shRNA targeting UBE3A-ATS to restore paternally derived UBE3A gene expression in Angelman syndrome

Abstract

Provided herein are compositions and methods for activating expression from the paternally inherited allele of UBE3A in Angelman syndrome using viral vector delivery of short hairpin RNA. Provided herein are compositions and methods for reducing or eliminating expression of UBE3A-ATS in Angelman syndrome using viral vector delivery of short hairpin RNA.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 317,154, filed March 7, 2022, the entire contents of which are incorporated herein by reference.

[0002] Government Assistance Notification This invention was made with government support under Grant No. 1R01HD094953 awarded by the National Institutes of Health. The United States Government has certain rights in the invention.

[0003] Technical Field The present disclosure relates to compositions and methods for activating expression from the paternally derived allele of UBE3A in subjects with Angelman syndrome using short hairpin RNA.

[0004] Citation of sequence listing A sequence listing corresponding to the sequences described herein is provided. [Background technology]

[0005] background Angelman syndrome (AS) is a neurodevelopmental disorder that affects 1 in 15,000 people. Individuals with AS have developmental delay, severe cognitive impairment, ataxic gait, frequent seizures, attention deficits, speech deficits, and a characteristically playful attitude. Neuronal cells derived from induced pluripotent stem cells (iPSCs) from AS patients exhibit depolarized resting membrane potentials, delayed action potential development, and reduced spontaneous synaptic activity. Fink, JJ, TM Robinson, ND Germain, CL Sirois, KA Bolduc, AJ Ward, F. Rigo, SJ Chamberlain and ES Levine (2017). “Disrupted neuronal maturation in Angelman syndrome-derived induced pluripotent stem cells.” Nat Commun 8: 15038. AS affects a relatively large patient population, with registries enrolling over 3,000 patients and approximately 250 new cases diagnosed each year. Individuals with AS require lifelong care.

[0006] AS is caused by loss of function of the maternal copy of the gene UBE3A, which encodes an E3 ubiquitin ligase. This loss-of-function mutation can occur by any genetic mutation in the maternal allele. UBE3A is expressed exclusively from the maternal allele in neurons. All individuals with AS have a normal, paternally derived UBE3A allele that is epigenetically silenced in cis in neurons by a long non-coding RNA called UBE3A antisense transcript (UBE3A-ATS) (Rougeulle, et al., 1997, Nat Genet 17, 14-15; Chamberlain and Brannan, 2001, Genomics, 73, 316-322). Reactivation of the paternal allele has been shown to restore UBE3A protein expression and alleviate behavioral disorders in AS mouse models. Restoration of UBE3A expression in humans, especially in infants, is expected to ameliorate disease. Summary of the Invention

[0007] overview Provided herein is a novel treatment for Angelman syndrome that replaces or enhances the missing maternally derived UBE3A by inhibiting the silencing of paternally derived UBE3A and allowing expression from the native control elements of paternally derived UBE3A. Increased expression of UBE3A in neurons is achieved by terminating the transcription of UBE3A-ATS. Overexpression of UBE3A is prevented because the native control elements control expression. This approach can improve AS symptoms with a single treatment, eliminating the need for multiple treatments.

[0008] Provided herein is a polynucleotide sequence comprising: [ka] (SEQ ID NO: 2). An expression vector is provided comprising SEQ ID NO: 2. In one embodiment, the expression vector is an adeno-associated virus (AAV) vector or a lentivirus vector. A pharmaceutical composition comprising the above is provided.

[0009] Provided herein is a polynucleotide encoding an shRNA comprising a nucleotide sequence at least 85%, at least 90%, at least 95%, or 100% complementary to an RNA encoded by any of SEQ ID NOs: 3-489. In one embodiment, the polynucleotide is SEQ ID NO: 2. In one embodiment, the shRNA causes activation or increased expression of paternally derived UBE3A. In one embodiment, the shRNA causes decreased expression of paternally derived UBE3A-ATS. An expression vector comprising the shRNA is provided. In one embodiment, the expression vector is an adeno-associated virus (AAV) vector or a lentivirus vector. A pharmaceutical composition comprising the above is provided.

[0010] Provided herein is a method for treating Angelman syndrome, comprising administering a polynucleotide of SEQ ID NO:2 to a patient in need thereof.In one embodiment, the polynucleotide of SEQ ID NO:2 encodes an shRNA that causes the expression of paternally derived UBE3A-ATS to be reduced.In one embodiment, the polynucleotide of SEQ ID NO:2 encodes an shRNA that causes the expression of paternally derived UBE3A gene to be activated or increased.

[0011] Provided herein is a method of treating Angelman syndrome, comprising administering to a patient in need thereof a polynucleotide encoding an shRNA comprising a nucleotide sequence at least 85%, at least 90%, at least 95%, or 100% complementary to an RNA encoded by any of SEQ ID NOs: 3-489. In one embodiment, the polynucleotide is SEQ ID NO: 2. In one embodiment, the shRNA causes activation or increased expression of paternally derived UBE3A. In one embodiment, the shRNA causes decreased expression of paternally derived UBE3A-ATS.

[0012] In one embodiment, SEQ ID NO:2 codes for shRNA that can inhibit the silencing of paternally derived UBE3A. In one embodiment, SEQ ID NO:2 is contained in an expression vector. In one embodiment, the expression vector is an adeno-associated virus (AAV) vector or a lentivirus vector. In one embodiment, a method for inhibiting the silencing of paternally derived UBE3A gene by RNA antisense transcript encoded by SEQ ID NO:1 is provided, comprising administering to a patient in need thereof an amount of SEQ ID NO:2 that is effective for cleaving the RNA antisense transcript encoded by SEQ ID NO:1.

[0013] In one aspect, a method for inhibiting silencing of the paternally derived UBE3A gene by an RNA antisense transcript encoded by SEQ ID NO:1 is provided, the method comprising administering to a patient in need thereof an amount of shRNA comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95%, or 100% complementary to an RNA encoded by any of SEQ ID NOs:3-489, and is effective for cleaving the RNAb antisense transcript encoded by SEQ ID NO:1.

[0014] In one embodiment, the shRNA provided herein is encoded by a portion of SEQ ID NO:2, e.g., the portion having the bolded nucleotides, which is truncated by one, two, three or four nucleotides on either side of the bolded nucleotides.Similarly, in one embodiment, the shRNA provided herein can include a portion of SEQ ID NO:2, e.g., the portion having the italicized nucleotides, which is truncated by one, two or three nucleotides on either side of the italicized nucleotides.

[0015] In one embodiment, the polynucleotide sequence is as shown below: [ka] (SEQ ID NO: 506), wherein nnnnnnnn can be CTCGAG (SEQ ID NO: 490), TCAAGAG (SEQ ID NO: 491), TTCG (SEQ ID NO: 492) or GAAGCTTG (SEQ ID NO: 493).

[0016] In one embodiment, a polynucleotide sequence is provided comprising a first portion, a second portion and a third portion, wherein the first portion comprises any of SEQ ID NOs: 3-489, the second portion comprises any of SEQ ID NOs: 490, 491, 492 or 493, and the third portion comprises a respective nucleotide sequence complementary to the nucleotide sequences of SEQ ID NOs: 3-489. [Brief description of the drawings]

[0017] [Figure 1] Figure 1 shows the chromosomal mutations in Angelman syndrome. [Diagram 2] FIG. 2 shows a diagram of the paternally derived UBE3A gene. [Diagram 3] Figure 3 shows the genomic location of the shRNA targets (solid bubbles). UCSC Genome Browser view of the 15q11-q13 region that contains the imprinted SNHG14 / UBE3A locus (dashed bubbles). Location of the shRNA targets within the UBE3A-ATS region (ATS-shRNA2) and the SNORD115 snoRNA cluster. [Figure 4] Figure 4 is a bar graph showing qRT-PCR analysis of Angelman syndrome iPSC-derived neurons after treatment with SNHG14-targeting shRNAs (551-2, ATS shRNA1-4, ATS shRNA2_3G) or a non-targeting control shRNA (SCRAM). Two shRNAs, 551-2 and ATS shRNA2, knocked down UBE3A-ATS and activated paternally derived UBE3A. [Diagram 5]Figure 5 is a bar graph showing qRT-PCR analysis of Angelman syndrome iPSC-derived neurons after treatment with either SNHG14-targeting shRNA (ATS shRNA2), non-targeting control shRNA (SCRAM), or no treatment (UTC). ATS shRNA2 knocked down UBE3A-ATS and activated paternally derived UBE3A. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0018] Detailed Description UBE3A is a gene encoding an E3 ubiquitin ligase. The genomic coordinates of UBE3A are hg19 chr15:25, 582, 381-25, 684, 175. There are three normal isoforms of UBE3A: isoform 1 (accession number X98032), isoform 2 (accession number X98031), and isoform 3 (accession number X98033). In nerve cells (neurons), UBE3A is expressed only from the maternal allele. The paternally derived UBE3A allele is epigenetically silenced by the long non-coding RNA UBE3A antisense transcript (UBE3A-ATS), encoded by SEQ ID NO:1. The genomic coordinates of UBE3A-ATS are hg19 chr15:25, 223, 730-25, 664, 609 in the positive strand. The following genomic coordinates are of particular note: hg19 chr15:25, 522, 751-25, 591, 391 (plus strand).

