WO2009126153A1

WO2009126153A1 - Telomerase suppressor (lela1) compositions and methods for diagnosis and treatment of cancer in a mammalian subject

Info

Publication number: WO2009126153A1
Application number: PCT/US2008/059800
Authority: WO
Inventors: Tatiana Dracheva; Jin Jen; Ping Yang; Sinchita Roy Chowdhuri
Original assignee: The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
Priority date: 2008-04-09
Filing date: 2008-04-09
Publication date: 2009-10-15

Abstract

Methods for diagnosing or predicting a risk factor for cancer, such as lung carcinoma or early age onset lung carcinoma, are provided. The method provides detecting a LELA1 gene (CCDC36 or LOC339834), LELA1 gene product, or LELA1 polypeptide. A method for treating cancer in a mammalian subject is provided which comprises administering to the subject a LELA1 polypeptide, or a mimetic, analog, or derivative thereof, in an amount effective to reduce or eliminate cancer in the mammalian subject or to prevent its occurrence or recurrence.

Description

TELOMERASE SUPPRESSOR (LELAl) COMPOSITIONS AND METHODS FOR DIAGNOSIS AND TREATMENT OF CANCER IN A MAMMALIAN SUBJECT

FIELD

[0001] The invention generally relates to genetics and oncology. The invention relates to methods for diagnosing or predicting a risk factor for cancer, such as renal cell carcinoma, lung carcinoma and early age onset lung carcinoma. The method provides detecting a LELAl gene (CCDC36, LOC339834), LELAl gene product, or LELAl polypeptide. The invention further relates to a method for treating cancer in a mammalian subject which comprises administering to the subject a LELAl polypeptide, or a mimetic, analog, or derivative thereof, in an amount effective to reduce or eliminate cancer in the mammalian subject or to prevent its occurrence or recurrence

BACKGROUND

[0002] Lung cancer is one of the most common cancers and the leading cause of cancer death worldwide. In general, most patients develop lung cancer between the ages 60-70 but about 10 percent are diagnosed at extreme ages, either before age 50 or after age 80. Cigarette smoking is a major risk factor contributing to lung cancer. However, only a small portion of smokers ever develop the disease and some affected patients are non-smokers. These facts indicate that genetic background of the individual plays a role in lung cancer development. Indeed, studies have suggested the presence of a genetic factor contributing to lung cancer onset.

[0003] Cancer is the result in the occurrence of multiple factors. Mutations may occur in proto-oncogenes that cause cellular proliferation to increase. Mutations also may occur in tumor suppressors whose normal function is to regulate cellular proliferation. Mutations in DNA repair enzymes impair the ability of the cell to repair damage before proliferating. Tumor suppressor genes are normal genes whose absence (loss or inactivation) can lead to cancer. Tumor suppressor genes encode proteins that slow cell growth and division. Cancer arises when there is a mutation in both alleles. Human chromosome band 3p21.3 has been shown to undergo overlapping homozygous deletions in several SCLC and NSCLC lines; candidates of TSGs have been located in this critical region in several human cancers, further defining a TSG region. The evidence shows that genes in the 3p21 region are involved in regulation of the telomerase- mediated cellular immortality pathway in lung cancer and renal cancer cells. It has also been shown that 3p deletion occurs more frequently in the lung tumor tissues of patients who smoke. A need exists in the art to identify genetic components contributing to lung cancer in patients, particularly the ability to identify and treat lung adenocarcinomas in patients who were diagnosed with the disease before age 50.

SUMMARY

[0004] The invention generally relates to methods for diagnosing a risk factor for renal cell carcinoma, lung carcinoma and early age onset lung carcinoma. The method provides detecting a LELAl (CCDC36; LOC339834) gene, LELAl gene product, or LELAl polypeptide. CCDC36 (LOC339834) gene was named "LELAl" for "loss in early onset lung adenocarcinoma 1." The invention further relates to a method for treating cancer, e.g., renal cell carcinoma, lung carcinoma and early age onset lung carcinoma in a mammalian subject comprising administering the subject a LELAl polypeptide, or a mimetic, analog, or derivative thereof, in an amount effective to reduce or eliminate cancer in the mammalian subject or to prevent its occurrence or recurrence.

BRIEF DESCRIPTION OF THE DRAWINGS

[0005] Figure IA shows expression of LELAl gene in lung cancers with loss of heterozygosity (LOH) and renal cell carcinoma (RCC +Ch. 3 cells) with no tTERT expression.

[0006] Figure IB shows in situ hybridization demonstrating the expression of genes, vWF (positive control), LELAl, and MGC35097 (adjacent to LELAl and serving as negative control).

[0007] Figure 2 shows frequent LELAl promoter methylation in primary lung tumors and lung cancer cell lines.

[0008] Figure3 shows demethylation of LELAl led to re-expression LELAl and reduced expression of hTERT in lung cancer cell lines. [0009] Figure 4 shows LELAl overexpression suppresses hTERT.

[0010] Figure 5A shows the nucleotide sequence of human LELAl (CCDC36; NM_178173).

[0011] Figure 5B shows the amino acid sequence of human LELAl (CCDC36;

NP_835467).

DETAILED DESCRIPTION

[0012] The invention generally relates to methods for diagnosing a risk factor for cancer in a mammalian subject. The cancer can be renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma. The method provides detecting a LELAl ("loss in early age lung adenocarcinoma"; CCDC36, LOC339834) gene, LELAl gene product, or LELAl polypeptide and identifying the gene, gene product, or polypeptide in an active or inactivate state. The invention further relates to a method for treating cancer in a mammalian subject which comprises administering the subject a LELAl polypeptide, or a mimetic, analog, or derivative thereof, in an amount effective to reduce or eliminate cancer in the mammalian subject or to prevent its occurrence or recurrence. The method for treating cancer can further comprise treating the mammalian subject with a pharmaceutical composition that activates expression of the LELAl gene or activates the LELAl polypeptide.

[0013] Telomerase reactivation is an important event during neoplastic transformation and occurs in most human cancers. Through a combination of genetic and gene expression analyses, a small region on chromosome 3p21 was identified that is lost at 82% in lung adenocarcinomas from patients who were diagnosed at ages before 50 but only lost in 30% tumors of patients who were diagnosed at age 80 or later. See Table 2. This region overlaps with a previously reported telomerase suppressor locus in renal cell carcinoma cells carrying a wild type chromosome 3. Based on gene expression, a novel gene, LELAl ("loss in early age lung adenocarcinoma") was identified. LELAl is inactivated in -50% of primary lung cancers and -70% of lung cancer cell lines via methylation at the 5'CpG island of the gene. Functionally, increased expression of LELAl either by exogenous expression or reactivation after 5'-azacytosine treatment reduced hTERT message levels in lung cancer cell lines and reduced hTERT activity. Genetically, six unique somatic and germline changes were identified among 19 early age onset patients but none in patients who had cancer at later ages. These results suggest that loss of LELAl contributes to early lung cancer onset through hTERT dysregulation.

[0014] It is to be understood that this invention is not limited to particular methods, reagents, compounds compositions or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a cell" includes a combination of two or more cells, and the like.

[0015] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. In describing and claiming the present invention, the following terminology will be used.

[0016] "Biological samples" refers to any tissue or liquid sample having genomic DNA or other nucleic acids (e.g., mRNA) or proteins. It refers to samples of cells with a normal complement of chromosomes as well as samples of cells suspected of malignancy.

[0017] "Patient", "vertebrate subject" or "mammalian subject" are used herein and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals. Animals include all vertebrates, e.g., mammals and non-mammals, such as sheep, cows, dogs, cats, avian species, chickens, amphibians, reptiles, osteichthyes, or chondrichthyes.

[0018] "Treating" refers to any indicia of success in the treatment or amelioration or prevention of the disease, condition, or disorder, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with a disease, condition or disorder as described herein. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject. "Treating" or "treatment" using the methods of the present invention includes preventing the onset of symptoms in a subject that can be at increased risk of a disease or disorder associated with a disease, condition or disorder as described herein, but does not yet experience or exhibit symptoms, inhibiting the symptoms of a disease or disorder (slowing or arresting its development), providing relief from the symptoms or side-effects of a disease (including palliative treatment), and relieving the symptoms of a disease (causing regression). Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease or condition.

[0019] "Concomitant administration" of a known drug with a compound of the present invention means administration of the drug and the compound at such time that both the known drug and the compound will have a therapeutic effect or diagnostic effect. Such concomitant administration can involve concurrent (i.e., at the same time), prior, or subsequent administration of the drug with respect to the administration of a compound of the present invention. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence, and dosages of administration for particular drugs and compounds of the present invention.

[0020] In general, the phrase "well tolerated" refers to the absence of adverse changes in health status that occur as a result of the treatment and would affect treatment decisions.

[0021] "Synergistic interaction" refers to an interaction in which the combined effect of two or more agents is greater than the algebraic sum of their individual effects.

