Compositions and methods for restoring paternal UBE3A gene expression in human happy puppet syndrome

Abstract

The present application relates to compositions and methods for restoring paternal UBE3A gene expression in human happy puppet syndrome. The present invention relates to compositions and methods for activating paternal genetic allele expression of UBE3A in human happy puppet syndrome (Angelman's Syndrome) neurons using viral vector delivery of short hairpin RNA, ribozyme, and / or micro RNA.

Description

[0001] This application is a divisional application of an application filed on September 20, 2019, with application number 201980076741.9, and invention name “Compositions and methods for restoring paternal UBE3A gene expression in human happy puppet syndrome”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims priority to U.S. Provisional Patent Application No. 62 / 734,435, filed on September 21, 2018, the entire contents of which are fully incorporated herein by reference. Technical Field

[0004] The present disclosure relates to compositions and methods for using short hairpin RNA, ribozymes and / or microRNA to activate expression of the paternally inherited allele of UBE3A in human Angelman Syndrome neurons. Background of the Invention

[0006] Happy Marionette Syndrome (AS) is a neurodevelopmental disorder that affects ~1 / 15,000 individuals. Individuals with AS have developmental delay, severe cognitive impairment, ataxic gait, frequent seizures, short attention span, absent speech, and characteristic happy demeanor. Neurons derived from induced pluripotent stem cells (iPSCs) from AS patients exhibit depolarized resting membrane potentials, delayed action potential development, and reduced spontaneous synaptic activity. AS affects a relatively large patient population; a contact registry of >3,000 patients has been established, and ~250 new AS diagnoses are made each year. Individuals with AS require lifelong care.

[0007] AS is caused by loss of function of the maternal copy of UBE3A, a gene encoding an E3 ubiquitin ligase. This loss-of-function mutation can be caused by any type of genetic mutation in the maternal allele. UBE3A is expressed exclusively by the maternal allele in neurons. All individuals with AS have a normal paternal UBE3A allele that is epigenetically silenced by a long noncoding RNA called the UBE3A antisense transcript (UBE3A-ATS). In a mouse model of AS, reactivation of the paternal allele was shown to restore UBE3A protein expression and alleviate behavioral deficits. Restoration of UBE3A expression in humans holds promise for disease amelioration, particularly if restored in infants.

[0008] There is no cure for Happy Marionette syndrome, however, pharmaceutical companies and academic laboratories are pursuing two approaches to cure this disease. The first is to use antisense oligonucleotides (ASOs) to cut UBE3A-ATS and activate paternal UBE3A. The second approach is AAV-mediated gene therapy to introduce the UBE3A gene back into the patient. The ASOs mentioned above cannot cross the blood-brain barrier and require repeated injections into the spinal cord throughout life. Gene therapy that introduces the UBE3A gene back into neurons lacks the ability to regulate UBE3A mRNA and protein levels, and requires the selection of a single protein isoform from a total of three protein isoforms, where the function of the individual isoforms remains uncertain. This may lead to overexpression of UBE3A, which may also lead to another related disease, Dup15q syndrome, as well as the absence of important protein isoforms.

[0009] Brief Summary of the Invention

[0010] The present invention provides a new method for treating AS by gene therapy by inhibiting the silencing of paternal UBE3A and expressing paternal UBE3A from its native regulatory elements. Increased UBE3A expression in neurons can improve the effects of UBE3A-ATS on the neurons listed above. All three protein isoforms can be expressed using endogenous regulatory elements that normally control their levels. Therefore, overexpression is prevented due to the use of native regulatory elements. This approach can improve AS symptoms with a single treatment and thereby avoid repeated spinal injections required for ASOs.

[0011] In one aspect, the present invention provides a polynucleotide comprising a first nucleotide sequence encoding a short hairpin RNA (shRNA), a ribozyme or a microRNA, wherein the shRNA, the ribozyme or the microRNA is capable of inhibiting the silencing of a paternal UBE3A.

[0012] In another aspect, the present invention provides an expression vector comprising a polynucleotide having a first nucleotide sequence encoding shRNA, ribozyme or microRNA and a promoter, wherein the shRNA, ribozyme or microRNA is capable of inhibiting the silencing of paternal UBE3A.

[0013] In another aspect, the present invention provides a viral particle comprising a polynucleotide having a first nucleotide sequence encoding a shRNA, a ribozyme or a microRNA, wherein the shRNA, the ribozyme or the microRNA is capable of inhibiting the silencing of a paternal UBE3A.

[0014] In another aspect, the present invention provides a pharmaceutical composition comprising a polynucleotide having a first nucleotide sequence encoding shRNA, ribozyme or microRNA or a viral particle comprising the polynucleotide and a pharmaceutically acceptable carrier, wherein the shRNA, ribozyme or microRNA is capable of inhibiting the silencing of paternal UBE3A.

[0015] In another aspect, the present invention provides a method for treating Happy Puppet Syndrome, comprising administering a therapeutically effective amount of a viral particle or a pharmaceutical composition thereof to a patient in need thereof, wherein the viral particle comprises a polynucleotide having a first nucleotide sequence encoding a shRNA, a ribozyme or a microRNA, wherein the shRNA, the ribozyme or the microRNA is capable of inhibiting the silencing of the paternal UBE3A.

[0016] In another aspect, the present invention provides a method for activating or increasing the expression of the paternal UBE3A gene, the method comprising administering a therapeutically effective amount of a viral particle or a pharmaceutical composition thereof to a patient in need thereof, the viral particle comprising a polynucleotide having a first nucleotide sequence encoding a shRNA, a ribozyme or a microRNA, wherein the shRNA, the ribozyme or the microRNA is capable of inhibiting the silencing of the paternal UBE3A.

[0017] In another aspect, the present invention provides a method for inhibiting paternal UBE3A gene silencing caused by RNA antisense transcripts encoded by SEQ ID NO: 1, the method comprising administering a therapeutically effective amount of a viral particle or a pharmaceutical composition thereof to a patient in need thereof, the viral particle comprising a polynucleotide having a first nucleotide sequence encoding a short hairpin RNA (shRNA), a ribozyme or a microRNA, wherein the shRNA, the ribozyme or the microRNA is capable of inhibiting the silencing of the paternal UBE3A.

[0018] In another aspect, the present invention provides shRNAs, ribozymes or microRNAs encoded by the polynucleotides described herein and capable of inhibiting silencing of paternal UBE3A.

[0019] The present invention also provides the following items:

[0020] 1. A polynucleotide comprising a first nucleotide sequence encoding a short hairpin RNA (shRNA), a ribozyme or a microRNA, wherein the shRNA, the ribozyme or the microRNA is capable of inhibiting the silencing of a paternal UBE3A.

[0021] 2. The polynucleotide according to item 1, wherein the first nucleotide sequence encodes the shRNA.

[0022] 3. The polynucleotide according to item 2, wherein the shRNA comprises a second nucleotide sequence that is at least 85%, at least 90%, at least 95% or 100% complementary to the RNA encoded by SEQ ID NO: 5 or any one of SEQ ID NOs: 9-508.

[0023] 4. The polynucleotide according to item 2 or 3, wherein the first nucleotide sequence is SEQ ID NO: 2.

[0024] 5. The polynucleotide according to item 1, wherein the first nucleotide sequence encodes the ribozyme.

[0025] 6. The polynucleotide according to item 5, wherein the ribozyme comprises a second nucleotide sequence that is at least 85%, at least 90%, at least 95% or 100% complementary to the RNA encoded by any one of SEQ ID NO: 6 or SEQ ID NO: 7.

[0026] 7. The polynucleotide according to item 5, wherein the first nucleotide sequence is SEQ ID NO: 3 or SEQ ID NO: 4.

[0027] 8. The polynucleotide according to item 1, wherein the first nucleotide sequence encodes the microRNA.

[0028] 9. The polynucleotide according to item 8, wherein the microRNA comprises a second nucleotide sequence that is at least 85%, at least 90%, at least 95% or 100% complementary to the RNA encoded by SEQ ID NO: 5 or any one of SEQ ID NOs: 9-508.

[0029] 10. The polynucleotide according to item 8 or 9, wherein the first nucleotide sequence is SEQ ID NO: 8.

[0030] 11. The polynucleotide according to any one of items 1 to 10, wherein the shRNA, ribozyme or microRNA increases the expression of paternal UBE3A.

[0031] 12. A polynucleotide according to any one of items 1-11, wherein the paternal UBE3A comprises the nucleotide sequence of SEQ ID NO: 509.

[0032] 13. The polynucleotide according to any one of items 1 to 12, wherein the silencing of the paternal UBE3A is caused by the RNA transcript encoded by SEQ ID NO: 1.

[0033] 14. An expression vector comprising the polynucleotide according to any one of items 1 to 13 and a promoter.

[0034] 15. The expression vector according to item 14, wherein the promoter is a U6 promoter.

[0035] 16. The expression vector according to item 14 or 15, wherein the polynucleotide is a DNA polynucleotide.

[0036] 17. The expression vector according to any one of items 14-16, wherein the expression vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

[0037] 18. A viral particle comprising the polynucleotide according to any one of items 1-13.

[0038] 19. The viral particle according to item 18, wherein the particle is an AAV particle or a lentiviral particle.

[0039] 20. The viral particle according to item 19, wherein the particle is an AAV particle with obligate neurotropism.

[0040] 21. The viral particle according to item 19, wherein the particle is an AAV9 particle or an AAV10 particle.

[0041] 22. A pharmaceutical composition comprising the polynucleotide according to any one of items 1 to 13 or the viral particle according to any one of items 18 to 21 and a pharmaceutically acceptable carrier.

