Oligonucleotide compositions and methods thereof

Abstract

In particular, the present disclosure provides various oligonucleotide technologies, including chiral-controlled oligonucleotide compositions and manufacturing technologies for such oligonucleotide compositions. In some embodiments, the method is a method for treating or preventing Huntington's disease in a subject in need thereof; a method for allele-specific knockdown of mutant Huntington's transcripts in a subject; a method for delaying the onset and / or reducing the severity of at least one symptom of Huntington's disease in a subject with Huntington's disease; a method for reducing the expression, level, amount, and / or activity of a mutant Huntington's gene or its gene product; and / or a method for preparing a medicament for the treatment of Huntington's disease, wherein the method involves the use of the oligonucleotides described herein administered in the doses described herein. In some embodiments, the present disclosure provides doses, dosages, and formulations of the oligonucleotides described herein.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Patent Application Nos. 63 / 397,284, filed August 11, 2022, 63 / 408,427, filed September 20, 2022, and 63 / 517,529, filed August 3, 2023, each of which is incorporated by reference in its entirety.

[0002] Field Among other things, the present disclosure provides techniques for treating Huntington's disease. In some embodiments, provided herein are methods of administering WVE-003 to ameliorate Huntington's disease, reduce mHTT RNA, or reduce mHTT protein in a human subject in need thereof. In certain instances, the methods are useful for ameliorating at least one symptom of Huntington's disease. Such symptoms of Huntington's disease include, but are not limited to, brain atrophy, muscle atrophy, neurodegeneration, loss of motor control, difficulty swallowing, difficulty speaking, anxiety, and depression. [Background technology]

[0003] background Oligonucleotides are useful in therapeutic, diagnostic, research, and nanomaterial applications. The use of naturally occurring nucleic acids (e.g., unmodified DNA or RNA) as therapeutic agents can be limited, for example, due to their instability to extracellular and intracellular nucleases and / or their poor cellular penetration and distribution. Currently, acceptable treatment options for neurodegenerative diseases, such as Huntington's disease, are lacking. It is an object herein to provide methods for treating such diseases. Summary of the Invention [Means for solving the problem]

[0004] overview WVE-003, sometimes referred to as WV-21405, is mG*SmUn001RmUmGn001RmA*ST*SC*ST*SG*ST*RA*SG*SC*SA*SG*Rm5Ceon001RAeoGeon001Rm5Ceo*STeo (In the formula, m represents a 2'-OMe modification to the nucleoside; *S represents an Sp phosphorothioate bond; m5Ceo represents 5-methyl 2'-O-methoxyethyl C; n001R represents an Rp n001 bond, where the n001 bond is [ka] having the structure eo represents a 2'-OCH2CH2OCH3 modification to the nucleoside; and *R represents Rp phosphorothioate bond) It has the following structure.

[0005] As typically used in referring to oligonucleotides, unless otherwise specified, the nucleosides are DNA nucleosides and the linkages are natural phosphate linkages.

[0006] As described herein, WVE-003 may be provided in various forms, including as a pharmaceutically acceptable salt, such as a sodium salt.

[0007] Among other things, the present disclosure encompasses the recognition that structural elements of HTT (huntingtin) oligonucleotides, such as base sequence, chemical modifications (e.g., sugar, base, and / or internucleotide linkage modifications and their pattern), and / or stereochemistry (e.g., the stereochemistry of backbone chiral centers (chiral internucleotide linkages) and / or their pattern), can significantly affect HTT oligonucleotide properties and / or activity, such as protein binding properties, stability, toxicity, delivery, allele-specific knockdown of HTT transcripts, etc. In some embodiments, the present disclosure demonstrates that HTT oligonucleotides and compositions thereof with controlled structural elements can provide unexpected therapeutic properties and activity. In some embodiments, the present disclosure provides technologies useful for treating Huntington's disease in a subject, such as HTT oligonucleotides, pharmaceutical compositions, drug products, doses, dosages, etc., particularly WVE-003. In some embodiments, the subject has an HTT allele or transcript that contains an expanded CAG repeat region and is fully complementary to the base sequence of WVE-003. As used herein, a nucleic acid or nucleotide sequence can be considered fully complementary to another nucleic acid (e.g., WVE-003) or nucleotide sequence if the nucleic acid or nucleotide sequence contains a sequence that is the same length as the other nucleic acid or nucleotide sequence and is fully complementary to the entire nucleotide sequence of the other nucleic acid (e.g., WVE-003). In some embodiments, the expanded CAG repeat comprises 36 or more CAG repeats. In some embodiments, the subject comprises an HTT transcript associated with Huntington's disease, which transcript is fully complementary to the nucleotide sequence of WVE-003. In some embodiments, the subject comprises an HTT transcript associated with Huntington's disease, which transcript is fully complementary to the nucleotide sequence of WVE-003 at SNP rs362273. In some embodiments, the subject comprises an HTT transcript that is fully complementary to the nucleotide sequence of WVE-003 and includes an expanded CAG repeat region. In some embodiments, the subject comprises an HTT transcript that is perfectly complementary to the base sequence of WVE-003 at SNP rs362273 and includes an expanded CAG repeat region.In some embodiments, the subject comprises an HTT nucleic acid, e.g., an HTT gene, encoding an HTT transcript associated with Huntington's disease, wherein the transcript is perfectly complementary to the nucleotide sequence of WVE-003. In some embodiments, the subject comprises an HTT nucleic acid, e.g., an HTT gene, encoding an HTT transcript associated with Huntington's disease, wherein the transcript is perfectly complementary to the nucleotide sequence of WVE-003 at SNP rs362273. In some embodiments, the subject comprises an HTT nucleic acid, e.g., an HTT gene, encoding an HTT transcript that is perfectly complementary to the nucleotide sequence of WVE-003 and includes an expanded CAG repeat region. In some embodiments, the subject comprises an HTT nucleic acid, e.g., an HTT gene, encoding an HTT transcript that is perfectly complementary to the nucleotide sequence of WVE-003 and includes an expanded CAG repeat region at SNP rs362273. In some embodiments, the subject has an A mutation at SNP rs362273. As will be understood by those skilled in the art, an HTT transcript having an A mutation at SNP rs362273 may be perfectly complementary to the base sequence of WVE-003, where A is complementary to T at WVE-003. In some embodiments, the A mutation at SNP rs362273 is on the same chromosome as the expanded CAG repeat region of HTT. In some embodiments, the subject has a non-A mutation at SNP rs362273. In some embodiments, the non-A mutation at SNP rs362273 is not on the same chromosome as the expanded CAG repeat region of HTT. In some embodiments, the SNP rs362273 mutation is C. In some embodiments, the subject is heterozygous for the A mutation at SNP rs362273. In some embodiments, the subject is heterozygous for the A mutation at SNP rs362273, where A mutation is on the same chromosome as the expanded CAG repeat region of HTT. In some embodiments, the subject is homozygous for the A mutation of SNP rs362273. In some embodiments, the subject is homozygous for the A mutation of SNP rs362273 and has the CAG repeat region expanded by HTT. In some embodiments, one chromosome of the subject has the CAG repeat region expanded by HTT.In some embodiments, both chromosomes of the subject independently have an expanded CAG repeat region in HTT.

[0008] In some embodiments, Huntington's disease (HD) is a debilitating, ultimately fatal, autosomal dominant neurological disorder characterized by cognitive decline, psychosis, and chorea. In some embodiments, HD damages brain neurons over time, affecting thinking, emotions, and movement. HD is reportedly caused by the expansion of a cytosine-adenine-guanine (CAG) triplet repeat in the huntingtin (HTT) gene, resulting in the production of a mutant HTT protein. In some embodiments, accumulation of mutant HTT leads to progressive loss of neurons in the brain. In some embodiments, wild-type, or healthy, HTT protein is critical for neuronal function, and its inhibition can have long-term adverse consequences. In some embodiments, approximately 30,000 people in the United States have symptomatic HD, and an additional 200,000 are at genetic risk for the disease. Currently, no approved disease-modifying therapies are available.

[0009] In some embodiments, Huntington's disease (HD) is reportedly caused by a cytosine-adenine-guanine (CAG) repeat expansion in the huntingtin (HTT) gene, resulting in the production of mutant HTT (mHTT) protein. In some embodiments, mHTT is reportedly the cause of the disease, but preclinical studies suggest that loss of wild-type HTT (wtHTT) may also contribute to neuronal dysfunction. In some embodiments, wtHTT protein is reportedly critical for neuronal function; suppression of wtHTT can have long-term adverse consequences.

[0010] In some embodiments, the ability to selectively reduce mHTT protein production while retaining adequate wtHTT levels (also known as allele-specific knockdown) reportedly holds great promise for the treatment of HD. In some embodiments, as confirmed herein, administration of oligonucleotides as described herein can result in a greater reduction in the level, expression, and / or activity of mHTT transcripts and / or proteins relative to wtHTT transcripts and / or proteins in individual subjects and / or populations of subjects.

[0011] In some embodiments, allele-specific knockdown exploits the association between single nucleotide polymorphisms (SNPs) and genetic mutations to specifically target errors in genetic disorders, including Huntington's disease (HD).

[0012] In some embodiments, the disclosure provides oligonucleotides, compositions, and methods for allele-specific knockdown of mHTT transcripts, wherein the allele-specific knockdown (also referred to herein as allele-specific suppression, allele-selective approaches, allele-selective knockdown or suppression, etc.) preferentially reduces the level, expression, and / or activity of mHTT transcripts (e.g., containing expanded CAG repeat regions) and / or their products (e.g., mHTT protein) relative to wild-type HTT and / or its products (e.g., wild-type HTT protein) (e.g., the expression, level, and / or activity of wild-type HTT is not significantly reduced, is not reduced, remains the same, or is increased).

[0013] In some embodiments, a decrease in the level, expression, and / or activity of mHTT transcripts results in or is associated with a decrease in the level, expression, and / or activity of mHTT protein, including, but not limited to, the formation, number, and / or size of aggregates (clusters) of mHTT protein. In some embodiments, the mHTT protein comprises an expanded polyglutamine (polyQ) tract, for example, translated from a CAG repeat expansion.

[0014] In some embodiments, the disclosure relates to methods of treating and / or preventing Huntington's disease in a subject (e.g., a patient, such as a human patient) in need thereof; methods of allele-specific knockdown of mutant huntingtin transcripts in a subject; and / or methods of reducing the severity and / or delaying the onset of one or more symptoms of Huntington's disease. In some embodiments, the methods comprise administering to the subject a therapeutically effective amount of an HTT oligonucleotide or an HTT oligonucleotide composition. In some embodiments, the disclosure relates to methods of treating, ameliorating, or slowing the onset or progression of Huntington's disease, comprising administering to the subject a compound comprising an HTT oligonucleotide or an HTT oligonucleotide composition, wherein the oligonucleotide is complementary to a mutant huntingtin allele at a position on the allele that contains a single nucleotide polymorphism (SNP) site, and wherein, upon administration of the compound to the subject, selective reduction of the mutant huntingtin allele treats, ameliorate, and / or slows the onset and / or progression of Huntington's disease. In some embodiments, the present disclosure relates to a method of ameliorating a symptom of Huntington's disease, comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to a human subject in need thereof.

[0015] In some embodiments, the HTT oligonucleotide is WVE-003. In some embodiments, the HTT oligonucleotide composition is a composition comprising WVE-003. In some embodiments, the WVE-003 composition is a chiral-controlled oligonucleotide composition.

[0016] In some embodiments, the WVE-003 composition is a chiral (e.g., stereochemically pure) HTT oligonucleotide composition capable of mediating allele-specific reduction of mHTT transcript levels, expression, and / or activity. WVE-003 can selectively target one isoform of HTT SNP rs362273 (sometimes referred to as SNP3). WVE-003 is fully complementary to the sequence (and surrounding sequence) of the SNP it targets. As described herein, in various embodiments, WVE-003 is fully complementary to mtHTT transcripts. In some embodiments, WVE-003 is fully complementary to mtHTT transcripts, but not to wtHTT transcripts. In some embodiments, mtHTT transcripts are associated with, or are more strongly associated with, a condition, disorder, or disease, such as Huntington's disease, than wtHTT transcripts. In some embodiments, mtHTT comprises 36, 37, 38, 39, 40, 45, 50 or more CAG repeats. As one of skill in the art will appreciate, complementarity to a transcript can be determined using the sequence of the gene from which the transcript is transcribed.

[0017] In some embodiments, WVE-003 can be used as a disease-modifying agent to treat subjects with Huntington's disease (HD). It can be provided as a stereochemically pure antisense oligonucleotide (ASO) that selectively targets mutant forms of the huntingtin (mHTT) gene transcript. In some embodiments, the WVE-003 composition is a stereochemically pure oligonucleotide composition that recognizes the disease-associated (e.g., mutant) allele of SNP rs362273 in the huntingtin gene, is effective in reducing the level, expression, and / or activity of the mHTT gene (or its gene product), and has the ability to mediate allele-specific knockdown of the mutant huntingtin (mHTT) gene.

[0018] In some embodiments, the present disclosure provides methods for reducing the level, expression, and / or activity of the mHTT gene (or its gene product), comprising administering to a subject an oligonucleotide at a dose or according to a dosage regimen as described herein. In some embodiments, the level of mHTT transcript is reduced. In some embodiments, the level of mHTT protein is reduced. In some embodiments, the level, expression, and / or activity of the wtHTT gene is not significantly reduced. In some embodiments, the determination is performed using a sample from the subject. In some embodiments, the subject in various methods herein is a human. In some embodiments, the determination is performed using a sample from a human subject susceptible to or suffering from a condition, disorder, or disease associated with mutant HTT (e.g., Huntington's disease). In some embodiments, the determination is performed using a patient sample. A "patient sample" is any biological specimen from a patient. The term sample includes, but is not limited to, biological fluids such as blood, serum, plasma, urine, cerebrospinal fluid (CSF), tears, saliva, lymph, dialysate, lavage fluid, semen, and / or other liquid samples, as well as cells and tissues of biological origin. In some preferred embodiments, the determination is performed using a CSF sample. In some embodiments, the determination is performed using a plasma sample. In some embodiments, the determination is performed using a blood sample. In some embodiments, the determination is performed at one or more suitable time points as will be understood by those skilled in the art, for example, about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or about 3, 4, 5, 6, 7, or 8 weeks, or about 3, 4, 5, or 6 months or more after administration of a dose, and before administration of the next dose, if any. In some embodiments, the determination is performed after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses have been administered. In some embodiments, the determination is for an individual subject.In some embodiments, the determination is performed on a population of subjects, for example, as is typically performed in a clinical trial or clinical application. In some embodiments, the population is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000, or more subjects in size. In some embodiments, the size is about 10 or more subjects. In some embodiments, the size is about 20 or more subjects. In some embodiments, the size is about 36 subjects. In some embodiments, the size is about 30 or more subjects. In some embodiments, the size is about 40 or more subjects. In some embodiments, the size is about 50 or more subjects. In some embodiments, the size is about 100 or more subjects. In some embodiments, the size is about 200 or more subjects. In some embodiments, the size is about 500 or more subjects. In some embodiments, the size is about 1000 or more subjects. In some embodiments, the reduction is about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more. In some embodiments, it is at least about 10% or more. In some embodiments, it is at least about 12% or more. In some embodiments, it is at least about 20% or more. In some embodiments, it is at least about 30% or more. In some embodiments, it is at least about 40% or more. In some embodiments, it is at least about 50% or more. As one of skill in the art will understand, in some embodiments, the mHTT gene or its product contains a sequence that is the same as or fully complementary to the WVE-003 sequence and contains an expanded CAG repeat (or a sequence encoded thereby), and the wild-type HTT gene or its product does not contain a sequence that is the same as or fully complementary to the WVE-003 sequence in HTT or rs362273 and contains fewer CAG repeats (or a sequence encoded by said repeats) (e.g., is not considered an expanded CAG repeat).

[0019] In some embodiments, the disclosure relates to any of a variety of methods involving the use of an HTT oligonucleotide or composition. In some embodiments, the HTT oligonucleotide is WVE-003. In some embodiments, the composition is a composition comprising WVE-003. In some embodiments, the composition is a WVE-003 composition, wherein substantially all of the oligonucleotide in the composition is WVE-003 (as one of skill in the art will appreciate, some other oligonucleotides may be present as impurities, but their levels can, and typically are, controlled in the provided compositions).

[0020] In some embodiments, the HTT oligonucleotide, e.g., WVE-003, or composition is administered or delivered to a subject at a dose of about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10-160 mg, about 10-150 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg.

[0021] As used herein, amounts of the present disclosure, e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, 10-170 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg, are typically equivalent amounts of oligonucleotide in free acid form, unless expressly noted otherwise. In some embodiments, WVE-003 is administered or delivered to a subject at a dose equivalent to about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10-160 mg, about 10-150 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg of WVE-003 free acid form. In some embodiments, the dose is equivalent to about 10 mg of WVE-003 free acid form. In some embodiments, the dose is equivalent to about 20 mg of WVE-003 free acid form. In some embodiments, the dose is equivalent to about 30 mg of WVE-003 free acid form. In some embodiments, the dose is equivalent to about 60 mg of WVE-003 free acid form. In some embodiments, the dose is equivalent to about 90 mg of WVE-003 free acid form. In some embodiments, the dose is equivalent to about 120 mg of WVE-003 free acid form. In some embodiments, the dose is equivalent to about 150 mg of WVE-003 free acid form.

[0022] In some embodiments, an HTT oligonucleotide or composition is administered or delivered to a subject at a dose of about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg. In some embodiments, WVE-003 is administered or delivered to a subject at a dose equivalent to about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg of WVE-003 free acid form. As described herein, WVE-003 may be administered or delivered in various forms, including pharmaceutically acceptable salt forms. In some embodiments, the composition administered to a subject comprises one or more forms of WVE-003, e.g., one or more pharmaceutically acceptable salt forms. In some embodiments, the composition is a liquid composition. In some embodiments, WVE-003 is dissolved in a suitable liquid, e.g., water or a suitable buffer as described herein, such as buffered saline, cerebrospinal fluid (CSF), or artificial cerebrospinal fluid (aCSF).

[0023] Techniques useful for preparing oligonucleotides and their products, e.g., WVE-003, are as described herein. In some embodiments, the oligonucleotide, e.g., WVE-003, is administered as a chiral-controlled oligonucleotide composition as described herein. In some embodiments, each chiral internucleotide linkage independently has a stereochemical purity of about 97%, 98%, 99%, or more. In some embodiments, the stereochemical purity of an oligonucleotide, e.g., WVE-003, in a composition can be represented by the product of the stereochemical purity (or stereochemical selectivity) at each chiral internucleotide linkage. In some embodiments, the stereochemical purity / stereochemical selectivity of a chiral internucleotide linkage can be determined by the stereochemical purity / stereochemical selectivity upon formation of the corresponding dimer comprising that chiral internucleotide linkage and the two nucleosides to which it is linked. In some embodiments, the stereochemical purity / stereochemical selectivity of a chiral internucleotide linkage can be determined by the stereochemical purity / stereochemical selectivity during oligonucleotide synthesis when the chiral internucleotide linkage is formed (e.g., by determining the product after the formation of the chiral internucleotide linkage but before the formation of the next internucleotide linkage). In some embodiments, the stereochemical purity / stereochemical selectivity of a chiral internucleotide linkage in a product oligonucleotide, such as WVE-003, is considered to be the stereochemical purity / stereochemical selectivity observed during dimer formation or oligonucleotide synthesis. In some embodiments, the stereochemical purity / stereochemical selectivity observed during dimer formation or oligonucleotide synthesis are the same or very close to each other. Those skilled in the art will understand that the stereochemical purity / stereochemical selectivity of each chiral internucleotide linkage can vary, but according to various techniques provided, it is consistently high (e.g., about 97%, 98%, 99% or more) for each chiral internucleotide linkage. In some embodiments, each chiral internucleotide linkage independently has a stereochemical purity of about 97%, 98%, 99% or more.In some embodiments, at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 internucleotide linkages independently have a stereochemical purity of about 98%, 99% or greater. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 internucleotide linkages independently have a stereochemical purity of about 99% or greater. In some embodiments, no more than 1, 2, 3, 4, or 5 independently have a stereochemical purity of less than about 98%. In some embodiments, no more than 1, 2, 3, 4, or 5 independently have a stereochemical purity of less than about 98%, but each is still independently greater than about 97%. In some embodiments, the stereochemical purity of WVE-003 is about 75% or greater. In some embodiments, the stereochemical purity of WVE-003 is about 80% or greater (e.g., as the product of the stereochemical purities of the corresponding dimers). In some embodiments, the stereochemical purity of WVE-003 is about 81% or greater (e.g., as the product of the stereochemical purity of the corresponding dimer). In some embodiments, the stereochemical purity of WVE-003 is about 82% or greater (e.g., as the product of the stereochemical purity of the corresponding dimer). In some embodiments, the stereochemical purity of WVE-003 is about 83% or greater (e.g., as the product of the stereochemical purity of the corresponding dimer). In some embodiments, the stereochemical purity of WVE-003 is about 84% or greater (e.g., as the product of the stereochemical purity of the corresponding dimer). In some embodiments, the stereochemical purity of WVE-003 is about 85% or greater (e.g., as the product of the stereochemical purity of the corresponding dimer). In some embodiments, the amount of oligonucleotide administered includes the amount of stereoisomers of the oligonucleotide (e.g., to the extent that they cannot be separated during manufacture). In some embodiments, the amount of oligonucleotide administered does not include other impurities, such as short oligonucleotides found during oligonucleotide synthesis (to the extent that the purification method used is capable of removing such impurities, as will be understood by those of skill in the art).

[0024] As those skilled in the art will understand, oligonucleotide can be administered in various forms, including one or more pharmaceutically acceptable salt forms.For example, in some embodiments, oligonucleotide is administered as the oligonucleotide and / or its salt dissolved in suitable solution, for example, water or suitable buffer system.In some embodiments, an amount of oligonucleotide includes all forms of oligonucleotide, and when expressed in weight units (for example, mg), this weight includes the weight of all forms of oligonucleotide, provided that all are converted to the weight of acid form (for example, for WVE-003, the free acid with molecular weight of 7257.94 is used). Alternatively or additionally, the amount of oligonucleotide may be expressed in molar units, which in some embodiments includes the number of moles of oligonucleotide in any form (e.g., about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg of WVE-003 corresponds to about 1.4, about 2.8, about 4.1, about 5.5, about 6.9, about 8.3, about 9.6, about 10.9, about 12.4, about 13.8, about 15.2, about 16.5, about 17.9, about 19.3, about 20.7, about 22.0, or about 23.1 umol of WVE-003).

[0025] As described herein, multiple doses may be administered at various suitable frequencies as described herein, for example, about every 3 weeks, about every 4 weeks, about every 8 weeks, about every 12 weeks, about once a month, about once every two months, or about once a quarter. In some embodiments, each dose is independently about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg of the free acid form of WVE-003. In some embodiments, the HTT oligonucleotide or composition is administered to a subject at a monthly dosage of about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg. In some embodiments, the HTT oligonucleotide or composition is administered to a subject at a dosage of about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg every 8 weeks. In some embodiments, the HTT oligonucleotide or composition is administered to a subject at a dosage of about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg every 12 weeks. In some embodiments, the provided technology provides long-lasting effects. In some embodiments, the dose is administered every two months or less frequently. In some embodiments, the dose is administered four times a year or less frequently.

[0026] In some embodiments, the present disclosure relates to methods of treating and / or preventing Huntington's disease in a subject (e.g., a patient, such as a human patient) in need thereof; methods of allele-specific knockdown of mutant Huntington's transcripts in a subject; and / or methods of reducing the severity and / or delaying the onset of one or more symptoms of Huntington's disease, comprising administering multiple doses of WVE-003, each of the doses independently being about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg of the free acid form of WVE-003. In some embodiments, in the methods of the disclosure, the amount of each dose remains constant (e.g., each dose the patient receives is about 30 mg; or each dose the patient receives is about 60 mg; or each dose the patient receives is about 90 mg; or each dose the patient receives is about 120 mg); in some embodiments, the amount of each dose varies over time (e.g., the patient may be maintained at a particular dose for one or more doses, and later the amount of each dose may be increased or decreased due to efficacy and / or safety or other issues). In some embodiments, multiple doses are administered, each independently about 30 mg WVE-003 free acid form. In some embodiments, the multiple doses are administered approximately monthly or approximately once every four weeks. In some embodiments, the doses are administered approximately monthly or approximately once every four weeks, except for the approximately two months or approximately eight weeks between the second and fourth doses. In some embodiments, the multiple doses are administered approximately once every eight weeks. For example, in some embodiments, the method comprises administering multiple doses approximately once every 8 weeks, each of which is independently equivalent to about 30 mg of WVE-003 in the free acid form. In some embodiments, the multiple doses are administered approximately once every 12 weeks. For example, in some embodiments, the method comprises administering multiple doses approximately once every 12 weeks, each of which is independently equivalent to about 30 mg of WVE-003 in the free acid form.In some embodiments, each dose is administered as a pharmaceutical composition as described herein. In some embodiments, the dose is administered at least about twice. In some embodiments, the dose is administered at least about three times. In some embodiments, the dose is administered at least about four times. In some embodiments, the interval between a dose and the immediately following dose is independently about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more. In some embodiments, the interval between each dose and the immediately following dose is independently about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months or more. In some embodiments, the interval is about 1 week. In some embodiments, the interval is about 2 weeks. In some embodiments, the intervals are about 3 weeks. In some embodiments, the intervals are about 4 weeks. In some embodiments, the intervals are about 5 weeks. In some embodiments, the intervals are about 6 weeks. In some embodiments, the intervals are about 7 weeks. In some embodiments, the intervals are about 8 weeks. In some embodiments, the intervals are about 9 weeks. In some embodiments, the intervals are about 10 weeks. In some embodiments, the intervals are about 9 weeks. In some embodiments, the intervals are about 11 weeks. In some embodiments, the intervals are about 9 weeks. In some embodiments, the intervals are about 12 weeks. In some embodiments, the intervals are about 1 month. In some embodiments, the intervals are about 2 months. In some embodiments, the intervals are about 3 months or longer. In some embodiments, the intervals are about 3, 4, 5, 6, 7, 8, or 9 months. In some embodiments, the intervals are about 3 months. In some embodiments, the intervals are about 6 months. In some embodiments, the intervals are about 9 months. In some embodiments, each interval is independently about 1 week or longer. In some embodiments, each interval is independently about 2 weeks or more, hi some embodiments, each interval is independently about 3 weeks or more.In some embodiments, each interval is independently about 4 weeks or longer. In some embodiments, each interval is independently about 5 weeks or longer. In some embodiments, each interval is independently about 6 weeks or longer. In some embodiments, each interval is independently about 7 weeks or longer. In some embodiments, each interval is independently about 8 weeks or longer. In some embodiments, each interval is independently about 9 weeks or longer. In some embodiments, each interval is independently about 10 weeks or longer. In some embodiments, each interval is independently about 11 weeks or longer. In some embodiments, each interval is independently about 12 weeks or longer. In some embodiments, each interval is independently about 1 month or longer. In some embodiments, each interval is independently about 2 months or longer. In some embodiments, each interval is independently about 3 months or longer. In some embodiments, each interval is independently about 3, 4, 5, 6, 7, 8, or 9 months. In some embodiments, each interval is independently about 1 month. In some embodiments, each interval is independently about 2 months. In some embodiments, each interval is independently about 3 months. In some embodiments, each interval is independently about 6 months. In some embodiments, each interval is independently about 9 months. In some embodiments, the intervals are all about the same. In some embodiments, one or more intervals are longer than the others. In some embodiments, one or more intervals are shorter than the others. In some embodiments, the dose administration frequency for multiple doses or all doses is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more. In some embodiments, the dose administration frequency for multiple doses or all doses is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or more. In some embodiments, one or more loading doses are administered. In some embodiments, no loading dose is administered.

[0027] In some embodiments, the use of WVE-003 in various methods is being tested in a clinical trial called the SELECT-HD Phase 1b / 2a trial. SELECT-HD is a Phase 1b / 2a multicenter, randomized, double-blind, placebo-controlled study evaluating the safety, tolerability, PK, pharmacodynamics (PD), and clinical efficacy of WVE-003 in adult patients with early-onset Huntington's disease (HD) who carry the targeted single nucleotide polymorphism (SNP) rs362273 (SNP3). Period 1 of the study evaluates a single ascending dose (SAD) of WVE-003. Period 2 evaluates a multiple ascending dose (MAD) of WVE-003. In some embodiments, subjects have early-onset Huntington's disease. In some embodiments, subjects are 25 years of age or older. In some embodiments, subjects are 60 years of age or younger.

[0028] In some embodiments, the oligonucleotide, e.g., WVE-003, or composition is administered intrathecally. In some embodiments, the intrathecal administration is intraspinal administration. In some embodiments, the methods described herein comprise administering an HTT oligonucleotide, e.g., WVE-003, or composition thereof, intrathecally in an amount described herein, e.g., about 10, about 20, about 30, about 40, about 50, about 60, about 70, about 80, about 90, about 100, about 110, about 120, about 130, about 140, about 150, or about 168 mg of the oligonucleotide (e.g., WVE-003) free acid form equivalent. In some embodiments, the methods described herein include administering an HTT oligonucleotide (e.g., WVE-003) or composition thereof intrathecally, each in an amount independently described herein, e.g., about 10, about 20, about 30, about 60, about 90, about 120, about 150, or about 168 mg of the oligonucleotide (e.g., WVE-003) free acid form equivalent, at a dose interval described herein, e.g., monthly.

