Compositions and methods for treating PCDH19-associated disorders

Abstract

Provided herein is an antisense oligonucleotide against a target region of the protocadherin 19 (PCDH19) gene.Antisense oligonucleotides or compositions comprising the same can be administered to subjects with PCDH19-related disorders, such as epilepsy, schizophrenia, or autism, to treat, alleviate, or prevent symptoms of PCDH19-related disorders.Therefore, compositions and methods useful for treating PCDH19-related disorders are also provided herein.

Description

[Technical field]

[0001] Related Applications This application claims priority to U.S. Provisional Application No. 63 / 248,803, entitled "Compositions and methods for the treatment of PCDH19 related disorders," filed on September 27, 2021, and U.S. Provisional Application No. 63 / 333,840, entitled "Compositions and methods for the treatment of PCDH19 related disorders," filed on April 22, 2022, the entire contents of which are incorporated by reference herein in their entireties.

[0002] The present invention relates to antisense oligonucleotides complementary to target regions of the human PCDH19 gene, its pre-mRNA transcripts, and / or its mRNA transcripts.Embodiments of the present invention also relate to compositions and methods for treating, alleviating at least one symptom, or preventing a PCDH19-associated disorder, such as epilepsy, schizophrenia, or autism. [Background technology]

[0003] Protocadherin 19 (PCDH19) is a cell surface protein expressed in neurons that may play a role in cell-cell adhesion. Mutations in the PCDH19 gene are associated with neurological disorders such as epilepsy, autism, and schizophrenia. Interestingly, PCDH19-associated early infantile epileptic encephalopathy 9 (EIEE9) is an X-linked disorder that affects heterozygous females but not hemizygous males. Mutations in PCDH19, although rare, may also affect mosaic male carriers. Although people with PCDH19 mutations can be diagnosed by genetic screening, there are currently no known therapies for treating PCDH19-associated disorders such as epilepsy. Thus, there is a need for new compositions and methods for treating these diseases. Summary of the Invention

[0004] In one aspect, the invention provides a compound comprising a single-stranded oligonucleotide 10-80 nucleosides in length, the single-stranded oligonucleotide having a nucleobase sequence that includes a portion of 10 contiguous nucleobases that has at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, or 100%) complementarity to an equal length portion of a target region of the human PCDH19 gene, its pre-mRNA transcript, and / or its mRNA transcript.

[0005] In certain embodiments, the target region is within the nucleotide sequence set forth in SEQ ID NO: 1 or a variant thereof having at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In certain embodiments, the single-stranded oligonucleotide hybridizes to a PCDH19 pre-mRNA transcript or mRNA transcript.

[0006] In some embodiments, the mRNA transcript or pre-mRNA transcript may comprise at least one PCDH19 mutation. The mutation may be, for example, a mutation encoding N340S, E307K, V441E, Q85X, N557K, D594H, S671X, L677fsx717, I119fsX122, and P364fsX375. The mutation may be a mutation selected from Table 1. The oligonucleotide may selectively hybridize to the mRNA transcript or pre-mRNA transcript comprising at least one PCDH19 mutation over the wild-type mRNA transcript or pre-mRNA transcript. Thus, the oligonucleotide may be an allele-specific oligonucleotide.

[0007] In some embodiments, the oligonucleotide consists of 12 to 40 (e.g., 15 to 30, e.g., 16 to 22, e.g., 15, 16, 17, 18, 19, 20, 21, or 22) nucleobases.

[0008] In some embodiments, the oligonucleotide comprises a gap segment comprising linked deoxyribonucleosides, a 5' wing segment comprising linked nucleosides, and a 3' wing segment comprising linked nucleosides. The gap segment can comprise a region of at least 10 contiguous nucleobases having at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, or 100%) complementarity to an equal length portion of a target region of a pre-mRNA transcript or mRNA transcript of the human PCDH19 gene located between the 5' wing segment and the 3' wing segment. Each of the 5' wing segment and the 3' wing segment comprises at least two linked nucleosides, and at least one nucleoside of each wing segment comprises an alternative nucleoside.

[0009] In some embodiments, the oligonucleotide comprises at least one alternative internucleoside linkage. At least one alternative internucleoside linkage can be a phosphorothioate internucleoside linkage. At least one alternative internucleoside linkage can be a 2'-alkoxy internucleoside linkage. At least one alternative internucleoside linkage can be an alkylphosphate internucleoside linkage.

[0010] In some embodiments, the oligonucleotide comprises at least one alternative nucleobase. The alternative nucleobase may be 5'-methylcytosine, pseudouridine, or 5-methoxyuridine.

[0011] In some embodiments, the oligonucleotide comprises at least one alternative sugar moiety, which can be a 2'-OMe modified sugar moiety or a bicyclic sugar moiety.

[0012] In some embodiments, the oligonucleotide further comprises a ligand conjugated to the 5' or 3' end of the oligonucleotide via a monovalent or branched divalent or trivalent linker.

[0013] In some embodiments, the oligonucleotide comprises a region complementary to at least 15 (eg, 15, 16, 17, 18, 19, 20, or 21) consecutive nucleotides of the PCDH19 gene.

[0014] In some embodiments, the oligonucleotide comprises a sequence set forth in any one of SEQ ID NOs: 2-385.

[0015] In another aspect, the invention provides a pharmaceutical composition comprising an oligonucleotide of any of the above embodiments and a pharma- ceutically acceptable carrier or excipient.

[0016] In another aspect, the invention provides a composition comprising an oligonucleotide of any of the above embodiments and a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.

[0017] In another aspect, the present invention provides a method of treating, preventing, or delaying the progression of a PCDH19-associated disorder in a subject in need thereof by administering to the subject an oligonucleotide, pharmaceutical composition, or composition of any of the above embodiments in an amount and for a duration sufficient to treat, prevent, or delay the progression of the PCDH19-associated disorder.

[0018] Also provided herein is the use of an oligonucleotide of the invention in the manufacture of a medicament for treating, preventing, or delaying the progression of a PCDH19-associated disorder.

[0019] In another aspect, the invention provides a method for inhibiting transcription of PCDH19 in a cell of a subject having a PCDH19-associated disorder by contacting the cell with an oligonucleotide, pharmaceutical composition, or composition of any of the above embodiments in an amount and for a period of time sufficient to obtain degradation of the mRNA transcript of the PCDH19 gene, such that the oligonucleotide inhibits expression of the PCDH19 gene in the cell.

[0020] The cells may be contacted in vivo or ex vivo.

[0021] In another aspect, the invention provides a method for reducing the level and / or activity of PCDH19 in a cell of a subject having a PCDH19-associated disorder by contacting the cell with an oligonucleotide, pharmaceutical composition, or composition of any of the above embodiments in an amount and for a period of time sufficient to reduce the level and / or activity of PCDH19 in the cell.

[0022] The cells may be contacted in vivo or ex vivo.

[0023] In some embodiments of the above aspects, the subject is a human. The subject may be male. The subject may be female.

[0024] In some embodiments of the above aspects, the cell is a cell of the central nervous system.

[0025] In some embodiments of the above aspects, the PCDH19-associated disorder is selected from the group consisting of epilepsy, schizophrenia, and autism.

[0026] In some embodiments of the above aspects, the PCDH19-associated disorder is epilepsy. The epilepsy can be epileptic early infantile encephalopathy 9.

[0027] In some embodiments of the above aspects, the subject has a mutation in at least one allele of the PCDH19 gene. The mutation can be heterozygous (e.g., if the subject is female). The mutation can be hemizygous (e.g., if the subject is male). If the subject is male, the subject can have a mosaic mutation, in which a subset of cells contain the mutation. The mutation can be a missense or nonsense mutation. The mutation can be a frameshift mutation. The mutation can be an insertion or deletion.

[0028] In some embodiments of the above aspects, the oligonucleotide selectively reduces expression of an allele that contains the mutation compared to an allele that does not contain the mutation.

[0029] In some embodiments of the above aspects, the oligonucleotide reduces expression of the allele that contains the mutation and the allele that contains the wild-type sequence.

[0030] In some embodiments of the above aspects, the treatment reduces one or more symptoms of a PCDH19-associated disorder. The one or more symptoms of a PCDH19-associated disorder may be selected from the group consisting of prolonged seizures, frequent seizures, behavioral and developmental delays, movement and balance problems, orthopedic conditions, speech and language delay problems, growth and nutrition problems, sleep disorders, chronic infections, sensory integration disorders, autonomic nervous system disorders, and sweating. [Brief description of the drawings]

[0031] Exemplary embodiments of the present disclosure are herein described, by way of non-limiting example only, with reference to the following drawings:

[0032] [Figure 1-1] Relative PCDH19 mRNA expression in HEK293 cells 48 hours after transfection with 0.2 nM (gray bars) and 2.0 nM (black bars) of an exemplary antisense oligonucleotide (ASO). Data for PCDH19 mRNA is normalized to GAPDH mRNA and expressed relative to the pooled mean of mock-transfected cells and cells transfected with Ahsa1 ASO, which does not cross-react with PCDH19. Data are shown as mean and standard deviation calculated from n=4 separate experiments. ASO numbering (abscissa values) corresponds to the ASO numbering shown in Table 2. [Figure 1-2]Relative PCDH19 mRNA expression in HEK293 cells 48 hours after transfection with 0.2 nM (gray bars) and 2.0 nM (black bars) of an exemplary antisense oligonucleotide (ASO). Data for PCDH19 mRNA is normalized to GAPDH mRNA and expressed relative to the pooled mean of mock-transfected cells and cells transfected with Ahsa1 ASO, which does not cross-react with PCDH19. Data are shown as mean and standard deviation calculated from n=4 separate experiments. ASO numbering (abscissa values) corresponds to the ASO numbering shown in Table 2.

[0033] SEQ ID NO: 1 shows the nucleotide sequence of the human PCDH19 gene of the NCBI reference sequence NC_000023.11 (region 100291644.100410273). Exemplary ASOs of the invention are shown in SEQ ID NOs: 2-385 (Table 2).

[0034] definition For convenience, the meanings of some terms and phrases used in the specification, examples, and appended claims are provided below. Unless otherwise stated or implied from the context, the following terms and phrases include the meanings provided below. The definitions are provided to help explain certain embodiments and are not intended to limit the claimed technology, since the scope of the technology is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the technology belongs. If there is an apparent discrepancy between the usage of a term in the art and its definition provided herein, the definition provided herein shall prevail.

[0035] In this application, unless otherwise clear from the context, (i) the term "a" may be understood to mean "at least one," (ii) the term "or" may be understood to mean "and / or," and (iii) the terms "including" and "comprising" may be understood to encompass the listed elements or steps, whether presented alone or together with one or more additional elements or steps.

[0036] As used herein, the terms "about" and "approximately" refer to values ​​within 10% above or below the stated value. For example, the term "about 5 nM" indicates a range of 4.5 to 5.5 nM.

[0037] The term "at least" before a number or series of numbers is understood to include the number adjacent to the term "at least" and all subsequent numbers or integers that may be logically included, as is clear from the context. For example, the number of nucleotides in a nucleic acid molecule must be an integer. For example, "at least 18 nucleotides of a 21-nucleotide nucleic acid molecule" means that 18, 19, 20, or 21 nucleotides have the indicated property. When at least is present before a series or range, it is understood that "at least" can modify each of the numbers in the series or range.

[0038] As used herein, "less than" or "less than" is understood as the value adjacent to the phrase, logically from the context, up to 0, and the logically lower value or integer. For example, an oligonucleotide having "3 or less mismatches to the target sequence" has 3, 2, 1, or 0 mismatches to the target sequence. When "less than" is present before a series of numbers or ranges, it is understood that "less than" can modify each of the numbers in the series or range.

[0039] Throughout this specification and the claims which follow, unless the context requires otherwise, the word "comprise" and variations such as "comprises" and "comprising" are understood to mean the inclusion of a stated integer or step or group of integers or steps but not to the exclusion of any other integer or step or group of integers or steps.

[0040] As used herein, the term "administration" refers to administration of a composition (e.g., a compound described herein or a preparation containing a compound) to a subject or system. Administration to an animal subject (e.g., a human) can be by any suitable route, such as those described herein.

[0041] As used herein, "combination therapy" or "administered in combination / co-administered" means that two (or more) different agents or treatments are administered to a subject as part of a defined treatment regimen for a particular disease or condition. The treatment regimen specifies the dosage and periodicity of administration of each agent such that the effects of the separate agents on the subject overlap. In some embodiments, the delivery of two or more agents is simultaneous or concurrent, and the agents may be co-formulated. In some embodiments, the two or more agents are not co-formulated, but are administered sequentially as part of a prescribed regimen. In some embodiments, the co-administration of two or more agents or treatments is such that the reduction in symptoms or other parameters associated with the disease is greater than that observed when one agent or treatment is delivered alone or in the absence of the other agent or treatment. The effect of the two treatments may be partially additive, fully additive, or greater than additive (e.g., synergistic). Sequential or substantially simultaneous administration of each therapeutic agent can be performed by any suitable route, including, but not limited to, oral, intravenous, intramuscular, and direct absorption through mucosal tissue. The therapeutic agents can be administered by the same route or by different routes. For example, a first therapeutic agent of the combination can be administered by intravenous injection, while a second therapeutic agent of the combination can be administered orally.

[0042] As used herein, the terms "PCDH19" and "protocadherin 19" refer to a calcium-dependent cell adhesion protein that is primarily expressed in the developing brain. PCDH19 may have an amino acid sequence from any vertebrate or mammalian source, including, but not limited to, human, bovine, chicken, rodent, mouse, rat, porcine, ovine, primate, monkey, and guinea pig, unless otherwise specified. The term also refers to fragments and variants of native PCDH19 that maintain at least one in vivo or in vitro activity of native PCDH19. The term encompasses the full-length unprocessed precursor form of PCDH19 as well as the mature form resulting from post-translational cleavage of the signal peptide. PCDH19 is encoded by the PCDH19 gene. Nucleic acid sequences of exemplary human PCDH19 genes are set forth in NCBI reference numbers NC_000023.11, NG_021319.1, NM_001105243.1, NM_001184880.1, and NM_020766.2. The term "PCDH19" also refers to naturally occurring variants of wild-type PCDH19 proteins, such as proteins having at least 85% identity (e.g., 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9% or more identity) to the amino acid sequence of wild-type human PCDH19.

[0043] The term "PCDH19" as used herein also refers to a specific polypeptide expressed in cells due to naturally occurring DNA sequence variation of PCDH19 gene, such as single nucleotide polymorphism in PCDH19 gene.A number of SNPs in PCDH19 gene have been identified and can be found, for example, in NCBI dbSNP (see, for example, www.ncbi.nlm.nih.gov / snp).

[0044] As used herein, "target sequence" refers to a contiguous portion of the nucleotide sequence of an mRNA molecule formed during transcription of the PCDH19 gene, including mRNA that is a product of RNA processing of a primary transcript. In one embodiment, the target portion of the sequence is at least long enough to serve as a substrate for oligonucleotide-directed (e.g., antisense oligonucleotide (ASO)-directed) cleavage at or near that portion of the nucleotide sequence of an mRNA molecule formed during transcription of the PCDH19 gene. The target sequence can be, for example, about 9-36 nucleotides in length, e.g., about 15-30 nucleotides in length, e.g., about 18-22 nucleotides in length. For example, the target sequence may be about 15-30 nucleotides in length, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 1 The length may be 9-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 nucleotides. Ranges and lengths intermediate to the above listed ranges and lengths are also considered part of the invention.

[0045] "G", "C", "A", "T" and "U" each generally represent naturally occurring nucleotides containing guanine, cytosine, adenine, thymidine and uracil, respectively, as bases. However, it is understood that the term "nucleotide" can also refer to alternative nucleotides or surrogate replacement moieties, as further detailed below. Those skilled in the art are well aware that guanine, cytosine, adenine and uracil can be substituted by other moieties without substantially changing the base pairing properties of an oligonucleotide containing a nucleotide with such a replacement moiety. For example, but not limited to, a nucleotide containing inosine as a base can base pair with a nucleotide containing adenine, cytosine or uracil. Thus, a nucleotide containing uracil, guanine or adenine can be substituted, for example, by a nucleotide containing inosine in the nucleotide sequence of an oligonucleotide featured in the present invention. In another example, any adenine and cytosine anywhere in the oligonucleotide can be substituted with guanine and uracil, respectively, to form a GU wobble base pair with a target mRNA. Sequences containing such replacement moieties are suitable for the compositions and methods featured in the invention.

[0046] The terms "nucleobase" and "base" include purine (e.g., adenine and guanine) and pyrimidine (e.g., uracil, thymine, and cytosine) moieties present in nucleosides and nucleotides that form hydrogen bonds during nucleic acid hybridization. In the context of the present invention, the term nucleobase also encompasses alternative nucleobases that may differ from naturally occurring nucleobases but are functional during nucleic acid hybridization. In this context, "nucleobase" refers to both naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine, and hypoxanthine, as well as alternative nucleobases. Such variants are described, for example, in Hirao et al (2012) Accounts of Chemical Research vol.45 page 2055 and Bergstrom (2009) Current Protocols in Nucleic Acid Chemistry Suppl.37 1.4.1.

[0047] The term "nucleoside" refers to a monomeric unit of an oligonucleotide or polynucleotide having a nucleobase and a sugar moiety. Nucleosides can include naturally occurring nucleosides as well as alternative nucleosides such as those described herein. The nucleobase of a nucleoside can be a naturally occurring nucleobase or an alternative nucleobase. Similarly, the sugar moiety of a nucleoside can be a naturally occurring sugar or an alternative sugar.

[0048] The term "alternative nucleoside" refers to a nucleoside having an alternative sugar or alternative nucleobase, such as those described herein.

[0049] In some embodiments, the nucleobase moiety is modified by changing the purine or pyrimidine to a modified purine or pyrimidine, e.g., a substituted purine or substituted pyrimidine, e.g., an "alternate nucleobase" selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiozolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uridine, 5-bromouridine, 5-thiazolo-uridine, 2-thio-uridine, pseudouridine, 1-methylpseudouridine, 5-methoxyuridine, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine, and 2-chloro-6-aminopurine.

[0050] Nucleobase moieties may be designated by the letter code for each corresponding nucleobase, e.g., A, T, G, C, or U, and each letter may optionally include alternative nucleobases of equivalent function. In some embodiments, e.g., for gapmers, 5-methylcytosine LNA nucleosides may be used.

[0051] "Sugar" or "sugar moiety" includes naturally occurring sugars having a furanose ring. Sugar also includes "sugar substitutes," defined as structures that can replace the furanose ring of a nucleoside. In certain embodiments, sugar substitutes are non-furanose (or 4'-substituted furanose) rings or ring systems or open systems. Such structures can include simple changes to the natural furanose ring, e.g., a six-membered ring, or can be more complex, as in the case of the acyclic systems used in peptide nucleic acids. Sugar substitutes can also include sugar surrogates in which the furanose ring is replaced with another ring system, such as, for example, a morpholino or hexitol ring system. Sugar moieties useful for preparing oligonucleotides having a motif include, but are not limited to, β-D-ribose, β-D-2'-deoxyribose, substituted sugars (such as 2', 5' and bis-substituted sugars), 4'-S-sugars (such as 4'-S-ribose, 4'-S-2'-deoxyribose and 4'-S-2'-substituted ribose), bicyclic sugar surrogates (such as bicyclic sugars derived from 2'-O-CH2-4' or 2'-O-(CH2)2-4' bridged ribose) and sugar surrogates (such as when the ribose ring is replaced with a morpholino or hexitol ring system). The type of heterocyclic base and internucleoside linkage used at each position is variable and is not a determining factor of the motif. In most nucleosides having surrogate sugar moieties, the heterocyclic nucleobase is generally maintained to allow hybridization.

[0052] As used herein, a "nucleotide" refers to a monomeric unit of an oligonucleotide or polynucleotide that includes a nucleoside and an internucleoside linkage. An internucleoside linkage may or may not include a phosphate linkage. Similarly, a "linked nucleoside" may or may not be linked by a phosphate linkage. Many "alternative internucleoside linkages" are known in the art, including, but not limited to, phosphate linkages, phosphorothioate linkages, and boronophosphate linkages. Alternative nucleosides include bicyclic nucleosides (BNAs) (e.g., locked nucleosides (LNAs) and constrained ethyl (cEt) nucleosides), peptide nucleosides (PNAs), phosphotriesters, phosphorothioates, phosphoramidates, and other variants of the phosphate backbone of natural nucleosides, including those described herein.

[0053] As used herein, "alternative nucleotide" refers to a nucleotide having an alternative nucleoside or sugar and an internucleoside linkage that may include alternative nucleoside or sugar linkages.

[0054] The terms "oligonucleotide" and "polynucleotide" as used herein are defined as molecules that contain two or more covalently linked nucleosides as generally understood by those skilled in the art. Such covalently linked nucleosides can also be referred to as nucleic acid molecules or oligomers. The term "oligonucleotide" refers to short polynucleotides (typically 100 or less linked nucleosides). Oligonucleotides are generally made in the laboratory by solid-phase chemical synthesis followed by purification. When referring to the sequence of an oligonucleotide, reference is made to the sequence or order of the nucleobase moieties of the covalently linked nucleotides or nucleosides or modifications thereof. The oligonucleotides of the present invention may be artificial, chemically synthesized, and typically purified or isolated. Oligonucleotides are also intended to include compounds with (i) one or more furanose moieties replaced by furanose derivatives or any structure, cyclic or acyclic, that can be used as a covalent attachment point for the base moiety, (ii) one or more phosphodiester linkages, modified as in the case of phosphoramidate or phosphorothioate linkages, or completely replaced by suitable linkages as in the case of formacetal or riboacetal linkages, and / or (iii) one or more linked furanose-phosphodiester linkages, replaced by any structure, cyclic or acyclic, that can be used as a covalent attachment point for the base moiety. The oligonucleotides of the present invention may include one or more alternative nucleosides or nucleotides, including, for example, those described herein. It is also understood that oligonucleotides include compositions that lack sugar moieties or nucleobases, but can still pair with or hybridize to target sequences.

[0055] In the context of this disclosure, the terms "compound" and "oligonucleotide" may be used interchangeably. However, falling within the scope of the term "compound" may be a compound that includes or comprises an oligonucleotide disclosed herein and further includes or comprises one or more additional moieties.

[0056] A "chimeric" oligonucleotide or "chimera", in the context of this invention, is an oligonucleotide that contains two or more chemically distinct regions, each composed of at least one monomeric unit, i.e., in the case of oligonucleotides, a nucleotide or nucleoside. Chimeric oligonucleotides also include "gapmers".

