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WO2021053582A1 - Vecteurs crispr à auto-inactivation tout-en-un - Google Patents

Vecteurs crispr à auto-inactivation tout-en-un Download PDF

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WO2021053582A1
WO2021053582A1 PCT/IB2020/058683 IB2020058683W WO2021053582A1 WO 2021053582 A1 WO2021053582 A1 WO 2021053582A1 IB 2020058683 W IB2020058683 W IB 2020058683W WO 2021053582 A1 WO2021053582 A1 WO 2021053582A1
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nucleic acid
cas9
aav
sequence encoding
crispr
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Seshidhar Reddy Police
Yanfei Yang
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CRISPR Therapeutics AG
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CRISPR Therapeutics AG
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
    • C12N2710/00011—Details
    • C12N2710/14011—Baculoviridae
    • C12N2710/14041—Use of virus, viral particle or viral elements as a vector
    • C12N2710/14044—Chimeric viral vector comprising heterologous viral elements for production of another viral vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • 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

  • the present disclosure relates to self-inactivating vectors encoding a targeting nuclease, methods of preparing said vectors, and methods of using said vectors.
  • RNA-guided clustered regularly interspersed short palindromic repeats have emerged as powerful genome modification tools due to their simplicity, target design plasticity, and multiplex targeting capacity.
  • CRISPR nucleases can increase the number of non-specific (off target) cleavage events and/or can elicit immune responses. Thus, it would be beneficial to limit the duration of nuclease expression following delivery.
  • FIG. 1 presents an image of a gel comprising long range PCR products of various SIN vectors.
  • Lane 1 1011CMV non-SIN control vector produced using HEK 293 T cells showing intact genome;
  • Lane 3 Kb ladder;
  • Lane 4 1014Ck8e- AcrA5 - SIN vector produced using HEK293T cell line expressing anti-CRISPR protein AcrIIA5;
  • Lane 5 1041CK8e-Bac SIN vector produced using baculovirus-Sf9 system showing intact genome.
  • nucleic acid comprising a sequence encoding a CRISPR nuclease and a sequence encoding a guide RNA (gRNA), wherein the sequence encoding the CRISPR nuclease comprises at least one intron, and the at least one intron comprises a binding site that is recognized by the CRISPR nuclease and gRNA.
  • gRNA guide RNA
  • the sequence encoding the CRISPR nuclease is codon optimized for expression in eukaryotic cells of interest.
  • the CRISPR nuclease is linked to at least one nuclear localization signal.
  • the intron in the sequence encoding the CRISPR nuclease is a mammalian intron, an engineered intron, or an artificial intron.
  • the binding site in the intron is about 17 nucleotides to about 23 nucleotides in length and is followed by a protospacer adjacent motif (PAM) sequence.
  • PAM protospacer adjacent motif
  • the sequence encoding the CRISPR nuclease is operably linked to a Pol II promoter, and the sequence encoding a guide RNA is operably linked to a Pol III promoter.
  • the CRISPR nuclease is a Cas9 nuclease or a variant having at least 90% sequence identity to the Cas9 nuclease.
  • the Cas 9 nuclease is Staphylococcus aureus Cas9, Neisseria meningitidis Cas9, or Campylobacter jejuni Cas9.
  • the sequence encoding the CRISPR nuclease and the sequence encoding the gRNA are flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).
  • AAV adeno-associated virus
  • Another aspect of the present disclosure provides a baculovirus expression vector comprising the nucleic acid as described above.
  • a further aspect of the present disclosure encompasses a recombinant AAV particle comprising the nucleic acid as described above and at least one capsid protein.
  • Still another aspect of the present disclosure provides a method for producing the nucleic acid as described above, wherein the method comprises introducing into a packaging cell a recombinant baculovirus comprising the sequence encoding the CRISPR nuclease, which is interrupted by the intron, and the sequence encoding the guide RNA, which are flanked by the AAV ITRs.
  • a functional CRISPR nuclease is not produced in the packaging cell.
  • the packaging cell produces AAV particles comprising the nucleic acid described above that is encapsidated by at least one AAV capsid protein.
  • the method further comprises expressing AAV replication (Rep) and capsid (Cap) proteins in the packaging cell.
  • a packaging recombinant baculovirus sequence encoding AAV Rep and Cap proteins can be introduced into the packaging cell.
  • the packaging cell comprises sequence encoding AAV Rep and Cap proteins stably integrated into its genome, wherein said sequence encoding AAV Rep and Cap proteins is operably linked to an inducible promoter.
  • the method further comprises introducing into the packaging cell a helper recombinant baculovirus comprising sequence encoding AAV packaging components.
  • the recombinant baculovirus encoding the CRISPR nuclease and gRNA is derived from Autographa califomica multiple nucleopolyhedrovirus (AcMNPV).
  • the packaging cell is a Spodoptera frugiperda Sf9 cell.
  • a further aspect of the present disclosure encompasses a method for temporally limiting expression of a CRISPR nuclease in eukaryotic cells, wherein the method comprises introducing into the eukaryotic cells the nucleic acid described above or the recombinant AAV particle described above.