[0022] "Chronic" administration refers to administration of the agent(s) in a continuous mode as opposed to an acute mode, so as to maintain the initial therapeutic effect (activity) for an extended period of time. "Intermittent" administration is treatment that is not consecutive without interruption, but rather is cyclic in nature.

[0023] "Administering", "introducing," "delivering," "placement," and "transplanting" are used interchangeably herein and refer to the placement of cells of the invention into a subject by a method or route which results in at least partial localization of the regenerative cells at a desired site. The cells can be administered by any appropriate route that results in delivery to a desired location in the subject where at least a portion of the cells or components of the cells remain viable. The period of viability of the cells after administration to a subject can be as short as a few hours, e.g., twenty- four hours, to a few days, to as long as several years.

[0024] "Inhibitors," "activators," and "modulators" of LELAl gene expression or LELAl polypeptide activity molecules of the invention (genes their associated gene products in cells) are used to refer to inhibitory, activating, or modulating molecules, respectively, identified using in vitro and in vivo assays for binding or signaling, e.g., ligands, agonists, antagonists, and their homologs and mimetics. The term "modulator" includes inhibitors and activators. Inhibitors are agents that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of LELAl gene or other genes, e.g., antagonists. Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of genes, e.g., agonists. Modulators include agents that, e.g., alter the interaction of gene or gene product with: proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like. Modulators include genetically modified versions of naturally-occurring activated ligands, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like. Such assays for inhibitors and activators include, e.g., applying putative modulator compounds to a cell expressing a receptor and then determining the functional effects on receptor signaling. Samples or assays comprising activated receptors that are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples (untreated with inhibitors) can be assigned an activity value of 100%. Inhibition of activated samples is achieved when the activity value relative to the control is about 80%, optionally 50% or 25-0%. Activation of sample is achieved when the activity value relative to the control is 110%, optionally 150%, optionally 200-500%, or 1000-3000% higher.

[0025] The ability of a molecule to bind to LELAl polypeptide can be determined, for example, by the ability of the putative activator to bind to LELAl polypeptide immunoadhesin coated on an assay plate. Specificity of binding can be determined by comparing binding to a molecule other than LELAl polypeptide.

[0026] "Test compound" refers to any compound tested as a modulator of LELAl polypeptide. The test compound can be any small organic molecule, or a biological entity, such as a protein, e.g., an antibody or peptide, a sugar, a nucleic acid, e.g., an antisense oligonucleotide, RNAi, or a ribozyme, or a lipid. Alternatively, test compound can be modulators that are genetically altered versions of LELAl protein. Typically, test compounds will be small organic molecules, peptides, lipids, or lipid analogs.

[0027] A "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).

[0028] A "naturally-occurring" polypeptide or protein refers to a polypeptide molecule having an amino acid sequence that occurs in nature (e.g., encodes a natural protein).

[0029] "Gene" and "recombinant gene" refer to nucleic acid molecules which include an open reading frame encoding a LELAl polypeptide, or mimetic, analog or derivative thereof, preferably a vertebrate, mammalian, bovine, human, avian reptilian, amphibian, osteichthyes, or chondrichthyes peptide, and can further include non-coding regulatory sequences, and introns. [0030] An "isolated" or "purified" polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized. In one aspect, the language "substantially free" means a preparation of a LELAl polypeptide, or mimetic, analog or derivative thereof, having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non- LELAl polypeptide (also referred to herein as a "contaminating protein"). When the LELAl polypeptide, or mimetic, analog or derivative thereof, or biologically active portion thereof, is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation. Aspects of the invention include isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.

[0031] A "non-essential" amino acid residue is a residue that can be altered from the wild-type sequence of the LELAl polypeptide, or mimetic, analog or derivative thereof, without abolishing or more preferably, without substantially altering a biological activity, whereas an "essential" amino acid residue results in such a change. For example, amino acid residues that are conserved among the LELAl polypeptide, or mimetic, analog or derivative thereof, those present in the domain of LELAl polypeptide necessary for activity to activate or inhibit telomerase activity, are predicted to be particularly not amenable to alteration.

[0032] A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art. These families include amino acids with basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), beta-branched side chains (e.g., threonine, valine, isoleucine) and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). Thus, a predicted nonessential amino acid residue in a LELAl polypeptide, or mimetic, analog or derivative thereof, is preferably replaced with another amino acid residue from the same side chain family. Alternatively, in another aspect, mutations can be introduced randomly along all or part of a LELAl polypeptide coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for LELAl polypeptide biological activity to identify mutants that retain activity. Following mutagenesis of a LELAl polypeptide, or mimetic, analog or derivative thereof, the encoded polypeptide can be expressed recombinantly and the activity of the protein can be determined.

[0033] "Biologically active," when used in conjunction with LELAl polypeptide refers to a LELAl polypeptide that affects telomerase gene expression or telomerase activity in a manner substantially similar to that of full length LELAl polypeptide in a mammalian subject.

[0034] A biologically active portion of LELAl polypeptide can be a polypeptide which is, for example, 10, 25, 50, 100, 200, or more, amino acids in length. Biologically active portions of a LELAl polypeptide e can be used as targets for developing agents which modulate a LELAl polypeptide activity as described herein.

[0035] Calculations of homology or sequence identity (the terms are used interchangeably herein) between sequences are performed as follows. To determine the percent identity of two amino acid sequences, or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). In a preferred aspect, the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, 90%, 100% of the length of the reference sequence (e.g., when aligning a second sequence to the LELAl amino acid sequence, or mimetic, analog or derivative thereof, at least 10, preferably at least 20, more preferably at least 50, even more preferably at least 100 amino acid residues of the two sequences are aligned. The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid "identity" is equivalent to amino acid or nucleic acid "homology"). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment of the two sequences.

[0036] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. In a preferred aspect, the percent identity between two amino acid sequences is determined using the Needleman and Wunsch ((1970) J. MoI. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossum 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6. In yet another preferred aspect, the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6. A particularly preferred set of parameters (and the one that should be used if the practitioner is uncertain about what parameters should be applied to determine if a molecule is within a sequence identity or homology limitation of aspects of the invention) are a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0037] The percent identity between two amino acid or nucleotide sequences can be determined using the algorithm of E. Meyers and W. Miller ((1989) CABIOS, 4:11-17) which has been incorporated into the ALIGN program (version 2.0), using a PAM 120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.

[0038] The nucleic acid and protein sequences described herein can be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences. Such searches can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J. MoI. Biol. 215:403-10. BLAST nucleotide searches can be performed with the NBLAST program, score=100, wordlength=12 to obtain nucleotide sequences homologous to nucleic acid molecules encoding a modified LELAl polypeptide, or mimetic, analog or derivative thereof, of aspects of the invention. BLAST protein searches can be performed with the XBLAST program, score=50, wordlength=3 to obtain amino acid sequences homologous to LELAl polypeptide of aspects of the invention. To obtain gapped alignments for comparison purposes, Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25:3389-3402. When utilizing BLAST and Gapped BLAST programs, the default parameters of the respective programs (e.g., XBLAST and NBLAST) can be used. See http://www.ncbi.nlm.nih.gov.

[0039] Particular LELAl polypeptide, or mimetic, analog or derivative thereof, in aspects of the present invention have an amino acid sequence sufficiently identical or substantially identical to the amino acid sequence of the LELAl polypeptide. "Sufficiently identical" or "substantially identical" is used herein to refer to a first amino acid or nucleotide sequence that contains a sufficient or minimum number of identical or equivalent (e.g., with a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences have a common structural domain or common functional activity. For example, amino acid or nucleotide sequences that contain a common structural domain having at least about 60%, or 65% identity, likely 75% identity, more likely 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity are defined herein as sufficiently or substantially identical. In one aspect the LELAl polypeptide, or mimetic, analog, or derivative thereof, can result from alternatively- spliced transcripts of the LELAl gene.

[0040] A "purified preparation of cells", as used herein, refers to, in the case of plant or animal cells, an in vitro preparation of cells and not an entire intact plant or animal. In the case of cultured cells or microbial cells, it consists of a preparation of at least 10% and more preferably 50% of the subject cells.

[0041] "Pharmaceutically acceptable carrier (or medium)", which can be used interchangeably with "biologically compatible carrier or medium", refers to reagents, cells, compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other complication commensurate with a reasonable benefit/risk ratio. As described in greater detail herein, pharmaceutically acceptable carriers suitable for use in the present invention include liquids, semi-solid (e.g., gels) and solid materials (e.g., cell scaffolds). As used herein, the term biodegradable describes the ability of a material to be broken down (e.g., degraded, eroded, dissolved) in vivo. The term includes degradation in vivo with or without elimination (e.g., by resorption) from the body. The semisolid and solid materials can be designed to resist degradation within the body (nonbiodegradable) or they can be designed to degrade within the body (biodegradable, bioerodable). A biodegradable material can further be bioresorbable or bioabsorbable, i.e., it can be dissolved and absorbed into bodily fluids (water-soluble implants are one example), or degraded and ultimately eliminated from the body, either by conversion into other materials or breakdown and elimination through natural pathways.