[0057] The oligonucleotides can be of any length that allows for specific degradation of the desired target RNA via the RNase H mediated pathway, and can be about 10-30 base pairs in length, e.g., about 15-30 base pairs in length, or about 16-22 (e.g., 18-20) base pairs in length, e.g., about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 base pairs in length, e.g., about 15-30, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-40, 18-50, 18-60, 18-70, 18-80, 18-90, 18-19, 18-21 ...18, 18-21, 18-30, 18-40, 18-50, 18-60, 0, 18-29, 18-28, 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, The length may be in the range of 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22 base pairs. Ranges and lengths intermediate to the above listed ranges and lengths are also considered part of the invention.

[0058] As used herein, the term "oligonucleotide comprising a nucleobase sequence" refers to an oligonucleotide that comprises a chain of nucleotides or nucleosides described by a sequence referenced using standard nucleotide nomenclature.

[0059] The term "contiguous nucleobase region" refers to a region of an oligonucleotide that is complementary to a target nucleic acid. This term may be used interchangeably herein with the term "contiguous nucleotide sequence" or "contiguous nucleobase sequence". In some embodiments, all nucleotides of an oligonucleotide are present in a contiguous nucleotide or nucleoside region. In some embodiments, an oligonucleotide comprises a contiguous nucleotide region and may optionally comprise a nucleotide linker region that may be used to attach additional nucleotides or nucleosides, such as functional groups, to the contiguous nucleotide sequence. The nucleotide linker region may or may not be complementary to the target nucleic acid. In some embodiments, the internucleoside linkages present between the nucleotides of the contiguous nucleotide region are all phosphorothioate internucleoside linkages. In some embodiments, the contiguous nucleotide region comprises one or more sugar-modified nucleosides.

[0060] The term "gapmer" as used herein refers to an oligonucleotide that includes a region of an RNase H recruiting oligonucleotide (the gap) flanked at 5' and 3' by regions that include one or more affinity enhancing alternative nucleosides (wings or flanks). Various gapmer designs are described herein. Headmers and tailmers are oligonucleotides capable of recruiting RNase H where one of the wings is missing, i.e., only one of the ends of the oligonucleotide includes an affinity enhancing alternative nucleoside. For headmers, the 3' wing is missing (i.e., the 5' wing includes the affinity enhancing alternative nucleoside) and for tailmers, the 5' wing is missing (i.e., the 3' wing includes the affinity enhancing alternative nucleoside). A "mixed winged gapmer" refers to a gapmer in which the wing region comprises at least one alternative nucleoside, such as at least one DNA nucleoside or at least one 2'-substituted alternative nucleoside, such as 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA (MOE), 2'-amino-DNA, 2'-fluoro-RNA, 2'-F-ANA nucleoside, or a bicyclic nucleoside (e.g., a locked nucleoside or a constrained ethyl (cEt) nucleoside). In some embodiments, a mixed winged gapmer has one wing that comprises an alternative nucleoside (e.g., 5' or 3') and the other wing (3' or 5', respectively) comprises a 2'-substituted alternative nucleoside.

[0061] The term "linker" or "linking group" refers to a connection between two atoms that connects one chemical group or segment of interest to another chemical group or segment of interest through one or more covalent bonds. The conjugate moiety can be attached to the oligonucleotide directly or through a linking moiety (e.g., a linker or tether). The linker serves to covalently connect a third region, e.g., the conjugate moiety, to the oligonucleotide (e.g., at the end of region A or C). In some embodiments of the present invention, the conjugate or oligonucleotide conjugate of the present invention may optionally include a linker region located between the oligonucleotide and the conjugate moiety. In some embodiments, the linker between the conjugate and the oligonucleotide is biocleavable. Phosphodiester-containing biocleavable linkers are described in more detail in WO 2014 / 076195, which is incorporated herein by reference.

[0062] As used herein, unless otherwise indicated, the term "complementary" when used to describe a first nucleotide or nucleoside sequence relative to a second nucleotide or nucleoside sequence refers to the ability of an oligonucleotide or polynucleotide comprising the first nucleotide or nucleoside sequence to hybridize and form a duplex structure with an oligonucleotide or polynucleotide comprising the second nucleotide sequence under certain conditions, as understood by those skilled in the art. Such conditions can be, for example, stringent conditions, which can include 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mM EDTA, 50°C, or 70°C for 12-16 hours, followed by washing (see, for example, "Molecular Cloning: A Laboratory Manual", Sambrook, et al. (1989) Cold Spring Harbor Laboratory Press). Other conditions, such as physiologically relevant conditions that may be encountered inside an organism, can be applied. One skilled in the art can determine the most appropriate set of conditions for testing the complementarity of two sequences according to the ultimate use of the hybridized nucleotide or nucleoside.

[0063] As used herein, a "complementary" sequence may also include or be formed entirely of non-Watson-Crick base pairs and / or base pairs formed from non-natural and alternative nucleotides or nucleosides, so long as the above requirements for ability to hybridize are met. Such non-Watson-Crick base pairs include, but are not limited to, G:U wobble base pairs or Hoogsteen base pairs. Complementary sequences between oligonucleotides and target sequences described herein include base pairing of an oligonucleotide or polynucleotide containing a first nucleotide or nucleoside sequence to an oligonucleotide or polynucleotide containing a second nucleotide or nucleoside sequence over the entire length of one or both nucleotide or nucleoside sequences. Such sequences may be referred to herein as "fully complementary" to each other. However, when a first sequence is referred to herein as being "substantially complementary" with respect to a second sequence, the two sequences may be fully complementary, or may form one or more, but generally no more than 5, 4, 3, or 2 mismatched base pairs upon hybridization for a duplex of up to 30 base pairs, while retaining the ability to hybridize under conditions most relevant to their ultimate use, e.g., inhibition of gene expression via the RNase H-mediated pathway. "Substantially complementary" may also refer to a polynucleotide that is substantially complementary to a continuous portion of an mRNA of interest (e.g., an mRNA encoding PCDH19). For example, if a sequence is substantially complementary to an uninterrupted portion of an mRNA encoding PCDH19, then the polynucleotide is complementary to at least a portion of the PCDH19 mRNA.

[0064] As used herein, the term "complementarity region" refers to a region on an oligonucleotide that is substantially complementary to all or a portion of a gene, primary transcript, sequence (e.g., a target sequence, e.g., a PCDH19 nucleotide sequence), or processed mRNA so as to disrupt expression of an endogenous gene (e.g., PCDH19). When the complementary region is not completely complementary to the target sequence, mismatches may occur in the internal or terminal regions of the molecule. In general, mismatches are most tolerated in the terminal regions, e.g., within 5, 4, 3, or 2 nucleotides of the 5' and / or 3' ends of the oligonucleotide.

[0065] "Percent (%) sequence identity" with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence after aligning the sequences and introducing gaps as necessary to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be accomplished in a variety of ways that are within the capabilities of those skilled in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms necessary to achieve maximum alignment over the entire length of the sequences being compared. For example, percent sequence identity values ​​can be generated using the sequence comparison computer program BLAST. By way of illustration, the percent sequence identity of a given nucleic acid or amino acid sequence A to, with, or against a given nucleic acid or amino acid sequence B (which can alternatively be expressed as a given nucleic acid or amino acid sequence A having a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence B) is calculated as follows: 100×(fraction X / Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in a programmatic alignment of A and B, and where Y is the total number of nucleic acids in B. It will be understood that if the length of nucleic acid or amino acid sequence A is not equal to the length of nucleic acid or amino acid sequence B, then the percent sequence identity of A to B will not equal the percent sequence identity of B to A.

[0066] As used herein, "hybridization" refers to the pairing of substantially complementary strands of nucleic acid, such as between an antisense oligonucleotide of the present invention and a PCDH19 nucleotide sequence, such as an mRNA or pre-mRNA transcript. One mechanism of pairing involves hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonds, between complementary nucleobases of a strand of nucleic acid. For example, adenine and thymine or uracil are complementary nucleotides that pair through the formation of hydrogen bonds. Hybridization can occur under a variety of circumstances. As used herein, the reference to "specifically hybridize" means that an antisense oligonucleotide hybridizes to a target region in one PCDH19 allele, such as a mutant PCDH19 allele, and does not hybridize to the same target region in another PCDH19 allele, such as a wild-type PCDH19 allele.

[0067] As used herein, "agent that reduces the level and / or activity of PCDH19" refers to any polynucleotide agent (e.g., oligonucleotide, e.g., ASO) that reduces the level or inhibits the expression of PCDH19 in a cell or subject. As used herein, the phrase "inhibits expression of PCDH19" includes inhibition of expression of mutant or variant forms of PCDH19 gene that encodes PCDH19 protein, in addition to inhibition of any PCDH19 gene (e.g., mouse PCDH19 gene, rat PCDH19 gene, monkey PCDH19 gene, or human PCDH19 gene, etc.). Thus, the PCDH19 gene can be a wild-type PCDH19 gene, a mutant PCDH19 gene (e.g., an insertion, deletion, nonsense mutation, missense mutation, frameshift mutation), or a transgenic PCDH19 gene in the context of a genetically engineered cell, cell group, or organism.

[0068] "Reducing the activity of PCDH19" means reducing the level of an activity (e.g., ion channel function) associated with PCDH19. The activity level of PCDH19 can be measured using any method known in the art (e.g., using standard biophysical methods).

[0069] "Reducing the level of PCDH19" means decreasing the amount of PCDH19 in a cell or a subject, for example, by administering an oligonucleotide to the cell or subject. The level of PCDH19 can be measured using any method known in the art (e.g., by measuring the level of PCDH19 mRNA or the level of PCDH19 protein in a cell or subject).

[0070] As used herein, the term "inhibitor" refers to any agent that reduces the level and / or activity of a protein (e.g., PCDH19). Non-limiting examples of inhibitors include polynucleotides (e.g., oligonucleotides, e.g., ASOs). As used herein, the term "inhibit" is used interchangeably with "reduce," "silence," "downregulate," "suppress," and other similar terms, and includes any level of inhibition.

[0071] As used herein, the phrase "contacting a cell with an oligonucleotide", such as an oligonucleotide, includes contacting a cell by any possible means. Contacting a cell with an oligonucleotide includes contacting a cell with an oligonucleotide in vitro or contacting a cell with an oligonucleotide in vivo. Contacting can be performed directly or indirectly. Thus, for example, the oligonucleotide can be physically contacted with the cell by the individual performing the method, or the oligonucleotide agent can be placed in a situation that allows or causes it to subsequently contact the cell.

[0072] The contacting of cells in vitro or ex vivo can be carried out, for example, by incubating the cells with oligonucleotide. The contacting of cells in vivo can be carried out, for example, by injecting the oligonucleotide into or near the tissue where the cells are located, or by injecting the oligonucleotide agent into another area, for example, the bloodstream or subcutaneous space, so that the agent then reaches the tissue where the cells to be contacted are located. For example, the oligonucleotide can contain and / or be bound to a ligand, for example, GalNAc3, that directs the oligonucleotide to the site of interest, for example, the liver. A combination of in vitro, ex vivo, and in vivo contact methods is also possible. For example, the cells can also be contacted with the oligonucleotide in vitro and then transplanted into a subject.

[0073] In one embodiment, contacting a cell with an oligonucleotide includes "introducing" or "delivering" the oligonucleotide to a cell by promoting or achieving uptake or absorption into the cell. Absorption or uptake of the ASO can occur via unassisted diffusion or active cellular processes, or by auxiliary agents or devices. Introduction of the oligonucleotide into a cell can be in vitro, ex vivo, and / or in vivo. For example, for in vivo introduction, the oligonucleotide can be injected into a tissue site or administered systemically. In vitro introduction into a cell includes methods known in the art, such as electroporation and lipofection. Further approaches are described herein below and / or known in the art.

[0074] As used herein, "lipid nanoparticles" or "LNPs" are vesicles that contain a lipid layer that encapsulates pharma- ceutically active molecules, such as nucleic acid molecules, e.g., oligonucleotides. LNPs refer to stable nucleic acid-lipid particles. LNPs typically contain cationic lipids, non-cationic lipids, and lipids that prevent particle aggregation (e.g., PEG-lipid conjugates). LNPs are described, for example, in U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, and 8,058,069, the entire contents of which are incorporated herein by reference.

[0075] As used herein, the term "liposome" refers to a vesicle composed of amphiphilic lipids arranged in at least one bilayer (e.g., one bilayer or multiple bilayers). Liposomes include unilamellar and multilamellar vesicles with a membrane formed from a lipophilic material and an aqueous interior. The aqueous portion contains the oligonucleotide composition. The lipophilic material isolates the aqueous interior from the aqueous exterior, which typically does not contain the oligonucleotide composition, but may in some instances. Liposomes also include "sterically stabilized" liposomes, which as used herein refers to liposomes that contain one or more specialized lipids that, when incorporated into the liposome, result in an extended circulation life compared to liposomes lacking such specialized lipids.

[0076] A "micelle" is defined herein as a particular type of molecular assembly in which amphiphilic molecules are arranged in a spherical structure such that all the hydrophobic parts of the molecules are directed inward, leaving the hydrophilic parts in contact with the surrounding aqueous phase. The reverse arrangement exists when the environment is hydrophobic.

[0077] As used herein, the term "antisense" refers to a nucleic acid, including an oligonucleotide or polynucleotide, that is sufficiently complementary to all or a portion of a gene, primary transcript, or processed mRNA to disrupt the expression of an endogenous gene (e.g., PCDH19). A "complementary" polynucleotide is one that can base pair according to standard Watson-Crick complementarity rules. Specifically, purines base pair with pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. It is understood that two polynucleotides can hybridize to each other even if they are not completely complementary to each other, so long as each has at least one region that is substantially complementary to the other.

[0078] As used herein, the terms "effective amount", "therapeutically effective amount" and "sufficient amount" of an agent that reduces the level and / or activity of PCDH19 described herein (e.g., in a cell or a subject) refer to an amount sufficient to produce beneficial or desired results, including clinical results, when administered to a subject, including a human, and thus "effective amount" or its synonyms vary depending on the context in which it is applied. For example, in the context of treating a PCDH19-associated disorder, it is the amount of an agent that reduces the level and / or activity of PCDH19 that is sufficient to achieve a therapeutic response compared to the response obtained without administering the agent that reduces the level and / or activity of PCDH19. The amount of a given agent that reduces the level and / or activity of PCDH19 described herein that corresponds to such an amount varies depending on various factors, such as the given agent, pharmaceutical formulation, route of administration, type of disease or disorder, identity (e.g., age, sex, and / or weight) or host of the subject to be treated, but can nevertheless be routinely determined by one of ordinary skill in the art. Also, as used herein, the "therapeutically effective amount" of the agent that reduces the level and / or activity of PCDH19 of the present disclosure is an amount that produces a beneficial or desired result in a subject compared to a control. As defined herein, the therapeutically effective amount of the agent that reduces the level and / or activity of PCDH19 of the present disclosure can be easily determined by those skilled in the art by routine methods known in the art. Dosage regimens can be adjusted to obtain optimal therapeutic response.

[0079] A "prophylactically effective amount," as used herein, is intended to include an amount of oligonucleotide that is sufficient to prevent or ameliorate a disease or one or more symptoms of a disease when administered to a subject having or predisposed to having a PCDH19-associated disorder. Ameliorating a disease includes slowing the course of the disease or reducing the severity of a disease that subsequently develops. A "prophylactically effective amount" may vary depending on the oligonucleotide, the manner in which the agent is administered, the degree of risk of the disease, and the medical history, age, weight, family history, genetic makeup, type of prior or concomitant treatment (if any), and other individual characteristics of the subject being treated. A prophylactically effective amount also refers to, for example, an amount of an agent that reduces the level and / or activity of PCDH19 (e.g., in a cell or subject) as described herein, and refers to an amount that, when administered to a subject, including a human, is sufficient to delay the onset of a PCDH19-associated disorder as described herein by at least 120 days, e.g., at least 6 months, at least 12 months, at least 2 years, at least 3 years, at least 4 years, at least 5 years, at least 10 years, or more, compared to the expected onset.

[0080] A "therapeutically effective amount" or a "prophylactically effective amount" also includes an amount of oligonucleotide (either administered in a single dose or in multiple doses) that produces some desired local or systemic effect at a reasonable benefit / risk ratio applicable to any treatment. The oligonucleotides used in the methods of the invention may be administered in an amount sufficient to produce a reasonable benefit / risk ratio applicable to such treatment.

[0081] As used herein, the term "subject identified as having a PCDH19-related disorder" refers to a subject identified as having a molecular or pathological state, disease or symptom of a PCDH19-related disorder, such as identification of a PCDH19-related disorder or a symptom thereof, or to identification of a subject having or suspected of having a PCDH19-related disorder who may benefit from a particular treatment regimen.

[0082] As used herein, "PCDH19-associated disorder" refers to a class of genetic diseases or disorders characterized by abnormal function of PCDH19. PCDH19-associated disorders include, for example, schizophrenia, autism, and epilepsy. For example, epilepsy can be epileptic encephalopathy, including early infantile epileptic encephalopathy, such as early infantile epileptic encephalopathy 9 (EIEE9).

[0083] "Determining the level of a protein" refers to detecting the protein or the mRNA encoding the protein, either directly or indirectly, by methods known in the art. "Directly determining" refers to performing a process to obtain a physical entity or value (e.g., performing an assay or test on a sample, or "analyzing a sample," as that term is defined herein). "Indirectly determining" refers to receiving a physical entity or value from another party or source (e.g., a third party laboratory that obtains the physical entity or value directly). Methods for measuring protein levels generally include, but are not limited to, Western blotting, immunoblotting, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), immunoprecipitation, immunofluorescence, surface plasmon resonance, chemiluminescence, fluorescence polarization, phosphorescence, immunohistochemistry, matrix-assisted laser desorption / ionization time-of-flight (MALDI-TOF) mass spectrometry, liquid chromatography (LC) mass spectrometry, microcytometry, microscopy, fluorescence-activated cell sorting (FACS), and flow cytometry, as well as assays based on protein properties, including but not limited to enzyme activity or interaction with other protein partners. Methods for measuring mRNA levels are known in the art.

[0084] "Level" refers to the level or activity of a protein, or of an mRNA encoding a protein (e.g., PCDH19), optionally compared to a reference. The reference can be any useful reference as defined herein. A "reduced level" or "elevated level" of a protein refers to a decrease or increase in protein levels compared to a reference (e.g., about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 150%, about 200%, about 300%, about 400%, about 500% or more decrease or increase; a decrease or increase of about 10%, about 15%, about 20%, about 30%, about 40%, about 50% or more compared to a reference). By "protein level" is meant a decrease or increase of more than about 0.01-fold, about 0.02-fold, about 0.1-fold, about 0.3-fold, about 0.5-fold, about 0.8-fold or less; or an increase of about 1.2-fold, about 1.4-fold, about 1.5-fold, about 1.8-fold, about 2.0-fold, about 3.0-fold, about 3.5-fold, about 4.5-fold, about 5.0-fold, about 10-fold, about 15-fold, about 20-fold, about 30-fold, about 40-fold, about 50-fold, about 100-fold, about 1000-fold or more. Protein levels can be expressed as mass / volume (e.g., g / dL, mg / mL, μg / mL, and ng / mL) or as a percentage of total protein or mRNA in the sample.

[0085] The term "pharmaceutical composition" as used herein refers to a composition containing a compound described herein, formulated with a pharmaceutically acceptable excipient, and preferably manufactured or sold with the approval of a government regulatory agency as part of a therapeutic regimen for the treatment of a disease in a mammal. Pharmaceutical compositions can be formulated, for example, for oral administration in unit dosage form (e.g., as a tablet, capsule, caplet, gelcap, or syrup); for topical administration (e.g., as a cream, gel, lotion, or ointment); for intravenous administration (e.g., as a particulate emboli-free sterile solution in a solvent system suitable for intravenous use); for intrathecal injection; for intraventricular infusion; for intraparenchymal infusion; or in any other pharmaceutically acceptable formulation.

[0086] As used herein, "a pharma- ceutically acceptable excipient" refers to any ingredient (e.g., a vehicle capable of suspending or dissolving an active compound) that has substantially non-toxic and non-inflammatory properties in a subject, other than the compounds described herein. Excipients may include, for example, anti-adherents, antioxidants, binders, coatings, compression aids, disintegrants, dyes (colorants), emollients, emulsifiers, fillers (diluents), film formers or coatings, flavors, fragrances, glidants (agents that promote flow), lubricants, preservatives, printing inks, adsorbents, suspending or dispersing agents, sweeteners, and hydration water. Exemplary excipients include, but are not limited to, butylated hydroxytoluene (BHT), calcium carbonate, calcium phosphate (dibasic), calcium stearate, croscarmellose, cross-linked polyvinylpyrrolidone, citric acid, crospovidone, cysteine, ethylcellulose, gelatin, hydroxypropylcellulose, hydroxypropylmethylcellulose, lactose, magnesium stearate, maltitol, mannitol, methionine, methylcellulose, methylparaben, microcrystalline cellulose, polyethylene glycol, polyvinylpyrrolidone, povidone, pregelatinized starch, propylparaben, retinyl palmitate, shellac, silicon dioxide, sodium carboxymethylcellulose, sodium citrate, sodium starch glycolate, sorbitol, starch (corn), stearic acid, sucrose, talc, titanium dioxide, vitamin A, vitamin E, vitamin C, and xylitol.

[0087] As used herein, the term "pharmaceutically acceptable salt" refers to any pharmaceutically acceptable salt of any of the compounds described herein. For example, pharmaceutically acceptable salts of any of the compounds described herein include those that are suitable for use in contact with human and animal tissues without undue toxicity, irritation, or allergic response, within the scope of sound medical judgment, which is commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art. For example, pharmaceutically acceptable salts are described in Berge et al., J. Pharmaceutical Sciences 66:1-19, 1977 and Pharmaceutical Salts: Properties, Selection, and Use, (Eds. P.H. Stahl and C.G. Wermuth), Wiley-VCH, 2008. Salts can be prepared in situ during the final isolation and purification of the compounds described herein, or separately by reacting the free base group with a suitable organic acid.

