WO2023185861A1 - Acide nucléique guide ciblant ube3a-ats et ses utilisations - Google Patents

Acide nucléique guide ciblant ube3a-ats et ses utilisations Download PDF

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WO2023185861A1
WO2023185861A1 PCT/CN2023/084417 CN2023084417W WO2023185861A1 WO 2023185861 A1 WO2023185861 A1 WO 2023185861A1 CN 2023084417 W CN2023084417 W CN 2023084417W WO 2023185861 A1 WO2023185861 A1 WO 2023185861A1
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sequence
promoter
nucleic acid
vector genome
raav vector
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Xing Wang
Jinhui Li
Hui Yang
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Huidagene Therapeutics Co., Ltd.
Huidagene Therapeutics (Singapore) Pte. Ltd.
Center For Excellence In Brain Science And Intelligence Technology, Chinese Academy Of Sciences
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    • C12N9/22—Ribonucleases RNAses, DNAses
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
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    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
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    • C12Y203/00—Acyltransferases (2.3)
    • C12Y203/02—Aminoacyltransferases (2.3.2)

Definitions

  • the disclosure contains an electronic sequence listing ( “xxx. xml” created on XX, by software “WIPO Sequence” according to WIPO Standard ST. 26) , which is incorporated herein by reference in its entirety. Wherever a sequence is an RNA sequence, the T in the sequence shall be deemed as U.
  • Angelman syndrome is a rare neurodevelopmental disorder caused by loss of function mutations in maternally expressed UBE3A. No gene-specific treatment is available for patients so far. Although intact and transcriptionally active, paternally inherited UBE3A is silenced by elongation of antisense long noncoding RNA UBE3A-ATS in neurons.
  • the disclosure herein is not limited to specific advantages, in an aspect, the disclosure provides a guide nucleic acid comprising:
  • napRNAn nucleic acid programmable RNA nuclease
  • the disclosure provides a system or composition comprising:
  • a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, comprising:
  • napRNAn nucleic acid programmable RNA nuclease
  • the disclosure provides a recombinant adeno-associated virus (rAAV) vector genome comprising:
  • napRNAn nucleic acid programmable RNA nuclease
  • rAAV vector genome is adapted to be encapsulated into a recombinant AAV particle.
  • the disclosure provides a recombinant AAV (rAAV) particle comprising the rAAV vector genome of the disclosure.
  • rAAV recombinant AAV
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the system or composition of the disclosure or the rAAV particle of the disclosure and a pharmaceutically acceptable excipient.
  • the disclosure provides a method for preventing or treating a disease or disorder associated with human UBE3A-ATS in a subject in need thereof, comprising administering to the subject the system or composition of the disclosure, the rAAV particle of the disclosure, or the pharmaceutical composition of the disclosure, wherein the napRNAn modifies the human UBE3A-ATS, and wherein the modification of the human UBE3A-ATS treats the disease or disorder.
  • Nucleic acid programmable RNA nuclease for example, Cas13, is capable of cleaving a target RNA as guided by a guide nucleic acid (e.g., a guide RNA) comprising a guide sequence targeting the target RNA.
  • a guide nucleic acid e.g., a guide RNA
  • the target RNA is eukaryotic.
  • the guide nucleic acid comprises a scaffold sequence responsible for forming a complex with the napRNAn, and a guide sequence that is intentionally designed to be responsible for hybridizing to a target sequence of the target RNA, thereby guiding the complex comprising the napRNAn and the guide nucleic acid to the target RNA.
  • an exemplary dsRNA is depicted to comprise a 5’ to 3’ single DNA strand and a 3’ to 5’ single DNA strand.
  • an exemplary RNA transcript may be transcribed using the 3’ to 5’ single DNA strand as a synthesis template, and thus the 3’ to 5’ single DNA strand is referred to as a “template strand” or a “antisense strand” .
  • the RNA transcript so transcribed has the same primary sequence as the 5’ to 3’ single DNA strand except for the replacement of T with U, and thus the 5’ to 3’ single DNA strand is referred to as a “coding strand” or a “sense strand” .
  • An exemplary guide nucleic acid is depicted to comprise a guide sequence and a scaffold sequence.
  • the guide sequence is designed to hybridize to a part of the RNA transcript (target RNA) , and so the guide sequence “targets” that part. And thus, that part of the target RNA based on which the guide sequence is designed and to which the guide sequence may hybridize is referred to as a “target sequence” .
  • the guide sequence is 100% (fully) reversely complementary to the target sequence.
  • the guide sequence is reversely complementary to the target sequence and contains a mismatch with the target sequence (as exemplified in 7) .
  • the double-strand sequence of a dsDNA may be represented with the sequence of its 5’ to 3’ single DNA strand conventionally written in 5’ to 3’ direction /orientation.
  • a nucleic acid sequence e.g., a DNA sequence, an RNA sequence
  • 5’ to 3’ direction /orientation e.g., a DNA sequence, an RNA sequence
  • the dsDNA may be simply represented as 5’-ATGC-3’.
  • the RNA transcript (target RNA) transcribed from the dsDNA then has a sequence of 5’-AUGC-3’.
  • the guide sequence of a guide nucleic acid is designed to have a sequence of 5’-GCAU-3’ that is fully reversely complementary to the target RNA.
  • symbol “t” is used to denote both T in DNA and U in RNA (See “Table 1: List of nucleotides symbols” , the definition of symbol “t” is “thymine in DNA/uracil in RNA (t/u) ” ) .
  • such a guide sequence would be set forth in GCAT but marked as a RNA sequence.
  • RNA sequence As used herein, if a DNA sequence, for example, 5’-ATGC-3’ is transcribed to an RNA sequence, with each dT (deoxythymidine, or “T” for short) in the primary sequence replaced with a U (uridine) and other dA (deoxyadenosine, or “A” for short) , dG (deoxyguanosine, or “G” for short) , and dC (deoxycytidine, or “C”for short) replaced with A (adenosine) , G (guanosine) , and C (cytidine) , respectively, for example, 5’-AUGC-3’, it is said in the disclosure that the DNA sequence “encodes” the RNA sequence.
  • the term “activity” refers to a biological activity.
  • the activity includes enzymatic activity, e.g., catalytic ability of an effector.
  • the activity can include nuclease activity, e.g., RNA nuclease activity, RNA endonuclease activity.
  • the term “complex” refers to a grouping of two or more molecules.
  • the complex comprises a polypeptide and a nucleic acid interacting with (e.g., binding to, coming into contact with, adhering to) one another.
  • the term “complex” can refer to a grouping of a guide nucleic acid and a polypeptide (e.g., a napRNAn, such as, a Cas13 polypeptide) .
  • the term “complex” can refer to a grouping of a guide nucleic acid, a polypeptide, and a target sequence.
  • the term “complex” can refer to a grouping of a target RNA-targeting guide nucleic acid, a napRNAn, and optionally, a target RNA.
  • guide nucleic acid refers to any nucleic acid that facilitates the targeting of a napRNAn (e.g., a Cas13 polypeptide) to a target sequence (e.g., a sequence of a target RNA) .
  • a guide nucleic acid may be designed to include a sequence that is complementary to a specific nucleic acid sequence (e.g., a sequence of a target RNA) .
  • a guide nucleic acid may comprise a scaffold sequence facilitating the guiding of a napRNAn to the target RNA.
  • the guide nucleic acid is a guide RNA.
  • nucleic acid 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.
  • guide RNA is used interchangeably with the term “CRISPR RNA (crRNA) ” , “single guide RNA (sgRNA) ” , or “RNA guide”
  • guide sequence is used interchangeably with the term “spacer sequence”
  • sinaffold sequence is used interchangeably with the term “direct repeat sequence” .
  • the guide sequence is so designed to be capable of hybridizing to a target sequence.
  • the term “hybridize” , “hybridizing” , or “hybridization” refers to a reaction in which one or more polynucleotide sequences react to form a complex that is stabilized via hydrogen bonding between the bases of the polynucleotide sequences. The hydrogen bonding may occur by Watson Crick base pairing, Hoogstein binding, or in any other sequence specific manner.
  • a polynucleotide sequence capable of hybridizing to a given polynucleotide sequence is referred to as the “complement” of the given polynucleotide sequence.
  • the hybridization of a guide sequence and a target sequence is so stabilized to permit an effector polypeptide (e.g., a napRNAn) that is complexed with a nucleic acid comprising the guide sequence or a function domain associated (e.g., fused) with the effector polypeptide to act (e.g., cleave, deaminize) on the target sequence or its complement (e.g., a sequence of a target RNA or its complement) .
