CA2283636A1

CA2283636A1 - Target genes for allele-specific drugs

Info

Publication number: CA2283636A1
Application number: CA002283636A
Authority: CA
Inventors: David Housman; Fred D. Ledley; Vincent P. Stanton, Jr.
Original assignee: Individual
Current assignee: Nuvelo Inc
Priority date: 1997-03-20
Filing date: 1998-03-19
Publication date: 1998-09-24
Also published as: WO1998041648A3; EP0973935A2; AU6764398A; WO1998041648A2

Abstract

This disclosure concerns genetic targets which have been found to be useful for allele specific anti-tumor therapy. The strategy for such therapy involves the steps of: (1) identification of alternative alleles of genes coding for proteins essential for cell viability or cell growth and the loss of one of these alleles in cancer cells due to loss of heterozygosity (LOH) and (2) the development of inhibitors with high specificity for the single remaining alternative allele of the essential gene retained by the tumor cell after LOH. Particular categories of appropriate target genes are described, along with specific exemplary genes within those categories and methods of using such target genes.

Description

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WO 98/.11648 PCT/US98/OS419 TARGET GENES FOR ALLELE-SPECIFIC DRUGS
BACKGROUND OF THE INVENTION
This invention is concerned with the field of treatment of proliferative disorders, including malignant and nonmalignant diseases, and with transplantation.
Specifically, this invention is concerned with target genes for drugs that are useful for treating such diseases by providing allele-specific inhibition of essential cell functions.
The following information is provided to assist the understanding of the reader, none of that information is admitted to be prior art to the present invention.
The treatment of cancer is one of the most heavily investigated areas in biomedical research today. Although many anticancer drugs have been and continue to be discovered, there remains the immense problem of developing drugs that will be specifically toxic to cancer cells without killing normal cells and causing toxic, often permanent, damage to vital organs or even death. One common measure of the clinical usefulness of any anticancer drugs is its therapeutic index: the ratio of the median lethal dose (LD50) to the median effective dose (ED50) of the drug.
With some cancer therapeutics this ratio is in the range of 4-6, or even 2-4, indicating a high risk of toxic side effects to the patient. Indeed, most anticancer drugs are associated with a high incidence of adverse drug events. The poor therapeutic index of most anticancer drugs not only limits the clinical efficacy of these drugs for the treatment of cancer, but limits their usefulness for treating many non-malignant, proliferative disorders.
A strategy for the development of anticancer agents having a high therapeutic SUBSTITUTE SHEET (RULE 26) index is described in Housman, International Application PCT/US/94 08473 and Housman, INHIBITORS OF ALTERNATIVE ALLELES OF GENES
ENCODING PROTEINS VITAL FOR CELL VIABILITY OR CELL GROWTH
AS A BASIS FOR CANCER THERAPEUTIC AGENTS, U.S. Patent 5,702,890, S issued December 30, 1997, which are hereby incorporated by reference in their entireties. As further described below, the method involves the identification of genes essential to cell growth or viability which are present in two or more allelic forms in normal somatic cells of a cancer patient and which undergo loss of heterozygosity in a cancer. Treatment of a cancer in an individual who is IO heterozygous with an allele specific inhibitor targeted to the single allele of an essential gene which is present in a cancer will inhibit the growth of the cancer cells. In contrast, the alternative allele present in non-cancerous cells (which have not undergone loss of heterozygosity) is able to express active product which supplies the essential gene function, so that the normal cells can survive and/or 15 grow.
Cancer cells from an individual almost invariably undergo a loss of genetic material (DNA) when compared to normal cells. Frequently, this deletion of genetic material includes the loss of one of the two alleles of genes for which the normal somatic cells of the same individual are heterozygous, meaning that there 20 are differences in the sequence of the gene on each of the parental chromosomes.
The loss of one allele in the cancer cells is referred to as "loss of heterozygosity"
(LOH). Recognizing that almost all, if not all, varieties of cancer undergo LOH, and that regions of DNA loss are often quite extensive, the genetic content of deleted regions in cancer cells was evaluated and it was found that genes essential 25 for cell viability or cell growth are frequently deleted, reducing the cancer cell to only one copy. In this context, the term "deleted" refers to the loss of one of two copies of a chromosome or sub-chromosomal segment. Further investigation demonstrated that the loss of genetic material from cancer cells sometimes results SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98I05419 in the selective loss of one of two alleles of a certain essential gene at a particular locus or loci on a particular chromosome.
Based on this analysis, a therapeutic strategy for the treatment of cancer was developed, which will produce agents characterized by a high therapeutic index.
The strategy includes: (1) identification of genes that are essential (or conditionally essential) for cell survival or growth; (2) identification of common alternative alleles of these genes; (3) identification of the absence of one of these alleles in cancer cells due to LOH and (4) development of specific inhibitors of the single remaining allele of the essential gene retained by the cancer cell, but not the alternative allele.
SUMMARY OF THE INVENTION
The utilization of inhibitors of alternative alleles, such as in the strategy described in Housman, supra, requires the provision of suitable target genes in order to identify such inhibitors and to implement corresponding diagnostic or therapeutic methods. Thus, as described below, the present invention identifies useful groups of genes which provide suitable target genes and further provides exemplary genes within those groups.
Additionally, the present inventors determined that LOH occurs not only in cancers, but also in non-cancerous proliferative disorders, though the location and frequency of LOH differs in different diseases, and established a method by which such non-cancerous proliferative disorders can be treated. Noncancer proliferative disorders include, for example, atherosclerotic plaques, premalignant metaplastic or dysplastic lesions, benign tumors, endometriosis, and polycystic kidney disease.
In each disease, the administration of such an inhibitor would have cytotoxic or antiproliferative effects on the abnormally proliferating cells that exhibited LOH
and contained only the sensitive allele of the target gene, but would not be toxic to SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 normal cells that contain also the alternative allele.
In addition, it was found that specific inhibitors of alternative alleles of an essential gene would be useful in managing transplantation in instances where the alleles in a donor bone marrow differ from the alleles in the recipient. For example, administration of an inhibitor of an allele that was present in a donor bone marrow but not the recipient could be used to treat graft-versus-host disease, suppressing proliferation of the donor marrow without toxicity to the recipient.
Alternatively, an inhibitor of an allele that is present in the recipient but not the donor bone marrow could be used to enhance engraftment by preferentially creating space in the recipient bone marrow for the graft without inhibiting proliferation of the engrafted donor marrow.
In this context, a "gene" is a sequence of DNA present in a cell that directs the expression of a "biologically active" molecule or "gene product" , most commonly by transcription to produce RNA ("RNA transcript") and translation to produce protein ("protein product"). Both RNA and protein may undergo secondary modifications such as those induced by reacting with other constituents of the cell which are also recognized as gene products. The gene product is most commonly a RNA molecule or protein, or a RNA or protein that is subsequently modified by reacting with, or combining with, other constituents of the cell. Such modifications may result, for example, in the modification of proteins to form glycoproteins, lipoproteins, and phosphoproteins, or other modifications known in the art. RNA may be modified by complexing with proteins, polyadenylation, or splicing. The term "gene product" refers to any product directly resulting from transcription of a gene. In particular this includes partial, precursor, and mature transcription products (i. e. , RNA), and translation products with or without further processing, such as lipidation, phosphorylation, glycosylation, or combinations of such processing (i. e. , polypeptides).
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/0~419 The term "target gene" refers to a gene where the gene, its RNA transcript, or its protein product are specifically inhibited or potentially inhibited by a drug.
In references herein to genes or alleles, the term "encoding" refers to the entire gene sequence, including both coding and non-coding sequences unless clearly indicated 5 otherwise.
The term "allele" refers to one specific form of a gene within a cell or within a population, the specific form differing from other forms of the same gene in the sequence of at least one, and frequently more than one, variant sites within the sequence of the gene. The sequences at these variant sites that differ between different alleles are termed "variances", "polymorphisms", or "mutations". The term "alternative allele", "alternative form", or "allelic form" refers to an allele that can be distinguished from other alleles by having distinct variances at at least one, and frequently more than one, variant site within the gene sequence.
It is recognized in the art that variances occur in the human genome at approximately one in every 100-500 bases. At most variant sites there are only two alternative variances, wherein the variances involve the substitution of one base for another or the insertion/deletion of a short gene sequence. Within a gene there may be several variant sites. Alternative alleles can be distinguished by the presence of alternative variances at a single variant site, or a combination of several different variances at different sites. In this invention, inhibitors targeted to a specific allelic form or subset of the allelic forms of a gene can be targeted to a specific variance in a selected variant site, or to an allele comprised of a set of variances at different sites: In most but not all cases, the target specificity is based on a nucleotide or amino acid change at a single variance site.
The term "proliferative disorder" refers to various cancers and disorders characterized by abnormal growth of somatic cells leading to an abnormal mass of SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98105419 tissue which exhibits abnormal proliferation, and consequently, the growth of which exceeds and is uncoordinated with that of the normal tissues. The abnormal mass of cells is referred to as a "tumor", where the term tumor can include both localized cell masses and dispersed cells, The term "cancer" refers to a neoplastic growth and is synonymous with the terms "malignancy", or "malignant tumor".
The treatment of cancers and the identification of anticancer agents is the concern of particularly preferred embodiments of the aspects of the present invention.
Other abnormal proliferative diseases include "nonmalignant tumors", and "dysplastic" conditions including, but not limited to, leiomyomas, endometriosis, benign prostate hypertrophy, atherosclerotic plagues, and dysplastic epithelium of lung, breast, cervix, or other tissues. Drugs used in treating cancer and other non-cancer proliferative disorders commonly aim to inhibit the proliferation of cells and are commonly referred to as antiproliferative agents.
"Loss of heterozygosity", "LOH", or "allele loss" refers to the loss of one of the alleles of a gene from a cell or cell lineage previously having two alleles of that gene. Normal cells contain two copies of each gene, one inherited from each parent. When these two genes differ in their gene sequence, the cell is said to be "heterozygous". The term heterozygous indicates that a cell contains two different allelic forms of a particular gene and thus indicates that the allelic forms differ at at least one sequence variance site. When one allele is lost in a cell, that cell and its progeny cells, comprising its cell lineage, become "hemizygous"
for that gene or "partially hemizygous" for a set of genes, and heterozygosity is lost.
LOH occurs in all cancers and is a common characteristic of non-malignant, proliferative disorders. In general, many different genes wilt be affected by loss of heterozygosity in a cell which undergoes loss of heterozygosity. In many cancers 10-40 % of all of the genes in the human genome (there are estimated to be 60,000-100,000 different genes in the genome) will exhibit LOH. In the context of this invention, these terms refer preferably to loss of heterozygosity of a gene SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTIUS98/05419 which has a particular sequence variance in normal somatic cells of an individual such that there is loss of heterozygosity with respect to that particular sequence variance. Also preferably, these terms refer to loss of heterozygosity of a particular sequence variance that is recognized by an inhibitor that will inhibit one allele of the gene present in normal cells of the individual, but not an alternative allele.
Preferably, loss of heterozygosity occurs before clonal or oligoclonal expansion of cells associated with a condition or disease, for example, cancer or non-cancer proliferative disorder. Cancer is a "clonal" disorder, meaning that all of the cells in the cancer or tumor are the progeny, or lineage, of a single cell which undergoes malignant transformation. Since cancer is cIonal, any loss of heterozygosity or allele loss that occurs during the process of malignant transformation will be uniformly present throughout the lineage of the initial transformed cell. This results in the cancer cells uniformly and consistently having only one allelic form of the gene which is present in two allelic forms in normal cells.
Some of the non-malignant proliferative conditions that exhibit LOH are "oligoclonal", meaning that unlike cancers and most benign tumors, there are multiple, independently arising clonal populations, with discrete LOH events in each of the individual clones. The alleles subject to LOH may vary from one clone to another. Therefore treatment of these conditions preferably utilizes inhibitors of at least two allelic forms. Thus, methods relating to such disorders can utilize alternative alleles of one gene and/or allelic forms of additional genes.
Certain noncancer, proliferative disorders are considered to be precursors for cancer. Such disorders progressively exhibit LOH until a single cell within the lesion caused by abnormal proliferation undergoes transformation and clonal expansion to form a cancer. Because LOH occurs in the precancerous condition, SUBSTITUTE SHEET (RULE 26) the present invention provides a method for preventing cancer by administering drugs that are selectively toxic to cells in which LOH involving a gene that is essential for cell survival or proliferation creates a genetic difference between cancer cells and normal cells. Since certain cancers are predictably associated with a high frequency of LOH in certain locations, for example segments of chromosomes 7,8,10,11,13,16, and 18 in prostate cancer, administration of an allele-specific drug that inhibits one allele that is within such a region, in a patient who is heterozygous for alternative forms of the gene, would kill cells that undergo LOH before cancer occurs. Preferably, in the context of this invention, LOH refers to loss of an allelic form of an essential gene in cells that are involved in cancer or noncancer proliferative disorders, which has sequence variants in a population of interest, in an individual whose normal somatic cells are heterozygous for sequence variants of that gene.
As pointed out above, an important aspect of methods for treating cancer or noncancer proliferative disorders utilizing LOH of essential genes is the identification of suitable essential genes for use as target genes. In accord with that requirement, this invention identifies certain useful groups or categories of essential genes, and provides, as examples, specific genes within those categories which are found to be suitable as targets for allele specific inhibitors, in particular for killing cancer cells or reducing the proliferation of cells in cancer or noncancer proliferative disorders. Thus, the present invention provides suitable target genes and methods of utilizing those genes in allele specific or variance specific targeting. Such targets are essential genes, which can include conditionally essential genes. As further described below, suitable target genes include those essential genes which encode gene products necessary for maintaining the level of a cellular constituent within the levels required for cell survival or proliferation, or which encode a gene product required for cell proliferation. If the level of activity of an essential gene product is reduced, the level of the corresponding cellular SUBSTITUTE SHEET (RULE 2fi) WO 98/.11648 PCT/US98/05419 constituent will not be properly maintained or the cell will be unable to perform the cellular functions required for cell proliferation. Confirmation that such a gene undergoes LOH in a neopiastic condition, e. g. , a cancer, and that there are at least two alleles of the gene in the population that differ in one or more variant positions, indicates that the gene is a useful potential target gene in this invention for the identification of allele specific inhibitors and in other aspects of the invention.
Certain useful groups of target genes are described in which the essential genes have been grouped according to the type of essential cellular function in which the gene products are involved. Thus, the gene product of each of the individual genes within each of the categories or subcategories is itself essential to the cell.
In particular, the categories of genes, or cell functions shown in Table 1(in the Detailed Description below) provide appropriate target genes. Particular exemplary target genes are also identified in Tables 1 and 2 and the Examples (including a GenBank accession number (or other sequence identifier as recognized by those skilled in the art) identifying the gene and providing a known sequence) which can be used for identifying allele specific inhibitors and for use in other aspects of this invention. Preferably the gene has the LOH frequency and at least one sequence variance in the gene has a heterozygosity rate in a population as indicated as preferable below, and occurs at only a single locus in the human genome.
An "essential" gene or gene product is one which is crucial to cell growth or viability. The terms "essential", "vital for cell viability or growth", or "essential for cell survival and proliferation" have the same meaning. A gene is essential if inhibition of the function of such a gene or gene product will kill the cell or inhibit its growth as determined by methods known in the art. Growth inhibition can be monitored as a reduction or preferably a cessation of cell proliferation.
SUBSTITUTE SHEET (RULE 26) WO 98/x1648 PCTlUS98/05419 Essentially can be demonstrated in a variety of different ways known in the art.
Examples include, among others, generation of growth conditional mutants and identification of the affected genes, replacement of active genes with inactive mutants, cell fusion gene complementation analysis (see, e. g. , John Wasmuth, 5 "Chinese Hamster Cell Protein Synthesis Mutants", Ch. 14 in Molecular Cell enetics, Michael Gottesman, ed. Wiley, New York, 1985), and insertion of genetic suppressor elements leading to growth arrest (Pestov & Lau, 1994, Proc.
Natl. Acad. Sci. USA 91:12549-12533). Other ways include the identification of conditionally lethal mutants, e. g. , temperature sensitive mutants and determination 10 of the affected gene, genetic disruption of the gene by homologous recombination or other methods in organisms ranging from yeast to mice, inhibition of the gene by antisense oligonucleotides or ribozymes, and identification of the target of known cytotoxic drugs and other inhibitors. As further discussed below, the essentiality of a gene can depend on the conditions to which the cell is exposed.
Thus, unless otherwise indicated, the term "essential gene" includes both "generally essential genes" and "conditionally essential genes" . "Generally essential genes" are those which are strictly essential for cell survival or growth, or which are essential under the conditions to which the cell is normally exposed.
Typically such conditions are the normal in vivo conditions or in vitro conditions which approximately replicate those in vivo conditions. Thus, in the methods described here utilizing essential genes, the method is carried out in conditions such that the gene product is required.
In connection with the determination of gene essentiality, it is generally recognized that the demonstration of essentiality of a gene in one organism is strongly suggestive that the homologous gene will be essential in another organism.
This is especially true for genes which have relatively high levels of sequence conservation across a broad range of organisms. Thus, the identification of essential genes in prokaryotes or in lower eukaryotes such as yeast is indicative of SUBSTITUTE SHEET (RULE 26) the identification of corresponding homologous essential genes or gene classes in higher eukaryotes such as humans. Therefore, studies of essential genes for non-human organisms provides useful information on likely human essential genes;
an example is the Stanford Saccharomyces cerevisiae Database: http://genome-WWW
Stanford.edulcgr-bin/dbrun/SacchDB which provides a catalog of essential genes in yeast. It should be recognized, however, that not all essential genes from lower organisms will have recognized homologues in humans. It should also be recognized that the essential genes for a particular organism will generally not be restricted to those for which homology can be shown to essential genes in other organisms. Thus, genes may be essential in humans that are not essential in lower organisms.
In addition to generally essential genes, it is also recognized in the art that environmental factors can cause certain genes to be essential that are not essential under other conditions (including usual culture conditions). For example, certain genes involved in intermediary metabolism are not essential if the cell or organism is supplemented with high concentrations of a particular nutrient or chemical entity, but if that nutrient or chemical entity is absent or present at low levels, the gene product is essential. In another example, the administration of a drug that inhibits one or more functions within the cell can cause other functions to be essential that are not essential in the absence of the drug. In another example, subjecting a cell to harsh physical agents, such as radiation, can cause certain genes to be essential that are not essential under normal conditions. Such genes are essential under certain conditions associated with the therapy of cancer.
The demonstration that such genes are present in the population in more than one allelic form and are subjected to loss of heterozygosity in cancer or noncancer proliferative disorders makes such genes targets for allele specific drugs for the treatment of such disorders.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98I05419 Thus, a gene is said to be "conditionally essential" if it is essential for cell survival or proliferation in a specific environmental condition caused by the presence or absence of specific environmental constituents, pharmaceutical agents, including small molecules or biologicals, or physical factors such as radiation.
S The term "cellular constituent" refers to chemical entities that comprise the substance of a living cell. In preferred embodiments, the cellular constituent is a protein or modified protein. Also, in preferred embodiments, the cellular constituent is an inorganic ion, an organic compound such as a lipid, carbohydrate, amino acid, organic acid, nucleoside, DNA, or RNA, or modified form of the preceding formed by the reaction of two constituents of the cell.
In another embodiment, the constituent may comprise a structural element of the cell such as a membrane or cytoskeleton. In the preferred embodiment of this invention, cellular constituent refers to chemical entities, including compounds but also including simple ions, which are required for survival or proliferation of a human cell.
Certain cellular constituents of a cell are synthesized by the cell while others are not synthesized by the cell but are taken into the cell from its environment.
Within the cell, constituents engage in various reactions to form new constituents by intermediary metabolism, are modified to form new constituents, and are preferentially compartmentalized in particular structures within the cell including, but not limited to, the nucleus, mitochondria, cytoplasm, or vesicles. Certain constituents are also specifically eliminated by the cell, or specific compartments within the cell, by degradation or excretion. In connection with cellular constituents, the term "maintaining the level" refers to maintaining the amount of the chemical entity normally associated with a specific cellular compartment or compartments and involves the action of various cellular processes, including synthesis, production, compartmentalization, transport, modification, combining SUBSTITUTE SHEET (RULE 26) WO 98Lt1648 PCT/US98/05419 of two or more constituents, polymerization, elimination, degradation, and excretion. It is recognized in the art that the failure to maintain the level of certain cellular constituents within normal levels results in cell death, for example, cell death may result from inappropriate levels of proteins, DNA, or RNA, inappropriate levels of inorganic ions, inappropriate levels of organic compounds required for energy or other metabolic processes, or inappropriate intracellular structure. These examples are meant to be illustrative of the understanding of the meaning of the terms to those skilled in the art and not limiting.
In addition to the useful functional groups of essential genes described above, the present invention also provides useful groups of essential genes which are advantageous for allele specific targeting due to the genes undergoing LOH at certain frequencies in a disorder or other conditions and/or by having at least two allelic forms of the gene which appear in the population at particularly useful frequencies.
Thus, it is found that essential genes which undergo LOH in at least 10 % of cases of a human cancer, and which exist in at least two allelic forms in a human population are advantageous targets. Preferably, the gene undergoes LOH in at least 20 % of cases of a disorder, more preferably in at least 30 % , still more preferably in at least 40 % , and most preferably in at least 50 % of such cases.
The LOH frequencies for a large number of different genetic markers for particular proliferative disorders are known in the art, and are used as indicators of the LOH frequency for neighboring essential genes. A number of LOH
markers are provided in Fig. 3 (Loss of Heterozygosity Table). In one aspect of this invention, those essential genes which are located within about 20 megabases, more preferably within about 10 megabases, and most preferably within about 5 megabases of an identified marker or tumor suppressor gene which undergoes SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US9810~419 LOH in at least 10, 20, 30, 40, or 50 % of cases of a proliferative disorder, are particularly useful as they will undergo LOH at similar frequencies as the marker gene.
The relative locations of a marker and an essential gene can also be described by S genetic, rather than physical, map distances, therefore, in preferred embodiments, an essential gene of this invention is preferably within about 20 centimorgans, more preferably within about 15 centimorgans, still more preferably within about centimorgans, and most preferably within about S centimorgans of such an LOH marker or tumor suppressor gene. In preferred embodiments, the target 10 gene is located near a reported marker which undergoes LOH at a frequency of at least 10, 20, 30, 40, or 50 % for a proliferative disorder. A number of such markers and the associated chromosomal locations are provided in Fig. 3. Even more preferably, essential genes which map to a locus bracketed by two such markers are appropriate potential target genes, as the essential gene very probably will also undergo LOH at similar high frequencies. Preferably both markers undergo LOH at frequencies of at least 10, 20, 30, 40, or 50% of cases of a cancer. Thus, confirmation that an essential gene, for example, a gene from one of the functional groups described above, or one of the particular exemplary genes, maps close to a marker as just described, indicates that the gene is an appropriate potential target. Identification of one or more sequence variances in that gene and/or in the corresponding gene products allows screening or design of such inhibitors for potential treatment.
A useful way to determine the frequency of loss of heterozygosity for a tumor cell based on the physical position of the gene on chromosomes within the human genome has been described by Vogelstein et al., 1989, Science 244:207-211.
These authors describe a measure of allele loss termed Fractional Allele Loss (FAL) which quantifies the extent of LOH in cancer based on LOH determinations SUBSTITUTE SHEET (RULE 26) over each informative chromosomal arm. FAL is determined by dividing the number of informative chromosomal arms which undergo LOH by the total number of informative chromosomal arms, i. e. , each chromosome/arm with at least one heterozygous locus in normal cells. Examples of such FAL
S determinations are provided by Vogelstein et al., 1989 (FAL= 0.20 in colon cancer), and Cliby et al., 1993, Cancer Research 53:2393-2398 (FAL= 0.17 for low grade ovarian cancers, 0.40 for high grade ovarian cancers, 0.35 for ail ovarian cancers).
These data indicate that genes on the chromosomal segment or 10 chromosomal arm that is commonly lost in a cancer or non-cancer proiiferative disorder are potential target genes. In preferred embodiments, the target gene is located on a chromosomal arm which is reported in the art or shown herein to contain a locus or loci which undergoes LOH at a frequency of at least 15, preferably at least 20%, still more preferably at least 25 % , and most preferably at 15 least 30, 40, or SO% in a proliferative disorder. As noted above, the frequency of LOH for a chromosomal arm is often utilized in calculating an average fraction of allele loss (FAL). Thus, a high LOH frequency for an arm or portion of an arm indicates that particular genes in the relevant chromosomal region will also undergo LOH at a comparable frequency, and thus define useful target genes.
Preferably the target genes are those which are located on particular chromosomal arms which commonly undergo tumor-related LOH. In particular, these human chromosomal arms include lp, lq, 3p, Sq, 6p, 6q, 7q, 8p, 9p, 9q, lOq, llp, llq, 13q, 16q, 17p, 17q, 18p, 18q, and 22q. It is recognized that the LOH frequency is not uniform for all positions along an arm of a particular chromosome, however such LOH frequencies provide a strong indicator for LOH frequency at a potential target gene. Thus, mapping of an essential gene to these chromosomal arms or to high frequency LOH regions on these arms indicates that the gene is a potential target. Confirmation of the LOH of the particular gene and of the presence of at SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 least one sequence variance, and therefore of individuals heterozygous for such variances, indicates that the gene can be used for the identification of inhibitors targeting allelic forms of the gene which have a particular variance or variances and in the other aspects of this invention.
The term "high frequency LOH chromosomal region" refers to a chromosomal region which undergoes LOH at a frequency as indicated above, and include high frequency LOH chromosomal arms (at least 15 % FAL), regions within the genetic or physical map distances indicated above of a chromosomal marker or tumor suppressor gene which undergoes LOH at a frequency as indicated above (at least 10%).
In connection with the location of a potential target gene with respect to a marker or tumor suppressor gene, the term "proximity" means that the target gene is located within a genetic or physical map distance of the reference gene or marker as stated above.
The present invention is aimed, in part, at treating cancer or proliferative disorders of any type in which LOH of an essential gene occurs at a frequency as indicated above. For example, this includes but is not limited to cancers and noncancer proliferative disorders provided in Tables 2 and 3 and Figure 3, or otherwise described herein. Table 2 and Fig. 3 describe a number of cancers for which LOH at substantial frequencies has been described in the art. Therefore, identification of an essential gene which maps to the LOH regions for a particular proliferative disorder, as described by genetic or physical mapping or by residence on a chromosomal arm or smaller region of an arm which is shown to undergo LOH, at high frequency in a proliferative disorder, identifies a potential target gene. Identification of sequence variances in that gene, such that normal somatic cells of individuals in a population are heterozygous for a variance and thus SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98I05419 contain two different alleles, confirms that the gene is a potential target.
The target gene, its RNA transcript or protein product can then be used as targets for allele-specific inhibitors for treating the proliferative disorder or other uses as described in the aspects of this invention.
A further indication of useful target genes is provided by tumor-specific LOH
of essential genes associated with tumor suppressor genes. LOH in certain cancers or noncancer proliferative disorders is frequently associated with specific chromosomal arms. This association is believed to be due, in many cases, to the presence of tumor suppressor genes located on those particular chromosomal arms, the loss of which eliminates the tumor suppressor function and contributes to the transformation of the cell. Consequently, essential genes which map near such a tumor suppressor gene are potential target genes for this invention.
Preferably, the essential gene maps within a physical or genetic map distance as described above for LOH markers. As for the above categorization aspect, the LOH for a particular gene preferably is at least 10, 20, 30, 40, or 50% for a tumor, such as the cancers and types of cancers identified in Tables 2 and 3 and in Fig. 3. It should be noted that tumor suppressor genes themselves are rarely essential for cell survival or proliferation and not likely to be preferred targets for this invention.
Another group of essential genes which are potentially useful as target genes are those which are present in the population in at least two alternative forms or alleles containing one or more sequence variations, where the alternate forms occur at frequencies such that at least 10 % of a population is heterozygous (i. e. , have two alternative forms of the gene), preferably so that at least 20% , more preferably at least 30% , and most preferably at least 40% are heterozygous.
The term "heterozygote frequency" refers to the fraction of individuals in a population who have two alternative forms of a gene, or particular variances within a gene, in SUBSTITUTE SHEET (RULE 26) WO 98/.ti648 PCT/US98/05419 their normal, somatic cells and are therefore heterozygous.
The term "allele frequency" refers to the fraction (or frequency of occurrence) of a specific allele as compared to all alleles in a population. It is recognized in the art that the heterozygote frequency and allele frequency are related and, for certain alleles, can be described by Hardy Weinberg equilibrium calculations. It will also be recognized that sequence variances that occur at high frequency in the population are commonly not deleterious to the health of the individuals who carry these genes and are commonly not disease genes or mutations that are associated with disease.
Methods for determining the heterozygote frequency or allele frequency or determining the number of individuals who are heterozygous for specific variances are known in the art, including but not limited to methods such as restriction fragment length polymorphism, hybridization of sequence specific nucleic acid probes to DNA or RNA sequences which include a sequence variance site, DNA
sequencing, or mass spectrometry of amplified sequence fragments containing a sequence variance site. Methods that are useful for the discovery of genetic variances can also be used including, but not limited to, methods such as methods such as the SSCP technique (see Example 28), Enzymatic Mutation Detection technique (see Example 29), Denaturing Gradient Gel Electrophoresis, or sequencing. identification of such genes which have sequence variances that are common in the general population and for which 10 % , 20 % , 30 % , or 50 % of the population are heterozygous for that gene provides genes which are particularly likely to be useful target genes for allele specific inhibition in this invention.
Confirmation that the gene undergoes LOH at a useful frequency in a proliferative disorder, preferably in at least 10, 20, 30, 40, or 50% of cases of such a disorder indicates that the gene is useful as a potential target for identifying allele specific inhibitors for the treatment of proliferative disorders and in other aspects of this SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 invention.
Exemplary genes described herein are shown to contain numerous sequence variances which are present in human populations. While some sequence variances and alleles are common throughout diverse human populations, it is recognized in the art that the allele frequency of different genes will vary in different populations. For example, allele frequencies have been shown to differ between populations comprised of individuals of different races, populations comprised of individuals from different countries, populations comprised of individuals from different regions, populations comprised of individuals with common ethnic background, and even populations comprised of individuals from different religions. Alleles that are common in one population, may be rare in another. While the allele frequency of any particular gene may vary in different populations, the genes that are described below are those that occur such that at least 1 % or 5 % of a population is heterozygous for the sequence variance, preferably so that at least 10 % or 20 % , more preferably at least 30 % , and most preferably at least 40 % are heterozygous in a specific population that may be treated with inhibitors to treat cancer or other proliferative disorder in that population. Once a specific variance is identified in a certain gene, the allele frequency in any specific population can be easily determined using methods known in the art including the use of allele-specific hybridization probes, sequencing, or specific PCR reactions.
In this regard, "population" refers to a geographically, ethnically, or culturally defined group of individuals, or a group of individuals with a particular disease or a group of individuals that have proliferative diseases that may be treated by the present invention. Thus, in most cases a population will preferably encompass at least ten thousand, one hundred thousand, one million, ten million, or more individuals, with the larger numbers being more preferable. In special SUBSTITUTE SHEET (RULE 26) WO 98/41648 PCTlUS98/05419 circumstances, diseases will occur with high frequency in specific geographical regions or within specific familial, racial, or cultural groups, and a relevant population may usefully be considered to be a smaller group.
In the context of this invention, an alternative allele, or other reference to an 5 appropriate target for the inhibitors of this invention refers to a form of a gene which differs in base sequence from at least one other allele or allelic form of the same gene. Usually, though not necessarily, the allelic forms of a gene will differ by, at most, several bases and may have only a single base difference (i.e., a single sequence variance). The allelic forms, however, are ones which contain at 10 least one sequence variance which appears in somatic cells of a population at an appreciable frequency, such that preferably at least 1 % , more preferably at least 5 % , still more preferably at least 10 % , and most preferably at least 20 %
of the population are heterozygous for that specific sequence variance. This advantageously allows the convenient identification of potential patients, because 15 an appreciable fraction of the population, and therefore also of the cancer patients will be heterozygous for sequence variances of the specific gene. In the context of this invention, different alleles need not result in different observable phenotypes under normal conditions. Preferably, a particular sequence variance produces no phenotypic effect on the physical condition of an individual having that variance 20 until the variance is targeted by an allele specific inhibitor.
In connection with allele specific inhibitors and the methods of this invention, the terms "allelic form" or "alternative form of the target gene" or "sequence variance within the target gene" refer to either or both of the gene or a product of that gene including the RNA transcript or protein product. Thus, a particular inhibitor may act in an allele specific manner (which will often be variance specific) at any of those levels and preferably the inhibitor is targeted to a particular sequence variance of the specific allelic form.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 As indicated above, two different allelic forms of a gene will have at least a one nucleotide difference in the nucleotide sequence of the gene. The difference can be of a variety of different types, including base substitution, single nucleotide insertion or deletion, multiple nucleotide insertion or deletion, and combinations of such differences. Thus, two allelic forms are sequence variants and will have at least one sequence variance, which refers to the sequence difference, between the allelic forms. However, there may also be more than one sequence variance between two allelic forms. The location of a sequence variance in a gene sequence is a "sequence variance site." This description applies to both the DNA and RNA
sequences, and similarly applies to a polypeptide sequence encoded by the gene, differences in the amino acid sequence of the polypeptide, and the location in the polypeptide chain of the sequence differences. As a particular gene may have more than one sequence variance site, more than two allelic forms may exist in a population, for example, see Fig. 1 for exemplary target summaries showing multiple sequence variance sites.
Sequence variances can involve a difference in the sequence in which any of the four bases: adenine, guanine, thymidine (uracil in the context of RNA), or cytosine are substituted with another of the four bases or a change in the length of the sequence. Different classes of variances are recognized in the art.
"Deletions" are variances in which one or more bases are missing from the sequence. "Insertions" are variances in which one or more bases are inserted into the sequence. It will be evident that the terms deletion and insertion refer to the variance in one sequence relative to another. "Transitions" are variances that involve substitution of one purine for the other or one pyrimidine for the other.
"Transversions" are variances that involve substitution of a purine for a pyrimidine or a pyrimidine for a purine. Certain sequence variances can interfere with the normal function of the gene or its gene product and can be associated with disease; such variances are commonly referred to as mutations. Most SUBSTITUTE SHEET (RULE 26) variances present in human populations are not associated with disease and are "normal" variants of the gene; such variances are commonly referred to as polymorphisms. In the present invention, specific variances are described from each of the classes described above in genes that are essential for cell survival or proliferation that can be the targets for allele-specific inhibitors for the treatment of cancer or noncancer proliferative disorders.
This invention provides inhibitors which are specific for at least one, but not all, allelic forms of a gene that encodes a gene product essential to cell growth or cell viability, for genes belonging to the specified categories of genes. The inhibitor may be active on the gene or gene product including the RNA transcript, protein product, or modifications thereof. Exposure to the inhibitor inhibits proliferation or kills cells which have undergone LOH of genes that are not inhibited by the drug and contain only an allelic form of the essential gene, its RNA
transcript, or its protein product against which the inhibitor is targeted. Normal cells which contain two alternative alleles of the target genes, one of which is not inhibited by the specific inhibitor, are spared from the toxic effects of the inhibitor because the remaining activity of the allele which is not inhibited by the inhibitor is adequate to permit continued cell viability and growth. This differential effect of the inhibitor on cells with LOH of a targeted gene (e. g. , a cancer cell) and normal cells accounts for the high therapeutic index of the inhibitors of this invention for the treatment of cancer or non-cancerous, proliferative disorders characterized by LOH. Toxicity of the inhibitor to normal cells is therefore low, compared to most currently available anticancer and antiproliferative agents.
Thus, in accord with the strategy and target genes indicated above and described in the Detailed Description of the Preferred Embodiments, in a first aspect the invention provides methods for identifying inhibitors potentially useful for treatment of a proliferative disorder, e. g. , cancer. Such inhibitors are active on SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 specific allelic forms of target genes as identified herein. The method involves determining at least two allelic forms of such a gene encoding an essential gene product, and testing a potential allele specific inhibitor to determine whether the potential inhibitor is active on, e. g. , inhibits expression of, at least one of the allelic forms, but not all of those forms. If the potential inhibitor inhibits only a subset of the allelic forms of the particular essential gene, then it is an allele specific inhibitor. Preferably the difference in activity of the inhibitor for different allelic forms is between allelic forms which have a sequence variance at a particular site.
In many, or even most, cases an allele specific inhibitor discriminates between two allelic forms due to a particular single sequence variance between the allelic forms of the target gene. For example, ribozymes which target a single sequence variance site will preferentially cleave only one of the sequence variants for a particular single nucleotide variance. In this case, sequence variances at other sites will generally not affect the cleavage. In the Detailed Description of the Invention specific examples of proteins, small molecules, and oligonucleotides providing allele specific inhibition based on single sequence variances are described. Thus, in preferred embodiments an allele specific inhibitor discriminates between two allelic forms by discriminating a single sequence variance. As previously indicated, inhibitors can be targeted to either the nucleic acid or a polypeptide (where a nucleotide change results in an amino acid change).
In particular embodiments, the allele specific inhibitor will recognize more than one linked sequence variances within a specific allele.
An "allele specific inhibitor" or "variance specific inhibitor" is a drug or inhibitor that inhibits the activity of one alternative allele of a gene to a greater degree than at least one other alternative allele. The difference in activity is commonly determined by the dose or level of a drug required to achieve a quantitative degree SUBSTITUTE SHEET (RULE 26) WO 981.11648 PCT/US98/0~419 of inhibition. A commonly used measure of activity is the IC50 or concentration of the drug required to achieve a 50 % reduction in the measured activity of the target gene. Preferably an allele specific inhibitor will have at least twice the activity on the target allelic form than on a non-target allelic form, more preferably at least 5 times, still more preferably at least 10 times, and still more preferably at least 50 times, and most preferably at least 100 times. This can also be expressed as the sensitivities of the different allelic forms to the inhibitor.
Thus, for example, it is equivalent to state that the target allelic form is most preferably at least 100 times as sensitive to the inhibitor as a non-target allelic form. The activity of an inhibitor can be measured either in vitro or in vivo, in assay systems that reconstitute the in vivo system, or in systems incorporating selected elements of the complete biological system. For use in inhibiting cells containing only the target allelic form rather than cells containing at least one non-targeted allelic form, the difference in activity is preferably sufficient to reduce the proliferation rate or survival rate of the cells having only the targeted allelic form to no more than one half of the proliferation rate or survival rate of cells having at least one non-targeted allelic form. More preferably, the fraction is no more than 1/5 or 1/10, and still more preferably no more than I/20, 1/50, 1/100, or even lower.
In a related aspect, the invention provides inhibitors potentially useful for tumor, e. g. , cancer treatment, or treatment of other proliferative disorders. Such inhibitors are active on a specif c allele of a gene which has at least two different alleles encoding an essential gene product in one of the target gene categories above. Such inhibitors can, for example, be identified by the above screening methods.
In a related aspect, the invention provides methods for producing inhibitors active on such specific allelic forms of belonging to one of the above categories genes by SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTlUS98/05419 identifying a gene encoding an essential gene product which has alternative allelic forms in a non-tumor cell and which undergoes LOH in a tumor cell, screening to identify an inhibitor which is active on at least one but less than all of the alleles of the gene, and synthesizing the inhibitor in an amount sufficient to produce a 5 therapeutic effect when administered to a patient suffering from a tumor in which tumor cells have only the allele on which the inhibitor is active.
In the context of this invention, the term "active on an allelic form" or "allele specific inhibitor" or "specifc for an allelic form" indicates that the relevant inhibitor inhibits an allele having a particular sequence to a greater extent 10 (preferably Z 2x) than an allele having a sequence which differs in a particular manner. Thus, for alleles for which a particular base position is identified, the inhibitor has a higher degree of inhibition when a certain base is in the specified position then when at least one different base is in that position. This means that for substitution at a particular base position, at least two of the possible allelic 15 forms differ in sensitivity to an inhibitor. Usually, however, for a specific sequence variance site, the site will be occupied by one of only two bases.
Further, if an inhibitor acts at the polypeptide level, and any of three bases may be present at a particular position in a coding sequence but only one of the substitutions results in an amino acid change, then the activity of the inhibitor 20 would be expected to be the same for the two forms producing the same amino acid sequence but different for the form having the different amino acid sequence.
Other types of examples can also occur.
The term "less active" indicates that the inhibitor will inhibit growth of or kill a cell containing only the allelic form of a gene on which the inhibitor is more active 25 at concentrations at which it does not significantly inhibit the growth of or kill a cell containing only an allelic form on which the inhibitor is less active.
SUBSTITUTE SHEET (RULE 26) The term "drug" or "inhibitor" refers to a compound or molecule which, when brought into contact with a gene, its RNA transcript, or its gene product which the compound inhibits, reduces the rate of a cellular process, reduces the level of a cellular constituent, or reduces the level of activity of a cellular component or process. This description is meant to be illustrative of the understanding of the meaning of the term to those skilled in the art and not limiting. Thus, the term generally indicates that a compound has an inhibitory effect on a cell or process, as understood by those skilled in the art. Examples of inhibitory effects are a reduction in expression of a gene product, reduction in the rate of catalytic activity of an enzyme, and reduction in the rate of formation or the amount of an essential cellular component. The blocking or reduction need not be complete, in most cases, for the inhibitor to have useful activity. Thus, in the present invention, "inhibitors" are targeted to genes, their RNA transcript, or their protein product that are essential for cell viability or proliferation. Such inhibitors would have the effect of inhibiting essential functions, leading to loss of cell viability or inhibition of cell proliferation. In preferred embodiments, such inhibitors cause cell death or stop cell proliferation. In preferred embodiments of this invention, inhibitors specifically include a molecule or compound capable of inhibiting one or more, but not all, alleles of genes, their RNA transcript, or their protein product that are essential for cell survival or proliferation. The terms "inhibitor of a gene"
or "inhibitor of an allele" as used herein include inhibitors acting on the level of the gene, its gene product, its RNA transcript, its protein product, or modifications thereof and is explicitly not limited to those inhibitors or drugs that work on the gene sequence itself.
Several types of inhibitors are generally recognized in the art. A
"competitive"
inhibitor is one that binds to the same site on the gene, its RNA transcript or gene product as a natural substrate or cofactor that is required for the action of the gene or gene product, and competitively prevents the binding of that substrate. An SUBSTfTUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 "allosteric" inhibitor is one that binds to a gene or gene product and alters the activity of the gene or gene product without preventing binding of a substrate or cofactor. Inhibition can also involve reducing the amount of the gene, RNA
transcript, or its protein product, and thus the total amount of activity from the gene in the cell. Such inhibition can occur by action at any of a large number of different process points, including for example by inhibiting transcription or translation, or by inducing the elimination of the gene, its RNA transcript, or its protein product where elimination may involve either degradation of the target or egress or export from the compartment in which it is active and the process of excretion or export. Inhibition can also be achieved by modifying the structure of the target, interfering with secondary modifications, or interfering with cofactors or other ancillary components which are required for its activity. Inhibitors can be comprised of small molecules or polymeric organic compounds including oligopeptides or oligonucleotides.
The term "active on a gene" or "targeted to a gene" indicates that an inhibitor exerts its inhibitory effect in a manner which is preferentially linked with the characteristic properties of a gene, its RNA transcript or its gene product.
Such properties include, for example, the nucleotide sequence of the gene or transcribed RNA, the amino acid sequence or post-translational modifications of the protein product, the structural conformation of a protein, or the configuration of a protein or RNA with other cellular constituents (RNA, protein, cofactors, substrates, etc.) required for activity. Thus, in general these terms indicate that the inhibitor acts on the gene, its RNA transcript, its protein product, its gene product, or modifications thereof, or on a reaction or reaction pathway necessarily involving such a gene product to a greater extent than on genes or gene products generally.
A "reduction of the level of activity" of a gene product or allele product refers to a decrease in the functional activity provided by that product. This can be due to SUBSTITUTE SHEET (RULE 26) any of a variety of direct causes, including for example, a reduction in the amount of a biologically active molecule present, a change in the structure or modifications of normally active molecules to produce inactive or less active molecules, blockage of a reaction in which the product participates, and blockage of a reaction pathway in which the product necessarily participates.
In another related aspect the invention provides methods for treating a patient suffering from a proliferative disorder in which an essential gene from one of the above categories has undergone loss of heterozygosity. The method involves administering a therapeutic amount of an allele specific inhibitor of such an essential gene to a patient whose normal somatic cells are heterozygous for that gene but whose tumor cells contain only a single allelic form of the gene. The inhibitor is active on the specific allele of the gene present in the tumor cells.
A "therapeutic effect" results, to some extent, in a measurable response in the treated disease or condition. Thus, a therapeutic effect can include a cure, or a lessening of the growth rate or size of a lesion such as a tumor, or an increase in the survival time of treated patients compared to controls, among other possible effects.
The term "therapeutic amount" means an amount which, when administered to a mammal, e. g. , a human, suffering from a disease or condition, produces a therapeutic effect.
In preferred embodiments of this treatment method, the method also involves determining whether the normal cells of the patient are heterozygous for the particular essential gene and determining whether tumor cells of the patient contain only a single allelic form of that gene. The determining may be performed on a variety of normal cells, such as blood or normal tissue, and on tumor cells.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTN598/0~419 Either or both of the normal cells and tumor cells may be cultured prior to the determination. The determination may also be carried out using cells retrieved from a frozen or preserved tissue specimen, e. g. , from pathological specimens of a patient's tumor and/or normal tissue preserved in a pathology laboratory.
Also, the determining may be performed using a variety of techniques, which may, for example include one of more of: hybridization with an allele specific oligonucleotide probe, hybridization to a gridded set of oligonucleotides, restriction fragment length polymorphism, denaturing gradient gel electrophoresis, heteroduplex analysis, single strand conformation polymorphism, ligase chain reaction, nucleotide sequencing, primer extension, dye quenching, sequence specific enzymatic or chemical cleavage, mass spectroscopy, and other methods known in the art.
In a related aspect, the invention provides a method for preventing the development of cancer. The method involves administering to a patient having a precancerous condition or an early stage cancer or cancers an allele specific inhibitor targeted to an allele of an essential gene for which the normal somatic cells of the patient are heterozygous and which has undergone LOH in cells involved in the precancerous condition. In a case where the cells of the precancerous condition are not clonal from a single cell, the method involves subsequently administering to the patient a second allele specific inhibitor in an amount su~cient to inhibit and preferably kill cells with LOH in which an allele not targeted by the first inhibitor is the only remaining allele of the gene.
In most cases, the second allele specific inhibitor will target the alternative allele of the gene targeted by the first inhibitor. However, the second inhibitor can also target an allele of a second essential gene which has undergone LOH. The second gene may have undergone LOH in the same deletion that affected the first gene due to their proximity on a chromosome, though this is not essential. Additionally, in other cases, allele specific inhibition of one of the alleles of each of 3, 4, or even SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 more target genes can be utilized in a serial manner (where the patient is heterozygous for each targeted gene). In this case the different target genes need not be tightly linked so that LOH of the various genes does not necessarily occur together. By using the serial inhibition of an allele of each of the target genes, it 5 is possible to inhibit and preferably kill the full population of precancerous cells in which LOH has occurred. Thus, the net effect is essentially the same as if allele specific inhibitors of each of the two alternative alleles of one essential gene had been used.
In the context of the administration of multiple allele specific inhibitors, the terms 10 "serial" or "subsequently" indicates that the administration of two or more inhibitors is sufficiently temporally separated so that normal somatic cells remain functional and are therefore able to survive and/or proliferate. Those skilled in the art will recognize that the required time will depend on various factors, such as clearance rate, type and extent of the effect of an inhibitor on normal cells, and 15 additive cellular toxicity, and that appropriate timing can be routinely determined for particular selections of compounds.
In another related aspect, the invention provides a method for identifying a potential patient for treatment with an inhibitor active on a specific allele of an essential gene from one of the above categories. The method involves identifying 20 a patient having a proliferative disorder characterized by LOH, e. g. , a cancer, whose normal somatic cells are heterozygous for the essential gene and determining whether tumor cells in the patient contain only a single allele of the gene. Thus, if the patient is normally heterozygous and the neoplastic cells contain only a single allele of the gene, then the patient is a potential patient for 25 treatment with the inhibitor.
With respect to identifying patients with precancerous or oligoclonal proliferative SUBSTfTUTE SHEET (RULE 26) WO 98/.ti648 PCT/US98/05419 diseases characterized by LOH, and selecting appropriate allele or variance-specific inhibitors for such patients, in some cases it may not be practical to obtain samples of all proliferative lesions for LOH assays.. For example, atherosclerotic plaques in the aorta cannot routinely be sampled by biopsy, and dysplastic lesions in the cervix, colon, or bronchus can be multifocal. Therefore, allele specific inhibitors can be selected for such conditions based on previously established patterns of LOH for the condition, and on specific testing for heterozygosity in a given patient. Characteristic patterns of LOH involving specific chromosomes or chromosomal regions have been reported in the art (by Vogelstein's group and others) for premalignant changes in the colon, such as adenomatous polyps, polyps with dysplasia and polyps with carcinoma in situ (pre-invasive cancer) (Fearon, E.
and B. Vogelstein). These studies demonstrate LOH on chromosomes Sq, 17p, and 18q in the earliest lesions. Similar studies have been performed for other premalignant conditions. It will be evident to one skilled in the art that similar studies can be readily performed on other conditions characterized by LOH
using retrospective analysis of tissue from pathological specimens. The optimal regions for allele or variance specific targeting will be those which are affected by LOH in a high fraction of lesions and in a high fraction of patients. Preferably, at least 40% of lesions will have LOH for a specific target gene, more preferably 60, 80, or 90 % , and most preferably 100 % . However, it is not necessary that 100 %
of lesions show LOH for a successful treatment by allele specific inhibitors because 2,3,4, or even more inhibitors can be used in a combined approach to target an ever higher fraction of lesions, and because substantial therapeutic benefit may be achieved by inhibiting the proliferation of less than 100% of lesions.
In a related aspect, the invention provides a method for treating a patient having a proliferative disorder, e. g. , suffering from a cancer. The patient's normal somatic cells are heterozygous for an essential gene from one of the above categories, but the patient's cancer cells, or other abnormally proliferating cells, SUBSTfTUTE SHEET (RULE 26) WO 98/.tl648 PCT/US98/0~419 have only a single allelic form of the gene. This method combines the identification and treatment methods described in the preceding aspects.
In another aspect, the invention provides a method for identifying a potential patient undergoing transplantation for treatment with an inhibitor active on a S specific allele of an essential gene from one of the above categories. The method involves identifying a patient undergoing an allogenic transplantation in which the tissue of the donor contains at Ieast one form of an essential gene that is different from those of the recipient. In a preferred aspect of this invention the donor or recipient is homozygous for an alternative form of an essential gene that differs from those present in the other. The term "homozygous" means that the two alleles of a gene present in somatic cells contain the same allele or alleles with identical sequence at at least one variant position that determines the activity of an allele specific drug. Such identification then allows methods of treating such patients by targeting the differing variances or allelic forms.
The term "allogenic" transplantation refers to transplantation of a tissue or cell fro the same species which contains different surface antigens than the recipient.
In contrast, an "autologous" transplantation is one in which the patient receives their own tissues (commonly bone marrow) that contain identical surface antigens.
The surface antigens are commonly those referred to as "histocompatibility"
antigens or "HLA" antigens which allow the immune system to recognize the patient's own tissues from foreign tissue. In an allogenic transplant, the antigens on the donor tissue are different from those of the recipient. This can lead to an immune response in which the antigens on the transplanted tissue stimulate the patient's immune system to destroy or reject the transplanted tissue. Alternatively, in bone marrow transplantation, the antigens on the patient's normal tissue can stimulate the immune system constituted from the donor tissue to destroy the patient's normal tissues. This is termed "graft versus host disease" (GVH).
SUBSTITUTE SHEET (RULE 26) In a related aspect, the invention provides a method for treating graft versus host disease in allogenic transplantation in which an allele specific inhibitor is used to inhibit proliferation of donor cells, e.g., to inhibit stimulation of the donor immune system. In preferred embodiments, the allele specific inhibitor is selected by identifying alternative variances or allelic forms of an essential gene that are present in the donor tissues but not the recipient. Therapy with a variance or allele specific inhibitor or inhibitors that recognizes both alleles of the essential gene that are present in the donor, but not both alleles of the same gene that are present in the recipient, can be used to suppress the immune response against the patient's tissues (GVH) without toxicity to these tissues. Most commonly, the donor tissue would be homozygous for a variance in the essential gene and the recipient would be homozygous to an alternative nucleotide or amino acid at a specificity determining site of variance. However, alternative combinations can also be used which result in at least one allelic form being present in the recipient which is not present in the donor cells, for example the donor could be homozygous and the recipient could be heterozygous for different allelic forms.
As in other aspects described, a plurality of target genes can also be utilized.
In another aspect, the invention provides a method for enhancing engraftment of an allogenic bone marrow transplant in which an allele specific inhibitor is used to kill or suppress the patient's own bone marrow, providing "space" for engraftment of the donor cells within the marrow cavity. In preferred embodiments, the allele specific inhibitor is selected by identifying alternative forms of an essential gene that are present in the recipient but not the donor marrow. Therapy with an allele specific (generally a variance specific) inhibitor that recognizes both forms of the essential gene that are present in the recipient, but not both forms of the same gene that are present in the recipient, can be used to suppress the patient's own marrow without toxicity to the transplanted cells. It will be recognized by those in the art that this method can be used to reduce the SUBSTITUTE SHEET (RULE 26) frequency of chimerism and increase the rate of success in engrafting an allogenic marrow.
"Chimerism" refers to a transplantation that is incomplete, leading to the proliferation of bone marrow progenitor cells derived from both the donor and recipient. Chimerism is generally an undesirable outcome that commonly results in gradual elimination of the graft due to competition with the patient's own cells.
Allele specific inhibitors can be used to treat or prevent chimerism by selectively killing or suppressing proliferation of the patient's own cells without toxicity to the donor cells.
In another aspect, the invention provides a method for treating cancer in a patient receiving allogenic or autologous transplantation in which an allele specific inhibitor is used to kill or inhibit the growth of cancer cells without toxicity to the transplanted marrow. In one embodiment, in an autologous transplantation the allele specific inhibitor is selected to recognize one alternative allele of an essential gene remaining in the cancer cell due to LOH in patients who are heterozygous with two different alternative forms of the essential gene in their normal cells and in the autologous bone marrow graft. Treatment with such a drug will enable continuing therapy of cancer without suppression of the transplanted marrow.
In an alternative embodiment, in an allogenic transplantation, therapy with an allele specific inhibitor that recognizes the one form of the essential gene that is present in cancer cells due to LOH in the recipient, but not an alternative form or forms of the same gene that are present in the recipient's normal cells and in the donor cells can be used to treat the cancer in the patient without toxicity to the transplanted cells. It will be recognized by those in the art that such therapy will enable more effective cancer therapy during and after transplantation. Moreover, such therapy would preserve the function of the immune system which is an important element in effective cancer therapy.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 In a related aspect, the invention can be used ex vivo during autologous transplantation to eliminate malignant cells from the transplanted marrow. The principle of autologous bone marrow transplantation is that bone marrow can be harvested from a patient prior tb high dose radiation or chemotherapy that would 5 normally be lethal to the bone marrow. Following such therapy, the patient can then be treated by reimplantation of their own marrow cells to reconstitute the bone marrow and hematopoietic functions. An important limitation of this procedure is that bone marrow harvested prior to such therapy often contains many malignant cells, and that implantation of the harvested bone marrow often 10 results in reseeding of the patient's malignancy. Various techniques for "purging"
