UBE3A antisense therapeutics

Abstract

The present invention provides compositions useful for knocking down overexpression of UBE3A and treating conditions associated with Dup15q syndrome, which may include antisense oligonucleotides, short oligonucleotides (ASOs) that are complementary to UBE3A transcripts and hybridize in vivo, preventing or inhibiting successful translation of UBE3A mRNA into protein. The ASOs include anti-UBE3A gapmer-oligos that include a central DNA portion flanked by RNA wings, where the gapmers hybridize to UBE3A pre-mRNA or mRNA, and the double-stranded hybrids recruit RNaseH, which cleaves or digests UBE3A pre-mRNA or mRNA, preventing expression of UBE3A protein. Thus, the ASOs prevent expression of UBE3A protein, and treatment with compositions that include the ASOs of the present disclosure may be effective for knocking down overexpression of UBE3A.

Description

[Technical field]

[0001] Technical Field The present disclosure relates to treatments for neurological disorders.

[0002] Sequence Listing This application contains a Sequence Listing that was submitted via EFS-Web in ASCII format and is incorporated herein by reference in its entirety. The ASCII formatted Sequence Listing was created on February 17, 2022, is titled "QSTA-036-01WO-Sequence-Listing", and is 44048 bytes in size. [Background technology]

[0003] background Ubiquitin ligase proteins, such as E3 ligase E6-associated protein (E6AP, also known as UBE3A), have been implicated in neurological and neurodevelopmental disorders. For example, E6AP is encoded by the UBE3A gene, the expression of which is regulated via genetic imprinting. Loss of E6AP expression leads to the development of Angelman syndrome, typically characterized by impaired speech and motor development, as well as seizures. Conversely, copy number variations (CNVs) of UBE3A can be associated with overexpression of E6AP and consequently the development of autism spectrum disorders (ASD).

[0004] In some clinical conditions, a portion of chromosome 15 is duplicated. This Dup15q syndrome occurs most commonly in one of two forms, an extra dicentric isochromosome 15 or an interstitial duplication in chromosome 15. Dup15q syndrome is characterized by hypotonia, gross and fine motor delays, intellectual disability, autism spectrum disorder (ASD), and epilepsy (including infantile spasms). It is believed that an increase in the copy number of methylated maternal 15q duplications leads to increased protein expression, and that overexpression of UBE3A is associated with severity in Dup15q. Here, an increase in the number of maternal alleles is believed to be the primary driver of Dup15q pathology. Summary of the Invention [Means for solving the problem]

[0005] Abstract The present invention provides compositions for treating disorders associated with CNVs in the UBE3A gene. Specifically, the present disclosure provides antisense oligonucleotides that are useful for knocking down overexpression of UBE3A to treat seizures, hypotonia, motor delays, intellectual disability, disorders exhibiting seizures, and autism spectrum disorder (ASD) occurring in subjects affected by Dup15q syndrome. The compositions of the present invention include antisense oligonucleotides that are complementary to UBE3A transcripts and hybridize in vivo. The ASO prevents translation of UBE3A mRNA into protein. Specifically, a preferred embodiment includes an anti-UBE3A gapmer-oligo that includes a central DNA portion flanked by RNA wings. When the gapmer hybridizes to UBE3A pre-mRNA or mRNA, the hybrid duplex recruits RNaseH, which cleaves or digests the UBE3A pre-mRNA or mRNA, preventing expression of UBE3A protein. Since the ASO prevents the expression of the UBE3A protein, treatment with a composition comprising the ASO of the present disclosure can be effective in knocking down the overexpression of UBE3A. Thus, the composition of the present disclosure is useful for treating Dup15q syndrome and its symptoms.

[0006] The oligonucleotides of the present disclosure are designed to bind to a specific target in the RNA used in the synthesis of ubiquitin ligase protein. The binding of the oligonucleotide prevents protein synthesis and downregulates the expression of ubiquitin ligase. Specifically, the oligonucleotides of the present invention have a sequence that is substantially or completely complementary to one of the targets specified on the ubiquitin protein ligase E3A pre-mRNA or mRNA. That is, the oligonucleotides are antisense to the specified target. When the antisense oligonucleotide (ASO) hybridizes to its target RNA, it forms a double-stranded ASO:RNA duplex, which recruits an enzyme (RNase H) that degrades a portion of the double-stranded duplex. Degrading the ASO:RNA duplex depletes the cell of E6AP mRNA, which reduces the amount of E6AP synthesized by the cell.

[0007] Thus, when a composition containing an oligonucleotide that is antisense to a specified target in E6AP pre-mRNA or mRNA is administered to a patient, the composition reduces expression of E6AP that might otherwise result from copy number variations in UBE3A or from chromosome 15 q11.2-q13.1 duplication syndrome, known as Dup15q syndrome.

[0008] In certain aspects, the present disclosure provides a composition for treating Dup15q. Such a composition comprises a synthetic antisense oligonucleotide (ASO) that inhibits the expression of a ubiquitin ligase protein. Preferably, the protein is ubiquitin protein ligase E3A. The ASO hybridizes to a complementary region in the transcript of the UBE3A gene. The sequence of the bases in the ASO may have at least 80% identity with one of SEQ ID NOs: 1-219, preferably one of SEQ ID NOs: 1-40, and more preferably one of SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164 169, 174, 175, 178, 179, 213, and 214. In some embodiments, the sequence of bases in the ASO is at least 90%, 95%, or 100% identical to one of SEQ ID NOs: 1-219, 1-40, or 146, 155, 156, 158, 159, 161, 164 169, 174, 175, 178, 179, 213, and 214, and the oligonucleotide is capable of hybridizing to UBE3A pre-mRNA or mRNA and inducing RNase cleavage thereof.

[0009] In some embodiments, the oligonucleotide comprises two RNA wings flanking a central region of at least 10 DNA bases, preferably about 12 bases. At least one of the two wings of the ASO comprises a modified RNA base. Each modified RNA base may be selected from the group consisting of 2'-O-methoxyethyl RNA and 2'-O-methyl RNA. The ASO may comprise at least about 20 bases, preferably between about 15 and about 25 bases. In certain embodiments, the ASO has a backbone comprising a plurality of phosphorothioate linkages. The ASOs provided herein comprise a central region of 10-12 bases and a flanking region of 4-5 bases.

[0010] Preferred ASOs have sequences that have been screened and determined to not meet a threshold match for any non-target transcripts in humans. Optionally, the ASO has a sequence that has zero mismatches with homologous segments in the non-human primate genome and about five or less mismatches in homologous segments in the rodent genome.

[0011] In certain embodiments, the compositions of the invention comprise a plurality of ASOs, each having a base sequence at least about 80% identical to one of SEQ ID NOs: 1-219, wherein each of the ASOs has a gapmer structure comprising a central DNA segment flanked by RNA wings. In certain preferred embodiments, the compositions comprise a plurality of ASOs, each having a base sequence at least about 80% identical to one of SEQ ID NOs: 1-40, and more preferably one of SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213, and 214, wherein each of the ASOs has a gapmer structure comprising a central DNA segment flanked by RNA wings. Each oligonucleotide may have a base sequence having at least about a 90% (or 95%, or 100%) match to one of SEQ ID NOs: 1-219 (preferably 1-40 and more preferably 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213, and 214) and having bases linked only by phosphorothioate linkages, the oligonucleotide further comprising a core of 10 DNA bases flanked by a 5' wing and a 3' wing, each of which comprises five consecutive 2' modified RNA bases.

[0012] In some embodiments, each oligonucleotide has a base sequence that matches one of SEQ ID NOs: 1-219, at least a majority of the interbase linkages comprise phosphorothioate linkages, and the oligonucleotide further comprises a core of 10 DNA bases flanked by a 5' wing and a 3' wing, each of the 5' wing and the 3' wing comprising five consecutive 2'-O-methoxyethyl (2'-MOE) 2'-MOE RNA bases. In a preferred embodiment, each oligonucleotide has a base sequence that matches one of SEQ ID NOs: 1-40, at least a majority of the interbase linkages comprise phosphorothioate linkages, and the oligonucleotide further comprises a core of 10 DNA bases flanked by a 5' wing and a 3' wing, each of the 5' wing and the 3' wing comprising five consecutive 2' MOE RNA bases. In a more preferred embodiment, each oligonucleotide has a base sequence matching one of SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213, and 214, at least a majority of the interbase linkages comprise phosphorothioate linkages, and the oligonucleotide further comprises a core of 10 DNA bases flanked by a 5' wing and a 3' wing, each of which comprises five consecutive 2' MOE RNA bases.

