Oligonucleotide compositions and methods thereof

Abstract

In particular, the disclosure provides various oligonucleotide techniques, including chirally controlled oligonucleotide compositions, and techniques for making such oligonucleotide compositions. In some embodiments, the methods are methods of treating or preventing Huntington's disease in a subject in need thereof; a method for allele-specific knock-down of a mutant Huntingtin transcript in a subject; methods for delaying the onset of Huntington's disease and / or reducing the severity of at least one symptom of Huntington's disease in a subject suffering from Huntington's disease; a method of reducing expression, level, amount and / or activity of a mutant Huntingtin gene or gene product thereof; and / or a method of preparing a medicament for treating Huntington's disease, wherein the method involves the use of the oligonucleotides described herein administered at doses described herein. In some embodiments, the disclosure provides doses, administration, and formulations of the oligonucleotides described herein.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to U.S. Provisional Application Nos. 63 / 397,284, filed on August 11, 2022, 63 / 408,427, filed on September 20, 2022, and 63 / 517,529, filed on August 3, 2023, the entire contents of each of which are incorporated herein by reference. Technical Field

[0003] In particular, the present disclosure provides technologies for treating Huntington's disease. In some embodiments, provided herein are methods of administering WVE-003 to improve Huntington's disease, reduce mHTT RNA, or reduce mHTT protein in a human subject in need thereof. In some cases, the methods can be used to improve 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, uncontrolled movements, difficulty swallowing, difficulty speaking, anxiety, and depression. Background Art

[0004] Oligonucleotides can be used for treatment, diagnosis, research and nanomaterial applications. Naturally occurring nucleic acids (e.g., unmodified DNA or RNA) may be limited for the purposes of treatment, for example, because naturally occurring nucleic acids are unstable to extracellular and intracellular nucleases and / or the cell penetration and distribution of naturally occurring nucleic acids are poor. Currently lacking an acceptable option for treating neurodegenerative diseases such as Huntington's disease. The purpose of this paper is to provide a method for treating such a disease. Summary of the Invention

[0005] WVE-003 (also known as WV-21405) has the following structure: mG*SmUn001R mU mGn001R mA*ST*SC*ST*SG*ST*RA*SG*SC*SA*SG*R m5Ceon001RAeoGeon001R m5Ceo*STeo, where:

[0006] m represents 2′-OMe modification of the nucleoside;

[0007] *S represents Sp phosphorothioate linkage;

[0008] m5Ceo represents 5-methyl 2′-O-methoxyethyl C;

[0009] n001R represents an Rp n001 linkage, where the n001 linkage has structure;

[0010] eo represents a 2'-OCH2CH2OCH3 modification to a nucleoside; and

[0011] *R represents Rp phosphorothioate linkage.

[0012] As typically used in oligonucleotide descriptions, if not otherwise indicated, the nucleosides are DNA nucleosides and the linkages are natural phosphate linkages.

[0013] As described herein, WVE-003 can be provided in various forms, including pharmaceutically acceptable salt forms, such as the sodium salt.

[0014] In particular, the present disclosure encompasses the recognition that structural elements of HTT (huntingtin) oligonucleotides, such as base sequence, chemical modifications (e.g., modifications of sugars, bases and / or internucleotide linkages and their patterns), and / or stereochemistry [e.g., stereochemistry of backbone chiral centers (chiral internucleotide linkages) and / or their patterns] can have a significant effect on HTT oligonucleotide properties and / or activities (e.g., protein binding characteristics, 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 activities. In some embodiments, the present disclosure provides techniques, such as HTT oligonucleotides, pharmaceutical compositions, pharmaceutical products, dosages, administration, etc., that can be used to treat Huntington's disease in a subject, particularly those of WVE-003. In some embodiments, the subject has an HTT allele or transcript that comprises an expanded CAG repeat region and is fully complementary to the base sequence of WVE-003. As used herein, a nucleic acid or base sequence can be considered to be fully complementary to another nucleic acid (e.g., WVE-003) or base sequence if it comprises a sequence having the same length as another nucleic acid or base sequence and fully complementary to the complete base sequence of another nucleic acid (e.g., WVE-003) or base sequence. 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, wherein the transcript is fully complementary to the base sequence of WVE-003. In some embodiments, the subject comprises an HTT transcript associated with Huntington's disease, wherein the transcript is fully complementary to the base sequence of WVE-003 at SNP rs362273. In some embodiments, the subject comprises an HTT transcript that is fully complementary to the base sequence of WVE-003 and comprises an expanded CAG repeat region. In some embodiments, the subject comprises an HTT transcript that is fully complementary to the base sequence of WVE-003 at SNP rs362273 and comprises an expanded CAG repeat region. In some embodiments, the subject comprises an HTT nucleic acid, such as an HTT gene, encoding an HTT transcript associated with Huntington's disease, wherein the transcript is completely complementary to the base sequence of WVE-003. In some embodiments, the subject comprises an HTT nucleic acid, such as an HTT gene, encoding an HTT transcript associated with Huntington's disease, wherein the transcript is completely complementary to the base sequence of WVE-003 at SNPrs362273. In some embodiments, the subject comprises an HTT nucleic acid, such as an HTT gene, encoding an HTT transcript that is completely complementary to the base sequence of WVE-003 and comprises an expanded CAG repeat region.In some embodiments, the subject comprises an HTT nucleic acid, such as an HTT gene, which encodes an HTT transcript that is completely complementary to the base sequence of WVE-003 at SNP rs362273 and comprises an expanded CAG repeat region. In some embodiments, the subject has the A variant of SNPrs362273. As will be understood by those skilled in the art, an HTT transcript having the A variant of SNP rs362273 can be completely complementary to the base sequence of WVE-003, wherein the A is complementary to the T in WVE-003. In some embodiments, the A variant of SNPrs362273 is on the same chromosome as the expanded CAG repeat region in HTT. In some embodiments, the subject has a variant of SNP rs362273 that is not A. In some embodiments, the variant of SNP rs362273 that is not A is not on the same chromosome as the expanded CAG repeat region in HTT. In some embodiments, the variant of SNP rs362273 is C. In some embodiments, the subject is heterozygous for the A variant of SNP rs362273. In some embodiments, the subject is heterozygous for the A variant of SNPrs362273, wherein the A variant is on the same chromosome as the CAG repeat region amplified in HTT. In some embodiments, the subject is homozygous for the A variant of SNP rs362273. In some embodiments, the subject is homozygous for the A variant of SNP rs362273 and has the CAG repeat region amplified in HTT. In some embodiments, one chromosome of the subject has the CAG repeat region amplified in HTT. In some embodiments, two chromosomes of the subject independently have the CAG repeat region amplified in HTT.

[0015] In some embodiments, Huntington's disease (HD) is a debilitating and ultimately fatal autosomal dominant neurological disorder characterized by cognitive decline, mental illness, and chorea. In some embodiments, HD causes nerve cells in the brain to deteriorate over time, affecting thinking ability, mood, and movement. HD is reportedly caused by amplified cytosine-adenine-guanine (CAG) triplet repeats in the huntingtin (HTT) gene, which leads to the production of mutant HTT protein. In some embodiments, the accumulation of mutant HTT leads to the gradual loss of neurons in the brain. In some embodiments, wild-type or healthy HTT protein is essential for neuronal function, and inhibition may have harmful long-term consequences. In some embodiments, approximately 30,000 people in the United States have symptomatic HD, and more than 200,000 people are at risk of inheriting the disease. There are currently no approved disease-modifying therapies available.

[0016] In some embodiments, Huntington's disease (HD) is reported to be caused by a cytosine-adenine-guanine (CAG) repeat expansion in the huntingtin (HTT) gene, which results in the production of mutant HTT (mHTT) protein. In some embodiments, although mHTT is reported to be the cause of the disease, preclinical studies have shown that loss of wild-type HTT (wtHTT) may also lead to neuronal damage. In some embodiments, wtHTT protein is reported to be critical for neuronal function; inhibition of wtHTT may have harmful long-term consequences.

[0017] In some embodiments, the ability to selectively reduce the production of mHTT protein while retaining adequate wtHTT levels (also described as allele-specific knockdown) has been reported to hold great promise for HD treatment. In some embodiments, as demonstrated herein, administration of oligonucleotides as described herein can reduce the level, expression, and / or activity of mHTT transcripts and / or proteins more than the level, expression, and / or activity of wtHTT transcripts and / or proteins in individual subjects and / or populations of subjects.

[0018] 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).

[0019] In some embodiments, the present disclosure provides oligonucleotides, compositions and methods for allele-specific knockdown of mHTT transcripts, wherein the allele-specific knockdown (also referred to as allele-specific inhibition, allele-selective methods, allele-selective knockdown or inhibition, etc.) preferentially reduces the level, expression and / or activity of the mHTT transcript (e.g., comprising an expanded CAG repeat region) and / or its product (e.g., mHTT protein) relative to wild-type HTT and / or its product (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).

[0020] In some embodiments, the reduction in the level, expression and / or activity of the mHTT transcript results in or is associated with a reduction in the level, expression and / or activity of the mHTT protein, including but not limited to the formation, number and / or size of aggregates (condensates) of the mHTT protein. In some embodiments, the mHTT protein comprises an extended polyglutamine (poly-Q) stretch, for example, translated from a CAG repeat expansion.

[0021] In some embodiments, the disclosure relates to: a method of treating and / or preventing Huntington's disease in a subject (e.g., a patient, such as a human patient) in need thereof; a method of allele-specifically knocking down mutant huntingtin transcripts in a subject; and / or a method for reducing the severity of one or more symptoms of Huntington's disease and / or delaying its onset. In some embodiments, the method comprises the step of administering to the subject a therapeutically effective amount of an HTT oligonucleotide or an HTT oligonucleotide composition. In some embodiments, the disclosure relates to: a method of treating, ameliorating Huntington's disease, or slowing the onset or progression of Huntington's disease, the method 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 comprising a single nucleotide polymorphism (SNP) site on the allele, wherein the compound administered to the subject treats, improves Huntington's disease, and / or slows the onset and / or progression of Huntington's disease by selectively reducing the mutant huntingtin allele. In some embodiments, the disclosure relates to a method of ameliorating symptoms of Huntington's disease, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to a human subject in need thereof.

[0022] 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 chirality-controlled oligonucleotide composition.

[0023] In some embodiments, the WVE-003 composition is a chirally controlled (e.g., stereopure) HTT oligonucleotide composition that is capable of mediating an allele-specific reduction in the level, expression, and / or activity of an mHTT transcript. WVE-003 can selectively target an isoform of the HTT SNP rs362273 (sometimes referred to as SNP3). WVE-003 is fully complementary to the SNP sequence (and surrounding sequence) to which it targets. As described herein, in various embodiments, WVE-003 is fully complementary to the mtHTT transcript. In some embodiments, WVE-003 is fully complementary to the mtHTT transcript and is not fully complementary to the wtHTT transcript. In some embodiments, the mtHTT transcript is associated with or is more associated with a condition, disorder, or disease (such as Huntington's disease) than the wtHTT transcript. In some embodiments, mtHTT comprises 36, 37, 38, 39, 40, 45, 50, or more CAG repeats. As will be appreciated by those skilled in the art, complementarity with the transcript can be assessed using the genetic sequence from which the transcript is transcribed.

[0024] In some embodiments, WVE-003 can be used as a disease-modifying agent for treating subjects with Huntington's disease (HD). It can be provided as a stereopure antisense oligonucleotide (ASO) that selectively targets mutant forms of the huntingtin (mHTT) gene transcript. In some embodiments, the composition of WVE-003 is a stereopure oligonucleotide composition that recognizes a disease-associated (e.g., mutant) allele of SNP rs362273 in the huntingtin gene, effectively reduces the level, expression, and / or activity of the mHTT gene (or its gene product), and is capable of mediating allele-specific knockdown of the mutant huntingtin (mHTT) gene.

[0025] In some embodiments, the present disclosure provides a method for reducing the level, expression and / or activity of the mHTT gene (or its gene product), comprising administering an oligonucleotide to a subject at a dose as described herein or according to a dosage regimen. In some embodiments, the level of mHTT transcripts 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, a sample from a subject is used for evaluation. In some embodiments, the subject in the various methods herein is human. In some embodiments, 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) is used for evaluation. In some embodiments, a patient sample is used for evaluation. A "patient sample" is any biological sample from a patient. The term sample includes, but is not limited to, biological fluids such as blood, serum, plasma, urine, cerebrospinal fluid (CFS), 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, a CSF sample is used for evaluation. In some embodiments, a plasma sample is used for evaluation. In some embodiments, a blood sample is used for evaluation. In certain embodiments, assess at one or more suitable time points as understood by those skilled in the art, for example, after about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 days of administration, or about 3, 4, 5, 6, 7 or 8 weeks, or about 3, 4, 5 or 6 or more months, and before using next dosage if any. In certain embodiments, assess after using about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 dosage. In certain embodiments, assessment result is for individual subjects. In certain embodiments, subject colony is assessed, for example, as typically carried out in clinical trials or clinical applications. In some embodiments, the population is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 or more subjects. 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, the reduction is at least about 10% or more. In some embodiments, the reduction is at least about 12% or more. In some embodiments, the reduction is at least about 20% or more. In some embodiments, the reduction is at least about 30% or more. In some embodiments, the reduction is at least about 40% or more. In some embodiments, the reduction is at least about 50% or more. As will be appreciated by those skilled in the art, in some embodiments, the mHTT gene or its product comprises a sequence that is identical or completely complementary to the WVE-003 sequence and contains expanded CAG repeats (or a sequence encoded thereby), and the wild-type HTT gene or its product does not contain a sequence that is identical or completely complementary to the WVE-003 sequence in HTT or at rs362273 and contains fewer CAG repeats (e.g., not considered to be expanded CAG repeats) (or a sequence encoded by these repeats).

[0026] In some embodiments, the disclosure relates to any of a variety of methods related to the use of HTT oligonucleotides or compositions. 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, and substantially all of the oligonucleotides in the composition are WVE-003 (as will be appreciated by those skilled in the art, some other oligonucleotides may be present as impurities, the levels of which can and typically are controlled in the provided compositions).

[0027] In some embodiments, an 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.

[0028] As used herein, unless expressly indicated otherwise, the amounts of the 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 to the free acid form of the oligonucleotide. 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 dosage is equivalent to about 60 mg of WVE-003 free acid form. In some embodiments, the dosage is equivalent to about 90 mg of WVE-003 free acid form. In some embodiments, the dosage is equivalent to about 120 mg of WVE-003 free acid form. In some embodiments, the dosage is equivalent to about 150 mg of WVE-003 free acid form.

[0029] In some embodiments, the 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 the free acid form of WVE-003. As described herein, WVE-003 can be administered or delivered in various forms, including various pharmaceutically acceptable salt forms. In some embodiments, the composition administered to a subject comprises one or more forms of WVE-003, such as 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, such as water or a suitable buffer as described herein, such as buffered saline, cerebrospinal fluid (CSF), artificial cerebrospinal fluid (aCSF), and the like.

[0030] Useful techniques for preparing oligonucleotides such as WVE-003 and their products 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 stereopurity of about 97%, 98%, 99% or higher. In some embodiments, the stereopurity of the oligonucleotide (e.g., WVE-003) in the composition can be represented by the product of the stereopurity (or stereoselectivity) at each chiral internucleotide linkage. In some embodiments, the stereopurity / stereoselectivity of the chiral internucleotide linkage can be assessed by the stereopurity / stereoselectivity during the formation of a corresponding dimer comprising the chiral internucleotide linkage and the two nucleosides to which it is bonded. In some embodiments, when forming a chiral internucleotide linkage, the stereopurity / stereoselectivity of the chiral internucleotide linkage can be assessed by the stereopurity / stereoselectivity during oligonucleotide synthesis (e.g., the product assessment after the chiral internucleotide linkage is formed but before the next internucleotide linkage is formed). In some embodiments, the stereopurity / stereoselectivity of the chiral internucleotide linkage in the product oligonucleotide (e.g., WVE-003) is considered to be the stereopurity / stereoselectivity observed in dimer formation or during oligonucleotide synthesis. In some embodiments, the stereopurity / stereoselectivity observed in dimer formation or during oligonucleotide synthesis is the same as or very close to each other. It is understood by those skilled in the art that the stereopurity / stereoselectivity of each chiral internucleotide linkage can be different, although according to various provided techniques, it is always higher (e.g., about 97%, 98%, 99% or higher) at each chiral internucleotide linkage. In some embodiments, each chiral internucleotide linkage independently has a stereopurity of about 97%, 98%, 99% or higher. 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 stereopurity of about 98%, 99% or higher. In some embodiments, at least 1, 2, 3, 4, 5, 6, or 7 internucleotide linkages independently have a stereopurity of about 99% or greater. In some embodiments, no more than 1, 2, 3, 4, or 5 independently have a stereopurity of less than about 98%. In some embodiments, no more than 1, 2, 3, 4, or 5 independently have a stereopurity of less than about 98%, but each is still independently greater than about 97%. In some embodiments, the stereopurity of WVE-003 is about 75% or greater. In some embodiments, the stereopurity of WVE-003 is about 80% or greater (e.g., as the product of the stereopurities of the corresponding dimers). In some embodiments, the stereopurity of WVE-003 is about 81% or greater (e.g., as the product of the stereopurities of the corresponding dimers).In some embodiments, the stereopurity of WVE-003 is about 82% or greater (e.g., as the product of the stereopurities of the corresponding dimers). In some embodiments, the stereopurity of WVE-003 is about 83% or greater (e.g., as the product of the stereopurities of the corresponding dimers). In some embodiments, the stereopurity of WVE-003 is about 84% or greater (e.g., as the product of the stereopurities of the corresponding dimers). In some embodiments, the stereopurity of WVE-003 is about 85% or greater (e.g., as the product of the stereopurities of the corresponding dimers). 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 manufacturing). In some embodiments, the amount of oligonucleotide administered does not include other impurities, such as shorter oligonucleotides observed during oligonucleotide synthesis (to the extent that the purification method utilized can remove such impurities, as will be understood by one skilled in the art).

[0031] As will be appreciated by those skilled in the art, oligonucleotides can be administered in a variety of forms, including one or more pharmaceutically acceptable salt forms. For example, in some embodiments, oligonucleotides are administered as oligonucleotides and / or salts thereof dissolved in a suitable solution (e.g., water or a suitable buffer system). In some embodiments, the amount of oligonucleotide includes all forms of the oligonucleotide, and when expressed in weight (e.g., mg), such weight includes the weight of all forms of the oligonucleotide, but all converted to the weight of the acid form (e.g., for WVE-003, the free acid with a molecular weight of 7257.94 is used). Alternatively or additionally, the amount of oligonucleotide can be expressed in moles, which in some embodiments includes moles of all forms of oligonucleotide (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 WVE-003).

[0032] As described herein, multiple doses can be administered at various suitable frequencies, such as 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, as described herein. In some embodiments, each dose is independently equivalent to 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 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 per month. 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 per 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 technology provided provides long duration. In some embodiments, the dosage is administered every two months or at a lower frequency. In some embodiments, the dosage is administered quarterly or at a lower frequency.

[0033] In some embodiments, the disclosure relates to: a method of treating and / or preventing Huntington's disease in a subject (e.g., a patient, such as a human patient) in need thereof; a method of allele-specific knockdown of mutant huntingtin transcripts in a subject; and / or a method for reducing the severity of and / or delaying the onset of one or more symptoms of Huntington's disease, wherein the method comprises administering WVE-003 in multiple doses, each dose independently equivalent to 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, in the methods of the present disclosure, the amount of each dose remains constant (e.g., each dose received by the patient is about 30 mg; or each dose received by the patient is about 60 mg; or each dose received by the patient is about 90 mg; or each dose received by the patient is about 120 mg). In some embodiments, the amount of each dose varies over time (e.g., a patient may be maintained at a specific dose for one or more doses, and later, due to efficacy and / or safety or other issues, the amount of each dose may be increased or decreased). In some embodiments, multiple doses are administered, and each dose is independently about 30 mg of WVE-003 free acid form. In some embodiments, the multiple doses are administered approximately monthly or approximately every 4 weeks. In some embodiments, the doses are administered approximately monthly or approximately every 4 weeks, except for approximately two months or approximately eight weeks between the second and fourth doses. In some embodiments, the multiple doses are administered approximately every 8 weeks. For example, in some embodiments, the method comprises administering multiple doses approximately every 8 weeks, wherein each dose is independently equivalent to about 30 mg of WVE-003 free acid form. In some embodiments, the multiple doses are administered approximately every 12 weeks. For example, in some embodiments, the methods comprise administering multiple doses approximately once every 12 weeks, wherein each dose is independently equivalent to about 30 mg of WVE-003 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 2 times. In some embodiments, the dose is administered at least about 3 times. In some embodiments, the dose is administered at least about 4 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, 12 or more months.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, 12, or more months. In some embodiments, the interval is about 1 week. In some embodiments, the interval is about 2 weeks. In some embodiments, the interval is about 3 weeks. In some embodiments, the interval is about 4 weeks. In some embodiments, the interval is about 5 weeks. In some embodiments, the interval is about 6 weeks. In some embodiments, the interval is about 7 weeks. In some embodiments, the interval is about 8 weeks. In some embodiments, the interval is about 9 weeks. In some embodiments, the interval is about 10 weeks. In some embodiments, the interval is about 9 weeks. In some embodiments, the interval is about 11 weeks. In some embodiments, the interval is about 9 weeks. In some embodiments, the interval is about 12 weeks. In some embodiments, the interval is about 1 month. In some embodiments, the interval is about 2 months. In some embodiments, the interval is about 3 or more months. In some embodiments, the interval is about 3, 4, 5, 6, 7, 8, or 9 months. In some embodiments, the interval is about 3 months. In some embodiments, the interval is about 6 months. In some embodiments, the interval is about 9 months. In some embodiments, each interval is independently about 1 week or more. In some embodiments, each interval is independently about 2 weeks or more. In some embodiments, each interval is independently about 3 weeks or more. In some embodiments, each interval is independently about 4 weeks or more. In some embodiments, each interval is independently about 5 weeks or more. In some embodiments, each interval is independently about 6 weeks or more. In some embodiments, each interval is independently about 7 weeks or more. In some embodiments, each interval is independently about 8 weeks or more. In some embodiments, each interval is independently about 9 weeks or more. In some embodiments, each interval is independently about 10 weeks or more. In some embodiments, each interval is independently about 11 weeks or more. In some embodiments, each interval is independently about 12 weeks or more. In some embodiments, each interval is independently about 1 month or more. In some embodiments, each interval is independently about 2 months or more. In some embodiments, each interval is independently about 3 months or more. 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, all intervals are approximately the same. In some embodiments, one or more intervals are longer than the others.In some embodiments, one or more intervals are shorter than other intervals. In some embodiments, the dosage frequency of multiple 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 or more months. In some embodiments, the dosage frequency of multiple or all doses is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months. In some embodiments, one or more loading doses are administered. In some embodiments, no loading dose is administered.

[0034] In some embodiments, WVE-003 is tested for use in various approaches in a clinical trial designated 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 effects of WVE-003 in adult patients with early-onset Huntington's disease (HD) who harbor the targeted single nucleotide polymorphism (SNP) rs362273 (SNP3). Phase 1 of this study evaluated single ascending doses (SAD) of WVE-003. Phase 2 evaluated multiple ascending doses (MAD) of WVE-003. In some embodiments, the subjects have early-onset Huntington's disease. In some embodiments, the subjects are 25 years of age or older. In some embodiments, the subjects are 60 years of age or younger.

[0035] In some embodiments, the oligonucleotide (e.g., WVE-003) or composition is administered intrathecally. In some embodiments, intrathecal administration is interspinal administration. In some embodiments, the methods described herein comprise administering an HTT oligonucleotide (e.g., WVE-003) or composition thereof intrathecally and in an amount described herein, such as 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. In some embodiments, the methods described herein comprise intrathecally and each independently administering an HTT oligonucleotide (e.g., WVE-003) or composition thereof in an amount described herein and at a dosage interval described herein (e.g., monthly), e.g., an amount equivalent to 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.

[0036] In some embodiments, a therapeutically effective amount of an HTT oligonucleotide or a salt 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 thereof, or an HTT oligonucleotide composition, is an amount 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 (e.g., about 30, about 60, about 90, about 120, about 150, or about 168 mg of the free acid form).

[0037] In some embodiments, the methods described herein comprise the step of administering a therapeutically effective amount of an HTT oligonucleotide or an HTT oligonucleotide composition. In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is or comprises WVE-003.