  • the intron Upon expression of the nucleic acid, the intron is excised and a CRISPR nuclease is produced, and the CRISPR nuclease complexes with the guide RNA to form a CRISPR system, wherein the CRISPR system cleaves the target genomic locus in the eukaryotic cells leading to an edited genomic locus, and the CRISPR system cleaves the intron in the sequence encoding the CRISPR nuclease in the nucleic acid, thereby inactivating the nucleic acid
  • the nucleic acid or the recombinant AAV particle is inactivated within about 1-3 days after being introduced into the eukaryotic cells, thereby temporally limiting expression of the CRISPR nuclease.
  • the present disclosure provides self-inactivating (SIN) CRISPR system vectors and methods of using the SIN CRISPR system vectors.
  • the SIN CRISPR system vectors comprise nucleic acid sequence encoding a CRISPR nuclease and nucleic acid sequence encoding a guide RNA (gRNA), wherein the nucleic acid sequence encoding the CRISPR nuclease comprises at least one intron.
  • gRNA guide RNA
  • the intron Upon expression of the vector in eukaryotic cells, the intron is spliced out such that a functional CRISPR nuclease is produced, which complexes with the gRNA to form a CRISPR system that cleaves the target locus and also cleaves the intron in the SIN vector, thereby limiting expression of the CRISPR nuclease.
  • the SIN CRISPR system vectors disclosed herein are recombinant AAV vectors. Moreover, all of the sequences needed for genome editing and self-inactivation are provided within one vector (z.e., all-in-one SIN vector).
  • recombinant baculovirus expression vectors for generating the SIN AAV vectors disclosed herein, wherein the recombinant baculovirus expression vectors comprise nucleotide sequence encoding any of the SIN CRISPR system vectors disclosed herein. Also provided herein are methods for generating the SIN AAV vectors in a baculovirus expression system, wherein the intron in the nucleotide sequence encoding the CRISPR nuclease is not recognized and not excised such that a functional CRISPR nuclease is not generated in the baculovirus expression system.
  • One aspect of the present disclosure encompasses self-inactivating (SIN) CRISPR system vectors.
  • the SIN CRISPR system vectors provide a CRISPR system for genome editing, and also are able to self-cleave, thereby temporally limiting expression of the CRISPR system in eukaryotic cells.
  • a CRISPR system comprises a guide RNA (gRNA) to target the system to a specific DNA sequence and a nuclease that cleaves the targeted DNA sequence.
  • the gRNA drives sequence recognition and specificity of the CRISPR system through Watson- Crick base pairing with a ⁇ 20 nucleotide (nt) target sequence in the target locus, wherein the target sequence is adjacent to a specific short DNA motif referred to as a protospacer adjacent motif (PAM).
  • PAM protospacer adjacent motif
  • the gRNA forms an RNA-duplex and stem-loop structure that is bound by the CRISPR nuclease to form the catalytically active ribonucleoprotein (RNP) CRISPR system.
  • RNP catalytically active ribonucleoprotein
  • the SIN CRISPR system vectors disclosed herein comprise a nucleic acid sequence encoding a CRISPR nuclease and a nucleic acid sequence encoding a gRNA, wherein the nucleic acid sequence encoding the CRISPR nuclease comprises at least one intron.
  • the intron in the sequence encoding the CRISPR nuclease comprises a binding site (also called a SIN site) that is recognized and bound by the CRISPR system.
  • the intron is spliced out of the sequence encoding the CRISPR nuclease, thereby producing a functional CRISPR nuclease, which complexes with the gRNA to form a CRISPR system.
  • the CRISPR system 1) is directed to and cleaves the target genomic locus for genome editing, and 2) is targeted to and cleaves the intron in the vector, thereby inactivating the vector in eukaryotic cells.
  • all the sequences needed for genome editing and inactivation of the vector are present within the same vector.
  • the self-inactivating vector disclosed herein is an all-in-one SIN vector.
  • the SIN CRISPR system vectors disclosed herein are designed to be generated in baculovirus expression systems. During propagation of a SIN CRISPR system vector in a baculovirus expression system, the intron in the sequence encoding the CRISPR nuclease is not recognized and a functional CRISPR nuclease is not generated. Thus, the SIN CRISPR system vector is not cleaved and not inactivated in the baculovirus expression system, but the SIN CRISPR vector system can be propagated in the baculovirus expression system.
  • the SIN vectors disclosed herein comprise a nucleic acid sequence encoding a CRISPR nuclease.
  • a nuclease is an enzyme that introduces a break in a double stranded nucleic acid sequence.
  • the break can be double stranded or single stranded.
  • the CRISPR nuclease introduces a double stranded break by cleaving both strands of the double stranded nucleic acid sequence.
  • the CRISPR nuclease can be a nickase in which one of the two nuclease domains within the CRISPR protein is inactivated such that the CRISPR nickase cleaves one strand of the double stranded nucleic acid sequence.
  • the CRISPR nuclease can be naturally occurring, a variant thereof, or a modified or engineered version thereof.
  • the CRISPR nuclease can be modified or engineered to have altered activity, specificity, and/or stability.
