiatric symptoms. The scale is designed to provide a standardized and comprehensive measure of disease severity, progression, and treatment response.

[0362] 1. Motor Assessment: Motor assessment in UHDRS includes the Total Motor Score (TMS), which is calculated by summing up the scores of various motor items. Each motor item is scored from 0 to 4, with higher scores indicating greater severity of impairment. The TMS ranges from 0 to 124.

[0363] 2. Cognitive Assessment: Cognitive assessment typically involves administering standardized neuropsychological tests. Scores on these tests are compared to normative data to assess the participant's cognitive abilities. Symbol Digit Modalities Test (SDMT) and Stroop Word Reading Test will be used for cognitive testing.

[0364] 3. Behavioral Assessment: Behavioral assessment involves rating scales for various psychiatric symptoms associated with HD, such as depression, anxiety, irritability, and apathy. Each item is scored based on the severity of symptoms, often using Likert-type scales ranging from 0 to 4 or 0 to 3.

[0365] 4. Independence Scale (IS): The Independence Scale assesses the participant's level of independence in various aspects of daily life, with each item scored from 0 to 4. The scores for each item are summed to obtain the total Independence Scale score, ranging from 0 to 100.

[0366] 5. Functional Capacity Assessment: The Functional Assessment section of the UHDRS includes items that assess the participant's ability to perform activities of daily living (ADLs). Each item is scored from 0 to 4, with higher scores indicating greater impairment. The Total Functional Capacity (TFC) score is derived from these items, ranging from 0 to 13. Total Functional Capacity (TFC): TFC is calculated based on the scores from the Functional Assessment section. It ranges from 0 to 13, with higher scores indicating better functional capacity.

[0367] Composite UHDRS (cUHDRS)

[0368] The cUHDRS is derived from the following key components of the UHDRS:

[0369] 1. Total Motor Score (TMS): Assesses motor dysfunction, with scores ranging from 0 (no impairment) to 124 (severe impairment).

[0370] 2. Symbol Digit Modalities Test (SDMT): Evaluates cognitive function, specifically processing speed. Higher scores indicate better cognitive function.

[0371] 3. Stroop Word Reading Test: Measures cognitive flexibility and executive function. Higher scores indicate better cognitive performance.

[0372] 4. Total Functional Capacity (TFC): Assesses overall functional ability, with scores ranging from 0 (completely dependent) to 13 (fully independent).

[0373] The cUHDRS score is calculated using a specific formula that integrates these components, weighted to reflect their relative importance in tracking disease progression. Although the exact formula can vary slightly depending on the study or clinical context, it typically involves normalizing and summing the scores from each component. A decline of 1.2 points is a clinically meaningful functional decline.

[0374] Calculation of cUHDRS:

[0375] cUHDRS score calculation: [(TFC - 10.4) / l.9 - (TMS - 29.7)14.9 + (SDMT - 28.4) / l 1.3 + (SWR- 66.1) / 20.1] + 10

[0376] Huntington's Disease Integrated Staging System (HD-ISS)

[0377] The HD-ISS is an approach to classifying the progression of Huntington's disease (HD). HD-ISS uses the following objective measures:• Genetic: Presence of the CAG repeat expansion in the huntingtin gene.• Biomarkers: Measurable changes in the brain before symptoms appear, such as brain atrophy.• Clinical signs: Observable motor and cognitive symptoms.• Functional decline: Deterioration in daily activities.

[0378] By considering these aspects, the HD-ISS creates a more comprehensive picture of HD progression and identifies individuals at different stages of the disease, even before they experience physical symptoms (FIG. 9).

[0379] Symbol Digit Modalities Test (SDMT)

[0380] The SDMT is used to assess divided attention, visual scanning, tracking and motor speed. Using a reference key, the examinee has 90 seconds to pair specific numbers with given geometric figures. Because examinees can give either written or spoken responses, the test is well suited for use with individuals who have motor disabilities or speech disorders. Because it involves only geometric figures and numbers, the SDMT is relatively culture free as well and can be administered to individuals who do not speak English. It takes approximately 5 minutes to complete the entire test. Scoring involves summing the number of correct substitutions within the 90 second interval (max = 110). Domains measured by SDMT are the following:• Visual scanning: Quickly finding the symbols among the list.• Working memory: Holding the code key in mind while the participant scans the symbols.• Processing speed: How fast the participant can match the symbol to the number and write it down.