[0029] In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition elicits a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is an amount (e.g., about 30, about 60, about 90, about 120, about 150, or about 168 mg of the free acid form) sufficient to treat, prevent, and / or delay the onset of Huntington's disease (e.g., at least one symptom of Huntington's disease) when administered to a subject suffering from or susceptible to Huntington's disease.

[0030] In some embodiments, the methods described herein comprise administering a therapeutically effective amount of an HTT oligonucleotide or an HTT oligonucleotide composition, hi some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is or comprises WVE-003.

[0031] In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is capable of mediating a clinically significant amount of allele-specific knockdown of a mutant HTT transcript. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition does not significantly reduce the amount of wild-type huntingtin transcript or its gene product. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition does not reduce the amount of wild-type huntingtin transcript or its gene product to a level associated with clinically manifested adverse events or side effects.

[0032] In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is sufficiently low to prevent or reduce the occurrence and / or reduce the severity of at least about one adverse event mediated by and / or associated with administration of the oligonucleotide or oligonucleotide composition to a subject. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is sufficient to mediate a clinically significant amount of allele-specific knockdown of a mutant HTT transcript in a subject, but is sufficiently low to prevent or reduce the occurrence and / or reduce the severity of at least about one adverse event mediated by and / or associated with administration of the oligonucleotide or oligonucleotide composition to a subject.

[0033] In some embodiments, the adverse event is an adverse effect. In some embodiments, the adverse event is mild, moderate, severe, or critical. In some embodiments, the adverse event is fever, headache, vomiting, or tachycardia. In some embodiments, the adverse event is, is measured by, or relates to an increase in pro-inflammatory markers (e.g., C-reactive protein and complement), a prolongation of aPTT, thrombocytopenia, a change in liver enzymes (e.g., AST and ALT), or a change in renal biomarkers (e.g., BUN, creatinine).

[0034] In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is sufficient to achieve a clinically significant peak plasma concentration of the HTT oligonucleotide or oligonucleotide composition. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 30 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 60 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 90 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 120 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 150 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 168 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is 30±25% mg. In some embodiments, the therapeutically effective amount of the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition is 60±25% mg. In some embodiments, the therapeutically effective amount of the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition is 90±25% mg. In some embodiments, the therapeutically effective amount of the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition is 120±25% mg. In some embodiments, the therapeutically effective amount of the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition is 150±25% mg. In some embodiments, the therapeutically effective amount of the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition is 168±25% mg.In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 30 mg, about 60 mg, about 90 mg, about 120 mg, or about 150 mg (independent of body weight, as the total central nervous system mass is similar in most adult humans). In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 30 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 60 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 90 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 120 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 150 mg. In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is about 168 mg. In some embodiments, the HTT oligonucleotide is WVE-003.

[0035] In some embodiments, the disclosure provides a method comprising administering to a subject an HTT oligonucleotide or HTT oligonucleotide composition in an amount equivalent to about 30, about 60, about 90, about 120, about 150, or about 168 mg of the oligonucleotide in free acid form.

[0036] In some embodiments, the present disclosure provides a method comprising administering to a subject an HTT oligonucleotide or HTT oligonucleotide composition at a dose of about 30, about 60, about 90, about 120, about 150, or about 168 mg, wherein the oligonucleotide is WVE-003. In some embodiments, the dose is administered about once a month or about every four weeks. In some embodiments, the dose is administered about once every two months or about every eight weeks. In some embodiments, the dose is administered about once every three months or about every 12 weeks. In some embodiments, the time between the first and second doses is about two months or about eight weeks, and each subsequent dose is administered about once a month or about every four weeks. In some embodiments, the time between the first and second doses is about three months or about twelve weeks, and each subsequent dose is administered about once every two months or about every eight weeks. In some embodiments, each dose is administered about once every two months or about every eight weeks. In some embodiments, each dose is administered approximately every two months or approximately every 12 weeks. In some embodiments, the doses are administered over a period of at least about 12 weeks. In some embodiments, the doses are administered over a period of at least about 16 weeks. In some embodiments, the doses are administered over a period of at least about 3 months. In some embodiments, the doses are administered over a period of at least about 4 months. In some embodiments, the patient is maintained on the same dose or a constant dose (e.g., each dose the patient receives is 30 mg; or each dose the patient receives is 60 mg; or each dose the patient receives is 90 mg; or each dose the patient receives is 120 mg). In some embodiments, the patient receives multiple doses of different amounts. For example, the patient receives one or more initial doses of a certain amount of HTT oligonucleotide or HTT oligonucleotide composition, and the patient's subsequent doses may be increased or decreased due to efficacy, safety, or other issues.

[0037] Among other things, the present disclosure recognizes the problem of providing oligonucleotides with high efficacy and reduced toxicity, and methods for using the same. In some embodiments, the present disclosure provides oligonucleotides, e.g., WVE-003, with reduced toxicity, as well as compositions and methods. In some embodiments, the provided technology provides reduced toxicity levels when about the same or equivalent amounts of oligonucleotide are delivered and / or when about the same or equivalent levels of desired effect and / or efficacy are achieved. In some embodiments, at about the same or equivalent or lower toxicity levels, more oligonucleotide, e.g., WVE-003, can be delivered and / or a higher level of desired effect and / or efficacy is achieved. In some embodiments, the reference oligonucleotide is a non-selective oligonucleotide. In some embodiments, the reference oligonucleotide has a different structure compared to WVE-003. In some embodiments, the reference oligonucleotide has a different structure but the same base sequence compared to WVE-003. In some embodiments, the reference oligonucleotide contains different modifications and / or patterns. In some embodiments, the reference oligonucleotide contains different internucleotide linkages and / or patterns. In some embodiments, the reference oligonucleotide contains a different bond stereochemistry or pattern. In some embodiments, the reference oligonucleotide is provided as a stereochemically random composition. In some embodiments, the reference oligonucleotide is provided as a chiral controlled oligonucleotide composition. In some embodiments, the reference oligonucleotide has low purity. In some embodiments, the reference oligonucleotide has low stereochemical purity. In some embodiments, the reference oligonucleotide is WV-1092. In some embodiments, the reference oligonucleotide is WV-2603.

[0038] In some embodiments, the present disclosure provides oligonucleotide compositions and methods that result in a reduced immune response. In some embodiments, the present disclosure recognizes that various toxicities induced by oligonucleotides may be related to cytokine and / or complement activation.

[0039] In some embodiments, the HTT oligonucleotide composition (e.g., WVE-003) is chirally controlled (e.g., stereochemically pure). In particular, a stereochemically random HTT oligonucleotide preparation contains multiple individual chemical entities that differ from each other, for example, in the stereochemistry of individual backbone chiral centers (e.g., phosphorothioates) within the HTT oligonucleotide chain. Due to the lack of control over the stereochemistry of backbone chiral centers, a stereochemically random HTT oligonucleotide preparation results in an uncontrolled (or stereochemically random) composition containing an indeterminate level of HTT oligonucleotide stereoisomers. Although these stereoisomers may have the same base sequence and / or chemical modification, they are different chemical entities at least largely because their backbone stereochemistry is different, and they may have different properties, such as activity, toxicity, distribution, etc., as demonstrated herein. In particular, the present disclosure provides chirally controlled compositions that are or include a specific stereoisomeric HTT oligonucleotide of interest; in contrast to non-chirally controlled compositions, chirally controlled compositions include a controlled level of a specific stereoisomeric HTT oligonucleotide, or in chirally controlled compositions, a controlled level of all oligonucleotides in the composition, or a controlled level of all oligonucleotides in the composition that share a specific base sequence (e.g., that of WVE-003), share a common bond phosphorus stereochemical configuration pattern (e.g., that of WVE-003). In some embodiments, a controlled level of all oligonucleotides sharing the same configuration as a form of WVE-003 is WVE-003. In some embodiments, the diastereomeric purity of WVE-003 is about or exceeds the controlled level.

[0040] In some embodiments, the controlled level is at least about 10%. In some embodiments, the controlled level is at least about 20%. In some embodiments, the controlled level is at least about 25%. In some embodiments, the controlled level is at least about 30%. In some embodiments, the controlled level is at least about 40%. In some embodiments, the controlled level is at least about 50%. In some embodiments, the controlled level is at least about 60%. In some embodiments, the controlled level is at least about 70%. In some embodiments, the controlled level is at least about 75%. In some embodiments, the controlled level is at least about 80%. In some embodiments, the controlled level is at least about 85%. In some embodiments, the controlled level is at least about 90%. In some embodiments, the controlled level is at least about 95%. In some embodiments, the controlled level is at least about 96%. In some embodiments, the controlled level is at least about 97%. In some embodiments, the controlled level is at least about 98%. In some embodiments, the regulated level is at least about 99%.

[0041] In some embodiments, the therapeutically effective amount of the HTT oligonucleotide, HTT oligonucleotide composition, or WVE-003 is about 30, about 60, about 90, about 120, about 150, or about 168 mg.

[0042] In some embodiments, the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition is or comprises WVE-003, which is a chiral controlled HTT oligonucleotide composition capable of mediating allele-specific reduction in mHTT transcript levels, expression and / or activity.

[0043] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 10 to about 168 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0044] In some embodiments, the HTT gene of a human subject is (A) heterozygous at the CAG repeat (wherein one allele contains a deleterious CAG repeat expansion and the other allele does not), and (B) heterozygous at the SNP targeted by the HTT oligonucleotide, where the HTT oligonucleotide has the ability to distinguish between two alleles of the same SNP (e.g., because of differences in the sequences of the SNP alleles); and (C) the deleterious CAG repeat is on the same chromosome as the SNP allele targeted by the HTT oligonucleotide; and such a configuration is said to be such that the CAG repeat expansion and the mutant SNP allele are in phase, and the HTT gene or mutation thereof can be described as being amenable to allele-specific knockdown.

[0045] In some embodiments, the mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated is a mutant HTT gene comprising a deleterious mutation (e.g., a CAG repeat expansion), where the deleterious mutation is on the same chromosome as (e.g., in phase with or in phase with) a particular allele of a SNP targeted by a particular HTT oligonucleotide (e.g., WVE-003), and where targeting of the SNP allele with the oligonucleotide also targets the deleterious mutation.

[0046] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 30 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0047] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 60 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0048] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 90 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0049] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 120 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0050] In some embodiments, the subject is determined to express an mtHTT transcript that is fully complementary to the sequence of the administered oligonucleotide, including at the SNP site (e.g., rs362273). In some embodiments, the subject is determined to have an mtHTT allele that is identical to or includes a sequence that is fully complementary to the sequence of the administered oligonucleotide, including at the SNP site (e.g., rs362273). (As one skilled in the art will understand, it may be appropriate to consider U and T to be the same) in the administered oligonucleotide. In some embodiments, the subject is homozygous for the SNP site and / or wild-type or mutant HTT. In some embodiments, the subject is heterozygous for the SNP site and / or wild-type or mutant HTT. In some embodiments, the subject expresses an mtHTT transcript that is fully complementary to the administered oligonucleotide and a wtHTT transcript that is not fully complementary to the administered oligonucleotide. In some embodiments, the subject has an mtHTT allele that contains the same sequence as, or is fully complementary to, the sequence of the administered oligonucleotide (as one of skill in the art will understand, it may be appropriate to consider U and T the same), and a wtHTT allele that does not contain the same sequence as, or is fully complementary to, the sequence of the administered oligonucleotide (as one of skill in the art will understand, it may be appropriate to consider U and T the same). In some embodiments, the transcript, protein, and / or activity level of the mtHTT allele is further reduced compared to wtHTT. An mtHTT allele that is fully complementary to the administered oligonucleotide, and a wtHTT transcript that is not fully complementary to the administered oligonucleotide. Various techniques are available and can be utilized in accordance with the present disclosure to detect and / or determine whether specific isoforms of a SNP are on the same allele or transcript.

[0051] In some embodiments, the present disclosure provides a method for determining whether a subject's treatment is suitable for administration of WVE-003 (or a salt form thereof), comprising: i) determining the presence of a mutant allele versus a wild-type allele in patients with the target single nucleotide polymorphism (SNP) rs362273 (SNP3); ii) comparing the level of the mutant allele in patients with the target single nucleotide polymorphism (SNP) rs362273 (SNP3) of step i) with one or more reference samples or reference values; iii) determining whether a subject is likely to be or is suitable for treatment with WVE-003, where the subject is suffering from or at risk of developing a neurodegenerative disorder, such as Huntington's disease; and iv) administering to the subject an effective amount of WVE-003. The present invention provides a method comprising:

[0052] In some embodiments, the present disclosure provides for the use of a phasing assay to detect the presence of a mutant allele versus a wild-type allele in patients with the target single nucleotide polymorphism (SNP) rs362273 (SNP3) to determine whether treatment with WVE-003 is appropriate.

[0053] Various techniques can be used to determine whether a certain SNP allele is on the same chromosome as a disease-related sequence, for example, the CAG repeat expansion of HTT. Typically, when the SNP allele and the CAG repeat expansion are on the same chromosome, the HTT oligonucleotide that targets this SNP allele can also "target" the disease-related CAG repeat expansion, thereby reducing the expression, level and / or activity of the HTT allele with its associated mutation.In this way, for example, HTT oligonucleotide can be used to treat HTT-related disorders such as Huntington's disease.Therefore, the HTT oligonucleotide that targets SNP can preferentially reduce the expression, level and / or activity of the mutant allele of HTT compared to the wild-type allele.

[0054] Humans, among other organisms, are diploid, and phasing techniques are desirable for determining the linkage of alleles of genetic loci on the same or different chromosomes. The corresponding chromosomal sequences are known as haplotypes. The process of determining which alleles are on which chromosomes is known as phasing, haplotype phasing, or haplotyping. Phasing information is useful in a variety of other applications in patient stratification, forensics, and the treatment of HTT-related diseases and disorders, such as Huntington's disease. For more general information regarding phasing, see, e.g., Twehey et al. 2011 Nat. Rev. Genet. 12:215-223; and Glusman et al. 2014 Genome Med. 6:73.

[0055] Phasing data can be important in allele-specific therapy for diseases such as Huntington's disease. In some diseases, genetic lesions such as deleterious repeats, deletions, insertions, inversions, or other mutations, such as expanded CAG repeats in mutant (and disease-associated) HTT alleles, have been identified. In some patients, one allele of a gene such as HTT may contain a disease-associated mutation at a certain locus, while the other allele is normal, wild-type, or otherwise has no or low disease association. In some embodiments, allele-specific therapy can target an HTT allele containing a disease-associated mutation at a specific locus, such as a CAG repeat expansion (or an expanded CAG chain), but by targeting a different locus on the mutant allele rather than directly targeting that locus. As a non-limiting example, allele-specific therapy can target an allele that contains a disease-associated mutation at a genetic locus by targeting a different locus of the same allele, such as a SNP (single nucleotide polymorphism) in the same gene.

[0056] For example, some disease-related genetic lesions may be difficult to target, or in other cases, may not be easily suitable for targeting. For example, some genes, such as mutant HTT, contain repeats (e.g., trinucleotide or tetranucleotide repeats); in some cases, such as Huntington's disease, a small number of repeats is not disease-related, but an abnormally large number of repeats, i.e., repeat expansion, is disease-related. Because repeats exist in both wild-type alleles and mutant alleles, it may be difficult to directly target disease-related repeats. However, if a certain SNP mutation exists on the same allele as the disease-related repeat expansion, but does not exist in the wild-type allele, the SNP mutation can be used to target allele-specific therapy, which targets the mutant allele but not the wild-type allele.

[0057] As a non-limiting example, phasing data for an individual indicates whether a particular SNP is in phase with a lesion (e.g., on the same chromosome or transcript) and therefore whether that SNP can be targeted with a therapeutic nucleic acid. A therapeutic agent can then target the mutant gene while not targeting the wild-type allele. Obtaining phasing data to target only the mutant allele can be particularly useful in cases where expression of the wild-type allele is essential.

[0058] As another non-limiting example, phasing information is useful when an individual is known to have both a wild-type allele and a mutant allele at each of two loci on the same gene. The phasing information will reveal whether both copies of the gene each have one mutant allele, or whether one copy of the gene has two mutations while the other is wild-type at both alleles.

[0059] In some embodiments, the present disclosure provides various methods for, among other things, phasing loci on a nucleic acid template. As a non-limiting example, the present disclosure provides methods for phasing a locus, such as a genetic lesion (such as an inversion, fusion, deletion, insertion, or other mutation) on a chromosome and another locus (such as a SNP), where the two loci can be in the same gene or in different genes.

[0060] In a non-limiting example, an exemplary patient may have Huntington's disease, which is associated with a mutation in the huntingtin gene (HTT) that contains an excessive number of repeats (e.g., a repeat expansion) of the sequence CAG. In some embodiments, the patient may be under consideration for treatment with an allele-specific therapeutic agent (e.g., an antisense oligonucleotide or an RNAi agent) that recognizes a specific allelic variation, such as a SNP, at a locus in the HTT gene (which is outside the repeat expansion). If phasing reveals that the same chromosome in the patient contains both the repeat expansion and the specific allelic variation (e.g., an SNP) at the locus recognized by the allele-specific therapeutic agent, then the patient is eligible for treatment with the allele-specific therapeutic agent.

[0061] Various phasing methods are described in, but not limited to, WO 2018 / 022473; and Berger et al. 2015 Res. Comp. Mol. Biol. 9029:28-29; Castel et al. 2015 Genome Biol. 16:195; Castel et al. 2016 phASER: Long range phasing and haplotypic expression from RNA sequencing, doi:http: / / dx.doi.org / 10.1101 / 039529; Delaneau et al. 2012 Nat. Methods 9:179-181; Garg et al. 2016 Read-Based Phasing of Related Individuals; Hickey et al. 2011 Genet. Select. Evol. 43:12; Kuleshov et al. 2014 Nat. Biotech. 32:261-266; Laver et al. 2016 Nature Scientific Reports | 6:21746 | DOI:10.1038 / srep21746; O'Connell et al. 2014 PLoS ONE 10:e1004234; Regan et al. 2015 PLoS ONE 10:e0118270; Roach et al. 2011 Am. J. Hum. Genet. 89:382-397; and Yang et al. 2013 Bioinformatics 29:2245-2252. In some embodiments, phasing can utilize sequencing, particularly sequencing capable of generating long single reads.

[0062] One example of a phasing assay that can be suitably used to detect mutant alleles in patients is allele-specific PCR or allele-specific long-range PCR. Such an assay, using allele-specific primers, can detect mutations in nucleic acid sequences in the presence of wild-type mutations. Allele-specific PCR is a technique for selectively amplifying and detecting mutations in nucleic acid sequences present in a PCR reaction mixture. Allele-specific PCR uses at least one "allele-specific primer." The term "allele-specific" primer generally refers to a primer whose extension occurs in a PCR reaction only when a specific mutation in a nucleic acid sequence is present in the reaction mixture. In other words, the allele-specific primer is designed to distinguish between nucleic acid mutations and selectively amplify nucleic acid templates containing the mutation to be detected.

[0063] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 150 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0064] In some embodiments of the methods described herein, the nucleic acid sequence is detected by a suitable method, such as quantitative amplification and / or nucleic acid sequencing. Quantitative amplification methods are disclosed, for example, in U.S. Patent Nos. 5,210,015; 5,804,375; 6,127,155; 6,180,349; 6,033,854; and 5,972,602, as well as, for example, Holland et al., Proc. Natl. Acad. Sci. 88:7276-7280 (1991); Gibson et al., Genome Research 6:995-1001 (1996); DeGraves et al., Biotechniques 34(1):106-10, 112-5 (2003); Deiman B, et al., Mol. Biotechnol. 20(2):163-79 (2002). Amplification can be monitored in "real time." Although standard Sanger dideoxy or other conventional nucleotide sequencing methods can be used, sequencing can be particularly effective when using high-throughput sequencing, such as "next-generation sequencing" methods such as HiSeq™, MiSeq™, or Genome Analyzer (each available from Illumina), SOLiD™ or Ion Torrent™ (each available from Life Technologies), and 454™ sequencing (from Roche Diagnostics). For example, high-throughput sequencing allows for rapid sequencing of a genome or large portions of a genome through parallel sequencing reactions using multiple templates and multiple primers.For example, International Publication Nos. WO 03 / 004690, WO 03 / 054142, WO 2004 / 069849, WO 2004 / 070005, WO 2004 / 070007, WO 2005 / 003375, WO 00 / 06770, WO 00 / 27521, WO 00 / 58507, WO 01 / 23610, WO 01 / 57248, WO 01 / 57249, WO 02 / 061127, ...61128, WO 03 / 061129, WO 03 / 062130, WO 03 / 062140, WO 03 / 062150, WO 03 / 062160, WO 03 / 062171, WO 03 / 062182, WO 03 / 062193, WO 03 / 062194, WO 03 / 062195, WO 03 / 062196, WO 03 See Publication No. 03 / 016565, WO 03 / 048387, WO 2004 / 018497, WO 2004 / 018493, WO 2004 / 050915, WO 2004 / 076692, WO 2005 / 021786, WO 2005 / 047301, WO 2005 / 065814, WO 2005 / 068656, WO 2005 / 068089, WO 2005 / 078130, and Seo, et al, Proc. Natl Acad. Sci. USA (2004) 101:5488-5493. In some embodiments, amplicon is sequenced by one of the methods selected from base incorporation method, for example, pyrosequencing method (US Pat. No. 6,274,320, US Pat. No. 6,258,568 and US Pat. No. 6,210,891); hydrogen ion detection method (ISFET) (for example, US Pat. No. 8,262,900) or dye terminator detection method (US Pat. No. 7,835,871, US Pat. No. 8,244,479, US Pat. No. 8,315,817 and US Pat. No. 8,412,467).Deep sequencing technology and equipment (for example, technology and equipment that can read digital sequence) can also be used. Without limitation, examples of instruments include the GS line of instruments (454 Life Sciences, Branford, Conn.); ION PROTON™ and PGM™ (Life Technologies, Grand Island, NY); HISEQ™ and MISEQ™ (Illumina, San Diego, Calif.), or any improvements and modifications thereof.In some embodiments, the sequencing technology is or includes long-read sequencing. In some embodiments, the long-read sequencing covers two or more sequence elements, for example, in some embodiments, one is a SNP and the other is a mutation (e.g., a point mutation, a CAG repeat, etc.).

[0065] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 168 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0066] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 30 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0067] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 60 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0068] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 90 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0069] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 120 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0070] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 150 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0071] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 168 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0072] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 30 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0073] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 60 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0074] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 90 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0075] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 120 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0076] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 150 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0077] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 168 mg, thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0078] In some embodiments, the disclosure relates to a method of delaying the onset of and / or reducing the severity of symptoms of Huntington's disease in a subject with Huntington's disease who has a mutant HTT gene that includes a mutation where allele-specific knockdown of the mutant HTT gene is indicated, the method comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 30 mg.

[0079] In some embodiments, the disclosure relates to a method of delaying the onset of and / or reducing the severity of symptoms of Huntington's disease in a subject with Huntington's disease who has a mutant HTT gene that includes a mutation where allele-specific knockdown of the mutant HTT gene is indicated, the method comprising administering to the subject WVE-003 at a dose of about 60 mg.

[0080] In some embodiments, the disclosure relates to a method of delaying the onset of and / or reducing the severity of symptoms of Huntington's disease in a subject with Huntington's disease who has a mutant HTT gene that includes a mutation where allele-specific knockdown of the mutant HTT gene is indicated, the method comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 90 mg.

[0081] In some embodiments, the disclosure relates to a method of delaying the onset of and / or reducing the severity of symptoms of Huntington's disease in a subject with Huntington's disease who has a mutant HTT gene that includes a mutation where allele-specific knockdown of the mutant HTT gene is indicated, the method comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 120 mg.

[0082] In some embodiments, the disclosure relates to a method of delaying the onset of and / or reducing the severity of symptoms of Huntington's disease in a subject with Huntington's disease who has a mutant HTT gene that includes a mutation where allele-specific knockdown of the mutant HTT gene is indicated, the method comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 150 mg.

[0083] In some embodiments, the disclosure relates to a method of delaying the onset of and / or reducing the severity of symptoms of Huntington's disease in a subject with Huntington's disease who has a mutant HTT gene that includes a mutation where allele-specific knockdown of the mutant HTT gene is indicated, the method comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 168 mg.

[0084] As described herein, amounts of WVE-003 typically include any form of WVE-003 administered, but are all calculated in terms of the acid form.

[0085] In some embodiments, the present disclosure relates to the method of any of the preceding embodiments, wherein the subject is administered a steroid at least about 1 month prior to the first dose of WVE-003.

[0086] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the subject is administered WVE-003 approximately monthly for at least about four months.

[0087] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the subject is administered WVE-003 approximately monthly for at least about 8 months.

[0088] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the subject is administered the oligonucleotide approximately monthly for at least about 12 months.

[0089] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the subject is administered the oligonucleotide approximately monthly for at least about 16 months.

[0090] In some embodiments, the present disclosure relates to the method of any of the preceding embodiments, wherein the subject is administered the oligonucleotide approximately monthly for at least about 48 months.

[0091] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the oligonucleotide is delivered intrathecally.

[0092] In some embodiments, the present disclosure relates to a method of any of the preceding embodiments, further comprising identifying a mutation in the HTT gene of interest that is amenable to allele-specific knockdown of the mutant HTT gene or its gene product transcript.

[0093] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation.

[0094] In some embodiments, the method includes a lyophilization step (eg, freeze-drying or freeze-drying under vacuum).

[0095] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation that is reconstituted from a lyophilized (e.g., freeze-dried) preparation of the oligonucleotide.

[0096] In some embodiments, the present disclosure relates to lyophilized (eg, freeze-dried) preparations of WVE-003.

[0097] In some embodiments, the present disclosure relates to the method of any of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation, and the liquid formulation comprises the oligonucleotide, sodium chloride, and water.

[0098] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation, and the liquid formulation is reconstituted with a sodium chloride solution from a lyophilized preparation.

[0099] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation, and the liquid formulation is reconstituted with 0.9% sodium chloride solution from a lyophilized preparation.

[0100] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation, and the liquid formulation is reconstituted with a sterile sodium chloride solution from a lyophilized preparation.

[0101] In some embodiments, the present disclosure relates to a method as in any of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation, and the liquid formulation is reconstituted with 0.9% sterile sodium chloride from a lyophilized preparation.

[0102] In some embodiments, the lyophilized preparation of WVE-003 is a dry solid (or a dried solid).

[0103] In some embodiments, the lyophilized preparation of WVE-003 is a dry powder.

[0104] In some embodiments, the lyophilized preparation of WVE-003 is a dry powder prepared by lyophilization of a liquid formulation of WVE-003 in water.

[0105] In some embodiments, the lyophilized preparation of WVE-003 is a dry powder contained in a single-use, clear glass vial.

[0106] In some embodiments, the lyophilized preparation of WVE-003 is approximately 20 mg of dry powder in a vial.

[0107] In some embodiments, the lyophilized preparation of WVE-003 is a dry powder in a 10 mL vial.

[0108] In some embodiments, the lyophilized preparation of WVE-003 is approximately 20 mg of dry powder in a 10 mL vial.

[0109] In some embodiments, the subject is administered a steroid prior to the first dose of WVE-003.

[0110] In some embodiments, the subject is administered a steroid at least about one month prior to the first dose of WVE-003.

[0111] In some embodiments, the subject is administered a dose of WVE-003 approximately once a month or approximately every four weeks.

[0112] In some embodiments, the subject is administered a dose of WVE-003 approximately once every two months or approximately every eight weeks.

[0113] In some embodiments, the subject is administered a dose of WVE-003 approximately once a month or approximately every four weeks, except that the time between the first and second doses is about two months or about eight weeks, and each subsequent dose is administered approximately once a month or approximately every four weeks.

[0114] In some embodiments, the subject is administered a dose of WVE-003 approximately every two months or approximately every eight weeks, except that the time between the first and second doses is about three months or about twelve weeks, and each subsequent dose is administered approximately every two months or approximately every eight weeks.

[0115] In some embodiments, the subject is administered WVE-003 approximately every 8 weeks for at least about 16 weeks.

[0116] In some embodiments, the subject is administered WVE-003 approximately every 12 weeks for at least about 12 weeks.

[0117] In some embodiments, the subject is administered WVE-003 approximately monthly for at least about four months.

[0118] In some embodiments, the subject is administered WVE-003 approximately monthly for at least about 8 months.

[0119] In some embodiments, the subject is administered WVE-003 approximately monthly for at least about 12 months.

[0120] In some embodiments, the subject is administered WVE-003 approximately monthly for at least about 16 months.

[0121] In some embodiments, the subject is administered WVE-003 approximately monthly for at least about 48 months.

[0122] In some embodiments, the subject is administered a steroid prior to the first dose of the HTT oligonucleotide or HTT oligonucleotide composition.

[0123] In some embodiments, the subject is administered a steroid at least about one month prior to the first dose of the HTT oligonucleotide or HTT oligonucleotide composition.

[0124] In some embodiments, the subject is administered hydrocortisone and / or acetaminophen within 24 hours of administration of the HTT oligonucleotide or HTT oligonucleotide composition.

[0125] In some embodiments, the subject is administered an HTT oligonucleotide or HTT oligonucleotide composition approximately monthly for at least about 8 months.

