DETAILED DESCRIPTION OF THE EMBODIMENTS

[0039] For convenience, the meaning of certain terms and phrases employed in the specification, examples, and appended claims are provided below:

[0040] Non-essential gene—a gene whose function is non-essential to the viability of the cell, either because it is dispensible for cell metabolism or due to the existence of one or more other genes which functionally overlap with it.

[0041] Molecule—In the present invention a molecule can be in one embodimenta chemical reagent, in another embodiment a nucleic acid, in another embodiment a drug, in another embodiment a nucleic acid, in another ambodiment ribozymes, in another embodiment RNA aptamers, in another embodiment peptide aptamers.

[0042] Drug—the the term drug refers to any molecule (see above) which has a therpeutic efficency, and could serve to treat or to relief a diseased condition.

[0043] Non-lethal mutation—a mutation within a non-essential gene or a defect within an essential gene which is partial and thus leaves the cell viable.

[0044] Gene of interest—a specific gene which is either non-essential for viability or an essential gene carrying a non-lethal mutation. Its function may be known or unknown. An example without being limited is the hypoxantine-guanine phosphoribosyl transferase enzyme.

[0045] Synthetic or synergistic lethality—a lethal cell phenotype which is the result of either the synergistic incapacitation of two genes, or due to the overexpression of one gene on the background of the incapacitation of the gene of interest and vice versa. Either one of these two conditions may also require the overexpression and/or underexpression of other gene (s). The incapacitation of activity may be full or only partial. The incapacitation may be as a result of a resident mutation, or due to an externally inserted element, such as a truncated cDNA, an antisense cDNA molecule or a chemical reagent.

[0046] Survival plasmid—a gene vehicle/vector which carries either a functioning copy or a dominant-negative mutant of a gene of interest. The plasmid is not incorporated into the genome of the cell, and yet can autonomously replicate within the cell (i. e. an episome). The episome includes an origin of DNA replication which may be of viral or mammalian origin, and a nuclear antigen gene. The plasmid is spontaneously gradually lost from the cell population.

[0047] Genetic suppressor element (GSE)—a nucleic acid capable of suppressing genetic expression in a dominant-negative fashion. Examples of GSEs are antisense cDNA, truncated sense cDNA, DNA encoding RNAi, duplexes of 21-nucleotide RNAs and other forms of synthetic duplex RNAs with overhanging 3′ ends. These either encode antisense RNAs, dsRNAs or RNAs having inverted repeats (both belonging to the RNAi type), or constitute synthetic small interfering RNAs (siRNAs), RNA aptamers, ribozymes, peptide aptamers, or truncated polypeptides.

[0048] Modulators of gene expression—a group of DNA/RNA/polypeptide molecules which affect gene expression of the host cells. These DNA molecules are either GSEs or overexpressed wild-type genes.

[0049] Vector vehicles for modulators of gene expression—a group of vectors containing among others: episomal mammalian expression vectors, retroviral vectors, other RNA-based viral vectors, DNA viral vectors and chimeric transposable element vectors an example without being limited is a chimeric EBV-based pisomal plasmid.

[0050] Plurality of molecules can be used interchngeably with a chemical library and/or a genetic library—in one embodiment without limitation it is a group of different Chemical Reagents which may comprise synthetic as well as natural compounds. This definition also includes, but is not limited to, in one embodiment to drug compounds, in another embodiment to synthetic antisense DNA oligonucleotides which may also be modified (phosphorothioate antisense oligodeoxynucleotides, chimeric oligodeoxynucleotides, etc.), in another embodiment to ynthetic small interfering RNAs. In another embodiment it is a genetic library which is a group of vectors containing random DNA fragments from a given species and cloned appropriate hosts.

[0051] Gene of interest—refer in this invention to a gene which serve as a basis for screening other genes, chemicals or drugs due to an interaction with the gene.

[0052] Reporter gene—the reported genes serve for the determination of the palsmid of the invention. In one embodiment the products of the reporter genes are fluorescent products. An example without limitation is a GFP gene.

[0053] Transfection/transfected/transfecting as used herein, the term “transfection” means the introduction of a nucleic acid, e.g., an expression vector or synthetic single or double-stranded DNA/RNA, into a recipient cell by nucleic acid-mediated gene transfer. “Transformation”, as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA, RNA or peptide.

[0054] In one embodiment there is provided a method for screening molecule which have a synthetic lethal property when in combination with a gene of interest carrying a non-lethal mutation for example, without being limited, a gene for HPTR1-hypoxantin-guanine phosphoribosyl tarnsferase, the method comprising the steps of: i. transfecting a first reporter gene which can be for example without limitation a variant GFP gene into a non yeast eukaryotic cell such as mammalian cells having a genome comprising a gene of interest which carries a non-lethal mutation, or a genome which is null for the gene of interest; ii. selecting clones stably expressing the first reporter gene; iii. Introducing into the cells according to the methods described in the Example section a survival plasmid comprising a functioning copy of the gene of interest, a second reporter gene selectable marker, which could be without limitation another variant of GFP gene, an origin of DNA replication and a nuclear antigen gene essential for replication of the plasmid within the cells, wherein the survival plasmid is autonomously replicating and spontaneously lost from the cells; vi. growing the cells under normal conditions which are well known to anyone who is skilled in the art in the presence of a selection compound such as without limitation Hygromycin which selects for the selectable marker; vii.

[0055] selecting cell clones stably expressing the second reporter gene and the functioning copy of the gene of interest the selection is performed, without limitation, by a flurescent light microscopy and/or using a fluorescence plate reader ; viii. removing selection for the selectable marker, and adding molecules destined for screening of their ability to impose selective pressure enforcing retention of the unstable survival plasmid. ix. determining survival plasmid retention in cells, according to the method which is well explained in the Examples section and is based on calculation of the ratio of the emission and/or the excitation of the fluorescent products of the reporter gene thus identifying a molecule having a synthetic lethal property when in combination with non-lethal mutated gene of interest.

[0056] It should be noted in this respect that the details, the examples and the conditions of each component of the above described embodiment may be applied, without limitation to the other embodiments described below. Further details without being limited may be found in the Examples method.