[0381] The rate of cognitive decline in HD, as measured by SDMT, varies depending on several factors, including the stage of the disease and individual variability. However, some general observations have been made in the literature regarding the rate of decline over time:• Pre -symptomatic and Early Stages

[0382] Subtle Decline Individuals who carry the HD gene mutation but have not yet shown overt motor symptoms (pre -symptomatic) orthose in the very early stages of the disease often show a subtle but measurable decline in SDMT performance overtime.

[0383] Annual Decline: Studies have reported that in the pre -symptomatic and early stages, the SDMT score may decline by approximately 1 to 2 points per year. This decline becomes more pronounced asindividuals approach the onset of motor symptoms. Therefore, an average change of 1.1 -1.9 points depending on stage of the disease is considered a minimal clinically significant change.

[0384] Volumetric MRI (vMRI)

[0385] Volumetric MRI protocols for studying Huntington's disease (HD) primarily aim to assess structural changes in the brain, particularly in regions known to be affected by the disease such as the striatum and cortex. A volumetric MRI protocol is designed to provide high-resolution images of the brain that can be used for detailed volumetric analysis of specific brain regions. The most notable changes observed in vMRI studies of HD are the progressive atrophy of the striatum, particularly the caudate nucleus and putamen. Over time, additional brain regions such as the cortex and white matter also show atrophy, correlating with disease progression. The rate of atrophy in HD is higher in individuals with more severe clinical symptoms and higher CAG repeat lengths. Baseline striatal volume has been shown to predict subsequent clinical decline, making it a potential biomarker for disease progression.

[0386] Accelerated volume loss in the brain regions most affected by HD, the caudate and putamen, is detected in asymptomatic individuals with expanded CAG repeats. This volume loss becomes more pronounced as the disease progresses, with higher CAP scores (indicating greater disease burden) and lower functional capacity correlating with steeper declines. vMRI’s sensitivity surpasses other imaging modalities in differentiating between various HD stages, making it a strong candidate for clinical trials (Dominguez 2013). At the time of clinical motor diagnosis, striatal volumes are markedly reduced compared to age-matched normal volumes or 30-50%.

[0387] Studies on diagnosed patients estimate annual volume loss rates of 2-3% for the caudate and 1-3% for the putamen. It is also suggested that putamen degeneration might begin even earlier in the disease course with a slightly higher pre-diagnosis loss rates in this region compared to the caudate. In the later-stage HD patients a potential plateauing of the caudate's decline is seen that accompanies a consistent volume loss rates observed over multiple follow-up periods. This characteristic makes vMRI a potentially reliable tool for monitoring disease progression and treatment response throughout the course of HD.

[0388] Stroop Word Reading Test (SWRT)

[0389] The Stroop Word Reading Test is often used in cognitive assessments, including for HD patients, as it measures processing speed, attention, and cognitive control - areas frequently impacted by the disease. In HD, the SWRT can help assess the progressive cognitive decline, particularly in executive function and the ability to inhibit automatic responses. The SWRT is often used to track cognitive deterioration overtime (Table 28).

[0390] 1. Processing Speed: HD leads to bradykinesia, which extends to cognitive functions, slowing down the ability to read words quickly.

[0391] 2. Executive Dysfunction: One of the hallmark symptoms of HD is executive dysfunction, making it hard for patients to switch between tasks and inhibit automatic responses.

[0392] 3. Difficulty with Inhibition: In the later stages, HD patients have trouble inhibiting dominant responses, leading to poor performance on the more challenging Stroop conditions, such as color word interference.

[0393] The SWRT is usually part of the Unified Huntington's Disease Rating Scale Cognitive Assessment. SWRT typically consists of three parts: (1) Word Reading Task (Baseline Task), (2) Color Naming Task (Color Naming Baseline), and (3) Color Word Interference Task (Critical Task).Table 28. Summary of SWRT Performance by HD StageTable 29. Summary of Clinical Trial Assessments* Participants will be in study until the last enrolled participant reaches 12 months of treatment or for a maximum of 24 months. Visits may not be applicable to all participants. CSF samples will be assessed for PK, mHTT protein, NfL and other HD markers.Abbreviations: AE = adverse event; BMI = body mass index; C-SSRS = Columbia Suicide Severity Rating Scale; ECG = electrocardiogram; EoS = end of study; ET = early termination; HD = Huntington disease; HTT = huntingtin; mHTT = mutant huntingtin; mRNA = messenger ribonucleic acid; PD = pharmacodynamic; PI = principal investigator; PK = pharmacokinetic; CGI-I= Clinician Global Impression -Improvement; CGI-S=Clinician Global Impression - Severity Scale; Patient Global Impression of Change = PGI-C; Stroop Word Reading Test = SWRT.**Drug accountability procedures, compliance and dosing instruction card: At each study visit, study drug will be dispensed, counted with compliance calculated and reinforcement on dosing instructions.