[0013] In a related aspect, the present invention provides a method for treating Dup15q syndrome, comprising delivering one of the compositions of the present disclosure to a subject in need thereof, for example, to downregulate overexpression of UBE3A. The therapeutic oligonucleotides of the present disclosure may have a gapmer structure that includes a central DNA segment flanked by modified RNA wings. Such therapeutic oligonucleotides may include two wings that flank a central region of DNA bases (e.g., of about 10-14 DNA bases, e.g., a central region of about 12 DNA bases). Preferably, at least one end of the oligonucleotide includes any number or combination of modified RNA bases, e.g., 2'-O-Methoxyethyl RNA ("2'-MOE") and / or 2'-O-Methyl RNA ("2' O-Me"). Additionally, compositions of the present invention may be designed to target exon-exon junctions and differentially target cytoplasmic mRNA versus nuclear pre-mRNA. Thus, the ASOs of the present invention can be designed to interact with RNA before or after splicing, adding specificity and multifunctionality to the composition.

[0014] In various embodiments, the therapeutic oligonucleotide may be provided in a solution or carrier formulated for delivery via any suitable route, including, for example, intravenously or intrathecally. The oligonucleotide may be of any suitable length, for example, at least about 18 bases, and preferably between about 15 and about 25 bases. The oligonucleotide may have phosphorothioate linkages in its backbone. In a preferred embodiment, the oligonucleotide has a base sequence that has been screened and determined not to meet a threshold match for any length, non-coding RNA or other off-target sequence or transcript in humans. The oligonucleotide may have a base sequence with 0 mismatches to homologous segments in the non-human primate genome and about 5 or less mismatches in homologous segments in the rodent genome.

[0015] When the composition is delivered to cells in vitro, the cells exhibit a dose-dependent knockdown of UBE3A. The oligonucleotide may be a gapmer having a base sequence with at least about 90% match with one of SEQ ID NOs: 1-219 and with at least some phosphorothioate linkages. The linkages may be all phosphorothioate or a mixture of phosphorothioate and phosphodiester linkages. The oligonucleotide may further have a core of 12 DNA bases flanked by 5' and 3' wings, each of which includes 4 consecutive 2' modified RNA bases. Preferably, the oligonucleotide has a base sequence that matches one of SEQ ID NOs: 1-219 with a structure having bases linked by phosphorothioate linkages and a core DNA base flanked by 5' and 3' wings. The number of RNA bases in the wings and DNA bases in the core segment may be 5-10-5 or 4-12-4, or a similar suitable pattern. The 5' and 3' wings may each contain several 2'-MOE RNA bases. For example, the oligonucleotide may have four consecutive 2'-MOE RNA bases in each wing and 12 core DNA bases ("4-12-4" structure), with phosphorothioate linkages throughout the core DNA segment, and a mixture of phosphorothioate and phosphodiester linkages in the wings. Alternatively, the oligonucleotide may have five consecutive 2'-MOE RNA bases in each wing and 10 core DNA bases ("5-10-5" structure), with phosphorothioate linkages throughout the core DNA segment, and a mixture of phosphorothioate and phosphodiester linkages in the wings. The 5' and 3' wings may also be of different lengths in the same ASO (for example, "4-11-5" or "5-11-4" structure).

[0016] In combination embodiments, the invention provides compositions comprising multiple copies of multiple distinct therapeutic gapmers (each according to the above descriptions) in a suitable formulation or carrier.

[0017] An aspect of the present disclosure relates to the use of antisense oligonucleotides (ASOs) for the manufacture of a medicament for treating Dup15q syndrome. In such an embodiment, the ASO has at least about 75% identity, and more preferably at least about 90% identity, e.g., 95% or 100% identity, to one of SEQ ID NOs: 1-219. A preferred embodiment uses an ASO that is between about 15-25 bases in length, preferably between about 18-22, or between about 19-21, inclusive. In general, reference to an "ASO" includes many copies of a substantially identical molecule. Thus, an "ASO" can be any number, e.g., hundreds of thousands, or millions of copies of the indicated ASO. In a preferred embodiment, the ASO is 20 bases in length and has the sequence of one of SEQ ID NOs: 1-219, and is used in the manufacture of a medicament for the treatment of Dup15q syndrome. The ASO may be provided in any suitable format, such as lyophilized in a tube or in solution in a tube, such as a microcentrifuge tube or test tube. A preferred embodiment of the use targets a transcript of the UBE3A gene. One or more (e.g., 2, 3, 4, or 5 or more) ASOs may be used in the manufacture of a medicament. The one or more ASOs may hybridize to a target in the UBE3A pre-mRNA or mRNA. In certain embodiments, the sequence of bases in the ASO is at least about 90% identical to one of SEQ ID NOs: 1-219. In other embodiments, the ASO may have a gapmer structure with a central DNA segment flanked by RNA wings, such as a central region of 12 DNA bases with four modified RNA bases on either side of the central region. Each modified RNA base may be a 2'-MOE. Preferably, the backbone of the ASO has multiple phosphorothioate linkages. Thus, the ASO may initially be in a form suitable for mixing into a formulation suitable for injection or delivery by pump.For example, the ASO (thousands or millions or more copies of one ASO) may be lyophilized in a tube or in solution at a known amount, molality, or concentration. The ASO may be dissolved or diluted into a pharma- ceutically acceptable composition with a carrier (e.g., solvent and / or excipient) containing the ASO, and loaded into an IV bag, syringe, or pump. The medicament may be made using more than one ASO, for example, 2, 3, 4, or 5, or any combination of more than this. The base in the composition of the present invention may be a modified or wobble base, which may be used to increase the range and efficacy of the composition of the present invention. In one example, the ASO for use in the present invention may include a methylated base (e.g., 5-methylcytosine, 5-methyluracil (thymine), etc.).

[0018] The compositions of the present invention can be formulated to accommodate sequential administration. For example, the formulation can provide a dosage to be administered at two or more separate times, and optionally with two or more different ASOs, to take advantage of the optimal therapeutic window and avoid potential competition between ASOs. Furthermore, the compositions of the present invention, whether administered sequentially or not, can interact with one or more targets, depending on the composition of the ASO involved. For example, an ASO can contain targeted mismatches that allow it to interact with multiple targets (both within and across mRNA and pre-mRNA species), thus allowing its associated treatment to affect transcripts from more than one gene copy. The compositions of the present invention can also be delivered in a time-release format and / or contain adjuvants to increase serum half-life. [Brief description of the drawings]

[0019] BRIEF DESCRIPTION OF THE DRAWINGS [Figure 1] FIG. 1 shows a composition for treating Dup15q syndrome.

[0020] [Diagram 2] FIG. 2 shows an oligonucleotide having a gapmer structure (ASO).

[0021] [Diagram 3] FIG. 3 shows the results from a screen of 40 UBE3A exonic ASOs.

[0022] [Figure 4] Figure 4 shows the results showing the dose response of 10 ASO candidates.

[0023] [Diagram 5] FIG. 5 shows results from a screen of human exonic ASOs with mouse homology.

[0024] [Figure 6] Figure 6 shows a table summarizing the qPCR readout of UBE3A knockdown (expressed as percent UBE3A knockdown) for certain screened ASOs of the invention.

[0025] [Figure 7] Figure 7 shows a table summarizing the qPCR readout of UBE3A knockdown (expressed as percent UBE3A knockdown) for certain screened ASOs of the invention.

[0026] [Figure 8] Figure 8 shows a table summarizing the qPCR readout of UBE3A knockdown (expressed as percent UBE3A knockdown) for certain screened ASOs of the invention.

[0027] [Figure 9] Figure 9 shows a table summarizing the qPCR readout of UBE3A knockdown (expressed as percent UBE3A knockdown) for certain screened ASOs of the invention.

[0028] [Figure 10] FIG. 10 shows UBE3A ASO dose-response regulation of target expression for two lead candidate example ASOs and their PO-modified daughter molecules in Dup15q patient fibroblasts (top) or mouse embryonic fibroblasts (bottom).

[0029] [Figure 11] FIG. 11 shows dose response plots showing EC50s for the same two example lead candidate ASOs from FIG.

[0030] [Figure 12] Figure 12 shows dose-response data for the lead all-PS backbone ASO candidate of the present invention targeting UBE3A exons.

[0031] [Figure 13] Figure 13 shows dose-response data for the lead all-PS backbone ASO candidate of the present invention targeting the UBE3A intron.

[0032] [Figure 14] Figure 14 shows dose response data of the lead all-PS backbone ASO candidate of the present invention with 100% mouse homology for rodent in vivo efficacy testing.

[0033] [Figure 15] Figure 15 shows dose response data of PO modified daughter lead ASO candidates with 100% mouse homology for rodent in vivo efficacy testing.

[0034] [Figure 16] Figure 16 shows dose response data of the PO-modified daughter lead ASO candidates of the present invention for human clinical candidate testing.

[0035] [Figure 17] FIG. 17 shows a Western blot of one particular candidate lead UBE3A ASO and three PO modified daughter molecules with identical ASO sequences.

[0036] [Figure 18] FIG. 18 shows quantification of UBE3A protein knockdown for the ASOs in FIG.

[0037] [Figure 19] Figure 19 provides a table summarizing the UBE3A protein knockdown results of lead all-PS backbone ASO candidates targeting UBE3A.