[0019] UBE3A-ATS / Ube3a-ATS (human / mouse) is an antisense DNA strand transcribed as part of a large transcript called SNHG14 (SNORNA HOST GENE 14) at the UBE3A locus. Human UBE3A-ATS, as part of SNHG14, is expressed exclusively from the paternally derived allele in the central nervous system (CNS). The transcript is approximately 600 kb long, starting at SNRPN and extending through most of UBE3A. SNHG14 (Small Nuclear RNA Host Gene 14) encodes a non-coding RNA and belongs to the lncRNA class. SNHG14 is located within the Prader-Willi critical region and produces a long spliced ​​maternally imprinted RNA that initiates at one of several promoters of the SNRPN (small nuclear ribonucleoprotein polypeptide N) gene. This transcript serves as a host RNA for two C / D box small nucleolar RNA clusters, SNORD116 and SNORD115. See Runte et al, 2001, Hum Mol Genet 10, 2687-2700. This RNA extends into the ubiquitin protein ligase E3A (UBE3A) gene and is thought to regulate the imprinted expression of UBE3A in the brain. The promoter of SNRPN is the Prader-Willi syndrome imprinting center (PWS-IC), and approximately 35 kb upstream of the PWS-IC is the Angelman syndrome imprinting center (AS-IC). These two regions are thought to regulate the expression of the entire SNHG14 transcript.

[0020] SNURF / SNRPN is a bicistronic gene encoding two protein-coding transcripts, SNURF and SNRPN. Both SNURF and SNRPN proteins are localized to the cell nucleus. SNRPN is a small nuclear ribonucleoprotein, and the function of SNURF is unknown. The SNRPN / SNURF-initiated transcript also encodes the SNHG14 transcript. Within the introns of SNHG14 are sequences of several C / D box snoRNAs. Box C / D small nucleolar RNAs (SNORDs) are a well-defined family of small non-coding RNAs that exert their regulatory function by an antisense-based mechanism. Most C / D box snoRNAs function in non-RNA methylation.

[0021] Many orphan snoRNAs are generated from two large imprinted chromosomal domains located in humans at 15q11q13 and 14q32. See for example Figure 3. As mentioned above, the imprinted human 15q11q13 region (also known as the Prader-Willi syndrome (PWS) / Angelman syndrome (AS) locus or the SNURF-SNRPN domain) contains several paternally expressed protein-coding genes, as well as a large number of paternally expressed neurospecific snoRNA genes organized as two major repetitive DNA arrays: the SNORD116 and SNORD115 clusters, consisting of 29 and 47 related gene copies, respectively.

[0022] SNORD115 encodes a small nucleolar RNA (snoRNA) that is clustered with several dozen other similar snoRNAs on chromosome 15. These genes are mostly located within introns of the SNHG14 transcript, are paternally imprinted, and originate from the PWS / AS region.

[0023] The compositions and methods described herein are described to target UBE3A-ATS and inactivate paternally derived UBE3A alleles. Effective inhibition of UBE3A-ATS by short hairpin RNA (shRNA) described herein results in a decrease in the expression level of UBE3A-ATS and a concomitant increase in the expression level of paternally derived UBE3A alleles.

[0024] In one embodiment, the compositions and methods described herein relate to the treatment or prevention of AS. A patient in need of such treatment or prevention has AS or is at risk of developing AS. As used herein, the term "patient in need thereof" includes any mammal in need of these treatment or prevention methods, including humans. The subject may be male or female. In certain aspects, a patient in need of AS treated with the methods and compositions provided herein may show improvement in anxiety, learning, balance, motor function, and / or seizures, or the method may return the resting membrane potential of a neuron to about -70mV, improve action potential development delay, increase spontaneous synaptic activity, and improve further changes in neuronal phenotype related to rheobase, action potential properties (e.g., shape), membrane current, synaptic potential, and / or ion channel conductance.

[0025] In one embodiment, the polynucleotide comprises a first nucleotide sequence that encodes a short hairpin RNA (shRNA) that results in reduced expression of the UBE3A-ATS sequence (SEQ ID NO:1). For example, a portion of the shRNA described herein may be complementary to the RNA sequence encoded by SEQ ID NO:1 or a sequence contained therein. In one embodiment, the shRNA described herein is an RNA polynucleotide encoded by the first nucleotide sequence. The polynucleotide encompassing the first nucleotide sequence may be a DNA polynucleotide suitable for cloning into a suitable vector (e.g., a plasmid) for culture and subsequent production of viral particles. As a result, the viral particle may comprise a DNA polynucleotide having a nucleotide coding sequence in a form suitable for infection. Thus, the first nucleotide sequence may be a DNA sequence cloned into a plasmid for viral particle production or may be a DNA sequence packaged into the viral particle. Because retroviruses carry nucleotide coding sequences in the form of RNA polynucleotides, retroviral particles (e.g., lentiviruses) comprise an RNA polynucleotide that comprises the first nucleotide sequence as a corresponding RNA sequence.

[0026] Disclosed herein are novel shRNAs that cleave UBE3A-ATS and reduce the expression of UBE3A-ATS, thereby activating the paternally inherited copy of UBE3A in neurons. This provides the UBE3A gene product to cell types that are deficient in Angelman syndrome. The genomic sequence of LNCAT has a potential search space of about 60 kb where potential shRNA targets may be found. However, not all predicted sequences actually reduce UBE3A-ATS and restore UBE3A. Therefore, as shown in certain examples herein, it is difficult to predict which sequences will or will not function. See, for example, FIG. 4.

[0027] The first nucleotide sequence encodes an shRNA. For example, the first nucleotide sequence can be SEQ ID NO:2. [ka] The first nucleotide sequence may also be a modified SEQ ID NO:2 in which the bolded nucleotides in SEQ ID NO:2 are replaced with any of SEQ ID NOs:4-489 and the italicized nucleotides in SEQ ID NO:2 are replaced with nucleotides complementary to the nucleotides in SEQ ID NOs:4-489. As used herein, "target" refers to an operative RNA polynucleotide that can undergo hybridization to a nucleotide sequence via hydrogen bonding, such as a nucleotide sequence transcribed from a nucleotide sequence in the large genomic sequence of UBE3A-ATS. Hybridization of an operative RNA polynucleotide to a nucleotide sequence transcribed from a nucleotide sequence with the large genomic sequence of UBE3A-ATS may result in a greater reduction in the expression level of UBE3A-ATS in the presence of the operative RNA polynucleotide compared to the expression level of UBE3A-ATS in the absence of the operative RNA polynucleotide. In one embodiment, the operative RNA polynucleotide comprises a nucleotide sequence of an shRNA that is complementary to an RNA sequence encoded within the large genomic sequence of UBE3A-ATS. For example, the shRNA comprises a nucleotide sequence complementary to an RNA sequence encoded by SEQ ID NO:3 and SEQ ID NOs:4-489. Thus, an operative RNA polynucleotide refers to the operative portion of an shRNA after the shRNA has been assimilated by the target organism and processed into a functional state.

[0028] "Reduced expression" refers to a reduction or blocking of expression or activity of UBE3A-ATS and does not necessarily indicate a complete loss of expression or activity. Mechanisms that reduce target expression include hybridization of an agonistic RNA polynucleotide with a target sequence or sequences transcribed from a sequence or sequences within the larger genomic UBE3A-ATS sequence (SEQ ID NO:1), the result or effect of hybridization being target degradation or target occupancy with the concomitant cessation of cellular machinery including, for example, transcription or splicing.