[0057] In this aspect of the invention, molecule such as a chemical reagent for example induced synthetic lethality (i. e. chemical synthetic lethality) identifies biochemical inhibitors or drugs whose lethal effect is dependent on the presence of a whole or partial inactivation of a specific cellular gene (i. e. gene of interest).

[0058] In an alternate embodiment of this aspect of the invention, there is provided a method for screening a chemical library comprising a plurality of molecule types in mammalian cells having a genome, in order to identify a molecule type having a gene-specific lethal property in the cell. The cellular gene of interest may be either deficient, or overexpressed in its normal/mutant form. In this specification, expression of the gene of interest includes all of these possibilities.

[0059] In another embodiment there is provided a method for screening a cDNA molecule, which has a synthetic lethal property when in combination with a gene of interest carrying a non-lethal mutation, the method comprising the steps of: i. transfecting a first reporter gene into mammalian cells having a genome comprising a gene of interest which carries a non-lethal mutation; ii. selecting clones stably expressing the first reporter gene; iii. introducing into the cells a survival plasmid comprising a functioning copy of the gene of interest, a second reporter gene, a selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the plasmid within the cells, wherein the plasmid is spontaneously lost from the cells; iv. growing the cells in the presence of a selection compound which selects for the selectable marker; v. selecting cell clones stably expressing the second reporter gene and the functioning copy of the gene of interest; vi. incorporating the cDNA molecule—into a vector vehicle containing a second selectable marker gene so as to obtain a vector vehicle-cDNA molecule. The vehicle is according to one embodiment an episomal mammalian expression vector vii. transfecting cells with vector vehicles-cDNA molecules while removing selection for the first selectable marker, and instituting selection for pools of cells expressing the second selectable marker gene. viii. determining survival plasmid retention in cells, thus identifying a cDNA having a synthetic lethal property when in combination with non lethal mutated gene of interest.

[0060] In a further embodiment of this aspect of the invention, there is provided a method for screening a collection of DNA molecules selected from the group consisting of antisense cDNA, truncated cDNA, DNA encoding RNAi, full-length cDNA, genomic DNA, or any other DNA form in order to identify among them one or more modulators of gene function which are synergistically lethal to a mammalian cell, the cell having a genome which expresses a non-lethal mutant gene of interest.

[0061] Cells maintaining the survival plasmid due to expression of a GMPS GSE, exhibit a high tpzGFP expression, allowing their isolation from the total cell population by FACS sorting. Putative GSE-containing episomes were recovered from the low molecular weight DNA fraction. Individual GSE-containing episome clones were retransfected into the recipient cell system, and pools of secondary transfectants displaying a high tpzGFP to sphGFP ratio over time were spotted by periodic readings with a microplate fluorescence reader. Retention of the HPRT1 survival plasmid is dependent on continued selection with G418 for the GSE-expressing episomal vector. As expected from GSEs, irrespective whether their polarity was sense or antisense, they had a dominant-negative effect on the activity of the GMPS endogenous gene. Noteworthy, GSE-containing clones exhibiting as much as 52-73% of the normal GMPS activity were still picked up by our screen (Table2). Similarily, the dose response of IMPDH inhibitors such as mycophenolic acid, mizoribine, and ribavirin, indicate that partial inhibition of IMPDH enzyme activity is accompanied by subsaturated tpzGFP fluorescence levels, reflecting a lower requirment for the survival plasmid encoded HPRT1 enzyme.

[0062] As can be seen in the Examples section, using the method described here, one could identify a gene (GMPS) synthetic lethal to a gene of interest (HPRT1). In comparison to the yeast screen (1), this method combines the “mutagenesis” together with the rescue of the synthetic lethal GSEs into a single step. This system allowed isolation of dominant-negative mutants, which should be useful on their own. To the best of our knowledge these mutants are the first dominant-negative suppressors to be described for the GMPS gene.

[0063] Initially, the GSE methodology was used to isolate dominant-negative mutants of single given genes, based on the ability of such constructs to disrupt a drug-induced apoptotic response, or to abrogate cell transformation. A further extension to this basic theme has been the use of random libraries of either antisense RNA alone or GSEs for isolation of apoptotic genes via actual selection for survival. Because of the high complexity of random GSE libraries, where each cDNA is usually represented by at least tens of antisense and truncated sense short DNA fragments, enrichment steps for cDNAs of interest have been employed prior to the construction of such libraries. As might have been anticipated, some of the apoptotic genes selected by this approach turned out to be tumor suppressors. The present report demonstrates that incorporation of the GSE method together with a FACS sorting step, into a genetic synthetic lethality screen leads to isolation of dominant-negative mutants for a gene of interest. Clearly, this approach should enable the isolation of new survival/antiapoptotic genes, some of which may turn out to be dominant oncogenes, such as Bcl2, Survivin and others.

[0064] To accomplish a genetic synthetic lethality screen at the multi-gene level, the usage of either an enriched cDNA pool, and/or the employment of several rounds of sorting by FACS (in which DNA from GSE containing plasmids is reintroduced, after amplification in bacteria, into recipient cells and subject to decay and selection for fluorescent cells by FACS), is than performed.

[0065] Enrichment for human cDNAs, serving as the source for making the GSE library, could be performed by any one of a number of methods, such as subtractive hybridization, differential display, or assaying for gene activity by DNA microarrays. In comparison to a GSE expressed by a retroviral vector present usually as one copy per cell, our method has the advantage of the employment of a multicopy episomal vector, whose GSE activity is not subject to modulation by neighboring chromosomal sequences.

[0066] The genetic synthetic lethality screen need not be confined to the use of antisense or truncated cDNAs. Other GSEs such as without limitation libraries of ribozymes, RNA aptamers, peptide aptamers or synthetic small interfering RNAs may be used by applying the same methods.