[0038] [Figure 20] Figure 20 provides a table summarizing UBE3A protein knockdown results of lead all-PS backbone ASO candidates with 100% mouse homology for rodent in vivo efficacy testing.

[0039] [Figure 21] Figure 21 provides a table summarizing the UBE3A protein knockdown results of PO-modified daughter lead ASO candidates with 100% mouse homology for rodent in vivo efficacy testing.

[0040] [Figure 22] Figure 22 provides a table summarizing the UBE3A protein knockdown results of PO-modified daughter lead ASO candidates for human clinical candidates.

[0041] [Diagram 23] Figure 23 provides data showing knockdown of UBE3A transcripts in human NGN2 stem cell-derived neurons using the UBE3A lead candidate ASO of the invention.

[0042] [Figure 24] Figure 24 provides data showing knockdown of UBE3A transcripts in human primary neurons using the UBE3A lead candidate ASO of the invention.

[0043] [Diagram 25]Figure 25 provides data showing knockdown of UBE3A transcripts in non-human primate primary fibroblast cultures using the UBE3A lead ASO candidate of the invention.

[0044] [Figure 26] Figure 26 provides data showing knockdown of UBE3A transcripts in mouse primary cortical neurons using the UBE3A lead candidate ASO of the invention.

[0045] [Figure 27] Figure 27 provides data showing knockdown of UBE3A transcripts using rat primary cortical neurons (where cells were harvested after 4 days for qPCR) using the UBE3A lead ASO candidate of the invention.

[0046] [Figure 28] Figure 28 provides data showing knockdown of UBE3A transcripts in rat primary cortical neurons (where cells were harvested after 8 days for qPCR) using the UBE3A lead ASO candidate of the invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0047] Detailed Description FIG. 1 shows a composition 101 for treating Dup15q syndrome. The composition 101 comprises an antisense oligonucleotide 107 that hybridizes to a target segment 115 in an mRNA 117 or pre-mRNA. The RNA 117 encodes a ubiquitin ligase protein (e.g., ubiquitin protein ligase ESA). The segment 115 of the RNA 117 that includes the target is at least about 75% complementary to one of SEQ ID NOs: 1-219. Hybridization of the ASO 107 to the segment 115 of the RNA 117 prevents translation of the mRNA into UBE3A protein. Preferably, the sequence of bases in the oligonucleotide has at least 80% identity, more preferably at least about 90% identity, to one of SEQ ID NOs: 1-219. In certain embodiments, the sequence of bases in the oligonucleotide is at least about 90% identical to one of SEQ ID NOs: 1-219, wherein the oligonucleotide hybridizes to UBE3A pre-mRNA or mRNA and induces RNase H cleavage thereof.

[0048] Oligonucleotide 107 hybridizes to segment 115 in mRNA 117 because oligonucleotide 107 is substantially or completely antisense to target segment 115 of mRNA 117. In this aspect, the composition includes an antisense oligonucleotide (ASO). Composition 101 includes an ASO that binds to target RNA with base pair complementarity and exerts various effects based on the chemical structure and design of the ASO. Various mechanisms commonly used in preclinical models and human clinical trial development of neurological diseases can be used. These mechanisms include RNA target degradation via recruitment of RNase H enzyme; alternative splicing modification to include or exclude exons, and miRNA inhibition to inhibit miRNA binding to its target.

[0049] A preferred embodiment of the present disclosure includes an ASO that hybridizes to UBE3A pre-mRNA or mRNA and recruits RNase H enzyme. The RNase H enzyme cleaves the RNA, which downregulates the expression of UBE3A protein. Thus, the oligonucleotide 107 of the present disclosure addresses UBE3A CNV as a target for Dup15q syndrome. The present disclosure is based on the insight that data suggests that one of the most common genetic variants associated with autism spectrum disorder (ASD) is a duplication of chromosome 15 q11.2-q13.1 (Dup15q syndrome). The chromosome 15q11.2-q13.1 region contains the imprinted Prader-Willi / Angelman syndrome critical region (PWACR), as well as several genes crucial for brain development and synaptic function, such as ubiquitin protein ligase E3A (UBE3A), small nuclear ribonucleoprotein polypeptide N (SNRPN), and three GABAA receptor genes (GABRB3, GABRA5, and GABRG3). Dup15q syndrome is classified into two main types of 15q11.2-13.1 duplications: (1) isodicentric chromosome 15 (idic(15)), which results in two additional maternally derived copies on the supernumerary chromosome that includes 15p and the proximal region of 15q11 (most commonly resulting in four copies of the region); or (2) interstitial 15q duplication, in which an extra copy of the 15q11.2-q13.1 region occurs on the same chromosomal arm, typically resulting in three copies of the region and an overall milder phenotype. Hogart, 2010, The comorbidity of autism with the genomic disorders of chromosome 15q11.2-13, Neurobiol Dis 38:181-91, incorporated by reference. Increased copy number of methylated maternal 15q duplications leads to changes in gene and protein expression, and overexpression of UBE3A is associated with severity in Dup15q, where increased number of maternal alleles is thought to be the primary driver of Dup15q pathology.See Scoles, 2011, Increased copy number for methylated maternal 15q duplications leads to changes in gene and protein expression in human cortical samples, Mol Autism 2:19 and Baker, 2020, Relationships between UBE3A and SNORD116 expression and features of autism in chromosome 15 imprinting disorders, Translational Psychiatry 10:362, both of which are incorporated by reference. Thus, a composition comprising a UBE3A ASO is administered to a subject to treat Dup15q syndrome.

[0050] Thus, the present disclosure provides the use of an antisense oligonucleotide (ASO) for the manufacture of a medicament for treating Dup15q syndrome in a patient. In this use, the ASO has at least about 75% identity, and more preferably at least 90% identity, e.g., 95% or higher identity, with one of SEQ ID NOs: 1-219. A preferred embodiment uses an ASO that is between about 15-25 bases in length, preferably between about 18-22 bases inclusive. In general, reference to an "ASO" includes many copies of a substantially identical molecule. Thus, an "ASO" may be hundreds of thousands or even millions of copies of a defined ASO. In a preferred embodiment, the ASO is 20 bases in length and has a sequence of one of SEQ ID NOs: 1-219, and is used in the manufacture of a medicament for the treatment of Dup15q syndrome. The ASO may be provided in any suitable format, such as, for example, lyophilized in a tube or in solution in a tube (e.g., a microcentrifuge tube or test tube). A preferred embodiment of the use targets UBE3A. One or more (e.g., 2, 3, 4, or 5 or more) ASOs may be used in the manufacture of a medicament. The one or more ASOs may hybridize to a target in UBE3A mRNA. In certain embodiments of the use, the sequence of bases in the ASO is at least about 90% identical to one of SEQ ID NOs: 1-219. In embodiments of the use, the ASO may have a gapmer structure having a central DNA segment flanked by RNA wings (e.g., a central region of 10-12 DNA bases with 4-5 modified RNA bases on either side of the central region). Each modified RNA base may be 2'-MOE RNA, 2'-O-methyl RNA, or other suitable sugar. Preferably, the backbone of the ASO has multiple phosphorothioate linkages (either exclusively or also including phosphodiester linkages), e.g., most or all of the sugar linkages may be phosphorothioate in embodiments of the use. The ASO may initially be in a form suitable for mixing into a formulation suitable for introduction by injection.For example, the ASO (thousands or millions or more copies of one ASO) may be lyophilized in a tube or in a solution at a known amount, molality, or concentration. The ASO may be dissolved or diluted into a pharma- ceutically acceptable composition in which the carrier (e.g., solvent and / or excipient) comprises the ASO and can be loaded into an IV bag, syringe, or vial. The medicament may be made using any combination of more than one ASO, for example, 2, 3, 4, or 5, or more.

[0051] Any ASO described in the above embodiments of use can be included in the composition of the present disclosure. A preferred embodiment of the composition of the present disclosure comprises one or more therapeutic oligonucleotides, each having at least 80% identical base sequence to one of SEQ ID NOs: 1-219, wherein each of the therapeutic oligonucleotides has a gapmer structure comprising a central DNA segment flanked by modified RNA wings, wherein the therapeutic oligonucleotides are provided in a solution or carrier formulated for injection.

