EP1248860A2 - Criblage synthetique letal a code barre pour identification de cibles de medicaments - Google Patents

Criblage synthetique letal a code barre pour identification de cibles de medicaments

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Publication number
EP1248860A2
EP1248860A2 EP01942675A EP01942675A EP1248860A2 EP 1248860 A2 EP1248860 A2 EP 1248860A2 EP 01942675 A EP01942675 A EP 01942675A EP 01942675 A EP01942675 A EP 01942675A EP 1248860 A2 EP1248860 A2 EP 1248860A2
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European Patent Office
Prior art keywords
mutation
cells
library
gene
cell
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EP01942675A
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German (de)
English (en)
Inventor
Charles M. Brenner
Daniel D. Shoemaker
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Thomas Jefferson University
Rosetta Inpharmatics LLC
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Thomas Jefferson University
Rosetta Inpharmatics LLC
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Publication of EP1248860A2 publication Critical patent/EP1248860A2/fr
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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
    • C12N15/01—Preparation of mutants without inserting foreign genetic material therein; Screening processes therefor
    • 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

Definitions

  • the present invention relates to methods of using synthetic lethal screening techniques to identify drug targets.
  • the methods of the present invention use "barcoded" libraries of cells, where the library consists of a collection of different mutant strains, each mutant strain bearing a knock-out mutation of a different gene. Each mutant strain also has a unique DNA identifier tag, or "barcode,” to allow for quick and convenient identification of the clone and its mutation.
  • barcode DNA identifier tag
  • TS Gs tumor suppressor genes
  • the traditional yeast synthetic lethal screens use a "plasmid shuffling" strategy (Sikorski RS, Boeke, 1991, Methods Enzymol., 194:302-18, incorporated by reference in its entirety).
  • the first step involves constructing a strain where the target gene has been deleted. This gene is then re-introduced into the cell on a low-copy plasmid that also contains a marker (e.g., ADE2) that can be used to select for the plasmid or detect the loss of the plasmid.
  • the strain is then mutagenized and allowed to grow in the absence of selection for the plasmid.
  • Another approach for performing synthetic lethal screens in yeast involves generating a conditional lethal mutant for a target gene. The strain bearing such a mutation is then mutagenized and screened for second-site mutations that specifically exacerbate its temperature sensitivity. This approach has successfully been used to identify proteins involved in the translocation step of protein secretion (Boisrame A, et al, 1999, Mol Gen Genet, 261(4-5):601-9, incorporated by reference in its entirety).
  • mutant hunts in synthetic lethal screens are limited by the mutagenic agent that is being used to generate the mutations. For example, not all genes can be disrupted using methyl methane sulfonate, which non-specifically methylates DNA.
  • the present invention relates to methods of using synthetic lethal screening techniques to identify drug targets.
  • the methods of the present invention entail the use of "barcoded" libraries of cells, where the library consists of a collection of different mutant clones, each mutant clone bearing a knock-out mutation of a different gene. Additionally, each mutant clone has a unique DNA identifier tag, or "barcode,” to allow for quick and convenient identification of the clone and its mutation.
  • the library of mutant clones is used as a panel of "secondary mutations,” against which the effects of a "primary mutation” can be assessed.
  • the "primary mutation” is a knock-out mutation induced in a particular target gene in each of the clones comprising the barcoded library.
  • the interaction of the primary mutation with each of the secondary mutations present in the library can be determined.
  • the library is allowed to grow. After several doublings of the library, those clones harboring secondary mutations which interact with the primary mutation causing a decrease in the growth rate of said clones will be selected against, i.e., present at a lower concentration relative to clones not harboring such interacting secondary mutations. Because each mutated clone is tagged (barcoded), the relative abundance of each clone can be easily determined by assaying for each of the tags.
  • Missing tags represent those clones that harbor a "synthetic lethal" secondary mutation, i.e., a mutation that interacts with a primary mutation resulting in decreased rate of growth of the cell harboring both the primary and the secondary mutation. Because the library of mutants is tagged and characterized, identification of under-represented tags in the library after introduction of the primary mutation is tantamount to identifying a gene product which, if knocked out, causes a decreased growth rate when combined with the primary mutation. Such gene products are excellent candidates for use in drug screening protocols designed to identify agents capable of inhibiting the growth of cells harboring the primary mutation. This is so because the screen of the invention identifies genes that, if knocked out, cause decreased growth rates of cells also harboring the primary mutation.
