WO2018009869A1 - Cellules métaboliquement compétentes, leurs procédés de fabrication, et utilisations - Google Patents

Cellules métaboliquement compétentes, leurs procédés de fabrication, et utilisations Download PDF

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WO2018009869A1
WO2018009869A1 PCT/US2017/041202 US2017041202W WO2018009869A1 WO 2018009869 A1 WO2018009869 A1 WO 2018009869A1 US 2017041202 W US2017041202 W US 2017041202W WO 2018009869 A1 WO2018009869 A1 WO 2018009869A1
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genetically engineered
cas9
engineered cell
cell
family
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Christopher Dillon VULPE
Amin SOBH
David Michael FAULKNER
Abderrahmane TAGMOUNT
Michael FASULLO
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University of Florida
University of Florida Research Foundation Inc
Research Foundation of the State University of New York
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University of Florida
University of Florida Research Foundation Inc
Research Foundation of the State University of New York
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    • 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
    • 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
    • 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
    • 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
    • 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
    • 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
    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
    • C12N15/1034—Isolating an individual clone by screening libraries
    • C12N15/1079—Screening libraries by altering the phenotype or phenotypic trait of the host
    • 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
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)

Definitions

  • This invention was created using funds received from Grant Number Project #2017- 10, awarded by the Johns Hopkins Bloomberg School of Public Health, Center for Alternatives to Animal Testing.
  • Described in some aspects herein are genetically engineered cells that can contain one or more modulated metabolic genes, wherein the expression of the one or more modulated metabolic genes can be greater than the expression in an unmodified control cell.
  • the genetically engineered cells can be an immortalized cell.
  • the one or more modulated genes can be a cytochrome P450, phase I , or phase I I gene.
  • the genetically engineered cell can be metabolically competent.
  • the genetically engineered cell can include in some aspects one or more sgRNAs for one or more metabolic genes.
  • the he genetically engineered cell can include a Cas9.
  • the Cas9 can be inactive. l
  • the Cas9 can be operatively linked to a VP64 transcriptional activator.
  • the genetically engineered cell can include one or more Cas9 transcriptional activators.
  • the one or more CAs9 transcriptional activators can be a VP64 transcriptional activator, a MS2-p65-HSF1 co-activation complex, a MS2 RNA aptamer sgRNA, and combinations thereof.
  • the one or more Cas9 transcriptional activators can form a synergistic activation mediator.
  • the one or more modulated metabolic genes can be selected from the group set forth in Table 1 or any combination thereof.
  • Also described herein in some aspects are methods of producing a genetically engineered cell as described herein, where the method can include the step of enhancing transcription of one or more metabolic genes in a cell using synergistic activation mediator CRISPR-Cas9, wherein transcription of one or more metabolic genes can be increased as compared to an unmodified control cell.
  • assays can include the steps of exposing a genetically engineered cell as described herein with a test chemical or compound for a period of time and measuring a physiologic characteristic of the genetically engineered cell.
  • the assay can further include the step of determining the toxicity of the test chemical or compound from the measured physiologic characteristic.
  • Fig. 1 shows a diagram of CAs9 based transcription activation.
  • Fig. 2 shows a diagram of an approach to activate cytochrome P450 or other metabolic gene(s) in a cell line for high-throughput screening (HTS) assays.
  • CRISPR metabolic activation of genes (C-MAGIC) components can be expressed in a reporter cell line.
  • Targeting sgRNA(s) can be provided to the reporter cell line by expression, infection, or transfection.
  • the targeted metabolic genes can then be expressed.
  • Metabolic competence of modified reporter cell lines can be optionally assessed.
  • the metabolically competent engineered cell lines can then be used in a HTS assay.
  • Fig. 3 shows a process flow diagram for generating and utilizing metabolically competent cells for an HTS via CRISPR based transcription activation.
  • Fig. 4 shows a graph demonstrating RT-qPCR results after transient transfection for the sgRNA target of CYP1 A2 and evaluation activation of gene expression of CYP1 A2 and various Phase I genes including CYP3A4, CYP3A5, CYP2E1 , CYP2B6..
  • 293T refers to the HEK293T cell line control which did not receive any treatment or transfection.
  • Pooh refers to a transfection conducted in which sgRNAs CYP1A2, CYP2B6, CYP2E1 , CYP3A4, UGT1A6, dCas9-VP84 and MS2-P65-HSF1 were pooled and administered to HEK293T cells simultaneously in a single transfection.
  • Pool2 refers to the same pooling of the same components as Pooh , except that sgRNAs for CYP1A2, CYP2B6, CYP2E1 , CYP3A4, and UGT1A6 with slightly different sequences were used.
  • Pool3 refers to the same pooling of the same components as Pooh and Pool2, except that sgRNAs for CYP1A2, CYP2B6, CYP2E1 , CYP3A4, and UGT1A6 with slightly different sequences were used.
  • SAM refers to a control treatment wherein the HEK293T cells were transfected with the dCas9, and MS2- p65-HSF1 plasmids, but no sgRNA plasmids for any metabolic genes.
  • Fig. 5 shows a graph demonstrating RT-qPCR results after transient transfection for the sgRNA target of CYP3A4 and evaluation of activation of gene expression of CYP3A4 and various Phase I genes including CYP3A4, CYP3A5, CYP2E1 , CYP2B6..
  • 293T refers to the HEK293T cell line control which did not receive any treatment or transfection.
  • Pooh refers to a transfection conducted in which sgRNAs CYP1A2, CYP2B6, CYP2E1 , CYP3A4, UGT1A6, dCas9-VP64 and S2-P65-HSF1 were pooled and administered to HEK293T cells simultaneously in a single transfection.
  • Pool2 refers to the same pooling of the same components as Pooh , except that sgRNAs for CYP1A2, CYP2B6, CYP2E1 , CYP3A4, and UGT1A6 with slightly different sequences were used.