[0126] In some embodiments, the subject is administered an HTT oligonucleotide or HTT oligonucleotide composition approximately monthly for at least about 12 months.

[0127] In some embodiments, the subject is administered an HTT oligonucleotide or HTT oligonucleotide composition approximately monthly for at least about 16 months.

[0128] In some embodiments, the subject is administered an HTT oligonucleotide or HTT oligonucleotide composition approximately monthly for at least about 48 months.

[0129] In some embodiments, the method further comprises identifying a mutation in the subject's HTT gene for which an allele-specific reduction in mHTT transcript level, expression and / or activity is indicated.

[0130] In some embodiments, the method further comprises identifying a mutation in the HTT gene of interest that is amenable to allele-specific knockdown of mutant HTT transcripts.

[0131] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an HTT oligonucleotide or HTT oligonucleotide composition of the present disclosure (eg, WVE-003) and a pharmaceutically acceptable carrier.

[0132] In some embodiments, the present disclosure provides pharmaceutical compositions comprising the HTT oligonucleotides or HTT oligonucleotide compositions (e.g., WVE-003) of the present disclosure, which can be utilized in pharmaceutical compositions by combining the oligomeric compounds with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate-buffered saline (PBS). PBS is a suitable diluent for use in compositions to be delivered parenterally. Thus, in certain embodiments, the methods described herein employ pharmaceutical compositions comprising the HTT oligonucleotides or HTT oligonucleotide compositions (e.g., WVE-003) of the present disclosure and a pharmaceutically acceptable diluent. In certain embodiments, the pharmaceutically acceptable diluent is PBS. In certain embodiments, the pharmaceutically acceptable diluent is artificial CSF (aCSF).

[0133] In certain embodiments, the pharmaceutical composition is both administered directly into the CSF (eg, IT and / or ICV injection and / or infusion) and administered systemically.

[0134] In some embodiments, the present disclosure provides a method for allele-specific knockdown of a target HTT transcript, the method comprising administering an HTT oligonucleotide composition of the present disclosure. In some embodiments, the present disclosure provides a method for reducing the level of an HTT transcript or its product, the method comprising administering an HTT oligonucleotide composition of the present disclosure. A method for treating Huntington's disease, the method comprising administering to a subject susceptible to or suffering from Huntington's disease a composition described in the present disclosure.

[0135] In some embodiments, the present disclosure provides a method of treating Huntington's disease, the method comprising administering to a subject susceptible to or suffering from Huntington's disease a composition comprising any of the HTT oligonucleotides disclosed herein.

[0136] In some embodiments, the present disclosure provides methods for treating Huntington's disease, comprising: (a) administering to a subject susceptible to or suffering from Huntington's disease a composition comprising any of the HTT oligonucleotides disclosed herein; and (b) administering to the subject an additional therapy capable of preventing, treating, ameliorating, or slowing the progression of Huntington's disease. In some embodiments, the present disclosure provides methods comprising administering WVE-003 to a subject, wherein the subject is determined to have a genetic sequence that is identical to or completely complementary to the nucleotide sequence of WVE-003. In some embodiments, the present disclosure provides methods comprising administering WVE-003 to a subject, wherein the subject is determined to have a genetic sequence that includes a sequence that is identical to or completely complementary to the nucleotide sequence of WVE-003 and an expanded CAG repeat, or a sequence encoding such an expanded CAG repeat. In some embodiments, the present disclosure provides methods comprising administering WVE-003 to a subject, wherein the subject is determined to have a gene sequence encoding a transcript that contains an expanded CAG repeat in HTT and is perfectly complementary to the base sequence of WVE-003. In some embodiments, the present disclosure provides methods comprising administering WVE-003 to a subject, wherein the subject is determined to express an HTT transcript that contains an expanded CAG repeat and is perfectly complementary to the base sequence of WVE-003.

[0137] The present disclosure also provides various formulations of HTT oligonucleotides or HTT oligonucleotide compositions, any of which can be used in any of the methods described herein.

[0138] In some embodiments, any of the methods described herein reduces the level, expression and / or activity of mutant HTT (eg, transcripts and / or its gene products) by at least about 5%.

[0139] In some embodiments, any of the methods described herein reduces the level, expression and / or activity of mutant HTT (eg, transcripts and / or its gene products) by at least about 10%. [Brief explanation of the drawings]

[0140] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] Illustrative flow diagram of the WVE-003 drug substance manufacturing process, including in-process controls. [Figure 2] An exemplary manufacturing process flow diagram for WVE-003 drug product. Abbreviations: DS: Drug Substance; IPC: In-Process Control; IPT: In-Process Testing. [Figure 3] Exemplary schematic of study design. Abbreviations: DEC = Dose Escalation Committee; MAD: Multiple Ascending Dose; N: Number of Patients; PxCx: Period x Cohort x; SAD: Single Ascending Dose. If aP1C2 is not performed, then P2C1 will enroll 6 new patients (2:1 active:placebo). [Figure 4] Blinded CSF PK data comparing doses of WVE-003 with doses of WVE-120101 (WV-1092) and WVE-120102 (WV-2603). [Figure 5A] The provided technology can reduce mHTT protein levels without reducing wtHTT protein levels in human subjects. The figures show the specific percent changes from baseline in CSF mHTT and wtHTT protein by Day 85 in certain subjects administered a single dose of placebo (aCSF), 30 mg of WVE-003, or 60 mg of WVE-003, respectively. As demonstrated, single doses of 30 mg and 60 mg of WVE-003 resulted in sustained mHTT reductions in several human subjects (A). A repeated measures mixed model was used to plot geometric mean ratios relative to baseline. Two bars (representing 95% CI) are presented for each time point: solid lines for WVE-003 (30 mg or 60 mg), and dashed lines for placebo. [Figure 5B]The provided technology can reduce mHTT protein levels without reducing wtHTT protein levels in human subjects. Figures 1A and 1B show the specific percent changes from baseline in CSF mHTT and wtHTT protein by day 85 in certain subjects administered a single dose of placebo (aCSF), 30 mg of WVE-003, or 60 mg of WVE-003, respectively. As demonstrated, in some human subjects, single doses of 30 mg and 60 mg of WVE-003 did not reduce wtHTT protein levels (B). A repeated measures mixed model was used to display the geometric mean ratios relative to baseline. Two bars (representing 95% CI) are presented for each time point: solid lines for WVE-003 (30 mg or 60 mg), and dashed lines for placebo. [Figure 5C] The provided technology can reduce mHTT protein levels without reducing wtHTT protein levels in human subjects. Figures 1 and 2 show the specific percent changes from baseline in CSF mHTT and wtHTT protein by Day 85 in specific subjects receiving placebo (aCSF), a single 30 mg dose of WVE-003, or a single 60 mg dose of WVE-003, respectively. A mean reduction of 22% (median reduction of 30%) in mHTT was observed for the pooled single 30 mg and 60 mg doses (C). A repeated measures mixed model was used to display the geometric mean ratios relative to baseline. Two bars (representing 95% CI) are presented for each time point: solid lines for WVE-003 (30 mg or 60 mg), and dashed lines for placebo. [Figure 5D]The provided technology can reduce mHTT protein levels without reducing wtHTT protein levels in human subjects. Figures 1 and 2 show the specific percent changes from baseline in CSF mHTT and wtHTT protein by Day 85 in specific subjects receiving placebo (aCSF), a single 30 mg dose of WVE-003, or a single 60 mg dose of WVE-003, respectively. For the pooled single 30 mg and 60 mg doses, on the other hand, no reduction in wtHTT was observed (D). A repeated measures mixed model was used to display the geometric mean ratios relative to baseline. Two bars (representing 95% CI) are presented for each time point: solid lines for WVE-003 (30 mg or 60 mg), and dashed lines for placebo. [Figure 6] Illustrative schematic of study design. DETAILED DESCRIPTION OF THE INVENTION

[0141] definition As used herein, the following definitions shall apply unless otherwise indicated. For purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th Ed. In addition, general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999, and "March's Advanced Organic Chemistry", 5th Ed., Ed.: Smith, M.B. and March, J., John Wiley & Sons, New York: 2001.

[0142] As used herein in this disclosure, unless the context clearly indicates otherwise, (i) the terms "a" or "an" may be understood to mean "at least one"; (ii) the term "or" may be understood to mean "and / or"; (iii) the terms "comprising," "comprise," "including" (whether or not used in conjunction with "limited to"), and "include" (whether or not used in conjunction with "limited to") may be understood to encompass the listed element or step, whether presented alone or with one or more additional elements or steps; (iv) the term "another" may be understood to mean at least an additional / second one or more; and (v) when ranges are provided, the endpoints are included.

[0143] Unless otherwise specified, descriptions of oligonucleotides and their components (e.g., base sequence, sugar modification, internucleotide linkage, bond phosphorus stereochemistry, etc.) are from 5' to 3'. Unless otherwise specified, the oligonucleotides described herein may be provided and / or utilized in salt form, particularly pharmaceutically acceptable salt form. As one of skill in the art will understand upon reading this disclosure, in some embodiments, oligonucleotides may be provided as salts, such as, but not limited to, sodium or potassium salts. As one of skill in the art will understand, in some embodiments, individual oligonucleotides in a composition may be considered to be of the same chemical constitution and / or structure, even though a particular such oligonucleotide may be in one or more different salt forms at a particular moment within such composition (e.g., a liquid composition) (and an oligonucleotide chain, which may be dissolved, may exist as an anionic form, for example, when in a liquid composition). For example, one skilled in the art will understand that at a given pH, an individual internucleotide bond along an oligonucleotide chain may be in the acid (H) form or one of several possible salt forms (e.g., a sodium salt or a salt of another cation, depending on which ions may be present in the formulation or composition), and that to the extent that the acid form (e.g., with all cations replaced by H, if present) is the same chemical constitution and / or structure, it may be appropriate to consider such individual oligonucleotides to be of the same chemical constitution and / or structure.

[0144] Approximately: As used herein, the term "about" or "approximately" in connection with a number can be understood to allow for standard deviation as would be understood by one of ordinary skill in the art. In some embodiments, the term "about" or "approximately" in connection with a number generally includes numbers that fall within 5%, 10%, 15%, or 20%, 25%, or 30% (greater or lesser than that number) in either direction of that number, unless otherwise specified or otherwise clear from the context.

[0145] Dose administration regimen: As used herein, a "dose administration regimen" or "treatment regimen" refers to a set of unit doses (typically two or more doses) administered individually, typically spaced apart, to a subject. In some embodiments, a given therapeutic agent has a recommended dose administration regimen that may involve one or more doses. In some embodiments, a dose administration regimen includes multiple doses, each separated by the same amount of time; in some embodiments, a dose administration regimen includes multiple doses with at least two different time intervals between the individual doses. In some embodiments, all doses within a dose administration regimen are the same unit dose size. In some embodiments, different doses within a dose administration regimen are different sizes. In some embodiments, a dose administration regimen includes a first dose of a first dose size, followed by one or more additional doses of a second dose size that is different from the first dose size. In some embodiments, a dose administration regimen includes a first dose of a first dose size, followed by one or more additional doses of a second dose size that is the same as the first dose size.

[0146] Pharmaceutical composition: As used herein, the term "pharmaceutical composition" refers to an active agent formulated with one or more pharmaceutically acceptable carriers. In some embodiments, the active agent is present in a unit dose size suitable for administration in a treatment regimen that exhibits a statistically significant probability of achieving a controlled therapeutic effect when administered to a relevant population.

[0147] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit-to-risk ratio.

[0148] Pharmaceutically acceptable carrier: As used herein, the term "pharmaceutically acceptable carrier" means a pharmaceutically acceptable material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, or solvent, that encapsulates a material, involved in the transport or delivery of a compound of interest from one organ or part of the body to another. Each carrier must be "acceptable" in the sense of being compatible with the other ingredients of the formulation and not deleterious to the subject.

[0149] Pharmaceutically acceptable salt: As used herein, the term "pharmaceutically acceptable salt" refers to a salt of such a compound that is suitable for use in a pharmaceutical context, i.e., a salt that is suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic reaction, etc., within the scope of sound medical judgment, and that is commensurate with a reasonable risk-to-benefit ratio. Pharmaceutically acceptable salts are well known in the art. For example, SM Berge, et al., describe pharmaceutically acceptable salts in detail in J. Pharmaceutical Sciences, 66:1-19 (1977). In some embodiments, provided compounds contain two or more acidic groups; for example, provided oligonucleotides may contain two or more acidic groups (e.g., in natural phosphate linkages and / or modified internucleotide linkages). In some embodiments, pharmaceutically acceptable salts of such compounds, or salts in general, contain two or more cations, which may be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or salt generally), each acidic group having sufficient acidity is independently present in its salt form (e.g., in an oligonucleotide comprising a natural phosphate linkage and a phosphorothioate internucleotide linkage, each of the natural phosphate linkage and the phosphorothioate internucleotide linkage is independently present in its salt form). In some embodiments, a pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide. In some embodiments, a pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide, wherein each acidic linkage, e.g., each natural phosphate linkage and phosphorothioate internucleotide linkage, is present in sodium salt form (all sodium salts).

[0150] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a provided compound or composition is administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes, in this disclosure. In some embodiments, the subject may be suffering from and / or susceptible to a disease, disorder, and / or condition.

[0151] Substantially: As used herein, the term "substantially" refers to the qualitative condition of exhibiting the full or nearly full extent or degree of a desired characteristic or property. Those skilled in the art of biology will understand that biological and chemical events rarely, if ever, proceed to completion and / or perfection or achieve or avoid absolute results. Thus, the term "substantially" is used herein to capture the potential lack of perfection inherent in many biological and / or chemical events.

[0152] Suffering from: An individual "suffering from" a disease, disorder, and / or condition has been diagnosed with the disease, disorder, and / or condition and / or exhibits one or more symptoms thereof.

[0153] Susceptible to: An individual who is "susceptible to" a disease, disorder, and / or condition is an individual who has an increased risk of developing the disease, disorder, and / or condition compared to members of the public. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not have been diagnosed with the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition may not exhibit symptoms of the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will develop the disease, disorder, and / or condition. In some embodiments, an individual who is susceptible to a disease, disorder, and / or condition will not develop the disease, disorder, and / or condition.

[0154] Systemic: The phrases "systemic administration," "systemically administered," "peripheral administration," and "peripherally administered," as used herein, have their art-recognized meanings to refer to administration of a compound or composition such that it enters the recipient's entire body.

[0155] Therapeutic Agent: As used herein, the phrase "therapeutic agent" refers to any agent that has a therapeutic effect and / or induces a desired biological and / or pharmacological effect upon administration to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, prevent, delay the onset of, reduce the severity of, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition.

[0156] Therapeutically effective amount: In some embodiments, the term "therapeutically effective amount" refers to an amount of a substance (e.g., a therapeutic agent, composition, and / or formulation) that elicits a desired biological response when administered as part of a treatment regimen. In some embodiments, a therapeutically effective amount of a substance is an amount that, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, is sufficient to treat, diagnose, prevent, and / or delay the onset of the disease, disorder, and / or condition. As one of ordinary skill in the art would understand, the effective amount of a substance can vary depending on factors such as the desired biological endpoint, the substance to be delivered, the target cell or tissue, etc. For example, the amount of a compound in a formulation effective to treat a disease, disorder, and / or condition is an amount that alleviates, ameliorates, relieves, inhibits, prevents, delays the onset of, reduces the severity of, and / or reduces the incidence of, one or more symptoms or characteristics of the disease, disorder, and / or condition. In some embodiments, a therapeutically effective amount is administered in a single dose; in some embodiments, multiple unit doses are required to deliver a therapeutically effective amount. In some embodiments, the single dose is an infusion, which can take up to an hour or more.

[0157] Treat: As used herein, the terms "treat," "treatment," or "treating" refer to any method used to partially or completely alleviate, ameliorate, relieve, inhibit, prevent, delay the onset, reduce the severity, and / or reduce the incidence of one or more symptoms or characteristics of a disease, disorder, and / or condition. Treatment may be administered to a subject who does not exhibit signs of the disease, disorder, and / or condition. In some embodiments, treatment may be administered to a subject who exhibits only early signs of a disease, disorder, and / or condition, for example, to reduce the risk of developing a pathology associated with the disease, disorder, and / or condition.

[0158] Chirality-controlled oligonucleotide composition: As used herein, the terms "chirality-controlled (stereocontrolled or stereodefined) oligonucleotide composition," "chirality-controlled (stereocontrolled or stereodefined) nucleic acid composition," and the like refer to a composition comprising multiple oligonucleotides (or nucleic acids, chirality-controlled oligonucleotides, or chirality-controlled nucleic acids) (specific types of oligonucleotides) that share 1) a common base sequence, 2) a common backbone bond pattern, 3) a common backbone chiral center pattern, and 4) a common backbone phosphorus modification pattern, where the multiple oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotide linkages (chirality-controlled internucleotide linkages, whose chiral bond phosphorus is Rp or Sp, rather than a random Rp and Sp mixture as in non-chirality-controlled internucleotide linkages). The level of multiple oligonucleotides (or nucleic acids) in a chirality-controlled oligonucleotide composition is non-random (predetermined, controlled). Chirality-controlled oligonucleotide compositions are typically prepared through the stereoselective formation of one or more chiral internucleotide linkages, for example, by chirality-controlled oligonucleotide preparation (e.g., using a chiral auxiliary as exemplified in the present disclosure, compared to non-chirality-controlled (stereochemically random, non-stereoselective, racemic) oligonucleotide synthesis, such as conventional phosphoramidite-based oligonucleotide synthesis, which does not use a chiral auxiliary or chiral catalyst to intentionally control stereoselectivity). Chirality-controlled oligonucleotide compositions are enriched for a plurality of oligonucleotides compared to substantially racemic preparations of oligonucleotides having a common base sequence, a common backbone bond pattern, and a common backbone phosphorus modification pattern.In some embodiments, a chiral controlled oligonucleotide composition comprises a plurality of oligonucleotides of a particular oligonucleotide type defined by 1) base sequence; 2) backbone bond pattern; 3) backbone chiral center pattern; and 4) backbone phosphorus modification pattern, wherein the oligonucleotides are enriched for a particular oligonucleotide type compared to a substantially racemic preparation of oligonucleotides having the same base sequence, backbone bond pattern, and backbone phosphorus modification pattern. As those skilled in the art will readily appreciate, such enrichment can be characterized by a higher level of the linked phosphorus having a desired configuration at each chiral controlled internucleotide linkage compared to a substantially racemic preparation. In some embodiments, each chiral controlled internucleotide linkage independently has a diastereomeric purity with respect to its chiral linked phosphorus of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, each independently has a diastereomeric purity of at least 90%. In some embodiments, each independently has a diastereomeric purity of at least 95%. In some embodiments, each independently has a diastereomeric purity of at least 97%. In some embodiments, each independently has a diastereomeric purity of at least 98%. In some embodiments, multiple oligonucleotides have the same chemical constitution. In some embodiments, multiple oligonucleotides have the same chemical constitution and stereochemistry and are structurally identical.

[0159] In some embodiments, multiple oligonucleotides in a chiral-controlled oligonucleotide composition share the same base sequence, the same nucleobase, sugar, and internucleotide linkage modifications, if present, and independently the same stereochemistry (Rp or Sp) at the linking phosphorus chiral center of one or more chiral-controlled internucleotide linkages, although the stereochemistry at a particular linking phosphorus chiral center may differ. In some embodiments, about 0.1% to 100% (e.g., about 1% to 100%, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5%, 10%, or 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the plurality of oligonucleotides. In some embodiments, about 0.1% to 100% (e.g., about 1% to 100%, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5% of all nucleotides in a chiral controlled oligonucleotide composition sharing a common base sequence. %, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%) of the plurality of oligonucleotides.In some embodiments, about 0.1% to 100%, (e.g., about 1% to 100%, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 10 ... %, 50% to 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the plurality of oligonucleotides.In some embodiments, all nucleotides in a chiral controlled oligonucleotide composition, or all oligonucleotides in a composition that share a common base sequence (e.g., among multiple oligonucleotides or oligonucleotide types), or all oligonucleotides in a composition that share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern (e.g., among multiple oligonucleotides or oligonucleotide types), or all oligonucleotides in a composition that share a common base sequence, a common base modification pattern, a common sugar modification pattern, a common internucleotide linkage type pattern, and / or a common internucleotide linkage modification pattern (e.g., among multiple oligonucleotides or oligonucleotide types), or all oligonucleotides in a composition that share the same sequence. About 0.1% to 100% (e.g., about 1% to 100%, 5% to 100%, 10% to 100%, 20% to 100%, 30% to 100%, 40% to 100%, 50% to 100%, 60% to 100%, 70% to 100%, 80% to 100%, 90% to 100%, 95% to 100%, 50% to 90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 5 ... 0%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%, or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the plurality of oligonucleotides. In some embodiments, the percentage is at least (DP). NCI(wherein DP is a percentage selected from 85% to 100%, and NCI is the number of chiral-controlled internucleotide linkages). In some embodiments, DP is at least 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, DP is at least 85%. In some embodiments, DP is at least 90%. In some embodiments, DP is at least 95%. In some embodiments, DP is at least 96%. In some embodiments, DP is at least 97%. In some embodiments, DP is at least 98%. In some embodiments, DP is at least 99%. In some embodiments, DP reflects the diastereopurity of the linkage phosphorus chiral center of a chiral-controlled internucleotide linkage. In some embodiments, the diastereopurity of the linkage phosphorus chiral center of an internucleotide linkage may typically be assessed using an appropriate dimer comprising such an internucleotide linkage and two nucleoside units linked by the internucleotide linkage. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1 to 50 (e.g., about 1 to 10, 1 to 20, 5 to 10, 5 to 20, 10 to 15, 10 to 20, 10 to 25, 10 to 30, or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) chiral internucleotide linkages.In some embodiments, the plurality of oligonucleotides has between about 0.1% and 100% (e.g., about 1% and 100%, 5% and 100%, 10% and 100%, 20% and 100%, 30% and 100%, 40% and 100%, 50% and 100%, 60% and 100%, 70% and 100%, 80% and 100%, 90% and 100%, 95% and 100%, 50% and 90%, about 5%, 10%, 1 5%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%, or at least 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% of the chiral internucleotide linkages share the same stereochemistry. In some embodiments, each chiral internucleotide linkage is a chiral-controlled internucleotide linkage, and the composition is a completely chiral-controlled oligonucleotide composition. In some embodiments, not all chiral internucleotide linkages are chiral-controlled internucleotide linkages, and the composition is a partially chiral-controlled oligonucleotide composition. In some embodiments, the chiral-controlled oligonucleotide composition comprises a predetermined level of distinct oligonucleotide or nucleic acid types. For example, in some embodiments, a chiral controlled oligonucleotide composition comprises one oligonucleotide type at a predetermined level (e.g., as described above). In some embodiments, a chiral controlled oligonucleotide composition comprises two or more oligonucleotide types, each independently at a predetermined level. In some embodiments, a chiral controlled oligonucleotide composition comprises multiple oligonucleotide types, each independently at a predetermined level. In some embodiments, a chiral controlled oligonucleotide composition is a composition of oligonucleotides of a certain oligonucleotide type, and the composition comprises multiple oligonucleotides of that oligonucleotide type at a predetermined level.

[0160] Detailed Description of Specific Embodiments In some embodiments, the present disclosure relates to, inter alia, oligonucleotide compositions, oligonucleotide drug products, and methods (e.g., WVE-003, oligonucleotide compositions thereof, chiral-controlled oligonucleotide compositions thereof, or therapeutically effective amounts thereof, methods of manufacture thereof, methods of treatment thereof, etc.) for the treatment of Huntington's disease (HD) or symptoms thereof.

[0161] In some embodiments, the present disclosure relates to WVE-003, and compositions and methods thereof.

[0162] In some embodiments, the present disclosure relates to WVE-003 or compositions thereof, wherein the WVE-003 or composition is administered according to any of the various dosage regimens described herein.

[0163] In some embodiments, the dosage regimen relates to the size of individual doses of the oligonucleotide, oligonucleotide composition, chiral controlled oligonucleotide composition, or therapeutically effective amount of the oligonucleotide, oligonucleotide composition, chiral controlled oligonucleotide composition; and / or the interval between multiple or successive doses thereof; and / or the total length or duration over which a subject receives one or more doses thereof; and / or the detailed formulation thereof.

[0164] Huntingtin (HTT) In some embodiments, the present disclosure provides technologies, e.g., oligonucleotides, compositions, methods, etc., related to the huntingtin (HTT) gene or products (transcripts, proteins, e.g., various mutations of huntingtin protein, etc.) encoded thereby. In some embodiments, the present disclosure provides technologies, including HTT oligonucleotides and compositions and methods thereof, for the treatment of Huntington's disease. In some embodiments, HTT comprises one or more mutations. In some embodiments, such mutations are associated with a decrease in the biological function of the huntingtin protein in subjects suffering from and / or susceptible to Huntington's disease. In some embodiments of Huntington's disease, one or both alleles of HTT are mutant.

[0165] In some embodiments, the huntingtin (HTT) gene or its product, or a variant or portion thereof, may be referred to as HTT.

[0166] In some embodiments, HTT refers to a gene or its gene product (including but not limited to, a nucleic acid, including but not limited to, DNA or RNA, or a wild-type or mutant protein encoded thereby) from any species, including HTT, HD, IT15, Huntingtin, Huntingtin, or LOMARS; External It may also be known as ID: OMIM:613004, MGI:96067, HomoloGene:1593, GeneCards:HTT; Species: Human: Entrez:3064; Ensembl:ENSG00000197386; UniProt:P42858; RefSeq(mRNA):NM_002111; RefSeq(protein):NP_002102; Location(UCSC):Chr4:3.04-3.24 Mb; Species: Mouse: Entrez:15194; Ensembl:ENSMUSG00000029104; UniProt:P42859; RefSeq(mRNA):NM_010414; RefSeq(protein):NP_034544; Location(UCSC):Chr5:34.76-34.91 Mb. Additional HTT sequences, including variants thereof, from human, mouse, rat, monkey, etc., are readily available to one of skill in the art. In some embodiments, the HTT is human or mouse HTT, which may be wild-type or mutant.

[0167] In some embodiments, the HTT protein is unmodified or modified, hi some embodiments, the HTT protein has any one or more modifications of N6-acetyllysine at position 9, N6-acetyllysine at position 176, N6-acetyllysine at position 234, N6-acetyllysine at position 343, phosphoserine at position 411, phosphoserine at position 417, phosphoserine at position 419, phosphoserine at position 432, N6-acetyllysine at position 442, phosphoserine at position 640, phosphoserine at position 643, phosphoserine at position 1179, phosphoserine at position 1199, phosphoserine at position 1870, and / or phosphoserine at position 1874.

[0168] Without wishing to be bound by any particular theory, the present disclosure notes that mutations in HTT (e.g., CAG repeat expansions) have been reported to be key factors in diseases and disorders such as Huntington's disease.

[0169] In some embodiments, mutant HTT is referred to as mHTT, muHTT, m HTT, mu HTT, MU HTT, etc., where m or mu indicates mutant. In some embodiments, wild-type HTT is referred to as wild-type HTT, wtHTT, wt HTT, WT HTT, WTHTT, etc., where wt indicates wild-type. In some embodiments, mutant HTT comprises an expanded CAG repeat region (as those skilled in the art will understand, the number of CAG repeats can vary from subject to subject; for example, some embodiments comprise 36 or more CAG repeats). In some embodiments, mutant HTT comprises mutant alleles of one or more SNPs (the alleles are on the same DNA strand or chromosome as the expanded CAG repeat region). In some embodiments, wild-type HTT in a population is reported to frequently have about or no more than about 18 CAG repeats, and mutant HTT is reported to frequently have about or more than about 43 CAG repeats. In some embodiments, heterozygotes for SNP rs362273 are reportedly identified in about 71% of individuals in a population. In some embodiments, SNP3 is reported to be associated with mHTT in about 40-45% of HD patients in a population.

[0170] In some embodiments, the mutant HTT comprises both an expanded CAG repeat region and a mutant allele of a specific SNP on the same chromosomal strand. In some embodiments, in such cases, the mutant allele of a specific SNP is targeted by an HTT oligonucleotide (e.g., WVE-003), and when the mutant allele is on the same chromosome as the harmful CAG repeat expansion, the oligonucleotide has the ability to target the harmful HTT allele and mediate allele-specific knockdown.

[0171] In some embodiments, human HTT is referred to as hHTT. In some embodiments, mutant HTT is referred to as mHTT. In some embodiments, when a mouse is utilized, mouse HTT may be referred to as mHTT, as will be understood by those skilled in the art.

[0172] In some embodiments, the oligonucleotide capable of mediating allele-specific knockdown of a mutant HTT gene or its gene product is WVE-003.

[0173] Huntington's disease Compositions comprising one or more HTT oligonucleotides described herein can be used to treat Huntington's disease or its symptoms.

[0174] Huntington's disease (HD) is a neurodegenerative disorder reportedly caused by mutations in the HTT (huntingtin) gene. Alterations in this single, widely expressed gene reportedly result in a progressive neurodegenerative disorder with many characteristic symptoms.