  • the present invention relates to the identification of genes differentially required for the survival of mammalian cells missing a target gene.
  • the target gene may be, as a non-limiting example, any of a class of genes including tumor suppressor genes and mutator genes, the function of which is absent or reduced in cancer cells.
  • This invention relates to use of synthetic lethal screening to identify genes that are more important to the growth or survival of cells missing a particular target gene as compared to how important those genes are for the growth or survival of wild-type cells with the target gene. This information is used to rationalize a drug target in mammalian cells of defined genotype.
  • a synthetic lethal screen might identify genes X, Y and Z in that cell, each of which have a more deleterious phenotype as double mutants with the target gene mutants than as single mutants.
  • genes X, Y and Z or their homologs in mammals, these gene products would be rationalized as drug targets for the elimination of cells missing the target gene.
  • this invention relates to the use of barcoded libraries and oligonucleotide arrays to perform synthetic lethal screens.
  • non-lethal mutation is a mutation with little or no effect on cell viability or growth
  • the combined mutations are considered to be synthetically lethal mutations, i.e., the combination of the two mutations is detrimental to the viability of a cell bearing that combination.
  • the primary mutation is the mutation of the target gene, i.e., the gene for which one wishes to identify secondary mutations which are synthetically lethal.
  • the mutations present in the strains comprising the mutation library are the secondary mutations.
  • the methods of the present invention provide a way of determining which of the secondary mutations present in a library are synthetic lethal mutations with respect to a given primary mutation. knock-out mutation
  • a knock out mutation of a gene causes the loss of function of the mutated gene. This is generally the result of a disruption or deletion of the gene.
  • a barcoded mutation library is a library comprised of different mutant strains of cells, each strain bearing a unique DNA tag. This DNA tag is referred to as the barcode.
  • the DNA tags are identifiable by, for example, utilizing the polymerase chain reaction to amplify the tags and then hybridizing the amplified tags to a DNA micro-array comprised of sequences complementary to each tag.
  • An example of such a library is a collection of yeast deletion mutants, each mutant having a different open reading frame (ORF) deleted and having in its genome one or more unique DNA tags.
  • the present invention provides methods of detecting synthetic lethality caused by the interaction of a primary and a secondary mutation.
  • a preferred method comprises the steps of:
  • step (b) incubating the cells of step (a) under conditions which would, in the absence of step (a), allow the cells to grow;
  • step (c) comparing the growth of each cell of step (b) that has the primary mutation with the growth of a control cell without the primary mutation but containing said secondary mutation,
  • step (d) identifying any cell in step (b) that exhibits a decreased rate of growth as compared to the rate of growth of a control cell without the primary mutation but containing said secondary mutation as containing a secondary mutation that causes a decreased rate of growth when combined with the primary mutation in a cell;
  • step (e) determining in which gene the secondary mutation that causes a decreased rate of growth when combined with the primary mutation identified in step (d) resides.
  • the method of the invention comprises the additional step of
  • step (f) isolating the gene in which the secondary mutation that causes a decreased rate of growth when combined with the primary mutation identified in step (d) resides.
  • the method further comprises the step of:
  • step (g) isolating the human homolog of the gene isolated in step (f).
  • the invention also provides a barcoded deletion mutant library comprised of different mutant cells, each mutant cell having a different deletion mutation, wherein between 25% and 100%, 50% and 95%, or 60% and 90% of the cells comprising the library also have a primary mutation, wherein said primary mutation is a mutation of the same gene in each of the different mutant cells containing said primary mutation.
  • yeast cells are used according to the methods of the invention.
  • the following sections describe aspects of the claimed methods as they relate to the use of a yeast model system.
  • Any library of mutants may be used as the panel of potential secondary mutations in which the primary mutation is induced according to the methods of the invention.
  • a gene may be disrupted or mutated in yeast.
  • an entire gene and create a null allele, in which no portion of the gene is expressed.
  • a deletion allele may be constructed comprising only a portion of the gene which is not sufficient for gene function, which can be constructed, for example, by inserting a nonsense codon into the sequence of the gene such that translation of the mutant mRNA transcript ends prematurely.
  • Alleles may also be made containing point mutations, individually or in combination, that reduce or abolish gene function. Such methods are well known in the art.
  • an allele can be conditional for function or expression.
