WO2023184108A1

WO2023184108A1 - Crispr-cas13 system for treating ube3a-associated diseases

Info

Publication number: WO2023184108A1
Application number: PCT/CN2022/083478
Authority: WO
Inventors: Hui Yang; Xing Wang; Jinhui Li
Original assignee: Huigene Therapeutics Co., Ltd.; Center For Excellence In Brain Science And Intelligence Technology, Chinese Academy Of Sciences
Priority date: 2022-03-28
Filing date: 2022-03-28
Publication date: 2023-10-05
Also published as: WO2023185861A1

Abstract

Provided is an CRISPR-Cas13 system and a method of treating UBE3A-associated with the same.

Description

CRISPR-CAS13 SYSTEM FOR TREATING UBE3A-ASSOCIATED DISEASES

REFERENCE TO RELATED APPLICATIONS

The instant application is related to International Patent Application No. PCT/CN2021/079821, filed on March 9, 2021, and PCT/CN2021/121926, filed on September 29, 2021, and the entire contents of each of the above-referenced application, including any sequence listing and drawings, are incorporated herein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on [**] , is named [**] _SL. txt and is [**] bytes in size.

BACKGROUND OF THE INVENTION

Angelman Syndrome (AS) is a severe neurodevelopmental disorder, with incidence approximately 1/12000-1/20000 worldwide, which is characterized by seizure disorder, severe motor, cognitive deficits, and language disability. AS is mainly due to loss of maternally inherited UBE3A (ubiquitin protein ligase E3A) , which is a ubiquitin–protein ligase targeting a series of protein substrates for degradation. Each of the substrates contributes to only a subset of UBE3A function, but none of them can explain all the phenotypic changes in AS patients. In neurons, maternally inherited UBE3A gene is the only active allele, because paternal UBE3A gene is selectively imprinted by an antisense RNA transcript called UBE3A-ATS (also named SNHG14) . Transcprition of UBE3A-ATS extends to UBE3A gene locus and results in early termination and then degradation of UBE3A transcripts. 75%of AS cases are due to deletion of the maternal chromosomal region 15q11.2–q13. The rest cases are attributable to maternal UBE3A mutation, paternal uniparental disomy, or imprinting defects.

There is currently no specific therapy for the treatment of AS. Given that the paternally inherited UBE3A in AS patients is intact and the associated promoter is transcriptionally active, activating the paternal copy of UBE3A by knocking-down UBE3A-ATS transcript is a promising therapeutic strategy. Genetic restoration of Ube3a by perturbing Ube3a-ATS dramatically correct the neurobehavioral phenotypes in AS mouse models. Antisense oligonucleotides (ASO) have been developed to reactivate the expression of Ube3a by inhibiting Ube3a-ATS in mouse models and is now in Phase 1/2 clinical trials, but this treatment requires frequent lumbar administrations. A more efficient therapy strategy is therefore needed.

SUMMARY OF THE INVENTION

One aspect of the invention provides a recombinant lentiviral or adeno-associated virus (AAV) vector genome, comprising:

(1) a Cas13 coding sequence encoding a Cas13 polypeptide,

(i) wherein 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,

(ii) wherein 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: 46 and has a non-conserved substitution at Y672 and/or Y676 (e.g., Y672A and/or Y676A substitution (s) ) of SEQ ID NO: 46, 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: 46 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity (collateral cleavage activity) of SEQ ID NO: 46; and,

(2) a single guide RNA (sgRNA) or a sgRNA coding sequence encoding the sgRNA, the sgRNA comprises:

(A) a spacer sequence substantially complementary to a target RNA sequence on a UBE3A-ATS transcript; and,

(B) a direct repeat (DR) sequence capable of forming a complex with the Cas13 polypeptide,

wherein the complex specifically cleaves the UBE3A-ATS transcript at or near the target RNA sequence when the sgRNA guides the Cas13 polypeptide to the target RNA sequence; optionally wherein the sgRNA or sgRNA coding sequence is 3’ or 5’ to the Cas13 coding sequence.

In some embodiments, 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;

optionally, 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) .

In some embodiments, the vector genome further comprises a 5’ AAV ITR sequence and a 3’ AAV ITR sequence.

In some embodiments, 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; optionally, the 5’ AAV ITR sequence has the polynucleotide sequence of SEQ ID NO: 31, and/or the 3’ AAV ITR sequence has the polynucleotide sequence of SEQ ID NO: 32.

In some embodiments, the vector genome further comprises a promoter operably linked to the Cas13 coding sequence.

In some embodiments, the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter; optionally, wherein the promoter comprises a promoter 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 β-actin promoter, an elongation factor 1αshort (EFS) promoter, a β glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken β-actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1α-subunit (EF1α) promoter, a ubiquitin C (UBC) promoter, a prion promoter, a neuron-specific enolase (NSE) , a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a platelet-derived growth factor (PDGF) promoter, a platelet-derived growth factor B-chain (PDGF-β) promoter, a synapsin (Syn) promoter, a synapsin 1 (Syn1) promoter, a methyl-CpG binding protein 2 (MeCP2) promoter, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) promoter, a metabotropic glutamate receptor 2 (mGluR2) promoter, a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a β-globin minigene nβ2 promoter, a preproenkephalin (PPE) promoter, an enkephalin (Enk) promoter, an excitatory amino acid transporter 2 (EAAT2) promoter, a glial fibrillary acidic protein (GFAP) promoter, and a myelin basic protein (MBP) promoter.

In some embodiments, the promoter comprises a Syn1 promoter, such as a Syn1 promoter having the polynucleotide sequence of SEQ ID NO: 25.

In some embodiments, 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.

In some embodiments, the polyA signal sequence comprises a SV40 polyA signal, or a variant thereof; optionally, the SV40 polyA signal comprises the polynucleotide sequence of SEQ ID NO: 29.

In some embodiments, the sgRNA coding sequence is operably linked to a promoter; optionally wherein the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter; optionally 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 β-actin promoter, an elongation factor 1α short (EFS) promoter, a β glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken β-actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1α-subunit (EF1α) promoter, a ubiquitin C (UBC) promoter, a prion promoter, a neuron-specific enolase (NSE) , a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a platelet-derived growth factor (PDGF) promoter, a platelet-derived growth factor B-chain (PDGF-β) promoter, a synapsin (Syn) promoter, a synapsin 1 (Syn1) promoter, a methyl-CpG binding protein 2 (MeCP2) promoter, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) promoter, a metabotropic glutamate receptor 2 (mGluR2) promoter, a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a β-globin minigene nβ2 promoter, a preproenkephalin (PPE) promoter, an enkephalin (Enk) promoter, an excitatory amino acid transporter 2 (EAAT2) promoter, a glial fibrillary acidic protein (GFAP) promoter, and a myelin basic protein (MBP) promoter; optionally wherein the promoter is an RNA pol III promoter.

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

In some embodiments, (1) the sgRNA comprises one spacer sequence directly linked to one DR sequence (e.g., SEQ ID NO: 3) ; (2) the sgRNA comprises one spacer sequence flanked by two DR sequences (e.g., each of SEQ ID NO: 3) ; or (3) the sgRNA 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; optionally, the sgRNA 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) ,

wherein each of the spacer sequence is independently substantially complementary to a distinct target RNA sequence on the UBE3A-ATS transcript, and each capable of directing the Cas13 polypeptide to cleave respective the distinct target RNA sequence.

In some embodiments, 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.

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

In some embodiments, the target RNA sequence comprises a stench of contiguous nucleotides of the RNA counterpart of NG_002690.1 or NC_000073.7; optionally 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 70-92 or the RNA counterpart thereof.

In some embodiments, the spacer sequence is independently selected from any one of SEQ ID NOs: 10-15 and 47-69, or a variant thereof differing from any one of SEQ ID NOs: 10-15 and 47-69 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 sgRNA to form a Cas13-sgRNA complex targeting the target RNA sequences to cleave the target RNA.

In some embodiments, the UBE3A-ATS transcript is associated with a disease or disorder, such as ALS (amyotrophic lateral sclerosis) .

In some embodiments, the vector genome comprises an ITR-to-ITR polynucleotide (such as SEQ ID NO: 33) comprising, from 5’ to 3’ :

(a) an optional 5’ ITR from AAV2 (such as SEQ ID NO: 31) ;

(b) a Syn1 promoter (such as SEQ ID NO: 25) ;

(c) a Kozak sequence (such as SEQ ID NO: 26) ;

(d) a first NLS coding sequence (such as one encoding SEQ ID NO: 27) ;

(e) 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;

(f) a second NLS coding sequence (such as one encoding SEQ ID NO: 27) ;

(g) an optional coding sequence encoding a 3xFlag sequence (e.g., SEQ ID NO: 28) ;

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

(i) a U6 promoter (such as SEQ ID NO: 30) ;

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

(k) a spacer coding sequence encoding a first spacer sequence specific for UBE3A-ATS transcript (such as SEQ ID NO: 4) ;

(l) a second DR DNA coding sequence encoding a second DR (such as SEQ ID NO: 3) ; and,

(m) an optional 3’ ITR from AAV2 (such as SEQ ID NO: 32) ;

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 to the ITR-to-ITR polynucleotide;

optionally, the ITR-to-ITR polynucleotide further comprises a linker sequence between any two adjacent sequence elements of (a) – (m) ;

optionally, the 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) ;

optionally, the 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; and

optionally, the 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.

Another aspect of the invention provides a recombinant AAV vector genome comprising, consisting essentially of, or consisting of:

(1) 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,

wherein the polynucleotide encodes

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

(b) a variant thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 46 and having a non-conserved substitution at Y672 and/or Y676 (e.g., Y672A and/or Y676A substitution (s) ) of SEQ ID NO: 46, 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: 46 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity of SEQ ID NO: 46; and,

(2) a sg RNA coding sequence encoding a sgRNA, the sgRNA comprises:

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

wherein the complex specifically cleaves the UBE3A-ATS transcript with substantially the same (e.g., at least about 80%, 90%, 95%, 99%or more) guide RNA-specific nuclease activity as SEQ ID NO: 46 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity of SEQ ID NO: 46,

at or near the target RNA sequence when the sgRNA guides the Cas13 polypeptide to the target RNA sequence; optionally wherein the sgRNA coding sequence is 3’ or 5’ to the Cas13 coding sequence.

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

Another aspect of the invention provides a recombinant lentiviral or AAV particle comprising the vector genome as described herein.

In some embodiments, 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.

In some embodiments, the capsid serotype is AAV. PHP. eB.

Another aspect of the invention provides a recombinant AAV particle comprising the vector genome as described herein, encapsidated in a capsid with a serotype of AAV. PHP. eB.

Another aspect of the invention provides a pharmaceutical composition comprising the vector genome as described herein, or the particle as described herein, and a pharmaceutically acceptable excipient.

Another aspect of the invention provides a method of treating a disease or disorder associated with UEB3A in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the vector genome as described herein, the particle as described herein, or the pharmaceutically composition as described herein, wherein the vector genome or the particle specifically down-regulate the expression of the UEB3A causative of the disease or disorder.

In some embodiments, the administrating comprises contacting a cell with the therapeutically effective amount of the vector genome as described herein, the particle as described herein, or the pharmaceutically composition as described herein.

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

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

In some embodiments, the administrating comprises intracerebroventricular administration.

In some embodiments, the subject is a human.

In some embodiments, the level of UEB3A-ATS transcript in the cell is decreased in comparison to a cell having not been contacted with the vector genome as described herein, the particle as described herein, or the pharmaceutically composition as described herein.

In some embodiments, the level of UBE3A-ATS transcript 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 transcript 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 invention provides a guide RNA (gRNA) (e.g., a single guide RNA) comprising:

(B) optionally, a direct repeat (DR) sequence capable of forming a complex with a Cas13 polypeptide, wherein the complex specifically cleaves the UBE3A-ATS transcript at or near the target RNA sequence when the sgRNA guides the Cas13 polypeptide to the target RNA sequence.

In some embodiments, the target RNA sequence comprises a stench of contiguous nucleotides of the RNA counterpart of NG_002690.1 or NC_000073.7; optionally 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 NOs: 16-21 and 70-92 or the RNA counterpart thereof.

In some embodiments, the gRNA comprises two or more identical or different spacer sequences, each flanked by two the DR sequence.

In some embodiments, the gRNA comprises two different spacer sequences (e.g., spacer 1 and spacer 2) separating three of the DR sequences (e.g., DR-spacer 1-DR-spacer 2-DR) .

In some embodiments, the gRNA comprises one or more spacer sequences each independently selected from any one of SEQ ID NOs: 10-15 and 47-69, or a variant thereof differing from any one of SEQ ID NOs: 10-15 and 47-69 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 sgRNA to form a Cas13-sgRNA complex targeting the respective target sequences to cleave the target sequences.

In some embodiments, 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: 46 and has a non-conserved substitution at Y672 and/or Y676 (e.g., Y672A and/or Y676A substitution (s) ) of SEQ ID NO: 46, 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: 46 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity of SEQ ID NO: 46.

Another aspect of the invention provides a cell or a progeny thereof, comprising the vector genome as described herein, the particle as described herein, or the gRNA as described herein.

Another aspect of the invention provides a kit comprising the vector genome as described herein, the particle as described herein, the gRNA as described herein, or the cell or a progeny thereof as described herein.

Another aspect of the invention provides a method of preparing the recombinant AAV particle as described herein, the method comprising:

a) introducing into an AAV packaging system a nucleic acid encoding the vector genome as described herein or the RNA sequence transcribed therefrom, and a coding sequence for the AAV capsid as described herein, for a period of time sufficient to package the vector genome or the transcribed RNA into the AAV capsid to produce the recombinant AAV particle, and

b) harvesting the recombinant AAV particle; and, optionally,

c) isolating or purifying the harvested recombinant AAV particle.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 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.

FIG. 2A is a schematic (not to scale) illustration of an exemplary lentivirus vector genome encoding hfCas13e. 1-sgRNA as well as the control. FIG. 2B is a schematic (not to scale) illustration of an exemplary AAV vector genome encoding hfCas13e. 1-sgRNA as well as the control.