[0067] In another embodiment there is provided a method for screening a drug which has a synthetic lethal property when in combination with a gene of interest carrying a non-lethal mutation, the method comprising the steps of: i. transfecting a first reporter gene into a non-yeast eukaryotic cells having a genome comprising a gene of interest which carries a non-lethal mutation; ii. selecting clones stably expressing the first reporter gene; iii. introducing into the cells a survival plasmid comprising a functioning copy of the gene of interest, a second reporter gene, a selectable marker, which according to one embodiment is a dominant selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the plasmid within the cells, wherein the plasmid is spontaneously lost from the cells; iv. growing the cells in the presence of a selection compound which selects for the selectable marker; v.

[0068] selecting cell clones stably expressing the second reporter gene and the functioning copy of the gene of interest; vi. adding the drug for screening of its ability to impose selective pressure for the retention of the spontaneously lost survival plasmid to the cell clones of step v. vii. determining survival plasmid retention in cells, thus identifying a drug having a synthetic lethal property when in combination with non lethal mutated gene of interest.

[0069] In another embodiment there is provided a method for screening a chemical agent which which have a synthetic lethal property when in combination with a gene of interest carrying a non-lethal mutation, the method comprising the steps of: i. transfecting a first reporter gene into mammalian cells having a genome comprising a gene of interest which carries a non-lethal mutation; ii. selecting clones stably expressing the first reporter gene; iii. introducing into the cells a survival plasmid comprising a functioning copy of the gene of interest, a second reporter gene, a selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the plasmid within the cells, wherein the plasmid is spontaneously lost from the cells; iv. growing the cells in the presence of a selection compound which selects for the selectable marker; v. adding the chemical agent for screening of its ability to impose selective pressure for the retention of the spontaneously lost survival plasmid to the cell clones of step V. vi. selecting cell clones stably expressing the second reporter gene and the functioning copy of the gene of interest; vii. Determining survival plasmid retention in cells which survive, thus identifying a chemical agent which has a synthetic lethal property when in combination with non lethal mutated gene of interest.

[0070] In another embodiment there is provided a method for screening a library comprising a plurality of molecules in order to identify molecule/s having a synthetic lethal property when in combination with a gene of interest carrying a non-lethal mutation, the method comprising the steps of: i. transfecting a first reporter gene into mammalian cells having a genome comprising a gene of interest which carries a non-lethal mutation; ii. selecting clones stably expressing the first reporter gene; iii. introducing into the cells a survival plasmid comprising a functioning copy of the gene of interest, a second reporter gene, a selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the plasmid within the cells, wherein the plasmid is spontaneously lost from the cells; vi. growing the cells in the presence of a selection compound which selects for the selectable marker; v. selecting cell clones stably expressing the second reporter gene and the functioning copy of the gene of interest; vi. adding the library comprising a plurality of molecules for screening of its ability to impose selective pressure for the retention of the spontaneously lost survival plasmid to the cell clones of step V. vii. determining survival plasmid retention in cells, thus identifying a molecule/s within a library having a synthetic lethal property when in combination with non lethal mutated gene of interest.

[0071] The invention enables also a method of screening a molecule, or a gene or a drug that are synthetic lethal when in combination mutant or normal gene of interest which is overexpressed as is the situation without limitation with oncogenes such as without being limited RAS.

[0072] In another embodiment there is provided a method for screening molecule which have a synthetic lethal property when in combination with a mutant or normal gene of interest which is overexpressed, the method comprising the steps of: i. transfecting a first reporter gene into mammalian cells having a genome comprising a mutant or normal gene of interest which is overexpressed, ii. selecting clones stably expressing the first reporter gene; iii.

[0073] introducing into the cells a survival plasmid comprising a dominant-negative mutant of the gene of interest, a second reporter gene, selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the plasmid within the cells, wherein the survivsal plasmid is autonomously replicating and spontaneously lost from the cells; vi.

[0074] growing the cells in the presence of a selection compound which selects for the selectable marker; vii. selecting cell clones stably expressing the second reporter gene and the dominant-negative mutant of the gene of interest; viii. removing selection for the selectable marker, and adding molecules destined for screening of their ability to impose selective pressure enforcing retention of the unstable survival plasmid. ix. determining survival plasmid retention in cells, thus identifying a molecule having a synthetic lethal property when in combination with the a mutant or normal gene of interest which is overexpressed.

[0075] In another embodiment there is provided a method for screening a cDNA molecule, which have a synthetic lethal property when in combination with a mutant or normal gene of interest which is overexpressed, the method comprising the steps of: i. transfecting a first reporter gene into a mammalian cells having a genome comprising a mutant or normal gene of interest which is overexpressed; ii. selecting clones stably expressing the first reporter gene;

[0076] iii. introducing into the cells a survival plasmid comprising a dominant-negative mutant of the gene of interest, a second reporter gene, a selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the plasmid within the cells, wherein the plasmid is spontaneously lost from the cells; iv.growing the cells in the presence of a selection compound which selects for the selectable marker; v. selecting cell clones stably expressing the second reporter gene and the dominant-negative mutant of the gene of interest; vi.

[0077] incorporating the cDNA molecule—into a vector vehicle containing a second selectable marker gene so as to obtain a vector vehicle-cDNA molecule. vii. transfecting cells with vector vehicles-cDNAs molecules while removing selection for the first selectable marker, and instituting selection for pools of cells expressing the second selectable marker gene. viii.

[0078] determining survival plasmid retention in cells, thus identifying a cDNA having a synthetic lethal property when in combination with the a mutant or normal gene of interest which is overexpressed.

[0079] In another embodiment there is provided a method for screening a drug which have a synthetic lethal property when in combination with a mutant or normal gene of interest which is overexpressed, the method comprising the steps of: i. transfecting a first reporter gene into a non-yeast eukaryotic cells having a genome comprising a mutant or normal gene of interest which is overexpressed; ii. selecting clones stably expressing the first reporter gene; iii. introducing into the cells a survival plasmid comprising a dominant-negative mutant of the gene of interest, a second reporter gene, a selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the plasmid within the cells, wherein the survival plasmid is spontaneously lost from the cells; iv. growing the cells in the presence of a selection compound which selects for the selectable marker; v. selecting cell clones stably expressing the second reporter gene and the dominant-negative mutant of the gene of interest; vi. adding the drugs destined for screening their ability to impose selective pressure enforcing retention of the spontaneously lost survival plasmid; vii. determining survival plasmid retention in cells, thus identifying a drug having a a synthetic lethal property when in combination with the mutant or normal gene of interest which is overexpressed.