[0029] Without wishing to be bound by a particular theory, the shRNA herein may inhibit the silencing of paternally derived UBE3A by: (1) cleaving the RNA transcript encoded by SEQ ID NO:1; (2) reducing the steady-state level (i.e., the baseline level in homeostasis) of the RNA transcript encoded by SEQ ID NO:1; and (3) terminating the transcription of SEQ ID NO:1. For example, the cleavage and reduction of the steady-state level of the RNA transcript encoded by SEQ ID NO:1 may occur via a mechanism involving an RNA-induced silencing complex (RISC). The shRNA may utilize a RISC. When the vector carrying the genomic material of the shRNA is integrated into the host genome, the shRNA genomic material is transcribed into pri-microRNA in the host. The pri-microRNA is processed into pre-shRNA by ribonucleases such as Drosha and transported out of the nucleus. The pre-shRNA is processed by endoribonucleases such as Dicer to form small interfering RNA (siRNA). The siRNA is loaded into RISC, the sense strand is degraded, and the antisense strand acts as a guide for RISC to the complementary sequence of the mRNA. RISC cleaves the mRNA if the base sequence is perfectly complementary, and inhibits the translation of the mRNA if the base sequence is incompletely complementary. Thus, the shRNA encoded by the first nucleic acid sequence increases the expression of paternally derived UBE3A by decreasing the steady-state level of UBE3A-ATS RNA.

[0030] As used herein, the term "nucleic acid" refers to a molecule composed of monomeric nucleotides. Examples of nucleic acids include ribonucleic acid (RNA), deoxyribonucleic acid (DNA), single-stranded nucleic acid, double-stranded nucleic acid, small interfering ribonucleic acid (siRNA), and short hairpin RNA (shRNA). "Nucleotide" refers to a nucleoside having a phosphate group covalently attached to the sugar moiety of the nucleoside. "Oligonucleotide" or "polynucleotide" refers to a polymer of linked nucleotides, each of which may be modified or unmodified and is independent of the others.

[0031] As used herein, "short hairpin" includes conventional stem-loop shRNAs that form precursor microRNAs (pre-miRNAs). "shRNA" also includes microRNA-incorporated shRNAs (miRNA-based shRNAs), where the guide and passenger strands of the miRNA duplex are incorporated into a pre-existing (or natural) miRNA or into an engineered or synthetic (designed) miRNA. Once transcribed, conventional shRNAs (i.e., not miR-451 shRNA mimics) form primary miRNAs (pri-miRNAs), or structures that closely resemble natural pri-miRNAs. The pri-miRNAs are then processed by Drosha and its coenzymes into pre-shRNAs. Thus, the term "shRNA" includes pri-miRNA (shRNA-mir) molecules and pre-shRNA molecules.

[0032] "Stem-loop structure" refers to a nucleic acid having a secondary structure that includes a region of nucleotides (the stem portion) that is known or predicted to form a double strand or duplex, and is connected on one side by a region of primarily single-stranded nucleotides (the loop portion). It is known in the art that the loop portion may be at least 4 nucleotides long, 6 nucleotides long (e.g., the underlined sequence in SEQ ID NO:2), 8 nucleotides long, or more. As used herein, the terms "hairpin" and "fold-back" structures are used to refer to stem-loop structures. Such structures are well known in the art, and the terms are used consistently with their known meaning in the art. For example, CTCGAG (SEQ ID NO:490), TCAAGAG (SEQ ID NO:491), TTCG (SEQ ID NO:492), and GAAGCTTG (SEQ ID NO:493) are suitable stem-loop structures. As known in the art, secondary structures do not require exact base pairing. Thus, the stem may include one or more base mismatches or bulges. Alternatively, the base pairs may be perfect, i.e., without mismatches. In one embodiment, a polynucleotide sequence is provided as follows: [ka] (SEQ ID NO: 506), wherein nnnnnnnn may be CTCGAG (SEQ ID NO: 490), TCAAGAG (SEQ ID NO: 491), TTCG (SEQ ID NO: 492) or GAAGCTTG (SEQ ID NO: 493). In one embodiment, a polynucleotide sequence is provided comprising a first portion, a second portion and a third portion, wherein the first portion comprises any of SEQ ID NOs: 3-489, the second portion comprises any of SEQ ID NOs: 490, 491, 492 or 493; and the third portion comprises a nucleotide sequence complementary to the respective nucleotide sequence of SEQ ID NOs: 3-489.

[0033] In one embodiment, shRNA may include modified shRNA, including, but not limited to, shRNA with improved in vivo stability. Modified shRNA includes molecules with nucleotide analogs, including molecules with additions, deletions and / or substitutions in nucleobases, sugars or backbones; cross-linked molecules; or other chemically modified molecules. Modified nucleotide(s) may be part of the shRNA molecule or may be the entire shRNA molecule. For example, shRNA molecules may be modified or may include modified nucleic acids at their 5'-end, 3'-end, or both regions, and / or within the guide strand, passenger strand, or both strands, and / or within the nucleotides that overhang the 5'-end, 3'-end, or both. (See Crooke, U.S. Patent Nos. 6,107,094 and 5,898,031; Elmen et al., U.S. Patent Publication Nos. 2008 / 0249039 and 2007 / 0191294; Manoharan et al., U.S. Patent Publication No. 2008 / 0213891; MacLachlan et al., U.S. Patent Publication No. 2007 / 0135372; and Rana, U.S. Patent Publication No. 2005 / 0020521, the entire contents of which are incorporated herein by reference).

[0034] The shRNA herein comprises a nucleotide sequence complementary to an RNA nucleotide sequence transcribed from within the entire genome UBE3A-ATS sequence (SEQ ID NO: 1) and inhibits the silencing of paternally derived UBE3A by UBE3A-ATS. In one embodiment, the shRNA comprises a nucleotide sequence complementary to an RNA sequence encoded by SEQ ID NO: 4-489. In one embodiment, the shRNA comprises a nucleotide sequence complementary to an RNA sequence encoded by SEQ ID NO: 3 (5'-GATATCACCTTACAGAAATTA-3', UBE3A-ATS artificial / synthetic target). In one embodiment, the shRNA is encoded by the nucleotide sequence of SEQ ID NO: 2. In one embodiment, the nucleotide sequence contained in the shRNA and complementary to the RNA nucleotide sequence transcribed from the UBE3A-ATS gene is 17-21 nucleotides in length. The complementary nucleotides may be contiguous or may be interspersed with non-complementary nucleotides. In one embodiment, the complementary nucleotide sequence is 21 nucleotides in length, as shown by the bolded sequence of SEQ ID NO: 2. The shRNA can comprise a nucleotide sequence in which 17, 18, 19, 20 or 21 nucleotides are complementary to SEQ ID NO: 3 or nucleotides 4-489. The 17, 18, 19, 20 or 21 complementary nucleotides can be contiguous or can be interspersed with non-complementary nucleotides. The total length of the shRNA, including the loop, can be 40-50 nucleotides long, such as 44-48 nucleotides long, such as 48 nucleotides long.

[0035] The method of determining whether a sequence can specifically hybridize to a target nucleic acid is well known in the art. In one embodiment, the shRNA polynucleotide provided herein comprises a nucleic acid sequence that can specifically hybridize to the RNA sequence transcribed from UBE3A-ATS (SEQ ID NO: 1).

[0036] The shRNA may comprise an RNA polynucleotide comprising a region of 17-21 linked nucleotides complementary to an RNA target sequence, where the RNA polynucleotide region is at least 85% complementary over its entire length to an equal length region of the UBE3A-ATS RNA nucleic acid sequence. In one embodiment, the RNA polynucleotide region is at least 90%, at least 95%, or 100% complementary over its entire length to an equal length region of the UBE3A-ATS RNA nucleic acid sequence.

[0037] The shRNA may comprise a nucleotide sequence that is at least 85% complementary to and equal in length to the RNA sequence encoded by SEQ ID NO:3 or any of SEQ ID NOs:4-489. The shRNA may comprise a nucleotide sequence that is at least 90% complementary to and equal in length to the RNA sequence encoded by SEQ ID NO:3 or any of SEQ ID NOs:4-489. The shRNA may comprise a nucleotide sequence that is at least 95% complementary to and equal in length to the RNA sequence encoded by SEQ ID NO:3 or any of SEQ ID NOs:4-489. The shRNA or microRNA may comprise a nucleotide sequence that is 100% complementary to and equal in length to the RNA sequence encoded by SEQ ID NO:3 or any of SEQ ID NOs:4-489.

[0038] In one embodiment, the shRNA is a single-stranded RNA polynucleotide. In one embodiment, the RNA polynucleotide is a modified RNA polynucleotide. Percent complementarity is used herein in the conventional sense to mean the base pairing between adenine and thymine, adenine and uracil (RNA), and guanine and cytosine.

[0039] Non-complementary nucleobases between the shRNA and the UBE3A-ATS nucleotide sequence can be tolerated as long as the shRNA maintains the ability to specifically hybridize to the UBE3A-ATS nucleotide sequence. Furthermore, the shRNA may hybridize on one or more segments of the UBE3A-ATS nucleotide sequence, where no intervening or adjacent segments are involved in the hybridization event (e.g., loop structures, mismatch structures, or hairpin structures).