[0042] 23. A method for treating Happy Puppet Syndrome, comprising administering a therapeutically effective amount of the viral particle according to any one of items 18 to 21 or the pharmaceutical composition according to item 22 to a patient in need thereof.

[0043] 24. A method for activating or increasing the expression of a paternal UBE3A gene, the method comprising administering a therapeutically effective amount of the viral particle according to any one of items 18 to 21 or the pharmaceutical composition according to item 22 to a patient in need thereof.

[0044] 25. A method for inhibiting paternal UBE3A gene silencing caused by RNA antisense transcripts encoded by SEQ ID NO: 1, the method comprising administering to a patient in need thereof an amount of the viral particle according to any one of items 18 to 21 or the pharmaceutical composition according to item 22, the amount being effective for cleaving the RNA antisense transcripts encoded by SEQ ID NO: 1.

[0045] 26. The method according to any one of items 23-25, wherein the viral particle or the pharmaceutical composition is administered to the brain of the patient.

[0046] 27. The method according to any one of items 23-25, wherein the viral particles or the pharmaceutical composition are administered to neurons of the patient.

[0047] 28. A method according to any one of items 23-27, wherein the patient is a human.

[0048] 29. The method according to any one of items 23-28, wherein the shRNA, ribozyme or microRNA inhibits the silencing of the paternal UBE3A gene caused by the RNA antisense transcript encoded by the SEQ ID NO: 1.

[0049] 30. The method according to any one of items 23-29, wherein the shRNA, ribozyme or microRNA terminates the transcription of a polynucleotide comprising the sequence of SEQ ID NO: 1.

[0050] 31. The method according to any one of items 23-30, wherein the shRNA, ribozyme or microRNA reduces the level of the RNA antisense transcript encoded by SEQ ID NO: 1.

[0051] 32. The method according to any one of items 23-31, wherein the shRNA, ribozyme or microRNA cleaves the RNA antisense transcript encoded by SEQ ID NO: 1.

[0052] 33. The polynucleotide according to any one of items 1 to 13, the viral particle according to any one of items 18 to 21 or the pharmaceutical composition according to item 22 for use in treating Happy Puppet Syndrome, for activating paternal UBE3A or for inhibiting silencing of the paternal UBE3A gene caused by an RNA antisense transcript encoded by SEQ ID NO: 1.

[0053] 34. Use of the polynucleotide described in any one of items 1 to 13, the viral particle described in any one of items 18 to 21, or the pharmaceutical composition described in item 22 in the preparation of a medicament for treating Happy Puppet Syndrome, for activating paternal UBE3A, or for inhibiting the silencing of the paternal UBE3A gene caused by the RNA antisense transcript encoded by SEQ ID NO: 1.

[0054] 35. A shRNA, ribozyme or microRNA, which is encoded by the polynucleotide according to any one of items 1 to 13. BRIEF DESCRIPTION OF THE DRAWINGS

[0056] Figure 1 Chromosomal mutations in Happy Marionette syndrome are shown.

[0057] Figure 2 A schematic representation of the paternal UBE3A gene is shown.

[0058] Figure 3A Hammerhead ribozyme cleaves UBE3A-ATS in vitro. Hammerhead ribozyme and UBE3A-ATS DNA were transcribed in vitro using PCR. The resulting ribozyme and UBE3A-ATS RNA were incubated in vitro (cell-free and vector-free), and cleavage was visualized on an agarose gel.

[0059] Figure 3B Hammerhead ribozymes are shown to cleave UBE3A-ATS in human AS iPSC-derived neurons.

[0060] Figure 4 Shown are shRNA knockdown of UBE3A-ATS and activation of paternal UBE3A. RT-qPCR was performed in AS iPSC-derived neurons transduced with lentivirus to express shRNA targeting UBE3A-ATS. One of the three shRNAs knocked down both UBE3A-ATS and activated paternal UBE3A.

[0061] Figure 5 AS neurons transduced with AAV9 particles carrying 551 shRNA2 are shown. The AAV vector also carries EGFP, allowing the neurons to emit green fluorescence after transduction. These neurons were transduced with 1X10 5 viral genomes / cell transduction.

[0062] Figure 6 The plasmid map of pAV-U6-GFP, the AAV vector 551 used for cloning shRNA2, is shown. DETAILED DESCRIPTION OF THE INVENTION

[0064] UBE3A

[0065] UBE3A is a gene encoding an E3 ubiquitin ligase. The genomic coordinates of UBE3A on the negative strand are hg19chr15:25,582,381-25,684,175. There are three normal isoforms of UBE3A: isoform 1 (accession number X98032); isoform 2 (accession number X98031); and isoform 3 (accession number X98033). In neurons, UBE3A is expressed exclusively by the maternal allele. The paternal UBE3A allele is epigenetically silenced by a long noncoding RNA called UBE3A antisense transcript (UBE3A-ATS), encoded by SEQ ID NO:1. The genomic coordinates of UBE3A-ATS on the positive strand are hg19 chr15:25,223,730-25,664,609. The following genomic coordinates are of particular interest: hg19 chr15:25,522,751-25,591,391 on the plus strand.

[0066] The compositions and methods of the present invention are intended to target the UBE3A antisense transcript (UBE3A-ATS) to relieve the silencing of the paternal UBE3A allele. Effective inhibition of UBE3A-ATS by short hairpin RNA (shRNA), ribozymes or microRNA can lead to a decrease in the expression level of UBE3A-ATS and a concomitant increase in the expression level of the paternal UBE3A allele.

[0067] In several aspects of the invention, the compositions and methods herein relate to the treatment or prevention of AS. In certain aspects of any of the foregoing embodiments of the method for treating a patient or human being in need, the patient or human being in need suffers from AS or is at risk of developing AS. As used herein, the term "patient in need" includes any mammal in need of these treatment or prevention methods, especially including humans. The subject can be male or female. In certain aspects, patients in need of AS treated according to the methods provided herein can show improvements in anxiety, learning, balance, motor function and / or epileptic seizures, or the method can restore the resting membrane potential of neurons to about -70mV, improve delayed action potential development, increase spontaneous synaptic activity, and can improve additional changes in neuronal phenotypes related to base strength, action potential characteristics (e.g., shape), membrane current, synaptic potential, ion channel conductance, etc.

[0068] shRNA, ribozymes, microRNAs, and coding sequences

[0069] According to one aspect of the present invention, the polynucleotide comprises a first nucleotide sequence encoding a short hairpin RNA (shRNA), a ribozyme or a microRNA, which results in reduced expression of the UBE3A-ATS sequence SEQ ID NO: 1. For example, a portion of the shRNA, ribozyme or microRNA may be complementary to the RNA sequence encoded by SEQ ID NO: 1 or a sequence contained therein.

[0070] ShRNA, ribozyme and microRNA are RNA polynucleotides encoded by the first nucleotide sequence. The polynucleotide comprising the first nucleotide sequence can be a DNA polynucleotide suitable for cloning into an appropriate vector (e.g., plasmid) for cultivation and subsequent production of viral particles. Then, the viral particles can include a DNA polynucleotide with a nucleotide coding sequence in a form suitable for infection. Therefore, the first nucleotide sequence can be a DNA sequence cloned into a plasmid for viral particle production or encapsulated in the viral particles. Because the retrovirus carries the nucleotide coding sequence in the form of RNA polynucleotides, the retroviral particles (e.g., slow virus) include RNA polynucleotides, and the RNA polynucleotides include the first nucleotide sequence as the corresponding RNA sequence.

[0071] The first nucleotide sequence may encode an shRNA. For example, the first nucleotide sequence may be SEQ ID NO: 2. The first nucleotide sequence may also be a modified SEQ ID NO: 2 in which the bold nucleotides in SEQ ID NO: 2 are replaced by any one of SEQ ID NO: 9-508, and the italic nucleotides in SEQ ID NO: 2 are replaced by nucleotides complementary to those in SEQ ID NO: 9-508.

[0072] The first nucleotide sequence may encode a ribozyme. For example, the first nucleotide sequence may be SEQ ID NO: 3. The first nucleotide sequence may be SEQ ID NO: 4.

[0073] The first nucleotide sequence may encode a microRNA. For example, the first nucleotide sequence may be SEQ ID NO: 8. The first nucleotide sequence may also be a modified SEQ ID NO: 8 in which the bold nucleotides in SEQ ID NO: 8 are replaced by any one of SEQ ID NO: 9-508, and the italic nucleotides in SEQ ID NO: 8 are replaced by nucleotides complementary to those in SEQ ID NO: 9-508. The italic nucleotides and the nucleotides complementary to those in SEQ ID NO: 9-508 may be less than 100% complementary.

[0074] As used herein, "target" means an effective RNA polynucleotide that is capable of hybridizing with a nucleotide sequence (such as a nucleotide sequence transcribed from a nucleotide sequence in a larger genomic sequence of UBE3A-ATS) by hydrogen bonding. The hybridization of an effective RNA polynucleotide with a nucleotide sequence transcribed from a nucleotide sequence in a larger genomic sequence of UBE3A-ATS can result in a decrease in the expression level of UBE3A-ATS in the presence of an effective RNA polynucleotide compared to the expression level of UBE3A-ATS in the absence of an effective RNA polynucleotide. Preferably, the effective RNA polynucleotide comprises a nucleotide sequence of an shRNA, ribozyme or microRNA complementary to an RNA sequence encoded in a larger genomic sequence of UBE3A-ATS. For example, the shRNA or microRNA comprises a nucleotide sequence complementary to an RNA sequence encoded by SEQ ID NO: 5 and SEQ ID NO: 9-508, and the ribozyme comprises a nucleotide sequence complementary to an RNA sequence encoded by SEQ ID NO: 6 or SEQ ID NO: 7. Thus, an effective RNA polynucleotide refers to the effective portion of the shRNA, ribozyme or microRNA after the shRNA, ribozyme or microRNA is assimilated into the target organism and processed into a functional state.