  • the CRISPR nuclease encoded by the vectors disclosed herein is a Cas9 nuclease or a variant/version thereof having at least 85%, at least 90%, at least 92%, at least 94%, at least 96%, at least 98%, or at least 99.9% sequence identity with a Cas9 nuclease.
  • the Cas9 nuclease encoded by said vectors generally is a small Cas9 protein, e.g., the Cas9 nuclease contains less than about 1200 amino acids (aa).
  • the Cas9 nuclease can be Staphylococcus aureus Cas9 (SauCas9; 1053 aa), Neisseria meningitidis Cas9 (NmeCas9; 1082 aa), or Campylobacter jejuni Cas9 (CjeCas9; 984 aa).
  • the Cas9 nuclease can b Azospirillum B510 Cas9 (1168 aa), Campylobacter lari CF89-12 Cas9 (1103 aa), Corynebacter diphtheriae Cas9 (1084 aa), Eubacterium ventriosum Cas9 (1107 aa), Gluconacetobacter diazotrophicus Cas9 (1150 aa), Lactobacillus farciminis Cas9 (1126 aa), Neisseria cinerea Cas9 (1082 aa), Nitratitr actor salsuginis DSM 16511 Cas9 (1132 aa), Parvibaculum lavamentivorans Cas9 (1037 aa), Roseburia intestinalis Cas9 (1128 aa), Sphaerochaeta globus Cas9 (1179 aa), Streptococcus pasteurianus Cas9 (1130 aa), Streptococcus thermophilus CRISPR
  • the CRISPR nuclease can be engineered by one or more amino acid substitutions, deletions, and/or insertions to have improved targeting specificity, improved fidelity, altered PAM specificity, decreased off-target effects, and/or increased stability.
  • Non-limiting examples of one or more mutations that improve targeting specificity, improve fidelity, and/or decrease off-target effects include N497A, R661A, Q695A, K810A, K848A, K855A, Q926A, K1003A, R1060A, and/or D1135E (with reference to the numbering system of SpyCas9).
  • the CRISPR nuclease generally is linked to at least one nuclear localization signal (NLS) at the or within about 50 amino acids of N-terminal end, at or within about 50 amino acids of the C-terminal end, or both.
  • NLSs are well known in the art.
  • the NLS can be the SV40 Large T-antigen NLS, nucleoplasmin NLS, c-Myc NLS, or derivatives thereof.
  • the linkage between the CRISPR nuclease and the NLS can be a direct or it can be indirect via an intervening linker sequence. Suitable linker sequences are well known in the art.
  • the sequence encoding the CRISPR nuclease is codon optimized for expression in eukaryotic cells of interest.
  • the sequence can be codon optimized for expression in human cells. Codon optimization programs are widely available.
  • the sequence encoding the CRISPR nuclease in the vectors disclosed herein is operably linked to a promoter sequence for expression in the cells of interest.
  • the sequence encoding the CRISPR nuclease is operably linked to a Pol II promoter.
  • the Pol II promoter can be a constitutively active promoter, for example, a cytomegalovirus (CMV) promoter, CMV immediate early promoter (CMVIE), a CAG promoter (a hybrid comprising the CMV enhancer fused to the chicken beta-actin promoter), actin promoters, elongation factor (EF)-l alpha promoter, SV40 early promoter, mouse mammary tumor virus long terminal repeat promoter, adenovirus major late promoter (Ad MLP), herpes simplex virus (HSV) promoter, Rous sarcoma virus (RSV) promoter, synthetic promoters, hybrid promoters, and the like.
  • CMV cytomegalovirus
  • CMVIE CMV immediate early promoter
  • CAG promoter a hybrid comprising the CMV enhancer fused to the chicken beta-actin promoter
  • actin promoters elongation factor (EF)-l alpha promoter
  • SV40 early promoter SV40 early promoter
  • the promoter can be a tissue-specific promoter that is active selectively or preferentially in particular cell populations over other cell populations.
  • tissue-specific promoter include muscle-specific promoters such as muscle creatine kinase (MCK), CK8e, or C5-12 promoters, CNS-specific promoters such as Synapsin 1, CaMKII alpha, GAD67, GAD65, or VGAT promoters, liver-specific promoter such as albumin promoter, lung- specific promoter such as SP-B, endothelial cell-specific promoter such as ICAM, hematopoietic cell-specific promoter such as IFN beta or CD45, and osteoblast-specific promoter such as OG-2.
  • the promoter can be an inducible promoter that responds to the presence of a specific compound, such as tetracycline.
  • the nucleic acid sequence encoding the CRISPR nuclease can also be operably linked to polyadenylation signal (e.g ., SV40 late polyadenylation signal, rabbit globin polyadenylation signal), enhancer sequences, and/or transcriptional termination signal (e.g., woodchuck hepatitis virus post-transcriptional regulatory element).
  • polyadenylation signal e.g ., SV40 late polyadenylation signal, rabbit globin polyadenylation signal
  • enhancer sequences e.g., SV40 late polyadenylation signal, rabbit globin polyadenylation signal
  • transcriptional termination signal e.g., woodchuck hepatitis virus post-transcriptional regulatory element
  • the sequence encoding the CRISPR nuclease is interrupted by at least one intron.