[0052] FIG. 2 shows an oligonucleotide 207 having a gapmer structure. The oligonucleotide 207 includes two wings (first wing 215 and second wing 216) flanking a central region 221 of about 10-12 DNA bases. In preferred embodiments, the wings 215, 216 are all or primarily RNA bases, while the central region 221 is all or primarily DNA bases. Preferably, the wings are all RNA bases (modified or unmodified) and the central region is all DNA bases. In some embodiments, each wing consists of five RNA bases, all or most of which are modified RNA bases (e.g., where each modified RNA base is selected from the group consisting of 2'-O-methoxyethyl RNA and 2'-O-methyl RNA). The modified RNA bases may include substitutions on the 2' hydroxyl group of the ribose sugar. 2'-O-methoxyethyl ("2'-MOE") modified sugars may be included in the RNA bases. Oligonucleotide 207 preferably contains at least about 15 bases, and may contain between about 15 and about 25 bases. In some embodiments, oligonucleotide 207 has a backbone that contains multiple phosphorothioate linkages. One or any number of phosphorothioate linkages may be included in the backbone of a segment of DNA (e.g., central region 221 of oligonucleotide 207). Oligonucleotide 207 may contain one or any number of phosphorothioate linkages. For example, every backbone linkage in oligonucleotide 207 may be phosphorothioate, or most, or about half, may be phosphorothioate. Additionally, other modifications to the phosphodiester backbone may be present.

[0053] The composition 101 may be formulated for delivery. Thus, the oligonucleotide 107 may initially be in a form suitable for mixing into a formulation suitable for introduction into a syringe, bag, or injection pump. For example, the oligonucleotide 107 (thousands or millions or more copies of one oligonucleotide 107) may be lyophilized in a tube or in a solution at a known molality or concentration. The oligonucleotide 107 may be dissolved or diluted into a pharma- ceutically acceptable composition in which a carrier (e.g., a solvent or excipient) comprises the oligonucleotide 107 and loaded into an IV bag, syringe, or vial. As described, the composition 101 includes at least one oligonucleotide 107 having a sequence defined by comparison with one of SEQ ID NOs: 1-219. Thus, the compositions of the present disclosure are defined and illustrated by a specified target.

[0054] Specifically, oligonucleotide 107 hybridizes to an mRNA encoding UBE3A protein along a segment of the mRNA that is at least about 75% complementary to one of SEQ ID NOs: 1-219, thereby preventing translation of said mRNA into UBE3A protein. This is achieved when said oligonucleotide has at least about 75% identity, preferably at least about 90% or 95% or 100% identity, to one of SEQ ID NOs: 1-219. In certain embodiments, said oligonucleotide has the sequence of one of SEQ ID NOs: 1-219, but those skilled in the art will understand that an oligonucleotide having 90% or preferably 95% identity to a complementary target will still tend to hybridize to said target in a sequence-specific manner. The formation of a double-stranded structure is energetically well favored through Watson-Crick base pairing and base stacking, which allows the double-stranded structure to tolerate about one mismatched base pair in every 10 or so. Thus, under moderately stringent physiological conditions in cells, 95% identity should be effective, especially if the oligonucleotide has a gapmer structure with at least some modified RNA bases or phosphorothioate backbone linkages to protect the oligonucleotide from enzymatic degradation.

[0055] Indeed, a feature and benefit of the compositions of the present disclosure is that the targets (SEQ ID NOs: 1-219) have been substantially screened to exclude sequences whose complements exist in molecules other than UBE3A transcripts. For example, the sequences have been screened against a database of RNA transcripts, including long non-coding RNAs (IncRNAs), and the first sequence that matches a non-target sequence has been excluded. Thus, when administered to a patient, an ASO having a sequence of SEQ ID NOs: 1-219 should have minimal chance of hybridizing to a non-target sequence. Thus, in a preferred embodiment, oligonucleotide 107 has a base sequence that has been screened and determined not to meet a threshold match for any off-target coding RNA or long non-coding RNA in humans. A composition or use that meets the above-mentioned criteria should not bind to off-target substances (e.g., IncRNAs) or other off-target RNA transcripts in vivo, because the sequences contained therein have been screened against a database of IncRNAs and other RNA transcripts. The sequences of the present disclosure have been screened for target specificity. Preferably, oligonucleotide 107 has a base sequence that has zero mismatches to homologous segments in the human or non-human primate genome and about 5 or less mismatches in homologous segments in the rodent genome.

[0056] When the composition is delivered to a cell, the cell exhibits a dose-dependent knockdown of UBE3A.

[0057] Figure 3 shows the results from a screen of 40 UBE3A exonic ASOs (along with one control fibroblast cell line; results are obtained 48 hours after treatment). The results shown correspond to SEQ ID NOs: 1 to 40. In the figure, the bars of the ASOs tested in the concentration response (CR) are marked with a circle.

[0058] 4 shows results showing the dose response (at six concentrations each) of ten ASO candidates SEQ ID NOs: 14, 17, 4, 7, 8, 18, 21, 26, 34, and 35 designed according to embodiments of the present disclosure (a DNA core region of about 12 bases linked by 20 base, 2'-O modified RNA through the ASO and RNA wings with phosphorothioate linkages). All ten ASOs reduced UBE3A expression in a dose-dependent manner compared to controls (cells treated with vehicle only and untreated "cells only" condition).

[0059] Because nucleic acid hybridization is somewhat tolerant of mismatches, an oligonucleotide 107 having a base sequence that is at least 90% a match to one of SEQ ID NOs: 1-219, and having bases linked only by phosphorothioate linkages, wherein the oligonucleotide 107 has a central segment of DNA bases flanked by 5' and 3' wings (e.g., the 5' and 3' wings each have a 4-12-4 structure containing 4 consecutive 2' modified RNA bases flanked by 12 DNA bases, or a 5-10-5 structure, or the like), can be found to exhibit dose-dependent knockdown according to the pattern shown in the chart. In some embodiments, oligonucleotide 107 specifically matches one of SEQ ID NOs: 1-219 (more preferably one of SEQ ID NOs: 1-40, or more preferably SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213, or 214) and has bases linked by phosphorothioate linkages (optionally with some phosphodiester linkages), said oligonucleotide 107 having a core of 12 DNA bases flanked by a 5' wing and a 3' wing, each of which has a base sequence including four consecutive 2'-MOE RNA bases.

[0060] Figure 5 shows results from a screen of mouse exonic Ube3a ASOs and human exonic ASOs with mouse homology in mouse fibroblasts. The human ASOs screened included SEQ ID NOs: 1, 4, 5, 9, 15, 16, 21, 25, 28, and 29. The results tend to indicate that it is possible to design ASOs against human targets that have homologous targets in rodent models.

[0061] Since these compositions are effective in knocking down the expression of UBE3A, the compositions of the present disclosure can be used to treat Dup15q syndrome in patients. The methods of the present disclosure include administering any of the compositions of the present disclosure to a patient in need thereof, thereby treating or alleviating Dup15q syndrome.

[0062] The compositions of the present disclosure may be tested on in vitro samples of living neurons. For example, the in vitro neurons may contain optogenetic constructs that provide neuronal activation under light stimulation (e.g., modified algal channel rhodopsin that causes neurons to fire in response to light) and optical reporters of neuronal activity (modified archaeal rhodopsin that emits light proportional to neuronal membrane potential, resulting in a signal of neuronal activity). The in vitro neurons may be assayed in a fluorescent microscopy instrument and, if necessary, treated with a neurostimulating composition that causes neurons to fire in a predictable manner. Any suitable optogenetic construct, optogenetic microscope, or neurostimulating composition may be used. For example, suitable optogenetic constructs include those described in U.S. Pat. No. 9,594,075 (incorporated by reference). Suitable optogenetic microscopes include those described in U.S. Pat. No. 10,288,863 (incorporated by reference).

[0063] The disclosed method and composition can be advantageously used to deliver the therapeutic oligonucleotide 107 described herein to neurons in vivo in subjects with Dup15q syndrome. Any suitable delivery approach can be used, including, for example, systemic delivery (e.g., by injection) or local delivery (e.g., subcutaneous, intrathecal, or by implantation of a delayed release device). The disclosed method can include delivering the disclosed composition once, several times over several days or weeks, or every few months (e.g., about 3 or 4 times a year).

[0064] Oligonucleotides of the disclosure (e.g., gapmer, ASO, or therapeutic oligonucleotide 107 in composition 101) may have a sequence defined with reference to one of the sequences shown in Table 1. For example, oligonucleotides of the disclosure may have a sequence that is at least about 75%, 80%, 90%, 95%, or completely identical to one of SEQ ID NOs: 1-219 as shown in Table 1. Certain preferred embodiments for UBE3A include those in Table 1 designated as SEQ ID NOs: 1-40.

[0065] Furthermore, as described in the following examples, the present inventors screened the ASOs of the present invention.Based on the data obtained, the ASOs corresponding to SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213 and 214 were identified as lead candidate ASOs based on single dose and dose-response efficacy, sequence motif liability and off-target alignment analysis.These ASOs showed the highest in vitro efficacy, the lowest off-target alignment and limited sequence motif concern. Thus, in certain aspects, preferred ASOs against UBE3A according to the present invention include ASOs having sequences that are at least about 75%, 80%, 90%, 95%, or completely identical to sequences corresponding to SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164 169, 174, 175, 178, 179, 213 and 214.

