WO2023025103A1 - Système crispr-cas13 pour le traitement de maladies associées au sod1 - Google Patents

Système crispr-cas13 pour le traitement de maladies associées au sod1 Download PDF

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WO2023025103A1
WO2023025103A1 PCT/CN2022/114021 CN2022114021W WO2023025103A1 WO 2023025103 A1 WO2023025103 A1 WO 2023025103A1 CN 2022114021 W CN2022114021 W CN 2022114021W WO 2023025103 A1 WO2023025103 A1 WO 2023025103A1
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promoter
seq
sequence
cas13
vector genome
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PCT/CN2022/114021
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Hui Yang
Xing Wang
Mingliang BAI
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Huigene Therapeutics Co., Ltd.
Center For Excellence In Brain Science And Intelligence Technology, Chinese Academy Of Sciences
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Priority claimed from PCT/CN2021/121926 external-priority patent/WO2022068912A1/fr
Priority claimed from PCT/CN2022/083476 external-priority patent/WO2023024504A1/fr
Application filed by Huigene Therapeutics Co., Ltd., Center For Excellence In Brain Science And Intelligence Technology, Chinese Academy Of Sciences filed Critical Huigene Therapeutics Co., Ltd.
Publication of WO2023025103A1 publication Critical patent/WO2023025103A1/fr

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    • C12N15/1137—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing against enzymes
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • ALS Amyotrophic lateral sclerosis
  • fALS familial ALS
  • SOD1 Cu/Zn superoxide dismutase 1
  • rAAV adeno-associated virus
  • sgRNA single guide RNA
  • sgRNA coding sequence encoding the sgRNA
  • the complex specifically cleaves the SOD1 mRNA at or near the target RNA sequence when said sgRNA guides said Cas13 polypeptide to the target RNA sequence;
  • sgRNA or sgRNA coding sequence is 3' or 5' to the Cas13 coding sequence or the RNA counterpart thereof.
  • the rAAV vector genome further comprises a first coding sequence for a first nuclear localization sequence (NLS, such as SEQ ID NO: 66) 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: 66) or NES fused C-terminal to said Cas13 polypeptide;
  • NLS nuclear localization sequence
  • NES nuclear export signal
  • the rAAV vector genome further comprises a coding sequence for one or more copies (e.g., 3 tandem copies) of an epitope tag, such as an HA tag, fused (e.g., C-terminally) to the Cas13 polypeptide (and the C-terminal NLS or NES, if present) .
  • an epitope tag such as an HA tag
  • the rAAV vector genome further comprises a 5' AAV ITR sequence and a 3' AAV ITR sequence.
  • the 5' and the 3' AAV ITR sequences are both wild-type AAV ITR sequences from AAV1, AAV2, AAV3A, AAV3B, AAV4, AAV5, AAV6, AAV7, AAVrh74, AAV8, AAV9, AAV10, AAV11, AAV12, AAV13, AAV-DJ, AAV PHP.
  • said 5' AAV ITR sequence has the polynucleotide sequence of SEQ ID NO: 71, and/or said 3' AAV ITR sequence has the polynucleotide sequence of SEQ ID NO: 72.
  • the rAAV vector genome further comprises a promoter operably linked to the Cas13 coding sequence.
  • the promoter is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter; 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 ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken ⁇ -actin (CBA) promoter
  • the promoter comprises a Cbh promoter, such as a Cbh promoter having the polynucleotide sequence of SEQ ID NO: 63, or a Cba promoter, such as a Cba promoter having the polynucleotide sequence of SEQ ID NO: 62.
  • the rAAV 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.
  • polyA polyadenylation
  • the polyA signal sequence comprises a bovine growth hormone polyadenylation signal (bGH polyA) , or a variant thereof; optionally, said bGH polyA comprises the polynucleotide sequence of SEQ ID NO: 67.
  • bGH polyA bovine growth hormone polyadenylation signal
  • 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 ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a
  • the RNA pol III promoter is U6 (such as SEQ ID NO: 68) , H1, 7SK, or a variant thereof.
  • said sgRNA comprises one spacer sequence directly linked to one DR sequence (e.g., SEQ ID NO: 6) ; (2) said sgRNA comprises one spacer sequence flanked by two DR sequences (e.g., each of SEQ ID NO: 6) ; 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: 6)
  • each of said spacer sequence is independently substantially complementary to a distinct target RNA sequence on said SOD1 mRNA, and each capable of directing said Cas13 polypeptide to cleave respective said distinct target RNA sequence.
  • the DR sequence comprises (1) SEQ ID NO: 6; (2) a sequence having at least 90%, 92%, 94%, 95%, 96%, 98%, or 99%identity to SEQ ID NO: 6; (3) a sequence having at most 1, 2, 3, 4, or 5 nucleotide differences from SEQ ID NO: 6; or (4) a sequence having substantially the same secondary structure as that of SEQ ID NO: 6.
  • the DR sequence comprises (1) SEQ ID NO: 6; (2) a sequence having substantially the same secondary structure as that of SEQ ID NO: 6; or (3) a variant of SEQ ID NO: 6 differeing from SEQ ID NO: 6 by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides without substantially reducing the ability of the complex to cleave the SOD1 mRNA.
  • each said DR sequence comprises, consists essentially of, or consists of SEQ ID NO: 6.
  • the target RNA sequence comprises a stench of contiguous nucleotides of SEQ ID NO: 73; 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 SEQ ID NO: 73, such as, any one of SEQ ID NO: 39-54.
  • the target RNA sequence comprises a stench of contiguous nucleotides of SEQ ID NO: 73; optionally 15-60, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, such as, any one of SEQ ID NO: 39-54.
  • the target RNA sequence comprises at least 15 contiguous nucleotides of any one of SEQ ID NO: 39-54.
  • the spacer sequence is independently selected from any one of SEQ ID NOs: 23-38, or a variant thereof differing from any one of SEQ ID NOs: 23-38 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 spacer sequence independently comprises (1) any one of SEQ ID NOs: 23-38; (2) at least 15 contiguous nucleotides of any one of SEQ ID NOs: 23-38; or (3) a variant of any one of SEQ ID NOs: 23-38 differing from any one of SEQ ID NOs: 23-38 by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides without substantially reducing the ability of the complex to cleave the SOD1 mRNA.
  • said SOD1 mRNA is associated with a disease or disorder, such as ALS (amyotrophic lateral sclerosis) .
  • the rAAV vector genome comprises an ITR-to-ITR polynucleotide (such as SEQ ID NO: 75) comprising, from 5' to 3':
  • a 5' ITR from AAV2 (such as SEQ ID NO: 71) ;
  • a Cbh promoter e.g., one comprising an enhancer sequence (such as SEQ ID NO: 64) and an intron sequence (such as SEQ ID NO: 65) ; optionally, the Cbh promoter comprises SEQ ID NO: 63) ;
  • a first NLS coding sequence (such as one encoding SEQ ID NO: 66) ;
  • a Cas13 polynucleotide (such as SEQ ID NO: 3 or 5 except the start codon ATG) encoding the Cas13 polypeptide of SEQ ID NO: 2 or 4 except the first amino acid M;
  • a second NLS coding sequence (such as one encoding SEQ ID NO: 66) ;
  • an bGH polyA signal sequence (such as SEQ ID NO: 67) ;
  • a U6 promoter such as SEQ ID NO: 68
  • a first direct repeat (DR) DNA coding sequence encoding a first DR (such as SEQ ID NO: 6) ;
  • (k) a first spacer coding sequence encoding a first spacer sequence specific for SOD1 mRNA (such as SEQ ID NO: 27 or 29) ;
  • a second spacer coding sequence encoding a second spacer sequence specific for SOD1 mRNA (such as SEQ ID NO: 27 or 29) ;
  • said ITR-to-ITR polynucleotide further comprises a linker sequence between any two adjacent sequence elements of (a) - (o) ;
  • sequence elements of (b) to (h) that are 5' to the sequence elements of (i) to (n) are relocated 3' to the sequence elements of (i) to (n) .