[0040] In one embodiment, the shRNAs provided herein, or specific portions thereof, are at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementary to the UBE3A-ATS RNA nucleotide sequence, UBE3A-ATS region, UBE3A-ATS segment, or specific portions thereof. Percent complementarity of shRNAs to UBE3A-ATS nucleotide sequences can be determined using routine methods.

[0041] For example, 18 bases out of 20 bases can be complementary to the UBE3A-ATS region and thus can specifically hybridize, showing 90% complementarity.In this example, the remaining non-complementary nucleobases can be clustered or interspersed with complementary nucleobases, and do not need to be contiguous with each other or with complementary nucleobases.Thus, an 18-base long shRNA with four non-complementary nucleobases flanking two regions that are completely complementary to the target nucleotide sequence can have 77.8% overall complementarity with the target nucleotide sequence, and thus falls within the scope of the invention described herein. The percent complementarity of a region of the UBE3A-ATS nucleotide sequence to an shRNA can be routinely determined using the BLAST program (basic local alignment search tool) and the PowerBLAST program 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, for example, with the Gap program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, Madison Wis.) using the algorithm of Smith and Waterman (Adv. Appl. Math., 1981, 2, 482 489) with default settings.

[0042] In one embodiment, the shRNA provided herein, or a specified portion thereof, is fully complementary (i.e., 100% complementary) to a specified portion of the UBE3A-ATS nucleotide sequence, or its transcription product of SEQ ID NO: 1. For example, the shRNA can be fully complementary to the UBE3A-ATS nucleotide sequence, or a region, or a segment or sequence thereof. As used herein, "fully complementary" means that each nucleobase of the shRNA can be precisely base-paired with the corresponding RNA nucleobase transcribed from the UBE3A-ATS nucleotide sequence.

[0043] In one embodiment, the shRNA provided herein can comprise a portion of SEQ ID NO:2, for example, the bold nucleotide, and is shortened by 1, 2, 3 or 4 nucleotides at either end of the bold nucleotide.Similarly, in one embodiment, the shRNA provided herein can comprise a portion of SEQ ID NO:2, for example, the italic nucleotide, and is shortened by 1, 2 or 3 nucleotides at either end of the italic nucleotide.For example, [ka]

[0044] Similarly, an effective concentration or dose of shRNA can inhibit silencing of paternally derived UBE3A by UBE3A-ATS 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%, or at least 100%.

[0045] An effective concentration or dose of shRNA can stop transcription of UBE3A-ATS 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%, or at least 100%.

[0046] An effective concentration or dose of shRNA can reduce the steady-state level of UBE3A-ATS 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%, or at least 100%.

[0047] An effective concentration or dose of shRNA cleaves UBE3A-ATS and reduces it 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%, or at least 100%.

[0048] An effective concentration or dose of shRNA can reduce expression of UBE3A-ATS 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%, or at least 100% and induce expression of paternally derived 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%, or at least 100%.

[0049] As used herein, the terms "UBE3A-ATS" and "Ube3A-ATS" may be used interchangeably without capitalization to refer to a specific species or ortholog. "UBE3A" and "Ube3A" may be used interchangeably without capitalization to refer to a specific biological species or ortholog. Additionally, "UBE3A", "UBE3A (italics)", "Ube3A" and "Ube3A (italics)" are used interchangeably without italics to refer to nucleic acids or proteins, unless specifically noted to the contrary.

[0050] Viral Vectors A "vector" is a replicon, such as a plasmid, phage or cosmid, into which a DNA or RNA segment may be inserted and which results in the replication of the inserted segment. Generally, a vector is capable of replication when associated with appropriate control elements. Suitable vector backbones include those commonly used in the art, such as, for example, plasmids, plasmids containing viral genomes, viruses or artificial chromosomes. The term "vector" includes cloning and expression vectors as well as viral and insertion vectors.

[0051] As will be appreciated by those of skill in the art, the term "viral vector" is used broadly to refer to a nucleic acid molecule (e.g., a transfer plasmid) that generally comprises viral nucleic acid elements that facilitate the transfer of a nucleic acid sequence into a cell and / or the transfer of a nucleic acid molecule into the viral particle, where it mediates the transfer of the nucleic acid sequence into the genome of a cell.

[0052] Viral vectors mainly comprise structural and / or functional genetic elements derived from viruses. It is desired that viral vectors are non-toxic, non-immunogenic, easy to manufacture, and protect DNA or RNA and efficiently deliver it to target cells. According to the compositions and methods described herein, viral vectors can comprise DNA encoding one or more shRNAs described herein. In one embodiment, viral vectors are lentiviral vectors or adeno-associated viral (AAV) vectors.

[0053] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Exemplary lentiviruses include, but are not limited to, HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Viridans virus (VMV); Caprine arthritis-encephalitis virus (CAEV); Equine infectious anemia virus (EIAV); Feline immunodeficiency virus (FIV); Bovine immunodeficiency virus (BIV); and Simian immunodeficiency virus (SIV). As used herein, the term "lentivirus" includes lentiviral particles. Lentiviruses are introduced into dividing cells and epigenetic cells.

[0054] The term "lentiviral vector" refers to a viral vector (e.g., a viral plasmid) that contains structural and functional genetic elements, including long terminal repeats (LTRs), primarily derived from a lentivirus, or portions thereof. A lentiviral vector is a hybrid vector (e.g., in the form of a transfer plasmid) that contains retroviral, e.g., lentiviral sequences for reverse transcription, replication, integration, and / or packaging of nucleic acid sequences (e.g., coding sequences). The term "retroviral vector" refers to a viral vector (e.g., a transfer plasmid) that contains structural and functional genetic elements, or portions thereof, primarily derived from a retrovirus.

[0055] Adenoviral vectors are designed to be directly administered to living subjects.Unlike retroviral vectors, most of the genome of adenoviral vectors is not integrated into the chromosome of host cells.Instead, the gene introduced into cells using adenoviral vectors is maintained in the nucleus as an extrachromosomal element (episomal) and persists for a long period of time.Adenoviral vectors can transduce dividing and non-dividing cells in many different tissues in vivo, such as airway epithelial cells, endothelial cells, hepatocytes, and various tumors (Trapnell, Advanced Drug Delivery, Reviews, 12 (1993) 185-199).

[0056] The term "adeno-associated virus" (AAV) refers to a small ssDNA virus that infects humans and other primates, is not known to cause disease, and elicits only a very mild immune response. As used herein, the term "AAV" is meant to include AAV particles. AAV can infect dividing and non-dividing cells and integrate its genome into the genome of the host cell. These characteristics make AAV an attractive candidate for a viral vector for gene therapy, but the cloning capacity of the vector is relatively limited. In one embodiment, the vector used is derived from an adeno-associated virus (i.e., an AAV vector). There are more than 30 naturally occurring AAV serotypes. Many natural variants of the AAV capsid exist, allowing AAVs to be identified and used with properties that are specifically suited to a particular type of target cell. AAV viruses can be engineered by conventional molecular biology techniques, and these particles can be optimized for cell-specific delivery of shRNA DNA sequences, minimized immunogenicity, tuned stability and particle life span, efficient degradation, precise delivery to the nucleus, etc.

[0057] An "expression vector" refers to a vector that contains a regulatory region. Numerous vectors and expression systems are commercially available, such as from Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.) and Invitrogen / Life Technologies (Carlsbad, Calif.). The expression vector may be a viral expression vector derived from a particular virus.

[0058] The vectors provided herein can also include, for example, an origin of replication, a scaffold attachment region (SAR), and / or a marker. The marker gene can confer a selectable phenotype to the host cell. For example, a marker can confer biocide resistance, such as resistance to antibiotics (e.g., kanamycin, G418, bleomycin, hygromycin). The expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Examples of such tag sequences include green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, FIag, and the like. (商標) Tag sequences, such as tags (Kodak, New Haven, Conn.), are typically expressed as fusions with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, such as at the carboxyl or amino terminus.

[0059] Further expression vectors can also include, for example, segments of chromosomal, non-chromosomal and synthetic DNA sequences.Suitable vectors include plasmids such as pLK0.1 puro derivatives, SV40 and RP4; phage DNA, such as many derivatives of phage 1, for example NM989 and other phage DNA, such as M13 and filamentous single-stranded phage DNA, vectors useful in eukaryotic cells, for example vectors useful in insect or mammalian cells, vectors derived from combinations of plasmids and phage DNA, such as plasmids modified to adopt phage DNA or other expression control sequences, etc.