[0080] In another embodiment there is provided a method for screening a library comprising a plurality of molecules which have a synthetic lethal property when in combination with a mutant or normal gene of interest which is overexpressed, the method comprising the steps of: i. transfecting a first reporter gene into mammalian cells having a genome comprising a mutant or normal gene of interest which is overexpressed; ii. selecting clones stably expressing the first reporter gene; iii. introducing into the cells a survival plasmid comprising a dominant-negative mutant of the gene of interest, a second reporter gene, a selectable marker, an origin of DNA replication and a nuclear antigen gene essential for replication of the plasmid within the cells, wherein the plasmid is spontaneously lost from the cells; vi. growing the cells in the presence of a selection compound which selects for the selectable marker; v. selecting cell clones stably expressing the second reporter gene and the dominant-negative mutant of the gene of interest; vi. adding the library comprising a plurality of molecules in order to identify those that impose selective pressure enforcing the retention of the spontaneously lost survival plasmid. vii. determining survival plasmid retention in cells, thus identifying at least one molecule within a library having a synthetic lethal property when in combination with the mutant or normal gene of interest which is overexpressed.

[0081] In another embodiment there is provided a kit for screening a library containing a plurality of molecule types in mammalian cells having a genome, in order to identify a the molecule having a gene-specific lethal property in the cell, comprising: an integration plasmid comprising a first reporter gene; a survival plasmid compatible with a mammalian cell comprising a functional copy of a gene of interest or a dominant-negative mutant of a gene of interest, a reporter gene, a dominant selectable marker gene, an origin of DNA replication and a nuclear antigen essential for replication of the survival plasmid, the survival plasmid being spontaneously lost from the cell.

[0082] In another embodiment there is provided a kit for screening a group of DNA molecules in order to identify among them one or more modulators of gene expression which are synergistically lethal to a mammalian cell together with a gene of interest, comprising: an integration plasmid comprising a first reporter gene; a survival plasmid compatible with a mammalian cell comprising a functional copy of a gene of interest or a dominant-negative mutant of a gene of interest, a reporter gene, a dominant selectable marker gene, an origin of DNA replication and a nuclear antigen gene essential for replication of the survival plasmid, the survival plasmid being spontaneously lost from the cell; and a vector vehicle containing a second dominant selectable marker gene and carrying either a human GSE library or a wild-type cDNA library.

[0083] In another embodiment there is provided a survival plasmid compatible with a mammalian cell comprising a functional copy of a gene of interest, a reporter gene, a dominant selectable marker gene, an origin of DNA replication and a nuclear antigen essential for replication of the episome, the episome being spontaneously lost from the cell, wherein the product of the reporter gene is a mutant green fluorescent protein (GFP).

[0084] In another embodiment there is provided a survival plasmid compatible with a mammalian cell comprising a dominant-negative mutant of a gene of interest, a reporter gene, a dominant selectable marker gene, an origin of DNA replication, and a nuclear antigen gene essential for replication of the episome, the episome being spontaneously lost from the cell, wherein the product of the reporter gene is a mutant green fluorescent protein (GFP).

[0085] In this aspect of the invention, synthetic lethality imposed by either a GSE or by an overexpressed full-length cDNA identifies a gene function or functional links between genes.

[0086] The method of the invention differs from the synthetic lethality screen previously described in yeast in the following respects:

[0087] (1) Synthetic lethality as disclosed in yeast is recognized by the visible color of yeast colonies grown on agar within petri dishes. The majority of colonies exhibit the appearance of white sectors within red colonies, while a synthetic lethal condition prevents the appearance of white sectors in a primarily red colony. The accumulation of red pigment is enabled by the reporter gene acting together with other genes. In contrast, the method of the invention involves the seeding of human/mammalian cells into microtiter plates, and the periodic measurement in a fluorescent plate reader of the double-label fluorescent ratio of two fluorescent proteins. Retention over time of a high ratio in the readings of a fluorescent variant encoded by the survival plasmid to a second fluorescent variant produced from a chromosomally integrated gene, indicates selection for maintenance of the survival plasmid and thus a synthetic lethality condition. The fluorescence is a direct product of the reporter gene. (2) Synthetic lethality is imposed in yeast by randomly mutagenizing the whole yeast genome with a chemical mutagen, thus leading to random gene inactivation. In contrast, in the present invention, synthetic lethality is achieved by either a chemical inhibitor (chemical synthetic lethality) or a genetic incapacitation via a full-length cDNA/GSE (genetic synthetic lethality). The latter involves overexpressing full-length sense cDNA libraries or GSE libraries, either one of which is incorporated into episomal plasmids, retroviral vectors, other RNA-or DNA-viral vectors, or chimeric transposable elements. Identification of the gene which is synthetic lethal with the gene of interest is performed in yeast by first isolating those colonies in which the red pigment was maintained and no white sectors appear. Those colonies putatively harbor a chromosomally mutated gene which is synthetic lethal with the gene of interest. Those yeast colonies are transfected by a normal yeast genomic library incorporated into a yeast multi-copy plasmid. Those transformants transfected by and expressing a wild-type copy of the chromosomally mutated gene, no longer sustain a synthetic lethality condition, and therefore no longer need to retain the survival plasmid. Those few colonies are recognized by the appearance of white sectors, from which the plasmid DNA is extracted, transformed into bacteria and further analyzed for the identity of the yeast gene insert by standard recombinant DNA methods.