  • rAAV recombinant AAV vector genome comprising, consisting essentially of, or consisting of:
  • SEQ ID NO: 75 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,
  • polynucleotide encodes a Cas13 polypeptide of
  • sgRNA coding sequence encoding a sgRNA comprises:
  • the complex specifically cleaves the SOD1 mRNA with substantially the same (e.g., at least about 80%, 90%, 95%, 99%or more) guide RNA-specific nuclease activity as the complex of the Cas13 polypeptide of SEQ ID NO: 1 and said sgRNA and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity of the complex of the Cas13 polypeptide of SEQ ID NO: 1 and said sgRNA,
  • the sgRNA coding sequence is 3' or 5' to the Cas13 coding sequence.
  • the rAAV vector genome is SEQ ID NO: 75, or the polynucleotide at least 95%or 99%identical thereto.
  • Another aspect of the invention provides a recombinant AAV (rAAV) viral particle comprising the rAAV vector genome as described herein.
  • rAAV recombinant AAV
  • the rAAV viral 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 rAAV vector genome.
  • the capsid serotype is AAV9-M8, comprising a VP1 sequence of SEQ ID NO: 82.
  • Another aspect of the invention provides a recombinant AAV (rAAV) viral particle comprising the rAAV vector genome as described herein, encapsidated in a capsid with a serotype of AAV9-M8.
  • rAAV recombinant AAV
  • Another aspect of the invention provides a pharmaceutical composition
  • a pharmaceutical composition comprising the rAAV vector genome as described herein, or the rAAV viral 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 SOD1 in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the rAAV vector genome as described herein, the rAAV 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 said SOD1 causative of the disease or disorder.
  • the administrating comprises contacting a cell with the therapeutically effective amount of the rAAV vector genome as described herein, the rAAV viral particle as described herein, or the pharmaceutically composition as described herein.
  • the cell is located in the CNS of the subject.
  • the disease or disorder is ALS.
  • the administrating comprises intrathecal administration.
  • the subject is a human.
  • the expression of SOD1 in the cell is decreased in comparison to a cell having not been contacted with the rAAV vector genome as described herein, the rAAV viral particle as described herein, or the pharmaceutically composition as described herein.
  • the expression of SOD1 is decreased in the subject by 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 expression of SOD1 in the subject prior to administration.
  • gRNA guide RNA
  • gRNA guide RNA
  • a direct repeat (DR) sequence capable of forming a complex with a Cas13 polypeptide, wherein the complex specifically cleaves the SOD1 mRNA at or near the target RNA sequence when said gRNA guides said Cas13 polypeptide to the target RNA sequence.
  • DR direct repeat
  • the target RNA sequence comprises a stench of contiguous nucleotides of SEQ ID NO: 73; 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 SEQ ID NO: 73, such as, any one of SEQ ID NO: 39-54.
  • the target RNA sequence comprises a stench of contiguous nucleotides of SEQ ID NO: 73; optionally 15-60, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, such as, 30 contiguous nucleotides of SEQ ID NO: 73, such as, any one of SEQ ID NO: 39-54.
  • the target RNA sequence comprises at least 15 contiguous nucleotides of any one of SEQ ID NO: 39-54.
  • the gRNA comprises two or more identical or different spacer sequences, each flanked by two said DR sequence.
  • the gRNA comprises 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 comprises one or more spacer sequences each independently selected from any one of SEQ ID NOs: 23-38, or a variant thereof differing from any one of SEQ ID NOs: 23-38 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 comprises one or more spacer sequences each comprising (1) any one of SEQ ID NOs: 23-38; (2) at least 15 contiguous nucleotides of any one of SEQ ID NOs: 23-38; or (3) a variant of any one of SEQ ID NOs: 23-38 differing from any one of SEQ ID NOs: 23-38 by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides without substantially reducing the ability of the complex to cleave the SOD1 mRNA.
  • the DR sequence comprises (1) SEQ ID NO: 6; (2) a sequence having at least 90%, 92%, 94%, 95%, 96%, 98%, or 99%identity to SEQ ID NO: 6; (3) a sequence having at most 1, 2, 3, 4, or 5 nucleotide differences from SEQ ID NO: 6; or (4) a sequence having substantially the same secondary structure as that of SEQ ID NO: 6.
  • the DR sequence comprises (1) SEQ ID NO: 6; (2) a sequence having substantially the same secondary structure as that of SEQ ID NO: 6; or (3) a variant of SEQ ID NO: 6 differeing from SEQ ID NO: 6 by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides without substantially reducing the ability of the complex to cleave the SOD1 mRNA.
  • the Cas13 polypeptide comprises a Cas13a polypeptide, a Cas13b polypeptide, a Cas13c polypeptide, a Cas13d polypeptide, a Cas13e polypeptide, or a Cas13f polypeptide.
  • the Cas13 polypeptide comprises
  • composition comprising the gRNA as described herein.
  • composition further comprises the Cas13 polypeptide as described herein.
  • Another aspect of the invention provides a cell or a progeny thereof, comprising the AAV vector genome as described herein, the rAAV viral particle as described herein, the gRNA as described herein, or the composition as described herein.
  • kits comprising the AAV vector genome as described herein, the rAAV viral particle as described herein, the gRNA as described herein, the composition as described herein, or the cell or a progeny thereof as described herein.
  • Another aspect of the invention provides a method of preparing the rAAV particle as described herein, the method comprising:
  • FIG. 1 is a schematic (not to scale) illustration of a construct expressing hfCas13f v1 and sgRNA-hSOD1 with mCherry as a reporter as well as a contruct expression hSOD1-targeting shRNA.
  • FIG. 2 shows a graph of in vitro knockdown of hSOD1 mRNA by CRISPR-hfCas13f v1 system normalized to the negative controls as 100%.
  • the hSOD1 mRNA levels were detected by RT-qPCR.
  • N 3/group.
  • Data were represented as mean ⁇ SEM. *, P ⁇ 0.05; **, P ⁇ 0.01 (unpaired two-tailed Student's t-test) . n.s. -non-significant.
  • FIG. 3 is a schematic (not to scale) illustration of a construct expressing hfCas13f v1 and sgRNA5&7-hSOD1 with mCherry as a reporter.
  • FIG. 4 shows a graph of in vitro knockdown of hSOD1 mRNA by CRISPR-hfCas13f v1 system normalized to the negative controls as 100%.
  • the hSOD1 mRNA levels were detected by RT-qPCR.
  • N 3/group.
  • Data were represented as mean ⁇ SEM. **, P ⁇ 0.01 (unpaired two-tailed Student's t-test) .
  • FIG. 5a is a schematic (not to scale) illustration of a construct expressing hfCas13f v2 and sgRNA [hSOD1] with mCherry as a reporter;
  • FIG. 5b shows a graph of in vitro knockdown of hSOD1 mRNA by CRISPR-hfCas13f v2 system normalized to the negative controls as 100%.
  • the hSOD1 mRNA levels were detected by RT-qPCR.
  • N 3-4/group. Data were represented as mean ⁇ SEM. *, P ⁇ 0.05; ***, P ⁇ 0.001 (unpaired two-tailed Student's t-test) . n.s. -non-significant.
  • FIG. 6 is a schematic (not to scale) illustration of an exemplary AAV vector genome, AAV9-M8-hfCas13f v2-sgRNA [hSOD1] 5&7 as well as the negative control.
  • FIG. 7 is a schematic illustration of treatment of a SOD1-mutation model mouse with AAV9-M8 deliverying hfCas13f v2-sgRNA [hSOD1] 5&7 decreasing hSOD1 expression and subsequent detections.