[0394] Example 13: Phase 1 Clinical Trial - Part C Extension

[0395] Part C Extension is a double-blind placebo-controlled parallel design study of two dose levels of Compound of Structure B and placebo of individuals with early-stage HD (HD-ISS Stage 1, 2, or mild Stage 3) for 84 days followed by a 12-month extension of active treatment where all participants receive either a low or high dose of Compound of Structure B in a blinded fashion (see Example 11 for Part C and below, and FIG. 10A). The objectives of the study include evaluating safety, pharmacokinetic, and pharmacodynamic parameters including mutant HTT (mHTT) protein, HTT mRNA, and PMS1 mRNA.

[0396] Part C ft) Non-interventional observational part. No dose administration planned (28 days).

[0397] Part C fit) A single dose of Compound of Structure B or placebo once a day (QD) on Days 1 to 28 (inclusive); 28 doses total.

[0398] Treatment Cycle 2 (optional*): A single dose of Compound of Structure B or placebo QD on Days 29 to 56 (inclusive); 28 doses total.

[0399] Treatment Cycle 3 (optional*): A single dose of Compound of Structure B or placebo QD on Days 57 to 84 (inclusive); 28 doses total.

[0400] * = Treatment Cycles 2 and 3 are optional, per principal investigator (PI) discretion. Patients received instructions on whether to continue treatment in Cycles 2 and 3 on Day 28 and 56, respectively. Only patients who received treatment in Cycle 2 were considered for treatment in Cycle 3.

[0401] Part C fit) Extension'. Participants that completed Part C (ii) Cycle 3 were offered to continue study drug dosing in the Part C (ii) Extension. All participants will receive blinded active treatment (Compound of Structure B) during this Extension study for 12 months. Demographic and baseline characteristics of participants are described in Table 30.Table 30. Demographic and baseline characteristics in Part C Extension*cUHDRS: composite Unified Huntington’s Disease Rating Scale

[0402] At Day 84, patients receiving Compound of Structure B demonstrated dose-dependent reductions of mHTT protein in blood including 62% at the 9 mg dose (FIG. 10B). Compound of Structure B has been generally well tolerated at both dose levels tested (3 mg and 9 mg) and the rate of TEAEs were similar across placebo, 3 mg, and 9 mg groups. There were no clinically significant changes in laboratory results, electrocardiogram (ECG), or vital signs. Two severe TEAEs were reported: (1) headache (possibly related) that resolved within 10 days and (2) psychosis (unlikely related) that resolved within 3 days. In addition, two moderate TEAEs were reported: (1) headache (possibly related) that resolved within 3 days and (2) fall (unrelated). Treatment with Compound of Structure B also resulted in dose-dependent HTT mRNA (mean reduction of 62% at the 9 mg dose as shown in FIG. 13A) and PMS1 mRNA (mean reduction of 26% at the 9 mg dose as shown in FIG. 13B) reduction and demonstrated linear and dose-proportional PK profile reaching steady state in 5-7 days in HD patients (FIG. 12) in addition to favorable blood brain barrier (BBB) penetration (Table 31). The overall safety profile has been favorable (no sign of dose-related toxicity), supporting the continued clinical development. There were no signs of group effects on Neurofilament Light (NfL) increase as shown in FIG. 14. Additional clinical and pharmacodynamics measures are being or to be evaluated (FIG. 15).Table 31. Compound of Structure B Exposure in HD patients*Compound of Structure B Exposure in cynomolgus monkeys: 0.68 Kp, uu** Compound of Structure B Exposure in Mice: 0.61 Kp, uu (Note: there was a 1: 1 reduction of mHTT protein in the plasma and in the brain in the mouse study, see FIGs. 5C and 5F.)