[0038] In some embodiments, the therapeutically effective amount of an HTT oligonucleotide, or a salt 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, the therapeutically effective amount of an HTT oligonucleotide, or a salt thereof, or an HTT oligonucleotide composition does not significantly reduce the amount of a wild-type huntingtin transcript or its gene product. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide, or a salt thereof, or an HTT oligonucleotide composition does not reduce the amount of a wild-type huntingtin transcript or its gene product to a level associated with a clinically manifest adverse event or side effect.

[0039] In some embodiments, the 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, the 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.

[0040] In certain embodiments, adverse events are adverse effects. In certain embodiments, adverse events are mild, moderate, severe or serious adverse events. In certain embodiments, adverse events are: fever, headache, vomiting, or tachycardia. In certain embodiments, adverse events are or proinflammatory markers (e.g., C-reactive protein and complement) increase, aPTT prolongation, thrombocytopenia, liver enzyme changes (e.g., AST and ALT) or kidney biomarker changes (e.g., BUN, creatinine), measured by, or associated with.

[0041] In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or its salt form or an HTT oligonucleotide composition is sufficient to achieve a clinically significant maximum plasma concentration of the HTT oligonucleotide or oligonucleotide composition. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or its salt form or an HTT oligonucleotide composition is about 30 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or its salt form or an HTT oligonucleotide composition is about 60 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or its salt form or an HTT oligonucleotide composition is about 90 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or its salt form or an HTT oligonucleotide composition is about 120 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or its salt form or an HTT oligonucleotide composition is about 150 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or its salt form or an HTT oligonucleotide composition is about 168 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or its salt form or an HTT oligonucleotide composition is 30 ± 25% mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or its salt form or an HTT oligonucleotide composition is 60 ± 25% mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or a salt form thereof, or an HTT oligonucleotide composition is 90±25% mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or a salt form thereof, or an HTT oligonucleotide composition is 120±25% mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or a salt form thereof, or an HTT oligonucleotide composition is 150±25% mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or a salt form thereof, or an HTT oligonucleotide composition is 168±25% mg. In some embodiments, the 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 (regardless of body weight, as the total volume of the central nervous system of most adults is similar). In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or a salt form thereof, or an HTT oligonucleotide composition is about 30 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or a salt form thereof, or an HTT oligonucleotide composition is about 60 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or a salt thereof, or an HTT oligonucleotide composition is about 90 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or a salt thereof, or an HTT oligonucleotide composition is about 120 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or a salt thereof, or an HTT oligonucleotide composition is about 150 mg. In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or a salt thereof, or an HTT oligonucleotide composition is about 168 mg. In some embodiments, the HTT oligonucleotide is WVE-003.

[0042] In some embodiments, the present disclosure provides methods comprising administering to a subject an amount of about 30, about 60, about 90, about 120, about 150, or about 168 mg of an HTT oligonucleotide or HTT oligonucleotide composition equivalent to the free acid form of the oligonucleotide.

[0043] In some embodiments, the present disclosure provides methods comprising administering to a subject a dose of about 30, about 60, about 90, about 120, about 150, or about 168 mg of an HTT oligonucleotide or HTT oligonucleotide composition, wherein the oligonucleotide is WVE-003. In some embodiments, the dose is administered approximately once a month or approximately every four weeks. In some embodiments, the dose is administered approximately once every two months or approximately every eight weeks. In some embodiments, the dose is administered approximately once every three months or approximately every twelve weeks. In some embodiments, the time between the first dose and the second dose is about 2 months or about 8 weeks, and each subsequent dose is administered approximately once a month or approximately every four weeks. In some embodiments, the time between the first dose and the second dose is about 3 months or about 12 weeks, and each subsequent dose is administered approximately once every two months or approximately every 8 weeks. In some embodiments, each subsequent dose is administered approximately once every two months or approximately every 8 weeks. In some embodiments, each subsequent dose is administered approximately once every two months or approximately every 12 weeks. In some embodiments, the dose is administered for at least about 12 weeks. In some embodiments, the dose is administered for at least about 16 weeks. In some embodiments, the dose is administered for at least about 3 months. In some embodiments, the dose is administered for at least about 4 months. In some embodiments, the patient maintains the same dose or a constant dose (e.g., each dose received by the patient is 30 mg; or each dose received by the patient is 60 mg; or each dose received by the patient is 90 mg; or each dose received by the patient is 120 mg). In some embodiments, the patient receives multiple doses of different amounts. For example, a patient can receive one or more initial doses of a specific amount of an HTT oligonucleotide or an HTT oligonucleotide composition, and the subsequent doses received by the patient can be increased or decreased due to efficacy, safety, or other issues.

[0044] In particular, the present disclosure recognizes the challenge of providing oligonucleotides with high efficacy and low toxicity and methods of using the same. In some embodiments, the present disclosure provides oligonucleotides with reduced toxicity, such as WVE-003, as well as compositions and methods. In some embodiments, the provided technology provides a reduced level of toxicity when delivering approximately the same or comparable amounts of oligonucleotides, and / or when achieving approximately the same or comparable levels of desired effects and / or effects. In some embodiments, more oligonucleotides, such as WVE-003, can be delivered, and / or a higher level of desired effects and / or effects can be achieved at approximately the same, comparable, or lower levels of toxicity. 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 the same base sequence but a different structure compared to WVE-003. In some embodiments, the reference oligonucleotide comprises different modifications and / or patterns thereof. In some embodiments, the reference oligonucleotide comprises different internucleotide linkages and / or patterns thereof. In some embodiments, the reference oligonucleotide comprises different linkage stereochemistries or patterns thereof. In some embodiments, the reference oligonucleotide is provided as a stereostochastic composition. In certain embodiments, the reference oligonucleotide composition is provided as an oligonucleotide composition controlled by chirality. In certain embodiments, the reference oligonucleotide has a lower purity. In certain embodiments, the reference oligonucleotide has a lower stereo purity. In certain embodiments, the reference oligonucleotide is WV-1092. In certain embodiments, the reference oligonucleotide is WV-2603.

[0045] In some embodiments, the present disclosure provides oligonucleotide compositions and methods with reduced immune responses.In some embodiments, the present disclosure recognizes that various toxicities induced by oligonucleotides may be associated with cytokine and / or complement activation.

[0046] In some embodiments, the HTT oligonucleotide composition (e.g., WVE-003) is chirality controlled (e.g., stereopure). In particular, the stereorandom HTT oligonucleotide formulation contains a plurality of different chemical entities that differ from one another, for example, in the stereochemical structures of the individual backbone chiral centers (e.g., phosphorothioate) within the HTT oligonucleotide chain. Without controlling the stereochemistry of the backbone chiral centers, the stereorandom HTT oligonucleotide formulation provides an uncontrolled (or stereorandom) composition comprising an undetermined level of HTT oligonucleotide stereoisomers. Even though these stereoisomers may have the same base sequence and / or chemical modification, they are different chemical entities, at least approximately due to their different backbone stereochemistry, and they can have different properties, as demonstrated herein, such as activity, toxicity, distribution, etc. In particular, the present disclosure provides chirality-controlled compositions that are or contain a specific stereoisomer of a target HTT oligonucleotide; compared to a composition in which chirality is not controlled, the chirality-controlled composition comprises a controlled level of a specific stereoisomer of the HTT oligonucleotide, or comprises a controlled level of all oligonucleotides of the composition in the chirality-controlled composition, or a controlled level of all oligonucleotides of the composition sharing a specific base sequence (e.g., the base sequence of WVE-003) sharing a common pattern of linkage phosphorus stereochemistry (e.g., the common pattern of WVE-003). In some embodiments, the controlled level of all oligonucleotides sharing the same composition as a form of WVE-003 is WVE-003. In some embodiments, the diastereomeric purity of WVE-003 is about or greater than the controlled level.

[0047] 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%.

[0048] 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 controlled level is at least about 99%.

[0049] In some embodiments, the therapeutically effective amount of an HTT oligonucleotide, an HTT oligonucleotide composition, or WVE-003 is about 30, about 60, about 90, about 120, about 150, or about 168 mg.

[0050] In some embodiments, the HTT oligonucleotide or salt form thereof, or the HTT oligonucleotide composition is or comprises WVE-003, which is a chiral-controlled HTT oligonucleotide composition capable of mediating allele-specific reduction in the level, expression, and / or activity of an mHTT transcript.

[0051] In some embodiments, the disclosure relates to a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the steps of administering WVE-003 (or a salt thereof) to the subject at a dose of 10 mg to about 168 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of symptoms of Huntington's disease is reduced.

[0052] In some embodiments, the HTT gene of the human subject: (A) is heterozygous at a CAG repeat (wherein one allele contains the deleterious CAG repeat expansion and the other allele does not), and (B) is heterozygous at a SNP targeted by an HTT oligonucleotide, where the HTT oligonucleotide is capable of distinguishing between two alleles of the same SNP (e.g., due to differences in the sequence 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 an arrangement is referred to as the CAG repeat expansion and the mutant SNP allele being in phase, and the HTT gene or mutation thereof can be described as being suitable for allele-specific knockdown.

[0053] In some embodiments, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene is: a mutant HTT gene comprising a deleterious mutation (e.g., a CAG repeat expansion), wherein the deleterious mutation is on the same chromosome as (e.g., is in phase or in the same phase as) a specific allele of the SNP targeted by a specific HTT oligonucleotide (e.g., WVE-003), wherein the SNP allele targeted by the oligonucleotide also targets the deleterious mutation.

[0054] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 30 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0055] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 60 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of symptoms of Huntington's disease is reduced.

[0056] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the steps of administering WVE-003 (or a salt form thereof) to the subject at a dose of about 90 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of symptoms of Huntington's disease is reduced.

[0057] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 120 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0058] In some embodiments, the subject is determined to express mtHTT transcripts that are 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 a mtHTT allele that comprises a sequence identical to the sequence of the administered oligonucleotide, including at the SNP site (e.g., rs362273) (as understood by those skilled in the art, U and T may be considered to be identical) or fully complementary to the sequence of the administered oligonucleotide, including at the SNP site (e.g., rs362273). 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 mtHTT transcripts that are fully complementary to the administered oligonucleotide, and wtHTT transcripts that are not fully complementary to the administered oligonucleotide. In some embodiments, the subject has an mtHTT allele comprising a sequence identical to the sequence of the administered oligonucleotide (as understood by those skilled in the art, U and T may be appropriately considered to be identical) or fully complementary to the sequence of the administered oligonucleotide; and a wtHTT allele not comprising a sequence identical to the sequence of the administered oligonucleotide (as understood by those skilled in the art, U and T may be appropriately considered to be identical) or fully complementary to the sequence of the administered oligonucleotide. In some embodiments, the level of transcript, protein and / or activity of the mtHTT allele is more reduced than the level of wtHTT. The mtHTT allele is fully complementary to the administered oligonucleotide, and the wtHTT transcript is not fully complementary to the administered oligonucleotide. Various techniques are available and can be used according to the present disclosure to detect and / or determine whether a specific isoform of a SNP is on the same allele or transcript.

[0059] In some embodiments, the present disclosure provides a method for determining the suitability of a subject for treatment with WVE-003 (or a salt form thereof), the method comprising the steps of:

[0060] i) determining the presence of mutant alleles relative to wild-type alleles in patients with the targeted single nucleotide polymorphism (SNP) rs362273 (SNP3);

[0061] ii) comparing the level of the mutant allele in the patient having the targeted single nucleotide polymorphism (SNP) rs362273 (SNP3) of step i) with one or more reference samples or reference values;

[0062] iii) determining whether the subject may be or is suitable for treatment with WVE-003, wherein the subject has or is at risk of developing a neurodegenerative disorder, such as Huntington's disease; and

[0063] iv) administering an effective amount of WVE-003 to the subject.

[0064] In some embodiments, the present disclosure provides for the use of a phasing assay to detect the presence of a mutant allele relative to a wild-type allele in a patient with the targeted single nucleotide polymorphism (SNP) rs362273 (SNP3) to determine suitability for treatment with WVE-003.

[0065] Various techniques can be used to determine whether a particular SNP allele is on the same chromosome as a disease-associated sequence (e.g., a CAG repeat expansion of HTT). Typically, if the SNP allele and the CAG repeat expansion are on the same chromosome, an HTT oligonucleotide targeting the SNP allele can also "target" the CAG repeat expansion associated with the disease, thereby reducing the expression, level, and / or activity of the HTT allele with the mutation associated with the disease. In this way, for example, HTT oligonucleotides can be used to treat disorders associated with HTT, such as Huntington's disease. Therefore, HTT oligonucleotides targeting SNPs can preferentially reduce the expression, level, and / or activity of the mutant allele of HTT compared to the wild-type allele.

[0066] Humans, like other organisms, are diploid, and for phasing techniques, it is necessary to determine the linkage of alleles at loci on the same or different chromosomes. The sequences on the corresponding chromosomes are called haplotypes. The process of determining which alleles are located on which chromosome is called phasing, haplotype phasing, or haplotype typing. Phasing information can be used for patient stratification, identification, and treatment of various other HTT-related diseases and disorders (such as Huntington's disease). For other general information on phasing, see, for example: Twehey et al. 2011 Nat. Rev. Genet. [Natural Genetics Review] 12: 215-223; and Glusman et al. 2014 Genome Med. [Genome and Medicine] 6: 73.

[0067] Phased data can be very important in allele-specific therapies for diseases such as Huntington's disease. In certain diseases, genetic damage has been identified, such as deleterious duplications, deletions, insertions, inversions or other mutations, such as expanded CAG repeat expansions in mutant (and disease-associated) HTT alleles. In some patients, one allele of a gene such as HTT may contain a disease-associated mutation at a genetic locus, while the other allele is normal, wild-type, or otherwise unrelated to the disease or less relevant to the disease. In some embodiments, allele-specific therapies can target HTT alleles that contain disease-associated mutations, but not the corresponding wild-type alleles. In some embodiments, allele-specific therapies can target HTT alleles that contain disease-associated mutations (such as CAG repeat expansions (or expanded CAG segments)) at a specific locus, but not by directly targeting the locus, but by targeting a different locus on the mutant allele. As a non-limiting example, allele-specific therapy can target an allele containing a disease-associated mutation at a locus by targeting a different locus in the same allele, such as a SNP (single nucleotide polymorphism) in the same gene.

[0068] As a non-limiting example, some genetic lesions associated with a disease may be difficult to target or otherwise not easy to target. As a non-limiting 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 amount of repeats is not associated with the disease, but an abnormally large amount of repeats or repeat expansion is associated with the disease. Because repeats are present on both wild-type and mutant alleles, it may be difficult to directly target the repeats associated with the disease. However, if a specific SNP variant is present on the same allele as the repeat expansion associated with the disease, but not on the wild-type allele, the SNP variant can be used to target an allele-specific therapy that targets the mutant allele rather than the wild-type allele.

[0069] As a non-limiting example, individual phasing data indicates whether a particular SNP is in phase with the lesion (e.g., on the same chromosome or transcript) and therefore can be targeted with a therapeutic nucleic acid. The therapeutic agent can then target the mutant gene without targeting the wild-type allele. If the wild-type allele must be expressed, it would be particularly useful to obtain phasing data only for the mutant allele.

[0070] As another non-limiting example, if an individual is known to have a wild-type and mutant allele for each of two genetic loci on the same gene, then phasing information is useful. The phasing information will reveal whether the two copies of the gene each have one mutant allele, or whether one copy of the gene has two mutations while the other copy is wild-type for both alleles.

[0071] In certain embodiments, the present disclosure especially proposes the various methods for phase separation of genetic loci on nucleic acid templates. As a non-limiting example, the present disclosure proposes a method for phase separation of a genetic locus (such as a genetic damage (such as an inversion, fusion, deletion, insertion or other mutation)) and another genetic locus (such as a SNP) on a chromosome; The two genetic loci can be in the same gene or in different genes.

[0072] In a non-limiting example, an example patient may have Huntington's disease, which is linked to a mutation in the huntingtin gene (HTT) that comprises an excess of repeats (e.g., a repeat expansion) of the sequence CAG. In some embodiments, it may be considered to treat the patient with an allele-specific therapeutic (e.g., an antisense oligonucleotide or an RNAi agent) that recognizes a specific allelic variant of a genetic locus in the HTT gene (outside of the repeat expansion), such as, for example, a SNP. If phasing reveals that the same chromosome of the patient contains both a repeat expansion and a specific allelic variant of a genetic locus (e.g., a SNP) recognized by the allele-specific therapeutic, then the patient is eligible for treatment with the allele-specific therapeutic.

[0073] Various methods for phasing are known in the art, including but not limited to those described in 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: Longrange 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 RelatedIndividuals[Read-based phasing of related individuals]; Hickey et al. 2011 Genet. Select. Evol. [Genetic Selection Evolution] 43:12; Kuleshov et al. 2014 Nat. Biotech. [Nature Biotechnology] 32:261-266; Laver et al. 2016 Nature Scientific Reports [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. [American Journal of Human Genetics] 89:382-397; and Yang et al. 2013 Bioinformatics [Bioinformatics] 29:2245-2252. In some embodiments, sequencing, particularly sequencing that can generate long single reads, can be used for phasing.

[0074] An example of a phased assay that can be suitable for detecting mutant alleles in patients is allele-specific PCR or allele-specific long-range PCR, which can be performed using allele-specific primers to detect mutations in nucleic acid sequences in the presence of wild-type variants of the sequence. Allele-specific PCR is a technique in which selective amplification and detection are present in variants of nucleic acid sequences in PCR reaction mixtures. Allele-specific PCR employs at least one "allele-specific primer." The term "allele-specific" primer generally refers to a primer that extends in a PCR reaction only when a specific variant of a nucleic acid sequence is present in a reaction mixture. In other words, allele-specific primers are designed in such a way that they distinguish variants of nucleic acid and selectively amplify a nucleic acid template comprising a variant to be detected.

[0075] In some embodiments, the disclosure relates to a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 150 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of symptoms of Huntington's disease is reduced.

[0076] In some embodiments of the methods described herein, nucleic acid sequences are detected by suitable methods, such as quantitative amplification and / or nucleic acid sequencing. Methods for quantitative amplification are disclosed in, for example, U.S. Patent Nos. 5,210,015; 5,804,375; 6,127,155; 6,180,349; 6,033,854; and 5,972,602, and in, 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 sequencing or other older nucleotide sequencing methods can be used, sequencing can be particularly efficient when high-throughput sequencing is used, such as "next-generation sequencing" methods such as HiSeq. TM , MiSeq TM or genome analyzer, SOLiD TMor Ion Torrent TM (each available from Life Technologies) and 454 TM Sequencing (from Roche Diagnostics). For example, in high-throughput sequencing, parallel sequencing reactions using multiple templates and multiple primers allow for rapid sequencing of genomes or large portions of a genome. See for example WO 03 / 004690, WO 03 / 054142, WO 2004 / 069849, WO 2004 / 070005, WO2004 / 070007, WO 2005 / 003375, WO 00 / 06770, WO 00 / 27521, WO 00 / 58507, WO 01 / 23610、WO01 / 57248、WO 01 / 57249、WO 02 / 061127、WO 03 / 016565、WO 03 / 048387、WO 2004 / 018497、WO2004 / 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, the amplicons are sequenced by one of the following methods: base incorporation methods, such as pyrosequencing (U.S. Pat. Nos. 6,274,320, 6,258,568, and 6,210,891); hydrogen ion detection (ISFET) (e.g., U.S. Pat. No. 8,262,900), or dye terminator detection (U.S. Pat. Nos. 7,835,871, 8,244,479, 8,315,817, and 8,412,467). Deep sequencing techniques and instruments (e.g., techniques and instruments capable of digital sequence readout) can also be employed. Examples of instruments include, but are not limited to, the GS instrument family (454 Life Sciences, Branford, Conn.); the ION PROTON * and PGM TM (Life Technologies, Grand Island, NY); HISEQ * and MISEQ *Or any improvements and modifications thereof. In some embodiments, the sequencing technology is or includes long read sequencing. In some embodiments, 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.).

[0077] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 168 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0078] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 30 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0079] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 60 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of symptoms of Huntington's disease is reduced.

[0080] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the steps of administering WVE-003 (or a salt form thereof) to the subject at a dose of about 90 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of symptoms of Huntington's disease is reduced.

[0081] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 120 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0082] In some embodiments, the disclosure relates to a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 150 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of symptoms of Huntington's disease is reduced.

[0083] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 168 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0084] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 30 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0085] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 60 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of symptoms of Huntington's disease is reduced.

[0086] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the steps of administering WVE-003 (or a salt form thereof) to the subject at a dose of about 90 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of symptoms of Huntington's disease is reduced.

[0087] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 120 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0088] In some embodiments, the disclosure relates to a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 150 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of symptoms of Huntington's disease is reduced.

[0089] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 168 mg, such that the subject's disease progression is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0090] In some embodiments, the disclosure relates to a method of delaying the onset of Huntington's disease and / or reducing the severity of symptoms of Huntington's disease in a subject having Huntington's disease, wherein the subject has a mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the step of administering WVE-003 (or a salt form thereof) to the subject at a dose of about 30 mg.

[0091] In some embodiments, the disclosure relates to a method of delaying the onset of Huntington's disease and / or reducing the severity of symptoms of Huntington's disease in a subject having Huntington's disease, wherein the subject has a mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the step of administering WVE-003 to the subject at a dose of about 60 mg.

[0092] In some embodiments, the disclosure relates to a method of delaying the onset of Huntington's disease and / or reducing the severity of symptoms of Huntington's disease in a subject having Huntington's disease, wherein the subject has a mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the step of administering WVE-003 (or a salt form thereof) to the subject at a dose of about 90 mg.

[0093] In some embodiments, the disclosure relates to a method of delaying the onset of Huntington's disease and / or reducing the severity of symptoms of Huntington's disease in a subject having Huntington's disease, wherein the subject has a mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the step of administering WVE-003 (or a salt form thereof) to the subject at a dose of about 120 mg.

[0094] In some embodiments, the disclosure relates to a method of delaying the onset of Huntington's disease and / or reducing the severity of symptoms of Huntington's disease in a subject having Huntington's disease, wherein the subject has a mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the step of administering WVE-003 (or a salt form thereof) to the subject at a dose of about 150 mg.

[0095] In some embodiments, the disclosure relates to a method of delaying the onset of Huntington's disease and / or reducing the severity of symptoms of Huntington's disease in a subject having Huntington's disease, wherein the subject has a mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the step of administering WVE-003 (or a salt form thereof) to the subject at a dose of about 168 mg.

[0096] As described herein, the amount of WVE-003 typically includes all forms of WVE-003 administered, but that amount that is fully converted to the acid form.

[0097] In some embodiments, the disclosure relates to the method of any one of the preceding embodiments, wherein the subject is administered a steroid at least about one month prior to the first dose of WVE-003.

[0098] In some embodiments, the disclosure relates to the method of any one of the preceding embodiments, wherein WVE-003 is administered to the subject approximately monthly for at least about 4 months.

[0099] In some embodiments, the disclosure relates to: the method of any one of the preceding embodiments, wherein WVE-003 is administered to the subject approximately monthly for at least about 8 months.

[0100] In some embodiments, the disclosure relates to: the method of any one of the preceding embodiments, wherein the oligonucleotide is administered to the subject approximately monthly for at least about 12 months.

[0101] In some embodiments, the disclosure relates to: the method of any one of the preceding embodiments, wherein the oligonucleotide is administered to the subject approximately monthly for at least about 16 months.

[0102] In some embodiments, the disclosure relates to: the method of any one of the preceding embodiments, wherein the oligonucleotide is administered to the subject approximately monthly for at least about 48 months.

[0103] In some embodiments, the disclosure relates to: the method of any one of the preceding embodiments, wherein the oligonucleotide is delivered intrathecally.

[0104] In some embodiments, the disclosure relates to: a method as described in any of the preceding embodiments, wherein the method further comprises a step of confirming that the subject has a mutation in the HTT gene, which mutation is suitable for allele-specific knockdown of the mutant HTT gene or its gene product transcript.

[0105] In some embodiments, the disclosure relates to: the method of any one of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation.

[0106] In some embodiments, the method includes a lyophilization step (eg, freeze drying or freeze drying under vacuum).

[0107] In some embodiments, the disclosure relates to the method of any one of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation reconstituted from a lyophilized (eg, freeze-dried) preparation of the oligonucleotide.

[0108] In some embodiments, the disclosure relates to a lyophilized (eg, freeze-dried) formulation of WVE-003.