  • FIG. 8 shows the high transduction efficiency of rAAV9-M8 particles encoding hfCas13f v2-sgRNA [hSOD1] 5&7 in spinal neurons. Scale bars, 100 ⁇ m.
  • FIG. 9 shows the reduced expression of hSOD1 protein detecting with Western Blot after intrathecal injection of AAV9-M8-hfCas13f v2-sgRNA [hSOD1] 5&7 into SOD1-G93A mice.
  • FIG. 10 shows the reduced expression of hSOD1 detected using immunostaining after intrathecal injection into SOD1-G93A mice. Spinal cords were harvest at 4 weeks post injection. Scale bars, 100 ⁇ m.
  • FIG. 11a and 11b show the survival curve and age of death of different groups of mice;
  • FIG. 11c shows the motor performance indicated by grip strength of mice measured at indicated days;
  • FIG. 11d shows the weight of mice measured at indicated days.
  • Data were represented as mean ⁇ SEM. *, P ⁇ 0.05; **, P ⁇ 0.01; ***, P ⁇ 0.001.
  • the invention described herein provides CRISPR-Cas13 systems and methods for treatment of SOD1-assocaited diseases, such as, Amyotrophic lateral sclerosis (ALS) .
  • SOD1-assocaited diseases such as, Amyotrophic lateral sclerosis (ALS) .
  • the invention described herein provides CRISPR-Cas13 systems for knocking down the expression of SOD1 gene by cleaving SOD1 mRNA including mutated SOD1 mRNA, so as to prevent the expression of disease-associated mutated SOD1 protein.
  • Such a system can be delivered by AAV vectors and intrathecal injection to subjects in need.
  • Exemplary constructs of the invention have demonstrated the efficacy to knockdown SOD1 mRNA and protein levels, especially in vivo, and improve the disease phenotype of the mouse disease models, thus opening the door for gene therapy to treat SOD1-assocaited diseases, such as, ALS.
  • the Cas13 polypeptide herein may comprise a Cas13a polypeptide, a Cas13b polypeptide, a Cas13c polypeptide, a Cas13d polypeptide, a Cas13e polypeptide, or a Cas13f polypeptide.
  • 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.
  • HEPN prokaryotes nucleotide-binding
  • 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) .
  • 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.
  • the invention described herein provides compositions and methods of use of engineered Cas13 (e.g., hfCas13f v1, hfCas13f v2) proteins with designed gRNAs to treat SOD1-associated diseases (e.g., ALS) .
  • engineered Cas13 e.g., hfCas13f v1, hfCas13f v2
  • gRNAs e.g., ALS
  • the Cas13 protein used herein is the Cas13 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 parental (or reference) Cas13;
  • an endonuclease catalytic domain e.g., a HEPN domain
  • the Cas13 is a Cas13f, such as SEQ ID NO: 1.
  • the parental (or reference) Cas13 is a parental (or reference) Cas13f, such as SEQ ID NO: 1.
  • 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.
  • an endonuclease catalytic domain e.g., an RXXXXH domain
  • 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.
  • the endonuclease catalytic domain is a HEPN domain, optionally a HEPN domain comprising an RXXXXH motif.
  • the RXXXXH motif comprises a R ⁇ N/H/K/Q/R ⁇ X 1 X 2 X 3 H sequence.
  • X 1 is R, S, D, E, Q, N, G, or Y
  • X 2 is I, S, T, V, or L
  • X3 is L, F, N, Y, V, I, S, D, E, or A.
  • the RXXXXH motif is an N-terminal RXXXXH motif comprising an RNXXXH sequence, such as an RN ⁇ Y/F ⁇ ⁇ F/Y ⁇ SH sequence.
  • the N-terminal RXXXXH motif has a RNYFSH sequence.
  • the N-terminal RXXXXH motif has a RNFYSH sequence.
  • 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.
  • the C-terminal RXXXXH motif has a RN (A/K) ALH sequence.
  • the C-terminal RXXXXH motif has a RAFFHH or RRAFFH sequence.
  • the region comprises, consists essentially of, or consists of 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: 1.
  • 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) .
  • the stretch is about 16 or 17 residues.
  • substantially all, except for up to 1, 2, or 3, charged and polar residues within the stretch are substituted.
  • a total of about 7, 8, 9, or 10 charged and polar residues within the stretch are substituted.
  • 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.
  • the N-terminal two residues are VF, and the C-terminal 2 residues are ED, and the restriction enzyme is BpiI.
  • the one or more charged or polar residues comprise N, Q, R, K, H, D, E, Y, S, and T residues.
  • the one or more charged or polar residues comprise R, K, H, N, Y, and/or Q residues.
  • one or more Y residue (s) within the stretch is substituted.
  • the one or more Y residues (s) correspond to Y666 and/or Y677 of parental (or reference) Cas13f. 1 (SEQ ID NO: 1) .
  • the charge-neutral short chain aliphatic residue is Ala (A) .
  • the mutation comprises, consists essentially of, or consists of:
  • 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 parental (or reference) Cas13f (such as SEQ ID NO: 1) , and exhibits less than about 25 or 27.5%collateral effect of parental (or reference) Cas13f (such as SEQ ID NO: 1) ;
  • a Cas13f mutation e.g., D160A, Q163A, D642A, L631A, P667A, H638A, T647A, D762A, L634A, L641A, V670A, A763V, T161A, R157A
  • 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, L634
  • 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 parental (or reference) Cas13f (such as SEQ ID NO: 1) , and exhibits less than about 25 or 27.5%collateral effect of parental (or reference) Cas13f (such as SEQ ID NO: 1) ; and/or
  • 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.
  • 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 parental (or reference) Cas13 towards the target RNA.
  • 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 parental (or reference) Cas13 towards the non-target RNA.
  • the Cas13 preserves at least about 80-90%of the guide sequence-specific endonuclease cleavage activity of the parental (or reference) Cas13 towards the target RNA, and lacks at least about 95-100%of the guide sequence-independent collateral endonuclease cleavage activity of the parental (or reference) Cas13 towards the non-target RNA.
  • the Cas13 protein further comprises a nuclear localization signal (NLS) sequence or a nuclear export signal (NES) .
  • NLS nuclear localization signal
  • NES nuclear export signal
  • the Cas13 protein comprises an N-and/or a C-terminal NLS.
  • the Cas13 protein comprises, consists essentially of, or consisting of SEQ ID NO: 2 or 4.
  • the Cas13 protein used herein comprises a Cas13 polypeptide, wherein said Cas13 coding sequence comprises:
  • Y666 and/or Y677 e.g., Y666A and/or Y677
  • a recombinant adeno-associate virus (rAAV) 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 SOD1 mRNA) , 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 said Cas13 polypeptide.
  • a target gene transcript such as, an SOD1 mRNA
  • rAAV adeno-associated virus
  • variants of SEQ ID NO: 2 or 4 that is at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99%identical to SEQ ID NO: 1 and has a non-conserved substitution at Y666 and/or Y677 (e.g., Y666A and/or Y677A substitution (s) ) , 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: 1 and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity (collateral cleavage activity) of SEQ ID NO: 1; and,
  • sgRNA single guide RNA
  • sgRNA coding sequence encoding the sgRNA
  • the complex specifically cleaves the SOD1 mRNA at or near the target RNA sequence when said sgRNA guides said Cas13 polypeptide to the target RNA sequence.
  • the Cas13 coding sequence or the RNA counterpart thereof and the sgRNA or the coding sequence thereof can be placed in 5'-3' direction or 3'-5' direction.
  • ITR sequences are important for initiation of viral DNA replication and circularization of adeno-associated virus genomes.
  • secondary structures e.g., stems and loops formed by palindromic sequences
  • Such sequence elements include the RBE sequence (Rep binding element) , RBE' sequence, and the trs (terminal resolution sequence) .
