# Patent

## Title
Modulation of UBE3A-ATS expression

## Abstract

Certain embodiments are directed to methods and compounds for inhibiting UBE3A-ATS, the endogenous antisense transcript of ubiquitin protein ligase E3A (UBE3A). Such methods and compounds are useful for inducing expression of paternal UBE3A in cells and animals.

## Background
CROSS REFERENCED TO RELATED APPLICATIONS
This application is a U.S. National Phase filing under 35 U.S.C. §371 claiming priority to International Serial No. PCT/US2013/047701 filed Jun. 25, 2013, which claims priority to U.S. Provisional Application 61/772,925, filed Mar. 5, 2013, U.S. Provisional Application 61/755,617, filed Jan. 23, 2013, U.S. Provisional Application 61/750,939, filed Jan. 10, 2013, U.S. Provisional Application 61/738,959, filed Dec. 18, 2012, and U.S. Provisional Application 61/664,083, filed Jun. 25, 2012, each of which is incorporated herein by reference in its entirety.


STATEMENT OF GOVERNMENT SUPPORT
This invention was made with government support under R01HD037283 awarded by National Institute of Health. The government has certain rights in the invention.


SEQUENCE LISTING
The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled BIOL0204USASEQ_ST25.txt created Dec. 17, 2014 which is approximately 2.75 MB in size. The information in the electronic format of the sequence listing is incorporated herein by reference in its entirety.
FIELD
Certain embodiments are directed to methods and compounds for inhibiting UBE3A-ATS, the endogenous antisense transcript of ubiquitin protein ligase E3A (UBE3A). Such methods and compounds are useful for inducing expression of paternal UBE3A in cells and animals.
BACKGROUND
UBE3A is maternally expressed in neurons and encodes an E3 ubiquitin ligase named E6-AP. An endogenous antisense transcript of UBE3A, termed UBE3A-ATS, has been identified in humans and mice. UBE3A-ATS functions to suppress paternal Ube3a expression. (Meng et al., Hum Mol Genet. 21:3001-12, 2012).
Angelman syndrome (AS) is a neurodevelopmental disorder mostly attributed to deficiency of maternal UBE3A at 15q11.2, whereas paternal UBE3A is subject to genomic imprinting and silencing in neurons. Patients of Angelman syndrome suffer from developmental delay, speech impairment and seizures. Therapies for Angelman syndrome are limited and mainly focus on symptomatic management. (Williams, C. A. et al., Genet. Med., 12: 385-395, 2010).
Recently, topoisomerase inhibitors currently used in cancer treatment were found to “unsilence” paternal Ube3a expression in both a neuronal culture system and mice. (Huang, H. S. et al., Nature, 481: 185-189, 2012). However, the exact mechanism of unsilencing paternal Ube3a expression remains unknown and topoisomerase inhibitors are fraught with safety concerns because they are known to be non-specific and capable of inducing DNA damage, such as single and double-strand breaks.
SUMMARY
Several embodiments provided herein relate to the discovery that antisense compounds targeting UBE3A-ATS induce paternal expression of UBE3A. Several embodiments are drawn to methods and compounds for inducing paternal expression of UBE3A using antisense compounds targeting UBE3A-ATS within an unexpected hotspot located at a region of UBE3A-ATS upstream from the region that overlaps with UBE3A. In certain embodiments, the hotspot is located between an upstream region of UBE3A-ATS that includes the sequence of at least one small nucleolar RNA (snoRNA), such as HBII-52 or MBII-52, and a downstream region of UBE3A-ATS that is complementary to UBE3A (i.e. antisense to UBE3A). Prior to the present discovery manifest in several embodiments described herein, it was uncertain whether UBE3A-ATS could be targeted successfully with antisense compounds and even more unclear where to target within UBE3A-ATS.
The discovery of a hotpot region of UBE3A-ATS described herein was unpredictable in view of several reports teaching that sense transcript expression can be upregulated by targeting antisense transcripts at regions that either overlap or do not overlap with the sense transcripts. For example, on one hand it has been shown that antisense compounds designed to a region of the natural antisense transcript BDNF-AS that overlaps with BDNF increases BDNF mRNA levels. (Madharessi et al., Nat Biotechnol. 30:453-459, 2012). Similar findings have been reported with p21 and Oct4 antisense transcripts (Morris et al., PLoS Genet. 4:e1000258, 2008, Hawkins and Morris, Transcription 1:165-175, 2010). On the other hand, it has been shown that antisense compounds targeting natural antisense transcripts at non-overlapping regions with sense transcripts can upregulate sense transcript expression. (Faghihi et al., PLoS ONE 5:e13177, 2010).



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 shows Western Blot analysis using an anti-YFP antibody, which confirmed that treatment with ASO increased paternal Ube3a protein expression.
FIG. 2 shows Western Blot analysis using an anti-YFP antibody, which confirmed that treatment with ASO increased paternal Ube3a protein expression.



DETAILED DESCRIPTION
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention, as claimed. Herein, the use of the singular includes the plural unless specifically stated otherwise. As used herein, the use of “or” means “and/or” unless stated otherwise. Furthermore, the use of the term “including” as well as other forms, such as “includes” and “included”, is not limiting. Also, terms such as “element” or “component” encompass both elements and components comprising one unit and elements and components that comprise more than one subunit, unless specifically stated otherwise.
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited in this application, including, but not limited to, patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
It is understood that the sequence set forth in each SEQ ID NO described herein is independent of any modification to a sugar moiety, an internucleoside linkage, or a nucleobase. As such, antisense compounds defined by a SEQ ID NO may comprise, independently, one or more modifications to a sugar moiety, an internucleoside linkage, or a nucleobase. Antisense compounds described by Isis Number (Isis No) indicate a combination of nucleobase sequence and motif.
Definitions
Unless specific definitions are provided, the nomenclature utilized in connection with, and the procedures and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those well known and commonly used in the art. Standard techniques may be used for chemical synthesis, and chemical analysis. Where permitted, all patents, applications, published applications and other publications, GENBANK Accession Numbers and associated sequence information obtainable through databases such as National Center for Biotechnology Information (NCBI) and other data referred to throughout in the disclosure herein are incorporated by reference for the portions of the document discussed herein, as well as in their entirety.
Unless otherwise indicated, the following terms have the following meanings:
“2′-O-methoxyethyl” (also 2′-MOE and 2′-O(CH2)2—OCH3) refers to an O-methoxy-ethyl modification at the 2′ position of a furanose ring. A 2′-O-methoxyethyl modified sugar is a modified sugar.
“2′-MOE nucleoside” (also 2′-O-methoxyethyl nucleoside) means a nucleoside comprising a 2′-MOE modified sugar moiety.
“2′-substituted nucleoside” means a nucleoside comprising a substituent at the 2′-position of the furanosyl ring other than H or OH. In certain embodiments, 2′ substituted nucleosides include nucleosides with bicyclic sugar modifications.
“3′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 3′-most nucleotide of a particular antisense compound.
“5′ target site” refers to the nucleotide of a target nucleic acid which is complementary to the 5′-most nucleotide of a particular antisense compound.
“5-methylcytosine” means a cytosine modified with a methyl group attached to the 5 position. A 5-methylcytosine is a modified nucleobase.
“About” means within ±7% of a value. For example, if it is stated, “the compounds affected at least about 70% inhibition of UBE3A-ATS”, it is implied that UBE3A-ATS levels are inhibited within a range of 63% and 77%. Similarly, if it is stated, “the compounds affected at least about 20% induction of paternal UBE3A expression”, it is implied that UBE3A levels are induced by a range within 13% and 27%.
“Animal” refers to a human or non-human animal, including, but not limited to, mice, rats, rabbits, dogs, cats, pigs, and non-human primates, including, but not limited to, monkeys and chimpanzees.
“Antisense activity” means any detectable or measurable activity attributable to the hybridization of an antisense compound to its target nucleic acid. In certain embodiments, antisense activity is a decrease in the amount or expression of a target nucleic acid or protein encoded by such target nucleic acid.
“Antisense compound” means an oligomeric compound that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding. Examples of antisense compounds include single-stranded and double-stranded compounds, such as, antisense oligonucleotides, siRNAs, shRNAs, ssRNAs, and occupancy-based compounds.
“Antisense inhibition” means reduction of target nucleic acid levels in the presence of an antisense compound complementary to a target nucleic acid compared to target nucleic acid levels in the absence of the antisense compound.
“Antisense mechanisms” are all those mechanisms involving hybridization of a compound with target nucleic acid, wherein the outcome or effect of the hybridization is either target degradation or target occupancy with concomitant stalling of the cellular machinery involving, for example, transcription or splicing.
“Antisense oligonucleotide” means a single-stranded oligonucleotide having a nucleobase sequence that permits hybridization to a corresponding region or segment of a target nucleic acid.
“Base complementarity” refers to the capacity for the precise base pairing of nucleobases of an antisense oligonucleotide with corresponding nucleobases in a target nucleic acid (i.e., hybridization), and is mediated by Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen binding between corresponding nucleobases.
“Bicyclic sugar moiety” means a modified sugar moiety comprising a 4 to 7 membered ring (including but not limited to a furanosyl) comprising a bridge connecting two atoms of the 4 to 7 membered ring to form a second ring, resulting in a bicyclic structure. In certain embodiments, the 4 to 7 membered ring is a sugar ring. In certain embodiments the 4 to 7 membered ring is a furanosyl. In certain such embodiments, the bridge connects the 2′-carbon and the 4′-carbon of the furanosyl.
“Bicyclic nucleic acid” or “BNA” or “BNA nucleosides” means nucleic acid monomers having a bridge connecting two carbon atoms between the 4′ and 2′position of the nucleoside sugar unit, thereby forming a bicyclic sugar. Examples of such bicyclic sugar include, but are not limited to A) α-L-Methyleneoxy (4′-CH2—O-2′) LNA, (B) β-D-Methyleneoxy (4′-CH2—O-2′) LNA, (C) Ethyleneoxy (4′-(CH2)2—O-2′) LNA, (D) Aminooxy (4′-CH2—O—N(R)-2′) LNA and (E) Oxyamino (4′-CH2—N(R)—O-2′) LNA, as depicted below.