FIG. 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) . The levels of Ube3a and Ube3a-ATS transcripts were detected by RT-qPCR. N = 3/group. *, P<0.05; ***, P<0.001. FIG. 3B shows the ex vivo reactivation of Ube3a in AS mouse primary neurons resulting from the knocking-down of Ube3a-ATS transcript by hfCas13e. 1-sg9-14 system (AS+sg9-14) compared to the control (WT+NT) . The expression of Ube3a protein was detected by Western Blot. Tissues for Western Blot were harvested at 4 weeks post dosing. N = 3/group. FIG. 3C shows statistic quantification of FIG. 3B. N = 3/group. *, P<0.05; ***, P<0.001.

FIG. 4A shows a schematic illustration of timeline of assays.

FIG. 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. The levels of Ube3a and Ube3a-ATS transcripts were detected by RT-qPCR. Tissues for RT-qPCR were harvested at 4 weeks post dosing. N = 3/group. *, P<0.05; ***, P<0.001.

FIG. 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. The levels of Ube3a and Ube3a-ATS transcripts were detected by RT-qPCR. Tissues for RT-qPCR were harvested at 4 weeks post dosing. N = 3/group. *, P<0.05; ***, P<0.001.

FIG. 4D 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 Blot. Tissues for Western Blot were harvested at 4 weeks post dosing. N = 3/group.

FIG. 4E shows statistic quantification of FIG. 4D. N = 3/group. *, P<0.05; ***, P<0.001.

FIG. 4F shows the expression of paternal Ube3a in the cortex and hippocampus of treated mice compared to control. The levels of Ube3a protein in the cortex and hippocampus were detected by Western Blot. Tissues for Western Blot were harvested at 18 weeks post dosing. N = 3/group.

FIG. 4G shows statistic quantification of FIG. 4F. N = 3/group. *, P<0.05; ***, P<0.001.

FIG. 5A is a schematic illustration of timeline of behavioral tests.

FIG. 5B shows body weight of female mice measured bi-weekly over 18 weeks.

FIG. 5C shows the results of hindlimb clasping test. N >8/group. *, P<0.05; ***, P<0.001.

FIG. 5D shows the results of open-field test. N >8/group. *, P<0.05; ***, P<0.001.

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

FIG. 5F-G shows the results of beam-walking test. N >8/group. *, P<0.05; ***, P<0.001.

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

DETAILED DESCRIPTION OF THE INVENTION

1. Overview

The invention described herein provides CRISPR-Cas13 systems and methods for treatment of UBE3A-assocaited diseases, such as, Angelman Syndrome (AS) .

Specifically, the invention described herein provides an CRISPR-Cas13 system for upregulating the expression of UBE3A gene by cleaving UBE3A-ATS transcript (e.g., UBE3A-ATS pre-mRNA) . Such a system can be delivered by lentiviral and AAV vectors and intracerebroventricular injection to subjects in need. Exemplary constructs of the invention have demonstrated efficacy to knockdown UBE3A-ATS transcript level and upregulate UBE3A protein levels, both ex vivo and in vivo, and improve the disease phenotype of the mouse disease models, thus opening the door for gene therapy to treat UBE3A-assocaited diseases, such as, AS.

2. Representative Cas13 effector enzymes

Wile-type or native Class 2 type VI enzymes or Cas13, while offering tremendous opportunity to knock down target gene products (e.g., mRNA) for gene therapy, their use is inherently limited by the co-called collateral cleavage activity that poses significant risk of cytotoxicity.

Specifically, in Class 2 type VI systems, a spacer (or guide RNA) sequence non-specific (independent) RNA cleavage, referred to as “ (off-target) collateral cleavage, ” is conferred by the higher eukaryotes and prokaryotes nucleotide-binding (HEPN) domain in Cas13 after target RNA binding. Binding of its cognate target ssRNA complementary to the bound gRNA causes substantial conformational changes in Cas13 effector enzyme, leading to the formation of a single, composite catalytic site for guide-sequence independent “collateral” RNA cleavage, thus converting Cas13 into a sequence non-specific ribonuclease. This newly formed highly accessible active site would not only degrade the target RNA in cis if the target RNA is sufficiently long to reach this new active site, but also degrade non-target RNAs in trans based on this promiscuous RNase activity (collateral cleavage activity) .

Most RNAs appear to be vulnerable to this promiscuous RNase activity of Cas13, and most (if not all) Cas13 effector enzymes possess this collateral cleavage activity. It has been shown recently that the collateral cleavage activitys by Cas13-mediated knockdown exist in mammalian cells and animals, suggesting that clinical application of Cas13-mediated target RNA knock down will face significant challenge in the presence of collateral cleavage activity.

Thus, to use Cas13 enzymes for specifically knocking down a target RNA in gene therapy, it is evident that this collateral cleavage activity must be tightly controlled to prevent unwanted spontaneous cellular toxicity.

Works have been done to provide engineered Cas13 (e.g., hfCas13e. 1, hfCas13f v1, hfCas13f v2, hfCas13f v3) proteins with decreased or eliminated undesired collateral cleavage activity while maintaining the desired on-target cleavage activity (or “cleavage activity” for short) . See PCT/CN2021/079821 and PCT/CN2021/121926, incorporated herein by reference in their entireties.

The invention described herein provides compositions and methods of use of engineered Cas13 (e.g., hfCas13e. 1, hfCas13f v1, hfCas13f v2, hfCas13f v3) proteins with designed gRNAs to treat UBE3A-associated diseases (e.g., AS) .

In some embodiments, the Cas 13 protein used herein:

(1) comprises a mutation in a region spatially close to an endonuclease catalytic domain (e.g., a HEPN domain) of the corresponding wild-type Cas13;

(2) substantially preserves (e.g., retains at least 50%, 60%, 70%, 72.5%, 75%, 80%, 85%, 87.5%, 90%, 95%, 96%, 97%, 97.5%, 98%, 99%or more of) cleavage activity of the wild-type Cas13 towards a target RNA complementary to the guide sequence; and,

(3) substantially lacks (e.g., retains less than 50%, 40%, 35%, 30%, 27.5%, 25%, 22.5%, 20%, 17.5%, 15%, 12.5%, 10%, 7.5%, 5%, 4%, 3%, 2.5%, 2%, 1%or less of) collateral cleavage activity of the wild-type Cas13 towards a non-target RNA that does not bind to the guide sequence.

In some embodiments, the Cas13 is a Cas13e, such as SEQ ID NO: 46, or the Cas13 is a Cas13f, such as SEQ ID NO: 93. In some embodiments, the wild-type Cas13 is a wild-type Cas13e, such as SEQ ID NO: 46, or the wild-type Cas13 is a wild-type Cas13f, such as SEQ ID NO: 93.

In some embodiments, the region includes residues within 130, 125, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acids from any residues of the endonuclease catalytic domain (e.g., an RXXXXH domain) in the primary sequence of the Cas13e; or the region includes residues within 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, or 10 amino acids from any residues of the endonuclease catalytic domain (e.g., an RXXXXH domain) in the primary sequence of the Cas13f.

In some embodiments, the region includes residues more than 100, 110, 120, or 130 residues away from any residues of the endonuclease catalytic domain in the primary sequence of the Cas13, but are spatially within 1-10 or 5

of a residue of the endonuclease catalytic domain.

In some embodiments, the endonuclease catalytic domain is a HEPN domain, optionally a HEPN domain comprising an RXXXXH motif.

In some embodiments, the RXXXXH motif comprises a R {N/H/K/Q/R} X ₁X ₂X ₃H sequence.

In some embodiments, in the R {N/H/K/Q/R} X ₁X ₂X ₃H sequence, X ₁ is R, S, D, E, Q, N, G, or Y; X ₂ is I, S, T, V, or L; and X3 is L, F, N, Y, V, I, S, D, E, or A.

In some embodiments, the RXXXXH motif is an N-terminal RXXXXH motif comprising an RNXXXH sequence, such as an RN {Y/F} {F/Y} SH sequence.

In some embodiments, the N-terminal RXXXXH motif has a RNYFSH sequence.

In some embodiments, the N-terminal RXXXXH motif has a RNFYSH sequence.

In some embodiments, the RXXXXH motif is a C-terminal RXXXXH motif comprising an R {N/A/R} {A/K/S/F} {A/L/F} {F/H/L} H sequence.

In some embodiments, the C-terminal RXXXXH motif has a RN (A/K) ALH sequence.

In some embodiments, the C-terminal RXXXXH motif has a RAFFHH or RRAFFH sequence.

In some embodiments, the region comprises, consists essentially of, or consists of (i) residues corresponding to residues between residues 1-194, 2-187, 227-242, 620-775, or 634-755 of SEQ ID NO: 46; or (ii) residues corresponding to the HEPN1 domain (e.g., residues 1-168) , Helical1 domain, Helical2 domain (e.g., residues 346-477) , and the HEPN2 domain (e.g., residues 644-790) of SEQ ID NO: 93.

In some embodiments, the mutation comprises, consists essentially of, or consists of substitutions, within a stretch of 15-20 consecutive amino acids within the region, (a) one or more charged, nitrogen- containing side chain group, bulky (such as F or Y) , aliphatic, and/or polar residues to a charge-neutral short chain aliphatic residue (such as A, V, or I) ; (b) one or more I/L to A substitution (s) ; and/or (c) one or more A to V substitution (s) .

In some embodiments, the stretch is about 16 or 17 residues.

In some embodiments, substantially all, except for up to 1, 2, or 3, charged and polar residues within the stretch are substituted.

In some embodiments, a total of about 7, 8, 9, or 10 charged and polar residues within the stretch are substituted.

In some embodiments, the N-and C-terminal 2 residues of the stretch are substituted to amino acids the coding sequences of which contain a restriction enzyme recognition sequence.

In some embodiments, the N-terminal two residues are VF, and the C-terminal 2 residues are ED, and the restriction enzyme is BpiI.

In some embodiments, the one or more charged or polar residues comprise N, Q, R, K, H, D, E, Y, S, and T residues.

In some embodiments, the one or more charged or polar residues comprise R, K, H, N, Y, and/or Q residues.

In some embodiments, one or more Y residue (s) within the stretch is substituted.

In some embodiments, the one or more Y residues (s) correspond to Y672 and/or Y676 of wild-type Cas13e. 1 (SEQ ID NO: 46) .

In some embodiments, the one or more Y residues (s) correspond to Y666 and/or Y677 of wild-type Cas13f. 1 (SEQ ID NO: 93) .

In some embodiments, the charge-neutral short chain aliphatic residue is Ala (A) .

In some embodiments, the mutation comprises, consists essentially of, or consists of:

(a) substitutions within 1, 2, 3, 4, or 5 of the stretches of 15-20 consecutive amino acids within the region;

(b) a mutation corresponds to a Cas13e mutation (e.g., that of Example 1, 2, or 5 of PCT/CN2021/121926) that retains at least about 75%of guide RNA-specific cleavage of wild-type Cas13e (such as SEQ ID NO: 46) , and exhibits less than about 25%collateral effect of wild-type Cas13e (such as SEQ ID NO: 46) ;

(c) a mutation corresponds to the M1V4, M2V2, M2V3, M2V4, M5V1, M6V2, M6V3, M6V4, M7V1, M7V2, M7V3, M7-Y55A, M7-Y61A, M11V1, M12V3, M15V1, M15V2, M15-Y643A, M15-Y647A, M16V1, M16V2, M17V2, M18V2, M18V3, M19V2, M19V3, or M19-IA mutation in PCT/CN2021/121926 of Cas13e mutation;

(d) a mutation corresponds to a Cas13e mutation (e.g., that of Example 5 of PCT/CN2021/121926) that retains between about 25-75%of guide RNA-specific cleavage of wild-type Cas13e (such as SEQ ID NO: 46) , and exhibits less than about 25%collateral effect of wild-type Cas13e (such as SEQ ID NO: 46) ;

(e) a mutation corresponds to the M17YY (hfCas13e. 1) , M8V4, M9V1, M11V2, M11V3, M13V1, M13V2, M13V3, M15V3, or M20V2 mutation of Cas13e mutation;

(f) a mutation corresponds to a Cas13f mutation (e.g., D160A, Q163A, D642A, L631A, P667A, H638A, T647A, D762A, L634A, L641A, V670A, A763V, T161A, R157A) that retains at least about 75%of guide RNA-specific cleavage of wild-type Cas13f (such as SEQ ID NO: 93) , and exhibits less than about 25 or 27.5%collateral effect of wild-type Cas13f (such as SEQ ID NO: 93) ;

(g) a mutation corresponds to the F10S6, F38S12, F38S11, F7V2, F10V1, F10V4, F40V2, F40V4, F44V2, F10S19, F10S21, F10S24, F10S26, F10S27, F10S33, F10S34, F10S35, F10S36, F10S45, F10S46, F10S48, F10S49, F40S22, F40S23, F40S26, F40S27, or F40S36 mutation of Cas13f mutation in Examples 12 and 13 of PCT/CN2021/121926, or a combination thereof; optionally, the Cas13f mutation comprises (A) a combination of any one, two, or more (e.g., 3, 4, or 5 more) mutations selected from a group consisting of D160A, Q163A, D642A, L631A, P667A, H638A, T647A, D762A, L634A, L641A, V670A, A763V, T161A, R157A (such as F10S6 (i.e., D160A) + F38S12 (i.e., D642A) ) with F40S23 (i.e., Y666A and Y677A) ; (B) any combination mutations selected from a group consisting of Cas13f v2 (i.e., F40S23, i.e., Y666A and Y677A) + L631A&H638A, Cas13f v2 + L631A&L641A, Cas13f v2 + L631A&D642A, Cas13f v2 + D160A&L631A, Cas13f v2 + H638A&L641A, Cas13f v2 + H638A&D642A, Cas13f v2 +L641A&D642A, and Cas13f v2 + D160A&D642A; (C) a Y666A/Y677A double mutation in combination with 1, 2, or 3 additional mutations selected from D160A, L641A, and D642A; or (D) a Y666A/Y677A/D160A/D642A mutation.