[0066] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5] [Table 1-6] [Table 1-7] [Table 1-8]

[0067] A preferred combination embodiment of the present disclosure includes a composition for treating Dup15q syndrome. The composition includes: a first oligonucleotide that hybridizes to an mRNA encoding a UBE3A protein along a segment of the mRNA that is at least about 90% complementary to one of SEQ ID NOs: 1-40; and optionally, a second oligonucleotide that hybridizes to an mRNA encoding a UBE3A protein along a segment of the mRNA that is at least about 90% complementary to a different one of SEQ ID NOs: 1-40. In a preferred combination embodiment, each of the therapeutic oligonucleotides may have a gapmer structure that includes a central DNA segment flanked by modified RNA wings.

[0068] A more preferred combination embodiment of the present disclosure includes a composition for treating Dup15q syndrome comprising an mRNA encoding a UBE3A protein along a segment of the mRNA that is at least about 90% complementary to one of SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213, and 214; and optionally a second oligonucleotide that hybridizes to the mRNA encoding a UBE3A protein along a segment of the mRNA that is at least about 90% complementary to one of SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213, and 214.

[0069] Either or both wings may contain modified RNA bases. For example, both wings may contain four consecutive RNA bases with 2'-O-methoxyethyl ribose modifications. The entirety of each oligonucleotide may be connected via phosphodiester or phosphorothioate linkages or others, as will be apparent to those skilled in the art. Most preferably, at least the terminal linkages are non-standard (i.e., non-phosphodiester, e.g., phosphorothioate). More preferably, most or all of the linkages within the RNA wings are non-standard (e.g., phosphorothioate). Preferably, the therapeutic oligonucleotides are provided in lyophilized or solution form for dilution or reconstitution at the clinic for delivery. That is, at least 1000 to millions of copies of the first oligonucleotide and, optionally, at least 1000 to millions of copies of the second oligonucleotide are packaged in one or more tubes, lyophilized or in solution. This preferred combination embodiment of the composition may prove to have unexpected benefits as an antisense therapeutic agent for the treatment of Dup15q syndrome. An embodiment of the present disclosure includes oligonucleotides comprising locked nucleic acid (LNA) antisense oligonucleotides targeting UBE3A that can downregulate overexpression of UBE3A. The present invention provides oligonucleotides of 10-30 nucleotides in length, which have at least 90% complementarity (e.g., 100% complementarity) with a UBE3A target nucleic acid and comprise a contiguous nucleotide sequence of 10-30 nucleotides in length that can inhibit overexpression of UBE3A in vivo. Oligonucleotide 107 can be 100% identical to one of SEQ ID NOs: 1-219, or preferably one of SEQ ID NOs: 1-40, or one of SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213, and 214.In certain aspects, oligonucleotide 107 may be at least 90%, 95%, 98%, or 99% identical to one of SEQ ID NOs: 1-219, or preferably one of SEQ ID NOs: 1-40, or one of SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213, and 214.

[0070] Embodiments include pharma- ceutically acceptable salts of an antisense oligonucleotide according to the invention, or a conjugate according to the invention.

[0071] The invention provides a pharmaceutical composition comprising an antisense oligonucleotide of the invention or a conjugate of the invention, and a pharma- ceutically acceptable diluent, solvent, carrier, salt and / or adjuvant.

[0072] The present invention provides an antisense oligonucleotide of the invention or a conjugate of the invention or a pharmaceutical salt or composition of the invention for use in medicine.

[0073] The present invention provides an antisense oligonucleotide of the present invention or a conjugate of the present invention or a pharmaceutical salt or composition of the present invention for use in treating or preventing or alleviating Dup15q syndrome.The present invention provides the use of an antisense oligonucleotide of the present invention or a conjugate of the present invention or a pharmaceutical salt or composition of the present invention for the preparation of a medicament for treating or preventing or alleviating Dup15q syndrome.

[0074] Oligonucleotides are generally made in the laboratory by solid phase chemical synthesis, followed by purification and isolation.When referring to the sequence of the oligonucleotide, reference is made to the sequence or order of the nucleobase moieties of covalently linked nucleotides or nucleosides, or to their modification.The oligonucleotides of the present invention can be artificial, i.e., chemically synthesized, and typically are purified or isolated.The oligonucleotides of the present invention can include one or more modified nucleosides or nucleotides (e.g., 2' sugar modified nucleosides).

[0075] The modified nucleotides may be independently selected from the group consisting of deoxy-nucleotides, 3'-terminal deoxy-thymine (dT) nucleotides, 2'-O-methyl modified nucleotides, 2'-fluoro modified nucleotides, 2'-deoxy modified nucleotides, locked nucleotides, unlocked nucleotides, conformationally restricted nucleotides, constrained ethyl nucleotides, abasic nucleotides, 2'-amino modified nucleotides, 2'-O-allyl modified nucleotides, 2'-C-alkyl modified nucleotides, 2'-hydroxy (hydroxl) modified nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, nucleotides containing unnatural bases, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, nucleotides containing 5'-phosphate mimetics, glycol modified nucleotides, and 2'-O-(N-methylacetamide) modified nucleotides, and combinations thereof.

[0076] The nitrogenous bases of the ASO can be naturally occurring nucleobases such as adenine, guanine, cytosine, thymidine, uracil, xanthine and hypoxanthine, as well as non-naturally occurring variants such as substituted purines or substituted pyrimidines such as nucleobases selected from isocytosine, pseudoisocytosine, 5-methylcytosine, 5-thiazolo-cytosine, 5-propynyl-cytosine, 5-propynyl-uracil, 5-bromouracil 5-thiazolo-uracil, 2-thio-uracil, 2'-thio-thymine, inosine, diaminopurine, 6-aminopurine, 2-aminopurine, 2,6-diaminopurine and 2-chloro-6-aminopurine.

[0077] The nucleobase moieties can be represented by the letter code of each corresponding nucleobase (e.g., A, T, G, C, or U), where each letter can optionally include modified nucleobases of equivalent function. For example, in the exemplified oligonucleotides, the nucleobase moieties are selected from A, T, G, C, and 5-methylcytosine. Optionally, for LNA gapmers, 5-methylcytosine LNA nucleosides can be used.

[0078] The oligonucleotide 107 of the present disclosure is capable of downregulating (inhibiting) the expression of UBE3A. In some embodiments, the antisense oligonucleotide of the present invention is capable of regulating the expression of the target by inhibiting or downregulating the target. Preferably, such regulation results in at least 20% inhibition of expression compared to the normal expression level of the target, more preferably at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% inhibition compared to the normal expression level of the target.

[0079] The antisense oligonucleotides (ASOs) of the disclosure may reduce the levels of a target nucleic acid (e.g., via RNase H cleavage) or may reduce (or alter the functionality of) a target nucleic acid, e.g., via modulation of pre-mRNA splicing.

[0080] The oligonucleotide 107 of the present disclosure may include one or more nucleotides with modified sugar moieties (i.e., modifications of the sugar moiety compared to the ribose sugar moiety found in DNA and RNA). Many nucleosides with modifications of the ribose sugar moiety have been made primarily for the purpose of improving certain properties of the oligonucleotide (e.g., affinity and / or nuclease resistance). Such modifications include those in which the ribose ring structure is modified, for example, by replacement with a hexose ring (HNA), or a bicyclic ring (which typically has a bridge between the C2 and C4 carbons on the ribose ring (LNA)), or an unlinked ribose ring (e.g., UNA) that typically lacks a bond between the C2 and C3 carbons. Modified nucleosides also include nucleosides in which the sugar moiety is replaced with a non-sugar moiety (e.g., in the case of peptide nucleic acid (PNA)), or morpholino nucleic acid.

[0081] Sugar modifications also include modifications made through changing the substituents on the ribose ring to groups other than hydrogen or to the 2'-OH group naturally found in DNA and RNA nucleosides. Substituents can be introduced, for example, at the 2', 3', 4' or 5' position.

[0082] The oligonucleotide may contain one or more locked nucleic acid (LNA) bases. LNA may include 2'-modified nucleosides that contain a biradical linkage (also referred to as "2'-4' bridge") between the C2' and C4' of the ribose sugar ring of the nucleoside, which restricts or locks the conformation of the ribose ring. These nucleosides are also referred to in the literature as bridged nucleic acids or bicyclic nucleic acids (BNA). Locking of the ribose conformation is associated with enhanced affinity of hybridization (duplex stabilization) when LNA is incorporated into an oligonucleotide with respect to a complementary RNA or DNA molecule. This can be routinely determined by measuring the melting temperature of the oligonucleotide / complement duplex. Exemplary, but non-limiting, LNA nucleosides are disclosed in: WO 99 / 014226, WO 00 / 66604, WO 98 / 039352, WO 2004 / 046160, WO 00 / 047599, WO 2007 / 134181, WO 2010 / 077578, WO 2010 / 036698, WO 2007 / 090071, WO 2009 / 006478, WO 2011 / 156202, WO 2008 / 154401, WO 2009 / 067647, and WO 2008 / 150729 (all incorporated by reference).