  • the sequence encoding the CRISPR nuclease can comprise one intron and two exons.
  • the sequence encoding the CRISPR nuclease can be interrupted by two or more introns.
  • the intron can be a mammalian intron (e.g., present in a mammalian protein-coding gene), an engineered (e.g., chimeric or hybrid) intron, or an artificial intron, provided the intron comprises the elements required for splicing.
  • Essential splicing elements include a donor site (5' end of the intron), a branch site (near the 3' end of the intron), and an acceptor site (3' end of the intron).
  • Non-limiting examples of suitable introns include 1) a chimeric intron comprising 5 -donor site from the first intron of the human b-globin gene and the branch and 3 '-acceptor site from the intron of an immunoglobulin gene heavy chain variable region (e.g ., pCI-Neo vector); 2) a hybrid intron comprising an adenovirus splice donor and an immunoglobulin G splice acceptor (Choi et al., Mol Cell Biol, 1991,
  • the intron can be 1) listed above.
  • the length of the intron can and will vary. In general, the intron can range in length from about 50 bp to about 250 bp. In certain embodiments, the length of the intron can range from about 50 bp to about 100 bp, from about 100 bp to about 150 bp, from about 150 bp to about 200 bp, or from about 200 bp to about 250 bp.
  • the intron can be inserted anywhere in the sequence encoding the CRISPR nuclease.
  • the intron can be introduced at the codon coding for Asn 580 of a Cas9 nuclease. Means for combining heterologous sequences are well known in the art.
  • the intron is engineered to contain a binding site that can be recognized by the CRISPR nuclease and gRNA encoded by the vector.
  • the binding site in the intron comprises a target sequence that is followed by a PAM sequence specific for the CRISPR nuclease, wherein the gRNA of the CRISPR system can hybridize to the complement of the target sequence in the intron.
  • the target sequence in the intron can range in length from about 17 nucleotides (nt) to about 23 nt, from about 18 nt to about 22 nt, or from about 19 nt to about 21 nt. In specific embodiments, the target sequence can be about 20 nt.
  • the length of the PAM sequence can range from about 2 nt to about 10 nt.
  • SauCas9 recognizes a 5 nt PAM (NGRRT)
  • NmeCas9 recognizes a 8 nt PAM (NNNNGATT)
  • CjeCa9 recognized a 8 nt PAM (NNNNRYAC), wherein N is A, C, G, or T; R is A or G; and Y is C or T.
  • NGRRT 5 nt PAM
  • NNNNGATT 8 nt PAM
  • CNNRYAC CjeCa9 recognized a 8 nt PAM
  • the SIN vectors disclosed herein also comprise a nucleic acid sequence encoding a gRNA.
  • a gRNA comprises a CRISPR repeat sequence (crRNA) comprising a spacer or guide sequence that hybridizes the complement of a target sequence, and a trans activating crRNA (tracrRNA) sequence.
  • crRNA CRISPR repeat sequence
  • tracrRNA trans activating crRNA
  • a portion of the crRNA sequence base pairs with a portion of the tracrRNA sequence to form a duplex, and the rest of the tracrRNA sequence can form secondary structure, e.g., at least one stem-loop structure(s), that mediates binding of gRNA to the CRISPR nuclease.
  • the crRNA can comprise an optional spacer extension sequence at the 5’ end, and the tracrRNA can comprise an optional tracrRNA extension sequence at the 3’ end.
  • a gRNA can be a single molecule (i.e., single molecule gRNA or sgRNA) or can comprise two separate molecules (e.g., crRNA and tracrRNA).
  • the gRNAs encoded by the vectors disclosed herein are single molecule gRNAs.
  • the spacer sequence at the 5’ end of the gRNA is a sequence that defines the target sequence of the target nucleic acid (e.g., a target genomic locus or the target intron sequence).
  • the target sequence is followed by a PAM sequence and the target sequence is cleaved by the CRISPR system.
  • the “target nucleic acid” is a double- stranded molecule; one strand comprises the target sequence and is referred to as the “PAM strand,” and the other complementary strand is referred to as the “non-PAM strand.”
  • PAM strand the target sequence that is located in the non-PAM strand of the target nucleic acid.
  • the spacer sequence of a gRNA interacts with a target nucleic acid in a sequence-specific manner via hybridization (i.e., base pairing).
  • the nucleotide sequence of the spacer thus varies depending on the target sequence of the target locus.
  • the spacer sequence of the gRNA can range in length from about 15 nt to about 25 nt. In various embodiments, the spacer sequence can range in length from about 16 nt to about 24 nt, from about 17 nt to about 23 nt, from about 18 nt to about 22 nt, from about 19 nt to about 21 nt. In some embodiments, the spacer sequence is 20 nt long. In general, the spacer sequence has at least about 90%, at least about 95%, or at least about 99% sequence identity to the target sequence in the target nucleic acid. In certain embodiments, the spacer sequence has 100% sequence identity to the target sequence.