[0075] "Reduced expression" refers to a reduction or blockage of the expression or activity of UBE3A-ATS, and does not necessarily refer to a complete elimination of expression or activity. Mechanisms for reducing target expression include hybridization of an effective RNA polynucleotide to one or more target sequences transcribed from one or more sequences within the larger genomic UBE3A-ATS sequence (SEQ ID NO: 1), wherein the result or effect of the hybridization is target degradation or target occupancy, with subsequent arrest of cellular machinery involved, for example, in transcription or splicing.

[0076] Without wishing to be bound by a particular theory, the shRNA, ribozyme and microRNA of the present invention can inhibit the silencing of paternal UBE3A by: (1) cleaving the RNA transcript encoded by SEQ ID NO: 1; (2) reducing the steady-state level of the RNA transcript encoded by SEQ ID NO: 1 (i.e., the baseline level when in steady state); and (3) terminating the transcription of SEQ ID NO: 1. For example, cleavage and reduction of the steady-state level of the RNA transcript encoded by SEQ ID NO: 1 can occur through a mechanism involving the RNA-induced silencing complex (RISC). Both shRNA and microRNA can utilize RISC. After the vector carrying the genomic material of the shRNA or microRNA is integrated into the host genome, the shRNA or microRNA genomic material is transcribed into pri-microRNA in the host. The pri-microRNA is processed into pre-shRNA or pre-microRNA, respectively, by ribonucleases such as Drosha and exported from the nucleus. The pre-shRNA or pre-microRNA is processed by endoribonucleases such as Dicer to form small interfering RNA (siRNA) or microRNA, respectively. For siRNA, siRNA is loaded into RISC, wherein the sense strand is degraded and the antisense strand serves as the guide for guiding RISC to the complementary sequence in mRNA. For microRNA, the single strand from microRNA is loaded into RISC, serving as the guide for guiding RISC to the complementary sequence in mRNA. When the sequence has complete complementarity, RISC cuts mRNA, and when the sequence has incomplete complementarity, RISC represses the translation of mRNA. In another example, the steady-state level of RNA transcripts encoded by SEQ ID NO:1 can be cut and reduced by the mechanism involving ribozymes. The vector of the genomic material carrying ribozymes is integrated into the host genome, and it is transcribed into RNA in the host. RNA forms a secondary structure with a catalytic domain and a region complementary to the target mRNA. When ribozymes are combined with target mRNA, the catalytic domain cracks the target mRNA. Transcription of SEQ ID NO: 1 can be terminated by a torpedo mechanism, in which 5'-3' and 3'-5' exonucleases (e.g., XRN2) attach to the cleaved, uncapped ends of the target RNA being transcribed. The 5'-3' exonuclease captures the polymerase and dissociates the polymerase from the DNA. Thus, shRNAs, ribozymes, and microRNAs encoded by the first nucleic acid sequence can increase the expression of paternal UBE3A by reducing the steady-state level of UBE3A-ATS RNA.

[0077] As used herein, the term "nucleic acid" refers to a molecule comprising 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), short hairpin RNA (shRNA) and microRNA (miRNA). "Nucleotide" means a nucleoside having a phosphate group covalently attached to the sugar portion of a nucleoside. "Oligonucleotide" or "polynucleotide" means a polymer of connected nucleotides, each of which can be modified or unmodified independently of one another.

[0078] As used herein, "short hairpin RNA (shRNA)" includes conventional stem-loop shRNA, which forms a precursor microRNA (pre-miRNA). "shRNA" also includes shRNA embedded in microRNA (shRNA based on miRNA), in which the guide strand and passenger strand of the miRNA duplex are integrated into an existing (or natural) miRNA or into a modified or synthetic (designed) miRNA. When transcribed, conventional shRNA (i.e., not a miR-451 shRNA mimic) forms a primary miRNA (primary miRNA, pri-miRNA) or a structure very similar to natural pri-miRNA. The pri-miRNA is then processed into a pre-shRNA by Drosha and its cofactors. Therefore, the term "shRNA" includes pri-miRNA (shRNA-mir) molecules and pre-shRNA molecules.

[0079] "Stem-loop structure" refers to a nucleic acid having a secondary structure comprising a nucleotide region (stem portion) of a double strand or duplex connected by a region (loop portion) that is mainly a single-stranded nucleotide, which is known or predicted to be formed on one side. As known in the art, the loop portion is at least 4 nucleotides long, preferably 6 nucleotides long (e.g., the underlined sequence in SEQ ID NO:2). The terms "hair clip" and "fold-back" structure are also used to refer to the stem-loop structure in this article. Such a structure is well known in the art, and the use of the term is consistent with its known meaning in the art. As known in the art, the secondary structure does not require accurate base pairing. Therefore, the stem can include one or more base mispairings or protrusions. Alternatively, the base pairing can be accurate, i.e., does not include any mispairing.

[0080] In some embodiments, shRNAs useful in the present invention include, but are not limited to, modified shRNAs, including shRNAs with enhanced in vivo stability. Modified shRNAs include molecules containing nucleotide analogs, including those with additions, deletions, and / or substitutions in nucleobases, sugars, or backbones; and cross-linked or otherwise chemically modified molecules. One or more modified nucleotides may be within a portion of the shRNA molecule, or throughout the entire shRNA molecule. For example, the shRNA molecule may be modified, or contain modified nucleic acids in a region at its 5' end, its 3' end, or both, and / or in a guide strand, a passenger strand, or both, and / or in a nucleotide protruding from the 5' end, the 3' end, or both. (See, Crooke, U.S. Pat. Nos. 6,107,094 and 5,898,031; Elmen et al., U.S. Publication Nos. 2008 / 0249039 and 2007 / 0191294; Manoharan et al., U.S. Publication No. 2008 / 0213891; MacLachlan et al., U.S. Publication No. 2007 / 0135372; and Rana, U.S. Publication No. 2005 / 0020521; all of which are hereby incorporated by reference.)

[0081] In the present invention, the shRNA comprises a nucleotide sequence complementary to an RNA nucleotide sequence transcribed from a complete genomic UBE3A-ATS sequence (SEQ ID NO: 1), and inhibits the silencing of UBE3A-ATS to paternal UBE3A. In another embodiment, the shRNA comprises a nucleotide sequence complementary to an RNA sequence encoded by SEQ ID NO: 9-508. In a more specific embodiment, the shRNA comprises a nucleotide sequence complementary to an RNA sequence encoded by SEQ ID NO: 5. In a specific embodiment, the shRNA is encoded by the nucleotide sequence of SEQ ID NO: 2. In an embodiment of the present invention, the length of the nucleotide sequence contained in the shRNA and complementary to the RNA nucleotide sequence transcribed from the UBE3A-ATS gene is 17-21 nucleotides. The complementary nucleotides may be continuous or may be interspersed with non-complementary nucleotides. In some embodiments, the length of the complementary nucleotide sequence is 21 nucleotides, as indicated by the bold sequence in SEQ ID NO: 2. The shRNA may comprise a nucleotide sequence in which 17, 18, 19, 20 or 21 nucleotides are complementary to nucleotides in SEQ ID NO: 5 or 9-508. The 17, 18, 19, 20 or 21 complementary nucleotides may be continuous or may be interspersed with non-complementary nucleotides. The total length of the shRNA including the loop may be 40-50 nucleotides, preferably 44-48 nucleotides, more preferably 48 nucleotides.

[0082] As used herein, the term "ribozyme" refers to an RNA molecule that functions like an enzyme or a molecule composed of a protein comprising the RNA molecule, and is also referred to as an RNA enzyme or catalytic RNA. It has been found that ribozymes catalyze chemical reactions with RNA molecules having a clear tertiary structure and have catalytic or autocatalytic properties. Some ribozymes cleave themselves or other RNA molecules to inhibit activity, and other ribozymes catalyze the aminotransferase activity of ribosomes. Such ribozymes may include hammerhead ribozymes, VS ribozymes, hairpin ribozymes, etc. In the present invention, the ribozyme comprises a nucleotide sequence complementary to an RNA nucleotide sequence transcribed from a complete genomic UBE3A-ATS sequence (SEQID NO: 1), and inhibits the silencing of UBE3A-ATS to paternal UBE3A. In another embodiment, the ribozyme comprises a nucleotide sequence complementary to an RNA sequence encoded by SEQ ID NO: 6 and / or SEQ ID NO: 7. In another embodiment, the ribozyme is encoded by a nucleotide sequence of SEQ ID NO: 3 or 4. In SEQ ID NOs: 3 and 4, the bold sequence is complementary to SEQ ID NO: 6 or 7, and the underlined sequence represents the catalytic region of the ribozyme.

