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WO2018164948A1 - Vecteurs comprenant des commutateurs dépendant de cpf1 autodirigés - Google Patents

Vecteurs comprenant des commutateurs dépendant de cpf1 autodirigés Download PDF

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WO2018164948A1
WO2018164948A1 PCT/US2018/020619 US2018020619W WO2018164948A1 WO 2018164948 A1 WO2018164948 A1 WO 2018164948A1 US 2018020619 W US2018020619 W US 2018020619W WO 2018164948 A1 WO2018164948 A1 WO 2018164948A1
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vector
sequence
cpfl
transgene
protospacer
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Guocai ZHONG
Michael Farzan
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Scripps Research Institute
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Scripps Research Institute
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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
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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
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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
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
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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]
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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
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—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
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    • C12N2310/00—Structure or type of the nucleic acid
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    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
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    • C12N2830/00—Vector systems having a special element relevant for transcription

Definitions

  • CRISPR based genome editing is guided by a short CRISPR RNA (crRNA) which targets the Cas9 DNase activity to genomic sequences complementary to the crRNA and preceded by a short protospacer-adjacent motif (PAM).
  • crRNA CRISPR RNA
  • PAM protospacer-adjacent motif
  • Cpfl crRNA a T-rich PAM distal from a staggered DNase cut site.
  • the mature Cpfl crRNA is composed of a 5' scaffold region (also described as a 5' handle or a direct repeat), and a 3' guide region.
  • Cas9 relies on RNase III to excise crRNAs from a CRISPR array.
  • FnCpfl Francisella novidica Ul 12
  • FnCpfl has its own RNase activity that can excise crRNA from a bacterial CRISPR array.
  • FnCpfl does not efficiently edit mammalian genomes, and it is not known whether the RNase activity of any Cpfl is functional in mammalian cells.
  • the invention provides vectors or expression constructs that contain a transgene and a polynucleotide switch.
  • the polynucleotide switch in the vectors harbors (a) a first sequence segment that contains a Cpfl DNase target sequence having (1) a protospacer that is approximately 23 ⁇ 2 nucleotides in length and (2) a protospacer-adjacent motif (PAM) that is located 5' to the protospacer, and (b) a second sequence segment that encodes an RNA transcript that contains (1) a scaffold RNA containing a sequence 4-10 nucleotides in length and its reverse complement and (2) a guide RNA (gRNA) sequence that is substantially identical to the protospacer and is approximately 23 ⁇ 2 nucleotides in length.
  • the polynucleotide switch is capable of undergoing a self-directed DNA modification that switches on or switches off the transgene in the vectors.
  • the scaffold RNA in the RNA transcript is capable of being cleaved by a Type V CRISPR effector protein (e.g., a Cpfl enzyme such as LbCpf 1 or AsCpf) in a mammalian cell, resulting in the generation of a mature crRNA of the CRISPR effector protein.
  • the generated crRNA is capable of targeting the CRISPR effector protein to the Cpfl DNase target sequence to cleave the vector within the protospacer.
  • the encoded RNA transcript contains two or more scaffold RNAs.
  • Some vectors of the invention can additionally contain an RNA polymerase II (Pol II) promoter.
  • the second sequence segment is operably linked to the promoter for the transcript to be expressed from the RNA polymerase II (Pol II) promoter.
  • the transgene contains at least one open reading frame (ORF).
  • the encoded scaffold RNA contains one or more structural elements selected from (a) a sequence 4-10 nucleotides in length comprising UCUAC and a reverse complement comprising GUAGA, (b) a U nucleotide at the first unpaired position 5' of the sequence 4-10 nucleotides in length, (c) a U nucleotide at the first unpaired position 3' of the sequence 4-10 nucleotides in length, (d) a U nucleotide at the first unpaired position 5' of the reverse complement, (e) a U nucleotide at the first unpaired position 3' of the reverse complement, and (f) a trinucleotide AAU at a position fewer than 5 nucleotides 5' of said sequence 4-10 nucleotides in length.
  • the encoded scaffold RNA can further contain a CU dinucleotide, an AU dinucleotide or an AAG trinucleotide between the sequence 4-10 nucleotides in length and its reverse complement.
  • the PAM is a tetranucleotide containing at least two thymidine (T) nucleotides.
  • the sequence of the guide RNA is identical to that of the protospacer. For some vectors, cleavage of the protospacer in a cell by a Cpfl present therein will switch off expression of the transgene.
  • cleavage of the protospacer in a cell by a Cpfl present therein will switch on expression of the transgene.
  • concentration of the transcript expressed from the vector will be reduced when the corresponding Cpfl enzyme is present.
  • Some vectors of the invention contain a promoter (e.g., a Pol II promoter) that harbors the protospacer.
  • the protospacer comprises or partially overlaps a TFIIB recognition element (BRE), a TATA box, an Initiator (Inr), a downstream promoter element (DPE), a splice acceptor AG dinucleotide, a splice donor GU dinucleotide, an ATG start codon trinucleotide, or an internal ribosomal entry site (IRES).
  • Some vectors of the invention contain two or more Cpfl DNase target sequences.
  • each Cpfl DNase target sequence can contain (1) a protospacer that is approximately 23 ⁇ 2 nucleotides in length and (2) a protospacer-adjacent motif (PAM) that is located 5' to the protospacer.
  • PAM protospacer-adjacent motif
  • the protospacers of the two or more Cpfl DNase target sequences are not identical to each other.
  • the ORF in the transgene encodes an amino acid sequence that is substantially identical to the amino acid sequence of at least a portion of a human protein. In some embodiments, the ORF encodes an amino acid sequence that is substantially identical to the Fc region of an antibody. In some embodiments, the ORF encodes an amino acid sequence other than an antibody Fc region that is substantially identical to one or more immunoadhesins or antibodies known in the art. In some embodiments, the ORF encodes a sequence encoding at least a portion of one or more known cellular proteins such as cellular receptors, other cell surface molecules, enzymes, cytokines, chemokines, costimulatory molecules, interleukins, and physiologically active polypeptide factors.
  • the ORF encodes at least a portion of a chimeric antigen receptor (CAR).
  • Some preferred vectors of the invention are based on or derived from an adeno-associated virus (AAV) vector or a retroviral vector.
  • Some vectors of the invention can additionally contain an inducible expression cassette that encodes a Type V CRISPR effector protein (e.g., a Cpfl enzyme).
  • the invention provides mammalian cells into which one or more vectors of the present invention have been introduced.
  • the invention provides pharmaceutical compositions that contain at least one vector of the present invention.
  • the invention provides methods of switching on or switching off expression of a transgene. These methods typically entail (a) administering a vector of the invention to a subject, and (b) administering either a Cpfl enzyme or a polynucleotide capable of expressing a Cpfl enzyme to the subject.
  • FIG. 1 shows that LbCpfl and AsCpfl have RNase activities in mammalian cells
  • the crRNA recognized by LbCpfl (SEQ ID NO:20) and AsCpfl (SEQ ID NO:21) are represented.
  • a 19-20 nucleotide scaffold RNA region in the crRNAs (SEQ ID NOs: 18 and 19, respectively) is followed by a 23-base guide RNA (gRNA) complementary to the Cpfl DNase target sequence
  • gRNA 23-base guide RNA
  • Cpfl recognizes an appropriate scaffold RNA region present in the 3' UTR of an mRNA encoding GLuc, the message is cleaved and GLuc expression is halted
  • Plasmids encoding LbCpfl, AsCpfl or vector alone were cotransfected with GLuc-expressing plasmids bearing the indicated scaffold variants, and GLuc activity was measured.
  • a small ' ⁇ ' preceding the scaffold RNA indicates replacement of the initial AAUU sequence with UUAA.
  • a large ' x ' indicates that the scaffold sequence has been randomized.