  • a single molecule gRNA can comprise, in the 5' to 3' direction, an optional spacer extension sequence, a spacer sequence, a minimum crRNA sequence, a single-molecule guide linker, a minimum tracrRNA sequence, a 3’ tracrRNA sequence and an optional tracrRNA extension sequence.
  • the optional tracrRNA extension may comprise elements that contribute additional functionality (e.g, stability) to the guide RNA.
  • the guide linker links the minimum CRISPR repeat and the minimum tracrRNA sequence to form a hairpin structure.
  • the optional tracrRNA extension may comprise one or more hairpins.
  • the overall length of a single molecule gRNA can range from about 80 nt to about 250 nt.
  • the gRNA in some embodiments, can comprise one or more uracil residues at the 3’ end of the gRNA sequence.
  • the gRNA may comprise one (U), two (UU), three (UUU), four (UUUU) or more uracils at the 3’ end of the gRNA sequence.
  • the gRNA comprises 5, 6, 7, or 8 uracils at the 3’ end of the gRNA sequence.
  • the gRNA comprises 1 to 8, 2 to 8, 3 to 8, or 4 to 8 uracils at the 3’ end of the gRNA sequence.
  • the nucleic acid sequence encoding the gRNA in the vectors disclosed herein is operably linked to a promoter sequence for expression in the cells of interest.
  • the sequence encoding the gRNA is operably linked to a Pol III promoter.
  • suitable Pol III promoters include mammalian U6, U3, HI, and 7SK promoters.
  • the nucleic acid sequence encoding the gRNA can be operably linked to a Pol III transcription termination sequence (e.g ., a short run of T residues).
  • sequence encoding the gRNA can be in the same orientation or in the opposite orientation as the sequence encoding the CRISPR nuclease (and any operably linked sequence) in the SIN vectors disclosed herein.
  • the nucleic acid sequences encoding the CRISPR nuclease and the gRNA are flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).
  • AAV adeno-associated virus
  • ITRs inverted terminal repeats
  • the SIN vectors disclosed herein are linear vectors comprising 5’ and 3’ ITRs.
  • the SIN vectors disclosed herein are recombinant AAV (rAAV) vectors.
  • the 5’ and 3’ ITRs flanking the CRISPR system coding sequence can be derived from any natural or recombinant AAV serotype.
  • the 5’ and 3’ ITRs can be derived from the same or different AAV serotypes.
  • Non-limiting examples of suitable serotypes include AAV1, AAV10, AAV106.1/hu.37, AAV11, AAV114.3/hu.40, AAV 12, AAV127.2/hu.41, AAV127.5/hu.42, AAV128.1/hu.43, AAV128.3/hu.44, AAV130.4/hu.48, AAV145.1/hu.53, AAV145.5/hu.54, AAV145.6/hu.55, AAV16.12/hu.ll, AAV16.3, AAV16.8/hu.lO, AAV161.10/hu.60, AAV161.6/hu.61, AAVl-7/rh.48, AAVl-8/rh.49, AAV2, AAV2.5T, AAV2-15/rh.62, AAV223.1, AAV223.2, AAV223.4, AAV223.5, AAV223.6, AAV223.7, AAV2-3/rh.61, AAV
  • rAAV particles also called virions
  • rAAV particles comprising any one of the SIN CRISPR system nucleic acid vectors described above in section (I) that is encapsidated by at least one AAV capsid (Cap) protein.
  • Cap AAV capsid
  • the Cap proteins can be wildtype AAV Cap proteins or can be variant AAV Cap proteins that may have altered and/or enhanced tropism towards one or more cell types.
  • the rAAV particles can be generated as described below in section (IV).
  • a further aspect of the present disclosure comprises recombinant baculovirus expression vectors comprising sequence encoding any one of the SIN CRISPR system nucleic acid vectors described above in section (I).
  • the recombinant baculovirus expression vector comprises the nucleic acid sequence encoding the CRISPR nuclease, which is interrupted by the intron, and the nucleic acid sequence encoding the guide RNA, wherein the CRISPR system coding sequences are flanked by AAV ITRs.
  • the baculovirus expression vectors disclosed herein are derived from Autographa californica multiple nucleopolyhedrovirus (AcMNPV).
  • Still another aspect of the present disclosure encompasses methods for producing the SIN CRISPR system vectors described above in section (I).
  • the methods comprise introducing into packaging cells one of the recombinant baculovirus vectors described above in section (III).
  • the method further comprises expressing AAV replication (Rep) and capsid (Cap) proteins in the packaging cell.
  • a packaging recombinant baculovirus sequence encoding AAV Rep and Cap proteins is introduced into the packaging cell, such that the packaging cells expresses said AAV Rep and Cap proteins.
  • the packaging cell comprises sequence encoding AAV Rep and Cap proteins stably integrated into its genome. The integrated sequence encoding said AAV Rep and Cap proteins is operably linked to an inducible promoter, such that upon induction of the promoter, the packaging cell expresses said AAV Rep and Cap proteins.
  • the method further comprises introducing into the packaging cell a helper recombinant baculovirus comprising sequence encoding AAV packaging components.