[0403] Methods:

[0404] Quantification of mHTT and Total HTT protein in whole blood

[0405] Analysis of mHTT and total HTT protein levels in whole blood samples collected in K2EDTA tubes and stored at -80°C was performed by Aptuit (Verona) Sri, an Evotec Company laboratory in Verona, Italy. Samples were analyzed using validated assays, proprietary to Aptuit (Verona) Sri., using the Mesoscale Discovery (MSD) platform. The objective of this work was to determine the concentration of mutant Huntingtin (mHTT) and total Huntingtin (tHTT) in human whole blood lysate at baseline and multipletimepoints after treatment to determine the pharmacodynamic effect of Compound of Structure B on the HTT protein regulation.

[0406] Quantitative HTT splicing assays

[0407] The purpose of these assays was to quantify the expression levels of endogenously expressed HTT transcripts and the transcripts of a non -canonical splice variant of HTT, relative to the housekeeping gene GUSB. The highly expressed HTT mRNA was quantified based on amplification across two locations, exons 51-52 (HTT E51E52, or Total HTT) and across exons 49 and exon 50 (HTT E49E50, or HTT1). The non-canonical splice variant of HTT possesses the inclusion of an intron between exons 49 and 50 (termed HTT E49bE50, or HTT2) which includes an in-frame translational termination codon that triggers nonsense-mediated decay. In contrast to the canonical HTT, the HTT2 isoform is endogenously expressed at very low levels in normal subjects and quantified based on alternative splicing at a cryptic splice junction (E49b) between exon 49 and exon 50. The investigational interventional treatment was expected to produce a dosedependent increase in the non-canonical HTT2 mRNA variant and a corresponding decrease in both the HTT1 and total HTT mRNAs.

[0408] Whole blood was collected in RNA Paxgene tubes and RNA was extracted using the PAXgene Blood miRNA Kit (Catalog # 763134), manufacturer’s protocol. Extracted RNA was normalized to 10 ng / pl, and 5 pl was used as input for the cDNA synthesis using the ThermoFisher Vilo IV cDNA synthesis kit (catalog #: 11766500) (total 50 ng input). Two microliters of cDNA were used as the template in a 22 pl ddPCR reaction. Three ddPCR reactions were run that contain primers and probes to detect one of the three targets (HTT1 / HTT2 / Total HTT) and the reference internal control GUSB to normalize for the ratio of target to internal control. All samples were run using triplicate PCR reactions. Following the reaction set up, the ddPCR reaction was partitioned into nanoliter-sized water-in-oil droplets using the QX200 Droplet Generator and subsequently amplified by PCR. Amplified samples were then quantified on the QX200 Droplet Reader, which flows the droplets in single file past a two-color optical detection system to determine which droplets are positive for the targets being assayed. Using the QuantaSoft software, Poisson statistics were applied to the data to determine the copy number of the target being assayed and internal control in each sample. The average concentration (copies / pL) of the HTT target and GUSB across the three replicates and ratio of targetGUSB are reported.Table 32. Primers and Probes for HTT Splicing Assay

[0409] Quantitative PMS1 splicing assay

[0410] The purpose of this assay was to quantify the expression levels of endogenously expressed canonical PMS1 transcript relative to the housekeeping control HPRT1 in whole blood using digital droplet PCR (ddPCR). Expression of canonical PMS1 was quantified based on amplification across exon 5 and 6 with an isoform specific probe over the exon junction (PMS1_E5E6). The investigational interventional treatment was predicted to lead to a dose-dependent decrease in canonical PMS1 (PMS1_E5E6) mRNA.

[0411] Whole blood was collected in RNA PAXgene tubes and RNA was extracted using the PAXgene blood RNA kit (Qiagen, Cat. # 762174) according to the manufacturer’s protocol. 500 ng extracted RNA was used as input for first strand cDNA synthesis using SuperScript IV VILO (Thermo Fisher, Cat. # 11756) in a 20 pL reaction. cDNA was diluted 1: 10 in water and 5 pL was used as template in a 25 pL ddPCR reaction. After mixing the template, ddPCR master mix, and primers / probes, the ddPCR reaction was partitioned into nanoliter-sized water-in-oil droplet using the Automated Droplet Generator (Bio-Rad) and subsequently amplified by PCR. Amplified samples were then quantified on the QX600 Droplet Reader (Bio-Rad), which flows the droplets in single file past a six-color optical detection system to determine which droplets are positive for the targets being assayed. The ddPCR reactions were run in duplex with primers and probes to detect the PMS1 isoform and the reference housekeeping control HPRT1. All samples were run in technical triplicates. The data was analyzed to determine copy numbers using the QX Manager software from Bio-Rad. The mean concentration of canonical PMS1 and HPRT1 across the three replicates is reported.Table 33. Primers and Probes for PMS1 Splicing Assay

