Guiding nucleic acids targeting UBE3A-ATS and uses thereof

Abstract

Provided are guide nucleic acids targeting human UBE3A-ATS, systems or compositions or rAAV vector genomes comprising the guide nucleic acids, and methods of using the systems or compositions or rAAV vector genomes to treat human UBE3A-ATS related diseases and disorders.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of and priority to PCT patent application No. PCT / CN2022 / 083478, entitled “CRISPR-CAS13 SYSTEM FOR TREATINGUBE3A-ASSOCIATED DISEASES,” filed on March 28, 2022, which patent application (including any sequence listing and drawings) is incorporated herein by reference in its entirety.

[0003] References to Electronic Sequence Listings

[0004] The present disclosure contains an electronic sequence listing ("xxx.xml", created on XX by "WIPOSequence" software according to WIPO Standard ST.26), which is incorporated herein by reference in its entirety. Where the sequence is an RNA sequence, T in the sequence should be considered as U. Background Art

[0005] Angelman syndrome (AS) is a rare neurodevelopmental disorder caused by loss-of-function mutations in maternally expressed UBE3A. To date, no gene-specific therapy is available for patients. Despite being intact and transcriptionally active, paternally inherited UBE3A is silenced in neurons by elongation of the antisense long noncoding RNA UBE3A-ATS.

[0006] Citation or identification of any document in this disclosure is not an admission that such document is available as prior art to the present disclosure. Summary of the invention

[0007] Against the above background, the present disclosure provides certain advantages over the prior art. Although the disclosure herein is not limited to specific advantages, in one aspect, the present disclosure provides a guide nucleic acid comprising:

[0008] (1) a scaffold sequence capable of forming a complex with a nucleic acid programmable RNA nuclease (napRNAn), and

[0009] (2) a guide sequence capable of hybridizing to a target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS.

[0010] In another aspect, the present disclosure provides a system or composition comprising:

[0011] (1) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid comprises:

[0012] (a) a scaffold sequence capable of forming a complex with a nucleic acid programmable RNA nuclease (napRNAn), and

[0013] (b) a guide sequence capable of hybridizing to a target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS; and

[0014] (2) The napRNAn or a polynucleotide encoding the napRNAn.

[0015] In yet another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector genome, the rAAV vector genome comprising:

[0016] (a) a first polynucleotide sequence comprising a first sequence encoding a first guide nucleic acid, the first guide nucleic acid comprising:

[0017] (1) a scaffold sequence capable of forming a complex with a nucleic acid programmable RNA nuclease (napRNAn), and

[0018] (2) a first guide sequence capable of hybridizing to a first target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS; and

[0019] (b) a second polynucleotide sequence, wherein the second polynucleotide sequence comprises a sequence encoding the napRNAn,

[0020] The rAAV vector genome is suitable for encapsulation into recombinant AAV particles.

[0021] In yet another aspect, the present disclosure provides a recombinant AAV (rAAV) particle comprising the rAAV vector genome of the present disclosure.

[0022] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the system or composition of the present disclosure or the rAAV particle of the present disclosure, and a pharmaceutically acceptable excipient.

[0023] In yet another aspect, the present disclosure provides a method for preventing or treating a disease or disorder associated with human UBE3A-ATS in a subject in need thereof, the method comprising administering to the subject a system or composition of the present disclosure, an rAAV particle of the present disclosure, or a pharmaceutical composition of the present disclosure, wherein the napRNAn modifies the human UBE3A-ATS, and wherein the modification of the human UBE3A-ATS treats the disease or disorder.

[0024] The details of one or more embodiments of the present disclosure are set forth in the following description. Other features or advantages of the present disclosure should be clear from the following drawings and detailed description of several embodiments and from the claims. It should be understood that, unless otherwise indicated, any aspect or embodiment of the present disclosure may be combined with any other aspect or embodiment of the present disclosure to constitute another embodiment explicitly or implicitly disclosed herein.

[0025] definition

[0026] The present disclosure will describe specific embodiments, but the present disclosure is not limited thereto, but only by the claims. Unless otherwise defined, all technical terms and scientific terms used herein have the same meanings as those commonly understood by ordinary technicians in the field to which the present disclosure belongs. Unless otherwise specified, the terms described below are generally understood in their ordinary meanings.

[0027] Overview

[0028] Nucleic acid programmable RNA nuclease (napRNAn), such as Cas13, is capable of cutting target RNA under the guidance of a guide nucleic acid (e.g., guide RNA) comprising a guide sequence targeting a target RNA. In some embodiments, the target RNA is eukaryotic.

[0029] Without wishing to be bound by theory, in some embodiments, the guide nucleic acid comprises a scaffold sequence responsible for forming a complex with the napRNAn, and a guide sequence intentionally designed to be responsible for hybridizing with a target sequence of a target RNA, thereby directing the complex comprising the napRNAn and the guide nucleic acid to the target RNA.

[0030] refer to Figure 6 , exemplary dsRNA is described as comprising a 5' to 3' single DNA strand and a 3' to 5' single DNA strand. According to conventional transcription processes, exemplary RNA transcripts can be transcribed using the 3' to 5' single DNA strand as a synthetic template, so the 3' to 5' single DNA strand is referred to as the "template strand" or "antisense strand". The RNA transcript so transcribed has the same primary sequence as the 5' to 3' single DNA strand (except that T is replaced by U), so the 5' to 3' single DNA strand is referred to as the "coding strand" or "sense strand".

[0031] Exemplary guide nucleic acids are depicted as comprising a guide sequence and a scaffold sequence. The guide sequence is designed to hybridize with a portion of the RNA transcript (target RNA), so the guide sequence "targets" that portion. Thus, the portion of the target RNA on which the guide sequence is designed and to which the guide sequence can hybridize is referred to as the "target sequence." In some embodiments, the guide sequence is 100% (completely) reverse complementary to the target sequence. In some other embodiments, the guide sequence is reverse complementary to the target sequence and contains mismatches (such as Figure 7 exemplified in ).

[0032] Typically, the double-stranded sequence of dsDNA can be represented by the sequence of its 5' to 3' single DNA strand, and conventionally, written in a 5' to 3' direction / orientation. Typically, nucleic acid sequences (e.g., DNA sequences, RNA sequences) are written in a 5' to 3' direction / orientation.

[0033] For example, for a dsDNA having a 5' to 3' single DNA strand of 5'-ATGC-3' and a 3' to 5' single DNA strand of 3'-TACG-5', the dsDNA can be simply represented as 5'-ATGC-3'. Normally, the RNA transcript (target RNA) transcribed from the dsDNA then has a sequence of 5'-AUGC-3'.

[0034] In order to hybridize with the target RNA, in one embodiment, the guide sequence of the guide nucleic acid is designed to have a 5'-GCAU-3' sequence that is completely reverse complementary to the target RNA. According to WIPO's electronic sequence table standard ST.26, the symbol "t" is used to represent both T in DNA and U in RNA (see "Table 1: List of nucleotide symbols", the definition of the symbol "t" is "thymine in DNA / uracil in RNA (t / u)"). Therefore, in the sequence table according to ST.26, such a guide sequence will be shown as GCAT, but marked as an RNA sequence.

[0035] the term

[0036] As used herein, if a DNA sequence, e.g., 5'-ATGC-3', is transcribed into an RNA sequence (e.g., 5'-AUGC-3') in which each dT (deoxythymidine, or "T" for short) in the primary sequence is replaced with U (uridine), and the other dA (deoxyadenosine, or "A" for short), dG (deoxyguanosine, or "G" for short), and dC (deoxycytidine, or "C" for short) are replaced with A (adenosine), G (guanosine), and C (cytidine), respectively, it is said in the present disclosure that the DNA sequence "encodes" the RNA sequence.

[0037] As used herein, the term "activity" refers to biological activity. In some embodiments, the activity includes enzymatic activity, such as the catalytic ability of the effector. For example, the activity can include nuclease activity, such as RNA nuclease activity, RNA endonuclease activity.

[0038] As used herein, the term "complex" refers to the grouping of two or more molecules. In some embodiments, the complex comprises polypeptides and nucleic acids that interact with each other (e.g., binding, contacting, adhering). As used herein, the term "complex" may refer to the grouping of guide nucleic acids and polypeptides (e.g., napRNAn, such as Cas13 polypeptides). As used herein, the term "complex" may refer to the grouping of guide nucleic acids, polypeptides, and target sequences. As used herein, the term "complex" may refer to the grouping of guide nucleic acids, napRNAn, and optionally target RNAs targeting target RNAs.

[0039] As used herein, the term "guide nucleic acid" refers to any nucleic acid that helps to target napRNAn (e.g., Cas13 polypeptide) to a target sequence (e.g., a sequence of a target RNA). The guide nucleic acid can be designed to include a sequence complementary to a specific nucleic acid sequence (e.g., a sequence of a target RNA). The guide nucleic acid can include a scaffold sequence that helps to guide napRNAn to the target RNA. In some embodiments, the guide nucleic acid is a guide RNA.

[0040] As used herein, the terms "nucleic acid," "polynucleotide," and "nucleotide sequence" are used interchangeably to refer to a polymeric form of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs or modifications thereof.

[0041] As used in the context of CRISPR-Cas technology (e.g., CRISPR-Cas13 technology), the term "guide RNA" is used interchangeably with the term "CRISPR RNA (crRNA)", "single guide RNA (sgRNA)" or "RNA guide", the term "guide sequence" is used interchangeably with the term "spacer sequence", and the term "scaffold sequence" is used interchangeably with the term "direct repeat sequence".

[0042] As described herein, the guide sequence is designed to be able to hybridize with the target sequence. As used herein, the term "hybridization (hybridize, hybridizing or hybridization)" refers to a reaction in which one or more polynucleotide sequences react to form a complex, which is stabilized via hydrogen bonds between the bases of the polynucleotide sequence. Hydrogen bonds can occur by Watson Crick base pairing, Hoogstein binding or in any other sequence-specific manner. The polynucleotide sequence that can hybridize with a given polynucleotide sequence is referred to as the "complementary sequence" of a given polynucleotide sequence. As used herein, the hybridization of a guide sequence and a target sequence is so stable that the functional domain associated with (e.g., fusion) of an effector polypeptide (e.g., napRNAn) or the effector polypeptide compounded with a nucleic acid comprising a guide sequence acts on (e.g., cutting, deamination) a target sequence or its complementary sequence (e.g., a sequence of a target RNA or its complementary sequence).

[0043] For the purpose of hybridization, in some embodiments, the guide sequence is complementary or reverse complementary to the target sequence. As used herein, the term "complementary" refers to the ability of the nucleobase of the first polynucleotide sequence (such as a guide sequence) to base pair with the nucleobase of the second polynucleotide sequence (such as a target sequence) by traditional Watson Crick base pairing. Two complementary polynucleotide sequences can be non-covalently bound under appropriate temperature and solution ionic strength conditions. In some embodiments, the first polynucleotide sequence (e.g., a guide sequence) has 100% (complete) complementarity with the second nucleic acid (e.g., a target sequence). In some embodiments, if the first polynucleotide sequence (e.g., a guide sequence) has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% complementarity with the second nucleic acid, the first polynucleotide sequence is complementary to the second nucleic acid. As used herein, the term "substantially complementary" refers to a polynucleotide sequence (e.g., a guide sequence) having a certain degree of complementarity with a second polynucleotide sequence (e.g., a target sequence). In some embodiments, the level of complementarity enables a first polynucleotide sequence (e.g., a guide sequence) to hybridize with a second polynucleotide sequence (e.g., a target sequence) with sufficient affinity to allow an effector polypeptide (e.g., napRNAn) complexed with a nucleic acid comprising a first polynucleotide sequence or a first polynucleotide sequence or a functional domain associated with (e.g., fused to) the effector polypeptide to act (e.g., cleavage, deamination) on a target sequence or its complementary sequence (e.g., a sequence of a target RNA or its complementary sequence). In some embodiments, a guide sequence that is substantially complementary to a target sequence has less than 100% complementarity with the target sequence. In some embodiments, a guide sequence that is substantially complementary to a target sequence has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the target sequence.

[0044] As used herein, the term "sequence identity" is related to sequence homology. Homology comparisons can be performed by eye, or more usually with the aid of readily available sequence comparison programs. These commercially available computer programs can calculate the homology percentage (%) between two or more sequences, and can also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.

[0045] Sequence homology can be generated by any of many computer programs known in the art such as BLAST or FASTA, etc. A suitable computer program for performing this comparison is the GCG Wisconsin Bestfit package (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research [nucleic acid research] 12: 387). Examples of other software that can perform sequence comparisons include, but are not limited to, BLAST packages (see Ausubel et al., 1999 ibid.-Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol. [Molecular Biology], 403-410) and GENEWORKS comparison tool suite. Both BLAST and FASTA can be used for offline and online searches (see Ausubel et al., 1999 ibid., pp. 7-58 to pp. 7-60). The percentage (%) of sequence homology can be calculated on a continuous sequence, that is, one sequence is aligned with other sequences, and each amino acid or nucleotide in one sequence is directly compared with the corresponding amino acid or nucleotide in the other sequence, one residue at a time. This is called an "ungapped" alignment. Typically, such ungapped alignments are performed only on multiple residues of a relatively short number. Although this is a very simple and reliable method, it does not take into account that, for example, in a pair of sequences that are originally identical, an insertion or deletion can cause the subsequent amino acid residues to no longer align, which may cause the % homology to be greatly reduced when a global alignment is performed. Therefore, most sequence comparison methods are designed to produce an optimal alignment that takes into account possible insertions and deletions, so as not to unduly impair the overall homology or identity score. This is achieved by inserting "gaps" in the sequence alignment in an attempt to maximize local homology or identity. However, these more complex methods assign a "gap penalty" to each gap that occurs in the alignment so that, for the same number of identical amino acids, a sequence alignment with as few gaps as possible - reflecting a higher relatedness between the two compared sequences - can achieve a higher score than a sequence alignment with many gaps. Typically, an "affine gap cost" is used, which charges a relatively high cost for the presence of a gap, and charges a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. Of course, high gap penalties can produce optimal alignments with fewer gaps. Most alignment programs allow gap penalties to be modified. However, it is preferred to use the default values ​​when using such software for sequence comparisons. For example, when using the GCG Wisconsin Bestfit package, the default gap penalties for amino acid sequences are: -12 for gaps, -4 for each gap extension.Therefore the calculation of maximum % homology first needs to produce the best comparison under the situation that gap penalty is taken into account.A new tool called BLAST 2Sequences can also be used to compare protein sequences and nucleotide sequences (referring to FEMS Microbiol Lett. [FEMS Microbiology Express] 1999 174 (2): 247-50; FEMS Microbiol Lett. [FEMS Microbiology Express] 1999 177 (1): 187-8 and (National Center for Biotechnology Information (National Center for Biotechnology) website in the National Institutes of Health (National Institutes for Health) website).Although the final homology % can be measured according to identity, the comparison process itself is typically not based on all or nothing pairwise comparison.On the contrary, a scaled similarity score matrix is ​​usually used, which assigns scores to each pairwise comparison based on chemical similarity or evolutionary distance.An example of such a matrix commonly used is the BLOSUM62 matrix-the default matrix of the BLAST program package.GCG The Wisconsin program typically uses public default values ​​or a custom symbol comparison table if provided (see the user manual for further details). For some applications, it is preferred to use the public default values ​​of the GCG software package, or in the case of other software, use the default matrix, such as BLOSUM62. Alternatively, an algorithm similar to CLUSTAL is used in DNASIS. TMThe multiple alignment feature in the Hitachi software is used to calculate the homology percentage (Higgins DG and Sharp PM (1988), Gene [Gene] 73 (1), 237-244). Once the software has produced the best alignment, the % homology, preferably the % sequence identity, can be calculated. Typically, as part of the sequence comparison, the software performs this calculation and generates a numerical result. The sequence can also have a deletion, insertion or substitution of amino acid residues that produces a silent change and produces a functionally equivalent substance. Intentional amino acid substitutions can be made based on the similarity of amino acid properties (such as the polarity, charge, solubility, hydrophobicity, hydrophilicity, and / or amphipathic nature of the residue), and it is therefore useful to group amino acids together in functional groups. Amino acids can be grouped together based solely on their side chain properties. However, it would also be more useful to include mutation data. For structural reasons, the groups of amino acids derived may therefore be conservative. These groups can be described in the form of a Venn diagram (Livingstone CD and Barton GJ (1993) "Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation" Comput. Appl. Biosci. 9:745-756) (Taylor WR (1986) "The classification of amino acid conservation" J. Theor. Biol. 119; 205-218). Conservative substitutions can be made, for example, according to the following table, which describes the generally accepted Venn diagram groupings of amino acids.