  • an effector polypeptide e.g., a napRNAn
  • a function domain associated e.g., fused
  • the guide sequence is complementary or reversely complementary to a target sequence.
  • complementary refers to the ability of nucleobases of a first polynucleotide sequence, such as a guide sequence, to base pair with nucleobases of a second polynucleotide sequence, such as a target sequence, by traditional Watson-Crick base-pairing. Two complementary polynucleotide sequences are able to non-covalently bind under appropriate temperature and solution ionic strength conditions.
  • a first polynucleotide sequence (e.g., a guide sequence) comprises 100% (fully) complementarity to a second nucleic acid (e.g., a target sequence) .
  • a first polynucleotide sequence (e.g., a guide sequence) is complementary to a second polynucleotide sequence (e.g., a target sequence) if the first polynucleotide sequence comprises 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 second nucleic acid.
  • substantially complementary refers to a polynucleotide sequence (e.g., a guide sequence) that has a certain level of complementarity to a second polynucleotide sequence (e.g., a target sequence) .
  • the level of complementarity is such that the first polynucleotide sequence (e.g., a guide sequence) can hybridize to the second polynucleotide sequence (e.g., a target sequence) with sufficient affinity to permit an effector polypeptide (e.g., a napRNAn) that is complexed with the first polynucleotide sequence or a nucleic acid comprising the first polynucleotide sequence or a function domain associated (e.g., fused) with the effector polypeptide to act (e.g., cleave, deaminize) on the target sequence or its complement (e.g., a sequence of a target RNA or its complement) .
  • an effector polypeptide e.g., a napRNAn
  • a guide sequence that is substantially complementary to a target sequence has less than 100%complementarity to 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.
  • sequence identity is related to sequence homology. Homology comparisons may be conducted by eye, or more usually, with the aid of readily available sequence comparison programs. These commercially available computer programs may calculate percent (%) homology between two or more sequences and may also calculate the sequence identity shared by two or more amino acid or nucleic acid sequences.
  • Sequence homologies may be generated by any of a number of computer programs known in the art, for example BLAST or FASTA, etc.
  • a suitable computer program for carrying out such an alignment is the GCG Wisconsin Bestfit package (University of Wisconsin, U.S.A; Devereux et al., 1984, Nucleic Acids Research 12: 387) .
  • Examples of other software than may perform sequence comparisons include, but are not limited to, the BLAST package (see Ausubel et al., 1999 ibid-Chapter 18) , FASTA (Atschul et al., 1990, J. Mol. Biol., 403-410) and the GENEWORKS suite of comparison tools.
  • BLAST and FASTA are available for offline and online searching (see Ausubel et al., 1999 ibid, pages 7-58 to 7-60) .
  • Percentage (%) sequence homology may be calculated over contiguous sequences, i.e., one sequence is aligned with the other sequence 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 over a relatively short number of residues.
  • gaps penalties assign “gap penalties” 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 higher relatedness between the two compared sequences-may achieve a higher score than one with many gaps.
  • “Affinity gap costs” are typically used that charge a relatively high cost for the existence of a gap and a smaller penalty for each subsequent residue in the gap. This is the most commonly used gap scoring system. High gap penalties may, of course, produce optimized alignments with fewer gaps. Most alignment programs allow the gap penalties to be modified. However, it is preferred to use the default values when using such software for sequence comparisons.
  • the default gap penalty for amino acid sequences is -12 for a gap and -4 for each extension.
  • Calculation of maximum %homology therefore first requires the production of an optimal alignment, taking into consideration gap penalties.
  • a new tool, called BLAST 2 Sequences is also available for comparing protein and nucleotide sequences (see FEMS Microbiol Lett. 1999 174 (2) : 247-50; FEMS Microbiol Lett. 1999 177 (1) : 187-8 and the website of the National Center for Biotechnology information at the website of the National Institutes for Health) .
  • the final %homology may be measured in terms of identity, the alignment process itself is typically not based on an all-or-nothing pair comparison.
  • a scaled similarity score matrix is generally used that assigns scores to each pair-wise comparison based on chemical similarity or evolutionary distance.
  • An example of such a matrix commonly used is the BLOSUM62 matrix-the default matrix for the BLAST suite of programs.
  • GCG Wisconsin programs generally use either the public default values or a custom symbol comparison table, if supplied (see user manual for further details) .
  • the public default values for the GCG package or in the case of other software, the default matrix, such as BLOSUM62.
  • percentage homologies may be calculated using the multiple alignment feature in DNASIS TM (Hitachi Software) , based on an algorithm, analogous to CLUSTAL (Higgins D G &Sharp P M (1988) , Gene 73 (1) , 237-244) .
  • %homology preferably %sequence identity.
  • the software typically does this as part of the sequence comparison and generates a numerical result.
  • the sequences may also have deletions, insertions or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent substance.
  • Deliberate amino acid substitutions may be made on the basis of similarity in amino acid properties (such as polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues) and it is therefore useful to group amino acids together in functional groups.
  • Amino acids may be grouped together based on the properties of their side chains alone. However, it is more useful to include mutation data as well.
  • the sets of amino acids thus derived are likely to be conserved for structural reasons. These sets may be described in the form of a Venn diagram (Livingstone C. D. and Barton G. J. (1993) “Protein sequence alignments: a strategy for the hierarchical analysis of residue conservation” Comput. Appl. Biosci.
  • polypeptide and “peptide” are used interchangeably herein to refer to polymers of amino acids of any length.
  • the polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids.
  • a protein may have one or more polypeptides.
  • the terms also encompass an amino acid polymer that has been modified; for example, by disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation, such as conjugation with a labeling component.
  • a “variant” is interpreted to mean a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties.
  • a typical variant of a polynucleotide differs in nucleic acid sequence from another, reference polynucleotide. Changes in the nucleic acid sequence of the variant may or may not alter the amino acid sequence of a polypeptide encoded by the reference polynucleotide. Nucleotide changes may result in amino acid substitutions, additions, deletions, fusions and truncations in the polypeptide encoded by the reference sequence, as discussed below.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination.
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polynucleotide or polypeptide may be a naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally. Non-naturally occurring variants of polynucleotides and polypeptides may be made by mutagenesis techniques, by direct synthesis, and by other recombinant methods known to skilled artisans.
  • upstream and downstream refer to relative positions within a single nucleotide (e.g., DNA) sequence in a nucleic acid.
  • a first sequence “upstream” of a second sequence means that the first sequence is 5’ to the second sequence
  • a first sequence “downstream” of a second sequence means that the first sequence is 3’ to the second sequence.
  • wild type has the meaning commonly understood by those skilled in the art to mean a typical form of an organism, a strain, a gene, or a feature that distinguishes it from a mutant or variant when it exists in nature. It can be isolated from sources in nature and not intentionally modified.
  • nucleic acid or polypeptide As used herein, the terms “non-naturally occurring” and “engineered” are used interchangeably and refer to artificial participation. When these terms are used to describe a nucleic acid or a polypeptide, it is meant that the nucleic acid or polypeptide is at least substantially freed from at least one other component of its association in nature or as found in nature.
  • the “cell” is understood to refer not only to a particular individual cell, but to the progeny or potential progeny of the cell. Because certain modifications may occur in succeeding generations due to either 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.
  • in vivo refers to inside the body of an organism
  • ex vivo or “in vitro” means outside the body of an organism.
  • the term “treat” , “treatment” , or “treating” is an approach for obtaining beneficial or desired results including clinical results.
  • the beneficial or desired clinical results include, but are not limited to, one or more of the following: alleviating one or more symptoms resulting from a disease, diminishing the extent of a disease, stabilizing a disease (e.g., preventing or delaying the worsening of a disease) , preventing or delaying the spread (e.g., metastasis) of a disease, preventing or delaying the recurrence of a disease, reducing recurrence rate of a disease, delay or slowing the progression of a disease, ameliorating a disease state, providing a remission (partial or total) of a disease, decreasing the dose of one or more other medications required to treat a disease, delaying the progression of a disease, increasing the quality of life, and/or prolonging survival.
  • treatment is a reduction of pathological consequence of
  • disease includes the terms “disorder” and “condition” and is not limited to those have been specifically medically defined.
  • reference to “not” a value or parameter generally means and describes “other than” a value or parameter.