[0175] Huntington's disease is reportedly a rare, progressive neurological disorder that results in motor, cognitive, and psychiatric disability and is always fatal. Bates G, Tabrizi S, Jones L, (editors). Huntington's Disease, 4th Edition. Oxford (UK): Oxford University Press; 2014. Because it is a genetically inherited disease, it can reportedly affect multiple family members across generations. Sturrock A et al. J Geriatr Psychiatry Neurol. 2010;23(4):243-259. While cognitive and psychiatric symptoms reportedly begin the disease, a clinical diagnosis of HD is usually based on the presence of chorea, the most visible symptom of the disease. Chorea is an abnormal involuntary movement disorder that reportedly occurs in 90% of patients, with approximately 70% experiencing moderate to severe symptoms. These physical symptoms reportedly can manifest at any age, but typically appear between the ages of 30 and 50. Bates G, Tabrizi S, Jones L, (editors). Huntington's Disease, 4th Edition. Oxford (UK): Oxford University Press; 2014. A physical examination, sometimes combined with a neurological examination, can reportedly determine whether the disease has begun. Life expectancy after symptom onset is reduced to approximately 15 to 20 years. Bates G, Tabrizi S, Jones L, (editors). Huntington's Disease, 4th Edition. Oxford (UK): Oxford University Press; 2014; and Sturrock A et al. J Geriatr Psychiatry Neurol. 2010;23(4):243-259. As the condition progresses, affected individuals reportedly become increasingly or completely dependent on others for care.Suicidal ideation is reportedly increased early in the disease, which appears to be related to perceived loss of independence, and subjects with HD have a significantly higher suicide rate when compared with the normal healthy adult population (138 per 100,000 per year and 12-13 per 100,000 per year, respectively). Bird TD. Am J Hum Genet. 1999;64(5):1289-1292; and Paulsen JS et al. Am J Psychiatry. 2005;162(4):725-731.

[0176] Some of the symptoms of HD can reportedly be managed with medications and therapies, including antipsychotics and drugs that act on the dopamine pathway to modulate movement disorders. Sturrock A et al. J Geriatr Psychiatry Neurol. 2010;23(4):243-259.

[0177] Huntington's disease is reportedly caused by a known mutation in a single gene, an expansion of a cytosine-adenine-guanine (CAG) triplet repeat in the huntingtin (HTT) gene. The Huntington's Disease Collaborative Research Group. Cell. 1993;72(6):971-983. Wild-type HTT protein is critical for neurodevelopment. Dragatsis I et al. Nat Genet. 2000;26(3):300-306. The purpose of wtHTT in adults is reportedly not fully understood, although some experiments indicate that it may play an important role in neuronal function. Dragatsis I et al. Nat Genet. 2000;26(3):300-306; Leavitt BR et al. J Neurochem. 2006;96(4):1121-1129; Rigamonti D et al. J Biol Chem. 2001;276(18):14545-14548; and Zhang Y et al. EMBO J. 2006;25(24):5896-5906. However, a CAG triplet repeat expansion in the HTT gene reportedly leads to the production of mHTT protein. Accumulation of this protein reportedly leads to progressive loss of neurons in the brain. Sturrock A et al. J Geriatr Psychiatry Neurol. 2010;23(4):243-259. Nonclinical studies have reportedly demonstrated therapeutic benefit in reducing mHTT protein levels as measured in CSF. DiFiglia et al. Proc Natl Acad Sci US A. 2007;104(43):17204-17209; and Kordasiewicz HB et al. Neuron. 2012;74(6):1031-1044. Thus, drugs that can silence mHTT gene transcripts while leaving the wild-type allele intact may slow, halt, or even reverse the progression of HD.Kay C et al. Clin Genet. 2014;86(1):29-36.

[0178] In some embodiments, the HD-associated mutation is an expansion of a CAG repeat region in the HTT gene, with larger expansions reportedly associated with increased disease severity and earlier age of onset. This mutation reportedly results in a variety of motor, emotional, and cognitive symptoms, and the formation of huntingtin aggregates in the brain.

[0179] CAG expansions reportedly result in polyglutamine (polyQ) expansions in the 350-kDa protein huntingtin (Huntington Disease Collaborative Research Group, 1993. Cell. 72:971-83). CAG repeat expansions have been reportedly associated with Huntington's disease. Longer sequences are reportedly associated with earlier disease onset. Individuals lacking one copy of huntingtin reportedly lack the HD phenotype, or individuals homozygous for the expansion have increased disease severity, suggesting that the mutation does not cause loss of function (Trottier et al., 1995, Nature Med., 10:104-110). Transcriptional deregulation and loss of function of transcriptional coactivator proteins have been reportedly implicated in the pathogenesis of HD. Mutant huntingtin has been reported to disrupt activator-dependent transcription, particularly early in the pathogenesis of HD (Dunah et al., 2002. Science 296:2238-2243).

[0180] In one report, gene profiling of human blood identified 322 mRNAs that showed significant changes in expression in HD blood samples compared with normal or presymptomatic individuals. Similarly, postmortem brain samples from the caudate nucleus of HD also showed substantial changes in the expression of marker genes, suggesting that the upregulation of genes in blood samples reflects disease mechanisms observed in the brain. Gene expression monitoring can provide a sensitive and quantitative method for monitoring disease progression, especially in the early stages of disease, in both animal models and human subjects (Borovecki et al., 2005, Proc. Natl. Acad. Sci. USA 102:11023-11028).

[0181] Huntington's disease is reported to be an autosomal dominant disorder, with onset typically occurring in middle age, although cases ranging from childhood onset to cases occurring after age 70 have been documented. Early age of onset is reportedly associated with paternal inheritance, with 70% of younger cases inherited from the father.

[0182] In some embodiments, Huntington's disease symptoms include affective, motor, and / or cognitive components. One symptom, chorea, is a distinctive feature of movement disorders and is defined as randomly distributed, sudden, and excessive spontaneous movements with irregular timing. This can range from barely noticeable to severe. Other frequently observed abnormalities include dystonia, rigidity, bradykinesia, oculomotor dysfunction, and tremor. Symptoms of voluntary movement disorders include fine motor incoordination, dysarthria, and dysphagia. Affective disorders or symptoms generally include depression and irritability, and cognitive components include subcortical dementia (Mangiarini et al. 1996. Cell 87:493-506). Changes in the HD brain are widespread and have been reported to include neuronal loss and gliosis, particularly in the cortex and striatum (Vonsattel and DiFiglia. 1998. J. Neuropathol. Exp. Neurol. 57:369-384).

[0183] In some embodiments, Huntington's disease phenotypes range from mild HTT to severe, depending on the length of the CAG repeat expansion.

[0184] Specific information regarding HTT and HTT-related conditions, disorders, or diseases and their symptoms can be found in the scientific literature, e.g., Kremer et al. 1994. NEJ Med. 330:1401; Kordasiewicz et al. 2012 Neuron 74:1031-1044; Carroll et al. 2011 Mol. Ther. 19:2178-2185; Warby et al. 2009 Am. J. Hum. Genet. 84:351-366; Pfister et al. 2009 Current Biol. 19:774-778; Kay et al. 2015 Mol. Ther. 23:1759-1771; Kay et al. 2014 Clin. Genet. 86:29-36; Lee et al. 2015 Am. J. Hum. Genet. 97:435-444; Skotte et al. 2014. PLOS ONE 9:e107434; Southwell et al. 2014. Mol. Ther. 22:2093-2106; Australian Patent Application Publication Nos. 2017276286 and 2007210038; European Patent Application Publication Nos. 3277814 and 3210633; International Patent Application Publication No. 2018145009; and US Patent Application Publication No. 20180273945.

[0185] Treatment of HTT-related conditions, disorders, or diseases In some embodiments, the present disclosure provides HTT oligonucleotides that target HTT (e.g., HTT oligonucleotides comprising an HTT target sequence or a sequence complementary to an HTT target sequence) and induce target-specific knockdown of HTT, including, for example, WVE-003. In some embodiments, WVE-003 is administered intrathecally in an amount of about 30, about 60, about 90, about 120, about 150, or about 168 mg and is administered as a liquid formulation (including, but not limited to, a solution in water, aCSF, aCSF reconstituted from a lyophilized preparation, or sodium chloride, or sodium chloride reconstituted from a lyophilized preparation). In some embodiments, the present disclosure provides HTT oligonucleotides that induce target-specific knockdown of HTT mediated by RNase H and / or RNA interference.

[0186] In some embodiments, the present disclosure provides methods for preventing and / or treating HTT-related conditions, disorders, or diseases using the provided HTT oligonucleotides and compositions thereof. In some embodiments, the HTT-related condition is Huntington's disease and / or one or more symptoms of Huntington's disease. In some embodiments, the present disclosure provides oligonucleotides and compositions thereof for use as pharmaceuticals, for example, for HTT-related conditions, disorders, or diseases. In some embodiments, the present disclosure provides oligonucleotides and compositions thereof for use in treating HTT-related conditions, disorders, or diseases. In some embodiments, the present disclosure provides oligonucleotides and compositions thereof for use in the manufacture of pharmaceuticals for treating HTT-related conditions, disorders, or diseases.

[0187] In some embodiments, the present disclosure provides a method for preventing, treating, or ameliorating an HTT-associated condition, disorder, or disease in a subject susceptible to or suffering from an HTT-associated condition, disorder, or disease, comprising administering to the subject a therapeutically effective amount of an HTT oligonucleotide or a pharmaceutical composition thereof.

[0188] In some embodiments, the present disclosure provides a method for treating or ameliorating an HTT-associated condition, disorder, or disease in a subject suffering from the condition, disorder, or disease, comprising administering to the subject a therapeutically effective amount of an HTT oligonucleotide or a pharmaceutical composition thereof. In some embodiments, the HTT oligonucleotide is WVE-003. In some embodiments, the therapeutically effective amount is about 30, about 60, about 90, about 120, about 150, or about 168 mg.

[0189] In some embodiments, the HTT-associated condition, disorder, or disease is Huntington's disease (HD), also known as Huntington's chorea, hi some embodiments, the HTT-associated condition, disorder, or disease is juvenile HD, akinesia-rigidity, or Westphal variant HD.

[0190] In some embodiments, the present disclosure provides methods for reducing HTT gene expression in a cell, comprising contacting the cell with an HTT oligonucleotide or a composition thereof. In some embodiments, the present disclosure provides methods for reducing HTT transcript levels in a cell, comprising contacting the cell with an HTT oligonucleotide or a composition thereof. In some embodiments, the present disclosure provides methods for reducing HTT protein levels in a cell, comprising contacting the cell with an HTT oligonucleotide or a composition thereof. In some embodiments, the provided methods selectively reduce levels of HTT transcripts and / or products encoded thereby that are associated with a condition, disorder, or disease.

[0191] HTT is reportedly expressed in all cells, with highest concentrations found in the brain and testes, and moderate amounts found in the liver, heart, and lungs. In various embodiments, the cells are in the brain, testes, liver, heart, or lungs.

[0192] In some embodiments, the present disclosure provides a method for reducing HTT gene expression in a mammal in need thereof, comprising administering to the mammal a nucleic acid-lipid particle comprising a provided HTT oligonucleotide or composition thereof.

[0193] In some embodiments, the present disclosure provides a method for in vivo delivery of an HTT oligonucleotide, the method comprising administering to a mammal an HTT oligonucleotide or a composition thereof.

[0194] In some embodiments, the mammal is a human. In some embodiments, the mammal develops, is suffering from, and / or is susceptible to an HTT-related condition, disorder, or disease.

[0195] In some embodiments, subjects with or suitable for treatment of an HTT-related condition, disorder, or disease, such as Huntington's disease (HD), can be identified or diagnosed by a medical professional. For example, for neurological conditions, disorders, or diseases, a physical examination may be followed by a thorough neurological examination. In some embodiments, the neurological examination may evaluate motor and sensory skills, neurological function, hearing and speech, vision, coordination and balance, mental status, and / or changes in mood or behavior. Exemplary symptoms of a neurological condition, disorder, or disease, such as Huntington's disease (HD), include: weakness in the arms, legs, feet, or ankles; slurred speech; difficulty dorsiflexing the front of the foot and toes; weakness or clumsiness in the hands; muscle paralysis; stiff muscles; involuntary jerking or writhing movements (writing difficulties). movement (chorea); involuntary persistent muscle contractures (dystonia); bradykinesia; loss of automatic movements; posture and balance problems; lack of flexibility; tingling in body parts; electric shock sensation with head movement; spasms of the arms, shoulders, and tongue; difficulty swallowing; difficulty breathing; difficulty chewing; partial or complete loss of vision; double vision; slow or abnormal eye movements; tremor; unsteady gait; fatigue; memory loss; dizziness; difficulty thinking or concentrating; difficulty reading or writing; misinterpretation of spatial relationships; disorientation; depression; anxiety; difficulty making decisions and making judgments; loss of impulse control; difficulty planning and carrying out routine tasks; aggression; irritability; social withdrawal; mood swings; dementia; changes in sleep habits; wandering; and / or changes in appetite.

[0196] In some embodiments, the symptoms of Huntington's disease are any of insoluble protein accumulation; huntingtin protein aggregate accumulation; neuronal aggregates in the striatum; changes in the size and number of neuronal intranuclear inclusions and other HD markers; altered regulation of DARPP-32 expression; striatal atrophy; striatal and cortical neurodegeneration; changes in blood glucose and / or insulin levels; or neuronal loss and gliosis, particularly in the cortex and striatum.

[0197] In some embodiments, the symptoms of Huntington's disease are any of behavioral and neuropathological abnormalities; altered rotarod performance in test animals; reduced weight loss; altered lifespan; behavioral disturbances; emotional, motor, and cognitive changes or impairments; depression; irritability; involuntary movements (chorea); choreiform movements; incoordination; randomly distributed, sudden, excessive spontaneous movements with irregular timing; bradykinesia; dystonia; seizures; rigidity; oculomotor dysfunction; tremor; fine motor coordination loss; dysarthria; dysphagia; subcortical dementia; progressive dementia; or psychiatric disorders.

[0198] In some embodiments, the provided oligonucleotides or compositions thereof prevent, treat, ameliorate, or slow the progression of an HTT-related condition, disorder, or disease, or at least one symptom of an HTT-related condition, disorder, or disease. In some embodiments, the provided oligonucleotides or compositions thereof prevent, treat, ameliorate, or slow the progression of an HTT-related condition, disorder, or disease, or two or more symptoms of an HTT-related condition, disorder, or disease. In some embodiments, a subject has been assessed for one or more markers and / or symptoms of an HTT-related condition, disorder, or disease, and the subject has subsequently been prescribed and / or administered an oligonucleotide or oligonucleotide administration (e.g., an oligonucleotide dose or dosage regimen) as described herein. In some embodiments, a subject has been assessed for one or more markers and / or symptoms of an HTT-related condition, disorder, or disease, and the subject has been administered an oligonucleotide or oligonucleotide administration as described herein. In some embodiments, the HTT-related condition, disorder, or disease is Huntington's disease.

[0199] In some embodiments, the disclosed method is for treating Huntington's disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an HTT oligonucleotide or a pharmaceutical composition thereof. In some embodiments, the HTT oligonucleotide is WVE-003.

[0200] In some embodiments, the subject has an allele or transcript that includes an expanded CAG repeat region and is perfectly complementary to the base sequence of WVE-003. In some embodiments, the HTT transcript that includes an expanded CAG repeat region is perfectly complementary to the base sequence of WVE-003. In some embodiments, the subject has an allele that does not include an expanded CAG repeat region and is perfectly complementary to the base sequence of WVE-003. In some embodiments, the subject has an HTT allele that does not include an expanded CAG repeat region and is not complementary to the base sequence of WVE-003 at rs362273. In some embodiments, the HTT transcript that does not include an expanded CAG repeat region is not perfectly complementary to the base sequence of WVE-003 at rs362273.

[0201] In some embodiments, provided are methods for reducing at least one symptom of Huntington's disease, the methods comprising administering a therapeutically effective amount of an HTT oligonucleotide or a pharmaceutical composition thereof to a subject.

[0202] In some embodiments, the present disclosure provides a method for treating and / or ameliorating one or more symptoms associated with an HTT-related condition, disorder, or disease in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of an HTT oligonucleotide or a composition thereof. In some embodiments, the present disclosure provides a method for reducing susceptibility to an HTT-related condition, disorder, or disease in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of an HTT oligonucleotide or a composition thereof. In some embodiments, the present disclosure provides a method for preventing or delaying the onset of an HTT-related condition, disorder, or disease in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of an HTT oligonucleotide or a composition thereof. In some embodiments, the present disclosure provides a method for treating and / or ameliorating one or more symptoms associated with an HTT-related condition, disorder, or disease in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle comprising an HTT oligonucleotide. In some embodiments, the present disclosure provides a method for reducing susceptibility to an HTT-associated condition, disorder, or disease in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of nucleic acid-lipid particles comprising an HTT oligonucleotide. In some embodiments, the present disclosure provides a method for preventing or delaying the onset of an HTT-associated condition, disorder, or disease in a mammal in need thereof, comprising administering to the mammal a therapeutically effective amount of nucleic acid-lipid particles comprising an HTT oligonucleotide. In some embodiments, the mammal is a human. In some embodiments, the mammal has developed, is suffering from, and / or is susceptible to an HTT-associated condition, disorder, or disease. In some embodiments, the HTT oligonucleotide is WVE-003. In some embodiments, the therapeutically effective amount is about 30, about 60, about 90, about 120, about 150, or about 168 mg.

[0203] In some embodiments, the present disclosure relates to compositions and methods involving specific doses of WVE-003 (or a salt form thereof) that are about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, or about 168 mg.

[0204] In some embodiments, about 30 mg is 30 mg ± 5%, about 60 mg is 60 mg ± 5%, about 90 mg is 90 mg ± 5%, about 120 mg is 120 mg ± 5%, about 150 mg is 150 mg ± 5%, and / or about 168 mg is 168 mg ± 5%.

[0205] In some embodiments, about 30 mg is 30 mg ± 10%, about 60 mg is 60 mg ± 10%, about 90 mg is 90 mg ± 10%, about 120 mg is 120 mg ± 10%, about 150 mg is 150 mg ± 10%, and / or about 168 mg is 168 mg ± 10%.

[0206] In some embodiments, about 30 mg is 30 mg ± 15%, about 60 mg is 60 mg ± 15%, about 90 mg is 90 mg ± 15%, about 120 mg is 120 mg ± 15%, about 150 mg is 150 mg ± 15%, and / or about 168 mg is 168 mg ± 15%.

[0207] In some embodiments, about 30 mg is 30 mg ± 20%, about 60 mg is 60 mg ± 20%, about 90 mg is 90 mg ± 20%, about 120 mg is 120 mg ± 20%, about 150 mg is 150 mg ± 20%, and / or about 168 mg is 168 mg ± 20%.

[0208] In some embodiments, about 30 mg is 30 mg ± 25%, about 60 mg is 60 mg ± 25%, about 90 mg is 90 mg ± 25%, about 120 mg is 120 mg ± 25%, about 150 mg is 150 mg ± 25%, and / or about 168 mg is 168 mg ± 25%.

[0209] In some embodiments, about 30 mg is 30 mg ± 30%, about 60 mg is 60 mg ± 30%, about 90 mg is 90 mg ± 30%, about 120 mg is 120 mg ± 30%, about 150 mg is 150 mg ± 30%, and / or about 168 mg is 168 mg ± 30%.

[0210] In some embodiments, the range of about 30 includes, but is not limited to, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2, 26.3, 26.4, 26.5, 26.6, 26.7, 26.8, 26.9, 27, 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28, 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 28.9, 28.10, 28.11, 28.12, 28.13, 28.14, 28.15, 28.16, 28.17, 28.18, 28.19, 28.20, 28.21, 28.22, 28.23, 28.24, 28.25, 28.26, 28.27, 28.28, 28.29, 28.30, 28.31, 28.32, 28.33, 28.34, 28.35, 28.36, 28.37, 28.38, 28.39, 28.40, 28.41, 28.42, 28.43, 28.44, 28.45, 28.46, 28.47, 28.48, 28.49, 28.50, 28.51, 8.5, 28.6, 28.7, 28.8, 28.9, 29, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30, 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32, 32.1, 32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9, 33, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 34, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6, 34.7, 34.8, 34.9, 35, 35.1, 35.2, 35.3, 35.4, These include 35.5, 35.6, 35.7, 35.8, 35.9, 36, 36.1, 36.2, 36.3, 36.4, 36.5, 36.6, 36.7, 36.8, 36.9, 37.0, 37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, and 38.7.

[0211] In some embodiments, the range includes, but is not limited to, about 30, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2, 26.3, 26.4, 26.5, 26.6, 26.7, 26.8, 26.9, 27, 27.1, 27.2, 27.3, 27.4, 27.5, 27 .6, 27.7, 27.8, 27.9, 28, 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 28.9, 29, 29.1, 29.2, 29.3, 29.4, 29.5, 29.6, 29.7, 29.8, 29.9, 30, 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31 .8, 31.9, 32, 32.1, 32.2, 32.3, 32.4, 32.5, 32.6, 32.7, 32.8, 32.9, 33, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 34, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6, 34.7, 34.8, 34.9, 35, 35.1, 35.2, 35.3, 35.4, 35.5, 35.6, 35.7, 35.8, 35.9, 36 , 36.1, 36.2, 36.3, 36.4, 36.5, 36.6, 36.7, 36.8, 36.9, 37.0, 37.1, 37.2, 37.3, 37.4, 37.5, 37.6, 37.7, 37.8, 37.9, 38, 38.1, 38.2, 38.3, 38.4, 38.5, 38.6, 38.7, 38.8, 38.9, 39, 39.1, 39.2, 39.3, 39.4, 39.5, 39.6, 39.7, 39.8, 39.9, and 40.

[0212] In some embodiments, the range includes, but is not limited to, about 30, 24, 24.1, 24.2, 24.3, 24.4, 24.5, 24.6, 24.7, 24.8, 24.9, 25, 25.1, 25.2, 25.3, 25.4, 25.5, 25.6, 25.7, 25.8, 25.9, 26, 26.1, 26.2, 26.3, 26 .4, 26.5, 26.6, 26.7, 26.8, 26.9, 27, 27.1, 27.2, 27.3, 27.4, 27.5, 27.6, 27.7, 27.8, 27.9, 28, 28.1, 28.2, 28.3, 28.4, 28.5, 28.6, 28.7, 28.8, 28.9, 29, 29.1, 29.2, 29.3, 29 .4, 29.5, 29.6, 29.7, 29.8, 29.9, 30, 30.1, 30.2, 30.3, 30.4, 30.5, 30.6, 30.7, 30.8, 30.9, 31, 31.1, 31.2, 31.3, 31.4, 31.5, 31.6, 31.7, 31.8, 31.9, 32, 32.1, 32.2, 32.3, 32.4 .4, 32.5, 32.6, 32.7, 32.8, 32.9, 33, 33.1, 33.2, 33.3, 33.4, 33.5, 33.6, 33.7, 33.8, 33.9, 34, 34.1, 34.2, 34.3, 34.4, 34.5, 34.6, 34.7, 34.8, 34.9, 35, 35.1, and 35.2.

[0213] In some embodiments, the present disclosure relates to compositions and methods involving specific doses of WVE-003 (or a salt form thereof) that are about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, or about 168 mg.

[0214] In some embodiments, the disclosure relates to compositions and methods involving a specific dose of WVE-003 (or a salt form thereof) that is about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, or about 168 mg, wherein the total amount of oligonucleotide in the dose is about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, or about 168 mg, respectively, and wherein substantially all of the oligonucleotide in the dose is WVE-003.

[0215] In some embodiments, the present disclosure provides highly pure WVE-003 preparations. Various techniques are available for determining WVE-003 purity. In some embodiments, purity is determined using Protocol A, as described herein. In some embodiments, the purity of the WVE-003 preparation is about 80% or greater. In some embodiments, it is about 81%. In some embodiments, it is about 82%. In some embodiments, it is about 83%. In some embodiments, it is about 84%. In some embodiments, it is about 85%. In some embodiments, it is about 86%. In some embodiments, it is about 87%. In some embodiments, it is about 88%. In some embodiments, it is about 89%. In some embodiments, it is about 90%. In some embodiments, it is greater than or equal to 90%. In some embodiments, various batches of WVE-003 have been found to be, for example, about 84%-88% pure, with a total of about 11%-16% impurities. In some embodiments, the impurities include nx deletion sequences; n-1 deletion sequences and phosphodiester modified full-length sequences and / or n+x addition sequences. Without wishing to be bound by any particular theory, the present disclosure notes that at least some of the impurities will have at least some activity (e.g., the ability to mediate allele-specific knockdown of a mutant HTT gene or gene product).

[0216] In some embodiments, WVE-003 is administered to a subject at a dose of about 30, about 60, about 90, about 120, about 150, or about 168 mg. In some embodiments, multiple doses of WVE-003 are administered to a subject. In some embodiments, multiple doses of WVE-003 are administered to a subject at regular intervals. In some embodiments, multiple doses of WVE-003 are administered to a subject approximately monthly (e.g., with about one month between doses). In some embodiments, multiple doses of WVE-003 are administered to a subject approximately once every two months (e.g., with about two months between doses). In some embodiments, multiple doses of WVE-003 are administered to a subject approximately once every eight weeks (e.g., with about eight weeks between doses). In some embodiments, multiple doses of WVE-003 are administered to a subject approximately once every 12 weeks (e.g., with about 12 weeks between doses). In some embodiments, each of the multiple doses is about the same, e.g., about 30 mg.

[0217] In some embodiments, multiple doses of WVE-003 are administered to a subject approximately once every four weeks (e.g., with about four weeks between doses). In some embodiments, multiple doses of WVE-003 are administered to a subject approximately once every eight weeks (e.g., with about eight weeks between doses). In some embodiments, multiple doses of WVE-003 are administered to a subject approximately once every 12 weeks (e.g., with about 12 weeks between doses).

[0218] In some embodiments, a subject is administered two or more doses of WVE-003, with the interval between any two of the doses being about one month or about four weeks. In some embodiments, a subject is administered two or more doses of WVE-003, with the interval between any two of the doses being about two months or about eight weeks. In some embodiments, a subject is administered two or more doses of WVE-003, with the interval between any two of the doses being about three months or about 12 weeks.

[0219] In some embodiments, a subject is administered two or more doses of WVE-003 approximately every month or approximately once every four weeks. In some embodiments, a subject is administered two or more doses of WVE-003 approximately once every two months or approximately once every eight weeks. In some embodiments, a subject is administered two or more doses of WVE-003 approximately once every three months or approximately once every 12 weeks.

[0220] In some embodiments, the subject is administered WVE-003 approximately monthly for at least about two months. In some embodiments, the subject is administered WVE-003 approximately once every two months for at least about two months.

[0221] In some embodiments, the subject is administered WVE-003 approximately monthly for at least about four months. In some embodiments, the subject is administered WVE-003 approximately once every two months for at least about four months.

[0222] In some embodiments, the subject is administered WVE-003 approximately monthly for at least about 8 months. In some embodiments, the subject is administered WVE-003 approximately once every two months for at least about 8 months.

[0223] In some embodiments, a subject is administered WVE-003 approximately once every four weeks for at least about eight weeks, hi some embodiments, a subject is administered WVE-003 approximately once every eight weeks for at least about 16 weeks.

[0224] In some embodiments, a subject is administered WVE-003 approximately once every four weeks for at least about 12 weeks. In some embodiments, a subject is administered WVE-003 approximately once every 12 weeks for at least about 12 weeks.

[0225] In some embodiments, a subject is administered WVE-003 approximately once every four weeks for at least about 16 weeks, hi some embodiments, a subject is administered WVE-003 approximately once every eight weeks for at least about 16 weeks.

[0226] In some embodiments, a subject is administered WVE-003 approximately once every 8 weeks for at least about 24 weeks. In some embodiments, a subject is administered WVE-003 approximately once every 12 weeks for at least about 24 weeks.

[0227] In some embodiments, administration of an HTT oligonucleotide to a subject or patient has the ability to mediate any one or more of: slowing the progression of Huntington's disease, delaying the onset of HD or at least one symptom thereof, improving one or more indicators of HD, and / or increasing the survival or lifespan of the subject or patient.

[0228] In some embodiments, slowing disease progression involves preventing or delaying clinically undesirable changes in one or more clinical parameters, such as those described herein, and / or in individuals susceptible to HD. Identifying slowing of disease progression in HD patients using one or more of the disease-determining tests described herein is well within the capabilities of a physician. In addition, it is understood that a physician may administer diagnostic tests other than those described herein to patients to determine the rate of disease progression in HD patients.

[0229] In some embodiments, delaying the onset of HD or its symptoms involves delaying one or more undesirable changes in one or more HD indicators that are negative for HD. A physician can determine an approximate expected age of onset for HD by using a family history of HD or a comparison with other HD patients (e.g., subjects or humans undergoing or in need of treatment for HD) with a similar genetic profile (e.g., CAG repeat number) to determine whether the onset of HD has been delayed.

[0230] In some embodiments, indicators of HD include parameters used by medical professionals, such as physicians, to diagnose HD or measure its progression, including, without limitation, genetic testing, hearing, eye movements, strength, coordination, chorea (rapid, jerky, involuntary movements), sensation, reflexes, balance, movement, mental status, dementia, personality disorder, family history, weight loss, and caudate nucleus degeneration, which is assessed by brain imaging techniques such as magnetic resonance imaging (MRI) or computed tomography (CT) scan.

[0231] In some embodiments, improvement in an HD index relates to the absence of an undesired change or the presence of a desirable change in one or more HD indexes. In one embodiment, improvement in an HD index is evidenced by the absence of a measurable change in one or more HD indexes. In another embodiment, improvement in an HD index is evidenced by a desirable change in one or more HD indexes.