[0109] In some embodiments, the disclosure relates to: the method of any one of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation, wherein the liquid formulation comprises the oligonucleotide, sodium chloride, and water.

[0110] In some embodiments, the disclosure relates to the method of any one of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation, wherein the liquid formulation is reconstituted from a lyophilized formulation with sodium chloride solution.

[0111] In some embodiments, the disclosure relates to the method of any one of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation, wherein the liquid formulation is reconstituted from a lyophilized formulation with 0.9% sodium chloride solution.

[0112] In some embodiments, the disclosure relates to the method of any one of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation, wherein the liquid formulation is reconstituted from a lyophilized formulation with sterile sodium chloride solution.

[0113] In some embodiments, the disclosure relates to the method of any one of the preceding embodiments, wherein the oligonucleotide is formulated as a liquid formulation, wherein the liquid formulation is reconstituted from a lyophilized formulation with 0.9% sterile sodium chloride solution.

[0114] In some embodiments, the lyophilized formulation of WVE-003 is a dry (or desiccated) solid.

[0115] In some embodiments, the lyophilized formulation of WVE-003 is a dry powder.

[0116] In some embodiments, the lyophilized formulation of WVE-003 is a dry powder prepared by lyophilizing a liquid formulation of WVE-003 in water.

[0117] In some embodiments, the lyophilized formulation of WVE-003 is a dry powder in single-use clear glass vials.

[0118] In some embodiments, the lyophilized formulation of WVE-003 is approximately 20 mg of dry powder in a vial.

[0119] In some embodiments, the lyophilized formulation of WVE-003 is a dry powder in 10 mL vials.

[0120] In some embodiments, the lyophilized formulation of WVE-003 is approximately 20 mg of dry powder in a 10 mL vial.

[0121] In some embodiments, the subject is administered a steroid prior to the first dose of WVE-003.

[0122] In some embodiments, the subject is administered a steroid at least about one month prior to the first dose of WVE-003.

[0123] In some embodiments, a dose of WVE-003 is administered to the subject about once a month or about every four weeks.

[0124] In some embodiments, a dose of WVE-003 is administered to the subject about once every two months or about every eight weeks.

[0125] In some embodiments, the subject is administered a dose of WVE-003 about once a month or about every four weeks, except that the time between the first and second doses is about 2 months or about 8 weeks, and each subsequent dose is administered about once a month or about every four weeks.

[0126] In some embodiments, the subject is administered a dose of WVE-003 about once every two months or about every 8 weeks, except that the time between the first and second doses is about 3 months or about 12 weeks, and each subsequent dose is administered about once every two months or about every 8 weeks.

[0127] In some embodiments, WVE-003 is administered to the subject approximately every 8 weeks for at least about 16 weeks.

[0128] In some embodiments, WVE-003 is administered to the subject approximately every 12 weeks for at least about 12 weeks.

[0129] In some embodiments, WVE-003 is administered to the subject approximately monthly for at least about 4 months.

[0130] In some embodiments, WVE-003 is administered to the subject approximately monthly for at least about 8 months.

[0131] In some embodiments, WVE-003 is administered to the subject approximately monthly for at least about 12 months.

[0132] In some embodiments, WVE-003 is administered to the subject approximately monthly for at least about 16 months.

[0133] In some embodiments, WVE-003 is administered to the subject approximately monthly for at least about 48 months.

[0134] In some embodiments, the subject is administered a steroid prior to the first dose of the HTT oligonucleotide or HTT oligonucleotide composition.

[0135] 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.

[0136] In some embodiments, hydrocortisone and / or acetaminophen is administered to the subject within 24 hours of administration of the HTT oligonucleotide or HTT oligonucleotide composition.

[0137] In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is administered to the subject approximately monthly for at least about 8 months.

[0138] In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is administered to the subject approximately monthly for at least about 12 months.

[0139] In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is administered to the subject approximately monthly for at least about 16 months.

[0140] In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is administered to the subject approximately monthly for at least about 48 months.

[0141] In some embodiments, the method further comprises the step of confirming that the subject has a mutation in the HTT gene suitable for an allele-specific reduction in the level, expression and / or activity of the mHTT transcript.

[0142] In some embodiments, the method further comprises the step of confirming that the subject has a mutation in the HTT gene that is suitable for allele-specific knockdown of the mutant HTT transcript.

[0143] 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.

[0144] In some embodiments, the present disclosure provides a pharmaceutical composition comprising an HTT oligonucleotide or HTT oligonucleotide composition (e.g., WVE-003) disclosed herein, which can be used in a pharmaceutical composition by combining such an oligomeric compound with a suitable pharmaceutically acceptable diluent or carrier. Pharmaceutically acceptable diluents include phosphate buffered saline (PBS). PBS is a diluent suitable for use in compositions to be delivered parenterally. Therefore, in certain embodiments, employed in the methods described herein is a pharmaceutical composition comprising an HTT oligonucleotide or HTT oligonucleotide composition (e.g., WVE-003) disclosed herein 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).

[0145] In certain embodiments, pharmaceutical compositions are administered directly into the CSF (eg, IT and / or ICV injection and / or infusion) and systemically.

[0146] In some embodiments, the present disclosure provides a method for allele-specific knockdown of a target HTT transcript, the method comprising the step of 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 the step of administering an HTT oligonucleotide composition of the present disclosure. A method for treating Huntington's disease, the method comprising the step of administering a composition described in the present disclosure to a subject susceptible to or suffering from Huntington's disease.

[0147] In some embodiments, the present disclosure provides a method for treating Huntington's disease, comprising the step of administering to a subject susceptible to or suffering from Huntington's disease a composition comprising any of the HTT oligonucleotides disclosed herein.

[0148] In some embodiments, the present disclosure provides a method for treating Huntington's disease, the method comprising (a) administering a composition comprising any of the HTT oligonucleotides disclosed herein to a subject susceptible to Huntington's disease or suffering from Huntington's disease, and (b) administering to the subject an additional treatment that can prevent, treat, ameliorate Huntington's disease, or slow the progression of Huntington's disease. In some embodiments, the present disclosure provides a method comprising administering WVE-003 to a subject, wherein the subject is determined to have a genetic sequence that is identical or completely complementary to the base sequence of WVE-003. In some embodiments, the present disclosure provides a method comprising administering WVE-003 to a subject, wherein the subject is determined to have a genetic sequence that comprises a sequence that is identical or completely complementary to the base sequence of WVE-003 and a sequence that is or encodes an expanded CAG repeat. In some embodiments, the present disclosure provides a method comprising administering WVE-003 to a subject, wherein the subject is determined to have a genetic sequence that encodes a transcript that comprises an expanded CAG repeat in HTT and is completely complementary to the base sequence of WVE-003. In some embodiments, the present disclosure provides a method comprising administering WVE-003 to a subject, wherein the subject is determined to express an HTT transcript comprising an expanded CAG repeat and being completely complementary to the base sequence of WVE-003.

[0149] 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.

[0150] In some embodiments, in any of the methods described herein, the level, expression and / or activity of mutant HTT (eg, transcript and / or gene product thereof) is reduced by at least about 5%.

[0151] In some embodiments, in any of the methods described herein, the level, expression and / or activity of mutant HTT (eg, transcript and / or gene product thereof) is reduced by at least about 10%. BRIEF DESCRIPTION OF THE DRAWINGS

[0152] Figure 1 .Exemplary flow chart of the WVE-003 drug substance manufacturing process including process controls.

[0153] Figure 2 An exemplary manufacturing process flow chart for the WVE-003 drug product. Abbreviations: DS: drug substance; IPC: in-process control; IPT: in-process testing.

[0154] Figure 3Exemplary schematic diagram of the study design. Abbreviations: DEC = Dose Escalation Committee; MAD: Multiple Ascending Dose; N: Number of patients; PxCx: x Period x Cohort; SAD: Single Ascending Dose. aIf P1C2 is not performed, 6 new patients (2:1 active:placebo group) will be enrolled in P2C1.

[0155] Figure 4 Blinded CSF PK data comparing doses of WVE-003 with doses of WVE-120101 (WV-1092) and WVE-120102 (WV-2603).

[0156] Figure 5 The provided technology can reduce mHTT protein levels in human subjects without reducing wtHTT protein levels. Shown are certain percentage changes in CSF mHTT and wtHTT protein relative to baseline by day 85 in certain subjects who were administered a single dose of placebo (aCSF), WVE-003 30 mg, or WVE-003 60 mg, respectively. As demonstrated, in several human subjects, single doses of 30 mg and 60 mg of WVE-003 resulted in a persistent reduction in mHTT (A) without reducing wtHTT protein levels (B). For the combined single 30 mg and 60 mg doses, an average reduction of 22% (30% median reduction) in mHTT was observed (C), while no reduction in wtHTT was observed (D). Using a mixed model for repeated measures, the geometric mean ratio to baseline is presented. For each time point, two bars (representing 95% CI) are presented: a solid line for WVE-003 (30 mg or 60 mg) and a dotted line for placebo.

[0157] Figure 6 . Schematic diagram of the study design.

[0158] definition

[0159] As used herein, unless otherwise indicated, the following definitions shall apply. For the purposes of this disclosure, chemical elements are identified according to the Periodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 75th edition. Additionally, the general principles of organic chemistry are described in "Organic Chemistry", Thomas Sorrell, University Science Books, Sausalito: 1999 and "March's Advanced Organic Chemistry", 5th edition, eds. Smith, MB and March, J., John Wiley & Sons, New York: 2001.

[0160] As used herein in this disclosure, unless the context clearly indicates otherwise, (i) the term "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 with "not limited to") and "include" (whether or not used with "not limited to") may be understood to cover the itemized components or steps, whether shown alone or with one or more other components or steps; (iv) the term "another" may be understood to mean at least one additional / second one or more; and (v) where a range is provided, the endpoints are included.

[0161] Unless otherwise indicated, the description of oligonucleotide and element thereof (for example, base sequence, sugar modification, internucleotide linkage, linkage phosphorus stereochemistry etc.) is from 5 ' to 3 '.Unless otherwise indicated, oligonucleotide as described herein can be provided and / or used with salt form (particularly, pharmaceutically acceptable salt form).As those skilled in the art will understand after reading this disclosure, in certain embodiments, oligonucleotide can be provided with salt form, but is not limited to, for example, sodium salt or potassium salt.As those skilled in the art will understand, in certain embodiments, the single oligonucleotide in compositions can be considered to have identical composition and / or structure, even in such compositions (for example, liquid compositions), especially, such oligonucleotide may be in different one or more salt forms (and when, for example, in liquid compositions, it can dissolve and oligonucleotide chain can exist in anionic form) at a specific time. For example, one skilled in the art will recognize that, at a given pH, individual internucleotide linkages along an oligonucleotide chain may be in the acid (H) form, or in one of a variety of possible salt forms (e.g., sodium salts or salts of different cations, depending on which ions may be present in the formulation or composition), and will understand that so long as their acid forms (e.g., replacing all cations, if any, with H) have the same conformation and / or structure, such individual oligonucleotides may appropriately be considered to have the same conformation and / or structure.

[0162] Approximately: As used herein, the terms "about" and "approximately" can be understood to allow for standard deviations, as would be understood by one of ordinary skill in the art. In some embodiments, the terms "about" or "approximately" in relation to numbers are generally used to include numbers that fall within 5%, 10%, 15%, 20%, 25%, or 30% of the number in either direction (greater than or less than), unless otherwise indicated or otherwise apparent from the context.

[0163] Dosing regimen: As used herein, a "dosing regimen" or "therapeutic regimen" refers to a group of unit doses (typically more than one) that are administered individually to a subject and typically administered over a period of time. In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, the dosing regimen comprises multiple doses, wherein each dose is separated from each other by a period of time of the same length; in some embodiments, the dosing regimen comprises multiple doses and at least two different time periods separating the individual doses. In some embodiments, all doses within a dosing regimen have the same unit dosage amount. In some embodiments, different doses within a dosing regimen have different amounts. In some embodiments, the dosing regimen comprises a first dose in a first dosage amount, followed by one or more additional doses in a second dosage amount that is different from the first dosage amount. In some embodiments, the dosing regimen comprises a first dose in a first dosage amount, followed by one or more additional doses in a second dosage amount that is the same as the first dosage amount.

[0164] 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 dosage suitable for administration in a therapeutic regimen that shows a statistically significant probability of achieving a controlled therapeutic effect when administered to a relevant population.

[0165] Pharmaceutically acceptable: As used herein, the phrase "pharmaceutically acceptable" refers to those compounds, materials, compositions, and / or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0166] 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 encapsulating material, which is involved in carrying or transporting the subject compound 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 injurious to the subject.

[0167] Pharmaceutically acceptable salts: As used herein, the term "pharmaceutically acceptable salts" refers to salts of compounds that are suitable for use in a pharmaceutical setting, i.e., salts that are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., within the scope of sound medical judgment, and commensurate with a reasonable benefit / risk 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 more than one acidic group, for example, provided oligonucleotides may contain two or more acidic groups (e.g., natural phosphate linkages and / or modified internucleotide linkages). In some embodiments, pharmaceutically acceptable salts (or generally, salts) of such compounds contain two or more cations, which may be the same or different. In some embodiments, in a pharmaceutically acceptable salt (or generally, a salt), each acidic group having sufficient acidity exists independently as a salt thereof (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 exists independently as a salt thereof). In some embodiments, the pharmaceutically acceptable salt of an oligonucleotide is a sodium salt of a provided oligonucleotide. In some embodiments, the 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) exists as a sodium salt (all sodium salts).

[0168] Subject: As used herein, the term "subject" or "test subject" refers to any organism to which a compound or composition provided in accordance with the present disclosure is administered, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. In some embodiments, the subject may be suffering from and / or susceptible to a disease, disorder, and / or condition.

[0169] Substantially: As used herein, the term "substantially" refers to a qualitative state of exhibiting an overall or nearly overall range or degree of a characteristic or property of interest. It will be understood by those of ordinary skill in the biological arts that biological and chemical phenomena rarely, if ever, reach completion and / or proceed to completion or achieve or avoid an absolute result. Thus, the term "substantially" is used herein to capture the inherent completeness that is potentially lacking in many biological and / or chemical phenomena.

[0170] Suffering from: An individual who is "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 of the disease, disorder, and / or condition.

[0171]

[00145] Susceptible to: An individual who is "susceptible" to a disease, disorder, and / or condition is one who has a higher risk of developing the disease, disorder, and / or condition than members of the general 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.

[0172] Systemic: As used herein, the phrases "systemic administration," "administering systemically," "peripheral administration," and "administering peripherally" have their art-understood meanings and refer to systemic administration of a compound or composition so that it enters a recipient.

[0173] Therapeutic agent: As used herein, the phrase "therapeutic agent" refers to an agent that has a therapeutic effect and / or elicits a desired biological and / or pharmacological effect when administered to a subject. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, alleviate, 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.

[0174] Therapeutically effective amount: In some embodiments, the term "therapeutically effective amount" means 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 sufficient to treat, diagnose, prevent, and / or delay the onset of a disease, disorder, and / or condition when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition. As one of ordinary skill in the art will appreciate, 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, and the like. For example, an effective amount of a compound in a formulation for treating a disease, disorder, and / or condition is an amount that alleviates, ameliorate, mitigate, 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. 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, a single dose is an infusion, which may take up to one or more hours.

[0175] Treatment: As used herein, the term "treat," "treatment," or "treating" refers to any method for partially or completely alleviating, ameliorating, alleviating, inhibiting, preventing, delaying the onset of, reducing the severity of, and / or reducing the incidence of one or more symptoms or features of a disease, disorder, and / or condition. Treatment can be administered to subjects who do not exhibit signs of a disease, disorder, and / or condition. In some embodiments, treatment can be administered to subjects who only exhibit early signs of a disease, disorder, and / or condition, for example, for the purpose of reducing the risk of developing pathology associated with the disease, disorder, and / or condition.

[0176] 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 a plurality of oligonucleotides (or nucleic acids, chirality-controlled oligonucleotides, or chirality-controlled nucleic acids) that share 1) a common base sequence, 2) a common backbone linkage pattern; 3) a common pattern of backbone chiral centers, and 4) a common backbone phosphorus modification pattern (a specific type of oligonucleotide), wherein the plurality of oligonucleotides (or nucleic acids) share the same stereochemistry at one or more chiral internucleotide linkages (chirality-controlled internucleotide linkages where the chiral linkage phosphorus is either Rp or Sp, rather than a random mixture of Rp and Sp as in achiral internucleotide linkages). The level of the plurality of oligonucleotides (or nucleic acids) in the chirality-controlled oligonucleotide composition is non-random (predetermined, controlled). Chirality-controlled oligonucleotide compositions are typically prepared by chirality-controlled oligonucleotide preparations to stereoselectively form one or more chiral internucleotide linkages (e.g., using chiral auxiliaries exemplified in the present disclosure to purposefully control stereoselectivity compared to achiral-controlled (stereorandom, non-stereoselective, racemic) oligonucleotide synthesis (such as traditional phosphoramidite-based oligonucleotide synthesis using achiral auxiliaries or chiral catalysts). The chirality-controlled oligonucleotide composition is enriched for the plurality of oligonucleotides relative to a substantially racemic preparation of oligonucleotides having a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern. In some embodiments, the chirality-controlled oligonucleotide composition comprises a plurality of oligonucleotides of a particular oligonucleotide type defined by: 1) base sequence; 2) backbone linkage pattern; 3) backbone chiral center pattern; and 4) backbone phosphorus modification pattern, wherein the chirality-controlled oligonucleotide composition is enriched for oligonucleotides of the particular oligonucleotide type relative to a substantially racemic preparation of oligonucleotides having the same base sequence, backbone linkage pattern, and backbone phosphorus modification pattern. As those of ordinary skill in the art readily appreciate, the feature of this enrichment is: compared with substantially racemic preparations, at the linkage place between the nucleotides of each chirality control, the linkage phosphorus of higher levels has desired configuration. In certain embodiments, the linkage between the nucleotides of each chirality control independently has at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% diastereomeric purity with respect to its chiral linkage phosphorus. In certain embodiments, each independently has at least 90% diastereomeric purity. In certain embodiments, each independently has at least 95% diastereomeric purity. In certain embodiments, each independently has at least 97% diastereomeric purity. In certain embodiments, each independently has at least 98% diastereomeric purity. In certain embodiments, the oligonucleotides in a variety have identical structures.In some embodiments, the oligonucleotides in a plurality have the same architecture and stereochemistry and are structurally identical.

[0177] In some embodiments, the plurality of oligonucleotides in a chirality-controlled oligonucleotide composition share the same base sequence, the same nucleobase, sugar, and internucleotide linkage modifications, if any, and independently the same stereochemistry (Rp or Sp) at the linked phosphorus chiral center of one or more chirality-controlled internucleotide linkages, although the stereochemistry of some linked phosphorus chiral centers may be different. In some embodiments, about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 5%, 10%-10% of all oligonucleotides in a chirality-controlled oligonucleotide composition share the same base sequence, the same nucleobase, sugar, and internucleotide linkage modifications, and independently the same stereochemistry (Rp or Sp) at the linked phosphorus chiral center of one or more chirality-controlled internucleotide linkages, although the stereochemistry of some linked phosphorus chiral centers may be different. , 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%) are the plurality of oligonucleotides. In some embodiments, about 0.1%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 5%-10% of all oligonucleotides in a chirality-controlled oligonucleotide composition that share 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%-100% (e.g., about 1%-100%, 5%-100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 100%-100% or more) of all oligonucleotides in a chirally controlled oligonucleotide composition that share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern. 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%-100% (e.g., about 1%-100%, 5%-10% of) all oligonucleotides in a chiral controlled oligonucleotide composition, or about 0.1%-100% (e.g., about 1%-100%, 5%-10% of) all oligonucleotides in a composition that share a common base sequence, or about 0.1%-100% (e.g., about 1%-100%) of all oligonucleotides in a composition that share a common base sequence, a common backbone linkage pattern, and a common backbone phosphorus modification pattern, or about 0.1%-100% (e.g., about 1%-100%) of 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) of all oligonucleotides in a composition that share the same architecture. -100%, 10%-100%, 20%-100%, 30%-100%, 40%-100%, 50%-100%, 60%-100%, 70%-100%, 80%-100%, 90%-100%, 95%-100%, 50%-90%, or about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 95%-100%, 50%-90%, or about 5%. In some embodiments, the percentage is at least (DP). NCI, wherein DP is a percentage selected from 85%-100%, and NCI is the number of chirality-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 diastereomeric purity of the chirality-controlled internucleotide linkage of the linked phosphorus chiral center. In some embodiments, the diastereomeric purity of the linked phosphorus chiral center of the internucleotide linkage can typically be assessed using an appropriate dimer comprising such an internucleotide linkage and two nucleoside units connected by the internucleotide linkage. In some embodiments, the plurality of oligonucleotides share the same stereochemistry at about 1-50 (e.g., about 1-10, 1-20, 5-10, 5-20, 10-15, 10-20, 10-25, 10-30, 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 is present in an amount ranging from 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%, about 5%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 95%, 100%, 50%, ... In some embodiments, the chiral internucleotide linkages are at least 0%, 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 chirality-controlled internucleotide linkage, and the composition is a fully chirality-controlled oligonucleotide composition. In some embodiments, not all chiral internucleotide linkages are chirality-controlled internucleotide linkages, and the composition is a partially chirality-controlled oligonucleotide composition.In certain embodiments, the oligonucleotide composition of chirality control comprises independent oligonucleotide or the nucleic acid type of predetermined level.For example, in certain embodiments, the oligonucleotide composition of chirality control is included in a kind of oligonucleotide type of (for example, as described above) at predetermined level.In certain embodiments, the oligonucleotide composition of chirality control comprises and exceeds a kind of oligonucleotide type, independently of one another at predetermined level.In certain embodiments, the oligonucleotide composition of chirality control comprises multiple oligonucleotide types, independently of one another at predetermined level.In certain embodiments, the oligonucleotide composition of chirality control is the composition of the oligonucleotide of oligonucleotide type, and said composition comprises the multiple oligonucleotide of the oligonucleotide type of predetermined level. DETAILED DESCRIPTION

[0178] In some embodiments, the present disclosure relates, inter alia, to oligonucleotide compositions, oligonucleotide drug products, and methods thereof for treating Huntington's disease (HD) or symptoms thereof (e.g., WVE-003, its oligonucleotide compositions, its chirality-controlled oligonucleotide compositions, or therapeutically effective amounts thereof, methods of manufacturing thereof, methods of treating thereof, etc.).

[0179] In some embodiments, the present disclosure relates to WVE-003 and compositions and methods thereof.

[0180] In some embodiments, the disclosure relates to WVE-003 or a composition thereof, wherein WVE-003 or the composition is administered according to any of the various dosing regimens described herein.

[0181] In some embodiments, the dosing regimen relates to: the amount of a single dose of an oligonucleotide, oligonucleotide composition, or chirality-controlled oligonucleotide composition, or a therapeutically effective amount of an oligonucleotide, oligonucleotide composition, or chirality-controlled oligonucleotide composition; and / or the intervals between multiple or consecutive doses thereof; and / or the total length or duration that a subject receives one or more doses thereof; and / or its specific formulation.

[0182] Huntingtin protein (HTT)

[0183] In some embodiments, the present disclosure provides technologies (e.g., oligonucleotides, compositions, methods, etc.) that relate to the Huntington's disease gene or its encoded products (transcripts, proteins (e.g., various variants of Huntington's disease), etc.). In some embodiments, the present disclosure provides technologies for treating Huntington's disease, including HTT oligonucleotides and compositions and methods thereof. In some embodiments, HTT comprises one or more mutations. In some embodiments, such mutations are associated with reduced biological function of Huntington's disease in subjects suffering from or susceptible to Huntington's disease. In some embodiments of Huntington's disease, one or both alleles of HTT are mutant.

[0184] In some embodiments, the huntingtin (HTT) gene or its product, or a variant or portion thereof, may be referred to as HTT.

[0185] In some embodiments, HTT refers to a gene or its gene product (including but not limited to nucleic acids, including but not limited to DNA or RNA, or wild-type or mutant proteins encoded thereby) from any species, which may also be referred to as: HTT, HD, IT15, huntingtin, Huntingtin or LOMARS; External ID: OMIM: 613004, MGI: 96067, Homologous gene (HomoloGene): 1593, GeneCards: HTT; Species: Homo sapiens: Entrez: 3064; Ensembl: ENSG00000197386; UniProt: P42858; RefSeq (mRNA): NM_002111; RefSeq (protein): NP_002102; Location (UCSC): Chr 4:3.04-3.24Mb; Species: Mouse: Entrez: 15194; Ensembl: ENSMUSG00000029104; UniProt: P42859; RefSeq (mRNA): NM_010414; RefSeq (protein): NP_034544; Position (UCSC): Chr 5:34.76-34.91Mb. Other HTT sequences from humans, mice, rats, monkeys, etc., including variants thereof, are readily available to those skilled in the art. In some embodiments, HTT is human or mouse HTT, which is wild type or mutant.