[0088] The compounds described herein may have ionizable groups so that they can be prepared as pharmaceutically acceptable salts. These salts may be acid addition salts, including inorganic or organic acids, or salts may be prepared from inorganic or organic bases in the case of the acidic form of the compounds described herein. In many cases, compounds are prepared or used as pharmaceutically acceptable salts prepared as addition products of pharmaceutically acceptable acids or bases. Suitable pharmaceutically acceptable acids and bases and methods for preparing suitable salts are well known in the art. Salts may be prepared from pharmaceutically acceptable non-toxic acids and bases, including inorganic and organic acids and bases. Representative acid addition salts include acetate, adipate, alginate, ascorbate, aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptonate, glycerophosphate, hemisulfate, heptonate, hexanoate, hydrobromide, hydrochloride, hydroiodide, and 2-hydroxy-ethanesulfonate. Representative alkali or alkaline earth metal salts include sodium, lithium, potassium, calcium, and magnesium, as well as non-toxic ammonium, quaternary ammonium, and amine cations, including, but not limited to, ammonium, tetramethylammonium, tetraethylammonium, methylamine, dimethylamine, trimethylamine, triethylamine, and ethylamine.

[0089] "Reference" means any useful reference used to compare the level or activity of a protein or mRNA. A reference can be any sample, standard, standard curve, or level used for comparison purposes. A reference can be a normal reference sample or a reference standard or level. A "reference sample" can be, for example, a control, a predefined negative control value such as a "normal control", or a previous sample taken from the same subject, a sample from a normal healthy subject such as a normal cell or normal tissue, a sample (e.g., cell or tissue) from a subject without a disease, a sample from a subject diagnosed with a disease but not yet treated with a compound described herein, a sample from a subject treated with a compound described herein, or a sample of a known normal concentration of a purified protein (e.g., any described herein). "Reference standard or level" means a value or number derived from a reference sample. A "normal control value" is a predefined value indicative of a non-disease state, e.g., a value expected in a healthy control subject. Typically, a normal control value is expressed as a range ("between X and Y"), a high threshold ("below X"), or a low threshold ("above X"). A subject having a measured value within the normal control value for a particular biomarker is typically referred to as "within the normal range" for that biomarker. A normal reference standard or level can be a value or number derived from a normal subject without a disease or disorder (e.g., PCDH19-associated disorder) and a subject treated with a compound described herein. In a preferred embodiment, the reference sample, standard, or level is matched to the sample, i.e., the subject sample, by at least one of the following criteria: age, weight, sex, disease stage, and overall health. A standard curve of purified protein within the normal reference range, e.g., any of the levels described herein, can also be used as a reference.

[0090] As used herein, the term "subject" refers to any organism to which a composition according to the invention may be administered, for example, for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Exemplary subjects include any animal (e.g., mammals such as mice, rats, rabbits, non-human primates, and humans). A subject may be a human or animal body that is seeking or in need of treatment, requesting treatment, currently undergoing treatment, will undergo treatment in the future, or is under the care of a professional trained in a particular disease or condition. A subject may be of any age, such as a newborn, neonate, infant, toddler, adolescent, or adult. A subject may be pre-born.

[0091] As used herein, the terms "treat", "treated" or "treating" refer to both therapeutic treatment and prophylactic or preventative measures, the purpose of which is to prevent or delay (alleviate) an undesired physiological condition, disorder, or disease, or to obtain a beneficial or desired clinical outcome. Beneficial or desired clinical outcomes include, but are not limited to, alleviation of symptoms; reduction in the extent of the condition, disorder, or disease; a stable (i.e., not worsening) state of the condition, disorder, or disease; a delay or slowing of the onset of the progression of the condition, disorder, or disease; an improvement or amelioration of the condition, disorder, or disease state, whether detectable or undetectable (whether partial or total); an improvement in at least one measurable physical parameter, not necessarily discernible by the subject; or an enhancement or amelioration of the condition, disorder, or disease. Treatment includes eliciting a clinically significant response without excessive levels of side effects. Treatment also includes prolonging survival compared to the expected survival in the absence of treatment.

[0092] As used herein, the terms "variant" and "derivative" are used interchangeably and refer to naturally occurring, synthetic, and semi-synthetic analogs of the compounds, peptides, proteins, or other substances described herein. Variants or derivatives of the compounds, peptides, proteins, or other substances described herein may retain or improve the biological activity of the original material.

[0093] The details of one or more embodiments of the invention are set forth in the description below. Other features, objects, and advantages of the invention will become apparent from the description and claims. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0094] According to an embodiment of the present invention, inhibition or depletion of PCDH19 level and / or activity in cells is effective in treating PCDH19-related disorders. Thus, the present invention features useful compositions and methods for treating PCDH19-related disorders, for example, in a subject in need thereof. The present invention features single-stranded oligonucleotides targeting the PCDH19 gene. The oligonucleotides (e.g., chemically modified oligonucleotides) can be administered to a subject with a PCDH19-related disorder (e.g., epilepsy, schizophrenia, and autism) to treat, reduce symptoms of, or prevent the PCDH19-related disorder. The oligonucleotides are antisense (e.g., at least partially complementary) to a target region of PCDH19 (e.g., PCDH19 mRNA, including pre-mRNA and processed mRNA). After administration, the oligonucleotides reduce the level, expression, and / or activity of PCDH19 (e.g., PCDH19 mRNA and / or protein), thereby providing a therapeutic effect to a subject with a PCDH19-related disorder.

[0095] PCDH19-Related Disorders PCDH19 is a cadherin family protein that is predominantly expressed in the developing brain. The gene encoding PCDH19 is located at position 22.1 on the long (q) arm of the X chromosome. Its function has not been fully elucidated, but it is thought to play a role in calcium-dependent cell adhesion. The full-length human processed PCDH19 mRNA is 9765 nucleotides long, with exon 2 alternatively spliced.

[0096] Exemplary human PCDH19 genes include those having the nucleotide sequences set forth in NCBI Reference Nos. NC_000023.11 (SEQ ID NO: 1), NG_021319.1, NM_001105243.1, NM_001184880.1, and NM_020766.2, as well as variants thereof, including variants containing single nucleotide polymorphisms, which are available from various publicly available databases (e.g., the Genome Aggregation Database, 2002). Database (gnomAD) (http: / / gnomad.broadinstitute.org / ). By way of example only, a PCDH19 variant may have at least or about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the sequence set forth in SEQ ID NO:1.

[0097] Mutations (e.g., missense or nonsense mutations) in PCDH19 have been associated with schizophrenia, autism, and certain forms of epilepsy, such as early infantile epileptic encephalopathy 9 (EIEE9). Because PCDH19 is located on the X chromosome, EIEE9 is associated with a unique X-linked inheritance pattern. X-linked mutations usually affect males, but not carrier females. However, EIEE9 only affects carrier females, while not harming inheriting males. EIEE9 is only associated with females, and it has been shown that male subjects with mosaic PCDH19 expression also suffer from cognitive impairment and / or epilepsy. Mosaicism involves the existence of two or more populations of cells that express different genotypes of PCDH19. In general, mutations arise (e.g., de novo) in certain cells and are only transmitted to daughter cells during division. Known mutations associated with PCDH19-related disorders include those encoding N340S, E307K, V441E, Q85X, N557K, D594H, S671X, L677fsx717, I119fsX122, and P364fsX375. Other known mutations associated with PCDH19-related disorders are listed in Table 1 below.

[0098] Subjects with one or more of these mutations can be treated with the compositions and methods described herein. Any of the PCDH19-associated disorders described herein (e.g., epilepsy, autism, and schizophrenia) can be treated by administering the oligonucleotides described herein (e.g., chemically modified oligonucleotides, e.g., ASOs) to reduce or eliminate symptoms of the disorder. [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6]

[0099] Oligonucleotide Agents The agents described herein that reduce the level and / or activity (e.g., abnormal activity) of PCDH19 in a cell can be, for example, polynucleotides, e.g., oligonucleotides. These agents reduce the level of activity (e.g., binding activity) associated with PCDH19, or an associated downstream effect, or reduce the level of PCDH19 in a cell or subject.

[0100] In some embodiments, the agent that reduces the level and / or activity of PCDH19 is a polynucleotide. In some embodiments, the polynucleotide is, for example, a single-stranded oligonucleotide that acts via an RNase H-mediated pathway. Oligonucleotides include DNA and DNA / RNA chimeric molecules, typically about 10-30 nucleotides in length, that recognize a polynucleotide target sequence or sequence portion through hydrogen bonding interactions with the nucleotide bases of the target sequence (e.g., PCDH19). The oligonucleotide molecule can reduce the expression level (e.g., protein level or mRNA level) of PCDH19. For example, the oligonucleotide includes an oligonucleotide that targets full-length PCDH19. In some embodiments, the oligonucleotide molecule recruits RNase H enzyme, resulting in target mRNA degradation.

[0101] In some embodiments, the oligonucleotide reduces the level and / or activity of a positive regulator of function. In other embodiments, the oligonucleotide increases the level and / or activity of an inhibitor of a positive regulator of function. In some embodiments, the oligonucleotide increases the level and / or activity of a negative regulator of function.

[0102] In some embodiments, the oligonucleotide reduces the level and / or activity or function of PCDH19. In some embodiments, the oligonucleotide inhibits expression of PCDH19. In other embodiments, the oligonucleotide enhances the degradation of PCDH19 and / or reduces the stability (i.e., half-life) of PCDH19 (e.g., mRNA). The oligonucleotide can be chemically synthesized.

[0103] Oligonucleotides include those having a region of complementarity (e.g., a contiguous nucleobase region) that is complementary to at least a portion of an mRNA formed upon expression of the PCDH19 gene. The region of complementarity can be about 30 or less nucleotides in length (e.g., about 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, or 10 or less nucleotides in length). Upon contact with a cell expressing the PCDH19 gene, the oligonucleotide can inhibit expression of the PCDH19 gene (e.g., a human, primate, non-primate, or avian PCDH19 gene) by at least about 10% (e.g., 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or 100%) as assayed, for example, by PCR or branched DNA (bDNA)-based methods, or by protein-based methods, such as immunofluorescence analysis using Western blotting or flow cytometry techniques.

[0104] Similarly, the region complementary to the target sequence may be 10-30 linked nucleosides long, e.g., 10-29, 10-28, 10-27, 10-26, 10-25, 10-24, 10-23, 10-22, 10-21, 10-20, 10-19, 10-18, 10-17, 10-16, 10-15, 10-14, 10-13, 10-12, 15-29, 15-28, 15-27, 15-26, 15-25, 15-24, 15-23, 15-22, 15-21, 15-20, 15-19, 15-18, 15-17, 18-30, 18-29, 18-28, The length of linked nucleosides may be 18-27, 18-26, 18-25, 18-24, 18-23, 18-22, 18-21, 18-20, 19-30, 19-29, 19-28, 19-27, 19-26, 19-25, 19-24, 19-23, 19-22, 19-21, 19-20, 20-30, 20-29, 20-28, 20-27, 20-26, 20-25, 20-24, 20-23, 20-22, 20-21, 21-30, 21-29, 21-28, 21-27, 21-26, 21-25, 21-24, 21-23, or 21-22. Ranges and lengths intermediate to the above recited ranges and lengths are also contemplated as part of the invention.

[0105] The oligonucleotide may comprise a region that is at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) complementary to a target region of the PCDH19 gene, for example, a portion of an equal length of a pre-mRNA transcript or mRNA transcript of PCDH19. The mRNA transcript or pre-mRNA transcript may contain a mutation (e.g., the subject is heterozygous (e.g., female) or hemizygous (e.g., male) for the mutation). The oligonucleotide may selectively target the mRNA or pre-mRNA that contains the mutation (e.g., allele-specific ASO). The oligonucleotide may target both the mutant allele and the wild-type allele.

[0106] Oligonucleotides can be synthesized by standard methods known in the art, as discussed further below, for example, by use of an automated DNA synthesizer, such as those commercially available from Biosearch, Applied Biosystems, Inc.

[0107] Oligonucleotide compounds can be prepared using solution phase or solid phase organic synthesis or both. Organic synthesis offers the advantage that oligonucleotides containing non-natural or alternative nucleotides can be easily prepared. The single-stranded oligonucleotides of the present invention can be prepared using solution phase or solid phase organic synthesis or both.

[0108] In one aspect, an oligonucleotide of the invention comprises a region of at least 10 (e.g., 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more) contiguous nucleobases that are at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) complementary to at least 10 contiguous nucleotides of the PCDH19 gene. In some embodiments, the oligonucleotide comprises a sequence that is complementary to at least 17 contiguous nucleotides, 19-23 contiguous nucleotides, 19 contiguous nucleotides, or 20 contiguous nucleotides of the PCDH19 gene. In this aspect, the sequence is substantially complementary to a sequence of an mRNA generated upon expression of the PCDH19 gene.

[0109] In some embodiments, the oligonucleotides of the invention, or contiguous nucleotide regions thereof, have a gapmer design or structure, also referred to herein simply as "gapmers." In a gapmer structure, the oligonucleotide comprises at least three distinct structural regions, a 5'-wing, a gap, and a 3'-wing, in a "5→3" orientation. In this design, the 5' and 3' wing regions (also referred to as flanking regions) comprise at least one alternative nucleoside adjacent to the gap region, and in some embodiments may comprise a continuous stretch of 2-7 alternative nucleosides, or a continuous stretch of alternative and DNA nucleosides (mixed wings comprising both alternative and DNA nucleosides). The length of the 5'-wing region may be at least two nucleosides long (e.g., at least 2, at least 3, at least 4, at least 5, or more nucleosides long). The length of the 3'-wing region may be at least two nucleosides long (e.g., at least 2, at least 3, at least 4, at least 5, or more nucleosides long). The 5' and 3' wing regions can be symmetric or asymmetric with respect to the number of nucleosides they contain, hi some embodiments, the gap region comprises about 10 nucleosides, flanked by 5' and 3' wing regions each comprising about 5 nucleosides, also referred to as a 5-10-5 gapmer.

[0110] As a result, the nucleosides of the 5' and 3' wing regions adjacent to the gap region are alternative nucleosides, such as 2' alternative nucleosides. The gap region comprises a contiguous stretch of nucleotides that can recruit RNase H when the oligonucleotide is duplexed with a PCDH19 target nucleic acid. In some embodiments, the gap region comprises a contiguous stretch of 5-16 DNA nucleosides. In other embodiments, the gap region comprises a region of at least 10 contiguous nucleobases having at least 80% (e.g., at least 85%, at least 90%, at least 95%, or at least 99%) complementarity to the PCDH19 gene. In some embodiments, the gapmer comprises a region complementary to at least 17 contiguous nucleotides, 19-23 contiguous nucleotides, or 19 contiguous nucleotides of the PCDH19 gene. The gapmer is complementary to the PCDH19 target nucleic acid and thus can be a contiguous nucleoside region of the oligonucleotide.

[0111] The 5' and 3' wing regions adjacent to the 5' and 3' ends of the gap region may comprise one or more affinity enhancing surrogate nucleosides. In some embodiments, the 5' wing and / or 3' wing comprise at least one 2'-O-methoxyethyl (MOE) nucleoside, preferably at least two MOE nucleosides. In some embodiments, the 5' wing comprises at least one MOE nucleoside. In some embodiments, both the 5' and 3' wing regions comprise MOE nucleosides. In some embodiments, all nucleosides within the wing regions are MOE nucleosides. In other embodiments, the wing regions can include both MOE nucleosides and other nucleosides (mixed wings), such as DNA nucleosides and / or non-MOE surrogate nucleosides, such as bicyclic nucleosides (BNAs) (e.g., LNA nucleosides or cET nucleosides), or other 2'-substituted nucleosides. In this case, the gap is defined as a contiguous sequence of at least five RNase H recruiting nucleosides (such as 5-16 DNA nucleosides) flanked on the 5' and 3' ends by affinity enhancing surrogate nucleosides, such as MOE nucleosides.

[0112] In other embodiments, the 5' and / or 3' wings comprise at least one BNA (e.g., at least one LNA nucleoside or cET nucleoside), preferably at least two bicyclic nucleosides. In some embodiments, the 5' wing comprises at least one BNA. In some embodiments, both the 5' wing region and the 3' wing region comprise BNAs. In some embodiments, all nucleosides in the wing region are BNAs. In other embodiments, the wing region can comprise both BNAs and other nucleosides (mixed wings), such as DNA nucleosides and / or non-BNA surrogate nucleosides, such as 2' substituted nucleosides. In this case, the gap is defined as a contiguous sequence of at least five RNase H recruiting nucleosides (e.g., 5-16 DNA nucleosides) flanked at the 5' and 3' ends by affinity enhancing surrogate nucleosides, such as BNAs, e.g., LNAs, e.g., β-D-oxy-LNAs.

[0113] The 5' flank or 5' wing attached to the 5' end of the gap region comprises, contains or consists of at least one alternative sugar moiety (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative sugar moieties). In some embodiments, the wing region comprises or consists of 1 to 7 alternative nucleobases, such as 2 to 6 alternative nucleobases, such as 2 to 5 alternative nucleobases, such as 2 to 4 alternative nucleobases, such as 1 to 3 alternative nucleobases, such as 1, 2, 3, or 4 alternative nucleobases. In some embodiments, the wing region comprises or consists of at least one alternative internucleoside linkage (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative internucleoside linkages).

[0114] The 3' flank or 3' wing attached to the 3' end of the gap region comprises, contains or consists of at least one alternative sugar moiety (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative sugar moieties). In some embodiments, the wing region comprises or consists of 1 to 7 alternative nucleobases, such as 2 to 6 alternative nucleobases, such as 2 to 5 alternative nucleobases, such as 2 to 4 alternative nucleobases, such as 1 to 3 alternative nucleobases, such as 1, 2, 3, or 4 alternative nucleobases. In some embodiments, the wing region comprises or consists of at least one alternative internucleoside linkage (e.g., at least three, at least four, at least five, at least six, at least seven, or more alternative internucleoside linkages).

[0115] In one embodiment, one or more or all of the alternative sugar moieties in the wing region are 2' alternative sugar moieties.

[0116] In further embodiments, the one or more 2' alternative sugar moieties in the wing region are selected from a 2'-O-alkyl-sugar moiety, a 2'-O-methyl-sugar moiety, a 2'-amino-sugar moiety, a 2'-fluoro-sugar moiety, a 2'-alkoxy-sugar moiety, an MOE sugar moiety, an LNA sugar moiety, an arabinonucleic acid (ANA) sugar moiety, and a 2'-fluoro-ANA sugar moiety.

[0117] In one embodiment of the invention, all of the alternative nucleosides in the wing region are bicyclic nucleosides, hi further embodiments, the bicyclic nucleosides in the wing region are independently selected from the group consisting of oxy-LNA, thio-LNA, amino-LNA, cET, and / or ENA, in either the β-D or α-L configuration, or combinations thereof.

[0118] In some embodiments, one or more of the alternative internucleoside linkages in the wing regions are phosphorothioate internucleoside linkages. In some embodiments, the phosphorothioate linkages are stereochemically pure phosphorothioate linkages. In some embodiments, the phosphorothioate linkages are Sp phosphorothioate linkages. In other embodiments, the phosphorothioate linkages are Rp phosphorothioate linkages. In some embodiments, the alternative internucleoside linkages are 2'-alkoxy internucleoside linkages. In other embodiments, the alternative internucleoside linkages are alkylphosphate internucleoside linkages.

[0119] The gap region can comprise, contain, or consist of at least 5-16 contiguous DNA nucleosides capable of recruiting RNase H. In some embodiments, all of the nucleosides in the gap region are DNA units. In further embodiments, the gap region can consist of a mixture of DNA and other nucleosides capable of mediating RNase H cleavage. In some embodiments, at least 50% of the nucleosides in the gap region are DNA, e.g., at least 60%, at least 70%, or at least 80%, or at least 90%.

[0120] The oligonucleotides of the present invention comprise a contiguous region that is complementary to a target nucleic acid. In some embodiments, the oligonucleotides may further comprise additional linked nucleosides located 5' and / or 3' to either the 5' and 3' wing regions. These additional linked nucleosides may be attached to the 5' end of the 5' wing region or the 3' end of the 3' wing region, respectively. The additional nucleosides may form part of a contiguous sequence that is complementary to the target nucleic acid in some embodiments, or may be non-complementary to the target nucleic acid in other embodiments.

[0121] The inclusion of additional nucleosides in either or both of the 5' and 3' wing regions may include 1, 2, 3, 4, or 5 additional nucleotides, which may be independently complementary or non-complementary to the target nucleic acid. In this regard, the oligonucleotides of the invention may, in some embodiments, include a contiguous sequence in which the additional nucleotides can modulate the target adjacent to the 5' and / or 3' ends. Such additional nucleosides may act as nuclease-sensitive biocleavable linkers, and may thereby be used to attach functional groups, such as conjugate moieties, to the oligonucleotides of the invention. In some embodiments, the additional 5' and / or 3' terminal nucleosides are linked with phosphodiester linkages and may be DNA or RNA. In another embodiment, the additional 5' and / or 3' terminal nucleosides are alternative nucleosides that may be included, for example, to enhance nuclease stability or to facilitate synthesis.

[0122] In other embodiments, the oligonucleotides of the invention utilize an "altimer" design and contain alternating 2'-fluoro-ANA and DNA regions that alternate every third nucleoside. Altimer oligonucleotides are described in more detail in Min, et al., Bioorganic & Medicinal Chemistry Letters, 2002, 12(18):2651-2654 and Kalota, et al., Nuc. Acid Res. 2006, 34(2):451-61, which are incorporated herein by reference.

[0123] In other embodiments, oligonucleotides of the invention utilize a "hemimer" design and contain a single 2'-modified wing segment adjacent to (either the 5' or 3' side of) the gap region. Hemimer oligonucleotides are described in more detail in Geary et al., 2001, J. Pharm. Exp. Therap., 296:898-904, which are incorporated herein by reference.

[0124] In some embodiments, the oligonucleotide has a nucleic acid sequence that has at least 50% (e.g., at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%) sequence identity to an equivalent length target sequence of PCDH19. In some embodiments, the oligonucleotide has a nucleic acid sequence that has at least 85% sequence identity to an equivalent length target sequence of PCDH19.

[0125] Although the ASO sequences are described as unmodified and / or unconjugated sequences, it will be understood that the nucleosides of the oligonucleotides of the invention, e.g., the oligonucleotides of the invention, may comprise any sequence that is an alternative nucleoside and / or conjugate, as described in more detail below.

[0126] Those skilled in the art are well aware that oligonucleotides having a structure of about 18-20 base pairs may be particularly effective in inducing RNase H-mediated degradation. However, it can be understood that shorter or longer oligonucleotides may also be effective. In the above embodiment, due to the nature of the oligonucleotide sequences provided herein, the oligonucleotides described herein may be included. It can be reasonably expected that shorter oligonucleotides minus just a few linked nucleosides at one or both ends may be similarly effective compared to the above oligonucleotides. Thus, oligonucleotides having a sequence of at least 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more consecutively linked nucleosides derived from one of the sequences provided herein, whose ability to inhibit expression of the PCDH19 gene differs from that of an oligonucleotide containing the complete sequence by no more than about 5, 10, 15, 20, 25, or 30% inhibition, are considered to be within the scope of the present invention.