[0042] This invention relies on routine techniques in the field of recombinant genetics. Basic texts disclosing the general methods of use in this invention include Sambrook et al., Molecular Cloning, A Laboratory Manual, 2ⁿ ed., 1989; Kriegler, Gene Transfer and Expression: A Laboratory Manual, 1990; and Ausubel et al., eds., Current Protocols in Molecular Biology, 1994; all of which are herein incorporated by reference for all purposes. PEPTIDES AND POLYPEPTIDES

[0043] Aspects of the invention provide isolated or recombinant polypeptides comprising an amino acid sequence having at least 95%, 96%, 97%, 98%, 99% or more sequence identity to LELAl polypeptide over a region of at least about 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1100 or more residues, or, the full length of the polypeptide, or, a polypeptide encoded by a nucleic acid of aspects of the invention. Aspects of the invention provide methods for preventing or treating cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma in a mammalian subject comprising administering to the mammalian subject a LELAl polypeptide, or mimetic, analog or derivative thereof. Aspects of the invention provide methods for preventing or treating early age onset lung cancer in a mammalian subject comprising administering to the mammalian subject an activator of LELAl polypeptide.

[0044] In one aspect, an aspects of the invention provides LELAl polypeptide, or mimetic, analog or derivative thereof, and the nucleic acids encoding them where one, some or all of the LELAl polypeptide, is replaced with substituted amino acids. In one aspect, an aspects of the invention provides methods to activate or inhibit LELAl activity or telomerase activity in cells and to treat cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma.

[0045] The LELAl polypeptide, or mimetic, analog or derivative thereof, in aspects of the invention can be expressed recombinantly in vivo after administration of nucleic acids, as described above, or, they can be administered directly, e.g., as a pharmaceutical composition.

[0046] LELAl polypeptide, or mimetic, analog or derivative thereof, in aspects of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo. The LELAl polypeptide in aspects of the invention can be made and isolated using any method known in the art. LELAl polypeptide in aspects of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers, Nucleic Acids Res. Symp. Ser. 215-223, 1980; Horn, Nucleic Acids Res. Symp. Ser. 225-232, 1980; Banga, A. K., Therapeutic Peptides and Proteins, Formulation, Processing and Delivery Systems Technomic Publishing Co., Lancaster, PA, 1995. For example, peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge, Science 269: 202, 1995; Merrifield, Methods Enzymol. 289: 3-13, 1997) and automated synthesis can be achieved, e.g., using the ABI 431A Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. [0047] The LELAl polypeptide, or mimetic, analog or derivative thereof, in aspects of the invention, as defined above, include all "mimetic" and "peptidomimetic" forms. The terms "mimetic" and "peptidomimetic" refer to a synthetic chemical compound which has substantially the same structural and/or functional characteristics of the polypeptides of aspects of the invention. The mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non- natural analogs of amino acids. The mimetic can also incorporate any amount of natural amino acid conservative substitutions as long as such substitutions also do not substantially alter the structure and/or activity of the mimetic. As with polypeptides of aspects of the invention which are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered. Thus, a mimetic composition is within the scope of the invention if, when administered to or expressed in a cell, it has a LELAl polypeptide activity.

[0048] In one aspect, the polypeptide or peptidomimetic composition can be a dominant-negative mutant within the scope of the invention if it can inhibit telomerase activity in cells. The dominant negative mutant can be a LELAl peptide or peptide mimetic that can inhibit telomerase activity in cells or treat cancer, or a nucleic acid composition, in the form of a DNA vector or gene therapy vector, that expresses a dominant-negative polypeptide that can treat cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma. The dominant negative mutant can bind to or interact with telomerase polypeptide. The dominant negative molecule can act, for example, by interfering with protein-protein interactions.

[0049] Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like. For example, a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds. Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N-hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N₅N'- diisopropylcarbodiimide (DIC). Linking groups that can be an alternative to the traditional amide bond ("peptide bond") linkages include, e.g., ketomethylene (e.g., — C(=O) — CH₂ — for — C(=O)— NH-), aminomethylene (CH₂-NH), ethylene, olefin (CH=CH), ether (CH₂-O), thioether (CH₂ — S), tetrazole (CN₄-), thiazole, retroamide, thioamide, or ester (see, e.g., Spatola (1983) in Chemistry and Biochemistry of Amino Acids, Peptides and Proteins, Vol. 7, pp 267- 357, "Peptide Backbone Modifications," Marcell Dekker, NY).

[0050] A polypeptide can also be characterized as a mimetic by containing all or some non-natural residues in place of naturally occurring amino acid residues. Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below. Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L- naphylalanine; D- or L-phenylglycine; D- or L- 2 thieneylalanine; D- or L-I, -2,3-, or A- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3-pyridinyl)- alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D- (trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluoro-phenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p-methoxy-biphenylphenylalanine; D- or L-2- indole(alkyl)alanines; and, D- or L-alkylainines, where alkyl can be substituted or unsubstituted methyl, ethyl, propyl, hexyl, butyl, pentyl, isopropyl, iso-butyl, sec-isotyl, iso-pentyl, or a non- acidic amino acids. Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.

[0051] Mimetics of acidic amino acids can be generated by substitution by, e.g., non- carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine. Carboxyl side groups {e.g., aspartyl or glutamyl) can also be selectively modified by reaction with carbodiimides (R' — N — C — N — R') such as, e.g., l-cyclohexyl-3(2-morpholin- yl- (4-ethyl) carbodiimide or l-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide. Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.

[0052] Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, or (guanidino)-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above. Nitrile derivative {e.g., containing the CN-moiety in place of COOH) can be substituted for aspargine or glutamine. Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.

[0053] Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, or ninhydrin, preferably under alkaline conditions. Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g., aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively. Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives. Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5-imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p- chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole. Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino- containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide. Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy guanidino, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline. Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide. Other mimetics include, e.g., those generated by hydroxylation of guanidino and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha- amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C- terminal carboxyl groups.

[0054] A component of a LELAl polypeptide, or mimetic, analog or derivative thereof, in aspects of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality. Thus, any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D-amino acid, but which can additionally be referred to as the R- or S-form.

[0055] Various chemical modifications will improve the stability, bioactivity and ability of the LELAl polypeptide, or mimetic, analog or derivative thereof, to cross the blood brain barrier to treat disease. One such modification is aliphatic amino terminal modification with a derivative of an aliphatic or aromatic acid, forming an amide bond. Such derivatives include, for example, CH₃CO, CH₃(CH₂)_nCO, C₆H₅CH₂CO and H₂N(CH₂)_nCO, wherein n=l-10. Another modification is carboxy terminal modification with a derivative of an aliphatic or aromatic amine/alcohol coupled to the LELAl polypeptide via an amide/ester bond. Such derivatives include those listed above. The LELAl polypeptide may also have both amino and carboxy terminal modifications, wherein the derivatives are independently selected from those listed above. The peptides may also be glycosylated, wherein either the alpha amino group or a D-Asn, or both, are modified with glucose or galactose. In another contemplated modification, selected backbone amide bonds are reduced (-NH-CH₂). Other modifications include N- methylation of selected nitrogens in the amide bonds and esters in which at least one of the acid groups on the peptide are modified as aromatic or aliphatic esters. Any combination of the above modifications is also contemplated.

[0056] Aspects of the invention also provide polypeptides that are "substantially identical" to an exemplary polypeptide of aspects of the invention. A "substantially identical" amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non-conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties. A conservative amino acid substitution, for example, substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine). One or more amino acids can be deleted, for example, from a LELAl polypeptide, or mimetic, analog or derivative thereof, of aspects of the invention, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal, or internal, amino acids which are not required for a LELAl activity or interaction can be removed.

[0057] The skilled artisan will recognize that individual synthetic residues and polypeptides incorporating these mimetics can be synthesized using a variety of procedures and methodologies, which are well described in the scientific and patent literature, e.g., Organic Syntheses Collective Volumes, Gilman, et al. (Eds) John Wiley & Sons, Inc., NY. Peptides and peptide mimetics of aspects of the invention can also be synthesized using combinatorial methodologies. Various techniques for generation of peptide and peptidomimetic libraries are well known, and include, e.g., multipin, tea bag, and split-couple-mix techniques; see, e.g., al- Obeidi, MoI. Biotechnol. 9: 205-223, 1998; Hruby, Curr. Opin. Chem. Biol. 1: 114-119, 1997; Ostergaard, MoI. Divers. 3: 17-27, 1997; Ostresh, Methods Enzymol. 267: 220-234, 1996. Modified peptides of aspects of the invention can be further produced by chemical modification methods, see, e.g., Belousov, Nucleic Acids Res. 25: 3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19: 373-380, 1995; Blommers, Biochemistry 33: 7886-7896, 1994.

[0058] A LELAl polypeptide, or mimetic, analog or derivative thereof, in aspects of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like. Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine-tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Amgen Inc., Seattle Wash.). The inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego Calif.) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification. For example, an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams, Biochemistry 34: 1787-1797, 1995; Dobeli, Protein Expr. Purif. 12: 404-14, 1998). The histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein. Technology pertaining to vectors encoding fusion proteins and application of fusion proteins are well described in the scientific and patent literature, see e.g., Kroll, DNA Cell. Biol, 12: 441-53, 1993.