[0083] As used herein, the terms "microRNA", "miRNA" and "miR" are used synonymously to refer to non-coding RNAs of about 17-21 nucleotides (nt) long derived from endogenous genes. MicroRNAs are processed from longer (about 75nt) hairpin-like precursors called pre-miRNAs. MicroRNAs are assembled in ribonucleoprotein complexes called miRNPs and recognize their targets by antisense complementarity. If the microRNA matches its target 100%, that is, the complementarity is complete, the target mRNA is cleaved, and the miRNA acts like siRNA. If the match is incomplete, that is, the complementarity is partial, the translation of the target mRNA is blocked. In an embodiment of the present invention, the length of the nucleotide sequence contained in the microRNA and complementary to the RNA nucleotide sequence transcribed from the UBE3A-ATS gene is 17-21 nucleotides. Complementary nucleotides may be continuous or may be interspersed with non-complementary nucleotides. In some embodiments, the length of the complementary nucleotide sequence is 21 nucleotides, as indicated by the bold sequence in SEQ ID NO:8. The microRNA may comprise a nucleotide sequence wherein 17, 18, 19, 20 or 21 nucleotides are complementary to the nucleotides in SEQ ID NO:5 or 9-508. 17, 18, 19, 20 or 21 complementary nucleotides may be continuous or may be interspersed with non-complementary nucleotides. The total length of the precursor microRNA including the loop may be 50-1000 nucleotides, preferably 59-67 nucleotides, more preferably 67 nucleotides.

[0084] In the present invention, the microRNA is designed to target one or more nucleotide sequences transcribed from one or more nucleotide sequences within the complete genomic UBE3A-ATS sequence (SEQ ID NO: 1) and inhibit the silencing of the paternal UBE3A by UBE3A-ATS. In another embodiment, the microRNA is designed to target one or more sequences transcribed from one or more sequences selected from SEQ ID NO: 9-508. In a more specific embodiment, the microRNA targets a sequence transcribed from SEQ ID NO: 5. In another embodiment, the microRNA is encoded by the nucleotide sequence of SEQ ID NO: 8, wherein the bold sequence is complementary to SEQ ID NO: 5 or 9-508.

[0085] Methods for determining whether a sequence can specifically hybridize to a target nucleic acid are well known in the art. In certain embodiments, the shRNA, ribozyme or microRNA polynucleotides provided herein comprise a nucleic acid sequence that can specifically hybridize to an RNA sequence transcribed from UBE3A-ATS SEQ ID NO:1.

[0086] The shRNA or microRNA may comprise an RNA polynucleotide comprising a region of 17-21 linked nucleotides complementary to an RNA target sequence, wherein the RNA polynucleotide region is at least 85% complementary to a region of equal length to a UBE3A-ATS RNA nucleic acid sequence over its entire length. In certain aspects, the RNA polynucleotide region is at least 90%, at least 95%, or 100% complementary to a region of equal length to a UBE3A-ATS RNA nucleic acid sequence over its entire length.

[0087] The shRNA or microRNA may comprise a nucleotide sequence that is at least 85% complementary to an RNA sequence encoded by SEQ ID NO:5 or any one of SEQ ID NOs:9-508 and has an equal length. The shRNA or microRNA may comprise a nucleotide sequence that is at least 90% complementary to an RNA sequence encoded by SEQ ID NO:5 or any one of SEQ ID NOs:9-508 and has an equal length. The shRNA or microRNA may comprise a nucleotide sequence that is at least 95% complementary to an RNA sequence encoded by SEQ ID NO:5 or any one of SEQ ID NOs:9-508 and has an equal length. The shRNA or microRNA may comprise a nucleotide sequence that is 100% complementary to an RNA sequence encoded by SEQ ID NO:5 or any one of SEQ ID NOs:9-508 and has an equal length.

[0088] The ribozyme may comprise an RNA polynucleotide comprising two linked nucleotide regions complementary to an RNA target sequence separated by a catalytic region, wherein the total non-catalytic region of the RNA polynucleotide is at least 85% complementary to an equal length region of a UBE3A-ATS RNA nucleotide sequence over its entire length. In certain aspects, the total non-catalytic region of the RNA polynucleotide is at least 90%, at least 95%, or 100% complementary to an equal length region of a UBE3A-ATS RNA nucleotide sequence over its entire length. In an embodiment of the present invention, the length of the nucleotide sequence contained in the ribozyme and complementary to the RNA nucleotide sequence transcribed from the UBE3A-ATS gene is 17-21 nucleotides. The complementary nucleotides may be interspersed with non-complementary nucleotides. In some embodiments, the length of the complementary nucleotide sequence is 21 nucleotides, as indicated by the bold sequences in SEQ ID NO:3-4. The ribozyme may comprise a nucleotide sequence in which 17, 18, 19, 20, or 21 nucleotides are complementary to the nucleotides in SEQ ID NO:6-7. 17, 18, 19, 20 or 21 complementary nucleotides may be interspersed with non-complementary nucleotides.The total length of the shRNA including the catalytic loop may be 50-150 nucleotides, preferably 57-65 nucleotides, more preferably 59 nucleotides.

[0089] The ribozyme may comprise a nucleotide sequence that is at least 85% complementary to and has an equal length to the RNA sequence encoded by SEQ ID NO: 6 or 7. The ribozyme may comprise a nucleotide sequence that is at least 90% complementary to and has an equal length to the RNA sequence encoded by SEQ ID NO: 6 or 7. The ribozyme may comprise a nucleotide sequence that is at least 95% complementary to and has an equal length to the RNA sequence encoded by SEQ ID NO: 6 or 7. The ribozyme may comprise a nucleotide sequence that is 100% complementary to and has an equal length to the RNA sequence encoded by SEQ ID NO: 6 or 7.

[0090] In some aspects, shRNA, ribozyme or microRNA is a single-stranded RNA polynucleotide. In some aspects, RNA polynucleotide is a modified RNA polynucleotide. Complementarity percentage is used in this article in a conventional sense to refer to the base pairing between adenine and thymine, adenine and uracil (RNA) and guanine and cytosine.

[0091] Non-complementary nucleobases between the shRNA, ribozyme or microRNA and the UBE3A-ATS nucleotide sequence can be tolerated if the shRNA, ribozyme or microRNA remains capable of specific hybridization with the UBE3A-ATS nucleotide sequence. In addition, the shRNA, ribozyme or microRNA can hybridize within one or more segments of the UBE3A-ATS nucleotide sequence such that intervening or adjacent segments are not involved in the hybridization event (e.g., loop structures, mismatches or hairpin structures).

[0092] In certain embodiments, the shRNA, ribozyme or microRNA provided herein, or a specified portion thereof, is 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary to a UBE3A-ATS RNA nucleotide sequence, a UBE3A-ATS region, a UBE3A-ATS segment or a specified portion thereof, or is at least 70%, 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% complementary. The percent complementarity of the shRNA, ribozyme or microRNA to the UBE3A-ATS nucleotide sequence can be determined using conventional methods.

[0093] For example, an shRNA, ribozyme or microRNA in which 18 of its 20 nucleobases are complementary to the UBE3A-ATS region and will therefore specifically hybridize, represents 90% complementarity. In this example, the remaining non-complementary nucleobases may be clustered or interspersed with complementary nucleobases and need not be adjacent to each other or to complementary nucleobases. Thus, an shRNA, ribozyme or microRNA of 18 nucleobases in length, with four non-complementary nucleobases flanked by two regions that are fully complementary to the target nucleotide sequence, has an overall complementarity of 77.8% with the target nucleotide sequence, and therefore falls within the scope of the present invention. The percentage of complementarity of shRNA, ribozyme or microRNA to a region of the UBE3A-ATS nucleotide sequence can be routinely determined using the BLAST program (Basic Local Alignment Search Tool) and 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). The percentage of homology, sequence identity or complementarity can be determined, for example, by 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).

[0094] In certain embodiments, the shRNA, ribozyme or microRNA provided herein, or a specified portion thereof, is fully complementary (i.e., 100% complementary) to a specified portion of a UBE3A-ATS nucleotide sequence or its transcription product of SEQ ID NO: 1. For example, the shRNA, ribozyme or microRNA may be fully complementary to a UBE3A-ATS nucleotide sequence or a region thereof or a segment thereof or a sequence thereof. As used herein, "fully complementary" means that each nucleobase of the shRNA, ribozyme or microRNA is capable of precise base pairing with a corresponding RNA nucleobase transcribed from a UBE3A-ATS nucleotide sequence.

[0095] An effective concentration or dosage of the shRNA, ribozyme or microRNA can inhibit silencing of the paternal 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%.

[0096] The effective concentration or dosage of the shRNA, ribozyme or microRNA can terminate the 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%.

[0097] An effective concentration or dose of the shRNA, ribozyme or microRNA 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%.

[0098] An effective concentration or dosage of the shRNA, ribozyme or microRNA can cleave UBE3A-ATS and reduce 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%.

[0099] The effective concentration or dosage of the shRNA, ribozyme or microRNA can reduce the 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 increase the expression of the 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% or at least 100%.

[0100] As used herein, the terms "UBE3A-ATS" and "Ube3A-ATS" are used interchangeably and are not capitalized to indicate any particular species or interspecies homologs. "UBE3A" and "Ube3A" are used interchangeably and are not capitalized to indicate any particular species or interspecies homologs. In addition, "UBE3A", "UBE3A", "Ube3A", and "Ube3A" are used interchangeably without italics to indicate nucleic acids or proteins, unless otherwise specifically indicated.

[0101] Viral vectors

[0102] A "vector" is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment can be inserted so as to cause replication of the inserted segment. Typically, a vector is capable of replication when associated with appropriate control elements. Suitable vector backbones include, for example, those conventionally used in the art, such as a plasmid, a plasmid containing a viral genome, a virus, or an artificial chromosome. The term "vector" includes cloning and expression vectors, as well as viral vectors and integrating vectors.

[0103] It will be apparent to those skilled in the art that the term "viral vector" is used broadly to refer to nucleic acid molecules that include viral nucleic acid elements that typically facilitate transfer of nucleic acid molecules into cells or viral particles (e.g., transfer plasmids), which mediate transfer of nucleic acid sequences and / or integration of nucleic acid sequences into the cellular genome.