[0232] In some embodiments, slowing disease progression may further include increasing survival of individuals with and / or susceptible to HD. In some embodiments, increasing survival refers to increasing survival of individuals with and / or susceptible to HD compared to the estimated survival based on HD progression and / or family history of HD. A physician can use one or more of the disease-defining tests described herein to predict the estimated survival of individuals with and / or susceptible to HD. In addition, a physician may use the family history of individuals with and / or susceptible to HD or comparison with other HD patients with similar genetic profiles (e.g., CAG repeat number) to predict expected survival.

[0233] In some embodiments, the present disclosure provides a method for inhibiting HTT expression in a cell, comprising: (a) contacting the cell with an HTT oligonucleotide; and (b) maintaining the cell produced in step (a) for a sufficient time to achieve degradation of mRNA transcripts of the HTT gene, thereby inhibiting expression of the HTT gene in the cell. In some embodiments, HTT expression is inhibited by at least 30%.

[0234] In some embodiments, the present disclosure provides a method for treating a condition, disorder, or disease mediated by HTT expression, comprising administering a therapeutically effective amount of an HTT oligonucleotide or composition thereof to a human suffering therefrom. In some embodiments, the administration causes a decrease in expression, activity, and / or level of HTT transcript. In some embodiments, the administration is accompanied by a decrease in expression, activity, and / or level of HTT transcript. In some embodiments, the administration is followed by a decrease in expression, activity, and / or level of HTT transcript.

[0235] In some embodiments, the present disclosure provides an HTT oligonucleotide for use in a subject to treat an HTT-associated condition, disorder, or disease. In some embodiments, the HTT-associated condition, disorder, or disease is Huntington's disease.

[0236] In some embodiments, provided methods reduce the amount and / or proportion of mHTT protein. In some embodiments, provided methods reduce the amount and / or proportion of mHTT protein in CSF. In some embodiments, provided methods increase the proportion of wtHTT protein. In some embodiments, provided methods increase the proportion of wtHTT protein in CSF. In some embodiments, provided methods do not reduce the amount of wtHTT protein or do not reduce the amount of wtHTT protein by more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, provided methods do not reduce the amount of wtHTT protein in CSF or do not reduce the amount of wtHTT protein by more than about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50%. In some embodiments, the amount and / or percentage of mHTT, wtHTT, and / or total HTT is independently determined after a suitable period of time (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 weeks, or 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 months) following administration of one or more independent doses of each as described herein. Certain techniques useful for determining mHTT, wtHTT, and / or total HTT protein are described in WO 2022 / 046723, which is incorporated herein by reference.

[0237] In some embodiments, a subject is administered an oligonucleotide, such as an HTT oligonucleotide (e.g., WVE-003), or a salt form thereof, or a composition thereof, and an additional agent and / or method, such as an additional therapeutic agent and / or method. In some embodiments, the oligonucleotide or composition thereof can be administered alone or in combination with one or more additional therapeutic agents and / or treatments. When administered in combination, each component can be administered at the same time or sequentially in any order at different times. In some embodiments, each component can be administered separately, but sufficiently close in time to impart the desired therapeutic effect. In some embodiments, the provided oligonucleotide and the additional therapeutic component are administered simultaneously. In some embodiments, the provided oligonucleotide and the additional therapeutic component are administered as a single composition. In some embodiments, at a given time, the subject is exposed to both the provided oligonucleotide and the additional component simultaneously.

[0238] Allele-specific knockdown of mutant HTT transcripts as a treatment for Huntington's disease In some embodiments, the treatment of Huntington's disease involves the use of an HTT oligonucleotide capable of mediating allele-specific knockdown of a mutant HTT transcript. In some embodiments, the present disclosure provides a method for the treatment of Huntington's disease, comprising administering an HTT oligonucleotide or a composition comprising an HTT oligonucleotide to a subject suffering from or susceptible to Huntington's disease. In particular, among other things, the present disclosure demonstrates that chiral-controlled oligonucleotides / chiral-controlled oligonucleotide compositions are unexpectedly effective in modulating allele-specific knockdown of a mutant HTT transcript compared to otherwise identical oligonucleotides / oligonucleotide compositions that are not chiral-controlled. In some embodiments, the term chiral-controlled may be considered equivalent to stereochemically pure or stereochemically defined.

[0239] In some embodiments, the treatment of Huntington's disease involves the use of HTT oligonucleotides, wherein the oligonucleotides are capable of providing allele-specific knockdown of mutant HTT transcripts. In some embodiments, the HTT oligonucleotides are capable of mediating allele-specific knockdown of mutant HTT transcripts containing mutations (e.g., CAG repeat expansions), wherein reducing the level, expression, and / or activity of mHTT is capable of treating, preventing, and / or ameliorating Huntington's disease or its symptoms, and / or delaying the severity or onset of Huntington's disease symptoms.

[0240] In some embodiments, a composition comprising an HTT oligonucleotide is useful for treating a huntingtin-related disorder of the central nervous system. In some embodiments, the present disclosure provides a method for treating a huntingtin-related disorder of the central nervous system, the method comprising administering a therapeutically effective amount of an HTT oligonucleotide to a subject suffering from a huntingtin-related disorder of the central nervous system. In some embodiments, the HTT oligonucleotide is administered to a subject suffering from and / or susceptible to a huntingtin-related disorder of the central nervous system outside the central nervous system (for example, but not limited to, intrathecally or intramuscularly), and the HTT oligonucleotide has the ability to cross the blood-brain barrier and enter the central nervous system. In some embodiments, the HTT oligonucleotide is administered directly to the central nervous system (for example, but not limited to, by intrathecally, intraventricularly, intracranially, etc.).

[0241] In some embodiments, in an HTT subject, the HTT gene or transcript has a CAG repeat expansion.

[0242] In some embodiments, a subject with HTT has a CAG repeat expansion. In some embodiments, allele-specific knockdown of mutant HTT transcripts can be used to eliminate or reduce the effects of such mutations by reducing the level, expression, and / or activity of the protein expressed from the mHTT transcript.

[0243] In some embodiments, a subject with HTT or suspected of having HTT is analyzed for HTT genotype prior to administration of a composition comprising an HTT oligonucleotide.

[0244] In some embodiments, a subject with HTT or suspected of having HTT is analyzed for an HTT phenotype prior to administration of a composition comprising an HTT oligonucleotide.

[0245] In some embodiments, subjects with HTT are analyzed for genotype and phenotype prior to administration of a composition comprising an HTT oligonucleotide, and the relationship between HTT genotype and HTT phenotype is determined.

[0246] In some embodiments, the subject is genetically confirmed to have Huntington's disease prior to administration of the composition comprising the HTT oligonucleotide.

[0247] In some embodiments, analyzing the HTT genotype or genetic confirmation of mHTT in a subject comprises determining whether the subject has one or more deleterious mutations in HTT.

[0248] In some embodiments, analyzing the HTT genotype or genetic confirmation of mHTT in a subject involves determining whether the subject has a CAG repeat expansion and a SNP that is the target of a particular HTT oligonucleotide on the same chromosome; in some embodiments, such analysis is referred to as phasing.

[0249] In some embodiments, the target nucleic acid sequence and the reference nucleic acid sequence differ at one or more sites, such as a mutation site, a single nucleotide polymorphism (SNP) site, etc. In some embodiments, the target nucleic acid sequence and the reference nucleic acid sequence contain differences at SNP sites. In some embodiments, a site in the target nucleic acid is perfectly complementary to a site in an oligonucleotide of the present disclosure, while the corresponding site in the reference nucleic acid is not complementary.

[0250] In some embodiments, analysis of the HTT genotype or genetic confirmation of mHTT informs the selection of compositions containing HTT oligonucleotides useful for treatment.

[0251] In some embodiments, an abnormal or mutant HTT gene or a portion thereof is removed or copied from a subject or one or more cells or tissues of the subject, and the abnormal or mutant HTT gene, or a portion thereof containing the abnormality or mutation, or a copy thereof, is inserted into a cell. In some embodiments, the cell can be used to test various compositions containing HTT oligonucleotides to predict whether such compositions may be useful as a treatment for the subject. In some embodiments, the cell is a myoblast or myotube.

[0252] In some embodiments, before a subject is treated with an HTT oligonucleotide, phasing is performed on the subject's genome to determine whether there is a CAG repeat expansion on the same chromosome as the SNP that is the target of the HTT oligonucleotide capable of mediating allele-specific knockdown.

[0253] In some embodiments, an HTT oligonucleotide capable of mediating allele-specific knockdown of a mutant HTT gene or its gene product and useful in methods for treating Huntington's disease is WVE-003.

[0254] WVE-003 In some embodiments, the present disclosure provides WVE-003, as well as preparations and compositions thereof. In some embodiments, the present disclosure provides techniques for manufacturing WVE-003. In some embodiments, the present disclosure provides techniques for determining and / or characterizing WVE-003. In some embodiments, the present disclosure provides techniques for determining the purity of WVE-003, e.g., Protocol A. In some embodiments, the present disclosure provides techniques for confirming the stereochemical identity of WVE-003 (or its stereoisomer (e.g., with respect to the chiral bond phosphorus)) using, e.g., IP-RP-UPLC (e.g., according to Protocol B). In some embodiments, the present disclosure provides techniques for determining the stereochemical purity of WVE-003 using, e.g., IP-RP-UPLC (e.g., according to Protocol B), dimer modeling, etc. In some embodiments, the present disclosure provides methods of using WVE-003. For example, in some embodiments, the present disclosure provides a method for treating Huntington's disease, comprising administering to a subject suffering from Huntington's disease an amount of WVE-003 as described herein.

[0255] In some embodiments, therapeutic uses of oligonucleotides include modulating the function of target mHTT RNA to reduce the production of disease-related mHTT protein. In some embodiments, the mechanism of action used by many oligonucleotides, including antisense oligonucleotides, is to promote the degradation of target mHTT RNA. In some embodiments, the use of phosphodiester bond modifications, such as phosphorothioate bonds, improves the stability, biodistribution, and cellular uptake of oligonucleotides. The use of chiral internucleotide linkages in oligonucleotide synthesis can create a chiral center at the linking phosphorus. The chiral linking phosphorus center can be in either the "Sp" or "Rp" configuration. Conventional stereochemically random oligonucleotide preparations with n chiral linking phosphorus result in 2 chiral linking phosphorus centers. nA mixture of 17 stereoisomers, each of which shares the same constitution but differs in stereochemistry along its backbone. In an oligonucleotide with 17 chiral phosphorus centers, the stereochemistry is 131,000 (2 17 ) stereoisomers, each of which is present at extremely low levels (approximately 1 / 131000).

[0256] As opposed to stereochemically random preparations, WVE-003 utilized herein is prepared stereoselectively. As described herein, in some embodiments, each chiral internucleotide linkage is independently formed with a diastereoselectivity of about 97% or greater (e.g., as measured through the preparation of suitable dimers). In some embodiments, most chiral internucleotide linkages are independently formed with a diastereoselectivity of about 98% or greater. In some embodiments, one or more, e.g., 1, 2, 3, 4, 5, 6, 7, 8, or more, chiral internucleotide linkages are independently formed with a diastereoselectivity of about 99% or greater. In some embodiments, the overall diastereoselectivity (as the product of the diastereoselectivities of all chiral internucleotide linkages) is about 80% or greater. In some embodiments, it is about 81% or greater. In some embodiments, it is about 82% or greater. In some embodiments, it is about 83% or greater. In some embodiments, it is about 84% or greater. In some embodiments, it is about 85% or greater.

[0257] In some embodiments, the oligonucleotide is WVE-003, a stereochemically defined oligonucleotide that can selectively target mHTT while leaving wtHTT relatively unaffected. WVE-003 specifically targets mHTT mRNA transcripts at the A variant of SNP rs362273 (SNP3). SNPs are single mutations that may be associated with a mutated gene in some cases. The most common SNP in the mHTT gene is SNP3, which has been reported to occur in approximately 40% to 45% of HD patients. Kay C et al. Clin Genet. 2014;86(1):29-36; Kay C et al. Mol Ther. 2015;23(11):1759-1771; Pfister EL et al. Curr Biol. 2009;19(9):774-778. Administration of WVE-003 may result in a selective reduction in mHTT protein levels by selectively targeting SNP3 mutations associated with pathogenic CAG expansions (≧36 repeats).

[0258] In some embodiments, WVE-003 is utilized as a disease-modifying agent to treat a subject with Huntington's disease (HD). In some embodiments, WVE-003 is a stereochemically pure antisense oligonucleotide (ASO) utilized to selectively target mutant forms of huntingtin (mHTT) gene transcripts.

[0259] In some embodiments, the HTT oligonucleotide, or a salt form thereof, is WVE-003 or a salt form thereof. In some embodiments, the oligonucleotide composition comprises WVE-003 or a salt form thereof.

[0260] The nucleotide sequence of WVE-003 is 5'-GUUGATCTGTAGCAGCAGCT-3'.

[0261] In some embodiments, WVE-003 is 5′-mG*SmUn001RmUmGn001RmA*ST*SC*ST*SG*ST*RA*SG*SC*SA*SG*Rm5Ceon001RAeoGeon001Rm5Ceo*STeo-3′, wherein *S represents an Sp phosphorothioate bond; *R represents an Rp phosphorothioate bond; mX represents 2'-O-methylribonucleoside; X represents a 2'-deoxyribonucleoside; Xeo represents 2'-O-(2-methoxyethyl)ribonucleoside; m5Ceo represents 2'-O-(2-methoxyethyl)-5-methylcytidine; n001R represents an Rp N-(1,3-dimethylimidazolidin-2-ylidene) phosphoramidate diester bond (PN); and The absence of "*R", "*S", or "n001R" between nucleosides may be described as representing a natural phosphate linkage (PO).

[0262] In some embodiments, WVE-003 is in the form of a sodium salt. In some embodiments, the sodium salt of WVE-003 is [P(S)]-2'-O-methyl-P-thioguanylyl-(3'→5')-[P(R)]-2'-O-methyl-P-deoxy-P-[(1,3-dimethylimidazolidin-2-ylidene)amino]uridylyl-(3'→5')-2'-O-methyluridylyl-(3'→5')-[P(R)]-2'-O-methyl-P-deoxy-P-[(1,3-dimethylimidazolidin-2-ylidene)amino]guanylyl-(3'→5')-[P(S)]-2'-O -Methyl-P-thioadenylyl-(3'→5')-[P(S)]-P-thiothymidylyl-(3'→5')-[P(S)]-2'-deoxy-P-thiocytidylyl-(3'→5')-[P(S)]-P-thiothymidylyl-(3'→5')-[P(S)]-2'-deoxy-P-thioguanylyl-(3'→5')-[P(R)]-P-thiothymidylyl-(3'→5')-[P(S)]-2'-deoxy-P-thioadenylyl-(3'→5')-[P(S)] -2'-Deoxy-P-thioguanylyl-(3'→5')-[P(S)]-2'-Deoxy-P-thiocytidylyl-(3'→5')-[P(S)]-2'-Deoxy-P-thioadenylyl-(3'→5')-[P(R)]-2'-Deoxy-P-thioguanylyl-(3'→5')-[P(R)]-2'-O-(2-methoxyethyl)-P-deoxy-P-[(1,3-dimethylimidazolidin-2-ylidene)amino]-5-methylcytidylyl-(3' →5')-2'-O-(2-methoxyethyl)adenylyl-(3'→5')-[P(R)]-2'-O-(2-methoxyethyl)-P-deoxy-P-[(1,3-dimethylimidazolidin-2-ylidene)amino]guanylyl-(3'→5')-[P(S)]-2'-O-(2-methoxyethyl)-P-thio-5-methylcytidylyl-(3'→5')-2'-O-(2-methoxyethyl)-5-methyluridine, pentadecasodium salt.

[0263] In some embodiments, the structure of WVE-003 sodium salt is presented as follows: As will be appreciated by those skilled in the art, in acid form, Na + Each of these is H + The zigzag line represents the bond between the 3' oxygen and the phosphorus of the internucleotide bond. [ka]

[0264] Specific information about WVE-003: Molecular formula (sodium salt form): C 236 H 308 N 86 Na 15 O 118 P 19 S 13 Molecular weight (sodium salt form): 7587.67 g / mol Molecular formula (free acid): C 236 H 323 N 86 O 118 P 19 S 13 Molecular weight (free acid): 7257.94g / mol WVE-003 has 19 internucleotide linkages, two of which are phosphodiester linkages, and 17 of which are stereochemically defined internucleotide linkages, 11 of which are Sp phosphorothioate diesters, two of which are Rp phosphorothioate diesters, and four of which are Rp N-(1,3-dimethylimidazolidin-2-ylidene) phosphoramidate diesters. This combination of internucleotide linkages can be illustrated by the following letter sequence: 5'-SnROnRSSSSSRSSSSRnROnRS-3', where "S," "R," "nR," and "O" represent Sp phosphorothioate diester, Rp phosphorothioate diester, Rp N-(1,3-dimethylimidazolidin-2-ylidene) phosphoramidate diester, and phosphodiester linkages, respectively.

[0265] In some embodiments, WVE-003 recognizes the disease-associated (e.g., mutant) allele of SNP rs362273 in the huntingtin gene, is effective in reducing the level, expression, and / or activity of the mHTT gene (or its gene product), and has the ability to mediate allele-specific knockdown of the mutant huntingtin (mHTT) gene.

[0266] In some embodiments, the potency and allele specificity of chiral-controlled WVE-003 compositions are superior to various stereochemically random oligonucleotide compositions.

[0267] For additional information regarding WVE-003, see, e.g., International Publication Nos. WO 2020 / 227691 and WO 2021 / 071788 (each of which is incorporated by reference in its entirety).

[0268] HTT Oligonucleotides and HTT Oligonucleotide Compositions In some embodiments, the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition (including but not limited to WVE-003) is capable of mediating allele-specific reduction in mHTT transcript levels, expression, and / or activity. In some embodiments, the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition is capable of mediating allele-specific knockdown of mutant HTT transcripts. In some embodiments, the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition is capable of mediating allele-specific reduction in mHTT transcript levels, expression, and / or activity. In some embodiments, the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition is chiral controlled.

[0269] In some embodiments, the HTT oligonucleotide, or a salt form thereof, or the HTT oligonucleotide composition is or comprises WVE-003. In some embodiments, the HTT oligonucleotide, or a salt form thereof, or the HTT oligonucleotide composition is capable of mediating allele-specific reduction in mHTT levels, expression, and / or activity and is chiral-controlled. When administered as described herein, WVE-003 is capable of mediating allele-specific knockdown of mutant HTT transcripts and may be a disease-modifying therapy for Huntington's disease. In some embodiments, the HTT oligonucleotide, or a salt form thereof, or the HTT oligonucleotide composition is capable of mediating allele-specific reduction in mHTT levels, expression, and / or activity and is chiral-controlled.

[0270] In some embodiments, the present disclosure provides methods of using huntingtin (HTT) oligonucleotides or HTT oligonucleotide compositions (eg, WVE-003) capable of mediating allele-specific knockdown of HTT transcripts.

[0271] In some embodiments, the present disclosure provides compositions and methods for allele-specific knockdown of HTT transcripts, wherein the allele-specific knockdown preferentially reduces the level, expression and / or activity of one or more alleles that contain a Huntington's disease-associated mutation.

[0272] In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is sufficient to mediate a clinically significant amount of allele-specific knockdown of a mutant HTT transcript in a subject.

[0273] In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is sufficiently low to prevent or reduce the occurrence and / or reduce the severity of at least about one adverse event mediated by administration of the oligonucleotide or oligonucleotide composition to a subject (e.g., when administered at higher doses). In some embodiments, a therapeutically effective amount of an HTT oligonucleotide, or a salt form thereof, or an HTT oligonucleotide composition is sufficient to mediate a clinically significant amount of allele-specific knockdown of a mutant HTT transcript in a subject, yet is sufficiently low to prevent or reduce the occurrence and / or reduce the severity of at least about one adverse event mediated by and / or associated with administration of the oligonucleotide or oligonucleotide composition to a subject (e.g., when administered at higher doses).

[0274] In some embodiments, the adverse event is an adverse effect. In some embodiments, the adverse event is mild, moderate, severe, or critical. In some embodiments, a serious adverse event is more severe than an adverse event classified as severe, moderate, or mild. In some embodiments, a serious adverse event is immediately life-threatening, requires inpatient hospitalization or an extension of an ongoing hospitalization, results in persistent or significant disability / incapacity, or is a congenital anomaly / birth defect not present at the time of screening. In some embodiments, the adverse event can be treated with hydrocortisone and / or acetaminophen.

[0275] In some embodiments, a severe adverse event is more severe than a moderate or mild adverse event. In some embodiments, a moderate adverse event is more severe than a mild adverse event. In some embodiments, the adverse event is fever, headache, vomiting, or tachycardia. In some embodiments, the adverse event is, is measured by, or is related to an increase in the inflammatory marker high-sensitivity C-reactive protein (hsCRP); an increase in complement factor Bb; or an increase in complement factor C3.

[0276] In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is an oligonucleotide or oligonucleotide composition that targets an HTT transcript and has the ability to regulate allele-specific knockdown of a mutant HTT transcript of the target transcript. In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is useful for preparing a medicament for treating Huntington's disease. In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is useful for treating Huntington's disease.

[0277] In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is useful for preparing a medicament for treating Huntington's disease, wherein the oligonucleotide is WVE-003, and the medicament is administered at a dose of about 30, about 60, about 90, about 120, about 150, or about 168 mg of the equivalent of the free acid form of WVE-003.

[0278] In some embodiments, the present disclosure provides methods of using HTT oligonucleotides or HTT oligonucleotide compositions. In some embodiments, the HTT oligonucleotides, or salt forms thereof, or HTT oligonucleotide compositions are capable of mediating allele-specific reduction in the level, expression, and / or activity of mHTT transcripts. In some embodiments, the HTT oligonucleotides, or salt forms thereof, or HTT oligonucleotide compositions are capable of mediating allele-specific knockdown of mutant HTT transcripts. In some embodiments, the HTT oligonucleotides, or salt forms thereof, or HTT oligonucleotide compositions are capable of mediating allele-specific reduction in the level, expression, and / or activity of mHTT transcripts. In some embodiments, the HTT oligonucleotides, or salt forms thereof, or HTT oligonucleotide compositions are chiral controlled.

[0279] In some embodiments, the HTT oligonucleotide, or a salt form thereof, or the HTT oligonucleotide composition is or comprises WVE-003. In some embodiments, the HTT oligonucleotide, or a salt form thereof, or the HTT oligonucleotide composition is capable of mediating allele-specific reduction in mHTT levels, expression, and / or activity and is chiral-controlled. In some embodiments, the HTT oligonucleotide, or a salt form thereof, or the HTT oligonucleotide composition is capable of mediating allele-specific reduction in mHTT levels, expression, and / or activity and is chiral-controlled.

[0280] The present disclosure recognizes the problem of providing HTT oligonucleotide compositions and methods of use with reduced toxicity. In some embodiments, the present disclosure provides HTT oligonucleotide compositions and methods with reduced toxicity. In some embodiments, the present disclosure provides HTT oligonucleotide compositions and methods with reduced immune response. In some embodiments, the present disclosure recognizes that various toxicities induced by HTT oligonucleotides are related to cytokine and / or complement activation. In some embodiments, the present disclosure provides HTT oligonucleotide compositions and methods with reduced or transient cytokine and / or complement activation. In some embodiments, the present disclosure provides HTT oligonucleotide compositions and methods with reduced complement activation via the alternative pathway. In some embodiments, the present disclosure provides HTT oligonucleotide compositions and methods with reduced complement activation via the classical pathway. In some embodiments, the present disclosure provides HTT oligonucleotide compositions and methods with reduced drug-induced vascular trauma. In some embodiments, the present disclosure provides HTT oligonucleotide compositions and methods with reduced injection site inflammation. In some embodiments, reduced toxicity can be assessed through one or more assays well known in the art, such as assessing the levels of full activation products, protein binding, etc.

[0281] Chiral controlled HTT oligonucleotides and compositions thereof In particular, the present disclosure relates to chiral controlled HTT oligonucleotides and oligonucleotides, such as WVE-003, and methods thereof.

[0282] In particular, the present disclosure embraces the recognition that, in contrast to chirally controlled HTT oligonucleotide compositions, stereochemically random HTT oligonucleotide preparations contain multiple individual chemical entities that differ from one another, for example, in the stereochemical configuration of individual backbone chiral centers within the HTT oligonucleotide chain. Due to the lack of control over the stereochemistry of the backbone chiral centers, stereochemically random HTT oligonucleotide preparations (e.g., random mixtures of diastereoisomers) result in uncontrolled (or stereochemically random) compositions containing an indeterminate level of HTT oligonucleotide stereoisomers. While these stereoisomers may have the same base sequence and / or chemical modifications, they are distinct chemical entities, likely due at least in part to their different backbone stereochemistry, and may have different properties, such as activity, toxicity, distribution, etc. In particular, the present disclosure provides chirally controlled compositions that are or contain a specific stereoisomeric HTT oligonucleotide (e.g., WVE-003) of interest; in contrast to non-chirally controlled compositions, chirally controlled compositions contain a controlled level of a specific stereoisomeric HTT oligonucleotide. In some embodiments, the level of a particular stereoisomer, e.g., WVE-003, in a chiral controlled oligonucleotide composition is enriched as described herein (e.g., in some embodiments, each chiral internucleotide linkage independently has a stereochemical purity of about 97%, 98%, 99% or greater).

[0283] In some embodiments, a particular stereoisomer may be defined by, for example, its base sequence, its backbone bonding pattern, its backbone chiral center pattern, and backbone phosphorus modification pattern, etc. As understood in the art, in some embodiments, base sequence may refer only to the sequence of bases and / or the identity and / or modification state of the nucleoside residues in the HTT oligonucleotide (e.g., of the sugar and / or base moieties, as compared to standard naturally occurring nucleotides such as adenine, cytosine, guanosine, thymine, and uracil) and / or the hybridization characteristics of such residues (i.e., the ability to hybridize with a particular complementary residue). In some embodiments, the present disclosure demonstrates that the improved properties (e.g., increased activity, decreased toxicity, etc.) achieved by the inclusion and / or location of a particular chiral structure within an HTT oligonucleotide can be comparable to, or even better than, those achieved by using chemical modifications, such as particular backbone linkages, residue modifications, etc. (e.g., by using particular types of modified phosphates [e.g., phosphorothioates, substituted phosphorothioates, etc.], sugar modifications [e.g., 2'-modifications, etc.], and / or base modifications [e.g., methylation, etc.]). In some embodiments, the present disclosure demonstrates that chirality-controlled HTT oligonucleotide compositions of HTT oligonucleotides (e.g., WVE-003, including various salt forms thereof) demonstrate unexpectedly high ability to mediate allele-specific knockdown of mutant HTT transcripts and are useful for treating and / or preventing Huntington's disease.

[0284] In some embodiments, the WVE-003 composition is a chirality-controlled oligonucleotide composition capable of mediating allele-specific reduction of mHTT transcript levels, expression, and / or activity. In some embodiments, the chirality-controlled WVE-003 composition is utilized to target human huntingtin pre-messenger ribonucleic acid (mRNA) to induce allele-specific knockdown of mutant HTT transcripts and restoration of huntingtin protein in subjects with Huntington's disease (HTT). WVE-003 is antisense to the HTT transcript and contains a base sequence capable of hybridizing thereto by complementary base pairing.

[0285] Applicants have developed technologies that enable the synthesis of linkage-modified nucleic acid therapeutics with precise control of the stereochemistry at each chiral linkage phosphorus position. This degree of control enables the rational design and synthesis of optimized, stereochemically pure oligonucleotides with improved pharmacological and toxicological properties. WVE-003 was developed and manufactured using certain such technologies.

[0286] In some embodiments, WVE-003 is utilized to target human huntingtin (HTT) pre-messenger ribonucleic acid (mRNA) to induce allele-specific knockdown of mutant HTT transcripts in subjects with Huntington's disease (HTT).

[0287] In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating allele-specific reduction of mHTT levels, expression, and / or activity in HTT (e.g., in mice, humans, etc.).

[0288] In some embodiments, the oligonucleotide composition comprises WVE-003. In some embodiments, the oligonucleotide composition is a chiral controlled oligonucleotide composition of WVE-003. In some embodiments, such a composition is a pharmaceutical composition of WVE-003.

[0289] As one of skill in the art will understand, an oligonucleotide, e.g., WVE-003, may be administered in one or more forms (e.g., an acid form, various salt forms, etc.). In some embodiments, the form is an acid form. In some embodiments, the form is a salt form. In some embodiments, the form is a sodium salt form. In some embodiments, the oligonucleotide, e.g., WVE-003, is administered as a salt form, e.g., a sodium salt form, optionally in solution. In some embodiments, a composition, e.g., a pharmaceutical composition, a chiral controlled oligonucleotide composition, etc., comprises one or more forms of WVE-003. As one of skill in the art will understand, in some embodiments, the amount of oligonucleotide, e.g., the amount of oligonucleotide administered, is a corresponding amount of any form, e.g., a particular form (e.g., an acid form) of any form administered (e.g., one or more salt forms (e.g., sodium salt form)). In some embodiments, multiple forms of the oligonucleotide may be present in the composition.