[0059] "Polypeptide" and "protein" as used herein, refer to amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isosteres, and can contain modified amino acids other than the 20 gene-encoded amino acids. The term "polypeptide" also includes peptides and polypeptide fragments, motifs and the like. The term also includes glycosylated polypeptides. The LELAl polypeptide, or mimetic, analog or derivative thereof, in aspects of the invention also include all "mimetic" and "peptidomimetic" forms, as described in further detail, below.

[0060] "Isolated" means that the material is removed from its original environment (e.g., the natural environment if it is naturally occurring). For example, a naturally occurring polynucleotide or polypeptide present in a living animal is not isolated, but the same polynucleotide or polypeptide, separated from some or all of the coexisting materials in the natural system, is isolated. Such polynucleotides could be part of a vector and/or such polynucleotides or polypeptides could be part of a composition, and still be isolated in that such vector or composition is not part of its natural environment. As used herein, an isolated material or composition can also be a "purified" composition, i.e., it does not require absolute purity; rather, it is intended as a relative definition. Individual nucleic acids obtained from a library can be conventionally purified to electrophoretic homogeneity. In alternative aspects, aspects of the invention provide nucleic acids which have been purified from genomic DNA or from other sequences in a library or other environment by at least one, two, three, four, five or more orders of magnitude.

Identification of Compounds for Treatment and Prophylaxis of Disease

(A) Identification of Bioactive Agents

[0061] Identifying bioactive agents that modulate LELAl activity, the information is used in a wide variety of ways. In one method, one of several cellular assays, e.g., a LELAl and/or telomerase gene expression assay, can be used in conjunction with high throughput screening techniques, to allow monitoring for antagonists or agonists of LELAl activity after treatment with a candidate agent. In one method, the candidate agents are added to cells.

[0062] "Candidate bioactive agent" or "drug candidate" or grammatical equivalents as used herein describes any molecule, e.g., protein, oligopeptide, small organic molecule, polysaccharide, polynucleotide, to be tested for bioactive agents that are capable of directly or indirectly altering LELAl activity. In one methods, the bioactive agents modulate LELAl activity. In a further embodiment of the method, the candidate agents induce an antagonist or agonist effect in a LELAl gene or telomerase gene expression assay, as further described below. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e., at zero concentration or below the level of detection.

[0063] Candidate agents encompass numerous chemical classes, though typically they are organic molecules, e.g., small organic compounds having a molecular weight of more than 100 and less than about 2,500 daltons. Candidate agents comprise functional groups necessary for structural interaction with proteins, particularly hydrogen bonding, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, for example, at least two of the functional chemical groups. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof. In a further embodiment, candidate agents are peptides. [0064] Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents can be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification to produce structural analogs.

[0065] In some embodiments, the candidate bioactive agents are proteins. By "protein" herein is meant at least two covalently attached amino acids, which includes proteins, polypeptides, oligopeptides and peptides. The protein can be made up of naturally occurring amino acids and peptide bonds, or synthetic peptidomimetic structures. Thus "amino acid", or "peptide residue", as used herein means both naturally occurring and synthetic amino acids. For example, homo-phenylalanine, citrulline and noreleucine are considered amino acids for the purposes of the methods herein. "Amino acid" also includes imino acid residues such as proline and hydroxyproline. The side chains can be in either the (R) or the (S) configuration. In further embodiments, the amino acids are in the (S) or (L) -configuration. If non-naturally occurring side chains are used, non-amino acid substituents can be used, for example to prevent or retard in vivo degradations.

[0066] In one method, the candidate bioactive agents are naturally occurring proteins or fragments of naturally occurring proteins. Thus, for example, cellular extracts containing proteins, or random or directed digests of proteinaceous cellular extracts, can be used. In this way libraries of procaryotic and eucaryotic proteins can be made for screening using the methods herein. The libraries can be bacterial, fungal, viral, and vertebrate proteins, and human proteins.

[0067] In some methods, the candidate bioactive agents are peptides of from about 5 to about 30 amino acids, typically from about 5 to about 20 amino acids, and typically from about 7 to about 15 being. The peptides can be digests of naturally occurring proteins as is outlined above, random peptides, or "biased" random peptides. By "randomized" or grammatical equivalents herein is meant that each nucleic acid and peptide consists of essentially random nucleotides and amino acids, respectively. Since generally these random peptides (or nucleic acids, discussed below) are chemically synthesized, they can incorporate any nucleotide or amino acid at any position. The synthetic process can be designed to generate randomized proteins or nucleic acids, to allow the formation of all or most of the possible combinations over the length of the sequence, thus forming a library of randomized candidate bioactive proteinaceous agents.

[0068] In some methods, the library can be fully randomized, with no sequence preferences or constants at any position. In other methods, the library can be biased. Some positions within the sequence are either held constant, or are selected from a limited number of possibilities. For example, in some methods, the nucleotides or amino acid residues are randomized within a defined class, for example, of hydrophobic amino acids, hydrophilic residues, sterically biased (either small or large) residues, towards the creation of nucleic acid binding domains, the creation of cysteines, for cross-linking, prolines for SH-3 domains, serines, threonines, tyrosines or histidines for phosphorylation sites, or to purines. In other methods, the candidate bioactive agents are nucleic acids, as defined above.

[0069] As described above generally for proteins, nucleic acid candidate bioactive agents can be naturally occurring nucleic acids, random nucleic acids, or "biased" random nucleic acids. For example, digests of procaryotic or eucaryotic genomes can be used as is outlined above for proteins.

[0070] In some methods, the candidate bioactive agents are organic chemical moieties.

(B) Animal Models

[0071] In one method, nucleic acids which encode LELAl proteins or their modified forms can also be used to generate either transgenic animals, including "knock-in" and "knock out" animals which, in turn, are useful in the development and screening of therapeutically useful reagents. A non-human transgenic animal (e.g., a mouse or rat) is an animal having cells that contain a transgene, which transgene is introduced into the animal or an ancestor of the animal at a prenatal, e.g., an embryonic stage. A transgene is a DNA which is integrated into the genome of a cell from which a transgenic animal develops, and can include both the addition of all or part of a gene or the deletion of all or part of a gene. In some methods, cDNA encoding a LELAl polypeptide can be used to clone genomic DNA encoding a LELAl polypeptide in accordance with established techniques and the genomic sequences used to generate transgenic animals that contain cells which either express (or overexpress) or suppress the desired DNA. Methods for generating transgenic animals, particularly animals such as mice or rats, have become conventional in the art and are described, for example, in U.S. Patent Nos. 4,736,866 and 4,870,009, each incorporated herein by reference in their entirety. Typically, particular cells would be targeted for a LELAl polypeptide transgene incorporation with tissue- specific enhancers. Transgenic animals that include a copy of a transgene encoding a LELAl polypeptide introduced into the germ line of the animal at an embryonic stage can be used to examine the effect of increased expression of the desired nucleic acid. Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with its overexpression or under expression of LELAl polypeptide, e.g., cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma. In accordance with this facet, an animal is treated with the reagent and a reduced incidence of the pathological condition, compared to untreated animals bearing the transgene, would indicate a potential therapeutic intervention for the pathological condition. Similarly, non-human homologues of a LELAl polypeptide can be used to construct a transgenic animal comprising a protein "knock out" animal which has a defective or altered gene encoding a LELAl polypeptide as a result of homologous recombination between the endogenous gene encoding a LELAl polypeptide and altered genomic DNA encoding the protein introduced into an embryonic cell of the animal. For example, cDNA encoding a LELAl polypeptide can be used to clone genomic DNA encoding the protein in accordance with established techniques. A portion of the genomic DNA encoding a LELAl polypeptide can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration. Typically, several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector (see, e.g., Thomas and Capecchi, Cell 51:503, 1987, incorporated herein by reference in its entirety, for a description of homologous recombination vectors). The vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see, e.g., Li et al., Cell 69:915, 1992, incorporated herein by reference in its entirety). The selected cells are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see, e.g., Bradley, in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987), pp. 113-152). A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal. Progeny harboring the homologously recombined DNA in their germ cells can be identified by standard techniques and used to breed animals in which all cells of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, for their ability to defend against certain pathological conditions and for their development of pathological conditions due to absence of a LELAl polypeptide, cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma.

[0072] Animal models exhibiting LELAl polypeptide related disorder or symptoms can be engineered by utilizing, for example, LELAl polypeptide sequences in conjunction with techniques for producing transgenic animals that are well known to those of skill in the art. For example, gene sequences can be introduced into, and overexpressed in, the genome of the animal of interest, or, if endogenous target gene sequences are present, they can either be overexpressed or, alternatively, can be disrupted in order to underexpress or inactivate target gene expression.

[0073] In order to overexpress a target gene sequence, the coding portion of the target gene sequence can be ligated to a regulatory sequence which is capable of driving gene expression in the animal and cell type of interest. Such regulatory regions will be well known to those of skill in the art, and can be utilized in the absence of undue experimentation.