[0186] In some embodiments, the HTT protein is unmodified or modified. In some embodiments, the HTT protein has any one or more of the following modifications: 9N6-acetyl lysine; 176N6-acetyl lysine; 234N6-acetyl lysine; 343N6-acetyl lysine; 411 phosphoserine; 417 phosphoserine; 419 phosphoserine; 432 phosphoserine; 442N6-acetyl lysine; 640 phosphoserine; 643 phosphoserine; 1179 phosphoserine; 1199 phosphoserine; 1870 phosphoserine; and / or 1874 phosphoserine.

[0187] Without wishing to be bound by any particular theory, the present disclosure indicates that mutations in HTT (eg, CAG repeat expansions) have been reported to be key factors in diseases and disorders such as Huntington's disease.

[0188] In some embodiments, mutant HTT is named mHTT, muHTT, m HTT, mu HTT, MU HTT, etc., where m or mu represents a mutant. In some embodiments, wild-type HTT is referred to as wild-type HTT, wtHTT, wt HTT, WT HTT, WTHTT, etc., where wt represents wild type. In some embodiments, mutant HTT comprises an amplified CAG repeat region (as understood by those skilled in the art, the number of CAG repeats may vary from subject to subject; for example, in some embodiments, 36 or more CAG repeats are included). In some embodiments, mutant HTT comprises a mutant allele of one or more SNPs (an allele on the same DNA chain or chromosome as the amplified CAG repeat region). In some embodiments, in a population, it is reported that wild-type HTT often has about or no more than about 18 CAG repeats, and it is reported that mutant HTT often has about or more than about 43 CAG repeats. In some embodiments, in a population, it is reported that heterozygosity of SNP rs362273 is identified in about 71% of individuals. In some embodiments, in a population, SNP3 is reported to be associated with mHTT in approximately 40%-45% of HD patients.

[0189] In some embodiments, the mutant HTT comprises an expanded CAG repeat region and a mutant allele of a specific SNP on the same chromosome chain. In some embodiments, where the mutant allele of a specific SNP is targeted by an HTT oligonucleotide (e.g., WVE-003) and the mutant allele is on the same chromosome as the deleterious CAG repeat expansion, the oligonucleotide is capable of targeting the deleterious HTT allele and mediating allele-specific knockdown.

[0190] In some embodiments, human HTT is referred to as hHTT. In some embodiments, mutant HTT is referred to as mHTT. In some embodiments, when mice are utilized, mouse HTT may be referred to as mHTT, as will be understood by those skilled in the art.

[0191] In some embodiments, the oligonucleotide capable of mediating allele-specific knockdown of a mutant HTT gene or its gene product is WVE-003.

[0192] Huntington's disease

[0193] Compositions comprising one or more HTT oligonucleotides described herein can be used to treat Huntington's disease or a symptom thereof.

[0194] Huntington's disease (HD) is a neurodegenerative disorder reportedly caused by mutations in the HTT (huntingtin) gene. Alterations in this ubiquitously expressed single gene have been reported to lead to a progressive neurodegenerative disorder with many characteristic symptoms.

[0195] Huntington's disease is reported to be a rare, progressive neurological disorder that causes motor, cognitive, and psychiatric disabilities and is always fatal. Bates G, Tabrizi S, Jones L, (eds). Huntington's Disease, 4th ed. Oxford (UK): Oxford University Press; 2014. Because it is a genetically inherited disease, it is reported to affect multiple family members across generations. Sturrock A et al. J Geriatr Psychiatry Neurol. 2010;23(4):243-259. Although cognitive and psychiatric symptoms may reportedly develop first, the clinical diagnosis of HD is usually based on the presence of chorea, which is one of the most visually obvious symptoms of the disease. Chorea is an abnormal, involuntary movement disorder that is reported to occur in 90% of subjects and is moderate to severe in approximately 70% of these subjects. These physical symptoms are reported to occur at any age but typically appear between the ages of 30 and 50. Bates G, Tabrizi S, Jones L, (eds). Huntington's Disease, 4th ed. Oxford (UK): Oxford University Press; 2014. A physical examination, sometimes combined with a neurological examination, reportedly can determine whether the onset of the disease has begun. Life expectancy after symptom onset is reduced to approximately 15 to 20 years. Bates G, Tabrizi S, Jones L, (eds). Huntington's Disease, 4th ed. Oxford (UK): Oxford University Press; 2014; and Sturrock A et al. J Geriatr Psychiatry Neurol. 2010;23(4):243-259. As symptoms progress, individuals reportedly become increasingly dependent or completely reliant on others for care. Suicidal ideation has been reported to increase in the early stages of the disease, possibly related to loss of sensory independence, and the suicide rate in HD patients is significantly higher than in 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.

[0196] It has been reported that some symptoms of HD can be managed with medications and therapies that modulate movement disorders, such as antipsychotics and drugs that affect dopamine pathways. Sturrock A et al. J Geriatr Psychiatry Neurol. 2010;23(4):243-259.

[0197] 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 neuronal development. Dragatsis I et al. Nat Genet. 2000;26(3):300-306. Although the purpose of wtHTT in adults is reportedly not fully understood, some studies suggest 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, it has been reported that expansion of the CAG triplet repeat in the HTT gene leads to the production of mHTT protein. The accumulation of this protein has been reported to lead to a gradual loss of neurons in the brain. Sturrock A et al. J Geriatr Psychiatry Neurol. 2010;23(4):243-259. In nonclinical studies, lowering the levels of mHTT protein as measured in CSF has been reported to be therapeutic. 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. Therefore, drugs that can silence the mHTT gene transcript while leaving the wild-type allele intact may be able to slow, halt, or even reverse the course of HD. Kay C et al. Clin Genet. 2014;86(1):29-36.

[0198] In some embodiments, the HD-associated mutation is an expansion of the CAG repeat region in the HTT gene, where larger expansions are reported to lead to greater disease severity and earlier age of onset. This mutation is reported to cause various motor, mood, and cognitive symptoms, and to lead to the formation of huntingtin protein aggregates in the brain.

[0199] CAG expansions have been reported to result in the expansion of polyglutamine (poly-Q) stretches in the huntingtin protein, a 350 kDa protein (Huntington's Disease Collaborative Study Group, 1993. Cell. 72:971-83). CAG repeat expansions have been reported to be associated with Huntington's disease. Longer repeats have been reported to be associated with earlier disease onset. The reported lack of an HD phenotype in individuals missing one copy of huntingtin, or increased disease severity in those homozygous for the expansion, suggests that the mutation does not result in loss of function (Trottier et al., 1995, Nature Med., 10:104-110). Transcriptional dysregulation and loss of function of transcriptional coactivators have been reported to be involved in HD pathogenesis. Mutant huntingtin has been shown to disrupt activator-dependent transcription early in HD pathogenesis (Dunah et al., 2002. Science 296:2238-2243).

[0200] In one report, gene profiling of human blood identified 322 mRNAs that showed significantly altered expression in HD blood samples compared to normal or presymptomatic individuals. Similar substantial changes in the expression of marker genes were observed in autopsy brain samples from the caudate nucleus of HD, suggesting that upregulation of genes in blood samples reflects disease mechanisms found in the brain. Monitoring gene expression could provide a sensitive and quantitative method to monitor disease progression, especially in the early stages of the disease in animal models and human subjects (Borovecki et al., 2005, Proc. Natl. Acad. Sci. USA 102: 11023-11028).

[0201] Huntington's disease is an autosomal dominant disorder with onset generally occurring in middle age, although onset has been documented ranging from childhood to over 70 years of age. Earlier age of onset has been reported to be associated with paternal inheritance, with 70% of adolescent cases being inherited through the father.

[0202] In some embodiments, the symptoms of Huntington's disease have emotional, motor and / or cognitive components. One symptom, chorea, is a characteristic of the movement disorder and is defined as excessive spontaneous movements that are irregular in time, randomly distributed and sudden. It can range from barely perceptible to severe. Other commonly observed abnormalities include dystonia, rigidity, bradykinesia, ocular motor dysfunction, tremor, etc. Autonomic movement disorders as symptoms include fine motor incoordination, dysarthria and dysphagia. Emotional disturbances typically include depression and irritability, and cognitive components include subcortical dementia (Mangiarini et al. 1996. Cell [Cell] 87: 493-506). It is reported that changes in the HD brain are very extensive and include neuronal loss and glial degeneration, especially in the cortex and striatum (Vonsattel and DiFiglia. 1998. J. Neuropathol. Exp. Neurol. [Journal of Neuropathology and Experimental Neurology] 57: 369-384).

[0203] In some embodiments, the Huntington's disease phenotype ranges from mild HTT to severe, depending on the length of the CAG repeat expansion.

[0204] Certain information regarding HTT and HTT-related conditions, disorders or diseases and their symptoms has been reported in the scientific literature, for example, 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 Publications AU2017276286 and AU 2007210038; European Patent Publications EP 3277814 and EP 3210633; International Patent Publication No. WO2018145009; and U.S. Patent Publication No. US20180273945.

[0205] Treatment of HTT-related conditions, disorders or diseases

[0206] In some embodiments, the present disclosure provides a kind of HTT oligonucleotide, which targets HTT (for example, an HTT oligonucleotide comprising an HTT target sequence or a sequence complementary to the HTT target sequence) and guides 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 an aqueous solution, or an aCSF solution, or an aCSF solution reconstituted from a lyophilized formulation, or a sodium chloride solution, or a sodium chloride solution reconstituted from a lyophilized formulation). In some embodiments, the present disclosure provides a kind of HTT oligonucleotide, which guides target-specific knockdown of HTT mediated by RNase H and / or RNA interference.

[0207] In some embodiments, the present disclosure provides methods for preventing and / or treating conditions, disorders, or diseases associated with HTT using provided HTT oligonucleotides and compositions thereof. In some embodiments, the condition associated with HTT 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, for example, medicaments for conditions, disorders, or diseases associated with HTT. In some embodiments, the present disclosure provides oligonucleotides and compositions thereof for use in treating conditions, disorders, or diseases associated with HTT. In some embodiments, the present disclosure provides oligonucleotides and compositions thereof for use in the manufacture of medicaments for treating conditions, disorders, or diseases associated with HTT.

[0208] In some embodiments, the present disclosure provides a method for preventing, treating, or ameliorating an HTT-related condition, disorder, or disease in a subject susceptible to or 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.

[0209] In some embodiments, the present disclosure provides a method of treating or ameliorating a condition, disorder, or disease associated with HTT 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.

[0210] In some embodiments, the condition, disorder, or disease associated with HTT is Huntington's disease (HD), also known as Huntington's chorea. In some embodiments, the condition, disorder, or disease associated with HTT is juvenile HD, myeloid or Westphal variant HD.

[0211] In some embodiments, the present disclosure provides a method for reducing HTT gene expression in a cell, the method comprising: contacting the cell with an HTT oligonucleotide or a composition thereof. In some embodiments, the present disclosure provides a method for reducing the level of HTT transcripts in a cell, the method comprising: contacting the cell with an HTT oligonucleotide or a composition thereof. In some embodiments, the present disclosure provides a method for reducing the level of HTT protein in a cell, the method comprising: contacting the cell with an HTT oligonucleotide or a composition thereof. In some embodiments, the methods provided selectively reduce the level of HTT transcripts and / or products encoded thereby associated with a condition, disorder, or disease.

[0212] HTT is reported to be expressed in all cells, with the highest concentrations found in the brain and testes, and intermediate levels in the liver, heart, and lungs. In various embodiments, the cell is in the brain, testes, liver, heart, or lungs.

[0213] In some embodiments, the present disclosure provides a method for reducing HTT gene expression in a mammal in need thereof, the method comprising administering to the mammal a nucleic acid-lipid particle comprising a provided HTT oligonucleotide or composition thereof.

[0214] In some embodiments, the present disclosure provides a method for delivering an HTT oligonucleotide in vivo, the method comprising administering an HTT oligonucleotide or a composition thereof to a mammal.

[0215] In some embodiments, the mammal is a human. In some embodiments, the mammal suffers from, is afflicted with, and / or is susceptible to a condition, disorder, or disease associated with HTT.

[0216] In some embodiments, a subject or subjects suitable for treatment of a condition, disorder, or disease associated with HTT, such as Huntington's disease (HD), can be identified or diagnosed by a healthcare professional. For example, for a neurological condition, disorder, or disease, a thorough neurological examination can be performed following a physical examination. In some embodiments, the neurological examination can assess motor and sensory skills, neurological function, hearing and speech, vision, coordination and balance, mental state, and / or emotional or behavioral changes. Example 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 lifting the front of the foot and toes; hand weakness or clumsiness; muscle paralysis; muscle stiffness; involuntary shaking or writing movements (chorea); involuntary, sustained muscle contractions (dystonia); slow movements; loss of spontaneous movement; decreased posture and balance; lack of flexibility; tingling in parts of the body; electric shock sensations that follow head movements; twitching 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; tremors; unsteady gait; fatigue; memory loss; dizziness; difficulty thinking or concentrating; difficulty reading or writing; misjudging spatial relationships; disorientation; depression; anxiety; difficulty making decisions and judgments; loss of impulse control; difficulty planning and performing familiar tasks; aggression; irritability; social withdrawal; mood swings; dementia; changes in sleep habits; confusion; and / or changes in appetite.

[0217] In some embodiments, the symptoms of Huntington's disease are any of the following: accumulation of insoluble protein; accumulation of huntingtin aggregates; neuronal aggregates in the striatum; changes in the size and number of neuronal nuclear inclusions and other markers of HD; changes in the 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.

[0218] In some embodiments, the symptoms of Huntington's disease are any of the following: behavioral and neuropathological abnormalities; altered rotarod performance in test animals; decreased weight loss; altered lifespan; behavioral disturbances; mood, motor and cognitive changes or disturbances; depression; irritability; involuntary movements (choreography); choreiform movements; impaired coordination; excessive spontaneous movements that are irregularly timed, randomly distributed and sudden; bradykinesia; dystonia; seizures; rigidity; oculomotor dysfunction; tremor; fine motor incoordination; dysarthria; dysphagia; subcortical dementia; progressive dementia; or a psychiatric disorder.

[0219] In some embodiments, the oligonucleotides provided, or compositions thereof, prevent, treat, improve, or slow the progression of a condition, disorder, or disease associated with HTT, or a condition, disorder, or disease associated with HTT. In some embodiments, the oligonucleotides provided, or compositions thereof, prevent, treat, improve, or slow the progression of a condition, disorder, or disease associated with HTT, or two or more symptoms of a condition, disorder, or disease associated with HTT. In some embodiments, a subject is assessed for one or more markers and / or symptoms of a condition, disorder, or disease associated with HTT, wherein the subject is subsequently prescribed and / or administered an oligonucleotide or oligonucleotide administration as described herein (e.g., a dose or dosage regimen of an oligonucleotide). In some embodiments, a subject is assessed for one or more markers and / or symptoms of a condition, disorder, or disease associated with HTT, wherein the subject has been administered an oligonucleotide or oligonucleotide administration as described herein. In some embodiments, the condition, disorder, or disease associated with HTT is Huntington's disease.

[0220] In some embodiments, the methods of the present disclosure are used to treat Huntington's disease in a subject, wherein the method comprises 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.

[0221] In some embodiments, the subject has an allele or transcript comprising an expanded CAG repeat region that is completely complementary to the base sequence of WVE-003. In some embodiments, the HTT transcript comprising an expanded CAG repeat region is completely complementary to the base sequence of WVE-003. In some embodiments, the subject has an allele that does not contain an expanded CAG repeat region and is completely complementary to the base sequence of WVE-003. In some embodiments, the subject has an HTT allele that does not contain 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 contain an expanded CAG repeat region is not completely complementary to the base sequence of WVE-003 at rs362273.

[0222] In some embodiments, methods are provided that alleviate at least one symptom of Huntington's disease, wherein the method comprises administering to a subject a therapeutically effective amount of an HTT oligonucleotide or a pharmaceutical composition thereof.

[0223] In some embodiments, the present disclosure provides a method for treating and / or ameliorating one or more symptoms associated with a condition, disorder, or disease associated with HTT in a mammal in need thereof, the method 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 a condition, disorder, or disease associated with HTT in a mammal in need thereof, the method 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 a condition, disorder, or disease associated with HTT in a mammal in need thereof, the method 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 a condition, disorder, or disease associated with HTT in a mammal in need thereof, the method 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 a condition, disorder, or disease associated with HTT in a mammal in need thereof, the method 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 preventing or delaying the onset of a condition, disorder, or disease associated with HTT in a mammal in need thereof, the method comprising administering to the mammal a therapeutically effective amount of a nucleic acid-lipid particle comprising an HTT oligonucleotide. In some embodiments, the mammal is a human. In some embodiments, the mammal suffers from and / or is suffering from a condition, disorder, or disease associated with HTT. 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.

[0224] In some embodiments, the present disclosure relates to compositions and methods associated with 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.

[0225] 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%.

[0226] 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%.

[0227] 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%.

[0228] 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%.

[0229] 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%.

[0230] 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%.

[0231] In some embodiments, 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 8.6, 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, 31.1 2, 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 and 38.7.

[0232] In some embodiments, about 30 includes, but is not limited to, 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 .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.

[0233] In some embodiments, about 30 includes, but is not limited to, 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 .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 and 35.2.

[0234] In some embodiments, the present disclosure relates to compositions and methods associated with 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.

[0235] In some embodiments, the disclosure relates to compositions and methods associated with 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, wherein the total amount of oligonucleotides 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 substantially all of the oligonucleotides in the dose are WVE-003.

[0236] In some embodiments, the present disclosure provides highly pure WVE-003 formulations. Various techniques can be used to assess WVE-003 purity. In some embodiments, purity is assessed using Protocol A as described herein. In some embodiments, the purity of the WVE-003 formulation is about 80% or greater. In some embodiments, the purity is about 81%. In some embodiments, the purity is about 82%. In some embodiments, the purity is about 83%. In some embodiments, the purity is about 84%. In some embodiments, the purity is about 85%. In some embodiments, the purity is about 86%. In some embodiments, the purity is about 87%. In some embodiments, the purity is about 88%. In some embodiments, the purity is about 89%. In some embodiments, the purity is about 90%. In some embodiments, the purity is about 90% or greater. In some embodiments, different batches of WVE-003 are found to be, for example, about 84%-88% pure, of which a total of about 11%-16% is impurities. In some embodiments, the impurities include nx deleted sequences; n-1 deleted sequences and phosphodiester; modified full-length sequences and / or n+x added sequences. Without wishing to be bound by any particular theory, the present disclosure indicates that at least some impurities will have at least some activity (e.g., the ability to mediate allele-specific knockdown of a mutant HTT gene or gene product).

[0237] 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 an interval of about 1 month between doses). In some embodiments, multiple doses of WVE-003 are administered to a subject approximately once every 2 months (e.g., with an interval of about 2 months between doses). In some embodiments, multiple doses of WVE-003 are administered to a subject approximately once every 8 weeks (e.g., with an interval of about 8 weeks between doses). In some embodiments, multiple doses of WVE-003 are administered to a subject approximately once every 12 weeks (e.g., with an interval of about 12 weeks between doses). In some embodiments, each of the multiple doses is approximately the same, e.g., about 30 mg.

[0238] In some embodiments, multiple doses of WVE-003 are administered to a subject approximately every 4 weeks (e.g., with an interval of approximately 4 weeks between doses). In some embodiments, multiple doses of WVE-003 are administered to a subject approximately every 8 weeks (e.g., with an interval of approximately 8 weeks between doses). In some embodiments, multiple doses of WVE-003 are administered to a subject approximately every 12 weeks (e.g., with an interval of approximately 12 weeks between doses).

[0239] In some embodiments, two or more doses of WVE-003 are administered to a subject, and the interval between any two of these doses is about one month or about four weeks. In some embodiments, two or more doses of WVE-003 are administered to a subject, and the interval between any two of these doses is about two months or about eight weeks. In some embodiments, two or more doses of WVE-003 are administered to a subject, and the interval between any two of these doses is about three months or about 12 weeks.

[0240] In some embodiments, two or more doses of WVE-003 are administered to a subject approximately once a month or approximately every 4 weeks. In some embodiments, two or more doses of WVE-003 are administered to a subject approximately once every 2 months or approximately every 8 weeks. In some embodiments, two or more doses of WVE-003 are administered to a subject approximately once every 3 months or approximately every 12 weeks.

[0241] In some embodiments, WVE-003 is administered to a subject approximately monthly for at least about 2 months. In some embodiments, WVE-003 is administered to a subject approximately once every two months for at least about 2 months.

[0242] In some embodiments, WVE-003 is administered to a subject approximately monthly for at least about 4 months. In some embodiments, WVE-003 is administered to a subject approximately once every two months for at least about 4 months.

[0243] In some embodiments, WVE-003 is administered to a subject approximately monthly for at least about 8 months. In some embodiments, WVE-003 is administered to a subject approximately once every two months for at least about 8 months.

[0244] In some embodiments, WVE-003 is administered to a subject approximately once every 4 weeks for at least about 8 weeks. In some embodiments, WVE-003 is administered to a subject approximately once every 8 weeks for at least about 16 weeks.

[0245] In some embodiments, WVE-003 is administered to a subject approximately once every 4 weeks for at least about 12 weeks. In some embodiments, WVE-003 is administered to a subject approximately once every 12 weeks for at least about 12 weeks.

[0246] In some embodiments, WVE-003 is administered to a subject approximately once every 4 weeks for at least about 16 weeks. In some embodiments, WVE-003 is administered to a subject approximately once every 8 weeks for at least about 16 weeks.

[0247] In some embodiments, WVE-003 is administered to a subject approximately once every 8 weeks for at least about 24 weeks. In some embodiments, WVE-003 is administered to a subject approximately once every 12 weeks for at least about 24 weeks.

[0248] In some embodiments, administration of an HTT oligonucleotide to a subject or patient can mediate any one or more of the following: 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 time or lifespan of the subject or patient.

[0249] In some embodiments, slowing disease progression involves preventing or delaying clinically undesirable changes in one or more clinical parameters in an individual suffering from and / or susceptible to HD, such as those described herein. It is well within the ability of a physician to identify a slowing of disease progression in an individual suffering from HD using one or more disease assessment tests described herein. Additionally, it should be understood that a physician may perform diagnostic tests on an individual in addition to those described herein to assess the rate of disease progression in an individual suffering from HD.

[0250] 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 unfavorable for HD. A physician can use a family history of HD or comparison with other HD patients (e.g., subjects or humans currently receiving or requiring HD treatment) with similar genetic characteristics (e.g., number of CAG repeats) to determine the approximate age at which HD onset is expected to occur to determine whether HD onset is delayed.

[0251] In some embodiments, indicators of HD include parameters used by medical professionals (such as physicians) to diagnose or measure the progression of HD and include, but are not limited to, genetic testing, hearing, eye movements, strength, coordination, chorea (rapid, jerky, involuntary movements), sensation, reflexes, balance, movement, mental status, dementia, personality disorders, family history, weight loss, and caudate nucleus degeneration. Degeneration of the caudate nucleus is assessed by brain imaging techniques such as magnetic resonance imaging (MRI) or computed tomography (CT) scans.

[0252] In some embodiments, improvement in an HD indicator involves the absence of an undesirable change or the presence of a desired change in one or more HD indicators. In one embodiment, improvement in an HD indicator is demonstrated by the absence of a measurable change in one or more HD indicators. In another embodiment, improvement in an HD indicator is demonstrated by a desired change in one or more HD indicators.

[0253] In some embodiments, the reduction in disease progression can further include an increase in the survival time of individuals suffering from and / or susceptible to HD. In some embodiments, the increase in survival time relates to an average increase in the survival of individuals suffering from and / or susceptible to HD relative to an approximate survival time based on HD progression and / or HD family history. A physician can use one or more disease assessment tests described herein to predict the approximate survival time of an individual suffering from and / or susceptible to HD. A physician can additionally use the family history of an individual suffering from and / or susceptible to HD or a comparison with other HD patients with similar genetic characteristics (e.g., number of CAG repeats) to predict expected survival time.