(h) a mutation corresponds to a Cas13f mutation (e.g., that of Example 12 of PCT/CN2021/121926) that retains between about 50-75%of guide RNA-specific cleavage of wild-type Cas13f (such as SEQ ID NO: 93) , and exhibits less than about 25 or 27.5%collateral effect of wild-type Cas13f (such as SEQ ID NO: 93) ; and/or

(i) a mutation corresponds to the F2V4, F3V1, F3V3, F3V4, F5V2, F5V3, F6V4, F7V1, F38V4, F40V1, F41V1, F41V3, F42V4, F43V1, F10S2, F10S11, F10S12, F10S18, F10S20, F10S23, F10S25, F10S28, F10S43, F10S44, F10S47, F10S50, F10S51, F10S52, F40S7, F40S9, F40S11, F40S21, F40S22, F40S24, F40S28, F40S29, F40S30, F40S35, or F40S37 or mutation of Cas13f mutation.

In some embodiments, the Cas13 preserves at least about 50%, 60%, 70%, 72.5%, 75%, 80%, 85%, 87.5%, 90%, 95%, 96%, 97%, 97.5%, 98%, 99%or more of the guide sequence-specific endonuclease cleavage activity of the wild-type Cas13 towards the target RNA.

In some embodiments, the Cas13 lacks at least about 70%, 72.5%, 75%, 77.5%, 80%, 82.5%, 85%, 87.5%, 90%, 92.5%, 95%, 96%, 97%, 98%, 99%, or 100%of the guide sequence-independent collateral endonuclease cleavage activity of the wild-type Cas13 towards the non-target RNA.

In some embodiments, the Cas13 preserves at least about 80-90%of the guide sequence-specific endonuclease cleavage activity of the wild-type Cas13 towards the target RNA, and lacks at least about 95-100%of the guide sequence-independent collateral endonuclease cleavage activity of the wild-type Cas13 towards the non-target RNA.

In some embodiments, the Cas13 protein further comprises a nuclear localization signal (NLS) sequence or a nuclear export signal (NES) .

In some embodiments, the Cas13 protein comprises an N-and/or a C-terminal NLS.

In some embodiments, the Cas13 protein comprises, consists essentially of, or consisting of SEQ ID NO: 1.

In some embodiments, the Cas13 protein used herein comprises a Cas13 polypeptide, wherein the Cas13 coding sequence comprises:

(a) the amino acid sequence of SEQ ID NO: 1 (hfCas13e. 1) , or

(b) a variant thereof that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 46 (Cas13e. 1 in US. App. No. 16/864, 982) and has a non-conserved substitution at Y672 and/or Y676 (e.g., Y672A and/or Y676A substitution (s) ) of SEQ ID NO: 46, 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: 46 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity (collateral cleavage activity) of SEQ ID NO: 46.

3. Polynucleotides and Vector Genomes

One aspect of the invention provides a recombinant lentiviral or adeno-associate virus vector genome, comprising (1) a Cas13 coding sequence encoding a Cas13 polypeptide of the invention (which substantially lacks collateral nuclease activity, but substantially retains cleavage activity of the original Cas13 protein from which such Cas13 polypeptide derives) ; and (2) a gRNA or a gRNA coding sequence encoding the gRNA, which targets a target gene transcript (such as, an UBE3A-ATS transcript) , wherein the gRNA comprises a spacer sequence substantially complementary to a target RNA sequence on a target RNA and a direct repeat (DR) sequence capable of forming a complex with the Cas13 polypeptide.

More specifically, one aspect of the invention provides a recombinant lentiviral or adeno-associated virus (AAV) vector genome, comprising:

(1) a Cas13 coding sequence encoding a Cas13 polypeptide,

(ii) wherein the Cas13 polypeptide comprises

(a) the amino acid sequence of SEQ ID NO: 1, or

Inverted Terminal Repeat (ITR) sequences are important for initiation of viral DNA replication and circularization of adeno-associated virus genomes. Within the ITR sequences, secondary structures (e.g., stems and loops formed by palindromic sequences) are important one or more ITR functions in viral replication and/or packaging. Such sequence elements include the RBE sequence (Rep binding element) , RBE’ sequence, and the trs (terminal resolution sequence) .

In certain embodiments, the rAAV vector genome comprises a 5’ AAV ITR sequence and/or a 3’ AAV ITR sequence.

In certain embodiments, the 5’ and/or 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.

In certain embodiments, the 5’ and the 3’ AAV ITR sequences are both wild-type AAV ITR sequences from AAV2.

In certain embodiments, the 5’ and/or 3’ ITR sequences are modified ITR sequences. For example, the most 5’ end or the most 3’ end of the wild-type ITR sequences (e.g., AAV5, 8, 9, PHP. eB, or DJ ITR sequences) may be deleted. The deletion can be up to 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 nucleotide.

In certain embodiments, up to 15 (such as exactly 15) nucleotides of the most 5’ end nucleotides, and/or up to 15 (such as exactly 15) nucleotides of the most 3’ end nucleotides, of the wild-type AAV2 ITR sequences may be deleted.

Thus the 5’ and/or 3’ modified ITR (s) may comprising up to 144, 143, 142, 141, 140, 139, 138, 137, 136, 135, 134, 133, 132, 131, 130, 129, 128, or 127-nt (such as 130 nucleotides) of the 145-nt wild-type AAV ITR sequences.

In certain embodiments, the modified ITR sequences comprise the RBE sequence, the RBE’s equence, and/or the trs of the wt ITR sequence.

In certain embodiments, the modified ITR sequences comprise both the RBE sequence and the RBE’ sequence.

In certain embodiments, the modified ITR sequences confer stability of the plasmids of the invention comprising the AAV vector genome (see below) in bacteria, such as stability during plasmid production.

In certain embodiments, the modified ITRs do not interfere with sequencing verification of the plasmids of the invention comprising the AAV vector genome.

In certain embodiments, the modified 5’ ITR sequence comprises a 5’ heterologous sequence that is not part of wild-type AAV 5’ ITR sequence. In certain embodiments, the modified 3’ ITR sequence comprises a 3’ heterologous sequence that is not part of wild-type AAV 3’ ITR sequence.

In certain embodiments, the modified 5’ ITR sequence comprises a 5’ heterologous sequence that is not part of wild-type AAV (e.g., wt AAV2) 5’ ITR sequence, and the modified 3’ ITR sequence comprises a 3’ heterologous sequence that is not part of wild-type AAV (e.g., wt AAV2) 3’ ITR sequence, wherein the 5’ heterologous sequence and the 3’ heterologous sequence are complementary to each other.

In certain embodiments, the 5’ heterologous sequence and the 3’ heterologous sequence each comprises a type II restriction endonuclease recognition sequence, such as recognition sequence for Sse8387I (CCTGCAGG) , or recognition sequence for PacI (TTAATTAA) .

In certain embodiments, the 5’ ITR comprises up to 141 nts of the most 3’ nucleotides of the 145-nt wt AAV2 5’ ITR (e.g., a deletion of 4 or more most 5’ end of the 145-nt wt AAV2 5’ ITR) .

In certain embodiments, the 5’ ITR comprises up to 130 nts of the most 3’ nucleotides of the 145-nt wt AAV2 5’ ITR (e.g., a deletion of 15 or more most 5’ end of the 145-nt wt AAV2 5’ ITR) .

In certain embodiments, the 3’ ITR comprises up to 141 nts of the most 5’ nucleotides of the 145-nt wt AAV2 3’ ITR (e.g., a deletion of 4 or more most 3’ end of the 145-nt wt AAV2 3’ ITR) .

In certain embodiments, the 3’ ITR comprises up to 130 nts of the most 5’ nucleotides of the 145-nt wt AAV2 3’ ITR (e.g., a deletion of 15 or more most 3’ end of the 145-nt wt AAV2 3’ ITR) .

In certain embodiments, the 5’ and 3’ ITR sequences are compatible for AAV production in mammalian-cell based on triple transfection.

In certain embodiments, the 5’ and 3’ ITR sequences are compatible for AAV production in insect cell (e.g., Sf9) based on baculovirus vector (see below) .

In certain embodiments, the 5’ and 3’ ITR sequences are compatible for AAV production in mammalian-cell based on HSV vectors (see below) .

In certain embodiments, the 5’ AAV ITR sequence comprises, consists essentially of, or consists of SEQ ID NO: 31.

In certain embodiments, the 3’ AAV ITR sequence comprises, consists essentially of, or consists of SEQ ID NO: 32.

In some embodiments, the Cas13 polynucleotide described herein is operably linked to a regulatory element (e.g., a promoter) in order to control the expression of the Cas13 polypeptide. In some embodiments, the promoter is ubiquitous. In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, the promoter is a cell-specific promoter. In some embodiments, the promoter is an organism-specific promoter, e.g., tissue-specific promoter.

Suitable promoters are known in the art and 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, retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a β-actin promoter, an elongation factor 1α short (EFS) promoter, a β glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken β-actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1α-subunit (EF1α) promoter, a ubiquitin C (UBC) promoter, a prion promoter, a neuron-specific enolase (NSE) , a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a platelet-derived growth factor (PDGF) promoter, a platelet-derived growth factor B-chain (PDGF-β) promoter, a synapsin (Syn) promoter, a synapsin 1 (Syn1) promoter, a methyl-CpG binding protein 2 (MeCP2) promoter, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) promoter, a metabotropic glutamate receptor 2 (mGluR2) promoter, a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a β-globin minigene nβ2 promoter, a preproenkephalin (PPE) promoter, an enkephalin (Enk) promoter, an excitatory amino acid transporter 2 (EAAT2) promoter, a glial fibrillary acidic protein (GFAP) promoter, a myelin basic protein (MBP) promoter. For example, a U6 promoter can be used to regulate the expression of a gRNA molecule described herein. In some embodiments, the elongation factor 1α short (EFS) promoter can be used to regulate the expression of Cas13 proteins described herein.

In certain embodiments, the promoter is a Syn1 promoter, such as a Syn1 promoter comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 25.

In certain embodiments, the rAAV vector genome of the invention further comprises a coding sequence for a nuclear localization sequence (NLS) fused N-terminal, C-terminal, and/or internally to the Cas13 polypeptide, and/or a coding sequence for a nuclear export signal (NES) fused N-terminal, C-terminal, and/or internally to the Cas13 polypeptide.

In certain embodiments, the rAAV vector genome of the invention comprises a first NLS coding sequence 5’ to the Cas13 polynucleotide, and/or a second NLS coding sequence 3’ to the Cas13 polynucleotide (e.g., comprising both the first and the second NLS coding sequences) .

In certain embodiments, the NLS, the first NLS, and the second NLS comprises, consists essentially of, or consists of SEQ ID NO: 27.

In certain embodiments, the rAAV vector genome of the invention further comprises a Kozak sequence or a functional variant thereof. In certain embodiments, the Kozak sequence is SEQ ID NO: 26; or a sequence comprising at most 1, 2, 3, or 4 nucleotide differences from SEQ ID NO: 26 other than the ATG start codon, if present, within the Kozak sequence, wherein the last three nucleotide is optionally ACC or GCC.

In certain embodiments, the rAAV vector genome of the invention further comprises a polyadenylation (polyA) signal sequence. In certain embodiments, the polyA signal sequence is selected from the group consisting of 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) , or a variant thereof. In certain embodiments, the polyA signal sequence is SV40 polyA signal sequence or a functional variant thereof (such as SEQ ID NO: 29) .

In certain embodiments, the expression cassette for transcribing a gRNA targeting the target gene transcript (e.g., UBE3A-ATS transcript) comprises an RNA pol III promoter, wherein the second transcription unit is 3’ to the Cas13 polynucleotide.

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

In certain embodiments, the gRNA coding sequence encodes a gRNA comprising one or more (e.g., 2 or 3) spacer sequences each substantially complementary to a target RNA sequence of a target RNA (e.g., UBE3A-ATS transcript) , and capable of directing the Cas13 polypeptide herein to cleave the target RNA. More detailed description for multiple spacer sequences and associated DR sequences are provided in a separate section below (incorporated herein by reference) .

In certain embodiments, the rAAV vector genome of the invention comprises an ITR-to-ITR polynucleotide (such as SEQ ID NO: 33) comprising, from 5’ to 3’ :

(a) an optional 5’ ITR from AAV2 (such as SEQ ID NO: 31) ;

(b) a Syn1 promoter (such as SEQ ID NO: 25) ;

(c) a Kozak sequence (such as SEQ ID NO: 26) ;

(d) a first NLS coding sequence (such as one encoding SEQ ID NO: 27) ;

(f) a second NLS coding sequence (such as one encoding SEQ ID NO: 27) ;

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

(i) a U6 promoter (such as SEQ ID NO: 30) ;

(m) an optional 3’ ITR from AAV2 (such as SEQ ID NO: 32) ;

In certain embodiments, the recombinant AAV (rAAV) vector genome comprises, consists essentially of, or consists of:

wherein the polynucleotide encodes

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

(2) a coding sequence for a single guide RNA (sgRNA) , the sgRNA comprises:

wherein the complex specifically cleaves the UBE3A-ATS transcript with substantially the same (e.g., at least about 80%, 90%, 95%, 99%or more) guide RNA-specific nuclease activity as SEQ ID NO: 46 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity of SEQ ID NO: 46, at or near the target RNA sequence when the sgRNA guides the Cas13 polypeptide to the target RNA sequence.

In certain embodiments, the rAAV vector genome is SEQ ID NO: 33, or the polynucleotide at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical thereto. In certain embodiments, the rAAV vector genome is SEQ ID NO: 33.

In some embodiments, the rAAV vector genome is present in a vector (e.g., a viral vector or a phage, such as an HSV vector, a baculovirus vector, or an AAV vector) . The vector can be a cloning vector, or an expression vector. The vectors can be plasmids, phagemids, Cosmids, etc. The vectors may include one or more regulatory elements that allow for the propagation of the vector in a cell of interest (e.g., a bacterial cell, insect cell, or a mammalian cell) . In some embodiments, the vector includes a nucleic acid encoding a single component of the CRISPR-Cas13 system described herein. In some embodiments, the vector includes multiple nucleic acids, each encoding a component of the CRISPR-Cas13 system described herein.

In one aspect, the present disclosure provides nucleic acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the nucleic acid sequences described herein, e.g., nucleic acid sequences (such as ITR-to-ITR sequences, for example, SEQ ID NO: 33) encoding the Cas13 protein and the gRNA as described herein.

In certain embodiments, the Cas13 polynucleotide sequence of the invention encodes amino acid sequences that are at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%identical to the amino acid sequences of the Cas13 protein herein (e.g., SEQ ID NO: 1) .