  • Pool3 refers to the same pooling of the same components as Pooh and Pool2, except that sgRNAs for CYP1A2, CYP2B6, CYP2E1 , CYP3A4, and UGT1A6 with slightly different sequences were used.
  • SAM refers to a control treatment wherein the HEK293T cells were transfected with the dCas9, and MS2- p65-HSF1 plasmids, but no sgRNA plasmids for any metabolic genes.
  • Fig. 6 shows a graph demonstrating RT-qPCR results after transient transfection for the sgRNA target of CYP2E1 and evaluation of activation of gene expression of CYP2E1 and other various Phase I and Phase II genes.
  • CYP255 indicates the gene for the CYP2B6 enzyme.
  • UGT1 a95 indicates the gene for the UGT1 a9 enzyme.
  • Fig. 7 shows a graph demonstrating RT-qPCR results after transient transfection for the sgRNA target of CYP2E1 and evaluation of gene expression of CYP2B6 and UGT1A6 and other Phase I genes and Phase II genes.
  • CYP255 indicates the gene for the CYP2B6 enzyme.
  • UGT1 a95 indicates the gene for the UGT1 a9 enzyme.
  • Fig. 8 shows a graph demonstrating RT-qPCR results from 293T-Cas9-vP64 cells transfected with the MS2-p65-HSF1 activator helper component and CYP2B6 or CYP2E1 sgRNA plasmids.
  • Fig. 9 shows a graph demonstrating RT-qPCR results from 293T-Cas9-vP64 cells transfected with the MS2-p65-HSF1 activator helper component and CYP3A4 sgRNA plasmid.
  • FIG. 10 shows a graph demonstrating RT qPCR results from 293T-CYP3A5/1 A1 /3A4 cells (a HEK293T cell line stably transfected with CYP3A4, CYP3A5, and CYP1 A1 ) and 293T-SAM cells (a HEK293T cell line stably transfected with dCas9-VP64 and MS2-P65- HSF 1) , transfected with CYP2B6, CY2E1 , CYP1 A2.
  • Embodiments of the present disclosure will employ, unless otherwise indicated, techniques of molecular biology, microbiology, nanotechnology, toxicology, pharmacology, physiology, organic chemistry, biochemistry, botany and the like, which are within the skill of the art. Such techniques are explained fully in the literature.
  • nucleic acid and polynucleotide generally refer to a string of at least two base-sugar-phosphate combinations and refers to, among others, single-and double-stranded DNA, DNA that is a mixture of single-and double-stranded regions, single- and double-stranded RNA, and RNA that is mixture of single- and double-stranded regions, hybrid molecules comprising DNA and RNA that may be single-stranded or, more typically, double-stranded or a mixture of single- and double-stranded regions.
  • polynucleotide as used herein refers to triple-stranded regions comprising RNA or DNA or both RNA and DNA.
  • the strands in such regions may be from the same molecule or from different molecules.
  • the regions may include all of one or more of the molecules, but more typically involve only a region of some of the molecules.
  • One of the molecules of a triple- helical region often is an oligonucleotide.
  • Polynucleotide and “nucleic acids” also encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including simple and complex cells, inter alia.
  • the term polynucleotide includes DNAs or RNAs as described above that contain one or more modified bases.
  • DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples are polynucleotides as the term is used herein.
  • Polynucleotide and “nucleic acids” also includes PNAs (peptide nucleic acids), phosphorothioates, and other variants of the phosphate backbone of native nucleic acids.
  • Natural nucleic acids have a phosphate backbone, artificial nucleic acids may contain other types of backbones, but contain the same bases.
  • DNAs or RNAs with backbones modified for stability or for other reasons are "nucleic acids" or "polynucleotide” as that term is intended herein.
  • RNA deoxyribonucleic acid
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • DNA deoxyribonucleic acid
  • RNA ribonucleic acid
  • RNA may be in the form of a tRNA (transfer RNA), snRNA (small nuclear RNA), rRNA (ribosomal RNA), mRNA (messenger RNA), anti-sense RNA, RNAi (RNA interference construct), siRNA (short interfering RNA), or ribozymes.
  • nucleic acid sequence and “oligonucleotide” also encompasses a nucleic acid and polynucleotide as defined above.
  • DNA molecule includes nucleic acids/polynucleotides that are made of DNA.
  • RNA molecule includes nucleic acids/polynucleotides that are made of RNA.
  • gene refers to a hereditary unit corresponding to a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a characteristic(s) or trait(s) in an organism.
  • the term “gene” encompasses specific nucleotide sequences of a genome that are transcribed into an RNA product and are not translated into a protein as well as those genomic sequences that are transcribed into an RNA product yet are translated into a protein.
  • locus refers to the position that a given gene or portion thereof occupies on a chromosome of a given species.
  • exogenous DNA or “exogenous nucleic acid sequence” or “exogenous polynucleotide” refers to a nucleic acid sequence that was introduced into a cell, organism, or organelle via transfection.
  • Exogenous nucleic acids originate from an external source, for instance, the exogenous nucleic acid may be from another cell or organism and/or it may be synthetic and/or recombinant. While an exogenous nucleic acid sometimes originates from a different organism or species, it may also originate from the same species (e.g. , an extra copy or recombinant form of a nucleic acid that is introduced into a cell or organism in addition to or as a replacement for the naturally occurring nucleic acid). Typically, the introduced exogenous sequence is a recombinant sequence.
  • the term "recombinant” generally refers to a non-naturally occurring nucleic acid, nucleic acid construct, or polypeptide.
  • Such non-naturally occurring nucleic acids may include natural nucleic acids that have been modified, for example that have deletions, substitutions, inversions, insertions, etc.