[0088] Identification of the genetic element which confers the synthetic lethal phenotype in human/mammalian cells of the present invention, on the other hand, does not require a further transfection with a normal gene library. This is because, unlike in the yeast method, gene incapacitation is not achieved by mutagenizing the endogenous resident cell genome but rather by an exogenous DNA element working either in a dominant-negative fashion or by overexpression of a wild-type cDNA. Accordingly, the external genetic element conferring the synthetic lethality is recovered by either one of two approaches, depending on the type of vector/vehicle used: (a) an episomal plasmid, chimeric RNA or DNA based viral replicons are rescued by Hirt supernatant-extract mediated bacterial transformation or purification of packaged virus-like particles, respectively; (b) chromosomally integrated GSE or a wild-type sense cDNA library incorporated into either a retroviral vector or a chimeric transposable element, are recovered by PCR on genomic DNA.

[0089] The availability of a large number of mutant human cell lines derived from genetic disorders on the one hand, and the ability to employ homologous recombination for gene disruption in somatic human cells on the other, constitutes a large reservoir of recipient cells and genes of interest.

[0090] The cells which may be used in the method of the invention are mammalian cells. Preferably they are human cells, but the same principle may be applied to e. g., rodent cells harboring a survival plasmid with the appropriate replication properties.

[0091] The survival plasmid contains a reporter gene so as to enable determination of the presence of the plasmid in the cells. The product of the reporter gene may be any detectable molecule, such as the following biosensors: luciferin (luciferase substrate); aequorin; Fluo-3/acetoxymethyl (esterase substrate); FDG (3-gal substrate); or CCF2 which is a p-lactamase substrate [J. E. Gonz lez and P. A. Negulescu, Curr. Opin. Biotechnol. 9,624 (1998)]. Preferably, the reporter gene encodes a fluorescent protein whose expression can be distinguished from that of a second fluorescent protein marking the cell number. Non overlapping excitation or emission spectra of the two fluorescent proteins allows for double-label fluorescence measurement.

[0092] Accordingly the cells are also made to incorporate in their genome a second reporter gene which indicates the number of cells. By comparing the signals obtained from the two reporter genes, a relative ratio between the number of survival plasmids and the number of cells may be determined.

[0093] The methods of the invention may be carried out using conventional systems for growing, scanning and sorting cells, such as microtiter plates, 96well. 384-well or other high-density microplates, a microplate fluorescent reader, and a fluorescent activated cell sorter (FACS). The methods are especially useful in high throughput screening, where automation allows for the rapid screening of large number of chemicals as well as the full spectrum of mammalian genes and their respective GSEs.

[0094] The present invention may be used in a number of applications:

[0095] The first aspect of the invention should prove advantageous in the search of drugs which synergize with particular gene deficiencies or gene status to cause cell lethality as well as identifying drug compounds having gene-specific lethal properties. A special application of this aspect would be to look for chemicals which kill either a benign or cancerous cell growth in a defined genetic milieu where the chemical is synthetic lethal with a particular gene, either in a deficient form (present in tumor suppressor genes) or overexpressed normal/mutated form (present in oncogenes).

[0096] The second aspect of the invention is useful in identifying human genes whose under or over-expression causes lethality of human cell lines with defined genetic abnormalities, be it either gene deficiency or overexpression of the normal/mutated gene form. Such genes are obviously potential targets for drugs aimed at eliminating the affected cells/tissue. The application of this approach to human tumor-derived cell lines, is particularly amenable to identification of targets for cancer therapy.

[0097] Above and beyond the identification of gene targets of therapeutic interest in defined genetic background, the invention should prove useful as a tool for basic research. In particular, the invention may enable researchers to rapidly screen large sets of gene products for functional interactions and helps define genetic pathways within the cell (2).

[0098] The method using rodent cells should be useful as a model for human genetic traits and responses in drug development and disease research. For example, mutant mice generated by either ectopic overexpression, homologous recombination or tagged random mutagenesis, supply a large source of recipient mutated mouse embryo fibroblasts which, together with the methods of the invention, will greatly facilitate research and development of new drugs and therapeutic strategies for human beings.

[0099] Also included in the invention are kits for synthetic lethality screening. One such kit in accordance with the first aspect of the invention would preferably include an episomal survival plasmid and an integrating vector, each carrying a reporter gene, for a chemical synthetic lethality screen. A kit in accordance with the second aspect of the invention would preferably include the above genetic elements together with a library of GSEs or sense cDNAs incorporated within an extrachromosomal vector.

[0100] The present invention describes the development and the feasibility of a genetic synthetic lethality screen in cultured human cells. The methodology is an extension of the chemical synthetic lethality screen, which was disclosed in U.S. patent application Ser. No. 09/931,444 . The invention shows that the chemicals used in the chemical screen can be replaced also by dominant-negative GSEs. Thus, the methods described herein is a general screening method which can apply to drugs, cDNA, different chemical compounds as well as plurality of molecules.

[0101] Because the method is based on identification of a lethal phenotype, it is particularly relevant to the search for human genes acting in the same essential pathway, or along two parallel ones. Therefore, such genetic synthetic lethality screens should have a major impact on human functional genomics. Moreover, the genetic synthetic lethality screen, when applied to human tumor cell lines having known primary genetic alterations, could lead to identification of new, perhaps even unexpected, secondary targets for cancer therapy. As targets identified by this approach would result in cellular synthetic lethality only when the primary tumor alteration is present, a high selectivity towards the tumor is insured.

EXAMPLES

Chemical Synthetic Lethality Screening Experimental Procedures

[0102] A. Construction of Plasmids

[0103] The plasmid pIS was constructed by replacing the BamHl fragment encoding CD20 from pCMV-CD20 (kind gift from S. van den Heuvel and E. Harlow) with a blunt-ended HindIII-BamHI fragment containing the coding sequence of sphGFP from the pGFPsph-b [R] vector (Packard Instruments).

[0104] The episomal HGPRT-tpzGFP survival plasmid was constructed by first cloning a HindIII-BamHI blunt-ended fragment encoding the tpzGFP and polyadenylation signal from the pGFPtpz-b [R] vector (Packard Instruments) into the HindIII site of pCEP4 (Invitrogen). The coding sequence of HGPRT was cloned into pcDNA3 (Invitrogen) and subsequently removed together with the CMV promoter by digestion with Bglll and BamHl. This fragment was then cloned into the BamHI site of the pCEP4-GFP vector. The final survival plasmid was produced by cloning the KpnI-BamHI fragment of the pCEP4-HGPRT-tpzGFP vector into the Kpnl BamHl site of pREP4 (Invitrogen), such that tpzGFP is under the influence of the RSV promoter.