  • the first three have LbCpfl (Lb) scaffold, and the other two have AsCpfl (As) scaffold,
  • e-f LbCpfl variants were assessed for their ability to edit an integrated gene when co- expressed with U6 promoter-driven crRNA, using (e) a T7E1 mismatch cleavage assay or (f) double-strand break (DSB)-induced gain-of-expression assay, depicted in Figure 3a.
  • H759 and K785 are proximal to the phosphate at 5' end of the Lb scaffold.
  • the first and second bases of the scaffold RNA, A(-20), A(-19), are also indicated.
  • Experiments shown are representative of two (panel c), three (panel e), or four (panels d and f) performed with nearly identical results.
  • Data points in panels c, d, and f represent mean ⁇ s.d. of three biological replicates.
  • FIG. 2 shows that crRNA excised from a Pol II-expressed RNA transcript can efficiently edit a mammalian genome
  • a An mRNA encoding GLuc with two Lb scaffold regions (sR) separated by a 23-base guide (gRl) can be cleaved by LbCpfl . If both scaffold RNA regions are cleaved, the GLuc message is degraded and the resulting crRNA can be loaded into LbCpfl to edit a reporter transgene.
  • U6 promoter-driven guide RNAs used in the subsequent panel are represented
  • the indicated U6-expressed crRNAs were co-expressed with LbCpfl in the presence of a DSB-induced FLuc (left) or GLuc (right) reporter gene, as depicted in Figure 5d. When expressed in tandem, both crRNAs are active, (g) A direct comparison of the efficiencies of Pol II and Pol Ill-expressed crRNA using a T7E1 mismatch cleavage assay.
  • Figure 3 shows assays and additional results for Figure 1.
  • a gene encoding EGFP and GLuc separated by a foot-and-mouth disease 2a protease (F2A) was integrated into the genome of 239T cells.
  • EGFP is initially encoded in the +1 frame, whereas F2A and GLuc are encoded in the +3 frame and the GLuc start methionine has been eliminated, so that only EGFP is expressed.
  • a frameshift is induced by nonhomologous end joining (NHEJ), inactivating EGFP expression.
  • NHEJ nonhomologous end joining
  • Figure 4 shows additional results for Figure 2 (panels a-c).
  • Figure 5 shows assays and additional results for Figure 2 (panels d-f).
  • Tandem crRNAs were expressed from a Pol-III (U6) promoter.
  • These crRNAs were assayed individually and in both tandem orders in Figure 2f. Experiments shown are representative of two with similar results. Data points in panels b and c represent mean ⁇ s.d. of three biological replicates.
  • FIG. 6 shows that the self-directed polynucleotide switch is capable of inactivating the vector containing it at the RNA level.
  • a first plasmid vector containing a Firefly luciferase transgene was co-transfected into 293T cells with an empty plasmid vector or a plasmid vector expressing either wild-type LbCpfl, RNase domain mutant LbCpfl H759A, or DNase domain mutant LbCpfl D832A.
  • the Firefly luciferase transgene was expressed as a single Pol II transcript, which either did not include a scaffold RNA and guide RNA (gRNA) (a), or did include a scaffold RNA and gRNA (b). Firefly luciferase (FLuc) expression is indicated in relative light units (RLU).
  • RLU relative light units
  • Figure 7 shows various strategies for a self-directed polynucleotide switch to inactivate the vector containing it at the DNA level.
  • the vectors shown here express Firefly luciferase from a Pol II promoter and express a second transcript containing a scaffold RNA and guide RNA (gRNA) from a U6 Pol III promoter.
  • the plasmid vector expressing both Firefly luciferase and the transcript containing the scaffold RNA and gRNA was co- trasnfected into 293T cells with an empty plasmid vector or a plasmid vector expressing either wild-type LbCpfl, RNase domain mutant LbCpfl H759A, or DNase domain mutant LbCpfl D832A.
  • the protospacer overlaps with the splice acceptor AG dinucleotide (SA) (a).
  • SA splice acceptor AG dinucleotide
  • the protospacer overlaps with the ATG start codon trinucleotide (b).
  • the Firefly luciferase expression vector contained two protospacers, the first protospacer overlapping with the SA site, and the second protospacer overlapping with the ATG start codon trinucleotide (c).
  • the protospacer resided in the coding region of the Firefly luciferase transgene (d). Multiple protospacer sequences within the Firefly luciferase transgene were evaluated (Fluc-gRl through Fluc- gR19). Firefly luciferase (FLuc) expression is indicated in relative light units (RLU).
  • Figure 8 shows the inactivation of a transgene in vivo.
  • Mice were inoculated in the left gastrocenemius muscle with 10 10 copies of an AAV vector encoding Firefly luciferase.
  • the vector contained a U6 promoter that expresses a transcript containing a scaffold RNA and a guide RNA (gRNA).
  • gRNA guide RNA
  • An otherwise-identical negative control vector was constructed that encoded Firefly luciferase but not a scaffold RNA and gRNA. The gRNA recognizes a protospacer sequence in the coding region of the Firefly luciferase transgene.
  • luciferase activity was measured using a Xenogen imager on days 8, 14, 21, and 28 post-injection of the animals that received the AAV vector lacking (a) or containing (b) the scaffold RNA and gRNA.
  • the luciferase signal from each mouse was quantified using the Xenoimager software for both groups of mice, which received the AAV vector lacking (c) or containing (d) the scaffold RNA and gRNA.
  • Cpfl is a Type V CRISPR-effector protein with greater specificity than Cas9 in genome-editing applications.
  • the present invention is predicated in part on the discoveries by the present inventors that some Cpfl proteins have RNase activities that can excise CRISPR RNAs (crRNAs) from a single Pol II-driven RNA transcript expressed in mammalian cells. Specifically, the inventors observed and assessed the utility of RNase activity of LbCpfl and AsCpfl for genome-editing applications. As detailed herein, it was found that AsCpfl and LbCpfl can excise multiple crRNAs from a single RNA transcript expressed from either a Pol-II or a Pol-III promoter.
  • Pol-II promoter allows regulated and tissue-specific control of crRNA expression, and more efficient expression of long transcripts. It was also found that Pol II-expressed crRNAs were consistently more efficient at mediating genome editing than those expressed from a Pol-III promoter. This observation may reflect in part the fact that only Pol-II transcripts are efficiently exported to the cytoplasm where they might more easily interact with recently translated Cpfl.
  • polynucleotide switches that are dependent on Cpfl enzymatic activities and expression vectors coexpressing such a polynucleotide switch and a target polypeptide of interest.
  • the Cpfl -based expression controlling systems of the invention depend on the RNase activity of Cpfl proteins in excising their own guide RNAs from mRNA transcripts made in mammalian cells.
  • Polynucleotide switches derived from such activities can be used as permanent off-switches for AAV delivered transgenes.
  • the polynucleotide switches and expression vectors of the invention have a number of useful properties that are advantageous over any polynucleotide switches that are currently employed in the art. First, they depend on a nuclease with limited off-target activity.
  • transgenes immediately halt transgene expression through one mechanism (by degrading the transgene mRNA), while permanently halting expression through another (by degrading the DNA transgene itself).
  • they can be engineered so that they are only active in cells carrying the exogenously introduced transgene. Additionally, once an off-switch of the invention has eliminated the transgene, it automatically turns itself off.
  • this invention is not limited to the particular methodology, protocols, and reagents described as these may vary. Unless otherwise indicated, the practice of the present invention employs conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry and immunology, which are within the skill of the art. Such techniques are explained fully in the literature. For example, exemplary methods are described in the following references, Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (3 rd ed., 2001); Brent et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc.
  • Protospacers are spacer sequences in CRISPR loci in a bacterium that were inserted into a CRISPR locus by invading viral or plasmid DNA.
  • Cas9 nuclease attaches to tracrRNAxrRNA which guides Cas9 to the invading protospacer sequence. But Cas9 will not cleave the protospacer sequence unless there is an adjacent PAM sequence.