[0104] Viral vectors include structural and / or functional genetic elements that are primarily derived from viruses. Desirably, viral vectors are nontoxic, non-immunogenic, easy to produce, and effectively protect and deliver DNA or RNA to target cells. According to the compositions and methods of the present invention, viral vectors may include DNA encoding shRNA, ribozymes and / or microRNA of the present invention. In a specific embodiment, the viral vector is a lentiviral vector or an adeno-associated virus (AAV) vector.

[0105] As used herein, the term "lentivirus" refers to a group (or genus) of complex retroviruses. Illustrative lentiviruses include, but are not limited to: HIV (human immunodeficiency virus; including HIV type 1 and HIV type 2); Vesnamedi virus (VMV) virus; caprine arthritis-encephalitis virus (CAEV); equine infectious anemia virus (EIAV); feline immunodeficiency virus (FIV); bovine immunodeficiency virus (BIV); and simian immunodeficiency virus (SIV). Lentiviruses transduce dividing and post-mitotic cells.

[0106] The term "lentiviral vector" refers to a viral vector (e.g., a viral plasmid) comprising structural and functional genetic elements derived primarily from a lentivirus, or portions thereof, including long terminal repeats (LTRs). A lentiviral vector is a hybrid vector (e.g., in the form of a transfer plasmid) comprising retroviral (e.g., lentiviral) sequences for reverse transcription, replication, integration, and / or packaging of a nucleic acid sequence (e.g., a coding sequence). The term "retroviral vector" refers to a viral vector (e.g., a transfer plasmid) comprising structural and functional genetic elements derived primarily from a retrovirus, or portions thereof.

[0107] Adenoviral vectors are designed to be administered directly to living subjects. Unlike retroviral vectors, most adenoviral vector genomes do not integrate into the chromosomes of host cells. Instead, genes introduced into cells using adenoviral vectors are maintained in the nucleus as extrachromosomal elements (episomes) that last for an extended period of time. Adenoviral vectors transduce dividing and non-dividing cells in many different tissues in vivo, including airway epithelial cells, endothelial cells, hepatocytes, and various tumors (Trapnell, 1993; Chuah et al., 2003).

[0108] The term adeno-associated virus (AAV) refers to a small ssDNA virus that infects humans and some other primate species, is known not to cause disease, and only causes a very mild immune response. AAV can infect both dividing cells and non-dividing cells, and its genome can be integrated into the genome of the host cell. These characteristics make AAV a very attractive candidate for producing viral vectors for gene therapy, although the cloning ability of the vector is relatively limited. In a preferred embodiment of the present invention, the vector used is derived from adeno-associated virus (i.e., AAV vector). More than 30 naturally occurring AAV serotypes are available. There are many natural variants in the AAV capsid, allowing identification and use of AAVs with properties that are particularly suitable for specific target cell types. AAV viruses can be engineered by conventional molecular biology techniques, so that these particles are optimized for cell-specific delivery of shRNA, ribozymes or microRNA DNA sequences, for minimizing immunogenicity, for regulating stability and particle life, for effective degradation, for precise delivery to the nucleus, etc. Become possible.

[0109] An "expression vector" is a vector that contains regulatory regions. Many vectors and expression systems are commercially available from companies such as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.). An expression vector may be a viral expression vector derived from a particular virus.

[0110] The vectors provided herein may also include, for example, an origin of replication, a scaffold attachment region (SAR) and / or a marker. The marker gene may confer a selectable phenotype to the host cell. For example, the marker may confer biocide resistance, such as resistance to antibiotics (e.g., kanamycin, G418, bleomycin or hygromycin). The expression vector may include a tag sequence that is designed to facilitate manipulation or detection (e.g., purification or positioning) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin or FIag. TM Tag (Kodak, New Haven, Conn.) sequences are usually expressed as fusions with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including the carboxyl or amino termini.

[0111] Additional expression vectors may also include segments of, for example, chromosomal, non-chromosomal, and synthetic DNA sequences. Suitable vectors include pLK0.1 puro, derivatives of SV40, and plasmids such as RP4; phage DNA, for example many derivatives of phage 1, such as NM989, and other phage DNA, such as M13 and filamentous single-stranded phage DNA, vectors that can be used in eukaryotic cells, such as vectors that can be used in insect or mammalian cells, vectors derived from a combination of plasmids and phage DNA, such as plasmids that have been modified to use phage DNA or other expression control sequences.

[0112] The vector may also include a regulatory region. The term "regulatory region" refers to a nucleotide sequence that affects the initiation and rate of transcription or translation and the stability and / or mobility of the transcription or translation product. Regulatory 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 start sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0113] As used herein, the term "operably connected" refers to the positioning of a sequence to be transcribed in a regulatory region and a nucleic acid so as to affect the transcription or translation of such a sequence. For example, in order to place a coding sequence under the control of a promoter, the translation start site of the translation reading frame of a polypeptide is generally located between 1 and about 50 nucleotides downstream of the promoter. However, a promoter may be located up to about 5,000 nucleotides upstream of the translation start site or up to about 2,000 nucleotides upstream of the transcription start site. A promoter generally includes at least a core (basic) 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 selection of a promoter to be included depends on several factors, including but not limited to efficiency, selectivity, inducibility, desired expression levels, and preferential expression of cells or tissues. It is a routine task for those skilled in the art to regulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.

[0114] The vector may also include other components or functions that further regulate gene delivery and / or gene expression or otherwise provide beneficial properties to the targeted cells. As described and illustrated in more detail below, such other components include, for example, components that affect binding or targeting cells (including components that mediate cell type or tissue specific binding); components that affect cell uptake of vector nucleic acid; components that affect the localization of polynucleotides in cells after uptake (such as agents that mediate nuclear localization); and components that affect polynucleotide expression. Such components may also include markers, such as detectable and / or selectable markers, which may be used to detect or select cells that have taken up the vector and express nucleic acids delivered by the vector. Such components may be provided as natural features of the vector (such as using certain viral vectors with components or functions that mediate binding and uptake), or the vector may be modified to provide such functions. Other vectors include those described by Chen et al.; BioTechniques, 34: 167-171 (2003). A large number of such vectors are known in the art and are generally available.

[0115] "Recombinant viral vector" refers to a viral vector comprising one or more heterologous gene products or sequences. Since many viral vectors exhibit size restrictions associated with packaging, heterologous gene products or sequences are usually introduced by replacing one or more parts of the viral genome. Such viruses may become replication-defective, requiring the provision of one or more missing functions in a trans manner during viral replication and encapsidation (e.g., by using a helper virus or a packaging cell line carrying gene products necessary for replication and / or encapsidation).

[0116] In some embodiments, the viral vector used in the present invention is at least 10 5 The concentration of viral genomes / cell was used.

[0117] The selection of appropriate promoters can be easily completed. The example of suitable promoter is RNA polymerase II or III promoter, such as U6 promoter. Other suitable promoters that can be used for gene expression include but are not limited to 763 base pairs of 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 regulatory sequence, PGK (phosphoglycerol kinase) promoter, alkaline phosphatase promoter; and animal transcription control region, the animal transcription control region shows tissue specificity and has been used in transgenic animals; myelin basic protein gene control region, the myelin basic protein gene control region is active in oligodendrocytes in the brain, and gonadotropin-releasing hormone gene control region, the gonadotropin-releasing hormone gene control region is active in the hypothalamus. Some proteins can be expressed using their natural promoters. Other elements that can enhance expression may also be included, such as enhancers or systems that lead to high levels of expression, such as tat genes and tar elements. The box can then be inserted into a vector, such as a plasmid vector, such as pLK0.1, pUC19, pUC118, pBR322 or other known plasmid vectors. See, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory press, (1989). The plasmid vector may also include a selective marker, such as a beta-lactamase gene for ampicillin resistance, as long as the marker polypeptide does not adversely affect the metabolism of the organism being treated. The box may also be incorporated into a nucleic acid binding portion in a synthetic delivery system (such as the system disclosed in WO 95 / 22618).

[0118] The coding sequences of shRNA, ribozymes and microRNAs can be cloned into viral vectors using any suitable genetic engineering techniques well known in the art, including but not limited to standard PCR, polynucleotide synthesis, restriction endonuclease digestion, ligation, transformation, plasmid purification and DNA sequencing techniques, such as Sambrook et al. (Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, NY (1989)), Coffin et al. (Retroviruses. Cold Spring Harbor Laboratory Press, NY (1997)) and "RNA Viruses: A Practical Approach" (Alan J. Cann, Ed., Oxford University Press, (2000)) described. In a preferred embodiment, the shRNA, ribozyme and microRNA DNA sequences contain flanking sequences at the 5' and 3' ends that are complementary to sequences cut by restriction endonucleases on plasmids and / or vectors. As is well known in the art, flanking sequences depend on the restriction endonucleases used during restriction digestion of plasmids and / or vectors. Therefore, those skilled in the art can select the flanking sequences at the 5' and 3' ends of the shRNA, ribozyme and microRNA DNA sequences accordingly. In another preferred embodiment, the target site can be cloned into a vector by nucleic acid fusion and exchange techniques currently known in the art, including Gateway, PCR in fusion, Cre-lox P and Creator.