  • the rAAV vector genome comprises a 5' AAV ITR sequence and/or a 3' AAV ITR sequence.
  • 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.
  • the 5' and the 3' AAV ITR sequences are both wild-type AAV ITR sequences from AAV2.
  • the 5' and/or 3' ITR sequences are modified ITR sequences.
  • 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
  • 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.
  • 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.
  • the 5' and/or 3' modified ITR 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.
  • the modified ITR sequences comprise the RBE sequence, the RBE' sequence, and/or the trs of the wt ITR sequence.
  • the modified ITR sequences comprise both the RBE sequence and the RBE' sequence.
  • 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.
  • the modified ITRs do not interfere with sequencing verification of the plasmids of the invention comprising the AAV vector genome.
  • the modified 5' ITR sequence comprises a 5' heterologous sequence that is not part of wild-type AAV 5' ITR sequence.
  • the modified 3' ITR sequence comprises a 3' heterologous sequence that is not part of wild-type AAV 3' ITR sequence.
  • 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
  • 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.
  • 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) .
  • a type II restriction endonuclease recognition sequence such as recognition sequence for Sse8387I (CCTGCAGG) , or recognition sequence for PacI (TTAATTAA) .
  • 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) .
  • 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) .
  • 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) .
  • 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) .
  • the 5' and 3' ITR sequences are compatible for AAV production in mammalian-cell based on triple transfection.
  • the 5' and 3' ITR sequences are compatible for AAV production in insect cell (e.g., Sf9) based on baculovirus vector (see below) .
  • the 5' and 3' ITR sequences are compatible for AAV production in mammalian-cell based on HSV vectors (see below) .
  • the 5' AAV ITR sequence comprises, consists essentially of, or consists of SEQ ID NO: 71.
  • the 3' AAV ITR sequence comprises, consists essentially of, or consists of SEQ ID NO: 72.
  • 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.
  • a regulatory element e.g., a promoter
  • the promoter is ubiquitous.
  • the promoter is a constitutive promoter.
  • the promoter is an inducible promoter.
  • the promoter is a cell-specific promoter.
  • the promoter is an organism-specific promoter, e.g., tissue-specific promoter.
  • Suitable promoters include, for example, a Cbh promoter, a Cba promoter, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken ⁇ -actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1 ⁇ -subunit (EF1 ⁇ ) promote
  • CBA
  • a U6 promoter can be used to regulate the expression of a gRNA molecule described herein.
  • the elongation factor 1 ⁇ short (EFS) promoter can be used to regulate the expression of Cas13 proteins described herein.
  • the promoter is a Cbh promoter, such as a Cbh promoter comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 63, or a Cba promoter, such as a Cba promoter comprising, consisting essentially of, or consisting of the polynucleotide sequence of SEQ ID NO: 62.
  • 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.
  • NLS nuclear localization sequence
  • NES nuclear export signal
  • 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) .
  • the NLS, the first NLS, and the second NLS comprises, consists essentially of, or consists of SEQ ID NO: 66.
  • the rAAV vector genome of the invention further comprises a Kozak sequence or a functional variant thereof.
  • the Kozak sequence is “GCCACC” ; or a sequence comprising at most 1, 2, 3, or 4 nucleotide differences from “GCCACC” other than the ATG start codon, if present, within the Kozak sequence, wherein the last three nucleotide is optionally ACC or GCC.
  • the rAAV vector genome of the invention further comprises a polyadenylation (polyA) signal sequence.
  • 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.
  • the polyA signal sequence is bgh-polyA signal sequence or a functional variant thereof (such as SEQ ID NO: 67) .
  • the expression cassette for transcribing a gRNA targeting the target gene transcript comprises an RNA pol III promoter, wherein said second transcription unit is 3' to the Cas13 polynucleotide.
  • the RNA pol III promoter is U6 (such as SEQ ID NO: 68) , H1, 7SK, or a variant thereof.
  • 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., SOD1 mRNA) , and capable of directing the Cas13 polypeptide herein to cleave said target RNA. More detailed description for multiple spacer sequences and associated DR sequences are provided in a separate section below (incorporated herein by reference) .
  • the rAAV vector genome of the invention comprises an ITR-to-ITR polynucleotide (such as SEQ ID NO: 75) comprising, from 5' to 3':
  • a 5' ITR from AAV2 (such as SEQ ID NO: 71) ;
  • a Cbh promoter e.g., one comprising an enhancer sequence (such as SEQ ID NO: 64) and an intron sequence (such as SEQ ID NO: 65) ; optionally, the Cbh promoter comprises SEQ ID NO: 63) ;
  • a first NLS coding sequence (such as one encoding SEQ ID NO: 66) ;
  • a Cas13 polynucleotide (such as SEQ ID NO: 3 or 5 except the start codon ATG) encoding the Cas13 polypeptide of SEQ ID NO: 2 or 4 except the first amino acid M;
  • a second NLS coding sequence (such as one encoding SEQ ID NO: 66) ;
  • an bGH polyA signal sequence (such as SEQ ID NO: 67) ;
  • a U6 promoter such as SEQ ID NO: 68
  • a first direct repeat (DR) DNA coding sequence encoding a first DR (such as SEQ ID NO: 6) ;
  • (k) a first spacer coding sequence encoding a first spacer sequence specific for SOD1 mRNA (such as SEQ ID NO: 27 or 29) ;
  • a second spacer coding sequence encoding a second spacer sequence specific for SOD1 mRNA (such as SEQ ID NO: 27 or 29) ;
  • said ITR-to-ITR polynucleotide further comprises a linker sequence between any two adjacent sequence elements of (a) - (o) ;
  • sequence elements of (b) to (h) that are 5' to the sequence elements of (i) to (n) are relocated 3' to the sequence elements of (i) to (n) .
  • the recombinant AAV (rAAV) vector genome comprises, consists essentially of, or consists of:
  • SEQ ID NO: 75 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,
  • polynucleotide encodes a Cas13 polypeptide of
  • sgRNA single guide RNA
  • the complex specifically cleaves the SOD1 mRNA with substantially the same (e.g., at least about 80%, 90%, 95%, 99%or more) guide RNA-specific nuclease activity as the complex of the Cas13 polypeptide of SEQ ID NO: 1 and said sgRNA and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity of the complex of the Cas13 polypeptide of SEQ ID NO: 1 and said sgRNA, at or near the target RNA sequence when said sgRNA guides said Cas13 polypeptide to the target RNA sequence.
  • guide RNA-specific nuclease activity as the complex of the Cas13 polypeptide of SEQ ID NO: 1 and said sgRNA and substantially no (e.g., at most 20%, 15%, 10%, 5%) collateral (guide RNA-independent) nuclease activity of the complex of the Cas13 polypeptide of SEQ ID NO: 1 and
  • the rAAV vector genome is SEQ ID NO: 75, 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.
  • the rAAV vector genome is SEQ ID NO: 75.
  • 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) .
  • the vector includes a nucleic acid encoding a single component of the CRISPR-Cas13 system described herein.
  • the vector includes multiple nucleic acids, each encoding a component of the CRISPR-Cas13 system described herein.
  • 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: 75) encoding the Cas13 protein and the gRNA as described herein.
  • nucleic acid sequences such as ITR-to-ITR sequences, for example, SEQ ID NO: 75
  • 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: 2 or 4) .
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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.
  • 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.
  • 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.
  • 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.
  • the nucleic acid can be codon-optimized for any prokaryotes (such as E.
  • 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. ) .
  • 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.
  • 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.
  • codon bias differs in codon usage between organisms
  • mRNA messenger RNA
  • tRNA transfer RNA
  • 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) .
  • 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.
  • one or more codons e.g., 1, 2, 3, 4, 5, 10, 15, 20, 25, 50, or more, or all codons
  • one or more codons in a sequence encoding a Cas correspond to the most frequently used codon for a particular amino acid.