In some embodiments, the nucleic acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is the same as the sequences described herein. In some embodiments, the nucleic acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 nucleotides, e.g., contiguous or non-contiguous nucleotides) that is different from the sequences described herein.

In related embodiments, the invention provides amino acid sequences having at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is the same as the sequences described herein. In some embodiments, the amino acid sequences have at least a portion (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues, e.g., contiguous or non-contiguous amino acid residues) that is different from the sequences described herein.

To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes) . In general, the length of a reference sequence aligned for comparison purposes should be at least 80%of the length of the reference sequence, and in some embodiments is at least 90%, 95%, or 100%of the length of the reference sequence. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences. For purposes of the present disclosure, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The Cas13 proteins described herein can be delivered or used as either nucleic acid molecules or polypeptides.

In certain embodiments, the nucleic acid molecule encoding the Cas13 protein, derivatives or functional fragments thereof are codon-optimized for expression in a host cell or organism. The host cell may include established cell lines (such as HeLa, 293, or 293T cells) or isolated primary cells. The nucleic acid can be codon optimized for use in any organism of interest, in particular human cells or bacteria. For example, the nucleic acid can be codon-optimized for any prokaryotes (such as E. coli) , or any eukaryotes such as human and other non-human eukaryotes including yeast, worm, insect, plants and algae (including food crop, rice, corn, vegetables, fruits, trees, grasses) , vertebrate, fish, non-human mammal (e.g., mice, rats, rabbits, dogs, birds (such as chicken) , livestock (cow or cattle, pig, horse, sheep, goat etc. ) , or non-human primates) . Codon usage tables are readily available, for example, at the “Codon Usage Database” available at www. kazusa. orjp/codon/, and these tables can be adapted in a number of ways. See Nakamura et al., Nucl. Acids Res. 28: 292, 2000 (incorporated herein by reference in its entirety) . Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, Pa. ) .

An example of a codon optimized sequence is in this instance a sequence optimized for expression in a eukaryote, e.g., humans (i.e. being optimized for expression in humans) , or for another eukaryote, animal or mammal as herein discussed; see, e.g., SaCas9 human codon optimized sequence in WO 2014/093622 (PCT/US2013/074667) . Whilst this is preferred, it will be appreciated that other examples are possible and codon optimization for a host species other than human, or for codon optimization for specific organs is known. In general, codon optimization refers to a process of modifying a nucleic acid sequence for enhanced expression in the host cells of interest by replacing at least one codon (e.g. about or more than about 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more codons) of the native sequence with codons that are more frequently or most frequently used in the genes of that host cell while maintaining the native amino acid sequence. Various species exhibit particular bias for certain codons of a particular amino acid. Codon bias (differences in codon usage between organisms) often correlates with the efficiency of translation of messenger RNA (mRNA) , which is in turn believed to be dependent on, among other things, the properties of the codons being translated and the availability of particular transfer RNA (tRNA) molecules. The predominance of selected tRNAs in a cell is generally a reflection of the codons used most frequently in peptide synthesis. Accordingly, genes can be tailored for optimal gene expression in a given organism based on codon optimization. Codon usage tables are readily available, for example, at the “Codon Usage Database” available at http: //www. kazusa. orjp/codon/and these tables can be adapted in a number of ways. See Nakamura, Y., et al. “Codon usage tabulated from the international DNA sequence databases: status for the year 2000” Nucl. Acids Res. 28: 292 (2000) . Computer algorithms for codon optimizing a particular sequence for expression in a particular host cell are also available, such as Gene Forge (Aptagen; Jacobus, PA) , are also available. In some embodiments, one or more codons (e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons) in a sequence encoding a Cas correspond to the most frequently used codon for a particular amino acid.

4. Guide RNA

In some embodiments, the CRISPR systems described herein include at least a gRNA. Such gRNA may be encoded by the same AAV vector genome encoding the Cas13 polypeptide. As used herein, gRNA, sgRNA, and CRISPR RNA (crRNA) are exchangeable.

Sequences for gRNAs from multiple CRISPR systems are generally known in the art, see, for example, Grissa et al. (Nucleic Acids Res. 35 (web server issue) : W52-7, 2007; Grissa et al., BMC Bioinformatics 8: 172, 2007; Grissa et al., Nucleic Acids Res. 36 (web server issue) : W145-8, 2008; and Moller and Liang, PeerJ 5: e3788, 2017; the CRISPR database at: crispr. i2bc. paris-saclayfr/crispr/BLAST/CRISPRsBlast. php; and MetaCRAST available at: github. com/molleraj/MetaCRAST) . All incorporated herein by reference.

In some embodiments, the gRNA includes a spacer (Spacer) sequence substantially complementary to a target RNA sequence on a target RNA (such as, a UBE3A-ATS transcript) and a direct repeat (DR) sequence capable of forming a complex with a Cas13 polypeptide as described herein, wherein the complex specifically cleaves the target RNA (such as, a UBE3A-ATS transcript) at or near the target RNA sequence when the sgRNA guides the Cas13 polypeptide to the target RNA sequence. The spacer sequence can recognize, bind, and/or hybridize to the target sequence via base-pairing due to the substantial complementarity between the spacer sequence and the target RNA sequence. The DR sequence is capable of forming a complex with a Cas13 polypeptide as described herein, and the complex specifically cleaves the target RNA (such as, a UBE3A-ATS transcript) at or near the target RNA sequence when the Cas13 polypeptide is guided to the target RNA sequence by the spacer sequence.

In some embodiments, the sgRNA comprises, consists essentially of, or consists of a direct repeat sequence linked to a spacer sequence, preferably at the 3’ -end of the spacer sequence.

In some embodiments, the sgRNA comprises, consists essentially of, or consists of one spacer sequence directly linked to one DR sequence, like DR-Spacer or Spacer-DR.

In some embodiments, the sgRNA comprises, consists essentially of, or consists of one spacer sequence indirectly linked to one DR sequence, like DR-Spacer-DR.

Two or more same or different spacer sequences that target the same or different target sequence of the same or different target RNAs may be used in combiantions. In some embodiments, the sgRNA comprises, consists essentially of, or consists of two or more spacer sequences. In some further embodiments, each of the two or more spacer sequences is flanked by two DR sequences, and optionally, with one DR sequence shared. For example, in the case that each of two spacer sequecnes is flanked by two DR sequences with one DR sequence of the two being shared, that would be like DR-Spacer-DR-Spacer-DR, where the DR in the middle is shared by two sgRNA of DR-Spacer-DR in tandem. Such a multiple Spacer structure as a larger single gRNA can also be considered and termed as a sgRNA array. Each of those spacer sequences can be independently substantially complementary to a distinct target RNA sequence on a target RNA (e.g, UBE3A-ATS transcript) , and each can be capable of directing a Cas13 polypeptide as described herein to cleave respective distinct target RNA sequence. Such a larger single gRNA comprising multiple spacer sequences and DR sequences can be considered as either one sgRNA as a whole or two or more sgRNAs in tandem each containing one spacer sequence for each and one or more DR sequences that, if applicable, are shared by two sgRNAs in tandem.

A cleavable or non-cleavable linker, such as an enzymatic restriction site, may be introduced between a DR sequence and a spacer sequence as needed.

In general, a Cas13 protein herein, forms a complex with a mature gRNA of DR-Spacer or Spacer-DR, which may be resulting from RNA processing of the gRNA array by the Cas13 protein, and the spacer sequence directs the complex to a sequence-specific binding with the target RNA that is substantially complementary to the spacer sequence, and/or hybridizes to the spacer sequence. The resulting complex comprises the Cas13 protein, and the mature crRNA bound to the target RNA.

The direct repeat sequences for the Cas13 systems are generally well conserved, especially at the ends, with, for example, a GCUG for Cas13e and GCUGU for Cas13 at the 5’ -end, reverse complementary to a CAGC for Cas13e and ACAGC for Cas13 at the 3’ end. This conservation suggests strong base pairing for an RNA stem-loop structure that potentially interacts with the protein (s) in the locus.

In some embodiments, the direct repeat sequence, when in RNA, comprises the general secondary structure of 5’ -S1a-Ba-S2a-L-S2b-Bb-S1b-3’ , wherein segments S1a and S1b are reverse complement sequences and form a first stem (S1) having 4 nucleotides in Cas13e and 5 nucleotides in Cas13; segments Ba and Bb do not base pair with each other and form a symmetrical or nearly symmetrical bulge (B) , and have 5 nucleotides each in Cas13e, and 5 (Ba) and 4 (Bb) or 6 (Ba) and 5 (Bb) nucleotides respectively in Cas13; segments S2a and S2b are reverse complement sequences and form a second stem (S2) having 5 base pairs in Cas13e and either 6 or 5 base pairs in Cas13; and L is an 8-nucleotide loop in Cas13e and a 5-nucleotide loop in Cas13.

In certain embodiments, S1a has a sequence of GCUG in Cas13e and GCUGU in Cas13.

In certain embodiments, S2a has a sequence of GCCCC in Cas13e and A/G CCUC G/Ain Cas13 (wherein the first A or G may be absent) .

In some embodiments, the direct repeat sequence comprises, consists essentially of, or consists of a nucleic acid sequence of SEQ ID NO: 3.

As used herein, “direct repeat sequence” may refer to either the direct repeat RNA sequence or the direct repeat DNA sequence encoding the direct repeat RNA sequence. Thus, when any direct repeate DNA sequence is referred to in the context of an RNA molecule, such as sgRNA, each T of the coding sequence is understood to represent a U. The same applies to the spacer sequence as well.

In some embodiments, the direct repeat sequence comprises, consists essentially of, or consists of a nucleic acid sequence having up to 1, 2, 3, 4, 5, 6, 7, or 8 nucleotides of deletion, insertion, or substitution of SEQ ID NO: 3. In some embodiments, the direct repeat sequence comprises, consists essentially of, or consists of a nucleic acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%of sequence identity with SEQ ID NO: 3 (e.g., due to deletion, insertion, or substitution of nucleotides in SEQ ID NO: 3) . In some embodiments, the direct repeat sequence comprises, consists essentially of, or consists of a nucleic acid sequence that is not identical to SEQ ID NO: 3 but can hybridize with a complement of SEQ ID NO: 3 under stringent hybridization conditions or can bind to a complement of SEQ ID NO: 3 under physiological conditions.

In certain embodiments, the deletion, insertion, or substitution does not change the overall secondary structure of that of SEQ ID NO: 3 (e.g., the relative locations and/or sizes of the stems and bulges and loop do not significantly deviate from that of the original stems, bulges, and loop) . For example, the deletion, insert, or substitution may be in the bulge or loop region so that the overall symmetry of the bulge remains largely the same. The deletion, insertion, or substitution may be in the stems 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 the two stems correspond to 4 total base changes) .

In certain embodiments, the deletion, insertion, or substitution results in a derivative DR sequence that may have ± 1 or 2 base pair (s) in one or both stems, have ± 1, 2, or 3 bases in either or both of the single strands in the bulge, and/or have ± 1, 2, 3, or 4 bases in the loop region.

In certain embodiments, any of the above direct repeat sequences that is different from SEQ ID NO: 3 retains the ability to function as a direct repeat sequence in the Cas13 proteins, as the DR sequence of SEQ ID NO:3.

In some embodiments, the direct repeat sequence comprises, consists essentially of, or consists of a nucleic acid having a nucleic acid sequence of SEQ ID NO: 3, with a truncation of the initial one, two, three, four, five, six, seven, or eight 3’ nucleotides.

In classic CRISPR systems, the degree of complementarity between a spacer sequence and its corresponding target sequence can be about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or 100%. In some embodiments, the degree of complementarity is 90-100%.

By “substantially complementary” as used herein, it means that the the degree of complementarity between a spacer sequence and its corresponding target sequence can be about 90-100%, such as, at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. The degree of complementarity can be calculated by 100%minus mismatches between spacer sequence /target sequence over their full length. For example, if a spacer sequence of 30 nt and a target sequence of 30 nt have two mismatches in the middle, their degree of complementarity is 100%-2 /30 = 93.3%. It is believed that at least 2 mismatches between the spacer sequence and the target RNA sequence can be tolerated for the Cas13 proteins herein without significantly decreasing cleavage efficiency.

In some embodiments, the spacer sequence is independently selected from any one of SEQ ID NOs: 10-15 and 47-69, or a variant thereof differing from any one of SEQ ID NOs: 10-15 and 47-69 by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides of deletion, insertion, or substitution without substantially diminishing the ability to direct the Cas13 polypeptide as described herein to bind to the sgRNA to form a Cas13-sgRNA complex targeting the respective target sequences to cleave the target sequences.

The gRNAs can be about 5, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 100, 125, 150, 175, 200 or more nucleotides in length. For example, for use in a Cas13 protein, the spacer can be between 10-60 nucleotides, 20-50 nucleotides, 25-45 nucleotides, 25-35 nucleotides, or about 27, 28, 29, 30, 31, 32, or 33 nucleotides.

To reduce off-target interactions, e.g., to reduce the interacting of a gRNA with a target sequence having low complementarity thereto, mutations can be introduced to the CRISPR systems so that the CRISPR systems can distinguish between target (or on-target) and off-target sequences that have greater than 80%, 85%, 90%, or 95%complementarity. In some embodiments, the degree of complementarity is from 80%to 95%, e.g., about 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, or 95% (for example, distinguishing between a target having 18 nucleotides from an off-target of 18 nucleotides having 1, 2, or 3 mismatches) . Accordingly, in some embodiments, the degree of complementarity between a spacer sequence and its corresponding target sequence is greater than 94.5%, 95%, 95.5%, 96%, 96.5%, 97%, 97.5%, 98%, 98.5%, 99%, 99.5%, or 99.9%. In some embodiments, the degree of complementarity is 100%.