the bone marrow of such malignant cells have been described. These methods are focused on selecting "normal" bone marrow stem cells or progenitor cells that are within the harvested tissue for selective reimplantation. The present invention provides for an improved method for purging bone marrow of malignant cells 15 using allele specific inhibitors of essential genes. The method involves identifying an essential gene with only one variant form remaining in the cancer cells due to LOH in patients who are heterozygous with two different alternative forms of the essential gene in their normal cells (and in the autologous bone marrow). The patient's bone marrow is then cultivated ex vivo using methods known in the art in 20 the presence of an allele specific inhibitor that inhibits the allele that is present in the cancer cells, but not the alternative allele that is present in the heterozygous normal bone marrow. This treatment will result in killing of cancer cells within the graft, enabling selective reimplantation of normal cells. It will be recognized that one or more drugs could be used simultaneously or sequentially in this 25 manner to achieve more efficient purging of cancer cells.
In another aspect, the present invention provides a method for sorting cells, for example for separating cancer cells from normal cells during an autologous bone marrow transplantation. The method utilizes a compound, preferably an antibody or SUBSTITUTE SHEET (RULE 26) WO 981.11648 PCT/US98/05419 antibody fragment, which specifically binds to at least one but less than all the products of alleles which occur in a population of a particular gene which encodes a cell surface protein. Such a binding compound is used to bind with cells which express a targeted allele. If cancer cells from a patient who is heterozygous for that S gene (having both a targeted allele and a non-targeted allele) have undergone LOH
of the particular gene such that only the non-targeted allele is present in the cancer cells, then the binding compound can be used to bind to normal cells and to pull them out from a mixture of normal and cancer cells. This separation is possible because the binding compound will bind to the protein from the targeted allele of the gene expressed in the normal cells, but will not recognize and will not bind to the cancer cells as there is no product of the targeted allele present on those cells. Use of this method thus allows the isolation of normal cells, which can then be reintroduced to the marrow in an autologous transplant following anticancer treatment of the patient, thereby avoiding the problem of reintroduction of cancer cells. In this method, the targeted gene need not be an essential gene, or have any particular function. All that is needed is that the gene product be accessible or can be made accessible to the allele specific binding compound and that there be alternative allelic forms of the gene present such that the products can be distinguished by allele specific binding compounds and that the gene have undergone LOH between the normal cells and the cancer cells. However, it is also recognized that this method can also be used to separate any sets of cells which express different allelic forms of a gene where the gene products are accessible to allele specific binding compounds.
In preferred embodiments, the binding compound is immobilized, such as on a solid support, or can be caused to leave solution, such as by precipitation or by sandwich binding of the binding compound with a second binding compound, so that the bound cells are directly removed from the mixture. In other embodiments, the binding compound allows the recognition of the targeted cell, such that the cells can SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTNS98/45419 be separated mechanically, for example using fluorescence activated cell sorting (FACS), or other cell sorting method as known to those skilled in the art.
Also in preferred embodiments, the binding compound is an antibody or antibody fragment which retains allele specific binding. Such antibodies can be readily obtained by conventional methods as polyclonal or monoclonal antibodies after isolation of an appropriate antigen.
In another aspect, the invention provides a method for inhibiting growth of or killing a cell containing only one allelic form of a gene by contacting the cell with an inhibitor active on that allelic form. The gene has at least two sequence variants in a population, and belongs to one of the categories of essential genes described below. The inhibitor is less active on at least one other allelic form of the gene.
In preferred embodiments of the above aspects in which an allele specific inhibitor is used to inhibit a cell or to treat a patient, a plurality of different inhibitors may be used. Preferably different inhibitors target a plurality of different variances in a single target gene, or target variances in different target genes, or both.
In particular embodiments a plurality of inhibitors is used simultaneously, in others there is serial administration using different inhibitors or different sets of inhibitors in separate administrations, which may be performed as a single set of administrations in which each set of inhibitors is administered once, or in multiple serial administrations in which each set of inhibitors is administered more than once. Such use of multiple inhibitors provides enhanced inhibition, which preferably includes killing, of the targeted cells. In addition, allele specific inhibitors as described can be used in conjunction with other treatments for diseases and conditions, including in conjunction with other chemotherapeutic agents such as other antineoplastic agents.
SUBSTITUTE SHEET (RULE 26) In a related aspect, an allele specific inhibitor can be used in conjunction with a conventional antiproliferative or chemotherapeutic agent or therapy, such therapies including radiation, immunotherapy, or surgery. In preferred embodiments the conventional therapy causes one or more genes within the cancer cell, or noncancer proliferative lesion, to be essential for cell survival that are would not be essential in the absence of said conventional therapy. For example, the treatment of cancer with radiation or alkylating agents makes e~cient DNA
repair essential for cell survival. In another example, depleting cancer cells of certain nutrients may make certain synthetic metabolic pathways essential. These examples are meant to be illustrative of the use of the present invention to those skilled in the art and not limiting. Further discussion and examples of the use of conditionally essential genes and their utilization in the methods of this invention are provided in the Detailed Description and the Examples.
In accord with the above aspects, in a further aspect the invention provides a pharmaceutical composition which includes at least one allele specific inhibitor.
In preferred embodiments the composition includes at least one allele specific inhibitor and a pharmaceutically acceptable carrier. Such carriers are known in the art and some commonly used carriers are described in the Detailed Description below. Also in preferred embodiments the composition includes two, three, or more allele specific inhibitors, and may also include a pharmaceutically acceptable carrier. In other preferred embodiments, the composition includes at least one allele specific inhibitor and another antineoplastic agent, which need not be an allele specific inhibitor. The embodiments of this aspect may also optionally include diluents and /or other components as are commonly used in pharmaceutical compositions or formulations. In embodiments having a plurality of allele specific inhibitors, the inhibitors may target a plurality of different variances of a single target essential gene, or may target sequence variances of a plurality of different essential genes or combinations thereof.
SUBSTITUTE SHEET (RULE 26) In accord with the use of pharmaceutical compositions, the present invention also provides a packaged pharmaceutical composition comprising an allele specific inhibitor as described above, bearing a Food and Drug Administration use indication for administration to a patient suffering from a cancer or suffering from S another proliferative disorder.
Determinations of essential gene heterozygosity and tumor cell LOH may be performed by a variety of methods, such as direct sequencing of known sequence variance sites and probe hybridization with variance specific probes. Thus, the invention also provides a nucleic acid probe at least 9, 12, 15 or 20 nucleotides in length, but preferably not more than 30 nucleotides, which will hybridize to a portion of a first allelic form of an essential gene in one of the above categories under specified hybridization conditions and not to a second allelic form under those hybridization conditions, the first and second allelic forms have a sequence variance within the complementary sequence. Preferably the probe is at least nucleotides in length and is perfectly complementary to a portion of the first allelic form which includes a sequence variance site. The probe hybridizes under stringent hybridization conditions to the portion of the first allelic form and not to the corresponding portion of the second allelic form. This means that the probe does not bind to the second allelic form to an extent which prevents identification of the preferential specific binding to the first allelic form. The thermodynamics of the probe hybridization can be predicted to maximize the desired differential hybridization, providing optimization for probe length, sequence, structural modifications, and modifications to hybridization conditions.
The invention also provides nucleic acid probes or primers adjacent to the site of a variance that can be used to amplify a sequence containing the variant position to determine which variance is present at that position. Such probes or primers can readily be designed based on the sequences provided in the corresponding database SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 sequence entry or otherwise determined. The method of determining the variance can involve allele specific hybridization, sequencing or analysis of the amplified fragment by mass spectroscopy, SSCP, gene sequence database analysis, capillary electrophoresis, bindase/resolvase systems, or other methods known in the art.
In S a preferred embodiment, the amplified sequence spans more than one variant position and the method used for determining the variances identifies which variances are present at each position and combinations of variances that are present on each allele.
In preferred embodiments of the above aspects, the specific target allelic form has 10 the characteristics as described above. Thus, for aspects in which the category of gene is specified, in preferred embodiments the gene belongs to a particular sub-category, for example, subcategories as specified in Table 1. Also in preferred embodiments, the gene is an identified target gene as listed in Table 1 or otherwise specified herein, including targeting utilizing the specified variances for exemplary 15 genes described herein, singly or in combination in an allelic form. Also in preferred embodiments, the target gene is an allelic form having characteristics as specified above, for example is a gene which has a high frequency of heterozygosity and/or occurs in a chromosomal region which undergoes LOH in a cancer at a frequency as specified above. For aspects in which the target gene has 20 a specified LOH frequency, the LOH frequency may be provided by published literature, inferred from the LOH of nearby genetic members, or independently determined, such as by the methods known in the art.
The use of conditionally essential genes for a number of applications is similar to the aspects above, but generally also involve an alteration of environment to make 25 the gene essential and also provides additional aspects. For a conditionally essential gene, the essentiality may, but need not be absolute. Instead, in this context, the term "essential" means that the gene confers a significant advantage, SUBSTITUTE SHEET (RULE 26) WO 98J41648 PCTlUS98/05419 such that the growth or survival of the non-targeted cells is preferably at least 2x, more preferably 3x, 4x, Sx, 10x, or more as compared to the targeted cells.
Thus, similar to the above, the invention provides a method for identifying an inhibitor potentially useful for treatment of cancer or other proliferative disorder.
The inhibitor is active on a conditionally essential gene, and the gene is subject to loss of heterozygosity in a cancer. The method includes identifying at least two alleles of a said gene which differ at at least one sequence variance site and testing a potential allele specific inhibitor to determine whether the potential inhibitor is active on at least one but less than all of the identified alleles. If the potential inhibitor inhibits expression of at least one but less than all of the alleles or reduces the level of activity of a product of at least one but less than all of the alleles, this indicates that the potential allele specific inhibitor is, in fact such an allele-specific inhibitor inhibitor.
In preferred embodiments of this and the various aspects described below, the conditionally essential gene is one of the exemplary genes presented in the table of conditionally essential genes or in the examples.
Similar to other types of target genes described above, the invention provides inhibitors, methods for producing inhibitors, pharmaceutical compositions, methods for identifying potential patients, probes, and primers which target or recognize alleles of a conditionally essential gene or utilize inhibitors which target such genes.
The invention also provides methods for preventing the development of cancer, methods for treating a patient suffering from a cancer, and methods for inhibiting growth of a cells as described above except that the targeted cells are subjected to an altered condition such that the gene becomes essential.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 In still another aspect, not requiring the use of allele specific inhibitors, but still utilizing information about sequence variance or allelic differences between normal somatic cells and cancer cells in a patient, the invention provides a method for selecting a patient for treatment with an antiproliferative treatment. The method includes the following steps: determining whether normal somatic cells in a potential patient are heterozygous for an essential or conditionally essential gene, where a first allelic form of the gene is more active than a second allelic form, and where a reduction in the activity of the gene in a cell increases the sensitivity of that cell to an antiproliferative treatment; and determining whether cancer cells from the patient have only the second allelic form of the gene. If the somatic cells are heterozygous and the cancer cells have only the second allelic form, this indicates that the patient is suitable for treatment with the antiproliferative treatment because the cancer cells will be more sensitive to the antiproliferative treatment. In preferred embodiments, the antiproliferative treatment is radiation or administration of a 1 S cytotoxic drug.
In a related aspect, the differences between the normal somatic cells and the cancer cells in a patient are used in a method for selecting an antiproliferative treatment for a patient suffering from a cancer. This method involves determining whether there will be a differential effect of the prospective treatment on the cancer cells as compared to the normal cells based on a differential response of the cancer cells due the presence in the cancer cells of only the less active form of a conditionally essential gene which is present in two alternative allelic forms with differing activities in the somatic cells. The method thus involves determining whether normal somatic cells in a potential patient are heterozygous for an essential or conditionally essential gene which reduces the sensitivity of cells to an andproliferative treatment. As noted, a first allelic form of the gene is more active than a second allelic form, and a reduction in the activity of the gene in a cell increases the sensitivity of that cell to the prospective antiproliferative treatment;
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98105419 and determining whether cancer cells of said patient have only the second, less active, allelic form of the gene. If these factors are present, this indicates that the proposed treatment is suitable for that patient.
In preferred embodiments of above aspects, a conventional therapy acts on a protein or other molecular target in the same pathway as the allele specific inhibitor. As an example, the antineoplastic drug hydroxyurea, which inhibits ribonucleotide reductase (RR), can be used in conjunction with an allele specific inhibitor of RR
subunit M 1 or M2 or another gene that encodes a product important in nucleotide synthesis. Similarly, the antiproliferative drug methotrexate inhibits the enzyme dihydrofolate reductase (DHFR), and can be used with allele specific inhibitors of DHFR that would result in a differential methotrexate effect on cancer tissues compared to normal proliferating tissues. Alternatively, methotrexate can be used with allele specific inhibitors of other genes important in folate metabolism to achieve an enhanced cancer cell specificity for methotrexate. Similarly, the anticancer drug 5-fluorouracil and related compounds can be administered together with an allele specific inhibitor of thymidylate synthase (TS) in a patient heterozygous for TS and with LOH at the TS gene in proliferating cells, e.g., cancer cells. Alternatively, an allele specific inhibitor of 5-FU degradation or metabolism can be administered with 5-FU. For example, the enzyme dihydropyrimidine dehydrogenase, which catalyzes the first and rate limiting step in 5-FU
catabolism would have the effect of potentiating S-FU action in cancer cells due to their lesser ability to metabolically inactivate 5-FU. One skilled in the art will readily recognize that similar methods can be used with other conditionally essential genes, including specific genes listed in the table of conditionally essential genes.
Some conditionally essential genes occur in active and less active, or nearly inactive allelic forms. Further, some cancer patients are heterozygous for active and less active forms in their normal tissues, but due to LOH, their cancer cells contain only SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTlUS98105419 the less active allelic form. As describe above, such patients can be identified by a diagnostic test of their normal cells and cancer cells. Such a test will identify which patients should be treated with a specific treatment, such as a particular drug or radiation treatment or other treatment. Such a therapy, which is not allele specific, would nonetheless have cancer specific effects due to the LOH-determined difference in the ability of the cancer cells to respond to the cytotoxic or cytostatic effects of therapy.
For example, patients with Ataxia Telangiectasia are homozygous for mutant alleles of the ATM gene. Such individuals are hypersensitive to radiation therapy or radiomimetic drugs. Heterozygotes for normal and mutant ATM are normal and have been estimated to account for 0.5-1% of the North American population, but, due to an increased risk of carer, may account for up to 5% of some cancers, for example, breast cancer. The ATM gene maps to chromosome l 1q23, a region frequently affected by LOH in breast and other cancers. In breast cancers arising in ATM heterozygotes in which the more active (normal) ATM allele is lost in cancer tissue due to LOH, treatment with radiation or radiomimetic drugs would be differentially toxic to cancer cells. It has been shown that ATM heterozygotes are less sensitive to such treatments than ATM mutant (less active) homozygotes.
Such use of an LOH diagnostic procedure to select appropriate antineoplastic therapy represents a change from the current procedures which are based solely on tissue origin, grade, and stage of cancer.
In such an approach, preferably the difference in activity between more active and less active allelic forms is at least 2x, more preferably at least 3x, 4x, or Sx, and most preferably at Ieast 6x, l Ox, or even more.
Preferably a target conditionally essential gene is one such that at least 0.1 % , 0.5 % , 1 % or 5 % , or the higher rates as stated above, of a population is SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 heterozygous for a particular sequence variance Additional specific genes within the categories or subcategories described which are potentially useful for allele specific therapy can be readily identified by those skilled in the art using the methods described herein and/or using information 5 available to those familiar with cellular genetics and tumor biology. In particular such genes can be identified and/or obtained by identifying essential genes, determining whether the gene contains sequence variants in a population, determining whether the gene undergoes LOH in one or more tumors or other proliferative disorders. Genes having these characteristics can then be used for 10 identifying allele specific inhibitors and evaluated for use in the other methods of this invention. Such procedures are routine, as is shown by the Detailed Description of the Preferred Embodiments below, including the Examples.
In preferred embodiments of the above methods and inhibitors involving particular target genes or classes or categories of genes, the inhibitor or potential inhibitor is 15 a ribozyme which is designed to specifically cleave a particular target allelic form of a gene (i. e. , a nucleotide sequence such as mRNA).
The ribozyme is designed to cleave the nucleotide (e. g. , RNA) sequence at a position in the nucleotide chain of the target allelic form at or near the position of a sequence variance. Usually the ribozyme will have a binding sequence which is 20 perfectly complementary to a target sequence surrounding the sequence variance site. Preferably, the ribozyme does not consist of only ribonucleotides, and therefore includes at least one nucleotide analog or modified linkage. In preferred embodiments the ribozyme has a hammerhead or hairpin motif, but may have other structural motifs as known to those skilled in the art..
25 The term "ribozyme" refers to a catalytic RNA molecule, including those SUBSTITUTE SHEET (RULE 26) WO 98Lt1648 PCT/US98/05419 commonly referred to as hammerhead ribozymes and hairpin ribozymes, generally having an endonuclease activity, but includes catalytic RNA molecules, catalytic DNA molecules (DNAzymes), and derivatives of such molecules unless indicated to the contrary. In particular, as understood by those skilled in the art, ribozymes may incorporate a variety of nucleotide analogs, modified linkages, and other modifications.
In connection with ribozymes, "target sequence" refers to a nucleotide sequence which includes a binding site and a cleavage site for a ribozyme. For use in this invention, preferably a gene having a ribozyme target sequence exists in two allelic forms in normal somatic cells of a patient. The two allelic forms differ in nucleotide sequence within the target sequence, i. e. , have a sequence variance within the target sequence.
Also in connection with ribozymes, the term "specifically cleaves" means that a particular ribozyme will cleave a target sequence to a greater extent than it will cleave a different sequence. For allele specific ribozymes, this means that for two allelic forms having a sequence variance in the target sequence, preferably the ribozyme will cleave one of the allelic forms more efficiently than the other.
Those skilled in the art will understand that the target discrimination can be provided by base differences within the ribozyme binding sequence of the substrate at or close to the cleavage site.
Similarly, in preferred embodiments the inhibitor or potential inhibitor is an oligonucleotide, e.g, an antisense oligonucleotide, preferably at least partially an oligodeoxyribonucleotide. The antisense oligonucleotide is complementary to a sequence which includes a sequence variance site. Usually, though not necessarily, the antisense oligonucleotide is perfectly complementary to a sequence of the target allelic form which includes a sequence variance site. The antisense SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98105419 oiigonucleotide preferably is at least twelve nucleotides, more preferably at least seventeen nucleotides in length. In some cases the antisense oligonucleotide may advantageously be longer, for example, at least 20, 25, or 30 nucleotides in length. Also in preferred embodiments, the oligonucleotide is no longer than 20, 25, 30, 35, 40, or 50 nucleotides The optimal length will depend on a number of factors, which may include the differences in binding free energy of the oligonucieotide to the target sequence as compared to binding to the non-target allelic form, i. e. , the non-target sequence variant, or the kinetics of nucleic acid hybridization. The oligonucleotide preferably contains at least one nucleic acid analog or modified linkage. Such complementary oligonucleotides may function in various ways, and those skilled in the art will know how to design the oligonucieotide accordingly. Such functional mechanisms include, but are not limited to direct blocking of transcription of a gene by binding to DNA (e. g.
, high affinity antisense, including triple helix), direct blocking of translation by binding to mRNA, RNaseH mediated cleavage of RNA or other RNAase mediated cleavage, and binding-induced conformational changes which block transcription or translation or alter the half life of mRNA. Triple-helix modes of action include the formation of a triple-helical structure between the two strands of genomic DNA and an antisense molecule, i. e. , anti-gene strategy, or between an RNA
molecule and an antisense oligonucleotide which loops back to contribute two of the three strands of the triple helix, or between an RNA and an antisense where the RNA provides two of the three strands of the triple helix.
The term "oligonucleotide" refers to a chain molecule comprising a plurality of covalently linked nucleotides as recognized in the art. The oligonucleotide preferably has about 200 or fewer backbone units corresponding to nucleotide subunits, more preferably about 100 or fewer, still more preferably about 80 or fewer, and most preferably about 50 or fewer. An oligonucleodde may be modified to produce an oligonucleotide derivative. Unless indicted otherwise the SUBSTITUTE SHEET (RULE 26) WO 98/x1648 PCTJUS98105419 term "oligonucleotide" includes "oligonucleotide derivatives" .
A large number of nucleic acid modifications are known in the art which may be used in the nucleic acid molecules of the present invention, thereby producing "nucleic acid derivatives" or "oligonucleotide derivatives" . Such modifications S can be used, for example, to enhance resistance to degradation by nucleases or to modify functional characteristics such as binding affinity. In preferred embodiments, the ribozyme, antisense oligonucleotide, or other nucleic acid molecule contains at least one modified linkage, including but not limited to phosphorothioate, phosphoramidate, methylphosphonate, morpholino-carbamate, and terminal 5'-5' or 3'-3' linkages. Also in preferred embodiments, the nucleic acid molecule contains at least one nucleotide analog. Such analogs include but are not limited to nucleotides modified at the 2' position of the ribose sugar, e. g. , 2'-O-alkyl (e.g., 2'-O-methyl or 2'-methyoxyethoxy) or allyl, 2'-halo, and 2'-amino substitutions, and/or on the base (e.g., C-5 propyne pyrimidines), and analogs which do not contain a purine or pyrimidine base, and includes the use of nucleotide analogs at the terminal positions of a nucleic acid molecule.
Preferably a 2'-O-alkyl analog is 2'-O-methyl; preferably a 2'-halo analog is 2'-F.
A specific embodiment of this invention is the use of hybrid oligonucleotides that contain within a linear sequence two different types of oligonucleotide modifications. In a particular embodiment, these modifications are used such that a segment of the oligonucleotide that hybridizes to the sequence variance is RNAase sensitive, but other segments are not RNAase sensitive.
Other modifications may also be used as are known in the art, such as those described in connection with antisense and triple helix in: Crooke & Bennett, 1996, Annual Rev. Pharm. and Toxicol. 36:107-129; Milligan et al., 1993, J.
Med. Chem. 36:1923-1937; Reynolds et al., 1994, Proc. Nat. Acad. Sci. USA
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 91:12433-12437; and McShan et al., 1992, J. Biol. Chem. 267-5712-5721, which are hereby incorporated by reference. An additional modification useful for delivery of oligonucleotides is complexation of oligonucleotides with nano-particles, as described in Schwab et al., 1994, Proc, Nat. Acad. Sci. USA
S 91:10460-10464. As described further below, oligonucleotides may be complexed with other components known in the art which provide protection and/or enhanced delivery for the oligonucleotides, and may be useful for either gene delivery or for delivery of non-coding oligonucleotides.
Thus, "derivatives of nucleic acid inhibitors" include modified nucleic acid molecules which may contain one or more of: one or more nucleotide analogs, including modifications in the sugar and/or the base, or modified linkages, base sequence modifications, and insertions or deletions, or combinations of the preceding. Other derivatives are also included as are known in the art.
Similarly, in preferred embodiments the inhibitor or potential inhibitor is an antibody, preferably a monoclonal antibody, which may be complexed or conjugated with one or more other components, or a fragment or derivative of such an antibody. It is recognized in the art that antibody fragments can be produced by cleavage or expression of nucleic acid sequences encoding shortened antibody molecule chains. Such fragments can be advantageously used due to their smaller size and/or by deletion of sites susceptible to cleavage. In addition, derivatives of antibodies can be produced by modification of the amino acid moieties by replacement or modification. Such modification can, for example, include addition or substitution or modification of a side chain or group.
Many modifications and biological effects of such modifications are known to those skilled in the art, and may be used in derivatives of antibodies in accord with those biological effects. Such effects can include, for example, increased resistance to peptidases, modified transport characteristics, and ability to carry a ligand or other SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTlUS98/0~419 functional moiety. In preferred embodiments, the antibody is a humanized antibody from a non-human animal, e. g. , a humanized mouse or rabbit antibody.
Many instances of monoclonal antibodies that distinguish protein differing by a single amino acid are known in the art.
5 An inhibitor may also be an oligopeptide or oligopeptide derivative. Such peptides may be natural or synthetic amino acid sequences, and may have modifications as described for antibodies above. In general, an oligopeptide will be between about 3 and 50 residues in length, preferably between about 4 and 30, more preferably between about 5 and 20 residues in length.
10 In other embodiments, the inhibitor is a small molecule, for example, a molecule of one of the structural types used for conventional anticancer chemotherapy.
By "small molecule" or "low molecular weight compound" is meant a molecule having a molecular weight of equal to or less than about 5000 daltons, and more preferably equal to or less than about 2000 daltons, and still more preferably equal 15 to or less than about 1000 daltons, and most preferably equal to or less that about 600 daltons. In other highly preferred embodiments, the small molecule is still smaller, for example less than about 500, 400, or 300 daltons. As well known in the art, such compounds may be found in compound libraries, combinatorial libraries, natural products libraries, and other similar sources, and may further be 20 obtained by chemical modification of compounds found in those libraries, such as by a process of medicinal chemistry as understood by those skilled in the art, which can be used to produce compounds having desired pharmacological properties.
In connection with the gene sequences or subsequences of gene sequences or 25 primer sequences as described herein, the sequences listed under the accession SUBSTITUTE SHEET (RULE 26) WO 981.11648 PCT/US98/05419 number are believed to be correct. However, the genes can be readily identified and the invention practiced even if one or more of the specified sequences contain a small number of sequence errors. The correct sequence can be confirmed by any of a variety of methods. For example, the sequence information provided herein and/or published information can be used to design probes for identifying and isolating a corresponding mRNA. The mRNA can be reverse transcribed to provide cDNA, which can be amplified by PCR. The PCR products can then by used for sequencing by standard methods. Alternatively, cDNA or genomic DNA
libraries can be screened with probes based on the disclosed or published gene sequences to identify corresponding clones. The inserts can then be sequenced as above. If complete sequence accuracy is desired, such accuracy can be provided by redundant sequencing of both DNA strands. Those skilled in the art will recognize that other strategies and variations can also be used to provide the sequence or subsequence for a particular gene.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Fig. 1 shows seventeen gene-specific Target Gene Smnmary Tables which show variances detected in some of the exemplary genes described as examples in the specification. Those genes are:
Sodium, potassium ATPase CTP synthetase Ribonucleotide reductase M1 subunit Thymidylate synthase SUBSTITUTE SHEET (RULE 2fi) Alanyl tRNA synthetase Cysteinyl tRNA synthetase Glutamyl-prolyl tRNA synthetase Glutaminyl tRNA synthetase Lysyl tRNA synthetase Threonyl tRNA synthetase Ribosomal protein S 14 Eukaryotic initiation factor SA
Replication protein A, 70 kD subunit Replication protein A, 32 kD subunit RNA Polymerase II, 220 kD subunit TATA associated factor )Ifi Dihydropyrimidine dehydrogenase These tables show, in the title, the name of each gene, its chromosome location and the Varia ID number. The horizontal section of the table displays, from left to right, the name of the primers used to amplify the polymorphic segment, the number of the polymorphic nucleotide (the numbering corresponds to the GenBank accession number reported in the central box under 'Sequence from:') and the two alternative sequences at the variant site. Then, under columns 1 - 36, the genotypes of 36 lymphoblastoid cell lines are given, followed by the frequency of heterozygotes ('het rate'), a 'Comments' section which describes any unusual aspects of the variances, a 'Location' section which reports the location of any variances and the inferred effect on amino acid sequence, if any, and a 'Race specific heterozygosity' section which reports frequency of heterozygotes in any racial groups with particularly high heteroxygosity levels. Below the 'Genotypes of 36 unrelated individuals' section the racial or ethnic identity of the subjects is shown (see legend in box at right: 'Ethnic & racial groups surveyed'). The sequence surrounding the variances is shown in the box at bottom left, with the SUBSTITUTE SHEET (RULE 26) WO 98/11648 PCTIUS98/0~419 location of the variant base marked in bold type.
Fig. 2 is a schematic showing the practical flow of the SSCP technique as used for exemplary target genes. This flow chart, in conjunction with the description of the SSCP technique in the Detailed Description, demonstrates how sequence variances of the exemplary genes were identified. In conjunction with published descriptions of the SSCP technique, one skilled in the art can thus readily use SSCP to identify sequence variances in other genes within the scope of this invention.
Fig. 3 is a table describing the extent and distribution of loss of heterozygosity throughout the genome for a number of cancers as reported in the literature.
The table is divided into 41 sections, one for each fo the chromosomal arms for which there is information about LOH frequency. (There is no information for the short arm [called the p arm] of chromosomes 13, 21 or 22, all of which are very short and contain mostly repetitive DNA.) In each of the 41 sections there is a list of polymorphic loci (sites) that have been tested for LOH in one or more cancer types.
The loci are ordered, to the extent that present information allows, from the telomeric end of the short arm of the chromosome to the centromere (p arm tables), or from the centromere to the telomeric end of the long arm of the chromosome (q arm tables). Many chromosomes have not yet been well studied for LOH, so the absence of data on LOH in a particular cancer type on a particular chromosome arm should not be construed as indicating no LOH. It may simply indicate no good LOH
studies have yet been published. The Loss of Heterozygosity Table is explained in detail below.
Column 1 Chromosomes, when stained with dyes such as giemsa, have alternating dark and Iight staining bands. These bands are the basis of chromosome nomenclature. Many of the markers used for LOH studies have been assigned to SUBSTITUTE SHEET (RULE 26) specific chromosome bands, or can be inferred as likely to belong to specific bands based on other information. The 'unknown' notation in this column indicates that the paper from which the data was obtained (column 7} did not provide chromosome band information. In such cases other information has generally been used to order the data; however the order of some markers remains uncertain.
Column 2 LOH studies are performed with specific DNA markers or probes (for Southern blotting) or with DNA primers (if polymerase chain reaction was used) from a specific site, or locus, on a chromosome. The name of the marker, locus or probe used to perform each LOH assay is given in the second column of the Table, under 'Marker'. In the Table the markers are listed in their likeliest order along the chromosome, from the telomere of the p arm to the centromere for the p arm tables, and from the centromere to the telomere of the q arm for the q arm tables.
Columns 3, 4 & S The total number of cancers evaluable for LOH at the specific marker shown in column 2 (in the paper cited in column 7) are shown in column 3, 'Total'. This is generally the number of patients that were heterozygous for the marker in their normal DNA. Column 4, 'Cases w/LOH', shows the number of patients with LOH at the DNA marker. Column 5, 'LOH Freq', is the quotient of column 4 divided by column 3, giving the fraction of patients with LOH at the indicated marker.
Column 6 The type of cancer studied is indicated under the heading 'Tumor Type'.
In some cases more detailed clinical information on cancer subtype or clinical stage is available in the paper cited in column 7.
Column 7 The literature citation, or 'Reference', from which the data was drawn.
The references are provided in a compact form consisting of journal abbreviation (see the list of journal abbreviations below), volume and page.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 Note Studies of allele loss in benign neoplasms or in non-neoplastic conditions are not included in this table.
Journal Abbreviations for Literature Cited in the Table 5 The abbreviations used in the Tables are as follows:
AJHG = American Journal of Human Genetics AJP = American Journal of Pathology B = Blood BJC = British Journal of Cancer 10 C or CA= Cancer CCG = Cancer Cytogenetics CGC = Cell Genetics and Cytogenetics CL = Cancer Letters CR = Cancer Research 15 CSurv = Cancer Surveys EJC = European Journal of Cancer G or GE = Genomics GCC = Genes, Chromosomes & Cancer GO = Gynecological Oncology 20 HG = Human Genetics HMG = Human Molecular Genetics IJC = International Journal of Cancer JAMA = Journal of the American Medical Association JJCR = Japanese Journal of Cancer Research (Gann) 25 JNCI = Journal of the National Cancer Institute JU = Journal of Urology SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/Q~419 Lan = Lancet LI = Laboratory Investigation N = Nature NEJM or NEJ = New England Journal of Medicine O = Oncogene PN or PNAS = Proceedings of the National Academy of Sciences S = Science This data base thus identifies sites and regions of LOH associated with the particular identified cancers, including high frequency LOH chromosomal arms as well as the identified smaller regions associated with the particular markers.
Both as indicated in the Summary and Detailed Description, LOH information such as this identifies essential genes mapping to those LOH regions as likely potential target genes because of the high probability that an essential gene in such a region undergoes LOH at frequencies similar to the marker. Such gene identif canon thus further identifies particular cancers which can potentially be treated with inhibitors targeting sequence variances in those essential genes.
The database provided shows information which is contained in published references dealing with cancer LOH. Those skilled in the art will recognize however that similar information can be readily obtained from the published literature in relation to other cancers and other neoplastic disorders. Thus this table demonstrates that one skilled in the art can readily identify regions of high frequency LOH for other such disorders and cancers, and can further readily identify essential genes which are potential targets for variance specific inhibition and the treatment of the corresponding condition and in other aspects of this invention.
Fig. 4 is a table summarizing the results in Fig. 3 by chromosome arm. Data for SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 all loci on each chromosome arm has been summed in a single statistic for LOH
frequency on that chromosome arm.
Fig. 5 is a Target Variances by Field Table, which summarizes information on DNA sequence variances in selected genes from the Target Gene Table (Table 1), and is organized into groups of related genes that parallel the fields in the Target Gene Table.
~ The heading at the top of each category of essential genes shows a number and a subcategory name. The number indicates which of the six principal categories of essential genes the subcategory belongs to (e.g. genes required for cell proliferation is category 1, genes required to maintain inorganic ions at levels compatible with cell growth or survival is category 2, etc.).
~ Below the heading is a sentence on 'Validation' which briefly refers to some of the data which shows that genes in the subcategory are essential.
Summary information on target gene variances is then listed, with five columns of data.
~ The first column gives the Variagenics gene ID number, which serves as a cross reference to the Target Variances Table (see below), where more detailed information on variances can be found.
~ The second column lists gene names. (The GenBank accession number in column 5 may be a more reliable way to identify genes.) ~ The third column lists the number of variances found. These variances were detected by a variety of experimental and informatics based procedures described in the examples. Many variances were detected by two independent methods (e.g. informatics based detection and T4 endonuclease VII detection). A molecular description of the variances is provided in the Target Variances Table (see below).
~ The fourth column lists the chromosome location of the target gene, if known. Knowledge of the chromosome location permits assessment of the SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 SS
cancers in which LOH would be expected to affect the target gene. (See the Loss of Heterozygosity Tables for a detailed listing of LOH by chromosome region. ) ~ The fifth column lists the GenBank accession number of the target gene.
S (Some of the genes specified in the Table do not yet have GenBank accession numbers. For example, genes encoding several human tltNA synthetases and ribosomal subunits have not yet been cloned, although their existence can be inferred from genetic and biochemical studies and from phylogeny.
Fig. 6 is identical to Fig. S, except that it concerns exemplary conditionally essential genes rather than generally essential genes.
Fig. 7 is a Target Variances Table shows molecular details of exemplary variances identified by Variagenics in exemplary target genes. There are six columns in the Table.
~ The first column gives the Variagenics gene ID number, which serves as a cross reference to the Target Variances by Field Table (see above), where information on gene location and GenBank accession number are provided.
After the ID number is a decimal point and then a list of one or more integers (on successive lines), which are the (arbitrary) numbers of the specific variances identified. Between one and 13 variances were identified per target gene. Information on different target genes is separated by dashed horizontal lines.
~ The second column lists the location of the variance - specifically the number of the nucleotide at which variation was observed. The nucleotide number refers to a cDNA sequence of the target gene which can be retrieved using the GenBank accession number provided in the Target Variances by Field Table.
~ The third column lists the two variant sequences identified at the specified SUBSTfTUTE SHEET (RULE 26) nucleotide. The variant nucleotides are bracketed and in bold font separated by a slash. Ten nucleotides of flanking sequence are provided on either side of the variance to localize the variant site unambiguously. (In the event of a conflict between the nucleotide number specified in column 2 and the sequence specified in column 3 the latter would rule as the correct sequence.) These variances were detected by a variety of experimental and informatics based procedures described in the examples. Many variances were detected by two independent methods (e.g. informatics based detection and T4 endonuclease VII detection).
~ The fourth and fifth columns (headed '# Varia 1' and '# Varia 2') provide the number of occurrences of variance 1 and 2, respectively, where variance 1 is the first and variance 2 the second of the bracketed nucleotides in column three. In both the fourth and fifrh columns there are two numbers.
The first number reports the number of occurrences of the variance.
'Occurrences' include ESTs identified during informatics based analysis, or variances identified experimentally by analysis of human cell lines, or both.
The second number, inside parentheses, reports the number of individuals in whom the occurrences were detected. An 'individual' means either a cell line (analyzed experimentally) or a cDNA library created from one individual (but from which many ESTs for the target gene may have been sequenced).
Thus if the first number is 15 and the second number is 11 then there were 15 occurrences of the variance (a combination of 15 ESTs and/or 15 experimentally identified alleles) in a total of 11 cDNA libraries and/or cell lines.
~ The fifth column provides annotation on the variances, particularly concerning the location of the variant site in the cDNA and the effect of the DNA sequence variance on the predicted amino acid sequence, if any. 5' UT = 5' untranslated region; 3' UT = 3' untranslated region; silent =
variance lies in coding region by does not affect predicted amino acid SUBSTfTUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 sequence; ND = analysis not done; Thr - > Asn = specific amino acid substitutions, inferred from the nucleotide sequence variance, are provided.
Similar information can be readily obtained for additional genes using the methods described or as known to those skilled in the art.
5 Figures 9-15 correlate with Example 31.
Fig. 9 is a bar graph showing the number of T24 human bladder cancer cells surviving 72 hours after transfection with antisense oligonucleotides. Anti-ras is an oligonucleotide known to have antiproliferative effects against T24 cells.
This oligonucleotide exhibits inhibition comparable to the anti-RPA70 oligonucieotide.
10 Anti-herpes and an oligonucleotide with a scrambled sequence are shown as controls. This experiment demonstrates that RPA70 is an essential protein.
Cells were plated in six well dishes 24 hr prior to the experiment and transfected at approximately 50-70 % confluency with various phosphorothioate oligomers at 400 nM. An oligomer:lipofectin ratio of 3 ug Lipofectin/ml Optimem/100 nM
15 Phosphorothioate oligomer was used for all transfections. Prior to transfection the cells were washed once with room temp Optimum (BRL) and then Lipofectin diluted into Optimem was added to the cells. After addition of the lipofectin the antisense oligomers were immediately added. After a five hour incubation the medium was removed from the cells and replete medium added. The cells were 20 allowed to recover, trypsinized, and cell number was determined at 72 hr by counting with a hemocytometer. Each bar represents two different determinations of cell number for each of three triplicate samples.
Fig. 10 is a Northern Blot demonstrating specific suppression of RPA70 mRNA
levels in two cell lines with opposite genotypes. RPA70 in Mia Paca II cells 25 matches the 13085 oligomer while RPA70 in T24 cells matches the 12781 SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 oligomer. The 13706 oligomer is a random sequence control. Cells were plated in P100 dishes transfected as described in figure legend 11. Twenty-four hours after the addition of the indicated oligomers, RNA was recovered from the cells by the SDS-Lysis method (Peppel, K and Baglioni, C. Biotechniques, Vol. 9, No. 6, pp 711-7131, 1990). For Northern Blots 5-10 ug RNA per well was loaded onto a formaldehyde gel, electrophoresed and transferred to BioRad Zeta Probe GT.
After baking (30 min at 80 C in a vac oven) the blot was probed for specific mRNA using a random primed 32P-labeled cDNA specific for RPA 70.
Fig. 11 is a Northern blot showing allele-specific Suppression of RPA 70 mRNA
in T24 and Mia Paca II cells. Cells were plated in P100 dishes, transfected, and RPA 70 mRNA levels measured as previously described. T24 cells contain the genotype targeted by oligomer 12781. Mia Paca II cells are homozygous for the variance targeted by oligomer 13085. 12781 is a 20 nucleotide long phosphorothioate oligomer which targets RPA70 in T24 cells. 13085 is an 18 nucleotide long phosphorothioate oligomer which targets RPA70 in Mia Paca II
cells. The lower half of the figure shows the EtBr stained gel of total RNA
probed by Northern Blot.
Fig. 12 is two graphs showing that the proliferation of two cell lines homozygous for different variant forms of the RPA70 gene is inhibited to a greater degree by matched oligonucleotides than by oligomers having a single base mismatch. Cell proliferation was measured by BrdU incorporation in cellular DNA.
Transfections were performed on consecutive days and BrdU incorporation measured 24 hours after the last transfection (see figure legend 9). Oligomer 12781 targets the variance contained in A549 cells and is mismatched relative to the genotype of Mia Paca II cells. Oligomer 13085 targets the variance contained in Mia Paca II
cells and is mismatched relative to the genotype of AS49 cells.
SUBSTITUTE SHEET (RULE 26) W O 98Lt 1648 PCT/U 598/05419 Fig. I3 is a graph showing Inhibition of BrdU incorporation in A549 cells by antisense oligonucleotides against the RPA 70 gene. Cells were transfected, as described previously, with a matched oligonucleotide (12781) or an oligonucleotide with one mismatch (13085). The oligonucleotide concentration was 400 nM with specific oligomer diluted with a random oiigonucleotide. Cell proliferation was measured by BrdU incorporation after two transfections.
Twenty-four hours after the first transfection the cells were transfected identically.
Twelve hours after the second transfection BrdU was added to the cells and BrdU
incorporation was assayed after a 12 hour incubation. BrdU incorporation was measured by ELISA (Boehringer Mannheim) with the following changes: Volumes were increased to assay BrdU incorporation in 6 well dishes. 1000 ~,1 of fix, ul of antibody, and 1000 ul of substrate. A portion of the samples were transferred to a 96 well dish (in triplicate) and read at 405 nm on a plate reader.
Fig. 14 is a graph showing antiproliferative/cytopathic effects of antisense oligonucleotides against the RPA70 gene in A549 cells. Cells were transfected on three consecutive days with a matched oligonucleotide (12781) or an oligonucleotide containing a one base mismatch (13085). Following the last transfection the cells were allowed to recover three days. Cell number was quantified by Sulforhodamine B staining (Molecular Probes). Volumes were increased to accommodate the assay in 6 well dishes. Fixation 1.25 ml, stain ul, solubilizer 1 ml. A portion of the samples were then transferred to a 96 well dish in triplicate and quantified by plate reader at 565 nm. All transfections were done with 400 nM oligomer by dilution of the specific oligomer with a random oligonucleotide to control for nonspecific oligonucleotide effects.
Fig. IS is a graph showing antiproliferative/cytopathic effects in Mia Paca II
cells by antisense oligonucleotides against the ltPA70 gene. Cells were transfected with a matched oligonucleotide (13085) or an oligomer with a one base mismatch SUBSTITUTE SHEET (RULE 2fi) (12781). Methods were identical to those described in figure legend 16.
Fig. 16 is a Northern blot showing suppression of Ribonucleotide Reductase (RR) mRNA by antisense oligomers. Mia Paca II cells were transfected and 24 hours later RR mRNA was measured by Northern Blot (for methods see figure legend 11). All oligomers have a phosphorothioate backbone throughout and are without modification. The lower half of each panel is a EtBr stained gel of the total RNA
probed. Oligomer 13704 is a scrambled random control oligomer. RR2410GA
targets the variance contained in Mia Paca Ii cells. Oligomer RR2410AG has two mismatches compared to the genotype of Mia Paca II cells. Oligomers RR1030 and RR1031 are negative control oligomers. They are targeted to a region of RR
which is not effective for mRNA down-regulation.
Fig. 17 shows a Northern blot which is a performed similarly to the experiments in Fig. 16. MDA-MB 468 cells were transfected and the level of RR mRNA
measured after 24 hours. 13706 is a scrambled random control oligomer.
2410AG targets the two variances contained in the MDA-MB 468 cells. Oligomer 2410GA contains two mismatches relative to the genotype of MDA-MB 468 cells.
Both 2410AG and 2410GA are identical to RR2410AG and RR2410GA, respectively.
Fig. 18 shows specific suppression of EPRS mRNA using hybrid oligomers. The sequences at the top provide the structures of the oligonucieotides. The graph at the bottom shows the relative specificity of oligonucleotides.
Fig. 19 is two blots showing specific suppression of EPRS mRNA using hybrid oligomers. A549 cells were transfected with the indicated concentrations of the hybrid oligomers (for structure see text). 14977 targets the two variances contained in A549 cells. 14971 contains two mismatches relative to the genotype SUBSTITUTE SHEET (RULE 26) of A549 cells.
Fig. 20 is a graph showing inhibition of mutant ras using antisense oligonucleotides specific for the mutant form, based on information available in Schwab et al., 1994, PNAS 91:10460-10464.
SUBSTITUTE SHEET (RULE 26) WO 981.11648 PCT/US98/05419 DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
I. Introduction All normal human cells have two copies of each autosomal chromosome (chromosomes I through 22); one copy is inherited from each parent. Each 5 chromosome pair thus contains two alleles for any gene. If a single allele of any gene pair is defective or absent, the surviving allele will continue to produce the encoded gene product. Generally, one allele of a gene pair is sufficient to carry on the normal functions of the cell. (Dominant genetic disorders in which mutations in one allele are sufficient to cause disease are generally those in which the mutation, 10 or gene product harboring the mutation, has a toxic effect on the cell.) Because humans are genetically heterogeneous, many of the paired alleles of genes of the somatic cells of an individual differ from one another in their gene sequence.
Typically both alleles are transcribed and ultimately translated into proteins used by the cell. In most cases, the sequence differences between two allelic forms of a gene 15 in an individual are small, usually differing by only one or a few base differences in sequence. The sequence differences may occur at a single variance site, or may constitute more than one variance site, i.e., two allelic forms in an individual may have more than one sequence variance distinguishing them.
When a cell is heterozygous, i.e., has at least one sequence variance, within the 20 transcribed sequence for a particular gene, each allele may encode a different mRNA, i.e., the mRNAs differ in base sequence. For base changes which are located within coding sequences, the effect of the nucleotide difference depends on whether the base change changes the amino acid which is encoded by the relevant codon. Many base changes do not change the coding sequence because they lie in 25 untranslated regions of the mRNA, outside of the mRNA in introns or intergenic sequences, or in a "wobble" position of a codon which changes the codon, but not SUBSTITUTE SHEET (RULE 26) the amino acid it encodes. As a result, the mRNAs encoded by two alleles may translate into the same protein or into forms of the same protein differing by one or more amino acids. An important aspect of the present invention is that many sequence variances that are targets for cancer therapy by the methods described here S are not mutations, are not functionally related to cancer, and may not, under normal environmental conditions, induce any function difference between the allelic forms of the gene or protein. Only in the circumstances described in this invention, namely genes that encode essential functions, the presence of variances with a sufficient population frequency, a su~cient frequency of LOH in cancers, do these genes, and the variant sequences within these genes, have utility for the therapy of cancer and other disorders through the discovery of variance-specific inhibitors.
Gene targets for a variance-specific inhibition strategy in this invention satisfy three criteria:
1. The target gene encodes a gene product, e.g., a RNA transcript or protein product essential for the growth or survival of cells.
2. The target gene is located within a chromosome region frequently deleted in cancer cells or cells of a noncancer, proliferative disorder.
3. The target gene exists in two alternative forms in the normal somatic cells of a patient having a cancer or noncancer proliferative disorder.
The allele specific therapy strategy for cancer and noncancer proliferative disorders utilizes the genetic differences between normal cells and neoplastic cells.
Thus, the first step in the therapeutic strategy is identifying genes which code for proteins or other factors essential to cell survival and growth that are lost through LOH
in tumor cells. Since many genes have been mapped to specific chromosomal regions, this identification can be readily performed by identifying such essential genes which are located in the chromosomal regions characteristically or frequently deleted in SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTJUS98/03419 different forms of human cancer or other tumors. Table 2, from the review conducted by Lasko et al., 1991, Ann. Rev. Genetics 25:281-314, summarizes results of numerous studies determining loss of heterozygosity in tumors, identifying specific tumor types. A much larger summary of tumor-related LOH is provided in Fig. 5.
Once regions of LOH are identified in the chromosomes of a patient's tumor cells, genes which map to the deleted chromosomal segments and are known to code for gene products essential for cell growth or survival are tested for DNA
sequence variances. The identification of a greater number of LOH sites affords a broader selection of target genes coding for essential proteins or other gene products and therefore of sequence variance sites for targeting.
Essential genes which have sequence variants in a population provide a set of target which are advantageous due to the presence of many patients heterozygous for a particular gene, so that the gene will provide a target in cases where the gene has undergone tumor-related LOH.
In accord with the description of target gene categories above, most advantageously a target gene is an essential gene which undergoes LOH in a tumor at a high frequency as described above and which has alternative allelic forms in a population at frequencies as described above. Such genes will provide many potentially treatable patients due to the conjunction of LOH and heterozygosity frequencies.
The most preferred target genes are those essential genes which have both a preferable rate of heterozygosity and a preferable frequency of LOH in a tumor or other proliferative condition in a population of interest. Also preferable is that the gene undergoes LOH in a plurality of different tumors or other conditions.
SUBSTITUTE SHEET (RULE 26) II. Essential Cellular Function and Essential Genes As indicated in the Summary above, the invention targets specific allelic forms of essential genes, which are also termed genes essential for cell growth or viability.
As used herein the term, "genes which code for a protein essential for the growth or survival or cells" or "genes which code for proteins or factors required for cell viability" or "essential genes" is meant to include those genes that express gene products (e.g., proteins) required for cell survival as well as those genes required for cell growth in actively dividing cell populations. These genes encode proteins which can be involved in any vital cell. An additional factor which applies to genes identified by any of the approaches described above is: a target gene or protein should be encoded by a single locus in man.
A large number of references have identified essential genes which constitute actual or potential targets for allele specific inhibition. The identification of essential genes can be approached in various ways.
1. What are the essential functions each cell must perform to sustain life, and what are the proteins responsible for performing those functions? This is a top down approach for identifying candidate genes whose essential role is then proven experimentally (see below). This approach enables essential genes to be categorized according to the essential cellular process or function which the gene product provides or of which the gene product is a necessary part. Table 1 shows such categories of essential genes and gene functions. In addition, the chromosomal location, where known, and gene product of certain example genes is provided. Thus, the categories of functions shown provide potential targets for the methods of this invention.
2. What genes have been proven essential for cell survival by mutagenesis or gene disruption experiments in cells of other organisms, such as hamster cells, mice, SUBSTITUTE SHEET (RULE 2fi) WO 98Lt1648 PCTlUS98/05419 flies, yeast, bacteria or other organisms? The idea of determining the necessity of specific genes for survival of an organism is well established in simple organisms such as bacteria and yeast. The consequences of gene disruption are easier to assess in these microorganisms that have a haploid genome because the haploid organism contains only one form of a particular single copy gene. A
particularly useful category of eukaryotic organisms are the yeasts, especially Saccharomyces cerevisae.
3. What are the protein targets of proven mammalian cytostatic and cytotoxic agents such as chemotherapy drugs and poisons?
4. What can be learned from genomics about the genes required for cell survival?
This analysis includes identification of the minimal gene set in simple prokaryotes, as well as sequence comparisons across widely divergent species.
5. Experimental testing of gene essentiality. As an example, antisense oligonucleotides can be used to down regulate candidate essential genes (identified by the four approaches listed above) and assess the effects on cell proliferation and survival. Application of an antisense approach to the identification of essential genes was described by Pestov & Lau, supra.
Once a gene coding for a protein or factor essential to cell viability is identified, its genomic DNA and cDNA sequences, if not previously established, can be ascertained and sequenced according to standard techniques known to those skilled in the art. See, for example, Sambrook, Fritsch and Maniatis, "Molecular Cloning, A Laboratory Manual," Cold Spring Harbor Press, Cold Spring Harbor, NY (1989).
Categories of essential genes Many essential genes function by encoding a gene product which is necessary for maintaining the level of a cellular constituent within the levels required for cell survival or proliferation. The survival and proliferation of cells within the body requires maintaining a state of homeostasis among many different cellular SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98105419 constituents. These may include, but are not limited to, specific proteins, nucleic acids, carbohydrates, lipids, organic ions, and inorganic ions, or cytoskeletal elements. The loss of homeostasis often results in cell death or apoptosis or inhibition of cell proliferation. Homeostasis in a living cell is dynamic, and 5 programed changes in homeostasis are required through the life cycle of the cell.
We have determined that those genes whose products are required for maintaining this homeostasis conducive to cell growth and survival are targets for anti-neoplastic e.g., anti-cancer, inhibitors as described in the methods herein. For example, many genes are involved in synthetic functions, allowing the cells to produce essential 10 cellular constituents including proteins, nucleic acids, carbohydrates, lipids, or organic ions or their components. Other genes are involved in the transport of essential constituents such as proteins, nucleic acids, carbohydrates, lipids, organic ions, or inorganic ions, or their components into the cell or among its internal compartments. Still other genes are involved in the chemical modification of 15 cellular constituents to form other constituents with specific activities.
Still other genes are involved in the elimination of specific cellular constituents such as proteins, nucleic acids, carbohydrates, lipids, organic ions, inorganic ions, or their components by metabolic degradation or transport out of the cell. The analysis is preferably carried out using genes which have been shown to be essential in human 20 cells or which are human homologs of genes which are essential in other organisms, preferably other eukaryotic organisms although useful essential data is also provided by prokaryotic essential genes.
A specific example are those genes that are involved in maintaining the amount and fidelity of DNA within a cell. This includes genes commonly considered to be 25 involved in "replication" and other functions; comprising genes involved in the synthesis (polymerization) of DNA sequences from its component elements, creating specific modifications of DNA, ensuring the proper compartmentalization of DNA during cell division (within the nucleus), and eliminating damaged DNA.
SUBSTITUTE SHEET (RULE 26) This also includes those genes involved in maintaining the amount of nucleosides that are the component elements of DNA by synthesis, salvage, or transport.
Another example are those genes that are involved in maintaining the amount of RNAs within a cell. This includes genes commonly considered to be involved in transcription and other functions; comprising genes required for the synthesis (polymerization) of linear RNA sequences from its component elements, ensuring proper compartmentalization of RNA within the cell, creating specific modification of the linear RNA molecule, and eliminating RNA. This also includes those genes involved in maintaining the amount of nucleosides that are the component elements of RNA by synthesis, salvage, or transport.
Another example are those genes that are involved in maintaining the amount of proteins within a cell. This includes those genes commonly considered to be part of "translation" and other functions;/ comprising genes required for transporting or synthesizing amino acids that are the component elements of proteins, synthesizing specific linear protein sequences from these amino acid elements, creating specific modifications of proteins including by not limited to the addition of specific nucleic acids, carbohydrates, lipids, or inorganic ions to the protein structure, ensuring the proper compartmentalization of synthesized proteins in the cell, and ensuring the proper elimination of proteins from the cell.
Another example are those genes that are involved in maintaining the amount of organic ions within the cell, including but not limited to amino acids, organic acids, fatty acids, nucleosides, and vitamins. This includes those genes that are required for transporting, or synthesizing organic ions, ensuring their proper compartmentalization within the cell, and ensuring proper elimination or degradation of these ions.
SUBSTITUTE SHEET (RULE 26) WO 98/11648 PCT/US98/0~419 Another example are those genes that are involved in maintaining the amount of inorganic ions within the cell. This includes those genes that are required for transporting inorganic ions, including but not limited to O, Na, K, Cl, Fe, P, S, Mn, Mg, Ca, H, P04 and Zn, ensuring their proper compartmentalization within the cell by binding or transporting these ions, and ensuring proper elimination from the cell.
Another example are those genes that are involved in maintaining the structures and integrity of the cell as described in Example 6 below.
The above groups of genes are shown in Table 1 below, which also points out useful subcategories of genes and lists particular exemplary target genes. This demonstrates that target genes can be grouped according to cellular function to provide classes of essential genes useful for allele specific targeting.
Additional target genes can be identified by routing methods, such as those described herein.
Confirmation of the essentiality of an additional gene in a specified gene category, and of the occurrence in normal somatic cells of sequence variances of the gene, and of the occurrence of LOH affecting the gene in a neoplastic disorder, establishes that the gene is a target gene potentially useful for identifying allele specific inhibitors and for other aspects of the invention. In addition, as described, target genes are useful in embodiments of certain aspects of the invention, e.g., transplantation and the use of essential or conditionally essential genes even in the absence of LOH.
Table 1 Gene Name GenBank Accession #
1) Genes Required For Cell Proliferation SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US9810~419 1.1 Genes that regulate cell division Cyclins, cyclin dependent kinases, regulators and effectors of cyclins and cyclin-dependent kinases 14-3-3 Protein TAU X56468 CCNA(G2/Mitotic-Specific Cyclin A ) X51688 CCNB 1 (G2/Mitotic-Specific Cyclin B 1 ) M25753 CCND 1 (G 1/S-Specific Cyclin D 1 ) M73554 CCND2(G1/S-Specific Cyclin D2) M90813 CCND3(GI/S-Specific Cyclin D3) M90814 Cell division control protein 16 018291 Cell division cycle 2, G 1 to S and G2 to M X05360 Cell division cycle 25A M81933 Cell division cycle 25B M81935 Cell division cycle 25C M34065 CeII division cycle 27 000001 Cell division-associated protein BIMB D79987 Cyclin A 1 (G2/Mitotic-Specific Cyclin A 1 ) 066838 Cyclin C (GI/S-Specific Cyclin C) M74091 Cyclin G1(G2/Mitotic-Specific Cyclin G) X77794 Cyclin G2 (G2/Mitotic-Specific Cyclin G) 047414 Cyclin H 011791 Cyclin H Assembly X87843 GSPT1(G1 to S phase transition 1) X17644 Mitotic MAD2 Protein 031278 RANBPI(RAN binding protein 1) D38076 Cell Division Protein Kinase 4 079269 CDC28 protein kinase 1 X54941 CDC28 protein kinase 2 X54942 M-Phase inducer phosphatase 2 M81934 M-phase phosphoprotein, mpp6 X98260 PPPIca(Protein phosphatase 1, catalytic subunit, alpha isoform)M63960 1.2 Genes that form structures of cell division including the centromere, kinetochore, kinesins, spindle pole body, chromatin assembly factors and their regulators CENP-F kinetochore protein 019769 Centromere autoantigen C M95724 SUBSTITUTE SHEET (RULE 26) WO 98l-11648 PCT/US98/05419 Centromere protein B (80kD) X05299 Centromere protein E (312kD) 215005 CHC 1 (Chromosome condensation 1 ) X 12654 Chromatin assembly factor-in p150 020979 subunit Chromatin assembly factor-in p60 020980 subunit Chromosome segregation gene homolog 033286 CAS