  • the spacer in the bacterial CRISPR loci will not contain a PAM sequence, and will thus not be cut by the nuclease. But the protospacer in the invading virus or plasmid will contain the PAM sequence, and will thus be cleaved by the Cas9 nuclease.
  • guideRNAs gRNAs are synthesized to perform the function of the tracrRNAxrRNA complex in recognizing gene sequences having a PAM sequence at the 3'-end (5'-end for Cpfl).
  • Protospacer adjacent motif is a 2-6 base pair DNA sequence immediately following the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system.
  • PAM is a component of the invading virus or plasmid, but is not a component of the bacterial CRISPR locus. Cas9 will not successfully bind to or cleave the target DNA sequence if it is not followed by the PAM sequence.
  • PAM is an essential targeting component (not found in bacterial genome) which distinguishes bacterial self from non-self DNA, thereby preventing the CRISPR locus from being targeted and destroyed by nuclease.
  • the canonical PAM is the sequence 5'-NGG-3' where "N” is any nucleobase followed by two guanine (“G”) nucleobases.
  • Guide RNAs gRNAs
  • the canonical PAM is associated with the Cas9 nuclease of Streptococcus pyogenes (designated SpCas9), whereas different PAMs are associated with the Cas9 proteins of the bacteria Neisseria meningitidis, Treponema denticola, and Streptococcus thermophilus.
  • 5'-NGA-3' can be a highly efficient non-canonical PAM for human cells, but efficiency varies with genome location. Attempts have been made to engineer Cas9s to recognize different PAMs to improve ability of CRISPR-Cas9 to do gene editing at any desired genome location.
  • Cas9 of Francisella novicida recognizes the canonical PAM sequence 5'-NGG-3', but has been engineered to recognize the PAM 5'-YG-3' (where "Y” is a pyrimidine), thus adding to the range of possible Cas9 targets.
  • the Cpfl nuclease of Francisella novicida recognizes the PAM 5'-TTN-3' or 5'-YTN-3'.
  • Cpfl refers to AsCpfl, LbCpfl, their functional derivatives or variants (e.g., the divergent LbCpfl exemplified herein), or any other Type V CRISPR effector protein.
  • AsCpfl from A cidaminococcus
  • LbCpfl from Lachnospiraceae
  • Cpfl proteins are RNA-guided nucleases, similar to Cas9. They recognize a T- rich protospacer-adjacent motif (PAM), TTTN, but on the 5' side of the guide. This makes Cpfl distinct from Cas9, which uses an NGG PAM on the 3' side.
  • PAM protospacer-adjacent motif
  • Cpfl makes is staggered. In AsCpfl and LbCpfl, it occurs 23 bp after the PAM on the targeted (+) strand and 19 bp on the other strand. Cpfl requires only a crRNA for activity and does not need a tracrRNA to also be present. Unless otherwise noted, Cpfl as described herein for the present invention also broadly encompasses any other Type V CRISPR effector proteins beyond the specifically exemplified AsCpfl and LbCpfl enzymes.
  • a Type V CRISPR effector protein refers to a CRISPR effector protein or enzyme that does not require a multiple-protein complex formation for its catalytic function and also does not require a tracrRNA(but instead is itself sufficient) for the maturation of its crRNA.
  • a "host cell” or “target cell” refers to a living cell into which a heterologous polynucleotide sequence is to be or has been introduced.
  • the living cell includes both a cultured cell and a cell within a living organism.
  • Means for introducing the heterologous polynucleotide sequence into the cell are well known, e.g., transfection, electroporation, calcium phosphate precipitation, microinjection, transformation, viral infection, and/or the like.
  • the heterologous polynucleotide sequence to be introduced into the cell is a replicable expression vector or cloning vector.
  • host cells can be engineered to incorporate a desired gene on its chromosome or in its genome.
  • host cells that can be employed in the practice of the present invention (e.g., CHO cells) serve as hosts are well known in the art. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (3 rd ed., 2001); and Brent et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003).
  • the host cell is a mammalian cell.
  • operably linked refers to functional linkage between genetic elements that are joined in a manner that enables them to carry out their normal functions.
  • a gene is operably linked to a promoter when its
  • a Cpfl -dependent polynucleotide switch sequence is operably linked to a transgene if its insertion into the 5'- UTR or 3'-UTR of the gene, as described herein, allows control of the transgene expression by Cpfl RNase digestion of mRNA transcript of the transgene.
  • a "substantially identical" nucleic acid or amino acid sequence refers to a polynucleotide or amino acid sequence which comprises a sequence that has at least 75%, 80% or 90% sequence identity to a reference sequence as measured by one of the well known programs described herein (e.g., BLAST) using standard parameters.
  • the sequence identity is preferably at least 95%, more preferably at least 98%, and most preferably at least 99%.
  • the subject sequence is of about the same length as compared to the reference sequence, i.e., consisting of about the same number of contiguous amino acid residues (for polypeptide sequences) or nucleotide residues (for polynucleotide sequences).
  • Polynucleotide sequences are no less substantially identical if they are composed of RNA or DNA, despite the chemical differences between RNA and DNA, and the presence of uracil in RNA instead of thymidine in DNA.
  • Sequence identity can be readily determined with various methods known in the art.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff & Henikoff, Proc. Natl. Acad. Sci. USA 89: 10915 (1989)).
  • Percentage of sequence identity is determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the percentage is calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • complementary refers to a nucleotide or nucleotide sequence that hybridizes to a given nucleotide or nucleotide sequence.
  • nucleotide A is complementary to T and vice versa
  • nucleotide C is complementary to G and vice versa.
  • nucleotide A is complementary to the nucleotide U and vice versa
  • nucleotide C is complementary to the nucleotide G and vice versa.
  • Reverse complement means a sequence that is complementary to another sequence, but in reverse order.
  • AATTGG is CC AATT.
  • AAUUGG is CCAAUU.
  • a cell has been "transformed” or “transfected” by exogenous or heterologous polynucleotide (or "a transgene” or “a target gene” as used interchangeably herein) when such polynucleotide has been introduced inside the cell.
  • the transforming polynucleotide may or may not be integrated (covalently linked) into the genome of the cell.
  • the transforming polynucleotide may be maintained on an episomal element such as a plasmid.
  • a stably transformed cell is one in which the transforming polynucleotide has become integrated into a chromosome so that it is inherited by daughter cells through chromosome replication. This stability is demonstrated by the ability of the eukaryotic cell to establish cell lines or clones comprised of a population of daughter cells containing the transforming polynucleotide.
  • a "clone” is a population of cells derived from a single cell or common ancestor by mitosis.
  • a "cell line” is a clone of a primary cell that is capable of stable growth in vitro for many generations.
  • a "vector” or “construct” is a non-naturally occurring nucleic acid with or without a carrier that can be introduced into a cell, or has been introduced into a cell.
  • Vectors that have been introduced into a cell include transfected plasmids and integrated DNA molecules, including those resulting from retroviral integration, integration of an AAV vector, and integration by homologous recombination.
  • Vectors capable of directing the expression of heterologous polynucleotide or transgene sequences encoding for one or more polypeptides are referred to as "expression vectors" or "expression constructs".
  • the cloned transgene sequence or open reading frame (ORF) is usually placed under the control of (i.e., operably linked to) certain regulatory sequences such as promoters, enhancers and polynucleotide switch sequences.
  • a transgene is any transcription unit contained within a vector.
  • the transgene may or may not encode a protein or proteins.
  • the transgene may encode one or more shRNA, miRNA, ribozyme, or protein.
  • AAV is adeno-associated virus, and may be used to refer to the naturally occurring wild-type virus itself or derivatives thereof. The term covers all subtypes, serotypes and pseudotypes, and both naturally occurring and recombinant forms, except where required otherwise.