[0105] B. Expression of Constructs in Cells

[0106] HGPRT-deficient HT1080 fibrosarcoma cells (W. F. Benedict, et al., Cancer Res. 44,3471 (1984)) were maintained in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% fetal calf serum (FCS) and 4 mM L-glutamine. This cell line has a generation time of about 24 hours, and a pseudo diploid chromosomal karyotype. Transfections were carried out using the calcium phosphate precipitation technique as previously described (T. Teitz et al., Proc. Natl. Acad. Sci. USA 84,8801 (1987)). For pIS, selection in G418 (Calbiochem) was carried out at 400 microg/ml, while maintenance was at 50 microg/ml. For the HGPRT-tpzGFP survival plasmid, selection in hygromycin B (Sigma) was at 150 microg/ml while maintenance was at 50 microg/ml. Selection and maintenance of the survival plasmid was also carried out in HAT medium (100 microM hypoxanthine, 0.4 microM aminopterin, 16 microM thymidine; Littlefield, J. W. Science 145,709 (1964)). GATA medium was DMEM with 10% dialyzed FCS, plus 3.5 microM guanine-HCI, 0.4 microM aminopterin, 16 microM thymidine and 35 microM adenine.

[0107] HATA medium was HAT medium plus 60 microM adenine. Concentrations of adenine were detenined empirically by plating Clone 12 cells into 96 well microplates in either HAT or GAT medium with varying concentrations of adenine. Loss of survival plasmid was followed on a microplate fluorescence reader and the concentration that was not toxic and allowed a rate of plasmid loss similar to spontaneous rates was chosen.

[0108] C. Fluorescent Scanning of Microtiter Plates

[0109] For fluorescent scanning, cells were trypsinized and distributed at 30,000 cells/well into 96 well microplates (TPP). Growth medium was changed twice a week and plates were grown for up to 75 days. Although cell populations were very dynamic due to shedding of large clumps, almost all wells contained viable, growing cells for the entire span of the experiment. Plates were prepared for scanning by replacement of medium in wells with Hank's balanced salt solution without phenol red. This procedure minimized background fluorescence from the growth medium while maintaining maximal viability. Plates were scanned with an FL600 microplate fluorescence reader using the KC4 software (Biotek Instruments). Excitation for sphGFP was at 380 nin with a bandpass of 20 nm, while emission was measured at 508 nm with a bandpass of 40 nm.

[0110] Excitation of tpzGFP was at 495 nm with a bandpass of 20 mn, while emission was measured at 535 nm with a bandpass of 40 nm. To avoid possible artifacts, all wells within a given experiment were assayed for the fluorescence of the two GFP mutants using fixed sensitivities. Integrated sphGFP was used as an internal control for the number of cells. This was achieved by dividing the relative fluorescence resulting from the episomal tpzGFP vector by the relative fluorescence for sphGFP for each well. This ratio was then divided by the average fluorescence ratio for cells maintained under hygromycin B or HAT selection, resulting in a value representing percent remaining fluorescence for each well as compared to wells maintained under continuous selection. The data points are an average for all wells and the calculated standard deviation. Cells were returned to growth medium immediately following scanning.

[0111] I. Establishment of a model system

[0112] In order to develop a synthetic lethality screening method in human cells, Epstein-Barr virus (EBV) based episomal vectors, which can replicate autonomously as a low copy number episome in human cells of diverse tissues. were selected as the basis for the survival plasmid (J. L. Yates, N. Warren and B. Sugden, Nature 313,812 (1985); U.S. Pat. No. 4,686,186, whose contents are incorporated herein). However, this vector is an imperfect episome because its retention in human cells requires continued selection for a dominant selectable marker gene built into the vector (D. Reisman, J. Yates and B. Sugden, Mol. Cell. Biol. 5,1822 (1985). M. P. Calos, Trends Genetics 12.463 (1996). N. Dafni and D. Canaani, unpublished results). In the design of the system for human cells, advantage was taken of this spontaneous gradual plasmid loss, by creating synthetic lethal conditions under which retention of the episomal plasmid is essential for viability.

[0113] As a model system for the establishment of the method, the biosynthetic pathway leading to the production of guanosine monophosphate (GMP) was chosen ( FIG. 1 ). This pathway has been thoroughly studied biochemically and is particularly amenable to a synthetic lethality screen. First of all, the gene of interest in the model system, hypoxanthine-guanine phosphoribosyltransferase (HGPRT), is non-essential for cell survival, since it works in salvage pathways by converting hypoxanthine and guanine to IMP and GMP, respectively. Secondly, immortalized HGPRT-deficient human cell lines are available. Also, a major advantage of this model system is that de novo GMP synthesis can be specifically blocked by certain chemical reagents ( FIG. 1 ). Accordingly, the HGPRT deficient human cells should become dependent on the HGPRT expressing “survival plasmid”, when the de novo pathway is blocked by aminopterin or mycophenolic acid (MPA), in the presence of the HGPRT precursors hypoxanthine and guanine, respectively.

[0114] II. Stable transfection of an internal control fluorescent marker. In order to establish synthetic lethality as a high throughput screening system based on fluorescent readout, it is essential to have an internal fluorescent reporter which normalizes the fluorescent reading from the episomal survival plasmid, relative to cell number. To this end, the sapphire-blue green fluorescent protein (sphGFP), a mutant form of the natural GFP from the jellyfish Aequara victoria, was chosen (U.S. Pat. No. 5,625,048, the contents of which are incorporated herein). The sphGFP coding region was cloned into a transcription unit driven by the strong CMV immediate-early promoter (U.S. Pat. No. 5,168,062, the contents of which are incorporated herein), and situated upstream of a neoR transcription unit, thus creating the vector pIS ( FIG. 2 a ). As a recipient cell line the HGPRT-deficient variant of the HT1080 fibrosarcoma cell line was chosen (W. F. Benedict, B. E. Weissman, C. Mark and E. J. Stanbridge, Cancer Res. 44, 3471 (1984).