[0127] The oligonucleotides described herein may function via nuclease-mediated degradation of target nucleic acids, and the oligonucleotides of the invention are capable of recruiting nucleases, particularly endonucleases, preferably endoribonucleases (RNases), such as RNase H. Examples of oligonucleotide designs that operate via a nuclease-mediated mechanism are oligonucleotides that typically include a region of at least five or six DNA nucleosides, which are flanked on one or both sides by affinity-enhancing surrogate nucleosides, such as gapmers, headmers, and tailmers.

[0128] RNase H activity of an oligonucleotide refers to its ability to recruit RNase H when in a duplex with a complementary RNA molecule. WO 01 / 23613 provides an in vitro method for determining RNase H activity that can be used to determine the ability to recruit RNase H. Typically, an oligonucleotide is considered to be capable of recruiting RNase H if, when provided with a complementary target nucleic acid sequence, it has an initial rate measured in pmol / l / min that is at least 5%, e.g., at least 10% or more than 20% of the initial rate determined when using an oligonucleotide that has the same base sequence as the modified oligonucleotide being tested but contains only DNA monomers with phosphorothioate linkages between all monomers in the oligonucleotide and using the method provided by Examples 91-95 of WO 01 / 23613 (herein incorporated by reference).

[0129] Additionally, the oligonucleotides described herein identify a site in the PCDH19 transcript that is susceptible to RNase H-mediated cleavage. Thus, the present invention further features an oligonucleotide that targets within this site. As used herein, an oligonucleotide is said to target within a specific site of an RNA transcript if the oligonucleotide promotes cleavage of the transcript anywhere within the specific site of the RNA transcript. Such oligonucleotides generally contain at least about 5-10 contiguous linked nucleosides from one of the sequences provided herein, linked to additional linked nucleoside sequences obtained from a region contiguous to the selected sequence in the PCDH19 gene.

[0130] Inhibitory oligonucleotides can be designed by methods well known in the art. Target sequences are generally about 10-30 linked nucleosides in length, although there is wide variation in the suitability of particular sequences within this range to direct cleavage of any given target RNA.

[0131] Oligonucleotides with sufficient homology to provide the necessary sequence specificity to uniquely degrade any RNA can be designed using programs known in the art.

[0132] Systematic testing of several designed species for optimizing inhibitory oligonucleotide sequences can also be carried out according to the teachings provided herein.Considerations when designing blocking oligonucleotides include, but are not limited to, biophysical, thermodynamic, and structural considerations, base preference at specific positions, and homology.The creation and use of inhibitory therapeutic agents based on non-coding oligonucleotides are also known in the art.

[0133] Although the various software packages and guidelines described herein provide guidance for the identification of optimal target sequences for any given gene target, an empirical approach can also be taken in which a "window" or "mask" of a given size (21 nucleotides as a non-limiting example) is literally or figuratively (including, for example, in silico) placed on the target RNA sequence to identify sequences within the size range that can serve as target sequences. By progressively moving the sequence "window" one nucleotide at a time upstream or downstream of the initial target sequence position, the next potential target sequence can be identified until a complete set of possible sequences is identified for any given target size selected. This process, combined with systematic synthesis and testing of the identified sequences (using assays described herein or known in the art) to identify the sequences that perform optimally, can identify RNA sequences that mediate the best inhibition of target gene expression when targeted with an oligonucleotide agent. Thus, while the sequences identified herein represent valid target sequences, it is contemplated that further optimization of inhibitory efficiency may be achieved by progressively "moving the window" by one nucleotide upstream or downstream of the given sequence to identify sequences with equal or better inhibitory properties.

[0134] It is further contemplated that for any sequence identified herein, further optimization may be achieved by systematically adding or removing linked nucleosides to generate longer or shorter sequences, and testing the generated sequences by shifting the target RNA up or down a longer or shorter size window from that point. Again, combining this approach to generate new candidate targets with testing the effectiveness of oligonucleotides based on the target sequence in inhibition assays as known in the art and / or as described herein can result in further improvements in the efficiency of inhibition.

[0135] Furthermore, such optimized sequences can be adjusted by the introduction of alternative nucleosides, sugar moieties, and / or internucleoside linkages described herein or known in the art, including alternative nucleosides, sugar moieties, and / or internucleoside linkages known in the art and / or discussed herein, for example, to further optimize the molecule as an expression inhibitor (e.g., to improve serum stability or circulating half-life, improve thermostability, enhance transmembrane delivery, target to a specific location or cell type, improve interaction with silencing pathway enzymes, increase release from endosomes). The oligonucleotide agents described herein can contain one or more mismatches to the target sequence. In one embodiment, the oligonucleotides described herein contain no more than three mismatches. When an oligonucleotide contains mismatches to a target sequence, it is preferred that the region of mismatch is not located in the center of the region of complementarity. When an oligonucleotide contains mismatches to a target sequence, it is preferred that the mismatch is limited to within the last five nucleotides from either the 5' or 3' end of the region of complementarity. For example, for a 30-linked nucleoside oligonucleotide agent, the contiguous nucleobase region that is complementary to a region of the PCDH19 gene generally does not contain any mismatches within the central 5-10 linked nucleosides. Methods described herein or known in the art can be used to determine whether an oligonucleotide containing mismatches to a target sequence is effective in inhibiting expression of the PCDH19 gene. Considering the effectiveness of oligonucleotides with mismatches in inhibiting expression of the PCDH19 gene is important, especially when a particular region of complementarity in the PCDH19 gene is known to have polymorphic sequence variation within the population.

[0136] The construction of vectors for the expression of polynucleotides for use in the present invention can be accomplished using conventional techniques that do not require detailed explanation to those skilled in the art. For the generation of an efficient expression vector, it is necessary to have regulatory sequences that control the expression of the polynucleotide. These regulatory sequences include promoter and enhancer sequences, are influenced by specific cellular factors that interact with these sequences, and are well known in the art. Alternative Oligonucleotides

[0137] In one embodiment, one or more of the linked nucleosides or internucleoside linkages of the oligonucleotide of the present invention are naturally occurring and do not include, for example, chemical modifications and / or conjugations known in the art and described herein. In another embodiment, one or more of the linked nucleosides or internucleoside linkages of the oligonucleotide of the present invention are chemically modified to enhance stability or other beneficial features. Without being bound by theory, it is believed that certain modifications can improve nuclease resistance and / or serum stability or reduce immunogenicity. For example, the oligonucleotide of the present invention may contain nucleotides (e.g., adenine, thymidine, guanosine, cytidine, uridine, or inosine) found naturally occurring in DNA or RNA, or may contain alternative nucleosides or internucleoside linkages having one or more chemical modifications to one or more components of the nucleotide (e.g., nucleobase, sugar, or phospholinker moiety). The oligonucleotides of the invention may be linked to each other via naturally occurring phosphodiester bonds or may contain alternative linkages (e.g., covalently linked via phosphorothioate (e.g., Sp phosphorothioate or Rp phosphorothioate), 3'-methylene phosphonate, 5'-methylene phosphonate, 3'-phosphoamidate, 2'-5' phosphodiester, guanidinium, S-methylthiourea, 2'-alkoxy, alkyl phosphate, or peptide bonds).

[0138] In certain embodiments of the present invention, substantially all nucleosides or internucleoside linkages of the oligonucleotide of the present invention are alternative nucleosides. In other embodiments of the present invention, all nucleosides or internucleoside linkages of the oligonucleotide of the present invention are alternative nucleosides. The oligonucleotide of the present invention in which "substantially all nucleosides are alternative nucleosides" is mostly, but not entirely, modified and can contain 5, 4, 3, 2, or 1 or less natural nucleosides. In yet other embodiments of the present invention, the oligonucleotide of the present invention can contain 5, 4, 3, 2, or 1 or less alternative nucleosides.

[0139] Nucleic acids featured in the invention can be synthesized and / or modified by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry", Beaucage, SLet al. (Edrs.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Alternative nucleotides and nucleosides include those having modifications such as terminal modifications, e.g., 5'-end modifications (phosphorylation, conjugation, backlinking) or 3'-end modifications (conjugation, DNA nucleotides, backlinking, etc.); base modifications, e.g., replacement with a stabilizing base, a destabilizing base, or a base that base pairs with an expanded repertoire of partners, removal of a base (abasic nucleotide), or conjugated base; sugar modifications (e.g., at the 2' or 4' position) or replacement of the sugar; and / or backbone modifications, including modification or replacement of the phosphodiester linkage. The nucleobase may also be an isonucleoside in which the nucleobase is moved from the C1 position of the sugar moiety to a different position (e.g., C2, C3, C4, or C5). Specific examples of oligonucleotide compounds useful in the embodiments described herein include, but are not limited to, alternative nucleosides that contain modified backbones or do not contain natural internucleoside linkages. Nucleotides and nucleosides with modified backbones include, among others, those that do not have a phosphorus atom in the backbone. For the purposes of this specification and as sometimes referenced in the art, alternative RNAs that do not have a phosphorus atom in the internucleoside backbone can also be considered to be oligonucleosides. In some embodiments, the oligonucleotide has a phosphorus atom in its internucleoside backbone.

[0140] Alternative internucleoside linkages include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates (including 3'-alkylene phosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-amino phosphoramidates and aminoalkyl phosphoramidates), thionophosphoramidates, thionoalkyl phosphonates, thionoalkyl phosphotriesters, and boronophosphates with the normal 3'-5' linkage, their 2'-5' linked analogs, and those with reverse polarity where adjacent pairs of nucleoside units are 3'-5' to 5'-3' or 2'-5' to 5'-2' linked.Various salts, mixed salts, and free acid forms are also included.

[0141] Representative United States patents which teach the preparation of such phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, and 5,321,131. , No. 5,399,676, No. 5,405,939, No. 5,453,496, No. 5,455,233, No. 5,466,677, No. 5,476,925, No. 5,519, No. 126, No. 5,536,821, No. 5,541,316, No. 5,550,111, No. 5,563,253, No. 5,571,799, No. 5,587,361; No. 5 ,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209, 6,239,265 , No. 6,277,603, No. 6,326,199, No. 6,346,614, No. 6,444,423, No. 6,531,590, No. 6,534,639, No. 6,608, Nos. 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Reissue Patent No. 39464 (the entire contents of each of which are incorporated herein by reference).

[0142] Alternative internucleoside linkages that do not contain a phosphorus atom have backbones formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatom or heterocyclic internucleoside linkages. These include morpholino linkages (formed in part from the sugar portion of the nucleoside), siloxane backbones, sulfide, sulfoxide and sulfone backbones, formylacetyl and thioformylacetyl backbones, methyleneformacetyl and thioformacetyl backbones, alkene-containing backbones, sulfamate backbones, methyleneimino and methylenehydrazino backbones, sulfonate and sulfonamide backbones, amide backbones, and others with mixed N, O, S and CH2 constituent moieties.

[0143] Representative U.S. patents which teach the preparation of the above oligonucleosides include U.S. Pat. Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470,967, 5,489,677, and 5,512,477. Nos. 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439 (the contents of each of which are incorporated herein by reference).

[0144] In other embodiments, suitable oligonucleotides include those in which both the sugar and the internucleoside linkage of the nucleotide units, i.e., the backbone, are replaced. The base units are maintained for hybridization with the appropriate nucleic acid target compound. Mimetics that have been shown to have excellent hybridization properties are called peptide nucleic acids (PNAs). In PNA compounds, the sugar of the nucleoside is replaced with an amide-containing backbone, particularly an aminoethylglycine backbone. The nucleobases are retained and are directly or indirectly linked to the aza nitrogen atom of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, the contents of each of which are incorporated herein by reference. Additional PNA compounds suitable for use in the oligonucleotides of the present invention are described, for example, in Nielsen et al., Science, 1991, 254, 1497-1500.

[0145] Some embodiments featured in the present invention include oligonucleotides with phosphorothioate backbones, and oligonucleotides with heteroatom backbones, particularly -CH2-NH-CH2-, -CH2-N(CH3)-O-CH2- [known as methylene (methylimino) or MMI backbones], -CH2-ON(CH3)-CH2-, CH2-N(CH3)-N(CH3)-CH2-, and -N(CH3)-CH2-CH2- [natural phosphodiester backbones are represented as -OPO-CH2 in the aforementioned U.S. Pat. No. 5,489,677], as well as the amide backbones of the above-referenced U.S. Pat. No. 5,602,240. In some embodiments, the oligonucleotides featured herein have the morpholino backbone structure of the above-referenced U.S. Pat. No. 5,034,506. In other embodiments, the oligonucleotides described herein comprise phosphorodiamidate moriphorino oligomers (PMOs) in which the deoxyribose moieties are replaced with monophasic rings and the charged phosphodiester intersubunit linkages are replaced with uncharged phosphorodiamidate linkages as described in Summerton, et al., Antisense Nucleic Acid Drug Dev. 1997:7-63,70.

[0146] Alternative nucleosides and nucleotides can also contain one or more substituted sugar moieties. Oligonucleotides, such as those featured herein, can include one of the following at the 2' position: OH, F, O-, S-, or N-alkyl, O-, S-, or N-alkenyl, O-, S-, or N-alkynyl, or O-alkyl-O-alkyl, where alkyl, alkenyl, and alkynyl are substituted or unsubstituted C1-C6. 10 Alkyl or C2-C 10 It can be alkenyl and alkynyl. Exemplary suitable modifications include -O[(CH2) n O] m CH3, -O(CH2) n OCH3, -O(CH2) n -NH2,

[0147] -O(CH2)n CH3, -O(CH2) n -ONH2 and -O(CH2) n -ON[(CH2) n CH3]2, where n and m are from 1 to about 10. In other embodiments, the oligonucleotide comprises at the 2' position: 10 The modifications include one of lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleaving group, reporter group, intercalator, group for improving the pharmacokinetic properties of oligonucleotides, or group for improving the pharmacodynamic properties of oligonucleotides, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O-CH2CHOCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chin. Acta, 1995, 78:486-504), i.e., an alkoxy-alkoxy group. MOE nucleosides confer several beneficial properties to oligonucleotides compared to unmodified oligonucleotides, including, but not limited to, improved nuclease resistance, improved pharmacokinetic properties, reduced nonspecific protein binding, reduced toxicity, reduced immunostimulatory properties, and enhanced target affinity.

[0148] Another exemplary alternative contains the group -O(CH2)2ON(CH3)2, also known as 2'-DMAOE, i.e., 2'-dimethylaminooxyethoxy, i.e., 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE, as described in the examples herein below, and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O-(CH2)2-O-(CH2)2-N(CH3)2. Further exemplary alternatives include 5'-Me-2'-F nucleotides, 5'-Me-2'-OMe nucleotides, 5'-Me-2'-deoxynucleotides (both R and S isomers in these three families); 2'-alkoxyalkyl; and 2'-NMA (N-methylacetamide).

[0149] Other alternatives include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the nucleosides and nucleotides of oligonucleotides, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Oligonucleotides can also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents which teach the preparation of such modified sugar structures include U.S. Patent Nos. 4,981,957, 5,118,800, 5,319,080, 5,359,044, 5,393,878, 5,446,137, 5,466,786, 5,514,785, 5,519,134, 5,567,811, 5,5 Nos. 76,427, 5,591,722, 5,597,909, 5,610,300, 5,627,053, 5,639,873, 5,646,265, 5,658,873, 5,670,633, and 5,700,920 (some of which are co-owned with this application), the entire contents of each of the foregoing being incorporated herein by reference.

[0150] The oligonucleotides of the invention can also include nucleobase (often referred to in the art simply as "base") alternatives (e.g., modifications or substitutions). Unmodified or natural nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Alternative nucleobases include other synthetic and natural nucleobases, such as 5-methylcytidine, 5-hydroxymethylcytidine, 5-formylcytidine, 5-carboxycytidine, pyrrolocytidine, dideoxycytidine, uridine, 5-methoxyuridine, 5-hydroxydeoxyuridine, dihydrouridine, 4-thiouridine, pseudouridine, 1-methyl-pseudouridine, deoxyuridine, 5-hydroxybutynyl-2'-deoxyuridine, xanthine, hypoxanthine, 7-deaza-xanthine, thienoguanine, 8-aza-7-deazaguanosine, 7-methylguanosine, 7-deazaguanosine, 6-aminomethyl-7-deazaguanosine, 8-aminoguanine, 2,2,7-trimethylguanosine, 8-methyladenine, 8-azidoadenine, 7-methyladenine, 7-deazaadenine, 3- These include deazaadenine, 2,6-diaminopurine, 2-aminopurine, 7-deaza-8-aza-adenine, 8-amino-adenine, thymine, dideoxythymine, 5-nitroindole, 2-aminoadenine, 6-methyl and other derivatives of adenine and guanine, 2-propyl and other derivatives of adenine and guanine, 2-thiouridine, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uridine and cytidine, 6-azouridine, cytidine and thymine, 4-thiouridine, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo, especially 5-bromo, 5-trifluoromethyl and other 5-substituted uridines and cytidines, 8-azaguanine and 8-azaadenine, and 3-deazaguanine.Further nucleobases include those disclosed in U.S. Patent No. 3,687,808, those disclosed in Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P. ed. Wiley-VCH, 2008, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, JL, ed. John Wiley & Sons, 1990 (which are disclosed in Englisch et al., (1991) Angewandte Chemie, International Edition, 30:613), and those disclosed in Sanghvi, Y S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, ST and Lebleu, B., Ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for improving the binding affinity of the oligonucleotide compounds featured in the present invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6, and 0-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine. 5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, YS, Crooke, ST and Lebleu, B., Eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are an exemplary base substitution, even more particularly when combined with a 2'-O-methoxyethyl sugar modification.

[0151] Representative U.S. patents which teach the preparation of certain of the above and other alternative nucleobases include, but are not limited to, U.S. Pat. Nos. 3,687,808, 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,594,121, 5,59 Nos. 6,091, 5,614,617, 5,681,941, 5,750,692, 6,015,886, 6,147,200, 6,166,197, 6,222,025, 6,235,887, 6,380,368, 6,528,640, 6,639,062, 6,617,438, 7,045,610, 7,427,672, and 7,495,088, the contents of each of which are incorporated herein by reference.

[0152] In other embodiments, the sugar moiety in a nucleotide can be a ribose molecule, optionally having a 2'-O-methyl, 2'-O-MOE, 2'-F, 2'-amino, 2'-O-propyl, 2'-aminopropyl, or 2'-OH modification.

[0153] The oligonucleotides of the present invention may contain one or more bicyclic sugar moieties. A "bicyclic sugar" is a furanosyl ring modified by a two-atom bridge. A "bicyclic nucleoside" ("BNA") is a nucleoside having a sugar moiety that includes a bridge connecting two carbon atoms of the sugar ring, thereby forming a bicyclic ring system. In certain embodiments, the bridge connects the 4'-carbon and the 2'-carbon of the sugar ring. Thus, in some embodiments, the agents of the present invention may include one or more locked nucleosides. A locked nucleoside is a nucleoside that has a modified ribose moiety and includes an extra bridge connecting the 2'-carbon and the 4'-carbon of the ribose moiety. In other words, a locked nucleoside is a nucleoside that includes a bicyclic sugar moiety that includes a 4'-CH2-O-2' bridge. This structure effectively "locks" the ribose into a 3'-end structural conformation. The addition of locked nucleosides to oligonucleotides has been shown to improve oligonucleotide stability in serum and reduce off-target effects (Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193). Examples of bicyclic nucleosides for use in the polynucleotides of the present invention include, but are not limited to, nucleosides that comprise a bridge between the 4' ribosyl ring atom and the 2' ribosyl ring atom. In certain embodiments, the polynucleotide agents of the present invention comprise one or more bicyclic nucleosides that comprise a 4'-2' bridge.Examples of such 4'-2' bridged bicyclic nucleosides include 4'-(CH2)-O-2' (LNA); 4'-(CH2)-S-2'; 4'-(CH2)2-O-2' (ENA); 4'-CH(CH3)-O-2' (also known as "constrained ethyl" or "cEt") and 4'-CH(CH2OCH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 7,399,845); 4'- C(CH3)(CH3)-O-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,283); 4'-CH2-N(OCH3)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,425); 4'-CH2-ON(CH3)2-2' (see, e.g., U.S. Patent Publication No. 2004 / 0171570); 4'-CH2-N(R)-O-2' (wherein R is H, C1-C. 12 alkyl, or a protecting group) (see, e.g., U.S. Pat. No. 7,427,672); 4'-CH2-C(H)(CH3)-2' (see, e.g., Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4'-CH2-C(=CH2)-2' (and analogs thereof; see, e.g., U.S. Pat. No. 8,278,426). The entire contents of each of the foregoing are incorporated herein by reference.

[0154] Additional representative U.S. patents and published U.S. patent applications that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, U.S. Pat. Nos. 6,268,490, 6,525,191, 6,670,461, 6,770,748, 6,794,499, 6,998,484, 7,053,207, 7,034,133, 7,084,125, 7,399,845, 7,421, and 7,525,191. Nos. 427,672, 7,569,686, 7,741,457, 8,022,193, 8,030,467, 8,278,425, 8,278,426, 8,278,283, U.S. Patent Application Publication Nos. 2008 / 0039618, and 2009 / 0012281, the entire contents of which are incorporated herein by reference.

[0155] Any of the foregoing bicyclic nucleosides can be prepared having one or more stereochemical sugar configurations, including, for example, α-L-ribofuranose and β-D-ribofuranose (see WO 99 / 14226).

[0156] The oligonucleotides of the invention may also be modified to include one or more constrained ethyl nucleosides. As used herein, a "constrained ethyl nucleoside" or "cEt" is a locked nucleoside that includes a bicyclic sugar moiety that includes a 4'-CH(CH3)-O-2' bridge. In one embodiment, the constrained ethyl nucleoside is in the S conformation, referred to herein as "S-cEt."

[0157] The oligonucleotides of the invention may also contain one or more "conformationally restricted nucleosides" ("CRNs"). CRNs are nucleoside analogs with a linker connecting the C2' and C4' carbons of ribose or the C3 and -C5' carbons of ribose. CRNs lock the ribose ring into a stable conformation and improve hybridization affinity to mRNA. The linker is long enough to place oxygens in optimal positions for stability and affinity, resulting in less puckering of the ribose ring.