HMG 1 (High-mobility group (nonhistoneD63874 chromosomal) protein 1) Minichromosome Maintenance (MCM7) D28480 Mitotic centromere-associated kinesin063743 RMSAI{Regulator of mitotic spindle assembly 1) L26953 SUPTSh(Chromatin structural protein homolog (SUPTSH)) Y12790 2) Genes Required to Maintain Inorganic Ions and Vitamins at Levels Compatible with Cell Growth or Survival 2.1 Transport of inorganic ions and vitamins across the plasma membrane and intracellular membranes Active transporters Uniporters PMCA1 (Calcium Pump) 015686 PMCA2 (Calcium Pump) M97260 PMCA3 (Calcium Pump) 015689 PMCA4 (Calcium Pump) M83363 ATP2b1 (Calcium-Transporting ATPase304027 Plasma Membrane) ATP2b2 (Calcium-Transporting ATPaseX63575 Plasma Membrane) ATP2b4 (Calcium-Transporting ATPaseM83363 Plasma Membrane) ATPSb (ATP Synthase Beta Chain, X03559 Mitochondria) Precursor ) Chloride Conductance Regulatory X91788 Protein ICLN

H-Erg (Potassium Channel Protein 004270 EAG) Nuclear Chloride Ion Channel Protein093205 (NCC27) SCNIb(Sodium Channel, Voltage-Gated, Type in, Beta L16242 Polypeptide) Two P-Domain K-~- Channel TWIK-1 033632 VDAC2 (Voltage-Dependent Anion-Selective Channel Protein L06328 2) Coupled transporters Symporters ATPIbI (Sodium/Potassium-Transporting X03747 ATPase Beta-1 Chain) SUBSTITUTE SHEET (RULE 26) ATPlb2 (Sodium/Potassium-Transporting M81181 ATPase Beta-2 Chain) Antiporters ATPase, Ca++ transporting, M25874 plasma membrane 4 ATPase, Ca++ transporting, L20977 plasma membrane 2 ATPase, Na+/K+ transporting, U16798 alpha 1 polypeptide ATPase, Na+/K+ transporting, X12910 alpha 3 polypeptide ATPase, Na+/K+ transporting, U 16799 beta 1 polypeptide ATPase, Na+/K+ transporting, U45945 beta 2 polypeptide Na+,K+ ATPase, I Subunit Na+,K+ ATPase, 2 alpha Na+,K+ ATPase, 3 beta U51478 SLC9al(Solute carrier family 9 M81768 (sodium/hydrogen exchanger)) Solute carrier family 4, M27819 anion exchanger, member i Solute carrier family 4, U62531 anion exchanger, member 2 Solute carrier family 9 X76180 (sodium/hydrogen exchanger), Passive transporters MaxiK Potassium Channel Beta Subunit U25138 Chloride Channel 2 X83378 Chloride Channel Protein (CLCN7) U88844 TRPCI (Transient Receptor Potential Channel 1) X89066 Potassium Channel Kv2.1 L02840 ATPSd(ATP synthase, H+ transporting, X63422 mitochondria) F1 complex, delta subunit) ATPSfI(ATP synthase, H+transporting, X60221 mitochondria) FO complex, subunit b) AT'PSo(ATP synthase, H+ transporting, X83218 mitochondria) F1 complex, O subunit) ETFa(Electron-transfer-flavoprotein, J04058 . alpha polypeptide (glutaric aciduria II)) ETFb(Electron-transfer-flavoprotein, X71129 beta polypeptide) Nadh-ubiquinone oxidoreductase 13 kd-B subunit U53468 Nadh-ubiquinone oxidoreductase L04490 39 kD subunit precursor SUBSTITUTE SHEET (RULE 26) WO 98/11648 PCTIUS9810~419 NADH-Ubiquinone oxidoreductase X61100 75 kD subunit precursor NADH-Ubiquinone oxidoreductase MFWE subunitX81900 NDUFV2(NADH dehydrogenase M22538 (ubiquinone) flavoprotein 2 (241cD)) Ubiquinol-cytochrome c reductase M36647 complex 1 I kD

ATP Synthase Alpha Chain D14710 NADH dehydrogenase-ubiquinone 065579 Fe-S protein 8, 23 lcDa subunit Vitamin transporters Ascorbic Acid (uncloned) Folate Binding Protein AF000380 Folate receptor 1 (adult) M28099 Nicotinamide (uncloned) Pantothenic Acid X92762 Riboflavin (uncloned) SCLI9A1 (Solute Carrier Family 19, Memberl) Solute carrier family 19 (folate transporter), member 1 019720 Thiamine, B6, B 12 (uncloned) Metal transporters ATP7b (Copper-Transporting ATPase 2) 003464 Ceruloplasmin (ferroxidase) MI3699 Ceruloplasmin receptor (Copper Transporter) Copper Transport Protein HAH1 070660 Molybdenum, Selenium, other Transporters (uncloned) Tranferrin Receptor (Iron Transporter) X01060 Zinc Transporter (uncloned) Soluble inorganic ion transporters Insoluble inorganic ion transporters Transporters of other essential small molecules Mitochondrial Import Receptor D13641 Subunit TOM20 2.2 Regulators of transport Sensors of ion levels 3) Genes Required to Maintain Organic Compounds at Levels Compatible with Cell Growth or Survival 3.1 Transporters of organic compounds Carbohydrate Transport Sugar Transport Glucose Transport SUBSTITUTE SHEET (RULE 26) WO 981.11648 PCT/US98/05419 GLUT 1 GDB:120627 GLUT4 M2074?