[0046] As used herein, the terms "polypeptide" and "peptide" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interrupted by non-amino acids. A protein may have one or more polypeptides. The term also encompasses amino acid polymers that have been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.

[0047] As used herein, "variant" is interpreted to mean a polynucleotide or polypeptide that is different from a reference polynucleotide or polypeptide, but retains basic properties. Typical variants of polynucleotides are different from another reference polynucleotide in nucleic acid sequence. Changes in the nucleic acid sequence of the variant may or may not change the amino acid sequence of the polypeptide encoded by the reference polynucleotide. As discussed below, nucleotide changes may result in amino acid substitutions, additions, deletions, fusions, and truncations in the polypeptide encoded by the reference sequence. Typical variants of polypeptides are different from another reference polypeptide in amino acid sequence. Usually, the differences are limited, so the sequences of the reference polypeptide and the variant are generally very similar and identical in many regions. Variants and reference polypeptides may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residues may or may not be amino acid residues encoded by the genetic code. The variant of a polynucleotide or polypeptide may be naturally occurring, such as an allelic variant, or it may be a variant known to be not naturally occurring. Non-naturally occurring variants of polynucleotides and polypeptides may be prepared by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to those skilled in the art.

[0048] As used herein, the terms "upstream" and "downstream" refer to the relative position within a sequence of individual nucleotides in a nucleic acid (e.g., DNA). Typically, a first sequence "upstream" of a second sequence means that the first sequence is 5' of the second sequence, while a first sequence "downstream" of a second sequence means that the first sequence is 3' of the second sequence.

[0049] As used herein, the term "wild type" has the meaning generally understood by those skilled in the art, i.e., refers to the typical form of an organism, strain, gene or characteristic, which, when it occurs in nature, distinguishes it from a mutant or variant. It can be isolated from a source in nature without being intentionally modified.

[0050] As used herein, the terms "non-naturally occurring" and "engineered" are used interchangeably and refer to human involvement. When these terms are used to describe a nucleic acid or polypeptide, it means that the nucleic acid or polypeptide is at least substantially free of at least one other component with which it is found in nature or in association with nature.

[0051] As used herein, "cell" should be understood to refer not only to a particular individual cell, but also to the progeny or potential progeny of a cell. Because certain modifications may occur in subsequent generations due to mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term.

[0052] As used herein, the term "in vivo" refers to the body of an organism, while the term "ex vivo" or "in vitro" refers to outside of an organism.

[0053] As used herein, the term "treatment" is a method for obtaining beneficial or desired results (including clinical results). For the purposes of this disclosure, beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms caused by the disease, reducing the extent of the disease, stabilizing the disease (e.g., preventing or delaying the deterioration of the disease), preventing or delaying the spread of the disease (e.g., metastasis), preventing or delaying the recurrence of the disease, reducing the recurrence rate of the disease, delaying or slowing the progress of the disease, improving the disease state, providing relief (partial or complete) of the disease, reducing the dosage of one or more other drugs required for the treatment of the disease, delaying disease progression, increasing quality of life and / or prolonging survival."Treatment" also encompasses the pathological results of reducing diseases (e.g., cancer). The method of this disclosure contemplates any one or more of these therapeutic aspects of treatment.

[0054] As used herein, the term "disease" includes the terms "disorder" and "condition", and is not limited to medically well-defined terms.

[0055] As used herein, reference to a value or parameter that is "not" generally means and describes a value or parameter that is "different from." For example, a method is not for treating cancer type X, meaning that the method can be used to treat cancers other than type X.

[0056] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0057] As used herein, the term "and / or" in phrases such as "A and / or B" is intended to include both A and B; A or B; A (alone); and B (alone). Similarly, the term "and / or" in phrases such as "A, B, and / or C" is intended to encompass each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).

[0058] As used herein, when the term "about" precedes a series of numbers (e.g., about 1, 2, 3), it is understood that each of the series of numbers is modified by the term "about" (i.e., about 1, about 2, about 3). The term "about XY" as used herein has the same meaning as "about X to about Y".

[0059] It should be understood that the embodiments of the present disclosure described herein include "consisting of" and / or "consisting essentially of" the embodiments.

[0060] It is also noted that the claims may be drafted to exclude any optional element. As such, this description is intended to serve as an antecedent basis for use of exclusive terminology such as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] An understanding of certain features and advantages of the present disclosure will be gained by referring to the following detailed description, which sets forth illustrative embodiments in which the principles of the present disclosure may be utilized, and to the accompanying drawings, in which:

[0062] Figure 1 Schematic diagram of treating AS patient neurons with the CRISPR-hfCas13e.1 system to reduce UBE3A-ATS transcripts and reactivate the expression of paternal UBE3A.

[0063] Figure 2 A is a schematic diagram of an exemplary lentiviral vector genome encoding hfCas13e.1-sgRNA and controls. Figure 2 B is a schematic diagram of an exemplary AAV vector genome encoding hfCas13e.1-sgRNA and controls.

[0064] Figure 3 A shows a graph of the in vitro knockdown efficiency of Ube3a-ATS transcripts in primary neurons of AS mice treated with the hfCas13e.1-sg9-14 system (AS+sg9-14) compared to controls (AS+NT). RNA levels of Ube3a and Ube3a-ATS transcripts were detected by RT-qPCR. N=3 / group. *, P<0.05; ***, P<0.001.

[0065] Figure 3 B shows the in vitro reactivation of Ube3a in primary neurons of AS mice caused by knockdown of Ube3a-ATS transcripts by the hfCas13e.1-sg9-14 system (AS+sg9-14) compared to the control (WT+NT). The expression of Ube3a (ubiquitin protein ligase E3A) protein was detected by Western Blot. Tissues for Western blotting were harvested 4 weeks after administration. N=3 / group.

[0066] Figure 3 C shows Figure 3 Statistical quantification of B. N = 3 / group. *, P < 0.05; ***, P < 0.001.

[0067] Figure 4 A shows a schematic diagram of the assay timeline.

[0068] Figure 4 B shows a graph of in vivo knockdown of Ube3a-ATS transcripts and restoration of paternal Ube3a transcripts in the cortex of treated AS mice compared to untreated AS mice. Levels of Ube3a and Ube3a-ATS transcripts were detected by RT-qPCR. Tissues for RT-qPCR were harvested 4 weeks after dosing. N=3 / group. *, P<0.05; ***, P<0.001.

[0069] Figure 4 C shows a graph of in vivo knockdown of Ube3a-ATS transcripts and restoration of paternal Ube3a transcripts in the hippocampus of treated AS mice compared to untreated AS mice. The levels of Ube3a and Ube3a-ATS transcripts were detected by RT-qPCR. Tissues for RT-qPCR were harvested 4 weeks after administration. N=3 / group. *, P<0.05; ***, P<0.001.

[0070] Figure 4 D shows the expression of paternal Ube3a in the cortex and hippocampus of treated AS mice compared to untreated AS mice. The levels of Ube3a protein in the cortex and hippocampus were detected by Western blotting. Tissues for Western blotting were harvested 4 weeks after dosing. N=3 / group.

[0071] Figure 4 E shows Figure 4 Statistical quantification of D. N = 3 / group. *, P < 0.05; ***, P < 0.001.

[0072] Figure 4 F shows the expression of paternal Ube3a in the cortex and hippocampus of treated mice compared to controls. The levels of Ube3a protein in the cortex and hippocampus were detected by Western blotting. Tissues for Western blotting were harvested 18 weeks after dosing. N=3 / group.

[0073] Figure 4 G shows Figure 4 Statistical quantification of F. N = 3 / group. *, P < 0.05; ***, P < 0.001.

[0074] Figure 5 A is a schematic diagram of the timeline of behavioral testing.

[0075] Figure 5 B shows body weights of female mice measured every two weeks for 18 weeks.

[0076] Figure 5 C shows the results of the hindlimb grasping test. N>8 / group. *, P<0.05; ***, P<0.001.

[0077] Figure 5 D shows the results of the open field test. N>8 / group. *, P<0.05; ***, P<0.001.

[0078] Figure 5 E shows the results of the dowel test. N>8 / group. *, P<0.05; ***, P<0.001.

[0079] Figure 5 FG show the results of the beam walking test. N>8 / group. *, P<0.05; ***, P<0.001.

[0080] Figure 5 H shows the results of the accelerating rotarod test. N>8 / group. *, P<0.05; ***, P<0.001.

[0081] Figure 6 is a schematic diagram showing an exemplary dsDNA, an exemplary RNA transcript transcribed from the dsDNA, an exemplary guide nucleic acid, and an exemplary napRNAn, wherein the guide sequence is reverse complementary to the target sequence.

[0082] Figure 7 is a schematic diagram showing an exemplary dsDNA, an exemplary RNA transcript transcribed from the dsDNA, an exemplary guide nucleic acid, and an exemplary napRNAn, wherein the guide sequence is reverse complementary to a target sequence and contains mismatches to the target sequence.

[0083] The drawings herein are for illustration purposes only and are not necessarily drawn to scale. DETAILED DESCRIPTION

[0084] Overview

[0085] The present disclosure provides tools and methods for treating UBE3A-ATS-related diseases such as Angelman syndrome (AS).

[0086] In particular, the present disclosure provides tools for restoring / upregulating paternal UBE3A gene expression by cleaving / degrading UBE3A-ATS transcripts (long non-coding RNA, lncRNA) that inhibit / silence the paternal UBE3A gene. Such tools can be delivered to subjects in need, for example, via lentiviral and AAV vectors and intracerebroventricular injection. Exemplary constructs of the present disclosure have demonstrated efficacy in knocking down UBE3A-ATS transcript levels and restoring UBE3A protein expression in vitro and in vivo, as well as improving disease phenotypes in mouse models, thereby opening the door to gene therapy for treating UBE3A-ATS-related diseases such as AS.

[0087] Guide nucleic acid

[0088] In one aspect, the present disclosure provides a guide nucleic acid comprising:

[0089] (1) a scaffold sequence capable of forming a complex with a nucleic acid programmable RNA nuclease (napRNAn), and

[0090] (2) a guide sequence capable of hybridizing to a target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS.

[0091] System or combination

[0092] In another aspect, the present disclosure provides a system or composition comprising:

[0093] (1) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid comprises:

[0094] (a) a scaffold sequence capable of forming a complex with a nucleic acid programmable RNA nuclease (napRNAn), and

[0095] (b) a guide sequence capable of hybridizing to a target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS; and

[0096] (2) The napRNAn or a polynucleotide encoding the napRNAn.

[0097] In some embodiments, the system is a complex comprising napRNAn complexed with a guide nucleic acid. In some embodiments, the complex further comprises human UBE3A-ATS hybridized to a target sequence.

[0098] rAAV vector genome

[0099] In yet another aspect, the present disclosure provides a recombinant adeno-associated virus (rAAV) vector genome, the rAAV vector genome comprising:

[0100] (a) a first polynucleotide sequence comprising a first sequence encoding a first guide nucleic acid, the first guide nucleic acid comprising:

[0101] (1) a scaffold sequence capable of forming a complex with a nucleic acid programmable RNA nuclease (napRNAn), and

[0102] (2) a first guide sequence capable of hybridizing to a first target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS; and

[0103] (b) a second polynucleotide sequence, wherein the second polynucleotide sequence comprises a sequence encoding the napRNAn,

[0104] The rAAV vector genome is suitable for encapsulation into recombinant AAV particles.

[0105] Multiple guide nucleic acids

[0106] The systems, compositions, or rAAV vector genomes of the present disclosure can comprise or encode one guide nucleic acid or encode more than one nucleic acid, e.g., for the purpose of improving cleavage efficiency against human UBE3A-ATS.

[0107] In some embodiments, the first polynucleotide sequence further comprises a second sequence encoding a second guide nucleic acid comprising:

[0108] (1) a scaffold sequence capable of forming a complex with napRNAn or the scaffold sequence capable of forming a complex with napRNAn, and

[0109] (2) a second guide sequence capable of hybridizing to a second target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS.

[0110] In some embodiments, the first polynucleotide sequence further comprises a third sequence encoding a third guide nucleic acid comprising:

[0111] (1) a scaffold sequence capable of forming a complex with napRNAn or the scaffold sequence capable of forming a complex with napRNAn, and

[0112] (2) capable of hybridizing to the third target sequence of human UBE3A-ATS to guide the complex to the third guide sequence of human UBE3A-ATS.

[0113] In some embodiments, the first polynucleotide sequence further comprises a fourth sequence encoding a fourth guide nucleic acid comprising:

[0114] (1) a scaffold sequence capable of forming a complex with napRNAn or the scaffold sequence capable of forming a complex with napRNAn, and

[0115] (2) capable of hybridizing to the fourth target sequence of human UBE3A-ATS to guide the complex to the fourth guide sequence of human UBE3A-ATS.

[0116] In some embodiments, the first polynucleotide sequence further comprises a fifth guide nucleic acid, a sixth guide nucleic acid, a seventh guide nucleic acid, etc.

[0117] In some embodiments using the same napRNAn, the scaffold sequences of the more than one guide nucleic acids can be the same or slightly different (e.g., differing by no more than 5, 4, 3, 2, or 1 nucleotides) to be compatible with the same napRNAn. In some embodiments using different napRNAn, the scaffold sequences of the more than one guide nucleic acids can be different to be compatible with different napRNAn.

[0118] Properties of the Guide Nucleic Acids of the Disclosure

[0119] In some embodiments, the guide nucleic acid (e.g., a guide nucleic acid, a first guide nucleic acid, a second guide nucleic acid, a third guide nucleic acid, a fourth guide nucleic acid, etc.; as elsewhere) is RNA. In some embodiments, the guide nucleic acid comprises a modification. In some embodiments, the guide nucleic acid is an unmodified RNA or a modified RNA. In some embodiments, the guide nucleic acid is a modified RNA containing modified ribonucleotides. In some embodiments, the guide nucleic acid is a modified RNA containing deoxyribonucleotides. In some embodiments, the guide nucleic acid is a modified RNA containing modified deoxyribonucleotides. In some embodiments, the guide nucleic acid comprises modified or unmodified deoxyribonucleotides and modified or unmodified ribonucleotides.

[0120] Mechanism of the present disclosure

[0121] Angelman syndrome (AS) is a severe neurodevelopmental disorder with an estimated prevalence of 1 / 12,000–1 / 20,000 worldwide, characterized by seizures and profound impairments in motor, cognitive, and language skills. AS is primarily attributed to the loss of the maternally inherited ubiquitin-protein ligase E3A (UBE3A), which mediates the targeted degradation of several protein substrates. Only the maternally inherited UBE3A allele is active in neurons due to the selective silencing of paternal UBE3A by its antisense transcript, UBE3A-ATS. UBE3A-ATS is a polyadenylated transcript transcribed from the SNHG14 small nucleolar RNA host gene 14 (SNHG14) that overlaps with the UBE3A locus, resulting in premature termination and degradation of the UBE3A transcript. Approximately 75% of AS cases are reported to be caused by deletions of the maternal chromosomal region 15q11.2–q13. The remaining cases can be attributed to other mutations in maternal UBE3A, paternal uniparental disomy, or imprinting defects.

[0122] There is currently no specific therapy available for the treatment of AS. However, the inventors discovered that activation of an intact, functional paternal copy of UBE3A can be achieved by eliminating the UBE3A-ATS long noncoding RNA (lncRNA) using a napRNAn (e.g., Cas13) guided by a guide nucleic acid targeting UBE3A-ATS, which may represent a promising potential therapeutic strategy.