  • the method is not used to treat cancer of type X means the method may be used to treat cancer of types other than X.
  • the term “and/or” in a phrase such as “A and/or B” is intended to include both A and B; A or B; A (alone) ; and B (alone) .
  • the term “and/or” in a phrase 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) .
  • 1 is a schematic illustration of treatment in neurons of AS patients with CRISPR-hfCas13e. 1 system decreasing UBE3A-ATS transcript and reactivating the expression of paternal UBE3A.
  • 2A is a schematic illustration of an exemplary lentivirus vector genome encoding hfCas13e. 1-sgRNA as well as the control.
  • 2B is a schematic illustration of an exemplary AAV vector genome encoding hfCas13e. 1-sgRNA as well as the control.
  • 3A shows a graph of ex vivo knockdown efficiency of Ube3a-ATS transcript in AS mouse primary neurons treated with hfCas13e. 1-sg9-14 systems (AS+sg9-14) compared to the control (AS+NT) .
  • Ube3a ubiquitin protein ligase E3A
  • 3C shows statistic quantification of 3B.
  • N 3/group. *, P ⁇ 0.05; ***, P ⁇ 0.001.
  • 4A shows a schematic illustration of timeline of assays.
  • 4B shows a graph of in vivo knockdown of Ube3a-ATS transcript and recovering of paternal Ube3a transcript in the cortex of treated AS mice compared with untreated AS mice.
  • 4C shows a graph of in vivo knockdown of Ube3a-ATS transcript and recovering of paternal Ube3a transcript in the hippocampus of treated AS mice compared with untreated AS mice.
  • FIG. 4D shows the expression of paternal Ube3a in the cortex and hippocampus of treated AS mice compared to untreated AS mice.
  • FIG. 4F shows the expression of paternal Ube3a in the cortex and hippocampus of treated mice compared to control.
  • 4G shows statistic quantification of 4F.
  • N 3/group. *, P ⁇ 0.05; ***, P ⁇ 0.001.
  • 5A is a schematic illustration of timeline of behavioral tests.
  • 5B shows body weight of female mice measured bi-weekly over 18 weeks.
  • 5C shows the results of hindlimb clasping test. N >8/group. *, P ⁇ 0.05; ***, P ⁇ 0.001.
  • 5D shows the results of open-field test. N >8/group. *, P ⁇ 0.05; ***, P ⁇ 0.001.
  • 5E shows the results of dowel test. N >8/group. *, P ⁇ 0.05; ***, P ⁇ 0.001.
  • 5F-G shows the results of beam-walking test. N >8/group. *, P ⁇ 0.05; ***, P ⁇ 0.001.
  • 5H shows the results of accelerating rotarod test. N >8/group. *, P ⁇ 0.05; ***, P ⁇ 0.001.
  • FIG. 6 is a schematic 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 reversely complementary to the target sequence.
  • FIG. 7 is a schematic 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 reversely complementary to the target sequence and contains a mismatch with the target sequence.
  • the disclosure provides tools and methods for treatment of UBE3A-ATS-assocaited diseases, such as, Angelman Syndrome (AS) .
  • AS Angelman Syndrome
  • the disclosure provides tools for restoring /upregulating the expression of parental UBE3A gene by cleaving /degrading UBE3A-ATS transcript (a long non-coding RNA, lncRNA) that inhibiting /silencing the parental UBE3A gene.
  • UBE3A-ATS transcript a long non-coding RNA, lncRNA
  • Such tools can be delivered, for example, by lentiviral and AAV vectors and intracerebroventricular injection to subjects in need.
  • Exemplary constructs of the disclosure have demonstrated efficacy to knockdown UBE3A-ATS transcript level and restore UBE3A protein expression, both ex vivo and in vivo, and improve disease phenotypes of mouse models, thus opening the door for gene therapy to treat UBE3A-ATS-assocaited diseases, such as, AS.
  • the disclosure provides a guide nucleic acid comprising:
  • napRNAn nucleic acid programmable RNA nuclease
  • the disclosure provides a system or composition comprising:
  • a guide nucleic acid or a polynucleotide encoding the guide nucleic acid, comprising:
  • napRNAn nucleic acid programmable RNA nuclease
  • the system is a complex comprising the napRNAn complexed with the guide nucleic acid.
  • the complex further comprises the human UBE3A-ATS hybridized with the target sequence.
  • the disclosure provides a recombinant adeno-associated virus (rAAV) vector genome comprising:
  • napRNAn nucleic acid programmable RNA nuclease
  • the system, composition, or rAAV vector genome of the disclosure may 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.
  • the first polynucleotide sequence further comprises a second sequence encoding a second guide nucleic acid comprising:
  • the first polynucleotide sequence further comprises a third sequence encoding a third guide nucleic acid comprising:
  • the first polynucleotide sequence further comprises a fourth sequence encoding a fourth guide nucleic acid comprising:
  • the first polynucleotide sequence further comprises a firth, a sixth, a seventh guide nucleic acid, and so on.
  • the scaffold sequences of the more than one guide nucleic acids may be the same or slightly different (e.g., different by no more than 5, 4, 3, 2, or 1 nucleotide) to be compatible to the same napRNAn. In some embodiments that different napRNAn are used, the scaffold sequences of the more than one guide nucleic acids may be different to be compatible to the different napRNAn.
  • 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, and so on; same as elsewhere) is an RNA.
  • the guide nucleic acid comprises a modification.
  • the guide nucleic acid is an unmodified RNA or modified RNA.
  • the guide nucleic acid is a modified RNA containing a modified ribonucleotide.
  • the guide nucleic acid is a modified RNA containing a deoxyribonucleotide.
  • the guide nucleic acid is a modified RNA containing a modified deoxyribonucleotide. In some embodiments, the guide nucleic acid comprises a modified or unmodified deoxyribonucleotide and a modified or unmodified ribonucleotide.
  • AS Angelman syndrome
  • UBE3A maternally inherited ubiquitin protein ligase E3A
  • UBE3A-ATS is a polyadenylated transcript processed from SNHG14 small nucleolar RNA host gene 14 (SNHG14) transcription and it overlaps with the UBE3A gene locus, resulting in early termination and degradation of UBE3A transcripts.
  • SNHG14 small nucleolar RNA host gene 14
  • Approximately 75%of AS cases are reportedly caused by deletions of the maternal chromosomal region 15q11.2–q13. The remaining cases are attributable to other mutations in maternal UBE3A, paternal uniparental disomy, or imprinting defects.
  • UBE3A-ATS long non-coding RNA lncRNA
  • napRNAn e.g., Cas13
  • the guide sequence of the guide nucleic acid is designed to be capable of hybridizing to the human UBE3A-ATS, thereby guiding the complex comprising the guide nucleic acid and the napRNAn to the human UBE3A-ATS.
  • said guiding the complex to the human UBE3A-ATS enables the napRNAn to specifically cleave the human UBE3A-ATS.
  • the specific cleavage of the human UBE3A-ATS leads to degradation of the human UBE3A-ATS and expression of a human UBE3A gene used to be inhibited by the human UBE3A-ATS.
  • the human UBE3A-ATS inhibits expression of a human UBE3A gene originated from father (parental) .
  • the human UBE3A protein expressed from the human UBE3A gene originated from father is functional.
  • the inhibition of expression of parental UBE3A gene by UBE3A-ATS is eliminated and the UBE3A protein expressed from the parental UBE3A gene is functional, it is reasonably expected that the syndromes of a subject caused by loss-of-function mutations in maternally expressed UBE3A can be alleviated.
  • Target site Design of the target sequence of the disclosure, Target site
  • the target sequence (e.g., the target sequence, the first target sequence, the second target sequence, the third target sequence, the fourth target sequence, and so on; same as elsewhere) is located such that the human UBE3A-ATS is specifically cleaved by the napRNAn.
  • the target sequence is located such that a mouse UBE3A-ATS is specifically cleaved by the napRNAn.
  • the target sequence is located such that both the human UBE3A-ATS and a mouse UBE3A-ATS is specifically cleaved by the napRNAn. That is, the target sequence is selected to be cross-reactive to both human and mouse.
  • UBE3A-ATS includes UBE3A-ATS as a long non-coding RNA, and any variants, derivatives, or ancestors thereof, including pre-UBE3A-ATS transcript, such as human pre-UBE3A-ATS transcript or mouse pre-UBE3A-ATS transcript, for example, the RNA counterpart or pre-transcript thereof of human UBE3A-ATS genome coding sequence, Accession No: NG_002690.1 incorporated herein by reference, the RNA counterpart or pre-transcript thereof of mouse UBE3A-ATS genome coding sequence, Accession No: NC_000073.7 incorporated herein by reference, or any transcripts or isoforms produced by alternative promoter usage, alternative splicing, and/or alternative initiation therefrom.