[0254] In some embodiments, the present disclosure provides a method for inhibiting HTT expression in a cell, the method comprising: (a) contacting the cell with an HTT oligonucleotide; and (b) maintaining the resulting cell in step (a) for a period of time sufficient to obtain degradation of the mRNA transcript 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%.

[0255] In some embodiments, the present disclosure provides a method of treating a condition, disorder, or disease mediated by HTT expression, comprising administering a therapeutically effective amount of an HTT oligonucleotide or a composition thereof to a human suffering from the condition, disorder, or disease. In some embodiments, administration results in a decrease in the expression, activity, and / or level of an HTT transcript. In some embodiments, administration is associated with a decrease in the expression, activity, and / or level of an HTT transcript. In some embodiments, administration is followed by a decrease in the expression, activity, and / or level of an HTT transcript.

[0256] In some embodiments, the present disclosure provides an HTT oligonucleotide for use in a subject to treat a condition, disorder, or disease associated with HTT. In some embodiments, the condition, disorder, or disease associated with HTT is Huntington's disease.

[0257] In some embodiments, the methods provided reduce the amount and / or percentage of mHTT protein. In some embodiments, the methods provided reduce the amount and / or percentage of mHTT protein in CSF. In some embodiments, the methods provided increase the percentage of wtHTT protein. In some embodiments, the methods provided increase the percentage of wtHTT protein in CSF. In some embodiments, the methods provided 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, the methods provided do not reduce the amount of wtHTT protein, or do not reduce the amount of wtHTT protein in CSF 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 assessed 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 each independent dose or doses as described herein. Certain useful techniques for assessing mHTT, wtHTT, and / or total HTT protein are described in WO 2022 / 046723 and incorporated herein by reference.

[0258] In some embodiments, an oligonucleotide, such as an HTT oligonucleotide (e.g., WVE-003), or a salt form thereof, or a composition thereof, and additional reagents and / or methods, such as additional therapeutic agents and / or methods, are administered to a subject. In some embodiments, the oligonucleotide or a composition thereof can be administered alone or in combination with one or more other therapeutic agents and / or treatments. When administered in combination, each component can be administered simultaneously or sequentially at different time points in any order. In some embodiments, each component can be administered separately but sufficiently closely in time to provide 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 composition. In some embodiments, at a certain time point, the subject to be administered is exposed to the provided oligonucleotide and the additional component simultaneously.

[0259] Allele-Specific Knockdown of Mutant HTT Transcripts as a Treatment for Huntington's Disease In some embodiments, treatment of Huntington's disease comprises 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 treating Huntington's disease comprising the step of administering an HTT oligonucleotide, or a composition comprising an HTT oligonucleotide, to a subject suffering from or susceptible to the disease. In particular, and especially, the present disclosure demonstrates that chirally-controlled oligonucleotides / chirally-controlled oligonucleotide compositions are unexpectedly effective in modulating allele-specific knockdown of mutant HTT transcripts compared to otherwise identical achiral controlled oligonucleotides / oligonucleotide compositions. In some embodiments, the term chirally-controlled may be equivalent to stereopure or stereodefined.

[0260] In some embodiments, treatment of Huntington's disease comprises the use of an HTT oligonucleotide, wherein the oligonucleotide is capable of providing allele-specific knockdown of a mutant HTT transcript. In some embodiments, the HTT oligonucleotide is capable of mediating allele-specific knockdown of a mutant HTT transcript comprising a mutation (e.g., a CAG repeat expansion), wherein reduction in the level, expression, and / or activity of mHTT can treat, prevent, and / or ameliorate Huntington's disease or a symptom thereof, and / or reduce the severity of Huntington's disease symptoms or delay their onset.

[0261] In some embodiments, the composition comprising HTT oligonucleotides can be used to treat huntingtin-related disorders 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, wherein the method includes administering a therapeutically effective amount of HTT oligonucleotides to a subject suffering from a huntingtin-related disorder of the central nervous system. In some embodiments, the HTT oligonucleotides are administered outside the central nervous system (as a non-limiting example, intrathecally or intramuscularly) to a subject suffering from and / or susceptible to a huntingtin-related disorder of the central nervous system, and the HTT oligonucleotides are able to enter the central nervous system through the blood-brain barrier. In some embodiments, the HTT oligonucleotides are administered directly to the central nervous system (as a non-limiting example, delivered via intrathecal, intraventricular, intracranial, etc.).

[0262] In some embodiments, in HTT subjects, the HTT gene or transcript has a CAG repeat expansion.

[0263] In some embodiments, the HTT subject has a CAG repeat expansion.In some embodiments, allele-specific knockdown of a mutant HTT transcript can be used to remove or reduce the effects of such a mutation by reducing the level, expression, and / or activity of the protein expressed by the mHTT transcript.

[0264] In some embodiments, prior to administering a composition comprising an HTT oligonucleotide, a subject with HTT or a subject suspected of having HTT is analyzed for HTT genotype.

[0265] In some embodiments, prior to administering a composition comprising an HTT oligonucleotide, the HTT phenotype of the HTT subject or a subject suspected of having HTT is analyzed.

[0266] In some embodiments, the genotype and phenotype of an HTT subject are analyzed to determine the relationship between the HTT genotype and the HTT phenotype prior to administering a composition comprising an HTT oligonucleotide.

[0267] In some embodiments, prior to administering a composition comprising an HTT oligonucleotide, the subject is genetically confirmed to have Huntington's disease.

[0268] In some embodiments, analysis of a subject's HTT genotype or genetic verification of mHTT comprises determining whether the subject has one or more deleterious mutations in HTT.

[0269] In some embodiments, analysis of a subject's HTT genotype or genetic verification of mHTT includes determining whether the subject has a CAG repeat expansion and a SNP targeted by a specific HTT oligonucleotide on the same chromosome; in some embodiments, such analysis is referred to as phasing.

[0270] In certain embodiments, the target nucleic acid sequence and the reference nucleic acid sequence are different at one or more sites, such as mutation sites, single nucleotide polymorphism (SNP) sites, etc. In certain embodiments, the target nucleic acid sequence and the reference nucleic acid sequence are included in the difference at the SNP site. In certain embodiments, the site in the target nucleic acid is fully complementary to the site in the oligonucleotide of the present disclosure, while the corresponding site in the reference nucleic acid is not.

[0271] In some embodiments, analysis of HTT genotype or genetic verification of mHTT informs the selection of compositions comprising HTT oligonucleotides that can be used therapeutically.

[0272] In some embodiments, an abnormal or mutant HTT gene or portion thereof is removed or replicated from a patient or one or more cells or one or more tissues of the patient, and the abnormal or mutant HTT gene or portion thereof comprising the abnormality or mutation, or a copy thereof, is inserted into a cell. In some embodiments, this cell can be used to test various compositions comprising an HTT oligonucleotide to predict whether such a composition can be used to treat a subject. In some embodiments, the cell is a myoblast or a myotubule.

[0273] In some embodiments, prior to a subject receiving treatment with an HTT oligonucleotide, phasing is performed on the subject's genome to determine whether the CAG repeat expansion is on the same chromosome as the SNP targeted by the HTT oligonucleotide capable of mediating allele-specific knockdown.

[0274] In some embodiments, the HTT oligonucleotide capable of mediating allele-specific knockdown of a mutant HTT gene or its gene product and useful in methods of treating Huntington's disease is WVE-003.

[0275] WVE-003

[0276] In some embodiments, the present disclosure provides WVE-003 and its formulations and compositions. In some embodiments, the present disclosure provides techniques for manufacturing WVE-003. In some embodiments, the present disclosure provides techniques for evaluating and / or characterizing WVE-003. In some embodiments, the present disclosure provides techniques for evaluating the purity of WVE-003, such as Scheme A. In some embodiments, the present disclosure provides techniques for confirming the stereochemical properties of WVE-003 (or its stereoisomers (e.g., with respect to chiral bonded phosphorus)) using, for example, IP-RP-UPLC (e.g., according to Scheme B). In some embodiments, the present disclosure provides techniques for evaluating the stereopurity of WVE-003 using, for example, IP-RP-UPLC (e.g., according to Scheme B), dimer modeling, etc. In some embodiments, the present disclosure provides methods for using WVE-003. For example, in some embodiments, the present disclosure provides methods for treating Huntington's disease, comprising administering an amount of WVE-003 as described herein to a subject suffering from Huntington's disease.

[0277] In some embodiments, the therapeutic use of oligonucleotides includes modulating the function of target mHTT RNA to reduce the production of disease-associated 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, modification of phosphodiester linkages (e.g., using phosphorothioate linkages) improves the stability, biodistribution, and cellular uptake of oligonucleotides. The use of chiral internucleotide linkages in oligonucleotide synthesis can produce chiral centers at the linkage phosphorus. The chiral linkage phosphorus center can have an "Sp" or "Rp" configuration. Conventional stereorandom formulations of oligonucleotides containing n chiral linkage phosphorus are 2 relative to the chiral linkage phosphorus center. n A mixture of stereoisomers wherein each stereoisomer shares the same composition but differs in stereochemistry along its backbone. For an oligonucleotide in which there are 17 chiral linked phosphorus centers, a stereorandom formulation can have over 131,000 (2 17) stereoisomers, each of which is present at very low levels (about 1 / 131000).

[0278] In contrast to stereorandom preparations, WVE-003 utilized herein is prepared stereoselectively. As described herein, in some embodiments, each chiral internucleotide linkage independently forms with a diastereoselectivity of about 97% or greater (e.g., as measured by a suitable dimer preparation). In some embodiments, a majority of chiral internucleotide linkages independently form 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 independently form 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, the overall diastereoselectivity is about 81% or greater. In some embodiments, the overall diastereoselectivity is about 82% or greater. In some embodiments, the overall diastereoselectivity is about 83% or greater. In some embodiments, the overall diastereoselectivity is about 84% or greater. In some embodiments, the overall diastereoselectivity is about 85% or greater.

[0279] In some embodiments, the oligonucleotide is WVE-003, which is a stereo-defined oligonucleotide that can selectively target mHTT, leaving wtHTT relatively unaffected. WVE-003 can specifically target the mHTT mRNA transcript at the A variant of SNP rs362273 (SNP3). A SNP is a single variation that can be associated with a mutated gene in some cases. One of the most common SNPs in the mHTT gene is SNP3, which has been reported to be present 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. By selectively targeting the SNP3 variant associated with pathogenic CAG expansions (≥36 repeats), administration of WVE-003 can result in a selective reduction in mHTT protein levels.

[0280] In some embodiments, WVE-003 is used as a disease-modifying agent for treating a subject with Huntington's disease (HD). In some embodiments, WVE-003 is a stereopure antisense oligonucleotide (ASO) that selectively targets mutant forms of the huntingtin (mHTT) gene transcript.

[0281] In some embodiments, the HTT oligonucleotide or a salt thereof is WVE-003 or a salt thereof. In some embodiments, the oligonucleotide composition comprises WVE-003 or a salt thereof.

[0282] The base sequence of WVE-003 is 5'-GUUGATCTGTAGCAGCAGCT-3'.

[0283] In some embodiments, WVE-003 can be described as: 5'-mG*SmUn001RmUmGn001RmA*ST*SC*ST*SG*ST*RA*SG*SC*SA*SG*Rm5Ceon001RAeoGeon001Rm5Ceo*STeo-3', wherein:

[0284] *S represents Sp phosphorothioate linkage;

[0285] *R represents Rp phosphorothioate linkage;

[0286] mX represents 2'-O-methylribonucleoside;

[0287] X represents 2'-deoxyribonucleoside;

[0288] Xeo stands for 2'-O-(2-methoxyethyl)ribonucleoside;

[0289] m5Ceo stands for 2′-O-(2-methoxyethyl)-5-methylcytidine;

[0290] n001R represents an Rp N-(1,3-dimethylimidazolidin-2-ylidene)phosphoramidate diester linkage (PN); and

[0291] "*R", "*S" or "n001R" between nucleosides does not represent a natural phosphate linkage (PO).

[0292] In some embodiments, WVE-003 is in the form of a sodium salt. In some embodiments, the sodium salt of WVE-003 can be described as:

[0293] [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-thioadenyl-(3′→5′)-[P(S)]-P-thiothymidyl-(3′→5′)-[P(S)]-2′-deoxy-P-thiocytidylyl-(3′→5′)-[P(S)]-P-thiothymidyl-(3′→5′)-[P(S)]-2′-deoxy-P-thioguanylyl-(3′→5′)-[P(R)]-P-thiothymidyl-(3′→5′)-[P(S)]-2′-deoxy-P-thioadenyl-(3′→5′)-[P(R)]-P-thiothymidyl-(3′→5′)-[P(S)]-2′-deoxy-P-thioadenyl-(3′→5′)-[P(R)]- 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′-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 pentasodium salt.

[0294] In some embodiments, the structure of the sodium salt of WVE-003 is as follows: As will be appreciated by those skilled in the art, in the acid form, each Na + H + The zig-zag line represents the linkage between the 3' oxygen and phosphorus in the internucleotide linkage.

[0295]

[0296] Some information about WVE-003:

[0297] Molecular formula (sodium salt form): C 236 H 308 N86 Na 15 O 118 P 19 S 13

[0298] Molecular weight (sodium salt form): 7587.67 g / mol

[0299] Molecular formula (free acid): C 236 H 323 N 86 O 118 P 19 S 13

[0300] Molecular weight (free acid): 7257.94 g / mol

[0301] WVE-003 has 19 internucleotide linkages, 2 of which are phosphodiester linkages; and 17 stereodefined internucleotide linkages, 11 of which are Sp phosphorothioate diesters, 2 of which are Rp phosphorothioate diesters, and 4 of which are Rp N-(1,3-dimethylimidazolidine-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 diesters, Rp phosphorothioate diesters, Rp N-(1,3-dimethylimidazolidine-2-ylidene) phosphoramidate diesters, and phosphodiester linkages, respectively.

[0302] In some embodiments, WVE-003 recognizes a disease-associated (e.g., mutant) allele of SNP rs362273 in the huntingtin gene, effectively reduces the level, expression and / or activity of the mHTT gene (or its gene product), and is capable of mediating allele-specific knockdown of the mutant huntingtin (mHTT) gene.

[0303] In some embodiments, the efficacy and allele specificity of the chirality-controlled WVE-003 compositions are superior to the efficacy and allele specificity of various stereorandom oligonucleotide compositions.

[0304] For additional information related to WVE-003, see, for example, WO 2020 / 227691 and WO 2021 / 071788, the entire contents of each of which are incorporated herein by reference. HTT Oligonucleotides and HTT Oligonucleotide Compositions

[0305] In some embodiments, the HTT oligonucleotide or its salt form or HTT oligonucleotide composition (including but not limited to WVE-003) can mediate an allele-specific decrease in the level, expression and / or activity of an mHTT transcript. In some embodiments, the HTT oligonucleotide or its salt form or HTT oligonucleotide composition can mediate an allele-specific knockdown of a mutant HTT transcript. In some embodiments, the HTT oligonucleotide or its salt form or HTT oligonucleotide composition can mediate an allele-specific decrease in the level, expression and / or activity of an mHTT transcript. In some embodiments, the HTT oligonucleotide or its salt form or HTT oligonucleotide composition is chirally controlled.

[0306] In some embodiments, the HTT oligonucleotide or salt form thereof or HTT oligonucleotide composition is or comprises WVE-003. In some embodiments, the HTT oligonucleotide or salt form thereof or HTT oligonucleotide composition is capable of mediating allele-specific reduction in the level, expression and / or activity of mHTT and is chirally controlled. When administered as described herein, WVE-003 is capable of mediating allele-specific knockdown of mutant HTT transcripts and potential disease-modifying therapy for Huntington's disease. In some embodiments, the HTT oligonucleotide or salt form thereof or HTT oligonucleotide composition is capable of mediating allele-specific reduction in the level, expression and / or activity of mHTT and is chirally controlled.

[0307] In some embodiments, the present disclosure provides methods of using a huntingtin (HTT) oligonucleotide or HTT oligonucleotide composition (eg, WVE-003) capable of mediating allele-specific knockdown of the HTT transcript of the HTT protein.

[0308] 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 comprising a Huntington's disease-associated mutation.

[0309] In some embodiments, the therapeutically effective amount of an HTT oligonucleotide or 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.

[0310] In some embodiments, the 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 a higher dose). In some embodiments, the 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 (e.g., when administered at a higher dose).

[0311] In certain embodiments, adverse event is an adverse effect.In certain embodiments, adverse event is slight, moderate, severe or serious adverse event.In certain embodiments, serious adverse event is more serious than being classified as severe, moderate or slight adverse event.In certain embodiments, serious adverse event is life-threatening immediately, needs hospitalization or extends existing hospitalization, causes lasting or significant disability / incapacity, or is non-existent congenital anomaly / birth defect when screening.In certain embodiments, can use hydrocortisone and / or acetaminophen treatment adverse event.

[0312] In some embodiments, a serious 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 associated with: 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.

[0313] In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition is an oligonucleotide or oligonucleotide composition that targets an HTT transcript and is capable of modulating allele-specific knockdown of a mutant HTT transcript of the target transcript. In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition can be used to prepare a medicament for treating Huntington's disease. In some embodiments, the HTT oligonucleotide or HTT oligonucleotide composition can be used to treat Huntington's disease.

[0314] In some embodiments, an HTT oligonucleotide or HTT oligonucleotide composition can be used to prepare a medicament for treating Huntington's disease, wherein the oligonucleotide is WVE-003 and the medicament is administered at a dose equivalent to about 30, about 60, about 90, about 120, about 150, or about 168 mg of WVE-003 free acid form.

[0315] In some embodiments, the present disclosure provides methods for using HTT oligonucleotides or HTT oligonucleotide compositions. In some embodiments, the HTT oligonucleotides or their salt forms or HTT oligonucleotide compositions are capable of mediating allele-specific reductions in the level, expression, and / or activity of mHTT transcripts. In some embodiments, the HTT oligonucleotides or their salt forms or HTT oligonucleotide compositions are capable of mediating allele-specific knockdown of mutant HTT transcripts. In some embodiments, the HTT oligonucleotides or their salt forms or HTT oligonucleotide compositions are capable of mediating allele-specific reductions in the level, expression, and / or activity of mHTT transcripts. In some embodiments, the HTT oligonucleotides or their salt forms or HTT oligonucleotide compositions are chirally controlled.

[0316] In some embodiments, the HTT oligonucleotide or salt form thereof or HTT oligonucleotide composition is or comprises WVE-003. In some embodiments, the HTT oligonucleotide or salt form thereof or HTT oligonucleotide composition is capable of mediating an allele-specific decrease in the level, expression and / or activity of mHTT and is chirally controlled. In some embodiments, the HTT oligonucleotide or salt form thereof or HTT oligonucleotide composition is capable of mediating an allele-specific decrease in the level, expression and / or activity of mHTT and is chirally controlled.

[0317] The present disclosure recognizes the challenges of providing low toxicity HTT oligonucleotide compositions and methods of use thereof. 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 responses. In some embodiments, the present disclosure recognizes that various toxicities induced by HTT oligonucleotides are associated with 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 that reduce complement activation via alternative pathways. In some embodiments, the present disclosure provides HTT oligonucleotide compositions and methods that reduce complement activation via traditional pathways. In some embodiments, the present disclosure provides HTT oligonucleotide compositions and methods with reduced drug-induced vascular damage. In some embodiments, the present disclosure provides HTT oligonucleotide compositions and methods with reduced injection site inflammation. In some embodiments, the reduced toxicity can be evaluated by one or more assays widely known and practiced by those of ordinary skill in the art (e.g., evaluating the level of fully activated product, protein binding, etc.).

[0318] Chirality-controlled HTT oligonucleotides and compositions thereof

[0319] In particular, the disclosure relates to chirality-controlled HTT oligonucleotides and oligonucleotides, such as WVE-003, and methods thereof.

[0320] In particular, the present disclosure encompasses the recognition that, compared to chirality-controlled HTT oligonucleotide compositions, stereorandom HTT oligonucleotide formulations contain multiple different chemical entities that differ from one another, for example, in the stereochemical structures of individual backbone chiral centers within the HTT oligonucleotide chain. Without controlling the stereochemistry of the backbone chiral centers, stereorandom HTT oligonucleotide formulations (e.g., random mixtures of diastereomers) provide uncontrolled (or stereorandom) compositions containing undetermined levels of HTT oligonucleotide stereoisomers. Even though these stereoisomers may have the same base sequence and / or chemical modifications, they are different chemical entities, at least approximately due to their different backbone stereochemistry, and they may have different properties, such as activity, toxicity, distribution, etc. In particular, the present disclosure provides chirality-controlled compositions that are or contain specific stereoisomers of a target HTT oligonucleotide (e.g., WVE-003); compared to compositions in which the chirality is not controlled, the chirality-controlled compositions contain controlled levels of specific stereoisomers of the HTT oligonucleotide. In some embodiments, the level of a particular stereoisomer of a chiral controlled oligonucleotide composition (e.g., WVE-003) is enriched as described herein (e.g., in some embodiments, each chiral internucleotide linkage independently has a stereopurity of about 97%, 98%, 99% or more).

[0321] In some embodiments, a specific stereoisomer can be defined, for example, by its base sequence, its backbone linkage pattern, its backbone chiral center pattern, and backbone phosphorus modification pattern, etc. As understood in the art, in some embodiments, base sequence can refer solely to the sequence of bases and / or to the identity and / or modification status of the nucleoside residues in the HTT oligonucleotide (e.g., the sugar and / or base components relative to standard naturally occurring nucleosides such as adenine, cytosine, guanosine, thymine, and uracil) and / or to the hybridization characteristics of such residues (i.e., the ability to hybridize to specific complementary residues). In some embodiments, the present disclosure demonstrates that the improved properties (e.g., improved activity, lower toxicity, etc.) achieved by including and / or positioning specific chiral structures within HTT oligonucleotides can be comparable to or even better than those achieved by using chemical modifications (e.g., specific backbone linkages, residue modifications, etc. (e.g., by using certain 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 HTT oligonucleotide compositions controlled by the chirality of HTT oligonucleotides (e.g., WVE-003, including various salt forms thereof) exhibit unexpectedly high ability to mediate allele-specific knockdown of mutant HTT transcripts and can be used to treat and / or prevent Huntington's disease.

[0322] In some embodiments, the composition of WVE-003 is a chirality-controlled oligonucleotide composition that is capable of mediating allele-specific reduction in the level, expression, and / or activity of mHTT transcripts. In some embodiments, the chirality-controlled WVE-003 composition is used to target human huntingtin protein pre-messenger RNA (mRNA) to induce allele-specific knockdown of mutant HTT transcripts and restoration of huntingtin protein in subjects with Huntington's disease (HTT). WVE-003 comprises a base sequence that can be antisense to the HTT transcript and hybridize thereto through complementary base pairing.

[0323] Applicants have developed technologies that enable the synthesis of linkage-modified nucleic acid therapeutics in which the stereochemistry at each chiral linkage phosphorus position is precisely controlled. This degree of control enables the rational design and synthesis of optimized, stereopure oligonucleotides with improved pharmacological and toxicological properties. WVE-003 was developed and manufactured using certain of these technologies.

[0324] In some embodiments, WVE-003 is used to target human huntingtin (HTT) pre-messenger RNA (mRNA) to induce allele-specific knockdown of mutant HTT transcripts in subjects with Huntington's disease (HTT).

[0325] In some embodiments, the present disclosure provides oligonucleotides, oligonucleotide compositions, and methods of use thereof for mediating allele-specific reduction in the level, expression, and / or activity of mHTT in HTT (eg, HTT of mouse, human, etc.).

[0326] In some embodiments, the oligonucleotide composition comprises WVE-003. In some embodiments, the oligonucleotide composition is a chirality-controlled oligonucleotide composition of WVE-003. In some embodiments, such a composition is a pharmaceutical composition of WVE-003.

[0327] As will be appreciated by those skilled in the art, an oligonucleotide (e.g., WVE-003) can be administered in one or more forms (e.g., 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, an oligonucleotide (e.g., WVE-003) is administered as a salt form (e.g., sodium salt form), optionally in solution. In some embodiments, a composition (e.g., a pharmaceutical composition, a chirally controlled oligonucleotide composition, etc.) comprises one or more forms of WVE-003. As will be appreciated by those skilled in the art, in some embodiments, the amount of an oligonucleotide (e.g., the amount of an oligonucleotide administered) is the corresponding amount of a particular form (e.g., acid form) of all forms (e.g., all forms administered (e.g., one or more salt forms (e.g., sodium salt form)). In some embodiments, multiple forms of oligonucleotides may be present in a composition.