  • the CRISPR systems described herein include at least a gRNA.
  • gRNA may be encoded by the same AAV vector genome encoding the Cas13 polypeptide.
  • guide RNA (gRNA) and CRISPR RNA (crRNA) are exchangeable.
  • the gRNA includes a spacer (Spacer) sequence substantially complementary to a target RNA sequence on a target RNA (such as, a SOD1 mRNA) 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 SOD1 mRNA) at or near the target RNA sequence when said sgRNA guides said 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 SOD1 mRNA) at or near the target RNA sequence when said Cas13 polypeptide is guided to the target RNA sequence by the spacer sequence.
  • target RNA such as, a SOD1 mRNA
  • the gRNA is a single guide RNA (sgRNA) .
  • sgRNA single guide RNA
  • the “sgRNA” refers to a guide RNA comprising both a spacer sequence and a direct repeat sequence in a single RNA sequence.
  • 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 (3' to the spacer sequence) .
  • the sgRNA comprises, consists essentially of, or consists of one spacer sequence directly linked to one DR sequence, like DR-Spacer or Spacer-DR.
  • the sgRNA comprises, consists essentially of, or consists of one spacer sequence indirectly linked to one DR sequence, like DR-Spacer-DR.
  • the sgRNA comprises, consists essentially of, or consists of two or more spacer sequences.
  • 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, SOD1 mRNA) , and each can be capable of directing a Cas13 polypeptide as described herein to cleave respective distinct target RNA sequence.
  • a target RNA e.g, SOD1 mRNA
  • 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.
  • 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.
  • 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
  • S1a has a sequence of GCUG in Cas13e and GCUGU in Cas13.
  • S2a has a sequence of GCCCC in Cas13e and A/G CCUC G/A in Cas13 (wherein the first A or G may be absent) .
  • the DR sequence comprises (1) SEQ ID NO: 6; (2) a sequence having substantially the same secondary structure as that of SEQ ID NO: 6; or (3) a variant of SEQ ID NO: 6 differeing from SEQ ID NO: 6 by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides without substantially reducing the ability of the complex to cleave the SOD1 mRNA.
  • the phrase “without substantially reducing the ability of the complex to cleave the SOD1 mRNA” means that the ability of the complex of the same Cas13 polypeptide and the same gRNA but with the DR sequence variant of SEQ ID NO: 6 to cleave the SOD1 mRNA is reduced by no more than 30%, 25%, 20%, 15%, 10%, or 5%as compared to the complex of the same Cas13 polypeptide and the same gRNA but with the parental DR sequence of SEQ ID NO: 6.
  • the direct repeat sequence comprises, consists essentially of, or consists of a nucleic acid sequence of SEQ ID NO: 6.
  • direct repeat sequence may refer to either the direct repeat RNA sequence or the direct repeat DNA sequence encoding the direct repeat RNA sequence.
  • 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.
  • 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: 6.
  • 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: 6 (e.g., due to deletion, insertion, or substitution of nucleotides in SEQ ID NO: 6) .
  • the direct repeat sequence comprises, consists essentially of, or consists of a nucleic acid sequence that is not identical to SEQ ID NO: 6 but can hybridize with a complement of SEQ ID NO: 6 under stringent hybridization conditions or can bind to a complement of SEQ ID NO: 6 under physiological conditions.
  • the deletion, insertion, or substitution does not change the overall secondary structure of that of SEQ ID NO: 6 (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) .
  • 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) .
  • 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.
  • any of the above direct repeat sequences that is different from SEQ ID NO: 6 retains the ability to function as a direct repeat sequence in the Cas13 proteins, as the DR sequence of SEQ ID NO: 6.
  • the direct repeat sequence comprises, consists essentially of, or consists of a nucleic acid having a nucleic acid sequence of SEQ ID NO: 6, with a truncation of the initial one, two, three, four, five, six, seven, or eight 3' nucleotides.
  • 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%.
  • substantially complementary means that 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 spacer sequence is independently selected from any one of SEQ ID NOs: 23-38, or a variant thereof differing from any one of SEQ ID NOs: 23-38 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 spacer sequence comprises (1) any one of SEQ ID NOs: 23-38; (2) at least 15 contiguous nucleotides of any one of SEQ ID NOs: 23-38; or (3) a variant of any one of SEQ ID NOs: 23-38 differing from any one of SEQ ID NOs: 23-38 by up to 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides without substantially reducing the ability of the complex to cleave the SOD1 mRNA.
  • the phrase “without substantially reducing the ability of the complex to cleave the SOD1 mRNA” means that the ability of the complex of the same Cas13 polypeptide and the same gRNA but with the spacer sequence variant of any one of SEQ ID NOs: 23-28 to cleave the SOD1 mRNA is reduced by no more than 30%, 25%, 20%, 15%, 10%, or 5%as compared to the complex of the same Cas13 polypeptide and the same gRNA but with the parental spacer sequence of any one of SEQ ID NOs: 23-28.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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) .
  • 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 (a sgRNA array, such as, DR-Spacer-DR-Spacer-DR) or separate sgRNAs.
  • a sgRNA array such as, DR-Spacer-DR-Spacer-DR
  • 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.
  • the Cas13 proteins may be delivered in complex with multiple RNA guides directed to different target RNAs.
  • 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
  • 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.
  • 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.
  • 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) .
  • the direct repeat length is 36 nucleotides.
  • 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.
  • 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) .
  • the gRNA comprises or is any one of SEQ ID NO: 7-22 and 55.
  • 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 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) .
  • 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.
  • the stimuli for the inducible systems include, e.g., electromagnetic radiation, sound energy, chemical energy, and/or thermal energy.
  • the transcription of gRNA can be modulated by a promoter that is a ubiquitous, tissue-specific, cell-type specific, constitutive, or inducible promoter.
  • the promoter is selected from a group consisting of a Cbh promoter, a Cba promoter, a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, a retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a ⁇ -actin promoter, an elongation factor 1 ⁇ short (EFS) promoter, a ⁇ glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (Ie) enhancer and/or promoter, a chicken ⁇ -actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1 ⁇ -subunit (EF1
  • the RNA pol III promoter is U6 (such as SEQ ID NO: 68) , H1, 7SK, or a variant thereof.
  • 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.
  • 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) .
  • FKBP small molecule two-hybrid transcription activations systems
  • LITE Light Inducible Transcriptional Effector
  • the sequences and the lengths of the gRNAs described herein can be optimized.
  • 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.
  • 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.
  • the gRNA has two or more aptamer sequences that are specific to the same adaptor proteins.
  • the two or more aptamer sequences are specific to different adaptor proteins.
  • the adaptor proteins can include, e.g., MS2, PP7, Q ⁇ , F2, GA, fr, JP501, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ⁇ kCb5, ⁇ kCb8r, ⁇ kCb12r, ⁇ kCb23r, 7s, and PRR1.
  • the aptamer is selected from binding proteins specifically binding any one of the adaptor proteins as described herein.
  • 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 SOD1 mRNA 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 Q ⁇ , F2, GA, fr, JP501, MS2, M12, R17, BZ13, JP34, JP500, KU1, M11, MX1, TW18, VK, SP, FI, ID2, NL95, TW19, AP205, ⁇ Cb5, ⁇ Cb8r, ⁇ Cb12r, ⁇ Cb23r, 7s and PRR1.
  • the bacteriophage coat protein is MS2.
  • 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.
  • the target RNA sequence is a part of the target RNA, for example, the target sequence is a stretch of 15-60, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, such as, 30, contiguous nucleotides of the target RNA.
  • the target RNA is associated with a condition or disease.
  • the systems described herein can be used to treat a condition or disease associated with the RNA (such as ALS) by targeting the RNA (e.g., SOD1 mRNA) .
  • 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 SOD1 mRNA) .