  • pre-UBE3A-ATS transcript such as human pre-UBE3A-ATS transcript or mouse pre-UBE3A-ATS transcript, for example, the RNA counterpart or pre-transcript thereof of human UBE3A-ATS genome coding sequence, Accession No: NG
  • the DNA counterpart of the human UBE3A-ATS (also named SNHG14) is set in forth in Genome sequence: NG_002690.1 at NCBI.
  • the DNA counterpart of the mouse UBE3A-ATS (also named Snhg14) is set in forth in Genome sequence: NC_000073.7 at NCBI.
  • the guide nucleic acid comprises the scaffold sequence 5’ or 3’ to the guide sequence (e.g., the guide sequence, the first guide sequence, the second guide sequence, the third guide sequence, the fourth guide sequence, and so on; same as elsewhere) .
  • the guide nucleic acid comprises the scaffold sequence 3’ to the guide sequence.
  • the scaffold sequence is fused to the guide sequence without a linker.
  • the guide nucleic acid comprises, from 5’ to 3’, one guide sequence and one scaffold sequence.
  • the guide nucleic acid comprises, from 5’ to 3’, one scaffold sequence, one guide sequence, and one scaffold sequence, wherein the scaffold sequences are the same or different.
  • the 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.
  • the 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.
  • the 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.
  • the target sequence is at least about 14 nucleotides in length, 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, 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 in a length of a numerical range between any of two preceding values, e.g., in a length of from about 16 to about 50 nucleotides.
  • the target sequence is about 30 nucleotides in length.
  • the target sequence comprises, consists essentially of, or consists of at least about 14 contiguous nucleotides of the 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, 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 contiguous nucleotides of the human UBE3A-ATS, or in a numerical range between any of two preceding values, e.g., from about 14 to about 50 contiguous nucleotides of the human UBE3A-ATS) .
  • the target sequence comprises, consists essentially of, or consists of about 30 contiguous nucleotides of the human UBE
  • the guide sequence is at least about 14 nucleotides in length, 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, 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 in a length of a numerical range between any of two preceding values, e.g., in a length of from about 16 to about 50 nucleotides.
  • the guide sequence is about 30 nucleotides in length.
  • the guide sequence is at least about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% (fully) , optionally about 100% (fully) , complementary to the target sequence; or wherein the guide sequence comprises no mismatch with the target sequence in 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 at the 5’ end of the guide sequence.
  • the guide sequence is 100% (fully) complementary to the target sequence.
  • the target sequence comprises, consists essentially of, or consists of about 30 contiguous nucleotides on a common region shared by the human UBE3A-ATS and the mouse UBE3A-ATS.
  • 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’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to any one of SEQ ID NOs: 16-21 and 58-80 or a 5’ or 3’ end 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.
  • the target sequence comprises the sequence of SEQ ID NO: 21.
  • 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’ end truncation thereof with 1, 2, 3, 4, 5, or 6, nucleotides truncated at the 5’ or 3’ end; or (2) a sequence having a sequence identity of at least about 70%, 75%, 80%, 85%, 90%, 95%, or 100%to any one of SEQ ID NOs: 10-15 and 35-57 or a 5’ or 3’ end 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: 10-15 and 35-57.
  • the guide sequence comprises the sequence of SEQ ID NO: 15.
  • the scaffold sequence is compatible with the napRNAn of the disclosure and is capable of complexing with the napRNAn.
  • the scaffold sequence may be a naturally occurring scaffold sequence identified along with the napRNAn, or a variant thereof maintaining the ability to complex with the napRNAn.
  • the ability to complex with the napRNAn is maintained as long as the secondary structure of the variant is substantially identical to the secondary structure of the naturally occurring scaffold sequence.
  • a nucleotide deletion, insertion, or substitution in the primary sequence of the scaffold sequence may not necessarily change the secondary structure of the scaffold sequence (e.g., the relative locations and/or sizes of the stems, bulges, and loops of the scaffold sequence do not significantly deviate from that of the original stems, bulges, and loops) .
  • nucleotide deletion, insertion, or substitution may be in a bulge or loop region of the scaffold sequence so that the overall symmetry of the bulge and hence the secondary structure remains largely the same.
  • the nucleotide deletion, insertion, or substitution may also be in the stems of the scaffold sequence so that the lengths of the stems do not significantly deviate from that of the original stems (e.g., adding or deleting one base pair in each of two stems correspond to 4 total base changes) .
  • the scaffold sequence has substantially the same secondary structure as the secondary structure of the sequence of SEQ ID NO: 3.
  • the scaffold sequence comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) 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 consecutive or not, compared to the sequence of SEQ ID NO: 3.
  • 80% e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
  • the scaffold sequence comprises the sequence of SEQ ID NO: 3.
  • the guide nucleic acid comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of any 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 consecutive or not, compared to the sequence of any of SEQ ID NOs: 4-9.
  • the first polynucleotide sequence comprises a promoter operably linked to the sequence encoding the guide nucleic acid.
  • the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter.
  • Suitable promoters include, for example, a Cbh promoter, a Cba promoter, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, a retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken ⁇ -actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1 ⁇ -subunit (EF1 ⁇
  • the promoter comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) 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 consecutive or not, compared to the sequence of SEQ ID NO: 30.
  • the first polynucleotide sequence comprises, from 5’ to 3’, a promoter of SEQ ID NO: 30, and a sequence encoding the guide nucleic acid of any one of SEQ ID NOs: 4-9.
  • the napRNAn is capable of forming a complex with the guide nucleic acid of the disclosure by complexing with the scaffold sequence of the guide nucleic acid and is thereby guided to the human UBE3A-ATS by the hybridization of the guide sequence of the guide nucleic acid to the target sequence of the human UBE3A-ATS.
  • the nuclease activity of the napRNAn functions to cleave the human UBE3A-ATS, leading to degradation of the human UBE3A-ATS and hence elimination of the inhibition of expression of parental human UBE3A gene by the human UBE3A-ATS.
  • any such napRNAn can be used with the guide nucleic acid of the disclosure, and when a napRNAn is selected, the scaffold sequence compatible to the napRNAn for complexing with the napRNAn can also be selected accordingly.
  • the scaffold sequence is generally conserved.
  • the 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) .
  • crRNA engineered CRISPR RNA
  • the Cas13 is a Cas13a (C2c2) , Cas13b (such as, Cas13b1, Cas13b2) , Cas13c, Cas13d, Cas13e, or Cas13f polypeptide.
  • Cas13e. 1 (Cas13X. 1) is the smallest known crRNA-guided RNA endonuclease, with only 775 amino acids, and a high-fidelity variant of this Cas, hfCas13e. 1 (hfCas13x. 1) , has been shown to exhibit high on-target activity with markedly lower collateral activity.
  • the napRNAn comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 1 or 34 or a 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.
  • 80% e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%
  • the napRNAn comprises the sequence of SEQ ID NO: 1.
  • the sequence encoding the napRNAn comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) 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.
  • the sequence encoding the napRNAn comprises the sequence of SEQ ID NO: 2.
  • the second polynucleotide sequence comprises a promoter operably linked to the sequence encoding the napRNAn.
  • the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter.
  • Suitable promoters include, for example, a Cbh promoter, a Cba promoter, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, a retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken ⁇ -actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1 ⁇ -subunit (EF1 ⁇
  • the promoter is a nerve cell (e.g., neuron) specific promoter.
  • the promoter is a human Syn1 promoter.
  • the second polynucleotide sequence comprises a Kozak sequence 5’ to the sequence encoding the napRNAn.
  • the second polynucleotide sequence comprises a sequence encoding a nuclear localization signal (NLS) 5’ and/or 3’ to the sequence encoding the napRNAn.
  • NLS nuclear localization signal
  • the second polynucleotide sequence comprises a sequence encoding a nuclear export signal (NES) 5’ and/or 3’ to the sequence encoding the napRNAn.
  • NES nuclear export signal
  • 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.
  • the NLS, the first NLS, and/or the second NLS is a SV40 NLS, a bpSV40 NLS, or a Nucleoplasmin NLS.
  • the second polynucleotide sequence comprises a WPRE sequence downstream of the sequence encoding the napRNAn.