[0328] In some embodiments, each composition comprising WVE-003 is a chirally pure or chirally controlled oligonucleotide composition of WVE-003.

[0329] In some embodiments, chirality-controlled oligonucleotide compositions are typically prepared by chirality-controlled oligonucleotide preparations to stereoselectively form one or more chiral internucleotide linkages (e.g., using chiral auxiliaries as exemplified in the present disclosure, to purposefully control stereoselectivity compared to achiral-controlled (stereorandom, non-stereoselective, racemic) oligonucleotide synthesis (such as traditional phosphoramidite-based oligonucleotide synthesis using achiral auxiliaries or chiral catalysts). The chirality-controlled oligonucleotide composition of oligonucleotides is enriched for a particular oligonucleotide (e.g., for a chirality-controlled oligonucleotide composition of WVE-003, enriched for WVE-003) relative to a substantially racemic preparation of oligonucleotides having the same base sequence and the same modifications. As will be readily understood by one of ordinary skill in the art, this enrichment is characterized by a higher level of the bonded phosphorus having a desired configuration at each chirality-controlled internucleotide linkage compared to a substantially racemic preparation. In some embodiments, each chiral-controlled internucleotide linkage independently has a diastereomeric purity of at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% relative to its chiral linkage phosphorus. 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.

[0330] As will be appreciated by those skilled in the art, a formulation typically comprises an oligonucleotide (e.g., WVE-003) and one or more other oligonucleotides and / or other impurities, as manufacturing processes rarely achieve 100% selectivity and / or purity (including but not limited to diastereomeric purity). Those skilled in the art will appreciate that the levels of such other oligonucleotides and / or impurities can and are appropriately controlled.

[0331] In some embodiments, the chirality-controlled oligonucleotide composition can consist essentially of the desired oligonucleotide (e.g., WVE-003), for example, the other oligonucleotides are impurities from the manufacture of this oligonucleotide. In some embodiments, the impurities include oligonucleotides that are similar but different from the desired oligonucleotide (e.g., one or more internucleotide linkages in the impurity can have an undesirable configuration; and / or the impurity can 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 indicates that it is possible that one or more impurities can be sufficiently similar to the desired oligonucleotide to mediate the desired activity (e.g., allele-specific knockdown of mHTT).

[0332] As described herein, in some embodiments, the purity (e.g., the purity level of WVE-003) is about or at least about 75%. In some embodiments, the purity is about or at least about 80%. In some embodiments, the purity is about or at least about 81%. In some embodiments, the purity is about or at least about 82%. In some embodiments, the purity is about or at least about 83%. In some embodiments, the purity is about or at least about 84%. In some embodiments, the purity is about or at least about 85%. In some embodiments, the purity is about or at least about 86%. In some embodiments, the purity is about or at least about 87%. In some embodiments, the purity is about or at least about 88%. In some embodiments, the purity is about or at least about 89%. In some embodiments, the purity is about or at least about 90%. In some embodiments, the purity is about or at least about 91%. In some embodiments, the purity is about or at least about 92%. In some embodiments, the purity is about or at least about 93%. In some embodiments, the purity is about or at least about 94%. In some embodiments, the purity is about or at least about 95%. In some embodiments, the purity is about or at least about 96%. In some embodiments, the purity is about or at least about 97%. In some embodiments, the purity is about or at least about 98%. In some embodiments, the purity is about or at least about 99%.

[0333] In some embodiments, the purity of WVE-003 in a formulation, composition, medicament, or the like can be determined using various suitable methods as described herein. In some embodiments, purity is assessed using chromatography with UV detection (e.g., UPLC-UV as described), and purity is measured as peak area % at a given wavelength (e.g., 260 nm). In some embodiments, purity is assessed as described in Scheme A. In some embodiments, purity is assessed as described in Scheme B. In some embodiments, purity is assessed by dimer modeling.

[0334] manufacture

[0335] In some embodiments, the present disclosure provides techniques for manufacturing oligonucleotides (e.g., WVE-003) that are particularly useful for stereoselective large-scale preparations. In some embodiments, the formulation of WVE-003 (e.g., WVE-003 API or drug product) is a solid. In some embodiments, it is a white to off-white powder. In some embodiments, the solubility of the prepared WVE-003 in water is 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 formulation in purified water is 6.0-8.0. Unless otherwise stated, 181181M is used herein. -1 cm -1The extinction coefficient is used, for example, to calculate the WVE-003 concentration / amount from UV at 260 nm.

[0336] In some embodiments, oligonucleotides (eg, WVE-003) are chemically synthesized using commercially available synthesizers in accordance with appropriate cGMP regulations.

[0337] The manufacture of WVE-003 (e.g., drug substance) is a multi-step process that includes solid phase oligonucleotide synthesis, cleavage of the crude protected oligonucleotide from the solid support, removal of protecting groups (deprotection), purification by preparative anion exchange (AEX) chromatography, concentration and desalting, filtration, lyophilization, and packaging. A flow chart depicting the manufacturing process of WVE-003 drug substance is provided in Figure 1 Shown in.

[0338] In some embodiments, the stereochemistry of the oligonucleotide (e.g., WVE-003) is established by controlling the starting materials and the synthetic process of the synthesis. For example, the use of phosphoramidites prepared from chiral auxiliaries (L)-DPSE, (D)-DPSE, and (L)-PSM during the coupling step ensures that the desired stereodefined Sp phosphorothioate diester, Rp phosphorothioate diester, and Rp N-(1,3-dimethylimidazolidin-2-ylidene) phosphoramidite diester linkages are obtained, respectively.

[0339] In some embodiments, the present disclosure provides methods for making various pharmaceutical products as described herein.

[0340] In some embodiments, the disclosure provides products prepared by the provided methods (e.g., crude / purified oligonucleotides from stereoselective preparations, crude compositions, purified compositions, formulated compositions, pharmaceutical compositions, drug substances, drug products, etc.).

[0341] In some embodiments, provided products have a certain purity as described herein. In some embodiments, provided products are suitable for therapeutic uses as described herein. In some embodiments, provided products achieve one or more properties and / or activities as described herein.

[0342] Certain methods that can be used to manufacture WVE-003 and various compositions and products thereof are described below as examples.Several batches of WVE-003 drug substance and / or drug product were manufactured.

[0343] As shown herein, the preparation of WVE-003 includes multiple cycles (e.g., as described below). In some embodiments, each cycle of introducing a stereo-defined phosphorothioate or stereo-defined phosphoramidate consists of the corresponding 5'-detritylation, coupling, capping of the exposed chiral auxiliary secondary amine (capping-1), thiolation or imidization, and capping of the unreacted 5'-hydroxyl group (capping-2), and each cycle of introducing a phosphodiester consists of 5'-detritylation, coupling, oxidation, and capping-2, and each coupling reaction is carried out by activating the appropriate phosphoramidite and reacting with the free 5'-hydroxyl group of the protected nucleoside or oligonucleotide fixed to the support. In some embodiments, after an appropriate number of cycles and final detritylation, the (L)-PSM chiral auxiliary and cyanoethyl phosphate protecting group are removed from the crude oligonucleotide by treatment on a column with diethylamine (DEA) in acetonitrile (ACN), and the oligonucleotide is purified by treatment with TEA. . The (L) and (D) DPSE chiral auxiliaries are removed by treatment with HF (triethylamine hydrofluoride) solution. The crude oligonucleotides are then cleaved from the solid support by treatment with ammonium hydroxide in a suitably sized rated pressure vessel. This reaction also affects the overall deprotection of the exocyclic amino groups (acetyl, benzoyl, and isobutyryl). In some embodiments, the resulting crude oligonucleotides are purified using anion exchange (AEX) chromatography, and the purified oligonucleotides are concentrated and desalted by tangential flow filtration, followed by filtration, lyophilization, and packaging to produce the WVE-003 drug substance. Certain steps in the manufacture of the WVE-003 drug substance are further described in detail below as examples. Certain key functional groups are described below.

[0344]

[0345]

[0346]

[0347] Cycle of phosphorothioate assembly

[0348]

[0349] Cycle of phosphoramidate assembly

[0350]

[0351] Phosphodiester assembly cycle

[0352]

[0353] Stage 1 - Synthesis

[0354] Oligonucleotide synthesis was performed on an automated oligonucleotide synthesizer on a controlled pore glass (CPG) solid support functionalized with 5'-ODMTr-2'-MOE-T. All reactions took place on the solid support of the column.

[0355] Detritylation

[0356] To begin the synthesis, the CPG-5′-ODMTr-2′-MOE-T solid support was subjected to acid-catalyzed removal of the DMTr protecting group from the 5′-hydroxyl group by treatment with 3% dichloroacetic acid (DCA) in toluene. Complete DMTr removal was ensured by online UV monitoring based on the watch command in the synthesis program. DMTr removal was performed in the same manner at the beginning of each synthesis cycle and after the final cycle. In each case, after detritylation, the support-bound material was washed with acetonitrile in preparation for the next step of the synthesis.

[0357]

[0358] Coupling

[0359] Extension of the growing oligonucleotide chain is achieved by reacting the 5'-hydroxyl group of the support-bound oligonucleotide with an excess solution of protected phosphoramidites in the presence of an activator (5-(ethylthio)-1H-tetrazole (ETT) or 1-cyanomethylimidazolium triflate (CMIMT) dissolved in acetonitrile). The phosphoramidites are dissolved in acetonitrile or isobutyronitrile or an 80:20 v / v acetonitrile:isobutyronitrile mixture. In particular, the present disclosure recognizes that ethyl acetate can be used to prepare phosphoramidite solutions for oligonucleotide formulations (e.g., WVE-003 formulations). In some embodiments, ethyl acetate can be used in place of isobutyronitrile. In some embodiments, the use of ethyl acetate reduces manufacturing costs and / or simplifies handling. In some embodiments, the phosphoramidites are 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.). In particular, the present disclosure recognizes that propylene carbonate can be used to prepare phosphoramidite solutions for oligonucleotide formulations (e.g., WVE-003 formulations). In some embodiments, propylene carbonate can be used to replace isobutyronitrile. In some embodiments, the use of propylene carbonate reduces manufacturing costs and / or simplifies operation. 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 phosphoramidite is 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 is determined by the oligonucleotide sequence. In order to introduce phosphodiester linkages, phosphoramidites and ETT activators are used. To introduce stereo-defined phosphorothioate or phosphoramidate linkages, (L)- or (D)-DPSE or (L)-PSM derived phosphoramidites and CMIMT activators are used, respectively. In either case, the phosphoramidite / activator solution is mixed online, pushed onto the synthesis column, and then recirculated on the column for the appropriate amount of time. Subsequently, the synthesis column is rinsed with acetonitrile to remove excess reagents.

[0360]

[0361] End-capping-1

[0362] The Capping 1 step is performed to protect the secondary amine generated by the ring opening of the oxazophosphine on the auxiliary group during the coupling step from reacting in the remainder of the synthesis. The secondary amine is capped by flowing Capping Reagent B ([Cap B]: acetic anhydride / 2,6-lutidine / acetonitrile (20:30:50, v:v:v)) through the synthesis column.

[0363]

[0364] Oxidation, thiolation, imidization

[0365] The newly created P(III) phosphite triester linkage is then processed in one of three ways:

[0366] Oxidation by treatment with iodine in pyridine:water (90:10, v:v) resulted in the formation of P(V) phosphodiester linkages.

[0367] Thiolation by treatment with xanthane hydride (XH) in pyridine:acetonitrile results in the formation of a stereodefined P(V) phosphorothioate triester linkage.

[0368] Imidization by treatment with 2-azido-1,3-dimethylimidazolium hexafluorophosphate (ADIH) in acetonitrile results in the formation of a stereodefined P(V)phosphoamidate triester linkage.

[0369] In each case, after each step, the synthesis column was flushed of excess reagents with acetonitrile.

[0370]

[0371] End-capping-2

[0372] Although the coupling reaction proceeds with very high yields, the yield is not quantitative. A small fraction of the 5'-hydroxyl groups available in any given cycle are unable to couple with the activated phosphoramidite. To prevent reactions during subsequent cycles, these sites are blocked by performing a capping-2 step using a capping reagent (1:1, v:v) (Capping Reagent A [Capping A]: N-methylimidazole / acetonitrile (20:80, v:v) and CapB). Thus, a 5'-O-acetylated ("capped") support-bound oligonucleotide sequence is formed. Excess capping reagent is washed from the column with acetonitrile.

[0373]

[0374] Oligonucleotide assembly and final steps

[0375] Independent repetition of these cycles using appropriately protected phosphoramidites allows assembly of the entire protected sequence with a DMTr protecting group at the 5'-terminal position. After addition of the last nucleotide in the sequence, the 5'-terminal DMTr group is removed during a final detritylation step.

[0376] Stage 2 - Cleavage and deprotection

[0377] A three-step process was used to remove the phosphate protecting group (L)-PSM and the (L)- and (D)-DPSE chiral auxiliaries from stereodefined phosphoroamidate and phosphorothioate triesters, cleave the crude oligonucleotide from the solid support, and remove the exocyclic nucleobase protecting groups.

[0378] Step 1: Removal of (L)-PSM chiral auxiliary and cyanoethyl groups using diethylamine treatment The crude oligonucleotide on the solid support was treated with a diethylamine solution in ACN, which simultaneously removed the cyanoethyl protecting group from the phosphotriester linkage and the (L)-PSM chiral auxiliary from the aminophosphotriester, generating a phosphodiester linkage and an aminophosphodiester linkage, respectively.

[0379] Treatment with diethylamine to remove the (L)-PSM chiral auxiliary and cyanoethyl groups:

[0380]

[0381] Step 2: Fluoride Treatment to Remove (L)- and (D)-DPSE Chiral Auxiliaries The crude oligonucleotides on the solid support are treated with a temperature-controlled solution of TEA·HF in a mixture of dimethyl sulfoxide (DMSO) and H O to effectively remove the chiral auxiliary. This process converts the stereodefined phosphorothioate triesters into stereodefined phosphorothioate diesters.

[0382] Removal of (L)- and (D)-DPSE chiral auxiliaries using fluoride treatment:

[0383]

[0384] Step 3: Cleavage and deprotection

[0385] The crude oligonucleotide is then cleaved from the solid support by treatment with ammonium hydroxide in an appropriately sized, pressure-rated vessel. This reaction effects global deprotection of exocyclic amino groups (acetyl, benzoyl, and isobutyryl).

[0386] Cleavage and deprotection reactions:

[0387]

[0388]

[0389] Note: M+ corresponds to an undefined salt mixture (e.g., in some embodiments, NH4 + 、Et3NH + 、Et2NH2 + ); the zigzag line represents the linkage between the 3′ oxygen and phosphorus in the following internucleotide linkages.

[0390] Stage 3: Purification by anion exchange chromatography

[0391] Purification of the crude oligonucleotide solution was accomplished by AEX chromatography. The crude oligonucleotide solution was loaded onto a purification column filled with TSK-GEL Super Q-5PW medium.

[0392] Purification was performed using a sodium hydroxide buffer eluent. A sodium chloride gradient was used to elute the oligonucleotide from the column. The elution profile was monitored by ultraviolet (UV) spectrophotometry. Fractions were collected and neutralized with sodium phosphate buffer. The simulated pool was evaluated by IP-RP-UPLC. The pool containing the oligonucleotide of the desired purity was subjected to the next step in the process.

[0393] Stage 4: Concentration and Desalting (Final UF / DF)

[0394] The oligonucleotide of ...

[0395] Stage 5: Filtration, freeze-drying and packaging

[0396] The oligonucleotide solution is filtered through a 0.2 micron filter and then placed in one or more freeze drying trays for lyophilization. After lyophilization, the final drug substance is isolated as a solid powder and packaged in sterile high-density polyethylene (HDPE) bottles, each of which is labeled and sealed in a Mylar foil bag and stored at -20°C.

[0397] Material Control

[0398] Starting materials used in the manufacture of active pharmaceutical ingredients

[0399] The following lists useful raw materials (solvents, reagents, and auxiliary materials) used in the manufacture of WVE-003 (eg, drug substance). In some embodiments, the quality of all raw materials is controlled by material specifications and / or certificates of analysis.

[0400] The starting materials used in the preparation of the WVE-003 drug substance include 15 phosphoramidites, a controlled pore glass (CPG) solid support, and 2-azido-1,3-dimethylimidazolium hexafluorophosphate (ADIH). The reactive exocyclic groups on the nucleobases are typically appropriately protected during oligonucleotide synthesis to render them unreactive, and the 5′-hydroxyl function is protected as 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 P(III) purity of the phosphoramidites is about 85% or greater, in many cases about 90% or greater, and in many cases about 95% or about 98% or greater (RP-HPLC (area %) at about 260 nm and / or by integration of 31 P NMR), about 97% or higher (by 31 P NMR integration), the moisture content is less than about 0.4% or in many cases less than 0.2% (w / w). In some embodiments, the controlled pore glass 5'-ODMTr-2'-OMOE T solid support is a white to off-white powder with a particle size of 120-200 mesh (analytical sieving), Pore ​​diameter (mercury intrusion method), 0.20-0.24g / cc Density (tap density), ≥0.8cc / g Pore volume (mercury intrusion method), ≥75m 2 In some embodiments, the 2-azido-1,3-dimethylimidazolium hexafluorophosphate salt has a purity of about 98.0% or greater (HPLC) and a nitrogen content of about 23.70% to 24.80% (elemental analysis).

[0401] Certain solvents, reagents, and auxiliary materials used in the manufacture of WVE-003 (e.g., drug substance)

[0402]

[0403]

[0404] Abbreviations: CoA = Certificate of Analysis; NF = National Formulary; CIP = Cleaning in Place; USP = United States Pharmacopoeia; ACS = American Chemical Society

[0405] Certain starting materials used in the manufacture of WVE-003 drug substance

[0406]

[0407]

[0408]

[0409]

[0410] Certain solvents useful in preparing phosphoramidite solutions for oligonucleotide formulations

[0411]

[0412] The stereochemistry of WVE-003 can be established by controlling the starting materials and the synthetic process. The stereodefined phosphoramidite starting materials used to make WVE-003 are prepared from appropriately protected nucleosides and chiral auxiliaries (L)- and (D)-DPSE and (L)-PSM:

[0413]

[0414] In some embodiments, by 31 The chemical purity of stereodefined phosphoramidites can be determined by P NMR. 31 PNMR, 1 H NMR and 13 The absolute stereo configuration is determined by a combination of C NMR. Since the stereochemical configuration of the protected nucleoside and the chiral auxiliary is fixed, the phosphitylation reaction may produce two diastereomers (trans and cis), of which the trans form typically predominates and the cis form exists as a trace impurity.

[0415] Some possible stereoisomers produced by coupling reactions of starting materials

[0416]

[0417] Throughout the manufacturing process of WVE-003 (e.g., drug substance), various in-process controls are applied, for example, as described below. After completion of the synthesis and cleavage and deprotection stages, 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 Scheme A). During purification, the purity and impurities of the selected simulated pool are evaluated by IP-RP-UPLC. Once the selected fractions are combined and concentrated, the desalination of the resulting solution is controlled by in-process conductivity (e.g., ≤50 μS / cm), pH (e.g., 6.7-7.3), and concentration measurements.

[0418] In some embodiments, the manufacture of the drug substance or drug product complies with one or more or all of the standards described herein. In some embodiments, the drug substance (e.g., WVE-003 or a salt form thereof) has a certain level of purity as described herein. In some embodiments, the WVE-003 pentadecasodium salt has a certain level of purity as described herein. In some embodiments, WVE-003 or a salt form thereof has a certain level of purity in a composition (e.g., a pharmaceutical composition). In some embodiments, WVE-003 or a salt form thereof has a certain level of purity in a drug product. In some embodiments, the salt form is WVE-003 pentadecasodium salt. In some embodiments, WVE-003 has a purity level 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% as determined by IP-RP-UPLC as described herein.

[0419] Multiple batches of WVE-003 drug substance have been manufactured. In some embodiments, the scale is greater than 20 mmol. In some embodiments, the scale is multiples of about 20 mmol. In some embodiments, the manufactured WVE-003 is utilized in clinical trials.

[0420] Characterization

[0421] In particular, the present disclosure provides techniques for characterizing and / or evaluating WVE-003 or compositions thereof (eg, WVE-003 formulations such as drug substances, drug products, etc.).

[0422] Products can be characterized by various techniques. For example, WVE-003 can be characterized by mass spectrometry. For one formulation, electrospray ionization mass spectrometry (ESI-MS) analysis was performed, and the theoretical average mass of WVE-003 (free acid form) was consistent with the experimentally determined value of 7257 Da.

[0423] In some embodiments, the sequence of WVE-003 was confirmed by ESI-MS / MS sequencing. For example, in one evaluation, at least one sequence-related fragment ion was observed for each of the 19 nucleobases in the sequence within 5 ppm of the calculated (expected) mass. The monoisotopic mass of the full-length oligonucleotide was also experimentally verified in ESI-MS / MS analysis (7253.3428 Da), and the identity and position of all 20 nucleobases were confirmed, thereby confirming the sequence of WVE-003.

[0424] Set up stereochemical properties by controlling the starting material for synthesis.The synthetic coupling of the phosphoramidite prepared from (L)-DPSE or (L)-PSM produces respectively the phosphorothioate diester linkage of Sp chirality and the phosphoramidite diester linkage of Rp chirality, and the synthetic coupling of the phosphoramidite prepared from (D)-DPSE or (D)-PSM produces the phosphorothioate diester linkage of Rp chirality and the phosphoramidite diester linkage of Sp chirality.For each coupled reaction, the consistent use of stereo-defined phosphoramidite makes it possible to control the stereochemical properties of every kind of phosphorothioate linkage.Can use according to the various technologies of this disclosure to confirm stereochemical properties.For example, can be measured by using several different technologies (these technology combinations are used) and by comparing with suitable standard (accurate information and confirmation about this key attribute can be provided) to confirm stereochemical properties.Available analytical techniques include NMR ( 1 H. 19 F. 31 P, multidimensional, etc.) and enzyme digestion. In some cases, NMR is performed in phosphate buffer (e.g., pH 7.0). 1 H. 19 F and 31 The results of P NMR and enzyme digestion of various preparations are consistent with the product structure. In some embodiments, NMR is related to water based on DSS standards. The following describes the observed data of certain experiments.

[0425] In some embodiments, WVE-003 drug substance was prepared at a concentration of 1 mM in 600 μL of a 100 atomic % D2O 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, indirect calibration was performed based on the DSS peak at 0 ppm. 1 H NMR spectroscopy. 1 The HNMR spectrum contains signals consistent with the WVE-003 structure, and the chemical shifts of a single region of the signals are consistent with the proposed structure. In some embodiments, WVE-003 (e.g., a prepared drug substance batch) is obtained. 31 P NMR spectra were recorded at 161.98 MHz for phosphorus. In some embodiments, external standards based on 1 Indirect calibration of the H NMR spectrum using a joint scale (where the DSS peak is set to 0 ppm) 31P NMR spectrum. In some embodiments, the sharp signal at approximately 2 ppm is derived from the phosphate buffer used. A total of 19 PS / PN / PO peaks are consistent with the stereo-defined mixed PO / PS / PN framework of the WVE-003 drug substance.

[0426]

[0427]

[0428] In some embodiments, the stereochemistry of WVE-003 is confirmed by enzymatic digestion assays.

[0429] In certain embodiments, stereochemical purity is presented as the percentage of the correct diastereomer associated with the stereochemical properties of distribution relative to a total diastereomeric mixture. This includes a small amount of other diastereomers that may exist. In WVE-003, each (L)- or (D)-DPSE phosphoramidite is very high in the diastereoselectivity of its stereo-defined phosphorothioate or phosphoramidate linkages, and the overall stereochemical purity of the oligonucleotide is the product of the diastereoselectivity combination of these 13 phosphorothioate linkages and 4 phosphoramidate linkages.

[0430] In certain embodiments, the diastereoselectivity of chiral linkage is assessed by preparing corresponding dimers. The synthesis conditions for producing each sequence dimer are identical to those applied to the whole molecule. These dimer units are separated and analyzed by RP-UPLC conditions, and Rp and Sp diastereoisomers are separated and quantitatively analyzed. In certain embodiments, the purity of WVE-003 sequence dimers and corresponding reference substances thereof are analyzed by ultra-high performance liquid chromatography (UPLC) and / or the quality confirmation of Rp and Sp diastereoisomers is analyzed by ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS). The analysis of these samples is completed on crude material to show the representative diastereoselectivity of linkage during the building-up process. The stereochemical purity of the various dimers analyzed is presented below. All sequence-specific dimers are synthesized using the same process conditions as WVE-003, accurately representing the internucleotide linkage of phosphorothioate and phosphoramidite.