w GLUT6 M95549 Solute carrier family 5 M95549 (sodium/glucose cotransporter) Solute carrier family 2 103810 (facilitated glucose transporter), member 2 Solute carrier family 2 M55531 (facilitated glucose transporter) member 5 Amino acid transport Solute carrier family 3 member 1 L 11696 System b,(Na+ independent) System y,(Na+ independent) ATRC 1 (Catioinc) OMIM 104615 LEUT(Leucine Transporter) OMIM 151310 SLC1A1(Solute Carrier Family 1, Member 1) OMIM 133550 Lipid or lipoprotein transport Nucleoside transport Other organic compounds transport Solute carrier family 16 L31801 (monocarboxylic acid transporters) 3.2 Genes required for maintenance of organic compounds at levels required for cell growth or survival Carbohydrate metabolism, including anabolism and catabolism ACOI (Aconitase 1 ) AC02(Aconitase 2, mitochondrial) U80040 Acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain M26393 Acy1-Coenzyme A dehydrogenase, C-4 to C-12 straight chain M16827 Acyl-Coenzyme A dehydrogenase, long chain M74096 Acyl-Coenzyme A dehydrogenase, very long chain D43682 aKGD (alpha ketoglutaratedehydrogenase) ALD-a (Aldolase) M11560 ALD-b (Aldolase) K01177 ALD-c (Aldolase) M21191 CS (Citrate Synthetase) OMIM 118950 Dihydrolipoamide S-succinyltransferase L37418 DLAT(Dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex)) DLD(Dihydrolipoamide dehydrogenase (E3 component of 103490 pyruvate dehydrogenase complex, 2-oxo-glutarate complex, branched chain keto acid dehydrogenase complex)) Elk (Oxoglutarate dehydrogenase) D10523 SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/0~419 E2k (Dihydrolipoamide S-succinyltransferase)D16373 E3 (Dihydrolipoyl Dehydrogenase) SEG_HUMDHL

ENO1(Enolase l,alpha) M14328 EN02(Enolase 2) X51956 EN03(Enolase 3) X55976 Enolase 2, (gamma, neuronal) M22349 Enolase 3, (beta, muscle) X16504 FH(Fumarate hydratase) M15502 G3PDH (Glyceraldehyde-3-Phosphate M17851 Dehydrogenase) G6PD (Glucose-6-Phosphate Dehydrogenase) Glucose-6-phosphate dehydrogenase X03674 HK1 (Hexokinase I) M75126 HK2 (Hexokinase 2) S70035 HK3 (Hexokinase 3) U51333 IDH1(Isocitrate dehydrogenase 1 (NADP+),OMIM 147700 soluble) IDH2(Isocitrate dehydrogenase 2 (NADP+),X69433 mitochondrial) MDH 1 (Malate dehydrogenase 1, NAD D55654 (soluble)) MDH2(Malate dehydrogenase 1, NAD OMIM 154100 (mitochondrial)) NAD(H)-specifc isocitrate dehydrogenaseU07681 alpha subunit Oxoglutarate dehydrogenase (lipoamide)D10523 PDHB (Pyruvate Dehydrogenase) J03576 PDHB(Pyruvate dehydrogenase (Iipoamide)M34479 beta) PDK4 (Pyruvate dehydrogenase kinase,U54617 isoenryme 4) PFKL(Phosphofructokinase) M 10036 PGI (Phosphoglucoisomerase) OMIM 172400 PGKa (Phosphoglyceromutase) Y00572 PGKb (Phosphoglyceromutase) K03201 PGM1 (Phosphoglyceromutase) M83088 PGM2 (Phosphoglyceromutase) OMiM 172000 PGM3 (Phosphoglyceromutase) OMIM 172100 PGM4 (Phosphoglyceromutase) OMIM 172110 Phosphofructokinase, muscle U24183 Phosphoglucomutase 1 M83088 Phosphoglycerate kinase 1 V00572 PK1 (Pyruvate Kinase) M15465 PK2 (Pyruvate Kinase) OMIM 179040 PK3 (Pyruvate Kinase) M23725 Pyruvate dehydrogenase kinase isoenzymeL42451 2 (PDK2) Pyruvate kinase, liver D10326 Pyruvate kinase, muscle M23725 SDH 1 (Succinate dehydrogenase, ironD 10245 sulphur (Ip) subunit) SDH2(Succinate dehydrogenase 2, flavoproteinD30b48 (Fp) subunit) TKT(Transketolase (Wernicke-KorsakoffL12711 syndrome)) TPI (Trisephosphate Isomerase) M10036 SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98105419 Amino Acid biosynthesis and processing Asparagine Synthetase SEG HUMASN

Aminoacylase-1 L07548 Aminoacylase-2 S67156 Fatty acid biosynthesis and processing ACAC (Acetyl CoA Carboxylase Beta) 019822 ACAC (Acetyl CoA Carboxylase) U 12778 ACADSB(Acyl-coA dehydrogenase) U 12778 Mevalonate kinase M88468 Phosphomevalonate kinase L77213 Alcohol biosynthesis and processing Other organic compounds biosynthesis and processing Aspartoacylase S67156 Ornithine decarboxylase 1 M16650 3.3 Genes required for catabolism, degradation and elimination of organic compounds Carbohydrate and Sugar Catabolism Amino acid Degradation Lipid or lipoprotein Degradation Short-acyl-CoA dehydrogenase M26393 Medium acyl-CoA dehydrogenase S75214 Long acyl-CoA dehydrogenase M74096 Isovalveryl CoA dehydrogenase M34192 2-methyl branched chain Nucleoside Degradation Adenosine Deaminase K00509 Purine-nucleoside phosphorylase K02574 Guanine Deaminase Xanthine Oxidase DI 1456 Degradation of other organic compounds 3.4 Genes Required to Modify Polypeptides, Lipids or Sugars by Addition, Removal or Modification of Chemical Groups to Form Compounds Necessary for Cell Growth or Survival Addition, removal or modifcation of sugar groups Glycosyltransferases Glycosylases ITM1 (Integral Transmembrane Protein) L38961 GFPT (Glutamine-Fructose-6-Phosphate M90516 Transaminase) Heparan 036601 Polypeptide N-Acetyltransferase 041514 Addition, removal or modification of methyl or other alkylgroups Acetyltransferase ACAA(Acetyl-Coenzyme A acyltransferase)X12966 Lysophosphatidic acid acyltransferase-alpha056417 SUBSTITUTE SHEET (RULE 26) WO 98/-11648 PCT/US98/0~419 Lysophosphatidic acid acyltransferase-beta056418 Farnesyltransferase FNTa (Farnesyltransferase Alpha Subunit)L00634 FNTb (Farnesyltransferase Beta Subunit)L00635 Myristoylation NMT1 (N-myristoyltransferase) Addition, removal or modification of sulfhydryl groups Addition, removal or modification of phosphate groups Calcineurin A S46622 Calcineurin B M30773 Calreticulin Precursor M84739 Phosphatase 2b M29551 PPP3ca(Protein phosphatase 3 , catalytic105480 subunit) SNK Interacting 2-28(Calcineurin 083236 B Subunit) Protein Kinase C

PRKCA(Protein kinase C, alpha) X52479 PRKCB 1 (Protein kinase C, beta 1 X06318 ) PRKCD(Protein kinase C, delta) L07861 PRKCM(Protein kinase C, mu) X75756 PRKCQ(Protein kinase C-theta) L01087 PRKCSH(Protein kinase C substrate 103075 SOK-H) Addition, removal or modification of iipid groups Geranylgeranyl Geranylgeranyltransferase (Type I Beta) L25441 GGTB (Geranylgeranyltransferase) Y08201 Geranylgeranyltransferase (Type II Beta-Subunit) X98001 3.5 Genes required for regulation of levels of organic ions Gdp Dissociation Inhibitors GDI Alpha (RAB GDP Dissociation Inhibitor Alpha) D45021 Rab Gdp (RAB GDP Dissociation Inhibitor Alpha) D13988 4) Genes Required to Maintain Cellular Proteins at Levels Compatible with Cell Growth or Survival Polypeptide precursor biosynthesis Amino acid biosynthesis and modification GOT(Glutamic-oxaloacetic transaminase 2) M22632 GOTI(Glutamic-oxaloacetic transaminase 1) M37400 PYCS(Pyrroline-5-carboxylate synthetase) X94453 Tyrosine aminotransferase X52520 Polypeptide precursor elimination Synthesis of components for polypeptide polymerization ppRS D32050 DARS

SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 FARS

SARS

YARS

Polypeptide polymerization Ribosome Subunits Ribosomal Protein L11 X79234 Ribosomal Protein L 12 L06505 Ribosomal Protein L17 X52839 Ribosomal Protein L18 L11566 Ribosomal Protein Ll8a X80822 Ribosomal Protein L19 X63527 Ribosomal Protein L21 014967 Ribosomal Protein L22 L21756 Ribosomal Protein L23 X53777 Ribosomal Protein L23a 043701 Ribosomal Protein L25 Ribosomal Protein L26 Ribosomal Protein L27 L19527 Ribosomal Protein L27a 014968 Ribosomal Protein L28 014969 Ribosomal Protein L29 010248 Ribosomal Protein L30 OMIM I 80467 Ribosomal Protein L31 Ribosomal Protein L32 X03342 Ribosomal Protein L35 012465 Ribosomal Protein L35a X52966 Ribosomal Protein L36a OMIM 180469 Ribosomal Protein L39 057846 Ribosomal Protein L4 L20868 Ribosomal Protein L41 Ribosomal Protein L44 SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 Ribosomal Protein L6 X69391 Ribosomal Protein L7 L16558 Ribosomal Protein L7a X52138 Ribosomal Protein L8 228407 Ribosomal Protein L9 009953 Ribosomal Protein P1 M17886 Ribosomal Protein SIO 014972 Ribosomal Protein S 11 X06617 Ribosomal Protein S 13 L01124 Ribosomal Protein S 14 Ribosomal Protein SIS J02984 Ribosomal Protein SISA X84407 Ribosomal Protein S16 M60854 Ribosomal Protein S17 M13932 Ribosomal Protein S 17A OMIM 180461 Ribosomal Protein S17B OMIM 180462 Ribosomal Protein S18 L06432 Ribosomal Protein S20 Ribosomal Protein S20A OMIM 180463 Ribosomal Protein S20B OMIM 180464 Ribosomal Protein S21 L04483 Ribosomal Protein S23 D14530 Ribosomal Protein S25 M64716 Ribosomal Protein S26 X69654 Ribosomal Protein S28 058682 Ribosomal Protein S29 L31610 Ribosomal Protein S3 014990 Ribosomal Protein S3A OMIM 180478 Ribosomal Protein S4 Ribosomal Protein S4X M58458 Ribosomal Protein S4Y M58459 Ribosomal Protein SS 014970 Ribosomal Protein S6 J03537 Ribosomal Protein S7 M77233 Ribosomal Protein S8 OMIM 600357 Ribosomal Protein S9 014971 Initiation of polypeptide polymerization eIF-2 (Eukaryotic initiation factor) L1916I

eIF-2-associated p67(Eukaryotic initiation013261 factor) eIF-2A(Eukaryotic initiation factor) J02645 eIF-2Alpha(Eukaryotic initiation factor)026032 eIF-2B(Eukaryotic initiation factor) 023028 eIF-2B-Gamma(Eukaryotic initiation factor)L40395 eIF-2Beta(Eukaryotic initiation factor) M29536 SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 eIF-3 p 110(Eukaryotic initiation factor) U46025 eIF-3 p36(Eukaryotic initiation factor) U39067 eIF-4A(Eukaryotic initiation factor) D21853 eIF-4C(Eukaryotic initiation factor) L18960 eIF-4E(Eukaryotic initiation factor) M15353 eIF-4Gamma(Eukaryotic initiation factor) 234918 e1F-5(Eukaryotic initiation factor) U49436 eIF-SA

Polypeptide elongation Eukaryotic peptide chain release factor subunit 1 X81625 P97(Eukaryotic initiation factor) U73824 eEFlA2(Eukaryotic elongation factor) X70940 eEFID(Eukaryotic elongation factor) 221507 eEF2(Eukaryotic elongation factor) X54166 eIF4A2 (Eukaryotic initiation factor) D30655 KIAA0031 (Elongation factor 2) D21163 KIAA0219{Putative translational activator C 18G6.OSC) D86973 Factor 1-Alpha 2(Eukaryotic translation elongation factor D30655 alpha 2) Termination of polypeptide polymerization Polypeptide folding Cis-Trans Isomerase M80254 DNAj Protein Homolog 1 X6242I

DNAj Protein Homolog 2 D13388 DNAJ Protein homolog HSJ1 X63368 Chaperone proteins T-Complex Aspartylglucosaminidase X55330 T-Complex 1, Alpha S70154 T-Complex 1, Epsilon D43950 T-Complex 1, Gamma X74801 T-Complex 1, Theta D13627 T-Complex l, Zeta M94083 Polypeptide Degradation Proteasome components and proteinases 26S Protease regulatory subunit 4 L02426 Alpha-2-Macroglobulin M11313 Calpain 1, Large X04366 CLPP(ATP-Dependent CLP protease proteolytic subunit) 250853 KIAA0123 (Mitochondria) processing peptidase alpha subunit) D50913 Proteasome Beta 6 D29012 Proteasome Beta 7 D38048 Proteasome C 13 U 17496 SUBSTITUTE SHEET (RULE 26) WO 98/-11648 PCTIUS98/0~419 Proteasome C2 D00759 Proteasome C7-1 D26599 Proteasome inhibitor hPI31 subunit D88378 Proteasome P112 D44466 Proteasome P27 AB003177 Proteasome P55 AB003103 Ubiquitin System Enzyme E2-17 Kd(Cyclin-selective ubiquitin carrierU73379 protein) ISOT-3(Ubiquitin carboxyl-terminal hydrolase U75362 T ) ORF (Ubiquitin carboxyl-terminal hydrolase 14) M68864 PGP(Ubiquitin carboxyl-terminal hydrolase isozymeX04741 L1) UBA52(Ubiquitin A-52 residue ribosomal protein S79522 fusion product 1) Ubiquitin carboxyl-terminal hydrolase 3 D80012 Ubiquitin carboxyl-terminal hydrolase isozyme M30496 Ubiquitin carboxyl-terminal hydrolase T X91349 Ubiquitin carrier protein (E2-EPF) M91670 Ubiquitin fusion-degradation protein (UFD1L) 064444 Ubiquitin Hydrolase X98296 Ubiquitin-conjugating enzyme E2I 045328 Polypeptide Transport SEC23(Protein transport protein SEC23) X97065 SEC23A(Protein transport protein SEC23) X97064 SEC7(Protein transport protein SEC7) X99688 SEC61 (Beta Subunit) L25085 Lipoprotein Transport LDLR (LDL receptor) 5) Genes Required to maintain Cellular Nucleotides at Levels Compatible with Cell Growth or Survival Genes Required to Maintain Cellular DNA with Fidelity and at Levels Compatible with Cell Growth or Survival DNA Precursor Biosynthesis Adenylate Kinase-2 039945 Adenylosuccinate synthetase X66503 Adenylosuccinate Lyase X65867 ADPRT (ADP-Ribosyltransferase) M32721 ADSL (Adenylosuccinate Iyase/AMP synthetase) X65867 ADSS (Adenylosuccinate Synthetase) X66503 CTP Synthetase CTPS(CTP synthetase} X52142 Cytidine Triphosphate Synthetase GARS (Phosphoribosylglycinamide synthetase) D32051 SUBSTITUTE SHEET (RULE 26) WO 98/1!648 PCT/US98/05419 GART (Phosphoribosylglycinamide formyltransferase) GART(Phosphoribosyiglycinamide formyltransferase, X54199 phosphoribosylglycinamide synthetase, - phosphoribosylaminoimidazole synthetase) GMP Synthetase U 10860 IMP Cyclohydrolase 037436 IMP dehydrogenase L 19709 IMPDH 1 (IMP (inosine monophosphate) dehydrogenase 1 ) 105272 IMPDH2(IMP (inosine monophosphate) dehydrogenase 2) 104208 Phosphoribosyl diphosphotransferase Phosphoribosylaminoimidazolecarboxamide formyltransferase Phosphoribosylformylglycinamide synthetase M32082 Phosphoribosylglycinamide carboxylase Phosphoribosylglycinamide-succinocarboxamide synthetase PPAT (Amidophoribosyltransferase) PPAT(Phosphoribosyl pyrophosphate amidotransferase) 000238 Ribonucleoside-diphosphate reductase MI chain X59543 Ribonucleoside-diphosphate reductase M2 chain X59618 Thymidine Kinase K02581 Thymidylate Synthase X02308 UMK(Uridine kinase) D78335 UMPK (Uridine monophosphate kinase) OMIM 191710 UMPS(CTridine monophosphate synthetase (orotate 103626 phosphoribosyl transferase and orotidine-5'-decarboxylase)) Uridine Phosphorylase X90858 DNA Precursor Elimination DNA Replication Origin Recognition Origin Recognition Complex ORC Regulators - DNA Polymerization DNA Polymerases Adprt (NAD(+) ADP- Ribosyltransferase) M18112 - DNA Polymerase Alpha-Subunit X06745 DNA Polymerase Delta 021090 SUBSTITUTE SHEET (F~ULE 26) WO 98141648 PCTlUS98/0~419 POLa(DNA Polymerase Alpha/Primase AssociatedL24559 Subunit) POLb(DNA Polymerase Beta Subunit) D29013 POLdl(Polymerase (DNA directed), Delta 1, M81735 Catalytic Subunit) POLd2(Polymerase (DNA directed), Delta 2) 021090 POLE(Polymerase (DNA directed)) OMIM 174762 POLg (DNA Polymerase Gamma Subunit) X98093 Terminal Transferase (DNA NucleotidylexotransferaseM11722 ) Accessory factors for DNA Polymerization Activator 1 36 Kd L07540 CDC46 (DNA Replication Licensing Factor) X74795 CDC47 (DNA Replication Licensing Factor CDC47)D55716 DNA Topoisomerase III 043431 DRAP1 (DNA Replication Licensing Factor MCM3)041843 KIAA0030 Gene (Cell Division Control ProteinX67334 19) KIAA0083 Gene (DNA Replication Helicase DNA2D42046 ) MCM3 (DNA Replication Licensing Factor MCM3)D38073 PCNA (Proliferating Cell Nuclear Antigen) J04718 PRIMI (DNA Primase 49 kD Subunit ) X74330 PRIM2 (DNA Primase) X74331 PRIM2a (DNA Primase 58 kD Subunit) X74331 PRIM2b (DNA Primase) OMIM 600741 RECa (Replication Protein A 14 kD Subunit) L07493 RFC1 {Replication Factor C (activator 1) L14922 I) RFC2 (Replication Factor C 2) M87338 RFC3 (Replication Factor C (activator I) L07541 3) RFC4 (Replication Factor C, 37-kD subunit) M87339 RFCS (Replication Factor C) OMIM 600407 RPA 1 (Replication protein A 1 (70kD)) M63488 RPA2 (Replication protein A2 (32kD)) J05249 RPA3 (Replication protein A3 ( l4kD)) L07493 TOP1 (DNA Topoisomerase I) J03250 TOP2a (Topoisomerase (DNA) II Alpha (170kD))J04088 TOP2b (Topoisomerase (DNA) II Beta (180kD)) 054831 DNA Helicases CHL1(CHLI-Related Helicase) 033833 DNA Helicase II M30938 Mi-2(Chromodomain-Helicase- DNA-Binding ProteinX86691 CHD-1 ) RECQL (ATP-Dependent DNA Helicase Q1) L36140 Smbp2 (DNA-Binding Protein SMUBP-2) L14754 DNA Packaging Proteins Histones H1(0) (Histone HSA) X03473 Histone H 1 d X57129 Histone Hlx D64142 SUBSTITUTE SHEET (RULE 26) WO 98/x1648 PCT/US98/05419 Histone H2a.1 U90551 Histone H2a.2 L 19779 Histone H2b.1 M60756 Histone H4 X60486 SLBP (Histone Hairpin-Binding Protein ) 271188 DNA Degradation DNA Repair Genes Required to Maintain Cellular RNA at Levels Compatible with Cell Growth or Survival RNA Precursor Biosynthesis RNA Precursor Elimination RNA Polymerization Initiation of polymerization TATA-binding Complex Small Nuclear RNA-Activating Complex, Polypeptide i, 43KD 247542 (SNAPC 1 ) Small Nuclear RNA-Activating Complex, Polypeptide 2, (SNAPC2) Small Nuclear RNA Activating Complex, Polypeptide 3, SOKD U71300 (SNAPC3) TAF2D(TBP-associated factor) U78525 TAFII100(TBP-associated factor) X95525 TAFII130(TBP-associated factor) U75308 TAFII20(TBP-associated factor) X84002 TAFII250(TBP-associated factor) D90359 TAFII28(TBP-associated factor) X83928 TAFII30(TBP-associated factor) U 13991 TAFII32(TBP-associated factor) U21858 TAFII40(TBP-associated factor) TAFII55(TBP-associated factor) U18062 TAFII80(TBP-associated factor) U31659 TBP(TATA Binding Protein) M55654 TMF1 (TATA Element Moduiatory Factor 1) Polymerization RpB 7,0 U52427 RPB 7.6 RPB 14.4 RNA Polymerise I subunits RNA polymerise I subunit hRPA39 AF008442 RNA Polymerise II subunits 13.6 Kd Polypeptide (DNA-Directed RNA Polymerise II 13.6 L37127 1cD Polypeptide) POLR2C(RNA polymerise II, polypeptide C (331cD)) J05448 SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTJUS9810~419 Polypeptide A (220kd) X63564 RNA Polymerase II 23k J04965 RNA polymerise II holoenzyme component (SRB7)046837 RNA polymerise II subunit (hsRPB 10) 037690 RNA polymerise II subunit (hsRPBB) 037689 RNA polymerise II subunit hsRPB4 085510 RNA polymerise II subunit hsRPB7 020659 RNA Polymerise II Subunit(DNA- Directed RNA 247727 Polymerises I, II, and III 7.3 kD polypeptide) TCEB1L(Transcription elongation factor B 247087 (SIII), polypeptide 1-like) RNA Polymerise III subunits RNA polymerise III subunit (RPC39) 093869 RNA polymerise III subunit (RPC62) 093867 RNA Elongation Elongation Factor 1-Beta X60489 Elongation Factor S-II M81601 Elongation TCEA ( 1 l OkD) OMIM 601425 TCEB (l8kD) TCEC (lSkDa) TFIIS (Transcription Elongation Factor IIS) 601425 E2F1 (E2F Transcription Factor) M96577 TFAP2A (Transcription Factor A2 Alpha) X95694 TFCP2 (Transcription Factor CP2) 001965 TFC 12 (Transcription Factor 12) M65209 PRKDC (Protein Kinase, DNA activated catalytic047077 subunit) Termination of RNA polymerization Factors that regulate RNA polymerization General factors TFIIA gamma subunit 014193 TFIIA delta TFIIB related factor hBRF (HBRF) 075276 TFIIE Alpha Subunit X63468 TFIIE Beta Subunit X63469 TFIIF, Beta Subunit X16901 GTF2F1 (TFIIF) X64037 GTF2F2 (TFIIF) X 16901 General Transcription Factor IIIA 020272 TFIIH(52 kD subunit of transcription factor)Y07595 SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTlUS98/05419 TFIIH(p89) TFIIH(p80) TFIIH(p62) 007595 TFIIH(p44) OMIM 601748 TFIIH(p34) OMIM 601750 Transcription Factor IIf(General transcriptionX64037 factor IIF, polypeptide 1 (74kD subunit)) Specific factors required for polymerization of essential genes BTF 62 kDSubunit (Basic transcription factorM95809 62 kD subunit) CAMP-dependent transcription factor ATF-4 M86842 CCAAT box-binding transcription factor 1 X92857 CRM1(Negative regulator CRM1) Y08614 Cyclic-AMP-dependent transcription factor X55544 GABPA(GA-binding protein transcription factor,013044 alpha subunit (60kD)) ISGF-3(Signal transducer and activator of M97935 transcription 1-alpha/beta) NFIX(Nuclear factor I/X (CCAAT-binding transcriptionL31881 factor)) NFYA(Nuclear transcription factor Y, alpha) M59079 NTF97(Nuclear factor p97) L38951 Nuclear factor I-B2 (NFIB2) 085193 Nuclear factor NF45 U 10323 Nuclear factor NF90 010324 POU2F1(POU domain, class 2, transcription X13403 factor I) Sp2 transcription factor M97190 TCF12(Transcription factor 12 (HTF4, helix-loop-helixM83233 transcription factors 4)) TCF3(Transcription factor 3 (E2A immunoglobulinM31523 enhancer binding factors E 12/E47)) TCF6L1(Transcription factor 6-like I) M62810 TF P65(Transcription factor p65) L19067 TFCOUP2(Transcription factor COUP 2 (a.k.a. X91504 ARPI)) Transcription factor IL-4 Stat 016031 Transcription Factor S-Ii (Transcription D50495 factor S-II-related protein) Transcription factor StatSb 048730 Transcription Factor L06633 Transcription factor (CBFB) L20298 RNA Processing Factors RNA splicing and other processing factors 9G8 Splicing Factor (Pre-mRNA Splicing factorL22253 SRP20) CC1.3(Splicing factor (CC1.3)) L10910 HnRNP F protein L28010 HNRPA2B1(Heterogeneous nuclear ribonucleoproteinsM29065 A2B1) HNRPG(Heterogeneous nuclear ribonucleoprotein223064 G) SUBSTITUTE SHEET (RULE 26) .. .... _... ....~.-~..._...-..~.~.:_._.__....~.~.

WO 98/.11648 PCTlUS98/05419 HNRPK(Heterogeneous nuclear ribonucleoproteinS74678 K) Pre-mRNA splicing factor helicase D50487 Pre-mRNA splicing factor SF2, P33 subunit M69040 Pre-mRNA splicing factor SRP20 L10838 Pre-mRNA splicing factor SRP75 L14076 PRP4(Serine/threonine-protein kinase PRP4)048736 PTB-Associated Splicing Factor X16850 Ribonucleoprotein A' X06347 Ribonucleoprotein AI X13482 Ribonucleoprotein C1/C2 M15841 RNP Protein, L (Heterogeneous nuclear ribonucleoproteinX16135 L) RNP-Specific C(LJI small nuclear ribonucleoproteinX12517 C ) SAP 145(Spliceosome associated protein 041371 ) SAP 61(Splicesomal protein) 008815 SC35(Splicing factor) L37368 SF3a120 X85237 SFRS2(Splicing factor, arginine/serine-richM90104 2) SFRSS(Splicing factor, arginine/serine-richAF020307 5) SFRS7(Splicing factor, arginine/serine-richL41887 7) Small nuclear ribonucleoprotein SM D1 J03798 SnRNP core protein Sm D2 015008 SnRNP core protein Sm D3 015009 SNRP70(LJ1 snRNP 70K protein) M22636 SNRPB(Small nuclear ribonucleoprotein polypeptidesJ04564 B and BI) SNRPE(Small nuclear ribonucleoprotein polypeptideM37716 E) SNRPN(Small nuclear ribonucleoprotein polypeptide041303 N) Splicing factor SF3a120 X85238 Splicing factor U2AF 35 kD subunit M96982 Splicing factor U2AF 65 kD subunit X64044 SRP30C(Pre-mRNA splicing factor SF2, p33 030825 subunit) SRP55-2(Pre-mRNA splicing factor SRP75) 030828 Transcription factor BTEB D31716 Transcription initiation factor TFIID 250 D90359 kD subunit RNA polyadenylation and cleavage Cleavage and polyadenylation specificity 037012 factor Cleavage stimulation factor, 3' pre-RNA, L02547 subunit 1, 50kD

Cleavage stimulation factor, 3' pre-RNA, 015782 subunit 3, 77kD

HNRNP Methyltransferase D66904 PABPL1(Poly(A)-binding protein-like 1) Y00345 Pap mRNA(Poly(A) Polymerase) X76770 RNA unwinding RNA Helicase SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 GU Protein (ATP-Dependent RNA helicase dead) 041387 KIAA0224 Gene(Putative ATP-dependent RNA helicase) D86977 RNA Helicase A L13848 RNA Helicase PI10 050553 Stel3(Nuclear RNA Helicase) 090426 RNA Degradation RNA modification RNA Transport 6) Genes Required to Maintain Integrity and Function of Cellular and Subcellular Structures 6.1 Genes Required to Move Proteins, Small Particles, and Other Ligands Across Membranes to Maintain their Concentration at Levels Compatible with Cell Growth or Survival Genes required to form coated pits and vesicles Clathrins AP47(Clathrin Coat Assembly AP47 ) D38293 AP50(Clathrin Coat Assembly Protein AP50) 036188 Cell Surface Protein (Clathrin Heavy Polypeptide-Like) X83545 Protein Cltb(Clathrin Light Chain B) M20470 Cltc (Clathrin Heavy Chain) 041763 6.2 Genes Required to Transmit Signals within Cells at Levels Compatible with Cell Growth or Survival Genes required to transmit signals from membranes Adenylate Cyclase Adenylate Cyclase D63481 Adenylate Cyclase, II X74210 Adenylate Cyclase,IV D25538 Genes required to transmit signals within cellular compartments 6.3 Genes Required to Maintain Cellular Energy Stores at Levels Compatible with Cell Growth or Survival Genes required to Produce ATP from catabolism of sugar Genes required for glycolysis (anaerobic and aerobic) Genes required for oxidative phosphorylation Complex I

MTND1 (Subunit NDI) OMIM 51600 MTND2 (Subunit ND2) OMIM S 1601 MTND3 (Subunit ND3) OMIM 51602 MTND4 (Subunit ND4) OMIM 51603 MTND4L (Subunit ND4L) OMIM 51604 MTNDS (Subunit NDS) OMIM 51605 MTND6 (Subunit ND6) OMIM 51606 Complex II

Complex III

Cytochrome b subunit Complex IV
SUBSTITUTE SHEET (RULE 26) CO1 (Cytochrome c Oxidase Subunit I) OMIM 516030 C02 (Cytochrome c Oxidase Subunit 2) AF035429 C03 (Cytochrome c Oxidase Subunit 3) Complex V

ATP Synthase Subunit ATPase 6 OMIM 516060 6.4 Genes Required to Transport or Dock Vesicles, Polypeptides or Other Solutes Moving Between Cellular Compartments at Rates and Levels Compatible with Cell Growth or Survival Transport to, from or within the cytoplasm Kinesins Kinesin Heavy Chain X65873 Kinesin Light Chain L04733 Syntaxin Syntaxin la L37792 Syntaxin lb U07158 Syntaxin 3 U32315 Syntaxin Sa U26648 Syntaxin 7 U77942 Transport to, from or within the endoplasmic reticulum CANX (Calnexin) M94859 ER Lumen Protein 1 M88458 ER Lumen Protein 2 X55885 Ribophorin I Y00281 Ribophorin II Y00282 Signal recognition particle receptor X06272 SRP Protein U20998 TIM17 preprotein translocase X97544 Transport to, from or within the Golgi apparatus Golgin-245 U31906 TGN46 (Traps-Golgi Network Integral MembraneX94333 Protein TGN38 Precursor ) Transport to, from or within the other membrane bound compartments Beta-Cop X82103 Coatomer Beta' Subunit X70476 Coatomer Delta Subunit X81198 Gp36b Glycoprotein (Vesicular integral-membraneU10362 protein VIP36 precursor) Homologue of yeast sec? M85169 Protein transport protein SEC 13 (ChromosomeL09260 3p25) SEC14 (S. Cerevisiae) D67029 Synaptic vesicle membrane protein VAT-1 U18009 Synaptobrevin-3 U64520 Synaptotagmin I M55047 Transmembrane(COP-coated vesicle membrane X92098 protein p24 precursor) SUBSTITUTE SHEET (RULE 26) WO 98Lt1648 PCT/US98/06419 Vacuolar-Type (Clathrin-coated vesicle/synaptic vesicle proton 271460 pump 116 kd subunit ) Transport to, from or within the nucleus Nuclear membrane constituents 140 kD Nucleolar phosphoprotein D21262 Autoantigen p542 L38696 Export protein Rael (RAE1) U84720 Heterogeneous nuclear ribonucleoproteinX79536 A I

Nuclear pore complex protein hnup153 225535 Nuclear pore complex protein NUP214 D14689 Nuclear pore glycoprotein p62 X58521 Nuclear Transport Factor 2 X07315 Nucleoporin 98 (MJP98) U41815 Ribonucleoprotein A M29063 Ribonucleoprotein B" U23803 Nuclear envelope & pore constituents Karyopherin Importin Alpha Subunit D89618 TRN (Transportin) U70322 6.5 Genes Required to Maintain Cell Shape and Motility at Levels Compatible with Cell Growth or Survival Cell structure genes (Cytoskeleton) Actin X04098 Beta-Centractin X82207 Capping Protein Alpha U03851 CFL1 (Cofilin, Non-Muscle Isoform) X95404 Desmin J03191 Dystrophin U26743 Gelsolin X04412 hOGG 1 (Myosin Light Chain Kinase) AB000410 IC Heavy Chain U31089 Itga2 (Integrin, Aipha 2 (CD49B, X17033 alpha 2 Subunit of VLA-2 receptor)) Itga3 (Integrin Alpha-3 Precursor) M59911 Keratin 19 Y00503 Keratin, Type II J00269 Lamin A M13451 LBR(Lamin B Receptor) L25931 Light Chain Alkali M22920 MacMarcks mRNA X70326 MAPIa (Microtubule-Associated ProteinU14577 lA) MAP2(Microtubule-Associated Protein U01828 2) MEG 1 (Protein-Tyrosine Phosphatase X79510 MEG 1 ) SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 Microtubule-Associated Protein TAU J03778 Suppresser Of Tubulin STU2 X92474 TUBg (Tubulin Gamma Chain) M61764 Tubulin Alpha-4 Chain X06956 USHlb (Myosin II Heavy Chain) 039226 Villin X 12901 Villin 2 (Ezrin) J05021 Genes required for cell motility Actin genes Actin Depolymerizing S65738 Capping (Actin Filament) M94345 Myosin genes MYH9(Myosin, Heavy Polypeptide 9, Non-Muscle) M31013 MYLS(Myosin Regulatory Light Chain 2) L03785 Myosin Heavy Chain 95F 090236 Myosin Heavy Chain IB D63476 Myosin IB 014391 Sh3p17(Myosin IC Heavy Chain) 061166 Sh3p18(Myosin IC Heavy Chain) 061167 KIAA0059(Dematin:Actin-Bundling Protein) D31883 TTN (Titin:Myosin Light Chain Kinase) X69490 6.6 Genes Required to Eliminate, Transform, Sequester or Otherwise Regulate Levels of Endogenous Cellular Toxins or Waste Substances at Levels Compatible with Cell Growth or Survival Organelles that transform or sequester toxic or waste substances Vacuoles ATP6c(Vacuolar H+ ATPase proton channel subunit) M62762 Lysosomes ATP6al (ATPase, H+ Transporting, LysosomalL09235 (Vacuolar Proton Pump), Alpha Polypeptide, 701cD) ATP6b1(ATPase, H+ transporting, lysosomaIM25809 (vacuolar proton pump), beta polypeptide, 56/58kD) ATP6d(ATPase, H+ transporting, lysosomal X69151 (vacuolar proton pump) 42kD) ATP6e(ATPase, H+ transporting, lysosomal X71490 (vacuolar proton pump) 3lkD) ATPase, H+ transporting, lysosomal (vacuolarX76228 proton pump) 3lkD