[0074] For underexpression of an endogenous target gene sequence, such a sequence can be isolated and engineered such that when reintroduced into the genome of the animal of interest, the endogenous target gene alleles will be inactivated. The engineered target gene sequence is introduced via gene targeting such that the endogenous target sequence is disrupted upon integration of the engineered target sequence into the animal's genome.

[0075] Animals of any species, including, but not limited to, mice, rats, rabbits, guinea pigs, pigs, micro-pigs, goats, and non-human primates, e.g., baboons, monkeys, and chimpanzees can be used to generate animal models of Wnt/β-catenin signaling related disorders or being a perpetually desired state of the Wnt/β-catenin signaling. (C) Nucleic Acid Based Therapeutics

[0076] Nucleic acids encoding LELAl polypeptides, or a mimetic, analog, or derivative thereof, can also be used in gene therapy. Broadly speaking, a gene therapy vector is an exogenous polynucleotide which produces a medically useful phenotypic effect upon the mammalian cell(s) into which it is transferred. A vector can or can not have an origin of replication. For example, it is useful to include an origin of replication in a vector for propagation of the vector prior to administration to a patient. However, the origin of replication can often be removed before administration if the vector is designed to integrate into host chromosomal DNA or bind to host mRNA or DNA. Vectors used in gene therapy can be viral or nonviral. Viral vectors are usually introduced into a patient as components of a virus. Nonviral vectors, typically dsDNA, can be transferred as naked DNA or associated with a transfer- enhancing vehicle, such as a receptor-recognition protein, lipoamine, or cationic lipid.

[0077] "Control sequences" or "regulatory sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. The control sequences that are suitable for prokaryotes, for example, include a promoter, optionally an operator sequence, a ribosome binding site, and possibly, other as yet poorly understood sequences. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

[0078] "Vector" refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of vector is a "plasmid", which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal vertebrate vectors). Other vectors (e.g., non-episomal vertebrate vectors) can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors"). In general, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. In the present specification, "plasmid" and "vector" can be used interchangeably as the plasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.

[0079] Viral vectors, such as retroviruses, adenoviruses, adenoassociated viruses and herpes viruses, are often made up of two components, a modified viral genome and a coat structure surrounding it (see generally Smith et al., Ann. Rev. Microbiol. 49:807-838, 1995, incorporated herein by reference in its entirety), although sometimes viral vectors are introduced in naked form or coated with proteins other than viral proteins. Most current vectors have coat structures similar to a wildtype virus. This structure packages and protects the viral nucleic acid and provides the means to bind and enter target cells. However, the viral nucleic acid in a vector designed for gene therapy is changed in many ways. The goals of these changes are to disable growth of the virus in target cells while maintaining its ability to grow in vector form in available packaging or helper cells, to provide space within the viral genome for insertion of exogenous DNA sequences, and to incorporate new sequences that encode and enable appropriate expression of the gene of interest. Thus, vector nucleic acids generally comprise two components: essential cis-acting viral sequences for replication and packaging in a helper line and the transcription unit for the exogenous gene. Other viral functions are expressed in trans in a specific packaging or helper cell line. [0080] Nonviral nucleic acid vectors used in gene therapy include plasmids, RNAs, antisense oligonucleotides (e.g., methylphosphonate or phosphorothiolate), polyamide nucleic acids, interfering RNA (RNAi), hairpin RNA, and yeast artificial chromosomes (YACs). Such vectors typically include an expression cassette for expressing a protein or RNA. The promoter in such an expression cassette can be constitutive, cell type- specific, stage-specific, and/or modulatable (e.g., by hormones such as glucocorticoids; MMTV promoter). Transcription can be increased by inserting an enhancer sequence into the vector. Enhancers are cis-acting sequences of between 10 to 300bp that increase transcription by a promoter. Enhancers can effectively increase transcription when either 5' or 3' to the transcription unit. They are also effective if located within an intron or within the coding sequence itself. Typically, viral enhancers are used, including SV40 enhancers, cytomegalovirus enhancers, polyoma enhancers, and adenovirus enhancers. Enhancer sequences from mammalian systems are also commonly used, such as the mouse immunoglobulin heavy chain enhancer.

[0081] Gene therapy vectors can be delivered in vivo by administration to an individual patient, typically by systemic administration (e.g., intravenous, intraperitoneal, intramuscular, subdermal, or intracranial infusion) or topical application. Alternatively, vectors can be delivered to cells ex vivo, such as cells explanted from an individual patient (e.g., lung cells, kidney cells, lymphocytes, bone marrow aspirates, tissue biopsy) or universal donor hematopoietic stem cells, msenchymal stem cells, or lung or kidney progenitor cells, followed by reimplantation of the cells into a patient, usually after selection for cells which have incorporated the vector.

(D) Viral Vectors For Lung or Kidney -Directed Gene Therapy [0082] Viruses have evolved mechanisms to enter host cells, translocate to the nucleus, express viral genes using cellular gene expression mechanisms and replicate. The DNA genome or the reverse-transcribed DNA product of the RNA genome of some viruses integrates into the host chromosomes, while other viral genes are expressed without integration. These characteristics are relevant in determining suitability of vectors specific applications. For example, for ex-vivo gene therapy, and in vivo gene therapy for lifelong inherited diseases, it is desirable that the transgene integrates into the host genome and is transmitted to the progeny of the transduced cell. Available space to accommodate relevant transgenes is also an important consideration. Immune response to the vector and repeatability of gene transfer are important issues for clinical applications. No single vector fulfills all these desirable criteria, but some of the currently available vectors are of sufficient safety and efficiency for going into clinical trials. A brief description of the commonly used viral vectors follows.

[0083] Examples of lung or kidney -specific promoters include, but are not limited to, hepatitis B virus promoters (Sandig et al., Gene Therapy 3: 1002-1009 (1996) and albumin gene promoters (Pinkert et al., Genes and Development, 1: 268-276 (1987); see also Guo et al., Gene Therapy, 3: 802-810 (1996) for other lung or kidney -specific promoter; lung specific surfactant protein C promoter. Zhuo et al., Transgenic Res. 15: 543-555, 2006; lung-specific promoter (Clara Cell 10; CClO) is expressed in both alveolar and airway epithelial cells; Auricchio et al., / Clin Invest. 110: 499-504, 2002; human lung-specific promoter, human surfactant protein C (hSP-C); Hartney et al., Am J Physiol Lung Cell MoI Physiol. 290: L105-L113, 2006; Thatte et al., Blood, 101: 4916-4922, 2003

[0084] Kidney- specific cadherin (Ksp-cadherin) is a tissue- specific member of the cadherin family that is expressed exclusively in the kidney and developing genitourinary tract; Bai, et al., Am J Physiol Renal Physiol. 283: F839-F851, 2002; Igarashi, Nephron Exp Nephrol. 94: el-e6, 2003; a promoter motif with homology to a purine-rich sequence responsible for the kidney- specific promoter activity of the rat CLC-Kl gene; Tanaka et al., Genomics 58: 281-292, 1999.

[0085] A preferred target for delivery of LELAl gene is lung tissue. Although ex vivo therapy {e.g., explanting lung cells followed by introduction of the polynucleotide expressing LELAl polypeptide and then transplantation back into the patient) is possible, gene delivery in vivo is particularly preferable. Surgery is performed to infuse the gene through a catheter into the pulmonary artery or vein. Ideally, less invasive practices such as intravenous injection are used.

[0086] Recombinant retroviruses. RNA genomes of retroviruses are reverse- transcribed into cDNA, which integrate into the host genome via viral integrase. Replication incompetent vectors are generated by replacing viral genes by the transgene, keeping the packaging signal (ψ) intact. The viral proteins are provided in trans by packaging cells.

[0087] Recombinant oncoretroviruses (e.g. Moloney's murine leukemia virus) require cell division to integrate, while immunoretroviruses (lenti viruses, e.g. HIV-I) are more efficient at transducing quiescent cells, such as lung cells or kidney cells. To broaden the range of target tissues, the native retroviral envelope can be replaced with alternative viral capsid proteins, such as the vesicular stomatitis virus G protein. Recombinant retroviral vectors are of relatively low immunogenicity and have low toxicity.

[0088] Adenoviral Vectors. Adenoviruses are large, linear DNA viruses that are highly efficient in delivering genes to both quiescent and dividing cells, and concentrate in the liver in many species. However, it is uncertain whether such hepatotropism exists for the human liver. As adenovectors are episomal, they are lost upon cell division. Humoral and cell-mediated host immune responses preclude readministration of adenovectors. Although the primary immune response is reduced by deleting all viral genes, the "gutted" vectors do not permit repeated gene transfer. Repeated gene transfer is possible by using different strains of non-human adenoviruses or by coexpressing an immunomodulatory gene (e.g. CTLA4-Ig or adenoviral E3). Preexisting immunity and innate immunity against adenoviral proteins are major concerns in the clinical application of adenovectors.