[0083] Pharmaceutically acceptable salts of the oligonucleotides of the present disclosure include those salts that retain the biological effectiveness and properties of free base or free acid, which are not biologically or otherwise undesirable.Said salts are formed with inorganic acids (e.g., hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, especially hydrochloric acid) and organic acids (e.g., acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, sulfonic acid, or salicylic acid).Furthermore, these salts can be prepared from adding inorganic or organic bases to said free acid.Salts obtained from inorganic bases include, but are not limited to, sodium salt, potassium salt, lithium salt, ammonium salt, calcium salt, magnesium salt. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines (including naturally occurring substituted amines), cyclic amines, and basic ion exchange resins (e.g., isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, lysine, arginine, N-ethylpiperidine, piperidine, polyamine resins).

[0084] Oligonucleotide 107 may mediate or promote nuclease-mediated degradation of UBE3A pre-mRNA or mRNA transcript. Nuclease-mediated degradation refers to an oligonucleotide that can mediate the degradation of a complementary nucleotide sequence when it forms a duplex with such a complementary nucleotide sequence. In some embodiments, the oligonucleotide may function via nuclease-mediated degradation of a target nucleic acid when the oligonucleotide of the present invention can recruit a nuclease, particularly an endonuclease, preferably an endoribonuclease (RNase) (e.g., RNase H). An example of an oligonucleotide design that functions via a nuclease-mediated mechanism is an oligonucleotide (e.g., a gapmer) that typically contains a region of at least 5 or 6 consecutive DNA nucleosides and is flanked on one or both sides by affinity-enhancing nucleosides. The RNase H activity of antisense oligonucleotide 107 refers to its ability to recruit RNase H when it is in a duplex with a complementary RNA molecule.

[0085] The antisense oligonucleotide 107 of the present invention, or a contiguous nucleotide sequence thereof, can be a gapmer (also referred to as a gapmer oligonucleotide or gapmer design). Antisense gapmers are generally used to inhibit target nucleic acids via RNase H-mediated degradation. A gapmer oligonucleotide comprises at least three distinct structural regions, a 5'-proximal, gap, and a 3'-proximal, FG-F', in a 5'→3' orientation. The "gap" region (G) comprises a stretch of contiguous DNA nucleotides to which the oligonucleotide can recruit RNase H. The gap region is flanked by a 5'-proximal region (F) comprising one or more sugar-modified nucleosides (advantageously high affinity sugar-modified nucleosides) and a 3'-proximal region (F') comprising one or more sugar-modified nucleosides (advantageously high affinity sugar-modified nucleosides). The one or more sugar modified nucleosides in regions F and F' enhance the affinity of the oligonucleotide for the target nucleic acid (i.e., are affinity enhancing sugar modified nucleosides). In some embodiments, the one or more sugar modified nucleosides in regions F and F' are 2' sugar modified nucleosides (e.g., high affinity 2' sugar modifications) (e.g., independently selected from LNA and 2'-MOE).

[0086] A mixed wing gapmer is an LNA gapmer in which one or both of regions F and F' comprise a 2'-substituted nucleoside (e.g., a 2'-substituted nucleoside independently selected from the group consisting of a 2'-O-alkyl-RNA unit, a 2'-O-methyl-RNA, a 2'-amino-DNA unit, a 2'-fluoro-DNA unit, a 2'-alkoxy-RNA, a 2'-MOE unit, an arabinonucleic acid (ANA) unit, a 2'-fluoro-ANA unit, or a combination thereof). In some embodiments in which at least one of regions F and F', or both regions F and F' comprise at least one LNA nucleoside, the remaining nucleosides of regions F and F' are independently selected from the group consisting of 2'-MOE and LNA. In some embodiments, at least one of regions F and F', or both regions F and F', comprise at least two LNA nucleosides, the remaining nucleosides of regions F and F' are independently selected from the group consisting of 2'-MOE and LNA. In some mixed wing embodiments, one or both of regions F and F' may further comprise one or more DNA nucleosides. Gapmer design is discussed in WO 2008 / 049085 and WO 2012 / 109395, both of which are incorporated by reference.

[0087] Table 2 provides examples of antisense oligonucleotides of the invention incorporating modified bases and other modifications as disclosed herein. As described, many non-standard nucleic acid monomers are commercially available from custom oligonucleotide manufacturers and are easily incorporated into the antisense oligonucleotides of the invention. These monomer units are described using well-known oligonucleotide synthesis nomenclature to indicate the non-standard monomer units, for example, as provided by Integrated DNA Technologies (Iowa, US). For example, in the sequences provided in Table 2, the non-standard monomer units are enclosed in forward slashes " / ", and an asterisk "*" between units indicates a PS linkage, while the absence of an asterisk indicates a PO linkage. Table 2 also provides the sequence numbers of the ASOs described above. [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]

[0088] Conjugation of oligonucleotide 107 to one or more non-nucleotide moieties may improve the pharmacology of the oligonucleotide, for example, by affecting the activity, cellular distribution, cellular uptake or stability of the oligonucleotide. In some embodiments, the conjugate moiety may modify or enhance the pharmacokinetic properties of the oligonucleotide by improving the cellular distribution, bioavailability, metabolism, excretion, permeability, and / or cellular uptake of the oligonucleotide. In particular, the conjugate may target the oligonucleotide to a specific organ, tissue or cell type, thereby enhancing the efficacy of the oligonucleotide in that organ, tissue or cell type. The conjugate may also serve to reduce the activity of the oligonucleotide in non-target cell types, tissues or organs, for example, off-target activity or activity in non-target cell types, tissues or organs.

[0089] In one embodiment, the non-nucleotide moiety (conjugate moiety) is selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substances, polymers, proteins, peptides, toxins (e.g., bacterial toxins), vitamins, viral proteins (e.g., capsids), or combinations thereof.

[0090] The oligonucleotide 107 of the present disclosure may be provided in a pharmaceutical composition comprising any of the aforementioned oligonucleotides and / or their oligonucleotide conjugates or salts, and a pharma- ceutically acceptable diluent, carrier, salt and / or adjuvant. Pharmaceutically acceptable diluents include ACSF (artificial cerebrospinal fluid), and pharma-ceutically acceptable salts include, but are not limited to, sodium and potassium salts. In some embodiments, the pharma-ceutically acceptable diluent is sterile phosphate buffered saline or sterile sodium carbonate buffer. In some preferred embodiments, diluents for clinical applications include Elliotts B solution and / or ACSF (artificial cerebrospinal fluid).

[0091] In some embodiments, the oligonucleotide of the present invention is in the form of a solution in a pharma- ceutically acceptable diluent (e.g., dissolved in PBS or sodium carbonate buffer). The oligonucleotide may be preformulated in the solution or, in some embodiments, in the form of a dry powder (e.g., a lyophilized powder) that can be dissolved in a pharma-ceutically acceptable diluent prior to administration. Suitably, for example, the oligonucleotide may be dissolved at a concentration of 0.1-100 mg / mL (e.g., 1-10 mg / mL). EXAMPLES

[0092] Working Example The following example provides an exemplary method for screening the ASO of the present invention.In the example, a series of ASOs are screened.Based on the data obtained, the ASOs corresponding to SEQ ID NOs: 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213 and 214 are identified as lead candidate ASOs based on dose-response efficacy, sequence motif responsibility and off-target alignment analysis.These ASOs show the highest in vitro efficacy, the lowest off-target alignment and limited sequence motif concern.However, other ASOs as described herein also function as described to knock down UBE3A for the treatment of various conditions.

[0093] Example 1 Single-dose screening of UBE3A ASO Forty UBE3A-targeting ASOs (SEQ ID NOs: 1-40) were screened in vitro by treating primary fibroblasts (10k plated per well of a 96-well plate) with 200 nM ASOs. ASOs were delivered by transfection using RNAi Max at 0.5 μL per well of a 96-well plate.

[0094] Data shown in Figure 3 are qPCR data of normalized relative UBE3A transcript expression of ASO-treated fibroblasts to vehicle. All samples were normalized to the second vehicle condition. The cells only condition (white) shows no change in UBE3A expression. UBE3A siRNA was used as a positive control and shows -80% knockdown of UBE3A transcript. Non-targeting siRNA was used as a negative control and shows no knockdown of UBE3A. The top graph shows data for UBE3A ASOs 001-020 (SEQ ID NOs: 1-20). The bottom graph shows data for UBE3A ASOs 021-040 (SEQ ID NOs: 21-40).

[0095] All cells were transfected with ASO 48 hours after plating. Cells were harvested for qPCR another 48 hours after ASO transfection. Actin was used as a normalization gene to UBE3A. Each bar represents three technical replicates and one biological replicate. The dots above a particular bar indicate the preferred ASOs identified within this set of 40 ASOs, and correspond to SEQ ID NOs: 4, 7, 8, 14, 17, 18, 21, 26, 34, and 35.