[0115] The pIS vector was transfected into these cells, stable clones were selected in G418, and examined with a fluorescent microscope. Clones with >99% fluorescing cells were chosen for further examination ( FIG. 3 ). These were seeded into 96-well microplates, and scanned for fluorescence intensity using a microplate fluorescent reader, as described in the Methods section. Compare to the recipient cells, or medium alone, up to 38-fold fluorescence enhancement was recorded for the sphGFP transformants (data not shown).

[0116] As expected, in the linear range of sphGFP reading, a close correlation was seen between the number of cells and the fluorescent intensity. The fluorescent intensity of these cells did not vary appreciably when removed from continuous G418 selection. Thus, the fluorescence levels obtained from the stably integrated sphGFP mutant gene are appreciable and can be easily detected as a mass population by a fluorescent microplate reader.

[0117] III. Generation of stable transfectants harboring the episomal survival plasmid

[0118] Survival plasmids containing a transcription unit for the human HGPRT cDNA (gene of interest) and a second GFP mutant gene, were constructed onto the backbone of the EBV-based pCEP4/pREP4 episomal vectors ( FIG. 2 b ), as described in the Methods section. These vectors replicate autonomously as episomes in human cells due to the EBV-oriP and EBNA-1 elements (J. L. Yates, N. Warren and B. Sugden, Nature 313,812 (1985)).

[0119] They also contain the hygromycin phosphotransferase dominant selectable marker. the bacterial colE1 origin of DNA replication and the (beta lactamase gene).

[0120] In order to identify the survival plasmid, a second GFP variant was incorporated, the topaz-green GFP mutant gene (tpzGFP), whose expression can be distinguished from the sphGFP mutant. TpzGFP has an excitation peak (514 nm) which does not overlap with that of sphGFP (395 nm), allowing for double-label fluorescence measurements. The corresponding emission peaks are 527 nm for tpzGFP and 511 nm for sphGFP. TpzGFP was cloned under the influence of the RSV promoter ( FIG. 2 b ). The human HGPRT cDNA was inserted in between the unique restriction sites HindIII and BamHI, so that it can be easily replaced by any human cDNA of interest ( FIG. 2 b ). This construct. a HGPRT-tpzGFP survival plasmid, was introduced into one cell clone, HIS4, which displayed stable expression of the integrated sphGFP reporter. Stable clones resistant to hygromycin B were selected for further study, as described in the Methods section.

[0121] Most hygromycin B resistant clones were also resistant to HAT medium, indicating expression of the HGPRT transcription unit. Scanning by fluorescence microscopy was used to select several clones that express the tpzGFP in >99% of their cells. The fluorescence resulting from these two GFP variant, one stably integrated into the genome (sphGFP) and one episomal (tpzGFP), could be distinguished by use of two different filter blocks ( FIG. 3 . D and G vs. E and H). Similar numbers of these cells were then plated into 96 well microplates and scanned by a fluorescent microplate reader. This scanning revealed up to a 140-fold tpzGFP fluorescence increase over HIS4 autofluorescence (data not shown). It can therefore be concluded that the double-label fluorescence from the sphGFP and tpzGFP can be readily distinguished at both the single cell level by fluorescence microscopy, as well as at the mass culture level when grown in microplates, by a fluorescence microplate reader.

[0122] IV. Spontaneous loss of the survival plasmid

[0123] It was next determined whether spontaneous loss of the survival plasmid could be detected by fluorescence measurements. It was imperative to show that in microtiter plates the expected plasmid loss occurs and could be detected. This is because an inherent feature of the proposed high throughput method is that scanning for genes or chemical reagents, that are synthetically lethal with a human gene of interest, will be performed on cell clones grown in microplate wells. The dynamics of cell division, and therefore the rate of the survival plasmid loss, could be very different in cells grown for long periods in microplates, as opposed to cells stimulated to divide by a regimen of periodic trypsinization, dilution and reseeding. Accordingly, measurement of fluorescence after removal of drug selection was carried out in cells continuously passaged in 90 mm plates as well as in cells grown in microtiter plates. Results from one isolate, Clone 12, carrying an integrated sphGFP gene and an episome-encoded tpzGFP are shown in FIG. 4 . Following removal of hygromycin B from the medium, the tpzGFP and sphGFP fluorescence ratios were monitored over time. The calculated ratio was normalized to readings taken at the same time point from cells kept continuously under hygromycin B selection. As shown in FIG. 4 , fluorescence from the survival plasmid marked with tpzGFP decayed rather quickly, so that after about one month, Clone 12 lost 80-90% of its initial fluorescence. Importantly, no significant difference in the rate of fluorescence decay could be detected between the cells maintained in microplate wells as opposed to those maintained by continuous passaging in petri dishes ( FIG. 4 ). Assuming that one of the major factors affecting EBV-based plasmid loss is the rate of cell division, it could be that besides the multilayer growth of these transformed cells, the cell shedding which was observed in the microplate wells may also contribute to the dynamics of cell division.

[0124] To test whether the gradual loss of tpzGFP fluorescence over time indeed reflets the loss of survival plasmid, two assays were conducted. In one, low molecular weight DNA present in Hirt supernatants (B. Hirt, J. Mol. Biol. 26,365 (1967) of Clone 12 cells was collected at various times after removal from hygromycin B selection. This DNA was used for bacterial transformation. It was found that the decrease of AmpR colonies correlated well with the loss of tpzGFP fluorescence over time (Table 1). In the second assay, a plasmid segregation assay (D. Reisman, J. Yates and B. Sugden, Mol. Cell. Biol. 5,1822 (1985) was carried out. At each time point after drug removal, cells were reseeded into petri dishes containing hygromycin B. It was found that the number of colonies able to grow in the presence of hygromycin B, did indeed decrease at later time points (data not shown).

Table

Monitoring of spontaneous survival plasmid loss by Hirt supernatant mediated bacterial transformation.