[0089] Recombinant Adeno-associated Virus (AAV). AAV is a single- stranded DNA virus of the papova family. The wildtype virus integrates preferentially on the ql3.4-ter arm of human chromosome 19 via the 145 bp inverted terminal repeats (ITR), but the sitespecificity is mostly lost in recombinant vectors. Recombinant AAV vectors remain episomally for long durations in non-dividing cells, but some integration can occur when DNA repair is impaired. AAV type 2-based vectors have been tested most frequently, but is relatively inefficient in transferring genes to lung cells or kidney cells in vivo. Pseudotyped vectors, e.g. AAV-2 vectors containing AAV-8 capsids are much more efficient in hepatic gene transfer. Other AAV strains and pseudotypes are being evaluated.

[0090] Recombinant Simian Virus 40 (SV40). SV40 is a small DNA virus of the papova family. Vectors are generated by replacing the T-antigen encoding region by a transgene. Additional viral genes can be deleted to increase the stuffing space. Recombinant SV40 vectors elicit little or no immune response and can be administered repeatedly. The vector integrates randomly into the host genome leading to long-term transgene expression. A 40-year follow-up of a large number of subjects who were infected inadvertently with wildtype SV40 contaminating polio vaccines showed no untoward effects associated with the infection.

(E) Non- Viral Nucleic Acid Delivery

[0091] The challenges of generating safe and efficient recombinant viral vectors have prompted the parallel development of methods for non-viral gene transfer. A brief review of these strategies follows.

[0092] Physical or receptor-mediated delivery of nucleic acids. Plasmid DNA or synthesized DNA or RNA oligonucleotides (ONs) can be introduced into the lung or kidney tissue by direct injection, ballistic delivery of DNA-coated particles or retrograde injection into the bile duct. In the hydrodynamic or hydroporation delivery method, the nucleic acid solution is administered rapidly in large volumes to cause hepatic congestion and transient increase of lung cell or kidney cell permeability. Although useful in laboratory studies, this method is unlikely to find clinical application. Cationic liposomes and polycations, are being evaluated for lung nucleic acid delivery. To target the nucleic acid to the lung or kidney, ligands for lung or kidney -specific receptors (e.g. asialoglycoprotein receptor) have been incorporated into gene transfer vehicles. Current research is directed toward incorporating peptides or chemicals (e.g. proteins of the Sendai virus) into the gene delivery vehicle for destabilization of the endocytotic vesicles, so that the nucleic acid is released into the cytosol. Nuclear localizing peptides may be incorporated into nanocapsule or nanoparticle carriers to promote gene translocation into the nucleus.

[0093] Transposon-mediated gene delivery system. Simple DNAs introduced into lung cells or kidney cells persist and express transgenes transiently. To promote transgene integration into the host genome, transposition-enabled plasmids are being tested. An active transposase reconstructed from an ancestral TcI -like fish element, mediates transposition by cutting at specific inverted repeat/direct repeats (IR/DRs) DNA and "pasting" the intervening DNA segment at a different genomic site. See, for example, U.S. Patent 6,489,458. Transposons for gene delivery contain the transgenes flanked by the IR/DRs that are sites for transposase action. The transposase may be expressed by transfection of a second plasmid. Alternatively, a single plasmid contains both the transposon and a second transcription unit expressing the transposase. Using the system in factor IX-deficient mice, it was possible to express the coagulation factor at 5-6% of normal levels, resulting in long-term amelioration of the coagulation defect.

[0094] Down-regulation of gene expression. Ribozymes, DNA ribonucleases, antisense RNA and, more recently, vectors expressing small hairpin containing inhibitory RNAs (shRNA) are being tested for inhibiting LELAl polypeptide for treatment of cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma.

[0095] Gene Repair Strategies. Site-directed gene repair has significant theoretical advantages over gene replacement, because the corrected gene remains in situ, whereby their physiological regulation is retained. In general, gene repair by cellular mechanisms is triggered by generating a mismatch using RNA-DNA chimeric oligonucleotides (ONs), single strand DNA ONs or triplex forming ONs. Short Fragment Homologous Recombination is also being explored. Recently, it has been reported that single stranded recombinant AAV vectors can be used to introduce single base substitutions in normal human cells. Despite the potential attractiveness of these approaches, their current efficiency is not high enough for clinical application.

[0096] Routes of gene transfer to the lung or kidney, include, but are not limited to, ex vivo, topical, or systemic. Isolation of kidney tissue, lung tissue, kidney cells or lung cells and insertion of a gene vector can occur by ex vivo transduction and transplantation back into the donor; Injection or ballistic insertion into tissues; lung-targeted (via receptors) or wide distribution; Injection into a local vessel or duct; hydroporation ("hydrodynamic delivery); sonoporation or electroporation; Viral and non-viral vectors can deliver LELAl genes to the mammalian subject

[0097] Gene therapy vehicles can be recombinant viruses; polycations (e.g., polyethyleneimine, polylysine); lipids, naked nucleic acids; charged liposomes; liposome- polycation-peptide complex, which can be complexed with nucleic acids only or with ligands, e.g. asialoglycoprotein plus nucleic acids; liposome-polycation complex; liposome-ligands (e.g. galactocerebroside), or viral proteins: ("virosomes")

[0098] An inherent issue in all randomly integrating vectors is the positional effect. Depending on the site of insertion, the transgene may not be expressed or may be silenced eventually. Furthermore, the enhancer present in the transcription unit can, potentially activate a neighboring oncogene, as illustrated by the occurrence of T-cell leukemia in two immunodeficient infants receiving bone marrow-based ex vivo gene therapy. A potential solution is to insert insulator sequences flanking the transcription unit, which prevent gene silencing, as well as prevent the activation of neighboring protooncogenes by enhancers in the transgene. Vector toxicity and immunological side-effects to vector proteins and the transgene products are other concerns affecting the choice of vectors.

[0099] "Liposomes" or "lipid vesicles" refer to substantially spherical structures made of materials having a high lipid content in which the lipids are organized in the form of lipid bilayers. Unilamellar vesicles have a single lipid bilayer surrounding an amorphous central cavity which can encapsulate an aqueous volume. Unilamellar vesicles can be prepared as either large unilamellar vesicles (LUVs; diameter greater than about lμ) or small unilamellar vesicles (SUVs; diameter less than about 0.2 μm). Multilamellar vesicles (MLVs) have many onion-like shells of lipid bilayers. Because of their high lipid content, MLVs have use for carrying certain small lipophilic molecules but have a low carrying capacity for aqueous material. Paucilamellar vesicles (PLVs) have about two-ten bilayers arranged in the form of substantially spherical shells separated by aqueous layers surrounding a central cavity free of lipid bilayers. PLVs can encapsulate both aqueous and hydrophobic material and thus can carry a wide variety of materials. Unilamellar vesicles composed of a single bilayer of phospholipids and/or glycolipids are the most commonly used lipid vesicles for modeling of cell membrane structures since phospholipids are the primary structural component of natural membranes, including the outer cell membrane. Liposomes (e.g., phospholipid vesicles), can be used as carrier vehicles for delivering biologically active materials to tissues using the methods of the invention. For reviews of phospholipid vesicle-mediated transfer of materials see Mannino, BioTechniques, 6:682 (1988); Litzinger, D. C. Biochim. et Biophys. Acta, 1113: 201 (1992). Methods for preparing liposomes as carrier vesicles for delivery of biologically active materials are known in the art (see, for example, U.S. Pat. No. 4,522,811).

[0100] "Locally" refers to administration, e.g., injection, directly into the tissue to be treated. Examples of local treatment include intrahepatic injection or hepatic vein injection.

[0101] "Locoregionally" or "regionally" refers to administration locally and and/or regionally. For example, the compound may be administered in the fluid surrounding the tissues and directly injected into the tissues, e.g., intraperitoneal treatment of lung cancer, intrapneumatic infusion, implants of drug release devices for the treatment of cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma.

[0102] "Microparticles" refer to particles which comprise LELAl polypeptide or nucleic acid, therapeutic agents or other substances which can be advantageously delivered using methods of the invention to the interior of a tissue, e.g., lung tissue or kidney tissue. The term refers to particles of about 0.1 μm to about 100 μm, about 0.5 μm to about 50 μm, 0.5 μm to about 20 μm in size, advantageously, particles of about 1 μm to about 10 μm in size, about 5 μm in size, or mixtures thereof. The microparticles may comprise macromolecules, gene therapy constructs, chemotherapeutic agents, or protein bound drugs, for example. Typically microparticles can be administered locally, locoregionally, or regionally, for example, to the lung tissue or kidney tissue or region surrounding the lung tissue or kidney tissue

[0103] "Nanoparticles" refer to particles which comprise LELAl polypeptide or nucleic acid, therapeutic agents or other substances which can be advantageously delivered using methods of the invention to the interior or a tissue, e.g., lung or kidney tissue. The term refers to particles of about 0.1 nm to about 1 μm, 1 nm to about 1 μm, about 10 nm to about 1 μm, about 50 nm to about 1 μm, about 100 nm to about 1 μm, about 250 to 900 nm in size, or, advantageously, about 600 to 800 nm. The nanoparticles may comprise macromolecules, gene therapy constructs, chemotherapeutic agents, or protein bound drugs. Typically, nanoparticles can be administered to a patient via local, locoregional, regional, or systemic administration. In one embodiment, the nanoparticles may comprise cross-linked gelatin.