[0290] In some embodiments, the compositions comprising WVE-003 are each chirally pure or chiral controlled oligonucleotide compositions of WVE-003.

[0291] In some embodiments, chiral-controlled oligonucleotide compositions are typically prepared from chiral-controlled oligonucleotide preparations by stereoselectively forming one or more chiral internucleotide linkages (e.g., using a chiral auxiliary as exemplified in the present disclosure, compared to non-chiral (stereochemically random, non-stereoselective, racemic) oligonucleotide syntheses, such as conventional phosphoramidite-based oligonucleotide syntheses that do not use a chiral auxiliary or chiral catalyst to purposefully control stereochemical selectivity). Chiral-controlled oligonucleotide compositions of oligonucleotides are enriched for a particular oligonucleotide compared to a substantially racemic oligonucleotide preparation having the same base sequence and the same modifications (e.g., a chiral-controlled oligonucleotide composition of WVE-003 is enriched for WVE-003). As one skilled in the art will readily appreciate, such enrichment can be characterized by a higher level of the linking phosphorus having the desired stereoconfiguration in each chiral-controlled internucleotide linkage compared to a substantially racemic preparation. In some embodiments, each chiral controlled internucleotide linkage independently has a diastereomeric purity about its chiral linking phosphorus of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some embodiments, the diastereomeric purity is at least 95%. In some embodiments, the diastereomeric purity is at least 96%. In some embodiments, the diastereomeric purity is at least 97%. In some embodiments, the diastereomeric purity is at least 98%. In some embodiments, the diastereomeric purity is about 99% or greater.

[0292] As one of skill in the art will understand, since manufacturing processes rarely achieve 100% selectivity and / or purity (including, but not limited to, diastereomeric purity), preparations typically include an oligonucleotide, e.g., WVE-003, and one or more other oligonucleotides and / or other impurities. One of skill in the art will understand that the levels of such other oligonucleotides and / or impurities can and will be appropriately controlled.

[0293] In some embodiments, the chiral-controlled oligonucleotide composition can consist essentially of a desired oligonucleotide (e.g., WVE-003), e.g., the other oligonucleotides are impurities from the production of that oligonucleotide. In some embodiments, the impurities include oligonucleotides that are similar to, but different from, the desired oligonucleotide (e.g., one or more internucleotide linkages in the impurity may have an undesired configuration; and / or the impurity may be shorter or longer than the desired oligonucleotide). In some embodiments, the purity levels of various preparations of WVE-003 are described herein. In some embodiments, the characteristics of the various impurities are as described herein. Without wishing to be bound by any particular theory, the present disclosure notes that it is possible that one or more impurities may be sufficiently similar to the desired oligonucleotide to mediate a desired activity (e.g., allele-specific knockdown of mHTT).

[0294] As described herein, in some embodiments, the purity (e.g., the purity level, e.g., the purity level of WVE-003) is about 75% or at least about 75%. In some embodiments, the purity is about 80% or at least about 80%. In some embodiments, the purity is about 81% or at least about 81%. In some embodiments, the purity is about 82% or at least about 82%. In some embodiments, the purity is about 83% or at least about 83%. In some embodiments, the purity is about 84% or at least about 84%. In some embodiments, the purity is about 85% or at least about 85%. In some embodiments, the purity is about 86% or at least about 86%. In some embodiments, the purity is about 87% or at least about 87%. In some embodiments, the purity is about 88% or at least about 88%. In some embodiments, the purity is about 89% or at least about 89%. In some embodiments, the purity is about 90% or at least about 90%. In some embodiments, the purity is about 91% or at least about 91%. In some embodiments, the purity is about 92% or at least about 92%. In some embodiments, the purity is about 93% or at least about 93%. In some embodiments, the purity is about 94% or at least about 94%. In some embodiments, the purity is about 95% or at least about 95%. In some embodiments, the purity is about 96% or at least about 96%. In some embodiments, the purity is about 97% or at least about 97%. In some embodiments, the purity is about 98% or at least about 98%. In some embodiments, the purity is about 99% or at least about 99%.

[0295] In some embodiments, the purity of WVE-003 in preparations, compositions, pharmaceutical preparations, etc. can be determined using various suitable methods as described herein. In some embodiments, purity is determined using chromatography and UV detection, e.g., UPLC-UV as described in, where purity is measured as % peak area at a given wavelength, e.g., 260 nm. In some embodiments, purity is determined as described in Protocol A. In some embodiments, purity is determined as described in Protocol B. In some embodiments, purity is determined by dimer modeling.

[0296] manufacturing In some embodiments, the present disclosure provides manufacturing techniques for oligonucleotides, e.g., WVE-003, that are particularly useful for stereoselective large-scale preparation. In some embodiments, the WVE-003 preparation, e.g., WVE-003 drug substance or drug product, is a solid. In some embodiments, it is a white to off-white powder. In some embodiments, the aqueous solubility of the prepared WVE-003 was determined to be at least 79.20 mg / mL as determined by UV spectrophotometry (e.g., at 260 nm). In some embodiments, the pH of the WVE-003 preparation in purified water is 6.0-8.0. Unless otherwise noted, the following is used herein to calculate WVE-003 concentration / amount from, e.g., UV at 260 nm: -1 cm -1 The extinction coefficient is used.

[0297] In some embodiments, the oligonucleotide, eg, WVE-003, is chemically synthesized using a commercially available synthesizer in compliance with appropriate cGMP regulations.

[0298] The manufacture of WVE-003, e.g., the drug substance, is a multistep process that includes solid-phase oligonucleotide synthesis, cleavage of the crude protected oligonucleotide from the solid support, removal of protecting groups (deprotection), preparative anion exchange (AEX) chromatographic purification, concentration and desalting, filtration, lyophilization, and packaging. A flow diagram depicting the WVE-003 drug substance manufacturing process is shown in Figure 1.

[0299] In some embodiments, the stereochemistry of an oligonucleotide, such as WVE-003, is established by the synthetic starting materials and synthetic process control. For example, the use of phosphoramidites prepared from chiral auxiliaries (L)-DPSE, (D)-DPSE, and (L)-PSM during the coupling step ensures the intended stereochemically defined Sp phosphorothioate diester, Rp phosphorothioate diester, and Rp N-(1,3-dimethylimidazolidin-2-ylidene) phosphoramidate diester linkages, respectively.

[0300] In some embodiments, the present disclosure provides methods of manufacturing various drug products as described herein.

[0301] In some embodiments, the present disclosure provides products (e.g., crude / purified oligonucleotides from stereoselective preparations, crude compositions, purified compositions, formulated compositions, pharmaceutical compositions, drug substances, drug products, etc.) from the provided processes. In some embodiments, the provided products are of a certain purity as described herein. In some embodiments, the provided products are suitable for therapeutic uses as described herein. In some embodiments, the provided products achieve one or more properties and / or activities as described herein.

[0302] Specific processes useful in the manufacture of WVE-003 and various compositions and products thereof are described below by way of example: Several batches of WVE-003 drug substance and / or drug product were manufactured.

[0303] As shown herein, the preparation of WVE-003 involves multiple cycles (e.g., as described below). In some embodiments, each cycle introducing a stereochemically defined phosphorothioate or a stereochemically defined phosphoramidate consists of 5'-detritylation, coupling, capping of the exposed chiral auxiliary secondary amine (capping-1), thiolation or imidization, respectively, and capping of the unreacted 5'-hydroxyl group (capping-2); each cycle introducing a phosphodiester consists of 5'-detritylation, coupling, oxidation, and capping-2; and each coupling reaction is carried out by activation of the appropriate phosphoramidite and reaction with the free 5'-hydroxyl group of a support-immobilized protected nucleoside or oligonucleotide. In some embodiments, after an appropriate number of cycles and final detritylation, the (L)-PSM chiral auxiliary and cyanoethyl phosphate protecting groups are removed from the crude oligonucleotide by on-column treatment with diethylamine (DEA) in acetonitrile (ACN), and the (L) and (D)DPSE chiral auxiliaries are removed by treatment with TEA·HF (triethylamine hydrogen fluoride) solution. The crude oligonucleotide is then cleaved from the solid support by treatment with ammonium hydroxide in an appropriately sized pressure-rated vessel. These reactions also achieve global deprotection of exocyclic amino groups (acetyl, benzoyl, and isobutyryl). In some embodiments, the resulting crude oligonucleotide is purified using anion exchange (AEX) chromatography, and the purified oligonucleotide is concentrated and desalted by tangential flow filtration followed by filtration, lyophilized, and packaged to yield the WVE-003 drug substance. Specific steps for preparing the WVE-003 drug substance are described in more detail below as examples. Certain key functional groups are described below.

[0304] [Table 1]

[0305] [ka]

[0306] Phosphorothioate assembly cycle [ka]

[0307] Phosphoroimidate assembly cycle [ka]

[0308] Phosphodiester Assembly Cycle [ka]

[0309] Step 1 - Synthesis Oligonucleotide synthesis is performed on a controlled pore glass (CPG) solid support functionalized with 5'-ODMTr-2'-MOE-T in an automated oligonucleotide synthesizer. All reactions occur on this solid support packed into a column.

[0310] Detritylation To begin the synthesis, the CPG-5'-ODMTr-2'-MOE-T solid support is subjected to acid-catalyzed removal of the DMTr protecting group from the 5'-hydroxyl by treatment with 3% dichloroacetic acid (DCA) in toluene. Complete DMTr removal is ensured by in-line UV monitoring based on the watch command of the synthesis program. DMTr removal is carried out in the same manner at the beginning of each synthesis cycle and after the last cycle. In both cases, after detritylation, the support-bound material is washed with acetonitrile in preparation for the next step in the synthesis. [ka]

[0311] Coupling Elongation of the growing oligonucleotide chain is achieved by reacting the 5'-hydroxyl group of the support-bound oligonucleotide with an excess of a solution of a protected phosphoramidite in the presence of an activator, either 5-(ethylthio)-1H-tetrazole (ETT) or 1-cyanomethylimidazolium triflate salt (CMIMT), dissolved in acetonitrile. The phosphoramidite is dissolved in acetonitrile, isobutyronitrile, or an 80:20 v / v mixture of acetonitrile and isobutyronitrile. Among other things, the present disclosure recognizes that ethyl acetate can be used to prepare phosphoramidite solutions for oligonucleotide preparations, such as WVE-003 preparations. In some embodiments, ethyl acetate can be used to replace isobutyronitrile. In some embodiments, the use of ethyl acetate reduces manufacturing costs and / or simplifies operations. In some embodiments, the phosphoramidite is dissolved in ethyl acetate or a mixture thereof. In some embodiments, the solvent is ethyl acetate. In some embodiments, the solvent is a mixture of ethyl acetate and acetonitrile (e.g., about 50:50 v / v ethyl acetate:acetonitrile, about 20:80 v / v ethyl acetate:acetonitrile, etc.). Among other things, the present disclosure recognizes that propylene carbonate can be utilized in preparing phosphoramidite solutions for oligonucleotide preparations, e.g., WVE-003 preparations. In some embodiments, propylene carbonate can be utilized to replace isobutyronitrile. In some embodiments, the utilization of propylene carbonate reduces manufacturing costs and / or simplifies operations. In some embodiments, the phosphoramidite is dissolved in propylene carbonate or a mixture thereof. In some embodiments, the solvent is propylene carbonate. In some embodiments, the solvent is a mixture of propylene carbonate and acetonitrile (e.g., about 50:50 v / v propylene carbonate:acetonitrile).In some embodiments, the phosphoramidites are dissolved in acetonitrile, ethyl acetate, a mixture of ethyl acetate and acetonitrile (e.g., about 20:80 v / v ethyl acetate:acetonitrile; about 50:50 v / v ethyl acetate:acetonitrile; etc.), or a mixture of propylene carbonate and acetonitrile (e.g., about 50:50 v / v propylene carbonate:acetonitrile). The phosphoramidite required for each coupling step depends on the oligonucleotide sequence. Phosphoramidites and ETT activators are used to introduce phosphodiester bonds. Phosphoramidites derived from (L)- or (D)-DPSE or (L)-PSM and CMIMT activators are used to introduce stereochemically defined phosphorothioate or phosphoramidate bonds, respectively. In either case, the phosphoramidite / activator solutions are mixed in-line and loaded onto a synthesis column, which is then recirculated for an appropriate length of time. The synthesis column is then flushed with acetonitrile to remove excess reagents. [ka]

[0312] Capping-1 The capping 1 step is performed to protect the secondary amine generated by oxazaphospholidine ring opening on the auxiliary group during the coupling step, making it unreactive during the remainder of the synthesis. The secondary amine is capped by running the synthesis column with capping reagent B ([Cap B]: acetic anhydride / 2,6-lutidine / acetonitrile (20:30:50, v:v:v). [ka]

[0313] Oxidation, thiolation, imidization The newly created P(III) phosphite triester bond is then treated in one of three ways: Oxidation by treatment with iodine in pyridine:water (90:10, v:v), which results in the generation of a P(V) phosphodiester bond. Thiolation by treatment with xanthan hydride (XH) in pyridine:acetonitrile results in the generation of stereochemically defined P(V) phosphorothioate triester linkages. Imidation by treatment with 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (ADIH) in acetonitrile results in the generation of a stereochemically defined P(V) phosphoroimidate triester linkage. In either case, after the step, excess reagent is flushed from the synthesis column with acetonitrile. [ka]

[0314] Capping-2 The coupling reaction proceeds in very high yields, but is not quantitative. A small percentage of the available 5'-hydroxy groups in any given cycle fail to couple with the activated phosphoramidite. To prevent reaction in subsequent cycles, these sites are blocked by a capping-2 step using a capping reagent (1:1, v:v) (capping reagent A [Cap A]:N-methylimidazole / acetonitrile (20:80, v:v) and Cap B). This results in the formation of a 5'-O-acetylated ("capped") support-bound oligonucleotide sequence. Excess capping reagent is washed from the column with acetonitrile. [ka]

[0315] Oligonucleotide Assembly and Final Steps By independently repeating the cycle using appropriate protected phosphoramidites, it is possible to assemble the entire protected sequence in which the DMTr protecting group is present at the 5'-terminal position. After adding the last nucleotide to the sequence, the 5'-terminal DMTr group is removed during a final detritylation step.

[0316] Step 2 - Cleavage and deprotection Removal of the phosphate protecting groups (L)-PSM and (L)- and (D)-DPSE chiral auxiliaries from the stereochemically defined phosphorimidate and phosphorothioate triesters, cleavage of the crude oligonucleotides from the solid support, and removal of the exocyclic nucleobase protecting groups is carried out in a three-step process.

[0317] Step 1: (L)-PSM chiral auxiliary and cyanoethyl removal by treatment with diethylamine The crude oligonucleotide on the solid support is treated with a solution of diethylamine in ACN, which simultaneously removes the cyanoethyl protecting group from the phosphotriester linkage and the (L)-PSM chiral auxiliary from the phosphoromidate triester to generate the phosphodiester and phosphoramidate diester linkages, respectively. (L)-PSM chiral auxiliary and cyanoethyl removal by treatment with diethylamine: [ka]

[0318] Step 2: (L)- and (D)-DPSE chiral auxiliary removal by fluoride treatment The removal of the chiral auxiliary occurs by treating the crude oligonucleotide on the solid support with a temperature-controlled solution of TEA·HF in a mixture of dimethyl sulfoxide (DMSO) and HO. This process converts the stereochemically defined phosphorothioate triesters into stereochemically defined phosphorothioate diesters. (L)- and (D)-DPSE chiral auxiliary removal by fluoride treatment: [ka]

[0319] Step 3: Cleavage and deprotection The crude oligonucleotide is then cleaved from the solid support by treatment with ammonium hydroxide in an appropriately sized pressure-rated vessel, a reaction that results in global deprotection of the exocyclic amino groups (acetyl, benzoyl, and isobutyryl). Cleavage and deprotection reactions: [ka] [ka]

[0320] Step 3: Purification by anion exchange chromatography Purification of the crude oligonucleotide solution is achieved by AEX chromatography: The crude oligonucleotide solution is loaded onto a purification column packed with TSK-GEL Super Q-5PW media.

[0321] Purification is performed using an eluent buffered with sodium hydroxide. The oligonucleotide is eluted from the column using a sodium chloride gradient. The elution profile is monitored by ultraviolet (UV) spectrophotometry. Fractions are collected and neutralized with sodium phosphate buffer. Mock pools are evaluated by IP-RP-UPLC. Pools containing oligonucleotides of the desired purity are subjected to the next step in the process.

[0322] Stage 4: Concentration and desalting (final UF / DF) The selected fraction pool is then concentrated and diafiltered with purified water to remove the purification buffer by tangential flow filtration (TFF) using a regenerated cellulose membrane cassette. The ultrafiltration / diafiltration (UF / DF) process proceeds as follows: the pH of the selected fraction pool is neutralized with hydrochloric acid or sodium hydroxide, then concentrated. The concentrated oligonucleotide is diafiltered with purified water and further concentrated for collection. The system is flushed with purified water, and the concentrated, desalted oligonucleotide is combined with the rinse to maximize yield, resulting in the final oligonucleotide solution.

[0323] Step 5: Filtration, freeze-drying and packaging The oligonucleotide solution is filtered through a 0.2 micron filter and then placed in one or more lyophilization trays for lyophilization. After lyophilization, the final drug substance is isolated as a solid powder, which is packaged in sterile high density polyethylene (HDPE) bottles, each of which is labeled, sealed in a Mylar foil pouch, and stored at -20°C.

[0324] Material Management Starting materials used in the manufacture of drug substances Useful raw materials (solvents, reagents and auxiliary materials) used in the manufacture of WVE-003, e.g., drug substance, are listed below: In some embodiments, the quality of all raw materials is controlled by material specifications and / or certificates of analysis.

[0325] Starting materials used in the preparation of the WVE-003 drug substance include 15 phosphoramidites, controlled pore glass (CPG) solid support, and 2-azido-1,3-dimethylimidazolinium hexafluorophosphate (ADIH). Reactive exocyclic groups on the nucleobases are typically appropriately protected to prevent their reaction during oligonucleotide synthesis, and the 5'-hydroxy functionality is protected as a 4,4'-dimethoxytrityl ether (DMTr). In some embodiments, the WVE-003 starting material is released based on a set of material specifications. In some embodiments, the phosphoramidites have purity levels of about 85% or greater, often about 90% or greater, and often about 95% or greater, or about 98% or greater (RP-HPLC at about 260 nm (area %) and / or 31 P NMR integral value), and P(III) purity of approximately 97% or more ( 31 P NMR integral) and has a water content of less than about 0.4%, or often less than 0.2% (w / w). In some embodiments, the controlled pore glass 5'-ODMTr-2'-OMOET solid support is a white to off-white powder, 120-200 mesh particle size (analytical sieve), 540-600 Å diameter (mercury porosimetry), 0.20-0.24 g / cc density (tapped density), ≥ 0.8 cc / g pore volume (mercury porosimetry), ≥ 75 m 2 / g surface area (mercury porosimetry) and DMT ligand assay of 70-80 μmol / g (spectrophotometric at 498 nm). In some embodiments, the 2-azido-1,3-dimethylimidazolinium hexafluorophosphate has a purity of about 98.0% or greater (HPLC) and a nitrogen content of about 23.70-24.80% (elemental analysis).

[0326] [Table 2]

[0327] [Table 3]

[0328] [Table 4]

[0329] [Table 5]

[0330] [Table 6]

[0331] The stereochemistry of WVE-003 can be established by controlling the synthetic starting materials and the synthetic process. Stereochemically defined phosphoramidite starting materials for the preparation of WVE-003 include appropriately protected nucleosides and chiral auxiliaries (L)- and (D)-DPSE and (L)-PSM: [ka] It is prepared from

[0332] In some embodiments, the chemical purity of the stereochemically defined phosphoramidite is 31 The absolute stereogenic configuration is determined by P NMR. 31 P NMR, 1 H NMR, and 13 The stereochemistry of the protected nucleoside and chiral auxiliary is fixed, so there are two possible diastereomers (trans and cis) that can result from the phosphitylation reaction, of which the trans form is typically predominant, with the cis form present as a minor impurity. Starting materials and specific possible stereoisomers resulting from coupling reactions [ka]

[0333] Throughout the manufacture of WVE-003, e.g., drug substance, various in-process controls are applied, for example, as described below. Upon completion of the synthesis and cleavage and deprotection steps, the presence of WVE-003 in the resulting crude oligonucleotide is identified by LC / MS (liquid chromatography mass spectrometry) (e.g., 7257.9 ± 3 Da), and its purity is quantified by IP-RP-UPLC (ion-pair reversed-phase ultra-performance liquid chromatography) (e.g., ≥ 84% according to Protocol A). During purification, select mock pools are evaluated for purity and impurities by IP-RP-UPLC. Once select fractions are pooled and concentrated, desalting of the resulting solution is controlled by in-process measurements of conductivity (e.g., ≤ 50 μS / cm), pH (e.g., 6.7-7.3), and concentration.

[0334] In some embodiments, the drug substance or drug product is manufactured to meet one or more or all of the criteria described herein. In some embodiments, the drug substance, e.g., WVE-003 or a salt form thereof, has a particular level of purity as described herein. In some embodiments, WVE-003 pentadeca sodium salt has a particular level of purity as described herein. In some embodiments, WVE-003 or a salt form thereof has a particular level of purity in a composition, e.g., a pharmaceutical composition. In some embodiments, WVE-003 or a salt form thereof has a particular level of purity in a drug product. In some embodiments, the salt form is WVE-003 pentadeca sodium salt. In some embodiments, WVE-003 has a level of purity of about 70%-90%, about 80%-90%, about 84%-90%, or about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% or greater as determined by IP-RP-UPLC as described herein.

[0335] Multiple lots of WVE-003 drug substance were manufactured. In some embodiments, the scale was greater than 20 mmol. In some embodiments, the scale was a multiple of about 20 mmol. In some embodiments, the manufactured WVE-003 was utilized in clinical trials.

[0336] Characterization Among other things, the present disclosure provides techniques for characterizing and / or assessing WVE-003 or compositions thereof, such as WVE-003 preparations, such as drug substances, drug products, and the like.

[0337] The product can be characterized by various techniques. For example, WVE-003 may be characterized by mass spectrometry. One preparation was analyzed by electrospray ionization mass spectrometry (ESI-MS). The theoretical average mass of WVE-003 (free acid form) is consistent with the experimentally determined value of 7257 Da.

[0338] In some embodiments, the sequence of WVE-003 is confirmed by ESI-MS / MS sequencing.For example, in one determination, at least one sequence-related fragment ion is observed for 19 nucleobases in the sequence, and the error of each calculated (predicted) mass is within 5 ppm.The monoisotopic mass of full-length oligonucleotide is also experimentally verified by ESI-MS / MS analysis (7253.3428Da), and the identity and position of all 20 nucleobases are verified, thereby confirming the sequence of WVE-003.

[0339] Stereochemical identity is established by controlling the synthetic starting materials. Synthetic coupling of phosphoramidites prepared from (L)-DPSE or (L)-PSM results in phosphorothioate diester bonds of Sp chirality and phosphoramidate diester bonds of Rp chirality, respectively, while those prepared from (D)-DPSE or (D)-PSM result in phosphorothioate diester bonds of Rp chirality and phosphoramidate diester bonds of Sp chirality. The consistent use of stereochemically defined phosphoramidites in each coupling reaction allows for control of the stereochemical identity of each phosphorothioate bond. Stereochemical identity can be confirmed using various techniques in this disclosure. For example, stereochemical identity can be confirmed by measurement using several different techniques, which, in combination with and compared to appropriate standards, provide precise information about and confirmation of this key attribute. Useful analytical techniques include NMR ( 1 H, 19 F, 31 These include NMR (P, multidimensional, etc.) and enzymatic digestion. In some cases, NMR is performed in phosphate buffer (e.g., pH 7.0). 1 H, 19 F and 31 Results, including P NMR and enzymatic digestion, are consistent with the product structure. In some embodiments, NMR is referenced to a water based DSS standard. Observed data from specific experiments are described below.

[0340] In some embodiments, WVE-003 drug substance was prepared at a concentration of 1 mM in 600 μL of 100 atom % DO solution containing 100 mM NaCl, 0.05 mM EDTA, and 10 mM phosphate buffer (pH 7.0). In some embodiments, spectra were recorded at 334 K. In some embodiments, sodium trimethylsilylpropanesulfonate (DSS) in phosphate buffer was used as an external reference standard. In some embodiments, 1The 1 H NMR spectra were indirectly calibrated based on the DSS peak at 0 ppm. 1 The H NMR spectrum contains signals consistent with the WVE-003 structure, and the chemical shifts of individual regions of the signals are consistent with the proposed structure. In some embodiments, WVE-003, e.g., the drug substance lot prepared 31 P NMR spectra were obtained and recorded at 161.98 MHz for phosphorus. 31 P NMR spectra were taken using an external standard with the DSS peak set to 0 ppm. 1 The H NMR spectrum was indirectly calibrated using a unified scale. In some embodiments, the sharp signal near 2 ppm arises from the phosphate buffer solution used. A total of 19 PS / PN / PO peaks are consistent with the stereochemically defined mixed PO / PS / PN backbone of the WVE-003 drug substance.

[0341] [Table 7]

[0342] In some embodiments, the stereochemical identity of WVE-003 was confirmed by enzymatic digestion assays.

[0343] In some embodiments, stereochemical purity is expressed as the percentage of the correct diastereomer associated with the assigned stereochemical identity relative to the total diastereomeric mixture, including minor amounts of other diastereomers that may be present. The diastereoselectivity of each (L)- or (D)-DPSE phosphoramidite relative to its stereochemically defined phosphorothioate or phosphoramidate linkages in WVE-003 is extremely high, and the overall stereochemical purity of the oligonucleotide is the combined product of the diastereoselectivities of these 13 phosphorothioate and 4 phosphoramidate linkages.

[0344] In some embodiments, the diastereoselectivity of the chiral linkage is determined by preparing the corresponding dimer. The synthetic conditions used to create each sequence dimer are identical to those applied to the entire molecule. These dimeric units are separated and analyzed using RP-UPLC conditions to separate and quantify the Rp and Sp diastereomers. In some embodiments, the WVE-003 sequence dimer and its respective references are analyzed for purity by ultra-performance liquid chromatography (UPLC) and / or for confirmation of the masses of the Sp and Rp diastereomers by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS). Analysis of these samples was completed on crude material, demonstrating representative diastereoselectivities of the linkages in this synthetic process. The stereochemical purity of the various dimers analyzed is presented below. All sequence-specific dimers were synthesized using the same process conditions as WVE-003 and accurately represent the phosphorothiolated and phosphoramidated internucleotide linkages.

[0345] [Table 8]

[0346] The stereochemical purity of WVE-003 can be attributed to the product of the stereochemical purity of each of the 13 phosphorothioate and 4 phosphoramidate linkages in the sequence.

[0347] [Table 9]

[0348] In some embodiments, as demonstrated herein, the diastereomeric purity of WVE-003 is about 82.7% based on dimer modeling. In some embodiments, UPLC analysis is used for dimer modeling. As demonstrated herein, all 17 bonds have a stereochemical purity of 97% or greater, 9 bonds have a stereochemical purity of 97% or greater, and 7 bonds have a stereochemical purity of 99% or greater. In some embodiments, as demonstrated herein, the average stereochemical purity of all bonds is 98.9%.

[0349] In some embodiments, the stereochemical identity is determined by UPLC as part of batch release using a reference standard, e.g., characterized by NMR, enzymatic digestion, etc. In some embodiments, the provided technology provides the reference standard. In some embodiments, the reference standard has purity (e.g., as determined by Protocol A) and / or stereochemical purity (e.g., as determined by Protocol B and / or dimer modeling as described herein).

[0350] The FTIR spectrum of WVE-003 was obtained with an attenuated total reflectance (ATR) sampling accessory, with the main absorbance band at 1636 cm -1 and 1600 cm -1 , the absorption peak is 2938 cm -1 , and a broad peak at 3199 cm -1 and 3339 cm -1 The FTIR results are consistent with the structure of WVE-003.

[0351] In the present disclosure, counterions may be analyzed. In some embodiments, sodium content is determined by ICP-OES. In some embodiments, a sodium content value of 4.5% was determined by ICP-OES for the WVE-003 drug substance, which is consistent with the theoretical sodium content value of 4.5% (w / w).

[0352] For various preparations, the WVE-003 drug substance is a white to off-white powder. The pH of the WVE-003 drug substance in purified water was found to be 6.0-8.0. In some embodiments, it is about 6.5-7.5. In some embodiments, it is about 6.4. In some embodiments, it is about 6.5. In some embodiments, it is about 6.6. In some embodiments, it is about 6.7. In some embodiments, it is about 6.8. In some embodiments, it is about 6.9. In some embodiments, it is about 7.0. In some embodiments, it is about 7.1. In some embodiments, it is about 7.2. In some embodiments, it is about 7.3. In some embodiments, it is about 7.4.

[0353] The molar extinction coefficient of WVE-003 drug substance is 181181 M at 260 nm in water. -1 cm -1 This was experimentally determined and used to convert the UV absorbance to a concentration, from which the amount can be calculated. This equates to an extinction coefficient of approximately 25.0 OD / mg.

[0354] In various embodiments, impurities are reduced to low levels as described herein. For example, in some embodiments, impurity levels are less than about 30%, about 25%, or about 20%, as described herein. In some embodiments, impurities include various oligonucleotide impurities. Impurities may be identified, characterized, and / or determined using various techniques, such as mass spectrometry, LC, UV, etc.