  • the target RNA is an mRNA (including pre-mRNA and mature mRNA) encoding a condition or disease associated protein.
  • a protein may be either a wild type protein or a mutated protein thereof, both may be naturally-occuring.
  • the term “mRNA” includes wild type mRNA and mutated mRNA thereof in vivo, both may be naturally-occuring, or any transcripts or isoforms produced by alternative promoter usage, alternative splicing, and/or alternative initiation therefrom.
  • the wild type mRNA encodes a wild type protein, and the mutanted mRNA encode a mutated protein.
  • the target RNA is an mRNA (including pre-mRNA and mature mRNA) encoding SOD1, i.e., SOD1 mRNA, such as human SOD1, i.e., human SOD1 mRNA, including wild type human SOD1 mRNA of SEQ ID NO: 73, Accession No: NM_000454.5 (it is understood that the sequence shown as NM_000454.5 in NCBI GenBank website recites the DNA version of the mRNA where “u” in the mRNA is replaced with “t” ) incorporated herein by reference, and any mutated mRNA of the wild type human SOD1 mRNA, which may be naturally-occuring and may or may not be pathogenic, or any transcripts or isoforms produced by alternative promoter usage, alternative splicing, and/or alternative initiation therefrom.
  • SOD1 mRNA such as human SOD1, i.e., human SOD1 mRNA, including wild type human SOD1 mRNA of SEQ ID NO
  • the function of the SOD1 protein expressed therefrom may be the same as that expressed from the wild type SOD1 mRNA, then the mutated SOD1 mRNA may not be pathogenic; when the mutation in such a mutated SOD1 mRNA is a missense mutation, leading to the expression of a misfunctional SOD1 protein mutant, then the mutated SOD1 mRNA may be pathogenic and would be desired to be corrected.
  • the target sequence is a part of human SOD1 mRNA, for example, the target sequence is a stretch of 15-60, or 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, or 60, such as, 30, contiguous nucleotides of SEQ ID NO: 73.
  • the target sequence (1) comprises or is any one of SEQ ID NO: 39-45; (2) comprises or is at least 15 contiguous nucleotides of any one of SEQ ID NO: 39-54; or (3) differs from any one of SEQ ID NO: 39-45 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: 39-45 over SEQ ID NO: 73.
  • 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) .
  • the complex further comprises the target RNA (such as a SOD1 mRNA) bound by the gRNA.
  • target RNA such as a SOD1 mRNA
  • a cell or a progeny thereof comprising the AAV vector genome as described herein, the rAAV viral particle as described herein, or the sgRNA as described herein.
  • the invention also provides a cell comprising any of the complex of the invention.
  • the cell is a prokaryote.
  • the cell is a eukaryote, such as, a mouse, monkey, or human cell.
  • 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.
  • 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.
  • the RNA is SOD1 mRNA.
  • SOD1 mRNA may include both wild type, normal SOD1 mRNA that is not pathogenic and mutated, abnormal SOD1 mRNA that is pathogenic, or the mutated, abnormal SOD1 mRNA only.
  • the mutated, abnormal SOD1 mRNA may be SOD1-G93A mRNA as tested in the animal model of Example 2 herein. It is the presence of such mutated, abnormal SOD1 mRNA that leads to the SOD1-associated diseases or disorders discussed herein.
  • an acceptable approach is to knock down both the normal and abnormal SOD1 mRNA, while the down-expression of overall SOD1 protein may be compensated or made up naturally by the other genes.
  • the disease or disorder associated with the RNA is SOD1-assocaited diseases, i.e., SOD1 gene-associated diseases.
  • the SOD1-assocaited disease is Amyotrophic lateral sclerosis (ALS) .
  • ALS Amyotrophic lateral sclerosis
  • a pharmaceutical composition comprising the rAAV vector genome as described herein, or the rAAV viral particle as described herein, and a pharmaceutically acceptable excipient.
  • a method of treating a disease or disorder associated with SOD1 in a subject in need thereof comprising administering to the subject a therapeutically effective amount of the rAAV vector genome as described herein, the rAAV 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 said SOD1 causative of the disease or disorder.
  • the administrating comprises contacting a cell with the therapeutically effective amount of the rAAV vector genome as described herein, the rAAV viral particle as described herein, or the pharmaceutically composition as described herein.
  • the cell is located in the CNS of the subject.
  • the disease or disorder is ALS.
  • the administration comprises intrathecal administration.
  • the subject is a human.
  • the subject is not a human.
  • the expression of SOD1 in the cell is decreased in comparison to a cell having not been contacted with the rAAV vector genome as described herein, the rAAV viral particle as described herein, or the pharmaceutically composition as described herein.
  • the expression of SOD1 is decreased in the subject by 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 expression of SOD1 in the subject prior to administration.
  • 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.
  • 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.
  • 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.
  • the modification may lead to increased transcription /translation /expression of the RNA product.
  • the modification may lead to decreased transcription /translation /expression of the RNA product.
  • 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) .
  • 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) .
  • 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.
  • fish such as salmon
  • bird such as poultry bird, including chick, duck, goose
  • reptile shellfish
  • shellfish e.g., oyster, claim, lobster, shrimp
  • a related aspect provides cells or progenies thereof modified by the methods of the invention using the CRISPR systems described herein.
  • the cell is modified in vitro, in vivo, or ex vivo.
  • the cell is a stem cell.
  • the cell is not an embryonic stem cell.
  • 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.
  • 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.
  • RNP ribonucleoprotein
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • AAV adeno-associated viruses
  • the proteins and one or more gRNAs can be packaged into one or more vectors, e.g., plasmids or viral vectors.
  • 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, ⁇ phage, T5 phage, T7 phage, T3 phage, ⁇ 29, M13, MS2, Q ⁇ , and ⁇ X174.
  • 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) .
  • the serotype is a AAV9 mutant, AAV9-M8 with a VP1 sequence of SEQ ID NO: 82, as described in PCT/CN2021/106935.
  • the vectors e.g., plasmids or viral vectors (e.g., AAV viral vectors)
  • the 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.
  • the administration is conducted by injection.
  • the AAV viral particle of the invention is delivered through intrathecal injection.
  • the delivery is by one intrathecal injection.
  • 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.
  • 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.
  • systemic e
  • Delivery may be either via a single dose, or multiple doses.
  • 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.
  • 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+10,
  • 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.
  • 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.
  • the delivery is via plasmids.
  • the dosage can be a sufficient number of plasmids to elicit a response.
  • 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.
  • 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.
  • the delivery is via nanoparticles or exosomes.
  • exosomes have been shown to be particularly useful in delivery RNA.
  • CRISPR-associated proteins are linked to the CRISPR-associated proteins.
  • the CRISPR-associated proteins and/or gRNAs are coupled to one or more CPPs to effectively transport them inside cells (e.g., plant protoplasts) .
  • 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.
  • 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 ⁇ 3 signal peptide sequence, polyarginine peptide Args sequence, Guanine rich-molecular transporters, and sweet arrow peptide.
  • Tat which is a nuclear transcriptional activator protein required for viral replication by HIV type 1
  • FGF Kaposi fibroblast growth factor
  • FGF Kaposi fibroblast growth factor
  • integrin ⁇ 3 signal peptide sequence integrin ⁇ 3 signal peptide sequence
  • polyarginine peptide Args sequence e.g., in et al., “Prediction of cell-penetrating peptides, ” Methods Mol.
  • 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.
  • kits 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.
  • 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.
  • kits comprising the AAV vector genome as described herein, the rAAV viral particle as described herein, the sgRNA as described herein, or the cell or a progeny thereof as described herein.
  • 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 SOD1 separated by DR sequences.
  • 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.
  • 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.
  • buffers may include one or more of PBS, HEPES, Tris, MOPS, Na 2 CO 3 , NaHCO 3 , NaB, or combinations thereof.