  • the helper recombinant baculovirus can encode E2A, E4, and VA genes.
  • the various recombinant baculoviruses can be introduced into the packaging cells by any suitable means, e.g., via transfection, electroporation, or the like.
  • the method further comprises culturing the packaging cells under suitable conditions such that the packaging cells produce rAAV particles comprising a SIN AAV nucleic acid vector encapsidated by Cap proteins.
  • the rAAV particles can be harvested and purified using conventional means (e.g., chromatography).
  • the packaging cell cells are insect cells.
  • suitable insect cells include those derived from Spodoptera frugiperda , e.g., Sf , Sf-21 cells, or derived from Trichoplusia ni , e.g., Tn-368, BTI-TN-5B1-4.
  • the packaging cells can be Sf9 cells.
  • Yet another aspect of the present disclosure encompasses methods for temporally limiting expression of a CRISPR nuclease in eukaryotic cells, wherein the methods comprise introducing into eukaryotic cells any of the SIN CRISPR system nucleic acids described above in section (I) or the rAAV particles comprising the SIN CRISPR system nucleic acids described above in section (II).
  • the intron in the RNA encoding the CRISPR nuclease is spliced out such that a functional CRISPR nuclease is produced.
  • the CRISPR nuclease complexes with the guide RNA to form a CRISPR system, wherein the CRISPR system cleaves the target genomic locus in the eukaryotic cell leading to an edited genomic locus, and the CRISPR system cleaves the intron in the nucleic acid sequence encoding the CRISPR nuclease in the SIN CRISPR system vector leading to degradation and inactivation of the SIN CRISPR system vector.
  • expression of the SIN CRISPR system vector is limited to the first several days after being introduced in to the eukaryotic cells.
  • the SIN CRISPR system nucleic acids or rAAV particles comprising the SIN CRISPR system nucleic acids can be introduced into the eukaryotic cells by a variety of methods.
  • suitable methods include transfection, nucleofection, electroporation, lipofection, sonoporation, and the like.
  • Cleavage of the target genomic locus can lead to insertion, deletion, and or substitution of at least nucleotide during cell mediated repair of the double stranded break (e.g., non-homologous end joining, NHEJ, or microhomology-mediated end joining, MMEJ), such that small deletions and insertions can occur at the cleavage site.
  • insertions and/or deletions also called indels
  • the resultant indels can reduce expression or lead to expression of a modified product from the targeted genomic locus, leading to a “knockdown.”
  • the method can further comprise introducing into the eukaryotic cells a donor template, wherein the donor template comprises a donor sequence that is flanked by sequence homologous to the target locus.
  • the donor sequence can be integrated into or exchange with sequence at the target genomic locus, such that exogenous or modified sequences can be integrated into the genome.
  • the donor sequence of the donor template can be a modified version of the target locus.
  • the donor sequence can be essentially identical to sequence at the target locus, but which comprises at least one nucleotide change.
  • the sequence at the target locus comprises at least one nucleotide change.
  • the nucleotide change can be an insertion of one or more nucleotides, a deletion of one or more nucleotides, a substitution of one or more nucleotides (e.g., modify a SNP), or combinations thereof.
  • the cell can produce a modified gene product from the targeted chromosomal sequence (i.e., target locus).
  • the nucleotide change can be a deletion of one or more nucleotides, such that the reading frame is interrupted and the cell no longer produces the gene product, thereby creating a “knockout” or “knockdown”.
  • the donor sequence of the donor polynucleotide can be an exogenous sequence.
  • an “exogenous” sequence refers to a sequence that is not native to the cell, or a sequence whose native location is in a different location in the genome of the cell.
  • the exogenous sequence can comprise protein coding sequence, which can be operably linked to an exogenous promoter control sequence such that, upon integration into the genome, the cell is able to express the protein coded by the integrated sequence.
  • the exogenous sequence can be integrated in-frame into the genome such that expression of the exogenous sequence is regulated by an endogenous promoter control sequence.
  • exogenous sequence integration of an exogenous sequence into a chromosomal sequence is termed a “knock in.”
  • the exogenous sequence can be a transcriptional control sequence, another expression control sequence, an RNA coding sequence, and so forth.
  • the length of the donor sequence can and will vary.
  • the donor sequence can vary in length from several nucleotides to hundreds of nucleotides to hundreds of thousands of nucleotides.
  • the donor template can be single stranded or double stranded, linear or circular.
  • the donor template can be RNA or DNA.
  • the donor template can be a single stranded DNA oligonucleotide.
  • the donor template can be double stranded DNA provided to the cell as part of a vector.
  • suitable vectors include plasmid vectors, DNA minicircles, adenovirus vectors, adeno-associated virus vectors, alphavirus vectors, baculovirus vectors, enterovirus vectors, Epstein Barr virus vectors, herpesvirus vectors, lentiviral vectors, papovavirus vectors, pestivirus vectors, poxvirus vectors; retroviral vectors, vaccinia virus, and combinations thereof.
  • the vector can be an adeno-associated virus (AAV) vector or a recombinant AAV vector, examples of which are detailed above in section (I)(d).