[0123] As described above, the guide sequence of the guide nucleic acid is designed to be able to hybridize with human UBE3A-ATS, thereby guiding the complex comprising the guide nucleic acid and napRNAn to human UBE3A-ATS. In some embodiments, the complex is guided to human UBE3A-ATS so that napRNAn can specifically cut human UBE3A-ATS. In some embodiments, the specific cutting of human UBE3A-ATS leads to the degradation of human UBE3A-ATS and the expression of human UBE3A genes that were previously inhibited by human UBE3A-ATS. In some embodiments, human UBE3A-ATS inhibits the expression of human UBE3A genes derived from the parent (paternal line). In some embodiments, the human UBE3A protein expressed from the human UBE3A gene derived from the parent is functional. Where the suppression of paternal UBE3A gene expression by the UBE3A-ATS is eliminated and the UBE3A protein expressed from the paternal UBE3A gene is functional, it is reasonable to expect that the syndrome in a subject caused by a loss-of-function mutation in maternally expressed UBE3A can be alleviated.

[0124] Design of target sequences and target sites disclosed herein

[0125] For purposes of the present disclosure, in some embodiments, a target sequence (e.g., a target sequence, a first target sequence, a second target sequence, a third target sequence, a fourth target sequence, etc.; same as elsewhere) is positioned such that human UBE3A-ATS is specifically cleaved by napRNAn.

[0126] To facilitate evaluation of the designed target sequence in a mouse model, in some embodiments, the target sequence is positioned so that the mouse UBE3A-ATS is specifically cleaved by napRNAn. In some embodiments, the target sequence is positioned so that both human UBE3A-ATS and mouse UBE3A-ATS are specifically cleaved by napRNAn. That is, the target sequence is selected to have cross-reactivity with both humans and mice.

[0127] As used herein, the term "UBE3A-ATS" includes UBE3A-ATS as a long non-coding RNA and any variants, derivatives or ancestors thereof, including pre-UBE3A-ATS transcripts, such as human pre-UBE3A-ATS transcripts or mouse pre-UBE3A-ATS transcripts, for example, the RNA counterpart of the human UBE3A-ATS genomic coding sequence or its pre-transcript (Accession No.: NG_002690.1, incorporated herein by reference), the RNA counterpart of the mouse UBE3A-ATS genomic coding sequence or its pre-transcript (Accession No.: NC_000073.7, incorporated herein by reference), or any transcript or isoform produced by alternative promoter usage, alternative splicing and / or alternative initiation thereof.

[0128] In some embodiments, the DNA counterpart of human UBE3A-ATS (also known as SNHG14) is shown as NCBI genome sequence NG_002690.1. In some embodiments, the DNA counterpart of mouse UBE3A-ATS (also known as Snhg14) is shown as NCBI genome sequence NC_000073.7.

[0129] Structure of the guide nucleic acids disclosed herein

[0130] In some embodiments, a guide nucleic acid comprises a scaffold sequence 5' or 3' to a guide sequence (e.g., a guide sequence, a first guide sequence, a second guide sequence, a third guide sequence, a fourth guide sequence, etc.; same as elsewhere). In some embodiments, a guide nucleic acid comprises a scaffold sequence 3' to a guide sequence. In some embodiments, a scaffold sequence is fused to a guide sequence without a linker.

[0131] In some embodiments, a guide nucleic acid comprises, from 5' to 3', a guide sequence and a scaffold sequence.

[0132] In some embodiments, a guide nucleic acid comprises from 5' to 3' a scaffold sequence, a guide sequence, and a scaffold sequence, wherein the scaffold sequences are the same or different.

[0133] In some embodiments, a guide nucleic acid comprises from 5' to 3' one scaffold sequence, one guide sequence, one scaffold sequence, and one guide sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

[0134] In some embodiments, a guide nucleic acid comprises from 5' to 3' one scaffold sequence, one guide sequence, one scaffold sequence, one guide sequence, and one scaffold sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

[0135] In some embodiments, a guide nucleic acid comprises from 5' to 3' one scaffold sequence, one guide sequence, one scaffold sequence, one guide sequence, one scaffold sequence, and one guide sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

[0136] The lengths of the target sequences and guide sequences disclosed herein

[0137] In some embodiments, the target sequence is at least about 14 nucleotides in length, such as about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides in length, or a range of values ​​between any two of the foregoing values ​​in length, such as about 16 to about 50 nucleotides in length. In some embodiments, the target sequence is about 30 nucleotides in length.

[0138] In some embodiments, the target sequence comprises, consists essentially of, or consists of at least about 14 consecutive nucleotides of human UBE3A-ATS (e.g., about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, In some embodiments, the target sequence comprises, consists essentially of, or consists of about 30 consecutive nucleotides of human UBE3A-ATS.

[0139] In some embodiments, the guide sequence is at least about 14 nucleotides in length, such as about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides in length, or a range of values ​​between any two of the foregoing values ​​in length, such as about 16 to about 50 nucleotides in length. In some embodiments, the guide sequence is about 30 nucleotides in length.

[0140] In some embodiments, the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (perfectly) complementary to the target sequence, optionally about 100% (perfectly) complementary; or wherein the guide sequence is at least about 100% complementary to the target sequence; or wherein the first 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23,24,25,26,27,28,29,30,31,32,33,34,35,36,37,38,39,40,41,42,43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69 or 70 nucleotides do not contain mismatches with the target sequence. It is generally believed that for the napRNAn (e.g., Cas13) disclosed herein, at least 2 mismatches between the guide sequence and the target sequence can be tolerated without significantly reducing the cleavage activity. In some embodiments, the guide sequence is 100% (completely) complementary to the target sequence.

[0141] Specific embodiments of the target sequence and guide sequence disclosed herein

[0142] In some embodiments, the target sequence comprises, consists essentially of, or consists of about 30 contiguous nucleotides over a common region shared by human UBE3A-ATS and mouse UBE3A-ATS.

[0143] In some embodiments, the target sequence comprises, consists essentially of, or consists of: (1) the sequence of any one of SEQ ID NOs: 16-21 and 58-80, or a 5' or 3' truncation thereof at the 5' or 3' end truncated by 1, 2, 3, 4, 5, or 6 nucleotides; or (2) a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 16-21 and 58-80, or a 5' or 3' truncation thereof at the 5' or 3' end truncated by 1, 2, 3, 4, 5, or 6 nucleotides; or (3) a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to any one of SEQ ID NOs: 16-21 and 58-80.

[0144] In some embodiments, the target sequence comprises the sequence of SEQ ID NO:21.

[0145] In some embodiments, the guide sequence comprises, consists essentially of, or consists of: (1) the sequence of any one of SEQ ID NOs: 10-15 and 35-57, or a 5' or 3' truncation thereof at the 5' or 3' end truncated by 1, 2, 3, 4, 5, or 6 nucleotides; or (2) a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100% sequence identity to any one of SEQ ID NOs: 10-15 and 35-57, or a 5' or 3' truncation thereof at the 5' or 3' end truncated by 1, 2, 3, 4, 5, or 6 nucleotides; or (3) a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to any one of SEQ ID NOs: 10-15 and 35-57.

[0146] In some embodiments, the guide sequence comprises the sequence of SEQ ID NO:15.

[0147] Specific embodiments of the scaffold sequence disclosed herein

[0148] For the purpose of the present disclosure, the support sequence is compatible with the napRNAn of the present disclosure, and can be compounded with the napRNAn. The support sequence can be a naturally occurring support sequence identified together with the napRNAn or a variant of the ability to be compounded with the napRNAn or the like. Generally, as long as the secondary structure of the variant is substantially the same as the secondary structure of the naturally occurring support sequence, the ability to be compounded with the napRNAn can be maintained. The nucleotide deletion, insertion or substitution in the primary sequence of the support sequence may not necessarily change the secondary structure of the support sequence (for example, the relative position and / or size of the stem, protrusion and ring of the support sequence will not significantly deviate from the relative position and / or size of the original stem, protrusion and ring). For example, nucleotide deletion, insertion or substitution can be in the protrusion or loop region of the support sequence, so that the overall symmetry of the protrusion and therefore the secondary structure remains roughly the same. Nucleotide deletion, insertion or substitution can also be in the stem of the support sequence, so that the length of the stem does not significantly deviate from the length of the original stem (for example, adding or missing a base pair in each of the two stems corresponds to a total of 4 base changes).

[0149] In some embodiments, the scaffold sequence has a secondary structure that is substantially identical to the secondary structure of the sequence of SEQ ID NO:3.

[0150] In some embodiments, the scaffold sequence comprises, consists essentially of, or consists of a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 3; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO: 3.

[0151] In some embodiments, the scaffold sequence comprises the sequence of SEQ ID NO:3.

[0152] Specific embodiments of the guide nucleic acid disclosed herein

[0153] In some embodiments, a guide nucleic acid comprises, consists essentially of, or consists of a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of any one of SEQ ID NOs: 4-9; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of any one of SEQ ID NOs: 4-9.

[0154] Regulation of the first polynucleotide sequence of the rAAV vector genome of the present disclosure

[0155] In some embodiments, the first polynucleotide sequence comprises a promoter operably linked to a sequence encoding a guide nucleic acid.

[0156] In some embodiments, the promoter is a ubiquitous promoter, a tissue-specific promoter, a cell-type-specific promoter, a constitutive promoter, or an inducible promoter.

[0157] Suitable promoters are known in the art and include, for example, Cbh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, retroviral Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1 alpha short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (le) enhancer and / or promoter, chicken β-actin (CBA) promoter or derivatives thereof such as CAG promoter, CB promoter, (human) elongation factor 1 alpha-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, Platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, synaptophysin (Syn) promoter, synaptophysin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, proenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter and myelin basic protein (MBP) promoter. In some embodiments, the promoter is a U6 promoter.

[0158] In some embodiments, the promoter comprises, consists essentially of, or consists of a sequence that has at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:30; or a sequence that has at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO:30.

[0159] In some embodiments, the first polynucleotide sequence comprises, from 5′ to 3′, a promoter of SEQ ID NO: 30 and a sequence encoding a guide nucleic acid of any one of SEQ ID NOs: 4-9.

[0160] napRNAn of the present disclosure

[0161] For the purposes of the present disclosure, napRNAn can form a complex with the guide nucleic acid of the present disclosure by complexing with the scaffold sequence of the guide nucleic acid, and thereby be guided to human UBE3A-ATS by hybridization of the guide sequence of the guide nucleic acid with the target sequence of human UBE3A-ATS. When napRNAn is guided to human UBE3A-ATS, the nuclease activity of napRNAn acts to cut human UBE3A-ATS, thereby causing human UBE3A-ATS degradation and thus eliminating human UBE3A-ATS inhibition of paternal human UBE3A gene expression. Generally, any such napRNAn can be used with the guide nucleic acid of the present disclosure, and when selecting napRNAn, a scaffold sequence compatible with napRNAn for complexing with napRNAn can also be selected accordingly. Scaffold sequences are generally conserved.

[0162] In some embodiments, napRNAn is a class 2 type VI CRISPR-associated protein (Cas13). Cas13, a class 2 type VI RNA endonuclease, can bind and cleave single-stranded RNA guided by an engineered CRISPR RNA (crRNA). RNA-targeted nuclease-based gene therapies are currently being explored as safer alternatives to Cas endonucleases that edit DNA because they carry a lower risk of introducing permanent genomic changes.

[0163] In some embodiments, Cas13 is Cas13a (C2c2), Cas13b (such as, Cas13b1, Cas13b2), Cas13c, Cas13d, Cas13e or Cas13f polypeptide. To date, Cas13e.1 (Cas13X.1) is the smallest known crRNA-guided RNA endonuclease, with only 775 amino acids, and the high-fidelity variant of Cas hfCas13e.1 (hfCas13x.1) has been confirmed to exhibit high on-target activity and significantly lower side cutting activity.

[0164] In some embodiments, the napRNAn comprises, consists essentially of, or consists of a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 1 or 34, or an N-terminal truncation thereof without the first N-terminal methionine, and retaining at least 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the guide sequence-specific RNA cleavage activity of the sequence of SEQ ID NO: 1 or 34.

[0165] In some embodiments, napRNAn comprises the sequence of SEQ ID NO:1.

[0166] In some embodiments, the sequence encoding the napRNAn comprises, consists essentially of, or consists of a sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 2, or a 5' end truncation thereof without the first 5'ATG codon, and wherein the napRNAn encoded by the sequence retains at least 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the guide sequence-specific RNA cleavage activity of the sequence of SEQ ID NO: 2.

[0167] In some embodiments, the sequence encoding napRNAn comprises the sequence of SEQ ID NO:2.

[0168] Regulation of the second polynucleotide sequence of the present disclosure

[0169] In some embodiments, the second polynucleotide sequence comprises a promoter operably linked to the sequence encoding napRNAn.

[0170] In some embodiments, the promoter is a broad spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter, or an inducible promoter.

[0171] Suitable promoters are known in the art and include, for example, the Cbh promoter, the Cba promoter, the pol I promoter, the pol II promoter, the pol III promoter, T7 promoter, U6 promoter, H1 promoter, retrovirus Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1α short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (Ie) enhancer and / or promoter, chicken β-actin (CBA) promoter or its derivatives such as CAG promoter, CB promoter, (human) elongation factor 1α-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, platelet-derived growth factor (PDGF) promoter, blood Platelet-derived growth factor B chain (PDGF-β) promoter, synapsin (Syn) promoter, synapsin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, myelin basic protein (MBP) promoter, HTT promoter, GRK1 promoter, CRX promoter, NRL promoter, and RCVRN promoter.

[0172] In some embodiments, the promoter is a neural cell (eg, neuron) specific promoter.

[0173] In some embodiments, the promoter is the human Syn1 promoter.

[0174] In some embodiments, the second polynucleotide sequence comprises a Kozak sequence 5' to the sequence encoding napRNAn.

[0175] In some embodiments, the second polynucleotide sequence comprises a sequence encoding a nuclear localization signal (NLS) 5' and / or 3' to the sequence encoding napRNAn.

[0176] In some embodiments, the second polynucleotide sequence comprises a sequence encoding a nuclear export signal (NES) 5' and / or 3' to the sequence encoding napRNAn.

[0177] In some embodiments, the second polynucleotide sequence comprises a sequence encoding a first NLS 5' to the sequence encoding napRNAn and a second sequence encoding a second NLS 3' to the sequence encoding napRNAn.

[0178] In some embodiments, the NLS, the first NLS and / or the second NLS is a SV40 NLS, a bpSV40 NLS, or a nucleoplasmin NLS.

[0179] In some embodiments, the second polynucleotide sequence comprises a WPRE sequence downstream of the sequence encoding napRNAn.

[0180] In some embodiments, the WPRE sequence is selected from the group consisting of a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), WPRE3 (shortened WPRE), and functional variants thereof (eg, functional truncations).

[0181] In some embodiments, the second polynucleotide sequence comprises a sequence encoding a polyadenylation (polyA) signal downstream of the sequence encoding napRNAn.

[0182] In some embodiments, the second polynucleotide sequence comprises a WPRE sequence downstream of a sequence encoding napRNAn, followed by a sequence encoding a polyadenylation (polyA) signal.

[0183] In some embodiments, the polyA signal is selected from the group consisting of a bovine growth hormone polyadenylation (bGH polyA) signal, a small polyA (SPA) signal, a human growth hormone polyadenylation (hGH polyA) signal, an SV40 polyA (SV40 polyA) signal, a rabbit beta globin polyA (rBG polyA) signal, and functional variants thereof (e.g., functional truncations).

[0184] In some embodiments, the polyA signal is an SV40 polyA signal.

[0185] In some embodiments, the second polynucleotide sequence comprises, from 5' to 3', a promoter, a Kozak sequence, a first sequence encoding a first NLS, a sequence encoding napRNAn, a second sequence encoding a second NLS, a WPRE sequence, and a sequence encoding a polyA signal.