  • An example of an allele that is conditional for function is a temperature sensitive mutation wherein the gene product is functional at one temperature (i.e., permissive temperature) but non- functional at a different temperature (i.e., non-permissive temperature), e.g., due to misfolding or mislocalization.
  • One of ordinary skill in the art can produce mutant alleles which may have only one or a few altered nucleotides but which encode inactive or temperature-sensitive proteins. Temperature-sensitive mutant yeast cells express a functional protein at permissive temperatures but do not express a functional protein at non-permissive temperatures.
  • An example of an allele that is conditional for expression is a chimeric gene where a regulated promoter controls the expression of the gene. Under one condition the gene is expressed and under another it is not. One may replace or alter the endogenous promoter of the gene with a heterologous or altered promoter that can be activated only under certain conditions. These conditional mutants only express the gene under defined experimental conditions. All of these methods are well known in the art. For example, see Stark, 1998, Methods in Microbiology 26:83-100; Garfinkel et al., 1998, Methods in Microbiology 26:101-118; and Lawrence & Rothstein, 1991, Methods in Enzymology 194:281-301.
  • a gene may have decreased
  • the expression of gene may be decreased by transforming yeast with an antisense molecule under the control of a regulated or constitutive promoter (see Nasr et al., 1995, Molecular & General Genetics 249:51-57).
  • a regulated or constitutive promoter see Nasr et al., 1995, Molecular & General Genetics 249:51-57.
  • Gene expression may also be decreased by inserting a sequence by homologous recombination into or next to the target gene wherein the sequence targets the mRNA or the protein for degradation. For instance, one can introduce a construct that encodes ubiquitin such that a ubiquitin fusion protein is produced. This protein will be
  • a target gene is completely disrupted in order to ensure that there is no residual function of the gene.
  • One can disrupt a gene by "classical” or PCR- based methods. The “classical” method of gene knockout is described by Rothstein, 1991.
  • PCR-based deletion method it is preferable to use a PCR-based deletion method because it is faster and less labor intensive.
  • the mutation library is a deletion mutation library and is barcoded.
  • Barcoded deletion strains have been generated by the Saccharomyces Genome Deletion Project and described in several publications (Winzeler EA, et al., 1999, Science
  • Saccharomyces Genome Deletion Project The goal of Saccharomyces Genome Deletion Project is to generate a complete set of yeast deletion strains.
  • a PCR-based gene deletion strategy (Baudin et al, 1993, Nucl. Acids Res. 21, 3329-3330; Wach et al., 1994, Yeast 10, 1793-1808, each incorporated by reference in its
  • each deletion was uniquely tagged with two independent 20mer sequences. The presence of the tags can be detected via hybridization to a high-density oligonucleotide array, enabling growth phenotypes of the resulting deletion strains to be analyzed in parallel. More than 10,000 strains are currently available (Research Genetics, Huntsville, Alabama; American Type Culture Collection, Manassas, NA).
  • the pool of mutants contains between 100 and 15,000 different mutant strains, between 1000 and 15,000 different mutant strains, between 1000 and 10,000 different mutant strains, or between 2000 and 8000 different mutant strains.
  • the library contains individual deletion strains for 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30% or 20% of the open reading frames in the yeast genome.
  • the methods of the invention can be used to identify secondary mutations for any given primary mutation of a target gene.
  • Preferred target genes include yeast homologs of any mammalian genes whose function is absent or reduced in cancer cells, e.g., tumor suppressor genes or mutator genes. Examples of such mammalian genes include p53, pl6, PTEN, NF-1, NF-2, DPC4, MTS1, retinoblastoma gene, APC and FHIT.
  • HNT2 the yeast homolog of the human tumor suppressor gene FHIT
  • the target gene K. Huebner, et al., 1999, Advances in Oncology 15:3-10; United States Patent No. 5,928,884, International Publication No. WO97/29119, incorporated by reference in its entirety.
  • the primary mutation would be a mutation that causes the reduction or loss of the HNT2 gene product or gene product function.
  • An example of such a primary mutation would be an HNT2 knock-out mutation, where the sequence of HNT2 is deleted and, optionally, replaced with another gene, for example, a marker gene such as URA3.
  • human homologs of the yeast genes associated with those secondary mutations are potential drug screening targets useful for the identification of compounds able to inhibit the growth of tumor cells containing reduced or non-existent levels of the tumor suppressor FHIT.