[0431] Summary of diastereoselectivity of WVE-003 drug substance model dimer

[0432] dimer %Sp %Rp A*SG 98.21 1.79 C*SA 97.35 2.65 C*ST 98.29 1.71 G*Rm5Ceo 1.52 98.48 G*SC 98.41 1.59 G*ST 98.47 1.53 Geon001Rm5Ceo 0.13 99.87 m5Ceo*STeo 99.84 0.16 m5Ceon001RAeo 0.16 99.84 mA*ST 99.77 0.23 mG*SmU 99.77 0.23 mGn001RmA 0.31 99.69 mUn001RmU 0.15 99.85 T*RA 1.48 98.52 T*SC 98.31 1.69 T*SG 98.28 1.72

[0433] The WVE-003 stereochemical purity can be attributed to the product of the stereochemical purity of each of the 13 phosphorothioate linkages and the 4 phosphoramidate linkages in the sequence:

[0434] dimer %purity A*SG 98.21 A*SG 98.21 C*SA 97.35 C*ST 98.29 G*Rm5Ceo 98.48 G*SC 98.41 G*ST 98.47 Geon001Rm5Ceo 99.87 m5Ceo*STeo 99.84 m5Ceon001RAeo 99.84 mA*ST 99.77 mG*SmU 99.77 mGn001RmA 99.69 mUn001RmU 99.85 T*RA 98.52 T*SC 98.31 T*SG 98.28 Stereochemical purity 82.69％

[0435] In some embodiments, as demonstrated herein, the diastereomeric purity of WVE-003 is approximately 82.7% by dimer modeling. In some embodiments, UPLC analysis is used for dimer modeling. As demonstrated herein, all 17 linkages are ≥97% stereopure, 9 linkages are ≥97% stereopure, and 7 linkages are ≥99% stereopure. In some embodiments, as demonstrated herein, the average stereochemical purity of all linkages is 98.9%.

[0436] In some embodiments, reference standards (e.g., standards characterized by NMR, enzymatic digestion, etc.) are used to determine stereochemical properties established by UPLC as part of batch release. In some embodiments, provided technology provides reference standards. In some embodiments, the reference standards have purity (e.g., as assessed by Protocol A) and / or stereopurity (e.g., as assessed by Protocol B and / or dimer modeling as described herein).

[0437] The FTIR spectrum of WVE-003 was obtained using an attenuated total reflectance (ATR) sampling accessory. The main absorption band is at 1636 cm -1 and 1600cm -1 The absorption peak is at 2938cm -1 and a broad peak at 3199 cm -1 and 3339cm -1 The FTIR results were consistent with the structure of WVE-003.

[0438] Counterions can be analyzed according to the present disclosure. In some embodiments, sodium content is assessed by ICP-OES. In some embodiments, a sodium content value of 4.5% has been determined for the WVE-003 drug substance by ICP-OES, which is consistent with a theoretical sodium content value of 4.5% (w / w).

[0439] For various formulations, WVE-003 drug substance is a white to off-white powder. The pH of WVE-003 drug substance in purified water was found to be 6.0-8.0. In some embodiments, the pH is about 6.5-7.5. In some embodiments, the pH is about 6.4. In some embodiments, the pH is about 6.5. In some embodiments, the pH is about 6.6. In some embodiments, the pH is about 6.7. In some embodiments, the pH is about 6.8. In some embodiments, the pH is about 6.9. In some embodiments, the pH is about 7.0. In some embodiments, the pH is about 7.1. In some embodiments, the pH is about 7.2. In some embodiments, the pH is about 7.3. In some embodiments, the pH is about 7.4.

[0440] The molar extinction coefficient of WVE-003 API in water at 260 nm was experimentally determined to be 181181M -1 cm -1 , and is used to convert UV absorbance into concentrations from which quantities can be calculated. It corresponds to an absorption coefficient of approximately 25.0 OD / mg.

[0441] In various embodiments, impurities are controlled to low levels as described herein. For example, in some embodiments, the impurity level is less than about 30%, about 25%, or about 20% as described herein. In some embodiments, the impurities include various oligonucleotide impurities. Various techniques can be used to identify, characterize, and / or evaluate impurities, such as mass spectrometry, LC, UV, etc.

[0442] The WVE-003 drug substance underwent GLP evaluation in an in vitro genotoxicity study and an in vivo micronucleus study. No toxicity was observed.

[0443] The various formulations of the WVE-003 drug substance have met the regulatory limits for residual solvents (including acetonitrile, toluene, and pyridine (ICH Class 2)) and / or elemental impurities (including various elements of ICH Class 1, 2A, and 3). The elemental impurities of the various formulations are within the limits.

[0444] The release specifications for WVE-003 drug substance and / or drug product can include one or more specifications described herein, such as appearance (e.g., visual, white to off-white powder), sequence identity (e.g., by MS / MS), molecular weight, stereochemistry (e.g., IP-RP-UPLC), purity (e.g., area % as determined by IP-RP-UPLC (e.g., Protocol A)), impurities (e.g., area % as determined 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.), assays (e.g., free acid, anhydrous, by UV), pH (e.g., 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., by USP <85> and / or Ph.Eur2.6.14), bioburden (total aerobic microorganisms, total yeast and mold count, etc., as measured by USP <61> and / or Ph.Eur2.6.12) etc.

[0445] In some embodiments, the identity of WVE-003 (e.g., drug substance) was determined by liquid chromatography-mass spectrometry (LC-MS). Samples and analytical reference materials were prepared in water and injected onto a Waters Acquity BEH C18 column. The analysis involved 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:

[0446]

[0447]

[0448] In some embodiments, for WVE-003, system suitability is confirmed when the molecular weight (deconvoluted mass) of the full-length product (FLP) after three initial injections of the analytical reference material should be 7258 ± 3 Da. In addition, the blank chromatogram should be free of other interfering peaks except for the solvent front and gradient offset.

[0449] In some embodiments, the identity of WVE-003, for example, as determined by sequencing of the drug substance, is determined by high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS / MS). A useful procedure is described below by way of example. Samples are prepared in water and injected directly into the mass spectrometer for MS / MS analysis. The analysis involves an isocratic LC method using 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:

[0450]

[0451]

[0452] 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.

[0453] In some embodiments, the sodium content of a WVE-003 formulation (e.g., drug substance) is determined by inductively coupled plasma optical emission spectroscopy (ICP-OES). Sample material in solution is introduced into a radiofrequency plasma via pneumatic nebulization, where an energy transfer process causes desolvation, atomization, and ionization. Sodium emits light during this process, 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, the sodium content is about 4%-5%. In some embodiments, the sodium content is about 4.3%-4.7%. In some embodiments, the sodium content is about 4.3%. In some embodiments, the sodium content is about 4.4%. In some embodiments, the sodium content is about 4.5%. In some embodiments, the sodium content is about 4.6%. In some embodiments, the sodium content is about 4.7%.

[0454] In some embodiments, the present disclosure provides techniques for assessing the purity and / or impurities of WVE-003 formulations or compositions. 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 (Protocol A) is described below with examples. Separation is provided using a gradient of mobile phase A (hexafluoroisopropanol [HFIP] and triethylamine [TEA] in water) and mobile phase B (50% acetonitrile in water). A summary of method parameters (Set A Parameters) is provided below with examples. In some embodiments, system suitability (system suitability criteria) is established by the absence of interfering peaks in the blank and acceptance criteria for retention time, peak area %, and %RSD of peak area for the initial and all injections of the WVE-003 analytical reference material. In some embodiments, all individual impurities are reported as ≥0.10 area % (as a function of the relative retention time of the main peak) and the total amount of all impurities is ≥0.10 area %. In some embodiments, the identity of WVE-003 is confirmed by comparing the relative retention time to the system suitability criteria. This method has been determined to be indicative of stability in method development studies.

[0455]

[0456] In some embodiments, the purity of a WVE-003 formulation (e.g., drug substance, drug product, etc.) is about 84% or greater (e.g., as assessed by Scheme A (% area)). In some embodiments, the purity is about 85% or greater. In some embodiments, the purity is about 86% or greater. In some embodiments, the purity is about 87% or greater. In some embodiments, the purity is about 88% or greater. In some embodiments, the purity is between about 80% and 90%. In some embodiments, the purity is between about 84% and 90%. In some embodiments, the purity is between about 84% and 88%. In some embodiments, the total impurities are about or no more than about 16% (e.g., as assessed by Scheme A (% area)). In some embodiments, the total impurities are about or no more than about 15%. In some embodiments, the total impurities are about or no more than about 14%. In some embodiments, the total impurities are about or no more than about 13%. In some embodiments, the total impurities are about or no more than about 12%. In some embodiments, the sum of WVE-003 and total impurities is about 99% to 101%.

[0457] In some embodiments, the measured value of a WVE-003 formulation (e.g., drug substance, drug product, etc.) is determined by UV spectrophotometry. A useful procedure is described herein by way of example. An amount of WVE-003 drug substance is accurately weighed and dissolved in water in a graduated glassware. The absorbance of the solution is determined at 260 nm, and the molar extinction coefficient (MEC; 181,181 M -1 cm -1 and 25.0 OD / mg) and convert this to the concentration of WVE-003 drug substance. <921> Method 1c) and correcting the weighed material by subtracting the sodium content. The measured value (%) is then determined as the ratio of the amount of WVE-003 measured by absorbance to the theoretical amount prepared based on the exact weight of the sample corrected for water and sodium content. In some embodiments, the measured value of a WVE-003 formulation (e.g., drug substance, drug product, etc.) is about 100% ± 10%. In some embodiments, the measured value is about 95%-105%. In some embodiments, the measured value is about 95%. In some embodiments, the measured value is about 96%. In some embodiments, the measured value is about 97%. In some embodiments, the measured value is about 98%.

[0458] In some embodiments, the water content of a WVE-003 formulation (e.g., drug substance, drug product, etc.) is determined by coulometric titration (Karl Fischer method). In some embodiments, system suitability is established by sufficient recovery from a certified control standard of sodium tartrate monohydrate of known water content. In some embodiments, the water content of a WVE-003 formulation (e.g., drug substance, drug product, etc.) is no more than about 15%, 10%, 5%, or 1% (w / w). In some embodiments, the water content is no more than about 15%. In some embodiments, the water content is no more than about 10%. In some embodiments, the water content is no more than about 9%. In some embodiments, the water content is no more than about 8%. In some embodiments, the water content is no more than about 7%. In some embodiments, the water content is no more than about 6%. In some embodiments, the water content is no more than about 5%. In some embodiments, the water content is no more than about 1%.

[0459] In some embodiments, residual solvent in WVE-003 formulations (e.g., drug substance, drug product, etc.) is quantified using gas chromatography with flame ionization detection (GC-FID). In some embodiments, samples are prepared by thorough mixing, weighing into crimped vials, and dissolving in solvent. The sample is then introduced via direct injection and quantified by comparison with an external standard as a limit test. Blanks and calibration verification are analyzed at appropriate intervals.

[0460] In some embodiments, the present disclosure provides techniques for confirming the stereochemical properties of WVE-003 or its diastereomers (e.g., with respect to one or more chiral-bound phosphorus centers). In some embodiments, the stereochemical properties of WVE-003 (e.g., in WVE-003 formulations, drug substances, drug products, etc.) are determined by IP-RP-UPLC using a Waters BEHC18 column. A useful procedure (Scheme B) is described below by way of example. In some embodiments, a gradient of mobile phase A (triethylamine acetate [TEAA] in water) and mobile phase B (50% acetonitrile in water) is used to provide separation of WVE-003 from closely related diastereomeric impurities. 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 the %RSD of the retention time and peak area of ​​the initial injection of the WVE-003 analytical reference material, the % difference between the peak area of ​​subsequent standards and the average of the initial injection of the standards, and the %RSD of the retention time of all standard injections. Method performance was also verified by demonstrating resolution of closely related diastereomers with reference samples. For stereochemical identification, stereochemical identity was confirmed if the % retention time of the major peak did not differ by more than 3% from that of the structurally characterized reference material.

[0461]

[0462]

[0463] In some embodiments, bacterial endotoxins are <85> In some embodiments, the bioburden (e.g., both the total aerobic microbial count (TAMC) and the total yeast and mold count (TYMC)) is determined according to USP <61> and / or as determined by Ph.Eur.2.6.12.

[0464] The WVE-003 drug substance is packaged in a suitable container (e.g., a sterile high-density polyethylene (HDPE) bottle with a polypropylene screw closure), labeled, and sealed in a protector (e.g., a polyester film foil pouch) that provides a gas / moisture barrier with a high level of abrasion and puncture resistance.

[0465] Various batches of WVE-003 formulations have been manufactured. Certain formulations are used in nonclinical studies, including GLP toxicology studies. Certain formulations are manufactured for clinical studies. The manufactured formulations are stable. In some embodiments, WVE-003 or a composition thereof is stored at about -20°C. In some embodiments, long-term storage conditions are at about -20°C, for example, ±5°C. In some embodiments, storage is at about 5°C, for example, ±3°C.

[0466] pharmaceutical products

[0467] In some embodiments, the present disclosure provides a WVE-003 drug product. In some embodiments, the WVE-003 drug product comprises a WVE-003 drug substance, such as a WVE-003 pentadecasodium salt manufactured using the above-described method. In some embodiments, the WVE-003 drug product consists of a WVE-003 drug substance (e.g., a WVE-003 pentadecasodium 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.1 type clear glass vial, The vials are sealed with a coated elastomeric rubber stopper and an aluminum top seal with a blue matte flip-top button. In some embodiments, the vials contain a single dose of WVE-003 as described herein. In some embodiments, the vials contain 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 WVE-003 free acid form. In some embodiments, the vials contain about 20 mg of WVE-003 (based on the free acid form unless otherwise stated). In some embodiments, a vial filled with 2.53 mL of a WVE-003 drug substance at a concentration of 8 mg / mL in water for injection (WFI) is lyophilized and backfilled with nitrogen NF after the lyophilization cycle is complete. In some embodiments, a 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 about 150 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 160 mg of WVE-003 drug substance. In some embodiments, a WVE-003 drug product vial contains about 170 mg of WVE-003 drug substance.In some embodiments, the present 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 present disclosure provides a pharmaceutical composition comprising WVE-003 pentadecasodium 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, prior to intrathecal administration, the WVE-003 drug substance is reconstituted in artificial cerebrospinal fluid (aCSF) and diluted to a sterile, preservative-free solution for the clinical site.

[0468] In some embodiments, the WVE-003 drug product contains no preservatives, inactive ingredients, or excipients.

[0469] In some embodiments, the WVE-003 drug product is a white to off-white solid with no visible material and is substantially free of particles after reconstitution. In some embodiments, the pH of 20 mg of 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 of WVE-003 in 2 mL of aCSF solution is about 6.4-7.2.

[0470] 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. Others skilled in the art will appreciate that other temperatures and / or pressures may also be utilized.

[0471] In the formulation, approximately 75 grams of WVE-003 drug substance was reconstituted with water for injection (Ph.Eur. / USP) to obtain an 8 mg / mL solution. Approximately 2.53 mL of this solution was lyophilized into a powder, yielding a nominal dose of 20 mg of drug per vial. Drug product batches can be prepared by combining multiple drug substance batches. In some embodiments, as an example, the total number of vials calculated from the amount of drug substance is as follows:

[0472] The purity, sodium content and water content of the raw material are 86%, 4.3% and 8% respectively.

[0473] The amount of drug substance (DS) used to manufacture the drug product (DP) batch was 75.04 g.

[0474] Total amount of oligonucleotides in a batch of DS = amount of DS (g) x 86% purity / 100% x (100% - 4.3% sodium content) / 100% x (100% - 8% water content) / 100% = 75.04 g x 0.86 x (0.957) x (0.92) x 1000 mg / g = 56,788.38 mg.

[0475] Total batch size of drug product at 20.24 mg / vial = DS batch amount / DP vial content = 56,788 mg / 20.24 mg = 2,805 vials.

[0476] In some embodiments, as an example, the total number of vials and batch size calculated from the amount of drug substance is as follows:

[0477] The purity, sodium content and water content of the raw material are 86%, 4.3% and 8% respectively.

[0478] The amount of drug substance (DS) used to manufacture the drug product (DP) batch was 75.04 g.

[0479] Total amount of oligonucleotides in a batch of DS = 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.

[0480] Total batch size of drug product at 20.24 mg / vial = DS batch amount / DP vial content = 56,596 mg / 20.24 mg = 2,824 vials.

[0481] In some embodiments, the batch size is calculated using the following formula:

[0482] Theoretical batch size (mL) = theoretical total DS in mg x (purity (%) determined by UPLC) / 100%) x [(100% - sodium content (%) - water content (%)) / 100%] ÷ concentration

[0483] For example, in one example, theoretical batch size (mL) = 75,000 mg x 0.86 x 0.877 ÷ 8.0 mg / mL = 7,070 mL.

[0484] Those skilled in the art will appreciate that, in each case, these exemplary methods can provide vials with approximately the same amount of WVE-003 and can provide for administration and delivery of approximately the same amount of WVE-003 to a patient.

[0485] Figure 2A flow chart of the WVE-003 drug product manufacturing process is provided as an example. WVE-003 drug substance (DS) containers were thawed at 2°C-8°C and then equilibrated at room temperature (RT). Using the purity factor, sodium content, and % moisture, the pure full-length product (FLP) was calculated as:

[0486]

[0487] In some embodiments, pure full-length product (FLP) is calculated as:

[0488]

[0489] Those skilled in the art will appreciate that in various circumstances (e.g., levels of purity, % sodium content, % moisture (water content), etc. as described herein, and methods for assessing purity, % sodium content (e.g., ICP-OES as described herein), % moisture (e.g., Karl Fischer method as described herein (e.g., USP <921> )) and other methods (e.g., UPLC as described herein), both calculations can provide approximately the same results. In cases where multiple batches of drug substance are to be combined, a purity correction factor is determined based on the amount of DS from each batch. Based on the weight of pure FLP, the batch size required to make an 8 mg / mL solution is calculated.

[0490] Add approximately 50% of the calculated batch size of WFI by weight to the formulation container. Weigh the DS containers and transfer the DS from each container to the formulation container. Use an appropriate WFI rinse to ensure complete removal of DS from the container. Dry the empty DS container and weigh it to calculate the total DS added to the formulation container.

[0491] The DS was thoroughly mixed and an in-process sample was taken to measure the concentration by ultraviolet (UV) assay. The remaining solution mass in the formulation was weighed. Based on the purity-corrected WVE-003 drug substance concentration and the solution mass results, the amount of WFI required to achieve a final WVE-003 drug substance concentration of 8.0 mg / mL was calculated as follows:

[0492]

[0493] The required amount of WFI is then slowly added directly to the formulation container and mixed to ensure a homogeneous solution. In-process samples are collected for appearance, concentration determined by UV spectrophotometry, and density, which should meet various specifications. This final compounded formulation undergoes filtration to reduce bioburden and can then be stored overnight at 2°C-8°C if necessary.

[0494] In some embodiments, a pre-filtration bioburden sample is obtained prior to sterile filtration of the compounded bulk solution. The final compounded formulation is sterile filtered through two 0.2 μm filters in series prior to filling. The filter integrity of the sterile filter unit is checked by the bubble point method before and after filtration.

[0495] The target fill weight is determined based on the target fill volume of 2.53 mL and the measured density. Regular fill checks are performed while filling, and filled vials with stoppers in the freeze-drying position are placed on the freeze-drying trays. The freeze-drying trays are aseptically transferred to the sterilized freeze dryer using a HEPA truck. In some embodiments, the freeze-drying cycle parameters are those provided below.

[0496]

[0497] After lyophilization is complete, the chamber pressure is increased to 9.6 psi to backfill the chamber with sterile nitrogen (N2). The stoppers are placed in place and the seals are applied via crimping before the items on the tray are removed from the shelf. The vials are then visually inspected for release, stability testing, and packaging or storage before sampling.

[0498] Process controls and in-process control tests and acceptance limits are applied during the manufacturing of the WVE-003 drug product. Primary controls are implemented at key steps during the manufacturing of the WVE-003 drug product via in-process concentration measurements by ultraviolet (UV) spectrophotometry. In-process concentration measurements are used to calculate the amount of WFI required to dilute the compounding solution to a final concentration of 8 mg / mL WVE-003 drug substance. Appropriate dilution is confirmed by measuring the concentration of the final bulk drug solution. Microbiological control is performed by measuring the pre-filtration bioburden and analyzing the pre- and post-filtration integrity of the sterilizing filter. Vials filled with drug product are regularly checked by weight during the filling process and 100% visually inspected prior to sampling for release, bulk packaging, and / or stability.

[0499] Release specifications for the WVE-003 drug product can include one or more specifications described herein, such as appearance (e.g., visual; white to off-white solid), appearance after reconstitution (e.g., visual), identity (e.g., retention time by LC-MS, IP-RP-UPLC, 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., % labeled amount; e.g., free acid, anhydrous, by UV), pH (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), osmolarity (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), dose uniformity (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 method, etc.), container closure integrity testing (e.g., USP <1207> In some embodiments, the WVE-003 drug product exhibits substantially the same or similar specifications, such as purity, impurities, pH, water content, etc., as the WVE-003 drug substance as described herein.

[0500] For example, in some embodiments, the purity of the WVE-003 drug product is about 84% or greater (e.g., as assessed by Scheme A (% area)). In some embodiments, the purity is about 85% or greater. In some embodiments, the purity is about 86% or greater. In some embodiments, the purity is about 87% or greater. In some embodiments, the purity is about 88% or greater. In some embodiments, the purity is between about 80% and 90%. In some embodiments, the purity is between about 84% and 90%. In some embodiments, the purity is between about 84% and 88%. In some embodiments, the total impurities are about or no more than about 16% (e.g., as assessed by Scheme A (% area)). In some embodiments, the total impurities are about or no more than about 15%. In some embodiments, the total impurities are about or no more than about 14%. In some embodiments, the total impurities are about or no more than about 13%. In some embodiments, the total impurities are about or no more than about 12%. In some embodiments, the total impurities are about or no more than about 11%. In some embodiments, the sum of WVE-003 and total impurities is about 99% to 101%.

[0501] In some embodiments, the assay value (% 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, as well as the molar extinction coefficient (MEC) and the experimentally measured purity of WVE-003 are measured and used to determine the purity-corrected amount of WVE-003 drug substance / vial (mg / vial), which is then compared to the expected amount in the vial (e.g., 20 mg) to obtain the % labeled amount. In some embodiments, the assay sample preparation and calculations are performed as follows:

[0502] Reconstitute the finished vial with 2.5 mL of water to obtain an 8 mg / mL solution. Stock Sample (0.1 mg / mL): Pipette 0.32 mL of the 8 mg / mL drug product (in duplicate) into a 25 mL volumetric flask, add water as needed, and mix thoroughly. Working Sample (0.02 mg / mL): Pipette 2.0 mL of each 0.1 mg / mL stock sample solution into a 10 mL volumetric flask. Dilute to volume with water and mix thoroughly.

[0503] Each working sample was analyzed with 1 reading (1 cm cuvette) at 260 nm (A260).

[0504] The % labeled amount (%LC) of WVE-003 from A260 was calculated as follows:

[0505]

[0506] DF = Dilution Factor. Determinations were averaged for final reporting.

[0507] In some embodiments, the % labeled amount is about 100% ± 10%. In some embodiments, the % labeled amount is about 95%-105%. In some embodiments, the % labeled amount is about 95%. In some embodiments, the % labeled amount is about 96%. In some embodiments, the % labeled amount is about 97%. In some embodiments, the % labeled amount is about 98%. In some embodiments, the % labeled amount is about 99%. In some embodiments, the % labeled amount is about 100%. In some embodiments, the % labeled amount is about 101%. In some embodiments, the % labeled amount is about 102%. In some embodiments, the % labeled amount is about 103%. In some embodiments, the % labeled amount is about 104%. In some embodiments, the % labeled amount is about 105%. In some embodiments, the % labeled amount is about 106%. In some embodiments, the % labeled amount is about 107%.

[0508] In some embodiments, the % labeled amount is about 108%. In some embodiments, the % labeled amount is about 109%. In some embodiments, the % labeled amount is about 110%.