[0060] A vector may also contain a regulatory region. The term "control region" refers to a nucleotide sequence that influences the initiation and rate of transcription or translation, as well as the stability and / or mobility of the transcription or translation product. Control regions include, but are not limited to, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0061] As used herein, the term "operably linked" refers to the positional relationship between a regulatory region in a nucleic acid and a sequence to be transcribed, and is positioned so as to affect the transcription or translation of such sequence. For example, to place a coding sequence under the control of a promoter, the translation initiation site of the translation reading frame of the polypeptide is generally positioned 1 to about 50 nucleotides downstream of the promoter. However, the promoter may be positioned about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription initiation site. A promoter usually includes at least a core (basal) promoter. A promoter may also include at least one control element, such as an enhancer sequence, an upstream element, or an upstream activation region (UAR). The choice of promoter to include will vary depending on several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell or tissue preferential expression. Regulation of expression of a coding sequence can be achieved by appropriate selection and positioning of promoters and other regulatory regions relative to the coding sequence.

[0062] Vectors may also include other components or functionalities that further regulate gene transfer and / or gene expression or confer beneficial properties on target cells. As described and illustrated in more detail below, such other components include, for example, components that affect binding or targeting to cells (including components that mediate cell type or tissue specific binding); components that affect uptake of vector nucleic acid by cells; components that affect localization of polynucleotides within cells after uptake (such as agents that mediate nuclear localization); and components that affect expression of polynucleotides. Such components may also include markers, such as detectable and / or selectable markers, that can be used to detect or select cells that have taken up and expressed the nucleic acid delivered by the vector. Such components may be provided as natural features of the vector (such as the use of certain viral vectors that have components or functionalities that mediate binding and uptake) or the vector may be modified to provide such functionality. Other vectors include those described in Chen et al., BioTechniques, 34: 167-171 (2003). Many such vectors are known in the art and are publicly available.

[0063] "Recombinant viral vector" refers to a viral vector that contains one or more heterologous gene products or sequences. Because of the size constraints associated with packaging of many viral vectors, the heterologous gene products or sequences are usually introduced by replacing one or more portions of the viral genome. Such viruses can be replication-defective and require the missing functions to be provided in trans during viral replication and encapsidation (e.g., by using a helper virus or packaging cell line carrying gene products required for replication and / or encapsidation).

[0064] In one embodiment, a viral vector as used herein can be, for example, at least 10 5 A concentration of up to 1000 viral genomes may be used.

[0065] Selection of an appropriate promoter can be readily accomplished. Examples of suitable promoters include RNA polymerase II or III promoters. For example, a candidate shRNA sequence can be selected from the RNA polymerase III promoters U6 or H1, or from the neuron-specific enolase (NSE), synapsin I (Syn), or Ca 2+ / CaM-activated protein kinase IIα (CaMKIIα) can be expressed under the control of a neuron-specific RNA polymerase II promoter.

[0066] Other suitable promoters that can be used for gene expression include, but are not limited to, the 763 base pair cytomegalovirus (CMV) promoter, Rous sarcoma virus (RSV) (Davis, et al., Hum Gene Ther 4:151 (1993)), SV40 early promoter region, herpes thymidine kinase promoter, metallothionein (MMT) gene control sequence, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and animal transcription control regions, which show tissue specificity and have been utilized in transgenic animals; myelin basic protein gene control region, which shows activity in oligodendrocyte cells of the brain; and gonadotropin releasing hormone gene control region, which shows activity in the hypothalamus. Certain proteins can be expressed using the native promoter. Other elements that can enhance expression can also be included, such as enhancers or systems that result in high levels of expression, such as the tat gene or tar elements. The assembly or cassette can then be inserted into a vector, e.g., a known plasmid vector, such as pLK0.1, pUC19, pUC118, pBR322, etc. See Sambrook, et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, (1989). The plasmid vector can contain a selectable marker, such as a β-lactamase gene for ampicillin resistance, provided that the marker polypeptide does not adversely affect the metabolism of the organism being treated. The cassette can also be bound to a nucleic acid binding site in a synthetic delivery system, such as the system described in WO 95 / 22618.

[0067] The coding sequence of shRNA is described in Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)), Coffin et al. (Retrovirus. Cold Spring Harbor Laboratory Press, NY (1997)) and "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, (2000)). In one embodiment, the shRNA DNA sequence contains flanking sequences at the 5' and 3' ends that are complementary to sequences on the plasmid and / or vector that are cleaved by a restriction endonuclease. As is well known in the art, the flanking sequences vary depending on the restriction endonuclease used during restriction digestion of the plasmid and / or vector. Therefore, those skilled in the art can appropriately select the flanking sequences at the 5' and 3' ends of the shRNA DNA sequence. In one embodiment, the target site can be cloned into the vector by nucleic acid fusion and exchange techniques currently known in the art, including Gateway, PCR in fusion, Cre-lox P, and Creator.

[0068] In one embodiment, the expression vector comprises a polynucleotide comprising a promoter and a first nucleotide sequence encoding an shRNA as described herein. In one embodiment, the promoter and the polynucleotide comprising the first nucleotide sequence are operably linked. In one embodiment, the promoter is a U6 promoter. In one embodiment, the first nucleotide sequence comprised in the expression vector can be SEQ ID NO: 2. In one embodiment, the first nucleotide sequence comprised in the expression vector can also be a modified SEQ ID NO: 2 in which the bolded nucleotides of SEQ ID NO: 2 are replaced by any of SEQ ID NOs: 4-489 and the italicized nucleotides of SEQ ID NO: 2 are replaced by nucleotides complementary to the nucleotides of SEQ ID NOs: 4-489. In one embodiment, the first nucleotide sequence comprised in the expression vector can be any of SEQ ID NOs: 490-497. In one embodiment, the polynucleotide comprising the first nucleotide sequence in the expression vector is a DNA polynucleotide. In one embodiment, the first nucleotide sequence of the expression vector is a DNA nucleotide sequence. The shRNA encoded by the first nucleotide sequence of the expression vector can be as described in any of the variations disclosed herein.

[0069] As described below, recombinant viral vector is transfected into packaging cell or cell line together with the elements required for packaging recombinant viral particles.The recombinant viral particles collected from transfected cell supernatant are used to infect target cell or organism to express shRNA.Transduced cell or organism is used for transient expression or selected for stable expression.

[0070] Viruses / virus particles Viral particles are used to deliver the coding nucleotide sequence of shRNA that targets UBE3A-ATS RNA.In this specification, the terms virus and viral particle are used interchangeably.Viral particles usually contain various viral components in addition to nucleic acid, and sometimes also host cell components.Nucleic acid sequence can be packaged in viral particles that can deliver shRNA nucleic acid sequence to target cells of patients who need it.

[0071] Viral particles can be produced by (a) introducing a viral expression vector into a suitable cell line; (b) culturing the cell line under appropriate conditions to allow production of viral particles; (c) harvesting the produced viral particles; and (d) optionally purifying the harvested infectious viral particles.

[0072] An expression vector containing a nucleotide sequence encoding one or more shRNAs herein can be introduced into an appropriate cell line for propagation or expression using well-known techniques readily available to those of skill in the art. These include microinjection of minute amounts of DNA into the nucleus of cells (Capechi et al, 1980, Cell 22, 479-488), CaPO4-mediated transfection (Chen and Okayama, 1987, Mol. Cell Biol. 7, 2745-2752), DEAE-dextran transfection, electroporation (Chu et al., 1987, Nucleic Acid Res. 15, 1311-1326), lipofection / liposome fusion (Feigner et al., 1987, Proc. Natl. Acad. Sci. USA 84, 7413-7417), and particle gun techniques (Yang et al., 1990, Proc. Natl. Acad. Sci. USA 87, Examples of techniques include, but are not limited to, gene guns, transduction, infection (e.g., infectious viral particles), and other techniques such as those found in Sambrook et al. (Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0073] In one embodiment, when the expression vector is defective, infectious particles can be produced in a complementing cell line or by using a helper virus that trans-supplies non-functional viral genes. For example, cell lines suitable for complementing adenoviral vectors include 293 cells (Graham et al., 1997, J. Gen. Virol. 36, 59-72) and PER-C6 cells, which are commonly used to complement E1 functions (Fallaux et al., 1998, Human Gene Ther. 9, 1909-1917). Other cell lines have been engineered to complement doubly defective adenoviral vectors (Yeh et al., 1996, J. Virol. 70, 559-565; Krougliak and Graham, 1995, Human Gene Ther. 6, 1575-1586; Wang et al., 1995, Gene Ther. 2, 775-783; Lusky et al., 1998, J. Virol. 72, 2022-2033; WO94 / 28152 and WO97 / 04119). Infectious viral particles can be recovered from the culture supernatant, but also from the cells after lysis, and, if necessary, further purified according to standard techniques (chromatography, e.g. ultracentrifugation on a caesium chloride gradient as described in WO 96 / 27677, WO 98 / 00524, WO 98 / 22588, WO 98 / 26048, WO 00 / 40702, EP 1016700 and WO 00 / 50573).