  • the WPRE sequence is selected from the group consisting of Woodchuck Hepatitis Virus (WHP) Posttranscriptional Regulatory Element (WPRE) , WPRE3 (a shortened WPRE) , and a functional variant (e.g., a functional truncation) thereof.
  • WP Woodchuck Hepatitis Virus
  • WPRE3 a shortened WPRE
  • a functional variant e.g., a functional truncation
  • the second polynucleotide sequence comprises a sequence encoding a polyadenylation (polyA) signal downstream of the sequence encoding the napRNAn.
  • polyA polyadenylation
  • the second polynucleotide sequence comprises, downstream of the sequence encoding the napRNAn, a WPRE sequence followed by a sequence encoding a polyadenylation (polyA) signal.
  • polyA polyadenylation
  • the polyA signal is selected from a group consisting of 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 a functional variant (e.g., a functional truncation) thereof.
  • bGH polyA bovine growth hormone polyadenylation
  • SPA small polyA
  • hGH polyA human growth hormone polyadenylation
  • SV40 polyA SV40 polyA
  • rBG polyA rabbit beta globin polyA
  • the polyA signal is a SV40 polyA signal.
  • 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.
  • the promoter comprises, consists essentially of, or consists of a sequence encoding a polypeptide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) 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 consecutive or not, compared to the sequence of SEQ ID NO: 25.
  • the Kozak sequence comprises, consists essentially of, or consists of a sequence encoding a polypeptide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 26; or a sequence having at most 1, 2, 3, or 4 nucleotide differences, whether consecutive or not, compared to the sequence of SEQ ID NO: 26.
  • the NLS, the first NLS, and/or the second NLS comprises, consists essentially of, or consists of a sequence encoding a polypeptide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) 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 consecutive or not, compared to the sequence of SEQ ID NO: 27.
  • the sequence encoding the polyA signal comprises, consists essentially of, or consists of a sequence encoding a polypeptide having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) 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 consecutive or not, compared to the sequence of SEQ ID NO: 29.
  • 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.
  • the rAAV vector genome comprises a 5’ inverted terminal repeat (ITR) sequence and a 3’ ITR sequence.
  • the 5’ ITR sequence and the 3’ ITR sequence are both 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 Clade to which any of the AAV1-AAV13 belong, or a functional variant (e.g., a functional truncation) thereof.
  • the 5’ ITR sequence comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 31.
  • the 3’ ITR sequence comprises, consists essentially of, or consists of a sequence having a sequence identity of at least about 80% (e.g., at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) to the sequence of SEQ ID NO: 32.
  • the rAAV vector genome comprises, from 5’ to 3’, the first polynucleotide sequence, and the second polynucleotide sequence.
  • the rAAV vector genome comprises, from 5’ to 3’, the second polynucleotide sequence, and the first polynucleotide sequence.
  • the disclosure provides a recombinant AAV (rAAV) particle comprising the rAAV vector genome of the disclosure.
  • a simple introduction of AAV for delivery may refer to “Adeno-associated Virus (AAV) Guide” (www. addgene. org/guides/aav/) .
  • Adeno-associated virus when engineered to delivery, e.g., a protein-encoding sequence of interest, may be termed as a (r) AAV vector, a (r) AAV vector particle, or a (r) AAV particle, where “r” stands for “recombinant” .
  • the genome packaged in AAV vectors for delivery may be termed as a (r) AAV vector genome, vector genome, or vg for short, while viral genome may refer to the original viral genome of natural AAVs.
  • the serotypes of the capsids of rAAV particles can be matched to the types of target cells.
  • Table 2 of WO2018002719A1 lists exemplary cell types that can be transduced by the indicated AAV serotypes (incorporated herein by reference) .
  • the rAAV particle comprising a capsid with a serotype suitable for delivery into nerve cells (e.g., neuron) .
  • the rAAV particle comprising a capsid with a serotype of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or AAV. PHP.
  • the serotype of the capsid is AAV9 or AAV. PHP. eB or a mutant thereof.
  • rAAV particles may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650) .
  • the vector titers are usually expressed as vector genomes per ml (vg/ml) .
  • the vector titer is above 1 ⁇ 10 9 , above 5 ⁇ 10 10 , above 1 ⁇ 10 11 , above 5 ⁇ 10 11 , above 1 ⁇ 10 12 , above 5 ⁇ 10 12 , or above 1 ⁇ 10 13 vg/ml.
  • RNA sequence as a vector genome into a rAAV particle
  • systems and methods of packaging an RNA sequence as a vector genome into a rAAV particle is recently developed and applicable herein. See PCT/CN2022/075366, which is incorporated herein by reference in its entirety.
  • sequence elements described herein for DNA vector genomes when present in RNA vector genomes, should generally be considered to be applicable for the RNA vector genomes 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 element in the DNA sequence is replaced with the corresponding element with a corresponding function in the RNA sequence or omitted because its function is unnecessary in the RNA sequence and/or an additional element necessary for the RNA vector genome is introduced.
  • dT is equivalent to U
  • dA is equivalent to A
  • a coding sequence e.g., as a sequence element of rAAV vector genomes herein, is construed, understood, and considered as covering and covers both a DNA coding sequence and an RNA coding sequence.
  • an RNA sequence can be transcribed from the DNA coding sequence, and optionally further a protein can be translated from the transcribed RNA sequence as necessary.
  • the RNA coding sequence per se can be a functional RNA sequence for use, or an RNA sequence can be produced from the RNA coding sequence, e.g., by RNA processing, or a protein can be translated from the RNA coding sequence.
  • a Cas13 coding sequence encoding a Cas13 polypeptide covers either a Cas13 DNA coding sequence from which a Cas13 polypeptide is expressed (indirectly via transcription and translation) or a Cas13 RNA coding sequence from which a Cas13 polypeptide is translated (directly) .
  • a gRNA coding sequence encoding a gRNA covers either a gRNA DNA coding sequence from which a gRNA is transcribed or a gRNA RNA coding sequence (1) which per se is the functional gRNA for use, or (2) from which a gRNA is produced, e.g., by RNA processing.
  • 5’-ITR and/or 3’-ITR as DNA packaging signals may be unnecessary and can be omitted at least partly, while RNA packaging signals can be introduced.
  • a promoter to drive transcription of DNA sequences may be unnecessary and can be omitted at least partly.
  • a sequence encoding a polyA signal may be unnecessary and can be omitted at least partly, while a polyA tail can be introduced.
  • DNA elements of rAAV DNA vector genomes can be either omitted or replaced with corresponding RNA elements and/or additional RNA elements can be introduced, in order to adapt to the strategy of delivering an RNA vector genome by rAAV particles.
  • the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the system or composition of the disclosure or the rAAV particle of the disclosure and a pharmaceutically acceptable excipient.
  • the pharmaceutical composition comprises the rAAV particle in a concentration selected from the group consisting of 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/
  • the pharmaceutical composition is an injection.
  • the volume of the injection is selected from the group consisting of 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 a volume of a numerical range between any of two preceding values, e.g., in a concentration of from about 10 microliters to about 750 microliters.
  • the disclosure provides a method for preventing or treating a disease or disorder associated with human UBE3A-ATS in a subject in need thereof, comprising administering to the subject the system or composition of the disclosure, the rAAV particle of the disclosure, or the pharmaceutical composition of the disclosure, wherein the napRNAn modifies the human UBE3A-ATS, and wherein the modification of the human UBE3A-ATS treats the disease or disorder.
  • the disease or disorder is Angelman syndrome (AS) .
  • the administrating comprises local administration or systemic administration.
  • the administrating comprises intrathecal administration, intramuscular administration, intravenous administration, transdermal administration, intranasal administration, oral administration, mucosal administration, intraperitoneal administration, intracranial administration, intracerebroventricular administration, or stereotaxic administration.
  • the administration is conducted by injection.
  • the subject is a human.
  • the level of the human UBE3A-ATS is decreased in the subject 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 the human UBE3A-ATS in the subject prior to the administration.
  • 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 a human population not suffering from the disease or disorder.
  • the dose of the rAAV particle for treatment of the disease or disorder may be either via a single dose, or multiple doses.
  • the actual dose may vary greatly depending upon a variety of factors, such as the vector choices, the target cells, organisms, tissues, the general conditions of the subject to be treated, the degrees of transformation/modification sought, the administration routes, the administration modes, the types of transformation/modification sought, etc.