[0355] WVE-003 drug substance was evaluated in GLP in vitro genotoxicity studies and in vivo micronucleus studies, and no toxicity was observed.

[0356] Various preparations of WVE-003 drug substance met the specified residual solvent limits, including elemental impurities including acetonitrile, toluene, and pyridine (ICH Class 2), and / or various elements such as ICH Class 1, 2A, and 3. Elemental impurities in various preparations were within the limits.

[0357] The release specifications for the WVE-003 drug substance and / or drug product may include one or more specifications described herein, e.g., appearance (e.g., visibly white to off-white powder), sequence identity (e.g., by MS / MS), molecular weight, stereochemical identity (e.g., by IP-RP-UPLC), purity (e.g., area % by IP-RP-UPLC (e.g., Protocol A)), impurities (e.g., area % by IP-RP-UPLC (e.g., Protocol A)), sodium content (e.g., by ICP-OES, etc.), water content (% w / w; e.g., USP <921> and / or Ph.Eur.2.5.12, etc.), assay (e.g., free acid, anhydrous, by UV), pH (e.g., of a solution in purified water; e.g., USP <791> and / or Ph.Eur.2.2.3, etc.), residual solvents (e.g., by gas chromatography), elemental impurities (e.g., by ICP-MS), bacterial endotoxins (e.g., USP <85> and / or Ph.Eur 2.6.14), bioburden (total aerobic microbial count, total yeast and mold count, etc. USP <61> ) and / or according to Ph.Eur 2.6.12), etc.

[0358] In some embodiments, the identity of WVE-003, e.g., the drug substance, is determined by liquid chromatography-mass spectrometry (LC-MS). Samples and analytical reference materials are prepared in water and injected onto a Waters Acquity BEH C18 column. The analysis involves a gradient of mobile phase A (hexafluoroisopropanol [HFIP] and triethylamine [TEA] in water) and mobile phase B (acetonitrile). A summary of the method parameters is provided below.

[0359] [Table 10]

[0360] In some embodiments, system suitability is confirmed for WVE-003 if the full-length product (FLP) molecular weight (deconvoluted mass) of the first three injections of analytical reference material is 7258±3 Da. Additionally, blank chromatograms show no obscured peaks other than the solvent front and gradient shift.

[0361] In some embodiments, the identity of WVE-003, e.g., by sequencing in a drug substance, is determined by high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS / MS). A useful procedure is described by way of example below: Samples are prepared in water and injected directly into the mass spectrometer for MS / MS analysis. The analysis involves an isocratic LC method with a mixture of mobile phase A (hexafluoroisopropanol [HFIP] and triethylamine [TEA] in LC-MS grade water) and mobile phase B (acetonitrile). A summary of the method parameters is provided below.

[0362] [Table 11]

[0363] In some embodiments, system suitability is confirmed when the m / z charge state used in the sequencing experiment is within ±1 Da of the theoretical value, the accurate full-length product mass is 7253.3302 ± 0.1000 amu, and the mass spectrum in MS mode matches the reference spectrum in the method.

[0364] In some embodiments, the sodium content of WVE-003 preparations, e.g., drug substance, is determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). Sample material in solution is introduced into a radio frequency plasma by air nebulization, where an energy transfer process causes desolvation, nebulization, and ionization. During this process, the sodium emits light, and the instrument measures the absorbance to quantify the sodium level. In some embodiments, the sodium content (anhydrous basis) is about 3.9-5.2% (w / w). In some embodiments, it is about 4-5%. In some embodiments, it is about 4.3%-4.7%. In some embodiments, it is about 4.3%. In some embodiments, it is about 4.4%. In some embodiments, it is about 4.5%. In some embodiments, it is about 4.6%. In some embodiments, it is about 4.7%.

[0365] In some embodiments, the present disclosure provides techniques for determining the purity and / or impurities of a WVE-003 preparation or composition. In some embodiments, the purity and impurities of the WVE-003 drug substance are determined by ion-pair reversed-phase UPLC (IP-RP-UPLC) using a Waters BEH C18 column. A useful procedure is described below as an example (Protocol A). A gradient of mobile phase A (hexafluoroisopropanol [HFIP] and triethylamine [TEA] in water) and mobile phase B (50% acetonitrile in water) is used to effect separation. A summary of the method parameters is provided below as an example (Set A Parameters). In some embodiments, system suitability is established by the absence of interfering peaks in the blank and acceptance criteria for retention time, peak area %, and %RSD for the peak areas of the initial injection and all injections of the WVE-003 analytical reference material (system suitability standard). In some embodiments, all individual impurities ≥ 0.10 area % and the sum of all impurities ≥ 0.10 area % as a function of relative retention time to the main peak are reported. In some embodiments, WVE-003 identity is confirmed by comparing relative retention times to system suitability standards. The method has been determined to be robust in method development studies.

[0366] [Table 12]

[0367] In some embodiments, the purity of a WVE-003 preparation, e.g., drug substance, drug product, etc., is about 84% or greater (e.g., as determined by Protocol A (% area)). In some embodiments, it is about 85% or greater. In some embodiments, it is about 86% or greater. In some embodiments, it is about 87% or greater. In some embodiments, it is about 88% or greater. In some embodiments, it is about 80%-90%. In some embodiments, it is about 84%-90%. In some embodiments, it is about 84%-88%. In some embodiments, the amount of total impurities is about 16% or not more than about 16% (e.g., as determined by Protocol A (% area)). In some embodiments, the amount of total impurities is about 15% or not more than about 15%. In some embodiments, the amount of total impurities is about 14% or not more than about 14%. In some embodiments, the amount of total impurities is about 13% or not more than about 13%. In some embodiments, the amount of total impurities is about 12% or not more than about 12%. In some embodiments, the sum of WVE-003 and total impurities is about 99% to 101%.

[0368] In some embodiments, the assay of a WVE-003 preparation, e.g., drug substance, drug product, etc., is determined by UV spectrophotometry. A useful procedure is described herein by way of example. An accurate amount of WVE-003 drug substance is weighed and dissolved in water in volumetric glassware. The solution absorbance is determined at 260 nm, and this value is converted to the molar extinction coefficient (MEC; 181,181 M -1 cm -1and 25.0 OD / mg) to convert to the concentration of WVE-003 drug substance. Subtract the water content of the sample from the weighed amount of material (e.g., Karl Fischer titration, USP <921> The assay is then corrected by subtracting the water content (by Method 1c) and sodium content. The assay (%) is then determined as the ratio of the amount of WVE-003 measured by absorbance to the theoretical amount of preparation based on the exact weight of the sample corrected for water and sodium content. In some embodiments, the assay of a WVE-003 preparation, e.g., drug substance, drug product, etc., is about 100% ± 10%. In some embodiments, it is about 95%-105%. In some embodiments, it is about 95%. In some embodiments, it is about 96%. In some embodiments, it is about 97%. In some embodiments, it is about 98%.

[0369] In some embodiments, the water content of a WVE-003 preparation, e.g., a drug substance, drug product, etc., is determined by coulometric titration (Karl Fischer). In some embodiments, system suitability is established by the required sufficient recovery from a certified sodium tartrate monohydrate reference standard of known water content. In some embodiments, the water content of a WVE-003 preparation, e.g., a drug substance, drug product, etc., does not exceed about 15%, 10%, 5%, or 1% (w / w). In some embodiments, it does not exceed about 15%. In some embodiments, it does not exceed about 10%. In some embodiments, it does not exceed about 9%. In some embodiments, it does not exceed about 8%. In some embodiments, it does not exceed about 7%. In some embodiments, it does not exceed about 6%. In some embodiments, it does not exceed about 5%. In some embodiments, it does not exceed about 1%.

[0370] In some embodiments, residual solvents in WVE-003 preparations, e.g., drug substance, drug product, etc., are quantified using gas chromatography-flame ionization detection (GC-FID). In some embodiments, samples are prepared by thoroughly mixing, weighing into a sealed vial, and dissolving in solvent. The sample is then introduced by direct injection, and quantification is performed as a limit test by comparison to an external standard. Blanks and calibration checks are analyzed at appropriate intervals.

[0371] In some embodiments, the present disclosure provides techniques for confirming the stereochemical identity of WVE-003 or its diastereomers (e.g., with respect to one or more chiral bond phosphorus centers). In some embodiments, for example, the stereochemical identity of WVE-003 in WVE-003 preparations, drug substances, drug products, etc., is determined by IP-RP-UPLC using a Waters BEH C18 column. A useful procedure is described below as an example (Protocol B). In some embodiments, separation of WVE-003 from closely related diastereomeric impurities is provided using a gradient of mobile phase A (triethylamine acetate [TEAA] in water) and mobile phase B (50% acetonitrile in water). A summary of the method parameters is provided below (Set B Parameters). In some embodiments, system suitability is established by the absence of interfering peaks in the blank and acceptance criteria for the %RSDs of retention times and peak areas for the initial injection of WVE-003 analytical reference material, the % difference between subsequent standard peak areas and the average of the initial injections of the standards, and the %RSDs of retention times for all standard injections. The performance of the method is also verified by demonstrating the resolution of a reference sample from closely related diastereomers. For stereochemical identification, stereochemical identity is confirmed if the % retention time difference of the main peak from the retention time of a structurally characterized reference material does not exceed 3%.

[0372] [Table 13]

[0373] In some embodiments, bacterial endotoxins are measured according to USP <85> and / or determined in accordance with Ph.Eur.2.6.14. In some embodiments, bioburden, e.g., both the total aerobic microbial count (TAMC) and the total yeast and mold count (TYMC), are determined in accordance with USP <61> and / or determined in accordance with Ph.Eur.2.6.12.

[0374] The WVE-003 drug substance will be packaged in a suitable container, e.g., a sterile high density polyethylene (HDPE) bottle with a polypropylene screw closure, labeled, and sealed in a protective device capable of providing gas / moisture barrier protection, e.g., a highly abrasion- and puncture-resistant Mylar foil pouch.

[0375] Various batches of WVE-003 preparations have been manufactured. Certain preparations have been utilized in non-clinical studies, including GLP toxicity studies. Certain preparations have been manufactured for clinical trials. The manufactured preparations are stable. In some embodiments, WVE-003 or compositions thereof are stored at about -20°C. In some embodiments, long-term storage conditions are about -20°C, e.g., ±5°C. In some embodiments, storage is at about 5°C, e.g., ±3°C.

[0376] Drug products In some embodiments, the disclosure provides a WVE-003 drug product. In some embodiments, the WVE-003 drug product comprises a WVE-003 drug substance, e.g., WVE-003 pentadeca sodium salt, manufactured using the process described above. In some embodiments, the WVE-003 drug product consists of the WVE-003 drug substance, e.g., WVE-003 pentadeca sodium salt, as a lyophilized solid in a vial. In some embodiments, the vial is a 10 mL vial. In some embodiments, the vial is a 10 mL USP / Ph.Eur. Type 1 clear glass vial sealed with a FluroTec®-coated elastomeric rubber stopper and an aluminum overseal with a blue, matte flip-off button. In some embodiments, the vial contains a single dose of WVE-003 as described herein. In some embodiments, the vial contains an amount of WVE-003 equivalent to about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg of the WVE-003 free acid form. In some embodiments, the vial contains about 20 mg WVE-003 (based on the free acid form unless otherwise specified). In some embodiments, upon completion of the lyophilization cycle, a vial filled with 2.53 mL of WVE-003 drug substance at a concentration of 8 mg / mL in water for injection (WFI) is lyophilized and backfilled with nitrogen gas, NF. In some embodiments, the WVE-003 drug product vial contains about 20 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 30 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 40 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 50 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 60 mg of WVE-003 drug substance.In some embodiments, a WVE-003 drug product vial contains about 70 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 80 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 90 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 100 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 110 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 120 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 130 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 140 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains approximately 150 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains approximately 160 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains approximately 170 mg of WVE-003 drug substance. In some embodiments, the disclosure provides a pharmaceutical composition comprising or delivering WVE-003 or a pharmaceutically acceptable salt form thereof and a pharmaceutically acceptable carrier. In some embodiments, the disclosure provides a pharmaceutical composition comprising WVE-003 pentadeca sodium salt and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition consists of WVE-003 drug substance and a pharmaceutically acceptable carrier. In some embodiments, the pharmaceutically acceptable carrier is aCSF. In some embodiments, each WVE-003 drug product vial contains a single dose of WVE-003 drug substance. In some embodiments, the WVE-003 drug substance is reconstituted and diluted in artificial cerebrospinal fluid (aCSF) as a sterile, preservative-free solution prior to intrathecal administration in the clinical setting.

[0377] In some embodiments, the WVE-003 drug product contains no preservatives, inactive ingredients, or excipients.

[0378] In some embodiments, the WVE-003 drug product is a white to off-white solid with no visible material upon reconstitution and is essentially free of particulate matter. In some embodiments, the pH of 20 mg WVE-003 in 2 mL of artificial cerebrospinal fluid (aCSF) solution is about 6.0-8.0. In some embodiments, the pH of 20 mg WVE-003 in 2 mL of aCSF solution is about 6.4-7.2.

[0379] In some embodiments, a freezing temperature of about −45° C., primary and secondary drying temperatures of about −14° C. and 25° C., and a pressure of 100 mTorr are utilized. One skilled in the art will recognize that other temperatures and / or pressures may also be utilized.

[0380] In the preparation, approximately 75 grams of WVE-003 drug substance is formulated with water for injection, Ph.Eur. / USP, to achieve an 8 mg / mL solution. Approximately 2.53 mL of this solution are lyophilized into a powder, resulting in a nominal 20 mg drug / vial. Drug product lots may be created by pooling multiple drug substance lots. In some embodiments, the total number of vials, calculated from the amount of drug substance, for example, is as follows: The purity, sodium content, and water content of the drug substance were 86%, 4.3%, and 8%, respectively. The amount of drug substance (DS) used in the manufacture of the drug product (DP) batch is 75.04 g. Total amount of oligonucleotide in DS for a batch = amount of DS (g) × 86% purity / 100% × (100% - 4.3% sodium content) / 100% × (100% - 8% water content) / 100% = 75.04 g × 0.86 × (0.957) × (0.92) × 1000 mg / g = 56,788.38 mg. Total batch size of drug product at 20.24 mg / vial = quantity of DS batch / DP vial content = 56,788 mg / 20.24 mg = 2,805 vials.

[0381] In some embodiments, by way of example, the total number of vials and batch volume calculated from the amount of drug substance is as follows: The purity, sodium content, and water content of the drug substance were 86%, 4.3%, and 8%, respectively. The amount of drug substance (DS) used in the manufacture of the drug product (DP) batch is 75.04 g. Total amount of oligonucleotide in DS for a batch = amount of DS (g) x 86% purity / 100% x (100% - 4.3% sodium content - 8% water content) / 100% = 75.04 g x 0.86 x 0.877 x 1000 mg / g = 56,596.67 mg. Total batch size of drug product at 20.24 mg / vial = quantity of DS batch / DP vial content = 56,596 mg / 20.24 mg = 2,824 vials.

[0382] In some embodiments, the batch volume is calculated using the following formula: Theoretical batch volume (mL) = theoretical total DS in mg × (purity by UPLC (%)) / 100%) × [(100% - sodium content (%) - water content (%)) / 100%] ÷ concentration For example, in one example, theoretical batch volume (mL) = 75000 mg x 0.86 x 0.877 ÷ 8.0 mg / mL = 7,070 mL.

[0383] Those skilled in the art will understand that, in various circumstances, these exemplary methods may provide approximately the same amount of WVE-003 in the vial, and may also provide approximately the same amount of WVE-003 for administration and delivery to the patient.

[0384] A flow diagram of the manufacturing process for WVE-003 drug product is provided as an example in Figure 2. WVE-003 drug substance (DS) containers are thawed at 2-8°C and subsequently equilibrated to room temperature (RT). Purity factor, sodium content, and % moisture are used to calculate the pure full-length product (FLP) as follows:

number

[0385] In some embodiments, the pure full-length product (FLP) is calculated as follows:

number

[0386] Those skilled in the art will be able to determine the levels as described herein for purity (e.g., purity, % sodium content, % moisture (water content), etc.) in various situations, and to determine the purity (e.g., UPLC as described herein), % sodium content (e.g., ICP-OES as described herein), % moisture (e.g., Karl Fischer as described herein (e.g., USP <921> It is understood that these two types of calculations may provide approximately the same results, such as the method described herein for determining DS). If multiple lots of drug substance are to be pooled, then a purity correction factor based on the amount of DS from each lot is determined. Depending on the pure FLP weight, the batch volume to produce an 8 mg / mL solution is calculated.

[0387] Add approximately 50% of the calculated batch volume of WFI by weight to the formulation vessel. Weigh the DS containers and transfer the DS from each container to the formulation vessel. Use appropriate WFI rinses to ensure all DS is removed from the containers. Calculate the total amount of DS added to the formulation vessel by drying and weighing the empty DS containers.

[0388] Mix the DS thoroughly and take an in-process sample for concentration determination by ultraviolet (UV) assay. Weigh the mass of the remaining solution in the formulation. Based on the purity-corrected WVE-003 drug substance concentration and the solution mass, calculate the amount of WFI needed to achieve a final WVE-003 drug substance concentration of 8.0 mg / mL as follows:

number

[0389] The required amount of WFI is then slowly added directly to the formulation vessel and mixed to ensure a homogeneous solution. In-process samples are collected for appearance, concentration by UV spectrophotometry, and density (which must meet various specifications). This final blended formulation is then subjected to bioburden reduction filtration, after which it may be stored overnight at 2-8°C if desired.

[0390] In some embodiments, a pre-filtration bioburden sample is taken, followed by sterile filtration of the compounded bulk solution. The final compounded formulation is sterile filtered through two 0.2 μm filters in sequence before packaging. Before and after filtration, the sterile filter unit is inspected for filter integrity by the bubble point method.

[0391] A target fill weight is determined based on a target fill volume of 2.53 mL and density measurements. Filling is performed with periodic fill volume inspections, and the stoppered, filled vials are placed in the lyo-position onto the lyophilization tray. A HEPA cart is used to aseptically transfer the lyophilization tray to the sterile lyophilizer. In some embodiments, the lyophilization cycle parameters are as provided below.

[0392] [Table 14]

[0393] After lyophilization is complete, the chamber pressure is increased to 9.6 psi and the chamber is backfilled with sterile nitrogen (N2). After the stoppers are fitted, the tray is removed from the shelf and the seal is applied by seaming. The vials are then visually inspected and then withdrawn for inspection for release, stability testing, packaging, or storage.

[0394] During the manufacturing of WVE-003 drug product, process and in-process control tests and critical limits are applied. Primary control of critical steps during the manufacturing of WVE-003 drug product is achieved using in-process concentration measurements by ultraviolet (UV) spectrophotometry. In-process concentration measurements are used to calculate the amount of WFI required to dilute the blended solution to a final concentration of 8 mg / mL WVE-003 drug substance. Measuring the concentration of the final drug substance solution confirms that the dilution is adequate. Microbial controls are achieved by measuring pre-filtration bioburden and analyzing the integrity of the sterile filters before and after filtration. Vials filled with drug product are subjected to periodic weight checks and 100% visual inspection during the filling process before being sampled for release, bulk packaging, and / or stability testing.

[0395] The release specifications for the WVE-003 drug product may include one or more specifications described herein, e.g., appearance (e.g., visual; white to off-white solid), appearance after reconstitution (e.g., visual), identity (e.g., by LC-MS, IP-RP-UPLC retention time, mass, etc.), purity (e.g., area % by IP-RP-UPLC (e.g., Protocol A)), impurities (e.g., area % by IP-RP-UPLC (e.g., Protocol A)), assay (e.g., % label; e.g., free acid, anhydrous, by UV), pH (e.g., after reconstitution: 20 mg vial reconstituted with 2 mL of aCSF diluent; e.g., USP <791> and / or Ph.Eur.2.2.3), osmolality (e.g., after reconstitution: reconstitute a 20 mg vial with 2 mL of aCSF diluent; e.g., USP <791> and / or Ph.Eur.2.2.3), bacterial endotoxins (e.g., USP <85> and / or Ph.Eur.2.6.14), sterility (e.g., USP <71> and / or Ph.Eur.2.6.1), uniformity of dose (e.g., USP <905> and / or Ph.Eur.2.9.40), particulate matter (e.g., USP <788> and / or Ph.Eur.2.9.19), reconstitution time (e.g., visual), water content (e.g., % w / w; e.g., USP <921> , Ph.Eur.2.5.12, Karl Fischer, etc.), container closure integrity test (e.g., USP <1207> ) etc. In some embodiments, the WVE-003 drug product demonstrates substantially the same or similar specifications, e.g., purity, impurities, pH, water content, etc., as the WVE-003 drug substance as described herein.

[0396] For example, in some embodiments, the purity of the WVE-003 drug product is about 84% or greater (e.g., as determined by Protocol A (% area)). In some embodiments, it is about 85% or greater. In some embodiments, it is about 86% or greater. In some embodiments, it is about 87% or greater. In some embodiments, it is about 88% or greater. In some embodiments, it is about 80%-90%. In some embodiments, it is about 84%-90%. In some embodiments, it is about 84%-88%. In some embodiments, the amount of total impurities is about 16% or not more than about 16% (e.g., as determined by Protocol A (% area)). In some embodiments, the amount of total impurities is about 15% or not more than about 15%. In some embodiments, the amount of total impurities is about 14% or not more than about 14%. In some embodiments, the amount of total impurities is about 13% or not more than about 13%. In some embodiments, the amount of total impurities is about 12% or not more than about 12%. In some embodiments, the total impurities content is about 11% or no more than about 11%. In some embodiments, the sum of WVE-003 and total impurities content is about 99%-101%.

[0397] In some embodiments, the assay (% labeled amount) of the WVE-003 drug product is determined by ultraviolet (UV) spectrophotometry. The test sample is reconstituted with water, then quantitatively transferred to a volumetric flask and diluted to volume with water. The absorbance of the solution at 260 nm is measured and used, along with the molar extinction coefficient (MEC), to experimentally measure WVE-003 purity and determine the purity-corrected amount of WVE-003 drug substance vial (mg / vial), which is then compared to the expected amount of the vial, e.g., 20 mg, to determine the % labeled amount. In some embodiments, assay sample preparation and calculations are performed as described below: Reconstitute the finished product vial with 2.5 mL of water to obtain an 8 mg / mL solution. Stock Samples (0.1 mg / mL): Pipet 0.32 mL of 8 mg / mL drug product into a 25 mL volumetric flask (in duplicate), add water to the appropriate amount, and mix well. Working Samples (0.02 mg / mL): Pipet 2.0 mL from each 0.1 mg / mL stock sample solution into a 10 mL volumetric flask. Dilute to volume with water and mix well. Each working sample is analyzed in one reading (1 cm cuvette) at 260 nm (A260). The WVE-003 percent labeled quantity (%LC) from A260 is calculated as follows:

number

[0398] In some embodiments, the % indicated amount is about 100% ± 10%. In some embodiments, it is about 95%-105%. In some embodiments, it is about 95%. In some embodiments, it is about 96%. In some embodiments, it is about 97%. In some embodiments, it is about 98%. In some embodiments, it is about 99%. In some embodiments, it is about 100%. In some embodiments, it is about 101%. In some embodiments, it is about 102%. In some embodiments, it is about 103%. In some embodiments, it is about 104%. In some embodiments, it is about 105%. In some embodiments, it is about 106%. In some embodiments, it is about 107%. In some embodiments, it is about 108%. In some embodiments, it is about 109%. In some embodiments, it is about 110%.

[0399] In some embodiments, the reconstitution time for WVE-003 drug product is determined visually after reconstitution with 2 mL of preservative-free sterile aCSF and gentle inversion until the cake is completely dissolved. A calibrated timer is used to determine the time it takes for the cake to completely reconstitute.

[0400] In some embodiments, dosage unit uniformity of WVE-003 drug product is determined by UV content uniformity method. Dosage unit uniformity of WVE-003 drug product is determined by reconstitution of 10 vials, and if necessary, testing an additional 20 vials. The assay (% labeled amount) is determined for each vial by UV spectrophotometry as described above. The assay (% labeled amount) results determined for each test vial are then used to calculate an Accepted Value (AV), as specified in the compendium.

[0401] In some embodiments, particulate matter in WVE-003 drug product is determined by reconstituting 10 vials with 10 mL purified water and then pooling the resulting solution for analysis. The pooled solution is then analyzed using a particle counter according to Test 1.B Light Obscuration Test to determine the number of particles 10 μm and larger and 25 μm and larger in the solution. Calibration verification is performed prior to analysis of test samples.

[0402] In some embodiments, the osmolality of the WVE-003 drug product (diluted in aCSF) is determined, e.g., according to USP <785> The specifications, when measured according to / Ph.Eur.2.2.35, are consistent with the reconstitution medium (e.g., aCSF) and correspond to the osmolality range of human cerebrospinal fluid.

[0403] In some embodiments, the WVE-003 drug product is stored at about 5°C, e.g., ±3°C. In some embodiments, it is stored at about 25°C, e.g., ±2°C. In some embodiments, it may be stored at about 40°C, e.g., ±2°C. In some embodiments, it is stored at 60% RH. In some embodiments, it is stored at 75% RH. The manufactured drug product is stable over a variety of time periods and / or temperatures. In some embodiments, the WVE-003 drug product is stable for about 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 months, or more. In some embodiments, the WVE-003 drug product is stable at about 5°C, e.g., ±3°C, for about 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 months, or more. In some embodiments, the WVE-003 drug product is stable for about 6, 9, 12, 15, 18, 21, 24 months or more at about 25°C, e.g., ±2°C (e.g., 60% RH ±5%). In some embodiments, the WVE-003 drug product is stable for about 1, 2, 3, 4, 5, 6 months or more at about 40°C, e.g., ±2°C (e.g., 75% RH ±5%).

[0404] In some embodiments, WVE-003 is provided in a solution composition, e.g., dissolved in aCSF. In some embodiments, the diluent to be used as a placebo and diluent for reconstitution of WVE-003 (drug product vial) is an artificial cerebrospinal fluid (aCSF) solution. In some embodiments, it is in a 20 mL USP / Ph.Eur. Type I clear glass vial sealed with a serum stopper and an aluminum overseal with a white frosted flip-off button. In some embodiments, the vial contains 20.8 mL of aCSF (which includes an overfill of 0.8 mL above the nominal fill volume of 20 mL). In some embodiments, the pH of the diluent, e.g., aCSF, is about 6.8-7.8. In some embodiments, it is about 7.2-7.4. The composition of the diluent is provided below:

[0405] [Table 15]

[0406] In some embodiments, the composition of the diluent is provided below:

[0407] [Table 16]

[0408] Formulation of WVE-003 In some embodiments, the present disclosure provides formulations of HTT oligonucleotides, such as WVE-003.

[0409] In some embodiments, the HTT oligonucleotide, or a salt form thereof, or the HTT oligonucleotide composition is capable of mediating allele-specific reduction of mHTT transcript level, expression, and / or activity. In some embodiments, the HTT oligonucleotide, or a salt form thereof, or the HTT oligonucleotide composition is WVE-003 or is WVE-003. In some embodiments, the HTT oligonucleotide, or a salt form thereof, or the HTT oligonucleotide composition is capable of mediating allele-specific knockdown of mutant HTT transcripts. In some embodiments, the present disclosure provides chiral-controlled HTT oligonucleotide compositions capable of mediating allele-specific knockdown of mutant HTT transcripts.

[0410] In some embodiments, the HTT oligonucleotide, or a salt form thereof, or HTT oligonucleotide composition is provided as a lyophilized powder that is reconstituted and diluted for administration. In some embodiments, the WVE-003 preparation or formulation is in the form of a white to off-white lyophilized solid. In some embodiments, the solubility of the WVE-003 drug substance in water is determined to be at least 79.20 mg / mL as determined by UV spectrophotometry. In some embodiments, the pH of the WVE-003 drug substance in purified water is in the range of 6.0 to 8.0.

[0411] In some embodiments, the HTT oligonucleotide, or salt form thereof, or HTT oligonucleotide composition is provided as a liquid formulation.

[0412] In some embodiments, WVE-003 is formulated as a lyophilized powder that is reconstituted and diluted for administration. In some embodiments, WVE-003 is formulated as a liquid formulation.

[0413] In some embodiments, the disclosure provides specific formulations of WVE-003. In some embodiments, the disclosure provides methods of using specific formulations of WVE-003 in the treatment of Huntington's disease at dosages of about 30, about 60, about 90, about 120, about 150, or about 168 mg.

[0414] In some embodiments, the HTT oligonucleotide is WVE-003.

[0415] In some embodiments, the present disclosure relates to a method of treating Huntington's disease in a subject in need thereof, wherein the subject is administered an HTT oligonucleotide formulated as a liquid formulation, which is reconstituted with a sodium chloride solution from a lyophilized preparation.

[0416] In some embodiments, the lyophilized preparation of WVE-003 is a dry powder.

[0417] In some embodiments, the lyophilized preparation of WVE-003 is a dry powder prepared by lyophilization of a liquid formulation of WVE-003 in water.

[0418] In some embodiments, the lyophilized preparation of WVE-003 is a dry powder in a vial.

[0419] In some embodiments, the lyophilized preparation of WVE-003 is approximately 20 mg of dry powder in a vial.

[0420] In some embodiments, the lyophilized preparation of WVE-003 is a dry powder in a 10 ml vial.