[0412] Quantification of Neurofilament Light (NfL) in plasma and cerebrospinal fluid (CSF)

[0413] Human plasma and CSF were collected and stored at -80°C. Sample analysis to determine the concentration of NfL was performed by Aptuit (Verona) Sri, an Evotec Company laboratory in Verona, Italy. Sample analysis was performed on the Quanterix SR-X platform using the NFLight v2 Advantage Kit and following the validated methods as described in the kit manual. The objective of this work was to determinethe concentration of NfL in human plasma and CSF at baseline and multiple timepoints after treatment to assess a potential effect of Compound of Structure B treatment on NfL levels compared to placebo treatment.

[0414] The examples and embodiments described herein are for illustrative purposes only and various modifications 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

CLAIMSWhat is claimed is:

1. A method of treating or preventing Huntington’s disease in a human subject in need thereof, comprising administering to the human 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,wherein the human subject has been genetically confirmed by DNA sequencing to have cytosine, adenine, and guanine (CAG) repeat length of at least 40 in an HTT gene.2 The method of claim 1, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject in need thereof for a treatment period of at least 28 days.3 A method of treating or preventing Huntington’s disease in a human subject in need thereof, comprising administering to the human 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:wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject in need thereof for a treatment period of at least 28 days.4 The method of any one of the preceding claims, wherein the human subject has a total motor score (TMS) of 6 or higher.5 The method of any one of the preceding claims, wherein the human subject has an independence scale (IS) score of 70 or higher.6 The method of any one of the preceding claims, wherein the human subject has a total functional capacity (TFC) score of 10 or higher.7 A method of treating or preventing Huntington’s disease in a human subject in need thereof, comprising administering to the human 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:wherein the human subject has one or more of:(i) a total motor score (TMS) of 6 or higher;(ii) has an independence scale (IS) score of 70 or higher; and / or(iii) a total functional capacity (TFC) score of 10 or higher.The method of any one of the preceding claims, wherein the amount of a mutant huntingtin (mHTT) is reduced in a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.The method of any one of the preceding claims, wherein the amount of mHTT 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 a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.A method of treating or preventing Huntington’s disease in a human subject in need thereof, comprising administering to the human 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:wherein the amount of a mutant huntingtin (mHTT) 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 a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.The method of any one of claims 3 to 10, wherein the human subject has been genetically confirmed by DNA sequencing to have cytosine, adenine, and guanine (CAG) repeat length of at least 40 in an HTT gene.

12. The method of any one of claims 7 to 11, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject in need thereof for a treatment period of at least 28 days.

13. The method of any one of the preceding claims, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject in need thereof for a treatment period of at least 56 days or at least 84 days.

14. The method of any one of the preceding claims, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject in need thereof for a treatment period of about 12 months or more than 12 months.

15. The method of any one of the preceding claims, wherein the amount of the mHTT protein is reduced by at least 10% in the blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of at least 84 days.

16. The method of any one of the preceding claims, wherein the treatment period is followed by a drug holiday period.

17. The method of claim 16, wherein the drug holiday period is about 14 days.

18. The method of any one of the preceding claims, wherein the human subject is between the ages of 25 and 70.

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

20. The method of any one of claims 1 to 18, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human 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.

21. The method of claim 19 or 20, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject at about 3 mg per dose.

22. The method of claim 19 or 20, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject at about 4 mg per dose.

23. The method of claim 19 or 20, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject at about 6 mg per dose.

24. The method of claim 19 or 20, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject at about 9 mg per dose.

25. The method of any one of the preceding claims, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject daily.

26. The method of any one of the preceding claims, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered to the human subject once a day.

27. The method of any one of the preceding claims, wherein the composition or the pharmaceutical composition comprising the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof is administered orally.

28. The method of any one of the preceding claims, wherein 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.

29. The method of claim 28, wherein the solid dosage form comprises a tablet or a capsule.

30. The method of any one of the preceding claims, wherein the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof penetrates a blood brain barrier (BBB) when administered to the human subject.

31. The method of any one of the preceding claims, wherein the human subject has Huntington’s Disease Integrated Staging System [HD-ISS] Stage 2 or 3 mild.

32. The method of any one of the preceding claims, wherein the amount of a 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 a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

33. The method of any one of the preceding claims, wherein the amount of a 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 a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of at least 28 days, at least 56 days, or at least 84 days.