[0158] Representative publications that teach the preparation of certain of the above CRNs include, but are not limited to, U.S. Patent Application Publication No. 2013 / 0190383 and WO 2013 / 036868, the contents of each of which are incorporated herein by reference in their entirety.

[0159] In some embodiments, the oligonucleotide of the present invention comprises one or more monomers that are UNA (unlocked nucleosides) nucleosides. UNA is an unlocked acyclic nucleoside in which any of the sugar bonds have been removed to form an unlocked "sugar" residue. In one example, UNA also encompasses monomers in which the C1'-C4' bond (i.e., the carbon-oxygen-carbon covalent bond between the C1' and C4' carbons) has been removed. In another example, the C2'-C3' bond (i.e., the carbon-carbon covalent bond between the C2' and C3' carbons) of the sugar has been removed (see Nuc. Acids Symp. Series, 52, 133-134 (2008) and Fluiter et al., Mol. Biosyst., 2009, 10, 1039, which are incorporated herein by reference).

[0160] Representative U.S. publications that teach the preparation of UNAs include, but are not limited to, U.S. Patent No. 8,314,227, and U.S. Patent Application Publication Nos. 2013 / 0096289, 2013 / 0011922, and 2011 / 0313020, the entire contents of which are incorporated herein by reference.

[0161] Ribose can also be modified with a cyclopropane ring to produce tricyclodeoxynucleic acid (tricycloDNA). The ribose moiety can be replaced with another sugar, such as 1,5-anhydrohexitol, threose to produce threose nucleosides (TNA), or arabinose to produce arabinonucleosides. The ribose molecule can also be replaced with a non-sugar, such as cyclohexene to produce cyclohexene nucleosides, or glycol to produce glycol nucleosides.

[0162] Potential stabilizing modifications to the termini of nucleoside molecules include N-(acetylaminocaproyl)-4-hydroxyprolinol (Hyp-C6-NHAc), N-(caproyl)-4-hydroxyprolinol (Hyp-C6), N-(acetyl-4-hydroxyprolinol (Hyp-NHAc), thymidine-2'-O-deoxythymidine (ether), N-(aminocaproyl)-4-hydroxyprolinol (Hyp-C6-amino), 2-docosanoyl-uridine-3''-phosphate, inverted base dT (idT), and the like. Disclosure of this modification can be found in WO 2011 / 005861.

[0163] Other alternative chemicals for the oligonucleotides of the present invention include 5' phosphates or 5' phosphate mimetics, such as the 5' terminal phosphate or phosphate mimetics of oligonucleotides. Suitable phosphate mimetics are disclosed, for example, in US Patent Application Publication No. 2012 / 0157511, the entire contents of which are incorporated herein by reference.

[0164] Exemplary oligonucleotides of the present invention include nucleosides with alternative sugar moieties, and may also include DNA or RNA nucleosides. In some embodiments, oligonucleotides include nucleosides with alternative sugar moieties and DNA nucleosides. The incorporation of alternative nucleosides into the oligonucleotides of the present invention may enhance the affinity of the oligonucleotide to a target nucleic acid. In this case, the alternative nucleoside may be referred to as an affinity enhancing alternative nucleotide.

[0165] In some embodiments, the oligonucleotide comprises at least one alternative nucleoside, e.g., at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, or at least 16 alternative nucleosides. In other embodiments, the oligonucleotide comprises 1-10 alternative nucleosides, e.g., 2-9 alternative nucleosides, e.g., 3-8 alternative nucleosides, e.g., 4-7 alternative nucleosides, e.g., 6 or 7 alternative nucleosides. In one embodiment, the oligonucleotide of the invention may comprise alternatives independently selected from these three types of alternatives (alternative sugar moieties, alternative nucleobases, and alternative internucleoside linkages), or combinations thereof. Preferably, the oligonucleotide comprises one or more nucleosides that comprise an alternative sugar moiety, e.g., a 2' sugar alternative nucleoside. In some embodiments, the oligonucleotides of the invention comprise one or more 2' sugar surrogate nucleosides independently selected from the group consisting of 2'-O-alkyl-RNA, 2'-O-methyl-RNA, 2'-alkoxy-RNA, 2'-O-methoxyethyl-RNA, 2'-amino-DNA, 2'-fluoro-DNA, arabinonucleic acid (ANA), 2'-fluoro-ANA, and BNA (e.g., LNA) nucleosides. In some embodiments, the one or more surrogate nucleosides are BNA.

[0166] In some embodiments, at least one of the alternative nucleosides is a BNA (e.g., an LNA), e.g., at least two, e.g., at least three, at least four, at least five, at least six, at least seven, or at least eight of the alternative nucleosides are BNAs. In still further embodiments, all of the alternative nucleosides are BNAs.

[0167] In further embodiments, the oligonucleotide comprises at least one alternative internucleoside linkage. In some embodiments, the internucleoside linkage in the contiguous nucleotide sequence is a phosphorothioate or boronophosphate internucleoside linkage. In some embodiments, all internucleoside linkages in the contiguous sequence of the oligonucleotide are phosphorothioate linkages. In some embodiments, the phosphorothioate linkage is a stereochemically pure phosphorothioate linkage. In some embodiments, the phosphorothioate linkage is an Sp phosphorothioate linkage. In other embodiments, the phosphorothioate linkage is an Rp phosphorothioate linkage.

[0168] In some embodiments, the oligonucleotide of the present invention comprises at least one alternative nucleoside that is 2'-MOE-RNA, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-MOE-RNA nucleoside units. In some embodiments, the 2'-MOE-RNA nucleoside units are connected by phosphorothioate linkages. In some embodiments, at least one of the alternative nucleosides is 2'-fluoro DNA, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10 2'-fluoro-DNA nucleoside units. In some embodiments, the oligonucleotide of the present invention comprises at least one BNA unit and at least one 2'-substitution modified nucleoside. In some embodiments of the present invention, the oligonucleotide comprises both 2' sugar modified nucleosides and DNA units. In some embodiments, the oligonucleotide of the present invention or a contiguous nucleotide region thereof is a gapmer oligonucleotide.

[0169] Ligand-conjugated oligonucleotides The oligonucleotides of the invention may be chemically linked to one or more ligands, moieties or conjugates that enhance the activity, cellular distribution or cellular uptake of the oligonucleotide.Such moieties include lipid moieties, e.g., cholesterol moieties (Letsinger et al., (1989) Proc. Natl. Acid. Sci. USA, 86:6553-6556), cholic acid (Manoharan et al. (1994) Biorg. Med. Chem., 4:1053-1060), thioethers, e.g., beryl-S-tritylthiol (Manoharan et al., (1992) Ann. NY Acad. Sci., 660:306-309; Manoharan et al., (1993) Biorg. Med. Chem. Let., 3:2765-2770), thiocholesterol (Oberhauser et al., (1992) Nucl. Acids Sci. USA, 86:6553-6556), cholic acid (Manoharan et al., ... Let., 4:1053-1060), thioesters, e.g., beryl-S-tritylthiol (Manoharan et al., (199 Res., 20:533-538), aliphatic chains such as dodecanediol or undecyl residues (Saison-Behmoaras et al., (1991) J. EMBO J, 10:1111-1118; Kabanov et al., (1990) FEBS Lett., 259:327-330; Svinarchuk et al., (1993), (1995) Biochimie, 75:49-54), phospholipids such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate (Manoharan et al., 36:3651-3654; Shea et al., (1990) Nucl. Acids, 20:533-538), Res., 18:3777-3783), polyamine or polyethylene glycol chains (Manoharan et al., (1995) Nucleosides & Nucleotides, 14:969-973), or adamantane acetic acid (Manoharan et al., (1995) Tetrahedron Lett., 36:3651-3654), palmityl moieties (Mishra et al., (1995) Biochim. Biophys. Acta, 1264:229-237), or octadecylamine or hexylamino-carbonyloxycholesterol moieties (Crooke et al., (1996) J. Pharmacol. Exp. Ther., 277:923-937).

[0170] In one embodiment, a ligand alters the distribution, targeting, or lifetime of an oligonucleotide agent into which it is incorporated. In some embodiments, a ligand provides enhanced affinity for a selected target, e.g., a molecule, a cell or cell type, a compartment, e.g., a cell or organ compartment, a tissue, an organ, or a region of the body, e.g., compared to a species in which such ligand is not present.

[0171] Ligands can include naturally occurring substances such as proteins (e.g., human serum albumin (HSA), low density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextran, pullulan, chitin, chitosan, inulin, cyclodextrin, N-acetylglucosamine, N-acetylgalactosamine, or hyaluronic acid); or lipids. Ligands can also be recombinant or synthetic molecules, such as synthetic polymers, such as synthetic polyamino acids. Examples of polyamino acids include polylysine (PLL), poly L-aspartic acid, poly L-glutamic acid, styrene-maleic anhydride copolymers, poly(L-lactide-co-glycolide) copolymers, divinyl ether-maleic anhydride copolymers, N-(2-hydroxypropyl)methacrylamide copolymers (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethanes, poly(2-ethylacrylic acid), N-isopropylacrylamide polymers, or polyphosphazines. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimeric polyamines, arginine, amidines, protamines, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or alpha-helical peptides.

[0172] The ligand can also include a targeting group, for example, a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid, or protein, such as an antibody that binds to a particular cell type, such as a kidney cell. The targeting group can be thyrotropin (thyroid stimulating hormone), melanotropin, lectin, glycoprotein, surfactant protein A, mucin carbohydrate, polyvalent lactose, polyvalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine polyvalent mannose, polyvalent fucose, glycosylated polyamino acids, polyvalent galactose, transferrin, bisphosphonate, polyglutamic acid, polyaspartic acid, lipid, cholesterol, steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0173] Other examples of ligands include dyes, intercalating agents (e.g., acridines), crosslinkers (e.g., psoralens, mitomycin C), porphyrins (TPPC4, texaphyrin, sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules, such as cholesterol, cholic acid, adamantane acetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O(hexadecyl)glycerol, geranyloxyhexyl groups, hexadecylglycerol, borneol, menthol, 1,3-propanediol, heptadecyl groups, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl)iso ... yl)cholenic acid, dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g., antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino, mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bis-imidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

[0174] Ligands can be proteins, e.g., glycoproteins, or peptides, e.g., molecules with specific affinity for co-ligands, or antibodies, e.g., antibodies that bind to specific cell types, such as hepatocytes. Ligands can also include hormones and hormone receptors. They can also include lipids, lectins, carbohydrates, vitamins, cofactors, non-peptide species such as multivalent lactose, multivalent galactose, N-acetyl-galactosamine, N-acetyl-glucosamine, multivalent mannose, or multivalent fucose.

[0175] The ligand can be a substance, e.g., a drug, that can increase cellular uptake of an oligonucleotide agent, e.g., by disrupting the cytoskeleton of a cell, e.g., by disrupting the microtubules, microfilaments, and / or intermediate filaments of the cell. The drug can be, e.g., taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0176] In some embodiments, the ligand attached to the oligonucleotide described herein acts as a pharmacokinetic modulator (PK modulator). PK modulators include lipophilic substances, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, and the like. Exemplary PK modulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacyl glycerides, phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, and the like. Oligonucleotides containing some phosphorothioate linkages are also known to bind to serum proteins, and therefore short oligonucleotides, for example, about 5-base, 10-base, 15-base, or 20-base oligonucleotides, containing multiple phosphorothioate linkages in the backbone, are also suitable for the present invention as ligands (e.g., as PK-modulating ligands). In addition, aptamers that bind serum components (e.g., serum proteins) are also suitable for use as PK-modulating ligands in the embodiments described herein.

[0177] The ligand-conjugated oligonucleotides of the invention can be synthesized by the use of an oligonucleotide having a pendant reactive functional group, such as that resulting from the attachment of a linking molecule to the oligonucleotide (described below). This reactive oligonucleotide can be reacted directly with a commercially available ligand, a synthesized ligand having any of a variety of protecting groups, or a ligand having a linking moiety attached thereto.

[0178] The oligonucleotides used in the conjugates of the present invention can be easily and routinely produced by the well-known technique of solid phase synthesis.The equipment for such synthesis is sold by several vendors, including, for example, Applied Biosystems (Foster City, Calif.).Any other means for such synthesis known in the art can additionally or alternatively be used.It is also known to use similar techniques to prepare other oligonucleotides (e.g., phosphorothioates and alkylated derivatives).

[0179] In the ligand-conjugated oligonucleotides of the invention, e.g., ligand molecules having sequence-specific linked nucleosides of the invention, the oligonucleotides and oligonucleosides can be assembled on a suitable DNA synthesizer utilizing standard nucleotide or nucleoside precursors, or nucleotide or nucleoside conjugate precursors that already have a linking moiety, ligand-nucleotide or nucleoside conjugate precursors that already have a ligand molecule, or non-nucleoside ligand-bearing building blocks.

[0180] When using a conjugate precursor that already has a linking moiety, synthesis of the sequence-specific linked nucleoside is typically completed and then the ligand molecule is reacted with the linking moiety to form the ligand-conjugated oligonucleotide. In some embodiments, the oligonucleotides or linked nucleosides of the invention are synthesized by automated synthesizers using phosphoramidites derived from the ligand-nucleoside conjugates, in addition to standard and non-standard phosphoramidites that are commercially available and commonly used in oligonucleotide synthesis.

[0181] Lipid conjugates In one embodiment, the ligand or conjugate is a lipid or lipid-based molecule.Such lipid or lipid-based molecule is preferably bound to serum protein, for example human serum albumin (HSA).HSA-binding ligand allows the distribution of conjugate to target tissue, for example non-renal target tissue of the body.Lipid or lipid-based ligand can be used to (a) improve the resistance of conjugate to degradation, (b) improve targeting or transport to target cell or cell membrane, and / or (c) regulate the binding to serum protein, for example HSA.

[0182] In another embodiment, the ligand is a moiety, e.g., a vitamin, that is taken up by a target cell, e.g., a proliferating cell. Exemplary vitamins include vitamins A, E, and K.

[0183] Cell permeabilization agents In another embodiment, the ligand is a cell-penetrating agent, preferably a helical cell-penetrating agent. Preferably, the agent is amphipathic. An exemplary agent is a peptide, such as tat or antennapedia. If the agent is a peptide, the peptide can be modified, including peptidyl mimetics, invertomers, non-peptide or pseudopeptide linkages, and the use of D-amino acids. The helical agent is preferably an α-helical agent, which preferably has a lipophilic phase and a lipophobic phase.

[0184] The ligand can be a peptide or peptidomimetic. Peptidomimetics (also referred to herein as oligopeptidomimetics) are molecules that can fold into defined three-dimensional structures similar to natural peptides. Attachment of peptides and peptidomimetics to oligonucleotide agents can affect the pharmacokinetic distribution of the oligonucleotide, for example, by enhancing cellular recognition and uptake. The peptide or peptidomimetic moiety can be about 5-50 amino acids in length, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids in length.

[0185] The peptide or peptidomimetic can be, for example, a cell penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., consisting mainly of Tyr, Trp, or Phe). The peptide moiety can be a dendrimeric peptide, a constrained peptide, or a cross-linked peptide. In another alternative, the peptide moiety can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF, which has the amino acid sequence AAVALLPAVLLALLAP. RFGF analogs containing hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP) can also be targeting moieties. The peptide moiety can be a "delivery" peptide that can carry large polar molecules, including peptides, oligonucleotides, and proteins, across cell membranes. For example, it has been found that sequences from the HIV Tat protein (GRKKRRQRRRPPQ) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWKK) can function as delivery peptides. The peptide or peptidomimetic can be encoded by a random sequence of DNA, such as peptides identified from a phage display library, or a one-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). An example of a peptide or peptidomimetic linked to an oligonucleotide agent via an incorporated monomer unit for cell targeting purposes is an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic. The peptide portion can range from about 5 amino acids to about 40 amino acids in length. The peptide portion can have structural modifications, such as to improve stability or direct conformational properties. Any of the structural modifications described below can be utilized.

[0186] RGD peptides for use in the compositions and methods of the present invention can be linear or cyclic, and can be modified (e.g., glycosylated or methylated) to facilitate targeting to specific tissues. RGD-containing peptides and peptidomimetics can include synthetic RGD mimetics in addition to D-amino acids. In addition to RGD, other moieties that target integrin ligands can be used. Some conjugates of this ligand target PECAM-1 or VEGF.

[0187] The cell-penetrating peptide can penetrate cells, for example, microbial cells, such as bacterial or fungal cells, or mammalian cells, such as human cells. The microbial cell-penetrating peptide can be, for example, an α-helical linear peptide (e.g., LL-37 or cecropin P1), a disulfide bond-containing peptide (e.g., α-defensin, β-defensin, or bactenecin), or a peptide containing only one or two predominant amino acids (e.g., PR-39 or indolicidin). The cell-penetrating peptide can also include a nuclear localization signal (NLS). For example, the cell-penetrating peptide can be a bipartite amphipathic peptide, such as MPG, derived from the fusion peptide domain of HIV-1 gp41 and the NLS of SV40 large T antigen (Simeoni et al., Nucl. Acid Res. 31:2717-2724, 2003).

[0188] Carbohydrate Conjugates In some embodiments of the compositions and methods of the present invention, the oligonucleotide further comprises a carbohydrate. Carbohydrate-conjugated oligonucleotides are advantageous for in vivo delivery of nucleic acids and compositions suitable for in vitro therapeutic use as described herein. As used herein, "carbohydrate" refers to either a compound that is a carbohydrate essentially composed of one or more monosaccharide units having at least six carbon atoms (which may be linear, branched or cyclic) and with an oxygen, nitrogen or sulfur atom bonded to each carbon atom, or a compound that has as a portion a carbohydrate moiety composed of one or more monosaccharide units each having at least six carbon atoms (which may be linear, branched or cyclic) and with an oxygen, nitrogen or sulfur atom bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides and oligosaccharides containing about 4, 5, 6, 7, 8 or 9 monosaccharide units) and polysaccharides such as starch, glycogen, cellulose and polysaccharide gums. Particular monosaccharides include sugars of C5 or higher (e.g., C5, C6, C7, or C8), and disaccharides and trisaccharides include sugars having two or three monosaccharide units (e.g., C5, C6, C7, or C8).

[0189] In one embodiment, the carbohydrate conjugate for use in the compositions and methods of the invention is a monosaccharide.

[0190] In some embodiments, the carbohydrate conjugate further comprises one or more additional ligands as described above, such as, but not limited to, a PK modulator and / or a cell penetrating peptide.

[0191] Additional carbohydrate conjugates (and linkers) suitable for use in the present invention include those described in WO 2014 / 179620 and WO 2014 / 179627, the entire contents of each of which are incorporated herein by reference.

[0192] Linker In some embodiments, the conjugates or ligands described herein can be attached to the oligonucleotide using a variety of linkers, which can be cleavable or non-cleavable.

[0193] The linker is typically a direct bond or an atom such as an oxygen atom or a sulfur atom, NR 8 , C(O), C(O)NH, SO, SO2, SO2NH, or units such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkynyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkynyl and the like, wherein one or more methylenes are selected from the group consisting of O, S, S(O), SO, N(R), S ... 8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocycle, 8 is hydrogen, acyl, aliphatic, or substituted aliphatic. In one embodiment, the linker is about 1-24 atoms, 2-24, 3-24, 4-24, 5-24, 6-24, 6-18, 7-18, 8-18 atoms, 7-17, 8-17, 6-16, 7-17, or 8-16 atoms.

[0194] A cleavable tether is one that is sufficiently stable outside a cell, but that is cleaved upon entry into a target cell to release the two moieties that the linker is holding together. In preferred embodiments, the cleavable tether is cleaved at least about 10-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, or more, or at least 100-fold faster in the target cell or under a first reference condition (which may, for example, be selected to replicate or represent intracellular conditions) than in the subject's blood, or under a second reference condition (which may, for example, be selected to replicate or represent conditions found in blood or serum).

[0195] Cleavable linking groups are sensitive to cleaving agents (cleaving agents) (e.g., pH, redox potential, or the presence of degradable molecules). Generally, cleaving agents are more prevalent or found at higher levels or activity within cells than in serum or blood. Examples of such degrading agents include redox agents that are selective for a particular substrate or have no substrate specificity (e.g., reducing agents such as oxidases or reductases or mercaptans present within cells that can degrade redox-cleavable linking groups by reduction), esterases; agents that can create endosomes or acidic environments, such as agents that result in a pH of 5 or less, general acids, peptidases (which can be substrate specific), and enzymes that can act as phosphatases to hydrolyze or degrade acid-cleavable linking groups.

[0196] The cleavable linking group, e.g., a disulfide bond, may be sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1 to 7.3. Endosomes have a more acidic pH, ranging from 5.5 to 6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand within the cell or into a desired compartment of the cell.

[0197] The linker can include a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the conjugate can vary depending on the cell to be targeted. For example, a liver targeting ligand can be linked to a cationic lipid via a linker that includes an ester group. Liver cells are rich in esterase, and therefore the linker is cleaved more efficiently in liver cells than in cell types that are not rich in esterase. Other cell types that are rich in esterase include lung, renal cortex, and testis cells.

[0198] Linkers containing peptide bonds can be used when targeting cell types that are rich in peptidases, such as hepatocytes and synovial cells.

[0199] In general, the suitability of a candidate cleavable linking group can be evaluated by testing the ability (or conditions) of a degrading agent to cleave the candidate linking group. It is also desirable to test the candidate cleavable linking group for its ability to resist cleavage in blood or when in contact with other non-target tissues. Thus, the relative susceptibility to cleavage between a first condition and a second condition can be determined, the first condition being selected to exhibit cleavage in target cells, and the second condition being selected to exhibit cleavage in other tissues or biological fluids, such as blood or serum. Evaluation can be performed in a cell-free system, in cells, in cell cultures, in organ or tissue cultures, or in whole animals. It can be useful to perform initial evaluations in a cell-free or culture conditions and confirm with further evaluations in whole animals. In preferred embodiments, useful candidate compounds are cleaved at least 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood or serum (or under in vitro conditions selected to mimic extracellular conditions).