  • nucleic acid sequences of different origin that are joined using molecular biology technologies
  • a nucleic acid sequences encoding a "fusion protein” e.g., a protein or polypeptide formed from the combination of two different proteins or protein fragments
  • the combination of a nucleic acid encoding a polypeptide to a promoter sequence where the coding sequence and promoter sequence are from different sources or otherwise do not typically occur together naturally (e.g, a nucleic acid and a constitutive promoter), etc.
  • Recombinant also refers to the polypeptide encoded by the recombinant nucleic acid.
  • Non-naturally occurring nucleic acids or polypeptides include nucleic acids and polypeptides modified by man.
  • polypeptides or "proteins” are as amino acid residue sequences. Those sequences are written left to right in the direction from the amino to the carboxy terminus. In accordance with standard nomenclature, amino acid residue sequences are denominated by either a three letter or a single letter code as indicated as follows: Alanine (Ala, A) , Arginine (Arg, R), Asparagine (Asn, N) , Aspartic Acid (Asp, D) , Cysteine (Cys, C) , Glutamine (Gin, Q), Glutamic Acid (Glu, E) , Glycine (Gly, G), Histidine (His, H) , Isoleucine (lie, I) , Leucine (Leu, L), Lysine (Lys, K), Methionine (Met, M) , Phenylalanine (Phe, F), Proline (Pro, P) , Serine (Ser, S), Threon
  • peptide refers to chains of at least 2 amino acids that are short, relative to a protein or polypeptide.
  • variant refers to a polypeptide that differs from a reference polypeptide, but retains essential properties.
  • a typical variant of a polypeptide differs in amino acid sequence from another, reference polypeptide. Generally, differences are limited so that the sequences of the reference polypeptide and the variant are closely similar overall and, in many regions, identical.
  • a variant and reference polypeptide may differ in amino acid sequence by one or more modifications (e.g. , substitutions, additions, and/or deletions).
  • a substituted or inserted amino acid residue may or may not be one encoded by the genetic code.
  • a variant of a polypeptide may be naturally occurring such as an allelic variant, or it may be a variant that is not known to occur naturally.
  • “functional variant” refers to a variant of a protein or polypeptide (e.g., a variant of a Cas9 protein) that can perform the same functions or activities as the original protein or polypeptide, although not necessarily at the same level (e.g., the variant may have enhanced, reduced or changed functionality, so long as it retains the basic function).
  • identity is a relationship between two or more polypeptide or nucleic acid sequences, as determined by comparing the sequences. In the art, “identity” also refers to the degree of sequence relatedness between polypeptides or nucleic acid sequences as determined by the match between strings of such sequences. “Identity” can be readily calculated by known methods, including, but not limited to, those described in (Computational Molecular Biology, Les , A. M. , Ed. , Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. I I . , Ed. , Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H.
  • plasmid refers to a non-chromosomal double- stranded DNA sequence including an intact "replicon” such that the plasmid is replicated in a host cell.
  • a vector may include a DNA molecule, linear or circular (e.g. plasmids), which includes a segment encoding a polypeptide of interest operatively linked to additional segments that provide for its transcription and translation upon introduction into a host cell or host cell organelles.
  • additional segments may include promoter and terminator sequences, and may also include one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, etc.
  • Expression vectors are generally derived from yeast or bacterial genomic or plasmid DNA, or viral DNA, or may contain elements of both.
  • promoter includes all sequences capable of driving transcription of a gene.
  • promoter as used herein can refer to a DNA sequence generally described as the 5' regulator region of a gene, located proximal to the start codon. The transcription of an adjacent gene sequence is initiated at the promoter region.
  • promoter also includes fragments of a promoter that are functional in initiating transcription of the gene.
  • promoter can encompass constitutive promoters and inducible promoters.
  • constitutive promoter is a promoter that allows for continual or ubiquitous transcription of its associated gene or polynucleotide. Constitutive promoters are generally are unregulated by cell or tissue type, time, or environment.
  • inducible promoter is a promoter that allows transcription of its associated gene or polynucleotide in response to a substance or compound (e.g. an antibiotic, or metal) , an environmental condition (e.g. temperature), developmental stage, or tissue type.
  • a substance or compound e.g. an antibiotic, or metal
  • an environmental condition e.g. temperature
  • developmental stage e.g. developmental stage, or tissue type.
  • wild-type is the average form of an organism, variety, strain, gene, protein, or characteristic as it occurs in a given population in nature, as distinguished from mutant forms that may result from selective breeding, recombinant engineering, and/or transformation with a transgene.
  • operatively linked indicates that the regulatory sequences useful for expression of the coding sequences of a nucleic acid are placed in the nucleic acid molecule in the appropriate positions relative to the coding sequence so as to effect expression of the coding sequence.
  • This same definition is sometimes applied to the arrangement of coding sequences and transcription control elements (e.g. promoters, enhancers, and termination elements) , and/or selectable markers in an expression vector.
  • expression as used herein describes the process undergone by a structural gene to produce an RNA molecule and/or polypeptide. It can refer to the combination of transcription and translation. Expression can refer to the "expression" of a nucleic acid to produce a RNA molecule and can also refers to "expression" of a polypeptide, indicating that the polypeptide is being produced via expression of the corresponding nucleic acid.
  • selectable marker refers to a gene whose expression allows one to identify cells that have been transformed or transfected with a vector containing the marker gene.
  • a recombinant nucleic acid may include a selectable marker operatively linked to a gene of interest and a promoter, such that expression of the selectable marker indicates the successful transformation of the cell with the gene of interest.
  • guide polynucleotide can refer to any polynucleotide sequence having sufficient complementarity with a target polynucleotide sequence to hybridize with the target sequence and direct sequence-specific binding of a CRISPR complex to the target sequence.
  • the degree of complementarity between a guide polynucleotide and its corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or more than about 50%, 60%, 75% , 80%, 85%, 90% , 95% , 97.5%, 99% , or more.