  • AAV adeno-associated virus
  • a variety of eukaryotic cells can be used in the methods disclosed herein.
  • the cells are mammalian cells.
  • the cells are human cells.
  • the cells can be in vitro (e.g ., cell line cells, cultured cells, primary cells).
  • the cells can be ex vivo cells isolated from an organism.
  • the cells can be in vivo cells within an organism.
  • the cells may be stem cells (e.g., embryonic stem cells, fetal stem cells, amniotic stem cells, or umbilical cord stem cells).
  • the stem cells may be adult stem cells isolated from bone marrow, adipose tissue, or blood.
  • the cells may be induced pluripotent stem cells (e.g, human iPSCs).
  • the cells may be hematopoietic stem and progenitor cells (HSPCs) or hematopoietic stem cells (HSCs).
  • HSPCs give rise to all blood cell types, including erythroid (erythrocytes or red blood cells (RBCs)), myeloid (monocytes and macrophages, neutrophils, basophils, eosinophils, megakaryocytes / platelets, and dendritic cells), and lymphoid (T-cells, B-cells, NK-cells).
  • Blood cells are produced by the proliferation and differentiation of a very small population of pluripotent HSCs that also have the ability to replenish themselves by self-renewal.
  • Bone marrow is the major site of hematopoiesis in humans and, under normal conditions, only small numbers of HSPCs can be found in the peripheral blood (PB).
  • cytokines in particular granulocyte colony-stimulating factor; G-CSF
  • G-CSF granulocyte colony-stimulating factor
  • myelosuppressive drugs used in cancer treatment, and compounds that disrupt the interaction between hematopoietic and BM stromal cells can rapidly mobilize large numbers of stem and progenitors into the circulation.
  • the cell surface glycoprotein CD34 is routinely used to identify and isolate HSPCs.
  • the cells may be mesenchymal stem cells (e.g ., multipotent stromal cells that can differentiate into a variety of cell types).
  • Mesenchymal stem cells are adult stem cells found in the bone marrow, or isolated from other tissues such as cord blood, peripheral blood, fallopian tube, and fetal liver and lung.
  • MSCs differentiate into multiple cell types including adipocytes, chondrocytes, osteocytes, and cardiomyocytes.
  • Mesenchymal stem cells are a distinct entity to the mesenchyme, embryonic connective tissue, which is derived from the mesoderm and differentiates to form hematopoietic stem cells (HPCs).
  • HPCs hematopoietic stem cells
  • the cells may be immune cells such as T cells, B cells, natural killer (NK) cells, NKT cells, mast cells, eosinophils, basophils, macrophages, neutrophils, or dendritic cells.
  • immune cells such as T cells, B cells, natural killer (NK) cells, NKT cells, mast cells, eosinophils, basophils, macrophages, neutrophils, or dendritic cells.
  • the cells may be primary cells isolated directly from human or animal tissue.
  • suitable primary cells include adipocytes, astrocytes, blood cells (e.g., erythroid, lymphoid), chondrocytes, endothelial cells, epithelial cells, fibroblasts, hair cells, hepatocytes, keratinocytes, melanocyte, myocytes, neurons, osteoblasts, skeletal muscle cells, smooth muscle cells, stem cells, or synoviocytes.
  • the cells can be (immortalized) mammalian cell line cells.
  • suitable mammalian cell lines include human embryonic kidney cells (HEK293, HEK293T); human cervical carcinoma cells (HELA); human lung cells (W138); human liver cells (Hep G2); human U2-OS osteosarcoma cells, human A549 cells, human A-431 cells, and human K562 cells; Chinese hamster ovary (CHO) cells, baby hamster kidney (BHK) cells; mouse myeloma NSO cells, mouse embryonic fibroblast 3T3 cells (NIH3T3), mouse B lymphoma A20 cells; mouse melanoma B16 cells; mouse myoblast C2C12 cells; mouse myeloma SP2/0 cells; mouse embryonic mesenchymal C3H-10T1/2 cells; mouse carcinoma CT26 cells, mouse prostate DuCuP cells; mouse breast EMT6 cells; mouse hepatoma Hepalclc7 cells; mouse
  • the terms “complementary” or “complementarity” refer to the association of double-stranded nucleic acids by base pairing through specific hydrogen bonds.
  • the base paring may be standard Watson-Crick base pairing (e.g., 5’-A G T C-3’ pairs with the complementary sequence 3’-T C A G-5’).
  • the base pairing also may be Hoogsteen or reversed Hoogsteen hydrogen bonding.
  • Complementarity is typically measured with respect to a duplex region and thus, excludes overhangs, for example.
  • Complementarity between two strands of the duplex region may be partial and expressed as a percentage (e.g., 70%), if only some (e.g., 70%) of the bases are complementary.
  • the bases that are not complementary are “mismatched.”
  • Complementarity may also be complete (i.e., 100%), if all the bases in the duplex region are complementary.