  • Pseudotyped AAV refers to an AAV that contains capsid proteins from one serotype and a viral genome including 5'-3' ITRs of a second serotype.
  • rAAV refers to recombinant adeno-associated viral particle or a recombinant AAV vector (or "rAAV vector”).
  • AAV virus or “AAV viral particle” refers to a viral particle composed of at least one AAV capsid protein (preferably by all of the capsid proteins of a wild-type AAV) and an encapsidated polynucleotide. If the particle comprises a heterologous polynucleotide (i.e., a polynucleotide other than a wild-type AAV genome such as a transgene to be delivered to a mammalian cell), it is typically referred to as "rAAV”.
  • rAAV heterologous polynucleotide
  • a retrovirus (e.g., a lentivirus) based vector or retroviral vector means that genome of the vector comprises components from the virus as a backbone.
  • the viral particle generated from the vector as a whole contains essential vector components compatible with the RNA genome, including reverse transcription and integration systems. Usually these will include the gag and pol proteins derived from the virus. If the vector is derived from a lentivirus, the viral particles are capable of infecting and transducing non-dividing cells. Recombinant retroviral particles are able to deliver a selected exogenous gene or polynucleotide sequence such as therapeutically active genes, to the genome of a target cell.
  • the present invention provides polynucleotide switches that are dependent on the RNase and DNase activities of a Type V CRISPR effector protein (e.g., a Cpfl enzyme). These switches and related expression vectors of the invention provide exceptional tools for controlling transgene expression. For example, there is currently no effective way to turn off expression of a transgene delivered by adeno-associated viral vectors (AAVs).
  • AAVs adeno-associated viral vectors
  • the off- switches of the invention allow immediate and permanent termination of AAV-mediated transgene expression. As exemplified herein, the off-switches can be used for terminating expression of an AAV-delivered transgene that expresses any antibody or other protein therapeutic, such that its safety more closely matches that of the protein therapeutic not expressed by a gene delivery vector.
  • the polynucleotide switches of the present invention can be used for switching on the expression of a transgene.
  • the present invention includes polynucleotide switches for turning on or turning off the expression of a transgene from a vector.
  • the polynucleotide switches of the invention contain a Cpfl DNase target sequence that can be targeted by a Type V CRISPR effector protein, e.g., Cpfl enzymes from two bacterial species, Lachnospiraceae bacterium (Lb) and Acidaminococus sp. (As) as exemplified herein.
  • the switches also contain a second sequence segment or polynucleotide motif that encodes a transcript that is capable of becoming the crRNA for the Cpfl enzymes.
  • polynucleotide switches of the invention contains a protospacer motif and a protospacer- adjacent motif (PAM) that is typically located 5' to the protospacer.
  • the protospacer motif can contain about 15 to about 30 nucleotides that are specifically targeted by the crRNA of a Cpfl enzyme disclosed herein.
  • the protospacer contains about 23 ⁇ 5 nucleotides, 23 ⁇ 4 nucleotides, 23 ⁇ 3 nucleotides, 23 ⁇ 2 nucleotides, 23 ⁇ 1 nucleotides or 23 nucleotides in length.
  • the protospacer used in the polynucleotide switches of the invention can contain a sequence identical to Guide 1 (SEQ ID NO: 9) or Guide 3 (SEQ ID NO: 11) exemplified herein.
  • the protospacer-adjacent motif (PAM) in the Cpfl DNase target sequence is a thymidine (T) - rich sequence motif. It typically contains 2-6 nucleotide residues in length. Unless otherwise specified, the PAM is located at the 5' of the protospacer in the Cpfl DNase target sequence. In various embodiments, the PAM can contain 2, 3, 4, 5, or 6 T nucleotides. In some embodiments, the employed PAM is a trinucleotide comprising two T residues, e.g., TTN. In some other embodiments, the employed PAM is a tetranucleotide comprising three T residues, e.g., TTTN.
  • the Cpfl DNase target sequence in some preferred polynucleotide switches of the invention can contain a PAM sequence of TTTA or TTTG as exemplified herein for sR-gRl or sR-gR3.
  • the Cpfl orthologs exemplified herein have RNase activity that can be used to excise its guide RNA from an mRNA transcript produced in mammalian cells.
  • the second sequence segment in the polynucleotide switches encodes an RNA transcript that harbors a scaffold RNA (or "Cpfl RNase target sequence") that mediates the cleavage of the transcript by a Type V CRISPR effector protein (e.g., Cpfl) RNase activity, as well as a gRNA that mediates the cleavage of its DNA target by Cpfl DNase activity.
  • the gRNA is flanked on either side by a scaffold RNA.
  • cleavage of both of the two scaffold RNA sequences located on either side of the gRNA by Cpfl generates a mature crRNA.
  • a transcript expressed by a vector can be processed into a mature crRNA by Cpfl .
  • the scaffold RNA sequence is generally 8-30 nucleotides in length.
  • Such scaffold RNAs contain a hairpin RNA structure that is formed from a sequence that is typically 4-10 (or 5-10) nucleotides in length and a reverse complement of that sequence that is also 4-10 (or 5-10) nucleotides in length.
  • the sequence 4-10 (or 5-10) nucleotides in length can also be termed "hairpin motif sequence" herein.
  • AAU trinucleotide motif Within the scaffold RNA, at a position fewer than 5 nucleotides 5' of the sequence 4-10 nucleotides in length, there is typically an AAU trinucleotide motif.
  • the scaffold RNA can contain from about 15 to about 25 nucleotides in length.
  • the scaffold RNA can contain about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides. In some preferred embodiments, the scaffold RNA contains about 19 or 20 nucleotides. In some embodiments, the 5' end of the scaffold RNA, including the sequence 4-10 nucleotides in length, contains the sequence: AAUUUCUACU (SEQ ID NO: 17).
  • functional scaffold RNAs for LbCfpl and AsCpfl are AAUUUCUACUAAGUGUAGAU (SEQ ID NO: 18) and
  • AAUUUCUACUCUUGUAGAU (SEQ ID NO: 19), respectively, as exemplified herein.
  • the scaffold RNA for a variant LbCpfl exemplified herein is
  • the scaffold RNAs of the switches can be substantially identical (e.g., at least 90%, 95, or 99% identical) to the scaffold RNAs shown in SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:22.
  • the scaffold RNA of the polynucleotide switch contains a sequence that is identical to SEQ ID NO: 18, SEQ ID NO: 19 or SEQ ID NO:22.
  • the gRNA can contain about 23 ⁇ 5 nucleotides.
  • the gRNA encoded by the second polynucleotide motif in the polynucleotide switches of the present invention can contain 23 ⁇ 4 nucleotides, 23 ⁇ 3 nucleotides, 23 ⁇ 2 nucleotides, 23 ⁇ 1 nucleotides or 23 nucleotides in length.
  • sequence of the gRNA is at least 75% identical to the protospacer sequence.
  • the gRNA has a sequence that is at least 80%, at least 85%, at least 90% or at least 95% identical to the sequence of the protospacer sequence in the same polynucleotide switch.
  • the gRNA encoded by the second sequence segment of the polynucleotide switches of the invention can have a sequence that is identical to any of the protospacer sequences exemplified in Figure 3, panel b (SEQ ID NOs:9-16). It is to be noted that as the PAM motif is located 5' of the protospacer, and the sequence of the protospacer is read from 5' to 3'. Consistent with this orientation, while the gRNA actually binds to the complementary strand of the protospacer, its sequence is denoted herein as being “identical” and not “complementary” to the sequence of the protospacer.
  • RNA and DNA sequences are said to be “identical,” despite chemical difference of ribose versus deoxyribose, and the inclusion of uracil (U) versus thymidine (T).
  • the switches of the present invention can generate more than one crRNA, and the crRNAs can target more than one protospacer within the vector.