[0200] Redox-cleavable linkers In one embodiment, the cleavable linking group is a redox cleavable linking group that is cleaved upon reduction or oxidation. An example of a reductive cleavable linking group is a disulfide linking group (-SS-). To determine whether a candidate cleavable linking group is a suitable "reductive cleavable linking group" or is suitable for use with, for example, a particular oligonucleotide moiety and a particular targeting agent, the methods described herein can be considered. For example, the candidate can be evaluated by incubation with dithiothreitol (DTT) or other reducing agents using reagents known in the art that reproduce the cleavage rate observed in cells, for example, target cells. The candidate can also be evaluated under conditions selected to reproduce blood or serum conditions. In one embodiment, the candidate compound is cleaved in blood at a maximum of about 10%. In other embodiments, useful candidate compounds are degraded at least about 2, 4, 10, 20, 30, 40, 50, 60, 70, 80, 90, or about 100 times faster in cells (or under in vitro conditions selected to mimic intracellular conditions) compared to blood (or under in vitro conditions selected to mimic extracellular conditions). The cleavage rate of the candidate compound can be determined using standard enzyme kinetic assays under conditions selected to mimic the intracellular medium and compared to conditions selected to mimic the extracellular medium.

[0201] Phosphate-Based Cleavable Linkers In another embodiment, the cleavable linker comprises a phosphate-based cleavable linker. The phosphate-based cleavable linker is cleaved by an agent that decomposes or hydrolyzes the phosphate group. An example of an agent that cleaves a phosphate group within a cell is an enzyme such as an intracellular phosphatase. An example of a phosphate-based linker is -OP(O)(OR k )-O-, -OP(S)(OR k )-O-, -OP(S)(SR k )-O-, -SP(O)(OR k )-O-, -OP(O)(OR k )-S-, -SP(O)(OR k )-S-, -OP(S)(OR k )-S-, -SP(S)(OR k )-O-, -OP(O)(R k )-O-, -OP(S)(R k )-O-, -SP(O)(R k )-O-, -SP(S)(R k )-O-, -SP(O)(R k )-S-, -OP(S)(R k )-S-. These candidates can be evaluated using methods similar to those described above.

[0202] Acid-cleavable linking group In another embodiment, the cleavable linker comprises an acid cleavable linking group. An acid cleavable linking group is a linking group that is cleaved under acidic conditions. In a preferred embodiment, the acid cleavable linking group is cleaved in an acidic environment having a pH of about 6.5 or less (e.g., about 6.0, 5.75, 5.5, 5.25, 5.0, or less) or by an agent such as an enzyme that can act as a general acid. In a cell, certain low pH organelles such as endosomes and lysosomes can provide a cleavage environment for the acid cleavable linking group. Examples of acid cleavable linking groups include, but are not limited to, hydrazones, esters, and esters of amino acids. Acid cleavable groups can have the general formula -C=NN-, C(O)O, or -OC(O). A preferred embodiment is where the carbon attached to the oxygen of the ester (alkoxy group) is an aryl group, a substituted alkyl group, or a tertiary alkyl group such as dimethylpentyl or t-butyl. These candidates can be evaluated using methods similar to those described above.

[0203] Ester-Based Linking Groups In another embodiment, the cleavable linker comprises an ester-based cleavable linker. The ester-based cleavable linker is cleaved by enzymes such as esterases and amidases in cells. Examples of ester-based cleavable groups include, but are not limited to, ester alkylene, alkenylene, and alkynylene groups. Ester cleavable linkers have the general formula -C(O)O- or -OC(O)-. These candidates can be evaluated using methods similar to those described above.

[0204] Peptide-Based Cleavage Groups In yet another embodiment, the cleavable linker comprises a peptide-based cleavable tether. Peptide-based cleavable tethers are cleaved by enzymes, such as peptidases and proteases, in cells. Peptide-based cleavable tethers are peptide bonds formed between amino acids to give rise to oligopeptides (e.g., dipeptides, tripeptides, etc.) and polypeptides. Peptide-based cleavable groups do not include amide groups (-C(O)NH-). Amide groups can be formed between any alkylene, alkenylene, or alkynylene. A peptide bond is a specific type of amide bond formed between amino acids to give rise to peptides and proteins. Peptide-based cleavable groups are generally limited to peptide bonds (i.e., amide bonds) formed between amino acids to give rise to peptides and proteins, and do not include the entire amide functionality. Peptide-based cleavable tethers have the general formula:

[0205] -NHCHR A C(O)NHCHR B C(O)-, where R A and R B are the R groups of two adjacent amino acids. These candidates can be evaluated using methods similar to those described above.

[0206] In one embodiment, the oligonucleotide of the present invention is conjugated to carbohydrate via a linker. The linker includes bivalent and trivalent branched linker groups. Linkers for oligonucleotide carbohydrate conjugates include, but are not limited to, those described in formulas 24-35 of WO 2018 / 195165.

[0207] Representative U.S. patents which teach the preparation of oligonucleotide conjugates include, but are not limited to, U.S. Pat. Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,077, 5,486,603, and the like. No. 5,512,439, No. 5,578,718, No. 5,608,046, No. 4,587,044, No. 4, No. 605,735, No. 4,667,025, No. 4,762,779, No. 4,789,737, No. 4,824,94 No. 1, No. 4,835,263, No. 4,876,335, No. 4,904,582, No. 4,958,013, No. 5 ,082,830, 5,112,963, 5,214,136, 5,082,830, 5,112,96 No. 3, No. 5,214,136, No. 5,245,022, No. 5,254,469, No. 5,258,506, No. 5 ,262,536, 5,272,250, 5,292,873, 5,317,098, 5,371,2 No. 41, No. 5,391,723, No. 5,416,203, No. 5,451,463, No. 5,510,475, No. No. 5,512,667, No. 5,514,785, No. 5,565,552, No. 5,567,810, No. 5,574,1 42, 5,585,481, 5,587,371, 5,595,726, 5,597,696, 5,599,923, 5,599,928, 5,688,941, 6,294,664, 6,320,017, 6,576,752, 6,783,931, 6,900,297, 7,037,646, and 8,106,022 (the entire contents of each of which are incorporated herein by reference).

[0208] Not all positions in a given compound need be uniformly modified, and in fact more than one of the aforementioned modifications can be incorporated into a single compound, or even into a single nucleoside within an oligonucleotide. The present invention also includes oligonucleotide compounds that are chimeric compounds. Chimeric oligonucleotides typically include at least one region in which the RNA is modified to confer improved resistance to nuclease degradation, increased cellular uptake, and / or improved binding affinity to the target nucleic acid to the oligonucleotide. Additional regions of the oligonucleotide can serve as substrates for enzymes capable of cleaving RNA:DNA. As an example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly enhancing the efficiency of oligonucleotide inhibition of gene expression. As a result, when chimeric oligonucleotides are used, comparable results can often be obtained with shorter oligonucleotides compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, optionally, associated nucleic acid hybridization techniques known in the art.

[0209] In certain instances, the nucleotides of the oligonucleotide can be modified with a non-ligand group. Many non-ligand molecules have been conjugated to oligonucleotides to enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide, and procedures for carrying out such conjugations are available in the scientific literature.Such non-ligand moieties include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm, 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053), thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765), thiocholesterol (Oberhauser et al., Nucl. Acids, 1997, 1:131-132), and the like. Res., 1992, 20:533), aliphatic chains, e.g., dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49), phospholipids, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777), polyamines or polyethylene glycol chains (Manoharan et al., al., Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. 1996:277-923).Representative US patents that teach the preparation of such oligonucleotide conjugates are listed above. A typical conjugation protocol involves the synthesis of an oligonucleotide with an amino linker at one or more positions of the sequence. The amino group is then reacted with the molecule to be conjugated using a suitable coupling or activating reagent. The conjugation reaction can be carried out in solution phase with the oligonucleotide still attached to the solid support or after cleavage of the oligonucleotide. Purification of the oligonucleotide conjugate by HPLC typically results in a pure conjugate.

[0210] Pharmaceutical Use The oligonucleotide compositions described herein are useful in the methods of the present invention and, without being bound by theory, are believed to exert their desired effects through their ability to modulate the levels, status, and / or activity of PCDH19, for example, by inhibiting the activity or levels of PCDH19 protein in mammalian cells.

[0211] One aspect of the invention relates to a method of treating a disorder associated with PCDH19 (e.g., epilepsy, schizophrenia, and autism) in a subject in need thereof. Another aspect of the invention includes reducing (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 97%, 99%, or 100%) the level of PCDH19 in a cell of a subject identified as having a PCDH19-associated disorder. Yet another aspect includes a method of inhibiting expression of PCDH19 in a cell of a subject. The method includes contacting the cell with an amount of an oligonucleotide effective to inhibit expression of PCDH19 in the cell, thereby inhibiting expression of PCDH19 in the cell.

[0212] Based on the above method, further aspects of the present invention include the oligonucleotide of the present invention, or a composition comprising such an oligonucleotide, for use in therapy, or for use as a medicament, or for use in treating a PCDH19-related disorder in a subject in need thereof, or for use in reducing the level of PCDH19 in a cell of a subject identified as having a PCDH19-related disorder, or for use in inhibiting the expression of PCDH19 in a cell in a subject. The use includes contacting a cell with an amount of the oligonucleotide effective to inhibit the expression of PCDH19 in the cell, thereby inhibiting the expression of PCDH19 in the cell. The embodiments described below in relation to the method of the present invention are also applicable to these further aspects.

[0213] The contacting of the cell with the oligonucleotide can be performed in vitro or in vivo. Contacting the cell with the oligonucleotide in vivo includes contacting a cell or a group of cells in a subject, such as a human subject, with the oligonucleotide. A combination of in vitro and in vivo methods of contacting the cell is also possible. Contacting the cell can be direct or indirect, as described above. Furthermore, contacting the cell can be achieved via a targeting ligand, including any ligand described herein or known in the art. In some embodiments, the targeting ligand is a carbohydrate moiety, such as a GalNAc3 ligand, or any other ligand that directs the oligonucleotide to a site of interest. The cell can include a cell of the central nervous system or a muscle cell.

[0214] Inhibiting expression of the PCDH19 gene includes any level of inhibition of the PCDH19 gene, for example, at least partial suppression of expression of the PCDH19 gene, for example, inhibition of at least about 20%. In certain embodiments, the inhibition is at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0215] Inhibiting the expression of PCDH19 gene includes inhibiting the expression of WT allele, mutant allele, or combination thereof. For example, the subject may have a mutation (e.g., nonsense, missense, and / or frameshift mutation) in one allele of PCDH19 gene. Since the gene is X-linked, the female subject is heterozygous and the male subject is hemizygous (the other copy is inactivated). The oligonucleotide may selectively target the mutant allele. The oligonucleotide may selectively target the WT allele. The oligonucleotide may target both alleles.

[0216] Expression of the PCDH19 gene can be assessed based on the level of any variable associated with PCDH19 gene expression, for example, PCDH19 mRNA level or PCDH19 protein level.

[0217] Inhibition can be assessed by a decrease in the absolute or relative levels of one or more of these variables compared to a control level, which can be any type of control level utilized in the art, such as a pre-administration baseline level, or a level determined from a similar subject, cell, or sample that is untreated or treated with a control (such as a buffer-only control or an inactive drug control).

[0218] In certain embodiments, surrogate markers can be used to detect inhibition of PCDH19. For example, effective treatment of a PCDH19-associated disorder can be understood to show a clinically relevant reduction in PCDH19, as indicated by acceptable diagnostic and monitoring criteria using agents that reduce PCDH19 expression.

[0219] In some embodiments of the methods of the invention, expression of the PCDH19 gene is inhibited by at least 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95%, or is inhibited to below the detection level of an assay. In certain embodiments, the methods include clinically relevant inhibition of expression of PCDH19, e.g., as indicated by a clinically relevant outcome following treatment of a subject with an agent that reduces expression of PCDH19.

[0220] Inhibition of expression of the PCDH19 gene may be evidenced by a decrease in the amount of mRNA expressed by a first cell or group of cells (such cells may be present, for example, in a sample from a subject) in which the PCDH19 gene is transcribed and which has been treated (e.g., by contacting the cells with an oligonucleotide of the invention, or by administering an oligonucleotide of the invention to a subject in which the cells are or were present) such that expression of the PCDH19 gene is inhibited, compared to a second cell or group of cells that is substantially identical to the first cell or group of cells but is not treated, or a second group of cells that is not treated (control cells that are not treated with an oligonucleotide targeting the gene of interest). The degree of inhibition may be expressed by the following formula:

number

[0221] In other embodiments, inhibition of expression of the PCDH19 gene may be assessed in terms of a reduction in a parameter functionally related to PCDH19 gene expression, such as PCDH19 protein expression or PCDH19 activity. PCDH19 gene silencing may be determined by any assay known in the art in any cell expressing either endogenous or heterologous PCDH19 from an expression construct.

[0222] Inhibition of expression of PCDH19 protein can be demonstrated by a decrease in the level of PCDH19 protein expressed by a cell or cell group (e.g., the level of protein expressed in a sample derived from a subject).As explained above, for the assessment of mRNA suppression, inhibition of protein expression level in treated cell or cell group can also be expressed as a percentage of protein level in control cell or cell group.

[0223] The control cell or cell group that can be used to evaluate the inhibition of the expression of PCDH19 gene includes a cell or cell group that has not yet been contacted with the oligonucleotide of the present invention.For example, the control cell or cell group can be derived from an individual subject (e.g., a human or animal subject) before the subject is treated with the oligonucleotide.

[0224] The level of PCDH19 mRNA expressed by a cell or group of cells can be determined using any method known in the art for assessing mRNA expression. In one embodiment, the expression level of PCDH19 in a sample is determined by detecting a transcribed polynucleotide or a portion thereof, such as the mRNA of the PCDH19 gene. RNA can be extracted from cells using RNA extraction techniques, including, for example, using acid phenol / guanidine isothiocyanate extraction (RNAzol B; Biogenesis), RNEASY™ RNA preparation kit (Qiagen), or PAXgene (PreAnalytix, Switzerland). Exemplary assay formats that utilize ribonucleic acid hybridization include nuclear run-on assays, RT-PCR, RNase protection assays, Northern blotting, in situ hybridization, and microarray analysis. Circulating PCDH19 mRNA can be detected using methods described in WO 2012 / 177906, the entire contents of which are incorporated herein by reference. In some embodiments, the expression level of PCDH19 is determined using a nucleic acid probe. The term "probe" as used herein refers to any molecule that can selectively bind to a specific PCDH19 sequence, such as an mRNA or polypeptide. Probes can be synthesized by those skilled in the art or derived from appropriate biological preparations. Probes can be specifically designed to be labeled. Examples of molecules that can be utilized as probes include, but are not limited to, RNA, DNA, proteins, antibodies, and organic molecules.

[0225] The isolated mRNA can be used in hybridization or amplification assays, including but not limited to Southern or Northern analysis, polymerase chain reaction (PCR) analysis, and probe arrays. One method for determining mRNA levels involves contacting the isolated mRNA with a nucleic acid molecule (probe) that can hybridize to PCDH19 mRNA. In one embodiment, the mRNA is immobilized on a solid surface and contacted with the probe, for example, by running the isolated mRNA on an agarose gel and transferring the mRNA from the gel to a membrane such as nitrocellulose. In another embodiment, the probe is immobilized on a solid surface and the mRNA is contacted with the probe, for example, in an AFFYMETRIX gene chip array. Those skilled in the art can easily adapt known mRNA detection methods for use in determining PCDH19 mRNA levels.

[0226] Alternative methods for determining the expression level of PCDH19 in a sample include, for example, RT-PCR (experimental embodiment described in Mullis, 1987, U.S. Pat. No. 4,683,202), ligase chain reaction (Barany (1991) Proc. Natl. Acad. Sci. USA 88:189-193), self-sustained sequence replication (Guatelli et al. (1990) Proc. Natl. Acad. Sci. USA 87:1874-1878), transcription amplification systems (Kwoh et al. (1989) Proc. Natl. Acad. Sci. USA 86:1173-1177), Q-beta replicase (Lizardi et al. (1988) Bio / Technology 6:1197), rolling circle replication (Lizardi et al. (1989) Bio / Technology 6:1197), and the like. These detection schemes involve the process of nucleic acid amplification and / or reverse transcriptase (to prepare cDNA), by PCR amplification using PCR primers such as PCR primers (e.g., TAQMAN™ System, et al., U.S. Pat. No. 5,854,033), or any other nucleic acid amplification method, followed by detection of the amplified molecules using techniques well known to those skilled in the art. These detection schemes are particularly useful for the detection of nucleic acid molecules when such molecules are present in very low numbers. In certain aspects of the present invention, the expression level of PCDH19 is determined by quantitative fluorogenic RT-PCR (i.e., TAQMAN™ System) or DUAL-GLO® Luciferase Assay.

[0227] The expression level of PCDH19 mRNA can be monitored using membrane blots (such as those used in hybridization analysis such as Northern, Southern, dot, etc.), or microwells, sample tubes, gels, beads, or fibers (or any solid support containing bound nucleic acid).See, for example, U.S. Patent Nos. 5,770,722, 5,874,219, 5,744,305, 5,677,195, and 5,445,934 (all of which are incorporated herein by reference).Determining the expression level of PCDH19 can also include using a nucleic acid probe in solution.

[0228] In some embodiments, the level of mRNA expression is assessed using branched DNA (bDNA) assays or real-time PCR (qPCR). The use of this PCR method is described and illustrated in the examples presented herein. Such methods can also be used for the detection of PCDH19 nucleic acid.

[0229] The level of PCDH19 protein expression can be determined by any method known in the art for measuring protein levels. Such methods include, for example, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), superdiffusion chromatography, liquid or gel precipitation reaction, absorption spectroscopy, colorimetric assay, spectrophotometric assay, flow cytometry, immunodiffusion (single or double), immunoelectrophoresis, Western blotting, radioimmunoassay (RIA), enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, electrochemiluminescence assay, etc. Such assays can also be used to detect proteins that indicate the presence or duplication of PCDH19 protein.

[0230] In some embodiments of the method of the present invention, oligonucleotide is administered to the subject so that the oligonucleotide is delivered to a specific site in the subject.The inhibition of PCDH19 expression can be evaluated by measuring the level or change in the level of PCDH19 mRNA or PCDH19 protein in a sample from a specific site in the subject.In certain embodiments, the method includes the clinically relevant inhibition of PCDH19 expression, for example, as shown by a clinically relevant outcome after the treatment of the subject with an agent that reduces the expression of PCDH19.

[0231] In other embodiments, the oligonucleotide is administered in an amount and for a time effective to result in a reduction (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100%) of one or more symptoms of a PCDH19-associated disorder, including, but not limited to, prolonged seizures, frequent seizures, behavioral and developmental delays, movement and balance problems, orthopedic conditions, speech and language delay problems, growth and nutrition problems, sleep disorders, chronic infections, sensory integration disorders, autonomic nervous system disruptions, and sweating.

[0232] Treatment of PCDH19-associated disorders may result in an improvement in the average survival time of an individual or a population of subjects treated by the present invention compared to a population of untreated subjects. For example, the individual survival time or the population average survival time is improved by more than 30 days (more than 60 days, 90 days, or 120 days). The improvement in the individual survival time or the population average survival time may be measured by any reproducible means. The improvement in the individual survival time may be measured, for example, by calculating the length of survival time for an individual after the initiation of treatment with a compound described herein. The improvement in the population average survival time may be measured, for example, by calculating the average length of survival time for an individual after the initiation of treatment with a compound described herein. The improvement in the individual survival time may also be measured, for example, by calculating the length of survival time for an individual after the completion of a first round of treatment with a compound described herein or a pharma- ceutically acceptable salt of the compound. The improvement in the population average survival time may also be measured, for example, by calculating the average length of survival time for a population after the completion of a first round of treatment with a compound described herein or a pharma- ceutically acceptable salt of the compound.

[0233] Treating PCDH19-associated disorders may also result in a reduction in the mortality rate of a population of treated subjects compared to an untreated population. For example, the mortality rate is reduced by more than 2% (e.g., more than 5%, 10%, or 25%). The reduction in the mortality rate of a population of treated subjects may be measured by any reproducible means, for example, by calculating the average number of disease-related deaths per unit time for a population after the start of treatment with a compound or a pharma- ceutically acceptable salt of a compound described herein. The reduction in the mortality rate of a population may also be measured, for example, by calculating the average number of disease-related deaths per unit time for a population after the completion of a first round of treatment with a compound or a pharma- ceutically acceptable salt of a compound described herein.

[0234] Delivery of oligonucleotides Delivery of the oligonucleotide of the present invention to a cell, for example, to a cell in a subject, such as a human subject, for example, to a cell in a subject in need, for example, to a cell in a subject having a PCDH19-associated disorder, can be achieved in several different ways. For example, delivery can be performed by contacting a cell with the oligonucleotide of the present invention in vitro or in vivo. In vivo delivery can also be performed directly by administering a composition comprising the oligonucleotide to a subject. These alternatives are further described below.

[0235] Generally, any method of delivering nucleic acid molecules (in vitro or in vivo) can be adapted for use with the oligonucleotides of the present invention (see, for example, Akhtar S. and Julian RL., (1992) Trends Cell.Biol.2(5):139-144, and WO 94 / 02595, which are incorporated herein by reference in their entirety). For in vivo delivery, factors to be considered for delivering oligonucleotide molecules include, for example, the biological stability of the delivered molecule, prevention of non-specific effects, and accumulation of the delivered molecule in the target tissue. Non-specific effects of oligonucleotides can be minimized by local administration, for example, by direct injection or implantation into tissue, or by local administration of a preparation. Local administration to the treatment site maximizes the local concentration of the drug, limits exposure of the drug to systemic tissues that may otherwise be harmed by or degrade the drug, and allows for a lower total dose of the oligonucleotide molecule to be administered.