As used herein, LNA compounds include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the sugar wherein each of the bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(R1)(R2)]n—, —C(R1)═C(R2)—, —C(R1)═N—, —C(═NR1)—, —C(═O)—, —C(═S)—, —O—, —Si(R1)2—, —S(═O)x— and —N(R1)—; wherein: x is 0, 1, or 2; n is 1, 2, 3, or 4; each R1 and R2 is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocycle radical, a substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.
Examples of 4′-2′ bridging groups encompassed within the definition of LNA include, but are not limited to one of formulae: —[C(R1)(R2)]n—, —[C(R1)(R2)]n—O—, —C(R1R2)—N(R1)—O— or —C(R1R2)—O—N(R1)—. Furthermore, other bridging groups encompassed with the definition of LNA are 4′-CH2-2′, 4′-(CH2)2-2′, 4′-(CH2)3-2′, 4′-CH2—O-2′, 4′-(CH2)2—O-2′, 4′-CH2—O—N(R1)-2′ and 4′-CH2—N(R1)—O-2′- bridges, wherein each R1 and R2 is, independently, H, a protecting group or C1-C12 alkyl.
Also included within the definition of LNA according to the invention are LNAs in which the 2′-hydroxyl group of the ribosyl sugar ring is connected to the 4′ carbon atom of the sugar ring, thereby forming a methyleneoxy (4′-CH2—O-2′) bridge to form the bicyclic sugar moiety. The bridge can also be a methylene (—CH2—) group connecting the 2′ oxygen atom and the 4′ carbon atom, for which the term methyleneoxy (4′-CH2—O-2′) LNA is used. Furthermore; in the case of the bicyclic sugar moiety having an ethylene bridging group in this position, the term ethyleneoxy (4′-CH2CH2—O-2′) LNA is used. α-L-methyleneoxy (4′-CH2—O-2′), an isomer of methyleneoxy (4′-CH2—O-2′) LNA is also encompassed within the definition of LNA, as used herein.
“Cap structure” or “terminal cap moiety” means chemical modifications, which have been incorporated at either terminus of an antisense compound.
“cEt” or “constrained ethyl” means a bicyclic sugar moiety comprising a bridge connecting the 4′-carbon and the 2′-carbon, wherein the bridge has the formula: 4′-CH(CH3)—O-2′.
“Constrained ethyl nucleoside” (also cEt nucleoside) means a nucleoside comprising a bicyclic sugar moiety comprising a 4′-CH(CH3)—O-2′ bridge.
“Chemically distinct region” refers to a region of an antisense compound that is in some way chemically different than another region of the same antisense compound. For example, a region having 2′-O-methoxyethyl nucleotides is chemically distinct from a region having nucleotides without 2′-O-methoxyethyl modifications.
“Chimeric antisense compounds” means antisense compounds that have at least 2 chemically distinct regions, each position having a plurality of subunits.
“Complementarity” means the capacity for pairing between nucleobases of a first nucleic acid and a second nucleic acid.
“Comply” means the adherence with a recommended therapy by an individual.
“Comprise,” “comprises” and “comprising” will be understood to imply the inclusion of a stated step or element or group of steps or elements but not the exclusion of any other step or element or group of steps or elements.
“Contiguous nucleobases” means nucleobases immediately adjacent to each other.
“Deoxyribonucleotide” means a nucleotide having a hydrogen at the 2′ position of the sugar portion of the nucleotide. Deoxyribonucleotides may be modified with any of a variety of substituents.
“Designing” or “Designed to” refer to the process of designing an oligomeric compound that specifically hybridizes with a selected nucleic acid molecule.
“Efficacy” means the ability to produce a desired effect.
“Expression” includes all the functions by which a gene's coded information is converted into structures present and operating in a cell. Such structures include, but are not limited to the products of transcription and translation.
“Fully complementary” or “100% complementary” means each nucleobase of a first nucleic acid has a complementary nucleobase in a second nucleic acid. In certain embodiments, a first nucleic acid is an antisense compound and a target nucleic acid is a second nucleic acid.
“Fully modified motif” refers to an antisense compound comprising a contiguous sequence of nucleosides wherein essentially each nucleoside is a sugar modified nucleoside having uniform modification.
“Gapmer” means a chimeric antisense compound in which an internal region having a plurality of nucleosides that support RNase H cleavage is positioned between external regions having one or more nucleosides, wherein the nucleosides comprising the internal region are chemically distinct from the nucleoside or nucleosides comprising the external regions. The internal region may be referred to as the “gap” and the external regions may be referred to as the “wings.”
“Gap-widened” means an antisense compound having a gap segment of 12 or more contiguous 2′-deoxyribonucleotides positioned between 5′ and 3′ wing segments having from one to six nucleotides having modified sugar moieties.
“Hybridization” means the annealing of complementary nucleic acid molecules. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense compound and a nucleic acid target. In certain embodiments, complementary nucleic acid molecules include, but are not limited to, an antisense oligonucleotide and a nucleic acid target.
“Immediately adjacent” means there are no intervening elements between the immediately adjacent elements.
“Individual” means a human or non-human animal selected for treatment or therapy.
“Induce”, “inhibit”, “potentiate”, “elevate”, “increase”, “decrease”, upregulate”, “downregulate”, or the like, generally denote quantitative differences between two states.
“Inhibiting the expression or activity” refers to a reduction, blockade of the expression or activity and does not necessarily indicate a total elimination of expression or activity.
“Internucleoside linkage” refers to the chemical bond between nucleosides.
“Lengthened” antisense oligonucleotides are those that have one or more additional nucleosides relative to an antisense oligonucleotide disclosed herein.
“Linked deoxynucleoside” means a nucleic acid base (A, G, C, T, U) substituted by deoxyribose linked by a phosphate ester to form a nucleotide.
“Linked nucleosides” means adjacent nucleosides linked together by an internucleoside linkage.
“Mismatch” or “non-complementary nucleobase” refers to the case when a nucleobase of a first nucleic acid is not capable of pairing with the corresponding nucleobase of a second or target nucleic acid.
“Modified internucleoside linkage” refers to a substitution or any change from a naturally occurring internucleoside bond (i.e. a phosphodiester internucleoside bond).
“Modified nucleobase” means any nucleobase other than adenine, cytosine, guanine, thymidine, or uracil. An “unmodified nucleobase” means the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
“Modified nucleoside” means a nucleoside having, independently, a modified sugar moiety and/or modified nucleobase.
“Modified nucleotide” means a nucleotide having, independently, a modified sugar moiety, modified internucleoside linkage, or modified nucleobase.
“Modified oligonucleotide” means an oligonucleotide comprising at least one modified internucleoside linkage, a modified sugar, and/or a modified nucleobase.
“Modified sugar” means substitution and/or any change from a natural sugar moiety.
“Monomer” refers to a single unit of an oligomer. Monomers include, but are not limited to, nucleosides and nucleotides, whether naturally occurring or modified.
“Motif” means the pattern of unmodified and modified nucleosides in an antisense compound.
“Natural sugar moiety” means a sugar moiety found in DNA (2′-H) or RNA (2′-OH).
“Naturally occurring internucleoside linkage” means a 3′ to 5′ phosphodiester linkage.
“Non-complementary nucleobase” refers to a pair of nucleobases that do not form hydrogen bonds with one another or otherwise support hybridization.
“Nucleic acid” refers to molecules composed of monomeric nucleotides. A nucleic acid includes, but is not limited to, ribonucleic acids (RNA), deoxyribonucleic acids (DNA), single-stranded nucleic acids, double-stranded nucleic acids, small interfering ribonucleic acids (siRNA), and microRNAs (miRNA).
“Nucleobase” means a heterocyclic moiety capable of pairing with a base of another nucleic acid.
“Nucleobase complementarity” refers to a nucleobase that is capable of base pairing with another nucleobase. For example, in DNA, adenine (A) is complementary to thymine (T). For example, in RNA, adenine (A) is complementary to uracil (U). In certain embodiments, complementary nucleobase refers to a nucleobase of an antisense compound that is capable of base pairing with a nucleobase of its target nucleic acid. For example, if a nucleobase at a certain position of an antisense compound is capable of hydrogen bonding with a nucleobase at a certain position of a target nucleic acid, then the position of hydrogen bonding between the oligonucleotide and the target nucleic acid is considered to be complementary at that nucleobase pair.
“Nucleobase sequence” means the order of contiguous nucleobases independent of any sugar, linkage, and/or nucleobase modification.
“Nucleoside” means a nucleobase linked to a sugar.
“Nucleoside mimetic” includes those structures used to replace the sugar or the sugar and the base and not necessarily the linkage at one or more positions of an oligomeric compound such as for example nucleoside mimetics having morpholino, cyclohexenyl, cyclohexyl, tetrahydropyranyl, bicyclo or tricyclo sugar mimetics, e.g., non furanose sugar units. Nucleotide mimetic includes those structures used to replace the nucleoside and the linkage at one or more positions of an oligomeric compound such as for example peptide nucleic acids or morpholinos (morpholinos linked by —N(H)—C(═O)—O— or other non-phosphodiester linkage). Sugar surrogate overlaps with the slightly broader term nucleoside mimetic but is intended to indicate replacement of the sugar unit (furanose ring) only. The tetrahydropyranyl rings provided herein are illustrative of an example of a sugar surrogate wherein the furanose sugar group has been replaced with a tetrahydropyranyl ring system. “Mimetic” refers to groups that are substituted for a sugar, a nucleobase, and/or internucleoside linkage. Generally, a mimetic is used in place of the sugar or sugar-internucleoside linkage combination, and the nucleobase is maintained for hybridization to a selected target.
“Nucleotide” means a nucleoside having a phosphate group covalently linked to the sugar portion of the nucleoside.
“Oligomeric compound” means a polymer of linked monomeric subunits which is capable of hybridizing to at least a region of a nucleic acid molecule.
“Oligonucleoside” means an oligonucleotide in which the internucleoside linkages do not contain a phosphorus atom.
“Oligonucleotide” means a polymer of linked nucleosides each of which can be modified or unmodified, independent one from another.
“Peptide” means a molecule formed by linking at least two amino acids by amide bonds. Without limitation, as used herein, “peptide” refers to polypeptides and proteins.
“Phosphorothioate linkage” means a linkage between nucleosides where the phosphodiester bond is modified by replacing one of the non-bridging oxygen atoms with a sulfur atom. A phosphorothioate linkage is a modified internucleoside linkage.
“Portion” means a defined number of contiguous (i.e., linked) nucleobases of a nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of a target nucleic acid. In certain embodiments, a portion is a defined number of contiguous nucleobases of an antisense compound
“Region” is defined as a portion of the target nucleic acid having at least one identifiable structure, function, or characteristic.
“Ribonucleotide” means a nucleotide having a hydroxy at the 2′ position of the sugar portion of the nucleotide. Ribonucleotides may be modified with any of a variety of substituents.
“Segments” are defined as smaller or sub-portions of regions within a target nucleic acid.
“Sites,” as used herein, are defined as unique nucleobase positions within a target nucleic acid.
“Specifically hybridizable” refers to an antisense compound having a sufficient degree of complementarity between an antisense oligonucleotide and a target nucleic acid to induce a desired effect, while exhibiting minimal or no effects on non-target nucleic acids under conditions in which specific binding is desired, i.e., under physiological conditions in the case of in vivo assays and therapeutic treatments. “Stringent hybridization conditions” or “stringent conditions” refer to conditions under which an oligomeric compound will hybridize to its target sequence, but to a minimal number of other sequences.
“Subject” means a human or non-human animal selected for treatment or therapy.
“Target” refers to a protein, the modulation of which is desired.
“Target gene” refers to a gene encoding a target.
“Targeting” means the process of design and selection of an antisense compound that will specifically hybridize to a target nucleic acid and induce a desired effect.
“Target nucleic acid,” “target RNA,” “target RNA transcript” and “nucleic acid target” all mean a nucleic acid capable of being targeted by antisense compounds.
“Target region” means a portion of a target nucleic acid to which one or more antisense compounds is targeted.
“Target segment” means the sequence of nucleotides of a target nucleic acid to which an antisense compound is targeted. “5′ target site” refers to the 5′-most nucleotide of a target segment. “3′ target site” refers to the 3′-most nucleotide of a target segment.
“UBE3A-ATS” and “Ube3A-ATS” can be used interchangeably without capitalization of their spelling referring to any particular species or ortholog.
“UBE3A” and “Ube3A” can be used interchangeably without capitalization of their spelling referring to any particular species or ortholog. Additionally, “UBE3A”, “UBE3A”, “Ube3A”, and “Ube3A” can be used interchangeably without italicization referring to nucleic acid or protein unless specifically indicated to the contrary.
“Unmodified” nucleobases mean the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U).
“Unmodified nucleotide” means a nucleotide composed of naturally occurring nucleobases, sugar moieties, and internucleoside linkages. In certain embodiments, an unmodified nucleotide is an RNA nucleotide (i.e. β-D-ribonucleosides) or a DNA nucleotide (i.e. β-D-deoxyribonucleoside).
“Validated target segment” is defined as at least an 8-nucleobase portion (i.e. 8 consecutive nucleobases) of a target region to which an antisense compound is targeted.
“Wing segment” means a plurality of nucleosides modified to impart to an oligonucleotide properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases.
Certain Embodiments
Certain embodiments are drawn to a method of inducing expression of paternal UBE3A in a cell comprising contacting the cell with an antisense compound targeted to UBE3A-ATS. In several aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO:1 or SEQ ID NO: 2.
Several embodiments provide a method of inducing expression of paternal UBE3A in a cell comprising contacting the cell with an antisense compound targeted to a region of UBE3A-ATS upstream from a region of UBE3A-ATS that is complementary to at least the 3′ end of a UBE3A pre-mRNA. Several embodiments provide a method of reducing UBE3A-ATS in a cell comprising contacting the cell with an antisense compound targeted to a region of UBE3A-ATS upstream from a region of UBE3A-ATS complementary to at least the 3′ end of a UBE3A pre-mRNA. In some aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 1. In the same aspect, the region complementary to at least the 3′ end of the UBE3A pre-mRNA can start at nucleobase 1032967 of SEQ ID NO: 1. In another aspect, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 2. In the same aspect, the region complementary to at least the 3′ end of the UBE3A pre-mRNA can start at nucleobase 513603 of SEQ ID NO: 2. In certain aspects of the foregoing methods, paternal UBE3A expression is induced and/or UBE3A-ATS is reduced in the cell without reducing levels of the neighboring small nucleolar RNAs (snoRNAs) Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52, some of are reported to be involved in Prader-Willi syndrome and are deleted in patients suffering from Prader-Willi syndrome (Sahoo, T. et al., Nat. Genet. 2008. 40: 719-21; Tsai, T. et al., Hum. Mol. Genet. 1999. 8: 1357-64). In several aspects, contacting a cell with an antisense compound targeted to a region of UBE3A-ATS upstream from a region of UBE3A-ATS that is complementary to at least the 3′ end of a UBE3A pre-mRNA induces expression of paternal UBE3A and/or reduces UBE3A-ATS in the cell without reducing Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52 levels by more than about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
Various embodiments are directed to a method of inducing expression of paternal UBE3A in a cell comprising contacting the cell with an antisense compound targeted to a first region of UBE3A-ATS, said first region flanked by (a) an upstream region and (b) a downstream region complementary to at least the 3′ end of a UBE3A pre-mRNA. Several embodiments are directed to a method of reducing UBE3A-ATS in a cell comprising contacting the cell with an antisense compound targeted to a first region of UBE3A-ATS, said first region flanked by (a) an upstream region and (b) a downstream region complementary to at least the 3′ end of a UBE3A pre-mRNA. In one aspect, the upstream region comprises the sequence of at least one snoRNA. In the same aspect, the snoRNA is HBII-52 or MBII-52. In certain aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO:1 and the first region of UBE3A-ATS consists of nucleobases at least 85% identical to nucleobases 997469 to 1032966 of SEQ ID NO: 1. In several aspects, the UBE3A pre-mRNA comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 5. In certain aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO:2 and the first region of UBE3A-ATS consists of nucleobases at least 85% identical to nucleobases 446213 through 513602 of SEQ ID NO: 2. In several aspects, the UBE3A pre-mRNA comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 6. In certain aspects of the foregoing methods, paternal UBE3A expression is induced and/or UBE3A-ATS is reduced in the cell without reducing levels of the neighboring small nucleolar RNAs (snoRNAs) Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52. In several aspects, contacting a cell with an antisense compound targeted to a first region of UBE3A-ATS, said first region flanked by (a) an upstream region and (b) a downstream region complementary to at least the 3′ end of a UBE3A pre-mRNA, induces expression of paternal UBE3A and/or reduces UBE3A-ATS in the cell without reducing Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52 levels by more than about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
Certain embodiments relate to a method of inducing expression of paternal UBE3A in a cell comprising contacting the cell with an antisense compound targeted to UBE3A-ATS within 35498 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO:1. Certain embodiments relate to a method of inhibiting UBE3A-ATS in a cell comprising contacting the cell with an antisense compound targeted to UBE3A-ATS within 35498 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO: 1. In one aspect, the region complementary to the UBE3A pre-mRNA consists of nucleobases 1032967 to 1110944 of SEQ ID NO:1. In certain aspects of the foregoing methods, paternal UBE3A expression is induced and/or UBE3A-ATS is reduced in the cell without reducing levels of the neighboring small nucleolar RNAs (snoRNAs) Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52. In several aspects, contacting a cell with an antisense compound targeted to UBE3A-ATS within 35498 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO:1, induces expression of paternal UBE3A and/or reduces UBE3A-ATS in the cell without reducing Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52 levels by more than about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
Certain embodiments relate to a method of inducing expression of paternal UBE3A in a cell comprising contacting the cell with an antisense compound targeted to UBE3A-ATS within 67390 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO: 2. Certain embodiments relate to a method of inhibiting UBE3A-ATS in a cell comprising contacting the cell with an antisense compound targeted to UBE3A-ATS within 67390 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO:2. In one aspect, the region complementary to the UBE3A pre-mRNA consists of nucleobases 513603 through 615382 of SEQ ID NO:2. In certain aspects of the foregoing methods, paternal UBE3A expression is induced and/or UBE3A-ATS is reduced in the cell without reducing levels of the neighboring small nucleolar RNAs (snoRNAs) Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52. In several aspects, contacting a cell with an antisense compound targeted to UBE3A-ATS within 67390 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO: 2, induces expression of paternal UBE3A and/or reduces UBE3A-ATS in the cell without reducing Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52 levels by more than about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
In several aspects of any of the foregoing embodiments, contacting the cell with the antisense compound reduces the level of UBE3A-ATS in the cell and/or induces expression of paternal UBE3A protein in the cell.
In several aspects of any of the foregoing embodiments, the cell is a cultured cell. In the same aspect, the cell is an animal.
Certain embodiments are drawn to a method of inducing expression of paternal UBE3A in an animal comprising administering to the animal an antisense compound targeted to UBE3A-ATS. In several aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO:1 or SEQ ID NO: 2.
Several embodiments relate to a method of inducing expression of paternal UBE3A and/or reducing UBE3A-ATS in an animal comprising administering to the animal an antisense compound targeted to a region of UBE3A-ATS upstream from a region of UBE3A-ATS complementary to at least the 3′ end of a UBE3A pre-mRNA. In some aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO:1. In the same aspect, the region complementary to at least the 3′ end of the UBE3A pre-mRNA can start at nucleobase 1032967 of SEQ ID NO: 1. In another aspect, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 2. In the same aspect, the region complementary to at least the 3′ end of the UBE3A pre-mRNA can start at nucleobase 513603 of SEQ ID NO: 2. In certain aspects of the foregoing methods, paternal UBE3A expression is induced and/or UBE3A-ATS is reduced in the animal without reducing levels of the neighboring snoRNAs Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52. In several aspects, administering an antisense compound targeted to a region of UBE3A-ATS upstream from a region of UBE3A-ATS that is complementary to at least the 3′ end of a UBE3A pre-mRNA induces expression of paternal UBE3A and/or reduces UBE3A-ATS in the animal without reducing Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52 levels by more than about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
Various embodiments are directed to a method of inducing expression of paternal UBE3A and/or reducing UBE3A-ATS in an animal comprising administering to the animal an antisense compound targeted to a first region of UBE3A-ATS, said first region flanked by (a) an upstream region and (b) a downstream region complementary to a UBE3A pre-mRNA. In one aspect, the upstream region comprises the sequence of least one small nucleolar RNA (snRNA). In the same aspect, the snoRNA is HBII-52 or MBII-52. In certain aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 1 and the first region of UBE3A-ATS consists of nucleobases at least 85% identical to nucleobases 997469 to 1032966 of SEQ ID NO: 1. In several aspects, the UBE3A pre-mRNA comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 5. In certain aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 2 and the first region of UBE3A-ATS consists of nucleobases at least 85% identical to nucleobases 446213 through 513602 of SEQ ID NO: 2. In several aspects, the UBE3A pre-mRNA comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 6. In certain aspects of the foregoing methods, paternal UBE3A expression is induced and/or UBE3A-ATS is reduced in the animal without reducing levels of the neighboring small nucleolar RNAs (snoRNAs) Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52. In several aspects, administering an antisense compound targeted to a first region of UBE3A-ATS, said first region flanked by (a) an upstream region and (b) a downstream region complementary to at least the 3′ end of a UBE3A pre-mRNA, induces expression of paternal UBE3A and/or reduces UBE3A-ATS in the animal without reducing Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52 levels by more than about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69% 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
Certain embodiments provide a method of inducing expression of paternal UBE3A and/or reducing UBE3A-ATS in an animal comprising administering to the animal an antisense compound targeted to UBE3A-ATS within 35498 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO: 1. In one aspect, the region complementary to the UBE3A pre-mRNA consists of nucleobases 1032967 to 1110944 of SEQ ID NO: 1. In certain aspects of the foregoing methods, paternal UBE3A expression is induced and/or UBE3A-ATS is reduced in the animal without reducing levels of the neighboring small nucleolar RNAs (snoRNAs) Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52. In several aspects, administering an antisense compound targeted to UBE3A-ATS within 35498 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO:1, induces expression of paternal UBE3A and/or reduces UBE3A-ATS in the animal without reducing Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52 levels by more than about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
Various embodiments relate to a method of inducing expression of paternal UBE3A and/or reducing UBE3A-ATS in an animal comprising administering to the animal an antisense compound targeted to UBE3A-ATS within 67390 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO: 2. In one aspect, the region complementary to the UBE3A pre-mRNA consists of nucleobases 513603 through 615382 of SEQ ID NO: 2. In certain aspects of the foregoing methods, paternal UBE3A expression is induced and/or UBE3A-ATS is reduced in the animal without reducing levels of the neighboring small nucleolar RNAs (snoRNAs) Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52. In several aspects, administering an antisense compound targeted to UBE3A-ATS within 67390 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO: 2, induces expression of paternal UBE3A and/or reduces UBE3A-ATS in the animal without reducing Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52 levels by more than about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52% 53%, 54% 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
In any of the aforementioned embodiments and aspects thereof, the antisense compound can comprise an oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the oligonucleotide is at least 85% complementary to a UBE3A-ATS nucleic acid sequence. In certain aspects, the oligonucleotide is at least 90%, at least 95%, or 100% complementary over its entire length to an equal length region of a UBE3A-ATS nucleic acid sequence. In certain aspects, the antisense compound or oligonucleotide is a single-stranded oligonucleotide. In several aspects, the oligonucleotide is a modified oligonucleotide. In the same aspect, the modified oligonucleotide can comprise at least one modified internucleoside linkage. Yet again in the same aspect, the modified internucleoside linkage can be a phosphorothioate internucleoside linkage. In several aspects, at least one nucleoside comprises a modified sugar. In the same aspect, the modified sugar is a bicyclic sugar comprising a bridge between the 4′ and the 2′ positions of the sugar, such as a bridge is selected from 4′-CH(CH3)—O-2′, 4′-CH2-2′, 4′-(CH2)2-2′, 4′-CH2—O-2′, 4′-(CH2)2—O-2′, 4′-CH2—O—N(R1)-2′ and 4′-CH2-N(R1)—O-2′- wherein each R1 is, independently, H, a protecting group or C1-C12 alkyl. Further in the same aspect, the bridge is 4′-CH(CH3)—O-2′.
In any of the preceding embodiments and aspects thereof including a modified sugar, the modified sugar can comprise a 2′-O-methoxyethyl group.
In any of the preceding embodiments and aspects thereof including a modified oligonucleotide, at least one nucleoside can comprise a modified nucleobase, such as a 5-methylcytosine.
In any of the foregoing embodiments and aspects thereof, the antisense compound or modified oligonucleotide induces expression of paternal UBE3A by at least 20%, at least 30%, at least 40%., at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, or at least 250%.
Certain embodiments are drawn to a method of treating an animal with an antisense compound targeted to UBE3A-ATS comprising selecting an animal in need thereof and administering to the animal an antisense compound targeted to UBE3A-ATS. In several aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 1 or SEQ ID NO: 2.
Several embodiments are directed to a method of treating an animal with an antisense compound targeted to UBE3A-ATS comprising selecting an animal in need thereof and administering to the animal an antisense compound targeted to a region of UBE3A-ATS upstream from a region of UBE3A-ATS complementary to at least the 3′ end of a UBE3A pre-mRNA. In some aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 1. In the same aspect, the region complementary to at least the 3′ end of the UBE3A pre-mRNA can start at nucleobase 1032967 of SEQ ID NO: 1. In another aspect, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 2. In the same aspect, the region complementary to at least the 3′ end of the UBE3A pre-mRNA can start at nucleobase 513603 of SEQ ID NO: 2.
Various embodiments relate to a method of treating an animal with an antisense compound targeted to UBE3A-ATS comprising selecting an animal in need thereof and administering to the animal an antisense compound targeted to a first region of UBE3A-ATS, said first region flanked by (a) an upstream region and (b) a downstream region complementary to at least the 3′ end of a UBE3A pre-mRNA. In one aspect, the upstream region comprises the sequence of at least one small nucleolar RNAs (snoRNA). In the same aspect, the snoRNA is HBII-52 or MBII-52. In certain aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 1 and the first region of UBE3A-ATS consists of nucleobases at least 85% identical to nucleobases 997469 to 1032966 of SEQ ID NO: 1. In several aspects, the UBE3A pre-mRNA comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 5. In certain aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO:2 and the first region of UBE3A-ATS consists of nucleobases at least 85% identical to nucleobases 446213 through 513602 of SEQ ID NO: 2. In several aspects, the UBE3A pre-mRNA comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 6.
Certain embodiments provide a method of treating an animal with an antisense compound targeted to UBE3A-ATS comprising selecting an animal in need thereof and administering to the animal an antisense compound targeted to UBE3A-ATS within 35498 nucleobases upstream from the start of the region complementary to a UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO: 1. In one aspect, the region complementary to the UBE3A pre-mRNA consists of nucleobases 1032967 to 1110944 of SEQ ID NO: 1.
Several embodiments relate to a method of treating an animal with an antisense compound targeted to UBE3A-ATS comprising selecting an animal in need thereof and administering to the animal an antisense compound targeted to UBE3A-ATS within 67390 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO: 2. In one aspect, the region complementary to the UBE3A pre-mRNA consists of nucleobases 513603 through 615382 of SEQ ID NO: 2.
In certain aspects of any of the foregoing embodiments drawn to a method of treating an animal, the animal has Angelman Syndrome. In certain aspects, the animal having Angelman Syndrome treated according to the methods provided herein show an improvement in anxiety, learning, balance, motor function, and/or seizures.
In certain aspects of any of the foregoing embodiments drawn to a method of treating an animal, administering an antisense compound provided treats the animal without reducing levels of the neighboring small nucleolar RNAs (snoRNAs) Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52. In several aspects, administering an antisense compound provided herein treats the animal without reducing Snrpn, MBII-85/HBII-85, and/or MBII-52/HBII-52 levels by more than about 0%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
Certain embodiments are drawn to an antisense compound targeted to UBE3A-ATS. In several aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 1 or SEQ ID NO: 2.
Several embodiments provide an antisense compound targeted to a region of UBE3A-ATS upstream from a region of UBE3A-ATS complementary to at least the 3′ end of a UBE3A pre-mRNA. In some aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 1. In the same aspect, the region complementary to at least the 3′ end of the UBE3A pre-mRNA can start at nucleobase 1032967 of SEQ ID NO: 1. In another aspect, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 2. In the same aspect, the region complementary to at least the 3′ end of the UBE3A pre-mRNA can start at nucleobase 513603 of SEQ ID NO: 2.
Various embodiments are directed to an antisense compound targeted to a first region of UBE3A-ATS, said first region flanked by (a) an upstream region and (b) a downstream region complementary to at least the 3′ end of a UBE3A pre-mRNA. In one aspect, the upstream region comprises the sequence of at least one small nucleolar RNAs (snoRNA). In the same aspect, the snoRNA is HBII-52 or MBII-52. In certain aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 1 and the first region of UBE3A-ATS consists of nucleobases at least 85% identical to nucleobases 997469 to 1032966 of SEQ ID NO: 1. In several aspects, the UBE3A pre-mRNA comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 5. In certain aspects, UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 2 and the first region of UBE3A-ATS consists of nucleobases at least 85% identical to nucleobases 446213 through 513602 of SEQ ID NO: 2. In several aspects, the UBE3A pre-mRNA comprises a nucleic acid sequence at least 85% identical to SEQ ID NO: 6.
Certain embodiments relate to an antisense compound targeted to UBE3A-ATS within 35498 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO: 1. In one aspect, the region complementary to the UBE3A pre-mRNA consists of nucleobases 1032967 to 1110944 of SEQ ID NO: 1.
Certain embodiments relate to an antisense compound targeted to UBE3A-ATS within 67390 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to the sequence of SEQ ID NO: 2. In one aspect, the region complementary to the UBE3A pre-mRNA consists of nucleobases 513603 through 615382 of SEQ ID NO: 2.
In several aspects of any of the foregoing embodiments, the antisense compound is capable of reducing the level of UBE3A-ATS in the cell and/or inducing expression of paternal UBE3A protein in the cell.
In any of the aforementioned embodiments and aspects thereof, the antisense compound can comprise an oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the oligonucleotide is at least 85% complementary to a UBE3A-ATS nucleic acid sequence. In certain aspects, the oligonucleotide is at least 90%, at least 95%, or 100% complementary over its entire length to an equal length region of a UBE3A-ATS nucleic acid sequence. In certain aspects, the antisense compound or oligonucleotide is a single-stranded oligonucleotide. In several aspects, the oligonucleotide is a modified oligonucleotide. In the same aspect, the modified oligonucleotide can comprise at least one modified internucleoside linkage. Yet again in the same aspect, the modified internucleoside linkage can be a phosphorothioate internucleoside linkage. In several aspects, at least one nucleoside comprises a modified sugar. In the same aspect, the modified sugar is a bicyclic sugar comprising a bridge between the 4′ and the 2′ positions of the sugar, such as a bridge is selected from 4′-CH(CH3)—O-2′, 4′-CH2-2′, 4′-(CH2)2-2′, 4′-CH2—O-2′, 4′-(CH2)2—O-2′, 4′-CH2—O—N(R1)-2′ and 4′-CH2-N(R1)—O-2′- wherein each R1 is, independently, H, a protecting group or C1-C12 alkyl. Further in the same aspect, the bridge is 4′-CH(CH3)—O-2′.
In any of the preceding embodiments and aspects thereof including a modified sugar, the modified sugar can comprise a 2′-O-methoxyethyl group.
In any of the preceding embodiments and aspects thereof including a modified oligonucleotide, at least one nucleoside can comprise a modified nucleobase, such as a 5-methylcytosine.
In any of the foregoing embodiments and aspects thereof, the antisense compound or modified oligonucleotide is capable of inducing expression of paternal UBE3A by at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 110%, at least 120%, at least 130%, at least 140%, at least 150%, at least 160%, at least 170%, at least 180%, at least 190%, at least 200%, at least 210%, at least 220%, at least 230%, at least 240%, or at least 250%.
Antisense Compounds
Oligomeric compounds include, but are not limited to, oligonucleotides, oligonucleosides, oligonucleotide analogs, oligonucleotide mimetics, antisense compounds, antisense oligonucleotides, and siRNAs. An oligomeric compound may be “antisense” to a target nucleic acid, meaning that is is capable of undergoing hybridization to a target nucleic acid through hydrogen bonding.
In certain embodiments, an antisense compound has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted. In certain such embodiments, an antisense oligonucleotide has a nucleobase sequence that, when written in the 5′ to 3′ direction, comprises the reverse complement of the target segment of a target nucleic acid to which it is targeted.
In certain embodiments, an antisense compound is 10-30 subunits in length. In certain embodiments, an antisense compound is 12 to 30 subunits in length. In certain embodiments, an antisense compound is 12 to 22 subunits in length. In certain embodiments, an antisense compound is 14 to 30 subunits in length. In certain embodiments, an antisense compound is 14 to 20 subunits in length. In certain embodiments, an antisense compound is 15 to 30 subunits in length. In certain embodiments, an antisense compound is 15 to 20 subunits in length. In certain embodiments, an antisense compound is 16 to 30 subunits in length. In certain embodiments, an antisense compound is 16 to 20 subunits in length. In certain embodiments, an antisense compound is 17 to 30 subunits in length. In certain embodiments, an antisense compound is 17 to 20 subunits in length. In certain embodiments, an antisense compound is 18 to 30 subunits in length. In certain embodiments, an antisense compound is 18 to 21 subunits in length. In certain embodiments, an antisense compound is 18 to 20 subunits in length. In certain embodiments, an antisense compound is 20 to 30 subunits in length. In other words, such antisense compounds are from 12 to 30 linked subunits, 14 to 30 linked subunits, 14 to 20 subunits, 15 to 30 subunits, 15 to 20 subunits, 16 to 30 subunits, 16 to 20 subunits, 17 to 30 subunits, 17 to 20 subunits, 18 to 30 subunits, 18 to 20 subunits, 18 to 21 subunits, 20 to 30 subunits, or 12 to 22 linked subunits, respectively. In certain embodiments, an antisense compound is 14 subunits in length. In certain embodiments, an antisense compound is 16 subunits in length. In certain embodiments, an antisense compound is 17 subunits in length. In certain embodiments, an antisense compound is 18 subunits in length. In certain embodiments, an antisense compound is 20 subunits in length. In other embodiments, the antisense compound is 8 to 80, 12 to 50, 13 to 30, 13 to 50, 14 to 30, 14 to 50, 15 to 30, 15 to 50, 16 to 30, 16 to 50, 17 to 30, 17 to 50, 18 to 22, 18 to 24, 18 to 30, 18 to 50, 19 to 22, 19 to 30, 19 to 50, or 20 to 30 linked subunits. In certain such embodiments, the antisense compounds are 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, or 80 linked subunits in length, or a range defined by any two of the above values. In some embodiments the antisense compound is an antisense oligonucleotide, and the linked subunits are nucleotides.
In certain embodiments antisense oligonucleotides may be shortened or truncated. For example, a single subunit may be deleted from the 5′ end (5′ truncation), or alternatively from the 3′ end (3′ truncation). A shortened or truncated antisense compound targeted to a UBE3A-ATS nucleic acid may have two subunits deleted from the 5′ end, or alternatively may have two subunits deleted from the 3′ end, of the antisense compound. Alternatively, the deleted nucleosides may be dispersed throughout the antisense compound, for example, in an antisense compound having one nucleoside deleted from the 5′ end and one nucleoside deleted from the 3′ end.
When a single additional subunit is present in a lengthened antisense compound, the additional subunit may be located at the 5′ or 3′ end of the antisense compound. When two or more additional subunits are present, the added subunits may be adjacent to each other, for example, in an antisense compound having two subunits added to the 5′ end (5′ addition), or alternatively to the 3′ end (3′ addition), of the antisense compound. Alternatively, the added subunits may be dispersed throughout the antisense compound, for example, in an antisense compound having one subunit added to the 5′ end and one subunit added to the 3′ end.
It is possible to increase or decrease the length of an antisense compound, such as an antisense oligonucleotide, and/or introduce mismatch bases without eliminating activity. For example, in Woolf et al. (Proc. Natl. Acad. Sci. USA 89:7305-7309, 1992), a series of antisense oligonucleotides 13-25 nucleobases in length were tested for their ability to induce cleavage of a target RNA in an oocyte injection model. Antisense oligonucleotides 25 nucleobases in length with 8 or 11 mismatch bases near the ends of the antisense oligonucleotides were able to direct specific cleavage of the target mRNA, albeit to a lesser extent than the antisense oligonucleotides that contained no mismatches. Similarly, target specific cleavage was achieved using 13 nucleobase antisense oligonucleotides, including those with 1 or 3 mismatches.
Gautschi et al. (J. Natl. Cancer Inst. 93:463-471, March 2001) demonstrated the ability of an oligonucleotide having 100% complementarity to the bcl-2 mRNA and having 3 mismatches to the bcl-xL mRNA to reduce the expression of both bcl-2 and bcl-xL in vitro and in vivo. Furthermore, this oligonucleotide demonstrated potent anti-tumor activity in vivo.
Maher and Dolnick (Nuc. Acid. Res. 16:3341-3358, 1988) tested a series of tandem 14 nucleobase antisense oligonucleotides, and a 28 and 42 nucleobase antisense oligonucleotides comprised of the sequence of two or three of the tandem antisense oligonucleotides, respectively, for their ability to arrest translation of human DHFR in a rabbit reticulocyte assay. Each of the three 14 nucleobase antisense oligonucleotides alone was able to inhibit translation, albeit at a more modest level than the 28 or 42 nucleobase antisense oligonucleotides.
Certain Antisense Compound Motifs and Mechanisms
In certain embodiments, antisense compounds have chemically modified subunits arranged in patterns, or motifs, to confer to the antisense compounds properties such as enhanced inhibitory activity, increased binding affinity for a target nucleic acid, or resistance to degradation by in vivo nucleases. Chimeric antisense compounds typically contain at least one region modified so as to confer increased resistance to nuclease degradation, increased cellular uptake, increased binding affinity for the target nucleic acid, and/or increased inhibitory activity. A second region of a chimeric antisense compound may confer another desired property e.g., serve as a substrate for the cellular endonuclease RNase H, which cleaves the RNA strand of an RNA:DNA duplex.
Antisense activity may result from any mechanism involving the hybridization of the antisense compound (e.g., oligonucleotide) with a target nucleic acid, wherein the hybridization ultimately results in a biological effect. In certain embodiments, the amount and/or activity of the target nucleic acid is modulated. In certain embodiments, the amount and/or activity of the target nucleic acid is reduced. In certain embodiments, hybridization of the antisense compound to the target nucleic acid ultimately results in target nucleic acid degradation. In certain embodiments, hybridization of the antisense compound to the target nucleic acid does not result in target nucleic acid degradation. In certain such embodiments, the presence of the antisense compound hybridized with the target nucleic acid (occupancy) results in a modulation of antisense activity. In certain embodiments, antisense compounds having a particular chemical motif or pattern of chemical modifications are particularly suited to exploit one or more mechanisms. In certain embodiments, antisense compounds function through more than one mechanism and/or through mechanisms that have not been elucidated. Accordingly, the antisense compounds described herein are not limited by particular mechanism.
Antisense mechanisms include, without limitation, RNase H mediated antisense; RNAi mechanisms, which utilize the RISC pathway and include, without limitation, siRNA, ssRNA and microRNA mechanisms; and occupancy based mechanisms. Certain antisense compounds may act through more than one such mechanism and/or through additional mechanisms.
RNase H-Mediated Antisense
In certain embodiments, antisense activity results at least in part from degradation of target RNA by RNase H. RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. It is known in the art that single-stranded antisense compounds which are “DNA-like” elicit RNase H activity in mammalian cells. Accordingly, antisense compounds comprising at least a portion of DNA or DNA-like nucleosides may activate RNase H, resulting in cleavage of the target nucleic acid. In certain embodiments, antisense compounds that utilize RNase H comprise one or more modified nucleosides. In certain embodiments, such antisense compounds comprise at least one block of 1-8 modified nucleosides. In certain such embodiments, the modified nucleosides do not support RNase H activity. In certain embodiments, such antisense compounds are gapmers, as described herein. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA-like nucleosides. In certain such embodiments, the gap of the gapmer comprises DNA nucleosides and DNA-like nucleosides.
Certain antisense compounds having a gapmer motif are considered chimeric antisense compounds. In a gapmer an internal region having a plurality of nucleotides that supports RNaseH cleavage is positioned between external regions having a plurality of nucleotides that are chemically distinct from the nucleosides of the internal region. In the case of an antisense oligonucleotide having a gapmer motif, the gap segment generally serves as the substrate for endonuclease cleavage, while the wing segments comprise modified nucleosides. In certain embodiments, the regions of a gapmer are differentiated by the types of sugar moieties comprising each distinct region. The types of sugar moieties that are used to differentiate the regions of a gapmer may in some embodiments include β-D-ribonucleosides, β-D-deoxyribonucleosides, 2′-modified nucleosides (such 2′-modified nucleosides may include 2′-MOE and 2′-O—CH3, among others), and bicyclic sugar modified nucleosides (such bicyclic sugar modified nucleosides may include those having a constrained ethyl). In certain embodiments, nucleosides in the wings may include several modified sugar moieties, including, for example 2′-MOE and bicyclic sugar moieties such as constrained ethyl or LNA. In certain embodiments, wings may include several modified and unmodified sugar moieties. In certain embodiments, wings may include various combinations of 2′-MOE nucleosides, bicyclic sugar moieties such as constrained ethyl nucleosides or LNA nucleosides, and 2′-deoxynucleosides.
Each distinct region may comprise uniform sugar moieties, variant, or alternating sugar moieties. The wing-gap-wing motif is frequently described as “X-Y-Z”, where “X” represents the length of the 5′-wing, “Y” represents the length of the gap, and “Z” represents the length of the 3′-wing. “X” and “Z” may comprise uniform, variant, or alternating sugar moieties. In certain embodiments, “X” and “Y” may include one or more 2′-deoxynucleosides. “Y” may comprise 2′-deoxynucleosides. As used herein, a gapmer described as “X-Y-Z” has a configuration such that the gap is positioned immediately adjacent to each of the 5′-wing and the 3′ wing. Thus, no intervening nucleotides exist between the 5′-wing and gap, or the gap and the 3′-wing. Any of the antisense compounds described herein can have a gapmer motif. In certain embodiments, “X” and “Z” are the same; in other embodiments they are different. In certain embodiments, “Y” is between 8 and 15 nucleosides. X, Y, or Z can be any of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30 or more nucleosides.
In certain embodiments, the antisense compound targeted to a UBE3A-ATS nucleic acid has a gapmer motif in which the gap consists of 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 linked nucleosides.
In certain embodiments, the antisense oligonucleotide has a sugar motif described by Formula A as follows:

(J)m-(B)n-(J)p-(B)r-(A)t-(D)g-(A)v-(B)w-(J)x-(B)y-(J)z 

    
    
        wherein:
        each A is independently a 2′-substituted nucleoside;
        each B is independently a bicyclic nucleoside;
        each J is independently either a 2′-substituted nucleoside or a 2′-deoxynucleoside;
        each D is a 2′-deoxynucleoside;
        m is 0-4; n is 0-2; p is 0-2; r is 0-2; t is 0-2; v is 0-2; w is 0-4; x is 0-2; y is 0-2; z is 0-4; g is 6-14;
    
    


provided that:

    
    
        at least one of m, n, and r is other than 0;
        at least one of w and y is other than 0;
        the sum of m, n, p, r, and t is from 2 to 5; and
        the sum of v, w, x, y, and z is from 2 to 5.
    
    


RNAi Compounds
In certain embodiments, antisense compounds are interfering RNA compounds (RNAi), which include double-stranded RNA compounds (also referred to as short-interfering RNA or siRNA) and single-stranded RNAi compounds (or ssRNA). Such compounds work at least in part through the RISC pathway to degrade and/or sequester a target nucleic acid (thus, include microRNA/microRNA-mimic compounds). In certain embodiments, antisense compounds comprise modifications that make them particularly suited for such mechanisms.
i. ssRNA Compounds
In certain embodiments, antisense compounds including those particularly suited for use as single-stranded RNAi compounds (ssRNA) comprise a modified 5′-terminal end. In certain such embodiments, the 5′-terminal end comprises a modified phosphate moiety. In certain embodiments, such modified phosphate is stabilized (e.g., resistant to degradation/cleavage compared to unmodified 5′-phosphate). In certain embodiments, such 5′-terminal nucleosides stabilize the 5′-phosphorous moiety. Certain modified 5′-terminal nucleosides may be found in the art, for example in WO/2011/139702.
In certain embodiments, the 5′-nucleoside of an ssRNA compound has Formula IIc:




wherein:

T1 is an optionally protected phosphorus moiety;
T2 is an internucleoside linking group linking the compound of Formula IIc to the oligomeric compound;
A has one of the formulas:




Q1 and Q2 are each, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(R3)(R4);
Q3 is O, S, N(R5) or C(R6)(R7);
each R3, R4 R5, R6 and R7 is, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl or C1-C6 alkoxy;
M3 is O, S, NR14, C(R15)(R16), C(R15)(R16)C(R17)(R18), C(R15)═C(R17), OC(R15)(R16) or OC(R15)(Bx2);
R14 is H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
R15, R16, R17 and R18 are each, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
Bx1 is a heterocyclic base moiety;
or if Bx2 is present then Bx2 is a heterocyclic base moiety and Bx1 is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
J4, J5, J6 and J7 are each, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
or J4 forms a bridge with one of J5 or J7 wherein said bridge comprises from 1 to 3 linked biradical groups selected from O, S, NR19, C(R20)(R21), C(R20)═C(R21), C[═C(R20)(R21)] and C(═O) and the other two of J5, J6 and J7 are each, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
each R19, R20 and R21 is, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
G is H, OH, halogen or O—[C(R8)(R9)]n—[(C═O)m—X1]j—Z;
each R8 and R9 is, independently, H, halogen, C1-C6 alkyl or substituted C1-C6 alkyl;
X1 is O, S or N(E1);
Z is H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or N(E2)(E3);
E1, E2 and E3 are each, independently, H, C1-C6 alkyl or substituted C1-C6 alkyl;
n is from 1 to about 6;
m is 0 or 1;
j is 0 or 1;
each substituted group comprises one or more optionally protected substituent groups independently selected from halogen, OJ1, N(J1)(J2), =NJ1, SJ1, N3, CN, OC(═X2)J1, OC(═X2)N(J1)(J2) and C(═X2)N(J1)(J2);
X2 is O, S or NJ3;
each J1, J2 and J3 is, independently, H or C1-C6 alkyl;
when j is 1 then Z is other than halogen or N(E2)(E3); and
wherein said oligomeric compound comprises from 8 to 40 monomeric subunits and is hybridizable to at least a portion of a target nucleic acid.
In certain embodiments, M3 is O, CH═CH, OCH2 or OC(H)(Bx2). In certain embodiments, M3 is O.
In certain embodiments, J4, J5, J6 and J7 are each H. In certain embodiments, J4 forms a bridge with one of J5 or J7.
In certain embodiments, A has one of the formulas:




wherein:

Q1 and Q2 are each, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy or substituted C1-C6 alkoxy. In certain embodiments, Q1 and Q2 are each H. In certain embodiments, Q1 and Q2 are each, independently, H or halogen. In certain embodiments, Q1 and Q2 is H and the other of Q1 and Q2 is F, CH3 or OCH3.
In certain embodiments, T1 has the formula:




wherein:

Ra and Rc are each, independently, protected hydroxyl, protected thiol, C1-C6 alkyl, substituted C1-C6 alkyl, C1-C6 alkoxy, substituted C1-C6 alkoxy, protected amino or substituted amino; and
Rb is O or S. In certain embodiments, Rb is O and Ra and Rc are each, independently, OCH3, OCH2CH3 or CH(CH3)2.
In certain embodiments, G is halogen, OCH3, OCH2F, OCHF2, OCF3, OCH2CH3, O(CH2)2F, OCH2CHF2, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—SCH3, O(CH2)2—OCF3, O(CH2)3—N(R10)(R11), O(CH2)2—ON(R10)(R11), O(CH2)2—O(CH2)2—N(R10)(R11), OCH2C(═O)—N(R10)(R11), OCH2C(═O)—N(R12)—(CH2)2—N(R10)(R11) or O(CH2)2—N(R12)—C(═NR13)[N(R10)(R11)] wherein R10, R11, R12 and R13 are each, independently, H or C1-C6 alkyl. In certain embodiments, G is halogen, OCH3, OCF3, OCH2CH3, OCH2CF3, OCH2—CH═CH2, O(CH2)2—OCH3, O(CH2)2—O(CH2)2—N(CH3)2, OCH2C(═O)—N(H)CH3, OCH2C(═O)—N(H)—(CH2)2—N(CH3)2 or OCH2—N(H)—C(═NH)NH2. In certain embodiments, G is F, OCH3 or O(CH2)2—OCH3. In certain embodiments, G is O(CH2)2—OCH3.
In certain embodiments, the 5′-terminal nucleoside has Formula IIe:




In certain embodiments, antisense compounds, including those particularly suitable for ssRNA comprise one or more type of modified sugar moieties and/or naturally occurring sugar moieties arranged along an oligonucleotide or region thereof in a defined pattern or sugar modification motif. Such motifs may include any of the sugar modifications discussed herein and/or other known sugar modifications.
In certain embodiments, the oligonucleotides comprise or consist of a region having uniform sugar modifications. In certain such embodiments, each nucleoside of the region comprises the same RNA-like sugar modification. In certain embodiments, each nucleoside of the region is a 2′-F nucleoside. In certain embodiments, each nucleoside of the region is a 2′-OMe nucleoside. In certain embodiments, each nucleoside of the region is a 2′-MOE nucleoside. In certain embodiments, each nucleoside of the region is a cEt nucleoside. In certain embodiments, each nucleoside of the region is an LNA nucleoside. In certain embodiments, the uniform region constitutes all or essentially all of the oligonucleotide. In certain embodiments, the region constitutes the entire oligonucleotide except for 1-4 terminal nucleosides.
In certain embodiments, oligonucleotides comprise one or more regions of alternating sugar modifications, wherein the nucleosides alternate between nucleotides having a sugar modification of a first type and nucleotides having a sugar modification of a second type. In certain embodiments, nucleosides of both types are RNA-like nucleosides. In certain embodiments the alternating nucleosides are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, the alternating modifications are 2′-F and 2′-OMe. Such regions may be contiguous or may be interrupted by differently modified nucleosides or conjugated nucleosides.
In certain embodiments, the alternating region of alternating modifications each consist of a single nucleoside (i.e., the pattern is (AB)xAy wherein A is a nucleoside having a sugar modification of a first type and B is a nucleoside having a sugar modification of a second type; x is 1-20 and y is 0 or 1). In certain embodiments, one or more alternating regions in an alternating motif includes more than a single nucleoside of a type. For example, oligonucleotides may include one or more regions of any of the following nucleoside motifs:
AABBAA;
ABBABB;
AABAAB;
ABBABAABB;
ABABAA;
AABABAB;
ABABAA;
ABBAABBABABAA;
BABBAABBABABAA; or
ABABBAABBABABAA;
wherein A is a nucleoside of a first type and B is a nucleoside of a second type. In certain embodiments, A and B are each selected from 2′-F, 2′-OMe, BNA, and MOE.
In certain embodiments, oligonucleotides having such an alternating motif also comprise a modified 5′ terminal nucleoside, such as those of formula IIc or IIe.
In certain embodiments, oligonucleotides comprise a region having a 2-2-3 motif. Such regions comprises the following motif:

-(A)2-(B)x-(A)2-(C)y-(A)3-

wherein: A is a first type of modified nucleoside;
B and C, are nucleosides that are differently modified than A, however, B and C may have the same or different modifications as one another;
x and y are from 1 to 15.
In certain embodiments, A is a 2′-OMe modified nucleoside. In certain embodiments, B and C are both 2′-F modified nucleosides. In certain embodiments, A is a 2′-OMe modified nucleoside and B and C are both 2′-F modified nucleosides.
In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(AB)xAy-(D)z 

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;
A is a first type of modified nucleoside;
B is a second type of modified nucleoside;
D is a modified nucleoside comprising a modification different from the nucleoside adjacent to it. Thus, if y is 0, then D must be differently modified than B and if y is 1, then D must be differently modified than A. In certain embodiments, D differs from both A and B.
X is 5-15;
Y is 0 or 1;
Z is 0-4.
In certain embodiments, oligonucleosides have the following sugar motif:

5′-(Q)-(A)x-(D)z 

wherein:

Q is a nucleoside comprising a stabilized phosphate moiety. In certain embodiments, Q is a nucleoside having Formula IIc or IIe;
A is a first type of modified nucleoside;
D is a modified nucleoside comprising a modification different from A.
X is 11-30;
Z is 0-4.
In certain embodiments A, B, C, and D in the above motifs are selected from: 2′-OMe, 2′-F, 2′-MOE, LNA, and cEt. In certain embodiments, D represents terminal nucleosides. In certain embodiments, such terminal nucleosides are not designed to hybridize to the target nucleic acid (though one or more might hybridize by chance). In certain embodiments, the nucleobase of each D nucleoside is adenine, regardless of the identity of the nucleobase at the corresponding position of the target nucleic acid. In certain embodiments the nucleobase of each D nucleoside is thymine.
In certain embodiments, antisense compounds, including those particularly suited for use as ssRNA comprise modified internucleoside linkages arranged along the oligonucleotide or region thereof in a defined pattern or modified internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region having an alternating internucleoside linkage motif. In certain embodiments, oligonucleotides comprise a region of uniformly modified internucleoside linkages. In certain such embodiments, the oligonucleotide comprises a region that is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide is uniformly linked by phosphorothioate internucleoside linkages. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate. In certain embodiments, each internucleoside linkage of the oligonucleotide is selected from phosphodiester and phosphorothioate and at least one internucleoside linkage is phosphorothioate.
In certain embodiments, the oligonucleotide comprises at least 6 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 8 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least 10 phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 6 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 8 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least 10 consecutive phosphorothioate internucleoside linkages. In certain embodiments, the oligonucleotide comprises at least one block of at least one 12 consecutive phosphorothioate internucleoside linkages. In certain such embodiments, at least one such block is located at the 3′ end of the oligonucleotide. In certain such embodiments, at least one such block is located within 3 nucleosides of the 3′ end of the oligonucleotide.
Oligonucleotides having any of the various sugar motifs described herein, may have any linkage motif. For example, the oligonucleotides, including but not limited to those described above, may have a linkage motif selected from non-limiting the table below:












5′ most linkage
Central region
3′-region







PS
Alternating PO/PS
6 PS


PS
Alternating PO/PS
7 PS


PS
Alternating PO/PS
8 PS









ii. siRNA Compounds
In certain embodiments, antisense compounds are double-stranded RNAi compounds (siRNA). In such embodiments, one or both strands may comprise any modification motif described above for ssRNA. In certain embodiments, ssRNA compounds may be unmodified RNA. In certain embodiments, siRNA compounds may comprise unmodified RNA nucleosides, but modified internucleoside linkages.
Several embodiments relate to double-stranded compositions wherein each strand comprises a motif defined by the location of one or more modified or unmodified nucleosides. In certain embodiments, compositions are provided comprising a first and a second oligomeric compound that are fully or at least partially hybridized to form a duplex region and further comprising a region that is complementary to and hybridizes to a nucleic acid target. It is suitable that such a composition comprise a first oligomeric compound that is an antisense strand having full or partial complementarity to a nucleic acid target and a second oligomeric compound that is a sense strand having one or more regions of complementarity to and forming at least one duplex region with the first oligomeric compound.
The compositions of several embodiments modulate gene expression by hybridizing to a nucleic acid target resulting in loss of its normal function. In some embodiments, the target nucleic acid is UBE3A-ATS. In certain embodiment, the degradation of the targeted UBE3A-ATS is facilitated by an activated RISC complex that is formed with compositions of the invention.
Several embodiments are directed to double-stranded compositions wherein one of the strands is useful in, for example, influencing the preferential loading of the opposite strand into the RISC (or cleavage) complex. The compositions are useful for targeting selected nucleic acid molecules and modulating the expression of one or more genes. In some embodiments, the compositions of the present invention hybridize to a portion of a target RNA resulting in loss of normal function of the target RNA.
Certain embodiments are drawn to double-stranded compositions wherein both the strands comprises a hemimer motif, a fully modified motif, a positionally modified motif or an alternating motif. Each strand of the compositions of the present invention can be modified to fulfil a particular role in for example the siRNA pathway. Using a different motif in each strand or the same motif with different chemical modifications in each strand permits targeting the antisense strand for the RISC complex while inhibiting the incorporation of the sense strand. Within this model, each strand can be independently modified such that it is enhanced for its particular role. The antisense strand can be modified at the 5′-end to enhance its role in one region of the RISC while the 3′-end can be modified differentially to enhance its role in a different region of the RISC.
The double-stranded oligonucleotide molecules can be a double-stranded polynucleotide molecule comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide molecules can be assembled from two separate oligonucleotides, where one strand is the sense strand and the other is the antisense strand, wherein the antisense and sense strands are self-complementary (i.e. each strand comprises nucleotide sequence that is complementary to nucleotide sequence in the other strand; such as where the antisense strand and sense strand form a duplex or double-stranded structure, for example wherein the double-stranded region is about 15 to about 30, e.g., about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs; the antisense strand comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense strand comprises nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof (e.g., about 15 to about 25 or more nucleotides of the double-stranded oligonucleotide molecule are complementary to the target nucleic acid or a portion thereof). Alternatively, the double-stranded oligonucleotide is assembled from a single oligonucleotide, where the self-complementary sense and antisense regions of the siRNA are linked by means of a nucleic acid based or non-nucleic acid-based linker(s).
The double-stranded oligonucleotide can be a polynucleotide with a duplex, asymmetric duplex, hairpin or asymmetric hairpin secondary structure, having self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a separate target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof. The double-stranded oligonucleotide can be a circular single-stranded polynucleotide having two or more loop structures and a stem comprising self-complementary sense and antisense regions, wherein the antisense region comprises nucleotide sequence that is complementary to nucleotide sequence in a target nucleic acid molecule or a portion thereof and the sense region having nucleotide sequence corresponding to the target nucleic acid sequence or a portion thereof, and wherein the circular polynucleotide can be processed either in vivo or in vitro to generate an active siRNA molecule capable of mediating RNAi.
In certain embodiments, the double-stranded oligonucleotide comprises separate sense and antisense sequences or regions, wherein the sense and antisense regions are covalently linked by nucleotide or non-nucleotide linkers molecules as is known in the art, or are alternately non-covalently linked by ionic interactions, hydrogen bonding, van der waals interactions, hydrophobic interactions, and/or stacking interactions. In certain embodiments, the double-stranded oligonucleotide comprises nucleotide sequence that is complementary to nucleotide sequence of a target gene. In another embodiment, the double-stranded oligonucleotide interacts with nucleotide sequence of a target gene in a manner that causes inhibition of expression of the target gene.
As used herein, double-stranded oligonucleotides need not be limited to those molecules containing only RNA, but further encompasses chemically modified nucleotides and non-nucleotides. In certain embodiments, the short interfering nucleic acid molecules lack 2′-hydroxy (2′-OH) containing nucleotides. In certain embodiments short interfering nucleic acids optionally do not include any ribonucleotides (e.g., nucleotides having a 2′-OH group). Such double-stranded oligonucleotides that do not require the presence of ribonucleotides within the molecule to support RNAi can however have an attached linker or linkers or other attached or associated groups, moieties, or chains containing one or more nucleotides with 2′-OH groups. Optionally, double-stranded oligonucleotides can comprise ribonucleotides at about 5, 10, 20, 30, 40, or 50% of the nucleotide positions. As used herein, the term siRNA is meant to be equivalent to other terms used to describe nucleic acid molecules that are capable of mediating sequence specific RNAi, for example short interfering RNA (siRNA), double-stranded RNA (dsRNA), micro-RNA (miRNA), short hairpin RNA (shRNA), short interfering oligonucleotide, short interfering nucleic acid, short interfering modified oligonucleotide, chemically modified siRNA, post-transcriptional gene silencing RNA (ptgsRNA), and others. In addition, as used herein, the term RNAi is meant to be equivalent to other terms used to describe sequence specific RNA interference, such as post transcriptional gene silencing, translational inhibition, or epigenetics. For example, double-stranded oligonucleotides can be used to epigenetically silence genes at both the post-transcriptional level and the pre-transcriptional level. In a non-limiting example, epigenetic regulation of gene expression by siRNA molecules of the invention can result from siRNA mediated modification of chromatin structure or methylation pattern to alter gene expression (see, for example, Verdel et al., 2004, Science, 303, 672-676; Pal-Bhadra et al., 2004, Science, 303, 669-672; Allshire, 2002, Science, 297, 1818-1819; Volpe et al., 2002, Science, 297, 1833-1837; Jenuwein, 2002, Science, 297, 2215-2218; and Hall et al., 2002, Science, 297, 2232-2237).
It is contemplated that compounds and compositions of several embodiments provided herein can target UBE3A-ATS by a dsRNA-mediated gene silencing or RNAi mechanism, including, e.g., “hairpin” or stem-loop double-stranded RNA effector molecules in which a single RNA strand with self-complementary sequences is capable of assuming a double-stranded conformation, or duplex dsRNA effector molecules comprising two separate strands of RNA. In various embodiments, the dsRNA consists entirely of ribonucleotides or consists of a mixture of ribonucleotides and deoxynucleotides, such as the RNA/DNA hybrids disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. The dsRNA or dsRNA effector molecule may be a single molecule with a region of self-complementarity such that nucleotides in one segment of the molecule base pair with nucleotides in another segment of the molecule. In various embodiments, a dsRNA that consists of a single molecule consists entirely of ribonucleotides or includes a region of ribonucleotides that is complementary to a region of deoxyribonucleotides. Alternatively, the dsRNA may include two different strands that have a region of complementarity to each other.
In various embodiments, both strands consist entirely of ribonucleotides, one strand consists entirely of ribonucleotides and one strand consists entirely of deoxyribonucleotides, or one or both strands contain a mixture of ribonucleotides and deoxyribonucleotides. In certain embodiments, the regions of complementarity are at least 70, 80, 90, 95, 98, or 100% complementary to each other and to a target nucleic acid sequence. In certain embodiments, the region of the dsRNA that is present in a double-stranded conformation includes at least 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 50, 75, 100, 200, 500, 1000, 2000 or 5000 nucleotides or includes all of the nucleotides in a cDNA or other target nucleic acid sequence being represented in the dsRNA. In some embodiments, the dsRNA does not contain any single stranded regions, such as single stranded ends, or the dsRNA is a hairpin. In other embodiments, the dsRNA has one or more single stranded regions or overhangs. In certain embodiments, RNA/DNA hybrids include a DNA strand or region that is an antisense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% complementarity to a target nucleic acid) and an RNA strand or region that is a sense strand or region (e.g, has at least 70, 80, 90, 95, 98, or 100% identity to a target nucleic acid), and vice versa.
In various embodiments, the RNA/DNA hybrid is made in vitro using enzymatic or chemical synthetic methods such as those described herein or those described in WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999. In other embodiments, a DNA strand synthesized in vitro is complexed with an RNA strand made in vivo or in vitro before, after, or concurrent with the transformation of the DNA strand into the cell. In yet other embodiments, the dsRNA is a single circular nucleic acid containing a sense and an antisense region, or the dsRNA includes a circular nucleic acid and either a second circular nucleic acid or a linear nucleic acid (see, for example, WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.) Exemplary circular nucleic acids include lariat structures in which the free 5′ phosphoryl group of a nucleotide becomes linked to the 2′ hydroxyl group of another nucleotide in a loop back fashion.
In other embodiments, the dsRNA includes one or more modified nucleotides in which the 2′ position in the sugar contains a halogen (such as fluorine group) or contains an alkoxy group (such as a methoxy group) which increases the half-life of the dsRNA in vitro or in vivo compared to the corresponding dsRNA in which the corresponding 2′ position contains a hydrogen or an hydroxyl group. In yet other embodiments, the dsRNA includes one or more linkages between adjacent nucleotides other than a naturally-occurring phosphodiester linkage. Examples of such linkages include phosphoramide, phosphorothioate, and phosphorodithioate linkages. The dsRNAs may also be chemically modified nucleic acid molecules as taught in U.S. Pat. No. 6,673,661. In other embodiments, the dsRNA contains one or two capped strands, as disclosed, for example, by WO 00/63364, filed Apr. 19, 2000, or U.S. Ser. No. 60/130,377, filed Apr. 21, 1999.
In other embodiments, the dsRNA can be any of the at least partially dsRNA molecules disclosed in WO 00/63364, as well as any of the dsRNA molecules described in U.S. Provisional Application 60/399,998; and U.S. Provisional Application 60/419,532, and PCT/US2003/033466, the teaching of which is hereby incorporated by reference. Any of the dsRNAs may be expressed in vitro or in vivo using the methods described herein or standard methods, such as those described in WO 00/63364.
Occupancy
In certain embodiments, antisense compounds are not expected to result in cleavage or the target nucleic acid via RNase H or to result in cleavage or sequestration through the RISC pathway. In certain such embodiments, antisense activity may result from occupancy, wherein the presence of the hybridized antisense compound disrupts the activity of the target nucleic acid. In certain such embodiments, the antisense compound may be uniformly modified or may comprise a mix of modifications and/or modified and unmodified nucleosides.
Target Nucleic Acids, Target Regions and Nucleotide Sequences
In certain embodiments, the UBE3A-ATS nucleic acid comprises the sequence set forth in the complement of GENBANK Accession No. NT_187035.1 truncated from nucleotides 66484120 to 67595063 (incorporated herein as SEQ ID NO: 1) or the sequence set forth in GENBANK Accession No. NT_026446.14 truncated from nucleotides 25068794 to 25684175 (incorporated herein as SEQ ID NO: 2). SEQ ID NO: 1 provides sequence from chromosome 7 of the mouse genome and SEQ ID NO: 2 provides sequence from chromosome 15 of the human genome. The mouse and human genomic loci corresponding to these SEQ ID NOs share a highly conserved genomic organization that includes a Snrpn gene, snoRNAs, and UBE3A-ATS in the same structural arrangement along their syntenic chromosomes. In addition to the highly conserved genomic map of the mouse and human syntenic loci, the genomic imprinting mechanism is also conserved at this loci; in mice and humans, most neurons express Ube3a only from the maternally-inherited allele. (Huang, H. S. et al., Nature, 481: 185-189, 2012)
In certain embodiments, UBE3A-ATS comprises a nucleic acid sequence at least 85%, at least 90%, at least 95%, or 100% identical to the complement of GENBANK Accession No. NT_187035.1 truncated from nucleotides 66484120 to 67595063 (incorporated herein as SEQ ID NO: 1) or GENBANK Accession No. NT_026446.14 truncated from nucleotides 25068794 to 25684175 (incorporated herein as SEQ ID NO: 2). In certain embodiments, UBE3A comprises a nucleic acid sequence at least 85%, at least 90%, at least 95%, or 100% identical to the reverse complement of nucleotides 1032967 to 1110944 of SEQ ID NO: 1 (incorporated herein as SEQ ID NO: 5) or the reverse complement of nucleotides 513603 to 615382 of SEQ ID NO: 2 (incorporated herein as SEQ ID NO: 6).
Targeting includes determination of at least one target segment to which an antisense compound hybridizes, such that a desired effect occurs. In certain embodiments, the desired effect is a reduction in UBE3A-ATS nucleic acid levels. In certain embodiments, the desired effect is induced expression of paternal UBE3A.
In certain embodiments, antisense compounds are targeted to UBE3A-ATS, which comprises a nucleic acid sequence at least 85%, at least 90%, at least 95%, or 100% identical to the complement of GENBANK Accession No. NT_187035.1 truncated from nucleotides 66484120 to 67595063 (incorporated herein as SEQ ID NO: 1) or GENBANK Accession No. NT_026446.14 truncated from nucleotides 25068794 to 25684175 (incorporated herein as SEQ ID NO: 2). In certain aspects, antisense compounds are targeted to a region of UBE3A-ATS upstream from the region of UBE3A-ATS complementary to at least the 3′ end of a UBE3A pre-mRNA. Stated differently, in certain aspects antisense compounds are targeted to a region of UBE3A-ATS that is upstream from the region of UBE3A-ATS overlapping or antisense to UBE3A. For example, the region of UBE3A-ATS complementary to at least the 3′ end of the UBE3A pre-mRNA can start at nucleobase 1032967 of SEQ ID NO: 1 or nucleobase 513603 of SEQ ID NO: 2. In certain aspects, the region of UBE3A-ATS complementary to at least the 3′ end of the UBE3A pre-mRNA can consist of nucleobases 1032967 to 1110944 of SEQ ID NO: 1 or nucleobases 513603 to 615382 of SEQ ID NO: 2.
In certain embodiments, antisense compounds are targeted to a first region of UBE3A-ATS that is flanked by (a) an upstream region and (b) a downstream region complementary to at least the 3′ end of a UBE3A pre-mRNA. Stated differently, in certain embodiments, antisense compounds are targeted to a first region of UBE3A-ATS that is flanked by (a) an upstream region and (b) a downstream region that overlaps or is antisense to UBE3A. In certain embodiments, the first region can consist of a nucleotide sequence at least 85%, at least 90%, at least 95%, or 100% identical to nucleobases 997469 to 1032966 of SEQ ID NO: 1 (the complement of GENBANK Accession No. NT_187035.1 truncated from nucleotides 66484120 to 67595063). In certain embodiments, the first region can consist of a nucleotide sequence at least 85%, at least 90%, at least 95, or 100% identical to nucleobases 446213 to 513602 of SEQ ID NO: 2 (GENBANK Accession No. NT_026446.14 truncated from nucleotides 25068794 to 25684175). In certain aspects, the downstream region complementary to at least the 3′ end of the UBE3A pre-mRNA can consist of nucleobases 1032967 to 1110944 of SEQ ID NO: 1 or nucleobases 513603 to 615382 of SEQ ID NO: 2.
In certain embodiments, antisense compounds are targeted to UBE3A-ATS within 35498 nucleobases upstream from the start of a region complementary to at least the 3′ end of the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85%, at least 90%, at least 95%, or 100% identical to the complement of GENBANK Accession No. NT_187035.1 truncated from nucleotides 66484120 to 67595063 (incorporated herein as SEQ ID NO: 1). In certain aspects, the region complementary to at least the 3′ end the UBE3A pre-mRNA consists of nucleobases 1032967 to 1110944 of SEQ ID NO: 1. In certain embodiments antisense compounds are targeted to UBE3A-ATS within 35498 nucleobases upstream from the region of UBE3A-ATS (SEQ ID NO:1) overlapping or antisense to UBE3A.
In certain embodiments, antisense compounds are targeted to UBE3A-ATS within 67390 nucleobases upstream from the start of a region complementary to the UBE3A pre-mRNA, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85%, at least 90%, at least 95%, or 100% identical to GENBANK Accession No. NT_026446.14 truncated from nucleotides 25068794 to 25684175. In certain aspects, the region complementary to the UBE3A pre-mRNA consists of nucleobases 513603 through 615382 of SEQ ID NO: 2. In certain embodiments, antisense compounds are targeted to UBE3A-ATS within 67390 nucleobases upstream from the region of UBE3A-ATS (SEQ ID NO: 2) overlapping or antisense to UBE3A.
In certain embodiments, antisense compounds target UBE3A-ATS immediately downstream of HBII-52 or MBII-52 snoRNA to the region overlapping or antisense to UBE3A. In several aspects, such antisense compounds target UBE3A-ATS between nucleobases 1 to 35499 of SEQ ID NO: 3. In several aspects, such antisense compounds target UBE3A-ATS between nucleobases 1 to 67391 of SEQ ID NO: 4.
There may be variation in activity (e.g., as defined by percent reduction of UBE3A-ATS nucleic acid levels or percent induction of paternal UBE3A expression) of the antisense compounds within an active target region. In certain embodiments, an induction of paternal UBE3A expression is indicative of inhibition of UBE3A-ATS expression.
Hybridization
In some embodiments, hybridization occurs between an antisense compound disclosed herein and an UBE3A-ATS. The most common mechanism of hybridization involves hydrogen bonding (e.g., Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding) between complementary nucleobases of the nucleic acid molecules.
Hybridization can occur under varying conditions. Stringent conditions are sequence-dependent and are determined by the nature and composition of the nucleic acid molecules to be hybridized.
Methods of determining whether a sequence is specifically hybridizable to a target nucleic acid are well known in the art. In certain embodiments, the antisense compounds provided herein are specifically hybridizable with UBE3A-ATS.
Complementarity
An antisense compound and a target nucleic acid are complementary to each other when a sufficient number of nucleobases of the antisense compound can hydrogen bond with the corresponding nucleobases of the target nucleic acid, such that a desired effect will occur (e.g., antisense inhibition of a target nucleic acid, such as a UBE3A-ATS nucleic acid).
Non-complementary nucleobases between an antisense compound and a UBE3A-ATS nucleic acid may be tolerated provided that the antisense compound remains able to specifically hybridize to a target nucleic acid. Moreover, an antisense compound may hybridize over one or more segments of a UBE3A-ATS nucleic acid such that intervening or adjacent segments are not involved in the hybridization event (e.g., a loop structure, mismatch or hairpin structure).
In certain embodiments, the antisense compounds provided herein, or a specified portion thereof, are, or are at least, 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to a UBE3A-ATS nucleic acid, a target region, target segment, or specified portion thereof. Percent complementarity of an antisense compound with a target nucleic acid can be determined using routine methods.
For example, an antisense compound in which 18 of 20 nucleobases of the antisense compound are complementary to a target region, and would therefore specifically hybridize, would represent 90 percent complementarity. In this example, the remaining noncomplementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be contiguous to each other or to complementary nucleobases. As such, an antisense compound which is 18 nucleobases in length having four noncomplementary nucleobases which are flanked by two regions of complete complementarity with the target nucleic acid would have 77.8% overall complementarity with the target nucleic acid and would thus fall within the scope of the present invention. Percent complementarity of an antisense compound with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment search tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol., 1990, 215, 403 410; Zhang and Madden, Genome Res., 1997, 7, 649 656). Percent homology, sequence identity or complementarity, can be determined by, for example, the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.), using default settings, which uses the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489).
In certain embodiments, the antisense compounds provided herein, or specified portions thereof, are fully complementary (i.e. 100% complementary) to a target nucleic acid, or specified portion thereof. For example, an antisense compound may be fully complementary to a UBE3A-ATS nucleic acid, or a target region, or a target segment or target sequence thereof. As used herein, “fully complementary” means each nucleobase of an antisense compound is capable of precise base pairing with the corresponding nucleobases of a target nucleic acid. For example, a 20 nucleobase antisense compound is fully complementary to a target sequence that is 400 nucleobases long, so long as there is a corresponding 20 nucleobase portion of the target nucleic acid that is fully complementary to the antisense compound. Fully complementary can also be used in reference to a specified portion of the first and/or the second nucleic acid. For example, a 20 nucleobase portion of a 30 nucleobase antisense compound can be “fully complementary” to a target sequence that is 400 nucleobases long. The 20 nucleobase portion of the 30 nucleobase oligonucleotide is fully complementary to the target sequence if the target sequence has a corresponding 20 nucleobase portion wherein each nucleobase is complementary to the 20 nucleobase portion of the antisense compound. At the same time, the entire 30 nucleobase antisense compound may or may not be fully complementary to the target sequence, depending on whether the remaining 10 nucleobases of the antisense compound are also complementary to the target sequence.
The location of a non-complementary nucleobase may be at the 5′ end or 3′ end of the antisense compound. Alternatively, the non-complementary nucleobase or nucleobases may be at an internal position of the antisense compound. When two or more non-complementary nucleobases are present, they may be contiguous (i.e. linked) or non-contiguous. In one embodiment, a non-complementary nucleobase is located in the wing segment of a gapmer antisense oligonucleotide.
In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases in length comprise no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a UBE3A-ATS nucleic acid, or specified portion thereof.
In certain embodiments, antisense compounds that are, or are up to 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleobases in length comprise no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 non-complementary nucleobase(s) relative to a target nucleic acid, such as a UBE3A-ATS nucleic acid, or specified portion thereof.
The antisense compounds provided also include those which are complementary to a portion of a target nucleic acid. As used herein, “portion” refers to a defined number of contiguous (i.e. linked) nucleobases within a region or segment of a target nucleic acid. A “portion” can also refer to a defined number of contiguous nucleobases of an antisense compound. In certain embodiments, the antisense compounds, are complementary to at least an 8 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 9 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 10 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least an 11 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 12 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 13 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 14 nucleobase portion of a target segment. In certain embodiments, the antisense compounds are complementary to at least a 15 nucleobase portion of a target segment. Also contemplated are antisense compounds that are complementary to at least a 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleobase portion of a target segment, or a range defined by any two of these values.
Identity
The antisense compounds provided herein may also have a defined percent identity to a particular nucleotide sequence, SEQ ID NO, or compound represented by a specific Isis number, or portion thereof. As used herein, an antisense compound is identical to the sequence disclosed herein if it has the same nucleobase pairing ability. For example, a RNA which contains uracil in place of thymidine in a disclosed DNA sequence would be considered identical to the DNA sequence since both uracil and thymidine pair with adenine. Shortened and lengthened versions of the antisense compounds described herein as well as compounds having non-identical bases relative to the antisense compounds provided herein also are contemplated. The non-identical bases may be adjacent to each other or dispersed throughout the antisense compound. Percent identity of an antisense compound is calculated according to the number of bases that have identical base pairing relative to the sequence to which it is being compared.
In certain embodiments, the antisense compounds, or portions thereof, are at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to one or more of the antisense compounds or SEQ ID NOs, or a portion thereof, disclosed herein.
In certain embodiments, a portion of the antisense compound is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
In certain embodiments, a portion of the antisense oligonucleotide is compared to an equal length portion of the target nucleic acid. In certain embodiments, an 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleobase portion is compared to an equal length portion of the target nucleic acid.
Modifications
A nucleoside is a base-sugar combination. The nucleobase (also known as base) portion of the nucleoside is normally a heterocyclic base moiety. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to the 2′, 3′ or 5′ hydroxyl moiety of the sugar. Oligonucleotides are formed through the covalent linkage of adjacent nucleosides to one another, to form a linear polymeric oligonucleotide. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside linkages of the oligonucleotide.
Modifications to antisense compounds encompass substitutions or changes to internucleoside linkages, sugar moieties, or nucleobases. Modified antisense compounds are often preferred over native forms because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for nucleic acid target, increased stability in the presence of nucleases, or increased inhibitory activity.
Chemically modified nucleosides may also be employed to increase the binding affinity of a shortened or truncated antisense oligonucleotide for its target nucleic acid. Consequently, comparable results can often be obtained with shorter antisense compounds that have such chemically modified nucleosides.
Modified Internucleoside Linkages
The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5′ phosphodiester linkage. Antisense compounds having one or more modified, i.e. non-naturally occurring, internucleoside linkages are often selected over antisense compounds having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Oligonucleotides having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In certain embodiments, antisense compounds targeted to a UBE3A-ATS nucleic acid comprise one or more modified internucleoside linkages. In certain embodiments, the modified internucleoside linkages are phosphorothioate linkages. In certain embodiments, each internucleoside linkage of an antisense compound is a phosphorothioate internucleoside linkage.
Modified Sugar Moieties
Antisense compounds can optionally contain one or more nucleosides wherein the sugar group has been modified. Such sugar modified nucleosides may impart enhanced nuclease stability, increased binding affinity, or some other beneficial biological property to the antisense compounds. In certain embodiments, nucleosides comprise chemically modified ribofuranose ring moieties. Examples of chemically modified ribofuranose rings include without limitation, addition of substitutent groups (including 5′ and 2′ substituent groups, bridging of non-geminal ring atoms to form bicyclic nucleic acids (BNA), replacement of the ribosyl ring oxygen atom with S, N(R), or C(R1)(R2) (R, R1 and R2 are each independently H, C1-C12 alkyl or a protecting group) and combinations thereof. Examples of chemically modified sugars include 2′-F-5′-methyl substituted nucleoside (see PCT International Application WO 2008/101157 Published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) or replacement of the ribosyl ring oxygen atom with S with further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a BNA (see PCT International Application WO 2007/134181 Published on Nov. 22, 2007 wherein LNA is substituted with for example a 5′-methyl or a 5′-vinyl group).
Examples of nucleosides having modified sugar moieties include without limitation nucleosides comprising 5′-vinyl, 5′-methyl (R or S), 4′-S, 2′-F, 2′-OCH3, 2′-OCH2CH3, 2′-OCH2CH2F and 2′-O(CH2)2OCH3 substituent groups. The substituent at the 2′ position can also be selected from allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, OCF3, OCH2F, O(CH2)2SCH3, O(CH2)2—O—N(Rm)(Rn), O—CH2—C(═O)—N(Rm)(Rn), and O—CH2—C(═O)—N(R1)—(CH2)2—N(Rm)(Rn), where each Rl, Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl.
As used herein, “bicyclic nucleosides” refer to modified nucleosides comprising a bicyclic sugar moiety. Examples of bicyclic nucleosides include without limitation nucleosides comprising a bridge between the 4′ and the 2′ ribosyl ring atoms. In certain embodiments, antisense compounds provided herein include one or more bicyclic nucleosides comprising a 4′ to 2′ bridge. Examples of such 4′ to 2′ bridged bicyclic nucleosides, include but are not limited to one of the formulae: 4′-(CH2)—O-2′ (LNA); 4′-(CH2)—S-2; 4′-(CH2)2—O-2′ (ENA); 4′-CH(CH3)—O-2′ (also referred to as constrained ethyl or cEt) and 4′-CH(CH2OCH3)—O-2′ (and analogs thereof see U.S. Pat. No. 7,399,845, issued on Jul. 15, 2008); 4′-C(CH3)(CH3)—O-2′ (and analogs thereof see published International Application WO/2009/006478, published Jan. 8, 2009); 4′-CH2—N(OCH3)-2′ (and analogs thereof see published International Application WO/2008/150729, published Dec. 11, 2008); 4′-CH2—O—N(CH3)-2′ (see published U.S. Patent Application US2004-0171570, published Sep. 2, 2004); 4′-CH2—N(R)—O-2′, wherein R is H, C1-C12 alkyl, or a protecting group (see U.S. Pat. No. 7,427,672, issued on Sep. 23, 2008); 4′-CH2—C—(H)(CH3)-2′ (see Chattopadhyaya et al., J. Org. Chem., 2009, 74, 118-134); and 4′-CH2—C(═CH2)-2′ (and analogs thereof see published International Application WO 2008/154401, published on Dec. 8, 2008).
Further reports related to bicyclic nucleosides can also be found in published literature (see for example: Singh et al., Chem. Commun., 1998, 4, 455-456; Koshkin et al., Tetrahedron, 1998, 54, 3607-3630; Wahlestedt et al., Proc. Natl. Acad. Sci. U.S.A., 2000, 97, 5633-5638; Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222; Singh et al., J. Org. Chem., 1998, 63, 10035-10039; Srivastava et al., J. Am. Chem. Soc., 2007, 129(26) 8362-8379; Elayadi et al., Curr. Opinion Invest. Drugs, 2001, 2, 558-561; Braasch et al., Chem. Biol., 2001, 8, 1-7; and Orum et al., Curr. Opinion Mol. Ther., 2001, 3, 239-243; U.S. Pat. Nos. 6,268,490; 6,525,191; 6,670,461; 6,770,748; 6,794,499; 7,034,133; 7,053,207; 7,399,845; 7,547,684; and 7,696,345; U.S. Patent Publication No. US2008-0039618; US2009-0012281; U.S. Patent Ser. Nos. 60/989,574; 61/026,995; 61/026,998; 61/056,564; 61/086,231; 61/097,787; and 61/099,844; Published PCT International applications WO 1994/014226; WO 2004/106356; WO 2005/021570; WO 2007/134181; WO 2008/150729; WO 2008/154401; and WO 2009/006478. Each 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 PCT international application PCT/DK98/00393, published on Mar. 25, 1999 as WO 99/14226).
In certain embodiments, bicyclic sugar moieties of BNA nucleosides include, but are not limited to, compounds having at least one bridge between the 4′ and the 2′ position of the pentofuranosyl sugar moiety wherein such bridges independently comprises 1 or from 2 to 4 linked groups independently selected from —[C(Ra)(Rb)]n—, —C(Ra)═C(Rb)—, —C(Ra)═N—, —C(═O)—, —C(═NRa)—, —C(═S)—, —O—, —Si(Ra)2—, —S(═O)x—, and —N(Ra)—;
wherein:
x is 0, 1, or 2;
n is 1, 2, 3, or 4;
each Ra and Rb is, independently, H, a protecting group, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, heterocycle radical, substituted heterocycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, OJ1, NJ1J2, SJ1, N3, COOJ1, acyl (C(═O)—H), substituted acyl, CN, sulfonyl (S(═O)2-J1), or sulfoxyl (S(═O)-J1); and
each J1 and J2 is, independently, H, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, acyl (C(═O)—H), substituted acyl, a heterocycle radical, a substituted heterocycle radical, C1-C12 aminoalkyl, substituted C1-C12 aminoalkyl or a protecting group.
In certain embodiments, the bridge of a bicyclic sugar moiety is —[C(Ra)(Rb)]n—, —[C(Ra)(Rb)]n—O—, —C(RaRb)—N(R)—O— or —C(RaRb)—O—N(R)—. In certain embodiments, the bridge is 4′-CH2-2′, 4′-(CH2)2-2′, 4′-(CH2)3-2′, 4′-CH2—O-2′, 4′-(CH2)2—O-2′, 4′-CH2—O—N(R)-2′ and 4′-CH2—N(R)—O-2′- wherein each R is, independently, H, a protecting group or C1-C12 alkyl.
In certain embodiments, bicyclic nucleosides are further defined by isomeric configuration. For example, a nucleoside comprising a 4′-2′ methylene-oxy bridge, may be in the α-L configuration or in the β-D configuration. Previously, α-L-methyleneoxy (4′-CH2—O-2′) BNA's have been incorporated into antisense oligonucleotides that showed antisense activity (Frieden et al., Nucleic Acids Research, 2003, 21, 6365-6372).
In certain embodiments, bicyclic nucleosides include, but are not limited to, (A) α-L-methyleneoxy (4′-CH2—O-2′) BNA, (B) β-D-methyleneoxy (4′-CH2—O-2′) BNA, (C) ethyleneoxy (4′-(CH2)2—O-2′) BNA, (D) aminooxy (4′-CH2—O—N(R)-2′) BNA, (E) oxyamino (4′-CH2—N(R)—O-2′) BNA, and (F) methyl(methyleneoxy) (4′-CH(CH3)—O-2′) BNA, (G) methylene-thio (4′-CH2—S-2′) BNA, (H) methylene-amino (4′-CH2—N(R)-2′) BNA, (I) methyl carbocyclic (4′-CH2—CH(CH3)-2′) BNA, (J) propylene carbocyclic (4′-(CH2)3-2′) BNA and (K) vinyl BNA as depicted below:







wherein Bx is the base moiety and R is independently H, a protecting group, C1-C12 alkyl or C1-C12 alkoxy.
In certain embodiments, bicyclic nucleosides are provided having Formula I:




wherein:

Bx is a heterocyclic base moiety;
-Qa-Qb-Qc- is —CH2—N(Rc)—CH2—, —C(═O)—N(Rc)—CH2—, —CH2—O—N(Rc)—, —CH2—N(Rc)—O— or —N(Rc)—O—CH2;
Rc is C1-C12 alkyl or an amino protecting group; and
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium.
In certain embodiments, bicyclic nucleosides are provided having Formula II:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Za is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl, acyl, substituted acyl, substituted amide, thiol or substituted thio.
In one embodiment, each of the substituted groups is, independently, mono or poly substituted with substituent groups independently selected from halogen, oxo, hydroxyl, OJc, NJcJd, SJc, N3, OC(═X)Jc, and NJeC(═X)NJcJd, wherein each Jc, Jd and Je is, independently, H, C1-C6 alkyl, or substituted C1-C6 alkyl and X is O or NJc.
In certain embodiments, bicyclic nucleosides are provided having Formula III:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Zb is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, substituted C1-C6 alkyl, substituted C2-C6 alkenyl, substituted C2-C6 alkynyl or substituted acyl (C(═O)—).
In certain embodiments, bicyclic nucleosides are provided having Formula IV:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
Rd is C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl;
each qa, qb, qc and qd is, independently, H, halogen, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl, C1-C6 alkoxyl, substituted C1-C6 alkoxyl, acyl, substituted acyl, C1-C6 aminoalkyl or substituted C1-C6 aminoalkyl;
In certain embodiments, bicyclic nucleosides are provided having Formula V:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
qa, qb, qc and qf are each, independently, hydrogen, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxy, substituted C1-C12 alkoxy, OJj, SJj, SOJj, SO2Jj, NJjJk, N3, CN, C(═O)OJj, C(═O)NJjJk, C(═O)Jj, O—C(═O)—NJjJk, N(H)C(═NH)NJjJk, N(H)C(═O)NJjJk or N(H)C(═S)NJjJk;
or qe and qf together are ═C(qg)(qh);
qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
The synthesis and preparation of the methyleneoxy (4′-CH2—O-2′) BNA monomers adenine, cytosine, guanine, 5-methyl-cytosine, thymine and uracil, along with their oligomerization, and nucleic acid recognition properties have been described (Koshkin et al., Tetrahedron, 1998, 54, 3607-3630). BNAs and preparation thereof are also described in WO 98/39352 and WO 99/14226.
Analogs of methyleneoxy (4′-CH2—O-2′) BNA and 2′-thio-BNAs, have also been prepared (Kumar et al., Bioorg. Med. Chem. Lett., 1998, 8, 2219-2222). Preparation of locked nucleoside analogs comprising oligodeoxyribonucleotide duplexes as substrates for nucleic acid polymerases has also been described (Wengel et al., WO 99/14226). Furthermore, synthesis of 2′-amino-BNA, a novel comformationally restricted high-affinity oligonucleotide analog has been described in the art (Singh et al., J. Org. Chem., 1998, 63, 10035-10039). In addition, 2′-amino- and 2′-methylamino-BNA's have been prepared and the thermal stability of their duplexes with complementary RNA and DNA strands has been previously reported.
In certain embodiments, bicyclic nucleosides are provided having Formula VI:




wherein:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently H, a hydroxyl protecting group, a conjugate group, a reactive phosphorus group, a phosphorus moiety or a covalent attachment to a support medium;
each qi, qj, qk and ql is, independently, H, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C1-C12 alkoxyl, substituted C1-C12 alkoxyl, OJj, SJj, SOJj, NJjJk, N3, CN, C(═O)OJj, C(═O)NJjJk, C(═O)Jj, O—C(═O)NJjJk, N(H)C(═NH)NJjJk, N(H)C(═O)NJjJk or N(H)C(═S)NJjJk; and
qi and qj or ql and qk together are ═C(qg)(qh), wherein qg and qh are each, independently, H, halogen, C1-C12 alkyl or substituted C1-C12 alkyl.
One carbocyclic bicyclic nucleoside having a 4′-(CH2)3-2′ bridge and the alkenyl analog bridge 4′-CH═CH—CH2-2′ have been described (Freier et al., Nucleic Acids Research, 1997, 25(22), 4429-4443 and Albaek et al., J. Org. Chem., 2006, 71, 7731-7740). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (Srivastava et al., J. Am. Chem. Soc., 2007, 129(26), 8362-8379).
As used herein, “4′-2′ bicyclic nucleoside” or “4′ to 2′ bicyclic nucleoside” refers to a bicyclic nucleoside comprising a furanose ring comprising a bridge connecting two carbon atoms of the furanose ring connects the 2′ carbon atom and the 4′ carbon atom of the sugar ring.
As used herein, “monocylic nucleosides” refer to nucleosides comprising modified sugar moieties that are not bicyclic sugar moieties. In certain embodiments, the sugar moiety, or sugar moiety analogue, of a nucleoside may be modified or substituted at any position.
As used herein, “2′-modified sugar” means a furanosyl sugar modified at the 2′ position. In certain embodiments, such modifications include substituents selected from: a halide, including, but not limited to substituted and unsubstituted alkoxy, substituted and unsubstituted thioalkyl, substituted and unsubstituted amino alkyl, substituted and unsubstituted alkyl, substituted and unsubstituted allyl, and substituted and unsubstituted alkynyl. In certain embodiments, 2′ modifications are selected from substituents including, but not limited to: O[(CH2)nO]mCH3, O(CH2)n—NH2, O(CH2)n—CH3, O(CH2)n—F, O(CH2)n—ONH2, OCH2C(═O)N(H)CH3, and O(CH2)nON[(CH2)n—CH3]2, where n and m are from 1 to about 10. Other 2′-substituent groups can also be selected from: C1-C12 alkyl, substituted alkyl, alkenyl, alkynyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, F, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving pharmacokinetic properties, or a group for improving the pharmacodynamic properties of an antisense compound, and other substituents having similar properties. In certain embodiments, modified nucleosides comprise a 2′-MOE side chain (Baker et al., J. Biol. Chem., 1997, 272, 11944-12000). Such 2′-MOE substitution have been described as having improved binding affinity compared to unmodified nucleosides and to other modified nucleosides, such as 2′-O-methyl, O-propyl, and O-aminopropyl. Oligonucleotides having the 2′-MOE substituent also have been shown to be antisense inhibitors of gene expression with promising features for in vivo use (Martin, Helv. Chim. Acta, 1995, 78, 486-504; Altmann et al., Chimia, 1996, 50, 168-176; Altmann et al., Biochem. Soc. Trans., 1996, 24, 630-637; and Altmann et al., Nucleosides Nucleotides, 1997, 16, 917-926).
As used herein, a “modified tetrahydropyran nucleoside” or “modified THP nucleoside” means a nucleoside having a six-membered tetrahydropyran “sugar” substituted in for the pentofuranosyl residue in normal nucleosides (a sugar surrogate). Modified THP nucleosides include, but are not limited to, what is referred to in the art as hexitol nucleic acid (HNA), anitol nucleic acid (ANA), mannitol nucleic acid (MNA) (see Leumann, Bioorg. Med. Chem., 2002, 10, 841-854) or fluoro HNA (F-HNA) having a tetrahydropyran ring system as illustrated below:




In certain embodiments, sugar surrogates are selected having Formula VII:




wherein independently for each of said at least one tetrahydropyran nucleoside analog of Formula VII:

Bx is a heterocyclic base moiety;
Ta and Tb are each, independently, an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound or one of Ta and Tb is an internucleoside linking group linking the tetrahydropyran nucleoside analog to the antisense compound and the other of Ta and Tb is H, a hydroxyl protecting group, a linked conjugate group or a 5′ or 3′-terminal group;
q1, q2, q3, q4, q5, q6 and q7 are each independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl or substituted C2-C6 alkynyl; and each of R1 and R2 is selected from hydrogen, hydroxyl, halogen, substituted or unsubstituted alkoxy, NJ1J2, SJ1, N3, OC(═X)J1, OC(═X)NJ1J2, NJ3C(═X)NJ1J2 and CN, wherein X is O, S or NJ1 and each J1, J2 and J3 is, independently, H or C1-C6 alkyl.
In certain embodiments, the modified THP nucleosides of Formula VII are provided wherein q1, q2, q3, q4, q5, q6 and q7 are each H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is other than H. In certain embodiments, at least one of q1, q2, q3, q4, q5, q6 and q7 is methyl. In certain embodiments, THP nucleosides of Formula VII are provided wherein one of R1 and R2 is fluoro. In certain embodiments, R1 is fluoro and R2 is H; R1 is methoxy and R2 is H, and R1 is methoxyethoxy and R2 is H.
In certain embodiments, sugar surrogates comprise rings having more than 5 atoms and more than one heteroatom. For example nucleosides comprising morpholino sugar moieties and their use in oligomeric compounds has been reported (see for example: Braasch et al., Biochemistry, 2002, 41, 4503-4510; and U.S. Pat. Nos. 5,698,685; 5,166,315; 5,185,444; and 5,034,506). As used here, the term “morpholino” means a sugar surrogate having the following formula:




In certain embodiments, morpholinos may be modified, for example by adding or altering various substituent groups from the above morpholino structure. Such sugar surrogates are referred to herein as “modified morpholinos.”