  • Optimal alignment may be determined with the use of any suitable algorithm for aligning sequences, non-limiting examples of which include the Smith- Waterman algorithm, the Needleman-Wunsch algorithm, algorithms based on the Burrows- Wheeler Transform (e.g. the Burrows Wheeler Aligner) , ClustalW, Clustal X, BLAT, Novoalign (Novocraft Technologies, ELAND (lllumina, San Diego, Calif.), SOAP (available at soap.genomics.org.cn), and Maq (available at maq.sourceforge.net) .
  • Burrows- Wheeler Transform e.g. the Burrows Wheeler Aligner
  • ClustalW ClustalW
  • Clustal X Clustal X
  • BLAT Novoalign
  • SOAP available at soap.genomics.org.cn
  • Maq available at maq.sourceforge.net
  • a guide polynucleotide (also referred to herein as a guide sequence and includes single guide sequences (sgRNA)) can be about or more than about 5, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75, 90, 100, 1 10, 1 12, 1 15, 120, 130, 140, or more nucleotides in length.
  • the guide polynucleotide can include a nucleotide sequence that is complementary to a target DNA sequence. This portion of the guide sequence can be referred to as the complementary region of the guide RNA.
  • the two are distinguished from one another by calling one the complementary region or target region and the rest of the polynucleotide the guide sequence or tracrRNA.
  • the guide sequence can also include one or more miRNA target sequences coupled to the 3' end of the guide sequence.
  • the guide sequence can include one or more MS2 RNA aptamers incorporated within the portion of the guide strand that is not the complementary portion.
  • guide sequence can include any specially modified guide sequences, including but not limited to those configured for use in synergistic activation mediator (SAM) implemented CRISPR (Nature 517, 583-588 (29 January 2015).
  • a guide polynucleotide can be less than about 150, 125, 75, 50, 45, 40, 35, 30, 25, 20, 15, 12, or fewer nucleotides in length.
  • the ability of a guide polynucleotide to direct sequence-specific binding of a CRISPR complex to a target sequence may be assessed by any suitable assay.
  • the components of a CRISPR system sufficient to form a CRISPR complex, including the guide polynucleotide to be tested may be provided to a host cell having the corresponding target sequence, such as by transfection with vectors encoding the components of the CRISPR sequence, followed by an assessment of preferential cleavage within the target sequence.
  • cleavage of a target polynucleotide sequence may be evaluated in a test tube by providing the target sequence, components of a CRISPR complex, including the guide polynucleotide to be tested and a control guide polynucleotide different from the test guide polynucleotide, and comparing binding or rate of cleavage at the target sequence between the test and control guide polynucleotide reactions.
  • Other assays are possible, and will occur to those skilled in the art.
  • a complementary region of the gRNA can be configured to target any DNA region of interest.
  • the complementary region of the gRNA and the gRNA can be designed using a suitable gRNA design tool. Suitable tools are known in the art and are available to the skilled artisan. Some such tools are discussed elsewhere herein. As such, the constructs described herein are enabled for any desired target DNA so long as it is CRISPR compatible according to the known requirements for CRISPR activation.
  • a guide polynucleotide can be selected to reduce the degree of secondary structure within the guide polynucleotide. Secondary structure may be determined by any suitable polynucleotide folding algorithm. Some programs are based on calculating the minimal Gibbs free energy.
  • mFold as described by Zuker & Stiegler ((1981) Nucleic Acids Res. 9, 133-148).
  • Another example folding algorithm is the online webserver RNAfold, developed at Institute for Theoretical Chemistry at the University of Vienna, using the centroid structure prediction algorithm (see e.g. Gruber et a/. , (2008) Cell 106: 23-24; and Carr & Church (2009) Nature Biotechnol. 27: 1 151 -1 162).
  • Homology-directed repair refers to a mechanism in cells to repair double- stranded and single stranded DNA breaks.
  • Homology-directed repair includes homologous recombination (HR) and single-strand annealing (SSA) (Lieber. (2010) Annu. Rev. Biochem. 79: 181 -21 1 ).
  • HR homologous recombination
  • SSA single-strand annealing
  • Other forms of HDR include single-stranded annealing (SSA) and breakage-induced replication, and these require shorter sequence homology relative to HR.
  • Homology- directed repair at nicks can occur via a mechanism distinct from HDR at double-strand breaks.
  • Error-prone DNA repair refers to mechanisms that can produce mutations at double- strand break sites.
  • the Non-Homologous-End-Joining (NHEJ) pathways are the most common repair mechanism to bring the broken ends together (Bleuyard et a/. , (2006) DNA Repair 5: 1 - 12) .
  • the structural integrity of chromosomes is typically preserved by the repair, but deletions, insertions, or other rearrangements are possible.
  • the two ends of one double- strand break are the most prevalent substrates of NHEJ (Kirik et a/. , (2000) EMBO J.
  • CRISPR can also be used to activate specific genes through CRISPR/synergistic activation mediator procedures. These procedures can utilize a guide polynucleotide that incorporates 2 MS2 RNA aptamers at the tetraloop and the stem- loop of the guide RNA such as that described in , but not limited to (Nature 517, 583-588 (29 January 2015).
  • binding refers to binding which occurs between such paired species as enzyme/substrate, receptor/agonist, antibody/antigen, and lectin/carbohydrate which may be mediated by covalent or non-covalent interactions or a combination of covalent and non-covalent interactions.
  • the binding which occurs is typically electrostatic, hydrogen- bonding, or the result of lipophilic interactions. Accordingly, “specific binding” occurs between a paired species where there is interaction between the two which produces a bound complex having the characteristics of an antibody/antigen or enzyme/substrate interaction.
  • the specific binding is characterized by the binding of one member of a pair to a particular species and to no other species within the family of compounds to which the corresponding member of the binding member belongs.
  • an antibody preferably binds to a single epitope and to no other epitope within the family of proteins.