  • multiple crRNAs can be generated by the RNase activity of Cpfl from a single transcript, in which each of the crRNAs is flanked on both sides by a scaffold RNA, i.e., one scaffold RNA on the 5' side and one scaffold RNA on the 3' side.
  • a scaffold RNA that is located between two gRNAs can be shared.
  • some polynucleotide switches of the invention can contain more than one Cpfl DNase target sequence.
  • each Cpfl DNase target sequence harbors a specific protospacer motif that is preceded at the 5' by a PAM.
  • the more than one Cpfl DNase target sequences in the polynucleotide switch can contain different protospacer sequences. These different Cpfl DNase target sequences can be used for switching on or switching off the expression of one or more transgenes or ORFs in the same vector.
  • the second sequence segment can encode a transcript containing one or more pre-crRNAs that respectively can contain distinct gRNAs, each targeting distinct protospacer sequences.
  • each of the encoded pre-crRNAs can contain a distinct gRNA, each flanked by a scaffold RNA sequence.
  • Such polynucleotide switches can be employed to redundantly target a vector, thus switching on or switching off a transgene.
  • the Cpfl -dependent polynucleotide switches of the invention are suitable for controlling expression of transgenes or ORFs in various applications. Some embodiments of the invention are directed to expression vectors that contain the polynucleotide switch that is operably fused to a transgene sequence.
  • the polynucleotide switch typically employs one or more guide RNA sequences that recognize rationally designed Cpfl DNase target sequences placed in or adjacent to the transgene.
  • the switch can be operably placed at the 3 '-untranslated region of a transgene on a viral vector (e.g., an AAV vector).
  • transgene is used herein to refer to a transcription unit of a vector, including a region that is transcribed into RNA from the vector template, and any operably -linked enhancer and promoter sequences.
  • the transgene can contain an open reading frame (ORF), which can be translated into a protein, or alternatively, can be a non-coding RNA, including, e.g., an antisense RNA, an shRNA, an miRNA, an aptamer, or a ribozyme.
  • transgene expression can be achieved by both an immediate degradation of its message RNA transcript and a permanent modification at one or more protospacer sites in the transgene DNA.
  • the control of transgene expression by the vectors of the present invention has several additional advantages.
  • Cpfl already shown to be more specific than the better known CRISPR effector Cas9, would only be active in the presence of the transgene, due to the absence of a scaffold RNA in cells that have not been transduced by the vector, so there would be no off-target effects in non-transduced cells. This is because the guide RNA, necessary for activation of the Cpfl DNase activity, is generated from the transgene, which is expressed by the vector.
  • the system is self-limiting, because after the transgene is eliminated, it no longer can be used to generate the guide RNA, thereby halting the generation of active Cpfl/crRNA complexes.
  • the off-target effects of this system are at an absolute minimum.
  • the system can also be self-limiting when Cpfl is provided transiently, either as protein, or as a polynucleotide that transiently expresses a Cpfl protein.
  • the transgene regulated by the polynucleotide switch of the present invention typically contains an ORF.
  • the ORF can encode any polypeptide of interest.
  • expression of the transgene can be switched off immediately (via Cpfl RNase cleavage of the mRNA transcript of the transgene) and permanently (via Cpfl DNase cleavage of the vector itself) as exemplified herein.
  • the transgene can be expressed in the same transcript that encodes the gRNA.
  • the transgene is expressed separately, e.g., as a different transcript from that encodes the gRNA.
  • expression of the gRNA and/or the transgene can be placed under the control of either a Pol II (e.g., CMV) or a Pol III (e.g., U6) promoter sequence.
  • the protospacer of the polynucleotide switch contains a sequence element that regulates the expression of a transgene.
  • the protospacer can be a region of a vector or a transgene that is not transcribed, the protospacer can be within a region of a transgene that is transcribed but not translated, or the protospacer can be within a region of a transgene that is translated.
  • the polynucleotide switch switches on or switches off transgene expression at the level of transcription. In some other embodiments, the polynucleotide switch switches on or switches off transgene expression at the level of RNA stability or transport. In some other embodiments, the polynucleotide switch switches on or switches off transgene expression at the level of translation.
  • both the gRNA and the transgene are expressed in a single transcript under the control of the same promoter, e.g., a Pol II promoter.
  • cleavage of the expression vector in or near the protospacer by the DNase activity of a Cpf 1 will lead to permanent termination of the expression of the transgene.
  • cleavage of the expression vector in or near the protospacer by the DNase activity of Cpfl switches on the expression of the transgene.
  • the protospacer of the polynucleotide switch is located in or near the coding region of the transgene.
  • the protospacer is located in an expression control sequence of the transgene, e.g., the promoter region.
  • the guide RNA in the second sequence segment of the polynucleotide switch is designed to be substantially identical (e.g., at least 80%, 90%, or 95% identical) to a chosen coding region or an expression control region of the transgene that is preceded by a T-rich PAM.
  • some expression vectors of the invention contain a promoter sequence (e.g., a RNA Pol II promoter sequence) that harbors the protospacer of the Cpfl DNase target sequence.
  • the protospacer contains promoter elements completely. In some other embodiments, the protospacer partially overlaps with promoter elements.
  • Such promoter elements which can be contained within or partially overlapped by protospacer, include, e.g., (i) a TFIIB recognition element (BRE), typically located at positions -37 to -32 5' of the first nucleotide of a transcript and often resembling the consensus sequence
  • BRE TFIIB recognition element
  • the protospacer contains or overlaps transcription factor binding sites.
  • some off-switch embodiments can include a protospacer that contains or overlaps the binding site of a transcriptional activator, whereas some on-switch embodiments can include a protospacer that contains or overlaps the binding site of a transcriptional repressor.
  • the protospacer contains or partially overlaps a splice acceptor site.
  • Splice acceptor sites typically contain the AG dinucleotide.
  • Canonical splice acceptor sites, but not all splice acceptor sites contain a polypyrimidine tract 5' of the AG dinucleotide.
  • a subset of splice acceptor sites contain the AG dinucleotide within a CAG trinucleotide.
  • the protospacer contains or partially overlaps an AG dinucleotide, CAG trinucleotide, or polypyrimidine tract.
  • the PAM overlaps the polypyrimidine tract.
  • the protospacer contains or partially overlaps a splice donor GU dinucleotide.
  • the protospacer sequence exists within the transgene at a position where Cpf 1 cleavage, and the repair of the cleaved protospacer, affects translation.
  • the protospacer includes or overlaps an ATG start codon.
  • the protospacer exists within an open reading frame (ORF).
  • ORF open reading frame
  • cleavage of the protospacer by Cpfl can switch on or switch off the expression of a protein by changing the reading frame of the encoded protein. Changing the reading frame can, e.g., result in a premature stop codon, or restore a reading frame without a premature stop codon.
  • the protospacer can be at a position that lies in any of the untranslated regions that are known to be involved in controlling mRNA translation, degradation, and/or localization. These include, e.g., stem-loop structures, alternative start codons and open reading frames, internal ribosome entry sites (IRESes), RNA instability elements, RNA stability elements, the woodchuck hepatitis virus post- transcriptional regulatory element (WPRE), and various other cis-acting elements that are bound by RNA-binding proteins.
  • the vectors can additionally harbor sequences corresponding to the 5'-ETS and ITS elements of the precursor RNA sequence.
  • the transgene is typically operably fused with the switch polynucleotide sequence on the expression construct.
  • the expression constructs can be recombinantly produced with many vectors well known in the art. These include viral vectors such as recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno-associated virus (AAV).
  • viral vectors such as recombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus, papilloma virus, or adeno-associated virus (AAV).
  • the vectors can be present in liposomes, e.g., neutral or cationic liposomes, such as DOSPA/DOPE, DOGS/DOPE or DMRIE/DOPE liposomes, and/or associated with other molecules such as DNA-anti-DNA antibody-cationic lipid (DOTMA/DOPE) complexes.