[0421] In some embodiments, the lyophilized preparation of WVE-003 is approximately 20 mg of dry powder in a 10 ml vial.

[0422] The reconstituted solution does not contain any preservatives; therefore, it must be administered without delay. If this is not possible, the solution must be stored at room temperature and administered within 4 hours.

[0423] Partially used, unused, or damaged vials must be discarded.

[0424] In some embodiments, the HTT oligonucleotide WVE-003 was analyzed in non-clinical and clinical trials.

[0425] In some embodiments, WVE-003 is provided in a solution. In some embodiments, WVE-003 is administered in a solution. In some embodiments, WVE-003 is dissolved in a suitable diluent. In some embodiments, the diluent is aCSF. In some embodiments, the WVE-003 composition is a WVE-003 preparation (e.g., drug substance, drug product, etc.) dissolved in aCSF.

[0426] HTT Oligonucleotide Dosage Regimen In some embodiments, the dosage regimen for the therapeutically effective amount of the oligonucleotide, oligonucleotide composition, chiral controlled oligonucleotide composition, or any of these is any dosage regimen described herein.

[0427] In some embodiments, the dosage regimen relates to the size of individual doses of the oligonucleotide, oligonucleotide composition, chiral controlled oligonucleotide composition, or therapeutically effective amount of the oligonucleotide, oligonucleotide composition, chiral controlled oligonucleotide composition; and / or the interval between multiple or successive doses thereof; and / or the total length or duration over which a subject receives one or more doses thereof; and / or the detailed formulation thereof.

[0428] In some embodiments, the dose regimen for WVE-003, an oligonucleotide composition of WVE-003, a chiral controlled oligonucleotide composition of WVE-003, or a therapeutically effective amount of ... chiral controlled oligonucleotide composition of WVE-003 is any dose regimen described herein. In some embodiments, the dosage regimen can include, but is not limited to, a specific amount (e.g., about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, and / or about 168 mg per dose, per dosage, and / or per administration) and / or a specific dosing interval (e.g., about once every 4 weeks, about once every 8 weeks, about once every 12 weeks, about once every month, about once every 2 months, etc.), and / or a specific dosing length or duration (e.g., at least about 1 month, about 2 months, etc.). (Dosage administration occurs over a period of time spanning about 1 month, about 3 months, about 4 months, about 5 months, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 18 months, about 24 months, about 36 months, about 48 months, and / or at least about 4 weeks, about 8 weeks, about 12 weeks, about 16 weeks, about 20 weeks, about 24 weeks, about 28 weeks, about 32 weeks, about 36 weeks, about 40 weeks, about 44 weeks, about 48 weeks, about 52, about 78 weeks, about 104 weeks, about 156 weeks, about 208 weeks, etc.) In some embodiments, the dosage regimen is any dosage regimen described herein. In some embodiments, the dosage regimen is or includes the dosage regimen described in the Examples.

[0429] In some embodiments, a dose of WVE-003 (e.g., about 30, about 60, about 90, about 120, about 150, or about 168 mg) is administered to a subject in need thereof on a regular schedule or dosing regimen.

[0430] In some embodiments, the dose of WVE-003 is administered approximately monthly. In some embodiments, the dose of WVE-003 is administered approximately once every two months. In some embodiments, the dose of WVE-003 is administered approximately once every eight weeks. In some embodiments, the dose of WVE-003 is administered approximately once every 12 weeks.

[0431] In some embodiments, doses of WVE-003 are administered approximately monthly for at least about three months. In some embodiments, doses of WVE-003 are administered approximately once every two months for at least about four months. In some embodiments, doses of WVE-003 are administered approximately once every eight weeks for at least about 16 weeks. In some embodiments, doses of WVE-003 are administered approximately once every 12 weeks for at least about 12 weeks.

[0432] In some embodiments, doses of WVE-003 are administered approximately monthly, with the first monthly dose being preceded by administration of WVE-003 and followed by a washout period of approximately 8 weeks (approximately 2 months). In some embodiments, doses of WVE-003 are administered approximately once every two months, with the first bimonthly dose being preceded by administration of WVE-003 and followed by a washout period of approximately 12 weeks (approximately 3 months). In some embodiments, doses of WVE-003 are administered approximately once every 8 weeks, with the first 8-weekly dose being preceded by administration of WVE-003 and followed by a washout period of approximately 12 weeks (approximately 3 months).

[0433] In some embodiments, a dose of WVE-003 is administered, followed by a washout period of about 8 weeks (about 2 months), followed by approximately monthly doses of WVE-003. In some embodiments, a dose of WVE-003 is administered, followed by a washout period of about 12 weeks (about 3 months), followed by approximately one dose of WVE-003 every two months. In some embodiments, a dose of WVE-003 is administered, followed by a washout period of about 12 weeks (about 3 months), followed by approximately one dose of WVE-003 every eight weeks.

[0434] In some embodiments, a lumbar puncture (spinal tap) procedure is performed to obtain CSF for analysis after administration of one or more doses of oligonucleotide.

[0435] In some embodiments, a lumbar puncture (spinal tap) procedure is performed to obtain CSF for analysis about one month (about four weeks) after the third monthly dose.

[0436] Specific Uses of HTT Oligonucleotides or HTT Oligonucleotide Compositions In some embodiments, the present disclosure provides a method for treating or preventing Huntington's disease, the method comprising administering to a subject an effective amount of an HTT oligonucleotide or composition thereof described herein (e.g., WVE-003).

[0437] In some embodiments, the HTT transcript is of the huntingtin gene or a variant thereof.

[0438] In some embodiments, HTT oligonucleotides can induce a pro-inflammatory response. In some embodiments, the present disclosure provides compositions and methods for reducing inflammation. In some embodiments, the present disclosure provides compositions and methods for reducing a pro-inflammatory response. In some embodiments, the present disclosure provides methods for reducing injection site inflammation using provided compositions. In some embodiments, the present disclosure provides methods for reducing drug-induced vascular trauma using provided compositions.

[0439] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression, and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally about monthly at a dose of 10 to about 168 mg, thereby slowing disease progression in the subject and thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression, and / or activity.

[0440] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally about monthly at a dose of about 30 mg, thereby slowing disease progression in the subject and thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0441] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally at a dose of about 60 mg approximately monthly, which will result in a delay in disease progression in the subject, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0442] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally at a dose of about 90 mg approximately monthly, which will result in a delay in disease progression in the subject, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0443] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally about monthly at a dose of about 120 mg, thereby slowing disease progression in the subject and thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0444] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally about monthly at a dose of about 150 mg, thereby slowing disease progression in the subject and thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0445] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally at a dose of about 168 mg approximately monthly, which will result in a delay in disease progression in the subject, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0446] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 30 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0447] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 60 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0448] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 90 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0449] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 120 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0450] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 150 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0451] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of about 168 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0452] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which an allele-specific reduction in mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally at a dose of 30 mg approximately monthly, which will result in a delay in disease progression in the subject, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0453] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally at a dose of 60 mg approximately monthly, thereby slowing disease progression in the subject and thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0454] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally at a dose of 90 mg approximately monthly, thereby slowing disease progression in the subject and thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0455] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally at a dose of 120 mg approximately monthly, which will result in a delay in disease progression in the subject, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0456] In some embodiments, the present disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally at a dose of 150 mg approximately monthly, thereby slowing disease progression in the subject and thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0457] In some embodiments, the disclosure relates to a method of treating a subject with Huntington's disease having a mutation in the HTT gene in which allele-specific reduction of mHTT level, expression and / or activity is indicated, comprising administering to the subject an HTT oligonucleotide or HTT oligonucleotide composition intrathecally at a dose of 168 mg approximately monthly, which will result in a delay in disease progression in the subject, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in mHTT transcript level, expression and / or activity.

[0458] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 30 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0459] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 60 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0460] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 90 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0461] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 120 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0462] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 150 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0463] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 168 mg, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0464] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 30 mg ± 5%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0465] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 60 mg ± 5%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0466] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 90 mg ± 5%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0467] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 120 mg ± 5%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0468] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 150 mg ± 5%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0469] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 168 mg ± 5%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0470] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 30 mg ± 10%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0471] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 60 mg ± 10%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0472] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 90 mg ± 10%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0473] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 120 mg ± 10%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0474] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 150 mg ± 10%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0475] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 168 mg ± 10%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0476] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 30 mg ± 15%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0477] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 60 mg ± 15%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0478] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 90 mg ± 15%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0479] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 120 mg ± 15%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0480] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 150 mg ± 15%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0481] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 168 mg ± 15%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0482] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 30 mg ± 20%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0483] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 60 mg ± 20%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0484] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 90 mg ± 25%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0485] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 120 mg ± 25%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0486] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 150 mg ± 25%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0487] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 168 mg ± 25%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0488] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 30 mg ± 30%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0489] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 60 mg ± 30%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0490] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 90 mg ± 30%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0491] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 120 mg ± 30%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0492] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 150 mg ± 30%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject.

[0493] In some embodiments, the disclosure relates to a method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is indicated, comprising administering to the subject WVE-003 (or a salt form thereof) at a dose of 168 mg ± 30%, thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in the subject....

Claims

1. 1. A method of treating a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of said mutant HTT gene is indicated, comprising administering to said subject a dose of about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, about 160 mg, or about 168 mg of WVE-003 (or a salt form thereof), thereby delaying the progression of Huntington's disease and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in said subject.

2. 1. A method of treating Huntington's disease in a subject having a mutant HTT gene comprising a mutation for which allele-specific knockdown of said mutant HTT gene is indicated, said method comprising administering to said subject a dose of about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, about 160 mg, or about 168 mg of WVE-003 (or a salt form thereof), thereby delaying disease progression and / or delaying the onset of Huntington's disease and / or reducing the severity of Huntington's disease symptoms in said subject.

3. 1. A method of delaying the onset of and / or reducing the severity of symptoms of Huntington's disease in a subject with Huntington's disease having a mutant HTT gene comprising a mutation for which allele-specific knockdown of the mutant HTT gene is appropriate, comprising administering to the subject a dose of about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, about 160 mg, or about 168 mg of WVE-003 (or a salt form thereof).

4. A method for treating Huntington's disease, comprising administering WVE-003 (or a salt form thereof) to a subject suffering from Huntington's disease, wherein WVE-003 is administered at a dose of about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, about 160 mg, or about 168 mg, and wherein the subject's HTT allele comprises an expanded CAG repeat region and is fully complementary to the base sequence of WVE-003.

5. 1. A method for treating Huntington's disease, comprising administering to a subject suffering from Huntington's disease a pharmaceutical composition comprising or delivering WVE-003 (or a salt form thereof), wherein WVE-003 is administered at a dose of about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, about 160 mg, or about 168 mg, and wherein the subject's HTT allele comprises an expanded CAG repeat region and is fully complementary to the base sequence of WVE-003.

6. A method comprising administering WVE-003 (or a salt form thereof) to a subject, wherein the subject is determined to have a gene sequence that is the same as or completely complementary to the base sequence of WVE-003, and optionally the subject is determined to have a gene sequence that is, or encodes, an expanded CAG repeat.

7. A method comprising administering WVE-003 (or a salt form thereof) to a subject, wherein the subject is determined to have a gene sequence that contains an HTT-expanded CAG repeat and encodes a transcript that is completely complementary to the base sequence of WVE-003.

8. A method comprising administering WVE-003 (or a salt form thereof) to a subject, wherein the subject is determined to express an HTT transcript that contains an expanded CAG repeat and is perfectly complementary to the base sequence of WVE-003.

9. 8. The method of any one of claims 5 to 7, wherein the subject is determined to have a gene sequence or transcript that is not identical to or not completely complementary to the base sequence of WVE-003 at rs362273, and optionally the gene sequence or transcript does not contain an expanded CAG repeat (or a sequence encoded thereby).

10. 10. The method of any one of claims 5 to 9, wherein WVE-003 is administered at a dose of about 30 mg, about 60 mg, about 90 mg, about 120 mg, about 150 mg, about 160 mg, or about 168 mg.

11. 11. The method of any one of claims 1 to 10, further comprising the step of identifying a mutation in the subject's HTT gene that is amenable to allele-specific knockdown of the mutant HTT gene or its gene product transcript.

12. The method of any one of claims 1 to 11, wherein WVE-003 is administered in salt form, optionally in sodium salt form.

13. 13. The method of any one of claims 1 to 12, wherein WVE-003 is formulated as a liquid formulation, optionally comprising WVE-003, sodium chloride and water, and / or wherein the liquid formulation is reconstituted from a lyophilized preparation.

14. 14. The method of any one of claims 1 to 13, wherein one or more pharmaceutically acceptable salt forms of WVE-003 are administered and / or the amount of WVE-003 includes an amount of one or more pharmaceutically acceptable salt forms, each of which amounts independently converts to an amount of the acid form.

15. 1. A method for treating Huntington's disease, comprising administering or delivering to a subject suffering from Huntington's disease WVE-003 at a dose of about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the equivalent amount of the free acid form of WVE-003; or 1. A method for preventing Huntington's disease, comprising administering or delivering to a subject suffering from Huntington's disease WVE-003 at a dose of about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the equivalent amount of the free acid form of WVE-003; or or a method comprising administering or delivering to a subject WVE-003 at a dose equivalent to about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the free acid form of WVE-003; or A method for reducing the activity, expression, and / or level of a mutant HTT gene or its gene product in a subject, comprising administering or delivering to said subject WVE-003 at a dose equivalent to about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the free acid form of WVE-003; or A method for preferentially knocking down repeat-expansion-containing HTT RNA transcripts relative to non-repeat-expansion-containing HTT RNA transcripts in a subject, comprising administering or delivering to the subject WVE-003 at a dose of about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the equivalent amount of the free acid form of WVE-003; or or a method of reducing the level of an HTT transcript comprising a CAG repeat expansion in a subject, comprising administering or delivering to the subject WVE-003 at a dose of about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the equivalent of the free acid form of WVE-003; or 1. A method of reducing the level of a product of an HTT transcript containing a CAG repeat expansion in a subject, comprising administering or delivering to the subject WVE-003 at a dose of about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the free acid form equivalent of WVE-003. In; WVE-003, mG*SmUn001RmUmGn001RmA*ST*SC*ST*SG*ST*RA*SG*SC*SA*SG*Rm5Ceon001RAeoGeon001Rm5Ceo*STeo (in the formula, m represents a 2'-OMe modification to the nucleoside; *S represents an Sp phosphorothioate bond; m5Ceo represents 5-methyl 2'-O-methoxyethyl C; n001R represents an Rp n001 bond, where the n001 bond is 【Chemical 1】 having the structure eo represents a 2'-OCH2CH2OCH3 modification to the nucleoside; and *R represents an Rp phosphorothioate bond.

16. 16. The method of any one of claims 1 to 15, wherein the dose of WVE-003 is administered in one or more forms, optionally wherein the dose of WVE-003 is administered in the form of one or more pharmaceutically acceptable salts, optionally one form being WVE-003 pentadeca sodium salt.

17. The method of any one of claims 1 to 16, wherein the dose of WVE-003 is administered in a pharmaceutical composition comprising WVE-003 and a pharmaceutically acceptable carrier.

18. 18. The method of any one of claims 1 to 17, wherein the dose of WVE-003 is administered in a pharmaceutical composition comprising or consisting of WVE-003 pentadeca sodium salt and a pharmaceutically acceptable carrier.

19. 19. The method of any one of claims 1 to 18, wherein each dose of WVE-003 is independently administered in the form of one or more pharmaceutically acceptable salts, optionally one form being WVE-003 pentadeca sodium salt.

20. 20. The method of any one of claims 1 to 19, wherein each dose of WVE-003 is administered independently in a pharmaceutical composition comprising WVE-003 and a pharmaceutically acceptable carrier.

21. 21. The method of any one of claims 1 to 20, wherein each dose of WVE-003 is independently administered in a pharmaceutical composition comprising or consisting of WVE-003 pentadeca sodium salt and a pharmaceutically acceptable carrier.

22. 1. A method for treating Huntington's disease, comprising administering to a subject suffering from Huntington's disease WVE-003 pentadeca sodium salt: 【Chemistry 2】 at a dose equivalent to about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the free acid form of WVE-003.

23. 1. A method for preventing Huntington's disease, comprising administering or delivering to a subject suffering from Huntington's disease WVE-003 pentadeca sodium salt at a dose equivalent to about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the free acid form of WVE-003.

24. or a method comprising administering or delivering to a subject WVE-003 pentadeca sodium salt at a dose equivalent to about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the free acid form of WVE-003; or A method of reducing the activity, expression, and / or level of a mutant HTT gene or its gene product in a subject, comprising administering or delivering to said subject WVE-003 pentadeca sodium salt at a dose equivalent to about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the free acid form of WVE-003; or A method for preferentially knocking down repeat-expansion-containing HTT RNA transcripts relative to non-repeat-expansion-containing HTT RNA transcripts in a subject, comprising administering or delivering to said subject WVE-003 pentadeca sodium salt at a dose of about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the equivalent amount of WVE-003 free acid form; or or a method of reducing the level of an HTT transcript comprising a CAG repeat expansion in a subject, comprising administering or delivering to the subject WVE-003 pentadeca sodium salt at a dose of about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the equivalent of the WVE-003 free acid form; or 1. A method of reducing the level of a product of an HTT transcript containing a CAG repeat expansion in a subject, comprising administering or delivering to the subject WVE-003 pentadeca sodium salt at a dose of about 10-200 mg (e.g., about 10-200 mg, about 10-190 mg, about 10-180 mg, about 10-170 mg, about 10 mg, about 20 mg, about 30 mg, 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, or about 168 mg) of the free acid form equivalent of WVE-003.

25. The method of any one of claims 1 to 24, wherein the product is a polypeptide, optionally a polypeptide comprising an expanded polyQ.

26. 26. The method of any one of claims 1 to 25, wherein two or more doses are administered, for example 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses.

27. 27. The method of any one of claims 1 to 26, wherein the dose of WVE-003 pentadeca sodium salt is administered in a pharmaceutical composition comprising or consisting of WVE-003 pentadeca sodium salt and a pharmaceutically acceptable carrier.

28. 28. The method of any one of claims 1 to 27, wherein each dose of WVE-003 pentadeca sodium salt is administered independently in a pharmaceutical composition comprising or consisting of WVE-003 and a pharmaceutically acceptable carrier.

29. 29. The method of any one of claims 22 to 28, wherein the pharmaceutically acceptable carrier is aCSF.

30. 30. The method of any one of claims 1 to 29, wherein the pH of the pharmaceutical composition is about 6 to 8, optionally the pH of the pharmaceutical composition is about 6.4 to 7.2, or about 7.3, or about 7.

4.

31. 30. The method of any one of claims 22 to 29, wherein the dose is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 170 mg of WVE-003 free acid form equivalent.

32. 30. The method of any one of claims 22-29, wherein each dose is independently about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 170 mg of the free acid form equivalent of WVE-003.

33. 33. The method of any one of claims 1-32, wherein two or more consecutive doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses) are independently administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or about every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer, or about every 1, 2, 3, or 4 quarter years.

34. 34. The method of any one of claims 1-33, wherein all doses are independently administered about every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks, or about every 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or longer, or about every 1, 2, 3, or 4 quarter years.

35. 35. The method of any one of claims 1-34, wherein the subject is administered WVE-003 approximately monthly for at least about 2, 4, 8, 12, 16, 24, or 48 months, or the subject is administered WVE-003 approximately once every 4 weeks for at least about 8, 12, or 16 weeks, or the subject is administered WVE-003 approximately once every 8 weeks for at least about 8 or 16 weeks.

36. 36. The method of any one of claims 1-35, wherein two or more consecutive doses (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses) of WVE-003, each independently equivalent to about 30 mg of the free acid form, are administered about every 8 weeks.

37. the subject has an expanded CAG repeat region in the HTT gene and / or expresses an HTT transcript that includes an expanded CAG repeat region; and / or 37. The method of any one of claims 1 to 36, wherein the expanded CAG repeat region comprises 36 or more CAG repeats, optionally 40 or more CAG repeats.

38. 38. The method of any one of claims 1 to 37, wherein the A mutation of rs362273 is on the same allele as the expanded CAG repeat region in the HTT gene.

39. 39. The method of any one of claims 1 to 38, wherein the expression of mutant HTT is reduced and / or mutant HTT protein levels are reduced and / or the level, expression and / or activity of mutant HTT transcripts or their gene products is reduced by at least about 5% or 10%.

40. 40. The method of any one of claims 1-39, wherein the level of mutant HTT transcript in cerebrospinal fluid is reduced by about 10%, 20%, 30%, 40%, 50% or more at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more weeks after the first dose.

41. 41. The method of any one of claims 1-40, wherein the level of the mutant HTT polypeptide in cerebrospinal fluid is reduced by about 10%, 20%, 30%, 40%, 50% or more at or about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more weeks after the first dose.

42. 42. The method of any one of claims 1 to 41, wherein the reduction in wtHTT transcript or polypeptide levels does not exceed about 10%, 20%, 30%, 40% or 50%.

43. 43. The method of any one of claims 1 to 42, wherein the method provides a reduction in the level of repeat expansion-containing HTT transcripts, measured as a percentage, that is at least 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 4, 5, 6, 7, 8, 9, or 10 times the reduction in the level of non-repeat expansion-containing HTT transcripts, measured as a percentage.

44. the reduction is about 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more; and / or the reduction is determined for each individual subject; and / or the reduction is determined for a population of subjects, optionally with a population size of about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 or more subjects, and / or the subjects in the population receive the same or different dosage regimens; and / or 44. The method of any one of claims 1 to 43, wherein one or more cerebrospinal fluid samples are utilized to determine the reduction.

45. 45. The method of any one of claims 1 to 44, wherein wild-type HTT transcript levels are not significantly reduced; and / or wild-type HTT protein levels are not significantly reduced; and / or total HTT transcript levels are not significantly reduced; and / or total HTT protein levels are not significantly reduced; and / or neurofilament light chain (NfL) levels in the CSF are not significantly increased.

46. the assessment is performed about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 days, or about 3, 4, 5, 6, 7, or 8 weeks, or about 3, 4, 5, or 6 months or more after administration of the dose, and before the next dose, if any, is administered; and / or 46. The method of any one of claims 1-45, wherein the determining is performed after administration of about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 doses.

47. 47. The method of any one of claims 1 to 46, wherein the onset and / or severity of Huntington's disease symptoms in the subject is delayed and / or reduced, and / or one or more functional assessments are improved in said subject.

48. 48. The method of any one of claims 1 to 47, wherein the improvement is compared to baseline, no WVE-003 administration, or administration of a reference composition, optionally said reference composition being equivalent to the administered WVE-003 composition but not comprising WVE-003.

49. 49. The method of any one of claims 1 to 48, wherein WVE-003 is administered intrathecally and / or by direct lumbar injection.

50. 50. The method of any one of claims 1-49, wherein the dose is administered as WVE-003 pentadeca sodium salt dissolved in aCSF, optionally wherein the dose is administered in 20 mL aCSF solution, and optionally wherein 20 mL CSF is withdrawn from the subject prior to administration of the dose.

51. 51. The method of any one of claims 1-50, wherein the subject is about 25 years of age or older, and / or the subject is about 60 years of age or older; and / or the subject has early-stage Huntington's disease.

52. 52. The method of any one of claims 1 to 51, wherein the subject is administered or exposed to an additional therapeutic agent.

53. 53. The method of any one of claims 1-52, wherein the subject is administered a steroid at least about 1 month prior to the first dose of WVE-003.

54. A composition comprising WVE-003.

55. A composition comprising WVE-003 or a composition thereof, wherein WVE-003 or said composition thereof is in solid form and / or is lyophilized.

56. A composition comprising WVE-003 or a composition thereof, wherein WVE-003 or said composition thereof is present in a vial in an amount of about 20 mg, optionally wherein said vial is nitrogen filled, and optionally wherein the amount of WVE-003 includes an amount of one or more pharmaceutically acceptable salt forms, each of which amounts independently converts to an amount of the acid form.

57. A composition comprising WVE-003 or a composition thereof, wherein WVE-003 or said composition thereof is diluted with a sodium chloride solution, optionally said solution being 0.9% sodium chloride.

58. A composition comprising WVE-003 or a composition thereof, the composition consisting essentially of WVE-003 or a composition thereof, sodium chloride, and water.

59. A composition comprising WVE-003 or a composition thereof, the composition consisting essentially of WVE-003 or a composition thereof and aCSF.

60. 60. The composition of any one of claims 54 to 59, wherein the form of WVE-003 in the composition is a pharmaceutically acceptable salt form and / or a WVE-003 pentadeca sodium salt, or wherein each form of WVE-003 in the composition is independently a salt form, optionally a pharmaceutically acceptable salt form and / or a WVE-003 pentadeca sodium salt.

61. 61. The composition of any one of claims 54 to 60, which is a drug substance and / or a drug product.

62. 62. The composition of any one of claims 54 to 61, wherein the composition is a liquid composition in which WVE-003 is dissolved, or wherein the composition is a pharmaceutical composition further comprising a pharmaceutically acceptable carrier, optionally wherein the pharmaceutically acceptable carrier is or comprises artificial cerebrospinal fluid (aCSF).

63. 63. The composition of any one of claims 54 to 62, wherein the composition is isotonic and / or the pH of the composition is about 6 to 8, optionally the pH of the composition is about 6.4 to 7.2 or about 7.3 or about 7.

4.

64. 64. The composition of any one of claims 54 to 63, which is a lyophilized WVE-003 powder.

65. 65. The composition of any one of claims 54 to 64, wherein the WVE-003 in the composition is in an amount equivalent to about 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, or 170 mg of WVE-003 free acid form.

66. 66. The composition of any one of claims 54 to 65, packaged in a vial.

67. 67. The composition of any one of claims 54 to 66, reconstituted and diluted with artificial cerebrospinal fluid (aCSF).

68. 68. The composition of any one of claims 54 to 67, wherein the composition is a WVE-003 drug product, optionally wherein the WVE-003 drug product is lyophilized WVE-003 pentadeca sodium salt.

69. 69. A vial comprising the composition of any one of claims 54 to 68, optionally filled with an inert gas, optionally wherein the inert gas is nitrogen.

70. 70. A syringe comprising the composition of any one of claims 54 to 69, optionally wherein the composition is a liquid composition and WVE-003 or WVE-003 pentadeca sodium salt is dissolved in aCSF.

71. 71. The syringe of claim 70, wherein the volume of the liquid composition is 20 mL.

72. 72. A syringe according to claim 70 or 71 containing a dose of WVE-003 according to any one of claims 1 to 71.

73. A method for producing a WVE-003 composition according to the methods described herein.

74. 74. The method of claim 73, comprising utilizing IP-RP-UPLC to determine the purity and / or impurities of the manufactured WVE-003 composition and releasing the preparation if the purity and / or impurities meet specified criteria.

75. 75. The method of claim 73 or 74, wherein the composition is a drug substance or drug product.

76. A method for shipping a WVE-003 preparation, comprising utilizing IP-RP-UPLC to determine the purity and / or impurities of said WVE-003 preparation, and shipping said preparation if said purity and / or impurities meet specified criteria; or Method for determining the purity of WVE-003 using IP-RP-UPLC.

77. 77. The method of any one of claims 74 to 76, wherein said IP-RP-UPLC utilizes one or more parameters described herein and / or one or more parameters of Set A.

78. Method for confirming the stereochemical identity of WVE-003 using IP-RP-UPLC.

79. 79. The method or composition of any one of claims 1-78, wherein the WVE-003 drug substance is manufactured by a process described herein, characterized by one or more methods described herein, shipped by one or more methods described herein, and / or stored by one or more methods described herein.

80. 80. The method or composition of any one of claims 1 to 79, wherein the WVE-003 drug substance is pentadeca sodium salt.

81. The WVE-003 drug product is manufactured by a process described herein, characterized by one or more methods described herein, shipped by one or more methods described herein, or stored by one or more methods described herein; or 81. The method or composition of any one of claims 1-80, wherein the pharmaceutical composition is manufactured by a process described herein, characterized by one or more methods described herein, shipped by one or more methods described herein, and stored by one or more methods described herein.

82. 82. The method or composition of any one of claims 1-81, wherein the purity of the composition is about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90% or more; the impurities in the composition do not exceed or are about 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10%; and / or the stereochemical purity of WVE-003 is about 80%, 83%, or more.

83. 83. The method or composition of any one of claims 1 to 82, wherein the purity and / or impurities are measured by IP-RP-UPLC using area % at 260 nm, optionally using % at 260 nm and the Set A parameters; and / or the amount of WVE-003 is 25.0 OD / mg as measured by UV at 260 nm.

84. 84. The method or composition of any one of claims 1-83, wherein the stereochemical identity of WVE-003 is confirmed by IP-RP-UPLC, optionally confirmed by IP-RP-UPLC according to Set B parameters, or an IP-RP-UPLC method for stereochemical identity as described herein.

85. 85. The method or composition of any one of claims 1-84, wherein the stereochemical purity is determined by dimer modeling and / or the stereochemical purity of WVE-003 is about 80%, 81%, 82%, 83%, 84%, or 85% or greater.

86. 86. The method or composition of any one of claims 1-85, wherein about is ±1%, ±2%, ±3%, ±4%, ±5%, ±6%, ±7%, ±8%, ±9%, or ±10%.

87. 434. The method or composition of any one of embodiments 1 to 433.

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