34. The method of any one of the preceding claims, wherein the amount of a canonical isoform of an HTT mRNA, a canonical isoform of a 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 a blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of at least 28 days, at least 56 days, or at least 84 days.

35. The method of claim 34, wherein the amount of the canonical isoform of the HTT mRNA is reduced by at least 50% in the blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of at least 84 days.

36. The method of claim 34, wherein the amount of the canonical isoform of the PMS1 mRNA is reduced by at least 20% in the blood sample of the human subject administered with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof for a treatment period of at least 84 days.

37. The method of any one of the preceding claims, wherein the human subject’s genome encodes a wild-type PMS1.

38. The method of any one of the preceding claims, wherein the human subject’s genome comprises an allele comprising a genetic variation in a PMS1 gene.

39. The method of claim 38, wherein the genetic variation is a non-synonymous coding variant.

40. The method of claim 38 or 39, wherein the genetic variation does not disrupt or modulate the PMS1 gene, or wherein the genetic variation is not a loss-of-function genetic variation.

41. The method of any one of claims 38 to 40, wherein 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 hgl9.

42. The method of any one of claims 38 to 41, wherein the allele comprising a genetic variation in a PMS1 gene encodes a variant PMS 1 comprising a mutation selected from the group consisting of a E59K mutation, a K433R mutation, a L524S mutation, and any combination thereof.

43. The method of any one of claims 38 to 42, wherein the human subject has been identified as having the genetic variation.

44. The method of claim 38, wherein the genetic variation disrupts or modulates the PMS1 gene.

45. The method of claim 38, wherein the human subject has been identified as not having the genetic variation.

46. The method of claim 44 or 45, wherein the genetic variation is a non-synonymous coding variant.

47. The method of any one of claims 44 to 46, wherein the genetic variation comprises chr2: 190660586 C> T, chr2: 190670391 >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 hgl9.

48. The method of any one of claims 44 to 47, wherein the allele comprising a genetic variation in a PMS1 gene encodes a variant PMS 1 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.

49. The method of any one of claims 38 to 48, wherein the human subject has been tested for a presence of the genetic variation with a genetic assay.

50. The method of any one of claims 38 to 49, wherein the human subject is heterozygous for the genetic variation.

51. The method of any one of claims 38 to 49, wherein the human subject is homozygous for the genetic variation.

52. The method of any one of claims 1 to 51, wherein the method delays onset or slows progression of the Huntington’s disease.

53. The method of any one of the preceding claims, wherein the Huntington’s disease is associated with expression level of activity level of a protein encoded by an HTT gene or a PMS1 gene.

54. The method of claim 53, wherein the Huntington’s disease is associated with a string of CAG repeats in the HTT gene.

55. The method of claim 53 or 54, wherein a pre-mRNA and / or an mRNA encoded by the HTT gene comprises the string of CAG repeats.

56. The method of any one of claims 53 to 55, wherein the Huntington’s disease is associated with an aberrant expansion of a string of CAG repeats in the HTT gene.

57. The method of any one of claims 53 to 56, wherein a pre-mRNA encoded by the HTT gene comprises the aberrant expansion of the string of CAG repeats.

58. The method of any one of claims 53 to 57, wherein the protein encoded by the HTT gene comprises a mutant HTT protein.

59. The method of any one of claims 53 to 58, wherein the Huntington’s disease 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.

60. The method of any one of the preceding claims, wherein the compound of structure B, or a pharmaceutically acceptable salt or a stereoisomer thereof binds to an HTT pre-mRNA, a PMS1 pre- mRNA, or both and modulates splicing of the HTT pre-mRNA, the PMS 1 pre-mRNA, or both at a splice site of the HTT pre-mRNA, the PMS 1 pre-mRNA, or both in a cell or cells of the human subject to produce a spliced product of the HTT pre-mRNA, the PMS1 pre-mRNA, or both.

61. The method of claim 60, wherein the HTT pre-mRNA, the PMS 1 pre-mRNA, or both comprise a splice site sequence.

62. The method of claim 60 or 61, wherein the expression level of a canonical isoform of a PMS1 mRNA encoded by the PMS 1 pre-mRNA is reduced by at least 20% in the cell contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

63. The method of any one of claims 60 to 62, wherein the expression level of a canonical isoform of an HTT mRNA encoded by the HTT pre-mRNA is reduced by at least 30% in the cell contacted with the compound of structure B, or the pharmaceutically acceptable salt or stereoisomer thereof.

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