[0236] When oligonucleotides are administered systemically for the treatment of disease, the oligonucleotides may contain alternative nucleobases, alternative sugar moieties, and / or alternative internucleoside linkages, or may be delivered using drug delivery systems, both of which act to prevent rapid degradation of the oligonucleotide in vivo by endo- and exonucleases. Modification of the oligonucleotide or pharmaceutical carrier may also allow targeting of the oligonucleotide composition to the target tissue and avoid undesirable off-target effects. Oligonucleotide molecules may be modified by chemical conjugation to lipophilic groups, such as cholesterol, to enhance cellular uptake and prevent degradation. In alternative embodiments, oligonucleotides may be delivered using drug delivery systems, such as nanoparticles, lipid nanoparticles, polyplex nanoparticles, lipoplex nanoparticles, dendrimers, polymers, liposomes, or cationic delivery systems. Positively charged cationic delivery systems facilitate binding of the oligonucleotide molecules (negatively charged) and also enhance interactions with the negatively charged cell membrane, allowing efficient uptake of the oligonucleotide by cells. Cationic lipids, dendrimers, or polymers can be either conjugated to oligonucleotides or induced to form vesicles or micelles that encase the oligonucleotides. The formation of vesicles or micelles further prevents the degradation of the oligonucleotides when administered systemically. Generally, any method of delivery of nucleic acids known in the art can be adapted to deliver the oligonucleotides of the present invention. Methods for making and administering cationic oligonucleotide complexes are well within the capabilities of those skilled in the art (see, for example, Sorensen, DR. et al. (2003) J. Mol. Biol 327:761-766; Verma, U N. et al., (2003) Clin. Cancer Res. 9:1291-1300; Arnold, AS et al., (2007) J. Hypertens. 25:197-205 (incorporated herein in their entirety by reference)).Some non-limiting examples of drug delivery systems useful for systemic delivery of oligonucleotides include DOTAP (Sorensen, D R., et al (2003), supra; Verma, U N et al., (2003), supra), oligofectamine "solid nucleic acid lipid particles" (Zimmermann, T S et al., (2006) Nature 441:111-114), cardiolipin (Chien, P Y. et al., (2005) Cancer Gene Ther. 12:321-328; Pal, A. et al., (2005) Int J. Oncol. 26:1087-1091), polyethylenimine (Bonnet M E. et al., (2008) Pharm. Res. Aug 16 Epub ahead of time), and ribozyme (R. print; Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD) peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines (Tomalia, D A. et al., (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H. et al., (1999) Pharm. Res. 16:1799-1804). In some embodiments, the oligonucleotides are complexed with cyclodextrins for systemic administration. Methods of administration and pharmaceutical compositions of oligonucleotides and cyclodextrins can be found in U.S. Patent No. 7,427,605, which is incorporated herein by reference in its entirety. In some embodiments, the oligonucleotides of the present invention are delivered by polyplex or lipoplex nanoparticles. Methods and pharmaceutical compositions for administering oligonucleotides and polyplex and lipoplex nanoparticles are disclosed in U.S. Pat.

[0237] Nos. 2017 / 0121454, 2016 / 0369269, 2016 / 0279256, 2016 / 0251478, 2016 / 0230189, 2015 / 0335764, 2015 / 0307554, 2015 / 0174549, 2014 / 0342003, 2014 / 0135376, and 2013 / 0317086.

[0238] Membrane molecular assembly delivery method The oligonucleotides of the present invention can also be delivered using various membrane molecular assembly delivery methods, including polymeric delivery devices, biodegradable microparticle delivery devices, or microcapsule delivery devices known in the art. For example, colloidal dispersion systems can be used for targeted delivery of the oligonucleotide agents described herein. Colloidal dispersion systems include macromolecular complexes, nanocapsules, microspheres, beads, and lipid-based systems including oil-in-water emulsions, micelles, mixed micelles, and liposomes. Liposomes are artificial membrane vesicles that are useful as in vitro and in vivo delivery vehicles. Large unilamellar vesicles (LUVs), ranging in size from 0.2 to 4.0 μm, have been shown to be capable of encapsulating a significant percentage of aqueous buffers containing large macromolecules. Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because liposomal membranes are structurally similar to biological membranes, when liposomes are applied to tissues, the liposomal bilayer fuses with the bilayer of the cell membrane. As the fusion of the liposome with the cell proceeds, the inner aqueous contents containing the oligonucleotide are delivered into the cell, and the oligonucleotide can specifically bind to the target RNA and mediate RNase H-mediated gene silencing. In some cases, the liposome is also specifically targeted, for example, to direct the oligonucleotide to a specific cell type. The composition of the liposome is usually a combination of phospholipids, usually in combination with steroids, especially cholesterol. Other phospholipids or other lipids can also be used. The physical properties of the liposome vary depending on pH, ionic strength, and the presence of divalent cations.

[0239] Liposomes containing oligonucleotides can be prepared by various methods. In one example, the lipid components of the liposome are dissolved in a detergent so that micelles are formed with the lipid components. For example, the lipid components can be amphipathic cationic lipids or lipid conjugates. The detergent can have a high critical micelle concentration and can be non-ionic. Exemplary detergents include cholate, CHAPS, octylglucoside, deoxycholate, and lauroyl sarcosine. The oligonucleotide preparation is then added to the micelles containing the lipid components. The cationic groups on the lipids interact with the oligonucleotides and condense around the oligonucleotides to form liposomes. After condensation, the detergent is removed, for example by dialysis, to obtain a liposome preparation of oligonucleotides.

[0240] If necessary, a carrier compound that aids in condensation can be added during the condensation reaction, for example, by controlled addition. For example, the carrier compound can be a polymer other than a nucleic acid (e.g., spermine or spermidine). The pH can also be adjusted to favor condensation.

[0241] Methods for producing stable polynucleotide delivery vehicles that incorporate polynucleotide / cationic lipid complexes as a structural component of the delivery vehicle are further described, for example, in WO 96 / 37194, the entire contents of which are incorporated herein by reference. Liposome formation has also been described by Feigner, PLet al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417; U.S. Pat. No. 4,897,355; U.S. Pat. al.,(1979)Biochim.Biophys.Acta 557:9;Szoka et al.,(1978)Proc.Natl.Acad.Sci.75:4194;Mayhew et al.,(1984)Biochim.Biophys.Acta 775:169;Kim et al.,(1983)Biochim.Biophys.Acta 728:339, and Fukunaga et al. al., (1984) Endocrinol. 115:757. Commonly used techniques for preparing lipid aggregates of appropriate size for use as delivery vehicles include sonication and freeze-thaw+extrusion (see, e.g., Mayer et al., (1986) Biochim. Biophys. Acta 858:161). When consistently small (50-200 nm) and relatively uniform aggregates are desired, microfluidization can be used (Mayhew et al., (1984) Biochim. Biophys. Acta 775:169). These methods are easily adapted for packaging oligonucleotide preparations into liposomes.

[0242] Liposomes are divided into two broad categories: cationic liposomes are positively charged liposomes that interact with negatively charged nucleic acid molecules to form stable complexes. The positively charged nucleic acid / liposome complexes bind to the negatively charged cell surface and are internalized in endosomes. The acidic pH within the endosomes causes the liposomes to rupture, releasing their contents into the cytoplasm (Wang et al. (1987) Biochem. Biophys. Res. Commun., 147:980-985).

[0243] pH-sensitive or negatively charged liposomes entrap nucleic acids rather than complexing with them. Because both the nucleic acid and the lipid are similarly charged, repulsion occurs rather than complexation. Nevertheless, some nucleic acids are entrapped in the aqueous interior of these liposomes. pH-sensitive liposomes have been used to deliver nucleic acids encoding the thymidine kinase gene to cell monolayers in culture. Expression of the exogenous gene has been detected in the target cells (Zhou et al. (1992) Journal of Controlled Release, 19:269-274).

[0244] One major type of liposome composition comprises phospholipids other than naturally occurring phosphatidylcholine. Neutral liposome compositions can be formed, for example, from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions are generally formed from dimyristoyl phosphatidylglycerol, and anionic fusogenic liposomes are primarily formed from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposome composition is formed, for example, from phosphatidylcholine (PC), such as soybean phosphatidylcholine (PC) and egg PC. Another type is formed from a mixture of phospholipids and / or phosphatidylcholine and / or cholesterol.

[0245] Other examples of methods for introducing liposomes into cells in vitro and in vivo include U.S. Pat. No. 5,283,185, U.S. Pat. No. 5,171,678, WO 94 / 00569, WO 93 / 24640, WO 91 / 16024, Feigner, (1994) J. Biol. Chem. 269(2550): Nabel, (1993) Proc. Natl. Acad. Sci. 90:11307; Nabel, (1992) Human Gene Ther. 3:649; Gershon, (1993) Biochem. 32:7143, and Strauss, (1992) EMBO J. 11:417.

[0246] Non-ionic liposomal systems, especially those containing non-ionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin.Non-ionic liposomal formulations containing NOVASOME™ I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and NOVASOME™ II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver cyclosporine-A to the dermis of mouse skin.The results showed that such non-ionic liposomal systems were effective in promoting the deposition of cyclosporine A into different layers of the skin (Hu et al., (1994) STP Pharma. Sci., 4(6): 466).

[0247] The liposomes may also be sterically stabilized liposomes, which contain one or more specialized lipids and provide an increased circulation lifespan compared to liposomes lacking such specialized lipids. An example of a sterically stabilized liposome is one in which a portion of the vesicle-forming lipid portion of the liposome is monosialoganglioside G M1or (B) are derivatized with one or more hydrophilic polymers, such as polyethylene glycol (PEG) moieties. Without being bound by any particular theory, it is believed in the art that the increased circulation half-life of these sterically stabilized liposomes, at least for those containing gangliosides, sphingomyelin, or PEG-derivatized lipids, is due to reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al., (1987) FEBS Letters, 223:42; Wu et al., (1993) Cancer Research, 53:3765).

[0248] Various liposomes containing one or more glycolipids are known in the art. Papahadjopoulos et al. (Ann. NY Acad. Sci., (1987), 507:64) report on monosialoganglioside G M1 reported the ability of (1) sphingomyelin and (2) ganglioside G to improve the blood half-life of liposomes. These findings are detailed by Gabizon et al. (Proc. Natl. Acad. Sci. USA (1988), 85:6949). U.S. Pat. No. 4,837,028 and WO 88 / 04924 (both by Allen et al.) report the ability of (1) sphingomyelin and (2) ganglioside G to improve the blood half-life of liposomes. M1 or galactocerebroside sulfate. U.S. Patent No. 5,543,152 (Webb et al.) discloses liposomes containing sphingomyelin. Liposomes containing 1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97 / 13499 (Lim et al.).

[0249] In one embodiment, cationic liposomes are used.Cationic liposomes have the advantage that they can fuse with cell membranes.Non-cationic liposomes cannot fuse with plasma membranes efficiently, but they can be taken up by macrophages in vivo and can be used to deliver oligonucleotides to macrophages.

[0250] Further advantages of liposomes include: Liposomes derived from natural phospholipids are biocompatible and biodegradable; Liposomes can incorporate a wide range of water-soluble and lipid-soluble drugs; Liposomes can protect oligonucleotides encapsulated in their internal compartment from metabolism and degradation (Rosoff, in "Pharmaceutical Dosage Forms", Lieberman, Rieger and Banker (Eds.), 1988, volume 1, p.245). Important considerations in the preparation of liposomal formulations are lipid surface charge, vesicle size, and water content of the liposomes.

[0251] The positively charged synthetic cationic lipid, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), can be used to form small liposomes that spontaneously interact with nucleic acids to form lipid-nucleic acid complexes that can fuse with the negatively charged lipids of tissue culture cell membranes, resulting in delivery of oligonucleotides (see, e.g., Feigner, PL et al., (1987) Proc. Natl. Acad. Sci. USA 8:7413-7417, and U.S. Pat. No. 4,897,355, for DOTMA and its use with DNA).

[0252] A DOTMA analog, 1,2-bis(oleoyloxy)-3-(trimethylammonia)propane (DOTAP), can be used in combination with phospholipids to form DNA-complexed vesicles. LIPOFECTIN™ (Bethesda Research Laboratories, Gaithersburg, MD) is an effective agent for the delivery of highly anionic nucleic acids to live tissue culture cells, containing positively charged DOTMA liposomes that spontaneously interact with negatively charged polynucleotides to form complexes. If sufficiently positively charged liposomes are used, the net charge on the resulting complex is also positive. The positively charged complexes thus prepared spontaneously attach to negatively charged cell surfaces, fuse with the plasma membrane, and efficiently deliver functional nucleic acids to, for example, tissue culture cells. Another commercially available cationic lipid, 1,2-bis(oleoyloxy)-3,3-(trimethylammonia)propane ("DOTAP") (Boehringer Mannheim, Indianapolis, Ind.), differs from DOTMA in that the oleoyl moieties are linked by ester rather than ether linkages.

[0253] Other reported cationic lipid compounds include those conjugated to a variety of moieties, including, for example, carboxyspermine conjugated to one of two types of lipids, and include compounds such as 5-carboxyspermylglycine dioctaoleoylamide ("DOGS") (TRANSFECTAM™, Promega, Madison, Wis.) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide ("DPPES") (see, e.g., U.S. Pat. No. 5,171,678).

[0254] Another cationic lipid conjugate involves derivatization of lipids with cholesterol ("DC-Chol") formulated into liposomes in combination with DOPE (see Gao, X. and Huang, L., (1991) Biochim. Biophys. Res. Commun. 179:280). Lipopolylysine, made by conjugating polylysine to DOPE, has been reported to be effective for transfection in the presence of serum (Zhou, X. et al., (1991) Biochim. Biophys. Acta 1065:8). For certain cell lines, these liposomes containing conjugated cationic lipids are said to exhibit lower toxicity and provide more efficient transfection than DOTMA-containing compositions. Other commercially available cationic lipid products include DMRIE and DMRIE-HP (Vical, La Jolla, Calif.) and Lipofectamine (DOSPA) (Life Technology, Inc., Gaithersburg, Md.). Other cationic lipids suitable for delivery of oligonucleotides are described in WO 98 / 39359 and WO 96 / 37194.

[0255] Liposomal formulations are particularly suitable for topical administration, and liposomes exhibit several advantages over other formulations. Such advantages include reduced side effects associated with high systemic absorption of administered drugs, increased accumulation of administered drugs in desired targets, and the ability to administer oligonucleotides to the skin. In some embodiments, liposomes are used to deliver oligonucleotides to epidermal cells, and also to enhance the penetration of oligonucleotides into dermal tissues (e.g., skin). For example, liposomes can be applied topically. Topical delivery of drugs formulated as liposomes to the skin has been demonstrated (e.g., Weiner et al., (1992) Journal of Drug Targeting, vol. 2405-410 and du Plessis et al., (1992) Antiviral Research, 18:259-265; Mannino, RJ and Fould-Fogerite, S., (1998) Biotechniques 6:682-690; Itani, T. et al., (1987) Gene 56:267-276; Nicolau, C. et al. (1987) Meth. Enzymol. 149:157-176; Straubinger, R. M. and Papahadjopoulos, D. (1983) Meth. Enzymol. 101:512-527; Wang, C. Y. and See Huang, L., (1987) Proc. Natl. Acad. Sci. USA 84:7851-7855).

[0256] Non-ionic liposomal systems, particularly those containing non-ionic surfactants and cholesterol, have also been tested to determine their usefulness in delivering drugs to the skin.Non-ionic liposomal formulations containing NOVASOME I (glyceryl dilaurate / cholesterol / polyoxyethylene-10-stearyl ether) and NOVASOME II (glyceryl distearate / cholesterol / polyoxyethylene-10-stearyl ether) were used to deliver drugs to the dermis of mouse skin.Such formulations containing oligonucleotides are useful for treating skin diseases.

[0257] Liposome targeting can also be based on, for example, organ specificity, cell specificity, and organelle specificity, and is known in the art. For liposome targeted delivery systems, lipid groups can be incorporated into the lipid bilayer of liposomes to keep targeting ligands in stable association with the liposome bilayer. Various linking groups can be used to connect lipid chains to targeting ligands. Further methods are known in the art, and are described, for example, in US Patent Publication No. 20060058255, whose linking groups are incorporated herein by reference.

[0258] Liposomes containing oligonucleotides can be made highly deformable. Such deformability can allow liposomes to penetrate through pores smaller than the average radius of the liposome. For example, transfersomes are yet another type of liposome, highly deformable lipid aggregates that are attractive candidates for drug delivery vehicles. Transfersomes can be described as lipid droplets that are so deformable that they can easily penetrate through pores smaller than the droplets. Transfersomes can be made by adding a surface edge activator, usually a surfactant, to a standard liposome composition. Transfersomes containing oligonucleotides can be delivered subcutaneously by infection, for example, to deliver oligonucleotides to keratinocytes in the skin. To pass through intact mammalian skin, lipid vesicles must pass through a series of micropores, each with a diameter of less than 50 nm, under the influence of a suitable transdermal gradient. Furthermore, due to lipid properties, these transfersomes can self-optimize (e.g., adapt to the shape of the pores in the skin), self-repair, and often self-inject to reach their targets without fragmentation. Transfersomes have been used to deliver serum albumin to the skin, and transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0259] Other formulations suitable for the present invention are described in U.S. Provisional Application No. 61 / 018,616, filed January 2, 2008, U.S. Provisional Application No. 61 / 018,611, filed January 2, 2008, U.S. Provisional Application No. 61 / 039,748, filed March 26, 2008, U.S. Provisional Application No. 61 / 047,087, filed April 22, 2008, and U.S. Provisional Application No. 61 / 051,528, filed May 8, 2008. International Application No. PCT / US2007 / 080331, filed October 3, 2007, also describes formulations suitable for the present invention.

[0260] Surfactants are widely applied in formulations such as emulsions (including microemulsions) and liposomes. The most common way to classify and rank the properties of the many different types of surfactants, both natural and synthetic, is by using the hydrophilic / lipophilic balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for classifying the different surfactants used in formulations (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0261] If the surfactant molecule does not ionize, it is classified as a nonionic surfactant. Nonionic surfactants are widely applied in pharmaceuticals and cosmetics and are usable over a wide range of pH values. Generally, their HLB values ​​range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers, such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated / propoxylated block polymers, are also included in this classification. Polyoxyethylene surfactants are the most common members of the nonionic surfactant classification.

[0262] If the surfactant molecule has a negative charge when dissolved or dispersed in water, the surfactant is classified as anionic.Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyltaurates and sulfosuccinates, and phosphates.The most important members of the anionic surfactant classification are alkyl sulfates and soaps.

[0263] If the surfactant molecule has a positive charge when dissolved or dispersed in water, the surfactant is classified as cationic.Cationic surfactants include quaternary ammonium salts and ethoxylated amines.Quaternary ammonium salts are the most used members of this classification.

[0264] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines, and phosphatides.

[0265] The use of surfactants in drug products, formulations, and emulsions has been reviewed (Rieger, in Pharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, NY, 1988, p. 285).

[0266] The oligonucleotide for use in the method of the present invention can also be provided as a micelle formulation.Micelle is a specific type of molecular assembly in which amphiphilic molecules are arranged in a globular structure, so that all the hydrophobic parts of the molecule are directed inward, and the hydrophilic parts remain in contact with the surrounding aqueous phase.When the environment is hydrophobic, the reverse arrangement exists.

[0267] Lipid nanoparticle-based delivery methods The oligonucleotides of the invention can be fully encapsulated in lipid formulations, such as lipid nanoparticles (LNPs), or other nucleic acid-lipid particles. LNPs exhibit extended circulatory life after intravenous (iv) injection and accumulate at distal sites (e.g., sites physically distant from the site of administration), making them extremely useful for systemic applications. LNPs include "pSPLPs," which include the encapsulated condensing agent-nucleic acid complexes described in WO 00 / 03683. The particles of the invention typically have an average diameter of about 50 nm to about 150 nm, more typically about 60 nm to about 130 nm, more typically about 70 nm to about 110 nm, and most typically about 70 nm to about 90 nm, and are substantially non-toxic. Furthermore, the nucleic acid, when present in the nucleic acid-lipid particles of the invention, is resistant to degradation by nucleases in aqueous solution. Nucleic acid-lipid particles and methods for their preparation are disclosed, for example, in U.S. Pat. Nos. 5,976,567, 5,981,501, 6,534,484, 6,586,410, 6,815,432, U.S. Publication No. 2010 / 0324120, and WO 96 / 40964.

[0268] In one embodiment, the lipid to drug ratio (mass / mass ratio) (e.g., lipid to oligonucleotide ratio) ranges from about 1:1 to about 50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, from about 4:1 to about 10:1, from about 5:1 to about 9:1, or from about 6:1 to about 9:1. Ranges intermediate to the above ranges are also considered part of the invention.

[0269] Non-limiting examples of cationic lipids include N,N-dioleyl-N,N-dimethylammonium chloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB), N-(I-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP), N-(I-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA), N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 1,2-dilinoleyloxy-N,N-dimethylaminopropane (DLen ... 1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP), 1,2-Dilinoleyloxy-3-(dimethylamino)acetoxypropane (DLin-DAC), 1,2-Dilinoleyloxy-3-morpholinopropane (DLin-MA), 1,2-Dilinoleoyl-3-dimethylaminopropane (DLin-DAP), 1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA), 1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP), 1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl), 1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl), 1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or 3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP), 3-(N,N-Dioleylamino)-1,2-propanediol (DOAP), 1,2-Dilinoleylxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA), 1,2-Dilinoleyloxy-N,N-dimethylaminopropane (DLinDMA), 2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) or analogs thereof, (3aR,5s,6aS)-N,N-Dimethyl-2,2-di((9Z,12Z) -octadeca-9,12-dienietetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine (ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31tetraen-19-yl 4-(dimethylamino)butanoate (MC3), 1,1'-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazine-1-iethylazanediediedodecan-2-ol (TEC G1), or mixtures thereof. The cationic lipid can, for example, constitute about 20 mol% to about 50 mol% or about 40 mol% of the total lipid present in the particle.

[0270] Ionic / non-cationic lipids include distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), dioleoyl-phosphatidylethanolamine (DOPE), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-( The lipid may be an anionic or neutral lipid, including, but not limited to, N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoyl phosphatidylethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1-trans PE, 1-stearoyl-2-oleoyl-phosphatidylethanolamine (SOPE), cholesterol, or mixtures thereof. The non-cationic lipid, for example, when cholesterol is included, may be about 5 mol% to about 90 mol%, about 10 mol%, or about 60 mol% of the total lipid present in the particle.

[0271] The conjugated lipid that inhibits particle aggregation can be, for example, a polyethylene glycol (PEG)-lipid, including, but not limited to, PEG-diacylglycerol (DAG), PEG-dialkyloxypropyl (DAA), PEG-phospholipid, PEG-ceramide (Cer), or mixtures thereof. PEG-DAA conjugates can be, for example, PEG-dilauryloxypropyl (C 12 ), PEG-dimyristyloxypropyl (C 14 ), PEG-dipalmityloxypropyl (C 16 ), or PEG-distearyloxypropyl (C 18 The conjugated lipid that prevents particle aggregation can be, for example, from 0 mol % to about 20 mol %, or about 2 mol % of the total lipid present in the particle.

[0272] In some embodiments, the nucleic acid-lipid particles further comprise cholesterol, for example, at about 10 mol % to about 60 mol % or about 50 mol % of the total lipid present in the particle. Combination therapy

[0273] The method of the present invention can be used alone or in combination with additional therapeutic agents (therapeutic agents), such as other agents that treat PCDH19-associated disorders or symptoms associated therewith, or in combination with other types of therapy for treating PCDH19-associated disorders. In combination therapy, the dosage of one or more therapeutic compounds can be reduced from the standard dosage when administered alone. For example, dosage can be empirically determined from drug combinations and permutations, or estimated by isobolographic analysis (e.g., Black et al., Neurology 65:S3-S6(2005)). In this case, the dosage of the compounds when combined should provide a therapeutic effect.