  • the primary mutation can be a mutation of any gene.
  • each individual mutation present in a yeast barcoded deletion mutation library may be used as a primary mutation to identify which, if any of the other mutations present in the library can provide synthetic lethality in combination with that primary mutation.
  • the screening of all possible combinations of two mutants present in a given collection of barcoded deletion mutants for synthetic lethality is also provided by the present invention.
  • the primary mutation of the target gene may be introduced into the yeast mutation library by any means known to one of skill in the art, including those methods provided in section 5.1.2.
  • the primary mutation is introduced into a high percentage of the cells present in the library, preferably greater than 30%, greater than 40%, greater than 50%, greater than 60%, greater than 70%, greater than 80%, greater than 90% or greater than 95% of the cells of the mutation library receive the primary mutation.
  • the goal is to generate enough independent isolates such that most or all of the possible double mutants are produced.
  • the use of a yeast mutation library provides several advantages.
  • the yeast genome readily undergoes homologous recombination, allowing for targeted mutation of the target gene.
  • yeast cells can be propagated as either haploid or diploid cells, so only one copy of the target gene need be mutated.
  • the primary mutation is introduced into the mutation library by mating the cells of the mutation library with a cell bearing the primary mutation.
  • the cells of the mutation library can be placed into separate mating reactions with cells bearing the primary mutation, as well as with control cells identical to the cells bearing the primary mutation, but lacking the primary mutation.
  • the cells bearing the primary mutation and the cells of the mutation library have different selectable markers, allowing ' for selection of diploid cells, i.e., cells with both selectable markers.
  • the control cell should also contain the same selectable marker as does the cell bearing the primary mutation.
  • the control cell corresponding to that double mutant is a cell containing only the secondary mutation.
  • the two selectable markers are URA3 and KanMX.
  • the URA3 gene is used to create the primary mutation by homologous recombination with the target gene, causing replacement of the target gene with the URA3 gene.
  • the mutations in the cells of the mutation library are produced by homologous recombination with the KanMx gene, rendering them resistant to G418.
  • selectable markers are available (Goldstein, A.L., et al., 1999, Yeast, 15(6):507-11).
  • diploids are selected for by incubating the mated cells in the presence of G418 and SC-URA media.
  • the diploid cells obtained from the above mating can then be incubated and made to undergo sporulation, producing haploid cells.
  • haploid cells Preferably, only those double mutant haploid cells with both the primary mutation and a secondary mutation are obtained.
  • the secondary mutations and the primary mutation are created by homologous recombination with different selectable markers, allowing for the selection of cells containing both markers and, consequently, both mutations.
  • Canl gene Bact, J.R., et al., 1979, Gene 8(l):121-33. Any cell with a functional copy of the Canl gene is sensitive to canavanine. Another example is the CYH2 gene; any cell with a copy of the CYH2 gene is sensitive to cycloheximide. Any diploid cell, which would have to contain a copy of the Canl gene since the diploid cell was produced by the mating of one cell with the Canl gene and one without the Canl gene, would be selected against in the presence of canavanine, leaving only haploid cells.
  • Another preferred method of introducing the primary mutation into the mutation library is by direct transformation. This approach offers the advantage of requiring viewer steps compared to the above-described mating strategy. Specifically, the genomic regions flanking the target gene are cloned onto each side of a selectable marker gene, such as the URA3 gene. This deletion construct is introduced into the mutation library by homologous recombination. After selection for the marker, only cells which have successfully integrated the selectable marker into the genome can survive. In yeast, over
  • a deletion construct can be generated that contains the selectable marker flanked by targeting homology to a gene not necessary for growth of the cell, such as the
  • HO endonuclease gene (YDL227c). This gene is a good control because the gene product is not required for normal vegetative growth (Baganz, F., et al., 1997, Yeast 13:1563-1573, incorporated by reference in its entirety).
  • the presence of synthetic lethality between the primary mutation and any of the mutation present in the mutation library can be detected by any means know to those of skill in the art.
  • the resulting double mutant haploid cells are allowed to reproduce.
  • the cells are grown under conditions and for sufficient time to allow for at least 5, at least 10, at least 15, 20, about 20, at least 20, at least 30, at least 40, at least 50, 60, about 60, or at least 60 population doublings. This competitive outgrowth period allows for the amplification of any differential growth rates of the double mutants.