HIGH THROUGHPUT ASSAYS FOR MODULATORS OF LELAl GENE PRODUCT

[0104] The compounds tested as modulators of LELAl activity can be any small organic molecule, or a biological entity, such as a protein, e.g., an antibody or peptide, a sugar, a nucleic acid, e.g., an antisense oligonucleotide, RNAi, or a ribozyme, or a lipid. Alternatively, modulators can be genetically altered versions of LELAl polypeptide, or a mimetic, analog, or derivative thereof. Typically, test compounds will be small organic molecules, peptides, lipids, and lipid analogs.

[0105] Essentially any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used. The assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g., in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma-Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.

[0106] In one preferred embodiment, high throughput screening methods involve providing a combinatorial small organic molecule or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries" or "ligand libraries" are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.

[0107] A combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents. For example, a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.

[0108] Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S. Patent 5,010,175, Furka, Int. J. Pept. Prot. Res. 37: 487-493, 1991 and Houghton et al, Nature 354: 84-88, 1991). Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No. 5,288,514), diversomers such as hydantoins, benzodiazepines and dipeptides (Hobbs et al, Proc. Nat. Acad. ScL USA 90: 6909-6913, 1993), vinylogous polypeptides (Hagihara et al., J. Amer. Chem. Soc. 114: 6568, 1992), nonpeptidal peptidomimetics with glucose scaffolding (Hirschmann et al., J. Amer. Chem. Soc. 114: 9217-9218, 1992), analogous organic syntheses of small compound libraries (Chen et al., J. Amer. Chem. Soc. 116: 2661, 1994), oligocarbamates (Cho et al., Science, 261: 1303, 1993), and/or peptidyl phosphonates (Campbell et al., J. Org. Chem. 59: 658, 1994), nucleic acid libraries (see Ausubel, Berger and Sambrook, all supra), peptide nucleic acid libraries (see, e.g., U.S. Patent 5,539,083), antibody libraries (see, e.g., Vaughn et al., Nature Biotechnology, 14: 309-314, 1996 and PCT/US96/10287), carbohydrate libraries (see, e.g., Liang et al., Science 274: 1520-1522, 1996 and U.S. Patent 5,593,853), small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).

[0109] Devices for the preparation of combinatorial libraries are commercially available (see, e.g., 357 MPS, 390 MPS, Advanced Chem Tech, Louisville KY, Symphony, Rainin, Woburn, MA, 433A Applied Biosystems, Foster City, CA, 9050 Plus, Millipore, Bedford, MA). In addition, numerous combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, NJ. , Asinex, Moscow, Ru, Tripos, Inc., St. Louis, MO, ChemStar, Ltd, Moscow, RU, 3D Pharmaceuticals, Exton, PA, Martek Biosciences, Columbia, MD, etc.).

[0110] Candidate compounds are useful as part of a strategy to identify drugs for treating disorders including, but not limited to, cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma. A test compound that binds to LELAl polypeptide or affects LELAl gene expression or telomerase gene expression is considered a candidate compound.

[0111] Screening assays for identifying candidate or test compounds that bind to LELAl polypeptide, or modulate the activity of LELAl polypeptide and/or telomerase proteins or polypeptides or biologically active portions thereof, are also included in the invention. The test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including, but not limited to, biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound" library method; and synthetic library methods using affinity chromatography selection. The biological library approach can be used for, e.g., peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small chemical molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997). Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al, Proc. Natl. Acad. ScL U.S.A. 90: 6909, 1993; Erb et al, Proc. Natl. Acad. ScL USA 91: 11422, 1994; Zuckermann et al, J. Med. Chem. 37: 2678, 1994; Cho et al, Science 261: 1303, 1993; Carrell et al., Angew. Chem. Int. Ed. Engl. 33: 2059, 1994; Carell et al, Angew. Chem. Int. Ed. Engl. 33: 2061, 1994; and Gallop et al, J. Med. Chem. 37: 1233, 1994. In some embodiments, the test compounds are activating variants of LELAl polypeptide.

[0112] Libraries of compounds can be presented in solution (e.g., Houghten, Bio/Techniques 13: All-All, 1992), or on beads (Lam, Nature 354: 82-84, 1991), chips (Fodor, Nature 364: 555-556, 1993), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al, Proc. Natl. Acad. ScL USA 89: 1865-1869, 1992) or on phage (Scott et al, Science 249: 386-390, 1990; Devlin, Science 249: 404-406, 1990; Cwirla et al, Proc. Natl. Acad. ScL USA 87: 6378-6382, 1990; and Felici, /. MoI. Biol. 222: 301-310, 1991).

[0113] The ability of a test compound to modulate the activity of LELAl polypeptide and/or telomerase or a biologically active portion thereof can be determined, e.g., by monitoring the inhibition or activation of telomerase activity in cells in the presence of the test compound. Modulating the activity of LELAl polypeptide or a biologically active portion thereof can be determined by measuring inhibition or activation of telomerase activity in cells. The binding assays can be cell-based or cell-free.

[0114] The ability of a test compound to modulate the activity of LELAl polypeptide and/or telomerase and telomerase activity in cells can be determined by one of the methods described herein or known in the art for determining LELAl polypeptide direct binding. In one embodiment, the ability of the LELAl polypeptide to bind to or interact with telomerase can be determined by monitoring telomerase activity in cells. Detection of the LELAl polypeptide telomerase activity in cells can include detection of the expression of a recombinant telomerase polypeptide or LELAl polypeptide that also encodes a detectable marker such as a FLAG sequence or a luciferase. This assay can be in addition to an assay of direct binding. In general, such assays are used to determine the ability of a test compound to affect LELAl inhibition of telomerase activity in cells.

[0115] In general, the ability of a test compound to affect LELAl polypeptide inhibition of telomerase activity in cells is compared to a control in which the LELAl polypeptide inhibition of telomerase activity is determined in the absence of the test compound. In some cases, a predetermined reference value is used. Such reference values can be determined relative to controls, in which case a test sample that is different from the reference would indicate that the compound binds to the molecule of interest (e.g., LELAl polypeptide and/or telomerase) or modulates expression (e.g., modulates, activates or inhibits telomerase activity in cells that has been treated with LELAl polypeptide or a mimetic, analog, or derivative thereof or by an inhibitor of telomerase activity). A reference value can also reflect the amount of LELAl polypeptide inhibition of telomerase activity observed with a standard (e.g., the affinity of antibody for LELAl polypeptide or telomerase polypeptide, or modulation of the activity of LELAl polypeptide and/or telomerase polypeptide). In this case, a test compound that is similar to (e.g., equal to or less than) the reference would indicate that compound is a candidate compound (e.g., binds to LELAl polypeptide or telomerase polypeptide to a degree equal to or greater than a reference compound or reference antibody).

[0116] This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.

[0117] In one embodiment the invention provides soluble assays using LELAl polypeptide and/or telomerase polypeptide, or a cell or tissue expressing LELAl gene product or telomerase gene product, either naturally occurring or recombinant. In another embodiment, the invention provides solid phase based in vitro assays in a high throughput format, where LELAl polypeptide and/or telomerase polypeptide is attached to a solid phase substrate via covalent or non-covalent interactions. Any one of the assays described herein can be adapted for high throughput screening.

[0118] In the high throughput assays of the invention, either soluble or solid state, it is possible to screen up to several thousand different modulators or ligands in a single day. This methodology can be used for LELAl polypeptide or telomerase polypeptide in vitro, or for cell- based or membrane-based assays comprising LELAl gene product or LELAl polypeptide and/or telomerase polypeptide In particular, each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator. Thus, a single standard microtiter plate can assay about 100 (e.g., 96) modulators. If 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or more than 100,000 different compounds are possible using the integrated systems of the invention.

[0119] For a solid state reaction, the protein of interest or a fragment thereof, e.g., an extracellular domain, or a cell or membrane comprising the protein of interest or a fragment thereof as part of a fusion protein can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage e.g., via a tag. The tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.

[0120] A number of tags and tag binders can be used, based upon known molecular interactions well described in the literature. For example, where a tag has a natural binder, for example, biotin, protein A, or protein G, it can be used in conjunction with appropriate tag binders (avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, etc.) Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).

[0121] Similarly, any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair. Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature. For example, in one common configuration, the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody. In addition to antibody- antigen interactions, receptor- ligand interactions are also appropriate as tag and tag-binder pairs. For example, agonists and antagonists of cell membrane receptors (e.g., cell receptor- ligand interactions such as toll-like receptors, transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I, 1993. Similarly, toxins and venoms, viral epitopes, hormones (e.g., opiates, steroids, etc.), intracellular receptors (e.g. which mediate the effects of various small ligands, including steroids, thyroid hormone, retinoids and vitamin D; peptides), drugs, lectins, sugars, nucleic acids (both linear and cyclic polymer configurations), oligosaccharides, proteins, phospholipids and antibodies can all interact with various cell receptors.