Combinations of modifications are also provided without limitation, such as 2′-F-5′-methyl substituted nucleosides (see PCT International Application WO 2008/101157 published on Aug. 21, 2008 for other disclosed 5′,2′-bis substituted nucleosides) and replacement of the ribosyl ring oxygen atom with S and further substitution at the 2′-position (see published U.S. Patent Application US2005-0130923, published on Jun. 16, 2005) or alternatively 5′-substitution of a bicyclic nucleic acid (see PCT International Application WO 2007/134181, published on Nov. 22, 2007 wherein a 4′-CH2—O-2′ bicyclic nucleoside is further substituted at the 5′ position with a 5′-methyl or a 5′-vinyl group). The synthesis and preparation of carbocyclic bicyclic nucleosides along with their oligomerization and biochemical studies have also been described (see, e.g., Srivastava et al., J. Am. Chem. Soc. 2007, 129(26), 8362-8379).
In certain embodiments, antisense compounds comprise one or more modified cyclohexenyl nucleosides, which is a nucleoside having a six-membered cyclohexenyl in place of the pentofuranosyl residue in naturally occurring nucleosides. Modified cyclohexenyl nucleosides include, but are not limited to those described in the art (see for example commonly owned, published PCT Application WO 2010/036696, published on Apr. 10, 2010, Robeyns et al., J. Am. Chem. Soc., 2008, 130(6), 1979-1984; Horváth et al., Tetrahedron Letters, 2007, 48, 3621-3623; Nauwelaerts et al., J. Am. Chem. Soc., 2007, 129(30), 9340-9348; Gu et al., Nucleosides, Nucleotides & Nucleic Acids, 2005, 24(5-7), 993-998; Nauwelaerts et al., Nucleic Acids Research, 2005, 33(8), 2452-2463; Robeyns et al., Acta Crystallographica, Section F: Structural Biology and Crystallization Communications, 2005, F61(6), 585-586; Gu et al., Tetrahedron, 2004, 60(9), 2111-2123; Gu et al., Oligonucleotides, 2003, 13(6), 479-489; Wang et al., J. Org. Chem., 2003, 68, 4499-4505; Verbeure et al., Nucleic Acids Research, 2001, 29(24), 4941-4947; Wang et al., J. Org. Chem., 2001, 66, 8478-82; Wang et al., Nucleosides, Nucleotides & Nucleic Acids, 2001, 20(4-7), 785-788; Wang et al., J. Am. Chem., 2000, 122, 8595-8602; Published PCT application, WO 06/047842; and Published PCT Application WO 01/049687; the text of each is incorporated by reference herein, in their entirety). Certain modified cyclohexenyl nucleosides have Formula X.




wherein independently for each of said at least one cyclohexenyl nucleoside analog of Formula X:
Bx is a heterocyclic base moiety;
T3 and T4 are each, independently, an internucleoside linking group linking the cyclohexenyl nucleoside analog to an antisense compound or one of T3 and T4 is an internucleoside linking group linking the tetrahydropyran nucleoside analog to an antisense compound and the other of T3 and T4 is H, a hydroxyl protecting group, a linked conjugate group, or a 5′- or 3′-terminal group; and
q1, q2, q3, q4, q5, q6, q7, q8 and q9 are each, independently, H, C1-C6 alkyl, substituted C1-C6 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, substituted C2-C6 alkynyl or other sugar substituent group.
As used herein, “2′-modified” or “2′-substituted” refers to a nucleoside comprising a sugar comprising a substituent at the 2′ position other than H or OH. 2′-modified nucleosides, include, but are not limited to, bicyclic nucleosides wherein the bridge connecting two carbon atoms of the sugar ring connects the 2′ carbon and another carbon of the sugar ring; and nucleosides with non-bridging 2′substituents, such as allyl, amino, azido, thio, O-allyl, O—C1-C10 alkyl, —OCF3, O—(CH2)2—O—CH3, 2′-O(CH2)2SCH3, O—(CH2)2—O—N(Rm)(Rn), or O—CH2—C(═O)—N(Rm)(Rn), where each Rm and Rn is, independently, H or substituted or unsubstituted C1-C10 alkyl. 2′-modified nucleosides may further comprise other modifications, for example at other positions of the sugar and/or at the nucleobase.
As used herein, “2′-F” refers to a nucleoside comprising a sugar comprising a fluoro group at the 2′ position of the sugar ring.
As used herein, “2′-OMe” or “2′-OCH3” or “2′-O-methyl” each refers to a nucleoside comprising a sugar comprising an —OCH3 group at the 2′ position of the sugar ring.
As used herein, “MOE” or “2′-MOE” or “2′-OCH2CH2OCH3” or “2′-O-methoxyethyl” each refers to a nucleoside comprising a sugar comprising a —OCH2CH2OCH3 group at the 2′ position of the sugar ring.
As used herein, “oligonucleotide” refers to a compound comprising a plurality of linked nucleosides. In certain embodiments, one or more of the plurality of nucleosides is modified. In certain embodiments, an oligonucleotide comprises one or more ribonucleosides (RNA) and/or deoxyribonucleosides (DNA).
Many other bicyclo and tricyclo sugar surrogate ring systems are also known in the art that can be used to modify nucleosides for incorporation into antisense compounds (see for example review article: Leumann, Bioorg. Med. Chem., 2002, 10, 841-854). Such ring systems can undergo various additional substitutions to enhance activity.
Methods for the preparations of modified sugars are well known to those skilled in the art. Some representative U.S. patents that teach the preparation of such modified sugars include without limitation, U.S. Pat. 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; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,670,633; 5,700,920; 5,792,847 and 6,600,032 and International Application PCT/US2005/019219, filed Jun. 2, 2005 and published as WO 2005/121371 on Dec. 22, 2005, and each of which is herein incorporated by reference in its entirety.
In nucleotides having modified sugar moieties, the nucleobase moieties (natural, modified or a combination thereof) are maintained for hybridization with an appropriate nucleic acid target.
In certain embodiments, antisense compounds comprise one or more nucleosides having modified sugar moieties. In certain embodiments, the modified sugar moiety is 2′-MOE. In certain embodiments, the 2′-MOE modified nucleosides are arranged in a gapmer motif. In certain embodiments, the modified sugar moiety is a bicyclic nucleoside having a (4′-CH(CH3)—O-2′) bridging group. In certain embodiments, the (4′-CH(CH3)—O-2′) modified nucleosides are arranged throughout the wings of a gapmer motif.
Modified Nucleobases
Nucleobase (or base) modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications can impart nuclease stability, binding affinity or some other beneficial biological property to antisense compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an antisense compound for a target nucleic acid. For example, 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278).
Additional modified nucleobases include 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl (—C≡C—CH3) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Heterocyclic base moieties can also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
In certain embodiments, antisense compounds targeted to UBE3A-ATS comprise one or more modified nucleobases. In certain embodiments, shortened or gap-widened antisense oligonucleotides targeted to UBE3A-ATS comprise one or more modified nucleobases. In certain embodiments, the modified nucleobase is 5-methylcytosine. In certain embodiments, each cytosine is a 5-methylcytosine.
Conjugated Antisense Compounds
Antisense compounds may be covalently linked to one or more moieties or conjugates which enhance the activity, cellular distribution or cellular uptake of the resulting antisense oligonucleotides. Typical conjugate groups include cholesterol moieties and lipid moieties. Additional conjugate groups include carbohydrates, phospholipids, biotin, phenazine, folate, phenanthridine, anthraquinone, acridine, fluoresceins, rhodamines, coumarins, and dyes.
Antisense compounds can also be modified to have one or more stabilizing groups that are generally attached to one or both termini of antisense compounds to enhance properties such as, for example, nuclease stability. Included in stabilizing groups are cap structures. These terminal modifications protect the antisense compound having terminal nucleic acid from exonuclease degradation, and can help in delivery and/or localization within a cell. The cap can be present at the 5′-terminus (5′-cap), or at the 3′-terminus (3′-cap), or can be present on both termini. Cap structures are well known in the art and include, for example, inverted deoxy abasic caps. Further 3′ and 5′-stabilizing groups that can be used to cap one or both ends of an antisense compound to impart nuclease stability include those disclosed in WO 03/004602 published on Jan. 16, 2003.
In certain embodiments, antisense compounds, including, but not limited to those particularly suited for use as ssRNA, are modified by attachment of one or more conjugate groups. In general, conjugate groups modify one or more properties of the attached oligonucleotide, including but not limited to pharmacodynamics, pharmacokinetics, stability, binding, absorption, cellular distribution, cellular uptake, charge and clearance. Conjugate groups are routinely used in the chemical arts and are linked directly or via an optional conjugate linking moiety or conjugate linking group to a parent compound such as an oligonucleotide. Conjugate groups includes without limitation, intercalators, reporter molecules, polyamines, polyamides, polyethylene glycols, thioethers, polyethers, cholesterols, thiocholesterols, cholic acid moieties, folate, lipids, phospholipids, biotin, phenazine, phenanthridine, anthraquinone, adamantane, acridine, fluoresceins, rhodamines, coumarins and dyes. Certain conjugate groups have been described previously, for example: cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let., 1994, 4, 1053-1060), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphatic chain, e.g., do-decan-diol or undecyl residues (Saison-Behmoaras et al., EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259, 327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res., 1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277, 923-937).
For additional conjugates including those useful for ssRNA and their placement within antisense compounds, see e.g., U.S. Application No. 61/583,963.
In Vitro Testing of Antisense Oligonucleotides
Described herein are methods for treatment of cells with antisense oligonucleotides, which can be modified appropriately for treatment with other antisense compounds.
Cells may be treated with antisense oligonucleotides when the cells reach approximately 60-80% confluency in culture.
One reagent commonly used to introduce antisense oligonucleotides into cultured cells includes the cationic lipid transfection reagent LIPOFECTIN (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotides may be mixed with LIPOFECTIN in OPTI-MEM 1 (Invitrogen, Carlsbad, Calif.) to achieve the desired final concentration of antisense oligonucleotide and a LIPOFECTIN concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another reagent used to introduce antisense oligonucleotides into cultured cells includes LIPOFECTAMINE (Invitrogen, Carlsbad, Calif.). Antisense oligonucleotide is mixed with LIPOFECTAMINE in OPTI-MEM 1 reduced serum medium (Invitrogen, Carlsbad, Calif.) to achieve the desired concentration of antisense oligonucleotide and a LIPOFECTAMINE concentration that may range from 2 to 12 ug/mL per 100 nM antisense oligonucleotide.
Another technique used to introduce antisense oligonucleotides into cultured cells includes electroporation.
Yet another technique used to introduce antisense oligonucleotides into cultured cells includes free uptake of the oligonucleotides by the cells.
Cells are treated with antisense oligonucleotides by routine methods. Cells may be harvested 16-24 hours after antisense oligonucleotide treatment, at which time RNA or protein levels of target nucleic acids are measured by methods known in the art and described herein. In general, when treatments are performed in multiple replicates, the data are presented as the average of the replicate treatments.
The concentration of antisense oligonucleotide used varies from cell line to cell line. Methods to determine the optimal antisense oligonucleotide concentration for a particular cell line are well known in the art. Antisense oligonucleotides are typically used at concentrations ranging from 1 nM to 300 nM when transfected with LIPOFECTAMINE. Antisense oligonucleotides are used at higher concentrations ranging from 625 to 20,000 nM when transfected using electroporation.
RNA Isolation
RNA analysis can be performed on total cellular RNA or poly(A)+mRNA. Methods of RNA isolation are well known in the art. RNA is prepared using methods well known in the art, for example, using the TRIZOL Reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer's recommended protocols.
EXAMPLES
Non-Limiting Disclosure and Incorporation by Reference
While certain compounds, compositions and methods described herein have been described with specificity in accordance with certain embodiments, the following examples serve only to illustrate the compounds described herein and are not intended to limit the same. Each of the references recited in the present application is incorporated herein by reference in its entirety.
Example 1
Antisense Inhibition of the Antisense Sequence of Ubiquitin Protein Ligase E3A (Ube3a-ATS) in Primary Neuronal Cells
Antisense oligonucleotides were designed targeting the mouse sequence immediately downstream of MBII-52 snoRNA to the beginning of Ube3a antisense sequence, described herein as nucleotides 997469 to 1110944 of SEQ ID NO: 1 and were tested for their effects on up-regulating the expression of the paternal allele of Ube3a in vitro.
The newly designed chimeric antisense oligonucleotides in Table 1 were designed as 5-10-5 MOE gapmers. The gapmers are 20 nucleosides in length, wherein the central gap segment comprises of ten 2′-deoxynucleosides and is flanked by wing segments on the 5′ direction and the 3′ direction comprising five nucleosides each. Each nucleoside in the 5′ wing segment and each nucleoside in the 3′ wing segment has a 2′-MOE modification. The internucleoside linkages throughout each gapmer are phosphorothioate (P═S) linkages. All cytosine residues throughout each gapmer are 5-methylcytosines.
Primary cultures of hippocampal and cortical neurons were obtained from P0-P2 Ube3a+/YFP offspring of C57BL/6 female mice crossed to Ube3a-YFP male mice (Dindot, S. V. et al., Hum. Mol. Genet. 2008. 17: 111-118). Chopped cortical hemispheres were digested with 0.25% trypsin and mechanically dissociated. Neurons were cultured in a Neurobasal Medium (Invitrogen, Carlsbad, Calif.) supplemented with B27 (Invitrogen) on plates coated with poly-D-lysine (Sigma-Aldrich, St Louis, Mo.). Half of the medium was changed at day 4 and the culture was further maintained for 3 more days at 37° C. in 5% CO2. Sets of cell culture were treated with antisense oligonucleotides by free uptake at a final concentration of 15 μM in the presence of Ara-C to inhibit glial proliferation. A set of cells treated with PBS was used as the untreated control.
Three days after antisense oligonucleotide treatment, cells were fixed with 4% paraformaldehyde for 1 hr. The cells were then washed three times with PBS, and blocked with 5% goat serum (Sigma-Aldrich, St Louis, Mo.) for 1 hr at room temperature. The cells were co-stained with anti-GFP (NB600-308, Novus Biologicals, Littleton, Colo.) at 1:2,500 dilution and anti-NeuN (MAB377, Millipore, Billerica, Mass.) at 1:500 dilution at 4° C. overnight in a humidified chamber with gentle agitation. After three washed in 0.5% Tween-20 in PBS, the cells were stained with secondary goat anti-rabbit antibody conjugated with Alexa Fluor 488 or goat anti-mouse antibody conjugated with Alexa Fluor 555 (Invitrogen, Carlsbad, Calif.) both at 1:1000 dilution. After washes, the plates were imaged with Texas Red and FITC channel by ImageXpressUltra confocal system (Molecular Device) with a 20× objective. The laser power was manually set for each plate to avoid fluorescence saturation in the samples. Nine images were taken from each well. The images were then processed with the MetaXpress software (Molecular Device) to quantify FITC intensity in those Texas Red positive cells. Typically 200-800 cells were scored per well and the signal intensity was averaged, and normalized to untransfected control cells.
Forty-three antisense oligonucleotides targeting unique sites within the region antisense to the Ube3a pre-mRNA at nucleotides 1032967 to 1110944 of SEQ ID NO: 1) were tested for their effect on inducing expression of Ube3a protein, as quantified by fluorescence. The average induction of the protein after treatment with these ASOs was 18% over the untreated control. A hundred and seventy six oligonucleotides targeting unique sites within the nucleotide region immediately downstream of MBII-52 snoRNA and upstream of the region antisense to the Ube3a pre-mRNA (nucleotides 997469 to 1032966 of SEQ ID NO: 1), designated in the Tables as the ‘hotspot’, were also tested for their effect on inducing expression of Ube3a-YFP protein. The average induction of the protein after treatment with these ASOs was 83% over the untreated control. The results of the complete screening of the antisense oligonucleotides are presented in Tables 1-3; each Table represents each screening plate utilized. ‘% induction’ is expressed as the percent induction of the protein over the untreated control. “Target start site” indicates the 5′-most nucleotide to which the gapmer is targeted on SEQ ID NO: 1. “Target stop site” indicates the 3′-most nucleotide to which the gapmer is targeted on SEQ ID NO: 1.













TABLE 1





Target
Target



%
SEQ


Start Site
Stop Site
Region
Sequence
ISIS No
upregulation
ID NO





















1003255
1003274
hotspot
GTTATACACACATATATTTT
592429
 0
 7


 


1003431
1003450
hotspot
TAGCTTAAACACACTTTTTC
592430
25
 8


 


1003601
1003620
hotspot
ATATGTTGGTCAGCTACTAC
592431
81
 9


 


1003801
1003820
hotspot
GTATATCCTTCCAGATCCTT
592432
157 
10


 


1003971
1003990
hotspot
TGAGTTCATTGGCACATTCA
592433
102 
11


 


1004144
1004163
hotspot
CTAGAAGGTGATATGAGGAT
592434
59
12


 


1004318
1004337
hotspot
ATAAATATTTCTGCATATTG
592435
 2
13


 


1004488
1004507
hotspot
CATGTACATCCCTATACCTG
592436
126 
14


 


1004658
1004677
hotspot
GACACTTTTCTTGCATAAAT
592437
67
15


 


1004833
1004852
hotspot
GTCAAAATAGTTATTTTGGG
592438
38
16


 


1005007
1005026
hotspot
AAAATTTTTATCTCAGCTAC
592439
 4
17


 


1005178
1005197
hotspot
TAAGAAGTAAGCAACTAGGG
592440
54
18


 


1005348
1005367
hotspot
AGCACATGCAATGATTAGAT
592441
95
19


 


1005518
1005537
hotspot
ACAATATATATTTTGAAACT
592442
 0
20


 


1005688
1005707
hotspot
CAAGATGGAGAATTCTTGGC
592443
 9
21


 


1005858
1005877
hotspot
TGAATATGCTTGACCCATAG
592444
161 
22


 


1006028
1006047
hotspot
TTCCTTTGTGTAAATGTACC
592445
72
23


 


1006245
1006264
hotspot
TCAAGCATACTTGGGCCTTC
592446
135 
24


 


1006420
1006439
hotspot
AACACCTGGCAGCTGCATAC
592447
97
25


 


1006590
1006609
hotspot
TGTTAGTGTCCCTATCCGTC
592448
158 
26


 


1006760
1006779
hotspot
CTGCAAGAACAGCATAGCAT
592449
89
27


 


1006930
1006949
hotspot
CAAAGGTCCAGGTTAGTTGA
592450
41
28


 


1007104
1007123
hotspot
TTTCAGGCCCCACCCTCGGG
592451
73
29


 


1007275
1007294
hotspot
AGGTCCAACATCTTTCTTTC
592452
140 
30


 


1007483
1007502
hotspot
AATATGCTGGTCAAGCCTTC
592453
97
31


 


1007699
1007718
hotspot
AATGCAGTTTGCTATGGCTC
592454
110 
32


 


1007869
1007888
hotspot
GGTTGTGAGCAACCATGTGG
592455
92
33


 


1008047
1008066
hotspot
ATTATATGATTGCATTTGAA
592456
12
34


 


1008230
1008249
hotspot
CATAGATGTTAAATTGTTAA
592457
 0
35


 


1008581
1008600
hotspot
ATGTGATCTTTTAAGATGTA
592459
13
36


 


1008751
1008770
hotspot
CATGAACAGTTATTCATTAT
592460
32
37


 


1009091
1009110
hotspot
GCTATTTTCCTAGGAGCATT
592461
68
38


 


1009262
1009281
hotspot
GAAGTGTTTTAAGGTTTTCA
592462
 0
39


 


1009604
1009623
hotspot
AACCCAGTAATACTGAGCGC
592464
92
40


 


1009790
1009809
hotspot
TGGATCCTCAGGTGCGACTC
592465
80
41


 


1010033
1010052
hotspot
GTACTGGTTAATTAATATTG
592466
 6
42


 


1010298
1010317
hotspot
ATTTCAAGTGTTATCCCCTT
592467
192 
43


 


1010468
1010487
hotspot
TATTGAAAATGTGAATCACT
592468
31
44


 


1010638
1010657
hotspot
GTTTTATCAGAATGGCTCTA
592469
138 
45


 


1010808
1010827
hotspot
CACCATTTATTAAAGATGTT
592470
36
46


 


1010978
1010997
hotspot
CCTGCAGAGGTATCTGAACC
592471
70
47


 


1011152
1011171
hotspot
TTAAATAAAGCCTTTATTAA
592472
 1
48


 


1011331
1011350
hotspot
TGAAACAGCTGAGCAATCTC
592473
108 
49


 


1011707
1011726
hotspot
TTTAACCACAATTGTTCTGG
592475
84
50


 


1011877
1011896
hotspot
TAGAGGTAGCCTTTCTGATG
592476
132 
51


 


1012050
1012069
hotspot
AGTCCCAGAGATCCTACCTG
592477
117 
52


 


1012220
1012239
hotspot
GTGTGTGCAAATGGAGGCTA
592478
55
53


 


1012404
1012423
hotspot
ATGGTAGTTGTAATTTTCGG
592479
93
54


 


1012579
1012598
hotspot
AAATGTGATCATTTTACTCT
592480
50
55


 


1012751
1012770
hotspot
GTTCTACTTTAATCACATTC
592481
114 
56


 


1012921
1012940
hotspot
GTAGTTGTCTTCAGACAAAT
592482
215 
57


 


1013122
1013141
hotspot
TGTTTACATCCCCAAATCTG
592483
191 
58


 


1013292
1013311
hotspot
GTGTAATGTGTGGCTGACTT
592484
153 
59


 


1013488
1013507
hotspot
GTGTATATGCTAGCTTGTGC
592485
132 
60


 


1013658
1013677
hotspot
TACCCATTCCTTGATCAATT
592486
138 
61


 


1013857
1013876
hotspot
TGTAGATAAAGAAATTATCT
592487
 0
62


 


1014197
1014216
hotspot
AGCCAACATTGTTTAACCAG
592488
180 
63


 


1014367
1014386
hotspot
TTGCCAGGCTTGTGCCAGAT
592489
196 
64


 


1014538
1014557
hotspot
AATAATATCTACTTGTGTCC
592490
80
65


 


1014708
1014727
hotspot
ATCCAATTTCTAGTTGAATC
592491
141 
66


 


1014878
1014897
hotspot
TACTTAAGTGATGTCACTGA
592492
132 
67


 


1015048
1015067
hotspot
TAATCTTGTGATATGGTATG
592493
101 
68


 


1015218
1015237
hotspot
GAGTGAAAATCAGTCTAACT
592494
76
69


 