  • the rAAV particle is administrated in a therapeutically effective dose.
  • the therapeutically effective dose of the rAAV particle may 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, 6.0E+12, 8.0E+12, 1.0E+13, 2.0E+13, 3.0E+13, 4.0E+13, 6.0E+13, 8.0E+13, 1.0E+14, 2.0E+14, 3.0E+14, 4.0E+14
  • the disclosure provides a cell or a progeny thereof, comprising the guide 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.
  • the cell is a eukaryote.
  • the cell is a human cell.
  • the cell is located in the CNS of the subject.
  • the cell is a human nerve cell (e.g., neuron) .
  • the disclosure provides a cell or a progeny thereof modified by the system or composition or complex of the disclosure, the rAAV particle of the disclosure, or the method of the disclosure.
  • the cell is a eukaryote.
  • the cell is a human cell.
  • the cell is located in the CNS of the subject.
  • the cell is a human nerve cell (e.g., neuron) .
  • the cell is not a human embryonic stem cell. In some embodiments, the cell is not a human germ cell.
  • the disclosure provides a kit comprising the guide nucleic acid of the disclosure, the system or composition of the disclosure, the rAAV vector genome of the disclosure, the rAAV particle of the disclosure, or the cell or a progeny thereof of the disclosure.
  • the kit further comprises an instruction to use the component (s) contained therein, and/or instructions for combining with additional component (s) that may be available or necessary elsewhere.
  • the kit further comprises one or more buffers that may be used to dissolve any of the component (s) contained therein, and/or to provide suitable reaction conditions for one or more of the component (s) .
  • buffers may include one or more of PBS, HEPES, Tris, MOPS, Na 2 CO 3 , NaHCO 3 , NaB, or combinations thereof.
  • the reaction condition includes a proper pH, such as a basic pH. In some embodiments, the pH is between 7-10.
  • any one or more of the kit components may be stored in a suitable container or at a suitable temperature, e.g., 4 Celsius degree.
  • a guide RNA (e.g., a single guide RNA) comprising a spacer sequence substantially complementary to a target sequence of a UBE3A-ATS.
  • the gRNA comprises a direct repeat (DR) sequence capable of forming a complex with a Cas13 polypeptide, wherein the complex specifically cleaves the UBE3A-ATS at or near the target sequence when the gRNA guides the Cas13 polypeptide to the target sequence.
  • DR direct repeat
  • Another aspect of the disclosure provides a recombinant lentiviral or adeno-associated virus (AAV) vector genome.
  • AAV adeno-associated virus
  • the recombinant lentiviral or adeno-associated virus (AAV) vector genome comprises a Cas13 coding sequence encoding a Cas13 polypeptide.
  • 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 an RNA counterpart thereof.
  • the Cas13 polypeptide comprises (a) the amino acid sequence of SEQ ID NO: 1, or (b) a variant thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 34 and has a non-conserved substitution at Y672 and/or Y676 (e.g., Y672A and/or Y676A substitution (s) ) of SEQ ID NO: 34, 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%) collateral (guide RNA-independent) nuclease activity (collateral cleavage activity) of SEQ ID NO: 34.
  • a variant thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%
  • the recombinant lentiviral or adeno-associated virus (AAV) vector genome comprises a single guide RNA (sgRNA) or a sgRNA coding sequence encoding the sgRNA, the sgRNA comprises:
  • the complex specifically cleaves the UBE3A-ATS at or near the target sequence when the sgRNA guides the Cas13 polypeptide to the target sequence.
  • the gRNA or gRNA coding sequence is 3’ or 5’ to the Cas13 coding sequence.
  • the vector genome further comprises a first coding sequence for a first nuclear localization sequence (NLS, such as SEQ ID NO: 27) or nuclear export signal (NES) fused N-terminal to the Cas13 polypeptide, and/or a second coding sequence for a second NLS (such as SEQ ID NO: 27) or NES fused C-terminal to the Cas13 polypeptide.
  • NLS nuclear localization sequence
  • NES nuclear export signal
  • the vector genome further comprises a coding sequence for one or more copies (e.g., 3 tandem copies) of an epitope tag, such as an 3xFLAG, fused (e.g., C-terminally) to the Cas13 polypeptide (and the C-terminal NLS or NES, if present) .
  • an epitope tag such as an 3xFLAG
  • the vector genome further comprises a 5’ AAV ITR sequence and a 3’ AAV ITR sequence.
  • the 5’ and the 3’ AAV ITR sequences are both wild-type AAV 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 Clade to which any of the AAV1-AAV13 belong, or a functional truncated variant thereof.
  • the 5’ AAV ITR sequence has the polynucleotide sequence of SEQ ID NO: 31.
  • the 3’ AAV ITR sequence has the polynucleotide sequence of SEQ ID NO: 32.
  • the vector genome further comprises a promoter operably linked to the Cas13 coding sequence.
  • the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter.
  • the promoter is selected from the group consisting of: a Cbh promoter, a Cba promoter, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, a retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie
  • EFS elongation factor 1
  • the vector genome further comprises a polyadenylation (polyA) signal sequence, such as a bovine growth hormone polyadenylation signal (bGH polyA) , a small polyA signal (SPA) , a human growth hormone polyadenylation signal (hGH polyA) , a SV40 polyA signal (SV40 polyA) , a rabbit beta globin polyA signal (rBG polyA) , and a functional truncation or variant thereof; or a corresponding polyA sequence.
  • a polyadenylation (polyA) signal sequence such as a bovine growth hormone polyadenylation signal (bGH polyA) , a small polyA signal (SPA) , a human growth hormone polyadenylation signal (hGH polyA) , a SV40 polyA signal (SV40 polyA) , a rabbit beta globin polyA signal (rBG polyA) , and a functional truncation or variant thereof; or a corresponding polyA sequence.
  • the polyA signal sequence comprises a SV40 polyA signal, or a variant thereof.
  • the SV40 polyA signal comprises the polynucleotide sequence of SEQ ID NO: 29.
  • the gRNA coding sequence is operably linked to a promoter.
  • the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter.
  • the promoter is selected from a group consisting of a Cbh promoter, a Cba promoter, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, a retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken ⁇ -actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1 ⁇ -subunit (EF1 ⁇
  • the RNA pol III promoter is U6 (such as SEQ ID NO: 30) , H1, 7SK, or a variant thereof.
  • the gRNA comprises one spacer sequence directly linked to one DR sequence (e.g., SEQ ID NO: 3) ; (2) the gRNA comprises one spacer sequence flanked by two DR sequences (e.g., each of SEQ ID NO: 3) ; or (3) the gRNA comprises two or more spacer sequences; and wherein each spacer sequence is flanked by two DR sequences each capable of forming a complex with the Cas13 polypeptide.
  • the gRNA comprises two spacer sequences flanked by three DR sequences to form a DR-spacer-DR-spacer-DR structure (e.g., each of SEQ ID NO: 3) .
  • each of the spacer sequence is independently substantially complementary to a distinct target sequence of the UBE3A-ATS, and each capable of directing the Cas13 polypeptide to cleave respective the distinct target sequence.
  • the DR sequence comprises (1) SEQ ID NO: 3; (2) a sequence having at least 90%, 92%, 94%, 95%, 96%, 98%, or 99%identity to SEQ ID NO: 3; (3) a sequence having at most 1, 2, 3, 4, or 5 nucleotide differences from SEQ ID NO: 3; or (4) a sequence having substantially the same secondary structure as that of SEQ ID NO: 3.
  • each the DR sequence comprises, consists essentially of, or consists of SEQ ID NO: 3.
  • the target sequence comprises a stench of contiguous nucleotides of the RNA counterpart of NG_002690.1 or NC_000073.7.
  • the target sequence comprises a stench of contiguous 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, contiguous nucleotides of the RNA counterpart of NG_002690.1 or NC_000073.7, such as, any one of SEQ ID NO: 16-21 and 58-80 or the RNA counterpart thereof.
  • the spacer sequence is independently selected from any one of SEQ ID NOs: 10-15 and 35-57, or a variant thereof differing from any one of SEQ ID NOs: 10-15 and 35-57 by up to 1, 2, 3, 4, 5 or 6 nucleotides without substantially diminishing the ability to direct the Cas13 polypeptide to bind to the gRNA to form a Cas13-gRNA complex targeting the target sequences to cleave the target RNA.
  • the UBE3A-ATS is associated with a disease or disorder, such as AS (Angelman syndrome) .