[0074] In one aspect, provided herein is a host cell comprising a nucleic acid molecule, vector, or infectious viral particle described herein. The term "host cell" should be understood broadly, without limitation regarding a particular tissue, organ, or isolated cell. Such cells may be cells of a unique type or a group of different types of cells, including cultured cell lines, primary cells, and expanded cells.

[0075] Thus, host cells include prokaryotic cells, lower eukaryotic cells such as yeast, other eukaryotic cells such as insect cells, plant cells, higher eukaryotic cells such as vertebrate cells, and, particularly preferably, mammalian (e.g., human or non-human) cells. Suitable mammalian cells include, but are not limited to, hematopoietic cells (pluripotent cells, stem cells, leukocytes, lymphocytes, monocytes, macrophages, APCs, dendritic cells, non-human cells, etc.), lung cells, tracheal cells, liver cells, epithelial cells, endothelial cells, muscle cells (e.g., skeletal, cardiac or smooth muscle) or fibroblast cells. For example, host cells can include E. coli, Bacillus subtilis, Listeria monocytogenes, Saccharomyces cerevisiae, BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandell feline kidney cell line), CV-1 cells (African monkey kidney cell line), COS (e.g., COS-7) cells, Chinese hamster ovary (CHO) cells, mouse NIH / 3T3 cells, HeLa cells, and Vero cells. Host cells also include complementing cells that can complement at least one defective function of the replication-defective vectors (e.g., defective adenoviral vectors) available herein, as cited above.

[0076] In one embodiment, the host cells can be encapsulated. Cell encapsulation techniques have been described previously (Tresco et al., 1992, ASAJO J. 38, 17-23; Aebischer et al., 1996, Human Gene Ther. 7, 851-860). For example, transfected or infected eukaryotic host cells can be encapsulated with a compound that forms a microporous membrane, and the encapsulated cells can be implanted in vivo. Capsules containing cells of interest can be prepared using hollow microporous membranes (e.g., Akzo Nobel Faser AG, Wuppertal, Germany; Deglon et al., 1996, Human Gene Ther. 7, 2135-2146) with appropriate molecular weight cutoffs to allow free passage of proteins and nutrients between the capsule interior and exterior while preventing contact between the implanted cells and the host cells.

[0077] Viral particles suitable for use herein include AAV particles and lentiviral particles.AAV particles carry the coding sequence of shRNA herein in the form of genomic DNA.On the other hand, lentiviral particles belong to the retrovirus class and carry the coding sequence of shRNA herein in the form of RNA.

[0078] Recombinantly engineered viral particles, such as AAV particles, artificial AAV particles, self-complementary AAV particles, and lentiviral particles, containing DNA (or RNA in the case of lentiviral particles) encoding shRNA targeting UBE3A-ATS RNA can be delivered to target cells to inhibit silencing of UBE3A by UBE3A-ATS. The use of AAV has become a common method of DNA introduction because it is relatively non-toxic, allows efficient gene transfer, and can be easily optimized for specific purposes. In one aspect, the selected AAV serotype has natural neurotropic properties. In one aspect, the AAV serotype is AAV9 or AAV10.

[0079] A suitable recombinant AAV can be produced by culturing a host cell that contains a nucleotide sequence encoding an AAV serotype capsid protein or fragment thereof as defined herein; a functional rep gene; a minigene consisting of, at a minimum, the AAV inverted terminal repeats (ITRs) and coding nucleotide sequence; and sufficient helper functions to allow packaging of the minigene into an AAV capsid protein. The components required for culturing the host cell to package the AAV minigene into an AAV capsid can be provided in trans to the host cell. Alternatively, any one or more of the required components (e.g., minigene, rep sequence, cap sequence, and / or helper functions) can be provided by a stable host cell that has been engineered to contain one or more of the required components using methods known to those of skill in the art.

[0080] Unless otherwise stated, the AAV inverted terminal repeats (ITRs), and other selected AAV components described herein, can be readily selected from any AAV serotype, including, but not limited to, AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAVRec3, or other known and unknown AAV serotypes. These ITRs or other AAV components can be readily isolated from AAV serotypes using techniques available to those of skill in the art. Such AAV can be isolated or obtained from academic, commercial, or public sources (e.g., American Type Culture Collection, Manassas, Va.). Alternatively, AAV sequences can be obtained by synthesis or other suitable means with reference to published sequences, such as those available in the literature or databases, e.g., GenBank, PubMed, etc.

[0081] Herein, the minigene, rep sequence, cap sequence, and helper functions required to produce rAAV can be delivered to the packaging host cell in the form of any genetic element that transfers the sequences carried therein. The selected genetic element can be delivered by any suitable method. The methods used to construct the embodiments herein are known to those skilled in the art of nucleic acid manipulation and include genetic engineering, recombinant engineering, and synthetic techniques. See, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, NY. Similarly, methods for producing rAAV virions are well known and the selection of an appropriate method is not limited. See, for example, K. Fisher et al, 1993 J. Viral., 70:520-532 and U.S. Patent No. 5,478,745. All references herein are incorporated by reference.

[0082] The selection of these and other common vectors and regulatory elements is routine, and many such sequences are available.See, for example, Sambrook et al. and the references cited therein, for example, pages 3.18-3.26 and 16.17-16.27, and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989.Of course, not all vectors and expression control sequences function equally to express all transgenes herein.However, one skilled in the art can select from among these and other expression control sequences.

[0083] Pharmaceutical Compositions and Therapeutic Treatments The virus containing the desired coding sequence of shRNA can be formulated for administration to a patient or human in need by any suitable means for administration. Such formulations include the use of pharmaceutically and / or physiologically acceptable vehicles or carriers, particularly suitable for administration to the brain, for example, by subcranial or spinal injection.Furthermore, one or more shRNAs can be administered herein in combination therapy.In combination therapy, different shRNAs can be administered simultaneously, separately, sequentially, and in any order.

[0084] The pharmaceutical compositions herein include carriers and / or diluents suitable for delivery by injection to humans or animals. Such carriers and / or diluents should generally be non-toxic at the dosages and concentrations used. They can be selected from those commonly used to formulate compositions for oral administration, either in unit or multiple dose form, or for direct injection by continuous infusion or periodic infusion. In one embodiment, they are isotonic, hypotonic or weakly hypertonic, with relatively low ionic strength, such as provided by sugars, polyhydric alcohols and isotonic saline solutions. Representative examples include sterile water, physiological saline (e.g., sodium chloride), bacteriostatic water, Ringer's solution, glucose or saccharose solution, Hank's solution, and other physiologically balanced salt solutions (see, for example, the latest edition of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins). The pH of the composition is appropriately adjusted and buffered to be suitable for human or animal use, e.g., physiological or slightly basic pH (between about pH 8 and about pH 9, particularly pH 8.5 is preferred). Suitable buffers include phosphate buffers (e.g., PBS), bicarbonate buffers and / or Tris buffers. In one embodiment, for example, the composition is formulated in 1 M sucrose, 150 mM NaCl, 1 mM MgCl2, 54 mg / l Tween 80, 10 mM Tris pH 8.5. In one embodiment, for example, the composition is formulated in 10 mg / ml mannitol, 1 mg / ml HSA, 20 mM Tris, pH 7.2, 150 mM NaCl. These compositions are stable at -70°C for at least 6 months.

[0085] The pharmaceutical composition herein may be in various forms, for example, solid (for example, powder, lyophilized form) or liquid (for example, aqueous solution).For solid compositions, preparation methods include, for example, vacuum drying and lyophilization, which obtains a powder of active agent and any additional desired components from a solution previously sterile filtered.If necessary, such a solution can be stored in a sterile ampoule, and can be reconstituted by adding sterile water, ready for injection.

[0086] Nebulized or aerosolized formulations are also suitable. Methods of intranasal administration are well known in the art, including administering droplets, sprays, or dry powder forms of the composition to the nasopharynx of the individual to be treated from a pressurized container or dispenser containing a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer (see, e.g., WO 95 / 11664). Enteric formulations, such as gastroresistant capsules and granules for oral administration, and suppositories for rectal or vaginal administration, are also suitable. For parenteral administration, the composition may contain an absorption enhancer that increases the pore size of the mucous membrane. Such absorption enhancers include sodium deoxycholate, sodium glycocholate, dimethyl-β-cyclodextrin, lauroyl-1-lysophosphatidylcholine, and other substances that are structurally similar to the phospholipid domains of the mucous membrane.