[0119] In some embodiments, the expression vector comprises a promoter and a polynucleotide comprising a first nucleotide sequence encoding a short hairpin RNA (shRNA), a ribozyme or a microRNA of the present invention. Preferably, the promoter and the polynucleotide comprising the first nucleotide sequence are operably linked. Preferably, the promoter is a U6 promoter. The first nucleotide sequence contained in the expression vector may be SEQ ID NO:2. The first nucleotide sequence contained in the expression vector may be SEQ ID NO:3. The first nucleotide sequence contained in the expression vector may be SEQ ID NO:4. The first nucleotide sequence contained in the expression vector may be SEQ ID NO:8. The polynucleotide comprising the first nucleotide sequence in the expression vector is preferably a DNA polynucleotide. The first nucleotide sequence of the expression vector is preferably a DNA nucleotide sequence. The shRNA, ribozyme or microRNA encoded by the first nucleotide sequence of the expression vector may be as described in any of the variations disclosed herein.

[0120] As discussed below, the recombinant viral vector is transfected into packaging cells or cell lines along with the elements required for packaging recombinant viral particles. The recombinant viral particles collected from the transfected cell supernatant are used to infect target cells or organisms to express shRNA, ribozymes or microRNAs. The transduced cells or organisms are used for transient expression or selected for stable expression.

[0121] Virus particles

[0122] Viral particles are used to deliver shRNA, ribozyme and microRNA encoding nucleotide sequences targeting UBE3A-ATS RNA. Viral particles typically include various viral components and sometimes host cell components in addition to one or more nucleic acids. Nucleic acid sequences can be packaged into viral particles that are capable of delivering shRNA, ribozyme and / or microRNA nucleic acid sequences to target cells in patients or humans in need.

[0123] Viral particles can be produced by: (a) introducing a viral expression vector into a suitable cell line; (b) culturing the cell line under suitable conditions to allow production of viral particles; (c) recovering the produced viral particles; and (d) optionally purifying the recovered infectious viral particles.

[0124] An expression vector containing a nucleotide sequence encoding the shRNA, ribozyme or microRNA of the present invention can be introduced into an appropriate cell line for proliferation or expression using well-known techniques readily available to those of ordinary skill in the art. These include, but are not limited to, microinjection of minute amounts of DNA into the nucleus (Capechi et al., 1980, Cell 22, 479-488), CaPO4-mediated transfection (Chen and Okayama, 1987, Mol. Cell Biol. 7, 2745-2752), DEAE-dextran-mediated transfection, electroporation (Chu et al., 1987, Nucleic Acid Res. 15, 1311-1326), lipofectamine transfection / lipofectamine fusion (Feigner et al., 1987, Proc. Natl. Acad. Sci. USA 84, 7413-7417), particle bombardment (Yang et al., 1990, Proc. Natl. Acad. Sci. USA 87, 9568-9572), gene guns, transduction, infection (e.g., with infectious viral particles), and other techniques, such as those described in Sambrook et al. (Molecular Biology). Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 2001).

[0125] When the vector of the present invention is defective, infectious particles are usually produced in a complementing cell line or by using a helper virus, which provides non-functional viral genes in trans. For example, suitable cell lines for complementation with adenoviral vectors include 293 cells (Graham et al., 1997, J. Gen. Virol. 36, 59-72) and PER-C6 cells (Fallaux et al., 1998, Human Gene Ther. 9, 1909-1917) commonly used for complementation with E1 function. Other cell lines have been engineered to complement double-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 or from the cells after lysis and optionally further purified according to standard techniques (chromatography, ultracentrifugation in cesium chloride gradients, as described, for example, in WO 96 / 27677, WO 98 / 00524, WO 98 / 22588, WO 98 / 26048, WO 00 / 40702, EP 1016700 and WO 00 / 50573).

[0126] The present invention also relates to host cells comprising the nucleic acid molecules, vectors or infectious viral particles of the present invention described herein. For the purposes of the present invention, the term "host cell" should be understood in a broad sense, without any limitation on a specific organism in a tissue, organ or isolated cell. Such cells can be a unique type of cell or a group of different types of cells, and include cultured cell lines, primary cells and proliferating cells.

[0127] Therefore, host cells include prokaryotic cells, lower eukaryotic cells such as yeast and other eukaryotic cells such as insect cells, plants and 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 (totipotent stem cells, leukocytes, lymphocytes, monocytes, macrophages, APCs, dendritic cells, non-human cells, etc.), pneumocytes, tracheal cells, hepatocytes, epithelial cells, endothelial cells, muscle (e.g., skeletal muscle, cardiac muscle or smooth muscle) cells or fibroblasts. Preferred host cells include Escherichia coli, Bacillus, Listeria, Saccharomyces, BHK (baby hamster kidney) cells, MDCK cells (Madin-Darby canine kidney cell line), CRFK cells (Crandell cat 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 capable of complementing at least one defective function of the replication-defective vector (e.g., adenovirus vector) of the present invention, such as those stated above.

[0128] The host cells of the present invention can be further encapsulated. Cell encapsulation technology has been previously described (Tresco et al., 1992, ASAJO J.38, 17-23; Aebischer et al., 1996, Human Gene Ther.7, 851-860). According to the specific embodiment, the eukaryotic host cells of transfection or infection are encapsulated with a compound forming a microporous membrane, and the encapsulated cells can be further implanted in vivo. The capsule containing the cells of interest can be prepared using a hollow microporous membrane (e.g., AkzoNobel Faser AG, Wuppertal, Germany; Deglon et al. 1996, Human Gene Ther.7, 2135-2146), which has a molecular weight cutoff suitable for allowing proteins and nutrients to pass freely between the inside and outside of the capsule while preventing the implanted cells from contacting the host cells.

[0129] Viral particles suitable for the present invention include AAV particles and lentiviral particles. AAV particles carry shRNA, ribozymes and microRNA coding sequences in the form of genomic DNA. On the other hand, lentiviral particles belong to the category of retroviruses and carry shRNA, ribozymes and microRNA coding sequences in the form of RNA.

[0130] Recombinant 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, ribozymes and / or microRNAs targeting UBE3A-ATS RNA can be delivered to target cells to inhibit silencing of UBE3A by UBE3A-ATS. The use of AAV is a common way of DNA delivery because it is relatively nontoxic, provides efficient gene transfer, and can be easily optimized for specific purposes. In one embodiment of the invention, the selected AAV serotype has a natural neurotropism. In another embodiment of the invention, the AAV serotype is AAV9 or AAV10.

[0131] Suitable recombinant AAV is produced by culturing a host cell comprising a nucleotide sequence encoding an AAV serotype capsid protein or a fragment thereof, as defined herein; a functional rep gene; a minigene consisting of at least an AAV inverted terminal repeat (ITR) and an encoding nucleotide sequence; and sufficient auxiliary functions to allow the minigene to be packaged into the AAV capsid protein. The components required for culturing in the host cell to package the AAV minigene into the AAV capsid can be provided to the host cell in trans. Alternatively, any one or more of the required components (e.g., minigenes, rep sequences, cap sequences, and / or auxiliary 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 skilled in the art.

[0132] Unless otherwise indicated, the AAV ITRs and other selected AAV components described herein can be easily selected from any AAV serotype, including but not limited to AAV1, AAV2, AAV3, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10 or other known and unknown AAV serotypes. These ITRs or other AAV components can be easily isolated from AAV serotypes using techniques available to those skilled in the art. Such AAVs 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 available in databases such as, for example, GenBank, PubMed, etc.

[0133] The minigenes, rep sequences, cap sequences and auxiliary functions required for producing the rAAV of the present invention can be delivered to the packaging host cell in the form of any genetic element that transfers the sequence carried thereon. The selected genetic element can be delivered by any suitable method. The method for constructing any embodiment of the present invention is known to technicians in nucleic acid manipulation and includes 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 virus particles are well known, and the selection of suitable methods is not a limitation of the present invention. See, for example, K. Fisher et al., 1993 J. Viral., 70: 520-532 and U.S. Patent No. 5,478,745, etc. These publications are incorporated herein by reference.

[0134] Selection of these and other common vectors and regulatory elements is routine, and many such sequences are available. See, e.g., Sambrook et al., and references cited therein, e.g., 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 will function equally well to express all transgenes of the present invention. However, one skilled in the art can make a selection among these and other expression control sequences without departing from the scope of the present invention.

[0135] Pharmaceutical compositions and therapeutic treatments

[0136] The viral particles comprising the desired coding sequence of coding shRNA, ribozyme and / or microRNA can be formulated for being applied to patients or humans in need by any suitable means of application. Such preparation includes the use of pharmaceutically and / or physiologically acceptable vehicles or carriers, particularly suitable for being applied to the vehicle or carrier of the brain (e.g., by subcranial or spinal injection). In addition, more than one shRNA, ribozyme and / or microRNA, or any combination thereof can be used in joint therapy. In joint therapy, different shRNA, ribozyme and / or microRNA can be used simultaneously, respectively, successively and in any order.

[0137] Suitably, the pharmaceutical composition of the present invention comprises a carrier and / or diluent suitable for delivery to a human or animal organism by injection. Such a carrier and / or diluent is nontoxic at the dosage and concentration used. It is selected from those that are generally used to prepare compositions for parenteral administration in unit dose or multiple dose form or for direct infusion by continuous or regular infusion. It is preferably isotonic, hypotonic or weakly hypertonic, and has a relatively low ionic strength, such as the ionic strength provided by sugar, polyols and isotonic saline solutions. Representative examples include sterile water, physiological saline (e.g., sodium chloride), bacteriostatic water, Ringer's solution, glucose or sucrose solution, Hank's solution and other physiologically balanced aqueous salt solutions (see, for example, the latest version of Remington: The Science and Practice of Pharmacy, A. Gennaro, Lippincott, Williams & Wilkins). The pH of the composition of the present invention is appropriately adjusted and buffered so as to be suitable for humans or animals, preferably at physiological pH or slightly alkaline pH (between about pH 8 and about pH 9, wherein pH 8.5 is particularly preferred). Suitable buffers include phosphate buffer (e.g., PBS), bicarbonate buffer, and / or Tris buffer. A particularly preferred composition is formulated with 1M sucrose, 150mM NaCl, 1mM MgCl2, 54mg / l Tween 80, 10mM Tris pH 8.5. Another preferred composition is formulated with 10mg / ml mannitol, 1mg / ml HSA, 20mM Tris, pH 7.2, and 150mM NaCl. These compositions are stable at -70°C for at least 6 months.