  • AS Angelman syndrome
  • the vector genome comprises an ITR-to-ITR polynucleotide (such as SEQ ID NO: 33) comprising, from 5’ to 3’ :
  • a first NLS coding sequence (such as one encoding SEQ ID NO: 27) ;
  • a Cas13 polynucleotide (such as SEQ ID NO: 2 except the start codon ATG) encoding the Cas13 polypeptide of SEQ ID NO: 1 except the first amino acid M;
  • a second NLS coding sequence (such as one encoding SEQ ID NO: 27) ;
  • an optional coding sequence encoding a 3xFlag sequence (e.g., SEQ ID NO: 28) ;
  • an optional SV40 polyA signal sequence (such as SEQ ID NO: 29) ;
  • a U6 promoter such as SEQ ID NO: 30
  • a first direct repeat (DR) coding sequence encoding a first DR sequence (such as SEQ ID NO: 3) ;
  • (k) a spacer coding sequence encoding a first spacer sequence specific for UBE3A-ATS (such as SEQ ID NO: 10 or 15) ;
  • polynucleotide 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 the ITR-to-ITR polynucleotide.
  • the ITR-to-ITR polynucleotide further comprises a linker sequence between any two adjacent sequence elements of (a) – (m) .
  • sequence elements of (b) to (h) that are 5’ to the sequence elements of (i) to (l) are relocated 3’ to the sequence elements of (i) to (l) .
  • sequence elements of (b) to (h) in 5’-3’ orientation are placed in an opposite order of from (h) to (b) in 5’-3’ orientation.
  • sequence elements of (i) to (l) in 5’-3’ orientation are placed in an opposite order of from (l) to (i) in 5’-3’ orientation.
  • AAV vector genome comprising, consisting essentially of, or consisting of:
  • SEQ ID NO: 33 or a polynucleotide at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%identical thereto,
  • a variant thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 34 and having a non-conserved substitution at Y672 and/or Y676 (e.g., Y672A and/or Y676A substitution (s) ) of SEQ ID NO: 34, 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%) collateral (guide RNA-independent) nuclease activity of SEQ ID NO: 34; and,
  • gRNA coding sequence encoding a gRNA comprises:
  • the complex specifically cleaves the UBE3A-ATS with 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%) collateral (guide RNA-independent) nuclease activity of SEQ ID NO: 34,
  • the gRNA coding sequence is 3’ or 5’ to the Cas13 coding sequence.
  • the vector genome is SEQ ID NO: 33, or the polynucleotide at least 95%or 99%identical thereto.
  • Another aspect of the disclosure provides a recombinant lentiviral or AAV particle comprising the vector genome of the disclosure.
  • the recombinant AAV particle comprises a capsid with a serotype of AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or AAV.
  • PHP. eB a member of the Clade to which any of the AAV1-AAV13 belong, or a functional truncated variant or a functional mutant thereof, encapsidating the vector genome.
  • the capsid serotype is AAV. PHP. eB or AAV9.
  • Another aspect of the disclosure provides a recombinant AAV particle comprising the vector genome of the disclosure, encapsidated in a capsid with a serotype of AAV. PHP. eB or AAV9.
  • Another aspect of the disclosure provides a pharmaceutical composition
  • a pharmaceutical composition comprising the vector genome of the disclosure, or the particle of the disclosure, and a pharmaceutically acceptable excipient.
  • Another aspect of the 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 the vector genome of the disclosure, the particle of the disclosure, or the pharmaceutically composition of the disclosure, wherein the vector genome or the particle specifically up-regulate the expression of the UBE3A causative of the disease or disorder.
  • the administrating comprises contacting a cell with the therapeutically effective amount of the vector genome of the disclosure, the particle of the disclosure, or the pharmaceutically composition of the disclosure.
  • the cell is located in the CNS of the subject.
  • the disease or disorder is Angelman Syndrome (AS) .
  • AS Angelman Syndrome
  • the administrating comprises intracerebroventricular administration.
  • the subject is a human.
  • the level of UBE3A-ATS transcript in the cell is decreased in comparison to a cell having not been contacted with the vector genome of the disclosure, the particle of the disclosure, or the pharmaceutically composition of the disclosure.
  • the level of UBE3A-ATS is decreased in the subject 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 prior to administration; and/or 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%, compared to the level of UBE3A protein in a subject not suffering from the disease or disorder.
  • Another aspect of the disclosure provides a cell or a progeny thereof, comprising the vector genome of the disclosure, the particle of the disclosure, or the gRNA of the disclosure.
  • kits comprising the vector genome of the disclosure, the particle of the disclosure, the gRNA of the disclosure, or the cell or a progeny thereof of the disclosure.
  • Another aspect of the disclosure provides a method of preparing the recombinant AAV particle of the disclosure, the method comprising:
  • the method comprises isolating or purifying the harvested recombinant AAV particle.
  • This example demonstrates the high ex vivo knockdown efficiency of UBE3A-ATS by the subject CRISPR-hfCas13e. 1 system via lentiviral delivery.
  • the subject CRISPR-hfCas13e. 1 system was composed of a hfCas13e. 1 protein (SEQ ID NO: 1, encoded by SEQ ID NO: 2) and a Ube3a-ATS-targeting sgRNA (one of “sgRNA [Ube3a-ATS] 9-14” or “sg9-14” hereinafter, SEQ ID NO: 4-9) consisting of a spacer sequence (one of spacer sequences 9-14 of SEQ ID NO: 10-15, respectively) , targeting the target sequence (one of target sequences 9-14 of SEQ ID NO: 16-21, respectively) of UBE3A-ATS, flanked by two direct repeat sequences (SEQ ID NO: 3) .
  • sgRNA [NT] a non-targeting-sgRNA
  • NT a non-targeting-sgRNA
  • SEQ ID NO: 22 consisting of a non-targeting spacer sequence (LacZ, SEQ ID NO: 23) flanked by two direct repeat sequences (SEQ ID NO: 3) was used in place of the sgRNA [Ube3a-ATS] .
  • the hfCas13e. 1 protein (SEQ ID NO: 1) and the sgRNA [Ube3a-ATS] or the sgRNA [NT] were encoded together with a EGFP reporter into a transgene plasmid as shown in 2A for the production of treatment or control lentiviral particles.
  • mice were housed in the in-house animal facility on 12h: 12h light/dark cycle with food and water ad libitum.
  • Ube3a knock-out (KO) mice were generated by Jiang and colleagues (YH Jiang et al. 1998, Neurons) .
  • AS mouse model were generated by crossing the Ube3a m+/p- heterogeneous females among Ube3a KO mice to C57BL/6 wild type (WT) males from Shanghai SLAC Laboratory Animal Co., Ltd. All experimental protocols were approved by the Animal Care and Use Committee of the Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China and HuidaGene Therapeutics Co., Ltd.
  • Treatment or control lentiviral transgene plasmids were constructed as shown in 2A, each of which was co-transfected with package plasmids and envelope plasmids into HEK293 cells to produce recombinant lentiviral particles delivering the CRISPR-hfCas13e. 1 system.
  • the HEK293 cell supernatant was filtered by 0.22 ⁇ m sterile Millex filter, and then collected for ultracentrifugation (27000rpm, CP90NX, Hitachi) , the lentiviral particles at the bottom of the tube after ultracentrifugation was resuspended in PBS buffer, and the titer of lentiviral vectors was determined by RT-qPCR with GAG and TET1 primers.
  • the copy numbers of GAG sequence were measured and normalized to the copy numbers of a housekeeping gene, TET1.
  • Cortex and hippocampus were dissected from E14-E16 embryos of AS and C57BL/6 WT mouse models and dissociated in digestion buffer with papain (Cat. No. LS003126, Worthington Bio Corp) .
  • the digestion buffer contained (in mM) : 161.0 NaCl, 5.0 KCl, 2.9 CaCl 2 , 5.0 HEPES, 5.5 glucose, 0.53 MgSO 4 , and 0.0056 phenol red, pH 7.4, with additional (in mM) 1.7 cysteine, 1.0 CaCl 2 , and 0.5 EDTA.
  • the tissues were digested for 30 minutes (min) at 37°C, and then plated on PDL (Cat. No.
  • the AS or WT primary neurons infected with the lentivirus above were lysated in SDS lysis buffer (Beyotime) containing 1x Protease inhibitor cocktail (Beyotime) . 40-60 ⁇ g of total proteins was loaded and separated by SDS-PAGE (Epizyme) and transferred to a PVDF membrane (Merk Millipore) . The membrane was blocked in 5%skim milk powder in TBST buffer (Epizyme) . The following primary antibodies are diluted and incubated with the membrane overnight: anti-Ube3a (1: 1000; Cat. No. A300-352A, Bethyl) , anti-Flag (1: 3000; Cat. NO.