Type VI CRISPR-Cas proteins have been demonstrated to employ more than one RNA guide, thus enabling the ability of these proteins, and systems and complexes that include them, to target multiple nucleic acids. In some embodiments, the CRISPR systems comprising the Cas13 protein, as described herein, include multiple RNA guides (e.g., two, three, four, five, six, seven, eight, nine, ten, fifteen, twenty, thirty, forty, or more RNA guides) . In some embodiments, the CRISPR systems described herein can include a single RNA strand or a nucleic acid encoding a single RNA strand, wherein the RNA guides are arranged in tandem. The single RNA strand can include multiple copies of the same RNA guide, multiple copies of distinct RNA guides, or combinations thereof. The multipe RNA guides may present as a larger single gRNA (asgRNA array, such as, DR-Spacer-DR-Spacer-DR) or separate sgRNAs. The processing capability of the Cas13 proteins described herein enables these proteins to be able to target multiple target RNAs (e.g., target mRNAs) without a loss of activity. In some embodiments, the Cas13 proteins may be delivered in complex with multiple RNA guides directed to different target RNAs. In some embodiments, the Cas13 protein, may be co-delivered with multiple RNA guides, each specific for a different target RNA. Methods of multiplexing using CRISPR-associated proteins are described, for example, in U.S. Pat. No. 9,790,490 B2, and EP 3009511 B1, the entire contents of each of which are expressly incorporated herein by reference.

In some embodiments, the spacer length of the gRNA herein can range from about 10-50 nucleotides, such as 15-50 nucleotides, 20-50 nucleotides, 25-50 nucleotide, or 19-50 nucleotides. In some embodiments, the spacer length is at least 16 nucleotides, at least 17 nucleotides, at least 18 nucleotides, at least 19 nucleotides, at least 20 nucleotides, at least 21 nucleotides, or at least 22 nucleotides. In some embodiments, the spacer length is from 15 to 17 nucleotides (e.g., 15, 16, or 17 nucleotides) , from 17 to 20 nucleotides (e.g., 17, 18, 19, or 20 nucleotides) , from 20 to 24 nucleotides (e.g., 20, 21, 22, 23, or 24 nucleotides) , from 23 to 25 nucleotides (e.g., 23, 24, or 25 nucleotides) , from 24 to 27 nucleotides, from 27 to 30 nucleotides, from 30 to 45 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, or 45 nucleotides) , from 30 or 35 to 40 nucleotides, from 41 to 45 nucleotides, from 45 to 50 nucleotides (e.g., 45, 46, 47, 48, 49, or 50 nucleotides) , or longer. In some embodiments, the spacer length is from about 15 to about 42 nucleotides. In an embodiment, the spacer length is 30 nucleotides.

In some embodiments, the direct repeat length of the gRNA herein is 15-36 nucleotides, is at least 16 nucleotides, is from 16 to 20 nucleotides (e.g., 16, 17, 18, 19, or 20 nucleotides) , is from 20-30 nucleotides (e.g., 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 nucleotides) , is from 30-40 nucleotides (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 nucleotides) , or is about 36 nucleotides (e.g., 33, 34, 35, 36, 37, 38, or 39 nucleotides) . In an embodiment, the direct repeat length is 36 nucleotides.

In some embodiments, the overall length of the gRNA is longer than any one of the spacer sequence lengths described herein in the length of one, two, three, or more DR sequences described herein. In some embodiments, the overall length of the gRNA is about 36, 72, 108, or more nucleotides longer than any one of the spacer sequence lengths described herein. For example, the overall length of the gRNA may be between 45-86 nucleotides, or 60-86 nucleotides, 62-86 nucleotides, or 63-86 nucleotides.

The gRNA sequences can be modified in a manner that allows for formation of a complex between the gRNA and the Cas13 protein herein and successful binding to the target, while at the same time not allowing for successful nuclease activity (i.e., without nuclease activity/without causing indels) . These modified gRNA sequences are referred to as “dead crRNAs, ” “dead gRNAs, ” or “dead spacer sequences. ” These dead guides or dead spacer sequences may be catalytically inactive or conformationally inactive with regard to nuclease activity. Dead spacer sequences are typically shorter than respective spacer sequences that result in active RNA cleavage. In some embodiments, dead gRNAs are 5%, 10%, 20%, 30%, 40%, or 50%, shorter than respective gRNAs that have nuclease activity. Dead spacer sequences of gRNAs can be from 13 to 15 nucleotides in length (e.g., 13, 14, or 15 nucleotides in length) , from 15 to 19 nucleotides in length, or from 17 to 18 nucleotides in length (e.g., 17 nucleotides in length) .

In some embodiments, the gRNA comprises any one of SEQ ID NO: 4-9.

Thus, in one aspect, the disclosure provides non-naturally occurring or engineered CRISPR systems including a Cas13 protein as described herein, and a gRNA, wherein the gRNA comprises a dead gRNA sequence whereby the gRNA is capable of hybridizing to a target sequence such that the CRISPR system is directed to a target RNA of interest in a cell without detectable nuclease activity (e.g., RNase activity) .

A detailed description of dead gRNAs is described, e.g., in International Publication No. WO 2016/094872, which is incorporated herein by reference in its entirety.

gRNAs can be generated as components of inducible systems. The inducible nature of the systems allows for spatio-temporal control of gene editing or gene expression. In some embodiments, the stimuli for the inducible systems include, e.g., electromagnetic radiation, sound energy, chemical energy, and/or thermal energy.

In some embodiments, the transcription of gRNA can be modulated by a promoter that is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter.

In some embodiments, 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 β-actin promoter, an elongation factor 1α short (EFS) promoter, a βglucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken β-actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1α-subunit (EF1α) promoter, a ubiquitin C (UBC) promoter, a prion promoter, a neuron-specific enolase (NSE) , a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a platelet-derived growth factor (PDGF) promoter, a platelet-derived growth factor B-chain (PDGF-β) promoter, a synapsin (Syn) promoter, a synapsin 1 (Syn1) promoter, a methyl-CpG binding protein 2 (MeCP2) promoter, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) promoter, a metabotropic glutamate receptor 2 (mGluR2) promoter, a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a β-globin minigene nβ2 promoter, a preproenkephalin (PPE) promoter, an enkephalin (Enk) promoter, an excitatory amino acid transporter 2 (EAAT2) promoter, a glial fibrillary acidic protein (GFAP) promoter, and a myelin basic protein (MBP) promoter; optionally wherein the promoter is an RNA pol III promoter.

In some embodiments, the transcription of gRNA can be modulated by inducible promoters, e.g., tetracycline or doxycycline controlled transcriptional activation (Tet-On and Tet-Off expression systems) , hormone inducible gene expression systems (e.g., ecdysone inducible gene expression systems) , and arabinose-inducible gene expression systems. Other examples of inducible systems include, e.g., small molecule two-hybrid transcription activations systems (FKBP, ABA, etc. ) , light inducible systems (Phytochrome, LOV domains, or cryptochrome) , or Light Inducible Transcriptional Effector (LITE) . These inducible systems are described, e.g., in WO 2016205764 and U.S. Pat. No. 8,795,965, both of which are incorporated herein by reference in the entirety.

The sequences and the lengths of the gRNAs described herein can be optimized. In some embodiments, the optimized length of an gRNA can be determined by identifying the processed form of crRNA (i.e., a mature crRNA) , or by empirical length studies for crRNA tetraloops.

The gRNAs can also include one or more aptamer sequences. Aptamers are oligonucleotide or peptide molecules have a specific three-dimensional structure and can bind to a specific target molecule. The aptamers can be specific to gene effectors, gene activators, or gene repressors. In some embodiments, the aptamers can be specific to a protein, which in turn is specific to and recruits and/or binds to specific gene effectors, gene activators, or gene repressors. The effectors, activators, or repressors can be present in the form of fusion proteins. In some embodiments, the gRNA has two or more aptamer sequences that are specific to the same adaptor proteins. In some embodiments, the two or more aptamer sequences are specific to different adaptor proteins. The adaptor proteins can include, e.g., MS2, PP7, Qβ, F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φkCb5, φkCb8r, φkCb12r, φkCb23r, 7s, and PRR1. Accordingly, in some embodiments, the aptamer is selected from binding proteins specifically binding any one of the adaptor proteins as described herein. In some embodiments, the aptamer sequence is a MS2 binding loop (5’ -ggcccAACAUGAGGAUCACCCAUGUCUGCAGgggcc-3’ . In some embodiments, the aptamer sequence is a QBeta binding loop (5’ -ggcccAUGCUGUCUAAGACAGCAUgggcc-3’) . In some embodiments, the aptamer sequence is a PP7 binding loop (5’ -ggcccUAAGGGUUUAUAUGGAAACCCUUAgggcc-3’ ) . A detailed description of aptamers can be found, e.g., in Nowak et al., “Guide RNA engineering for versatile Cas9 functionality, ” Nucl. Acid. Res., 44 (20) : 9555-9564, 2016; and WO 2016205764, which are incorporated herein by reference in their entirety.

The invention also encompasses methods for delivering multiple nucleic acid components, wherein each nucleic acid component is specific for a different target locus of interest thereby modifying multiple target loci of interest (for example, two different gRNA each targeting a different target sequence within the same UBE3A-ATS transcript may be employed in the construct of the invention) . The nucleic acid component of the complex may comprise one or more protein-binding RNA aptamers. The one or more aptamers may be capable of binding a bacteriophage coat protein. The bacteriophage coat protein may be selected from the group comprising Qβ, F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, φCb5, φCb8r, φCb12r, φCb23r, 7s and PRR1. In certain embodiments, the bacteriophage coat protein is MS2.

5. Target RNA and Target Sequence

The target RNA can be any RNA molecule of interest, including naturally-occurring and engineered RNA molecules. The target RNA can be an mRNA, a tRNA, a ribosomal RNA (rRNA) , a microRNA (miRNA) , an interfering RNA (siRNA) , a ribozyme, a riboswitch, a satellite RNA, a microswitch, a microzyme, or a viral RNA. In some embodiments, the target sequence is a part of the target RNA, for example, the target sequence is a stretch of 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, such as, 30, contiguous nucleotides of the target RNA.

In some embodiments, the target RNA is associated with a condition or disease.

In certain embodiments, the target RNA is an UBE3A-ATS transcript, including UBE3A-ATS pre-mRNA, such as human UBE3A-ATS pre-mRNA or mouse UBE3A-ATS pre-mRNA, for example, the RNA counterpart of human UBE3A-ATS genome coding sequence, Accession No: NG_002690.1 incorporated herein by reference, the RNA counterpart 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. In some embodiments, the target sequence is a part of the RNA counterpart of human and/or mouse UBE3A-ATS genome coding sequence, for example, the target sequence is 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, contiguous nucleotides of the RNA counterpart of human and/or mouse UBE3A-ATS genome coding sequence. It would be preferred if such a stretch can be found on both the RNA counterparts of human and mouse UBE3A-ATS genome coding sequence, which means that such a target sequence and therefore the sgRNA designed therefrom are cross-reactive to both human and mouse, thereby facilitating both research on mouse models and clinical treatment on human subjects.

In certain embodiments, the target sequence (1) is selected from SEQ ID NO: 16-21 and 70-92; or (2) differs from any one of SEQ ID NO: 16-21 and 70-92 by 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 more or less nucleotides 5’ or 3’ adjacent to any one of SEQ ID NO: 16-21 and 70-92 over the RNA counterpart of human and/or mouse UBE3A-ATS genome coding sequence.

Thus, in some embodiments, the systems described herein can be used to treat a condition or disease associated with the RNA (such as AS) by targeting the RNA (e.g., UBE3A-ATS transcript) . For instance, the target RNA associated with a condition or disease may be an RNA molecule that is overexpressed in a diseased cell (e.g., VEGFA mRNA overexpressed in a disease cell in wet AMD patient) . The target RNA may also be a toxic RNA and/or a mutated RNA (e.g., an mRNA molecule having a splicing defect or a mutation, such as, a UBE3A-ATS transcript) .

6. Complex and Cell

One aspect of the invention provides a complex of a Cas13 protein, such as CRISPR-Cas13 mutant complex, comprising (1) any of the Cas13 protein (e.g., a Cas13 mutants, homologs, orthologs, fusions, derivative, conjugates, or functional fragments thereof as described herein) , and (2) any of the gRNA described herein, each including a spacer sequence designed to be at least partially complementary to a target RNA, and a DR sequence compatible with the Cas13 protein (e.g., a Cas13 mutant, homologs, orthologs, fusions, derivatives, conjugates, or functional fragments thereof) .

In certain embodiments, the complex further comprises the target RNA (such as a UBE3A-ATS transcript) bound by the gRNA.

In an aspect of the invention, it is provided a cell or a progeny thereof, comprising the vector genome as described herein, the viral particle as described herein, or the sgRNA as described herein. In a related aspect, the invention also provides a cell comprising any of the complex of the invention. In certain embodiments, the cell is a prokaryote. In certain embodiments, the cell is a eukaryote, such as, a mouse, monkey, or human cell.

7. Therapeutic Applications

The CRISPR systems described herein can have various therapeutic applications. Such applications may be based on one or more of the abilities below, both in vitro and in vivo, of the subject Cas13, e.g., CRISPR-Cas13 systems.

In some embodiments, the CRISPR-Cas13 systems can be used to treat various diseases and disorders associated with RNA, for example, with overexpression of RNA or expression of abnormal RNA.

In some embodiments, the RNA is UBE3A-ATS transcript.

In some embodiments, the disease or disorder associated with the RNA is UBE3A-assocaited diseases.

In some embodiments, the UBE3A-assocaited disease is Amyotrophic lateral sclerosis (AS) .

In an aspect of the inventin, it is provided a pharmaceutical composition comprising the vector genome as described herein, or the viral particle as described herein, and a pharmaceutically acceptable excipient.

In an aspect of the inventin, it is provided 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 as described herein, the viral particle as described herein, or the pharmaceutically composition as described herein, wherein the rAAV vector genome or the rAAV viral particle specifically down-regulate the expression of the UBE3A causative of the disease or disorder.

In some embodiments, the administrating comprises contacting a cell with the therapeutically effective amount of the vector genome as described herein, the viral particle as described herein, or the pharmaceutically composition as described herein.

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

In some embodiments, the disease or disorder is AS.

In some embodiments, the administration comprises intrathecal administration.

In some embodiments, the subject is a human.

In some embodiments, the subject is not a human.

In some embodiments, the level of UEB3A-ATS transcript in the cell is decreased in comparison to a cell having not been contacted with the vector genome as described herein, the viral particle as described herein, or the pharmaceutically composition as described herein.

In certain embodiments, the methods of the invention can be used to introduce the CRISPR systems described herein into a cell and cause the cell and/or its progeny to alter the production of one or more cellular products, such as growth factor, antibody, starch, ethanol, or any other desired products. Such cells and progenies thereof are within the scope of the invention.