1015546
1015565
hotspot
GAGATTTTAGGATGTGTTTC
592495
40
70


 


1015716
1015735
hotspot
ACACTACTTTAGAGTGCACA
592496
181 
71


 


1015911
1015930
hotspot
ATTCTTTAAAATGTGAACTG
592497
28
72


 


1016081
1016100
hotspot
ATATCATCAAGGAAAATGTT
592498
14
73


 


1016262
1016281
hotspot
ATTAAATCCTAGGTTTGCTC
592499
68
74


 


1016432
1016451
hotspot
CTGCCAACCAAGAATTACTA
592500
158 
75


 


1016602
1016621
hotspot
AAGACATGGCTACAGACAAC
592501
177 
76


 


1016772
1016791
hotspot
GAGGAAACAGTTGTTATGAC
592502
53
77


 


1017123
1017142
hotspot
CAGCACCCAAGGAGCTGAAG
592504
52
78


 


1017650
1017669
hotspot
TTTAAGAGGCTGAAGAATTA
592507
42
79






















TABLE 2





Target
Target



%
SEQ


Start Site
Stop Site
Region
Sequence
ISIS No
upregulation
ID NO







1017820
1017839
hotspot
GGAAGTTTGTGGCATTGGTG
592508
95
 80


 


1018002
1018021
hotspot
TGATTGAGGATTCAAAACAA
592509
65
 81


 


1018172
1018191
hotspot
TTATTACAAGGGTCAAAAGA
592510
38
 82


 


1018342
1018361
hotspot
GTGCTAGCCAGTGTAGAGTT
592511
115 
 83


 


1018516
1018535
hotspot
TGCCAGATGGAATTGGTCTG
592512
66
 84


 


1018687
1018706
hotspot
CTAGTGAATGAATTGGATAA
592513
42
 85


 


1019053
1019072
hotspot
CAAACAAAAGCTTTCCACAT
592515
159 
 86


 


1019228
1019247
hotspot
CAAAGATGAAATATATTGGT
592516
44
 87


 


1019398
1019417
hotspot
CGAGTCCAACAATGAACCAA
592517
191 
 88


 


1019568
1019587
hotspot
CTCAATCTTCACAAAATGTT
592518
105 
 89


 


1019738
1019757
hotspot
ATTAAAATTAAACTGATCTC
592519
11
 90


 


1019929
1019948
hotspot
GTAGACAATAGTTCGAATAA
592520
52
 91


 


1020102
1020121
hotspot
ACCCTACCTCAACAAAGAGC
592521
78
 92


 


1020285
1020304
hotspot
AACAAGGAATATTATGTGGG
592522
61
 93


 


1020480
1020499
hotspot
TTTACATATACGAGCAATAA
592523
15
 94


 


1020650
1020669
hotspot
ACGAGCCAAAATGTGACATT
592524
60
 95


 


1020820
1020839
hotspot
TGTTGGAACTTGAATTACTA
592525
123 
 96


 


1021024
1021043
hotspot
GTGATCCTAAGTACTGAATC
592526
27
 97


 


1021212
1021231
hotspot
GAAGCCTCTTGCAATTAATC
592527
151 
 98


 


1021382
1021401
hotspot
AAGGAACAATTTCGACTCAG
592528
184 
 99


 


1021567
1021586
hotspot
TTTTGAGAAATCACTACTAC
592529
68
100


 


1021749
1021768
hotspot
AATGATTGGATAGCTTGTTT
592530
44
101


 


1021930
1021949
hotspot
CTGGAAAAAGTGCAATTTTT
592531
58
102


 


1022276
1022295
hotspot
CAACAGTGAAATGACAATTT
592532
28
103


 


1022446
1022465
hotspot
GGAAGAATCTAGAACCTTTC
592533
94
104


 


1022791
1022810
hotspot
TGCTCAATTTAAATAAAAGC
592535
22
105


 


1022961
1022980
hotspot
GAGACAGTGATTCTCATACA
592536
52
106


 


1023136
1023155
hotspot
TGTGGCTTTAGAATAAGCTG
592537
54
107


 


1023311
1023330
hotspot
GCATGTGTAAATCAAAGCTC
592538
66
108


 


1023510
1023529
hotspot
ACACTGGTACTATAATTTTT
592539
61
109


 


1023851
1023870
hotspot
GTCTTTTAACTATTAATAAA
592540
19
110


 


1024021
1024040
hotspot
CCCCGAAAATTTCTGCCACT
592541
135 
111


 


1024192
1024211
hotspot
ACTATGTGTGTGCACGCACG
592542
24
112


 


1024989
1025008
hotspot
GGTATGAACTCAGTTTTCTT
592543
110 
113


 


1025159
1025178
hotspot
TGTTTCTACATAACCAACTC
592544
100 
114


 


1025339
1025358
hotspot
AAATCCATCATGTTTTTATA
592545
32
115


 


1025509
1025528
hotspot
GTGTATGAAGACTCATCCTG
592546
114 
116


 


1025708
1025727
hotspot
GGAGTGGTTTCAACTTTTCT
592547
133 
117


 


1025898
1025917
hotspot
GTTTCCAAAACTCTTGCATC
592548
120 
118


 


1026088
1026107
hotspot
TGAGCTACATCCCTATCCCC
592549
118 
119


 


1026258
1026277
hotspot
ATAATTGAAATATTAACTCT
592550
 3
120


 


1026437
1026456
hotspot
ACCATACAATGAGCAGACTG
592551
108 
121


 


1026607
1026626
hotspot
AATTACTATCCTTTGAGAGG
592552
22
122


 


1026777
1026796
hotspot
TAAGTCACATTCTTTGTGTA
592553
29
123


 


1026947
1026966
hotspot
GATTGTTGCTATAGTTGCAG
592554
62
124


 


1027117
1027136
hotspot
TTGGGTTGAGATAAGTAGCT
592555
84
125


 


1027287
1027306
hotspot
GTTTAAAATGAACGACTTGT
592556
93
126


 


1027459
1027478
hotspot
TAGGAGGACCTGAGCAGGAG
592557
75
127


 


1027629
1027648
hotspot
CCAAGAAGGTAAATTCTGAA
592558
88
128


 


1027799
1027818
hotspot
GCCATACATATATCATTATT
592559
118 
129


 


1027971
1027990
hotspot
TTGAAATAATATACTTTGAA
592560
 3
130


 


1028485
1028504
hotspot
ATTGAAGTGGCAAGAGTGTA
592563
51
131


 


1028655
1028674
hotspot
TGTATTAACCTTATATACTG
592564
36
132


 


1029011
1029030
hotspot
CATCAAGATAATTTTACCTG
592566
23
133


 


1029188
1029207
hotspot
CTATCTTTGTCAAAAACACG
592567
70
134


 


1029389
1029408
hotspot
AACACTTTGTGTACATGTTT
592568
40
135


 


1029559
1029578
hotspot
TCCACAAAGCAATGAGTTCA
592569
85
136


 


1029736
1029755
hotspot
TCCAGAGCATTTCATCGCTG
592570
133 
137


 


1029908
1029927
hotspot
AAGACTTATTTTCTAAAACC
592571
 6
138


 


1030078
1030097
hotspot
TCCCTTGACTTGGATTATAC
592572
93
139


 


1030248
1030267
hotspot
AAATTAAGATTATTATGCTG
592573
 7
140


 


1030418
1030437
hotspot
CCTTCATTTTCACAAAGCCA
592574
150 
141


 


1030607
1030626
hotspot
GCAACTGATACCTTAATTCG
592575
93
142


 


1030777
1030796
hotspot
GAATAGAGTTTCTGGAATCA
592576
102 
143


 


1030986
1031005
hotspot
AGGAGGCCTTCAGAAGTGCG
592577
101 
144


 


1031156
1031175
hotspot
AAAATTCCTTTTTAGATTAA
592578
17
145


 


1031326
1031345
hotspot
ATTTACTCAGGTTATCTTCC
592579
81
146


 


1031505
1031524
hotspot
TCCACATAAACATGGAAGGA
592580
26
147


 


1031845
1031864
hotspot
ATCCCAACTCATAGACACCT
592582
134 
148


 


1032185
1032204
hotspot
TAGTAACTTCCATTTGCTGC
592583
118 
149


 


1032355
1032374
hotspot
AATCAACCTCTTTTAAAGAA
592584
 9
150


 


1032525
1032544
hotspot
AATTCATTTGCAATAACCTT
592585
41
151


 


1032695
1032714
hotspot
GATTGTCATGGAACAATACT
592586
64
152


 


1032873
1032892
hotspot
TTCAAATGATAATATAGATT
592587
10
153






















TABLE 3





Target
Target



%
SEQ


Start Site
Stop Site
Region
Sequence
ISIS No
upregulation
ID NO







 997638
 997657
hotspot
GAAATTCCCAAGAGTAGAAT
592398
 0
154


 


 997810
 997829
hotspot
GGCTCAGAATTGAAACCAAA
592399
142 
155


 


 997998
 998017
hotspot
ATACTAAAAATGTCATCTTC
592400
19
156


 


 998168
 998187
hotspot
CCAGCCTTGTTGGATATCAT
592401
147 
157


 


 998338
 998357
hotspot
TGAGGTTCAGTAAGAGCCCC
592402
178 
158


 


 998518
 998537
hotspot
GATCCATTTGTGTTAAGCTG
592403
111 
159


 


 998688
 998707
hotspot
AGGTATTTCGAGTGTGATTA
592404
224 
160


 


 998870
 998889
hotspot
TACCATAGAGAAACCTAATT
592405
83
161


 


 999040
 999059
hotspot
TGGGACTTAATGACCTTGGA
592406
151 
162


 


 999213
 999232
hotspot
GTCCCAGAAAAGAATCTCTC
592407
145 
163


 


 999393
 999412
hotspot
TCAGTCCAGCTCTTTAGTTC
592408
199 
164


 


 999733
 999752
hotspot
TTGGATCCTTAAAATTTTAG
592409
30
165


 


 999903
 999922
hotspot
GAATTTATTATTGCATGGTG
592410
122 
166


 


1000100
1000119
hotspot
GCATGAAATTGTCAAAGAAC
592411
104 
167


 


1000270
1000289
hotspot
AATGGATAATTCTGAAGTCT
592412
46
168


 


1000440
1000459
hotspot
AGCTCATAGTACCAGTGGCT
592413
145 
169


 


1000626
1000645
hotspot
CATGGTCATGAAAACAAGTA
592414
100 
170


 


1000796
1000815
hotspot
TAGTGACATAGTTTTATGGT
592415
62
171


 


1000971
1000990
hotspot
GTATACCATTCATATTTCTA
592416
188 
172


 


1001141
1001160
hotspot
AAGAAACACTGAGAGCCTGA
592417
142 
173


 


1001313
1001332
hotspot
ATTAGGCTTATTGATGTCTG
592418
127 
174


 


1001483
1001502
hotspot
AATACAATATTATTGCCATG
592419
36
175


 


1001830
1001849
hotspot
GATTAGGTGTGCTTTCAGAC
592421
157 
176


 


1002002
1002021
hotspot
CGGGTTAGAGGAAGAATTAT
592422
15
177


 


1002172
1002191
hotspot
TATAAGCCACAAACATCTTG
592423
148 
178


 


1002355
1002374
hotspot
TCATTGTGACAGTAAAGAAA
592424
106 
179


 


1002525
1002544
hotspot
TAACTGGGTTAATTTACTAC
592425
49
180


 


1002695
1002714
hotspot
TATGTAGGAACATTTTTAGT
592426
43
181


 


1002894
1002913
hotspot
ACCAGTTGATCATGTTTTAT
592427
95
182


 


1033418
1033437
antisense
TACAAGCTACAGATAACCTG
592348
32
183




to Ube3a








pre-mRNA






 


1037625
1037644
antisense
TGAGAGACATTTGTCTCTGG
592351
30
184




to Ube3a








pre-mRNA






 


1039051
1039070
antisense
ATTTCTACATGGTCATTCCT
592352
59
185




to Ube3a








pre-mRNA






 


1046085
1046104
antisense
AGATGTCTATACTAAGAAAC
592356
 2
186




to Ube3a








pre-mRNA






 


1047501
1047520
antisense
CAACTTAATTGCTTTTTGAA
592357
16
187




pre-mRNA








antisense






 


1050351
1050370
antisense
GATACATTATCATTGTTATA
592358
10
188




to Ube3a








pre-mRNA






 


1051751
1051770
antisense
TCTGGTTTTCTCAAGTTCAG
592359
49
189




to Ube3a








pre-mRNA






 


1053151
1053170
antisense
ACAGTTGATATGTGTGTGGC
592360
39
190




to Ube3a








pre-mRNA






 


1054551
1054570
antisense
GCTTAATTGTCCTTGAGACC
592361
41
191




to Ube3a








pre-mRNA






 


1055951
1055970
antisense
AGTGTCAGACCTACCTATTA
592362
36
192




to Ube3a








pre-mRNA






 


1057351
1057370
antisense
GGCGGGTTGTATTTTGAGAG
592363
 0
193




to Ube3a








pre-mRNA






 


1058771
1058790
antisense
GCAACAAGAACTTGATTTAA
592364
35
194




to Ube3a








pre-mRNA






 


1060171
1060190
antisense
GTAGGCGAGTAAATTAGAAT
592365
20
195




to Ube3a








pre-mRNA






 


1062997
1063016
antisense
ACTAGAAACCGAACTTGGCG
592366
31
196




to Ube3a








pre-mRNA






 


1064397
1064416
antisense
GGAATACATAAGATGAGTCA
592367
28
197




to Ube3a








pre-mRNA






 


1065809
1065828
antisense
AAAAAGTTTCTTTACTTAAC
592368
 0
198




to Ube3a








pre-mRNA






 


1067235
1067254
antisense
AGGACTGGTATTTAGTTTGT
592369
14
199




to Ube3a








pre-mRNA






 


1068635
1068654
antisense
CATGAAGTTTTAGAATGAAA
592370
 0
200




pre-mRNA








antisense






 


1070036
1070055
antisense
GCATATCCATTTTCAATAAA
592371
11
201




to Ube3a








pre-mRNA






 


1071436
1071455
antisense
AAAAAGGCATATTTTTATTT
592372
 0
202




to Ube3a








pre-mRNA






 


1072836
1072855
antisense
TACTTTCAGTCTTGTAATAC
592373
20
203




to Ube3a








pre-mRNA






 


1074250
1074269
antisense
GATTTCCAATATTCTTCATC
592374
10
204




to Ube3a








pre-mRNA






 


1075650
1075669
antisense
CCAAACAATTTTCAGATATA
592375
 1
205




to Ube3a








pre-mRNA






 


1077065
1077084
antisense
GGTATTTTCCTCAATTACAT
592376
23
206




to Ube3a








pre-mRNA






 


1078467
1078486
antisense
GCCACCATATGGTTGCTGGG
592377
147 
207




to Ube3a








pre-mRNA






 


1079867
1079886
antisense
CAGGCATAAGAGTCAGAAGC
592378
24
208




to Ube3a








pre-mRNA






 


1081267
1081286
antisense
TTGAGGAAGAGGGCTAATTT
592379
 2
209




to Ube3a








pre-mRNA






 


1084067
1084086
antisense
GTTTTCCAGTGAGTACCAGC
592380
 4
210




to Ube3a








pre-mRNA






 


1085491
1085510
antisense
TAACTAGAGGAATACCAACT
592381
 2
211




to Ube3a








pre-mRNA






 


1086900
1086919
antisense
GAAAGCATCCTTAGTTTCTC
592382
 9
212




to Ube3a








pre-mRNA






 


1088316
1088335
antisense
TTGCAGCATTAATTAAACAA
592383
 9
213




to Ube3a








pre-mRNA






 


1091119
1091138
antisense
GGCAATTAAATTCTACTTTT
592384
 1
214




to Ube3a








pre-mRNA






 


1093920
1093939
antisense
CTGGAGTTTGTGATGGTTGT
592386
 9
215




to Ube3a








pre-mRNA






 


1095320
1095339
antisense
AGGATATATTTTGGCAACTT
592387
 0
216




to Ube3a








pre-mRNA






 


1096720
1096739
antisense
TTTATCAGAAATGCCTGGGA
592388
 1
217




to Ube3a








pre-mRNA






 


1099541
1099560
antisense
GTCCATCTATCAATTTATTA
592389
22
218




to Ube3a








pre-mRNA






 


1100945
1100964
antisense
TTTAAATAGCTGATTATCTG
592390
 4
219




to Ube3a








pre-mRNA






 


1102346
1102365
antisense
CAAGAGATAATAGCTTAATT
592391
 6
220




to Ube3a








pre-mRNA






 


1103750
1103769
antisense
ATTAGTTGATACCACTCTTC
592392
23
221




to Ube3a








pre-mRNA






 


1105150
1105169
antisense
AGTGAAACCAGAGTAGTAGT
592393
 3
222




to Ube3a








pre-mRNA






 


1106554
1106573
antisense
CATGTGGTATATATAAAATG
592394
 0
223




to Ube3a








pre-mRNA






 


1107954
1107973
antisense
AGCCTAGACTAAGAGGCGAG
592395
 0
224




to Ube3a








pre-mRNA






 


1109354
1109373
antisense
GCTAAATATCAAAGCCCTAT
592396
 8
225




to Ube3a








pre-mRNA









Example 2
Antisense Inhibition of the Antisense Sequence of Ubiquitin Protein Ligase E3A (Ube3a-ATS) in Primary Neuronal Cells
Antisense oligonucleotides selected from the study described above were tested for their effects on inhibiting the expression of Ube3a-ATS mRNA in vitro.
Primary neuronal cultures from Ube3a-YFP paternal+/− mice were prepared, as described above. Cells were seeded on poly-D-lysine-coated 24-well plates at a density of 2.5×105 cells/cm2 in 500 μL of culture medium. At day 4, 400 μL medium was removed from sets of wells and 100 μL of fresh medium, along with 10 μM antisense oligonucleotide and 2 μM Ara-C were added. A set of cells treated with PBS was used as the untreated control. At day 7, the sets were washed with PBS twice and RNA isolation was conducted.
Total RNA was prepared with miRNAeasy Mini Kit (Qiagen, Valencia, Calif.). On-column DNase treatment was performed for all the samples. mRNA was purified from total RNA with oligo(dT) beads supplied in Illumina mRNA-seq Sample Preparation Kit, according to the manufacturer's instructions (Illumina, San Diego, Calif.). cDNA was then generated with SuperScript III First-Strand Synthesis System (Invitrogen) and q-PCR was performed using Applied Biosystems StepOnePlus Real-Time PCR System and SYBR Green Master Mix (Applied Biosystems, Carlsbad, Calif.).
The results are presented in Table 4, expressed as a percentage of the untreated control. The data indicates that treatment with antisense oligonucleotides targeted to the hotspot region that we identified results in marked inhibition of the Ube3a-YFP-ATS mRNA transcript and correspondingly induces expression of the paternal Ube3a-YFP transcript.







TABLE 4







% expression of mRNA transcript expression relative to the untreated control










% inhibition of
% induction of



Ube3a-YFP-ATS
Ube3a-YFP





ISIS 592467
88 ± 2
 71 ± 23


ISIS 592517
94 ± 5
128 ± 5 


ISIS 592528
81 ± 9
288 ± 71


ISIS 592404
94 ± 4
137 ± 12









Example 3
Dose-Dependent Antisense Inhibition of Ube3a-ATS and Up-Regulation of Paternal Ube3a in Primary Neuronal Cells
Antisense oligonucleotides selected from the study described above were tested for their effects on inhibiting the expression of Ube3a-ATS mRNA and up-regulating the expression of paternal Ube3a in vitro. The effect on snoRNA genes near the hotspot region and some of which have been shown to be involved in Prader-Willi syndrome (Sahoo, T. et al., Nat. Genet. 2008. 40: 719-21; Tsai, T. et al., Hum. Mol. Genet. 1999. 8: 1357-64) was also assessed.
Primary neuronal cultures from Ube3a-YFP paternal+/− mice were prepared, as described above, and transfected with 39 nM, 156 nM, 625 nM, 2,500 nM, or 10,000 nM concentrations of antisense oligonucleotide. The cells were separately treated with the topoisomerase inhibitor, Topotecan, at 1.17 nM, 4.69 nM, 18.75 nM, 75.00 nM, or 300.00 nM concentrations. Total RNA was prepared with RNeasy 96 Kit (Qiagen). On-column DNase treatment was performed for all the samples. qRT-PCR was performed using EXPRESS One-Step SuperScript qRT-PCR Kit (Qiagen) and EXPRESS One-Step SYBR GreenER Kit (Qiagen).
The results are presented in Tables 5 and 6, expressed as a percentage of the untreated control. The data indicates that treatment with antisense oligonucleotides targeted to the hotspot region that we identified resulted in marked inhibition of the Ube3a-YFP-ATS transcript and correspondingly induced expression of the paternal Ube3a-YFP transcript. Treatment with Topotecan also inhibited Ube3a-YFP-ATS transcript levels and induced the expression of Ube3a-YFP mRNA as expected. Several of ISIS antisense oligonucleotides inhibited Ube3a-YFP-ATS and up-regulated Ube3a-YFP mRNA levels.
The cells were also assessed for the induction of Ube3a-YFP protein, as quantified by fluorescence, using the method described in Example 1. The results are presented in Table 7, and demonstrate that treatment with antisense oligonucleotides targeted to the hotspot region that we identified resulted in an increase in Ube3a-YFP protein levels, as indicated by increased YFP fluorescence. Treatment with Topotecan also induced the expression of Ube3a-YFP protein levels, as expected. The expression levels of the MBII-85 and MBII-52 snoRNAs and Snrpn were also evaluated. The results are presented in Tables 8-10. The data indicates that treatment with antisense oligonucleotides targeted to the hotspot region resulted in minimal reduction in these snoRNA genes, the reduction of which is associated with Prader-Willi syndrome. By contrast, treatment with Topotecan reduced the levels of the snoRNA gene MBII-52, and Snrpn levels.