  • the expression constructs are based on retroviral vectors.
  • Some preferred embodiments of the invention can employ AAV vectors or adenoviral vectors for introducing into host cells the transgene that is operably linked to a polynucleotide switch of the invention.
  • reporter gene e.g., FLuc or GLuc
  • AAV vectors gRlT(+l)-FLuc(+3) and gR3T(+l)-GLuc(+3) which have a start codon in the functional +1 frame followed by a Cpfl DNase target sequence or guide RNA target sequence (gRT) and a reporter gene at +3 reading frame.
  • Cpfl makes double-strand cleavage at the gRT region, cellular non-homologous end joining system will repair the DNA double-strand break by introducing random length of insertion or deletion.
  • the reporter gene will be placed in frame with the +1 frame translational start codon and the reporter expression is activated ("switched on”).
  • Adeno-associated virus is a small, nonenveloped virus that was adapted for use as a gene transfer vehicle.
  • Adeno-associated virus vectors refer to recombinant adeno-associated viruses that are derived from nonpathogenic parvoviruses. They evoke essentially no cellular immune response, and produce transgene expression lasting months in most systems. Like adenovirus, adeno- associated virus vectors also have the capability to infect replicating and nonreplicating cells and are believed to be nonpathogenic to humans. Delivery of heterologous polynucleotide sequences via recombinant AAV can provide for safe, unobtrusive and sustained expression (> 2 years) of high levels of protein therapeutics.
  • adenoviral or retroviral based expression vector of the invention In order to construct an adenoviral or retroviral based expression vector of the invention, the transgene and an operably linked polynucleotide switch sequence are often inserted into the viral genome in the place of certain viral sequences to produce a viral construct that is replication-defective.
  • Methods for producing adenoviral and retroviral vectors are well-known in the art.
  • Suitable host or producer cells for producing recombinant retroviruses or retroviral vectors according to the invention are also well known in the art (e.g., 293T cells exemplified herein).
  • expression vectors of the invention that harbor a transgene sequence and an operably linked polynucleotide switch sequence can be readily constructed in accordance with methodologies known in the art of molecular biology in view of the exemplifications provided herein. See, e.g., Sambrook et al, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press (3 rd ed., 2001); Brent et al, Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (ringbou ed., 2003); and Freshney, Culture of Animal Cells: A Manual of Basic Technique, Wiley-Liss, Inc. (4 th ed., 2000).
  • the expression vectors are assembled by inserting into a suitable vector backbone the transgene harboring a heterologous polynucleotide or transgene of interest and a polynucleotide switch described herein, as well as sequences encoding, e.g., selection markers, and other optional elements.
  • a suitable vector backbone the transgene harboring a heterologous polynucleotide or transgene of interest and a polynucleotide switch described herein, as well as sequences encoding, e.g., selection markers, and other optional elements.
  • Many virus based expression vector systems well known in the art can be used in the invention. Widely used retroviral vectors include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), simian immunodeficiency virus (SIV), human immunodeficiency virus (HIV), and combinations thereof (see, e.g., Buchscher et al , J.
  • Adeno-associated viral vectors have also been used in many reported studies for gene therapy in research and clinical environment. See, e.g., Kaplitt et al, Lancet 369: 2097-105, 2007; Daya et al, Clin Microbiol Rev.
  • AAV based expression vectors for practicing the invention can be based on the pAAV-MCS construct that is available from Agilent Technoligies (Santa Clara, CA). Similarly, a number of retroviral vectors and compatible packing cell lines are available from Clontech (Mountain View, CA).
  • lentiviral based vectors examples include, e.g., pLVX-Puro, pLVX-IRES- Neo, pLVX-IRES-Hyg, and pLVX-IRES-Puro.
  • Corresponding packaging cell lines are also available, e.g., Lenti-X 293T cell line.
  • other retroviral based vectors and packaging systems are also commercially available.
  • MMLV based vectors pQCXIP, pQCXIN, pQCXIQ and pQCXIH include MMLV based vectors pQCXIP, pQCXIN, pQCXIQ and pQCXIH, and compatible producer cell lines such as HEK 293 based packaging cell lines GP2-293, EcoPack 2-293 and AmphoPack 293, as well as NIH/3T3 -based packaging cell line
  • RetroPack PT67 Any of these and other retroviral vectors and producer cell lines may be employed in the practice of the present invention.
  • Vectors with Cpfl -based polynucleotide switches can be readily employed in many clinical or therapeutic settings. For example, they can be included in various gene transfer vectors, thus preparing the gene transfer vector to subsequently be switched on or switched off in a subject upon the addition of Cpfl.
  • the polynucleotide switches of the invention can be used to terminate expression of a transgene that is introduced into a subject for gene therapies.
  • the polynucleotide switches can be used to switch on the expression of a transgene encoded by a gene therapy vector administered to a subject. In certain embodiments, it is preferable to eliminate vector- transduced cells within a subject, rather than merely switch off the transgene.
  • a polynucleotide switch of the present invention can be employed to switch on the expression of a suicide gene, e.g., a caspase, in a subject who received the vector.
  • the vectors of the invention have a number of advantages over currently known systems for controlling transgene expression in mammalian settings.
  • the vectors can contain a scaffold RNA for Cpfl RNAse activity in the 3 '-untranslated region of the transgene.
  • expression of the transgene can be inactivated immediately (by degrading its message RNA transcript) and permanently (by cleaving at one or more sites of the transgene DNA).
  • Certain off-switch embodiments of the present invention in which the scaffold RNA and gRNA components of the polynucleotide switch are included in the transgene, have at least two notable safety advantages that are relevant in human subjects and clinical settings.
  • the crRNA-dependent DNase activity of Cpfl would only be active in the presence of the transgene. Although there might be cells that receive Cpfl that were not previously transduced with the vector expressing the transgene, there would be no off-target effects in these cells without the transgene that received only Cpfl. This is because the crRNA, which is necessary for activation of the DNase activity of Cpfl, is generated from the transgene.
  • the system is self-limiting, because after Cpfl cleaves a protospacer resulting in the switching off of transgene expression, the transgene can no longer be processed to form a mature crRNA, precluding the DNase activity of Cpfl.
  • the off-target effects of this polynucleotide switch in a subject are at an absolute minimum.
  • Retroviral vectors or recombinant retroviruses are widely employed in gene transfer in various therapeutic or industrial applications. For example, gene therapy procedures have been used to correct acquired and inherited genetic defects, and to treat cancer or viral infection in a number of contexts. The ability to express artificial genes in humans facilitates the prevention and/or cure of many important human diseases, including many diseases which are not amenable to treatment by other therapies.
  • the invention accordingly provides methods or uses of the Cpfl -dependent switches or expression vectors in various clinical or industrial bioengineering context.
  • the vectors expressing a transgene can be transduced into host cells in various gene therapy and other clinical applications.
  • the transgene harbored by the vector can encode a therapeutic agent.
  • These constructs can be transferred, for example to treat cancer cells, to express immunomodulatory genes to fight viral infections, or to replace a gene's function as a result of a genetic defect.
  • the polynucleotide switches of the present invention can be used to optimize dosing of a therapeutic protein encoded by a transgene.
  • Cpfl can be provided in an amount that is sufficient to switch off transgene expression in some but not all of the vectors in a subject.
  • Cpfl can be provided in an amount that is sufficient to switch off some but not all copies of a vector present within a cell.
  • a transgene that is initially administered to a subject at a level that is too high can be switched off in some but not all transduced cells, in order to decrease the dose of the transgene to the subject.
  • Transgene expression by the vectors of the present invention can be switched on or switched off, in some or all transduced cells in a subject by administering to the subject a Cpfl protein (or a Cpfl -expressing polynucleotide construct) that can cleave both the expression vector and the mRNA transcript.
  • expression of the therapeutic agent can be temporarily terminated by degrading the mRNA transcript but not the expression vector itself.