  • a “gene,” as used herein, refers to a DNA region (including exons and introns) encoding a gene product, as well as all DNA regions which regulate the production of the gene product, whether or not such regulatory sequences are adjacent to coding and/or transcribed sequences. Accordingly, a gene includes, but is not necessarily limited to, promoter sequences, terminators, translational regulatory sequences such as ribosome binding sites and internal ribosome entry sites, enhancers, silencers, insulators, boundary elements, replication origins, matrix attachment sites, and locus control regions.
  • nuclease and “endonuclease” are used interchangeably herein, and refer to an enzyme that cleaves both strands of a double-stranded nucleic acid sequence.
  • nucleic acid and “polynucleotide” refer to a deoxyribonucleotide or ribonucleotide polymer, in linear or circular conformation, and in either single- or double-stranded form.
  • these terms are not to be construed as limiting with respect to the length of a polymer.
  • the terms can encompass known analogs of natural nucleotides, as well as nucleotides that are modified in the base, sugar and/or phosphate moieties (e.g., phosphorothioate backbones).
  • nucleotide refers to deoxyribonucleotides or ribonucleotides.
  • the nucleotides may be standard nucleotides (i.e., adenosine, guanosine, cytidine, thymidine, and uridine), nucleotide isomers, or nucleotide analogs.
  • a nucleotide analog refers to a nucleotide having a modified purine or pyrimidine base or a modified ribose moiety.
  • a nucleotide analog may be a naturally occurring nucleotide (e.g., inosine, pseudo uridine, etc.) or a non-naturally occurring nucleotide.
  • Non-limiting examples of modifications on the sugar or base moieties of a nucleotide include the addition (or removal) of acetyl groups, amino groups, carboxyl groups, carboxymethyl groups, hydroxyl groups, methyl groups, phosphoryl groups, and thiol groups, as well as the substitution of the carbon and nitrogen atoms of the bases with other atoms (e.g., 7-deaza purines).
  • Nucleotide analogs also include dideoxy nucleotides, 2’-0-methyl nucleotides, locked nucleic acids (LNA), peptide nucleic acids (PNA), and morpholinos.
  • sequence identity indicates a quantitative measure of the degree of identity between two sequences of substantially equal length.
  • the percent identity of two sequences, whether nucleic acid or amino acid sequences is the number of exact matches between two aligned sequences divided by the length of the shorter sequence and multiplied by 100.
  • An approximate alignment for nucleic acid sequences is provided by the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981). This algorithm can be applied to amino acid sequences by using the scoring matrix developed by Dayhoff, Atlas of Protein Sequences and Structure, M. O. Dayhoff ed., 5 suppl.
  • targeted deletion refers to a deletion of at least one nucleotide base pair at a specific site in a target nucleic acid by a gene editing system that is engineered to target the specific site.
  • target sequence refers to the specific sequence in a target nucleic acid to which the gene editing system is targeted.
  • treating refers to alleviating, ameliorating, or inhibiting the symptoms of a disease or disorder; reversing, inhibiting, or slowing the progression of a disease or disorder; and/or preventing or delaying the onset of a disease or disorder.
  • a chimeric intron from pCI-Neo vector was inserted at amino acid Asn 580 (AA A C) of 580) of the nucleotide sequence encoding SauCas9.
  • the chimeric intron was modified to contain a gRNA binding site (e.g., 21 nt followed by PAM specific for Cas9).
  • the Cas9 coding sequence was codon optimized and linked one NLS at each end.
  • a recombinant AAV vector was constructed by inserting the Cas9 coding sequence operably linked to a muscle-specific promoter (e.g., CK8e) and gRNA expression sequence operably linked to a Pol III promoter between the ITRs of AAV8 vector.
  • a muscle-specific promoter e.g., CK8e
  • All-in-one vector sequences were first cloned into pFastBac and transferred into bacmids in Bac-to-Bac DHlOBac cells. Similarly, bacmids containing the capsid and helper proteins were generated using Bac-to-Bac method.
  • Baculoviruses were generated in insect Sf9 cells.
  • AAV vector were generated by coinfection of Sf9 cells with the resulting baculoviruses.
  • the infected cells were harvested at 72 hours post-infection and lysed using a standard chemical method. The lysates were clarified by centrifugation and concentrated by tangential flow filtration.
  • the vectors were further purified using affinity chromatography and buffer exchange was performed by ultrafiltration using a hollow fiber module.
  • Nucleotide sequence verification of long-range PCR product confirmed intactness of genome of the all-in-one SIN AAV vector produced using the baculovirus-Sf9 method.

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Abstract

L'invention concerne des vecteurs de système CRISPR à auto-inactivation (SIN), des procédés de préparation desdits vecteurs, et des procédés d'utilisation desdits vecteurs. Les vecteurs de système CRISPR SIN comprennent une séquence d'acide nucléique codant pour un système CRISPR (c'est-à-dire, une nucléase CRISPR et un ARN guide), la séquence codant pour la nucléase CRISPR comprenant au moins un intron comprenant un site de liaison reconnu par le système CRISPR.
PCT/IB2020/058683 2019-09-18 2020-09-17 Vecteurs crispr à auto-inactivation tout-en-un Ceased WO2021053582A1 (fr)

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