[0186] In some embodiments, the promoter comprises, consists essentially of, or consists of a sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:25; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO:25.

[0187] In some embodiments, the Kozak sequence comprises, consists essentially of, or consists of a sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:26; or a sequence having at most 1, 2, 3, or 4 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO:26.

[0188] In some embodiments, the NLS, the first NLS and / or the second NLS comprises, consists essentially of, or consists of a sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%) sequence identity to the sequence of SEQ ID NO:27; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acid differences, whether contiguous or not, compared to the sequence of SEQ ID NO:27.

[0189] In some embodiments, the sequence encoding the polyA signal comprises, consists essentially of, or consists of a sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:29; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO:29.

[0190] In some embodiments, the second polynucleotide sequence comprises, from 5′ to 3′, a promoter of SEQ ID NO:25, a Kozak sequence of SEQ ID NO:26, a first sequence encoding a first NLS of SEQ ID NO:27, a sequence encoding napRNAn of SEQ ID NO:2, a second sequence encoding a second NLS of SEQ ID NO:27, and a sequence encoding a polyA signal of SEQ ID NO:29.

[0191] ITRs of the rAAV vector genome of the present disclosure

[0192] In some embodiments, the rAAV vector genome comprises a 5' inverted terminal repeat (ITR) sequence and a 3' ITR sequence.

[0193] In some embodiments, both the 5' ITR sequence and the 3' ITR sequence are wild-type ITR sequences from: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.eB, or a member of the evolutionary branch to which any one of AAV1-AAV13 belongs, or a functional variant thereof (e.g., a functional truncation).

[0194] In some embodiments, the 5' ITR sequence comprises, consists essentially of, or consists of a sequence that has at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:31.

[0195] In some embodiments, the 3' ITR sequence comprises, consists essentially of, or consists of a sequence that has at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:32.

[0196] Construction of the rAAV vector genome of the present disclosure

[0197] In some embodiments, the rAAV vector genome comprises, from 5' to 3', a first polynucleotide sequence and a second polynucleotide sequence.

[0198] In some embodiments, the rAAV vector genome comprises the second polynucleotide sequence and the first polynucleotide sequence from 5' to 3'.

[0199] rAAV particles of the present disclosure

[0200] In yet another aspect, the present disclosure provides a recombinant AAV (rAAV) particle, the rAAV particle comprising the rAAV vector genome of the present disclosure. A brief introduction to AAV for delivery can be found in the "Adeno-associated virus (AAV) Guide" (www.addgene.org / guides / aav / ).

[0201] Adeno-associated virus (AAV), when engineered for delivery of, for example, a protein coding sequence of interest, may be referred to as an (r)AAV vector, (r)AAV vector particle, or (r)AAV particle, where "r" stands for "recombinant". And the genome packaged in an AAV vector for delivery may be referred to as an (r)AAV vector genome, vector genome, or simply vg, while the viral genome may refer to the original viral genome of native AAV.

[0202] The serotype of the rAAV particle capsid can be matched to the type of target cell. For example, Table 2 of WO 2018002719A1 lists exemplary cell types that can be transduced by the indicated AAV serotypes (incorporated herein by reference).

[0203] In some embodiments, the rAAV particles include a capsid with a serotype suitable for delivery to neural cells (e.g., neurons). In some embodiments, the rAAV particles include a capsid of a serotype of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or AAV.PHP.eB, AAV1-AAV13, to which any one of the clades belongs, or a functional variant thereof (e.g., a functional truncation), which encapsulates the rAAV vector genome. In some embodiments, the serotype of the capsid is AAV9 or AAV.PHP.eB or a mutant thereof.

[0204] The general principles of rAAV particle production are known in the art. In some embodiments, rAAV particles can be produced using the triple transfection method (described in detail in US Pat. No. 6,001,650).

[0205] Vector titer is usually expressed as vector genome / ml (vg / ml). In some embodiments, the vector titer is greater than 1×10 9 , higher than 5×10 10 , higher than 1×10 11 , higher than 5×10 11 , higher than 1×1012 , higher than 5×10 12 or higher than 1×10 13 vg / ml.

[0206] As an alternative to packaging single-stranded (ss) DNA sequences as vector genomes of rAAV particles, systems and methods for packaging RNA sequences as vector genomes into rAAV particles have recently been developed and can be applied herein. See PCT / CN2022 / 075366, which is incorporated herein by reference in its entirety.

[0207] When the vector genome is RNA, such as in PCT / CN2022 / 075366, for the sake of simplicity of description and declaration, the sequence elements for the DNA vector genome described herein, when present in the RNA vector genome, should generally be considered to be applicable to the RNA vector genome, except that the deoxyribonucleotides in the DNA sequence are the corresponding ribonucleotides in the RNA sequence (e.g., dT is equivalent to U, and dA is equivalent to A) and / or the elements in the DNA sequence are replaced with corresponding elements having corresponding functions in the RNA sequence, or are omitted because their functions are not required in the RNA sequence, and / or additional elements necessary for the RNA vector genome are introduced.

[0208] As used herein, a coding sequence (e.g., as a sequence element of an rAAV vector genome herein) is interpreted, understood, and considered to cover and encompass both DNA coding sequences and RNA coding sequences. When it is a DNA coding sequence, an RNA sequence can be transcribed from the DNA coding sequence, and optionally a protein can be further translated from the transcribed RNA sequence as needed. When it is an RNA coding sequence, the RNA coding sequence itself can be a functional RNA sequence for use, or an RNA sequence can be generated from the RNA coding sequence (e.g., by RNA processing), or a protein can be translated from the RNA coding sequence.

[0209] For example, a Cas13 coding sequence encoding a Cas13 polypeptide covers a Cas13 DNA coding sequence from which the Cas13 polypeptide is expressed (indirectly via transcription and translation) or a Cas13 RNA coding sequence from which the Cas13 polypeptide is translated (directly).

[0210] For example, a gRNA coding sequence encoding a gRNA covers a gRNA DNA coding sequence from which the gRNA is transcribed or a gRNA RNA coding sequence that (1) is itself a functional gRNA for use or (2) from which the gRNA is generated (e.g., by RNA processing).

[0211] In some embodiments of the rAAV RNA vector genome, the 5'-ITR and / or 3'-ITR may not be necessary as DNA packaging signals and may be at least partially omitted, but RNA packaging signals may be introduced.

[0212] In some embodiments of the rAAV RNA vector genome, a promoter driving transcription of a DNA sequence may not be necessary and may be at least partially omitted.

[0213] In some embodiments of the rAAV RNA vector genome, the sequence encoding the polyA signal may be unnecessary and may be at least partially omitted, but a polyA tail may be introduced.

[0214] Similarly, other DNA elements of the rAAV DNA vector genome can be omitted or replaced with corresponding RNA elements and / or additional RNA elements can be introduced to accommodate strategies for delivering the RNA vector genome by rAAV particles.

[0215] Pharmaceutical compositions of the present disclosure

[0216] In yet another aspect, the present disclosure provides a pharmaceutical composition comprising the system or composition of the present disclosure or the rAAV particle of the present disclosure, and a pharmaceutically acceptable excipient.

[0217] In some embodiments, the pharmaceutical composition comprises rAAV particles at a concentration selected from about 1×10 10 vg / mL, 2×10 10 vg / mL, 3×10 10 vg / mL, 4×10 10 vg / mL, 5×10 10 vg / mL, 6×10 10 vg / mL, 7×10 10 vg / mL, 8×10 10 vg / mL, 9×10 10 vg / mL, 1×10 11 vg / mL, 2×10 11 vg / mL, 3×10 11 vg / mL, 4×10 11 vg / mL, 5×10 11 vg / mL, 6×10 11 vg / mL, 7×10 11 vg / mL, 8×10 11 vg / mL, 9×10 11 vg / mL, 1×10 12 vg / mL, 2×10 12vg / mL, 3×10 12 vg / mL, 4×10 12 vg / mL, 5×10 12 vg / mL, 6×10 12 vg / mL, 7×10 12 vg / mL, 8×10 12 vg / mL, 9×10 12 vg / mL, 1×10 13 vg / mL, or a concentration in a numerical range between any two of the foregoing values, such as about 9×10 10 vg / mL to about 8×10 11 The concentration of vg / mL.

[0218] In some embodiments, the pharmaceutical composition is an injectable solution.

[0219] In some embodiments, the volume of the injection solution is selected from about 1 microliter, 10 microliters, 50 microliters, 100 microliters, 150 microliters, 200 microliters, 250 microliters, 300 microliters, 350 microliters, 400 microliters, 450 microliters, 500 microliters, 550 microliters, 600 microliters, 650 microliters, 700 microliters, 750 microliters, 800 microliters, 850 microliters, 900 microliters, 950 microliters, 1000 microliters, and volumes in a numerical range between any two of the foregoing values, for example, a concentration of about 10 microliters to about 750 microliters.

[0220] How to use this disclosure

[0221] In yet another aspect, the present disclosure provides a method for preventing or treating a disease or disorder associated with human UBE3A-ATS in a subject in need thereof, the method comprising administering to the subject a system or composition of the present disclosure, an rAAV particle of the present disclosure, or a pharmaceutical composition of the present disclosure, wherein the napRNAn modifies the human UBE3A-ATS, and wherein the modification of the human UBE3A-ATS treats the disease or disorder.

[0222] In some embodiments, the disease or disorder is Angelman Syndrome (AS).

[0223] In some embodiments, administering comprises local administration or systemic administration.

[0224] In some embodiments, administration comprises intrathecal administration, intramuscular administration, intravenous administration, transdermal administration, intranasal administration, oral administration, mucosal administration, intraperitoneal administration, intracranial administration, intracerebroventricular administration, or stereotactic administration.

[0225] In some embodiments, the administering is performed by injection.

[0226] In some embodiments, the subject is a human.

[0227] In some embodiments, the level of human UBE3A-ATS in the subject is reduced by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85% or more compared to the level of UBE3A-ATS in the subject prior to said administration.

[0228] In some embodiments, the level of human UBE3A protein in the subject is at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, or more compared to the level of human UBE3A protein in a human or population not suffering from the disease or disorder.

[0229] The dosage of rAAV particles used to treat the disease or disorder may be a single dose or multiple doses. It will be appreciated by those skilled in the art that the actual dosage may vary widely depending on a variety of factors, such as vector selection, target cells, organisms, tissues, the general condition of the subject to be treated, the degree of transformation / modification sought, the route of administration, the mode of administration, the type of transformation / modification sought, etc.

[0230] In some embodiments, the rAAV particles are administered at a therapeutically effective dose. For example, a therapeutically effective dose of rAAV particles can be about 1.0E+8, 2.0E+8, 3.0E+8, 4.0E+8, 6.0E+8, 8.0E+8, 1.0E+9, 2.0E+9, 3.0E+9, 4.0E+9, 6.0E+9, 8.0E+9, 1.0E+10, 2.0E+10, 3.0E+10, 4.0E+10, 6.0E+10, 8.0E+10, 1.0E+11, 2.0E+11, 3.0E+11, 4.0E+11, 6.0E+11, 8.0E+11, 1.0E+12, 2.0E+12, 3.0E+12, 4.0E+12, 6 + 15, 2.0E+15, 3.0E+15, 4.0E+15, 6.0E+15, 8.0E+15, 1.0E+16, 2.0E+16, 3.0E+16, 4.0E+16, 6.0E+16, 8.0E+16, or 1.0E+17 vg, or a range between any two of these point values. vg represents the vector genome of the rAAV particle used for administration.

[0231] cell

[0232] In another aspect, the disclosure provides a cell or its offspring, the cell or its offspring comprising the guiding nucleic acid of the disclosure, the system or composition or complex of the disclosure, the rAAV vector genome of the disclosure, or the rAAV particle of the disclosure. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is located in the CNS of the subject. In some embodiments, the cell is a human neural cell (e.g., a neuron).

[0233] In yet another aspect, the disclosure provides a cell or its progeny, which is modified by a system or composition or complex of the disclosure, an rAAV particle of the disclosure, or a method of the disclosure. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is located in the CNS of the subject. In some embodiments, the cell is a human neural cell (e.g., a neuron).

[0234] In some embodiments, the cell is not a human embryonic stem cell. In some embodiments, the cell is not a human germ cell.

[0235] Reagent test kit

[0236] In yet another aspect, the present disclosure provides a kit comprising a guide nucleic acid of the present disclosure, a system or composition of the present disclosure, an rAAV vector genome of the present disclosure, an rAAV particle of the present disclosure, or a cell of the present disclosure or its progeny.

[0237] In some embodiments, the kit further includes instructions for using one or more components contained therein, and / or instructions for combining with additional one or more components that may be obtained or required elsewhere.

[0238] In some embodiments, the kit further comprises one or more buffers, which can be used to dissolve any of the components contained therein, and / or provide suitable reaction conditions for one or more of the components. Such buffers can include one or more of the following: PBS, HEPES, Tris, MOPS, Na2CO3, NaHCO3, NaB or a combination thereof. In some embodiments, the reaction conditions include a suitable pH, such as an alkaline pH. In some embodiments, the pH is between 7-10.

[0239] In some embodiments, any one or more of the kit components can be stored in a suitable container or stored at a suitable temperature, such as 4 degrees Celsius.

[0240] Further embodiments are described in the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the present disclosure.

[0241] The present disclosure provides the following additional embodiments.

[0242] One aspect of the present disclosure provides a guide RNA (gRNA) (e.g., a single guide RNA) comprising a spacer sequence that is substantially complementary to a target sequence of UBE3A-ATS. In some embodiments, the gRNA comprises a direct repeat (DR) sequence that can form a complex with a Cas13 polypeptide, wherein when the gRNA guides the Cas13 polypeptide to the target sequence, the complex specifically cuts UBE3A-ATS at or near the target sequence.

[0243] Another aspect of the present disclosure provides a recombinant lentiviral or adeno-associated viral (AAV) vector genome.

[0244] In some embodiments, the recombinant lentiviral or adeno-associated virus (AAV) vector genome comprises a Cas13 coding sequence encoding a Cas13 polypeptide. In some embodiments, the Cas13 coding sequence is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, 99.8%, 99.9% or 100% identical to SEQ ID NO: 2 or its RNA counterpart. In some embodiments, the Cas13 polypeptide comprises (a) the amino acid sequence of SEQ ID NO: 1, or (b) a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 34 and having a non-conservative substitution at Y672 and / or Y676 of SEQ ID NO: 34 (e.g., Y672A and / or Y676A substitution), wherein the variant has substantially the same (e.g., at least about 80%, 90%, 95%, 99% or more) guide RNA-specific nuclease activity (cleavage activity) as SEQ ID NO: 34, and substantially no (e.g., at most 20%, 15%, 10%, 5%) para-cleavage (guide RNA-independent) nuclease activity (para-cleavage activity) of SEQ ID NO: 34.

[0245] In some embodiments, the recombinant lentiviral or adeno-associated viral (AAV) vector genome comprises a single guide RNA (sgRNA) or an sgRNA coding sequence encoding an sgRNA, the sgRNA comprising:

[0246] (A) a spacer sequence that is substantially complementary to the target sequence of UBE3A-ATS; and

[0247] (B) Direct repeat (DR) sequence capable of forming a complex with Cas13 polypeptide.

[0248] In some embodiments, when the sgRNA guides the Cas13 polypeptide to the target sequence, the complex specifically cleaves UBE3A-ATS at or near the target sequence.

[0249] In some embodiments, the gRNA or gRNA encoding sequence is 3' or 5' to the Cas13 encoding sequence.

[0250] In some embodiments, the vector genome further comprises a first coding sequence of a first nuclear localization sequence (NLS, such as SEQ ID NO: 27) or a nuclear export signal (NES) fused to the N-terminus of the Cas13 polypeptide, and / or a second coding sequence of a second NLS (such as SEQ ID NO: 27) or NES fused to the C-terminus of the Cas13 polypeptide.