[0087] The composition may also include other pharmacologic acceptable excipients to provide desired pharmaceutical or pharmacodynamic properties, such as, for example, pH, osmolality, viscosity, transparency, color, sterility, stability, dissolution rate, and control or maintenance of release or absorption in the human or animal body of the formulation.For example, polymers such as polyethylene glycol can be used to obtain desirable properties, such as solubility, stability, half-life, and other pharmacologic advantageous properties (Davis et al., 1978, Enzyme Eng. 4, 169-173; Burnham et al., 1994, Am. J. Hosp. Pharm. 51, 210-218).Representative examples of stabilizing components include polysorbate 80, L-arginine, polyvinylpyrrolidone, trehalose, and combinations thereof. Other stabilizing components that are particularly suitable for plasmid-based compositions include hyaluronidase (which is believed to destabilize the extracellular matrix of the host cell, as described in WO 98 / 53853), protic compounds such as chloroquine, propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl-L-2-pyrrolidone or a derivative thereof, aprotic compounds such as dimethyl sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile (see EP 890 362), nuclease inhibitors such as actin G (WO 99 / 56784), magnesium (Mg 2+ ) (EP 998 945) and lithium (Li +) (WO 01 / 47563), and any derivatives thereof. The amount of cationic salt in the compositions herein is preferably in the range of about 0.1 mM to about 100 mM, more preferably about 0.1 mM to about 10 mM. Viscosity enhancers include sodium carboxymethylcellulose, sorbitol, dextran, and the like. The compositions may also include substances known in the art to promote penetration or transport across blood barriers or membranes of certain organs (e.g., antibodies against transferrin receptors; Friden et al., 1993, Science 259, 373-377). To facilitate administration to arterial cells, gel complexes of polylysine and lactose (Midoux et al., 1993, Nucleic Acid Res. 21, 871-878) or poloxamer 407 (Pastore, 1994, Circulation 90, 1-517) can be used.

[0088] The viral particles and pharmaceutical compositions can be administered to a patient in a therapeutically effective amount. As used herein, the term "therapeutically effective amount" refers to an amount sufficient to achieve a desired biological effect. For example, a therapeutically effective amount for treating Angelman syndrome is an amount sufficient to improve one or more symptoms of Angelman syndrome described herein (e.g., developmental delay, severe cognitive impairment, ataxic gait, frequent seizures, short attention span, aphasia, and characteristic happy attitude). In addition, AS iPSC-derived neurons show depolarization of resting membrane potential, delayed action potential development, and reduced spontaneous synaptic activity. Thus, a therapeutically effective amount for treating AS can restore the resting membrane potential of a neuronal cell to about -70mV, improve action potential onset delay, increase spontaneous synaptic activity, or improve further changes in the phenotype of a neuronal cell with respect to rheobase, action potential properties (e.g., shape), membrane current, synaptic potential, ion channel conductance, etc.

[0089] The appropriate dosage may vary according to known factors such as the pharmacodynamic properties of the particular active agent, the age, health and weight of the host organism; the condition to be treated, the nature and extent of symptoms, the type of concurrent treatment, the frequency of treatment, the need for prophylaxis or therapy, and / or the desired effect. The dosage is also calculated according to the particular route of administration selected. Further refinement of the calculations required to determine the appropriate dosage for treatment can be performed by the attending physician in light of the relevant circumstances. As a general guideline, compositions based on viral particles may be administered, for example, at least 10 per cell. 5 The composition may be formulated to administer a viral genome of 10 ...

[0090] The pharmaceutical composition herein can be enclosed in glass or plastic ampoules, disposable injection syringes or multiple dose vials.In all cases, the composition should be sterile and fluid enough to be easily squirted.It should be stable under the conditions of manufacture and storage, and preserved against the contaminating action of microorganisms such as bacteria and fungi.Sterile injection solution can be prepared by incorporating the required amount of active agent (e.g., infectious particles) with one or a combination of the components listed above, and then sterilizing by filtration.

[0091] The viral particles and pharmaceutical compositions herein can be administered by parenteral routes, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, e.g., intracerebral or intraventricular administration. In one embodiment, the viral particles or pharmaceutical compositions herein are administered intracerebrally or intraventricularly. In one embodiment, the viral particles or pharmaceutical compositions herein are administered intradermally.

[0092] In one embodiment, the viral particles and pharmaceutical compositions described above are administered to a subject by subcranial injection into the brain or spinal cord of a patient or human in need thereof. In one embodiment, the use of subcranial administration to the brain results in direct administration of the coding nucleotide sequence described herein to brain cells, including glia and neurons. As used herein, the term "neuron" refers to cells involved in or associated with brain function. This term can refer to any one type of neuron, including unipolar, bipolar, multipolar, and pseudounipolar. EXAMPLES

[0093] Generation of shRNA vectors and preparation of lentivirus Oligonucleotides encoding shRNAs were cloned into the pLKO.1-puro vector, which expresses small RNAs driven by the U6 promoter (Addgene plasmid #8453). Specifically, polynucleotides for generating shRNAs encompassed the specific 21 nucleotide sequence of interest and its reverse complement, separated by a loop sequence of CTCGAG, with a 5' flanking sequence of CCGG and a 3' flanking sequence of TTTTTG added for cloning into the plasmid vector. The following oligonucleotides encoding shRNAs as well as a scrambled shRNA control were used: [ka] Cloning was verified by Sanger sequencing. Lentiviral particles were produced from the cloned shRNA in HEK293T cells using second-generation lentiviral packaging plasmids (psPAX2, Addgene plasmid #12260; pMD2.G, Addgene plasmid #12259) and concentrated using a LentI-X Concentrator KIt (Takara). Lentiviral titers were estimated using a qPCR kit detecting the 5'LTR (Applied Biological Materials).

[0094] Stem cell culture and neural differentiation Angelman syndrome (AS) induced pluripotent stem cells (iPSCs) and human embryonic stem cells (hESCs) were maintained under feeder-free conditions on Matrigel-coated substrates (Corning) in mTeSR-plus medium (Stem Cell Technologies). iPSCs / hESCs were cultured in a humidified incubator at 37°C and 5% CO2. Cells were fed daily and passaged every 4–5 days with 0.5 mM EDTA. Glutamatergic neurons were generated from iPSCs / hESCs by doxycycline-inducible expression of the human neurogenin 2 (NGN2) transgene (Fernandopulle et al., 2018, Curr Protoc Cell Biol. 79(1): e51). Briefly, a doxycycline-inducible NGN2 construct was stably integrated into the safe harbor AAVS1 locus of AS iPSCs / hESCs using a pair of AAVS1-targeting TALENS, and then clonal cell lines were obtained. These iPSCs / hESCs were cultured in neural induction medium consisting of DMEM / F12, N2 supplement, non-essential amino acids (NEAA), L-glutamine (all Gibco products), and 2 μg / mL doxycycline for 3 days for neural induction. They were then plated for terminal maturation in cortical neuron medium consisting of DMEM / F12, Neurobasal medium, B27 supplement, penicillin / streptomycin (all Gibco products), BDNF (10 ng / mL), GDNF (10 ng / mL), NT-3 (10 ng / mL), and laminin (1 ug / mL). Human iPSC / ESC-derived NGN2-induced neurons (7–10 days after induction) were transduced with lentiviral particles at an MOI of 10.

[0095] Quantitative RT-PCR (qRT-PCR) analysis Neurons were harvested for RNA isolation and qRT-PCR 7 days after viral transduction. Total RNA was isolated from iPSC-derived neurons using RNA-STAT60 (AMS Biotechnology) according to the manufacturer's protocol. cDNA was generated using the High Capacity cDNA Reverse Transcription Kit (Life Technologies). Gene expression analysis was performed at least in triplicate. All qPCR assays were performed using TaqMan Gene Expression Assays (Life Technologies). Ct values ​​of each gene were normalized to the housekeeping gene GAPDH. Relative expression was calculated as 2^ relative to the calibrator sample. -ΔΔCt was quantified as:

[0096] Data Summary and Results AS iPSC-derived neurons were transduced with lentiviral particles to express selected shRNA sequences targeting SNHG14 long non-coding RNA. qRT-PCR was used to measure the expression of SNORD115 host genes UBE3A-ATS and UBE3A in neurons treated with SNHG14-shRNA versus neurons treated with non-targeting control shRNA (SCRAM). Figures 4 and 5 show qRT-PCR analysis of AS iPSC-derived neurons treated with SNHG14-targeting shRNAs (551-2, ATS shRNA1-4, ATS shRNA2_3G) or non-targeting control shRNA (SCRAM). Expression of UBE3A-ATS, UBE3A, and SNORD115 was normalized to the housekeeping gene GAPDH, and expression is shown relative to SCRAM-shRNA-treated neurons. Error bars are standard error of the mean, n=3 biological replicates. As shown in Figures 4 and 5, selected SNHG14 shRNAs effectively reduced UBE3A-ATS (55%-60% reduction) and SNORD115 (45%-50% reduction) RNA levels compared to SCRAM controls. This reduction in SNHG14 transcript levels was associated with a robust increase in UBE3A RNA (5- to 9-fold increase over SCRAM controls). ATS shRNA-1, ATS shRNA-3, and ATS shRNA-4 were predicted to reduce UBE3A-ATS and SNORD115 RNA levels and increase UBE3A expression, but had no such effect.