  • the reaction condition includes a proper pH, such as a basic pH. In certain embodiments, the pH is between 7-10.
  • any one or more of the kit components may be stored in a suitable container.
  • 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” .
  • 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.
  • 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.
  • 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.
  • recombinant AAVs may be produced using the triple transfection method (described in detail in U.S. Pat. No. 6,001,650) .
  • 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.
  • 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.
  • the subject rAAV viral particle is produced using a baculovirus expression system packaged in insect cells such as Sf9 cells.
  • 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) .
  • the vector titer is above 1 ⁇ 10 9 , above 5 ⁇ 10 10 , above 1 ⁇ 10 11 , above 5 ⁇ 10 11 , above 1 ⁇ 10 12 , above 5 ⁇ 10 12 , or above 1 ⁇ 10 13 vg/ml.
  • RNA sequence as a vector genome into a 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.
  • 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.
  • the deoxyribonucleotides in the DNA sequence are the corresponding ribonucleotides in the RNA sequence (e.g., dT is equivalent to U, and dA is equivalent to A) and/or the element in the DNA sequence is replaced with the corresponding element with a corresponding function in the RNA sequence or omitted because its function is unnecessary in the RNA sequence and/or an additional element necessary for the
  • 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.
  • an RNA sequence can be transcribed from the DNA coding sequence, and optionally further a protein can be translated from the transcribed RNA sequence as necessary.
  • the RNA coding sequence per se can be 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.
  • 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) .
  • a (e.g., Cas13, NLS) coding sequence 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
  • 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.
  • a (e.g., sgRNA) coding 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.,
  • 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.
  • promoters to drive transcription of DNA sequences would be unnecessary and can be omitted at least partly.
  • polyA signal sequence would be unnecessary and can be omitted, while a polyA tail can be introduced.
  • 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.
  • This example demonstrates the high in vitro knockdown efficiency of hSOD1 expression by the subject CRISPR-hfCas13f v1 and v2 system.
  • the subject CRISPR-hfCas13f v1 systems comprised a hfCas13f v1 protein (SEQ ID NO: 2, Cas13f. 1-Y677A mutant) and a single hSOD1-targeting sgRNA (one of “sgRNA [hSOD1] 1-7” or “sg1-7” hereinafter, SEQ ID NO: 7-13) comprising two Direct Repeats (each of SEQ ID NO: 6) and a Spacer (one of Spacer 1-7, SEQ ID NO: 23-29) targeting the target sequence (one of target sequences 1-7, SEQ ID NO: 39-45) of hSOD1 mRNA.
  • the subject CRISPR-hfCas13f v2 systems comprised a hfCas13f v2 protein (SEQ ID NO: 4, Cas13f. 1-Y666A, Y677A mutant) and a single hSOD1-targeting sgRNA (one of “sgRNA [hSOD1] 8-16” or “sg8-16” hereinafter, SEQ ID NO: 14-22) comprising two Direct Repeats (each of SEQ ID NO: 6) and a Spacer (one of Spacer 8-16, SEQ ID NO: 30-38) targeting the target sequence (one of target sequences 8-16, SEQ ID NO: 46-54) of hSOD1 mRNA.
  • SEQ ID NO: 4 Cas13f. 1-Y666A, Y677A mutant
  • SEQ ID NO: 14-22 comprising two Direct Repeats (each of SEQ ID NO: 6) and a Spacer (one of Spacer 8-16, SEQ ID NO: 30-38) targeting the target sequence
  • sgRNA [NT] As a negative control, a non-targeting-sgRNA ( “sgRNA [NT] ” or “NT” hereinafter, SEQ ID NO: 56) comprising two Direct Repeats (each of SEQ ID NO: 17) and a non-targeting-Spacer (LacZ, SEQ ID NO: 57) was used in place of the sgRNA [hSOD1] .
  • shRNAs targeting hSOD1 were also used.
  • the plasmid constructs for the CRISPR-hfCas13f v1 or v2 systems and the shRNAs along with a mCherry reporter are shown in Fig. 1, Fig. 3 and Fig. 5a.
  • the hfCas13f v1 encoding sequence in the constructs was flanked with two NLS encoding sequences encoding the same NLS (SEQ ID NO: 66) .
  • the Spacers were also used in combiations.
  • the hfCas13f v1 protein (SEQ ID NO: 2) and a sgRNA [hSOD1] containing two Spacers (5&7, SEQ ID NO: 55) were encoded together with a mCherry reporter (SEQ ID NO: 70) into the same plamid construct as shown in Fig. 3.
  • HEK293T cells were cultured in Dulbecco's modified eagle medium (DMEM) containing 10%fetal bovine serum (FBS) and penicillin/streptomycin, and maintained at 37°C with 5%CO 2 .
  • DMEM Dulbecco's modified eagle medium
  • FBS fetal bovine serum
  • penicillin/streptomycin penicillin/streptomycin
  • the cultured cells were then seeded onto 6-well plates- (8.0E+5 cells/well) and transfected with 2 ⁇ g treatment plasmids (encoding the hSOD1-targeting CRISPR-hfCas13f v1 or CRISPR-hfCas13f v2 system) or control plasmids (encoding shRNA or non-targeing CRISPR-hfCas13f v1 or non-targeing CRISPR-hfCas13f v2 system) using polyethylenimine (
  • hSOD1 mRNAs were normalized to the levels of a reference gene 18s ribosomal ribonucleic acid (18s rRNA) . Relative expression levels were calculated using the 2 - ⁇ Ct method. Each experiment was performed in triplicate and repeated three times.
  • CRISPR-hfCas13f v1 7 sgRNAs targeting mature hSOD1 mRNA (sg1-7) were designed.
  • hSOD1-targeting CRISPR-hfCas13f v1 systems with sg2-7 decreased human SOD1 mRNA level by >50%relative to the negative control, with the most active sgRNAs 5 and 6 found to reduce human SOD1 mRNA level by >90% (Fig. 2 and Table 1) .
  • the hSOD1-targeting CRISPR-hfCas13f v1 system with paired Spacers 5 and 7 decreased hSOD1 mRNA level by 98%relative to the negative control (Fig. 4 and Table 2) .
  • Sg5 and 7 were selected for use in combination instead of sg5 and 6 because the target sequences for sg5 and 6 are spatially close on the target hSOD1 mRNA.
  • CRISPR-hfCas13f v2 system could be used to reduce the expression of hSOD1 gene
  • another 9 sgRNAs targeting mature hSOD1 mRNA sg8-16
  • hSOD1-targeting CRISPR-hfCas13f v2 systems with sg10-16 decreased human SOD1 mRNA level by >90%relative to the negative control (Fig. 5b and Table 3) .
  • treatment and control transgene plasmids for a variant of AAV9-M8 packaging encoding hfCas13f v2 and sgRNA [hSOD1] 5&7 or sgRNA [NT] were constructed, respectively, as shown in Fig. 6.
  • Both the treatment and control rAAV9-M8 particles herein were produced 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 AAV9-M8 capsids to form the genomes inside the capsids, and together the genome and the capids constituted the rAAV9-M8 particles.
  • the HEK293T cells were cultured in competent DMEM medium, and the cells were plated 14-16 hrs before transfection of the plasmids. Shortly before transfection, the culture medium was replaced with fresh DMEM containing 2%FBS. PEI was used as the transfection reagent. The transfected HEK293T cells were harvested from the media at 72 hours post translation. The treatment and control rAAV9-M8 particles were purified from the cells by using iodixanol density gradient ultracentrifugation.
  • RT-qPCR was used with a pair of hfCas13f v2 primers specific for the hfCas13f v2 sequence on the genomes to detect the genome titer of any genomes packaged in the treatment and control rAAV9-M8 particles.