  • a control experiment is also be performed in which a collection of single mutant controls (i.e., cells bearing only the secondary mutation) is grown under the same conditions for the same amount of time.
  • the abundance of double mutant cells bearing each secondary mutation is determined.
  • the abundance of each double mutant is compared to the abundance of the single mutant control cell bearing the same secondary mutation. If a double mutant and its corresponding single mutant control grow at the same rate (i.e., there is little or no difference between the abundance of the double mutant and the abundance of the single mutant control), then no synthetic lethality between those two genes was detected, and the secondary mutation is not a synthetic lethal mutation with respect to the primary mutation.
  • the relative amounts of the double and corresponding single mutants are determined by detecting a DNA tag, or barcode, present in the mutation library.
  • a barcoded mutation library described above, allows for rapid and simple assessment of the population of mutation bearing cells. Because each member of a barcoded mutation library has a unique DNA tag, and because each tag is associated with a known deletion mutation, detection (or lack thereof) of a given DNA tag allows one to know which gene is deleted in the cell that bore that tag.
  • the tags can be detected by first amplifying and fluorescently labeling them via the polymerase chain reaction, followed by hybridization of the amplified products with a DNA microarray comprised of DNA molecules complementary to the DNA tags. Examples of suitable fluorescent labels include Cy3, Cy5 and fluorescein.
  • the amount of fluorescence detectable at a given location in the DNA microarray reveals the relative abundance of the cell bearing the tag complementary to the DNA at that location.
  • the tags in the single mutant control cells are amplified and labeled with a fluorophore different from the one used to label the amplified tags from
  • the double mutant cells When the differently labeled tags are simultaneously hybridized to the DNA microarray, the ratio of one fluor to the other detectable at each address of the DNA microarray provides a direct measure of the relative abundance of each double mutant with respect to its corresponding single mutant that was present in the culture from which the tags were amplified. Ratios close to 1 : 1 indicate that there is no difference in the growth
  • a ratio of single mutant fluorophore to double mutant fluorophore of greater than 2:1, greater than 3:1, greater than 5:1, greater than 8:1, or greater than 10:1 is considered to be an indicator of synthetic lethality in the screening methods of the invention.
  • the cells present at a lower abundance as compared to the other double mutant cells is identified as a cell bearing a primary mutation and a synthetic lethal secondary mutation.
  • Any library of mutants may be used as the panel of secondary mutations in which the primary mutation is introduced according to the methods of the invention.
  • Mutants strains comprising the mutation library may be constructed by any method known to those of skill in the art.
  • each mutant strain exhibits a reduction or absence of the expression of a different gene.
  • the reduced level of gene expression or activity can be generated by deleting or mutating at least one copy of the gene, by expressing a dominant _ negative form of a component of a cellular pathway of the gene, or by lowering the activity or abundance of the RNA encoded by the gene.
  • the activity or abundance of a gene encoded RNA can be lowered by means of a ribozyme, an anti-sense nucleic acid, a double- stranded RNA or an aptamer.
  • RNA abundance or activity are classes, ribozymes, antisense species, and RNA aptamers (Good et al., 1997, Gene Therapy 4:45-54). Controllable exposure of a cell to these entities permits controllable perturbation of RNA abundances.
  • Ribozymes are RNAs which are capable of catalyzing RNA cleavage reactions. (Cech, 1987, Science 236:1532-1539; PCT International Publication WO 90/11364, published October 4, 1990; Sarver et al., 1990, Science 247:1222-1225). "Hairpin” and "hammerhead” RNA ribozymes can be designed to specifically cleave a particular target mRNA. Rules have been established for the design of short RNA molecules with ribozyme activity, which are capable of cleaving other RNA molecules in a highly sequence specific way and can be targeted to virtually all kinds of RNA.
  • Ribozyme methods for underexpression of a gene involve inducing expression in a cell, etc. of such small RNA ribozyme molecules. (Grassi and Marini, 1996, Annals of Medicine 28:499-510; Gibson, 1996, Cancer and Metastasis Reviews 15:287-299).
  • Ribozymes can be routinely expressed in vivo in sufficient number to be catalytically effective in cleaving mRNA, and thereby modifying mRNA abundances in a cell. (Gotten et al., 1989, EMBO J. 8:3861-3866).
  • a ribozyme coding DNA sequence designed according to the previous rules and synthesized, for example, by standard phosphoramidite chemistry, can be ligated into a restriction enzyme site in the anticodon stem and loop of a gene encoding a tRNA, which can then be transformed into and expressed in a cell of interest by methods routine in the art.