  • Cas9 and Cas9 polypeptide are used interchangeably herein to refer to an enzyme (wild-type or recombinant) that can exhibit least endonuclease activity (e.g. cleaving the phosphodiester bond within a polynucleotide) guided by a CRISPR RNA (crRNA) bearing complementary sequence to a target polynucleotide.
  • Cas9 polypeptides are known in the art, and include Cas9 polypeptides from any of a variety of biological sources, including, e.g., prokaryotic sources such as bacteria and archaea.
  • Bacterial Cas9 includes, Actinobacteria (e.g., Actinomyces naeslundii) Cas9, Aquificae Cas9, Bacteroidetes Cas 9, Chlamydiae Cas9, Chloroflexi Cas9, Cyanobacteria Cas9, Elusimicrobia Cas9, Fibrobacteres Cas9, Firmicutes Cas9 (e.g., Streptococcus pyogenes Cas9, Streptococcus thermophilus Cas9, Listeria innocua Cas9, Streptococcus agalactiae Cas9, Streptococcus mutans Cas9, and Enterococcus faecium Cas9), Fusobacteria Cas9, Proteobacteria (e.g., Actinobacteria (e.g., Actinomyces naeslundii) Ca
  • Archaea Cas 9 includes Euryarchaeota Cas9 (e.g., Methanococcus maripaludis Cas9) and the like.
  • Cas9 and related polypeptides are known, and are reviewed in, e.g., Makarova et al. (201 1) Nature Reviews Microbiology 9:467-477, Makarova et al. (201 1) Biology Direct 6:38, Haft et al.
  • Cas9 polypeptides can be Francisella tularensis subsp. novicida Cas9, Pasteurella multocida Cas9, mycoplasma gallisepticum str.
  • Flavobacterium columnare Cas9 Fluviicola taffensis Cas9, Bacteroides coprophilus Cas9, mycoplasma mobile Cas9, lactobacillus farciminis Cas9, Streptococcus pasteurianus Cas9, Lactobacillus johnsonii Cas9, Staphlococcus pseudintermedius Cas9, filifactor alocis Cas9, Treponema denticola Cas9, Legionella pneumophila str. Paris Cas9, Sutterella wadsworthensis Cas9, and Corynebacter diptheriae Cas9.
  • Cas9 includes a Cas9 polypeptide of any Cas9 family, including any isoform of Cas9. Amino acid sequences of various Cas9 homologs, orthologs, and variants beyond those specifically stated or provided herein are known in the art and are publicly available, within the purview of those skill in the art, and thus within the spirit and scope of this disclosure.
  • inactive Cas9 refers to a Cas9 that lacks endonuclease activity.
  • corresponding to refers to the underlying biological relationship between these different molecules. As such, one of skill in the art would understand that operatively "corresponding to” can direct them to determine the possible underlying and/or resulting sequences of other molecules given the sequence of any other molecule which has a similar biological relationship with these molecules. For example, from a DNA sequence an RNA sequence can be determined and from an RNA sequence a cDNA sequence can be determined.
  • metabolic competent can refer to the manipulation, such as through genetic engineering, of the gene expression profile of a cell such that it expresses one or more metabolic related genes (including but not limited to those listed in Table 1 herein) at a different level (e.g. expressed at a greater lever or a reduced level) when compared to the unmodified cell.
  • gene (or protein) expression profile can refer to the gene (or protein) expression pattern observed across two or more genes in a cell, tissue, organ, and/or organism. In some instances, the gene (or protein) expression profile reflects the typical gene (or protein) for a type of cell, tissue, etc. across a population .
  • HTS High throughput screening
  • immortalized cells have different metabolic profiles from in vivo or in situ cells, which are typically the cells for which toxicity information is desired.
  • immortalized cells typically have poor, if any, expression of genes involved in metabolism of chemicals, such as cytochrome P450 enzymes.
  • metabolically competent cells that can be suitable for use in HTS assays, including but not limited to HTS toxicity assays.
  • the cells can be genetically engineered to express one or more cytochrome P450 enzyme genes.
  • the metabolically competent cells described herein can be generated using a SAM-mediated CRISPR-Cas9 system (or other CRISPR-Cas9 based transcriptional activation approach) to induce expression of one or more cytochrome P450 genes.
  • methods of screening chemicals and other compounds by exposing a metabolically competent cell or population thereof described herein with one or more chemicals or other compounds and measuring toxicity, metabolite production, and/or other relevant cellular endpoints of interest.
  • the cells and methods provided herein can improve existing in vitro screening tests by allowing for intracellular metabolic transformations and providing toxicity data, or other cellular endpoints of interest that require metabolic transformation that is more accurate and more reflective of the in vivo situation, and for the assessment of the role of cytochrome P450 allelic variation in xenobiotic metabolism toxicity and other biological activity.
  • the genetically engineered cells can include one or more modulated metabolic genes, where the expression of the modulated metabolic gene(s) is greater than the expression in an unmodified cell.
  • the modulated metabolic gene(s) can be, without limitation, any of the genes listed in Table 1 and any combination thereof.
  • the metabolic gene(s) can be a cytochrome P450 gene(s). Table 1 shows exemplary CYP450 and other phase I and I I metabolic genes related to chemical toxicity.