In certain embodiments, the methods and/or the CRISPR systems described herein lead to modification of the translation and/or transcription of one or more RNA products of the cells. For example, the modification may lead to increased transcription /translation /expression of the RNA product. In other embodiments, the modification may lead to decreased transcription /translation /expression of the RNA product.

In certain embodiments, the cell is a eukaryotic cell, such as a mammalian cell, including a human cell (a primary human cell or an established human cell line) . In certain embodiments, the cell is a non-human mammalian cell, such as a cell from a non-human primate (e.g., monkey) , a cow /bull /cattle, sheep, goat, pig, horse, dog, cat, rodent (such as rabbit, mouse, rat, hamster, etc) . In certain embodiments, the cell is from fish (such as salmon) , bird (such as poultry bird, including chick, duck, goose) , reptile, shellfish (e.g., oyster, claim, lobster, shrimp) , insect, worm, yeast, etc.

A related aspect provides cells or progenies thereof modified by the methods of the invention using the CRISPR systems described herein.

In certain embodiments, the cell is modified in vitro, in vivo, or ex vivo. In certain embodiments, the cell is a stem cell. In certain embodiments, the cell is not an embryonic stem cell.

8. Delivery

Through this disclosure and the knowledge in the art, the CRISPR systems described herein comprising a Cas13 protein (such as the Cas13 mutants herein) , or any of the components thereof described herein (Cas13 proteins, derivatives, functional fragments or the various fusions or adducts thereof, and gRNA) , nucleic acid molecules thereof, and/or nucleic acid molecules encoding or providing components thereof, can be delivered by various delivery systems such as vectors, e.g., plasmids and viral delivery vectors, using any suitable means in the art. Such methods include (and are not limited to) electroporation, lipofection, microinjection, transfection, sonication, gene gun, etc.

In an aspect of the invention, the CRISPR-Cas13 system can be delivered in the form of a ribonucleoprotein (RNP) complex comprising the gRNA and the Cas13 protein, optionally along with a donor DNA template or a vector encoding the donor DNA template.

In an aspect of the invention, the CRISPR-Cas13 system can be delivered in the form of one or more vectors comprising one or more polynucleotides encoding the gRNA and the Cas13 protein, optionally along with a donor DNA template or a vector encoding the donor DNA template, optionally wherein the one or more vectors are one or more viral vectors, optionally wherein the viral vector is a retroviral vector, a Herpes Simplex virus vector, an adenovirus vector, an adeno-associated virus (AAV) vector, or a lentiviral vector.

In an aspect of the invention, the CRISPR-Cas13 system can be delivered in the form of a mixture of the gRNA and an mRNA encoding the Cas13 protein, optionally along with a donor DNA template or a vector encoding the donor DNA template, optionally wherein the mixture is delivered as a lipid nanoparticle.

In certain embodiments, the CRISPR-associated proteins and/or any of the RNAs (e.g., gRNAs) and/or accessory proteins can be delivered using suitable vectors, e.g., plasmids or viral vectors, such as adeno-associated viruses (AAV) , lentiviruses, adenoviruses, retroviral vectors, and other viral vectors, or combinations thereof. The proteins and one or more gRNAs can be packaged into one or more vectors, e.g., plasmids or viral vectors. For bacterial applications, the nucleic acids encoding any of the components of the CRISPR systems described herein can be delivered to the bacteria using a phage. Exemplary phages, include, but are not limited to, T4 phage, Mu, λ phage, T5 phage, T7 phage, T3 phage, Φ29, M13, MS2, Qβ, and ΦX174.

In certain embodiments, the delivery is through AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, or AAV PHP. eB serotype viral vectors, a member of the Clade to which any of the AAV1-AAV13 belong, or a functional truncated variant or a functional mutant thereof (e.g, sharing significant sequence homology and spectrum of tropism as AAV5, 8, 9, PHP. eB, or DJ) . In an embodiment, the serotype is a AAV. PHP. eB 9 mutant.

In some embodiments, the vectors, e.g., plasmids or viral vectors (e.g., AAV viral vectors) , are delivered to the cell, tissue, or organ of interest by, e.g., intrathecal administration, intramuscular administration, intravenous administration, transdermal administration, intranasal administration, oral administration, mucosal administration, intraperitoneal administration, intracranial administration, intracerebroventricular administration, or stereotaxic administration. In certain embodiments, the administration is conducted by injection.

In certain embodiments, the AAV viral particle of the invention is delivered through intrathecal injection. In certain embodiments, the delivery is by one intrathecal injection. For example, under adequate anesthesia, an intrathecal injection of a therapeutically effective amount of the vector genomes (vg) of the invention in a suitable total volume is performed, using standard techniques for intrathecal surgery. In certain embodiments, the subject is given a short-term corticosteroid regimen of oral prednisone (or the equivalent) , before and/or after the intrathecal injection in need to treatment. A suitable volume for administration herein may be about 0.01 ml to about 20 ml (such as, about 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, 11, 11.5, 12, 12.5, 13, 13.5, 14, 14.5, 15, 15.5, 16, 16.5, 17, 17.5, 18, 18.5, 19, 19.5, or 20 ml, or within a range of any two of those point values) for nervous system administration and about 10ml to about 100ml (such as, about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 ml, or within a range of any two of those point values) for systemic (e.g., intravenous) administration.

Delivery may be either via a single dose, or multiple doses. One skilled in the art understands that the actual dosage to be delivered herein 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. A therapeutically effective dose of the rAAV vectors for use herein may be suitably about 1E+8 vg to about 1E+17 vg, where vg stands for vector genomes of rAAV vectors for administration.

For example, a therapeutically effective dose of the rAAV vectors for use herein 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, 6.0E+14, 8.0E+14, 1.0E+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 within a range of any two of the those point values.

In certain embodiments, the delivery is via adenoviruses, which can be at a single dose containing at least 1×10 ⁵ particles (also referred to as particle units, pu) of adenoviruses. In some embodiments, the dose preferably is at least about 1×10 ⁶ particles, at least about 1×10 ⁷ particles, at least about 1×10 ⁸ particles, and at least about 1×10 ⁹ particles of the adenoviruses. The delivery methods and the doses are described, e.g., in WO 2016205764 A1 and U.S. Pat. No. 8,454,972 B2, both of which are incorporated herein by reference in the entirety.

In some embodiments, the delivery is via plasmids. The dosage can be a sufficient number of plasmids to elicit a response. In some cases, suitable quantities of plasmid DNA in plasmid compositions can be from about 0.1 to about 2 mg. Plasmids will generally include (i) a promoter; (ii) a sequence encoding a nucleic acid-targeting CRISPR-associated proteins and/or an accessory protein, each operably linked to a promoter (e.g., the same promoter or a different promoter) ; (iii) optionally a selectable marker; (iv) an origin of replication; and (v) a transcription terminator downstream of and operably linked to (ii) . The plasmids can also encode the RNA components of a CRISPR complex, but one or more of these may instead be encoded on different vectors. The frequency of administration is within the ambit of the medical or veterinary practitioner (e.g., physician, veterinarian) , or a person skilled in the art.

In another embodiment, the delivery is via liposomes or lipofection formulations and the like, and can be prepared by methods known to those skilled in the art. Such methods are described, for example, in WO 2016205764 and U.S. Pat. Nos. 5,593,972; 5,589,466; and 5,580,859; each of which is incorporated herein by reference in its entirety.

In some embodiments, the delivery is via nanoparticles or exosomes. For example, exosomes have been shown to be particularly useful in delivery RNA.

Further means of introducing one or more components of the new CRISPR systems to the cell is by using cell penetrating peptides (CPP) . In some embodiments, a cell penetrating peptide is linked to the CRISPR-associated proteins. In some embodiments, the CRISPR-associated proteins and/or gRNAs are coupled to one or more CPPs to effectively transport them inside cells (e.g., plant protoplasts) . In some embodiments, the CRISPR-associated proteins and/or gRNA (s) are encoded by one or more circular or non-circular DNA molecules that are coupled to one or more CPPs for cell delivery.

CPPs are short peptides of fewer than 35 amino acids derived either from proteins or from chimeric sequences capable of transporting biomolecules across cell membrane in a receptor independent manner. CPPs can be cationic peptides, peptides having hydrophobic sequences, amphipathic peptides, peptides having proline-rich and anti-microbial sequences, and chimeric or bipartite peptides. Examples of CPPs include, e.g., Tat (which is a nuclear transcriptional activator protein required for viral replication by HIV type 1) , penetratin, Kaposi fibroblast growth factor (FGF) signal peptide sequence, integrin β3 signal peptide sequence, polyarginine peptide Args sequence, Guanine rich-molecular transporters, and sweet arrow peptide. CPPs and methods of using them are described, e.g., in

et al., “Prediction of cell-penetrating peptides, ” Methods Mol. Biol., 2015; 1324: 39-58; Ramakrishna et al., “Gene disruption by cell-penetrating peptide-mediated delivery of Cas9 protein and gRNA, ” Genome Res., 2014 June; 24 (6) : 1020-7; and WO 2016205764 A1; each of which is incorporated herein by reference in its entirety.

Various delivery methods for the CRISPR systems described herein are also described, e.g., in U.S. Pat. No. 8,795,965, EP 3009511, WO 2016205764, and WO 2017070605; each of which is incorporated herein by reference in its entirety.

In some embodiments, a Cas protein is delivered in the form of a rAAV particle packaging a Cas-encoding mRNA by means of a AAV packaging system capable of packaging an RNA as described in, for example, PCT/CN2022/075366.

9. Kits

Another aspect of the invention provides a kit, comprising any two or more components of the subject CRISPR/Cas system described herein comprising a Cas13 protein, such as the Cas13 mutants herein, derivatives, functional fragments or the various fusions or adducts thereof, gRNA, complexes thereof, vectors encompassing the same, or host encompassing the same.

In another aspect of the invention, it is provided herein a kit comprising the vector genome as described herein, the viral particle as described herein, the sgRNA as described herein, or the cell or a progeny thereof as described herein.

In certain embodiments, the kit comprises an rAAV vector genome or an rAAV particle described herein comprising a polynucleotide comprising a Cas13 coding sequence, as well as coding sequence for one or more sgRNA targeting UBE3A separated by DR sequences.

In certain embodiments, the kit further comprises an instruction to use the components encompassed therein, and/or instructions for combining with additional components that may be available elsewhere.

In certain embodiments, the kit further comprises one or more buffers that may be used to dissolve any of the components, and/or to provide suitable reaction conditions for one or more of the components. Such buffers may include one or more of PBS, HEPES, Tris, MOPS, Na ₂CO ₃, NaHCO ₃, NaB, or combinations thereof. In certain embodiments, the reaction condition includes a proper pH, such as a basic pH. In certain embodiments, the pH is between 7-10.

In certain embodiments, any one or more of the kit components may be stored in a suitable container.

10. Host Cells and AAV Production

In another aspect of the invention, it is provided a method of preparing the rAAV particle as described herein, the method comprising:

a) introducing into an AAV packaging system a nucleic acid encoding the vector genome as described herein or the RNA sequence transcribed therefrom, and a coding sequence for the AAV capsid as described herein, for a period of time sufficient to package the rAAV vector genome or the transcribed RNA into the AAV capsid to produce the rAAV particle, and

b) harvesting the rAAV particle; and, optionally,

c) isolating or purifying the harvested rAAV particle.

General principles of rAAV production are known in the art. See review in, for example, Carter (Current Opinions in Biotechnology, 1533-539, 1992) ; and Muzyczka, Curr. Topics in Microbial, and Immunol 158: 97-129, 1992, both incorporated herein by reference) . Various approaches are described in Ratschin et al (Mol. Cell. Biol. 4: 2072, 1984; Hermonat et al. (Proc. Natl. Acad. Sci. USA 81: 6466, 1984) ; Tratschin et al. (Mol. Cell. Biol. 5: 3251, 1985) ; McLaughlin et al. (J. Virol 62: 1963, 1988) ; and Lebkowski et al. (Mol. Cell. Biol 7: 349, 1988) , Samulski et al. (J. Virol 63: 3822-3828, 1989) ; U.S. 5,173,414; WO 95/13365 and U.S. 5,658,776; WO 95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441; WO 97/08298; WO 97/21825; WO 97/06243; WO 99/11764; Perrin et al. (Vaccine 13: 1244-1250, 1995; Paul et al. (Human Gene Therapy 4: 609-615, 1993) ; Clark et al. (Gene Therapy 3: 1124-1132, 1996; U.S. 5,786,211; U.S. 5,871,982; and U.S. 6,258,595.

AAV, when enginerred 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” . And the genome packaged in AAV vectors for delivery may be termed as a AAV vector genome, vector genome, or vg for short, while viral genome may refer to the original viral genome of natural AAVs.

AAV vector serotypes can be matched to target cell types. For example, Table 2 of WO2018002719A1 lists exemplary cell types that can be transduced by the indicated AAV serotypes (incorporated herein by reference) .

Packaging cell lines are used to form AAV vectors that can infect host cells. Such packaging cell lines include HEK293 and Sf9 cells.

AAV vectors used in gene therapy are usually generated by packaging cell lines that package vector genomes into AAV capsids to form AAV vectors. A vector gemome typically contains minimal viral sequences required for packaging, while the other viral sequences are replaced by an expression cassette encoding, e.g., a Cas protein and/or a sgRNA. The missing viral functions can be supplied in trans by the packaging cell lines, usually as a result of expression of these viral functions /proteins (such as the rep and cap genes for AAV) either as transgenes integrated into the packaging cells, or as transgenes on a second viral vector or expression vector introduced into the packaging cells.

For example, vector genomes typically only possess inverted terminal repeat (ITR) sequences from viral genomes, which are required for packaging. Vector genomes are packaged in a packaging cell line, which contains one or more helper plasmids encoding the other AAV genes, namely rep and cap, but lacking ITR sequences. The packaging cell line is also infected with adenovirus as a helper. The helper virus promotes replication of the vector genomes and expression of AAV genes from the helper plasmid. The helper plasmids are not packaged in significant amounts due to the lack of ITR sequences. Contamination with adenovirus can be reduced by, e.g., heat treatment to which adenovirus is more sensitive than AAV.