[0251] In some embodiments, the vector genome further comprises a coding sequence for one or more copies (e.g., 3 tandem copies) of an epitope tag such as 3xFLAG (and a C-terminal NLS or NES, if present) fused (e.g., C-terminally fused) to the Cas13 polypeptide.

[0252] In some embodiments, the vector genome further comprises a 5'AAV ITR sequence and a 3'AAV ITR sequence. In some embodiments, the 5'AAV ITR sequence and the 3'AAV ITR sequence are both wild-type AAV ITR sequences from the following: AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.eB, or a member of the clade to which any one of AAV1-AAV13 belongs, or a functional truncated variant thereof. In some embodiments, the 5'AAV ITR sequence has a polynucleotide sequence of SEQ ID NO: 31. In some embodiments, the 3'AAV ITR sequence has a polynucleotide sequence of SEQ ID NO: 32.

[0253] In some embodiments, the vector genome further comprises a promoter operably linked to the Cas13 coding sequence. In some embodiments, the promoter is a broad-spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter, or an inducible promoter. In some embodiments, the promoter is selected from: Cbh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, retrovirus Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1α short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (Ie) enhancer and / or promoter, chicken β-actin (CBA) promoter or its derivatives such as CAG promoter, CB promoter, (human) elongation factor 1α-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, Platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, synaptophysin (Syn) promoter, synaptophysin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, proenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter and myelin basic protein (MBP) promoter. In some embodiments, the promoter includes a Syn1 promoter, such as a Syn1 promoter having a polynucleotide sequence of SEQ ID NO: 25.

[0254] In some embodiments, the vector genome further comprises a polyadenylation (polyA) signal sequence, such as a bovine growth hormone polyadenylation (bGH polyA) signal, a small polyA (SPA) signal, a human growth hormone polyadenylation (hGH polyA) signal, a SV40 polyA (SV40 polyA) signal, a rabbit beta globin polyA (rBG polyA) signal, and functional truncations or variants thereof; or a corresponding polyA sequence.

[0255] In some embodiments, the polyA signal sequence comprises an SV40 polyA signal or a variant thereof. In some embodiments, the SV40 polyA signal comprises the polynucleotide sequence of SEQ ID NO:29.

[0256] In some embodiments, the gRNA coding sequence is operably linked to a promoter. In some embodiments, the promoter is a broad-spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter, or an inducible promoter.

[0257] In some embodiments, the promoter is selected from: Cbh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, retrovirus Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1 alpha short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (Ie) enhancer and / or promoter, chicken β-actin (CBA) promoter or its derivatives such as CAG promoter, CB promoter, (human) elongation factor 1 alpha-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, Platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, synaptophysin (Syn) promoter, synaptophysin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter and myelin basic protein (MBP) promoter. In some embodiments, the promoter is an RNA pol III promoter.

[0258] In some embodiments, the RNA pol III promoter is U6 (such as SEQ ID NO: 30), H1, 7SK, or a variant thereof.

[0259] In some embodiments, (1) the gRNA comprises a spacer sequence directly linked to a DR sequence (e.g., SEQ ID NO: 3); (2) the gRNA comprises a spacer sequence flanked by two DR sequences (e.g., each SEQ ID NO: 3); or (3) the gRNA comprises two or more spacers; and each spacer sequence is flanked by two DR sequences each capable of forming a complex with a Cas13 polypeptide. In some embodiments, the gRNA comprises two spacers flanked by three DR sequences to form a DR-spacer-DR-spacer-DR structure (e.g., each SEQ ID NO: 3). In some embodiments, each spacer sequence is independently substantially complementary to a unique target sequence of UBE3A-ATS, and each spacer sequence is capable of directing the Cas13 polypeptide to cut its own unique target sequence.

[0260] In some embodiments, the DR sequence comprises (1) SEQ ID NO:3; (2) a sequence that is at least 90%, 92%, 94%, 95%, 96%, 98% or 99% identical to SEQ ID NO:3; (3) a sequence that has at most 1, 2, 3, 4 or 5 nucleotide differences from SEQ ID NO:3; or (4) a sequence that has a secondary structure substantially identical to SEQ ID NO:3.

[0261] In some embodiments, each DR sequence comprises, consists essentially of, or consists of: SEQ ID NO:3.

[0262] In some embodiments, the target sequence comprises a continuous stretch of nucleotides of the RNA counterpart of NG_002690.1 or NC_000073.7. In some embodiments, the target sequence comprises a stretch of 20-50 or 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 or 50, such as 30, continuous nucleotides of the RNA counterpart of NG_002690.1 or NC_000073.7 (such as any one of SEQ ID NOs: 16-21 and 58-80 or their RNA counterparts).

[0263] In some embodiments, the spacer sequence is independently selected from any one of SEQ ID NOs: 10-15 and 35-57 or a variant thereof, which differs from any one of SEQ ID NOs: 10-15 and 35-57 by at most 1, 2, 3, 4, 5 or 6 nucleotides without substantially reducing the ability of the guide Cas13 polypeptide to bind to the gRNA to form a Cas13-gRNA complex that targets the target sequence to cleave the target RNA.

[0264] In some embodiments, UBE3A-ATS is associated with a disease or disorder such as AS (Angelman Syndrome).

[0265] In some embodiments, the vector genome comprises an ITR to ITR polynucleotide (such as SEQ ID NO: 33), which from 5' to 3' comprises:

[0266] (a) an optional 5' ITR from AAV2 (such as SEQ ID NO: 31);

[0267] (b) Syn1 promoter (such as SEQ ID NO: 25);

[0268] (c) Kozak sequence (such as SEQ ID NO: 26);

[0269] (d) a first NLS encoding sequence (such as a sequence encoding SEQ ID NO: 27);

[0270] (e) a Cas13 polynucleotide encoding the Cas13 polypeptide of SEQ ID NO: 1 (excluding the first amino acid M) (such as SEQ ID NO: 2 excluding the start codon ATG);

[0271] (f) a second NLS encoding sequence (such as a sequence encoding SEQ ID NO: 27);

[0272] (g) an optional coding sequence encoding a 3xFlag sequence (eg, SEQ ID NO: 28);

[0273] (h) an optional SV40 polyA signal sequence (such as SEQ ID NO: 29);

[0274] (i) U6 promoter (such as SEQ ID NO: 30);

[0275] (j) a first direct repeat (DR) coding sequence encoding a first DR sequence (such as SEQ ID NO: 3);

[0276] (k) a spacer coding sequence encoding a first spacer sequence specific for UBE3A-ATS (such as SEQ ID NO: 10 or 15);

[0277] (1) a second DR coding sequence encoding a second DR sequence (such as SEQ ID NO: 3); and

[0278] (m) an optional 3' ITR from AAV2 (such as SEQ ID NO: 32);

[0279] Or a polynucleotide that is at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identical to an ITR to ITR polynucleotide.

[0280] In some embodiments, the ITR to ITR polynucleotide further comprises a linker sequence between any two adjacent sequence elements of (a)-(m).

[0281] In some embodiments, the sequence elements of (b) to (h) that are 5' to the sequence elements of (i) to (l) are repositioned 3' to the sequence elements of (i) to (l).

[0282] In some embodiments, the sequence elements of (b) to (h) in the 5'-3' direction are placed in the reverse order of (h) to (b) in the 5'-3' direction.

[0283] In some embodiments, the sequence elements from (i) to (l) in the 5'-3' direction are placed in the reverse order of (l) to (i) in the 5'-3' direction.

[0284] Another aspect of the present disclosure provides a recombinant AAV vector genome comprising, consisting essentially of, or consisting of:

[0285] (1) SEQ ID NO:33, or a polynucleotide having at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9% identity thereto,

[0286] The polynucleotide encodes

[0287] (a) a Cas13 polypeptide of SEQ ID NO: 1, or

[0288] (b) a variant thereof having at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identity to SEQ ID NO: 34 and having a non-conservative substitution at Y672 and / or Y676 of SEQ ID NO: 34 (e.g., Y672A and / or Y676A substitution), wherein the variant has substantially the same (e.g., at least about 80%, 90%, 95%, 99% or more) guide RNA-specific nuclease activity as SEQ ID NO: 34 and substantially no (e.g., at most 20%, 15%, 10%, 5%) flanking (guide RNA-independent) nuclease activity of SEQ ID NO: 34; and

[0289] (2) a gRNA coding sequence encoding a gRNA, wherein the gRNA comprises:

[0290] (A) a spacer sequence that is substantially complementary to the target sequence of UBE3A-ATS; and

[0291] (B) a direct repeat (DR) sequence that forms a complex with the Cas13 polypeptide,

[0292] wherein when the gRNA guides the Cas13 polypeptide to the target sequence, the complex specifically cleaves UBE3A-ATS at or near the target sequence, has substantially the same (e.g., at least about 80%, 90%, 95%, 99% or more) guide RNA-specific nuclease activity as SEQ ID NO: 34, and has substantially no (e.g., at most 20%, 15%, 10%, 5%) side-cutting (guide RNA-independent) nuclease activity of SEQ ID NO: 34

[0293] In some embodiments, the gRNA coding sequence is 3' or 5' to the Cas13 coding sequence.

[0294] In some embodiments, the vector genome is SEQ ID NO: 33 or a polynucleotide at least 95% or 99% identical thereto.

[0295] Another aspect of the present disclosure provides a recombinant lentiviral or AAV particle comprising the vector genome of the present disclosure.

[0296] In some embodiments, the recombinant AAV particle comprises a capsid of a serotype of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or a member of the evolutionary branch to which any one of AAV.PHP.eB, AAV1-AAV13 belongs, or a functional truncated variant or functional mutant thereof, which encapsulates the vector genome.

[0297] In some embodiments, the capsid serotype is AAV.PHP.eB or AAV9.

[0298] Another aspect of the present disclosure provides a recombinant AAV particle, which comprises the vector genome of the present disclosure, and the vector genome is encapsulated in a capsid of the serotype AAV.PHP.eB or AAV9.

[0299] Another aspect of the present disclosure provides a pharmaceutical composition comprising the vector genome of the present disclosure, or the particle of the present disclosure, and a pharmaceutically acceptable excipient.

[0300] Another aspect of the present disclosure provides a method of treating a disease or disorder associated with UBE3A in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a vector genome of the present disclosure, a particle of the present disclosure, or a pharmaceutical composition of the present disclosure, wherein the vector genome or the particle specifically upregulates the expression of UBE3A that is causative of the disease or disorder.

[0301] In some embodiments, the administering comprises contacting the cell with a therapeutically effective amount of a vector genome of the disclosure, a particle of the disclosure, or a pharmaceutical composition of the disclosure.

[0302] In some embodiments, the cell is located in the CNS of the subject.

[0303] In some embodiments, the disease or disorder is Angelman Syndrome (AS).

[0304] In some embodiments, the administering comprises intracerebroventricular administration.

[0305] In some embodiments, the subject is a human.

[0306] In some embodiments, the level of UBE3A-ATS transcript is reduced in the cell compared to a cell that has not been contacted with a vector genome of the disclosure, a particle of the disclosure, or a pharmaceutical composition of the disclosure.

[0307] In some embodiments, the level of UBE3A-ATS in the subject is reduced by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or about 85% compared to the level of UBE3A-ATS in the subject before administration; and / or the level of UBE3A protein in a subject not suffering from the disease or disorder is reduced by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, or about 85%. In some embodiments, the level of UBE3A protein in the subject is at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, or about 135% as compared to the level of UBE3A protein in the subject.

[0308] Another aspect of the present disclosure provides a cell or its progeny, comprising the vector genome of the present disclosure, the particle of the present disclosure, or the gRNA of the present disclosure.

[0309] Another aspect of the present disclosure provides a kit comprising the vector genome of the present disclosure, the particle of the present disclosure, the gRNA of the present disclosure, or the cell of the present disclosure or its progeny.

[0310] Another aspect of the present disclosure provides a method for preparing the recombinant AAV particles of the present disclosure, the method comprising:

[0311] a) introducing a nucleic acid encoding a vector genome of the present disclosure or an RNA sequence transcribed therefrom and a coding sequence for an AAV capsid of the present disclosure into an AAV packaging system for a period of time sufficient to package the vector genome or the transcribed RNA into AAV capsids to produce recombinant AAV particles, and

[0312] b) Harvesting recombinant AAV particles.

[0313] In some embodiments, the method includes isolating or purifying the harvested recombinant AAV particles.

[0314] Example

[0315] The following examples are provided to further illustrate some embodiments of the present disclosure but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methods or techniques known to those skilled in the art may alternatively be used.

[0316] Example 1 Highly efficient in vitro knockdown of UBE3A-ATS by the CRISPR-hfCas13e.1 system

[0317] This example demonstrates the high in vitro knockdown efficiency of UBE3A-ATS by the CRISPR-hfCas13e.1 system of the present invention via lentiviral delivery.

[0318] Lentiviral transgenic plasmid construction:

[0319] The CRISPR-hfCas13e.1 system of the present invention is composed of hfCas13e.1 protein (SEQ ID NO: 1, encoded by SEQ ID NO: 2) and sgRNA targeting Ube3a-ATS (hereinafter "sgRNA [Ube3a-ATS] 9-14" or "sg9-14" SEQ ID NO: 4-9, one of), wherein the sgRNA targeting Ube3a-ATS is composed of a spacer sequence (one of the spacer sequences 9-14 of SEQ ID NO: 10-15, respectively) flanked by two direct repeat sequences (SEQ ID NO: 3), and a target sequence targeting UBE3A-ATS (one of the target sequences 9-14 of SEQ ID NO: 16-21, respectively).

[0320] As a negative control, a non-targeting sgRNA consisting of a non-targeting spacer sequence (LacZ, SEQ ID NO: 23) flanked by two direct repeat sequences (SEQ ID NO: 3) (hereinafter referred to as “sgRNA [NT]” or “NT”, SEQ ID NO: 22) was used instead of sgRNA [Ube3a-ATS].

[0321] The hfCas13e.1 protein (SEQ ID NO: 1) and sgRNA [Ube3a-ATS] or sgRNA [NT] were encoded together with the EGFP reporter into Figure 2 The transgenic plasmids shown in A were used to produce treatment or control lentiviral particles.

[0322] AS mouse model:

[0323] All mice were housed in an in-house animal facility with a 12 hr:12 hr light / dark cycle and free access to food and water. Ube3a knockout (KO) mice were generated by Jiang et al. (YH Jiang et al. 1998, Neurons). m+ / p-The AS mouse model was generated by crossing heterospecies females with C57BL / 6 wild-type (WT) males from Shanghai SLAC Laboratory Animal Co., Ltd. All experimental protocols were approved by the Animal Care and Utilization Committee of the Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China and HuidaGene Therapeutics Co., Ltd.

[0324] Preparation of recombinant lentiviral particles:

[0325] like Figure 2 As shown in A, treatment or control lentiviral transgenic plasmids were constructed, and each plasmid was co-transfected with packaging plasmids and envelope plasmids into HEK293 cells to produce recombinant lentiviral particles delivering the CRISPR-hfCas13e.1 system. Two days later, the HEK293 cell supernatant was filtered through a 0.22 μm sterile Millex filter and then collected for ultracentrifugation (27000 rpm, CP90NX, Hitachi), and the lentiviral particles at the bottom of the tube after ultracentrifugation were resuspended in PBS buffer, and the titer of the lentiviral vector was determined by RT-qPCR with GAG and TET1 primers. The copy number of the GAG ​​sequence was measured and normalized to the copy number of the housekeeping gene TET1.

[0326] RT-qPCR detection:

[0327] Total RNA was extracted from AS or C57BL / 6WT primary neurons using Trizol (Ambion) after lentiviral infection and then reverse transcribed into complementary DNA (HiScript Q RT SuperMix for qPCR, Vazyme, Biotech) for RT-qPCR detection. RT-qPCR reactions were followed by SYBR green probe (AceQ qPCR SYBR Green premix, Vazyme Biotech). Transcription levels of UBE3A-ATS and Ube3a mRNA were measured using Ube3a-ATS and Ube3a primers and normalized to the mRNA level of the housekeeping gene Gapdh.