[0509] In some embodiments, the reconstitution time of the WVE-003 drug product was determined visually after reconstitution with 2 mL of sterile, preservative-free aCSF and gentle inversion until the filter cake was completely dissolved. A calibrated timer was used to determine the time it took for the filter cake to be completely reconstituted.

[0510] In some embodiments, the uniformity of the WVE-003 drug product dosage unit is determined by UV content uniformity testing. The uniformity of the WVE-003 drug product dosage unit is determined by reconstituting ten vials, and if necessary, an additional twenty vials are tested. As described above, for each vial, an assay value (% labeled amount) is determined by UV spectrophotometry. The acceptance value (AV value) is then calculated using the assay value (% labeled amount) results determined in each test vial as specified in the summary.

[0511] In some embodiments, particulate matter in the WVE-003 drug product is determined by reconstituting 10 vials with 10 mL of purified water and then combining the resulting solutions for analysis. The combined solutions are then analyzed using a particle counter according to Test 1.B: Light Obscuration Test to determine the number of particles 10 μm or larger and 25 μm or larger in the solution. Calibration verification is performed prior to test sample analysis.

[0512] In some embodiments, the osmolarity of the WVE-003 drug product (diluted in aCSF) is specified (e.g., as per USP <785> / Ph.Eur.2.2.35 measurements) are consistent with reconstitution media (e.g., aCSF) and are consistent with the osmolarity range of human cerebrospinal fluid.

[0513] In some embodiments, the WVE-003 drug product is stored at about 5°C, for example, ±3°C. In some embodiments, it is stored at about 25°C, for example, ±2°C. In some embodiments, it can be stored at about 40°C, for example, ±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 various evaluated 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 or more months. In some embodiments, the WVE-003 drug product is stable at about 5°C, for example, ±3°C, for about 6, 9, 12, 15, 18, 21, 24, 27, 30, 33, 36 or more months. In some embodiments, the WVE-003 drug product is stable at about 25° C., e.g., ±2° C. (e.g., 60% RH ±5%) for 6, 9, 12, 15, 18, 21, 24, or more months. In some embodiments, the WVE-003 drug product is stable at about 40° C., e.g., ±2° C. (e.g., 75% RH ±5%) for 1, 2, 3, 4, 5, 6, or more months.

[0514] In some embodiments, WVE-003 is provided as a solution composition, for example, dissolved in aCSF. In some embodiments, the diluent used as a placebo and the 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.I type clear glass vial sealed with a serum stopper and an aluminum top seal with a white matte flip-top button. In some embodiments, the vial contains 20.8 mL of aCSF (including 0.8 mL overflow 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, the pH is about 7.2-7.4. The composition of the diluent is presented as follows:

[0515]

[0516]

[0517] USP = United States Pharmacopoeia, NF = National Formulary, Ph.Eur. = European Pharmacopoeia, JP = Japanese Pharmacopoeia, BP = British Pharmacopoeia, QS = Quantitative

[0518] In some embodiments, the composition of the diluent is presented as follows:

[0519]

[0520]

[0521] USP = United States Pharmacopoeia, NF = National Formulary, Ph.Eur. = European Pharmacopoeia, JP = Japanese Pharmacopoeia, BP = British Pharmacopoeia, QS = Quantitative, WFI = Water for Injection, N = Normal concentration

[0522] Formulations of WVE-003

[0523] In some embodiments, the present disclosure provides formulations of HTT oligonucleotides (eg, WVE-003).

[0524] In some embodiments, the HTT oligonucleotide or its salt form or HTT oligonucleotide composition is capable of mediating an allele-specific decrease in the level, expression and / or activity of an mHTT transcript. In some embodiments, the HTT oligonucleotide or its salt form or HTT oligonucleotide composition is or has WVE-003. In some embodiments, the HTT oligonucleotide or its salt form or HTT oligonucleotide composition is capable of mediating an allele-specific knockdown of a mutant HTT transcript. In some embodiments, the present disclosure provides a chirality-controlled HTT oligonucleotide composition that is capable of mediating an allele-specific knockdown of a mutant HTT transcript.

[0525] In some embodiments, the HTT oligonucleotide or its salt form or HTT oligonucleotide composition is provided as a lyophilized powder for reconstitution and dilution for administration. In some embodiments, the WVE-003 formulation or preparation 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 ranges from 6.0 to 8.0.

[0526] In some embodiments, the HTT oligonucleotide or a salt form thereof, or an HTT oligonucleotide composition is provided as a liquid formulation.

[0527] In some embodiments, WVE-003 has been formulated as a lyophilized powder for reconstitution and dilution for administration. In some embodiments, WVE-003 has been formulated as a liquid formulation.

[0528] In some embodiments, the present disclosure provides specific formulations of WVE-003. In some embodiments, the present disclosure provides methods of using a specific formulation of WVE-003 at a dose of about 30, about 60, about 90, about 120, about 150, or about 168 mg to treat Huntington's disease.

[0529] In some embodiments, the HTT oligonucleotide is WVE-003.

[0530] In some embodiments, the disclosure relates to a method of treating Huntington's disease in a subject in need thereof, wherein an HTT oligonucleotide formulated as a liquid formulation is administered to the subject, wherein the liquid formulation is reconstituted from a lyophilized formulation with sodium chloride solution.

[0531] In some embodiments, the lyophilized formulation of WVE-003 is a dry powder.

[0532] In some embodiments, the lyophilized formulation of WVE-003 is a dry powder prepared by lyophilizing a liquid formulation of WVE-003 in water.

[0533] In some embodiments, the lyophilized formulation of WVE-003 is a dry powder in a vial.

[0534] In some embodiments, the lyophilized formulation of WVE-003 is approximately 20 mg of dry powder in a vial.

[0535] In some embodiments, the lyophilized formulation of WVE-003 is a dry powder in 10 ml vials.

[0536] In some embodiments, the lyophilized formulation of WVE-003 is approximately 20 mg of dry powder in a 10 ml vial.

[0537] The reconstituted solution does not contain any preservatives; therefore, it should be administered without delay. If this is not possible, the solution should be stored at room temperature and administered within 4 hours.

[0538] Partially used, unused, or damaged vials should be disposed of.

[0539] In some embodiments, the HTT oligonucleotide WVE-003 is analyzed in nonclinical and clinical studies.

[0540] In some embodiments, WVE-003 is provided as a solution. In some embodiments, WVE-003 is administered as 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 formulation (e.g., a drug substance, a drug product, etc.) dissolved in aCSF.

[0541] Dosing regimen for HTT oligonucleotides

[0542] In some embodiments, the dosing regimen of the oligonucleotide, oligonucleotide composition, chirality-controlled oligonucleotide composition, or therapeutically effective amount of any thereof is any dosing regimen described herein.

[0543] In some embodiments, the dosing regimen relates to: the amount of a single dose of an oligonucleotide, oligonucleotide composition, or chirality-controlled oligonucleotide composition, or a therapeutically effective amount of an oligonucleotide, oligonucleotide composition, or chirality-controlled oligonucleotide composition; and / or the intervals between multiple or consecutive doses thereof; and / or the total length or duration that a subject receives one or more doses thereof; and / or its specific formulation.

[0544] In some embodiments, the dosing regimen of WVE-003, an oligonucleotide composition of WVE-003, a chirality-controlled oligonucleotide composition of WVE-003, or a therapeutically effective amount of WVE-003, an oligonucleotide composition of WVE-003, a chirality-controlled oligonucleotide composition of WVE-003, or a therapeutically effective amount of WVE-003, an oligonucleotide composition of WVE-003, a chirality-controlled oligonucleotide composition of WVE-003, or a therapeutically effective amount of WVE-003, an oligonucleotide composition of WVE-003, a chirality-controlled oligonucleotide composition of WVE-003, is any dosing regimen described herein. In some embodiments, the dosing regimen includes, 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 administration, 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 a month, about once every 2 months, etc.) and / or a specific dosing length or duration (e.g., dosing occurs over a time span of at least about 1 month, about 1 month, about 2 months, 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.

[0545] In some embodiments, 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.

[0546] In some embodiments, a dose of WVE-003 is administered approximately monthly. In some embodiments, a dose of WVE-003 is administered approximately once every 2 months. In some embodiments, a dose of WVE-003 is administered approximately once every 8 weeks. In some embodiments, a dose of WVE-003 is administered approximately once every 12 weeks.

[0547] In some embodiments, a dose of WVE-003 is administered approximately monthly for at least about three months. In some embodiments, a dose of WVE-003 is administered approximately every two months for at least about four months. In some embodiments, a dose of WVE-003 is administered approximately every eight weeks for at least about 16 weeks. In some embodiments, a dose of WVE-003 is administered approximately every twelve weeks for at least about twelve weeks.

[0548] In some embodiments, a dose of WVE-003 is administered approximately monthly, and WVE-003 is administered prior to the first monthly dose, followed by a washout period of approximately 8 weeks (approximately 2 months). In some embodiments, a dose of WVE-003 is administered approximately every 2 months, and WVE-003 is administered prior to the first 2-monthly dose, followed by a washout period of approximately 12 weeks (approximately 3 months). In some embodiments, a dose of WVE-003 is administered approximately every 8 weeks, and WVE-003 is administered prior to the first 8-weekly dose, followed by a washout period of approximately 12 weeks (approximately 3 months).

[0549] In some embodiments, a dose of WVE-003 is administered, followed by a washout period of about 8 weeks (about 2 months), followed by a dose of WVE-003 about monthly. In some embodiments, a dose of WVE-003 is administered, followed by a washout period of about 12 weeks (about 3 months), followed by a dose of WVE-003 about every 2 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 a dose of WVE-003 about every 8 weeks.

[0550] In some embodiments, following one or more doses of oligonucleotide, a lumbar puncture (spinal tap) procedure is performed to obtain CSF for analysis.

[0551] In some embodiments, about 1 month (about 4 weeks) after the third monthly dose, a lumbar puncture (spinal tap) procedure is performed to obtain CSF for analysis. Certain Methods of Use of HTT Oligonucleotides or HTT Oligonucleotide Compositions In some embodiments, the disclosure provides a method for treating or preventing Huntington's disease, wherein the method comprises the step of administering to a subject an effective amount of an HTT oligonucleotide described herein or a composition thereof (e.g., WVE-003).

[0552] In some embodiments, the HTT transcript is a huntingtin gene or a variant thereof.

[0553] In some embodiments, HTT oligonucleotides can trigger 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 the provided compositions. In some embodiments, the present disclosure provides methods for reducing drug-induced vascular damage using the provided compositions.

[0554] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally approximately monthly at a dose of 10 to about 168 mg, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0555] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally at a dose of about 30 mg approximately monthly, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0556] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally approximately monthly at a dose of about 60 mg, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0557] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally at a dose of about 90 mg approximately monthly, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0558] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally approximately monthly at a dose of about 120 mg, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0559] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally approximately monthly at a dose of about 150 mg, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0560] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally approximately monthly at a dose of about 168 mg, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0561] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 30 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0562] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 60 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0563] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 90 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0564] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 120 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0565] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 150 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0566] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of about 168 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0567] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally at a dose of 30 mg approximately monthly, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0568] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally at a dose of 60 mg approximately monthly, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0569] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally at a dose of 90 mg approximately monthly, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0570] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally at a dose of 120 mg approximately monthly, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0571] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally at a dose of 150 mg approximately monthly, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0572] In some embodiments, the disclosure relates to: a method for treating a subject with Huntington's disease, the subject having an HTT gene mutation that is suitable for allele-specific reduction in the level, expression and / or activity of mHTT, the method comprising administering an HTT oligonucleotide or an HTT oligonucleotide composition to the subject intrathecally at a dose of 168 mg approximately monthly, such that the subject's disease progression is delayed, thereby treating the subject, wherein the HTT oligonucleotide or HTT oligonucleotide is capable of mediating a reduction in the level, expression and / or activity of the mHTT transcript.

[0573] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 30 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0574] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 60 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0575] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 90 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0576] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 120 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0577] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 150 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0578] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 168 mg, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0579] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 30 mg±5%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0580] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 60 mg±5%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0581] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 90 mg±5%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0582] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 120 mg±5%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0583] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 150 mg±5%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0584] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 168 mg±5%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0585] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 30 mg±10%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0586] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 60 mg±10%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0587] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 90 mg±10%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0588] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 120 mg±10%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0589] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 150 mg±10%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0590] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 168 mg±10%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0591] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 30 mg±15%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0592] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 60 mg±15%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0593] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 90 mg±15%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0594] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 120 mg±15%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0595] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 150 mg±15%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0596] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 168 mg±15%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0597] In some embodiments, the disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 30 mg±20%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0598] In some embodiments, the present disclosure relates to: a method for treating a subject having a mutant HTT gene, wherein the mutant HTT gene comprises a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: administering WVE-003 (or a salt form thereof) to the subject at a dose of 60 mg±20%, such that the progression of Huntington's disease in the subject is delayed, and / or the onset of Huntington's disease is delayed, and / or the severity of the symptoms of Huntington's disease is reduced.

[0599] In some embodiments, the present disclosure relates to: a method for treating a subject having a ...

Claims

1. A method for treating a subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: Administration of WVE-003 (or a salt form thereof) to the subject 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 results in a delay in progression of Huntington's disease in the subject, and / or a delay in the onset of Huntington's disease, and / or a reduction in the severity of symptoms of Huntington's disease.

2. A method for treating Huntington's disease in a subject having a mutant HTT gene, the mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the following steps: Administration of WVE-003 (or a salt form thereof) to the subject 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 results in a delay in disease progression and / or a delay in the onset of Huntington's disease in the subject and / or a reduction in the severity of symptoms of Huntington's disease.

3. A method for delaying the onset of Huntington's disease and / or reducing the severity of symptoms of Huntington's disease in a subject having Huntington's disease, wherein the subject has a mutant HTT gene comprising a mutation suitable for allele-specific knockdown of the mutant HTT gene, wherein the method comprises the step of administering WVE-003 (or a salt form thereof) to the subject 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.

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 has an HTT allele comprising an expanded CAG repeat region and fully complementary to the base sequence of WVE-003.

5. A method for treating Huntington's disease, comprising administering or delivering a pharmaceutical composition comprising 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 has an HTT allele comprising an expanded CAG repeat region and having a base sequence completely complementary to that 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 genetic sequence that is identical or completely complementary to the base sequence of WVE-003, optionally wherein the subject is determined to have a genetic 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 genetic sequence encoding a transcript comprising an expanded CAG repeat in HTT and fully 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 comprising an expanded CAG repeat and being completely complementary to the base sequence of WVE-003.

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

10. The method of any one of claims 5-9, 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. The method of any of the preceding claims, wherein the method further comprises the step of confirming that the subject has a mutation in the HTT gene that is suitable for allele-specific knockdown of the mutant HTT gene or its gene product transcript.

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

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

14. The method of any one of the preceding claims, wherein one or more pharmaceutically acceptable salt forms of WVE-003 are administered, and / or wherein the amount of WVE-003 comprises an amount of one or more pharmaceutically acceptable salt forms that are each independently converted to an amount of the acid form.

15. A method for treating Huntington's disease, comprising administering or delivering WVE-003 to a subject suffering from Huntington's disease 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 WVE-003 free acid form; or A method for preventing Huntington's disease, comprising administering or delivering WVE-003 to a subject suffering from Huntington's disease 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 WVE-003 free acid form; or A method comprising administering or delivering WVE-003 to a subject 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 WVE-003 free acid form; or A method for reducing the activity, expression, and / or level of a mutant HTT gene or its gene product in a subject, the method comprising administering or delivering WVE-003 to the subject 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 WVE-003 free acid form; or A method for preferentially knocking down HTT RNA transcripts containing repeat expansions relative to HTT RNA transcripts without repeat expansions in a subject, the method comprising administering or delivering WVE-003 to the subject 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 WVE-003 free acid form; or A method for reducing the level of an HTT transcript comprising a CAG repeat expansion in a subject, the method comprising administering or delivering WVE-003 to the subject 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 WVE-003 free acid form; or A method for reducing the level of a product of an HTT transcript comprising a CAG repeat expansion in a subject, the method comprising administering or delivering WVE-003 to the subject 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 WVE-003 free acid form; Where WVE-003 is: mG*S mUn001R mU mGn001R mA*ST*SC*ST*SG*ST*RA*SG*SC*SA*SG*Rm5Ceon001RAeoGeon001Rm5Ceo*STeo, where: m represents 2′-OMe modification of the nucleoside; *S represents Sp phosphorothioate linkage; m5Ceo represents 5-methyl 2′-O-methoxyethyl C; n001R represents an Rp n001 linkage, where the n001 linkage has the following structure: eo represents a 2'-OCH2CH2OCH3 modification to a nucleoside; and *R represents Rp phosphorothioate linkage.

16. The method of any of the preceding claims, wherein the dose of WVE-003 is administered in one or more forms, optionally wherein the dose of WVE-003 is administered in one or more pharmaceutically acceptable salt forms, optionally wherein one form is WVE-003 pentadecasodium salt.

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

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

19. The method of any one of the preceding claims, wherein each dose of WVE-003 is independently administered in one or more pharmaceutically acceptable salt forms, optionally one of which is WVE-003 pentadecasodium salt.

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

21. The method of any one of the preceding claims, wherein each dose of WVE-003 is administered independently in a pharmaceutical composition comprising or consisting of WVE-003 pentadecasodium salt and a pharmaceutically acceptable carrier.

22. A method for treating Huntington's disease, comprising administering or delivering WVE-003 pentadecasodium salt to a subject suffering from Huntington's disease at a dose of: 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 WVE-003 free acid form.

23. A method for preventing Huntington's disease, comprising administering or delivering WVE-003 pentadecasodium salt to a subject suffering from Huntington's disease 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 WVE-003 free acid form.

24. A method comprising administering or delivering to a subject WVE-003 pentadecasodium 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 WVE-003 free acid form; or A method for reducing the activity, expression and / or level of a mutant HTT gene or its gene product in a subject, the method comprising administering or delivering to the subject WVE-003 pentadecasodium 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 WVE-003 free acid form; or A method for preferentially knocking down HTT RNA transcripts containing repeat expansions relative to HTT RNA transcripts without repeat expansions in a subject, the method comprising administering or delivering to the subject WVE-003 pentadecasodium 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 WVE-003 free acid form; or A method for reducing the level of an HTT transcript comprising a CAG repeat expansion in a subject, the method comprising administering or delivering to the subject WVE-003 pentadecasodium 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 WVE-003 free acid form; or A method for reducing the level of a product of an HTT transcript comprising a CAG repeat expansion in a subject, the method comprising administering or delivering to the subject WVE-003 pentadecasodium 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 WVE-003 free acid form.

25. The method of any one of the preceding claims, wherein the product is a polypeptide, optionally a polypeptide comprising amplified poly-Q.

26. The method of any one of the preceding claims, wherein two or more doses are administered, e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more doses.

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

28. The method of any one of the preceding claims, wherein each dose of WVE-003 pentadecasodium salt is administered independently in a pharmaceutical composition comprising or consisting of WVE-003 and a pharmaceutically acceptable carrier.

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

30. The method of any one of the preceding claims, wherein the pharmaceutical composition has a pH of about 6-8, optionally wherein the pharmaceutical composition has a pH of about 6.4-7.2 or about 7.3 or about 7.

4.

31. The method of any one of claims 22-29, wherein the dose is equivalent to 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.

32. The method of any one of claims 22-29, wherein each dose is independently equivalent to 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.

33. The method of any one of the preceding claims, wherein two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more) consecutive 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 or more months, or about every 1, 2, 3, or 4 quarters.

34. The method of any of the preceding claims, wherein all doses are administered independently 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 or more months, or about every 1, 2, 3, or 4 quarters.

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

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

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

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

39. The method of any of the preceding claims, wherein expression of mutant HTT is reduced and / or mutant HTT protein levels are reduced, and / or wherein the level, expression and / or activity of a mutant HTT transcript or its gene product is reduced by at least about 5% or 10%.

40. The method of any of the preceding claims, wherein the level of mutant HTT transcript in the 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. The method of any of the preceding claims, wherein the level of mutant HTT polypeptide in the 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. The method of any of the preceding claims, wherein the level of wtHTT transcript or polypeptide is reduced by no more than about 10%, 20%, 30%, 40% or 50%.

43. The method of any of the preceding claims, wherein the method provides a reduction in the level of the HTT transcript containing the repeat expansion as measured by 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-fold greater than the reduction in the level of the HTT transcript without the repeat expansion as measured by a percentage.

44. The method of any of the preceding claims, wherein the reduction is about 10%, 12%, 15%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% or more; and / or wherein said reduction is assessed in an individual subject; and / or wherein the reduction is assessed in a population of subjects, optionally wherein the population size is about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 500, 1000 or more subjects, and / or wherein the subjects in the population receive the same or different dosing regimens; and / or One or more cerebrospinal fluid samples are utilized for reduction assessment.

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

46. The method of any one of the preceding claims, wherein 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 or more months after administration of the dose, and before the next dose, if any; and / or wherein the assessment 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. The method of any one of the preceding claims, wherein the onset of Huntington's disease and / or the severity of symptoms of Huntington's disease in the subject is delayed and / or reduced, and / or wherein the subject improves in one or more functional assessments.

48. The method of any of the preceding claims, wherein improvement is compared to baseline, no administration of WVE-003, or administration of a reference composition, optionally wherein the reference composition is comparable to the administered WVE-003 composition but does not contain WVE-003.

49. The method of any of the preceding claims, wherein WVE-003 is administered intrathecally and / or by direct lumbar injection.

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

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

52. The method of any of the preceding claims, wherein the subject receives or is exposed to an additional therapeutic agent.

53. The method of any of the preceding claims, wherein the subject is administered a steroid at least about one 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 a composition thereof is in solid form and / or is lyophilized.

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

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

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

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

60. The composition of any of the preceding claims, wherein the form of WVE-003 in the composition is a pharmaceutically acceptable salt form and / or WVE-003 pentadecasodium salt, or each form of WVE-003 in the composition is independently a salt form, optionally a pharmaceutically acceptable salt form and / or WVE-003 pentadecasodium salt.

61. The composition of any of the preceding claims, wherein the composition is a drug substance and / or a drug product.

62. The composition of any of the preceding claims, 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. The composition of any of the preceding claims, wherein the composition is isotonic, and / or wherein the composition has a pH of about 6-8, optionally wherein the composition has a pH of about 6.4-7.2, or about 7.3, or about 7.

4.

64. The composition of any preceding claim, wherein the composition is lyophilized WVE-003 powder.

65. The composition of any of the preceding claims, wherein the amount of WVE-003 in the composition is 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. A composition as claimed in any one of the preceding claims, wherein the composition is packaged in a vial.

67. The composition of any of the preceding claims, wherein the composition is reconstituted and diluted in artificial cerebrospinal fluid (aCSF).

68. The composition of any preceding claim, wherein the composition is a WVE-003 drug product, optionally wherein the WVE-003 drug product is lyophilized WVE-003 pentadecasodium salt.

69. A vial comprising the composition of any preceding claim, optionally wherein the vial is filled with an inert gas, optionally wherein the inert gas is nitrogen.

70. A syringe comprising the composition of any preceding claim, optionally wherein the composition is a liquid composition wherein WVE-003 or WVE-003 pentadecasodium salt is dissolved in aCSF.

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

72. The syringe of any one of claims 70-71, wherein the syringe contains the dose of WVE-003 described in any one of the preceding claims.

73. A method for making a WVE-003 composition according to the method described in the specification.

74. The method of claim 73, comprising utilizing IP-RP-UPLC to assess the purity and / or impurities in the manufactured WVE-003 composition, and releasing the formulation if the purity and / or impurities meet certain standards.

75. The method of any one of claims 73-74, wherein the composition is a drug substance or a drug product.

76. A method for releasing a WVE-003 formulation, the method comprising evaluating the purity and / or impurities in the WVE-003 formulation using IP-RP-UPLC, and releasing the formulation if the purity and / or impurities meet certain standards; or A method for assessing the purity of WVE-003 using IP-RP-UPLC.

77. The method of any one of claims 74-76, wherein the IP-RP-UPLC utilizes one or more parameters described in the instructions and / or one or more parameters of Group A.

78. A method for confirming the stereochemical characteristics of WVE-003 using IP-RP-UPLC.

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

80. The method or composition of any preceding claim, wherein the WVE-003 drug substance is the pentasodium salt.

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

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

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

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

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

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

87. A method or composition as described in any one of embodiments 1-433.

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