TABLE 5







% inhibition of Ube3a-YFP-ATS transcript expression relative to the untreated control












ISIS No
39 nM
156 nM
625 nM
2,500 nM
10,000 nM















592401
0
0
40
68
91


592404
32
50
78
93
96


592408
29
40
49
67
75


592413
34
43
63
75
85


592482
0
16
55
86
94


592483
19
49
62
91
96


592489
0
33
62
85
93


592515
6
37
69
87
91


592517
12
66
71
89
96


592528
0
59
77
92
93


Small
1.17 nM
4.69 nM
18.75 nM
75.00 nM
300.00 nM


molecule







inhibitor







Topotecan
23
33
50
73
88
















TABLE 6







% Ube3a-YFP transcript expression relative to the untreated control












ISIS No
39 nM
156 nM
625 nM
2,500 nM
10,000 nM















592401
103
183
305
753
955


592404
151
260
499
890
810


592408
89
97
189
240
616


592413
136
145
254
556
764


592482
142
167
372
697
1277


592483
150
163
237
607
852


592489
149
188
215
514
646


592515
145
136
192
467
705


592517
151
189
237
528
821


592528
215
268
378
715
639


Small
1.17 nM
4.69 nM
18.75 nM
75.00 nM
300.00 nM


molecule 







inhibitor







Topotecan 
124
127
193
430
734
















TABLE 7







% Ube3a-YFP protein expression relative to the untreated control












ISIS No
39 nM
156 nM
625 nM
2,500 nM
10,000 nM















592401
95
102
109
154
193


592404
95
108
153
183
262


592408
100
96
105
134
173


592413
107
104
109
126
185


592482
95
110
130
135
201


592483
94
109
97
148
202


592489
102
105
104
132
177


592515
101
112
104
134
177


592517
97
104
121
137
211


592528
102
121
117
146
207


Small
1.17 nM
4.69 nM
18.75 nM 
75.00 nM 
300.00 nM


molecule







inhibitor







Topotecan
102
112
139
217
290
















TABLE 8







% MBII-85 transcript expression relative to the untreated control












ISIS No
39 nM
156 nM
625 nM
2,500 nM
10,000 nM















592401
96
114
118
122
125


592404
108
79
87
101
93


592408
83
66
81
75
81


592413
74
72
68
79
85


592482
121
91
81
106
88


592483
84
84
91
97
102


592489
74
78
100
94
69


592515
66
108
93
88
118


592517
54
73
94
74
98


592528
81
98
119
139
115


Small
1.17 nM
4.69 nM 
18.75 nM
75.00 nM
300.00 nM


molecule







inhibitor







Topotecan 
93
84
79
67
85
















TABLE 9







% MBII-52 transcript expression relative to the untreated control












ISIS No
39 nM
156 nM
625 nM
2,500 nM
10,000 nM















592401
98
138
106
112
86


592404
91
66
73
63
31


592408
94
62
97
89
75


592413
80
83
92
104
82


592482
107
94
87
95
63


592483
104
102
103
100
97


592489
106
98
101
99
87


592515
100
103
104
104
120


592517
109
95
106
95
92


592528
106
119
143
131
111


Small
1.17 nM
4.69 nM
18.75 nM
75.00 nM
300.00 nM


molecule







inhibitor







Topotecan
112
92
81
50
40
















TABLE 10







% Snrpn transcript expression relative to the untreated control












ISIS No
39 nM
156 nM
625 nM
2,500 nM
10,000 nM















592401
93
127
107
133
114


592404
76
88
99
102
92


592408
75
81
94
80
85


592413
83
74
78
88
78


592482
126
105
105
74
46


592483
115
115
99
108
103


592489
125
12
98
87
72


592515
101
114
101
95
103


592517
99
109
99
71
45


592528
122
108
109
114
111


Small
1.17 nM
4.69 nM 
18.75 nM
75.00 nM
300.00 nM


molecule







inhibitor







Topotecan 
102
99
87
71
54









Example 4
Comparison of the Effect of ASOs Targeting the Hotspot Region and ASOs Targeting within the Region Antisense to the Ube3a Pre-mRNA on the Up-Regulation of Paternal Ube3a Transcript in Primary Neuronal Cells
To evaluate the optimal region for targeting with antisense oligonucleotides resulting in up-regulation of paternal Ube3a gene transcript, treatment with ASOs targeting the hotspot region and treatment with ASOs targeting within the region antisense to the Ube3a pre-mRNA was assessed.
Primary neuronal cultures from Ube3a-YFP paternal+/− mice were prepared, as described above, and transfected with 15 μM concentration of antisense oligonucleotide. Total RNA was prepared with RNeasy 96 Kit (Qiagen). On-column DNase treatment was performed for all the samples. qRT-PCR was performed using EXPRESS One-Step SuperScript qRT-PCR Kit (Qiagen) and EXPRESS One-Step SYBR GreenER Kit (Qiagen). The results are presented in Table 10, expressed as a percentage of the untreated control.
The data indicates that treatment with antisense oligonucleotides targeting both regions resulted in marked inhibition of the Ube3a-YFP-ATS mRNA transcript. However, treatment with ASOs targeting the hotspot region resulted in markedly higher upregulation of paternal Ube3a-YFP transcript expression compared to treatment with ASOs targeting the region antisense to the Ube3a pre-mRNA.







TABLE 11







% expression levels compared to the untreated control

















% 
%



Target
Target


expression
expression
SEQ


Start
Stop

ISIS
of Ube3a-
of Ube3Pa-
ID


Site
Site
Region
No
YFP-ATS
YFP
NO
















997810
997829
hotspot
592399
4
624
155


997998
998017
hotspot
592400
7
259
156


998168
998187
hotspot
592401
3
936
157


998688
998707
hotspot
592404
7
1139
160


998870
998889
hotspot
592405
9
518
161


999040
999059
hotspot
592406
7
644
162


999213
999232
hotspot
592407
10
555
163


999393
999412
hotspot
592408
6
670
164


1000270
1000289
hotspot
592412
14
827
168


1000440
1000459
hotspot
592413
16
576
169


1000971
1000990
hotspot
592416
10
802
172


1001141
1001160
hotspot
592417
15
935
173


1001830
1001849
hotspot
592421
12
1042
176


1002172
1002191
hotspot
592423
13
624
178


1002355
1002374
hotspot
592424
20
871
179


1033418
1033437
hotspot
592348
9
704
183


1046085
1046104
antisense to
592356
15
288
186




Ube3a pre-








mRNA






1057351
1057370
antisense to
592363
9
158
193




Ube3a pre- 








mRNA






1058771
1058790 
antisense to
592364
16
315
194




Ube3a pre-








mRNA






1062997
1063016
antisense to
592366
18
163
196




Ube3a pre-








mRNA






1072836 
1072855 
antisense to
592373
20
131
203




Ube3a pre- 








mRNA






1078467
1078486
antisense to
592377
7
435
207




Ube3a pre- 








mRNA






1085491 
1085510 
antisense to
592381
16
158
211




Ube3a pre- 








mRNA






1086900 
1086919
antisense to
592382
16
94
212




Ube3a pre-








mRNA






1099541
1099560
antisense to
592389
12
296
218




Ube3a pre-








mRNA






1100945 
1100964
antisense to
592390
13
111
219




Ube3a pre- 








mRNA






1102346 
1102365
antisense to
592391
15
88
220




Ube3a pre-








mRNA






1103750
1103769 
antisense to
592392
4
100
221




Ube3a pre- 








mRNA






1109354
1109373
antisense to
592396
18
182
225




Ube3a pre- 








mRNA









Example 5
Antisense Inhibition of the Antisense Sequence of Ube3a-ATS In Vivo
Antisense oligonucleotides, selected from the in vitro screening study described above, were screened for in vivo efficacy in wild-type C57BL6 mice.
Antisense oligonucleotide administration was accomplished by intracerebroventricular bolus injection of 300 μg in the mice. The mice were euthanized 4 weeks after the administration and the level of Ube3a-ATS mRNA down-regulation was assessed using sections from the cortex, hippocampus, and spinal cord. RNA isolation and qRT-PCR was performed as described in Example 4. The expression of MBII-85, MBII-52, and Snrpn were also evaluated.
The data is presented in Tables 12-15, normalized to the house-keeping gene, GAPDH. The results indicate that treatment with antisense oligonucleotides targeted to the hotspot region that we identified resulted in inhibition of the Ube3a-ATS mRNA transcript. Moreover, MBII-85, MBII-52, and Snrpn expression was maintained following knockdown of Ube3a-ATS.







TABLE 12







% expression of Ube3a-ATS transcript 


expression relative to the untreated control












ISIS No
Cortex
Hippocampus
Spinal Cord







592401
35
44
48



592408
50
43
45



592413
38
45
67



592482
30
31
32



592489
28
36
52



592515
67
66
79



592517
39
43
44

















TABLE 13







% expression of MBII-85 transcript 


expression relative to the untreated control












ISIS No
Cortex
Hippocampus
Spinal Cord
















592401
82
104
98



592408
99
96
112



592413
121
99
90



592482
85
74
99



592489
98
105
99



592515
101
100
92



592517
78
82
94

















TABLE 14







% expression of MBII-52 transcript 


expression relative to the untreated control












ISIS No
Cortex
Hippocampus
Spinal Cord
















592401
90
103
92



592408
95
85
115



592413
103
103
91



592482
81
91
103



592489
135
118
124



592515
112
140
97



592517
92
103
98

















TABLE 15







% expression of Snrpn transcript 


expression relative to the untreated control












ISIS No
Cortex
Hippocampus
Spinal Cord







592401
92
90
92



592408
96
90
84



592413
97
97
80



592482
85
74
46



592489
96
88
74



592515
95
93
82



592517
88
87
49










Example 6
Antisense Inhibition of Ube3a-ATS in Ube3a-YFP Mice
Antisense oligonucleotides selected from the studies described above were screened for in vivo efficacy and tolerability in the Ube3a-YFP mouse model (Dindot, S. V. et al., Hum. Mol. Genet. 2008. 17: 111-118).
Antisense oligonucleotide was administered to the mice by intracerebroventricular bolus injection of 300 μg of ISIS 592401 or ISIS 592489. The mice were euthanized after 4 weeks of administration. Levels of Ube3a-YFP-ATS mRNA and Ube3a-YFP mRNA were assessed in the cortex, hippocampus and spinal cord. The assays were performed as described previously (Meng, L. et al., Hum. Mol. Genet. 2012. doi:10.1093/hmg/dds1).
RNA was isolated from the different brain sections and analyzed by qRT-PCR. The results are presented in Tables 16 and 17, normalized to GAPDH. The data indicates that ASO administration resulted in approximately 50-65% knockdown of Ube3a-YFP-ATS transcript in the brain and spinal cord. Correspondingly, there was an approximate 1.5-2-fold increase of Ube3a-YFP mRNA in ASO-treated mice. As shown in FIG. 1, Western Blot analysis was performed using an anti-YFP antibody and confirmed that treatment with ASO increased paternal Ube3a protein expression.
The expression of MBII-85, MBII-52, and Snrpn were also evaluated. As presented in Tables 18-20, MBII-85, MBII-52, and Snrpn expression was maintained following knockdown of Ube3a-YFP-ATS. Levels of the microglial marker AIF1 were also assessed as a measure of CNS toxicity. As presented in Table 21, there was negligible increase in Aif1 levels, indicating that ASO treatment was neurotolerable.







TABLE 16







% inhibition of Ube3a-YFP-ATS mRNA transcript


expression relative to untreated control













Cortex
Hippocampus
Spinal cord







ISIS 592401
48 ± 9 
59 ± 9
63 ± 12



ISIS 592489
59 ± 16
65 ± 5
64 ± 7 

















TABLE 17







% Ube3a-YFP mRNA transcript expression 


relative to untreated control (denoted as 100%)













Cortex
Hippocampus
Spinal cord







ISIS 592401
169 ± 7 
198 ± 22
176 ± 72



ISIS 592489
140 ± 31
156 ± 22
142 ± 22

















TABLE 18







% MBII-85 mRNA transcript expression 


relative to untreated control (denoted as 100%)













Cortex
Hippocampus
Spinal cord







ISIS 592401
107 ± 13
114 ± 3
108 ± 11



ISIS 592489
 73 ± 25
102 ± 5
109 ± 16

















TABLE 19







% MBII-52 mRNA transcript expression 


relative to untreated control (denoted as 100%)













Cortex
Hippocampus
Spinal cord







ISIS 592401
75 ± 1 
 99 ± 0
83 ± 13



ISIS 592489
95 ± 33
109 ± 9
91 ± 13

















TABLE 20







% Snrpn mRNA transcript expression 


relative to untreated control (denoted as 100%)













Cortex
Hippocampus
Spinal cord







ISIS 592401
103 ± 3 
95 ± 4
91 ± 7



ISIS 592489
 94 ± 12
90 ± 6
92 ± 5

















TABLE 21







% Aif1 mRNA transcript expression 


relative to untreated control (denoted as 100%)













Cortex
Hippocampus
Spinal cord







ISIS 592401
94 ± 4 
106 ± 7 
111 ± 5 



ISIS 592489
92 ± 11
112 ± 21
182 ± 30










Example 7
Antisense Inhibition of Ube3a-ATS in Ube3a-YFP Mice
Antisense oligonucleotides selected from the studies described above were screened for in vivo efficacy and tolerability in the Ube3a-YFP mouse model (Dindot, S. V. et al., Hum. Mol. Genet. 2008. 17: 111-118).
Antisense oligonucleotide was administered to the mice by intracerebroventricular bolus injection of 500 μg of ISIS 592401. The mice were euthanized after 4 weeks of administration. Levels of Ube3a-YFP-ATS mRNA, Ube3a-YFP mRNA and Ube3a protein were assessed in the cortex, hippocampus and spinal cord. The assays were performed as described previously (Meng, L. et al., Hum. Mol. Genet. 2012. doi:10.1093/hmg/dds1).
RNA was isolated from the different brain sections and analyzed by qRT-PCR. The results are presented in Tables 22 and 23, normalized to GAPDH. The data indicates that ASO administration resulted in approximately 65-85% knockdown of Ube3a-YFP-ATS transcript in the brain and spinal cord. Correspondingly, there was an approximate 2.5 to 4.5-fold increase of Ube3a-YFP mRNA in ASO-treated mice. As shown in FIG. 2, Western Blot analysis was performed using an anti-YFP antibody and confirmed that treatment with ASO increased paternal Ube3a protein expression.
The expression of MBII-85, MBII-52, and Snrpn were also evaluated. As presented in Tables 24-26, MBII-85, MBII-52, and Snrpn expression was minimally affected following knockdown of Ube3a-YFP-ATS. Levels of the microglial marker AIF1 were also assessed as a measure of CNS toxicity. As presented in Table 27, there was negligible increase in Aif1 levels, indicating that ASO treatment was neurotolerable.







TABLE 22







% inhibition of Ube3a-YFP-ATS mRNA transcript 


expression relative to untreated control













Cortex
Hippocampus
Spinal cord







ISIS 592401
84 ± 6
66 ± 4
69 ± 1

















TABLE 23







% Ube3a-YFP mRNA transcript expression 


relative to untreated control (denoted as 100%)













Cortex
Hippocampus
Spinal cord







ISIS 592401
445 ± 81
263 ± 8
284 ± 25

















TABLE 24







% MBII-85 mRNA transcript expression 


relative to untreated control (denoted as 100%)













Cortex
Hippocampus
Spinal cord







ISIS 592401
65 ± 183
98 ± 12
98 ± 7

















TABLE 25







% MBII-52 mRNA transcript expression 


relative to untreated control (denoted as 100%)













Cortex
Hippocampus
Spinal cord







ISIS 592401
72 ± 17
92 ± 8
89 ± 10

















TABLE 26







% Snrpn mRNA transcript expression 


relative to untreated control (denoted as 100%)













Cortex
Hippocampus
Spinal cord







ISIS 592401
102 ± 11
101 ± 3
95 ± 2

















TABLE 27







% Aif1 mRNA transcript expression 


relative to untreated control (denoted as 100%)













Cortex
Hippocampus
Spinal cord







ISIS 592401
104 ± 13
80 ± 8
113 ± 14










Example 8
Antisense Inhibition of Ube3a-ATS in Mice Having Maternal Deficiency in Ube3a
Antisense oligonucleotides selected from the studies described above will be screened for in vivo efficacy in a genetic mouse model of Angelman Syndrome described in Jiang, Y. et al., Neuron. 1998. 21: 799-811. The mice have a null mutation in the maternal Ube3a allele. A targeting vector replaces a 3 kb genomic DNA fragment containing exon 2 (299 bp; 3-302 bp in GENBANK Accession No. U82122) of the maternal Ube3a allele, thereby deleting one hundred of the most N terminal amino acids, shifting the reading frame, and inactivating all putative isoforms of maternal Ube3a. The mice have normal neuroanatomy but display a deficiency in context-dependent learning long-term potentiation (LTP), motor dysfunction, and inducible seizures similar to human Angelman Syndrome.
Antisense oligonucleotides described herein are administered to the mice by intracerebroventricular bolus injection at a dose range from 10 μg to 1 mg final concentration. The mice are euthanized and the levels of Ube3a-ATS mRNA down-regulation as well as Ube3a protein induction are assessed using hippocampus or whole brain sections.
Mice treated with ASOs are assessed by various phenotypic and behavioral assays for treatment or amelioration of the phenotypic defects. The open field assay as described in Miller, B. H. et al., PloS One. 2010. 5: e14458 is conducted to assess whether the hypoactive phenotype is treated by ASO administration. The marble burying assay as described in Njung'e, K. and Handley, S. L. Pharmacol. Biochem. Behav. 1991. 38: 63-67 is conducted to assess whether anxiety in the mice is treated by ASO administration. The rotarod assay and the dowel test as described in Chen, M. and Xu, R. BMC Neurosci. 2011. 12: 1 is conducted to assess for improvements in the balance and motor functions of the mice by ASO administration.
Mice treated with ASOs are tested for motor function using footprint analysis. The hindpaws of the mice are dipped into red waterproof ink and the mice are placed at an open end of a dark tunnel. The step length, step width, and left-right alternation coefficient are calculated as described in Clark, H. B. et al., J. Neurosci. 1997. 17: 7385-7395.
Mice treated with ASOs are tested for reduction in seizures by an assay involving scratching a plastic pen across a stainless steel cage, grating as rapidly and intensively as possible, until seizures occur or for a maximum of 45 sec. Each animal will be tested once between 5 and 10 weeks of age. EEG recordings will be performed, as described in Cattanach, B. M. et al., Mamm. Genome 1997. 8: 472-478.
Mice treated with ASOs are tested for improvement in context dependent learning with the fear conditioning assay, which uses a standard testing chamber with a stainless steel grated floor through which scrambled foot shock is administered. Freezing will be assessed and scored as either 1 (freezing posture) or 0 (non-freezing posture). The scores will be averaged over a 1 minute interval and converted into percentages.

## Claims

1. A method of treating Angelman syndrome in an animal comprising administering to an animal with Angelman syndrome an antisense compound targeted to UBE3A-ATS, wherein the antisense compound comprises an oligonucleotide consisting of 12 to 30 linked nucleosides, wherein the oligonucleotide is at least 85% complementary to a sequence within nucleobases 446213 to 513602 of SEQ ID NO: 2.

2. The method of claim 1, wherein UBE3A-ATS comprises a nucleic acid sequence at least 85% identical to SEQ ID NO:2.

3. The method of claim 1, wherein the oligonucleotide is at least 90% complementary over its entire length to an equal length region of a UBE3A-ATS nucleic acid sequence.

4. The method of claim 1, wherein the oligonucleotide is at least 95% complementary over its entire length to an equal length region of a UBE3A-ATS nucleic acid sequence.

5. The method of claim 1, wherein the oligonucleotide is 100% complementary over its entire length to an equal length region of a UBE3A-ATS nucleic acid sequence.

6. The method of claim 1, wherein the oligonucleotide is a single-stranded oligonucleotide.

7. The method of claim 6, wherein the oligonucleotide is a modified oligonucleotide.

8. The method of claim 7, wherein the modified oligonucleotide comprises at least one modified internucleoside linkage.

9. The method of claim 8, wherein the modified internucleoside linkage is a phosphorothioate internucleoside linkage.

10. The method of claim 7, wherein at least one nucleoside comprises a modified sugar.

11. The method of claim 10, wherein the modified sugar is a bicyclic sugar comprising a bridge between the 4′ and the 2′ positions of the sugar.

12. The method of claim 11, wherein the bridge is selected from 4′-CH(CH3)—O-2′, 4′-CH2-2′, 4′-(CH2)2-2′, 4′-CH2—O-2′, 4′-(CH2)2—O-2′, 4′-CH2—O—N(R1)-2′ and 4′-CH2-N(R1)—O-2′- wherein each R1 is, independently, H, a protecting group or C1-C12 alkyl.

13. The method of claim 12, wherein the bridge is 4′-CH(CH3)—O-2′.

14. The method of claim 12, wherein the bridge is selected from 4′-CH2—O-2′ and 4′-(CH2)2—O-2′.

15. The method of claim 10, wherein the modified sugar comprises a 2′-O-methoxyethyl group.

16. The method of claim 7, wherein at least one nucleoside comprises a modified nucleobase.