[0125] Clone 12 cells were plated at the beginning of the experiment in DMEM without hygromycin B. Cells were continuously passaged throughout the entire experiment. Low-molecular weight DNA present in Hirt supernatants was collected at the indicated time points. All Hirt supernatants were normalized by addition of 1 ng of a chloramphenicol-resistant plasmid prior to the beginning of cell extraction.

[0126] Each Hirt supernatant transformation of bacteria was plated both on chloramphenicol and ampicillin plates. Values in table were normalized to the number of colonies counted on the chloramphenicol plates. 1

[0127] V. Detection of chemical reagent induced synthetic lethality

[0128] These results demonstrated that, in the absence of selection, the HGPRT-tpzGFP survival plasmid is unstable in HGPRT-deficient HT1080 cells. Its loss or retention can be determined by measuring its normalized fluorescence in a microplate reader. It was then necessary to test whether these features would enable the tracing of a synthetic lethality condition. The biosynthesis of GMP from IMP via XMP can be efficiently blocked using MPA, which inhibits IMP dehydrogenase ( FIG. 1 ). Under these conditions, normal HGPRT-positive cells can use supplied guanine to produce GMP via the salvage pathway, and survive, while HGPRT-deficient cells die. Clone 12. as an inherently HGPRT-deficient cell line, must retain the HGPRT-tpzGFP survival plasmid in order to remain alive in this synthetic lethality situation. Indeed, when hygromycin B was removed from Clone 12 cells grown in GATA medium (medium supplemented with guanine, aminopterin, thymidine and adenine-see Methods section), spontaneous loss over time of the survival plasmid occurred, as traced by a decrease in tpzGFP fluorescence ( FIG. 6 ). It has been shown that under the specific conditions used, this loss is enabled due to a relative surplus of adenine, which besides serving as a precursor for AMP biosynthesis, allows GMP to be synthesized via the AMP-IMP-XMP pathway (data not shown, and see also Methods and FIG. 1 ).

[0129] In contrast, addition of MPA to the GATA medium at successively higher concentrations, caused increasing retention of the survival plasmid, which could be detected by an increase in tpzGFP to sphGFP fluorescence ratio. A dose response was observed which reached a plateau at fluorescence levels similar to those obtained when HAT selection is imposed ( FIG. 5 ). Moreover, as shown in FIG. 6 , when fluorescence was observed over time, MPA could cause retention of the survival plasmid for the entire time period, while cells without MPA continued to lose tpzGFP fluorescence. Importantly, almost identical patterns of decay or retention in the presence of MPA were found when cells were grown in medium plus 3 microM guanine, without aminopterin or adenine (data not shown).

[0130] IMP dehydrogenase (IMPDH) has two isoforms and is considered to be the rate-limiting enzyme in guanine nucleotide biosynthesis. Two basic types of drugs can effectively inhibit the enzyme: nucleoside analogs and non nucleoside inhibitors. MPA is of the second class and binds the NAD site within the enzyme. Nucleoside analog inhibitors bind to the IMP substrate site. Accordingly, it was asked whether one could detect synthetic lethality when nucleoside analog inhibitors were applied to Clone 12 cells. In this assay two drugs were screened, mizoribine and ribavirin, both in use against viral infections. Both drugs were tested on Clone 12 cells grown in 96 well microtiter plates in serial dilutions of the drugs as well as serial dilutions of guanine. The observed matrices of results with mizoribine and ribavirin are shown in FIGS. 7 and 8 , respectively. Both drugs caused retention of the survival plasmid, in a way that was dependent on the concentration of each drug as well as that of guanine. These matrices allowed sensitive measurement of the synthetic lethal effects imposed by these nucleoside analogs.

[0131] The next step was to test the synthetic lethality assay in a blind test. IMPDH inhibitors were screened. Clone 12 was seeded into 1200 wells in microtiter plates in medium lacking selection. MPA, ribavirin, mizoribine, and hygromycin B were added at random (together with guanine) to three wells. Alanosine, an inhibitor of adenylosuccinate synthase (an enzyme in the pathway converting IMP to AMP) was also added as a negative control. As expected, the survival plasmid was not retained in the presence of alanosine (data not shown). However, the presence of all three IMPDH inhibitors and hygromycin B was clearly detected as were two wells containing false positive cells ( FIG. 9 ). The calculated false positive rate for this experiment was {fraction (1/600)} and was similar to previous control experiments (data not shown).

[0132] These results demonstrate the feasibility of a synthetic lethality screen in cultured human cells, using a sensitive fluorescent assay allowing detection of synthetic lethality imposed with a chemical reagent.

[0133] VI. Rodent model of synthetic lethality

[0134] Autonomous replication of a survival plasmid in rodent cells may be conferred by either the EBV-based pREP/pCEP vectors described above, or by the polyoma virus origin of DNA replication together with the virus segment encoding the large T antigen (Z. Zhu et al., J. Virol. 51,170 (1984). We have found that the EBV-based survival plasmid described above can be used in mouse cells for chemical synthetic lethality screening much as described above with respect to the human system (our unpublished results).

Genetic Synthetic Lethality Screening Experimental Procedures

[0135] I. Construction of plasmids.

[0136] pIS was constructed by replacing the BamHI fragment encoding CD20 from pCMV-CD20 with a blunt-ended HindIII-BamHI fragment containing the coding sequence of sphGFP from the pGFPsph-b [R] vector (Packard Instruments). The dominant selectable marker encoding resistance to zeocin (zeo ^R )was initially excised (together with a synthetic bacterial promoter) from pVgRXR (Invitrogen) by PstI and SalI restriction enzymes. The zeo ^R gene was introduced into a mammalian transcription unit by substitution of the neo ^R gene present in pREP9 (Invitrogen) in between the Bgl II and Rsr II restriction sites. The generated plasmid product was cleaved by PflMI and BstEII restriction endonucleases, and the excised zeo ^R transcription unit, replaced a HindIII and XbaI flanked neo ^R gene present in the original pIS vector. The episomal HPRT1-tpzGFP survival plasmid was previously described.

[0137] II. Construction of truncated sense and antisense cDNA library.