[0328] Neuronal cultures and lentiviral infection:

[0329] Cortices and hippocampi were excised from E14-E16 embryos of AS and C57BL / 6WT mouse models and dissociated in digestion buffer containing papain (Cat. No. LS003126, Worthington Bio Corp.). The digestion buffer contained (in mM): 161.0 NaCl, 5.0 KCl, 2.9 CaCl2, 5.0 HEPES, 5.5 glucose, 0.53 MgSO4, and 0.0056 phenol red, pH 7.4, and additionally contained (in mM) 1.7 cysteine, 1.0 CaCl2, and 0.5 EDTA. The tissue was digested at 37°C for 30 minutes (min) and then plated onto PDL (Cat. No. P6407, Sigma-Aldrich) in neuronal culture medium (Cat. No. 21103049, Thermo Fisher Scientific) containing 2mM Glutamax-I (Cat. No. 35050-061, Thermo Fisher Scientific), 1x B27 (Cat. No. 17504-044, Thermo Fisher Scientific) and 5% FBS. Four hours later, AS or C57BL / 6WT primary neurons from digested tissues of AS or C57BL / 6WT mouse models were maintained in vitro in the same culture medium without FBS. On the second day after AS and WT primary neurons were maintained (DIV2), primary neurons were transfected in vitro with lentiviral particles for 24 hours, MOI (multiplicity of infection) = 10, and then half-changes of the culture medium were performed for three consecutive days. Subsequent experiments were performed 7–9 days after lentiviral infection.

[0330] Western blot analysis:

[0331] AS or WT primary neurons infected with the above lentivirus were lysed in SDS lysis buffer (Beyotime) containing 1x protease inhibitor cocktail (Beyotime). 40-60 μg of total protein was loaded and separated by SDS-PAGE (Epizyme) and transferred to PVDF membrane (Merk Millipore). The membrane was blocked in TBST buffer (Epizyme) containing 5% skim milk powder. The following primary antibodies were diluted and incubated overnight with the membrane: anti-Ube3a (1:1000; catalog number A300-352A, Bethyl), anti-Flag (1:3000; catalog number F1804, Sigma), anti-α-tubulin (1:5000, catalog number AF0001, Beyotime). After primary antibody incubation, the membrane was probed with goat anti-mouse HPR (1:5000; catalog number A9044, Sigma-Aldrich) or goat anti-rabbit HRP (1:5000; catalog number A0545, Sigma) secondary antibodies. Images were collected using Amersham Imager 600 (GE) and analyzed by ImageJ software.

[0332] result:

[0333] RT-qPCR results ( Figure 3 A) Shows that ex vivo transfection of lentiviral vectors encoding hfCas13e.1 protein and sgRNA [Ube3a-ATS] (sg9, sg10, sg11, sg12, sg13, or sg14) into AS primary neurons induced the expression of Ube3a-ATS, respectively, compared with the Ube3a-ATS RNA level in AS primary neurons transfected with hfCas13e.1-sg[NT]. RNA levels (right column) decreased by 85.1%, 69.2%, 67.4%, 77.8%, 83.1% and 81.3% (mean, n=3 replicates) and reached Ube3a mRNA levels of 109.2%, 127.6%, 3.9%, 15.9%, 93.6% and 107.3% (mean, n=3 replicates) compared to the mRNA levels of maternal Ube3a in C57BL / 6WT primary neurons transfected with hfCas13e.1-NT (left column).

[0334] WB results ( Figure 3B-3C) show that compared with the protein level of Ube3a in C57BL / 6WT primary neurons transfected with hfCas13e.1-NT, ex vivo transfection of lentiviral vectors encoding hfCas13e.1 protein and sgRNA [Ube3a-ATS] (sg9, sg10, sg11, sg12, sg13 or sg14) into AS primary neurons achieved 123.0%, 131.6%, 30.6%, 40.0%, 134.9% and 182.8% (mean, n = 3 replicates) of paternal Ube3a expression, respectively.

[0335] Thus, sg9, sg10, sg13, and sg14 significantly reduced Ube3a-ATS RNA levels compared to the AS model, and substantially maintained Ube3a mRNA levels and significantly increased UBE3A protein levels compared to the WT model. sg9 was selected for further evaluation in Examples 2 and 3.

[0336] Example 2 Highly efficient in vivo knockdown of UBE3A-ATS by the CRISPR-hfCas13e.1 system

[0337] In order to demonstrate the in vivo knockdown efficiency of the CRISPR-hfCas13e.1 system of the present invention in Example 1, the system was delivered to target cells of animals in vivo using the AAV-PHP.eB delivery system.

[0338] AAV-PHP.eB transgenic plasmid construction:

[0339] Similar to the treatment and control transgenic plasmids in Example 1, treatment and control transgenic plasmids encoding hfCas13e.1 and sg9 or sgNT for AAV-PHP.eB packaging were constructed, respectively. Figure 2 As shown in B.

[0340] Preparation of recombinant AAV-PHP.eB particles:

[0341] In this paper, the traditional three-plasmid transfection system was used with necessary modifications to co-transfect the corresponding transgenic plasmid, packaging plasmid and auxiliary plasmid into HEK293T cells at a mass ratio of 1:1:2 to produce treatment and control rAAV-PHP.eB particles. The transgenic plasmid was packaged with the AAV-PHP.eB capsid to form the genome within the capsid, and the genome and capsid were constructed together into AAV-PHP.eB particles.

[0342] Specifically, HEK293T cells were cultured in competent DMEM medium and the cells were plated 24 hours before plasmid transfection. Shortly before transfection, the medium was replaced with fresh DMEM containing 2% FBS. PEI-MAX was used as a transfection reagent. Transfected HEK293T cells were harvested from the culture medium 72 hours after translation. Treatment and control AAV-PHP.eB particles were purified from the cells by using iodixanol density gradient ultracentrifugation.

[0343] RT-qPCR was used with a pair of 5'-ITR primers specific for the 5'-ITR sequence on the genome to detect the genomic titer of any genome packaged in the treatment and control AAV-PHP.eB particles.

[0344] AS mouse model and intracerebroventricular injection

[0345] All mice were housed in an in-house animal facility with a 12 hr:12 hr light / dark cycle and free access to food and water. Ube3a knockout (KO) mice were generated by Jiang et al. (YH Jiang et al. 1998, Neurons). m+ / p- The AS mouse model was generated by crossing heterospecies females with C57BL / 6 wild-type (WT) males from Shanghai SLAC Laboratory Animal Co., Ltd. All experimental protocols were approved by the Animal Care and Utilization Committee of the Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China and HUIGENE THERAPEUTICS CO., LTD. Newborn AS mice were placed on ice for hypothermia anesthesia and injected with AAV-PHP.eB particles into the bilateral lateral ventricles at four sites. A total of 5×10 10 AAV particles (encoding Figure 2 hSyn1 promoter in B-hfCas13e.1-sg9 / NT) plus 2.5×10 9Reporter AAV particles (encoding CAG promoter-tdTomato reporter) were injected into each AS mouse within 24 hours after birth. Treated AS mice (AS+sg9): AS mice were injected with AAV-PHP.eB particles delivering hfCas13e.1-sg[Ube3a-ATS]9. Untreated AS mice (AS+NT): AS mice were injected with AAV-PHP.eB particles delivering hfCas13e.1-sg[NT]. Untreated WT mice (WT+NT): C57BL / 6WT mice were injected with AAV-PHP.eB particles delivering hfCas13e.1-sg[NT].

[0346] RT-qPCR detection:

[0347] Four weeks after injection, tdTomato expression in the brain was isolated from injected AS mice. + Area. Total RNA from the cortex and hippocampus was extracted and purified using Trizol (Ambion), and then reverse transcribed into complementary DNA (HiScript Q RTSuperMix for qPCR, Novazon Biotech) for RT-qPCR. The levels of Ube3a and UBE3A-ATS were detected by RT-qPCR using Taqman probes (Bestar qPCR Master Mix, DBI-2041, DBI) with Ube3a-ATS and Ube3a primers, and normalized to the mRNA level of the housekeeping gene GAPDH.

[0348] Western blot analysis:

[0349] Tdtomato positive brain regions were dissected and homogenized in SDS lysis buffer (Biyuntian) containing 1x protease inhibitor cocktail (Biyuntian). 40-60 μg of total protein was loaded and separated by SDS-PAGE (Epizyme) and transferred to PVDF membrane (Merk Millipore). The membrane was blocked in TBST buffer (Yazyme) containing 5% skim milk powder. The following antibodies were diluted and incubated overnight with the membrane: anti-Ube3a (1:1000; catalog number A300-352A, Bethel), anti-Flag (1:3000; catalog number F1804, Sigma), anti-a tubulin (1:5000, catalog number AF0001, Biyuntian). After primary antibody incubation, the membrane was probed with goat anti-mouse HPR (1:5000; catalog number A9044, Sigma-Aldrich) or goat anti-rabbit HRP (1:5000; catalog number A0545, Sigma) secondary antibodies. Images were collected using Amersham Imager 600 (GE) and analyzed by ImageJ software.

[0350] result:

[0351] RT-qPCR results ( Figure 4 B and Figure 4 C) shows that the in vivo efficiency of the CRISPR-hfCas13e.1 system containing sg9 delivered by AAV-PHP.eB is that the Ube3a-ATS RNA level of treated AS mice is reduced by 77.5% in the cortex and 76.6% in the hippocampus compared with untreated AS mice (right column), and the Ube3a mRNA level of treated AS mice is restored by 44.3% in the cortex and 17.6% in the hippocampus compared with untreated WT mice (left column).

[0352] WB results ( Figure 4 D-4G) showed that the hfCas13e.1-sg9 system induced 35.9% and 41.6% UBE3A expression in the cortex and hippocampus of treated AS mice, respectively, at 4 weeks, and 21.1% and 39.1% UBE3A expression in the cortex and hippocampus, respectively, at 18 weeks, compared with untreated WT mice.

[0353] All these results indicate that the hfCas13e.1-sg9 system is highly efficient in knocking down UBE3A-ATS and restoring paternal Ube3a expression.

[0354] Example 3 Effective improvement of the phenotype of AS mouse model by CRISPR-hfCas13e.1-sg9 system

[0355] AS mouse model and intracerebroventricular injection

[0356] All mice were housed in an in-house animal facility with a 12 hr:12 hr light / dark cycle and free access to food and water. Ube3a null mice were generated by Jiang et al. (YH Jiang et al. 1998, Neurons). m+ / p-The AS mouse model was generated by crossing C57BL / 6 wild-type (WT) males from Shanghai SLAC Laboratory Animal Co., Ltd. (paternal Ube3a deletion) with females. All experimental protocols were approved by the Animal Care and Utilization Committee of the Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China and HUIGENE THERAPEUTICS CO., LTD. Newborn AS mice were placed on ice for hypothermia anesthesia and injected with AAV-PHP.eB particles into the bilateral lateral ventricles at four sites. A total of 5×10 10 AAV particles (encoding Figure 2 hSyn1 promoter in B-hfCas13e.1-sg9 / NT) plus 2.5×10 9 Reporter AAV particles (encoding CAG promoter-tdTomato reporter) were injected into each AS mouse or C57BL / 6WT mouse within 24 hours after birth. Treated AS mice (AS+sg9): AS mice were injected with AAV-PHP.eB particles delivering hfCas13e.1-sg[Ube3a-ATS]9. Untreated AS mice (AS+NT): AS mice were injected with AAV-PHP.eB particles delivering hfCas13e.1-sg[NT]. Untreated WT mice (WT+NT): C57BL / 6WT mice were injected with AAV-PHP.eB particles delivering hfCas13e.1-sg[NT].

[0357] Behavioral testing

[0358] All behavioral experiments were performed blind to the animals' genotype and injection treatment. Mice were placed in the testing room for 30 min to acclimate before testing.

[0359] Hindlimb grasp test

[0360] At 7 weeks, each mouse was suspended 10 cm above a table by holding the tail for 20 seconds. Videos were recorded and the time was noted offline. Hindlimb grasping time was the total time spent grasping. Grasping was defined as behavior with incomplete opening of one or both hindlimbs.

[0361] Open field test

[0362] Each mouse was placed at the edge of a 40 cm × 40 cm open field box and allowed to explore for 15 minutes. The center area was a 20 cm × 20 cm square in the center of the arena. The travel distance was the total travel distance of each mouse in the arena, which was recorded and analyzed by a camera and EthoVision software (Noldus Wageningen). The center frequency means the number of times each mouse entered the center within 15 minutes.

[0363] Dowel test

[0364] A 1-meter-long dowel with a diameter of 9 mm was placed parallel to the ground at a height of more than 30 cm. The mouse was placed on the dowel individually, and the time it was on the dowel was recorded. The longest experimental record was 120 seconds.

[0365] Beam walking test

[0366] A 1 m long dowel with a diameter of 9 mm was placed parallel to the ground at a height of about 30 cm. There was a safety platform at the end of the dowel. After 2 days of training, the latency was quantified by measuring the time it took the mouse to walk across the dowel, and the number of foot slips was also counted.

[0367] Accelerating Rotarod Test

[0368] The test was performed on a rotating rod system, where the rod rotated from 5 rpm to 30 rpm in 5 minutes. The mice were trained for two days. Two trials were performed every day, with an interval of more than 1 hour between trials. On the test day, the mice were tested twice, and the time to stay on the rod until falling or performing two consecutive rotations was recorded. The average time of the two trials was calculated.

[0369] result:

[0370] After intracerebroventricular administration of the CRISPR-hfCas13e.1-sg9 / sg[NT] system on postnatal day 0, mice were weighed every two weeks and observed ( Figure 5 B) Compared with untreated AS mice injected with hfCas13e.1-sg[NT], AS female mice systemically treated with hfCas13e.1-sg9 showed reduced body weight, implying that the obese phenotype of the AS mouse model was significantly ameliorated by this treatment.

[0371] Increased center frequency ( Figure 5 D) Means that AS mice treated with the hfCas13e.1-sg9 system prefer to explore the central area than untreated AS mice.

[0372] In addition, the hindlimb grasp test, dowel test, beam walking test, and accelerating rotarod test ( Figure 5 C. Figure 5E-5H) demonstrated that AS mice treated with the hfCas13e.1-sg9 system performed better in motor coordination and balance compared with untreated AS mice.

[0373] In summary, the data presented in this article demonstrate that the CRISPR-hfCas13e.1 system of the present invention efficiently knocks down UBE3A-ATS in isolated AS primary neurons and in the brain of AS mouse models in vivo, and that intracerebroventricular administration of AAV-PHP.eB particles delivering the hfCas13e.1-sgRNA[Ube3a-ATS] system can rescue the abnormal behavior of AS mice, thus showing good prospects for treating AS and other UBE3A-ATS-related diseases in humans.

[0374] ***

[0375] Without departing from the scope and spirit of the present disclosure, various modifications and variations of the products, methods and uses of the present disclosure will be clear to those skilled in the art. Although the present disclosure has been described in conjunction with specific embodiments, it will be understood that further modifications can be made, and the disclosure claimed for protection should not be unduly limited to such specific embodiments. In fact, various modifications of the described manner for performing the present disclosure that are obvious to those skilled in the art are intended to fall within the scope of the present disclosure. The present application is intended to cover any changes, uses or adaptations of the present disclosure that generally follow the principles of the present disclosure, and includes such deviations from the present disclosure: it is within the known customary operations in the field to which the present disclosure belongs and is applicable to the basic features shown heretofore.