In some embodiments, recombinant AAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650) . Typically, the recombinant AAVs are produced by transfecting host cells with vector genomes (comprising a gene of interest) to be packaged into AAV particles in form of a transgene plasmid, an AAV helper function vector (also known as a packaging plasmid) , and an accessory function vector (also known as a helper plasmid) . An AAV helper function vector encodes the “AAV helper function” sequences (e.g., rep and cap) , which function in trans for productive AAV replication and encapsidation. Preferably, the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes) . The accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (e.g., “accessory functions” ) . The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1) , and vaccinia virus.

In some embodiments, the subject rAAV viral particle is produced using a baculovirus expression system packaged in insect cells such as Sf9 cells. See, for example, WO2007046703, WO2007148971, WO2009014445, WO2009104964, WO2013036118, WO2011112089, WO2016083560, WO2015137802, and WO2019016349, all incorporated herein by reference.

A simple introduction of AAV for delivery may also refer to “Adeno-associated Virus (AAV) Guide” (https: //www. addgene. org/guides/aav/) .

The vector titers are usually expressed as vector genomes per ml (vg/ml) . In certain embodiments, 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.

Instead of packaging a single strand (ss) DNA sequence as a vector genome of a AAV particle, systems and methods of packaging an RNA sequence as a vector genome into a AAV particle is recently developed and applicable herein. See PCT/CN2022/075366, which is incorporated herein by reference in its entirety.

When the vector genome is RNA as in, for example, PCT/CN2022/075366, for simplicity of description and claiming, 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 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.

As used herein, a coding sequence, e.g., as a sequence element of AAV vector genomes herein, is construed, understood, and considered as covering and covers both a DNA coding sequence and an RNA coding sequence. When it is a DNA 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. When it is an RNA coding sequence, the RNA coding sequence per se can be an RNA sequence for use (although it seems that the RNA coding sequence does not encode something) , or an RNA sequence can be produced from the RNA coding sequence, e.g., by RNA processing (although it seems that the RNA coding sequence does not encode something) , or a protein can be translated from the RNA coding sequence.

For example, a (e.g., Cas13, NLS) coding sequence (encoding a (e.g., Cas13, NLS) polypeptide) covers either a (e.g., Cas13, NLS) DNA coding sequence from which a (e.g., Cas13, NLS) polypeptide is expressed (indirectly via transcription and translation) or a (e.g., Cas13, NLS) RNA coding sequence from which a (e.g., Cas13, NLS) polypeptide is translated (directly) .

For example, a (e.g., sgRNA) coding sequence (encoding an RNA (e.g., a sgRNA) sequence) covers either a (e.g., sgRNA) DNA coding sequence from which an RNA sequence (e.g., a sgRNA sequence or array) is transcribed or a (e.g., sgRNA) RNA coding sequence (1) which per se is the RNA sequence (e.g., a sgRNA sequence or array) for use, or (2) from which a sgRNA sequence or array is produced, e.g., by RNA processing.

In some embodiments for RNA AAV vector geomes, 5’ -ITR and/or 3’ -ITR as DNA packaging signals would be unnecessary and can be omitted, while RNA packaging signals can be introduced.

In some embodiments for AAV RNA vector geomes, promoters to drive transcription of DNA sequences would be unnecessary and can be omitted at least partly.

In some embodiments for AAV RNA vector geomes, polyA signal sequence would be unnecessary and can be omitted, while a polyA tail can be introduced.

Similary, other DNA elements of AAV DNA vector genomes can be either omitted or replaced with corresponding RNA elements and/or new RNA elements can be introduced, in order to adapt to the strategy of delivering an RNA vector genome by rAAV particles.

EXAMPLES

Example 1 High Efficiency ex vivo Knockdown of Ube3a-ATS transcript by CRISPR-hfCas13e. 1 system

This example demonstrates the high ex vivo knockdown efficiency of Ube3a-ATS transcript by the subject CRISPR-hfCas13e. 1 system via lentiviral delivery.

Lentiviral Transgene Plasmid Construction:

The subject CRISPR-hfCas13e. 1 system comprised a hfCas13e. 1 protein (SEQ ID NO: 1) and a Ube3a-ATS-targeting sgRNA (one of “sgRNA [Ube3a-ATS] 9-14” or “sg9-14” hereinafter, SEQ ID NO: 4-9) comprising two Direct Repeats (SEQ ID NO: 3) and a Spacer (one of Spacer 9-14, SEQ ID NO: 10-15) targeting the target sequence (one of target sequences 9-14, SEQ ID NO: 16-21) of Ube3a-ATS transcript.

As a negative control, a non-targeting-sgRNA ( “sgRNA [NT] ” or “NT” hereinafter, SEQ ID NO: 22) comprising two Direct Repeats (SEQ ID NO: 3) and a non-targeting-Spacer (LacZ, SEQ ID NO: 23) 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 the same transgene plasmid as shown in FIG. 2A for the production of treatment or control lentiviral particles.

AS mouse model:

All 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 HUIGENE THERAPEUTICS CO., LTD.

Recombinant Lentivirus Particles Preparation:

Treatment or control lentiviral transgene plasmids were constructed as shown in FIG. 2A, each of which was co-transfected with package plasmids and envelope plasmids into HEK293 cells to produce recombinant lentiviral particles delivering the hfCas13e. 1 system. After two days, the HEK293 cell supernatant was filtered by 0.22 μm sterile Millex filter, and then collected for ultracentrifugation (27000rpm, CP90NX, Hitachi) , the lentiviral particles at the bottom of the tube after ultracentrifucation was resuspended in PBS buffer, and the titer of lentiviral vectors was determined by RT-qPCR with the following primers. The copy numbers of GAG sequence were measured and normalized to the copy numbers of a housekeeping gene, TET1.

GAG RT-qPCR primers:

Forward: 5’-GGAGCTAGAACGATTCGCAGTTA-3’ (SEQ ID NO: 34) ;

Reverse: 5’-GGTGTAGCTGTCCCAGTATTTGTC-3’ (SEQ ID NO: 35) .

TET1 RT-qPCR primers:

Forward: 5’-TGGACCTACCTGAAGTATGT-3’ (SEQ ID NO: 36) ;

Reverse: 5’-GGCTGCTATGGAGTTAATGA-3’ (SEQ ID NO: 37) .

RT-qPCR Detection:

Total RNA was extracted from AS or C57BL/6 WT primary neurons after lentivirus infection using Trizol (Ambion) and then reversely transcribed into complementary DNA (HiScript Q RT SuperMix for qPCR, Vazyme, Biotech) for RT-qPCR detection. RT-qPCR reactions were tracked by SYBR green probe (AceQ qPCR SYBR Green Master Mix, Vazyme, Biotech) . The transcription levels of Ube3a-ATS trancript and Ube3a mRNA were measured and normalized to the mRNA level of a housekeeping gene, Gapdh.

Ube3a-ATS RT-qPCR primers:

Forward: 5’-CCAATGACTCATGATTGTCCTG-3’ (SEQ ID NO: 38) ,

Reverse: 5’-GTGAGGCCTTCAACAATCTC-3’ (SEQ ID NO: 39) .

Ube3a RT-qPCR primers:

Forward: 5’-CAAAAGGTGCATCTAACAACTCA-3’ (SEQ ID NO: 40) ,

Reverse: 5’-GGGGAATAATCCTCACTCTCTC-3’ (SEQ ID NO: 41) .

Gapdh RT-qPCR primers:

Forward: 5’-CTCCCACTCTTCCACCTTCG-3’ (SEQ ID NO: 42) ,

Reverse: 5’-TAGGGCCTCTCTTGCTCAGT-3’ (SEQ ID NO: 43) .

Neuronal culture and lentivirus infection:

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℃, and then plated on PDL (Cat. No. P6407, Sigma-Aldrich) in neuronal medium (Cat. No. 21103049, Thermo Fisher Scientific) containing 2 mM 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/6 WT primary neurons from the digested tissues of AS or C57BL/6 WT mouse models were maintained ex vivo in the same medium without FBS. On day 2 after maintaining the AS and WT primary neurons (DIV2) , the primary neurons were transfected with the lentiviral particles for 24 hours with MOI (multiplicity of infection) =10 and then half-changing the medium was carried out for three consecutive days. The subsequent experiments were performed 7-9 days later after lentivirus infection.

Western blotting:

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 μg 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. F1804, Sigma) , anti-α tubulin (1: 5000; Cat. No. AF0001, Beyotime) . Following the primary antibody incubation, the membrane was probed with goat anti-mouse HPR (1: 5000; Cat. No. A9044, Sigma-Aldrich) or goat anti-rabbit HRP (1: 5000; Cat. No. A0545, Sigma) secondary antibody. Images were collected using AmershamImager600 (GE) and analyzed by ImageJ software.

Results:

RT-qPCR results show that the transfection of a lentiviral vector encoding both a hfCas13e. 1 protein and a sgRNA [Ube3a-ATS] (sg9, sg10, sg11 sg12, sg13, or sg14) into AS primary neurons ex vivo resulted in 85.1%, 69.2%, 67.4%, 77.8%, 83.1%, and 81.3% (Mean, n = 3 repeats) reduction of Ube3a-ATS transcript level, respectively, compared to the Ube3a-ATS transcript level in AS primary neurons transfected with hfCas13e. 1-sg [NT] , and achieved 109.2%, 127.6%, 3.9%, 15.9%, 93.6%, and 107.3%of Ube3a mRNA level (Mean, n = 3 repeats) , respectively, compared to the mRNA level of maternal Ube3a in C57BL/6 WT primary neurons transfected with hfCas13e. 1-NT (FIG. 3A) .

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

Example 2 High Efficiency In vivo Knockdown of Ube3a-ATS transcript by CRISPR-hfCas13e. 1 system

To demonstrate the in vivo knockdown efficiency of the subject CRISPR-hfCas13e. 1 system in Example 1, AAV. PHP. eB delivery system was used to deliver the system in vivo to target cells in animals.

AAV. PHP. eB Transgene Plasmid Contruction:

Similar to the treatment and control transgene plasmids in Example 1, 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 FIG. 2B.

Recombinant AAV. PHP. eB Particles Preparation:

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 capids constituted the AAV. PHP. eB particles.

Specifically, 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 titer of any genomes packaged in the treatment and control AAV. PHP. eB particles.

5’-ITR forward primer: 5’ -GGAACCCCTAGTGATGGAGTT-3’ (SEQ ID NO: 44) ;

5’-ITR reverse primer: 5’ -CGGCCTCAGTGAGCGA-3’ (SEQ ID NO: 45) .

AS mouse model and Intracerebroventricular injection

All 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. eB particles into bilateral ventricles at four sites. Total 5 × 10 ¹⁰ AAV particles (encoding hSyn1 promoter-hfCas13e. 1-sg9/NT in FIG. 2B) plus 2.5 × 10 ⁹ reporter AAV particles (encoding CAG promoter-tdTomato reporter) in 2 μl 0.9%NaCl was injected into each AS 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. 1-sg [Ube3a-ATS] 9. Untreated AS mice (AS+NT) : 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] .

RT-qPCR Detection:

4 weeks after the injection, 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-ATS transcripts were detected with RT-qPCR by Taqman probe (Bestar qPCR master mix, DBI-2041, DBI) and normalized to the mRNA level of a housekeeping gene, GAPDH.

Ube3a-ATS qPCR primers:

Forward: 5’-CCAATGACTCATGATTGTCCTG-3’ (SEQ ID NO: 38) ,

Reverse: 5’-GTGAGGCCTTCAACAATCTC-3’ (SEQ ID NO: 39) .

Ube3a qPCR primers:

Forward: 5’-CAAAAGGTGCATCTAACAACTCA-3’ (SEQ ID NO: 40) ,

Reverse: 5’-GGGGAATAATCCTCACTCTCTC-3’ (SEQ ID NO: 41) .

Gapdh qPCR primers:

Forward: 5’-CTCCCACTCTTCCACCTTCG-3’ (SEQ ID NO: 42) ,

Reverse: 5’-TAGGGCCTCTCTTGCTCAGT-3’ (SEQ ID NO: 43) .

Western blotting:

Tdtomato positive brain regions were dissected and homogenized in SDS lysis buffer (Beyotime) containing 1x Protease inhibitor cocktail (Beyotime) . 40-60 μg 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. AF0001, Beyotime) . Following the primary antibody incubation, the membrane was probed with goat anti-mouse HPR (1: 5000; Cat. No. A9044, Sigma-Aldrich) or goat anti-rabbit HRP (1: 5000; Cat. No. A0545, Sigma) secondary antibody. Images were collected using AmershamImager600 (GE) and analyzed by ImageJ software.

Results:

RT-qPCR results show that the in vivo knockdown efficiency of Ube3a-ATS transcript by the AAV. PHP. eB delivered CRISPR-hfCas13e. 1 system was 77.5 %in cortex and 76.6%in hippocampus of treated AS mice compared with untreated AS mice and achieved 44.3%in cortex and 17.6%in hippocampus of Ube3a mRNA level (Mean, n = 3 repeats) , respectively, compared to the mRNA level of maternal Ube3a in untreated WT mice (FIG. 4B and 4C) .

WB results show 35.9%and 41.6%expression of Ube3a induced by hfCas13e. 1-sg9 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 treated AS mice, compared with untreated WT mice (FIG. 4D-4G) .

All these results indicate that hfCas13e. 1-sg9 has great efficiency in knocking-down Ube3a-ATS transcript and recovering the expression of paternal Ube3a.

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

AS mouse model and Intracerebroventricular injection

All 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 FIG. 2B) plus 2.5 ×10 ⁹ reporter AAV particles (encoding CAG promoter-tdTomato reporter) in 2 μl 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. 1-sg [Ube3a-ATS] 9. Untreated AS mice (AS+NT) : 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] .

Behavioural Tests

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 Test

At 7 weeks, each mouse was suspended 10 cm above the table for 20 seconds by being holding the tail. Video was recorded and time was scored offline. Hindlimb clasping time was the total time spent on clasping. Clasping was defined by the behavior of incomplete splay with one or both hindlimbs.

Openfield Test

Each mouse was placed on the edge of 40 cm × 40 cm of openfield box and allowed to explore for 15 min. The centre area was 20 cm × 20 cm square in the centre 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.

Dowel test

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.