  • CYP1 1A1 cytochrome P450 family 1 1 , NG_007973.1 subfamily A, polypeptide 1
  • CYP1A1 cytochrome P450 family 1 , NG_008431 .2 subfamily A, polypeptide 1
  • CYP1A2 cytochrome P450 family 1 , NG_008431 subfamily A, polypeptide 2
  • CYP27A1 cytochrome P450 family 27, NC_000002.12 subfamily A, polypeptide 1
  • CYP3A5 cytochrome P450 family 3, AF355800.1 , subfamily A, polypeptide 5 AF355803.1
  • subfamily F polypeptide 1 1
  • CYP4F12 similar to cytochrome P450, family AB03513G.1
  • EPHX2 epoxide hydrolase 2 cytoplasmic AF233334.1 FM01 flavin containing monooxygenase AY879266.1
  • UGT1 A1 UDP glucuronosyltransferase 1 LC00551 .1 family, polypeptide A1
  • UGT2A1 UDP glucuronosyltransferase 2 AJQG6054.1 family, polypeptide A1
  • UGT2A3 UDP glucuronosyltransferase 2 BC130533.1 family, polypeptide A3
  • UGT2B10 UDP glucuronosyltransferase 2 BC1 13649.1 family, polypeptide B10
  • UGT2B17 UDP glucuronosyltransferase 2 U59209.1 family, polypeptide B17
  • UGT2B7 UDP glucuronosyltransferase 2 BC030974.1 family
  • polypeptide B7 UGT3A1 UDP glycosyltransferase 3 family
  • the genetically engineered cells can be any cell line, including but not limited to an immortalized cell line, that are generally known in the art, where the cell line has been genetically modified such that it expresses one or more metabolic genes at a level greater than an unmodified cell of the same cell line.
  • Immortalized cell lines that can be genetically engineered as described herein include, but are not limited to, HEK293, HepG2, Huh7, HepaRG, K562, HeLa, BEAS-2B and A549.
  • the genetically engineered cells can be derived from any tissue in an organism, including but not limited to, liver, heart, kidney, brain hematopoietic system, skin, spleen, lung, and intestine.
  • the genetically engineered cells can be generated using a SAM CRISPR-Cas9 system or similar CRISPR-Cas9 activation system.
  • the genetically engineered cells can contain one or more sgRNAs directed to one or more metabolic genes.
  • the genetically engineered cells can contain Cas9.
  • the Cas9 can be an inactive Cas9.
  • the inactive Cas9 can be a Cas9 that is operatively linked to a VP64 transcriptional activator.
  • the genetically engineered cells can contain one or more Cas9 transcriptional activators.
  • the transcriptional activator(s) can be, without limitation, VP64 transcriptional activator, a MS2-p65-HSF1 co-activation complex, a MS2 RNA aptamer sgRNA, and any combination thereof.
  • the transcriptional activators can form a synergistic activation mediator (SAM).
  • SAM synergistic activation mediator
  • any of these CRISPR related components can be present in the genetically engineered cell transiently (i.e. not integrated into the genome of the cell) or stably (i.e. integrated into the genome of the genetically engineered cell or stable episomal integration (e.g. episomal amplification from an SV40 origin of replication in a plasmid)).
  • some components may be transiently present in the genetically engineered cell while others may be stably present in the genetically engineered cell.
  • the method can include the step of enhancing the transcription of one or more metabolic genes in a cell using synergistic activation mediator (SAM) CRISPR-Cas9, where transcription of the one or more metabolic genes is increased as compared to an unmodified control cell.
  • SAM synergistic activation mediator
  • the metabolically competent cells can be made by transiently expressing one or more of the SAM CRISPR-Cas9 components. In other embodiments, the metabolically competent cells can be made by stably incorporating and expressing one or more of the SAM CRISPR-Cas9 system, such as the invariant components.
  • Transient introduction of all (or some) of the components allows for components needed to modulate metabolic gene expression immediately prior to each high throughput screening (HTS) assay.
  • HTS high throughput screening
  • Methods of transient introduction e.g. transfection or viral transduction or infection
  • RT-PCR reverse transcription polymerase chain reaction
  • quantitative RT-PCR or other suitable method, which will be appreciated by one of ordinary skill in the art.
  • Other assays can be used to measure gene transcription or other activity of the gene or gene product. These can be specific to the particular genes of interest. Standard assays for measuring particular gene activity will be instantly appreciated by those of skill in the art.
  • P450 activity can be measured using an EROD assays.
  • Stable introduction can be used to introduce some, such as the invariant SAM CRISPR-Cas9 components (e.g. not the sgRNA), or all of the components.
  • a DNA encoding the desired sgRNA can be stably incorporated into the cell.
  • Cells with stable integration of the desired components can be selected for using methods generally known in the art.
  • sgRNA can also be delivered transiently via a DNA vector that can express the sgRNA.
  • the sgRNA can be introduced transiently using any appropriate method.
  • RT-PCR reverse transcription polymerase chain reaction
  • Multiple metabolic genes can be activated simultaneously through the simultaneous expression of multiple sgRNAs in a suitable expression vector.
  • Such expression vectors will be appreciated by those of ordinary skill in the art.
  • multiple vectors each configured to express one or more sgRNAs can be transiently or stably introduced into a cell to achieve simultaneous activation of multiple metabolic genes.
  • multiple metabolic genes can be activated simultaneously by transient delivery of a pool of sgRNAs to the cell.
  • the pools can contain any desired group of sgRNAs selected from any provided and/or described herein.
  • IsgRNAs can be designed based of the gene and/or coding sequence information available to one of ordinary skill in the art, including that from the major publically available databases, including but not limited to GenBank. Algorithms and design considerations for sgRNAs are described elsewhere herein and available through publically accessible resources, companies, and the body of peer-reviewed literature.
  • Transient or stable C- MAGIC can be scalable to as many multi-well plates as needed for the assay described elsewhere herein.
  • the lentivirus transduction and plasmid construction can be appropriately scaled to provide sufficient cell number. It is not expected that CRISPR based activation will have any toxicity.
  • the expression of P450s in the cell line is not expected to interfere with any of the established endpoints.
  • the approaches described herein can be scalable and replicable.
  • stable cell lines in which a single or multiple metabolic enzymes are transcriptionally induced can be generated.
  • a particular stable cell line can be grown and multiplied to obtain a large number of cells that is enough to perform large-scale experiments or to perform multiple replicates of each experimental condition (exposure).
  • the stable cell line can be maintained in culture or cryopreserved in liquid nitrogen to be used whenever repeating an experiment is necessary.