[0376] Exemplary sequences

[0377] hfCas13e.1 (hfCas13e, hfCas13X, hfCas13X.1) amino acid sequence, SEQ ID NO: 1

[0378] MAQVSKQTSKKRELSIDEYQGARKWCFTIAFNKALVNRDKNDGLFVESLLRHEKYSKHDWYDEDTRALIKCSTQAANAKAEALRNYFSHYRHSPGCLTFTAEDELRTIMERAYERAIFECRRRETEVIIEFPSLFEGDRITTAGVVFFVSFFVERRVLDRLYGAVSGLKKNEGQYKLTRKALSMYCLKDSRFTKAWDKRVLLFRDILAQLGRIPAEAYEYYHGEQGDKKRANDNEGTNPKRHKDKFIEFALHYLEAQHSEICFGRRHIVREEAGAGDEHKKHRTKGKVVVDFSKKDEDQSYYISKNNVIVRIDKNAGPRSYRMGLNELKYLVLLSLQGKGDDAIAKLYRYRQHVENILDVVKVTDKDNHVFLPRFVLEQHGIGRKAFKQRIDGRVKHVRGVWEKKKAATNEMTLHEKARDILQYVNENCTRSFNPGEYNRLLVCLVGKDVENFQAGLKRLQLAERIDGRVYSIFAQTSTINEMHQVVCDQILNRLCRIGDQKLYDYVGLGKKDEIDYKQKVAWFKEHISIRRGFLRKKFWYDSKKGFAKLVEEHLESGGGQRDVGLDKKYYHIDAIGRFEGANPALYETLARDRLCLMMAQYFLGSVRKELGNKIVWSNDSIELPVEGSVGNEKSIVFSVSDYGKLYVLDDAEFLGRICEYFMPHEKGKIR A HTV A EKGFRAYNDLQKKCVEAVLAFEEKVVKAKKMSEKEGAHYIDFREILAQTMCKEAEKTAVNKVRRAFFHHHLKFVIDEFGLFSDVMKKYGIEKEWKFPVK

[0379] The coding sequence of hfCas13e.1 (hfCas13e, hfCas13X, hfCas13X.1), SEQ ID NO:2

[0380]

[0381] The hfCas13e.1 DR RNA sequence, SEQ ID NO:3

[0382] GCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGC

[0383] [sg9] gRNA sequence, SEQ ID NO:4

[0384] GCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGCGATAGGTATTTCGAGTGTGATTAAAGTAACGCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGC

[0385] [sg10] gRNA sequence, SEQ ID NO:5

[0386] GCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGCGTTCATGACATTGGGTTGTCATCATTGACCGCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGC

[0387] [sg11] gRNA sequence, SEQ ID NO:6

[0388] GCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGCTGTGCACCCTAGATGTCCACATTTCCTCTCGCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGC

[0389] [sg12] gRNA sequence, SEQ ID NO:7

[0390] GCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGCTACCTGATTATCTGTGTTTCTCTGCCTTCCGCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGC

[0391] [sg13] gRNA sequence, SEQ ID NO:8

[0392] GCTGGAGCAGCCCCGATTTGTGGGGTGATTACAGCGAATTCGGCTACATCTACTTGGATATCACCGCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGC

[0393] [sg14] gRNA sequence, SEQ ID NO: 9

[0394] GCTGGAGCAGCCCCGATTTGTGGGGTGATTACAGCTGGGCCTCAGCGTAATCCTATTGAGCATGAGCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGC

[0395] [sg9] spacer sequence, SEQ ID NO: 10

[0396] GATAGGTATTTCGAGTGTGATTAAAGTAAC

[0397] [sg10] Spacer sequence, SEQ ID NO: 11

[0398] GTTCATGACATTGGGTTGTCATCATTGACC

[0399] [sg11] spacer sequence, SEQ ID NO: 12

[0400] TGTGCACCCTAGATGTCCACATTTCCTCTC

[0401] [sg12] spacer sequence, SEQ ID NO: 13

[0402] TACCTGATTATCTGTGTTTCTCTGCCTTCC

[0403] [sg13] Spacer sequence, SEQ ID NO: 14

[0404] GAATTCGGCTACATCTACTTGGATATCACC

[0405] [sg14] Spacer sequence, SEQ ID NO: 15

[0406] TGGGCCTCAGCGTAATCCTATTGAGCATGA

[0407] [sg9] target sequence, SEQ ID NO: 16

[0408] GTTACTTTAATCACACTCGAAATACCTATC

[0409] [sg10] target sequence, SEQ ID NO: 17

[0410] GGTCAATGATGACAACCCAATGTCATGAAC

[0411] [sg11] target sequence, SEQ ID NO: 18

[0412] GAGAGGAAATGTGGACATCTAGGGGTGCACA

[0413] [sg12] target sequence, SEQ ID NO: 19

[0414] GGAAGGCAGAGAAACACAGATAATCAGGTA

[0415] [sg13] target sequence, SEQ ID NO: 20

[0416] GGTGATATCCAAGTAGATGTAGCCGAATTC

[0417] [sg14] target sequence, SEQ ID NO: 21

[0418] TCATGCTCAATAGGATTACGCTGAGGCCCA

[0419] Non-targeting gRNA sequence, SEQ ID NO: 22

[0420] GCTGGAGCAGCCCCGATTTGTGGGGTGATTACAGCTCCAGCACAGCTCTGCTGAAGTCATCATTGGCTGGAGCAGCCCCCGATTTGTGGGGTGATTACAGC

[0421] Non-targeting spacer sequence, SEQ ID NO: 23

[0422] TCCAGCACAGCTCTGCTGAAGTCATCATTG

[0423] Non-targeted target sequence, SEQ ID NO: 24

[0424] CAATGATGACTTCAGCAGAGCTGTGCTGGA

[0425] hSyn1 promoter of Cas, SEQ ID NO: 25

[0426] CTGCAGAGGGCCCTGCGTATGAGTGCAAGTGGGTTTTAGGACCAGGATGAGGCGGGGTGGGGGTGCCTACCTGACGACCGACCCCGACCCACTGGACAAGCACCCAACCCCCATTCCCCAAATTGCGCATCCCCTATCAGAGAGGGGGAGGGGAAACAGGATGCGGCGAGGCGCGTGCGCACTGCCAGCTTCAGCACCGCGGACAGTGCCTTCGCCCCCGCCTGGCGGCGCGCGCCACCGCCGCCTCAGCACTGAAGGCGCGCTGACGTCACTCGCCGGTCCCCCGCAAACTCCCCTTCCCGGCCACCTTGGTCGCGTCCGCGCCGCCGCCGGCCCAGCCGGACCGCACCACGCGAGGCGCGAGATAGGGGGGCACGGGCGCGACCATCTGCGCTGCGGCGCCGGCGACTCAGCGCTGCCTCAGTCTGCGGTGGGCAGCGGAGGAGTCGTGTCGTGCCTGAGAGCGCAGTCGAGAA

[0427] Kozak, SEQ ID NO:26

[0428] GCCACC

[0429] bpSV40 NLS amino acid sequence, SEQ ID NO:27

[0430] KRTADGSEFESPKKKRKV

[0431] 3×FLAG, SEQ ID NO:28

[0432] DYKDHDGDYKDHDIDYKDDDDK

[0433] SV40 - polyA, SEQ ID NO:29

[0434] AACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATC

[0435] U6 promoter, SEQ ID NO:30

[0436] GAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTGGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGAC

[0437] AAV2 5’ ITR, SEQ ID NO:31

[0438] CCTGCAGGCAGCTGCGCGCTCGCTCGCTCACTGAGGCCGCCCGGGCGTCGGGCGACCTTTGGTCGCCCGGCCTCAGTGAGCGAGCGAGCGCGCAGAGAGGGAGTGGCCAACTCCATCACTAGGGGTTCCT

[0439] AAV2 3’ ITR, SEQ ID NO:32

[0440] AGGAACCCCTAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCCGGGCGACCAAAGGTCGCCCGACGCCCGGGCTTTGCCCGGGCGGCCTCAGTGAGCGAGCGAGCGCGCAG

[0441] AAV9 - transgenic plasmid - hfCas13e.1 - sg9 ITR to ITR, SEQ ID NO:33

[0442]

[0443] Wild-type Cas13e.1, SEQ ID NO:34

[0444] MAQVSKQTSKKRELSIDEYQGARKWCFTIAFNKALVNRDKNDGLFVESLLRHEKYSKHDWYDEDTRALIKCSTQAANAKAEALRNYFSHYRHSPGCLTFTAEDELRTIMERAYERAIFECRRRETEVIIEFPSLFEGDRITTAGVVFFVSFFVERRVLDRLYGAVSGLKKNEGQYKLTRKALSMYCLKDSRFTKAWDKRVLLFRDILAQLGRIPAEAYEYYHGEQGDKKRANDNEGTNPKRHKDKFIEFALHYLEAQHSEICFGRRHIVREEAGAGDEHKKHRTKGKVVVDFSKKDEDQSYYISKNNVIVRIDKNAGPRSYRMGLNELKYLVLLSLQGKGDDAIAKLYRYRQHVENILDVVKVTDKDNHVFLPRFVLEQHGIGRKAFKQRIDGRVKHVRGVWEKKKAATNEMTLHEKARDILQYVNENCTRSFNPGEYNRLLVCLVGKDVENFQAGLKRLQLAERIDGRVYSIFAQTSTINEMHQVVCDQILNRLCRIGDQKLYDYVGLGKKDEIDYKQKVAWFKEHISIRRGFLRKKFWYDSKKGFAKLVEEHLESGGGQRDVGLDKKYYHIDAIGRFEGANPALYETLARDRLCLMMAQYFLGSVRKELGNKIVWSNDSIELPVEGSVGNEKSIVFSVSDYGKLYVLDDAEFLGRICEYFMPHEKGKIRYHTVYEKGFRAYNDLQKKCVEAVLAFEEKVVKAKKMSEKEGAHYIDFREILAQTMCKEAEKTAVNKVRRAFFHHHLKFVIDEFGLFSDVMKKYGIEKEWKFPVK

[0445] Additional human and mouse cross-reactive spacer and target sequences 15 - 37

[0446]

[0447] Human UBE3A-ATS (also known as SNHG14)

[0448] Genome sequence: NG_002690.1

[0449] Mouse Ube3a-ATS (also known as Snhg14)

[0450] Genome sequence: NC_000073.7

Claims

1. A guide nucleic acid, comprising: (1) a scaffold sequence capable of forming a complex with a nucleic acid programmable RNA nuclease (napRNAn), and (2) a guide sequence capable of hybridizing to a target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS.

2. A system or composition, comprising: (1) a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, wherein the guide nucleic acid comprises: (a) a scaffold sequence capable of forming a complex with a nucleic acid programmable RNA nuclease (napRNAn), and (b) a guide sequence capable of hybridizing to a target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS; and (2) The napRNAn or a polynucleotide encoding the napRNAn.

3. A recombinant adeno-associated virus (rAAV) vector genome, the rAAV vector genome comprising: (a) a first polynucleotide sequence comprising a first sequence encoding a first guide nucleic acid, the first guide nucleic acid comprising: (1) a scaffold sequence capable of forming a complex with a nucleic acid programmable RNA nuclease (napRNAn), and (2) a first guide sequence capable of hybridizing to a first target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS; and (b) a second polynucleotide sequence, wherein the second polynucleotide sequence comprises a sequence encoding the napRNAn, The rAAV vector genome is suitable for encapsulation into recombinant AAV particles.

4. The rAAV vector genome of claim 3, wherein the first polynucleotide sequence further comprises a second sequence encoding a second guide nucleic acid, the second guide nucleic acid comprising: (1) a scaffold sequence capable of forming a complex with napRNAn or the scaffold sequence capable of forming a complex with napRNAn, and (2) a second guide sequence capable of hybridizing to a second target sequence of human UBE3A-ATS to guide the complex to the human UBE3A-ATS.

5. The rAAV vector genome of claim 4, wherein the first polynucleotide sequence further comprises a third sequence encoding a third guide nucleic acid, the third guide nucleic acid comprising: (1) a scaffold sequence capable of forming a complex with napRNAn or the scaffold sequence capable of forming a complex with napRNAn, and (2) capable of hybridizing to the third target sequence of human UBE3A-ATS to guide the complex to the third guide sequence of human UBE3A-ATS.

6. The rAAV vector genome of claim 5, wherein the first polynucleotide sequence further comprises a fourth sequence encoding a fourth guide nucleic acid, the fourth guide nucleic acid comprising: (1) a scaffold sequence capable of forming a complex with napRNAn or the scaffold sequence capable of forming a complex with napRNAn, and (2) capable of hybridizing to the fourth target sequence of human UBE3A-ATS to guide the complex to the fourth guide sequence of human UBE3A-ATS.

7. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide nucleic acid (e.g., the guide nucleic acid, the first guide nucleic acid, the second guide nucleic acid, the third guide nucleic acid, the fourth guide nucleic acid, etc.) is RNA.

8. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein said directing the complex to the human UBE3A-ATS enables the napRNAn to specifically cleave the human UBE3A-ATS.

9. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the specific cleavage of the human UBE3A-ATS results in degradation of the human UBE3A-ATS and expression of the human UBE3A gene that was previously suppressed by the human UBE3A-ATS.

10. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the human UBE3A-ATS inhibits expression of a parentally derived human UBE3A gene.

11. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the human UBE3A protein expressed from the parentally derived human UBE3A gene is functional.

12. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the target sequence (e.g., the target sequence, the first target sequence, the second target sequence, the third target sequence, the fourth target sequence, etc.) is positioned such that the human UBE3A-ATS is specifically cleaved by the napRNAn.

13. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the target sequence is positioned such that mouse UBE3A-ATS is specifically cleaved by the napRNAn.

14. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the target sequence is positioned such that both the human UBE3A-ATS and the mouse UBE3A-ATS are specifically cleaved by the napRNAn.

15. The guide nucleic acid, system or composition, or rAAV vector genome of claim 17, wherein the DNA counterpart of human UBE3A-ATS (also known as SNHG14) is represented by NCBI genomic sequence NG_002690.

1.

16. The guide nucleic acid, system or composition, or rAAV vector genome of claim 18 or 19, wherein the DNA counterpart of mouse UBE3A-ATS (also known as Snhg14) is shown as NCBI genome sequence NC_000073.

7.

17. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide nucleic acid comprises the scaffold sequence 5' or 3' of the guide sequence (e.g., the guide sequence, the first guide sequence, the second guide sequence, the third guide sequence, the fourth guide sequence, etc.), and optionally the scaffold sequence 3'.

18. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide nucleic acid comprises from 5' to 3' a guide sequence and a scaffold sequence.

19. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide nucleic acid comprises from 5' to 3' a scaffold sequence, a guide sequence, and a scaffold sequence, wherein the scaffold sequences are the same or different.

20. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide nucleic acid comprises from 5' to 3' a scaffold sequence, a guide sequence, a scaffold sequence, and a guide sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

21. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide nucleic acid comprises from 5' to 3' a scaffold sequence, a guide sequence, a scaffold sequence, a guide sequence, and a scaffold sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

22. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide nucleic acid comprises from 5' to 3' a scaffold sequence, a guide sequence, a scaffold sequence, a guide sequence, a scaffold sequence, and a guide sequence, wherein the scaffold sequences are the same or different, and wherein the guide sequences are the same or different.

23. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the target sequence is at least about 14 nucleotides in length, such as about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides, or a numerical range in length between any two of the foregoing values, for example, from about 16 to about 50 nucleotides in length; optionally, wherein the target sequence is about 30 nucleotides in length.

24. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the target sequence comprises, consists essentially of, or consists of: 43,44,45,46,47,48,49,50,51,52,53,54,55,56,57,58,59,60,61,62,63,64,65,66,67,68,69,70,71,72,73,74,75,76,77,78,79,80,81,82,83,84,85,86,87,88,89,90,91,92,93,94,95,96,97,98,99,100,101,102,103,104,105 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more consecutive nucleotides, or a numerical range between any two of the foregoing values, for example, about 14 to about 50 consecutive nucleotides of said human UBE3A-ATS); optionally, wherein the target sequence comprises, consists essentially of, or consists of: about 30 consecutive nucleotides of said human UBE3A-ATS.

25. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide sequence is at least about 14 nucleotides in length, such as about 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75 , 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70 or more nucleotides in length, or a numerical range between any two of the aforementioned values ​​in length, for example, about 16 to about 50 nucleotides in length; optionally, wherein the guide sequence is about 30 nucleotides in length.

26. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% (perfectly) complementary to the target sequence, optionally about 100% (perfectly) complementary; or wherein the guide sequence is at least about 100% complementary to the target sequence; or wherein the guide sequence is at least about 100% complementary to the target sequence; 64, 65, 66, 67, 68, 69, or 70 nucleotides contain no mismatches to the target sequence.

27. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the target sequence comprises, consists essentially of, or consists of about 30 contiguous nucleotides over a common region shared by the human UBE3A-ATS and the mouse UBE3A-ATS.

28. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the target sequence comprises, consists essentially of, or consists of: (1) a sequence of any one of SEQ ID NOs: 16-21 and 58-80, or a 5' or 3' truncation thereof with 1, 2, 3, 4, 5 or 6 nucleotides truncated at the 5' or 3' end; or (2) a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to any one of SEQ ID NOs: 16-21 and 58-80, or a 5' or 3' truncation thereof with 1, 2, 3, 4, 5 or 6 nucleotides truncated at the 5' or 3' end; or (3) a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide differences, whether consecutive or not, compared to any one of SEQ ID NOs: 16-21 and 58-80.

29. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide sequence comprises, consists essentially of, or consists of: (1) a sequence of any one of SEQ ID NOs: 10-15 and 35-57 or a 5' or 3' truncation thereof at the 5' or 3' end truncated by 1, 2, 3, 4, 5 or 6 nucleotides; or (2) a sequence having at least about 70%, 75%, 80%, 85%, 90%, 95% or 100% sequence identity to any one of SEQ ID NOs: 10-15 and 35-57 or a 5' or 3' truncation thereof at the 5' or 3' end truncated by 1, 2, 3, 4, 5 or 6 nucleotides; or (3) a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleotide differences, whether consecutive or not, compared to any one of SEQ ID NOs: 10-15 and 35-57.

30. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide sequence comprises or is set forth as the sequence of SEQ ID NO:

15.

31. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the scaffold sequence has a secondary structure substantially identical to the secondary structure of the sequence of SEQ ID NO:

3.

32. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the scaffold sequence comprises, consists essentially of, or consists of: A sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 3; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO:

3.

33. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the guide nucleic acid comprises, consists essentially of, or consists of: A sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of any one of SEQ ID NOs: 4-9; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of any one of SEQ ID NOs: 4-9.

34. The rAAV vector genome of any of the preceding claims, wherein the first polynucleotide sequence comprises a promoter operably linked to the sequence encoding the guide nucleic acid.

35. The rAAV vector genome of any of the preceding claims, wherein the promoter is a broad-spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter, or an inducible promoter; optionally selected from: Cbh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, retrovirus Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1α short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (Ie) enhancer and / or promoter, chicken β-actin (CBA) promoter or its derivatives such as CAG promoter, CB promoter, (human) elongation factor 1α-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, Platelet-derived growth factor (PDGF) promoter, platelet-derived growth factor B chain (PDGF-β) promoter, synapsin (Syn) promoter, synapsin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, and myelin basic protein (MBP) promoter.

36. The rAAV vector genome of any of the preceding claims, wherein the promoter is a U6 promoter.

37. The rAAV vector genome of any of the preceding claims, wherein the promoter comprises, consists essentially of, or consists of: A sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:30; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO:

30.

38. The rAAV vector genome of any of the preceding claims, wherein the first polynucleotide sequence comprises, from 5′ to 3′, a promoter of SEQ ID NO: 30 and a sequence encoding a guide nucleic acid of any one of SEQ ID NOs: 4-9.

39. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the napRNAn is a Class 2, Type VI CRISPR-associated protein (Cas13).

40. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the Cas13 is a Cas13a (C2c2), Cas13b (such as, Cas13b1, Cas13b2), Cas13c, Cas13d, Cas13e or Cas13f polypeptide.

41. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the napRNAn comprises, consists essentially of, or consists of: A sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 1 or 34, or an N-terminal truncation thereof without the first N-terminal methionine, and retaining at least 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the guide sequence-specific RNA cleavage activity of the sequence of SEQ ID NO: 1 or 34.

42. The guide nucleic acid, system or composition, or rAAV vector genome of any of the preceding claims, wherein the sequence encoding the napRNAn comprises, consists essentially of, or consists of: A sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO: 2, or a 5' end truncation thereof without the first 5'ATG codon, and wherein the napRNAn encoded by the sequence retains at least 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) of the guide sequence-specific RNA cleavage activity of the sequence of SEQ ID NO:

2.

43. The rAAV vector genome of any of the preceding claims, wherein the second polynucleotide sequence comprises a promoter operably linked to the sequence encoding the napRNAn.

44. An AAV vector genome as described in any of the preceding claims, wherein the promoter is a broad-spectrum promoter, a tissue-specific promoter, a cell type-specific promoter, a constitutive promoter or an inducible promoter; optionally, wherein the promoter is selected from: Cbh promoter, Cba promoter, pol I promoter, pol II promoter, pol III promoter, T7 promoter, U6 promoter, H1 promoter, retrovirus Rous sarcoma virus LTR promoter, cytomegalovirus (CMV) promoter, SV40 promoter, dihydrofolate reductase promoter, β-actin promoter, elongation factor 1α short (EFS) promoter, β-glucuronidase (GUSB) promoter, cytomegalovirus (CMV) immediate early (Ie) enhancer and / or promoter, chicken β-actin (CBA) promoter or its derivatives such as CAG promoter, CB promoter, (human) elongation factor 1α-subunit (EF1α) promoter, ubiquitin C (UBC) promoter, prion promoter, neuron-specific enolase (NSE), neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, platelet-derived growth factor (PDGF) promoter, blood Platelet-derived growth factor B chain (PDGF-β) promoter, synapsin (Syn) promoter, synapsin 1 (Syn1) promoter, methyl-CpG binding protein 2 (MeCP2) promoter, Ca2+ / calmodulin-dependent protein kinase II (CaMKII) promoter, metabotropic glutamate receptor 2 (mGluR2) promoter, neurofilament light chain (NFL) promoter, neurofilament heavy chain (NFH) promoter, β-globin minigene nβ2 promoter, preproenkephalin (PPE) promoter, enkephalin (Enk) promoter, excitatory amino acid transporter 2 (EAAT2) promoter, glial fibrillary acidic protein (GFAP) promoter, myelin basic protein (MBP) promoter, HTT promoter, GRK1 promoter, CRX promoter, NRL promoter, and RCVRN promoter.

45. The rAAV vector genome of any of the preceding claims, wherein the promoter is a neural cell (eg, neuron) specific promoter.

46. ​​The rAAV vector genome of any of the preceding claims, wherein the promoter is the human Syn1 promoter.

47. The rAAV vector genome of any of the preceding claims, wherein the second polynucleotide sequence comprises a Kozak sequence 5' to the sequence encoding the napRNAn.

48. The rAAV vector genome of any of the preceding claims, wherein the second polynucleotide sequence comprises a sequence encoding an NLS 5' and / or 3' to the sequence encoding the napRNAn.

49. The rAAV vector genome of any of the preceding claims, wherein the second polynucleotide sequence comprises a sequence encoding a first NLS 5' to the sequence encoding the napRNAn and a second sequence encoding a second NLS 3' to the sequence encoding the napRNAn.

50. The rAAV vector genome of any of the preceding claims, wherein the NLS, the first NLS and / or the second NLS is a SV40 NLS, a bpSV40 NLS or a nucleoplasmin NLS.

51. The rAAV vector genome of any of the preceding claims, wherein the second polynucleotide sequence comprises a WPRE sequence downstream of the sequence encoding the napRNAn.

52. The rAAV vector genome of any of the preceding claims, wherein the WPRE sequence is selected from the group consisting of a woodchuck hepatitis virus (WHP) post-transcriptional regulatory element (WPRE), WPRE3 (shortened WPRE), and functional variants thereof (e.g., functional truncations).

53. The rAAV vector genome of any of the preceding claims, wherein the second polynucleotide sequence comprises a sequence encoding a polyadenylation (polyA) signal downstream of the sequence encoding the napRNAn.

54. The rAAV vector genome of any of the preceding claims, wherein the second polynucleotide sequence comprises a WPRE sequence downstream of the sequence encoding the napRNAn, followed by a sequence encoding a polyadenylation (polyA) signal.

55. The rAAV vector genome of any of the preceding claims, wherein the polyA signal is selected from the group consisting of a bovine growth hormone polyadenylation (bGH polyA) signal, a small polyA (SPA) signal, a human growth hormone polyadenylation (hGH polyA) signal, an SV40 polyA (SV40 polyA) signal, a rabbit beta globin polyA (rBG polyA) signal, and functional variants thereof (e.g., functional truncations).

56. The rAAV vector genome of any of the preceding claims, wherein the polyA signal is an SV40 polyA signal.

57. The rAAV vector genome of any of the preceding claims, wherein the second polynucleotide sequence comprises from 5' to 3' the promoter, the Kozak sequence, the first sequence encoding the first NLS, the sequence encoding the napRNAn, the second sequence encoding the second NLS, the WPRE sequence, and the sequence encoding the polyA signal.

58. The rAAV vector genome of any of the preceding claims, wherein the promoter comprises, consists essentially of, or consists of: A sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:25; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO:

25.

59. The rAAV vector genome of any of the preceding claims, wherein the Kozak sequence comprises, consists essentially of, or consists of: A sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:26; or a sequence having at most 1, 2, 3, or 4 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO:

26.

60. The rAAV vector genome of any of the preceding claims, wherein the NLS, the first NLS, and / or the second NLS comprises, consists essentially of, or consists of: A sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:27; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acid differences, whether contiguous or not, compared to the sequence of SEQ ID NO:

27.

61. The rAAV vector genome of any of the preceding claims, wherein the sequence encoding the polyA signal comprises, consists essentially of, or consists of: A sequence encoding a polypeptide having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:29; or a sequence having at most 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotide differences, whether contiguous or not, compared to the sequence of SEQ ID NO:

29.

62. The rAAV vector genome of any of the preceding claims, wherein the second polynucleotide sequence comprises from 5' to 3' the promoter of SEQ ID NO:25, the Kozak sequence of SEQ ID NO:26, the first sequence encoding the first NLS of SEQ ID NO:27, the sequence encoding the napRNAn of SEQ ID NO:2, the second sequence encoding the second NLS of SEQ ID NO:27, and the sequence encoding the polyA signal of SEQ ID NO:

29.

63. The rAAV vector genome of any of the preceding claims, wherein the rAAV vector genome comprises a 5' inverted terminal repeat (ITR) sequence and a 3' ITR sequence.

64. The rAAV vector genome of any of the preceding claims, wherein the 5' ITR sequence and the 3' ITR sequence are both wild-type ITR sequences from: a member of a branch to which any one of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.eB, or AAV1-AAV13 belongs, or a functional variant thereof (e.g., a functional truncation).

65. The rAAV vector genome of any of the preceding claims, wherein the 5' ITR sequence comprises, consists essentially of, or consists of: A sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:

31.

66. The rAAV vector genome of any of the preceding claims, wherein the 3' ITR sequence comprises, consists essentially of, or consists of: A sequence having at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the sequence of SEQ ID NO:

32.

67. The rAAV vector genome of any of the preceding claims, wherein the rAAV vector genome comprises the first polynucleotide sequence and the second polynucleotide sequence from 5' to 3'.

68. The rAAV vector genome of any of the preceding claims, wherein the rAAV vector genome comprises the second polynucleotide sequence and the first polynucleotide sequence from 5' to 3'.

69. A recombinant AAV (rAAV) particle comprising the rAAV vector genome of any of the preceding claims.

70. The rAAV particle of any of the preceding claims, wherein the rAAV particle comprises a capsid of a serotype of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or a member of an evolutionary branch to which any one of AAV.PHP.eB, AAV1-AAV13 belongs, or a functional variant thereof (e.g., a functional truncation), which encapsulates the rAAV vector genome.

71. The rAAV particle of any of the preceding claims, wherein the serotype of the capsid is AAV9 or AAV.PHP.eB.

72. A pharmaceutical composition comprising the system or composition of any of the preceding claims or the rAAV particles of any of the preceding claims and a pharmaceutically acceptable excipient.

73. The pharmaceutical composition of any of the preceding claims, wherein the pharmaceutical composition comprises the rAAV particles at a concentration selected from about 1×10 10 vg / mL, 2×10 10 vg / mL, 3×10 10 vg / mL, 4×10 10 vg / mL, 5×10 10 vg / mL, 6×10 10 vg / mL, 7×10 10 vg / mL, 8×10 10 vg / mL, 9×10 10 vg / mL, 1×10 11 vg / mL, 2×10 11 vg / mL, 3×10 11 vg / mL, 4×10 11 vg / mL, 5×10 11 vg / mL, 6×10 11 vg / mL, 7×10 11 vg / mL, 8×10 11 vg / mL, 9×10 11 vg / mL, 1×10 12 vg / mL, 2×10 12 vg / mL, 3×10 12 vg / mL, 4×10 12 vg / mL, 5×10 12 vg / mL, 6×10 12 vg / mL, 7×10 12 vg / mL, 8×10 12 vg / mL, 9×10 12 vg / mL, 1×10 13 vg / mL, or a concentration in a numerical range between any two of the foregoing values, such as about 9×10 10 vg / mL to about 8×10 11 The concentration of vg / mL.

74. A pharmaceutical composition as described in any one of the preceding claims, wherein the pharmaceutical composition is an injectable solution.

75. pharmaceutical composition as described in any one of the preceding claims, the volume of wherein the injection is selected from about 1 microliter, 10 microliters, 50 microliters, 100 microliters, 150 microliters, 200 microliters, 250 microliters, 300 microliters, 350 microliters, 400 microliters, 450 microliters, 500 microliters, 550 microliters, 600 microliters, 650 microliters, 700 microliters, 750 microliters, 800 microliters, 850 microliters, 900 microliters, 950 microliters, 1000 microliters, and the volume in the numerical range between any two of the aforementioned values, for example a concentration of about 10 microliters to about 750 microliters.

76. A method for preventing or treating a disease or disorder associated with human UBE3A-ATS in a subject in need thereof, the method comprising administering to the subject the system or composition of any of the preceding claims, the rAAV particle of any of the preceding claims, or the pharmaceutical composition of any of the preceding claims, wherein the napRNAn modifies the human UBE3A-ATS, and wherein the modification of the human UBE3A-ATS treats the disease or disorder.

77. The method of any one of the preceding claims, wherein the disease or disorder is Angelman Syndrome (AS).

78. The method of any of the preceding claims, wherein the administering comprises topical administration.

79. The method of any of the preceding claims, wherein the administration comprises intracerebroventricular administration.

80. The method of any one of the preceding claims, wherein the administration comprises systemic administration.

81. The method of any of the preceding claims, wherein the subject is a human.

82. The method of any of the preceding claims, wherein the level of human UBE3A-ATS in the subject is reduced by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, or more compared to the level of UBE3A-ATS in the subject before the administration.

83. The method of any of the preceding claims, wherein the level of human UBE3A protein in the subject is at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 100%, about 105%, about 110%, about 115%, about 120%, about 125%, about 130%, about 135%, or more compared to the level of human UBE3A protein in a human or population not suffering from the disease or disorder.

84. A cell or its progeny, comprising the guide nucleic acid of any of the preceding claims, the system or composition of any of the preceding claims, the rAAV vector genome of any of the preceding claims, or the rAAV particle of any of the preceding claims.

85. A cell or progeny thereof as described in any one of the preceding claims, wherein the cell is a eukaryotic cell.

86. A cell or progeny thereof as described in any one of the preceding claims, wherein the cell is a human cell.

87. A cell or progeny thereof as described in any one of the preceding claims, wherein the cell is a human neural cell (e.g., a neuron).

88. A kit comprising the guide nucleic acid of any of the preceding claims, the system or composition of any of the preceding claims, the rAAV vector genome of any of the preceding claims, the rAAV particle of any of the preceding claims, or the cell or progeny thereof of any of the preceding claims.

Images