  • an inducible promoter e.g., a glucocorticoid or a tetracycline response element
  • an inducible promoter e.g., a glucocorticoid or a tetracycline response element
  • tDNA genes i.e., genes encoding tRNAs
  • ribozymes can be routinely designed to cleave virtually any mRNA sequence, and a cell can be routinely transformed with DNA coding for such ribozyme sequences such that a controllable and catalytically effective amount of the ribozyme is expressed. Accordingly the abundance of virtually any RNA species in a cell can be perturbed.
  • activity of an RNA (preferable mRNA) species can be controllably inhibited by the controllable application of antisense nucleic acids.
  • An "antisense" nucleic acid as used herein refers to a nucleic acid capable of hybridizing to a sequence-specific (e.g., non-poly A) portion of the RNA, for example its translation initiation region, by virtue of some sequence complementarity to a coding and/or non-coding region.
  • the antisense nucleic acids of the invention are produced intracellularly by transcription of exogenous, introduced sequences in controllable quantities sufficient to perturb translation of the RNA.
  • antisense nucleic acids are of at least six nucleotides and to about 200 oligonucleotides).
  • the antisense nucleic acids of the invention comprise a sequence complementary to at least a portion of an RNA species.
  • absolute complementarity although preferred, is not required.
  • a sequence "complementary to at least a portion of an RNA,” as referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with a given RNA it may contain and still form a stable duplex (or triplex, as the case may be) with that RNA.
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • the amount of antisense nucleic acid that will be effective in the inhibiting translation of a geven RNA can be determined by standard assay techniques.
  • the antisense nucleic acids of the invention are controllably expressed intracellularly by transcription from an exogenous sequence.
  • a vector can be introduced in vivo such that it is taken up by a cell, within which cell the vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encoding the antisense nucleic acid.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Such vectors can be constructed by recombinant DNA technology methods standard in the art.
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian and insect cells.
  • Expression of the sequences encoding the antisense RNAs can be by any promoter known in the art to act in a cell of interest.
  • promoters can be inducible or constitutive.
  • promoters are controllable or inducible by the administration of an exogenous moiety in order to achieve controlled expression of the antisense oligonucleotide.
  • controllable promoters include but are not limited to the Tet promoter, the SN40 early promoter region
  • antisense nucleic acids can be designed to target virtually any mRNA sequence, and a cell can be routinely transformed with nucleic acids coding for such antisense sequences such that an effective and controllable amount of the antisense nucleic acid is expressed. Accordingly the translation of virtually any RNA species in a cell can be controllably perturbed.
  • RNA aptamers can be introduced into or expressed in a cell.
  • RNA aptamers are specific RNA ligands for proteins, such as for Tat and Rev RNA (Good et al., 1997, Gene Therapy 4:45-54) that can specifically inhibit their translation.
  • mutants can be grown to saturation in individual cultures of rich medium.
  • the resulting cultures can be pooled together and used for the screens of the invention.
  • the pool of mutants contains between 100 and 100,000 different mutant strains, between 1000 and
  • the library contains individual deletion strains for 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30% or
  • the mutation library is a deletion mutation library.
  • the mutation library is preferably barcoded. Barcoded deletion strains have been generated by the Saccharomyces Genome Deletion Project and described in several publications
  • 30 deletion was uniquely tagged with two independent 20mer sequences.
  • the presence of the tags can be detected via hybridization to a high-density oligonucleotide array, enabling growth phenotypes of the resulting deletion strains to be analyzed in parallel. More than
  • target genes include any mammalian genes whose function is absent or reduced in cancer cells, e.g., tumor suppressor genes or mutator genes. Examples of such mammalian genes include p53, pl6,
  • the primary mutation of the target gene may be introduced into the mammalian mutation library by any means known to one of skill in the art, including those provided in section 5.2.1 hereinabove.
  • the primary mutation is introduced into a high percentage of the cells present in the library, preferably greater than 30%, greater than 40%o, greater than 50%, greater than 60%, greater than 70%, greater than 80% or greater than 90%) of the cells of the mutation library receive the primary mutation.
  • the result of the primary mutation is a reduced or nonexistent level of target gene expression or activity.
  • the presence of synthetic lethality between the primary mutation and any of the mutation present in the mutation library can be detected by any means know to those of skill in the art.