[0097] It is understood that the above detailed description and the accompanying examples are merely illustrative and are not to be considered as limitations on the scope of the subject matter described herein, which is defined solely by the appended claims and their equivalents.Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art.Such changes and modifications can be made without departing from the spirit and scope thereof, including but not limited to chemical structures, substituents, derivatives, intermediates, synthesis, compositions, formulations, or methods of use.

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Claims

【Request 1】 【Chemistry 1】 (SEQ ID NO:2).

2. An expression vector comprising the polynucleotide sequence of claim 1.

3. The expression vector of claim 2 further comprising a promoter.

4. The expression vector of claim 3 , wherein the promoter is a neuron-specific promoter.

5. The neuron-specific promoter is selected from the group consisting of neuron-specific enolase (NSE), synapsin I (Syn), and Ca ２+ The expression vector of claim 4, wherein the gene is CaM-activated protein kinase IIα (CaMKIIα).

6. The expression vector of claim 3 , wherein the promoter is a U6 promoter or an H1 promoter.

7. The expression vector of claim 2 , wherein the expression vector is an adeno-associated virus (AAV) vector or a lentivirus vector.

8. 8. The expression vector of claim 7, wherein the expression vector is AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10.

9. 10. A pharmaceutical composition comprising the polynucleotide sequence of claim 1 and a pharma- ceutically acceptable carrier.

10. The pharmaceutical composition of claim 9 , wherein the polynucleotide sequence is contained within an expression vector.

11. The pharmaceutical composition of claim 10, wherein the expression vector is an AAV vector or a lentiviral vector.

12. A polynucleotide encoding an shRNA comprising a nucleotide sequence that is at least 85%, at least 90%, at least 95% or 100% complementary to an RNA encoded by any of SEQ ID NOs: 3-489.

13. The polynucleotide of claim 12, wherein the polynucleotide is SEQ ID NO:

2.

14. The polynucleotide of claim 12, wherein the shRNA causes activation or increased expression of paternally derived UBE3A.

15. The polynucleotide of claim 12, wherein the shRNA causes a decrease in expression of paternally derived UBE3A-ATS.

16. An expression vector comprising the polynucleotide of claim 12 and a promoter.

17. The expression vector of claim 14 , wherein the promoter is a neuron-specific promoter.

18. The neuron-specific promoter is selected from the group consisting of neuron-specific enolase (NSE), synapsin I (Syn), and Ca ２＋ 18. The expression vector of claim 17, wherein the gene is CaM-activated protein kinase IIα (CaMKIIα).

19. 17. The expression vector of claim 16, wherein the promoter is a U6 promoter or an H1 promoter.

20. 17. The expression vector of claim 16, wherein the expression vector is an adeno-associated virus (AAV) vector or a lentivirus vector.

21. 21. The expression vector of claim 20, wherein the expression vector is AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9 or AAV10.

22. 17. The expression vector of claim 16, wherein the polynucleotide is a DNA polynucleotide.

23. 13. A pharmaceutical composition comprising the polynucleotide sequence of claim 12 and a pharma- ceutically acceptable carrier.

24. 24. The pharmaceutical composition of claim 23, wherein the polynucleotide sequence is contained within an expression vector.

25. 25. The pharmaceutical composition of claim 24, wherein the expression vector is an AAV vector or a lentiviral vector.

26. 13. A method for treating Angelman syndrome, comprising administering to a patient in need thereof the polynucleotide of claim 1.

27. The method for treating Angelman syndrome according to claim 26, wherein the polynucleotide encodes an shRNA that causes a decrease in expression of paternally derived UBE3A-ATS.

28. 27. The method for treating Angelman syndrome according to claim 26, wherein the polynucleotide encodes an shRNA that causes activation or increased expression of the paternally derived UBE3A gene.

29. A method for treating Angelman syndrome, comprising administering the polynucleotide of claim 12 to a patient in need thereof.

30. The method for treating Angelman syndrome according to claim 29, wherein the polynucleotide encodes an shRNA that causes a decrease in expression of paternally derived UBE3A-ATS.

31. 30. The method for treating Angelman syndrome according to claim 29, wherein the polynucleotide encodes an shRNA that causes activation or increased expression of the paternally derived UBE3A gene.

32. A polynucleotide comprising sequence number 2 encoding an shRNA, wherein the shRNA is capable of inhibiting silencing of paternally derived UBE3A.

33. A method for inhibiting silencing of the paternally derived UBE3A gene by an RNA antisense transcript encoded by SEQ ID NO:1, comprising administering to a patient in need thereof a polynucleotide described in claim 1 encoding an shRNA in an amount effective to cleave the RNA antisense transcript encoded by SEQ ID NO:

1.

34. 34. The method of claim 33, wherein the polynucleotide is contained within an expression vector.

35. 35. The method of claim 34, wherein the expression vector is an AAV vector or a lentiviral vector.

36. 34. The method of claim 33, wherein the polynucleotide is administered to the brain of the patient.

37. 34. The method of claim 33, wherein the polynucleotide is administered to a neuron of the patient.

38. The method of claim 33, wherein the shRNA reduces or terminates transcription of a polynucleotide comprising the sequence of SEQ ID NO:

1.

39. The method of claim 33, wherein the shRNA reduces the level of an RNA antisense transcript encoded by SEQ ID NO:

1.

40. A method for inhibiting silencing of the paternally derived UBE3A gene by an RNA antisense transcript encoded by SEQ ID NO:1, comprising administering to a patient in need thereof a polynucleotide described in claim 12 encoding an shRNA in an amount effective to cleave the RNA antisense transcript encoded by SEQ ID NO:

1.

41. 41. The method of claim 40, wherein the polynucleotide is contained within an expression vector.

42. 42. The method of claim 41, wherein the expression vector is an AAV vector or a lentiviral vector.

43. 41. The method of claim 40, wherein the polynucleotide is administered to the brain of the patient.

44. 41. The method of claim 40, wherein the polynucleotide is administered to a neuron of the patient.

45. The method of claim 40, wherein the shRNA reduces or terminates transcription of a polynucleotide comprising the sequence of SEQ ID NO:

1.

46. The method of claim 40, wherein the shRNA reduces the level of an RNA antisense transcript encoded by SEQ ID NO:

1.

47. The polynucleotide of claim 1, for use in treating Angelman syndrome, for use in activating paternally derived UBE3A, or for use in inhibiting silencing of the paternally derived UBE3A gene by an RNA antisense transcript encoded by SEQ ID NO:

1.

48. The polynucleotide of claim 12, for use in treating Angelman syndrome, for use in activating paternally derived UBE3A, or for use in inhibiting silencing of the paternally derived UBE3A gene by an RNA antisense transcript encoded by SEQ ID NO:

1.

49. Use of the polynucleotide described in claim 1 in the manufacture of a medicament for the treatment of Angelman syndrome, for inhibiting activation of paternally derived UBE3A or silencing of the paternally derived UBE3A gene by an RNA antisense transcript encoded by SEQ ID NO:

1.

50. Use of the polynucleotide described in claim 12 in the manufacture of a medicament for the treatment of Angelman syndrome, for activating paternally derived UBE3A or inhibiting silencing of the paternally derived UBE3A gene by an RNA antisense transcript encoded by SEQ ID NO:

1.

51. An shRNA encoded by a portion of SEQ ID NO:2, which portion of SEQ ID NO:2 defines a first segment defined by bolded nucleotides, the first segment being shortened by one, two, three or four nucleotides at either end of the first segment, and a second segment defined by italicized nucleotides, the second segment being shortened by one, two or three nucleotides at either end of the italicized nucleotides.

52. The shRNA of claim 51, wherein the shRNA is encoded by SEQ ID NO:494, SEQ ID NO:495, SEQ ID NO:496, SEQ ID NO:497, SEQ ID NO:498, SEQ ID NO:499, SEQ ID NO:500 or SEQ ID NO:

501.

53. 【Chemical 2】 (SEQ ID NO:506), wherein nnnnnnnn can be CTCGAG (SEQ ID NO:490), TCAAGAG (SEQ ID NO:491), TTCG (SEQ ID NO:492) or GAAGCTTG (SEQ ID NO:493).

54. A polynucleotide sequence comprising a first portion, a second portion and a third portion, wherein the first portion comprises any of SEQ ID NOs:3-489, the second portion comprises any of SEQ ID NOs:490, 491, 492 or 493, and the third portion comprises a nucleotide sequence complementary to a nucleotide sequence of SEQ ID NOs:3-489, respectively.

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