  • Temporary shut-off at the RNA level can be achieved with engineered Cpfl variants maintaining only the RNAse activity as exemplified herein.
  • a Cpfl enzyme needs to be present to mediate cleavage of the protospacer in the vector administered to a subject undergoing treatment.
  • the Cpfl protein can be delivered to the patient, e.g., in case of adverse events, via one of several approaches. These include, e.g., (1) delivery by AAV with the original AAV transgene and activated when necessary by a morphilino or small molecules, (2) delivery by AAV to the site of transgene expression, for example the liver or a specific set of muscle cells, and (3) delivery as a mRNA message to the site of transgene expression.
  • the Cpfl protein or variant can be administered to the subject via a separate expression vector as exemplified herein.
  • an ORF present in the transgene in the expression vectors of the invention can encode any polypeptide of interest.
  • the ORF or transgene encodes a polypeptide that is at least 90% identical to one or more human proteins.
  • the ORF can encode a constant region of an antibody, e.g., the Fc of IgGl, IgG2, IgG3, or IgG4, or other constant regions such as CHI, the constant region of a kappa light chain, or the constant region of a lambda light chain.
  • the transgene operably inserted into the polynucleotide switch containing expression vectors of the invention encodes a portion or a fragment (e.g., an antigen-binding fragment) derived from one or more immunoadhesins or antibodies.
  • antibody-related molecules that are well characterized in the art, e.g., CD4-Ig, eCD4-Ig, PG9, PG16, PGT121, PGT128, 10-1074, PGT145, PGT151, CAP256, 2F5, 4E10, 10E8, 3BNC117, VRCOl, VRC07, VRC13, PGDM1400, PGV04, 2G12, bl2, N6, TR66, etanercept, abatacept, rilonacept, aflibercept, belatacept, romiplostim, efmoroctocog, eftrenonacog, asfotase alpha, muromonab-CD3, edrecolomab, capromab, ibritumomab, blinatumomab, abciximab, rituximab, basiliximab, infliximab
  • the transgene in the expression vectors of the invention can encode at least a chain or functional fragment derived from any of the other known cellular proteins such as cellular receptors, other cell surface molecules, enzymes, cytokines, chemokines, costimulatory molecules, interleukins, and physiologically active polypeptide factors.
  • these known cellular proteins include, e.g., CD4, TPST1, TPST2, TNFR II, CD28, CTLA-4, PD-1, PD-L1, PD-L2, 4-1BBL, 4-1BB, EPO, Factor VIII, Factor IX, alkaline phosphatase, hemoglobin, fetal hemoglobin, and RPE65.
  • the polypeptide expressed from the ORF in the expression vectors of the invention is at least part of a chimeric antigen receptor (CAR).
  • the expression vectors of the invention can be used in gene therapies for expression many therapeutic agents known in the art. These include factor VIII, factor IX, ⁇ -globin, low-density lipoprotein receptor, adenosine deaminase, purine nucleoside phosphorylase, sphingomyelinase, glucocerebrosidase, cystic fibrosis
  • transmembrane conductance regulator a-antitrypsin, CD- 18, ornithine transcarbamylase, argininosuccinate synthetase, phenylalanine hydroxylase, branched-chain a-ketoacid dehydrogenase, fumarylacetoacetate hydrolase, glucose 6-phosphatase, a-L-fucosidase, ⁇ - glucuronidase, a-L-iduronidase, galactose 1 -phosphate uridyltransferase, interleukins, cytokines, small peptides, and the like.
  • therapeutic agents or proteins of interest include, but are not limited to, insulin, erythropoietin, tissue plasminogen activator (tPA), urokinase, streptokinase, neutropoesis stimulating protein (also known as filgastim or granulocyte colony stimulating factor (G-CSF)), thrombopoietin (TPO), growth hormone, emoglobin, insulinotropin, imiglucerase, sarbramostim, endothelian, soluble CD4, and antibodies and/or antigen-binding fragments (e.g., FAbs) thereof (e.g., orthoclone OKT-e (anti-CD3), GPIIb/IIa monoclonal antibody), ciliary neurite transforming factor (CNTF), granulocyte macrophage colony stimulating factor (GM-CSF), brain-derived neurite factor (BDNF), parathyroid hormone(PTH)-like hormone, insulinotrophic hormone, insulin-
  • tPA tissue
  • expression vectors or polynucleotide switches of the present invention can be used to control expression of a transgene for regulating cell growth, differentiation or viability in cells transplanted into a subject.
  • the expression constructs used in these methods expresses a transgene operably linked to a polynucleotide switch of the invention.
  • the transgene can contain an ORF encoding a polypeptide that regulates the growth or other cellular processes of the cell.
  • the level of expression of the polypeptide can be readily switched on or off via the targeted digestion of the vector and also the
  • these methods can be used to prevent the growth of hyperplastic or tumor cells, or even the unwanted proliferation of normal cells.
  • the methods can also be used to induce the death of fat cells, to regulate growth and differentiation of stem cells, or to regulate an immune response.
  • the transgene to be expressed under the control of Cpfl- dependent polynucleotide switches of the present invention can encode a therapeutic polypeptide or agent noted above.
  • transfection of tumor suppressor gene p53 into human breast cancer cell lines has led to restored growth suppression in the cells (Casey et al, Oncogene 6: 1791-7, 1991).
  • the Rb protein can be employed similarly.
  • the transgene operably linked to a polynucleotide switch of the present invention can encode an enzyme.
  • the gene can encode a cyclin-dependent kinase (CDK).
  • Additional embodiments of the invention encompass expression in target cells of cell adhesion molecules, other tumor suppressors such as p21 and BRCA2, inducers of apoptosis such as Bax and Bak, other enzymes such as cytosine deaminases and thymidine kinases, hormones such as growth hormone and insulin, and interleukins and cytokines.
  • target cells are mammalian cells, e.g., cells of both human and non-human animals including vertebrates and mammals.
  • the target cells are cancer or tumor cells.
  • the target cells are stem cells.
  • the vector introduced into the cells can express a transgene that regulates differentiation, proliferation, or death (e.g., by apoptosis) of stem cells.
  • a stem cell therapy can include stem cells modified to include a vector containing a polynucleotide on-switch that expresses a transgene such as a suicide gene that promotes apoptosis, e.g., a caspase.
  • Stem cells suitable for practicing the invention include and are not limited to hematopoietic stem cells (HSC), embryonic stem cells, pluripotent stem cells, or mesenchymal stem cells.
  • the invention provides engineered mammalian cells which express a transgene that is operably fused to an polynucleotide switch described herein.
  • Cpfl -dependent polynucleotide switch of the present invention various mammalian cells can be employed for introducing a vector of the invention or by stably integrating the rDNA described herein into the host genome.
  • Vectors encoding Cpfl -dependent polynucleotide switches can be introduced into an appropriate host cell (e.g., a mammalian cell such as 293T cell, N2a cell or CHO cell) by any means known in the art.
  • the cells can transiently or stably express the introduced transgene.
  • mammalian cells are used in these embodiments of the invention.
  • Mammalian expression systems allow for proper post-translational modifications of expressed mammalian proteins to occur, e.g., proper processing of the primary transcript, glycosylation, phosphorylation and advantageously secretion of expressed product.
  • Suitable cells include cells rodent, cow, goat, rabbit, sheep, non-human primate, human, and the like).
  • Specific examples of cell lines include CHO, BHK, HEK293, N2a, VERO, HeLa, COS, MDCK, and W138.
  • any convenient protocol may be employed for in vitro or in vivo introduction of the vector into the host cell, depending on the location of the host cell.
  • the expression vector may be introduced directly into the cell under cell culture conditions permissive of viability of the host cell, e.g., by using standard transduction techniques.