  • the resulting double mutant haploid cells are allowed to reproduce.
  • the cells are grown under conditions and for sufficient time to allow for at least 5, at least 10, at least 15, 20, about 20, at least 20, at least 30, at least 40, at least 50, 60, about 60, or at least 60 population doublings. This competitive outgrowth period allows for the amplification of any differential growth rates of the double mutants.
  • a control experiment is also be performed in which a collection of single mutant controls (i.e., cells bearing only the secondary mutation) is grown under the same conditions for the same amount of time.
  • the abundance of double mutant cells bearing each secondary mutation is determined.
  • the abundance of each double mutant is compared to the abundance of the single mutant control cell bearing the same secondary mutation. If a double mutant and its corresponding single mutant control grow at the same rate (i.e., there is little or no difference between the abundance of the double mutant and the abundance of the single mutant control), then no synthetic lethality between those two genes was detected, and the secondary mutation is not a synthetic lethal mutation with respect to the primary mutation.
  • the relative amounts of the double and corresponding single mutants are determined by detecting a DNA tag, or barcode, present in the mutation library.
  • a barcoded mutation library described above, allows for rapid and simple assessment of the population of mutation bearing cells. Because each member of a barcoded mutation library has a unique DNA tag, and because each tag is associated with a known deletion mutation, detection (or lack thereof) of a given DNA tag allows one to know which gene is deleted in the cell that bore that tag.
  • the tags can be , detected by first amplifying and fluorescently labeling them via the polymerase chain reaction, followed by hybridization of the amplified products with a DNA microarray comprised of DNA molecules complementary to the DNA tags.
  • Suitable fluorescent labels include Cy3, Cy5 and fluorescein.
  • the use of a DNA microarray to detect tags present in a barcoded mutation library has been published (Winzeler, E.A., et al., 1999, 285:901-906, incorporated by reference in its entirety).
  • Such arrays can be produced by one of skill in the art according to established protocols (Marton, M.J., et al., 1998, Nat Med 4(11):1293-301) or obtained commercially (Affymetrix Inc., Santa Clara, CA).
  • Each address of the DNA microarray contains DNA complementary to a known DNA tag. After hybridization, the amount of fluorescence detectable at a given location in the DNA microarray reveals the relative abundance of the cell bearing the tag complementary to the
  • the tags in the single mutant control cells are amplified and labeled with a fluorophore different from the one used to label the amplified tags from the double mutant cells.
  • DNA microarray provides a direct measure of the relative abundance of each double mutant with respect to its corresponding single mutant that was present in the culture from which the tags were amplified. Ratios close to 1 : 1 indicate that there is no difference in the growth- rates of the single and double mutant, while ratios varying significantly from 1:1 indicate that the single and double mutants grow at different rates.
  • a ratio of single mutant fluorophore to double mutant fluorophore of greater than 2:1, greater than 3:1, greater than 5:1, greater than 8:1, or greater than 10:1 is considered to be an indicator of synthetic lethality in the screening methods of the invention.
  • the cells present at a lower abundance as compared to the other double mutant cells is identified as a cell bearing a primary mutation and a synthetic lethal secondary mutation.
  • the cells of the mutation library are C. elegans cells, 0 drosophila cells, or E. coli cells.
  • the bar-coded deletion strains used in our synthetic lethal screens were generated by the Saccharomyces Genome Deletion Project (Winzeler EA, et al., 1999, Science 285:901-6, Shoemaker, D., et al, 1996, Nature Genetics, 14, 450-456).
  • the goal of this project is to generate a complete set of yeast deletion strains.
  • a PCR-based gene deletion strategy was used to generate individual deletion strains for each of the -6,200 ORFs in the yeast genome. As part of the strain construction process, each deletion was uniquely tagged with two independent 20mer sequences (Winzeler EA, et al., 1999, Science 285:901-6).
  • the presence of the tags can be detected via hybridization to a high-density oligonucleotide array, enabling growth phenotypes of the resulting deletion strains to be analyzed in parallel. More than 10,000 strains are currently available through Research . Genetics and the ATCC (Research Genetics, Huntsville, Alabama; American Type Culture Collection, Manassas, NA). For the synthetic lethal experiment described below, a pool of 1,600 haploid strains (BY4739, MAT alpha leu2D0 lys2D0 ura3D0 CA ⁇ 1 KanMX + ) was used. These haploid alpha strains are resistant to the drug G418 and sensitive to the drug n canavanine.