[0096] 4 provides results showing the dose response of ten ASO candidates (SEQ ID NOs: 14, 17, 4, 7, 8, 18, 21, 26, 34, and 35) designed according to embodiments of the present disclosure (approximately 20 bases in length with a central region of approximately 10-12 bases DNA linked by RNA wings with 2'-O modified RNA and phosphorothioate linkages throughout the ASO) at six concentrations each. All ten ASOs reduced UBE3A expression in a dose-dependent manner compared to controls (cells treated with vehicle only and untreated "cells only" conditions).

[0097] Example 2 Single-dose screening of all UBE3A ASOs Using the method of Example 1, UBE3A-targeting ASOs (SEQ ID NOs: 1-80, 101-139, and 184-203) were screened in vitro by treating primary fibroblasts (10k plated per well of a 96-well plate) with 200 nM ASO. ASOs were delivered by transfection using RNAi Max at 0.5 μL per well of a 96-well plate.

[0098] All screened ASOs were designed according to embodiments of the present disclosure (i.e., approximately 20 bases in length with a DNA central region of approximately 10-12 bases flanked by RNA wings with 2'-O modified RNA and phosphorothioate linkages throughout the ASO).

[0099] The data shown in Figures 6-9 are presented as a summary table of qPCR readouts of UBE3A knockdown (expressed as percent UBE3A knockdown) for all 139 ASOs screened. All samples were normalized to either vehicle or cells only conditions. The tables of ASOs are categorized into UBE3A exon-targeted ASOs (Figures 6-7), UBE3A intron-targeted ASOs (Figure 8), and UBE3A ASOs with 100% homology to both human and mouse UBE3A transcripts (Figure 9) for downstream rodent proof-of-concept in vivo testing.

[0100] All cells were transfected with ASO 48 hours after plating. Cells were harvested another 48 hours after ASO transfection for qPCR. Actin was used as a normalization gene for UBE3A. Where appropriate, ASOs were screened in both control fibroblasts and fibroblasts derived from Dup15q patients (Figures 6-8). In Figure 9, for ASOs with mouse UBE3A homology, data are shown for duplicates.

[0101] Example 3 Dose-response screening of UBE3A lead ASO candidates Based on the data in Examples 1 and 2, candidate lead UBE3A-targeting ASOs were selected based on greater than 80-85% transcript knockdown in the primary single dose screen. For each candidate lead, a new ASO with identical sequence was synthesized with one to three phosphodiester (PO) backbone modifications (2'-MOE RNA-like wings at 3' and 5', respectively, with a total of four to five PO modifications (i.e., PS linkages replaced with PO linkages) per ASO). These modifications replace their corresponding PS linkages in the original lead ASO. The PO-modified ASOs are referred to in FIG. 10 as daughter ASOs.

[0102] These candidate leads were then tested for dose-response modulation of UBE3A transcript expression. For these experiments, either primary fibroblasts (10k plated per well of a 96-well plate) or mouse embryonic fibroblasts (15k plated per well) were plated in 96-well plates. ASOs were screened at six doses: 6.25nM, 12.5nM, 25nM, 50nM, 100nM, and 200nM. ASOs were delivered by transfection using RNAi Max at 0.5μL per well of a 96-well plate.

[0103] Figure 10 shows exemplary data of UBE3A ASO dose response of target expression for two example lead candidates and their PO-modified daughter molecules in Dup15q patient fibroblasts (top) or mouse embryonic fibroblasts (bottom). All samples were normalized to vehicle control.

[0104] Figure 11 plots dose response and shows EC50 for the same two example lead candidates from Figure 10. All cells were transfected with ASO 48 hours after plating. Cells were harvested another 48 hours after ASO transfection for qPCR. Actin was used as a normalization gene to UBE3A. Each data point represents two technical replicates and one biological replicate.

[0105] Example 4 Dose-response screening of UBE3A lead ASO candidates Candidate lead UBE3A-targeting ASOs were selected based on greater than 80-85% transcript knockdown in the primary single dose screen from Examples 1 and 2. For each candidate lead, a new ASO with identical sequence was synthesized with one to three PO backbone modifications, each at the 3' and 5', with 2'-MOE RNA-like wings (total of four to five PO modifications per ASO), as described in Example 3. All candidate leads were then tested for dose-response modulation of UBE3A transcript expression.

[0106] For these experiments, either primary fibroblasts (plated at 10k per well of a 96-well plate) or mouse embryonic fibroblasts (plated at 15k per well) were plated in 96-well plates. ASOs were screened at six doses: 6.25nM, 12.5nM, 25nM, 50nM, 100nM, and 200nM unless otherwise indicated. ASOs were delivered by transfection using RNAi Max at 0.5μL per well of a 96-well plate.

[0107] All samples were normalized to either vehicle or control conditions within each experiment. All cells were transfected with ASO 48 hours after plating. Cells were harvested an additional 48 hours after ASO transfection for qPCR. Actin was used as a normalization gene to UBE3A.

[0108] FIG. 12 shows the resulting dose-response data for lead all-PS scaffold candidates targeting UBE3A exons.

[0109] FIG. 13 shows the resulting dose response data for lead all-PS scaffold candidates targeting the UBE3A intron.

[0110] FIG. 14 shows the resulting dose response data of a lead all-PS scaffold candidate with 100% mouse homology for rodent in vivo efficacy testing.

[0111] FIG. 15 shows the resulting dose response data of PO modified daughter leads with 100% mouse homology for rodent in vivo efficacy testing.

[0112] FIG. 16 shows the resulting dose response data of the PO modified daughter leads for human clinical candidate testing.

[0113] Example 5 Protein knockdown of UBE3A using UBE3A ASO ASO-treated Dup15q patient fibroblasts were screened for UBE3A protein knockdown to determine efficacy and to help rank ASOs for downstream experiments.

[0114] Fibroblasts were plated at 10k per well in a 96-well plate. ASO treatment occurred 48 hours after plating. To allow for accumulation of protein knockdown, fibroblasts were harvested for Western blot analysis approximately 4.5 days after ASO treatment.

[0115] Figure 17 shows a Western blot of a particular candidate lead UBE3A ASO and three PO modified daughter molecules with the same ASO sequence. GFP-targeting ASO was used as a negative control. UBE3A expression was normalized to the housekeeping gene ACTIN and then to the vehicle condition.

[0116] Figure 18 shows quantification of UBE3A protein knockdown for the aforementioned samples. For UBE3A blots, exposure was 600s. For GAPDH, exposure was 15s. 5μg of protein was loaded per lane and high molecular weight transfer was used. UBE3A antibody: Rb-E6AP Antibody(Bethyl)-A300-351A(1:1000). Actin Antibody: Ms β-Actin-(Cell Signaling)-8H10D10(1:2000).

[0117] Example 6 Protein knockdown of UBE3A using UBE3A-targeting ASO ASO-treated Dup15q patient fibroblasts were screened for UBE3A protein knockdown to determine efficacy and to help rank ASOs for downstream experiments.

[0118] Figures 19-22 provide summary tables regarding UBE3A protein knockdown of candidate leads.

[0119] ASO-treated Dup15q patient fibroblasts were screened for UBE3A protein knockdown to determine efficacy and to help rank ASOs for downstream experiments. Fibroblasts were plated at 10k per well of a 96-well plate. ASO treatment occurred 48 hours after plating. To allow for accumulation of protein knockdown, fibroblasts were harvested for Western blot analysis approximately 4.5 days after ASO treatment. In all experiments, a GFP-targeting ASO was used as a negative control. UBE3A expression was normalized to the housekeeping gene ACTIN and then to vehicle conditions. For UBE3A blots, exposure was 600s. For GAPDH, exposure was 15s. 5μg of protein was loaded per lane and high molecular weight transfer was used. UBE3A antibody: Rb-E6AP Antibody(Bethyl)-A300-351A (1:1000). Actin Antibody: Ms β-Actin-(Cell Signaling)-8H10D10(1:2000).

[0120] FIG. 19 provides a table summarizing the UBE3A protein knockdown results of all lead PS scaffold candidates targeting UBE3A.

[0121] FIG. 20 provides a table summarizing UBE3A protein knockdown results of lead all-PS scaffold candidates with 100% mouse homology for rodent in vivo efficacy testing. (C)

[0122] FIG. 21 provides a table summarizing UBE3A protein knockdown results of PO modified daughter leads with 100% mouse homology for rodent in vivo efficacy testing.

[0123] FIG. 22 provides a table summarizing the UBE3A protein knockdown results of PO modified daughter leads for human clinical candidates.

[0124] Example 7 – Knockdown of UBE3A transcripts in human NGN2 stem cell-derived neurons using UBE3A lead candidates UBE3A is imprinted in neurons, a cell type that is crucial for the pathogenesis of Dup15q. To demonstrate that the ASO of the present invention is effective in disease-related human cell types, in this example, human induced pluripotent stem cell-derived neurons (differentiated via overexpression of the transcription factor NGN2 and small molecule inhibition of SMAD signaling) were treated with the UBE3A-targeting ASO of the present invention.