Free radical inactivation Superoxide Dismutase X02317 Maintenance of cellular redox potential at levels compatible with cell survival SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 Conditionally essential genes As indicated in the Summary, some genes are conditionally essential, meaning that they are essential for cell survival or proliferation only in certain circumstances.
Most commonly such circumstances are related to changes in the environment, 5 such as changes in the concentration of specific constituents such as nutrients, administration of pharmaceuticals (drugs), or physical elements affecting the cell.
In many cases the changes in the environment may be induced as part of a treatment regiment for cancer such as the administration of drugs or ionizing radiation. In the presence of such specific environmental changes or therapies, 10 genes with are not normally essential for cell survival or proliferation become essential and, consequently, targets for therapy under the present invention.
Therapy with inhibitors of conditionally essential genes involves administration of the inhibitor together with a chemical or physical elements that causes the target gene to be essential for cell survival or proliferation. The use of allele specific 15 inhibitors in the current invention allows specific killing of cancer cells with such chemical or physical agent since the gene function that is essential for the survival of cells (in the presence of the chemical or physical agent) is inhibited in the cancer cell but not in the normal cell.
This strategy begins with the identification of heterozygous alleles of genes coding 20 for proteins that are conditionally essential for cell viability or growth due to change in the chemical or physical environment. In one aspect of this invention, the gene targets of this application are responsible for mediating cell response to changes in the environment. Such environmental alterations include, for example, changes in the concentration of naturally occurring constituents such as amino 25 acids, sugars, lipids and inorganic and organic ions, as well as larger molecules such as hormones or antibodies, or changes in the partial pressure of oxygen or other gasses. The absence of a specific constituent in the environment makes the genes that are involved in synthesizing that nutrient within the cell essential, SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98105419 whereas if the constituent were present in the environment in sufficient quantities, such genes would not be essential. Alternatively, high concentrations of a specific constituent in the environment may make genes that are responsible for eliminating or detoxifying that constituent within the cell essential, wheras, if the constituent were absent or present in normal concentrations, such genes would not be essential. Changes thus may involve either an increase or a decrease in specific constituents of the environments including nutrients, inorganic, or organic materials .
In another aspect of this invention, the gene targets of this application are responsible for maintaining cell survival or proliferation in the presence of a drug or biological material. For example, a drug that inhibits one pathway for maintaining the level of a cellular constituent within levels required for cell survival or proliferation may make alternative pathways essential. In a specific embodiment, the inhibition of a synthetic pathway for a cellular constituent may make alternative synthetic pathways essential for cell survival or proliferation.
Alternatively, a drug that is toxic to the cell will make genes that are involved in the elimination, degradation, or excretion of the drug from the cell essential for continued survival or proliferation. It will be evident to those skilled in the art that anything which inhibits the ability of a cell to survive in the presence of a specific drug that is designed to be cytostatic or cytotoxic, will sensitize that cell to the effects of the drug. A "chemosensitizing" agent is one that inhibits a function in the cell that is conditionally essential due to the administration of a chemotherapeutic drug.
In another aspect of this invention, the gene targets of this application are responsible for maintaining cell survival or proliferation in response to external physical forces including, but not limited to, electromagnetic radiation of various amplitudes and wavelengths, including ionizing and nonionizing radiation and heating or cooling. In the presence of ionizing radiation, for example, genes that are SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 involved in DNA repair may be essential that are not essential in the absence of the external physical force. An agent that inhibits functions in the cell that are essential due to the adminitration of ionizing radition would be termed a "radiosensitizing"
agent.
In each instance, treatment of cancer or noncancer proliferative diseases may be achieved by identifying genes that are conditionally essential in the presence of specific environmental, pharmacological, or physical factors, determining whether such genes are subject to loss of heterozygosity, identifying alternative alleles in these genes and developing allele specific inhibitors of alternative forms of the gene.
The administration of such an inhibitor to a patient who has two alternative forms of the gene in normal cells but only one in the cancer cell due to LOH, together with the environmental, pharmacological or physical factors will result in an antiproliferative effect or killing of the cancer cell.
Different environmental, pharmacological, and physical changes in the environment that result in homeostatic or compensatory responses in which genes that are not normally essential for cell survival or proliferation become essential are known in the art. These are described in the following Table 2.
Table 2 1 Changes in the concentration of constituent in the environment o Change in nutritional environment v Change in hormonal environment o Change in the immunological environment o Presence or accumulation of toxic materials o Change in partial pressure of oxygen o Change in partial pressure of carbon dioxide.
o Change in partial pressure of other gasses including nitrous oxide 2. Administration of pharmaceuticals including small molecules, biologicals, nucleic acids, or antibodies.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/0~419 3. Physical changes v Electromagnetic radiation a Ionizing radiation including Alpha particles, Beta particles, Gamma radiation Non-ionizing radiation including infrared radiation, microwave radiation, other wavelengths v Temperature When LOH results in a difference in normal cell genotype vs. cancer cell genotype that affects a locus encoding a product affecting the cells' ability to survive in the presence of an environmental change, a pharmaceutical or biological agent, or a physical factor, there is an opportunity to exploit a therapeutic window between cancer cells and normal cells. Below we describe specific examples of genes that (1) affect cell responses to altered environments, (2) are located on chromosomes that undergo LOH in cancer and (3) exist in two or more variant forms. These examples have been selected to illustrate how the therapeutic strategy described in this application would work with a variety of different alterations in chemical or physical environment. Example 43 describes a gene (Dihydropyrimidine Dehydrogenase) that mediates response to an altered chemical environment (presence of the toxic chemical 5-floxuridine) by specifically transforming the chemical to an inactive metabolite. Example 39 describes a gene (Methylguanine methyltransferase) that mediates response to an altered chemical environment (presence of toxic chemicals such as nitrosourea or other alkylating agents) by removing methyl or alkyl adducts to DNA, the principal toxic lesion of these agents. Example 44 describes a set of genes (Fanconi Anemia genes A,B,C,D,E,F,G and H) which mediate response to an altered chemical environment (presence of chemicals which cause DNA
crosslinking, such as diepoxybutane, mitomycin C and cisplatinum) by repairing the crosslinks. Example 48 describes a set of genes (the DNA Dependent Protein Kinase Complex, including the DNA Dependent Protein Kinase catalytic subunit (DNA-PKcs), the DNA binding component (called Ku), made up of Ku-70 and Ku-86 kDa subunits, and the Ku-86 related protein Karp-1 ) that mediates repair of SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 double stranded DNA breaks, such as occurs after x-irradiation. Example 45 describes a gene (asparagine synthase) that mediates response to an altered nutritional environment (absence of extracellular asparagine) which can be produced by an enzyme such as asparaginase, which hydrolyzes serum asparagine. Example 49 describes the Ataxia Telangiectasia gene, which is involved in response to ionizing radiation and radiomimetic chemicals. Other detailed examples include methionine synthase (Ex. 46) and methylthioadenosine phosphorylase (Ex. 47).
Other examples include Poly (ADP) Ribose Polymerase (PARP), Glutathione-S-Transferase pi (GST-pi), NF-kappa B, Abl Kinase, 3-alkaylguanine alkyltransferase, N-methyipurine DNA glycosylase (hydrolyzes the deoxyribose N-glycosidic bond to excise 3-methyladenine and 7-methyiguanine from alkylating agent-damaged DNA
polymers), OGG-1, MDR-1.
The table below presents exemplary categories and exemplary specific genes along with the type of conditions which render the gene essential.
is Table 3: Categories of Conditionally Essential Genes Genes and proteins vital for cell survival or proliferation in the presence of an altered chemical or physical environment I. Genes required for adaptation to changes in the chemical environment 1. Adaptation to altered concentration of a naturally occuring small molecule A. Increased concentration of a naturally occuring small molecule i. Increased levels of amino acids l.Targets: amino acid degradation pathways SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 Increased intracellular levels of amino acids can damage cells.
One cause of such increased levels is failure to properly degrade amino acids into simpler compounds. Therefore an amino acid catabolizing enzyme can be a conditionally essential gene, particularly in the presence of elevated levels of the twenty amino acids commonly used in protein synthesis. Amino acid catabolic pathways are well described in textbooks and in the scientific literature.
ii. Increased levels of sugars or starches 2. Targets: mono, di and polysaccharide metabolic pathways S Galactose-1-phosphate uridyltransferase Galactose kinase UDPgalactose-4-epimerase Increased intracellular levels of sugars or starches can damage cells. One cause of increased levels is failure to properly degrade starches into simple compounds, as exemplified by diseases of impaired polysaccharide metabolism. Therefore a polysaccharide catabolizing enzyme can be a conditionally essential gene, specifically in the presence of elevated levels of particular polysaccharides. A second mechanism of damage arises in the context of impaired sugar metabolism. Thus enzymes that degrade sugars or starches to simpler compounds may be conditionally essential for cell health and consequently cell proliferation. An example is the enzymes of the Leloir pathway of galactose metabolism. Mutant copies of these proteins make cells conditionally sensitive to elevated concentrations of galactose. Thus enzymes that degrade sugars or starches to simpler compounds may be conditionally essential for cell proliferation.
iii. Increased levels of vitamins B. Decreased concentration of a naturally occuring small molecule SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 i. Decreased levels of amino acids 1.Targets: amino acid transporters Decreased intracellular levels of amino acids can impair protein synthesis and thereby slow or arrest cell division. One cause of such decreased levels is impairment of cellular uptake of amino acids, particularly amino acids that the cell is not actively synthesizing, whether essential (e.g. methionine) or nonessential (e.g. asparagine; see examples). Cells have a variety of mechanisms for amino acid uptake, including membrane anchored transporters. In the presence of decreased extracelluiar levels of amino acids the protein and other constituents of these transporters become conditionally more essential.
2. Targets: amino acid biosynthetic machinery a. Essential amino acids Methionine Synthase, essential for responding to decreased extracellular methionine. (GenBank U73338) b. Non-essential amino acid biosynthesis Asparagine Synthase, essential for responding to decreased extracellular asparagine. (GenBank M27396) Glutamine Synthetase, essential for responding to decreased extracellular glutamine. (GenBank Y00387) SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/0~419 Decreased intracellular levels of amino acids can impair protein synthesis and thereby slow or arrest cell division. One cause of such decreased levels is impairment of amino acid biosynthesis, particularly amino acids that the cell is not actively synthesizing, whether essential (e.g. methionine) or nonessential (e.g.
asparagine; see examples). Cells have a variety of well described biochemical pathways for biosynthesis of the 20 amino acids commonly used in proteins. These biosynthetic enzymes can be conditionally essential in the absence of adequate intracellular levels of amino acids. Specific examples of such conditionally essential genes are described in the Examples. However, other enzymes which catalyze reactions important for maintaining levels of amino acids adequate for protein synthesis in the presence of decreased extracellular concentrations are also useful.
3. Targets: transaminases In the presence of decreased extraceilular levels of amino acids cells must increase intracellular mechanisms for amino acid biosynthesis. One such mechanism is transfer of amino groups from nonessential to essential amino acids to compensate for insufficient quantities of essential amino acids. These reactions are catalyzed by transamin-aces, which therefore can become conditionally essential in environments characterized by decreased levels of extraceIlular amino acids.
ii. Decreased levels of sugars 1. Targets: sugar transporters 2. Targets: sugar metabolism machinery SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 Increased intracellular levels of sugars or starches can damage cells. One cause of such increased levels is failure to properly degrade starches into simple compounds, as exemplified by diseases of impaired polysaccharide metabolism. Therefore a sugar or poly-saccharide catabolizing enzyme can be a conditionally essential gene in the presence of elevated levels of particular sugars or polysaccharides.
2. Adaptation to presence of non-naturally occuring molecules A. Elimination of non-naturally occuring molecules i. Elimination by export Multidrug resistance gene/P glycoprotein (MDRl) (GenBank AF016535) Multidrug resistance associated proteins 1-5 (MRPs) (GenBank L05628) Cells have evolved specific mechanisms to export a variety of chemicals, including nonnatural chemicals such as cytotoxic drugs. MDR1 and MRP are exemplary ATP-dependent transmembrane drug-exporting pumps. Deficiency of these pumps is associated with increased sensitivity to a variety of cytotoxic drugs in vitro and in vivo. For example, mice lacking functional MRP are hypersensitive to the drug etoposide. Thus these pumps are important for cell survival in the presence of a variety of toxic drugs. Polymorphisms have been reported in MDR1 at amino acids 893 and 999. MDR also maps to a region of chromosome 7 which is frequently affected by LOH in prostate, ovarian breast and other cancers.

Multispecific organic anion transporters (MOATs) Other drug export proteins ii. Elimination by metabolic transformation 1. Specific metabolic transformation of drugs SUBSTITUTE SHEET (RULE 26) a. Inactivation of bleomycin Bleomycin hydrolase (GenBank U14426) Bleomycin hydrolase was discovered through its abililty to detoxify the anticancer glycopeptide bleomycin. Cells lacking bleomycin hydrolase are highly susceptible to bleomycin toxicty (for example pulmonary fibrosis) thus the gene is conditionally essential for cell growth and survival in the presence of bleomycin. Bleomycin hydrolase is a member of the cysteine protease papain superfamily. The protein is expresed in all tissues surveyed. 'The crystal structure of the closely related yeast bleomycin hydrolase has been determined. A common A/G
polymorphism has been described at nucleotide 1450 of the bleomycin hydrolase gene. It results in an isoleucine-valine variance at amino acid 443, part of the oligomerization domain of the homotetrameric enzyme. The Bleomycin hydrolase gene has been mapped to the proximal long arm of chromsome 17 (17q11.2), a site of frequent LOH in commonly occuring epithelial cancers such as breast and ovarian cancer.
b. Inactivation of pyrimidine analogs including 5-fluorouracil (5-FU) and 5-fluorouridine.
Dihydropyrimidine Dehydrogenase (DPD) (i= ureidopropionase (i - alanine synthetase DPD is described in the examples. The other two enzymes are responsible for the further metabolism of dihydro-5-fluorouracil, the metabolic product of DPD. In the absence of these enzymes toxic metabolites of 5-FU accumulate in cells.
c. Inactivation of of pyrimidine analogs including cytosine arabinoside and 5-azacytidine.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 lOS
Cytidine deaminase Cytidine deaminase (CDA) catalyzes hydrolytic deamination of cytidine or deoxycytidine. It can also deaminate cytotoxic cytosine nucleotide analogs such as cytosine arabinoside, rendering them nontoxic. Resistance to the cytotoxic effects of these drugs has been reported associated with increased expression of the CDA
gene. Thus CDA is a conditionally essential gene in the presence of cytotoxic cytosine nucleotide analogs.
S d. Inactivation of thiopurine drugs, including 6-mercaptopurine, 6-thioguanine and azathioprine.
Thiopurine methyltransferase (GenBank U12387) e. Inactivation or transformation of other drugs including, but not limited to, purine analogs, folate analogs, topoisomerase inhibitors and tubulin acting drugs via specific enzymatic modification.
2. General metabolic transformation of drugs a. Cytochrome P4S0 system.
CYPl CYP1A1 (GenBank K03191 ) CYP1A2 (GenBatilc MSSOS3) CYP2A6 (GenBank U33317) CYP2C9 (OMIM 601 I30) CYP2C19 (OMIM 124020) CYP2D6 (OMIM 124030) CYP2E1 (OMIM 124040) 3S CYP3A4 (GenBanic D00003) ' CYP3A5 SUBSTITUTE SHEET (RULE 26) CYPl l CYPi7 CYP4Bi The cytochrome P450s are a large gene family whose members metabolically transform and inactivate a wide variety of drugs, including cytotoxic drugs. Wide variation in P450 protein expression has been described, including null alleles. For example cytochrome P450 2D6 may be involved in the metabolism of ~25%
of all drugs. Between 5 and I 0% of all Caucasians are homozygous for completely inactive alleles of P450 2D6. In the presence of a toxic drug the P450 enzyme responsible for metabolizing the drug may be conditionally essential. For example, acute liver faillure has been reported in a patient treated with cyclophosphamide who was homozygous for the deficient CYP 2D6B allele. Liver failure was due to accumulations of a hepatotoxic 4-hydroxylated cyclophosphamide metabolite.
b. N-acetyltransferases c. Glucuronyltransferases d. Glutathione transferases Glutathione transferase alpha (GenBank AF020919) GIutathione transferase theta (OMIM 600436 & 600437) Glutathione transferase mu (OMIM 138350, 138380, 138380, 138333 & 138385) Glutathione transferase pi (GenBank X65032) A large number of drugs are are biotransformed into electrophilic intermediary compounds which are potentially harmful to cell constituents unless rendered harmless by conjugation with glutathione. Thus proteins of the GST system are conditionally essential for cell survival.
SUBSTITUTE SHEET (RULE 26) WO 98/11648 PCT/US98/0~419 B. Repair or prevention of damage by non-naturally occuring molecules i. Repair or prevention of damage by molecules that react with nucleic acids 1. Molecules that add alkyl or other groups to DNA
a. Targets: genes & gene products involved in repair of alkylating agent damage Methylguanine Methyltransferase (MGMT) (GenBank M29971 ) 3-allcylguanine alkyltransferase 3-methyladenine DNA glycosylase (GenBank M74905) MGMT is described in the examples. hOGGl is a DNA
glycosylase with associated lyase activity that excises this adduct and introduces a strand break. Cells lacking this protein are deficient in repair of oxidative damage and have high mutation rates. In conditions of high oxidative damage, including cellular aerobic metabolism, ionizing radiation and some chemotherapy drugs the hOGGI gene would be conditionally essential for DNA
repair. The human OGG1 gene maps to chromosome 3p25, a region of high frequency LOH in lung, kidney, head and neck and other cancers. Homozygous mutant mouse cells lacking 3-methyladenine DNA glycosylase have increased sensitivity to alkylation induced chromosome damage and cell killing.
2. Molecules that induce single or double stranded DNA
breaks (also relevant to survival in the presence of ionizing radiation; see below) a. Targets: genes & gene products involved in repair of double stranded DNA breaks DNA Dependent Protein Kinase (DNA-PK) and subunits Catalytic subunit of DNA-PK (GenBank U47077) DNA binding subunit of DNA-PK (Ku subunit) Ku-70 subunit (GenBank J04611) SUBSTITUTE SHEET (RULE 2fi) WO 98/.11648 PCT/US98/0~419 Ku-86 subunit (OMIM 1943641GenBank AF039597) Poly (ADP-ribose) polymerise (PARP) (GenBank M32721 ) b. Targets: genes & gene products that repair DNA cross-links induced by molecules such as Mitomycin C or diepoxybutane Fanconi Anemia genes Fanconi Anemia A gene (GenBank X99226) Fanconi Anemia B gene Fanconi Anemia C gene (GenBank X66894) Fanconi Anemia D gene Fanconi Anemia E gene Fanconi Anemia F gene Fanconi Anemia G gene Fanconi Anemia H gene 4. Targets: genes & gene products required for repair of DNA
damage caused by drugs such as, for example, 4-nitroquinoline -1-oxide, bromobenz(a)anthracene, benz(a)anthracene epoxide, 1-nitorpyridine-I-oxide, acetylaminofluorine and aromatic amides, benz(a)pyrene.
a. Nucleotide excision repair system ERCC-1 (GenBank M13194) ERCC2/XPD (GenBank X52222) ERCC3/XPB (GenBank M31899) ERCC4 (OMIM 133520) ERCCS (GenBank L20046) ERCC6 (GenBank L04791) b. Other DNA repair genes XPA (GenBank D14533) XPC (GenBank D21090) XPE (GenBank U18300) HHR23A (GenBank U21235) HHR23B (GenBank D21090) Uracil glycosylase (GenBank X52486) 3-methyladenine DNA glycosylase (GenBank M74905) ii. Repair of damage by chemicals that interact with proteins iii. Repair of damage by chemicals that interact with membranes SUBSTITUTE SHEET (RULE 26) WO 98Lt1648 PCT/US98/0~419 1. Free radical damage iv. Adaptation to molecules that alter the cellular redox state (such as pyrrolidinedithiocarbamate) 3. Adaptation to change in nutritional environment A. Decreased levels of nutrients.
B. Increased levels of nutrients.
4. Change in hormonal environment A. Decreased levels of hormones.
B. Increased levels of hormones.

5. Change in the itnmunological environment A. Introduction of new immune molecules (antibodies or antibody fragments) B. Introduction of immune regulatory molecules Fanconi anemia C
NF-kappa B (GenBank M58603) Cells lacking the Fanconi anemia C gene have been shown hypersensitive to interferon gamma in vitro. Cells lacking the RelA/p65 subunit of NF kappa B are essential for preventing Tumor Necrosis Factor alpha induced cell death. Other Fanconi anemia genes or other proteins of the NF-Kappa B system and its regulators, for example I kappa B, may also mediate sensitivity to immune system molecules, for example interferons, interleukins or TNF.
II. Changes in physical environment 1. Repair of damage caused by electromagnetic radiation SUBSTITUTE SHEET (RULE 26) WO 98/41648 PCT/US98/0~419 A. Repair of damage caused by ionizing radiation (Alpha particles, Beta particles, Gamma radiation) i. DNA-PK constitutents (see above) ii. Other proteins that repair DNA damage created by DNA-PK
XRCC4 (GenBank U40622) XRCCS/Ku80 (OMIM 194364) XRCC7 (GenBank L27425) iii. Other proteins that repair or protect from DNA damage Glutathione-S-transferase (alpha, theta, mu and pi proteins) Transfection of an exogenous Glutathione-S-transferase pi (GST-pi) gene is partially protective of cells treated with ionizing radiation.
Thus GST activity is conditionally essential for cells exposed to ionizing radiation. Similarly, any protein that is essential for the repair of radiation induced damage or for protection of cells from radiation induced damage is a conditionally essential gene. GST
activity can also affect radiation sensitivity in the presence of electron affinic drugs such as the nitroimidazoles.
I-kappa B alpha (GenBank M69043) Increased expression of exogenous I kappa B-alpha, an inhibitor of NF-kappa B, increases cell sensitivity to ionizing radiation. Thus is conditionally essential for cells exposed to ionizing radiation.
Other proteins of the NF kappa B pathway that affect radiosensitivity are likewise conditionally essential in the presence of ionizing radiation.
B. Non-ionizing radiation i. infrared radiation ii. ultra high frequency electromagnetic radiation (UHF) Glutathione S transferase system (see genes listed above) SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 UHF electromagnetic radiation of 434 Mhz will change resonance of the glutathione cycle resuiting in thiol depletion which increases radiosensiviry. UHF is therefore a radiosensitizing treatment, contingent on the status of the glutathione system.
iii. Other wavelenths of electromagnetic radiation 2. Temperature A. Heating 1. Heat shock proteins HSP70 (OMIM 138120) HSP27 (GenBank X54079) B. Cooling 2. Cold sensitive proteins 3. Change in redox environment, including change in partial pressure of gasses A. Change in partial pressure of oxygen i. Repair of damage from reactive oxygen species 8-oxoguanine DNA glycosylase (hOGGl) (GenBank U96710) The major mutagenic lesion caused by exposure to reactive oxygen species is 8-oxoguanine. hOGG 1 is a DNA glycosylase with associated lyase activity that excises this adduct and introduces a strand break. Cells lacking this protein are deficient in repair of oxidative damage and have high mutation rates. In conditions of high oxidative damage, including cellular aerobic metabolism, ionizing radiation and some chemotherapy drugs the hOGGI gene would be conditionally essential for DNA repair. The human OGG1 gene maps to chromosome 3p25, a region of high frequency LOH in lung, kidney, head and neck and other cancers.
Fanconi anemia genes (see above for list of 8 FA
complementation groups; FA genes also mediate sensitivity to oxygen) SUBSTITUTE SHEET (RULE 26) WO 98Li1648 PCT/US98/05419 B. Change in partial pressure of carbon dioxide.
C. Change in partial pressure of other gases.
In addition to being hypersensitive to ionizing radiation Ataxia-Telangiectasia cells are hypersensitive to the nitric oxide donor S-nitrosoglutathione (GSNO), as are cells from some radiosensitive individuals without ataxia. GSNO induces dose-dependent DNA
strand breakage; cell killing appears to be associated with formation of nitrite as the ultimate oxidation product of nitric oxide. Any protein important for response to damage induced by a dissolved gas is a conditionally essential gene in this category.
III. Identification of variances and alternative alleles.
A target gene of this invention must occur as alternative alleles in the population;
that is, the DNA sequence variance should either affect the gene sequence, RNA
sequence, or protein sequence of the gene or its gene products, which would facilitate the design of inhibitors of the protein product, or be a base difference anywhere within the genomic DNA sequence, including the promoter or intron regions. Such DNA sequence variance can be exploited to design inhibitors of transcription or translation which distinguish between two allelic forms of the targeted gene. Sequence variants that do not alter protein sequence can be targeted, for example, with antisense oligonucleotides or ribozymes.
The most elementary genetic variant, which is common in mammalian genomes, is the single nucleotide substitution. It has been estimated that the comparison of haploid genomes will reveal this type of variant every 300 to 500 nucleotides (Cooper, et al., Human Genetics, 69:201:205 (1985)).
Sequence variances are identified by testing DNA from multiple individuals from SUBSTITUTE SHEET (RULE 2fi) WO 98/.11648 PCT/US98/05419 the populations) to determine whether the DNA sequence for the target gene differs in different individuals. Many different methods for identifying gene sequence variances are known in the art, several of which are described in detail in the Examples noted below. These include, but are not limited to: ( 1 ) sequencing using methods such as Sanger sequencing which is commonly performed using automated methods (Example 37); (2) Single Strand Conformation Polymorphism (Example 28); (3) DGGE (Example 36); (4) Computational methods (Example 30); (5) Chemical cleavage, (6) HPLC; (7) Enzymatic Mutation Detection, (Example 29);
(8) Hybridization; (9) Hybridization arrays; and (10) Mass spectroscopy.
Often combinations of these methods are used. For example, methods such as SSCP, DGGE, or HPLC are useful in identifying whether amplified gene segments from two individuals are identical or contain a variance. These methods do not identify the location of the variant site within the linear sequence of the amplified gene segment, nor do these methods identify the specific nature of the variance, namely the alternative bases within the variant site. Methods such as Enzymatic Mutation Detection determines where the variant site is located within the sequence, but not the specific variance. Methods such as mass spectroscopy identify the specific variance, but not it location within the segment. Methods such as sequencing, computational analysis, and hybridization arrays can determine the location of the variance and specific sequence of the variance within the segment.
In addition, methods such as SSCP, DGGE, EMD, and chemical cleavage are useful for determining alleles containing more than one variant site, if such sites occur within a single amplified gene segment. For the purpose of this invention, methods have been used to identify novel variant sites within genes that are essential for cell survival or proliferation. With the above methods, the presence and type of variance are preferably confirmed, such as by sequencing PCR amplification products extending through the identified variance site.
SUBSTITUTE SHEET (RULE 26) WO 98/41648 1'CT/US98105419 IV. Loss of Hertozygosity Essential genes which are located in chromosomal regions which frequently undergo LOH in a tumor or other disease or condition provide advantageous targets, as the LOH of the chromosomal region indicates that the particular gene will also S undergo LOH at similar high frequency. Also, essential genes which undergo LOH
at high frequencies in a particular tumor, or in a range of tumor types provide advantageous targets, as a large number of patients will be potentially treatable due to the LOH of a particular essential gene.
Cancer cells, or more broadly cells associated with certain other proliferative conditions, are generally genetically different from normal somatic cells as a result of partial or complete chromosome loss, called loss of heterozygosity (LOH), which occurs at the earliest stages of these disorders. In cancer, as a result of such early chromosome loss, all the tumor cells in an individual exhibit the same pattern of LOH since the cancer results from clonal expansion of the progenitor cell with LOH. Losses of genes in LOH range from less than 5% of a chromosome, to loss of a chromosome arm, to loss of an entire chromosome. Generally only one chromosome copy is lost, making cancer cells partially hemizygous - i.e., they have only one allele of many genes. As a result of such allele loss, only the single remaining allele will be available to be expressed. Such loss of heterozygosity and other losses of genetic material in cancers is described in a variety of references, for example in Mitelman, F., Catalog of Chromosome Aberrations in Cancer, New York: Liss (1988); and Seizinger, et al., "Report of the committee on chromosome and gene loss in neoplasia," Cytogenet. Cell Genetics, 58:1080-1096 (1991). A
review of many published studies of LOH in cancer cells is provided in Lasko, Cavenee, and Nordenskjold, "Loss of Constitutional Heterozygosity in Human Cancer," Ann. Rev. Genetics, 25:281-314 ( 1991 ).
There is considered to be a causal relationship between LOH and the origin of SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/U598/0~419 cancer or other proliferative disorders. Loss of heterozygosity commonly involves chromosomes and chromosome segment that contain at least one tumor suppressor gene in addition to many other genes that may not have any function associated with cancer but are coincidentally located in the same region of the chromosome, measured in physical distance or genetic distance, as the tumor suppressor gene.
Tumor suppressor genes generally regulate cell proliferation or are involved in initiating programmed cell death when threshold level of damage occurs to the cell.
The loss of tumor suppressor gene function is believed to confer a growth advantage to cells undergoing LOH, because it allows them to evade these negative growth regulatory events. It is the loss of tumor suppressor genes, and the proliferative advantage associated with loss of tumor suppressor functions, that drives allele loss or loss of heterozygosity. Loss of tumor suppressor gene function requires inactivation of both gene copies. Inactivation is usually due to the presence of mutations on one gene copy and partial or complete loss of the chromosome, or chromosome region, containing the other gene copy. (Lasko et al., 1991, Annu.
Rev. Genet. 25:281-314) Several tumor suppressor genes have been cloned. They include, for example, TP53 on chromosome arm 17p, BRCAI on 17q, RB and BRCA2 on 13q, APC on Sq, DCC on 18q, VHL on 3p, and p16'N"4/MTSI on 9p. Many other, as yet uncloned, tumor suppressor genes are believed to exist based on LOH data;
research groups are currently working to identify new tumor suppressor genes at more than a dozen genomic regions characterized by high LOH in cancer cells, including generating detailed LOH maps which provide LOH information useful for this invention due to the ability to identify essential genes which map to these regions of LOH. While there is an extensive literature considering tumor suppressor genes as potential targets for anti-cancer therapy, these genes are, in general, not candidates for antiproliferative therapy under the present invention because most tumor suppressor genes are not essential for cell proliferation or survival. To the contrary, SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 it is the loss of tumor suppressor genes that enables the abnormal proliferation and survival of cancer cells.
The pattern of LOH for a particular cancer or tumor or other proliferative disorder is not merely random. Often, there is a characteristic pattern for each major cancer type. Certain regions, including segments of chromosomes 3, 9, 1 I, 13, and 17, are frequently lost in most major cancer types. Other regions, such as on chromosomes 1, 3, 5, 6, 7, 8, 9, 11, 13, 16, 17, 18, and 22, exhibit high frequency LOH in selected cancers. It is believed that the characteristic LOH patterns of different cancers reflects the locations) of tumor suppressor genes related to the development of the particular cancer or cancer type. Thus, essential genes located in regions which are characteristically associated with LOH for a particular cancer, or other tumor are particularly advantageous targets for inhibitors useful for treatment of that cancer or tumor because such genes will also characteristically undergo LOH at high frequency. The fact that certain cancers predictably undergo LOH in specific regions of the genome, and that LOH occurs before the clonal expansion of cancers in precancerous, abnormally proliferating tissue is potentially useful for preventing cancer with allele specific inhibitors of essential genes.
The treatment method described herein is applicable to proliferative disorders in which clonal proliferation occurs and in which the proliferating cells commonly undergo LOH. Another example of a disorder which has been characterized as a proliferative disorder is inflammatory pannus in arthritic joints. The demonstration of LOH associated with such a disorder will indicate that the allele specific treatment would be appropriate for the disorder. For the application of the general allele specific inhibition strategy to such conditions (e.g., selection of target gene and variance, identification of inhibitors, selection of composition and administration method appropriate for the condition and the inhibitor), the cells associated with the condition correspond with the tumor, e.g., cancer cells, for the SUBSTITUTE SHEET (RULE 26) WO 981.11648 PCT/US98/05419 methods described in the Summary above.
LOH has been described for such polyclonal or oligoclonal disease conditions, in particular for atherosclerosis (arteriosclerosis), for example in Hatzistamou et al., 1996, Biochem. Biophys. Res. Comm. 225:186-190. Using a limited set of markers located on 18 chromosomal arms (one marker per arm), it was found that 23% of atherosclerotic plaques exhibited LOH for at least one marker. This does not necessarily represent the maximum fraction of plaques which could potentially be treated with allele specific inhibitors because the study did not attempt to determine the sites of maximum LOH on each arm. LOH which is partial arm LOH not affecting the particular marker for that arm was not detected. In general, fine scale LOH studies (using closely spaced markers) have revealed more sites of high frequency LOH than coarser scale studies.
The LOH for alleles of essential genes in cancers forms the basis for the anticancer therapeutic strategy described in Housman, supra. When one allele of the essential gene is lost from the patient's cancer cells, the retained allele can be targeted with an allele specific inhibitor. Such an inhibitor will kill, or reduce or prevent the growth of cancer cells by abolishing the function of an essential gene. Normal cells, which retain both uninhibited and inhibited alleles, will survive or grow due to the expression of the uninhibited allele. This is clearly indicated because tumor cells having only one allelic form (after LOH) thrive, thus, normal cells will also function normally with one of two allelic forms inhibited.
A large number of high frequency LOH regions are identified in Fig. 5. if not previously known, this correlation can be determined routinely for one or more tumor types by mapping of essential genes to chromosomal regions which have been identified as having high frequency LOH, or by identifying essential genes which map to locations near markers which have been identified as undergoing high SUBSTITUTE SHEET (RULE 26) WO 98/.i1b48 PCT/US98/05419 frequency LOH in a tumor. As previously described, the LOH of a marker near an essential gene, or the bracketing of an essential gene by two markers which undergo LOH, is strongly indicative that the essential gene also undergoes LOH at a similar frequency.

Loss of Heterozygosity in Human Solid Tumors By Chromosome Arm Chromosome Region Tumor TXpe Chromosome Region Tumor Tune 1 p Breast carcinoma 1 Cutaneous melanoma 2 Uveal melanoma (metastastic) Medullary thyroid carcinoma:

Neuroblastoma 1 Pheochromocytoma: MEN2A
sporadic 1 q Breast carcinoma Gastric adenocarcinoma 4q Hepatocellular carcinoma 3p Breast carcinoma Cervical carcinoma Lung cancer:
small carcinoma non-small cell carcinoma carcinoma large cell carcinoma squamous cell adenocarcinoma Ovarian carcinoma Renal cell carcinoma: familial sporadic Testicular carcinoma SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 Sq Colorectal carcinoma 6q Ovarian carcinoma Hepatocellular carcinoma Primitive neuroectodermal tumor Renal cell carcinoma Testicular teratocarcinoma 9p Glioma 10 Glioblastoma multiforme 9q Bladder carcinoma l Oq Hepatocellular carcinoma Prostate cancer 11 p Adrenal adenoma 12q Gastric adenocarcinoma Adrenocortical carcinoma Bladder carcinoma Breast carcinoma Embryonal L rhabdomyosarcoma Hepatoblastoma Hepatocellular carcinoma Lung cancer:
squamous cell I carcinoma large cell carcinoma adenocarcinoma Ovarian carcinoma Pancreatic cancer Z Parathyroid tumors Pheochromocytoma Skin cancer squamous cell carcinoma 2 basal cell carcinoma Testicular cancer Wilms tumor 11 q Insulinoma Parathyroid tumors SUBSTITUTE SHEET (RULE 28) WO 98/.11648 PCT/US98/0~419 13q Adrenocortical adenoma 14 Colorectal carcinoma Breast carcinoma 14q Neuroblastoma Gastric carcinoma Hepatocellular carcinoma Lung cancer:
small cell carcinoma Neuroblastoma Osteosarcoma Retinoblastoma 17p Adrenocortical adenoma 1 16 Breast carcinoma Astrocytoma 16q Breast carcinoma Bladder carcinoma Hepatocellular carcinoma Breast carcinoma Primitive neuroectodermal Colorectal carcinoma tumor Lung cancer:

1 Prostate cancer small cell carcinoma squamous cell carcinoma adenocarcinoma Medulloblastoma Neurofibrosarcoma: NF1 Osteosarcoma Ovarian carcinoma Primitive neuroectodermal tumor Rhabdomyosarcoma 17q Breast carcinoma Neurofibroma: NF 1 22q Acoustic neurinoma 18 Renal cell carcinoma Colorectal carcinoma 18q Breast carcinoma Ependymoma Colorectal carcinoma Meningioma Neurofibroma V. Use of variance-specific inhibitors of essential genes to treat non-malignant, 20 proliferative conditions.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98105419 It was found in the present invention that noncancer proliferative disorders could also be targeted using such an allele specific strategy. Such conditions include, but are not limited to atherosclerotic plaques, abnormal tissue in arthritic joints, including pannus, benign tumors such as leiomyomas and meningiomas, and hyperplastic conditions such as benign prostatic hyperplasia. For most of these conditions there is evidence of a mono- or oligoclonal origin and evidence of LOH.
Such evidence includes the following:
t A recent study (Hatzistamou, J., Kiaris, H., Ergazaki, M., et al. ( 1996) Loss of heteroxygosity and microsatellite instability in human atherosclerotic plaques. Biochemical and Biophysical Research Communications 225: 186-190.) demonstrated that allele loss occurs in atheromatous plaques, which have long been viewed as benign neoplastic proliferations by some investigators (Benditt, E.P. and J.M. Benditt (1973) Evidence for a monoclonal origin of human atherosclerotic plaque. Proc. Natl. Acad.
Sci. U. S. A. 70: 1753-7). Each atheromatous plaque constitutes a separate independently arising primary lesion. Consequently, allele loss in individual atherosclerotic plaques will differ, with, for example, allele A
of a hypothetical essential gene lost in some plaques and allele A' in others. An inhibitor of allele A would be expected to kill (or arrest growth of) only about half of all the plaques with allele loss at the hypothetical locus - those plaques hemizygous for A. To kill the other half of the plaques with allele loss at the target locus would require an inhibitor of A' . Simultaneous use of inhibitors of A and A' would be highly toxic to diploid normal cells. However serial use of an inhibitor directed to allele A followed by an inhibitor directed to A' (perhaps repeating treatment for several cycles, or even indefinitely) would alternately abolish essential gene function in one half of all haploid plaque cells and then the other half, leading eventually to death or sustained inhibition of proliferation of all plaque cells. Normal cells would retain SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTIUS98I05419 50 % gene function in the presence of inhibitor (either from allele A or allele A'). This therapeutic approach is applicable to the eradication of any clonal proliferation of cells in which allele loss has rendered the cells partialy haploid.
~ LOH has been described in a wide variety of premalignant conditions such as metaplasia and dysplasia of colonic epithelium, breast epithelium, lung epithelium and cervical epithelium. Most studies have focused on metaplastic or dysplastic epithelium adjacent to cancer tissue, and have shown patterns of LOH similar to those in the adjacent malignant epithelium. Prophylactic ablation of such premalignant tissues could prevent the subsequent development of cancer.
In benign tumors such as leiomyomas and parathyroidomas, which frequently must be surgically removed, LOH has been well described. As with atherosclerotic plaques, these tumors are frequently multifocal and therefore the approach of serial inhibition of allele A followed by inhibition of allele A' would alternately abolish essential gene function in one half of all haploid tumor cells and then the other half, leading eventually to death or sustained inhibition of proliferation of all tumor cells.
~ LOH has been described in endometriosis, a proliferative condition associated with pain and infertility and frequently requiring surgical removel of endometrial tissue growing outside the uterine cavity. As with atherosclerotic plaques, there is only one study published to date and the frequency of LOH is low ( 15-18 % ), however the study examined only six chromosome arms; additional studies may lead to identification of regions of higher frequency LOH
LOH is apparently the necessary event in the development of cyts in some, and possibly all, forms of autosomal dominant polycystic kidney disease (ADPKD). (There are three forms, with ADPKD1 accounting for about SUBSTITUTE SHEET (RULE 26) WO 98/-11648 PCTIUS98/0~419 85 % of cases and ADPKD2 about 15 % of cases.) LOH has been demonstrated by genetic analysis of the cells lining cyst walls in kidneys of ADPKD1 patients: the cells have undergone LOH for markers flanking the ADPKD 1 gene. As a result the cyst cells lack functional ADPKD 1.
(Patients with ADPKD inherit one defective copy of an ADPKD gene from their parents.) Only about 20% of cysts were shown to have LOH
when studied with a few markers, but this likely reflects, at least to some extent, technical difficulties in obtaining pure populations of cyst cells for analysis. The extent of loss of heterozygosity in cyst cells has not been well studied; only several polymorphic markers in the vicinity of the ADPKD1 gene on chromosome 16p were tested in one study (Qian, F., Watnick, T.J., et al. The molecular basis of focal cyst formation in human autosomal dominant polycystic kidney disease type I. Cell 87:979-987, 1996.) Another study found one case of LOH on chromosome 3p, distant from the ADPKD gene. Future LOH studies may reveal more extensive LOH in ADPKD. Also, it is worth noting that, unlike malignancy where it is desirable to eradicate all disease cells, eradication of a fraction of the cysts in ADPKD would be expected to have a significant beneficial effect.
This is evident from the disparate clinical presentation of ADPKD, with varying numbers of cyts being associated with varying degrees of impairment of kidney function.
Other conditions in which LOH has been demonstrated include hamartomas in tuberous sclerosis patients, odontogenic keratocysts and pterygia (benign lesions of the corneoconjunctival limbus).
~ Other conditions in which there is evidence of clonal proliferation include inflammatory pannus in arthritic joints, benign prostatic hypernophy, and hereditary hemorrhagic telangiectasia. (Qian, F. and G.G. Germino.
"Mistakes Happen": Somatic Mutation and Disease. Am. J. Hum. Genet.
61: 1000-1005, 1997.) SUBSTITUTE SHEET (RULE 26) Thus, consistent with the Summary above, it was found that LOH occurs in many non-malignant neoplasias or tumors with subsequent clonal growth of cells which contain only one allelic form in individuals whose normal somatic cells are heterozygous for the particular essential gene. The essential gene can therefore be inhibited by an allele specific inhibitor, i. e. , a variance specific inhibitor. In some conditions, however, multiple, independently arising lesions in an individual are subjected to LOH in a disease or condition, e. g. , in the development of atherosclerotic plaques. For that example, in individuals heterozygous for a particular essential gene which undergoes LOH, this results in some atherosclerotic plaques in which cells have one of the allelic forms of an essential gene, and other plaques in which cells have the alternative form of the gene.
It was determined that such conditions can be treated using allele specific inhibitors despite the presence of both alleles in cells related to the condition.
There are two strategies for such therapy. The first is to serially administer different inhibitors targeted to the different allelic forms of the target gene. This can be accomplished by using inhibitors which target the alternative sequence variants of one sequence variance site. Simultaneous administration of inhibitors of both allelic forms of an essential gene would inhibit the cells which have undergone LOH at that gene, but would also inhibit the normal heterozygous cells of the individual. This treatment would inhibit essential functions in normal cells as well as cancer cells and have no advantage over the administration of conventional antiproliferative drugs, many of which are inhibitors of known essential functions. In contrast, administration of the first inhibitor targets the subset of cells which have only the first allelic form of an essential gene.
As described for the general strategy, this inhibitor will not significantly affect the growth or survival of the normal heterozygous somatic cells. This first administration is followed by administration of a second inhibitor; the second SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 inhibitor targets the cells which contain only the second allelic form of the gene, and again does not significantly affect the normal somatic cells. This process of ' alternating administration can be repeated as needed to achieve a desired therapeutic effect. In some cases many rounds of alternating administrations will be useful. Similarly, recurring, or even indefinitely continued alternating administrations will provide useful treatment. Likewise, these methods can incorporate the use of inhibitors targeted to specific alleles of a plurality, e.g., 2, 3, 4, or more different target genes.
In certain instances, even though the lesions in non-malignant diseases are not clonal, there may be systematic loss of one parental chromosome allowing effective therapy with only one variance-specific inhibitor. This would occur, for example, if there were an inherited or early embryonic mutation within a tumor suppressor gene on one parental chromosome, in which case any event which was associated with the elimination of the corresponding normal tumor suppressor gene on the other parental chromosome would lead to abnormal proliferation. In such cases a variance-specific inhibitor of an essential gene that was closely linked to the normal tumor suppressor gene would preferentially kill cells in the proliferating lesion.
VI. Characteristics of allele-specific inhibitors As indicated above "allele specific inhibitors" or " allele specific anti-neoplastic agents" represent a new approach to tumor therapy because they are lethal or significantly inhibit the growth only of tumor cells. The advantages of this approach include, first, lack of toxicity to the normal cells of the patient resulting in a therapeutic index greater than that of conventional tumor, e. g. , cancer ' 25 chemotherapy drugs, and second, it is not necessary that the inhibitors be targeted specifically to the tumor cells, as they can be administered systemically. As also described above, usually an allele specific inhibitor is specific for a single SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 sequence variance of an essential gene, though in some cases the inhibitor utilizes the joint effects of two or more sequence variances on a particular allele.
It is not necessary for the allele specific inhibitor to have absolute specificity.
Normal cells expressing equal amounts of two allelic forms of a gene product encoded by the essential gene will often show a reduction in gene activity when they take up the inhibitors of this invention, but should remain viable due to the activity of the protein encoded by the uninhibited allele. On the other hand, tumor cells expressing only one allele due to LOH, will respond to the inhibitors of this invention which are specifically directed to the remaining allele, with a greater reduction in gene activity. Growth of tumor cells exposed to the inhibitors of this invention will be inhibited due to the suppression of either the synthesis or the biological activity of the essential gene product.
Also, while a single gene has only two allelic forms in any given individual, the gene can have more than two allelic forms in a human population. Accordingly, inhibitors can be targeted to any of the alleles in the population. A
particular inhibitor will generally be targeted to a subset of the allelic forms; the members of the subset will have a particular sequence variance which provides the specific targeting. In some cases, however, the inhibitor will jointly target two, or possibly more sequence variances.
Once two or more alleles are identified for a target essential gene, inhibitors of high specificity for an allele can be designed or identified empirically.
Inhibitors that can be used in the present invention will depend on whether allelic variation at a target locus affects the amino acid sequence, the mRNA sequence, or the DNA in intron and promoter regions. If there is variation at the protein level, then classes of inhibitors would include low molecular weight drugs, oligopeptides and their derivatives, and antibodies, including modified or partial SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 antibody fragments or derivatives. For mRNA or DNA sequence variance the main class of inhibitors are complementary oligonucleotides and their derivatives and catalytic RNA molecules such as ribozymes, including modified ribozymes.
The generation of inhibitors of this invention can be accomplished by a number of methods. The preferred method for the generation of specific inhibitors of the targeted allelic gene product uses computer modeling of both the target protein and the specific inhibitor. Other methods include screening compound libraries or microorganism broths, empirical screening of libraries of peptides displayed on bacteriophage, and various immunological approaches.
Further, in the treatment of cancer patients, a therapeutic strategy includes using more than one inhibitor of this invention to inhibit more than one target. In this manner, inhibitors directed to different proteins essential to cell growth can be targeted and inhibited simultaneously. The advantage of this approach is to increase the specificity of the inhibition of proliferation of cancer cells, while at 1 S the same time maintaining a low incidence of side effects.
A. Targeted Drug Design.
Computer-based molecular modeling of target proteins encoded by the various alleles can be used to predict their three-dimensional structures using computer visualization techniques. On the basis of the differences between the three-dimensional structure of the alternate allelic forms of the proteins, determinants can be identified which distinguish the allelic forms. Novel low molecular weight inhibitors or oligopeptides can then be designed for selective binding to these determinants and consequent allele-specific inhibition. Descriptions of targeted drug design can be found, for example, in I. Kuntz, "Structure-Based Strategies ' 25 for Drug Design and Discovery," Science 257:1078-1082 (1992) and J.
Dixon, "Computer-Aided Drug Design: Getting the Best Results," Trends in Biotechnology 10:357-363 (1992). Specific applications of the binding of SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/0~419 molecules to receptors using computer modeling have been described in Piper et al. , "Studies Aided by Molecular Graphics of Effects of Structural Modifications on the Binding of Antifolate Inhibitors to Human Dihydrofolate Reductase,"
Proc Am. Assoc. Cancer Res. Annual Meeting 33:412 (1992); Hibert et al., "Receptor S 3D-Models and Drug Design," Therapie (Paris) 46:445-451 {1991)(serotonin receptor recognition sites). Computer programs that can be used to conduct three-dimensional molecular modeling are described in G. Klopman, "Multicase 1: A Hierarchical Computer Automated Structure Evaluation Program,"
Quantitative Structure-Activity Relationships, 11:176-184 (1992); Pastor et al., "The Edisdar Programs Rational Drug Series Design," Quantitative Structure-Activity Relationships, 10:350-358 (1991); Boils et al., "A Machine Learning Approach to Computer-Aided Molecular Design," J. Computer Aided Molecular Desig, 5:617-628 (1991); and Lawrence and Davis, "CLIX: A Search Algorithm for Finding Novel Ligands Capable of Binding Proteins of Known Three-1S Dimensional Structure," Proteins Structure Functional Genetics 12:31-41 (1992).
Low molecular weight inhibitors specific for each allelic protein form can be predicted by molecular modeling and synthesized by standard organic chemistry techniques. Computer modeling can identify oligopeptides which block the activity of the product of the target gene. Techniques for producing the identified oligopeptides are well known and can proceed by organic synthesis of oligopeptides or by genetic engineering techniques. R. Silverman, The Org~'c Chemistry of Drug Desissn and Drug Action, Academic Press (1992).
The inhibitors of this invention can be identified by selecting those compounds that selectively inhibit the growth of cells expressing one allelic form of a gene, 2S but do not inhibit the activity of the A allelic form.
B. Small Molecule Inhibitors SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 Low molecular weight inhibitors can be identified and generated by at least one of the following methods; (1) screening of small organic molecules present in ' microorganism fermentation broth for allele-specific activity; or (2) screening of compound libraries. Once a compound is identified which exhibits allele specific activity, derivatives of that compound can be obtained or produced in order to obtain compounds having superior properties, such as greater activity, greater specificity , or better administration related properties (e.g., solubility, toxicity, and others).
A small molecule for allele specific targeting, i. e. , variance specific targeting, to a polypeptide or protein target will generally have the following characteristics:
v Differential binding affinity for protein domains altered by the amino acid variance or uniform binding to the protein with differential effects due to subsequent interactions with variant residues.
v Inhibition of protein function following differential binding. Several mechanisms of inhibition are possible including:
competitive inhibition of active sites or critical allosteric sites, allosteric inhibition of protein function, altering compartmentalization or stability, and inhibition of quaternary associations.
o Favorable pharmaceutical properties, such as safety, stability, and kinetics.
In view of the art relating to identification of compounds that interact with particular features of a polypeptide or protein or protein complex, There are clear ' precedents for developing drugs, i. e. , inhibitors, that are variance-specific including drugs that are allosteric inhibitors of protein functions. Several lines of experimental evidence demonstrate that small molecule variance specific SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 inhibitors can be designed and constructed for particular targets.
Specifically:
o Several essential gene targets have been identified that contain variances within domains comprising the active site.
a It is possible to screen for ligands that recognize variant surface features.
Combinatorial methods using antibodies, peptides, or nucleic acids suggest that specific ligands can be selected for large fractions of the surface of any protein.
a There are many literature reports of single amino acid substitutions, within the active site as well as elsewhere within a protein, altering ligand specificity and drug action.
a Allosteric (noncompetitive) inhibition of protein function may be induced by binding ligands to many different surfaces of a protein. Ligands can cause allosteric inhibition by disturbing secondary, tertiary or quaternary (subunit-subunit} interactions of a protein. There is ample evidence that 1 S such effects can a induced by binding to sequences outside the active site and even in regions that are uninvolved in the normal catalytic or regulatory activity of a protein.
Each of these points is discussed in more detail below.
Variances located within domains comprising the active site.
Crystal structures are available for several of the exemplary targets or for homologous proteins that can allow prediction of tertiary structure. As noted, the protein variance in Replication Protein A occurs within the domain that is involved in binding DNA. The protein variance in CARS occurs within the domain involved in tRNA binding.
The proximity of the active site to these variances may be exploited by several different strategies:
SUBSTITUTE SHEET (RULE 26) WO 98/-11648 PCT/US98/0~419 o Competitive inhibitors can exert variance-specific effects by exhibiting differential affinities for variant active sites, thereby interfering with binding of the substrate or critical allosteric effectors.
o Competitive inhibitors may bind with equal affinity for the active site but exerting different effects on the structure or function of the variant domain.
o Allosteric inhibitors can exert variance-specific effects by binding differentially to variant forms of the active domain and distorting the structure or function of the active site.
Screening for ligands that recognize variant surface features.
Combinatorial libraries of antibodies, peptides, nucleic acids, or carbohydrates have been used to demonstrate that ligands can be identified that will bind to large fractions of the surface of any protein.
A library of 6.5 X 1010 antibody-bearing phage was screened for binding to various targets and contained antibodies against all targets tested.
Selex and Aptamer technologies involve selection of random oligonucleotides that bind to specific targets. Reports indicate that ligands with high affinity and specificity can be selected for diverse targets despite the limited chemical diversity of the nucleic acid-based ligands.
These studies demonstrate the ability to identify ligands for unique surface features using several different chemistries. Similarly, small molecule protein surface interaction can be screened; two broad approaches for identifying small molecule ligands can be distinguished:
' 25 o Combinatorial approaches coupled with methods for high-throughput screening provide a similar scope of opportunities as combinatorial methods focused on nucleic acids, peptides, or carbohydrates.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 o Rational design or focused combinatorial approaches based on biochemical, biophysical, and structural data about the target protein may be optimal when the crystal structure of the protein is known.. When the crystal structure of the target protein or its homologues are known it will S often be possible to model the topology and surface chemistry of the target in detail. These data are useful in optimizing the binding specificity or allosteric inhibitory function of the product through a series of iterative steps once a prototype binding ligand is identified. Structural modeling of the target can be particularly useful in optimizing the variance specificity of a ligand that binds to the target sequence.
Examples of single amino acid substitutions altering sensitivity to small molecules Many amino acid substitutions have been described in proteins that alter the specificity or function of small-molecule ligands. These substitutions are useful models for variance-specific interactions (e.g. interactions that are altered by the amino acid substitutions that distinguish variant forms of a protein.) There are clear precedents for variance-specific drug effects in humans.
Variance-specific interactions are observed in a wide variety of structurally and functionally heterogeneous proteins. Among these are variances in human proteins including:
o N-acetyl transferase 2 - variances affect acetylation of drugs including caffeine and arylamines;
o CYP2C19 - variances affect the hydroxylation of mephenytoin and related compounds;
v CYP2D6 - variances affect hydroxylation of debrisoquine and related compounds;
o glucose-6-phosphate dehydrogenase - variances account for sensitivity to primaquine and other drugs.
- SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 There are numerous examples of variance-specific drug effects in targets for antiviral and antimicrobial drugs. The most extensively characterized are those in HIV Reverse Transcriptase and ~3-lactamase. These data indicate that many different amino acid substitutions can alter drug effects. Moreover, while amino acid substitutions are classically distinguished as "conservative" or "non-conservative," it is evident from these data that many seemingly "conservative"
substitutions can have significant effects. For each of the types of amino acid substitution identified within the exemplary target genes, examples of the same amino acid substitution altering the interaction of small molecule drugs on a target protein is shown in one or more of the model systems.
Sites of allosteric inhibition Most drug development focuses on competitive inhibitors of protein action rather than noncompetitive, allosteric inhibitors. There is no a priori advantage to a competitive versus allosteric inhibitor except for the fact that medicinal chemistry often begins with candidate molecules derived from natural substrates or cofactors. There are, in fact, conceptual advantages to allosteric inhibitors since each protein may contain multiple allosteric sites, and allosteric inhibitors may be effective at lower concentrations (e.g. those equivalent to the substrate) since there is no need to compete with the substrate for binding.
Detailed crystallographic and other structural studies of a variety of enzymes show that the mechanism of allosteric inhibition commonly involves conformational changes (e.g. domain movements) far from the site of contact with the allosteric regulator. These data illustrate the cooperativity of protein structure, demonstrating how a small change in one region of a protein is amplified throughout the structure. Such cooperativity allows small molecules binding to various regions of a protein to have significant structural and SUBSTITUTE SHEET (RULE 26) functional effects.
One way to assess the probability of achieving allosteric effects from a variant sequence is to examine the distribution and nature of mutations that affect drug action in several well-characterized proteins. Another is to examine the distribution of epitopes for antibodies that bind to the surface of a protein and inhibit its function. Analyses of these types show that allosteric sites are widely dispersed within proteins and may comprise the majority of the protein's surface.
For example:
HN 1 reverse transcriptase (RTE is a heterodimer with p66 and p51 subunits.
The p66 subunit is 560 amino acids, and p51 is a 440 amino acid subfragment of p66. The three dimensional structure of HIV-1 RT has been solved by x-ray crystallography. Three HIV-1 RT structures have been published, including complexes with double stranded DNA at 3.0 A resolution and with the non-nucleoside inhibitors nevirapine (at 3.SA) and -APA (at 2.S.A).
Two classes of HIV-1 RT inhibitors have been developed. The first class comprises nucleoside analogues including AZT, ddI and ddC. The second class comprises non-nucleoside analogues belonging to several chemical groups, including TIBO, BHAP, HEPT, -APA, dipyridodiazepinone, pyridinone, and inophyllum derivatives, all of which bind the same hydrophobic pocket in HIV
RT. Many amino acid substitutions have been described that produce resistance to these drugs. Table S shows the location of selected mutations within HIV-1 RT that cause resistance to nucleoside analogues as well as the mechanism of inhibition postulated from physical-chemical experiments and structural data;
the list is not comprehensive.
Table 4 SUBSTITUTE SHEET (RULE 26) Location and postulated mechanism of amino acid substitutions which confer resistance to nucleoside analog inhibitors. trp266X - multiple substitutions.
Potential resistance mechanism Mutation Location Mutation Direct I n d I n d of creates effect i r a i r a mutation resistanceon c t c t to drugs)dNTP effects effect binding via by interactions with dNTP
binding site met411eu a4 AZT X

1ys65arg 3- 4 ddC,ddI, X

asp67asn 3- 4 loop AZT X

thr69asp 3- 4 loop ddC X

1ys70arg 3- 4 loop AZT X

1eu74va1 4 ddI X

va175thr ddI,ddA

glu89gly 5a ddI,ddA X

i1e135thr7- 8 loop ddI X

met184va19- 10 turnddI, ddC X X

thr215tyrlla AZT X X

thr215phella AZT X X

1ys219g1nllb AZT X X X

trp266X -thumb AZT

These data demonstrate that nucleoside analog resistance arises from mutations in multiple domains. Many of the mutations are located far from the dNTP binding sites. These changes inhibit drug function by altering the conformation of the target protein in a manner analogous to those conformational changes that may be induced by an allosteric inhibitor.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 Table 5 summarizes the mutations that alter the function of non-nucleoside inhibitor drugs Table 5 Location and postulated mechanism of amino acid substitutions which confer resistance to non-nucleoside analog inhibitors.
Mutation Effect Mutation confers resistance of Mutation location mutation to:

ala98gly Sb- 6 loop flexibilityPyridinone L-697661, Nevirapine leui00ileSb- 6 loop -branch Pyridinone L-697661, Nevirapine, TIBO 882913 lyslOlgluSb- 6 loop charge Pyridinone L-697661, Pyridinone L-697639, 1ys103asnSb- 6 loop charge Pyridinone L-697661, loss BHAP U-87201,Nevirapine va1106a1a6 less bulkyNevirapine, TIBO 882913 va1108i1e6 bulkier Pyridinone L-697661, Nevirapine g1uI381ys7- 8 Loop charge TIBO 882913 val i 9 charge Pyridinone L-697661 79asp va1179g1u9 charge Pyridinone L-697661 tyr181cys9 less bulkyPyridinone L-697661, BHAP U-87201, Nevirapine, TIBO 882913 tyr188cys10 less bulkyNevirapine tyr188his10 less bulkyTIBO 882913, g1y190g1u10 charge Nevirapine 1eu228phe12 bulkier BHAP U-90152 g1u233va113 charge BHAP U-87201 pro2361eu13- 14 loopflexibilityBHAP U-87201 1ys238thr14 charge BHAP U-87201 trp266X -thumb TIBO 882913 SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/0~419 It is evident from these examples that the substitutions which inhibit drug functions are distributed across several domains. Different inhibitory mechanisms have been postulated in domains throughout the protein, based on the three-dimensional structure of the protein. Most involve conformational disruption of the protein secondary and tertiary structure.
Thyrotropin receptor Naturally occurring antibodies against the thyrotropin receptor can cause activation of thyroid function (Grave's disease) or inhibition of thyroid function (Hashimoto's disease). The sites within the thyrotropin receptor that are targeted by these natural antibodies have been mapped in detail and have been tested with monoclonal antibodies. Most of the inhibitory antibodies do not interfere with binding of thyrotropin to its receptor, and thus, are allosteric rather than competitive inhibitors. Several independent classes of inhibitory antibodies have been identified that bind to epitopes within different domains of the receptor.
At least one of these epitopes is in a domain that is entirely unimportant for receptor activity and can be deleted by site-directed mutagenesis without disrupting the function of the receptor. These experiments provide an explicit precedent for achieving allosteric inhibitory effects from ligands that target widely dispersed sequences within the protein.
Thermus aquaticus DNA polymerase The inhibitory activity of 24 monoclonal antibodies to Thermus aquaticus DNA polymerase has been investigated. The antibodies recognized 13 non-overlapping epitopes. Antibody binding to eight epitopes was inhibitory. Inhibitory antibodies mapped to several distinct domains, including the 5' nuclease domain, the polymerase domain and the boundary region between the 5' nuclease and polymerase domains. Some antibodies recognized epitopes overlapping the DNA binding groove of the polymerase. Significantly, the inhibitory antibodies recognized epitopes constituting as much as 50% of the Taq polymerase surface, and the non-inhibitory antibodies a further ~25%.
SUBSTITUTE SHEET (RULE 26) WO 98.11648 PCTIU598/0~419 ~i-lactamase The ~i-lactamases are a diverse family of enzymes which catalyze the hydrolysis of the ~3-lactam ring of penicillin and cephalosporin antibiotics.
Interactions of these proteins with various small molecule drugs have been characterized in detail as the pharmaceutical industry has worked to develop chemically modified penicillins and cephalosporins to elude inactivation by ~i-lactamases. In addition, a ~i-lactamase inhibitor (clavulanic acid) has also been introduced into clinical use.
As each new drug has been introduced into wide use, mutant ~i-lactamases have emerged that are resistant to the drug. Over 190 ~3-lactamases have been described i0 with differential specif city for the various penicillins and cephalosporins. Many of these differ by only a few amino acids. Many different amino acid substitutions at various sites within the protein can change the substrate specificity of the enzyme.
kat G (Isoniazid resistance) The kat G protein of M. tuberculosis encodes a catalase-peroxidase enzyme that is one of two mycobacterial genes frequently altered in isoniazid resistant strains (the other is inhA). There are a wide variety of amino acid substitutions in katG associated with drug resistance distributed evenly across the 740 amino acids of the protein. The mechanism by which some of these substitutions inhibit katG function can be inferred from the structure of the homologous yeast and E. coli enzymes and knowledge of the catalytic function of the enzyme. For example, insertion of an Ile between positions 125 and 126 affects a conserved interhelical loop near the active site residues; substitutions at amino acid 275 and 315 are likely to affect the ligand access channel; substitutions at amino acid 463 may affect a N-terminal substrate binding site. Other substitutions occur in regions that are not directly related to the functional sites of the protein.
The examples described above demonstrate that small molecules can discriminate in activity between polypeptides or proteins which have one a single amino acid SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98105419 difference in sequence, i. e., a single amino acid sequence variance.
The application of small molecule inhibitor identification is specifically discussed in Example 39 below in connection with the methylguanine methyltransferase gene.
C. Antibody Inhibition.
Once an essential gene is identified and is determined to exist in two or more allelic forms which encode different proteins, antibodies can be raised against both allelic forms of the protein. The techniques for using a specific protein or an oligopeptide as an antigen to elicit antibodies which specifically recognize epitopes on the IO peptide or protein are well known. Preferably monoclonal antibodies (MABs) are used.
In one embodiment, the DNA sequence of the desired allelic form of the target gene can be cloned by insertion into an appropriate expression vector and translated into protein in a prokaryotic or eukaryotic host cell. The protein can be recovered and 1 S used as an antigen to elicit the production of specific antibodies. In another embodiment, the DNA of the desired allelic form of the target gene is amplified by PCR technology and is subsequently translated in vitro into protein to be used as the antigen to elicit the production of specific antibodies. A third embodiment is to use the DNA sequence of the alternative alleles as a basis for the generation of synthetic 20 peptides representing the amino acid sequence of the alleles for use as antigen to elicit the production of specific antibodies.
Antibodies can be generated either by standard monoclonal antibody techniques or - generated through recombinant based expression systems. See generally, Abbas, Lichtman, and Pober, ~:'ellular and Molecular Immunolog.Y, W.B. Saunders Co.
25 (1991). The term "antibodies" is meant to include intact antibody molecules of the SUBSTITUTE SHEET (RULE 26) WO 98/-11648 PCTlUS98/05419 IgD isotype as well as antibody fragments or derivatives, such as Fab and F(ab')2, which are capable of specifically binding to antigen. The antibodies so produced will preferentially bind only the protein produced in the allelic form which was used as an antigen to create the antibody. If the targeted protein is expressed on the cell surface, the antibody or antibody derivative can be tested as a therapeutic.
Antibody inhibitors are most effective when they are directed against cell surface proteins or receptors. If the essential protein produced by the targeted allele is not a cell surface protein or receptor, the development of antibody inhibitors may also require the use of a special antibody-delivery system to facilitate entry of the antibody into the tumor cells. The plasma membrane that surrounds all cells is designed to limit the entrance of most compounds. Entry is generally restricted to small, non-charged molecules (absence of charge allows them to slip through the fatty membrane) or to those factors that can penetrate the cell using existing, specialized import mechanisms. The introduction into cells of much larger molecules, such as specific antibodies, other proteins, or peptides, requires appropriate delivery systems such as are known in the art. Alternatively, the structure of the variable region of allele specific antibodies can be used as the basis for design of smaller allele specific inhibitory molecules.
D. Oligopeptides Oligopeptides can be demonstrated to have a very high degree of specificity in their interaction with functional polypeptides such as cellular enzymes, receptors or other polypeptides essential for cell viability. Methods for screening peptide sequences which have high specificity for binding to, and functional inhibition of, a specific polypeptide target have been well described previously. Scott, J.K. and Smith G.P., "Searching for Peptide Ligands with an Epitope Library," Science 249:386-390 (1990). These methods include the screening of M13 libraries by "phage display"
of polypeptide sequences as well as direct screening of peptides or mixtures of synthetic peptides for binding to or inhibition of the target functional polypeptide.
SU8STlTUTE SHEET (RULE 26) WO 98/.11648 PCTlUS98/05419 The oligopeptides of this invention can be synthesized chemically or through an appropriate gene expression system. Synthetic peptides can include both naturally occurring amino acids and laboratory synthesized, modified amino acids.
Also provided herein are functional derivatives of a polypeptide or protein.
By "functional derivative" is meant a "chemical derivative," "fragment,"
"variant,"
"chimera," or "hybrid" of the polypeptide or protein, which terms are defined below. A functional derivative retains at least a portion of the function of the protein, for example reactivity with a specific antibody, enzymatic activity or binding activity mediated through noncatalytic domains, which permits its utility in accordance with the present invention.
A "chemical derivative" of the complex contains additional chemical moieties not normally a part of the protein. Such moieties may improve the molecule's solubility, absorption, biological half life, and the like. The moieties may alternatively decrease the toxicity of the molecule, eliminate or attenuate any undesirable side effect of the molecule, and the like. Moieties capable of mediating such effects are disclosed in Remington's Pharmaceutical Sciences (1980).
Procedures for coupling such moieties to a molecule are well known in the art.
Covalent modifications of the protein or peptides are included within the scope of this invention. Such modifications may be introduced into the molecule by reacting targeted amino acid residues of the peptide with an organic derivatizing agent that is capable of reacting with selected side chains or terminal residues, as described below.
Cysteinyl residues most commonly are reacted with alpha-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to give carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues also are derivatized by reaction with bromotrifluoroacetone, chloroacetyl phosphate, N-SUBSTITUTE SHEET (RULE 26) alkylmaleimides, 3-vitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloro-mercuribenzoate, 2-chloromercuri-4-nitrophenol, or chIoro-7-nitrobenzo-2-oxa-1,3-diazole.
Histidyl residues are derivatized by reaction with diethylprocarbonate at pH
5.5-7.0 because this agent is relatively specific for the histidyl side chain. Para-bromophenacyl bromide also is useful; the reaction is preferably performed in 0.1 M sodium cacodylate at pH 6Ø
Lysinyl and amino terminal residues are reacted with succinic or other carboxylic acid anhydrides. Derivatization with these agents has the effect or reversing the charge of the lysinyl residues. Other suitable reagents for derivatizing primary amine containing residues include imidoesters such as methyl picolinimidate;
pyridoxal phosphate; pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;
O-methylisourea; 2,4 pentanedione; and transaminase-catalyzed reaction with glyoxylate.
Arginyl residues are modified by reaction with one or several conventional reagents, among them phenylglyoxal, 2,3-butanedione, 1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residues requires that the reaction be performed in alkaline conditions because of the high pKa of the guanidine functional group. Furthermore, these reagents may react with the groups of lysine as well as the arginine alpha-amino group.
Tyrosyl residues are well-known targets of modification for introduction of spectral labels by reaction with aromatic diazonium compounds or tetranitromethane.
Most commonly, N-acetylimidizol and tetranitromethane are used to form O-acetyl tyrosyl species and 3-vitro derivatives, respectively.
SUBSTITUTE SHEET (RULE 26) WO 98Lt1648 PCT/US98/05419 Carboxyl side groups (aspartyl or glutamyl) are selectively modified by reaction carbodiimide (R'-N-C-N-R') such as 1-cyclohexyl-3-(2-morpholinyl(4-ethyl) carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.
Furthermore, aspartyl and glutamyl residue are converted to asparaginyl and S glutaminyl residues by reaction with ammonium ions.
Glutaminyl and asparaginyl residues are frequently deamidated to the corresponding glutamyl and aspartyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Either form of these residues falls within the scope of this invention.
Derivatization with bifunctional agents is useful, for example, for cross-linking component peptides to each other or the complex to a water-insoluble support matrix or to other macromolecular carriers. Commonly used cross-linking agents include, for example, 1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylic acid, homobi-functional imidoesters, including disuccinimidyl esters such as 3,3'-dithiobis(succinimidylpropionate), and bifunctional maleimides such as bis-N-maleimido-1,8-octane. Derivatizing agents such as methyl-3-[p-azidophenyl) dithiolpropioimidate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light. Alternatively, reactive water-insoluble matrices such as cyanogen bromide-activated carbohydrates and the reactive substrates described in U.S. Patent Nos. 3,969,287; 3,691,016; 4,195,128;
4,247,642; 4,229,537; and 4,330,440 are employed for protein immobilization.
Other modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the alpha-amino groups of lysine, arginine, and histidine side chains (Creighton, T.E., Proteins:
Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, pp. 79-SUBSTITUTE SHEET (RULE 26) (1983)), acetylation of the Nterminal amine, and, in some instances, amidation of the C-terminal carboxyl groups.
Such derivatized moieties may improve the stability, solubility, absorption, biological half life, and the like. The moieties may alternatively eliminate or S attenuate any undesirable side effect of the protein complex and the like.
Moieties capable of mediating such effects are disclosed, for example, in Remin on's Pharmaceutical Sciences, 18th ed., Mack Publishing Co., Easton, PA (I990).
The term "fragment" is used to indicate a polypeptide derived from the amino acid sequence of the protein or polypeptide having a length less than the full-length polypeptide from which it has been derived. Such a fragment may, for example, be produced by proteolytic cleavage of the full-length protein. Preferably, the fragment is obtained recombinantly by appropriately modifying the DNA sequence encoding the proteins to delete one or more amino acids at one or more sites of the C-terminus, N-terminus, and/or within the native sequence.
Another functional derivative intended to be within the scope of the present invention is a "variant" polypeptide which either lack one or more amino acids or contain additional or substituted amino acids relative to the native polypeptide. The variant may be derived from a naturally occurring polypeptide by appropriately modifying the protein DNA coding sequence to add, remove, and/or to modify codons for one or more amino acids at one or more sites of the C-terminus, N-terminus, and/or within the native sequence.
A functional derivative of a protein or polypeptide with deleted, inserted and/or substituted amino acid residues may be prepared using standard techniques well-known to those of ordinary skill in the art. For example, the modified components of the functional derivatives may be produced using site-directed mutagenesis SU9STITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 techniques (as exemplified by Adelman et al., 1983, DNA 2:183) wherein nucleotides in the DNA coding the sequence are modified such that a modified coding sequence is modified, and thereafter expressing this recombinant DNA in a prokaryotic or eukaryotic host cell, using techniques such as those described above.
Alternatively, components of functional derivatives of complexes with amino acid deletions, insertions and/or substitutions may be conveniently prepared by direct chemical synthesis, using methods well-known in the art.
E. Complementary Oligonncleotides and Ribozymes Oligonucleotides or oligonucleotide analogs which interact with complementary sequences of cellular target DNA or RNA can be synthesized and used to inhibit or control gene expression at the levels of transcription or translation. The oligonucieotides of this invention can be either oIigodeoxyribonucleotides or oligoribonucleotides, or derivatives thereof, which are complementary to the allelic forms of the targeted essential gene or they can act enzymatically, such as ribozymes. Both antisense RNA and DNA can be used in this capacity as chemotherapeutic agents for inhibiting gene transcription or translation.
Trojan, J., et al., "Treatment and prevention of rat glioblastoma by immunogenic C6 cells expressing antisense insulin-like growth factor I RNA," Science 259:94-97 (1993).
Inhibitory complementary oligonucleotides may be used as inhibitors for cancer therapeutics because of their high specificity and lack of toxicity.
Included in the scope of the invention are oligoribonucleotides, including antisense RNA and DNA molecules and ribozymes that function to inhibit expression of an essential gene in an allele specific manner. Anti-sense RNA and DNA molecules act to directly block the translation of mRNA by binding to targeted mRNA and preventing protein translation or directing RNase mediated degradation of the mRNA. With respect to antisense DNA, oligodeoxyribonucleotides derived from the translation initiation site, e.g., between -10 and +10 regions of the relevant SUBSTITUTE SHEET (RULE 26) WO 981.11648 PCT/U598/0~419 nucleotide sequence, are preferred.
Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. The mechanism of ribozyme action involves sequence specific interaction of the ribozyme molecule to complementary target RNA, followed by a endonucleolytic cleavage. Within the scope of the invention are engineered hammerhead, hairpin, and other motif ribozyme molecules that catalyze sequence specific endonucleolytic cleavage of RNA sequences encoding a gene product essential for cell survival, growth, or vitality.
Specific ribozyme cleavage sites within any potential RNA target can initially be identified by scanning the target molecule for ribozyme cleavage sites, such as sites which include the following sequences, GUA, GUU and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for predicted structural features, such as secondary structure, that may render the oiigonucleotide sequence unsuitable. The suitability of candidate targets may also be evaluated by testing their accessibility to hybridization with complementary oligonucleotides, using ribonuclease protection assays. See, for example, Draper PCT WO
93/23569.
For the present invention, the target site will generally include a sequence variance site as described above.
Both anti-sense RNA and DNA molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA and DNA
molecules. See, for example, Draper, supra. hereby incorporated by reference herein. These include techniques for chemically synthesizing oligodeoxyribonucleotides well known in the art such as, for example, solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro and in vivo transcription of DNA sequences encoding the SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 antisense or ribozyme RNA molecule. Such DNA sequences may be incorporated into a wide variety of vectors which incorporate suitable RNA polymerase promoters such as the T7 or SP6 polymerase promoters. Alternatively, antisense or ribozyme cDNA constructs that synthesize antisense or ribozymes RNA
constitutively or inducibly, depending on the promoter used, can be introduced stably into cell lines.
Various modifications to the DNA molecules may be introduced as a means of increasing intracellular stability and half life. Possible modifications include but are not limited to the addition of flanking sequences of ribo- or deoxy-nucleotides IO to the 5' and/or 3' ends of the molecule or the use of phosphorothioate or methyl phosphonate rather than phosphodiesterase linkages within the oligodeoxyribonucleotide backbone. Modifications may also be made on the nucleotidic sugar or purine or pyrimidine base, such as 2'-O-alkyl (e.g., 2'-O-methyl), 2'-O-allyl, 2'-amino, or 2'-halo (e.g., 2'-F). A variety of other substitutions are also known in the art and may be used in the present invention.
More than one type of nucleotide modification may be used in a single modified oligonucleotide.
A specific application of generating inhibitors which are either complementary oligonucleotides or inhibitory oligopeptides is described in Holzmayer, Pestov, and Roninson, "Isolation of dominant negative mutants and inhibitory antisense RNA
sequences by expression selection of random DNA fragments," Nucleic Acids Research 20:711-717 (1992). In this study, genetic suppressor elements (GSEs) are identified by random DNA fragmentation and cloning in expression plasmids.
Preferred oligonucleotide inhibitors include oligonucleotide analogues which are resistant to degradation or hydrolysis by nucleases. These analogues include neutral, or nonionic, methylphosphonate analogues, which retain the ability to SUBSTITUTE SHEET (RULE 26) WO 98/-Ii648 PCT/US98/0~419 interact strongly with complementary nucleic acids. Miller and Ts'O, Anti-Cancer Drug Des. 2:11-128 (1987). Further oligonucleotide analogues include those containing a sulfur atom in place of the 3'-oxygen in the phosphate backbone, and oligonucleotides having one or more nucleotides which have modif ed bases and/or modified sugars. Particularly useful modifications include phosphorothioate linkages and 2'-modification (e.g., 2'-O-methyl, 2'-F, 2'-amino).
F. Gene Therapy Nucleic acid molecules encoding oligonucleotide or polypeptide inhibitors will also be useful in gene therapy (reviewed in Miller, Nature 357:455-460, (1992).
Miller indicates that advances have resulted in practical approaches to human gene therapy that have demonstrated positive initial results. An in vivo model of gene therapy for human severe combined immunodeficiency is described in Ferrari, et al., Science 251:1363-1366, (1991). The basic science of gene therapy is described in Mulligan, Science 260:926-931, (1993).
Some methods of delivery that may be used include:
a. complexation with lipids, b. transduction by retroviral vectors, c. localization to nuclear compartment utilizing nuclear targeting site found on most nuclear proteins, d. transfection of cells ex vivo with subsequent reimplantation or administration of the transfected cells, e. a DNA transporter system.
A nucleic acid sequence encoding an inhibitor may be administered utilizing an ex vivo approach whereby cells are removed from an animal, transduced with the nucleic acid sequence and reimplanted into the animal. The liver can be accessed by an ex vivo SUBSTITUTE SHEET (RULE 26) WO 98/-11648 PCT/US98/0~419 approach by removing hepatocytes from an animal, transducing the hepatocytes in vitro with the nucleic acid sequence and reimplanting them into the animal (e.g., as described for rabbits by Chowdhury et al, Science 254: 1802-1805, 1991, or in humans by Wilson, Hum. Gene Ther. 3: 179-222, 1992) incorporated herein by reference.
Many nonviral techniques for the delivery of a nucleic acid sequence encoding an inhibitor into a cell can be used, including direct naked DNA uptake (e.g., Wolff et al., Science 247: 1465-1468, 1990), receptor-mediated DNA uptake, e.g., using DNA coupled to asialoorosomucoid which is taken up by the asialoglycoprotein receptor in the liver (Wu and Wu, J. Biol. Chem. 262: 4429-4432, 1987; Wu et aL, J. Biol. Chem. 266: 14338-14342, 1991), and liposorne-mediated delivery (e.g., Kaneda et al., Expt. Cell Res. 173: 56-69, 1987; Kaneda et al., Science 243:

378, 1989; Zhu et al., Science 261: 209-211, 1993). Many of these physical methods can be combined with one another and with viral techniques;
enhancement of receptor-mediated DNA uptake can be effected, for example, by combining its use with adenovirus (Curiel et al., Proc. Natl. Acad Sci. USA 88: 8850-8854, 1991;
Cristiano et al., Proc. Natl. Acad. Sci. USA 90: 2122-2126, 1993).
In one preferred embodiment, an expression vector containing a sequence encoding a ribozyme or an antisense oligonucleotideis inserted into cells, the cells are grown in vitro and then infused in large numbers into patients.
The gene therapy may involve the use of an adenovirus containing a sequence encoding a ribozyme or an antisense oligonucleotide targeted to a tumor.
Expression vectors derived from viruses such as retroviruses, vaccinia virus, adenovirus, adeno-associated virus, herpes viruses, several RNA viruses, or bovine papilloma virus, may be used for delivery of nucleotide sequences into the targeted SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTIUS98/0~419 cell population (e.g., tumor cells). Methods which are well known to those skilled in the art can be used to construct recombinant viral vectors containing coding sequences. See, for example, the techniques described in Maniatis et. al., Molecular Toning: A Laboratory Manual; Cold Spring Harbor Laboratory, N.Y. (1989), and in Ausubel et. al., Current Protocols in Molecular Biology, Greene Publishing Associates and Wiley Interscience, N.Y. (1989). Alternatively, recombinant nucleic acid molecules encoding protein sequences can be used as naked DNA or in reconstituted system e.g., liposomes or other lipid systems for delivery to target cells (See e.g., Feigner et. al., Nature 337:387-8, 1989). Several other methods for the direct transfer of plasmid DNA into cells exist for use in human gene therapy and involve targeting the DNA to receptors on cells by complexing the plasmid DNA to proteins. See, Miller, supra.
In its simplest form, gene transfer can be performed by simply injecting minute amounts of DNA (e.g., a plasmid vector encoding an inhibitor) into the nucleus of a cell, through a process of microinjection. Capecchi MR, Cell 22:479-88 (1980).
The DNA can be part of a formulation which protects the DNA from degradation or prolongs the bioavailability or the DNA, for example by complexing the DNA
with a compound such as polyvinylpyrrolidone. Once recombinant genes are introduced into a cell, they can be recognized by the cells normal mechanisms for transcription and translation, and a gene product will be expressed. Other methods have also been used for introducing DNA into larger numbers of cells. These methods include: transfection, wherein DNA is precipitated with CaP04 and taken into cells by pinocytosis (Chen C. and Okayama H, Mol. Cell Biol. 7:2745-52 (1987));
electroporation, wherein cells are exposed to large voltage pulses to introduce holes into the membrane (Chu G. et al., Nucleic Acids Res., 15:1311-26 (1987));
lipofection/liposome fusion, wherein DNA is packaged into lipophilic vesicles which fuse with a target cell (Felgner PL., et al., Proc. Natl. Acad. Sci.
USAi 84:7413-7 (1987)); and particle bombardment using DNA bound to small SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/U598/05419 projectiles (Yang NS. et aL, Proc. Natl. Acad. Sci. 87:9568-72 (1990)).
Another method for introducing DNA into cells is to couple the DNA to chemically modified proteins.
It has also been shown that adenovirus proteins are capable of destabilizing S endosomes and enhancing the uptake of DNA into cells. The admixture of adenovirus to solutions containing DNA complexes, or the binding of DNA to polylysine covalently attached to adenovirus using protein crosslinking agents substantially improves the uptake and expression of the recombinant gene.
Curiel DT et al., Am. J. Respir. Cell. Mol. Biol., 6:247-S2 (1992).
As used herein "gene transfer" means the process of introducing a foreign nucleic acid molecule into a cell. Gene transfer is commonly performed to enable the expression of a particular product encoded by the gene. The product may include a protein, polypeptide, anti-sense DNA or RNA, or enzymatically active RNA. Gene transfer can be performed in cultured cells or by direct administration into animals.
1S Generally gene transfer involves the process of nucleic acid contact with a target cell by non-specific or receptor mediated interactions, uptake of nucleic acid into the cell through the membrane or by endocytosis, and release of nucleic acid into the cytoplasm from the plasma membrane or endosome. Expression may require, in addition, movement of the nucleic acid into the nucleus of the cell and binding to appropriate nuclear factors for transcription.
As used herein "gene therapy" is a form of gene transfer and is included within the definition of gene transfer as used herein and specifically refers to gene transfer to express a therapeutic product from a cell in vivo or in vitro. Gene transfer can be performed ex vivo on cells which are then transplanted into a patient, or can be 2S performed by direct administration of the nucleic acid or nucleic acid-protein complex into the patient.
SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 In another preferred embodiment, a vector having nucleic acid sequences encoding an allele specific inhibitor is provided in which the nucleic acid sequence is expressed only in specific tissue. Examples or methods of achieving tissue-specific gene expression are described in International Publication No. WO 93/09236, published May 13, 1993.
VII. Utility of allele-specific inhibitors of essential genes A. Conditions susceptible to therapy.
The fraction of all cancers could be treated with allele specific inhibitors directed against allele specific essential gene targets is a function of the frequency of the target allele and the frequency of LOH. The ideal target would be deleted in 100%
of alI major cancers and would exist in two allelic forms, each with an allele frequency of 0.5 so that half the population would be heterozygous. An inhibitor of one allele of such an ideal target would be a useful agent for 25% of all cancer patients. An inhibitor of the other allele of the same ideal target would be therapeutic for an additional 25% of all patients, making 50% of all patients treatable. The ideal target has so far not been identified, but we have identified many essential gene sequence variance targets which are deleted in 30-70% of several major cancers, and which are heterozygous in 25-50% of North Americans.
Allele specific inhibitors of both alleles of such targets would be expected to address 0.4 x 0.5 = 0.2 or 20% of the relevant cancer population. The relevant cancer population often includes breast, colon and lung cancer, which sum to 500,000 new cases per year in the United States. Thus a total available market of 100,000 patients is not unusual, and many targets would be expected to address markets of at least 50,000 patients.
The targets of this invention are suitable for treatment of many different cancers, which includes cancers of different types, as well as non-malignant proliferative SUBSTITUTE SHEET (RULE 26) disorders, as well as being suitable for use in other applications involving targeting - alternative allelic forms of a gene. The classification and nomenclature for a variety of benign and malignant tumors relevant to the present invention is shown in the following table (Table 6-1 from Robbins et al., Pathologic Basis of Disease, 3rd ed.
( 1984), however, the invention is not limited to these cancers or classifications.
Table 6 Tissue of Origin Benign Malignant I. Composed of one parenchyma) cell type Sarcomas A. Tumors of mesenchymal origin ( 1 ) Connective tissue and derivatives fibrous tissue fibroma fibrosarcoma myxomatous tissue myxoma myxocarmo fatty tisssue lipoma liposarcoma cartilage chondroma chondrasarcoma bone osteoma osteosarcoma osteogenic sarcoma (2) Endothelial & related tissues blood vessels hemangioma angiosarcoma capillary cavernous sclerosing hemangioendothelioma endotheliosarcoma, Kaposi's sarcoma lymph vessels lymphoangioma lymphangiosarcoma synovia synovioma (synoviosarcoma) mesothelium mesothelioma (mesotheliosarcoma) brain coverings meningioma glomus glomus tumor SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/0~419 ?endothelial or Ewing's tumor mesenchymal cells (3) Blood cells & related cells hematopoietic cells myelogenous leukemia monocytic leukemia lymphoid tissue malignant lymphomas lymphocytic leukemia plastocytoma (multiple myeloma) monocyte-macrophage histiocytosis X

Langerhans' cells ?histiocytic lymphoma (4) Muscle ?Hodgkin's disease smooth muscle leiomyoma leiomyosarcoma striated muscle rhabdomyoma rhabdomyosarcoma B. Tumors of epithelial Carcinomas origin stratified squamous squamous cell squamous cell or papilloma epidermoid carcinoma basal cells of skin or adnexia basal cell carcinoma skin adnexal glands sweat glands sweat gland adenomasweat gland carcinoma sebaceous gland sebaceous gland sebaceous gland adenoma carcinoma epithelial lining glands or ducts -well adenoma adenocarcinoma differentiated papillary adenomapapillary group cystadenoma adenocarcinoma cystadenocarcinoma poorly differentiated group medullary carcinoma undifferentiated carcinoma (simplex) respiratory tract bronckogenic carcinoma bronchial "adenoma"

neuroectoderm nevus melanoma (melanocarcinoma) renal epithelium renal tubular renal cell carcinoma adenoma (hypernephroma) SUBSTITUTE SHEET (RULE 26) liver cells liver cell adenomahepatocellular carcinoma bile duct bile duct adenomabile duct carcinoma (cholangiocarcinoma) urinary tract epithelium transitional cellpapillary carcinoma ' (transitional) papilloma transitional cell carcinoma squamous cell carcinoma placental eptithelium hydatiform mole choriocarcinoma testicular epithelium (germ seminoma cells) embryonal carcinoma II. More than one neoplastic cell type--_ mixed tumors---usually derived from mixed tumor of salivary malignant mixed tumor one germ layer gland origin of salivary gland origin salivary glands (pleiomorphic adenoma) renal anlage Wilms' tumor III. More than one neoplastic cell type derived from more than one germ layer---teratogenous totipotential cells in gonads or teratoma, dermoid cyst malignant teratoma and in teratocarcinoma embryonic rests Allele specific therapy can be targeted to essential genes which undergo LOH
in many different tumor types, including the tumors and tumor types described in the tables - 25 above, and in Figure 3.
For the treatment of patients suffering from a tumor using an allele specific inhibitor, SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US9810~419 the preferred method of preparation or administration will generally vary depending on the type of inhibitor to be used. Thus, those skilled in the art will understand that administration methods as known in the art will also be appropriate for the inhibitors of this invention.
B. Pharmaceutical Formulations and Modes of Administration The particular compound, antibody, antisense or ribozyme molecule that exhibits allele specific inhibitor activity can be administered to a patient either by itself, or in pharmaceutical compositions where it is mixed with suitable Garners or excipient(s).
In treating a patient exhibiting a disorder of interest, a therapeutically effective amount of a agent or agents such as these is administered. A therapeutically effective dose refers to that amount of the compound that results in amelioration of one or more symptoms or a prolongation of survival in a patient.
Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LDS° (the dose lethal to 50% of the population) and the EDS° (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LDS°/EDS°. Compounds which exhibit large therapeutic indices are preferred. The data obtained from these cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage of such compounds lies preferably within a range of circulating concentrations that include the EDS° with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
For any compound used in the method of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. For example, a dose can be formulated in animal models to achieve a circulating plasma concentration range that SUBSTITUTE SHEET (RULE 26) WO 98Lti648 PCT/US98105419 includes the ICso as determined in cell culture. Such information can be used to more accurately determine useful doses in humans. Levels in plasma may be measured, for example, by HPLC.
The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g. Fingl et.
al., in T a Pharmacological Basis of Therapeutics, 1975, Ch. 1 p.l). It should be noted that the attending physician would know how to and when to terminate, interrupt, or adjust administration due to toxicity, or to organ dysfunctions. Conversely, the attending physician would also know to adjust treatment to higher levels if the clinical response were not adequate (precluding toxicity). The magnitude of an administrated dose in the management of the oncogenic disorder of interest will vary with the severity of the condition to be treated and to the route of administration. The severity of the condition may, for example, be evaluated, in part, by standard prognostic evaluation methods.
Further, the dose and perhaps dose frequency, will also vary according to the age, body weight, and response of the individual patient. A program comparable to that discussed above may be used in veterinary medicine.
Depending on the specific conditions being treated, such agents may be formulated and administered systemically or locally. Techniques for formulation and administration may be found in ReminQ-ton's Pharmaceutical Sciences,18th ed., Mack Publishing Co., Easton, PA ( 1990). Suitable routes may include oral, rectal, transdermal, vaginal, transmucosal, or intestinal administration; parenteral delivery, including intramuscular, subcutaneous, intramedullary injections, as well as intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, or intraocular injections, just to name a few.
For injection, the agents of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks's solution, Ringer's solution, or physiological saline buffer. For such transmucosal administration, SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCTIL1S98/0~419 penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.
Use of pharmaceutically acceptable carriers to formulate the compounds herein disclosed for the practice of the invention into dosages suitable for systemic administration is within the scope of the invention. With proper choice of carrier and suitable manufacturing practice, the compositions of the present invention, in particular, those formulated as solutions, may be administered parenterally, such as by intravenous injection. The compounds can be formulated readily using pharmaceutically acceptable carriers well known in the art into dosages suitable for oral administration. Such carriers enable the compounds of the invention to be formulated as tablets, pills, capsules, liquids, gels, syrups, slurries, suspensions and the like, for oral ingestion by a patient to be treated.
Agents intended to be administered intracellularly may be administered using techniques well known to those of ordinary skill in the art. For example, such agents may be encapsulated into liposomes, then administered as described above.
Liposomes are spherical lipid bilayers with aqueous interiors. All molecules present in an aqueous solution at the time of liposome formation are incorporated into the aqueous interior.
The Iiposomal contents are both protected from the external microenvironment and, because Iiposomes fuse with cell membranes, are efficiently delivered into the cell cytoplasm. Additionally, due to their hydrophobicity, small organic molecules may be directly administered intraceilularly.
Pharmaceutical compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve its intended purpose. Determination of the effective amounts is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein. In addition to the active ingredients, these pharmaceutical SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. The preparations formulated for oral administration may be in the farm of tablets, dragees, capsules, or solutions.
The pharmaceutical compositions of the present invention may be manufactured in a manner that is itself known, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levitating, emulsifying, encapsulating, entrapping or lyophilizing processes.
Pharmaceutical formulations for parenteral administration include aqueous solutions of the active compounds in water-soluble form. Additionally, suspensions of the active compounds may be prepared as appropriate oily injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes. Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
Pharmaceutical preparations for oral use can be obtained by combining the active compounds with solid excipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as - sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carboxymethylcellulose, and/or polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate. Dragee cores are provided with suitable SUBSTITUTE SHEET (RULE 26) coatings. For this purpose, concentrated sugar solutions may be used, which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.
Pharmaceutical preparations which can be used orally include push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules can contain the active ingredients in admixture with filler such as lactose, binders such as starches, and/or lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added.
Factors specific for the delivery of antisense and ribozyme nucleic acids are known in the art, for example as discussed in Couture et al., WO 94/02595, which is hereby IS incorporated herein by reference. This reference also describes the synthesis of nucleic acid molecules having a variety of 2' modified nucleotides.
The references cited herein are incorporated by reference to the same extent as if each had been individually incorporated by reference. The invention is illustrated further by the following examples, which are not to be taken as limiting in any way.
The examples, individually, and together, further demonstrate that one skilled in the art would be able to practice each of the steps in developing useful pharmaceutical products as described in the invention. Generally, the development of such a product involves the following steps:
1. Select candidate target gene essential for cell survival or proliferation.
2. Determine chromosome location and LOH frequency.
3. Identify common variance in the normal population.
SUBSTITUTE SHEET (RULE 26) 4. Demonstrate antiproliferative effects from inhibition of candidate gene.
- 5. Design variance-specific inhibitor.
6. Achieve variance-specific antiproliferative effects in cancer cells.
EXAMPLES
Example 1. Genes required for Cell Proliferation Many genes are involved in the process of cell proliferation and are potential targets for anti-proliferative drugs in this invention. Dividing cells progress through a repeating cycle of four stages, each of which is critical to the proliferation process.
During the first phase, G1, cells ready the proteins they need to replicate their DNA, which occurs during S phase. Following S phase, cells enter G2, in which they prepare to divide into two daughter cells, each of which will contain the DNA content of the original cell. The final stage of the cell cycle is M phase, in which cells undergo mitosis. During mitosis, the cell nucleus disappears and the two sets of replicated chromosomes are separated to opposite sides of the cell. The cell then divides into two cells, the nucleus reforms in each new cell, and the cycle begins again. Cell proliferation is exceedingly complex and requires the precise coordination of many processes, including DNA synthesis, chromosome condensation and separation, and cell fission. In eukaryotic cells such as yeast, many of the proteins involved in cell division are encoded by essential genes, including those contributing to the duplication of the nucleus and the functions of microtubules, spindle pole bodies the centromere and the kinetochore.
A number of proteins are essential for cell proliferation. Proteins that are critical to - this process can be divided into two classes: (i) proteins that regulate cell division; (ii) proteins that form structures involved in cell division. Proteins that regulate cell ' division include, but are not limited to, proteins involved in the regulation of particular SUBSTITUTE SHEET (RULE 26) WO 98/x1648 PCT/iJS98/05419 steps in the division process, such as nuclear breakdown and the transition between the different stages of mitosis, as well as proteins regulating the initiation of mitosis, such as the cylins, cyclin-dependent kinases (CDKs), and the kinases and phosphatases that regulate CDKs. Cyciin B, the cyclin-dependent kinase cdc2, and the cdc25C
phosphatase are examples of proteins that regulate the initiation of mitosis.
Deletion of yeast homologs of these genes is lethal, verifying their critical role in regulating the entry into mitosis. (It has been established that many human genes which encode proteins involved in highly conserved cellular processes can substitute for their yeast counterparts, and vice versa. For example such conservation has been demonstrated for components of the transcriptional apparatus, as well as components of the translational apparatus.) Proteins that form structures involved in cell division include, but are not limited ta, those involved in the processes of chromosome condensation and separation.
Examples are tubulin and kinesin, which participate in the separation of chromosomes, and KIAA0165 and CDC37, involved in the spindle pole. Deletion of the yeast homolog of CDC37 is lethal.
Inhibiting the ability of a cell to divide induces, by definition, a cytostatic response, often followed by cell death. Colchicine and nocodazole are examples of drugs that inhibit microtubule function in vitro, thereby preventing chromosome separation and leading to cell cycle arrest during mitosis. Vinblastine and vincristine, which also inhibit microtubule function and therefore cell proliferation, have been used widely in the treatment of cancer.
Examples of genes that are involved in the process of cell proliferation, and are thus essential for cell survival or proliferation are shown in the accompanying table. Each of these genes has been disrupted in Saccharomyces cerevisiae and the mutant yeast shown to be nonviable.
SUBSTITUTE SHEET (RULE 26) Table: Genes Essential for Cell Proliferation in Yeast Gene Name Function of Gene Product APC 1 . Component of the anaphase promoting complex.

CAK1 cdk activating kinase, activates cdc28p CBF2, CBF3B, Essential constituents of the kinetochore CSEl CBFS, protein complex CTFI3, SKP1 Cbf3 (subunits a-d), a structural component of centromeres to which microtubules attach.

CDC 14 Protein tyrosine phosphatase that performs a function late in the cell cycle.

CDC I 5 Essential for late nuclear division CDC 16, CDC23, Part of anaphase promoting complex, required CDC27 for Clb2p degradation and metaphase-anaphase transition.

CDC28 Essential for mitosis CDC3I Calcium binding protein of spindle pole body (SPB), involved in SPB duplication CDC37 Required for spindle pole duplication and passage through START.

CDCS Protein kinase required for exit from mitosis, and operation of mitotic spindle.

CKS 1 Associated with cdc28p kinase CRM1 Chromosome region maintenance protein.

CSE I Probable kinetochore protein, interacts with cetromeric element CDEII.

CSE4 Required for chromosome segregation.

DBF4 Regulatory subunit for cdc7p protein kinase, required for G1/S transition.

DIS3 Involved in mitotic control.

DNA43 Required for S-phase initiation or completion.

DPB11 Involved in DNA replication and an S-phase checkpoint.

ESPI, KAKI Required for regulation of spindle body pole duplication.

IPL I Protein kinase involved in chromosome segregation.

KRRI Essential for cell division.

MEC 1 Checkpoint protein required for mitotic growth, DNA repair and recombination.

MIF2 Centromere protein required for chromosome segregation and s indle integrity SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 MOB 1 Required for normal cell cycle progression MPS 1 Protein kinase involved in spindle body pole duplication; also mitotic checkpoint NDC1 Required for spindle body pole duplication;
nuclear envelope component NNF 1 Nuclear envelope protein required for nuclear migration during mitosis.

NRK1 Protein kinase that interacts with cdc3lp NUF2 Component of spindle body pole required for nuclear division.

RFTI Involved in nuclear division.

SMC1, SMC2, Coiled coil proteins involved in chromosome SMC3 condensation and segregation; required for nuclear division.

SPC42, SPC97, Components of spindle pole body. The latter SPC98, SPI6 3 interact with microtubules, gamma tubulin & stu2p, respectively.

SPK1 Protein kinase with a checkpoint function in S and G2 STU1 Required for mitotic spindle assembly.

TEMI Involved in termination of M-phase.

It will be evident to one skilled in the art that many genes that express essential metabolic and homeostatic functions of the cell will also be essential for cell proliferation.
Example 2. Genes required to maintain inorganic ions at levels compatible with cell growth or survival.
Inorganic Ions are Essential for Cellular Life Inorganic ions are required for virtually all cellular processes: they are important for maintenance of cell shape and osmolality; they are prosthetic groups of a wide variety of enzymes; they are required for ATP production coupled to ion diffusion;
they mediate signal transduction both from intracellular and extracellular signals.
Hence maintenance of inorganic ions at physiological concentrations is essential for cell SUBSTfTUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/0~419 proliferation and cell survival. The importance of maintaining physiological ion concentrations is further demonstrated by the observation that deviation from normal levels leads to cytostatic or cytotoxic effects, as demonstrated by the effects of selectively poisoning ion channels or placing cells in hypotonic or hypertonic extracellular fluid.
Inorganic Ions Must be Transported Across Membranes Maintenance of ion concentrations at optimal concentrations within cells is complicated by the presence of membranes which, because of their hydrophobic interior, form a highly impermeable barrier to most polar molecules, including inorganic ions. Important cell membranes include the plasma membrane as well as the nuclear membrane, mitochondria) membranes, the endoplasmic reticulum and Golgi apparatus, lysosomes and vesicles of various types, all of which are essential for cell proliferation or survival. Therefore maintaining the concentration of essential polar molecules, including both organic and inorganic ions, at levels compatible with cell growth or survival requires specialized mechanisms for moving such ions across the plasma membrane and the various intracellular membrane bound compartments.
Vital components of the apparatus for maintaining ion concentrations at levels essential for cell survival include regulatory molecules that sense the concentration of ions in different cellular compartments and produce signals to increase or decrease the concentration of said ions to levels compatible with cell survival; proteins that actively or passively transport ions across membranes; and proteins that modify ions so they can be transported across membranes.
Membrane transport proteins can be divided into several categories depending on whether they require energy (provided either by ATP hydrolysis or by co-transport of ions such as sodium or protons down their electrochemical gradients), produce energy SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/05419 (ATP synthetases, which are usually coupled to proton diffusion) or are energy neutral.
Other categories of transporters include those that transport one or more solutes (one or more of which may be ions), gated vs. non-gated - i.e. open only transiently (ligand gated and voltage gated channels) or open continuously, allowing ions to move down their concentration and electrochemical gradients. Specific types of essential membrane transporters include uniports, which simply transport one solute from one side of the membrane to the other, and cotransports, in which the transport of one solute is dependent on the simultaneous or sequential transport of a second solute in the same direction (symport) or in the opposite direction (antiport).
Other inorganic ions, such as iron, are transported bound to carrier proteins (transferrin in the case of iron). Transport of the iron carrier protein involves a complex cycle that begins with binding of iron to transferrin, binding of the iron-transferrin complex to transfernn receptor, formation of coated pits, endocytosis of the transferrin-iron complex via the coated pits, release of iron from transferrin in endosomes upon acidification to pH 5, and then recycling of the transferrin receptor-apotransferrin complex to the surface of the cell where, at neutral pH, the apotransferrin is released from transferrin receptor into the extracellular fluid to bind more iron and participate in another cycle. Thus in the case of transferrin-mediated iron transfer there are a variety of specialized proteins which must interact in a coordinated manner for transport to occur effectively.
Some of the specific inorganic ions which must be transported across the both the plasma membrane and intracellular membranes are sodium, potassium, chloride, calcium, hydrogen, magnesium, manganese, phosphate, selenium, molybdenum, iron, copper, zinc, fluorine, iodine, chromium, silicon, tin and arsenic. Specific transporters have been identified for many of these solutes including sodium, potassium, chloride, protons, copper and iron among others.
SUBSTITUTE SHEET (RULE 26) WO 98/41648 PCT/US98/0~419 Regulation of ion concentrations at appropriate levels is often an energy-dependent process; intracellular and extracellular concentrations may differ by 10 fold or more (see Table).
Ion Concetrations Inside and Outside a Typical Mammalian Cell Intracellular Extraceilular Ion concentration concentration (m~ (m~
Cations Na+ ~ 5-15 145 K+ 140 5 Mg++ 30 1-2 Ca++ 1-2 2.5-5 Anions _ Cl - 4 ~ 110 Inhibitors of lon Transporting Proteins are Cytostatic or Cytotoxic Blocking import of essential cell nutrients, including inorganic ions, prevents cell growth and can lead to cell death. A well studied example is blockade of iron transport by inhibition of transferrin receptor. Dividing cells require iron, and transferrin receptor-mediated uptake of iron-transferrin complexes is the principal route for iron aquisition. Iron uptake requires multiple steps, including receptor binding, endocytosis via coated pits, acidification of endosomes and consequent release of iron from transferrin, followed by recycling of transferrin receptor-apotransferrin to the cell surface for another round of binding. Each step requires the coordinated function of a variety of proteins. Anti-transferrin receptor antibodies arrest cell growth by blocking iron uptake; antitumor effects have been demonstrated in vitro and in vivo with such antibodies.
Ion pumps are another class of proteins for which cytotoxic inhibitors have been SUBSTITUTE SHEET (RULE 26) WO 98/.11648 PCT/US98/0~419 identified. All animal cells contain a Na+, K+ pump which operates as an antiport, actively pumping Na+ out of the cell and K+ in against their concentration gradients.
In coupling the hydrolysis of ATP to the active transport of 3 Na+ out and 2 K+ into the cell the pump is electrogenic. The electrochemical gradients generated and maintained by the Na+,K+ pump are essential for regulation of cell volume and for the secondary, sodium-coupled active transport of a variety of organic and inorganic molecules including glucose, amino acids and Cap. Hence the sodium potassium pump plays an essential role in cellular physiology. More than one third of a typical animal cells energy requirement is expended in fueling this pump. (Alberts et al. Molecular Biology of the Cell, Garland Publishing, New York, 1983, p.291.) Ouabain is an inhibitor of the Na+, K+ ATPase. It binds to the catalytic alpha 1 subunit of sodium potassium ATPase and is a potent cytotoxic drug. Cells treated with ouabain swell and eventually burst as they are unable to maintain a balance of osmotic forces because they can no longer pump out Na+. See Example 11 for a more detailed description of the essential properties of the Na+, K+ ATPase. Amiloride is another cytotoxic drug;
it blocks the sodium-proton antiporter. Thus inhibition of proteins essential for maintaining physiologial levels of inorganic ions is toxic to cells.
lon Transporting Proteins are Evolutionarily Conserved and Essential in Other Species Many of the proteins required to maintain inorganic ions at physiologic levels are widely conserved in eukaryotes, reflecting an ancient and vital role. A number of gene disruption experiments in non-human cells demonstrate the importance of ion transponting proteins for cell growth and survival. For example in the yeast Saccharomyces Cerevisiae the gene encoding CDC1 protein, involved in maintaining ion homeostasis, has been disrupted resulting in non-viable yeast. Another essential yeast gene is PMA1, which encodes a H+ transporting P-type ATPase of the plasma membrane; activity of the encoded protien is rate limiting for growth at low pH.
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Claims

What we claim is:

1. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:
(a) determining at least two alleles of a said gene, wherein said gene encodes a product required for cell proliferation;
(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles;
wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

2. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:
(a) determining at least two alleles of a said gene, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival;
(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles;
wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

3. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:
(a) determining at least two alleles of a said gene, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival;
(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles;
wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

4. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:
(a) determining at least two alleles of a said gene, wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival;
(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles;
wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

5. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:
(a) determining at least two alleles of a said gene, wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival;
(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles;
wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

6. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:
(a) determining at least two alleles of a said gene, wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures;
(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles;

wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

7. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:
(a) determining at least two alleles of a said gene, wherein said gene is located on a high frequency LOH chromosomal region;
(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles;
wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

8. The method of claim 7, wherein said gene is located on a chromosomal arm which has a frequency of allele loss of at least 15 % in a cancer.

9. The method of claim 7, wherein said gene is located in proximity to a chromosomal marker which undergoes LOH at a frequency of at least 10 % in a cancer.

10. The method of claim 7, wherein said gene is located in proximity to a tumor suppressor gene which undergoes LOH at a frequency of at least 10 % in a cancer.

11. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a gene vital for cell growth or viability, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:
(a) determining at least two alleles of a said gene, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene;
(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles;
wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.

12. The method of claim 11, wherein said gene is located on a high frequency LOH chromosomal region.

13. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth, wherein said gene encodes a product required for cell proliferation, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

14. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

15. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

16. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth,wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

17. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth,wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

18. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth,wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

19. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth, wherein said gene is located on a high frequency LOH chromosomal arm region, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

20. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a gene vital for cell viability or cell growth, wherein said gene has at least two sequence variances which occur at frequences such that at least 10 % of a population is heterozygous for said gene, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.

21. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required for cell proliferation; and a pharmaceutically acceptable carrier or excipient.

22. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival; and a pharmaceutically acceptable carrier or excipient.

23. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival; and a pharmaceutically acceptable carrier or excipient.

24. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival; and a pharmaceutically acceptable carrier or excipient.

25. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival; and a pharmaceutically acceptable carrier or excipient.

26. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures; and a pharmaceutically acceptable carrier or excipient.

27. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene is located on a high frequency LOH chromosomal arm region; and a pharmaceutically acceptable carrier or excipient.

28. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of an essential gene in a population, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene; and a pharmaceutically acceptable carrier or excipient.

29. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:
(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required for cell proliferation;

(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and (c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in which cancerous cells have only the allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene.

30. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:
(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival;
(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and (c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in which cancerous cells have only the allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene.

31. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:
(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival;

(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and (c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in which cancerous cells have only the allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene.

32. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:
(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival;
(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and (c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in which cancerous cells have only the allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene.

33. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:
(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival;
(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and (c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in which cancerous cells have only the allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene.

34. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:
(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures;
(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and (c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in which cancerous cells have only the allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene.

35. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at least two alternative alleles, comprising the steps of:
(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene is located on a high frequency LOH
chromosomal arm region;
(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and (c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in which cancerous cells have only the allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene.

36. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a gene having at Least two alternative alleles, comprising the steps of:
(a) identifying a gene vital to cell viability or cell growth that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell, and wherein said gene has at least two sequence variances which occur at frequences such that at Least 10% of a population is heterozygous for said gene;
(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and (c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in which cancerous cells have only the allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene.

37. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of:
a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required for cell proliferation; and wherein cells of said precancerous condition have undergone LOH of said first gene.

38. The method of claim 37, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of:
b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

39. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of:
a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival; and wherein cells of said precancerous condition have undergone LOH of said first gene.

40. The method of claim 39, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of:
b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

41. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of:
a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival; and wherein cells of said precancerous condition have undergone LOH of said first gene.

42. The method of claim 41, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of:
b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

43. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of:
a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival; and wherein cells of said precancerous condition have undergone LOH of said fast gene.

44. The method of claim 43, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of:
b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

45. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of:
a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival; and wherein cells of said precancerous condition have undergone LOH of said first gene.

46. The method of claim 45, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of:
b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

47. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of:
a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene encodes a product required to maintain the integrity and function of cellular and subcellular structures;
and wherein cells of said precancerous condition have undergone LOH of said first gene.

48. The method of claim 47, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of:
b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

49. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of:
a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a first essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene is located on a high frequency LOH chromosomal arm region; and wherein cells of said precancerous condition have undergone LOH of said first gene.

50. The method of claim 49, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of:
b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

51. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of:
a. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of a fast essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells, and said first gene has at least two sequence variances which occur at frequences such that at least 10 % of a population is heterozygous for said gene; and wherein cells of said precancerous condition have undergone LOH of said first gene.

52. The method of claim 51, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of:
b. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different essential gene, and wherein said patient is heterozygous for each targeted essential gene and each targeted essential gene has undergone LOH in cells of said precancerous condition.

53. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of:
administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required for cell proliferation, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.
54. The method of claim 53, further comprising the steps of:
(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or (b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).
55. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of:
administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.
56. The method of claim 55, further comprising the steps of:
(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or (b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).
57. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of:
administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.
58. The method of claim 57, further comprising the steps of:
(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or (b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).
59. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of:
administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.
60. The method of claim 59, further comprising the steps of:
(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or (b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).
61. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of:
administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.
62. The method of claim 61, further comprising the steps of:
(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or {b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).
63. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of:
administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.
64. The method of claim 63, further comprising the steps of:
(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or (b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).
65. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of:
administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene is located on a high frequency LOH chromosomal arm region, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.
66. The method of claim 65, further comprising the steps of:
(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or (b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).
67. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a gene vital for cell growth or viability, comprising the step of:
administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene, said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.
68. The method of claim 67, further comprising the steps of:
(a) determining whether non-cancerous cells of said patient are heterozygous for a particular gene essential for cell growth or viability; or (b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).
69. A method of inhibiting growth of a cell comprising the step of:
administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required for cell proliferation, and wherein said inhibitor is less active on at least one other allele of said gene.
70. A method of inhibiting growth of a cell comprising the step of:
administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, and wherein said inhibitor is less active on at least one other allele of said gene.
71. A method of inhibiting growth of a cell comprising the step of:
administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival, and wherein said inhibitor is less active on at least one other allele of said gene.

72. A method of inhibiting growth of a cell comprising the step of:
administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival, and wherein said inhibitor is less active on at least one other allele of said gene.
73. A method of inhibiting growth of a cell comprising the step of:
administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival, and wherein said inhibitor is less active on at least one other allele of said gene.
74. A method of inhibiting growth of a cell comprising the step of:
administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures, and wherein said inhibitor is less active on at least one other allele of said gene.
75. A method of inhibiting growth of a cell comprising the step of:
administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene is located on a high frequency LOH chromosomal arm region, and wherein said inhibitor is less active on at least one other allele of said gene.
76. A method of inhibiting growth of a cell comprising the step of:

administering at least one inhibitor active on an allele of a gene vital for cell viability or growth, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene, and wherein said inhibitor is less active on at least one other allele of said gene.
77. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of:
identifying a patient heterozygous for a said gene encoding a product required for cell proliferation, wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.
78. The method of claim 77, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.
79. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of:
determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required for cell proliferation, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.

80. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of:
identifying a patient heterozygous for a said gene encoding a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.
81. The method of claim 80, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.
82. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of:
determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.
83. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of:

identifying a patient heterozygous for a said gene encoding a product required to maintain organic compounds at levels compatible with cell growth or survival;
wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.
84. The method of claim 83, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.
85. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of:
determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required to maintain organic compounds at levels compatible with cell growth or survival, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.
86. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of:
identifying a patient heterozygous for a said gene encoding a product required to maintain cellular proteins at levels compatible with cell growth or survival;
wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.

87. The method of claim 86, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.
88. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of:
determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required to maintain cellular proteins at levels compatible with cell growth or survival, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.
89. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of:
identifying a patient heterozygous for a said gene encoding a product required to maintain cellular nucleotides at levels compatible with cell growth or survival;
wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.
90. The method of claim 89, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.

91. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of:
determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required to maintain cellular nucleotides at levels compatible with cell growth or survival, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.
92. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of:
identifying a patient heterozygous for a said gene encoding a product required to maintain the integrity and function of cellular and subcellular structures;
wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.
93. The method of claim 91, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.
94. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of:
determining whether cancer cells in said patient have undergone LOH of a said gene encoding a product required to maintain the integrity and function of cellular and subcellular structures, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.
95. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of:
identifying a patient heterozygous for a said gene located on a high frequency LOH chromosomal arm region;
wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.
96. The method of claim 95, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.
97. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of:
determining whether cancer cells in said patient have undergone LOH of a said gene located on a high frequency LOH chromosomal arm region, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.
98. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the steps of:
identifying a patient heterozygous for a said gene which has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene;
wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.
99. The method of claim 98, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.
100. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a gene vital for cell growth or viability, wherein said patient is suffering from a cancer, said method comprising the step of:
determining whether cancer cells in said patient have undergone LOH of a said gene which has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.
101. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required for cell proliferation, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.

102. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required to maintain inorganic ions and vitamins at levels compatible with cell growth or survival, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.
103. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required to maintain organic compounds at levels compatible with cell growth or survival, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.
104. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required to maintain cellular proteins at levels compatible with cell growth or survival, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.
105. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required to maintain cellular nucleotides at levels compatible with cell growth or survival, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.
106. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene encodes a product required to maintain the integrity and function of cellular and subcellular structures, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.
107. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene is located on a high frequency LOH chromosomal arm region, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.
108. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a gene vital for cell growth or viability, wherein said gene has at least two sequence variances which occur at frequences such that at least 10% of a population is heterozygous for said gene, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.
109. The method, inhibitor, pharmaceutical composition, or nucleic acid probe of any of claims 1, 13, 21, 29, 37, 53, 69, 77, and 101, wherein said gene is selected from the group consisting of 14-3-3 Protein TAU, CCNA(G2/Mitotic-Specific Cyclin A), CCNB1(G2/Mitotic-Specific Cyclin B1), CCND1(G1/S-Specific Cyclin D1), CCND2(G1/S-Specific Cyclin D2), CCND3(G1/S-Specific Cyclin D3), Cell division control protein 16, Cell division cycle 2, G1 to S and G2 to M, Cell division cycle 25A, Cell division cycle 25B, Cell division cycle 25C, Cell division cycle 27, Ceil division-associated protein BIMB, Cyclin A1(G2/Mitotic-Specific Cyclin A1), Cyclin C
(G1/S-Specific Cyclin C), Cyclin G1(G2/Mitotic-Specific Cyclin G), Cyclin G2 (G2/Mitotic-Specific Cyclin G), Cyclin H, Cyclin H Assembly, GSPT1(G1 to S
phase transition 1), Mitotic MAD2 Protein, MRNP7, RANBP1(RAN binding protein 1), WEE1, Cell Division Protein Kinase 4, CDC28 protein kinase 1, CDC28 protein kinase 2, M-Phase inducer phosphatase 2, M-phase phosphoprotein, mpp6, PPPlca(Protein phosphatase 1, catalytic subunit, alpha isoform), STM7-LSB, CENP-F kinetochore protein, Centromere autoantigen C, Centromere protein B
(80kD), Centromere protein E (312kD), CHC1(Chromosome condensation 1), Chromatin assembly factor-I p150 subunit, Chromatin assembly factor-I p60 subunit, Chromosome segregation gene homolog CAS, HMG1(High-mobility group (nonhistone chromosomal) protein 1), Minichromosome Maintenance (MCM7), Mitotic centromere-associated kinesin, RMSA1(Regulator of mitotic spindle assembly 1), and SUPTSh(Chromatin structural protein homolog (SUPT5H)).
110. The method ,inhibitor, pharmaceutical composition, or nucleic acid probe of any of claims 2, 14, 22, 30, 39, 55, 70, 80, and 102, wherein said gene is selected from the group consisting of PMCA1 (Calcium Pump), PMCA2 (Calcium Pump), PMCA3 (Calcium Pump), PMCA4 (Calcium Pump), ATP2b1 (Calcium-Transporting ATPase Plasma Membrane), ATP2b2 (Calcium-Transporting ATPase Plasma Membrane), ATP2b4 (Calcium-Transporting ATPase Plasma Membrane), ATP5b (ATP Synthase Beta Chain, Mitochondrial Precursor), Chloride Conductance Regulatory Protein ICLN, H-Erg (Potassium Channel Protein EAG), Nuclear Chloride Ion Channel Protein (NCC27), SCN1b(Sodium Channel, Voltage-Gated, Type I, Beta Polypeptide), Two P-Domain K+ Channel TWIK-1, VDAC2 (Voltage-Dependent Anion-Selective Channel Protein 2), ATP1b1 (Sodium/Potassium-Transporting ATPase Beta-1 Chain), ATP1b2 (Sodium/Potassium-Transporting ATPase Beta-2 Chain), ATPase, Ca++ transporting, plasma membrane 4, ATPase, Ca++ transporting, plasma membrane 2, ATPase, Na+/K+ transporting, alpha 1 polypeptide, ATPase, Na+/K+ transporting, alpha 3 polypeptide, ATPase, Na+/K+ transporting, beta 1 polypeptide, ATPase, Na+/K+ transporting, beta 2 polypeptide, Na+,K+ ATPase, 1 Subunit, Na+,K+ ATPase, 2 alpha, Na+,K+
ATPase, 3 beta, SLC9a1(Solute carrier family 9 (sodium/hydrogen exchanger)), Solute carrier family 4, anion exchanger, member 1, Solute carrier family 4, anion exchanger, member 2, Solute carrier family 9 (sodium/hydrogen exchanger), Passive transporters, MaxiK Potassium Channel Beta Subunit, Chloride Channel 2, Chloride Channel Protein (CLCN7), TRPC1 (Transient Receptor Potential Channel 1), Potassium Channel Kv2.1, ATPSd(ATP synthase, H+ transporting, mitochondria) F1 complex, delta subunit), ATP5f1(ATP synthase, H+ transporting, mitochondria) F0 complex, subunit b), ATPSo(ATP synthase, H+ transporting, mitochondria) F1 complex, O subunit), ETFa(Electron-transfer-flavoprotein, alpha polypeptide (glutaric aciduria II)), ETFb(Electron-transfer-flavoprotein, beta polypeptide), Nadh-ubiquinone oxidoreductase 13 kd-B subunit, Nadh-ubiquinone oxidoreductase 39 kD
subunit precursor, NADH-Ubiquinone oxidoreductase 75 kD subunit precursor, NADH-Ubiquinone oxidoreductase MFWE subunit, NDUFV2(NADH
dehydrogenase (ubiquinone) flavoprotein 2 (24kD)), Ubiquinol-cytochrome c reductase complex 11 kD, ATP Synthase Alpha Chain, NADH dehydrogenase-ubiquinone Fe-S protein 8, 23 kDa subunit, Ascorbic Acid (transporter), Folate Binding Protein, Folate receptor 1 (adult), Nicotinamide (transporter), Pantothenic Acid transporter, Riboflavin (transporter), SCL19A1 (Solute Carrier Family 19, Member1), Solute carrier family 19 (folate transporter), member 1, Thiamine, B6, B12 (transporter), ATP7b (Copper-Transporting ATPase 2), Ceruloplasmin (ferroxidase), Ceruloplasmin receptor (Copper Transporter), Copper Transport Protein HAH1, Molybdenum, Selenium, Tranferrin Receptor (Iron Transporter), Zinc Transporter, and mitochondria) import receptor subunit TOM20.
111. The method, inhibitor, pharmaceutical composition, or nucleic acid probe of 3, 25, 23, 31, 41, 57, 71, 83, and 103, wherein said gene is selected from the group consisting of GLUT1, GLUT2, GLUT3, GLUT4, GLUT5, GLUT6, Solute carrier family 5 (sodium/glucose cotransporter), Solute carrier family 2 (facilitated glucose transporter), member 2, Solute carrier family 2 (facilitated glucose transporter) member 5, Solute carrier family 3 member 1, System b,(Na+ independent), System y,(Na+ independent), ATRC1(Catioinc), LEUT(Leucine Transporter), SLC1A1(Solute Carrier Family 1, Member 1), Solute carrier family 16 (monocarboxylic acid transporters), ACO1(Aconitase 1), ACO2(Aconitase 2, mitochondrial), Acyl-Coenzyme A dehydrogenase, C-2 to C-3 short chain, Acyl-Coenzyme A dehydrogenase, C-4 to C-12 straight chain, Acyl-Coenzyme A
dehydrogenase, long chain, Acyl-Coenzyme A dehydrogenase, very long chain, aKGD (alpha ketoglutaratedehydrogenase), ALD-a (Aldolase), ALD-b (Aldolase), ALD-c (Aldolase), CS (Citrate Synthetase), Dihydrolipoamide S-succinyltransferase, DLAT(Dihydrolipoamide S-acetyltransferase (E2 component of pyruvate dehydrogenase complex)), DLD(Dihydrolipoamide dehydrogenase (E3 component of pyruvate dehydrogenase complex, 2-oxo-glutarate complex, branched chain keto acid dehydrogenase complex)), Elk (Oxoglutarate dehydrogenase), E2k (Dihydrolipoamide S-succinyltransferase), E3 (Dihydrolipoyl Dehydrogenase), ENOl(Enolase 1,alpha), ENO2(Enolase 2), ENO3(Enolase 3), Enolase 2, (gamma, neuronal), Enolase 3, (beta, muscle), FH(Fumarate hydratase), G3PDH
(Glyceraldehyde-3-Phosphate Dehydrogenase), G6PD (Glucose-6-Phosphate Dehydrogenase), Glucose-6-phosphate dehydrogenase, HK1 (Hexokinase 1), HK2 (Hexokinase 2), HK3 (Hexokinase 3), IDH1(Isocitrate dehydrogenase 1 (NADP+), soluble), IDH2(Isocitrate dehydrogenase 2 (NADP+), mitochondrial), MDH1 (Malate dehydrogenase 1, NAD (soluble)), MDH2(Malate dehydrogenase 1, NAD (mitochondrial)), NAD(H)-specific isocitrate dehydrogenase alpha subunit, Oxoglutarate dehydrogenase (lipoamide), PDHB (Pyruvate Dehydrogenase), PDHB(Pyruvate dehydrogenase (lipoamide) beta), PDK4 (Pyruvate dehydrogenase kinase, isoenzyme 4), PFKL(Phosphofructokinase), PGI (Phosphoglucoisomerase), PGKa (Phosphoglyceromutase), PGKb (Phosphoglyceromutase), PGM1 (Phosphoglyceromutase), PGM2 (Phosphoglyceromutase), PGM3 (Phosphoglyceromutase), PGM4 (Phosphoglyceromutase), Phosphofructokinase, muscle, Phosphoglucomutase 1, Phosphoglycerate kinase 1, PK1 (Pyruvate Kinase), PK2 (Pyruvate Kinase), PK3 (Pyruvate Kinase), Pyruvate dehydrogenase kinase isoenzyme 2 (PDK2), Pyruvate kinase, liver, Pyruvate kinase, muscle, SDH1(Succinate dehydrogenase, iron sulphur (Ip) subunit), SDH2(Succinate dehydrogenase 2, flavoprotein (Fp) subunit), TKT(Transketolase (Wernicke-Korsakoff syndrome)), TPI (Trisephosphate Isomerase), Asparagine Synthetase, Aminoacylase-1, Aminoacylase-2, ACAC (Acetyl CoA Carboxylase Beta), ACAC
(Acetyl CoA Carboxylase), ACADSB(Acyl-coA dehydrogenase), Mevalonate kinase, Phosphomevalonate kinase, Aspartoacylase, Ornithine decarboxylase 1, Short-acyl-CoA
dehydrogenase, Medium acyl-CoA dehydrogenase, Long acyl-CoA
dehydrogenase, Isovalveryl CoA dehydrogenase, 2-methyl branched chain, Adenosine Deaminase, Purine-nucleoside phosphorylase, Guanine Deaminase, Xanthine Oxidase, ITM1 (Integral Transmembrane Protein), GFPT (Glutamine-(Glutamine-Fructose-6-Phosphate Transaminase), Heparan, Polypeptide N-Acetyltransferase, ACAA(Acetyl-Coenzyme A acyltransferase), Lysophosphatidic acid acyltransferase-alpha, Lysophosphatidic acid acyltransferase-beta, FNTa (Farnesyltransferase Alpha Subunit), FNTb (Farnesyltransferase Beta Subunit), NMT1 (N-myristoyltransferase), Calcineurin A, Calcineurin B, Calreticulin Precursor, Phosphatase 2b, PPP3ca(Protein phosphatase 3, catalytic subunit), SNK Interacting 2-28(Calcineurin B Subunit), Protein Kinase C, PRKCA(Protein kinase C, alpha), PRKCB1 (Protein kinase C, beta 1), PRKCD(Protein kinase C, delta), PRKCM(Protein kinase C, mu), PRKCQ(Protein kinase C-theta), PRKCSH(Protein kinase C substrate 80K-H), Geranylgeranyl, Geranylgeranyltransferase (Type I Beta), GGTB
(Geranylgeranyltransferase), Geranylgeranyltransferase (Type II Beta-Subunit), Gdp Dissociation Inhibitors, GDI Alpha (RAB GDP Dissociation inhibitor Alpha), and Rab Gdp (RAB GDP Dissociation Inhibitor Alpha).
112. The method, inhibitor, pharmaceutical composition, or nucleic acid probe of any of claims 4, 16, 24, 32, 43, 59, 72, 86, and 104, wherein said gene is selected from the group consisting of GOT(Glutamic-oxaloacetic transaminase 2), GOT1(Glutamic-oxaloacetic transaminase 1), PYCS(Pyrroline-5-carboxylate synthetase), Tyrosine aminotransferase, AARS, CARS, DARS, EPRS, FARS, GARS, HARS, IARS, KARS, LARS, MARS, NARS, QARS , RARS, SARS, TARS, VARS, WRS, YARS, Ribosomal Protein L11, Ribosomal Protein L12, Ribosomal Protein L17, Ribosomal Protein L18, Ribosomal Protein L18a, Ribosomal Protein L19, Ribosomal Protein L21, Ribosomal Protein L22, Ribosomal Protein L23, Ribosomal Protein L23a, Ribosomal Protein L25, Ribosomal Protein L26, Ribosomal Protein L27, Ribosomal Protein L27a, Ribosomal Protein L28, Ribosomal Protein L29, Ribosomal Protein L30, Ribosomal Protein L31, Ribosomal Protein L32, Ribosomal Protein L35, Ribosomal Protein L35a, Ribosomal Protein L36a, Ribosomal Protein L39, Ribosomal Protein L4, Ribosomal Protein L41, Ribosomal Protein L44, Ribosomal Protein L6, Ribosomal Protein L7, Ribosomal Protein L7a, Ribosomal Protein L8, Ribosomal Protein L9, Ribosomal Protein P1, Ribosomal Protein S10, Ribosomal Protein S11, Ribosomal Protein S13, Ribosomal Protein S14, Ribosomal Protein S15, Ribosomal Protein S15A, Ribosomal Protein S16, Ribosomal Protein S17, Ribosomal Protein S17A, Ribosomal Protein S17B, Ribosomal Protein S18, Ribosomal Protein S20, Ribosomal Protein S20A, Ribosomal Protein S20B, Ribosomal Protein S21, Ribosomal Protein S23, Ribosomal Protein S25, Ribosomal Protein S26, Ribosomal Protein S28, Ribosomal Protein S29, Ribosomal Protein S3, Ribosomal Protein S3A, Ribosomal Protein S4, Ribosomal Protein S4X, Ribosomal Protein S4Y, Ribosomal Protein S5, Ribosomal Protein S6, Ribosomal Protein S7, Ribosomal Protein S8, Ribosomal Protein S9, Initiation of polypeptide polymerization, eIF-2 (Eukaryotic initiation factor), eIF-2-associated p67(Eulcaryotic initiation factor), eIF-2A(Eukaryotic initiation factor), eIF-2Alpha(Eukaryotic initiation factor), eIF-2B(Eukaryotic initiation factor), eIF-2-2B-Gamma(Eukaryotic initiation factor), eIF-2Beta(Eukaryotic initiation factor), eIF-3 p110(Eulcaryotic initiation factor), eIF-3 p36(Eukaryotic initiation factor), eIF-4A(Eukaryotic initiation factor), eIF-4C(Eukaryotic initiation factor), eIF-4E(Eukaryotic initiation factor), eIF-4Gamma(Eukaryotic initiation factor), eIF-S(Eukaryotic initiation factor), eIF-SA, Eukaryotic peptide chain release factor subunit 1, P97(Eukaryotic initiation factor), eEF1A2(Eukaryotic elongation factor), eEF1D(Eukaryotic elongation factor), eEF2(Eukaryotic elongation factor), eIF4A2 (Eukaryotic initiation factor), KIAA0031(Elongation factor 2), KIAA0219(Putative translational activator C18G6.O5C), Factor 1-Alpha 2(Eukaryotic translation elongation factor 1 alpha 2), Cis-Trans Isomerase, DNAj Protein Homolog 1, DNAj Protein Homolog 2, DNAJ Protein homolog HSJ1, T-Complex, Aspartylglucosaminidase, T-Complex 1, Alpha, T-Complex 1, Epsilon, T-Complex 1, Gamma, T-Complex 1, Theta, T-Complex 1, Zeta, 26S Protease regulatory subunit 4, Alpha-2-Macroglobuiin, Calpain 1, Large, CLPP(ATP-Dependent CLP
protease proteolytic subunit), KIAA0123 (Mitochondrial processing peptidase alpha subunit), MMP7, Proteasome Beta 6, Proteasome Beta 7, Proteasome C13, Proteasome C2, Proteasome C7-1, Froteasome inhibitor hPI31 subunit, Proteasome P112, Proteasome P27, Proteasome P55, Enzyme E2-17 Kd(Cyclin-selective ubiquitin carrier protein), ISOT-3(Ubiquitin carboxyl-terminal hydrolase T), ORF
(Ubiquitin carboxyl-terminal hydrolase 14), PGP(Ubiquitin carboxyl-terminal hydrolase isozyme L1), UBA52(Ubiquitin A-52 residue ribosomal protein fusion product 1), Ubiquitin carboxyl-terminal hydrolase 3, Ubiquitin carboxyl-terminal hydrolase isozyme L3, Ubiquitin carboxyl-terminal hydrolase T, Ubiquitin carrier protein (E2-EPF), Ubiquitin fusion-degradation protein (UFD1L), Ubiquitin Hydrolase, Ubiquitin-conjugating enzyme E2I, SEC23(Protein transport protein SEC23), SEC23A(Protein transport protein SEC23), SEC7(Protein transport protein SEC7), SEC61 (Beta Subunit), and LDLR (LDL receptor).
113. The method, inhibitor, pharmaceutical composition, or nucleic acid probe of any of claims 5, 17, 25, 33, 45, 73, 89, and 105, wherein said gene is selected from the group consisting of Adenylate Kinase-2, Adenylosuccinate synthetase, Adenylosuccinate Lyase, DPRT (ADP-Ribosyltransferase), ADSL (Adenylosuccinate lyase/AMP synthetase), ADSS (Adenylosuccinate Synthetase), CAD PROTEIN, CTP Synthetase, CTPS(CTP synthetase), Cytidine Triphosphate Synthetase, GARS
(Phosphoribosylglycinamide synthetase), GART (Phosphoribosylglycinamide formyltransferase), GART(Phosphoribosylglycinamide formyltransferase, phosphoribosylglycinamide synthetase, phosphoribosylaminoimidazole synthetase), GMP Synthetase, IMP Cyclohydrolase, IMP dehydrogenase, IMPDH1(IMP(inosine monophosphate) dehydrogenase 1), IMPDH2(IMP (inosine monophosphate) dehydrogenase 2), Phosphoribosyl diphosphotransferase, Phosphoribosylaminoimidazolecarboxamide formyltransferase, Phosphoribosylformylglycinamide synthetase, Phosphoribosylglycinamide carboxylase, Phosphoribosylglycinamide-succinocarboxamide synthetase, PPAT
(Amidophoribosyltransferase), PPAT(Phosphoribosyl pyrophosphate amidotransferase), Ribonucleoside-diphosphate reductase M1 chain, Ribonucleoside-diphosphate reductase M2 chain, Thymidine Kinase, Thymidylate Synthase, UMK(Uridine kinase), UMPK (Uridine monophosphate kinase), UMPS(Uridine monophosphate synthetase (orotate phosphoribosyl transferase and orotidine-5'-decarboxylase)), Uridine Phosphorylase, DNA Origin Recognition Complex, ORC1, ORC2, ORC3, ORC4, ORC5, ORC6, ORC Regulators, CDC6, CDC7, CDC1, DNA Polymerization, DNA Polymerases, Adprt (NAD(+) ADP-Ribosyltransferase), DNA Polymerase Alpha-Subunit, DNA Polymerase Delta, POLa(DNA Polymerase Alpha/Primase Associated Subunit), POLb(DNA
Polymerase Beta Subunit), POLd1(Polymerase (DNA directed), Delta 1, Catalytic Subunit), POLd2(Polymerase (DNA directed), Delta 2), POLE(Polymerase (DNA
directed)), POLg (DNA Polymerase Gamma Subunit), Terminal Transferase (DNA
Nucleotidylexotransferase), Activator 1 36 Kd, CDC46 (DNA Replication Licensing Factor), CDC47 (DNA Replication Licensing Factor CDC47), DNA Topoisomerase III, DRAP1 (DNA Replication Licensing Factor MCM3), KIAA0030 Gene (Cell Division Control Protein 19), KIAA0083 Gene (DNA Replication Helicase DNA2 ), MCM3 (DNA Replication Licensing Factor MCM3), PCNA (Proliferating Cell Nuclear Antigen), PRIMl (DNA Primase 49 kD Subunit), PRIM2 (DNA Primase), PRIM2a (DNA Primase 58 kD Subunit), PRIM2b (DNA Primase), RECa (Replication Protein A 14 kD Subunit), RFC1 (Replication Factor C (activator 1)1), RFC2 (Replication Factor C 2), RFC3 (Replication Factor C (activator 1) 3), (Replication Factor C, 37-kD subunit), RFC5 (Replication Factor C), RPA1 (Replication protein A1 (70kD)), RPA2 (Replication protein A2 (32kD)), RPA3 (Replication protein A3 (14kD)), TOP1 (DNA Topoisomerase I), TOP2a (Topoisomerase (DNA) II Alpha (170kD)), TOP2b (Topoisomerase (DNA) II Beta (180kD)), CHL1(CHL1-Related Helicase), DNA Helicase II, Mi-2(Chromodomain-Helicase- DNA-Binding Protein CHD-1), RECQL (ATP-Dependent DNA Helicase Q1), Smbp2 (DNA-Binding Protein SMUBP-2), H1(0) (Histone H5A), Histone H1d, Histone H1x, Histone H2a.1, Histone H2a.2, Histone H2b.1, Histone H4, SLBP
(Histone Hairpin-Binding Protein), TATA-binding Complex, Small Nuclear RNA-Activating Complex, Polypeptide 1, 43KD (SNAPC1), Small Nuclear RNA-Activating Complex, Polypeptide 2, (SNAPC2), Small Nuclear RNA_Activating Complex, Polypeptide 3, 50KD (SNAPC3), TAF2D(TBP-associated factor), TAFII100(TBP-associated factor), TAFII130(TBP-associated factor), TAFII20(TBP-associated factor), TAFII250(TBP-associated factor), TAFII28(TBP-associated factor), TAFII30(TBP-associated factor), TAFII32(TBP-associated factor), TAFII40(TBP-associated factor), TAFII55(TBP-associated factor), TAFII80(TBP-associated factor), TBP(TATA Binding Protein), TMF1 (TATA Element Modulatory Factor 1), RPB 7.0, RPB 7.6, RPB 17, RPB 14.4, RNA polymerise I subunit hRPA39, 13.6 Kd Polypeptide (DNA-Directed RNA Polymerise II 13.6 kD
Polypeptide), POLR2C(RNA polymerise II, polypeptide C (33kD)), Polypeptide A
(220kd), RNA Polymerise II 23k, RNA polymerise II holoenzyme component (SRB7), RNA polymerise II subunit (hsRPB10), RNA polymerise II subunit (hsRPB8), RNA polymerise II subunit hsRPB4, RNA polymerise II subunit hsRPB7, RNA Polymerise II Subunit(DNA- Directed RNA Polymerises I, II, and III 7.3 kD polypeptide), TCEB1L(Transcription elongation factor B (SIII), polypeptide 1-like), RNA polymerise III subunit (RPC39), RNA polymerise III
subunit (RPC62), Elongation Factor 1-Beta, Elongation Factor S-II, TCEA
(110kD), TCEB1, TCEB (18kD), TCEB1L, TCEB3, TCEC (15kDa), TFIIS (Transcription Elongation Factor IIS), E2F1 (E2F Transcription Factor), TFAP2A (Transcription Factor A2 Alpha), TFCP2 (Transcription Factor CP2), TFC12 (Transcription Factor 12), PRKDC (Protein Kinase, DNA activated catalytic subunit), SUPT6H, TFIIA
gamma subunit, TFIIA delta, TFIIB related factor hBRF (HBRF), TFIIE Alpha Subunit, TFIIE Beta Subunit, TFIIF, Beta Subunit, GTF2F1 (TFIIF), GTF2F2 (TFIIF), General Transcription Factor IIIA, TFIIH(52 kD subunit of transcription factor), TFIIH(p89), TFIIH(p80), TFIIH(p62), TFIIH(p44), TFIIH(p34), Transcription Factor IIf(General transcription factor IIF, polypeptide 1 (74kD
subunit))Transcription Factor IIf(General transcription factor IIF, polypeptide 1 (74kD subunit)), BTF 62 kDSubunit (Basic transcription factor 62 kD subunit), CAMP-dependent transcription factor ATF-4, CCAAT box-binding transcription factor 1, CRM1(Negative regulator CRM1), Cyclic-AMP-dependent transcription factor ATF-1, GABPA(GA-binding protein transcription factor, alpha subunit (60kD)), ISGF-3(Signal transducer and activator of transcription 1-alpha/beta), NFIX(Nuclear factor I/X (CCAAT-binding transcription factor)), NFYA(Nuclear transcription factor Y, alpha), NTF97(Nuclear factor p97), Nuclear factor I-B2 (NFIB2), Nuclear factor NF45, Nuclear factor NF90, POU2F1(POU domain, class 2, transcription factor 1), Sp2 transcription factor, TCF12(Transcription factor 12 (HTF4, helix-loop-helix transcription factors 4)), TCF3(Transcription factor 3 (E2A
immunoglobulin enhancer binding factors E12/E47)), TCF6L1(Transcription factor 6-like 1), TF P65(Transcription factor p65), TFCOUP2(Transcription factor COUP
2 (a.k.a. ARP1)), Transcription factor IL-4 Stat, Transcription Factor S-II
(Transcription factor S-II-related protein), Transcription factor Stat5b, Transcription Factor, Transcription factor (CBFB), 9G8 Splicing Factor (Pre-mRNA Splicing factor SRP20), CC1.3(Splicing factor (CC1.3)), HnRNP F protein, HNRPA2B1(Heterogeneous nuclear ribonucleoproteins A2/B1), HNRPG(Heterogeneous nuclear ribonucleoprotein G), HNRPK(Heterogeneous nuclear ribonucleoprotein K), Pre-mRNA splicing factor helicase, Pre-mRNA
splicing factor SF2, P33 subunit, Pre-mRNA splicing factor SRP20, Pre-mRNA

splicing factor SRP75, PRP4(Serine/threonine-protein kinase PRP4), PTB-Associated Splicing Factor, Ribonucleoprotein A', Ribonucleoprotein A1, Ribonucleoprotein C1/C2, RNP Protein, L (Heterogeneous nuclear ribonucleoprotein L), RNP-Specific C(U1 small nuclear ribonucleoprotein C), SAP 145(Spliceosome associated protein ), SAP 61(Splicesomal protein), SC35(Splicing factor), SF3a120, SFRS2(Splicing factor, arginine/serine-rich 2), SFRS5(Splicing factor, arginine/serine-rich 5), SFRS7(Splicing factor, arginine/serine-rich 7), Small nuclear ribonucleoprotein SM
D1, SnRNP core protein Sm D2, SnRNP core protein Sm D3, SNRP70(U1 snRNP
70K protein), SNRPB(Small nuclear ribonucleoprotein polypeptides B and B1), SNRPE(Small nuclear ribonucleoprotein polypeptide E), SNRPN(Small nuclear ribonucleoprotein polypeptide N), Splicing factor SF3a120, Splicing factor 35 kD subunit, Splicing factor U2AF 65 kD subunit, SRP30C(Pre-mRNA splicing factor SF2, p33 subunit), SRP55-2(Pre-mRNA splicing factor SRP75), Transcription factor BTEB, Transcription initiation factor TFIID 250 kD subunit, Cleavage and polyadenylation specificity factor, Cleavage stimulation factor, 3' pre-RNA, subunit 1, 50kD, Cleavage stimulation factor, 3' pre-RNA, subunit 3, 77kD, HNRNP
Methyltransferase, PABPL1(Poly(A)-binding protein-like 1), Pap mRNA(Poly(A) Polymerase), RNA unwinding, RNA Helicase, GU Protein (ATP-Dependent RNA
helicase dead), KIAA0224 Gene(Putative ATP-dependent RNA helicase), RNA
Helicase A, RNA Helicase P110, and Ste13(Nuclear RNA Helicase).
114. The method, inhibitor, pharmaceutical composition, or nucleic acid probe of any of claims 6, 18, 26, 34, 47, 63, 92, and 106, wherein said gene is selected from the group consisting of AP47(Clathrin Coat Assembly AP47), AP50(Clathrin Coat Assembly Protein AP50), Cell Surface Protein (Clathrin Heavy Polypeptide-Like Protein ), Cltb(Clathrin Light Chain B), Cltc (Clathrin Heavy Chain), Adenylate Cyclase, Adenylate Cyclase, Adenylate Cyclase, II, Adenylate Cyclase,IV, Complex I, MTND1 (Subunit ND1), MTND2 (Subunit ND2), MTND3 (Subunit ND3), MTND4 (Subunit ND4), MTND4L (Subunit ND4L), MTND5 (Subunit ND5), MTND6 (Subunit ND6), Complex II, Complex III, Cytochrome b subunit, Complex IV, CO1 (Cytochrome c Oxidase Subunit I), CO2 (Cytochrome c Oxidase Subunit 2), CO3 (Cytochrome c Oxidase Subunit 3), Complex V, ATP Synthase Subunit ATPase 6, Kinesin Heavy Chain, Kinesin Light Chain, Syntaxin 1a, Syntaxin 1b, Syntaxin 3, Syntaxin 5a, Syntaxin 7, CANX (Calnexin), ER Lumen Protein 1, ER
Lumen Protein 2, Ribophorin I, Ribophorin II, Signal recognition particle receptor, SRP Protein, TIM17 preprotein translocase, Golgin-245, TGN46 (Traps-Golgi Network Integral Membrane Protein TGN38 Precursor), Beta-Cop, Coatomer Beta' Subunit, Coatomer Delta Subunit, Gp36b Glycoprotein (Vesicular integral-membrane protein VIP36 precursor), Homologue of yeast sec7, Protein transport protein (Chromosome 3p25), SEC14(S. Cerevisiae), Synaptic vesicle membrane protein VAT-1, Synaptobrevin-3, Synaptotagmin I, Transmembrane(COP-coated vesicle membrane protein p24 precursor), Vacuolar-Type (Clathrin-coated vesicle/synaptic vesicle proton pump 116 kd subunit), 140 kD Nucleolar phosphoprotein, Autoantigen p542, Export protein Rael (RAE1), Heterogeneous nuclear ribonucleoprotein A1, Nuclear pore complex protein hnup153, Nuclear pore complex protein NUP214, Nuclear pore glycoprotein p62, Nuclear Transport Factor 2, Nucleoporin 98 (NUP98), NUP88, Ribonucleoprotein A, Ribonucleoprotein B", Karyopherin, Importin Alpha Subunit, TRN (Transportin), Actin, Beta-Centractin, Capping Protein Alpha, CFL1 (Cofilin, Non-Muscle Isoform), Desmin, Dystrophin, Gelsolin, hOGG1(Myosin Light Chain Kinase), IC Heavy Chain, Itga2 (Integrin, Alpha 2 (CD49B, alpha 2 Subunit of VLA-2 receptor)), Itga3 (Integrin Alpha-3 Precursor), Keratin 19, Keratin, Type II, Lamin A, LBR(Lamin B Receptor), Light Chain Alkali, MacMarcks mRNA, MAP1a (Microtubule-Associated Protein 1A), MAP2(Microtubule-Associated Protein 2), MEG1(Protein-Tyrosine Phosphatase MEG1), Microtubule-Associated Protein TAU, Suppressor Of Tubulin STU2, TUBg (Tubulin Gamma Chain), Tubulin Alpha-4 Chain, USH1b (Myosin II Heavy Chain), Villin, Villin 2 (Ezrin), Actin Depolymerizing, Capping (Actin Filament), MYH9(Myosin, Heavy Polypeptide 9, Non-Muscle), MYL5(Myosin Regulatory Light Chain 2), Myosin Heavy Chain 95F, Myosin Heavy Chain IB, Myosin IB, Sh3p17(Myosin IC Heavy Chain), Sh3p18(Myosin IC Heavy Chain), KIAA0059(Dematin:Actin-Bundling Protein), TTN (Titin:Myosin Light Chain Kinase), ATP6c(Vacuolar H+ ATPase proton channel subunit), ATP6a1 (ATPase, H+ Transporting, Lysosomal (Vacuolar Proton Pump), Alpha Polypeptide, 70kD), ATP6b1(ATPase, H+ transporting, lysosomal (vacuolar proton pump), beta polypeptide, 56/58kD), ATP6d(ATPase, H+ transporting, lysosomal (vacuolar proton pump) 42kD), ATP6e(ATPase, H+ transporting, lysosomal (vacuolar proton pump) 31kD), ATPase, H+ transporting, lysosomal (vacuolar proton pump) 31kD, and Superoxide Dismutase.
115. A method for identifying an inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on a conditionally essential gene, and wherein said gene is subject to loss of heterozygosity in a cancer, said method comprising the steps of:
(a) determining at least two alleles of a said gene;
(b) testing a potential allele specific inhibitor to determine whether said potential allele specific inhibitor is active on at least one but less than all of said alleles;
wherein inhibition of expression of at least one but less than all of said alleles or reduction of the level of activity of a product of at least one but less than all of said alleles in the presence of said potential allele specific inhibitor is indicative that said potential allele specific inhibitor is a said inhibitor.
116. An inhibitor potentially useful for treatment of cancer, wherein said inhibitor is active on an allelic form of a conditionally essential gene, said gene has at least two alternative alleles in a population, and wherein said inhibitor targets at least one but less than all of said alternative alleles.
117. A pharmaceutical composition, comprising at least one allele specific inhibitor targeting at least one but less than all allelic forms of a conditionally essential gene in a population; and a pharmaceutically acceptable carrier or excipient.
118. A method for producing an inhibitor potentially useful for cancer treatment, wherein said inhibitor is active on at least one but less than all alternative alleles of a conditionally essential gene having at least two alternative alleles, comprising the steps of:
(a) identifying a conditionally essential gene that has alternative allelic forms in a noncancerous cell, wherein one of said alternative allelic forms is deleted in a cancer cell;
(b) screening to identify an inhibitor which inhibits said at least one but less than all of said at least two alternative alleles; and (c) synthesizing said inhibitor in an amount sufficient to produce a therapeutic effect when administered to a patient suffering from a cancer in whom cancerous cells have only an allele of said gene inhibited by said inhibitor and in whom normal cells are heterozygous for said gene and contain an allelic form not inhibited by said inhibitor.
119. A method for preventing the development of cancer in a patient having a precancerous condition, comprising the steps of:
a. subjecting cells of said precancerous condition to an altered condition such that a first conditionally essential becomes essential;
b. administering to said patient a therapeutic amount of a first allele specific inhibitor targeted to an allele of said first conditionally essential gene present in cells of said precancerous condition, wherein the normal somatic cells of said patient are heterozygous for said first gene, said inhibitor is active on at least one but less than all allelic forms of said gene present in a population and targets only one allelic form present in said normal somatic cells; and wherein cells of said precancerous condition have undergone LOH of said first gene.
120. The method of claim 119, wherein the cells of said precancerous condition are not clonal from a single cell, further comprising the step of:
c. serially administering to said patient at least one additional allele specific inhibitor, wherein each of said at least one additional allele specific inhibitors targets a different allele of a conditionally essential gene or an essential gene than is targeted by said first allele specific inhibitor, wherein said different allele may be a different allele of said first gene or an allele of a different gene, and wherein said patient is heterozygous for each targeted gene and each targeted gene has undergone LOH
in cells of said precancerous condition.
121. A method for treating a patient suffering from a cancer, wherein said patient is heterozygous for a conditionally essential gene, comprising the steps of:
a) subjecting cells of said cancer to altered conditions such that said gene is essential; and administering a therapeutic amount of an allele specific inhibitor active on at least one but less than all allelic forms of said gene present in a population, wherein said allele specific inhibitor inhibits only one allelic form of said gene present in said patient, and said only one allelic form of said gene is present in cancer cells in said patient.
122. The method of claim 121, further comprising the steps of:
(a) determining whether non-cancerous cells of said patient are heterozygous for a particular conditionally essential gene; or (b) determining whether cancerous cells of said patient have only one allele of said particular gene; or (c) both (a) and (b).

123. A method of inhibiting growth of a cell comprising the steps of:
a) subjecting said cell to conditions such that said gene is essential; and b) administering at least one inhibitor active on an allele of said conditionally essential gene, wherein said inhibitor is less active on at least one other allele of said gene.
124. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a conditionally essential gene, wherein said patient is suffering from a cancer, said method comprising the step of:
identifying a patient heterozygous for a said gene, wherein if said patient is heterozygous for said gene, then said patient is a potential patient for said treatment.
125. The method of claim 124, further comprising the step of determining whether cancer cells in said patient contain only a single allele of said gene, wherein if said cancer cells contain only a single allele of said gene, then said patient is a potential patient for said treatment.
126. A method of identifying a potential patient for treatment with an inhibitor active on at least one but less than all alleles of a conditionally essential gene, wherein said patient is suffering from a cancer, said method comprising the step of:
determining whether cancer cells in said patient have undergone LOH of a said gene, wherein if said cells have undergone LOH of said gene, then said patient is a potential patient for said treatment.
126. A nucleic acid probe at least 12 nucleotides in length which is perfectly complementary to a portion of a first allelic form of a conditionally essential gene, wherein said portion comprises a sequence variance site, and wherein said probe hybridizes under stringent hybridization conditions to said portion and not to a corresponding portion of a second allelic form having at least one different nucleotide at said sequence variance site.
127. A method for selecting a patient for treatment with an antiproliferative treatment, comprising the steps of:
a) determining whether normal somatic cells in a potential patient are heterozygous for an essential or conditionally essential gene, wherein a first allelic form of said gene is more active than a second allelic form, and wherein a reduction in the activity of said gene in a cell increases the sensitivity of said cell to a said antiproliferative treatment; and b) determining whether cancer cells of said patient have only said second allelic form of said gene, wherein if said somatic cells are heterozygous and said cancer cells have only said second allelic form, it is indicative that said patient is suitable for treatment with said antiproliferative treatment.
128. A method for selecting an antiproliferative treatment for a patient suffering from a cancer, comprising the steps of:
a) determining whether normal somatic cells in a potential patient are heterozygous for an essential or conditionally essential gene which reduces the sensitivity of cells to an antiproliferative treatment, wherein a first allelic form of said gene is more active than a second allelic form, and wherein a reduction in the activity of said gene in a cell increases the sensitivity of said cell to a said antiproliferative treatment; and b) determining whether cancer cells of said patient have only said second allelic form of said gene, wherein if said somatic cells are heterozygous for said gene and said cancer cells have only said second allelic form, it is indicative that said antiproliferative treatment is suitable for said patient.
129. The method of any of claims 115-129, wherein said gene is selected from the group consisting of:
galactose-1-phosphate uridyltransferase, galactose kinase, UDP galactose-4-epimerase, methionine synthase, asparagine synthase, glutamine synthetase, multidrug resistance gne/Pglycoprotein, multidrug resistance associated proteins 1-5, bleomycin hydrolase, dihydropyrimidine dehydrogenase, .beta.-ureidopropoinase, .beta.-alanine synthetase, cytidine deaminase, thiopurine methyltransferase, CYP1A1, CYP1A2, CYP2A6, CYP2A7, CYP2B6, CYP2B7, CYP2C8, CYP2C9, CYP2C17, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2F1, CYP3A3, CYP3A4, CYP3A5, CYP3A7, CYP4B1, CYP7, CYP11, CYP17, CYP19, CYP21, CYP27, glutathione transferase alpha, glutathione transferase theta, glutathione transferee mu, glutathione transferase pi, methylguanine methyltransferase, 3-alkylguanine alkyltransferase, 3-methyladenine DNA glucosylase, DNA dependent protein kinase, catalytic subunit of DNA-PK, DNA
binding subunit of DNA-PK Ku-70 or Ku-80 subunit, KARP-1, Poly(ADP-ribose) polymerase, Fanconi Anemia genes A, B, C, D, E, F, G, and H, ERCC-1, ERCC2/XPD, ERCC3/XPB, ERCC4, ERCC5, ERCC6, XPA, XPC, XPE, HHR23A, HHR23B, uracil glycosylase, 3-methyl adenine DNA glycosylase, NF-kappa B, XRCC4, XRCCS/Ku80, XRCC6, XRCC7, glutathione-X-transferase, I-kappa B alpha, HSP70, HSP27, and 9-oxoguanine DNA glycosylase.
131. A method for identifying a potential patient undergoing transplantation for treatment with an inhibitor active on at least one but less than all alleles of an essential gene, comprising the step of:
identifying a patient undergoing an allogenic bone marrow transplantation in which the donor tissue contains at least one alternative allele of an essential gene different from the alleles in somatic cells of said patient.

132. The method of claim 131, wherein said donor or said recipient is homozygous for an alternative allelic form of an essential gene that is not present in the other of said donor or said recipient.
133. A method for treating graft versus host disease in a patient receiving allogenic bone marrow transplantation, said method comprising the step of administering to said patient at least one allele specific inhibitor specific for at least one but less than all of the allelic forms of an essential gene in a population, wherein said inhibitor inhibits stimulation of the donor immune system, and cells of the said patient comprise an allelic form of said gene not present in the donor bone marrow.
134. The method of claim 133, wherein said allele specific inhibitor is selected by identifying at least one alternative alleles of an essential gene present in the donor tissues but absent in the normal somatic cells of said patient; and selecting a said inhibitor active on a said alternative allele of an essential gene present in said donor tissues but absent in the normal somatic cells of said patient.
135. The method of claim 134, wherein said at least one inhibitor recognizes both alleles of said essential gene that are present in said donor, but not both alleles of said gene that are present in said patient.
136. A method for enhancing engraftment of an allogenic bone marrow transplant, comprising the step of administering to a patient receiving said transplant an allele specific inhibitor which kills or suppresses the patient's bone marrow but not the donor bone marrow, thereby providing space for engraftment of the donor cells within the marrow cavity.
137. The method of claim 136, wherein the allele specific inhibitor is selected by identifying alternative alleles of an essential gene that are present in the recipient but not the donor marrow.
138. The method of claim 137, wherein said allele specific inhibitor recognizes both allelic forms of the essential gene that are present in the recipient, but not both allelic forms of the same gene that are present in the recipient.
139. A method for treating or preventing chimerism in allogenic bone marrow transplantation, comprising selectively killing or suppressing proliferation of the patient's own cells without toxicity to the donor cells by administering to a patient receiving said transplantation at least one allele specific inhibitor active on at least one but less than all alternative alleles of a gene vital for cell growth or viability, wherein said inhibitor targets the allelic form or forms of a gene in bone marrow of said patient but does not target at least one allelic form of said gene in the donor bone marrow.
140. A method for treating cancer in a patient receiving allogenic or autologous transplantation, comprising the step of administering to said patient at least one allele specific inhibitor which kills or inhibits the growth of cancer cells without toxicity to the transplanted marrow.
141. The method of claim 141, wherein said transplantation is autologous transplantation and said at least one allele specific inhibitor is selected to be active on the allele of an essential gene remaining in the cancer cells due to LOH in patients whose normal somatic cells are heterozygous for said essential gene, but not on the alternative allele of said gene present in said normal somatic cells, whereby said administration enables continuing therapy of cancer without suppression of the transplanted marrow.

142. The method of claim 140, wherein said transplantation is allogenic transplantation and said allele specific inhibitor recognizes both alleles of said essential gene that are present in the recipient, but not both forms of the said gene that are present in said patient.
143. A method for eliminating malignant cells from transplanted marrow during autologous transplantation of a patient heterozygous for an essential gene, comprising contacting cells from harvested autologous bone marrow ex vivo with at least one allele specific inhibitor active on at least one but less than all alternative alleles of said essential gene, wherein said inhibitor targets an allelic form of said gene present in cancer cells of said patient but does not target an alternative allele of said gene present in normal cells from said autologous bone marrow, wherein said gene has undergone LOH in cancer cells of said patient.
144. The method of claim 143, wherein said autologous bone marrow is harvested from said patient prior to high dose radiation or chemotherapy.
145. The method of claim 143, further comprising the steps of:
a. identifying one alternative allele of an essential gene remaining in the cancer cell due to LOH in patients who are heterologous with two different alternative forms of the essential gene in normal cells of the autologous bone marrow;
b. cultivating said autologous bone marrow ex vivo in the presence of an allele specific inhibitor that inhibits the allele that is present in the cancer cells, but not the heterologous allele that is present in the normal bone marrow.
146. The method of claim 143, wherein said autologous bone marrow is contacted with a plurality of said allele specific inhibitors.

147. A method for separating a first cell from a mixture of cells, comprising the steps of:
a) providing an allele specific binding compound which binds to at least one but less than all alleles of a gene, wherein a said allele of said gene expressed in said first cell is not expressed in other cells of said mixure of cells or is expressed in other cells in said mixture of cells and not in said first cell;
b) contacting said mixture of cells with said binding compound under conditions such that said binding compound binds to said allele and not to non-target alleles; and c) separating bound cells from unbound cells.
148. The method of claim 147, wherein said mixture of cells comprises normal somatic cells and cancer cells from a patient, said first cells are said normal somatic cells, and said first cells express a said allele deleted in said cancer cells due to LOH
of said gene, comprising separating said normal somatic cells from said cancer cells.
149. The method of claim 147, wherein said allele specific binding compound is an antibody or antibody fragment.
150. The method of claim 147, wherein said binding compound is attached to a solid support.