  • the targeting vector may be administered to the organism or host in a manner such that the vector is able to enter the host cell(s), e.g., via an in vivo or ex vivo protocol.
  • in vivo it is meant in the target construct is administered to a living body of an animal.
  • ex vivo it is meant that cells or organs are modified outside of the body. Such cells or organs are typically returned to a living body. Techniques well known in the art for the transfection of cells can be used for the ex vivo administration of vectors. The exact formulation, route of administration and dosage can be chosen empirically. See e.g.
  • DNA and RNA vectors can be delivered with cationic lipids (Goddard, et al, Gene Therapy, 4: 1231-1236, 1997; Gorman et al., Gene Therapy 4:983-992, 1997; Chadwick et al, Gene Therapy 4:937-942, 1997; Gokhale et al, Gene Therapy 4: 1289-1299, 1997; Gao and Huang, Gene Therapy 2:710-722, 1995), using viral vectors (Monahan et al, Gene Therapy 4:40-49, 1997; Onodera et al, Blood 91 :30-36, 1998), by uptake of "naked DNA", and the like.
  • the vectors or expression constructs of the invention can be introduced into the target cells via a liposome.
  • the physical properties of liposomes depend on pH, ion strength and the existence of divalent cations.
  • Pharmaceutical preparations or compositions are typically employed in the practice of the various therapeutic embodiments of the invention.
  • the pharmaceutical preparations contain a vector harboring the polynucleotide switch.
  • a transgene sequence is operably linked to the polynucleotide switch in the vector as described herein.
  • the pharmaceutical compositions of the invention can also contain a pharmaceutically acceptable carrier suitable for administration to a human or non-human subject.
  • the pharmaceutically acceptable carrier can be selected from pharmaceutically acceptable salts, ester, and salts of such esters.
  • the pharmaceutical compositions may be administered to a subject via any route including, but not limited to, intramuscular, buccal, rectal, intravenous or intracoronary routes.
  • LbCpfl DNase mutants inactivated GLuc as efficiently as wild-type LbCpfl.
  • RNase LbCpfl mutants retained their DNase activity when an appropriate crRNA was expressed from a U6 (Pol III) promoter.
  • H759A, K768A, and K785A LbCpfl variants, but not Cpfl DNase domain mutants D832A and E925A mediated efficient cleavage in the EGFP gene of an integrated DNA double-strand break (DSB) reporter construct (Fig. 3a), as indicated by a T7E1 mismatch cleavage assay (Fig. le).
  • GLuc expression was turned on by a polynucleotide switch of the present invention by cleavage of a protospacer in EGFP coding region and its repair in a manner that changed the reading frame, thereby switching on the expression of GLuc.
  • Wild-type LbCpfl efficiently introduced mutations in an integrated transgene encoding a green-fluorescent protein variant engineered for a short half-life (EGFPd4), as indicated by a T7E1 assay (Fig. 2b) and by loss of GFP fluorescence (Fig. 2c, Fig. 4b). Nearly identical results were obtained when gRl in the 3' UTR was replaced by a different guide RNA, gR3 (Fig. 4c and 4d). In both cases, LbCpfl inactivated more than half of all GFP expression, consistent with efficient editing of the integrated EGFPd4 gene.
  • wild-type LbCpfl coexpressed with the GLuc transcript also efficiently induced expression of firefly luciferase (FLuc) from a DSB-induced gain-of-expression reporter plasmid (Certo et al. Nat. Methods 8, 671-676, 2011) (Fig 2d; assay depicted in Fig. 5 a).
  • FLuc firefly luciferase
  • Fig 2d assay depicted in Fig. 5 a
  • This gain-of-expression was caused by the Cpfl DNase activity cleaving a protospacer, the repair of which resulted in a change of reading frame, thus switching on FLuc expression.
  • This experiment shows that our approach can be used to switch on the expression of a transgene.
  • RNA transcripts (Fig. 2e) in a DSB-induced gain-of-expression assay (depicted in Fig. 5d).
  • Two DSB reporter plasmids were used in this assay.
  • One reporter, gRlT(+l)-FLuc(+3) carries the target sequence for gRl and a DSB- responsive FLuc gene.
  • the other reporter, gR3T(+l)-GLuc(+3) carries the target sequence for gR3 and a DSB-responsive GLuc gene.
  • Example 3 Switching off transgene expression at RNA level
  • the same Pol II transcript can contain a transgene, a scaffold RNA and a guide RNA (gRNA). In such embodiments where all of these elements reside on the same transcript, transgene expression can be shut off at the RNA level.
  • gRNA guide RNA
  • the plasmid vector expressing both Firefly luciferase and the transcript containing the scaffold RNA and gRNA was co-trasnfected into 293T cells with an empty plasmid vector or a plasmid vector expressing either wild-type LbCpfl, RNase domain mutant LbCpfl H759A, or DNase domain mutant LbCpfl D832A.
  • the protospacer overlaps with the splice acceptor AG dinucleotide (SA) site (Fig. 7a).
  • SA splice acceptor AG dinucleotide
  • wild-type LbCpfl greatly diminished the expression of Firefly luciferase through a mechanism that includes cleavage of a protospacer overlapping the SA site.
  • the switching off of luciferase expression was abolished in the context of the DNase domain mutant, indicating that the effect is mediated by the cleavage of vector DNA.
  • the protospacer overlaps with the ATG start codon trinucleotide (Fig. 7b).
  • the Firefly luciferase expression vector contained two protospacers, with the first protospacer overlapping with the splice acceptor AG dinucleotide site and the second protospacer overlapping with the ATG start codon trinucleotide (Fig. 7c).
  • the scaffold RNA and gRNA were in the same Pol II transcript that expresses Firefly luciferase.
  • the wild-type LbCpfl efficiently inactivated the transgene.
  • the DNase domain mutant (H832A) LbCpfl also efficiently shut off expression, due to the RNase domain of LbCpfl cleaving the Firefly luciferase mRNA at a site proximal to the scaffold RNA.
  • the RNase domain mutant did not inactivate Firefly luciferase, since it could not process the scaffold and gRNA.
  • the protospacer resided in the coding region of the Firefly luciferase transgene (Fig 7d).
  • Fig 7d Nineteen of nineteen protospacer sequences within the Firefly luciferase coding region were functional as the protospacer for the self-directed inactivation of a transgene in the present invention.
  • mice were inoculated with 10 10 copies of an AAV vector encoding Firefly luciferase as a model transgene.
  • the same AAV vector that encoded Firefly luciferase also included a U6 promoter that expresses a transcript containing a scaffold RNA and a guide RNA (gRNA).
  • An otherwise-identical negative control vector was constructed that contained Firefly luciferase, but did not express a transcript containing a scaffold RNA and gRNA.
  • the gRNA was engineered to match a protospacer sequence in the coding region of the Firefly luciferase transgene.
  • a separate AAV vector encoding LbCpfl or a control AAV vector was administered to the same site. Luciferase activity was quantified using a Xenogen imager on days 8, 14, 21, and 28 post- injection (Fig. 8).

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Abstract

La présente invention concerne des commutateurs polynucléotidiques autodirigés pouvant activer ou désactiver l'expression d'un transgène dans des cellules de mammifère à partir d'un vecteur en présence de Cpf1. L'invention concerne également des procédés d'utilisation des commutateurs dépendant de Cpf1 et des vecteurs d'expression de l'invention dans la régulation de l'expression transgénique dans diverses applications cliniques ou industrielles.
PCT/US2018/020619 2017-03-09 2018-03-02 Vecteurs comprenant des commutateurs dépendant de cpf1 autodirigés Ceased WO2018164948A1 (fr)

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CN113897397A (zh) * 2021-09-30 2022-01-07 中南大学 一种基于DNAzyme调控基因编辑的方法

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CN113897397B (zh) * 2021-09-30 2024-04-02 中南大学 一种基于DNAzyme调控基因编辑的方法

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