[0274] In some embodiments, the oligonucleotide agent described herein can be used in combination with an additional therapeutic agent for treating PCDH19-related disorder.In some embodiments, the additional therapeutic agent can be an oligonucleotide (e.g., ASO) that hybridizes with the mRNA of the gene associated with PCDH19-related disorder.

[0275] In some embodiments, the second therapeutic agent is a chemotherapeutic agent (eg, a cytotoxic agent or other chemical compound useful in the treatment of a PCDH19-associated disorder).

[0276] In some embodiments, the second therapeutic agent is an anti-seizure agent.

[0277] The second agent can be a therapeutic agent that is a non-pharmacological treatment, for example, the second therapeutic agent is physical therapy.

[0278] In any of the combination embodiments described herein, the first and second therapeutic agents may be administered simultaneously or sequentially in any order. The first therapeutic agent may be administered immediately before or after the second therapeutic agent, or up to 1 hour, up to 2 hours, up to 3 hours, up to 4 hours, up to 5 hours, up to 6 hours, up to 7 hours, up to 8 hours, up to 9 hours, up to 10 hours, up to 11 hours, up to 12 hours, up to 13 hours, 14 hours, up to 16 hours, up to 17 hours, up to 18 hours, up to 19 hours, up to 20 hours, up to 21 hours, up to 22 hours, up to 23 hours, up to 24 hours, or up to 1-7 days, 1-14 days, 1-21 days, or 1-30 days before or after the second therapeutic agent.

[0279] Pharmaceutical Compositions The oligonucleotides described herein are preferably formulated into pharmaceutical compositions for administration to human subjects in a biologically compatible form suitable for administration in vivo.

[0280] The compounds described herein may be used in the form of free base, salt, solvate, and prodrug. All forms are within the scope of the methods described herein. According to the methods of the present invention, the compounds described, or their salts, solvates, or prodrugs, may be administered to a subject in various forms, as will be understood by those skilled in the art, depending on the route of administration selected. The compounds described herein may be administered, for example, orally, parenterally, intrathecally, intracerebroventricularly, intraparenchymal, buccal, sublingually, nasally, rectally, by patch, pump, or transdermal administration, and the pharmaceutical composition may be formulated accordingly. Parenteral administration includes intravenous, intraperitoneal, subcutaneous, intramuscular, transepithelial, nasal, pulmonary, intraspinal, intracerebroventricular, intraparenchymal, rectally, and topical modes of administration. Parenteral administration may be by continuous infusion over a selected period of time.

[0281] The compounds described herein may be administered at any time during life. The compounds may be administered to a newborn, neonate, infant, toddler, adolescent, or adult. The compounds may be administered before birth. The compounds may be administered immediately after birth.

[0282] The compounds described herein may be administered orally, for example, with an inert diluent or an assimilable edible carrier, or may be enclosed in hard or soft shell gelatin capsules, or may be compressed into tablets, or may be incorporated directly into dietary food. For oral therapeutic administration, the compounds described herein may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, and wafers. The compounds described herein may also be administered parenterally. Solutions of the compounds described herein may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, DMSO, and mixtures thereof with or without alcohol, as well as in oils. Under ordinary conditions of storage and use, these preparations may contain a preservative to prevent the growth of microorganisms. Conventional procedures and ingredients for the selection and preparation of suitable formulations are described, for example, in Remington's Pharmaceutical Sciences (2012, 22nd ed.) and The United States Pharmacopeia: The National Formulary (USP 41 NF 36), published in 2018. Pharmaceutical forms suitable for injection use include sterile injectable solutions or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. In all cases, the form must be sterile and fluid to the extent that it can be administered by syringe. Compositions for nasal administration can be conveniently formulated as aerosols, drops, gels, and powders. Aerosol formulations typically contain a solution or fine suspension of the active substance in a physiologically acceptable aqueous or non-aqueous solvent, and are usually provided in single or multiple doses in sterile form in a sealed container that can take the form of a cartridge or refill for use in a nebulizer device. Alternatively, the sealed container may be an integrated dispensing device, such as a single-dose nasal inhaler or an aerosol dispenser equipped with a metering valve intended for disposal after use. Where the dosage form comprises an aerosol dispenser, it will contain a propellant, which can be a compressed gas, such as compressed air, or an organic propellant, such as a fluorochlorohydrocarbon.Aerosol dosage forms can also take the form of a pump-atomizer.Compositions suitable for buccal or sublingual administration include tablets, lozenges, and pastilles, in which the active ingredient is formulated with a carrier such as sugar, acacia, tragacanth, gelatin, and glycerin.Compositions for rectal administration are conveniently in the form of suppositories containing a conventional suppository base such as cocoa butter.

[0283] The compounds described herein can be administered to animals, such as humans, alone or in combination with pharma- ceutically acceptable carriers described herein, the proportions being determined by the solubility and chemical properties of the compounds, the chosen route of administration, and standard pharmaceutical practice. Dosage

[0284] The dosage of the compositions described herein (e.g., compositions comprising oligonucleotides) may vary depending on many factors, such as the pharmacodynamic properties of the compound, the mode of administration, the age, health, and weight of the recipient, the nature and extent of symptoms, the frequency of treatment, the type of concomitant treatment (if any), and the clearance rate of the compound in the treated animal. The compositions described herein may be administered initially at a suitable dosage, which may be adjusted as necessary depending on clinical response. In some embodiments, the dosage of the compositions (e.g., compositions comprising oligonucleotides) is a prophylactically or therapeutically effective amount.

[0285] kit The invention also features a kit that includes (a) a pharmaceutical composition comprising an oligonucleotide agent that reduces the level and / or activity of PCDH19 in a cell or subject as described herein, and (b) a package insert that includes instructions for practicing any of the methods described herein. In some embodiments, the kit includes (a) a pharmaceutical composition comprising an oligonucleotide agent that reduces the level and / or activity of PCDH19 in a cell or subject as described herein, (b) an additional therapeutic agent, and (c) a package insert that includes instructions for practicing any of the methods described herein.

[0286] How to Choose an ASO Oligonucleotides suitable for use in ASO therapy may be selected using bioinformatics methods. The oligonucleotides may be 15-30 (e.g., 18-22, e.g., 19, 20, or 21) nucleotides in length. The oligonucleotides may have a GC content of about 40% to about 70% (e.g., 45%, 50%, 55%, 60%, 65%, or 70%). The oligonucleotides may contain no more than three (e.g., 2, 1, or 0) mismatches to human PCDH19. The oligonucleotides may contain at least 80% (e.g., 85%, 90%, 95%, 97%, 99%, and 100%) sequence identity to an equal length target of human PCDH19. The oligonucleotides may contain at least three (e.g., 4, 5, 6, 7, 8, 9, 10, or more) mismatches to non-PCDH19 transcripts. The oligonucleotides may not form dimers. The oligonucleotides may not form hairpins. The oligonucleotide may lack a poly-G run, such as GGGG.

[0287] ASO evaluation The activity of the antisense oligonucleotide of the present disclosure can be evaluated and confirmed using various techniques known in the art.For example, the ability of antisense oligonucleotide to inhibit PCDH19 expression can be evaluated in an in vitro assay to confirm that the antisense oligonucleotide is suitable for use in treating diseases or conditions associated with PCDH19 mutation.Mouse models can be used to not only evaluate the ability of antisense oligonucleotide to inhibit PCDH19 expression, but also to improve symptoms associated with PCDH19 mutation.

[0288] In one example, cells such as mammalian cells (e.g., CHO cells) transfected with PCDH19 and expressing this gene are also transfected with the antisense oligonucleotides of the present disclosure. Typically, PCDH19 contains a mutation, such as a missense mutation, a nonsense mutation, or a frameshift (e.g., insertion or deletion) mutation. In another example, a human neuronal cell line (e.g., SH-SY5Y) that naturally expresses native wild-type PCDH19 is used. Optionally, the genome of this cell is edited to contain a mutation, such that the resulting PCDH19 single cell is a disease-causing mutant. The level of PCDH19 mRNA can be assessed using qRT-PCR or Northern blot, as known in the art. The expression level of protein from PCDH19 can be assessed by Western blot on total cell lysates or fractions as described in Rizzo et al. (Mol Cell Neurosci.72:54-63,2016).

[0289] In certain instances, the activity of the antisense oligonucleotides of the present disclosure is evaluated and confirmed using stem cell modeling (for reference, see, e.g., Tidball and Parent Stem Cells 34:27-33, 2016; Parent and Anderson Nature Neuroscience 18:360-366, 2015). For example, human induced pluripotent stem cells (iPSCs) can be produced from somatic cells (e.g., skin fibroblasts or blood-derived hematopoietic cells) derived from a patient that has a PCDH19 mutation and exhibits an associated disease or condition (e.g., EIEE9). Optionally, genome editing can be used to revert the mutation to wild type to create an isogenic control cell line (Gaj et al. Trends Biotechnol 31, 397-405, 2013), which can also be used to determine the desired wild type level of activity for subsequent evaluation and comparison of oligonucleotides. Alternatively, genome editing can be used to introduce a mutation into the PCDH19 gene of a wild type control iPSC (e.g., a reference iPSC line). The mutation-containing iPSCs, and optionally isogenic controls, can then be differentiated into neurons, including excitatory neurons, using known techniques (see, for example, Kim et al. Front Cell Neurosci 8:109,2014; Zhang et al. 2013, Chambers et al. Nat Biotechnol 27,275-280,2009).The effect of the antisense oligonucleotide of the present invention on PCDH19 expression (assessed by PCDH19 mRNA or protein levels) and / or activity (e.g., assessed by calcium binding) can then be evaluated after exposure of the iPSCs to the antisense oligonucleotide of the present invention.

[0290] The level of PCDH19 expression (mRNA or protein) observed when cells expressing PCDH19 are exposed to the antisense oligonucleotide of the present disclosure is compared to the respective levels observed when cells expressing PCDH19 are exposed to a negative control antisense oligonucleotide to determine the level of inhibition provided by the antisense oligonucleotide of the present disclosure. Typically, the expression level of PCDH19 is reduced by at least or about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more. Thus, the antisense oligonucleotide of the present disclosure can be used to treat diseases or conditions associated with mutations in PCDH19.

[0291] The functional effect of administration of antisense oligonucleotides can also be evaluated by using mixing experiments such as those described in Pederick et al. Neuron 97:59-66, 2018, which is incorporated herein by reference in its entirety. K562 cells can be used because they do not aggregate in culture due to the lack of endogenous PCDH and classical cadherins. The cells can be engineered to express PCDH19 (either WT or mutant) in combination with another non-clustered PCDH, such as PCDH17 and / or PCDH10. In some specific examples, the cells can express both WT and mutant PCDH19. Two populations of fluorescently labeled K562 cells expressing the same or different combinations of PCDHs can be mixed in a 1:1 ratio. The resulting cell aggregates can be evaluated for the contribution of each population. Aggregates biased towards one population can indicate cell sorting by different adhesion specificities, whereas heterogeneous aggregates can indicate cell mixing by the same adhesion specificities. If the disease-causing variant causes sorting as opposed to mixing, cells containing the PCDH19 variant can be treated with an ASO (e.g., an allele-specific ASO) and the results of the cell sorting experiment can be re-evaluated. If the results then show mixing instead of sorting, the ASO can be considered successful in targeting and reducing expression of mutant PCDH19.

[0292] Also, mouse models can be used to evaluate and confirm the activity of the antisense oligonucleotides of the present disclosure.For example, knock-in or transgenic mouse models can be made using PCDH19 genes containing mutations in a manner similar to that previously described for SCN1A and SCN2A knock-in and transgenic mouse models (see, for example, Kearney et al. Neuroscience 102,307-317,2001; Ogiwara et al. J Neurosci 27:5903-5914,2007; Yu et al. Nat Neurosci 9:1142-1149,2006).In certain examples, knock-in or transgenic mice are made using PCDH19 genes that match specific antisense oligonucleotides.Mutant PCDH19 knock-in or transgenic mice can show phenotypes similar to EIEE9, including, for example, enhanced neuronal activity, spontaneous seizures, and heterogeneous focal seizure activity in electroencephalogram (EEG). Antisense oligonucleotides of the invention can then be assessed for their ability to inhibit expression of PCDH19 in these mice and to ameliorate any symptoms associated with PCDH19 mutations.

[0293] In another example, the level of PCDH19 mRNA and / or protein can be evaluated after administration of the antisense oligonucleotide of the present disclosure or a negative control antisense oligonucleotide to a mouse. In a particular example, the level of PCDH19 mRNA and / or protein is evaluated in the brain, particularly in neurons. The level of PCDH19 expression after administration of the antisense oligonucleotide of the present disclosure is compared to the respective levels observed when a negative control antisense oligonucleotide is administered to determine the level of inhibition caused by the antisense oligonucleotide of the present disclosure. Typically, the expression level of PCDH19 in the mouse (e.g., in the mouse brain) is reduced by at least or about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more.

[0294] In yet another example, the functional effect of administering the antisense oligonucleotide of the present disclosure is evaluated. For example, the number, severity, and / or type of seizures can be evaluated visually and / or by EEG. Neuronal excitability can also be evaluated, for example, by dissecting brain slices from mice administered the antisense oligonucleotide of the present disclosure or negative control antisense oligonucleotide and evaluating whole cell currents (e.g., using whole cell patch clamp technique). Similar neuronal excitability analysis can be performed with neurons isolated from mice and then cultured. Furthermore, mouse behavior, including gait characteristics, can be evaluated to determine the functional effect of administering the antisense oligonucleotide of the present disclosure.

[0295] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference in their entirety to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. In the event that a term in this application is found to be defined differently in a document incorporated herein by reference, the definition provided herein serves as the definition of that term.

[0296] Reference in this specification to any prior publication (or information derived therefrom), or to any known matter, is not intended to be, and should not be construed as, an acknowledgement or admission, or any form of suggestion, that the prior publication (or information derived therefrom) or known matter forms part of the common general knowledge in the field of endeavor to which this specification pertains.

[0297] While the invention has been described in relation to specific embodiments thereof, it will be understood that it is capable of further modifications, and this application is intended to cover any variation, use, or adaptation of the invention generally in accordance with the principles of the invention, including departures from the present disclosure which are known or customarily made within the art to which the invention pertains, and which fall within the scope of the claims, to the essential characteristics hereinbefore described. EXAMPLES

[0298] Example 1. Design and construction of ASOs Exemplary 15-, 16-, 17- and 20-mer antisense oligonucleotides (ASOs) were generated against the human PCDH19 gene, as shown in NCBI reference sequence: NC_000023.11 (SEQ ID NO: 1), located on the complementary strand within region 100291644.100410273) (SEQ ID NO: 1). The 5' end of the ASO target site was assigned to a position within the gene listed in Table 2 below. Nucleotide composition was determined and ASOs with GC content less than 20% or more than 80% were excluded from further analysis. Cross-reactivity of ASOs was calculated for all known PCDH19 mRNA and pre-mRNA transcripts from NCBI RefSeqDB and EnsemblDB to identify ASOs targeting as many different protein-coding transcripts as possible. Off-target ASOs (with up to two mismatches) were excluded from further analysis. Single nucleotide polymorphisms (SNPs) located at ASO target sites were identified based on the human PCDH19 gene (NC_000023.11; B. Region: Complement (100291644.100410273)), and ASO targeting sites with SNPs with minor allele frequency of 1% or more were excluded from further analysis. The remaining ASOs were further screened for cross-reactivity with non-human primates and dogs. From this analysis, 384 ASO candidates (SEQ ID NO:2 to SEQ ID NO:385) were selected for synthesis and in vitro screening, as shown in Table 2.

[0299] Example 2. Screening of ASOs by assessing inhibitory activity against PCDH19 Exemplary ASOs shown in Table 2 (SEQ ID NOs: 2-385) were screened using HEK293 cells (ATCC) seeded at a density of 30,000 cells per well in 96-well plates. Cells were transfected with 0.2 nM or 2.0 nM of each ASO using 0.4 μl / well of Lipofectamine 2000 transfection reagent (Invitrogen). 48 hours post-transfection, PCDH19 mRNA expression was quantified from cell lysates using the Quantigene singleplex branched DNA assay (ThermoFisher). PCDH19 expression was normalized to GAPDH and expressed relative to the pooled mean of mock-transfected cells and cells transfected with the Ahsa1 ASO, which does not cross-react with PCDH19. Data calculated from four separate experiments are shown in Table 2 (as mean and standard deviation) and Figure 1.

[0300] Example 3. Treatment of EIEE9 by administration of ASO. A female patient with EIEE9 is selected for ASO therapy. A 16-mer ASO targeting PCDH19 mRNA is synthesized with phosphorothioate linkages throughout and two MOE modifications on all sugar moieties. The ASO is dissolved in a suitable excipient compatible with human administration. A solution containing the dissolved ASO is injected into the patient's brain so that the ASO solution interacts with the target neurons in the brain. The ASO transfects the neurons and alters the translation of PCDH19 in the target cells, resulting in a reduction in PCDH19 protein. A quantitative assay is performed to measure the reduction in PCDH19 protein. The patient undergoes extensive regular testing to measure the relief of seizures and other symptoms associated with EIEE9 after administration of ASO therapy. [Table 2-1] [Table 2-2] [Table 2-3]

Table 2-4

Table 2-5

Table 2-6

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Table 2-16

Table 2-17

Table 2-18

Claims

1. A single-stranded oligonucleotide having a length of 10 to 80 nucleosides, the single-stranded oligonucleotide having a nucleobase sequence comprising a portion of 10 consecutive nucleobases that has at least 80% complementarity to an equal length portion of a target region of the human PCDH19 gene, its pre-mRNA transcript, and / or its mRNA transcript.

2. 2. The oligonucleotide of claim 1, wherein the target region is within the nucleotide sequence set forth in SEQ ID NO: 1 or a variant thereof having at least 80% or about 80% sequence identity thereto.

3. 2. The oligonucleotide of claim 1, wherein the mRNA transcript or pre-mRNA transcript comprises at least one PCDH19 mutation.

4. 4. The oligonucleotide of claim 3, wherein the at least one PCDH19 mutation is selected from the group consisting of mutations encoding N340S, E307K, V441E, Q85X, N557K, D594H, S671X, L677fsx717, I119fsX122, and P364fsX375, or the at least one PCDH19 mutation is a mutation selected from Table 1.

5. 4. The oligonucleotide of claim 3, wherein the oligonucleotide selectively hybridizes to the mRNA or pre-mRNA transcript comprising the at least one PCDH19 mutation over a wild-type mRNA or pre-mRNA transcript.

6. 2. The oligonucleotide of claim 1, wherein the oligonucleotide consists of 12 to 40 nucleobases, 16 to 30 nucleobases, or 18 to 22 nucleobases.

7. the oligonucleotide is (a) a gap segment comprising linked deoxyribonucleosides; (b) a 5' wing segment comprising linked nucleosides; (c) a 3' wing segment comprising the linked nucleosides; 2. The oligonucleotide of claim 1, wherein the gap segment comprises a region of at least 10 contiguous nucleobases having at least 80% complementarity to an equal length portion of the target region of the pre-mRNA transcript or the mRNA transcript of the human PCDH19 gene positioned between the 5' wing segment and the 3' wing segment, and wherein the 5' wing segment and the 3' wing segment each comprise at least two linked nucleosides, and at least one nucleoside of each wing segment comprises an alternative nucleoside.

8. 2. The oligonucleotide of claim 1, comprising at least one alternative internucleoside linkage, at least one alternative nucleobase, and / or at least one alternative sugar moiety.

9. 9. The oligonucleotide of claim 8, wherein the at least one alternative internucleoside linkage is selected from the group consisting of a phosphorothioate internucleoside linkage, a 2'-alkoxy internucleoside linkage, or an alkylphosphate internucleoside linkage.

10. 9. The oligonucleotide of claim 8, wherein the alternative nucleobase is 5'-methylcytosine, pseudouridine, or 5-methoxyuridine.

11. 9. The oligonucleotide of claim 8, wherein the alternative sugar moiety is a 2'-OMe modified sugar moiety or a bicyclic sugar moiety.

12. 2. The oligonucleotide of claim 1, wherein the oligonucleotide further comprises a ligand conjugated to the 5' or 3' end of the oligonucleotide via a monovalent or branched divalent or trivalent linker.

13. 2. The oligonucleotide of claim 1, wherein the oligonucleotide comprises a region complementary to at least 17 or at least 19 consecutive nucleotides of the PCDH19 gene.

14. The oligonucleotide of claim 1, wherein the oligonucleotide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 2 to 385.

15. A pharmaceutical composition comprising the oligonucleotide of any one of claims 1 to 14 and a pharmaceutically acceptable carrier.

16. 16. The pharmaceutical composition of claim 15, comprising a lipid nanoparticle, a polyplex nanoparticle, a lipoplex nanoparticle, or a liposome.

17. 15. A composition comprising the oligonucleotide of any one of claims 1 to 14 for treating, preventing or delaying the progression of a PCDH19-associated disorder in a subject in need thereof, or for inhibiting transcription of PCDH19 or reducing the level and / or activity of PCDH19 in cells of a subject having a PCDH19-associated disorder.

18. 18. The composition of claim 17, wherein the cell is a cell of the central nervous system.

19. 18. The composition of claim 17, wherein the PCDH19-associated disorder is selected from the group consisting of epilepsy, schizophrenia, and autism.

20. 18. The composition of claim 17, wherein the subject has a mutation in at least one allele of the PCDH19 gene.

21. 21. The composition of claim 20, wherein the oligonucleotide selectively reduces expression of the allele containing the mutation compared to an allele that does not contain the mutation, or wherein the oligonucleotide reduces expression of the allele containing the mutation and an allele containing a wild-type sequence.

22. 21. The composition of claim 20, wherein the mutation is a missense mutation, a nonsense mutation, or a frameshift mutation.

23. 18. The composition of claim 17, which alleviates one or more symptoms of the PCDH19-associated disorder.

24. 24. The composition of claim 23, wherein the one or more symptoms of the PCDH19-associated disorder are selected from the group consisting of prolonged seizures, frequent seizures, behavioral and developmental delays, movement and balance problems, orthopedic conditions, speech and language delay problems, growth and nutritional problems, sleep difficulties, chronic infections, sensory integration disorders, autonomic nervous system disturbances, and sweating.

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