  • sg9, sg10, sg13, and sg14 significantly decreased the Ube3a-ATS RNA level compared to AS model, and substantially maintained the Ube3a mRNA level and significantly increased UBE3A protein level compared to WT model.
  • sg9 was selected for further evaluation in Examples 2 and 3.
  • AAV-PHP. eB delivery system was used to deliver the system in vivo to target cells in animals.
  • treatment and control transgene plasmids for AAV-PHP. eB packaging encoding the hfCas13e. 1 and a sg9 or sgNT were constructed, respectively, as shown in 2B.
  • Both the treatment and control rAAV-PHP. eB particles herein were produced by using conventional triple-plasmid transfection system mutatis mutandis, by co-transfecting the respective transgene plasmids, packaging plasmids, and helper plasmids in a weight ratio of 1: 1: 2 into HEK293T cells.
  • the transgene plasmids were packaged by AAV-PHP.
  • eB capsids to form the genomes inside the capsids, and together the genome and the capsids constituted the AAV-PHP. eB particles.
  • the HEK293T cells were cultured in competent DMEM medium, and the cells were plated 24 hours before transfection of the plasmids. Shortly before transfection, the culture medium was replaced with fresh DMEM containing 2%FBS. PEI-MAX was used as the transfection reagent. The transfected HEK293T cells were harvested from the media at 72 hours post translation. The treatment and control AAV-PHP. eB particles were purified from the cells by using iodixanol density gradient ultracentrifugation.
  • RT-qPCR was used with a pair of 5’-ITR primers specific for the 5’-ITR sequence on the genomes to detect the genome titre of any genomes packaged in the treatment and control AAV-PHP. eB particles.
  • mice were housed in the in-house animal facility on 12h: 12h light/dark cycle with food and water adlibitum.
  • Ube3a knock-out (KO) mice were generated by Jiang and colleagues (YH Jiang et al. 1998, Neurons.
  • AS mouse model were generated by crossing the Ube3a m+/p- heterogeneous females to C57BL/6 wild type (WT) males from Shanghai SLAC Laboratory Animal Co., Ltd. All experimental protocols were approved by the Animal Care and Use Committee of the Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China and HUIGENE THERAPEUTICS CO., LTD.
  • Neonatal AS mice were placed on ice for hypothermia anesthesia and injected with AAV-PHP.
  • Untreated WT mice (WT+NT) : C57BL/6 WT mice injected with AAV-PHP. eB particles delivering hfCas13e. 1-sg [NT] .
  • tdTomato + regions of brains were isolated from injected AS mice.
  • Total RNA of cortex and hippocampus were extracted and purified with Trizol (Ambion) and then reverse transcribed into complementary DNA (HiScript Q RT SuperMix for qPCR, Vazyme, Biotech) for RT-qPCR.
  • the levels of Ube3a and UBE3A-ATSs were detected with RT-qPCR by Taqman probe (Bestar qPCR master mix, DBI-2041, DBI) with Ube3a-ATS and Ube3a primers and normalized to the mRNA level of a housekeeping gene, GAPDH.
  • Tdtomato positive brain regions were dissected and homogenized in SDS lysis buffer (Beyotime) containing 1x Protease inhibitor cocktail (Beyotime) . 40-60 ⁇ g of total proteins was loaded and separated by SDS-PAGE (Epizyme) and transferred to a PVDF membrane (Merk Millipore) . The membrane was blocked in 5%skim milk powder in TBST buffer (Epizyme) . The following antibodies were diluted and incubated with the membrane overnight: anti-Ube3a (1: 1000; Cat. No. A300-352A, Bethyl) , anti-Flag (1:3000; Cat. NO. F1804, Sigma) , anti-a-tubulin (1: 5000; Cat. No.
  • RT-qPCR results show that the in vivo efficiency of the AAV-PHP. eB delivered CRISPR-hfCas13e. 1 system comprising sg9 was 77.5 %reduction in cortex and 76.6%reduction in hippocampus of Ube3a-ATS RNA level (right columns) of the treated AS mice compared with the untreated AS mice, and 44.3%reinstatement in cortex and 17.6%reinstatement in hippocampus of Ube3a mRNA level (left columns) of the treated AS mice compared with the untreated WT mice.
  • the WB results show 35.9%and 41.6%expression of UBE3A induced by the hfCas13e. 1-sg9 system in cortex and hippocampus, respectively, at 4 weeks, and 21.1%and 39.1%expression of Ube3a in cortex and hippocampus, respectively, at 18 weeks, of the treated AS mice, compared with the untreated WT mice.
  • mice were housed in the in-house animal facility on 12h: 12h light/dark cycle with food and water ad libitum.
  • Ube3a deletion mice were generated by Jiang and colleagues (YH Jiang et al. 1998, Neurons. AS mouse model were generated by crossing the UBE3A m+/p- (deletion of paternal Ube3a) heterogenous females to C57BL/6 wildtype (WT) males from Shanghai SLAC Laboratory Animal Co., Ltd. All experimental protocols were approved by the Animal Care and Use Committee of the Institute of Neuroscience, Chinese Academy of Sciences, Shanghai, China and HUIGENE THERAPEUTICS CO., LTD.
  • Neonatal AS mice were placed on ice for hypothermia anesthesia and injected with AAV-PHP. eB particles into bilateral ventricles at four sites. Total 5 ⁇ 10 10 AAV particles (encoding hSyn1 promoter-hfCas13e. 1-sg9/NT in 2B) plus 2.5 ⁇ 10 9 reporter AAV particles (encoding CAG promoter-tdTomato reporter) in 2 ⁇ l 0.9%NaCl was injected into each AS or C57BL/6 WT mouse within 24 hours after birth using nanoject III (Warner Instruments) . Treated AS mice (AS+sg9) : AS mice injected with AAV-PHP. eB particles delivering hfCas13e.
  • Untreated AS mice AS mice injected with AAV-PHP. eB particles delivering hfCas13e. 1-sg [NT] .
  • Untreated WT mice WT+NT: C57BL/6 WT mice injected with AAV-PHP. eB particles delivering hfCas13e. 1-sg [NT] .
  • mice All behavioral experiments were performed blind to genotype and injection treatment of animals. Mice were placed in a test room for 30 minutes to acclimate to the environment before the test.
  • Hindlimb clasping time was the total time spent on clasping. Clasping was defined by the behavior of incomplete splay with one or both hindlimbs.
  • Each mouse was placed on the edge of 40 cm ⁇ 40 cm of open field box and allowed to explore for 15 min.
  • the center area was 20 cm ⁇ 20 cm square in the center of the arena.
  • the distance traveled was the total travel distance by each mouse in the arena, which was recorded and analyzed by camera and EthoVision software (Noldus Wageningen) .
  • Centre frequency means the number of center entries of each mouse for the 15 min.
  • a 1 m long dowel with a diameter of 9 mm was placed parallel to the ground at a height of above 30cm. Mice were individually placed on the dowel, and the time on the towel was recorded. The longest experimental record was 120 sec.
  • 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 safe platform at one end of the dowel. After 2 days of training, latency was quantified by measuring the time it took for the mouse to walk through the dowel, and also the number of foot slips were counted.
  • mice were trained for two days. Two trials were performed for each day with more than 1 hour inter-trial interval. On the test day, mice were given two trials, and the time of remaining on the rod until falling off or making two consecutive turns was recorded. The average time of two trials were calculated.
  • the increased center frequency ( 5D) means that the hfCas13e. 1-sg9 system treated AS mice preferred to explore in the central region than the untreated AS mice.
  • hindlimb clasping test demonstrated that the hfCas13e.
  • 1-sg9 system treated AS mice showed better performance in terms of motor coordination and balance compared with the untreated AS mice.

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Abstract

L'invention concerne un acide nucléique guide ciblant l'UBE3A-ATS humaine, un système ou une composition ou un génome de vecteur rAAV le comprenant, et une méthode l'utilisant pour traiter une maladie et des troubles associés à l'UBE3A-ATS humaine.
PCT/CN2023/084417 2022-03-28 2023-03-28 Acide nucléique guide ciblant ube3a-ats et ses utilisations WO2023185861A1 (fr)

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