[0138] The compositions of the invention may be in various forms, for example solid (e.g. powder, lyophilized form) or liquid (e.g. aqueous). In the case of solid compositions, preferred methods of preparation are vacuum drying and freeze drying, which produce a powder of the active agent plus any additional desired ingredients from a previously sterile filtered solution thereof. Such solutions may be stored in sterile ampoules, ready for reconstitution by adding sterile water for ready injection, if desired.

[0139] Atomization or aerosolized preparations also form a part of the present invention. The method of intranasal administration is well known in the art, including the drops, sprays or dry powder forms of the composition are applied to the nasopharynx of the individual to be treated from a pressurized container or a dispenser (it contains a suitable propellant, for example a gas such as carbon dioxide) or a nebulizer (see, for example, WO 95 / 11664). Enteric-coated preparations, such as gas-tolerant capsules and granules for oral administration, suppositories for rectal or vaginal administration also form a part of the present invention. For non-parenteral administration, the composition can also include an absorption enhancer that increases the mucosal pore size. Such absorption enhancers include sodium deoxycholate, sodium glycocholate, dimethyl-β-cyclodextrin, lauroyl-1-lysophosphatidylcholine and other substances with structural similarity to the phospholipid domain of the mucosa.

[0140] The composition can also include other pharmaceutically acceptable excipients for providing desired pharmaceutical or pharmacodynamic properties, including, for example, changing or maintaining the pH, osmotic pressure, viscosity, transparency, color, sterility, stability, dissolution rate of the formulation, changing or maintaining release or absorption into a human or animal organism. For example, polymers such as polyethylene glycol can be used to obtain desired solubility, stability, half-life and other pharmaceutically favorable 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, polyvinyl pyrrolidone, trehalose, and combinations thereof. Other stabilizing components particularly suitable for use in plasmid-based compositions include hyaluronidase (which is believed to destabilize the extracellular matrix of host cells, as described in WO 98 / 53853), chloroquine, protic compounds such as propylene glycol, polyethylene glycol, glycerol, ethanol, 1-methyl L-2-pyrrolidone or their derivatives, aprotic compounds such as dimethyl sulfoxide (DMSO), diethyl sulfoxide, di-n-propyl sulfoxide, dimethyl sulfone, sulfolane, dimethylformamide, dimethylacetamide, tetramethylurea, acetonitrile (see EP 890362), nuclease inhibitors such as actin G (WO 99 / 56784), and cationic salts such as magnesium (Mg 2+ ) (EP 998 945) and lithium (Li +) (WO01 / 47563), and any derivatives thereof. The amount of cationic salt in the composition of the present invention preferably ranges from about 0.1 mM to about 100 mM, and still more preferably from about 0.1 mM to about 10 mM. Viscosity enhancers include sodium carboxymethylcellulose, sorbitol, and dextrose. The composition may also contain substances known in the art that promote penetration or transport across the blood barrier or membrane of a particular organ (e.g., antibodies to transferrin receptors; Friden et al., 1993, Science 259, 373-377). A gel complex of polylysine and lactose (Midoux et al., 1993, Nucleic Acid Res. 21, 871-878) or poloxamer 407 (Pastore, 1994, Circulation 90, 1-517) may be used to facilitate administration in arterial cells.

[0141] Virus particles and pharmaceutical compositions can be applied to patients 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 happy puppet syndrome is sufficient to improve one or more symptoms of happy puppet syndrome as described herein (e.g., developmental delay, severe cognitive impairment, ataxic gait, frequent epileptic seizures, short attention span, speech loss, and characteristic happy behavior). In addition, AS iPSC-derived neurons show depolarized resting membrane potential, delayed action potential development, and spontaneous synaptic activity reduction. Therefore, the therapeutically effective amount for treating AS can restore the resting membrane potential of neurons to about -70mV, improve action potential development delay, increase spontaneous synaptic activity, or improve the other changes in the neuronal phenotypes related to base strength, action potential characteristics (e.g., shape), membrane current, synaptic potential, ion channel conductance, etc.

[0142] The appropriate dosage may vary according to known factors, such as the pharmacodynamic characteristics of the particular active agent, the age, health and weight of the host organism; the condition or conditions to be treated, the nature and extent of the symptoms, the kind of concurrent treatment, the frequency of treatment, the need for prevention or treatment and / or the desired effect. The dosage is also calculated according to the particular route of administration selected. The practitioner will routinely further refine the calculations required to determine the appropriate dosage for treatment according to the relevant circumstances. As a general guide, the viral particle-based composition can be administered in a dose of at least 10 5 The composition based on the vector plasmid can be prepared in a dosage form of between 1 μg and 100 mg, advantageously between 10 μg and 10 mg, and preferably between 100 μg and 1 mg. Administration can occur in a single dose, or in a dose repeated once or several times after a certain time interval.

[0143] The composition of the present invention can be encapsulated in an ampoule, a disposable syringe or a multiple dose vial made of glass or plastic. In all cases, the composition must be sterile and should be fluid to the extent that it is easy to inject. It must be stable under manufacturing and storage conditions and must prevent the contaminating effects of microorganisms such as bacteria and fungi. Sterile injectable solutions can be prepared by incorporating an active agent (e.g., infectious particles) into one of the components or a combination of components listed above in the required amount, followed by filtration sterilization.

[0144] The viral particles and pharmaceutical compositions of the present invention are preferably administered by parenteral routes, including intravenous, intraarterial, subcutaneous, intraperitoneal or intramuscular injection or infusion, intrathecal or intracranial, such as intracerebral or intraventricular administration. In one embodiment, the viral particles or pharmaceutical compositions are administered intracerebrally or intraventricularly. In another embodiment, the viral particles or pharmaceutical compositions are administered intrathecally.

[0145] In certain embodiments of the methods of the invention, 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 being in need. In a specific embodiment of the invention, subcranial administration into the brain results in the direct administration of the encoding nucleotide sequence of the invention to brain cells, including glia and neurons. As used herein, the term "neuron" refers to any cell in the brain or any cell associated with brain function. The term can refer to any type of neuron, including monopolar, bipolar, multipolar, and pseudo-monopolar. Example

[0146] Overall approach

[0147] Human embryonic stem cell (hESC) culture and neural differentiation

[0148] hESCs were cultured on irradiated mouse embryonic fibroblasts and maintained daily with hESC medium (DMEM / F12 containing knockout serum replacement, L-glutamine + β-mercaptoethanol, non-essential amino acids and basic fibroblast growth factor). hESCs were cultured at 37°C in a humidified incubator with 5% CO2. Cells were manually passaged every 5-7 days.

[0149] hESC is differentiated into neurons using a modified form of the monolayer protocol. 2 days after passage, neural induction is started by culturing cells in N2B27 culture medium (neural basal culture medium, 1% N2, 2% B27, 2mM L-glutamine, 0.5% penicillin / streptomycin, 1% insulin-transferrin-selenium). In the first 10 days of differentiation, N2B27 culture medium is supplemented with fresh Noggin (500ng / mL). About 14 days after starting neural induction, neural rosettes are artificially passaged onto poly-D-lysine and laminin coated plates using Stem Pro EZ passage tools. Neural progenitor cells are re-plated with high density in about 3 weeks of differentiation, switched to neural differentiation medium (NDM) in about 4 weeks of differentiation, and then sparsely plated for terminal differentiation in about 5 weeks of differentiation. NDM consists of neurobasal medium, 1% B27, 2mM L-glutamine, 0.5% penicillin-streptomycin, nonessential amino acids, 1μM ascorbic acid, 200μM cyclic AMP, 10ng / mL brain-derived neurotrophic factor, and 10ng / mL glial cell-derived neurotrophic factor. Neurons are maintained in culture until differentiation for 10 to 17 weeks. Protocols for hESC maintenance and neuronal differentiation have been previously described.

[0150] Generation of shRNA and ribozyme vectors

[0151] ShRNA and ribozyme are produced by annealing two complementary polynucleotides with the desired sequence. Specifically, the polynucleotides producing shRNA include a sequence of specific 21 nucleotides to be targeted and its reverse complementary sequence, separated by the loop sequence of CTCGAG, and 5' flanking sequence CCGG and 3' flanking sequence TTTTTG are added for cloning into a plasmid vector. The polynucleotides producing ribozyme include a sequence of specific 41 nucleotides and the same 5' flanking sequence and 3' flanking sequence for plasmid cloning. The annealed polynucleotides are connected to the pLK0.1 puro vector (Stewart et al. RNA 2003Apr; 9(4): 493-501). The shRNA and ribozyme sequences are under the control of the U6 promoter. The resulting plasmid is subjected to Sanger sequencing to confirm the correct insertion of the shRNA sequence.