  • HNT2 gene is the yeast homolog of human FHIT, a human tumor suppressor gene which is deleted in many solid tumors (K. Huebner, et al., 1999, Advances in Oncology 15:3-10; United States Patent No. 5,928,884, International Publication No. WO97/29119).
  • Our goal is to identify new anti-cancer drug targets by identifying mutations that are synthetic lethal with HNT2.
  • standard yeast genetic techniques were used to delete the HNT2 gene (YDR305c) in a haploid yeast strain (MATa canl hnt2DO::URA3).
  • a mating strategy was used to generate the all of the possible double mutants between hnt2 and the collection of 1,600 bar-coded deletion strains. This approach ensures that enough independent isolates can be tested to maintain the complexity of the library of bar-coded deletion mutants.
  • canavanine-resistance is recessive, canavanine selects against diploid cells that did not sporulate.
  • the calculated number of URA+, canavanine-resistant, G418-resistant cells is 8,000,000 x 30% sporulated x 4 spores/tetrad x 12.5% of the right genotype, or 1,200,000. 420,000 independent colonies resulting from the sporulated double mutant pool and 380,000 independent colonies from the sporulated single mutant pool were obtained.
  • the fact that the yield was 1/3 of the theoretical yield indicates that some cells are killed treatment with zymolyase and glass beads.
  • 400,000 diploids covers the 1, 600-fold complex library 250 times. 100 out of 100 of the resulting colonies had an identifiable mating type, proving that the protocol rapidly and efficiently generates haploid strains via mating and random spore analysis.
  • tags present in the control but missing from the hnt2 deletion strain following the 20 population doublings of competitive outgrowth These tags represent mutations that are synthetically lethal or synthetically less fit in combination with 15 the hnt2 deletion.
  • the "downtags" bar-codes are flanked by different common priming sites. These tags can be amplified with the two 18mer primers called 1DN and 3DN. Again, the 1DN primer has a Cy3 or Cy5 label on the 5' end.
  • the tags from the control cells were amplified with the Cy3 labeled primers and the tags from the hnt2 cells with the Cy5 labeled primers.
  • the amplified tags are mixed with blocking primers that bind to the common priming sites. This serves of the reduce the background during the hybridization.
  • This 1x3 glass slide contains oligonucleotides that are complementary tag sequences from each of the different deletion strains.
  • oligonucleotides that are complementary tag sequences from each of the different deletion strains.
  • This array contains the tags for each of the 6,200 deletion stains even though the current pool only contains 1,600 deletion strains. Similar chips can be obtained from Affymetrix Inc. (Santa Clara, CA).
  • the scanned image of the oligonucleotide array was then analyzed by a standard software package.
  • This program quantifies the signal intensity for each of the different tags in the both the Cy5 and Cy3 channel.
  • the program After normalizing the data, the program generates ratios for each of the different tags.
  • the final output is a list of the deletion strains in the pool along with the corresponding uptag and downtag ratios. Sorting the list by the ratios in ascending order identifies deletion strains that are synthetically lethal with HNT2. In this example, 1% of the 1,600 tagged deletion strains displayed significant growth differences in the hnt2 genetic background relative to the wild-type control.

Abstract

La présente invention concerne des méthodes qui font intervenir des techniques de criblage létal synthétique pour l'identification de cibles de médicaments. On utilise ici des bibliothèques de cellules « à code barre » composée d'une collection de différents clones mutants qui portent chacun une mutation par inactivation d'un gène différent. Chaque clone mutant comporte un identifiant ADN, ou « code barre », qui permet d'identifier rapidement et sans peine le clone et sa mutation. Grâce à une telle bibliothèque, on dispose d'un moyen rapide, quantitatif, sensible et simple d'identification de gènes qui interagissent avec un gène cible mutant. Les gènes ainsi identifiés constituent des cibles prometteuses pour le criblage de médicaments.
EP01942675A 2000-01-20 2001-01-18 Criblage synthetique letal a code barre pour identification de cibles de medicaments Withdrawn EP1248860A2 (fr)

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WO2022133734A1 (fr) * 2020-12-22 2022-06-30 Singleron (Nanjing) Biotechnologies, Ltd. Procédés et réactifs pour séquençage de transcriptome à haut débit pour criblage de médicament
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