  • induction of multiple metabolic enzymes in a reporter cell line using transient transfection is also scalable and replicable. Transient transfection of cells can be scaled- up by proportionally increasing the number of cultured cells, transfection reagents and quantity of the plasmids used. Also, the current transfection reagents can provide efficient transfection that can be consistently replicated.
  • CRISPR based activation can be compatible with currently used cell based assays and cell lines.
  • selection can be used to ensure a uniform population of cells expressing the sgRNA and CRISPR-Cas9 Fusion.
  • CRISPR vectors can alternatively be selected or modified to use an alternative selectable marker.
  • the infection efficiency is typically high and can be up to 100% so, in some embodiments, there may be no need for selection of transfected or transduced cells.
  • the transient CRISPR based activation approach can include a period of time (about 1 hour) to induce the expression of the metabolic genes.
  • a screening assay can include the step of exposing a genetically engineered cell as described herein with a test chemical or compound for a period of time and measuring a physiologic characteristic of the genetically engineered cell. A change in the physiologic characteristic due to the chemical or compound can be determined by comparing the measurement of the physiologic characteristic to one or more controls (e.g. negative controls and positive controls).
  • the physiologic characteristic is measured directly, such as by measuring apoptosis, production of a metabolite, production of another cellular product or other direct measurement of the cell. In other embodiments, the physiologic characteristic is measured indirectly such as via activation or inactivation of a reporter gene present in the cell line.
  • the toxicity of the chemical or compound is determined by measuring cellular ATP levels, mitochondrial membrane potential, plasma membrane integrity or the redox potential of the cell.
  • the gene expression of one or more reporter genes are measured to determine the biological effect of a chemical or compound. For example, a promoter- reporter assay (e.g. Estrogen Response Element (ERE) - luciferase) can be measured to determine biological activity of a compound.
  • a promoter- reporter assay e.g. Estrogen Response Element (ERE) - luciferase
  • CRISPR-Cas9 Modulating the expression of CYP450 genes and other metabolic enzymes can affect the response to a toxic exposure.
  • the CRISPR-Cas9 system was used to transiently activate metabolically important genes in a high throughput screening (HTS) cell- based assay.
  • Transcriptional activation using the CRISPR-Cas9 system is an innovative approach to induce the endogenous expression of metabolic enzymes in human cell lines, particularly for development of assays capable of sensing toxic exposure and response thereto.
  • a feature of the assay is the RNA-guided recruitment of Cas9 to specific regions of the genome.
  • CRISPR-Cas9 uses inactivate a target gene
  • fusion of a catalytically inactive Cas9 to a transcriptional activation domain is used to activate a target gene when expressed with a sgRNA that recruits the fusion complex to the gene promoter.
  • one tool for transcriptional activation using CRISPR-Cas9 is the synergistic activation mediator (SAM) system 2 .
  • SAM synergistic activation mediator
  • the system is comprised of multiple components: Protein components of the system are a catalytically inactive Cas9 nuclease fused to a VP64 transcriptional activator (dCas9-VP64) and a co-activation complex (MS2-p65-HSF1 ) .
  • the third component is a RNA molecule (sgRNA) that incorporates a gene-specific 20bp guide sequence in addition to MS2 RNA aptamers.
  • the synergistic activation mediator (SAM) CRISPR-Cas9 system or similar system can be used to activate the expression of metabolically important genes in human cell lines used in HTS (Figs. 1 and 2). This Example can demonstrate a metabolically competent environment for toxicity testing, and ultimately mimic tissue-specific metabolism in vitro while studying toxicity of certain chemicals.
  • transient transfections were used to provide the components for metabolic enzyme expression immediately prior to each HTS, which can be performed in about 30 min to 48 hours or more after transient transfection.
  • sgRNA constructs were designed and generated to transiently active transcription of CYP1 A2, CYP3A4, CYP3A5, CYP2E1 , CYP2B6, UGT1 a6, and UGT1 a9 in HEK293 cells. It will be appreciated that constructs to transiently activate the other genes listed in Table 1 will be able to be produced by one of ordinary skill in the art without undue experimentation in view of the design and production methods demonstrated in this Example and described elsewhere herein.
  • each guide strand was cloned individually in the lenti sgRNA(MS2)_zeocin plasmid (addgene# 61427) using the Golden-Gate sgRNA method described in detail in Konermann S et al. 2014 2 . After selection, bacteria were cultured flasks of liquid LB-Amp and on LB-Amp agar plates. Maxi Preps of plasmid were made from bacteria cultured in flasks of liquid LB-AMP using Qiagen Plasmid kits.
  • HEK293T cells were maintained in Dulbecco's Modified Eagle Medium (DMEM) with 10% (v/v) fetal bovine serum (FBS) and 1 % (v/v) Penicillin/Streptomycin. The day before transfection, cells were trypsinized and counted. Cells were seeded in a 24-well at about 1.25x10 s cells per well in about 0.5 ml of complete growth medium. Cell density was about 50-80% confluent on the day of transfection.
  • DMEM Dulbecco's Modified Eagle Medium
  • FBS fetal bovine serum
  • Penicillin/Streptomycin penicillin/Streptomycin
  • dCas9 plasmid Using a 24-well plate, about 0.5 ⁇ g of dCas9 plasmid, about 0.5 ⁇ g of SAM plasmid, and up to about 0.5 ⁇ g of sgRNA plasmid, not exceeding a total of about 1 .5 ⁇ g per well for a 24-well plate, was added to a volume of Optimem equal to 50 ⁇ for each well to be transfected.
  • This mixture and additional reagents were prepared as directed by the Lipofectamine 3000 kit (Thermofisher Cat. L3000015). Cells were then incubated at 37 °C in a C0 2 incubator.