  • hfCas13f v2 forward primer 5'-GAAATCGTGGAAGAACTGGTGG-3' (SEQ ID NO: 80) ;
  • hfCas13f v2 reverse primer 5'-ATAAGCGTAATCTGGAACATCGTA-3' (SEQ ID NO: 81) .
  • mice Male humanized SOD1-G93A mice (B6. Cg-Tg (SOD1*G93A) 1Gur/J) with incorporated human SOD1 gene bearing G93A disease-associated mutation were purchased from Jackson Laboratory (Jax: 004435) and female C57BL/6J mice (6-8 weeks) were purchased from the Shanghai SLAC Laboratory Animal Co. Ltd. and housed in the in-house animal facility on 12h: 12h light/dark cycle with food and water ad libitum. 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 mice were each injected slowly within 6-12 hours after birth using 10 ⁇ l syringes (1 inch needle, 30 degrees bevel; Hamilton Company, Reno, NV) via spinal cord into the lumbar of spinal cord with 3 ⁇ l of treatment or control rAAV9-M8 particles (P1: 6 ⁇ 10 11 vg/mL, 2 ⁇ 10 12 vg/mL, and 6 ⁇ 10 12 vg/mL) in phosphate-buffered saline (1 ⁇ PBS) buffer with 0.01%Non-ionic Surfactant, Thermo Fisher, and 0.25%Fast Green, Sigma, with a white line (spinal cord) down their back at 5 mm from the base of the tail observed.
  • the injection started almost parallel to the back when the needle punctured the skin, then the needle angle was increased to 50-60° to insert the needle into the vertebral column. After the injection, the neonatal mice were returned to the home cage.
  • mice age The body weight was recorded between 4 and 15 weeks (30 and ⁇ 105 days, respectively) of mice age.
  • Behavioral test namely, Grip Strength Test was conducted in a sound proof room with a neutral environment. After the test, all the mice were returned to their home cages. All the tests were conducted during the light phase of the light/dark cycle. Mice were given a 1-hour habituation period after transportation to the behavioral room before the test.
  • the test device was cleaned with 75 percent ethanol after a mouse finished a test. The test was performed at least once a week, according to the disease stage, using a grip-strength meter (47200, Ugo Basile) . The device was locked to a heavy base that allowed the device to remain still and parallel to the surface. To perform the test, mice were gently pulled back with a steady force until both paws released the bar. Peak tension in grams was recorded for six consecutive trials. The device was set on pounds force to achieve more sensitivity.
  • mice were euthanized with pentobarbital (100 mg/kg) and perfused with 20 mL ice-cold PBS and then sacrificed. The spinal cord and liver were then dissected. The spinal cord was divided into cervical, thoracic, and lumbar segments, and each segment divided into three pieces. One piece of spinal cord was used for immunostaining. The other two pieces of spinal cord and the liver were used for Western Blot analysis.
  • the spinal cord was dissected from the sacrificed mice, post-fixed overnight in 4%paraformaldehyde (PFA) at 4°C.
  • PFA paraformaldehyde
  • the spinal cord was cryoprotected using 30%sucrose/PBS solution overnight at 4°C, and washed with PBS and embedded in Tissue-Tek O. C. T. compound.
  • the spinal cord was sectioned at 30 ⁇ m using a Leica CM3050 S cryostat.
  • the spinal cord sections were stored in the freezing medium which contains 30%sucrose and 30%ethanediol, 11.36%Na 2 HPO 4 and 2.4%NaH 2 PO 4 .
  • the sections were washed three times with PBS and then incubated with PBS containing 0.1%Triton X-100 and 10%fetal bovine serum (FBS) for 1 hour at room temperature. Subsequently, the sections were incubated with the appropriate dilutions of primary antibodies in PBS with 0.1%Triton X-100 and 5%FBS overnight at 4°C. The sections were washed three times with PBS and then incubated with incubated with the appropriate dilutions of secondary antibodies in PBS with 0.1%Triton X-100 and 5%FBS for 1 h at room temperature. Sections were mounted on slides, dried at room temperature and cover-slipped in ProLong Gold antifade mounting medium, with DAPI (Invitrogen) .
  • DAPI Invitrogen
  • the spinal cord was dissected from the sacrificed mice and washed with ice-cold PBS and harvested in protein lysis buffer and 1 ⁇ protease inhibitor cocktail (Roche) .
  • the lysates were centrifuged at 12,000 rpm for 15 minutes at 4°C, and protein 10 ⁇ g was separated by denaturing with 4-20%Bis-Tris Plus Gels (Tanon) and transferred onto PVDF membranes (Millipore) .
  • Blots were blocked in 5%non-fat dry milk in Tris-buffered saline at pH 7.4 (20 mM Tris, 150 mM NaCl and distilled H 2 O) with 1%Tween (TBST) and then incubated with primary antibody overnight at 4°C.Blots were washed three times with TBST, and then incubated with anti-rabbit IgG horseradish peroxidase (HRP) -conjugated secondary antibody for 1 hour at room temperature. Blots were washed three times with TBST, and then viewed under the SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) .
  • SOD1 (rabbit, Abcam, no. Ab13498, dilution 1: 1000)
  • HA rabbit, Cell Signaling, no. 5017, dilution 1: 1000
  • Tubulin ⁇ (rabbit, Bioworld, AP0064, dilution 1: 1000)
  • HRP horseradish peroxidase
  • ImageJ https: //imagej. en. softonic. com
  • Tubulin ⁇ was also detected as a control.
  • the efficiency of AAV9-M8 transduction was first evaluated in the SOD1-G93A mouse model.
  • the models were injected intrathecally at postnatal day 1 (to be referred to as P1) with AAV9-M8-hfCas13f v2-SOD1 particles, which hfCas13f v2 was fused with triple Human influenza hemagglutinin (HA) tag (Fig. 7) .
  • the transduction efficiency was high in the neurons of the SOD1-G93A mouse models, with HA expressed in 85%lumbar spinal cord neurons of P1-injected ventral horn (indicated by immunoreactivity for neuronal marker, NeuN) and HA expressed in 60%motor neurons (indicated by immunoreactivity for motor neurons marker, ChAT) .
  • the transduction efficiency was low in the astrocytes of the SOD1-G93A mouse models (indicated by immunoreactivity for astrocytes marker, GFAP) , with HA expressed in 8%astrocytes and HA expressed in 1%microglia (indicated by immunoreactivity for microglial marker Iba1) (Fig. 8 and Table 4) . It was therefore well confirmed the desired neuron specificity of the AAV9-M8 delivery system.
  • hSOD1 protein expression by Western blot showed notable reduction in hSOD1 protein expression in the cervical and lumbar spinal cord of SOD1-G93A mice, 4 weeks after injection with AAV9-M8-hfCas13f v2-SOD1 into the lumbar spine. There was no effect on hSOD1 expression in peripheral organ liver that do not project axons into the spinal cord (Fig. 9) .
  • SOD1 expression within transduced motor neurons identified by HA-and choline acetyltransferase (ChAT) -expressing cells was reduced compared with the surrounding neurons that had not been transduced to express viral-encoded HA (Fig. 10) .
  • the data showed that the CRISPR-hfCas13f v2-sgRNA [SOD1] system could both in vitro and in vivo specifically knockdown the expression of human SOD1 gene and significantly improve the grip strength and extend the survival of the disease mice models, showing the promising prospect of treating SOD1-ALS in human.

Abstract

L'invention concerne des systèmes CRISPR-Cas13 et un procédé de traitement de maladies associées au SOD1 avec celui-ci.
PCT/CN2022/114021 2021-08-22 2022-08-22 Système crispr-cas13 pour le traitement de maladies associées au sod1 WO2023025103A1 (fr)

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PCT/CN2021/121926 WO2022068912A1 (fr) 2020-09-30 2021-09-29 Système crispr/cas13 modifié et ses utilisations
CNPCT/CN2022/083476 2022-03-28
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