[0125] Neurons were plated in 96-well plates at a density of 80,000 cells / well and treated with 100 nM UBE3A-targeted ASO. ASO was delivered to neurons cultured with Endoporter reagent at DIV (day in vitro) 21. Cells were harvested for qPCR 10 days after treatment at DIV 31. UBE3A lead candidate ASO and optimized lead candidate ASO were screened.

[0126] Figure 23 provides data summarizing this screening. As shown, many ASOs showed >80% knockdown of UBE3A transcripts in human neurons. UBE3A expression levels were normalized to β-tubulin transcript levels (a housekeeping gene used as a reference). All normalized expressions were then quantified relative to the first vehicle condition. Each bar represents three technical replicates and one biological replicate.

[0127] Example 8 –Knockdown of UBE3A transcripts in human primary neurons using UBE3A lead candidate ASO UBE3A is imprinted in neurons, a cell type that is crucial for the pathogenesis of Dup15q. To demonstrate that the ASO of the present invention is effective in relevant human cell types, human primary neurons (derived from a 19-week-old male fetus; obtained from Sciencell) were treated with UBE3A-targeted ASO. Neurons were plated in 96-well plates at a density of 30,000 cells / well and treated with 500 nM of UBE3A-targeted ASO. ASO was delivered gymnotically (without transfection reagent) at DIV 1. Cells were harvested for qPCR 6 days after ASO treatment. UBE3A lead candidate ASO and a subset of optimized lead candidate ASOs were screened.

[0128] Figure 24 provides results summarizing this screen. As shown, many ASOs show >60% knockdown of UBE3A transcripts in human primary neurons upon naked delivery. UBE3A expression levels were normalized to β-tubulin transcript levels (a housekeeping gene used as a reference). All normalized expressions were then quantified relative to the first vehicle condition. Each bar represents three technical replicates and one biological replicate.

[0129] Example 9 –Knockdown of UBE3A transcripts in non-human primate primary fibroblast cultures using UBE3A lead candidate ASO A UBE3A ASO with 100% homology to the corresponding sequence in cynomolgus non-human primates (NHPs) was selected for this assay. Lead ASO candidates were screened in vivo in NHPs to test for in vivo tolerability, toxicology, PK and PD.

[0130] To demonstrate that the ASO of the present invention is effective in relevant NHP cell types, NHP primary fibroblasts (Coriell) were transduced with UBE3A-targeted ASO. Fibroblasts were plated in 96-well plates at a density of 10,000 cells / well and treated with 200 nM UBE3A ASO. ASO was transfected into NHP fibroblasts using RNAi Max on DIV 2. Cells were harvested for qPCR 48 hours after ASO treatment. UBE3A lead candidate ASO and optimized lead candidates were screened.

[0131] Figure 25 provides results summarizing this screen. As shown, many ASOs show 80-90% knockdown of UBE3A transcripts. UBE3A expression levels were normalized to GAPDH, a housekeeping gene used as a reference. All normalized expression was then quantified relative to the first cells-only condition. Each bar represents two technical replicates and one biological replicate.

[0132] Example 10 –Knockdown of UBE3A transcripts in mouse primary cortical neurons using UBE3A lead candidates UBE3A is imprinted in neurons, a cell type crucial for the pathogenesis of Dup15q. Lead ASOs will be screened in vivo in mice to test for in vivo tolerability, toxicology, PK and PD.

[0133] The mouse model of Dup15q is useful for demonstrating proof of concept and efficacy in vivo in disease model systems. To demonstrate that the ASO of the present invention is effective in relevant mouse cell types, mouse primary cortical neurons (Brainbits) were treated with UBE3A ASO. Neurons were plated in 96-well plates at 9k per well and treated with 1 μM UBE3A ASO. ASOs were delivered naked on DIV 3. Cells were harvested for qPCR 8 days after ASO treatment (DIV 11). UBE3A lead candidates and optimized lead candidates were screened. Results from these screens are shown in FIG. 26. As shown, many ASOs show >60% knockdown of UBE3A transcripts with naked delivery, especially ASOs with 100% rat homology. UBE3A expression levels were normalized to β-tubulin (a housekeeping gene used as a reference). All normalized expression was then quantified against a second cell-only condition. Each bar represents two technical and one biological replicates.

[0134] Example 11 –Knockdown of UBE3A transcripts in rat primary cortical neurons using UBE3A lead candidates UBE3A is imprinted in neurons, a cell type crucial for the pathogenesis of Dup15q. Lead ASOs will be screened in vivo in rats to test for in vivo tolerability, toxicology, PK and PD.

[0135] To demonstrate that the ASO of the present invention is effective in relevant rat cell types, rat primary cortical neurons (Brainbits) were treated with UBE3A ASO as described herein.Neurons were plated in 96-well plates at 9k per well and treated with 3 μM UBE3A ASO.ASO was delivered naked on DIV 3.

[0136] Cells were harvested for qPCR 4 and 8 days after ASO treatment (DIV 7 and DIV 11, respectively). UBE3A lead candidates and optimized lead candidates were screened.

[0137] FIG. 27 provides results summarizing the screen after cells were harvested for qPCR after 4 days.

[0138] FIG. 28 provides results summarizing the screen after cells were harvested for qPCR after 8 days.

[0139] As shown in Figures 27-28, many ASOs show >60% knockdown of UBE3A transcripts with naked delivery, especially ASOs with 100% rat homology. UBE3A expression levels were normalized to β-tubulin (a housekeeping gene used as a reference). All normalized expression was then quantified against a second cell-only condition. Each bar represents two technical replicates and one biological replicate.

Claims

1. A composition comprising a synthetic antisense oligonucleotide (ASO) that inhibits expression of a ubiquitin ligase protein.

2. The composition of claim 1 , wherein the protein is ubiquitin protein ligase E3A.

3. The composition of claim 1 , wherein the ASO hybridizes to a complementary target in a transcript derived from the UBE3A gene.

4. The composition of claim 1, wherein the sequence of bases in the ASO has at least 80% identity to one of SEQ ID NOs: 1 to 219.

5. The composition of claim 1, wherein the sequence of bases in the ASO is at least 90% identical to one of SEQ ID NOs: 1-219, and the oligonucleotide is capable of hybridizing to UBE3A pre-mRNA or mRNA and inducing RNase H-mediated cleavage.

6. The composition of claim 1 , wherein the oligonucleotide comprises two wings flanking a central region of at least 10 DNA bases.

7. The composition of claim 6, wherein at least one wing of the ASO comprises a modified RNA base.

8. 8. The composition of claim 7, wherein each modified RNA base is selected from the group consisting of 2'-O-methoxyethyl RNA and 2'-O-methyl RNA.

9. The composition of claim 1 , wherein the ASO comprises at least about 15 bases.

10. The composition of claim 1, wherein the ASO comprises between about 15 and about 25 bases.

11. The composition of claim 1 , wherein the ASO has a backbone comprising a plurality of phosphorothioate linkages.

12. The composition of claim 1, wherein the ASO has a base sequence that has been screened and determined to not meet a threshold match for any non-target transcripts in humans.

13. The composition of claim 1, wherein the ASO has a base sequence with zero mismatches to homologous segments in the non-human primate genome and about five or less mismatches in homologous segments in the rodent genome.

14. The composition of claim 1, wherein the composition comprises a plurality of ASOs, each having a base sequence at least 80% identical to one of SEQ ID NOs: 1-40, 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213, and 214, and each of the ASOs has a gapmer structure comprising a central DNA segment flanked by RNA wings.

15. 3. The composition of claim 2, wherein the oligonucleotide has a base sequence having at least a 90% match with one of SEQ ID NOs: 1-219 and having bases linked only by phosphorothioate linkages, the oligonucleotide further comprising a core of 12 DNA bases flanked by a 5' wing and a 3' wing, each of the 5' wing and the 3' wing comprising four consecutive 2' modified RNA bases.

16. 3. The composition of claim 2, wherein the oligonucleotide has a base sequence matching one of SEQ ID NOs: 1-40, 146, 155, 156, 158, 159, 161, 164, 169, 174, 175, 178, 179, 213, and 214, and at least a majority of the interbase linkages comprise phosphorothioate linkages, and the oligonucleotide further comprises a core of 12 DNA bases flanked by a 5' wing and a 3' wing, each of the 5' wing and the 3' wing comprising 4 consecutive 2'-MOE RNA bases.

17. The composition of claim 1, wherein the ASO is conjugated to a member selected from the group consisting of carbohydrates, cell surface receptor ligands, drug substances, hormones, lipophilic substrates, polymers, proteins, peptides, toxins, vitamins, viral proteins, and combinations thereof.

18. A composition described in any one of claims 1 to 16, characterized in that the composition is administered to a subject having Dup15q syndrome, thereby knocking down expression of the UBE3A gene.

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