[0152] Lentivirus production and neuronal transduction

[0153] Lentiviral particles were made by transfecting 293FT cells with the second generation packaging system using Lipofectamine 3000. Viruses were concentrated using the Lenti-X Concentrator kit (Clontech), and viral titers were calculated using the qPCR Lentiviral Titering Kit (abm). Ten-week-old neurons were expressed at 1.3 cells / cm2 Neurons were plated at a density of 1:1 on laminin-coated plastic dishes and transduced with 10 viral particles / cell. Seven days after lentiviral transduction, RNA from neurons was harvested.

[0154] Quantitative RT-PCR

[0155] cDNA was synthesized using a high capacity cDNA reverse transcription kit (Thermo Fisher Scientific) according to the manufacturer's instructions. Quantitative RT-PCR was performed on Step One Plus (Thermo Fisher Scientific) using Taqman gene expression assays and Mastermix (ThermoFisher Scientific) for each expected target. Reactions were performed in technical duplicates, with the GAPDH endogenous control Taqman assay used as a housekeeping gene for normalization. ΔΔC t Methods Quantification of gene expression.

[0156] AAV production and neuronal transduction

[0157] 551shRNA2 and scrambled shRNA control were cloned into pAV-U6-GFP vector and packaged into viral particles by a commercial supplier (Vigene). Neurons were treated with 551shRNA2 AAV9 particles using at least 1x10 5 100 viral genomes / cell were transduced. Neurons were imaged 48 hours later to visualize the transduced neurons.

[0158] Example 1

[0159] In order to test the in vitro cleavage ability of ribozymes, ribozymes and target RNA sequences were transcribed in vitro and tested under controlled conditions. Specifically, primers including T7 sequences were used to amplify the genomic sequence of the expected target. The PCR product was cleaned up and concentrated, and then used in the in vitro transcription assay performed with T7 polymerase. DNA was used in the in vitro transcription reaction performed with T7 polymerase. RNA was column purified using MEGAclear kit (Ambion). Ribozyme RNA was prepared by annealing two complementary ribozyme polynucleotides. They were then end-filled with T4 polymerase, and cleaned up and concentrated. Ribozymes were diluted to an appropriate concentration (5pmol / μL) in ddH2O as a working storage solution. Ribozymes and target RNA were combined at 37 degrees Celsius for 30 minutes in the in vitro cleavage assay, and visualized on 2% agarose gel with ethidium bromide.

[0160] Hammerhead ribozymes, which are catalytic RNA molecules that hydrolyze phosphodiester bonds in RNA, were first tested. These ribozymes cleave rapidly and independently of other cellular mechanisms and have been tested in clinical trials for HIV and certain tumors. Four hammerhead ribozymes were designed to cleave nearby sequences targeted by functional ASOs. In vitro cleavage assays revealed that the ribozymes were active and could cleave the UBE3A-ATS sequence ( Figure 3A The two most active ribozymes, 551 Ribo 1 (SEQ ID NO: 3) and 551 Ribo 2 (SEQ ID NO: 4), were encoded by lentiviral vectors that were packaged into lentiviral particles and used to transduce mature AS iPSC-derived neurons. UBE3A-ATS levels were reduced by ∼40% ( Figure 3B ).

[0161] Example 2

[0162] shRNAs targeting similar sequences in the UBE3A-ATS gene were developed. The shRNAs exploit the RNA interference pathway to knock down RNA. Four shRNAs were designed by cloning them into lentiviral vectors, which were packaged into lentiviral particles and used to transduce mature AS iPSC-derived neurons. 551shRNA2 (SEQ ID NO: 2) achieved 40% knockdown of UBE3A-ATS, and 2.2-fold activation of the paternal UBE3A ( Figure 4 These studies have been replicated by cloning the same shRNA into an adeno-associated virus (AAV) vector and packaging it into AAV9 particles ( Figure 5 and Figure 6 ).

[0163] It should be understood that the foregoing detailed description and accompanying examples are merely illustrative and should not be taken as limiting the scope of the present invention, which is defined solely by the appended claims and their equivalents.

[0164] Various changes and modifications to the disclosed embodiments will be apparent to those skilled in the art. Such changes and modifications may be made without departing from the spirit and scope of the invention, including but not limited to those changes and modifications related to the chemical structures, substituents, derivatives, intermediates, syntheses, compositions, formulations or methods of use of the invention.

[0165] sequence

[0166] SEQ ID NO:1

[0167] Human UBE3A-ATS genome sequence.

[0168]

[0169]

[0170]

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

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[0180]

[0181]

[0182]

[0183]

[0184]

[0185]

[0186]

[0187]

[0188]

[0189]

[0190]

[0191] SEQ ID NO: 2 shRNA artificial / synthetic sequence.

[0192]

[0193] SEQ ID NO: 3

[0194] 551 Ribo 1 artificial / synthetic sequence.

[0195]

[0196] SEQ ID NO: 4

[0197] 551 Ribo 2 artificial / synthetic sequence.

[0198]

[0199] SEQ ID NO: 5

[0200] UBE3A-ATS artificial / synthetic target sequence.

[0201]

[0202] SEQ ID NO: 6

[0203] UBE3A-ATS artificial / synthetic sequence.

[0204]

[0205] SEQ ID NO: 7

[0206] UBE3A-ATS artificial / synthetic sequence.

[0207]

[0208] SEQ ID NO: 8

[0209] Artificial / synthetic sequences of microRNAs.

[0210]

[0211] SEQ ID NOs:9-508

[0212] UBE3A-ATS artificial / synthetic sequence.

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225] SEQ ID NO:509 Human UBE3A sequence.

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249]

[0250]

[0251]

[0252]

[0253]

[0254]

[0255]

[0256]

[0257]

[0258]

[0259]

[0260]

[0261]

[0262]

[0263]

[0264]

[0265]

[0266]

[0267]

[0268]

[0269]

[0270]

[0271]

Claims

1. A polynucleotide comprising a nucleotide sequence encoding a ribozyme, wherein the ribozyme is capable of inhibiting the silencing of a paternal UBE3A gene, and wherein the nucleotide sequence encoding the ribozyme consists of SEQ ID NO: 3 or SEQ ID NO:

4.

2. The polynucleotide of claim 1, wherein the ribozyme increases expression of the paternal UBE3A gene.

3. The polynucleotide of claim 1, wherein the paternal UBE3A gene comprises the nucleotide sequence of SEQ ID NO:

509.

4. The polynucleotide of claim 1, wherein silencing of the paternal UBE3A gene is caused by the RNA transcript encoded by SEQ ID NO:

1.

5. An expression vector comprising the polynucleotide according to any one of claims 1 to 4 and a promoter. The expression vector according to claim 5 , wherein the promoter is a U6 promoter.

7. The expression vector according to claim 5 or 6, wherein the polynucleotide is a DNA polynucleotide.

8. The expression vector according to any one of claims 5-7, wherein the expression vector is an adeno-associated virus (AAV) vector or a lentiviral vector.

9. A viral particle comprising the polynucleotide according to any one of claims 1 to 4.

10. The viral particle of claim 9, wherein the particle is an AAV particle or a lentiviral particle.

11. The viral particle of claim 10, wherein the particle is an AAV particle with obligate neurotropism.

12. The viral particle of claim 10, wherein the particle is an AAV9 particle or an AAV10 particle.

13. A pharmaceutical composition comprising the polynucleotide according to any one of claims 1 to 4, the expression vector according to any one of claims 5 to 8 or the virus particle according to any one of claims 9 to 12 and a pharmaceutically acceptable carrier.

14. Use of the expression vector of any one of claims 5-8, the viral particle of any one of claims 9-12, or the pharmaceutical composition of claim 13 in the preparation of a medicament for treating Happy Puppet Syndrome, wherein when the medicament is used, a therapeutically effective amount of the expression vector, the viral particle, or the pharmaceutical composition is administered to a patient in need thereof.

15. Use of the expression vector of any one of claims 5 to 8, the viral particle of any one of claims 9 to 12, or the pharmaceutical composition of claim 13 in the preparation of a medicament for activating or increasing the expression of the paternal UBE3A gene, wherein when the medicament is used, a therapeutically effective amount of the expression vector, the viral particle, or the pharmaceutical composition is administered to a patient in need thereof.

16. Use of the expression vector of any one of claims 5 to 8, the viral particle of any one of claims 9 to 12, or the pharmaceutical composition of claim 13 in the preparation of a medicament for inhibiting paternal UBE3A gene silencing caused by the RNA antisense transcript encoded by SEQ ID NO: 1, wherein when the medicament is used, the expression vector, the viral particle, or the pharmaceutical composition in an amount effective to cleave the RNA antisense transcript encoded by SEQ ID NO: 1 is administered to a patient in need thereof.

17. The use according to any one of claims 14 to 16, wherein the expression vector, the viral particle or the pharmaceutical composition is administered to the brain of the patient.

18. The use according to any one of claims 14 to 16, wherein the expression vector, the viral particle or the pharmaceutical composition is administered to neurons of the patient.

19. The use according to any one of claims 14 to 18, wherein the patient is a human.

20. The use according to any one of claims 14 to 19, wherein the ribozyme inhibits the silencing of the paternal UBE3A gene caused by the RNA antisense transcript encoded by SEQ ID NO:

1.

21. The use according to any one of claims 14 to 20, wherein the ribozyme terminates the transcription of a polynucleotide comprising the sequence of SEQ ID NO:

1.

22. The use according to any one of claims 14-21, wherein the ribozyme reduces the level of the RNA antisense transcript encoded by SEQ ID NO:

1.

23. The use according to any one of claims 14-22, wherein the ribozyme cleaves the RNA antisense transcript encoded by SEQ ID NO:

1.

24. A ribozyme encoded by the polynucleotide according to any one of claims 1-4.

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