  • the cDNA was used in cDNA as the template in the qPCR experiments, which were performed using a Bio-Rad CFX Connect Real-Time System using SsoFastTM EvaGreen® Supermix (Bio-Rad 1725201). Expression data was normalized (AACq) to either GAPDH or RPL19, and we compared the fold expression of enzyme transcripts against controls receiving Cas9 and SAM components but no sgRNA plasmids.
  • CYP gene expression was measured about 48 h post-transfection and is presented in terms of relative quantity of SAM-only control and normalized to expression of RPL19. Transcriptional activation of the desired genes in HEK293T cell lines using a transient transfection approach as indicated by RT-qPCR. In all cases it was observed that the CYP3A4 gene is effectively upregulated using CRISPRa.
  • the most effective guide (sgRNA) generated about a 65-fold increased expression of CYP3A4 expression over the control when the guide was transiently transfected. However, we do not believe that this is by no means the maximum expression increase that may be achieved with our system, further refinement of sgRNA sequences or transfection techniques will likely yield increased expression beyond what we have demonstrated here. References for Example 1
  • CRISPR-Cas9 Modulating the expression of CYP450 genes and other metabolic enzymes can affect the response to a toxic exposure.
  • the CRISPR-Cas9 system was used to stably activate metabolically important genes in a high throughput screening (HTS) cell- based assay, as indicated in Figure 10.
  • Transcriptional activation using the CRISPR-Cas9 system is an innovative approach to induce the endogenous expression of metabolic enzymes in human cell lines, particularly for development of assays capable of sensing toxic exposure and response thereto.
  • a feature of the assay is the RNA-guided recruitment of Cas9 to specific regions of the genome.
  • CRISPR-Cas9 uses inactivate a target gene
  • fusion of a catalytically inactive Cas9 to a transcriptional activation domain is used to activate a target gene when expressed with a sgRNA that recruits the fusion complex to the gene promoter.
  • one tool for transcriptional activation using CRISPR-Cas9 is the synergistic activation mediator (SAM) system 2 .
  • SAM synergistic activation mediator
  • the system is comprised of multiple components: Protein components of the system are a catalytically inactive Cas9 nuclease fused to a VP64 transcriptional activator (dCas9-VP64) and a co- activation complex (MS2-p65-HSF1 ) .
  • the third component is a RNA molecule (sgRNA) that incorporates a gene-specific 20bp guide sequence in addition to MS2 RNA aptamers.
  • the synergistic activation mediator (SAM) CRISPR-Cas9 system or similar system can be used to activate the expression of metabolically important genes in human cell lines used in HTS (Figs. 1 and 2). This Example can demonstrate a metabolically competent environment for toxicity testing, and ultimately mimic tissue-specific metabolism in vitro while studying toxicity of certain chemicals. Materials and Methods
  • HEK293T cells stably transfected with the SAM component were generated using lentiviral transduction. Briefly, Lentiviruses were produced for each of the SAM components and packaged by co-transfecting using lipofectamine 3000 and each of the following lentiviral plasmids: addegene# 61425 (dCas9-VP64), Addgene# 61426 (MS2-P65-HSF1 activator helper complex), or guide RNA plasmid (addegene#61427 harboring a cloned CYP activation gRNA), with the psPAX2 packaging and pMD2. G envelope plasmids.
  • the cell culture medium was replaced with fresh medium (DMEM, 10% FBS). After about 48 h of culture, the medium, which contained the lentivirus, was collected and then filtered using 0.45 ⁇ sterile syringe filters. The 293T cells were seeded in 6-well plates and infected with 250 ⁇ of the lentiviral solution in the presence of 8 ⁇ g/ml polybrene to enhance the infection rate. After about 24 h, the media was changed and the cells were allowed to grow for another 24 h before starting the selection against the appropriate antibiotics.
  • DMEM fetal bovine serum
  • 293T cells were transduced either with dCas9-VP64 alone to generate 293T- Cas9-VP64 (blasticin selection) or with both dCas9-VP64 and MS2-P65-HSF1 lentiviruses (blasticin and hygromycin B selection) to generate 293T-SAM.
  • 293T cells were co-transduced with the two previous components and with lentiviruses for three different CYP3A5 activation guides (blasticin, hygromycin B, and zeocin selection). Once the selection was completed, the successfully-transfected cells were transduced yet again with CY3A4 and CY1A1 lentiviruses (for all sgRNA guides). Transfections were performed using all three of the potential activation guides, as it was not known which guide sequences would prove to be the most effective for inducing expression in the stable-transfection cell lines. Transfections were performed sequentially to increase the likelihood of success. RT-qPCR was performed 48 hours after the final transfection.
  • Figs. 8-10 The results are demonstrated in Figs. 8-10. In all cases it was observed that the CYP3A4 gene is effectively upregulated using CRISPRa.
  • the most effective guide (sgRNA) generated about a 12-fold increase in expression (Figs. 8-9).
  • sgRNA The most effective guide
  • the gene expression level of the CYP2B6, CYP2E1 , and CYP3A4 was increased as compared to the control.
  • the CYP2B6, CYP1A2, CYP2E1 as well as the CYP3A4 and CYP3A5 expression was increased in the transfected 293T- CYP3A5/1 A1/3A4 cells when compared to the 293T-SAM cells.

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Abstract

La présente invention décrit des cellules génétiquement modifiées contenant un ou plusieurs gènes métaboliques modulés, où l'expression du(des) gène(s) métabolique(s) modulé(s) peut être supérieure à celle d'un témoin non modifié. L'invention concerne également des procédés de fabrication des cellules génétiquement modifiées utilisant le médiateur d'activation synergétique CRISPR-Cas9. L'invention concerne en outre des essais à haut débit qui peuvent utiliser les cellules génétiquement modifiées décrites par la présente invention.
PCT/US2017/041202 2016-07-07 2017-07-07 Cellules métaboliquement compétentes, leurs procédés de fabrication, et utilisations Ceased WO2018009869A1 (fr)

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