Beam-walking Test

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 footslips were couted.

Accelerating Rotarod Test

This test was performed on a rotarod system with the rod rotating from 5 r. p. m. to 30 r. p. m. over 5 min. 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.

Results:

After intracerebroventricular adminstration of CRISPR-hfCas13e. 1-sg9/sg [NT] system at postnatal day 0, the mice were weighed every two weeks, and it was observed that the hfCas13e. 1-sg9 treated AS female mice showed decreased body weight compared with the untreated AS mice injected with hfCas13e. 1-sg [NT] (FIG. 5B) , which means the phenotype of obesity in AS mouse model was significantly improved by the treatment.

The increased centre frequency as shown in FIG. 5D means that the hfCas13e. 1-sg9 treated AS mice prefered to explore in the central region than the untreated AS mice.

Also, hindlimb clasping test, dowel test, beam-walking test, and accelerating rotarod test demonstrated that the hfCas13e. 1-sg9 treated AS mice showed better performance in terms of motor coordination and balance compared with the untreated AS mice (FIG. 5C, 5E-5H) .

In summary, the data presented herein demonstrated that the subject CRISPR-hfCas13e. 1 system efficiently knocked-down Ube3a-ATS transcript in AS primary neurons ex vivo and in the brains of AS mouse models in vivo, and the intracerebroventricular administration of AAV. PHP. eB particles delivering hfCas13e. 1-sgRNA [Ube3a-ATS] system could rescure the abnormal behaviors of AS mice, showing the promising prospect of treating AS and other UBE3A-associated diseases in human.

Claims

A recombinant lentiviral or adeno-associated virus (AAV) vector genome, comprising:

(1) a Cas13 coding sequence encoding a Cas13 polypeptide,

(i) wherein said 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,

(ii) wherein said 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: 46 and has a non-conserved substitution at Y672 and/or Y676 (e.g., Y672A and/or Y676A substitution (s) ) of SEQ ID NO: 46, wherein said 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: 46 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity (collateral cleavage activity) of SEQ ID NO: 46; and,

(2) a single guide RNA (sgRNA) or a sgRNA coding sequence encoding the sgRNA, said sgRNA comprises:

(A) a spacer sequence substantially complementary to a target RNA sequence on a UBE3A-ATS transcript; and,

(B) a direct repeat (DR) sequence capable of forming a complex with said Cas13 polypeptide,

wherein the complex specifically cleaves the UBE3A-ATS transcript at or near the target RNA sequence when said sgRNA guides said Cas13 polypeptide to the target RNA sequence; optionally wherein the sgRNA or sgRNA coding sequence is 3’ or 5’ to the Cas13 coding sequence.
The vector genome of claim 1, further comprising 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 said Cas13 polypeptide, and/or a second coding sequence for a second NLS (such as SEQ ID NO: 27) or NES fused C-terminal to said Cas13 polypeptide;

optionally, 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) .
The vector genome of claim 1 or 2, further comprising a 5’ AAV ITR sequence and a 3’ AAV ITR sequence.
The vector genome of claim 3, wherein 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; optionally, said 5’ AAV ITR sequence has the polynucleotide sequence of SEQ ID NO: 31, and/or said 3’ AAV ITR sequence has the polynucleotide sequence of SEQ ID NO: 32.
The vector genome of any one of claims 1-4, further comprising a promoter operably linked to the Cas13 coding sequence.
The vector genome of claim 5, wherein the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter; optionally, wherein the promoter comprises a promoter 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 β-actin promoter, an elongation factor 1α short (EFS) promoter, a β glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken β-actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1α-subunit (EF1α) promoter, a ubiquitin C (UBC) promoter, a prion promoter, a neuron-specific enolase (NSE) , a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a platelet-derived growth factor (PDGF) promoter, a platelet-derived growth factor B-chain (PDGF-β) promoter, a synapsin (Syn) promoter, a synapsin 1 (Syn1) promoter, a methyl-CpG binding protein 2 (MeCP2) promoter, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) promoter, a metabotropic glutamate receptor 2 (mGluR2) promoter, a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a β-globin minigene nβ2 promoter, a preproenkephalin (PPE) promoter, an enkephalin (Enk) promoter, an excitatory amino acid transporter 2 (EAAT2) promoter, a glial fibrillary acidic protein (GFAP) promoter, and a myelin basic protein (MBP) promoter.
The vector genome of claim 6, wherein the promoter comprises a Syn1 promoter, such as a Syn1 promoter having the polynucleotide sequence of SEQ ID NO: 25.
The vector genome of any one of claims 1-7, further comprising 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 vector genome of claim 8, wherein the polyA signal sequence comprises a SV40 polyA signal, or a variant thereof; optionally, said SV40 polyA signal comprises the polynucleotide sequence of SEQ ID NO: 29.
The vector genome of any one of claims 1-9, wherein the sgRNA coding sequence is operably linked to a promoter; optionally wherein the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter; optionally 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 β-actin promoter, an elongation factor 1α short (EFS) promoter, a β glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken β-actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1α-subunit (EF1α) promoter, a ubiquitin C (UBC) promoter, a prion promoter, a neuron-specific enolase (NSE) , a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a platelet-derived growth factor (PDGF) promoter, a platelet-derived growth factor B-chain (PDGF-β) promoter, a synapsin (Syn) promoter, a synapsin 1 (Syn1) promoter, a methyl-CpG binding protein 2 (MeCP2) promoter, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) promoter, a metabotropic glutamate receptor 2 (mGluR2) promoter, a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a β-globin minigene nβ2 promoter, a preproenkephalin (PPE) promoter, an enkephalin (Enk) promoter, an excitatory amino acid transporter 2 (EAAT2) promoter, a glial fibrillary acidic protein (GFAP) promoter, and a myelin basic protein (MBP) promoter; optionally wherein the promoter is an RNA pol III promoter.
The vector genome of claim 10, wherein the RNA pol III promoter is U6 (such as SEQ ID NO: 30) , H1, 7SK, or a variant thereof.
The vector genome of any one of claims 1-11, wherein

(1) said sgRNA comprises one spacer sequence directly linked to one DR sequence (e.g., SEQ ID NO: 3) ;

(2) said sgRNA comprises one spacer sequence flanked by two DR sequences (e.g., each of SEQ ID NO: 3) ; or

(3) said sgRNA comprises two or more spacer sequences; and wherein each spacer sequence is flanked by two DR sequences each capable of forming a complex with said Cas13 polypeptide; optionally, said sgRNA 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)

wherein each of said spacer sequence is independently substantially complementary to a distinct target RNA sequence on said UBE3A-ATS transcript, and each capable of directing said Cas13 polypeptide to cleave respective said distinct target RNA sequence.
The vector genome of any one of claims 1-12, wherein 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.
The vector genome of claim 13, wherein each said DR sequence comprises, consists essentially of, or consists of SEQ ID NO: 3.
The vector genome of any one of claims 1-14, wherein the target RNA sequence comprises a stench of contiguous nucleotides of the RNA counterpart of NG_002690.1 or NC_000073.7; optionally 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 70-92 or the RNA counterpart thereof.
The vector genome of any one of claims 1-15, wherein the spacer sequence is independently selected from any one of SEQ ID NOs: 10-15 and 47-69, or a variant thereof differing from any one of SEQ ID NOs: 10-15 and 47-69 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 sgRNA to form a Cas13-sgRNA complex targeting the target RNA sequences to cleave the target RNA.
The vector genome of any one of claims 1-16, wherein said UBE3A-ATS transcript is associated with a disease or disorder, such as ALS (amyotrophic lateral sclerosis) .
The vector genome of any one of claims 1-17, comprising an ITR-to-ITR polynucleotide (such as SEQ ID NO: 33) comprising, from 5’ to 3’:

(a) an optional 5’ ITR from AAV2 (such as SEQ ID NO: 31) ;

(b) a Syn1 promoter (such as SEQ ID NO: 25) ;

(c) a Kozak sequence (such as SEQ ID NO: 26) ;

(d) a first NLS coding sequence (such as one encoding SEQ ID NO: 27) ;

(e) 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;

(f) a second NLS coding sequence (such as one encoding SEQ ID NO: 27) ;

(g) an optional coding sequence encoding a 3xFlag sequence (e.g., SEQ ID NO: 28) ;

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

(i) a U6 promoter (such as SEQ ID NO: 30) ;

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

(k) a spacer coding sequence encoding a first spacer sequence specific for UBE3A-ATS transcript (such as SEQ ID NO: 4) ;

(l) a second DR DNA coding sequence encoding a second DR (such as SEQ ID NO: 3) ; and,

(m) an optional 3’ ITR from AAV2 (such as SEQ ID NO: 32) ;

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 to said ITR-to-ITR polynucleotide;

optionally, said ITR-to-ITR polynucleotide further comprises a linker sequence between any two adjacent sequence elements of (a) – (m) ;

optionally, the 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) ;

optionally, the 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; and

optionally, the 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.
A recombinant AAV vector genome comprising, consisting essentially of, or consisting of:

(1) 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,

wherein said polynucleotide encodes

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

(b) a variant thereof at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 46 and having a non-conserved substitution at Y672 and/or Y676 (e.g., Y672A and/or Y676A substitution (s) ) of SEQ ID NO: 46, wherein said 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: 46 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity of SEQ ID NO: 46; and,

(2) a sg RNA coding sequence encoding a sgRNA, said sgRNA comprises:

(A) a spacer sequence substantially complementary to a target RNA sequence on a UBE3A-ATS transcript; and,

(B) a direct repeat (DR) sequence that forms a complex with said Cas13 polypeptide,

wherein the complex specifically cleaves the UBE3A-ATS transcript with substantially the same (e.g., at least about 80%, 90%, 95%, 99%or more) guide RNA-specific nuclease activity as SEQ ID NO: 46 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity of SEQ ID NO: 46,

at or near the target RNA sequence when said sgRNA guides said Cas13 polypeptide to the target RNA sequence; optionally wherein the sgRNA coding sequence is 3’ or 5’ to the Cas13 coding sequence.
The vector genome of claim 19, which is SEQ ID NO: 33, or the polynucleotide at least 95%or 99%identical thereto.
A recombinant lentiviral or AAV particle comprising the vector genome of any one of claims 1-18.
The recombinant AAV particle of claim 21, 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. 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 recombinant AAV particle of claim 21 or 22, wherein the capsid serotype is AAV. PHP. eB.
A recombinant AAV particle comprising the vector genome of claim 19 or 20, encapsidated in a capsid with a serotype of AAV. PHP. eB.
A pharmaceutical composition comprising the vector genome of any one of claims 1-20, or the particle of any one of claims 21-24, and a pharmaceutically acceptable excipient.
A method of treating a disease or disorder associated with UEB3A in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the vector genome of any one of claims 1-20, the particle of any one of claims 21-24, or the pharmaceutically composition of claim 25, wherein the vector genome or the particle specifically down-regulate the expression of said UEB3A causative of the disease or disorder.
The method of claim 26, wherein the administrating comprises contacting a cell with the therapeutically effective amount of the vector genome of any one of claims 1-20, the particle of any one of claims 21-24, or the pharmaceutically composition of claim 25.
The method of claim 27, wherein the cell is located in the CNS of the subject.
The method of any one of claims 26-28, wherein the disease or disorder is Angelman Syndrome (AS) .
The method of any one of claims 26-29, wherein the administrating comprises intracerebroventricular administration.
The method of any one of claims 26-30, wherein the subject is a human.
The method of any one of claims 26-31, wherein the level of UEB3A-ATS transcript in the cell is decreased in comparison to a cell having not been contacted with the vector genome of any one of claims 1-20, the particle of any one of claims 21-24, or the pharmaceutically composition of claim 25.
The method of claim 32, wherein the level of UBE3A-ATS transcript 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 transcript 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.
A guide RNA (gRNA) (e.g., a single guide RNA) comprising:

(A) a spacer sequence substantially complementary to a target RNA sequence on a UBE3A-ATS transcript; and,

(B) optionally, a direct repeat (DR) sequence capable of forming a complex with a Cas13 polypeptide, wherein the complex specifically cleaves the UBE3A-ATS transcript at or near the target RNA sequence when said sgRNA guides said Cas13 polypeptide to the target RNA sequence.
The gRNA of claim 34, wherein the target RNA sequence comprises a stench of contiguous nucleotides of the RNA counterpart of NG_002690.1 or NC_000073.7; optionally 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 NOs: 16-21 and 70-92 or the RNA counterpart thereof.
The gRNA of claim 34 or 35, comprising two or more identical or different spacer sequences, each flanked by two said DR sequence.
The gRNA of any one of claims 34-36, comprising two different spacer sequences (e.g., spacer 1 and spacer 2) separating three of said DR sequences (e.g., DR-spacer 1-DR-spacer 2-DR) .
The gRNA of any one of claims 34-37, comprising one or more spacer sequences each independently selected from any one of SEQ ID NOs: 10-15 and 47-69, or a variant thereof differing from any one of SEQ ID NOs: 10-15 and 47-69 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 sgRNA to form a Cas13-sgRNA complex targeting the respective target sequences to cleave the target sequences.
The gRNA of any one of claims 34-38, wherein 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.
The gRNA of any one of claims 34-39, wherein 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: 46 and has a non-conserved substitution at Y672 and/or Y676 (e.g., Y672A and/or Y676A substitution (s) ) of SEQ ID NO: 46, wherein said 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: 46 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity of SEQ ID NO: 46.
A cell or a progeny thereof, comprising the vector genome of any of claims 1-20, the particle of any one of claims 21-24, or the gRNA of any one of claims 34-40.
A kit comprising the vector genome of any of claims 1-20, the particle of any one of claims 21-24, the gRNA of any one of claims 34-40, or the cell or a progeny thereof of claim 41.
A method of preparing the recombinant AAV particle of any one of claims 21-24, the method comprising:

a) introducing into an AAV packaging system a nucleic acid encoding the vector genome of any one of claims 1-20 or the RNA sequence transcribed therefrom, and a coding sequence for the AAV capsid as defined in claim 22 or 23, for a period of time sufficient to package the vector genome or the transcribed RNA into the AAV capsid to produce the recombinant AAV particle, and

b) harvesting the recombinant AAV particle; and, optionally,

c) isolating or purifying the harvested recombinant AAV particle.