[0122] Synthetic polymers, such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.

[0123] Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids. Such flexible linkers are known to persons of skill in the art. For example, polyethylene glycol linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or heterofunctional linkages.

[0124] Tag binders are fixed to solid substrates using any of a variety of methods currently available. Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder. For example, groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups. Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, /. Am. Chem. Soc. 85: 2149-2154, 1963 (describing solid phase synthesis of, e.g., peptides); Geysen et al, J. Immun. Meth. 102: 259-274, 1987 (describing synthesis of solid phase components on pins); Frank & Doring, Tetrahedron 44: 6031-6040, 1988 (describing synthesis of various peptide sequences on cellulose disks); Fodor et al, Science 251: 767-777, 1991; Sheldon et al, Clinical Chemistry 39: 718-719, 1993; and Kozal et al, Nature Medicine 2: 753-759, 1996 (all describing arrays of biopolymers fixed to solid substrates). Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.

DIAGNOSTIC METHODS

[0125] Diagnosis or prognosis of early age-onset lung cancer. The invention provides a variety of methods for the diagnosis or prognosis of early age-onset lung cancer. In particular, A method of detecting an increased susceptibility to early age-onset lung cancer in an individual comprising analyzing a DNA sample from the individual for the presence of at least one single nucleotide polymorphism of a LELAl gene sequence, wherein the presence of the at least one single nucleotide polymorphism is indicative of an increased susceptibility to early age- onset lung cancer. In some methods, the at least one single nucleotide polymorphism is a single nucleotide polymorphism (SNP) in the LELAl gene. It is to be understood that "diagnosis or prognosis of early age-onset lung cancer" does not necessarily mean that the subject will develop early age-onset lung cancer but rather that the subject is, in a statistical sense, more likely to develop early age-onset lung cancer than an average member of the population. As used herein, "diagnosis or prognosis for early age-onset lung cancer" can exist if the subject has one or more genetic determinants (e.g., polymorphic variants or alleles) that can, either alone or in combination with one or more other genetic determinants, contribute to an increased risk of developing early age-onset lung cancer in some or all subjects. Ascertaining whether the subject has any such genetic determinants (i.e., genetic determinants that can increase the risk of developing early age-onset lung cancer in the appropriate genetic background) is included in the concept of diagnosing susceptibility to early age-onset lung cancer, as used herein. Such determination is useful, for example, for purposes of genetic counseling. Thus providing diagnostic information regarding early age-onset lung cancer includes providing information useful in genetic counseling, and the provision of such information is encompassed herein.

[0126] The sample itself will typically consist of cells (e.g., cells of the lung), tissue, and the like, removed from the subject. The subject can be an adult, child, fetus, or embryo. According to certain embodiments of the invention the sample is obtained prenatally, either from the fetus or embryo or from the mother (e.g., from fetal or embryonic cells in that enter the maternal circulation). The sample can be further processed before the detecting step. For example, DNA in the cell or tissue sample can be separated from other components of the sample, can be amplified, and the like. All samples obtained from a subject, including those subjected to any sort of further processing, are considered to be obtained from the subject.

[0127] In general, if the polymorphism is located in a gene, it can be located in a noncoding or coding region of the gene. If located in a coding region the polymorphism can, but frequently will not, result in an amino acid alteration. Such alteration can or can not have an effect on the function or activity of the encoded polypeptide. If the polymorphism is linked to, but not located within, a gene, it is preferred that the polymorphism is closely linked to the gene. For example, it is preferred that the recombination frequency between the polymorphism and the gene is less than approximately 20%, preferably less than approximately 10%, less than approximately 5%, less than approximately 1%, or still less.

[0128] According to certain preferred embodiments of any of the inventive methods described above, the gene can be coincident with a mapped or identified locus for early age-onset lung cancer, e.g., genes encoding LELAl. For example, according to various embodiments of the invention the gene can encode any of the molecules listed in the tables as shown herein. In a particular embodiment of the invention, discussed further below, the preferred genes encode a single nucleotide polymorphism (SNP) in the LELAl gene. The inventive methods also encompass genes coincident with early age-onset lung cancer susceptibility loci that have yet to be mapped or identified. By "coincident with" is meant either that the gene or a portion thereof falls within the identified chromosomal location or is located in close proximity to that location. In general, the resolution of studies identifying genetic susceptibility loci can be on the order of tens of centimorgans. According to certain embodiments of the invention "close proximity" refers to within 20 centimorgans of either side of the susceptibility locus, more preferably within 10 centimorgans of either side of the susceptibility locus, yet more preferably within 5 centimorgans of either side of the susceptibility locus. In general, susceptibility loci are designated by the chromosomal band positions that they span (e.g., 3p21 refers to chromosome 3, arm p, band 21; 3p20-21 refers to chromosome 3, arm p, bands 20-21 inclusive) and can be defined at higher resolution (e.g., 3p21.1). In general, the terms "coincident with" and "close proximity" can be interpreted in light of the knowledge of one of ordinary skill in the art.

[0129] Methods and reagents for identification and detection of polymorphisms. In general, polymorphisms of use in the practice of the invention can be initially identified using any of a number of methods well known in the art. For example, numerous polymorphisms are known to exist and are available in public databases, which can be searched as described. Alternately, polymorphisms can be identified by sequencing either genomic DNA or cDNA in the region in which it is desired to find a polymorphism. According to one approach, primers are designed to amplify such a region, and DNA from a subject suffering from early age-onset lung canceris obtained and amplified. The DNA is sequenced, and the sequence (referred to as a "subject sequence") is compared with a reference sequence, which is typically taken to represent the "normal" or "wild type" sequence. Such a sequence can be, for example, the human draft genome sequence, publicly available in various databases, or a sequence deposited in a database such as GenBank. In general, if sequencing reveals a difference between the sequenced region and the reference sequence, a polymorphism has been identified. Note that this analysis does not necessarily presuppose that either the subject sequence or the reference sequence is the "normal", most common, or wild type sequence. It is the fact that a difference in nucleotide sequence is identified at a particular site that determines that a polymorphism exists at that site. In most instances, particularly in the case of SNPs, only two polymorphic variants will exist at any location. However, in the case of SNPs, up to four variants can exist since there are four naturally occurring nucleotides in DNA. Other polymorphisms such as insertions can have more than four alleles.

[0130] Once a polymorphic site is identified, any of a variety of methods can be employed to detect the existence of any particular polymorphic variant in a subject. In general, a subject can have either the reference sequence or an alternate sequence at the site. The phrase "detecting a polymorphism" or "detecting a polymorphic variant" as used herein generally refers to determining which of two or more polymorphic variants exists at a polymorphic site, although "detecting a polymorphism" can also refer to the process of initially determining that a polymorphic site exists in a population. The meaning to be given to these phrases will be clear from the context as interpreted in light of the knowledge of one of ordinary skill in the art. For purposes of description, if a subject has any sequence other than a defined reference sequence (e.g. the sequence present in the human draft genome) at a polymorphic site, the subject can be said to exhibit the polymorphism. In general, for a given polymorphism, any individual will exhibit either one or two possible variants at the polymorphic site (one on each chromosome). (This can, however, not be the case if the individual exhibits one more chromosomal abnormalities such as deletions.)

[0131] Detection of a polymorphism or polymorphic variant in a subject (genotyping) can be performed by sequencing, similarly to the manner in which the existence of a polymorphism is initially established as described above. However, once the existence of a polymorphism is established a variety of more efficient methods can be employed. Many such methods are based on the design of oligonucleotide probes or primers that facilitate distinguishing between two or more polymorphic variants.

[0132] "Probes" or "primers", as used herein, typically refers to oligonucleotides that hybridize in a base-specific manner to a complementary nucleic acid molecule as decribed herein. Such probes and primers include polypeptide nucleic acids, as described in Nielsen et al., Science 254: 1497-1500, 1991. "primer" in particular generally refers to a single- stranded oligonucleotide that can act as a point of initiation of template-directed DNA synthesis using methods such as PCR (polymerase chain reaction), LCR (ligase chain reaction), and the like. Typically, a probe or primer will comprise a region of nucleotide sequence that hybridizes to at least about 8, more often at least about 10 to 15, typically about 20-25, and frequently about 40, 50 or 75, consecutive nucleotides of a nucleic acid molecule. In certain embodiments of the invention, a probe or primer comprises 100 or fewer nucleotides, preferably from 6 to 50 nucleotides, preferably from 12 to 30 nucleotides. In certain embodiments of the invention, the probe or primer is at least 70% identical to the contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence, preferably at least 80% identical, more preferably at least 90% identical, even more preferably at least 95% identical, or having an even higher degree of identity. In certain embodiments of the invention a preferred probe or primer is capable of selectively hybridizing to a target contiguous nucleotide sequence or to the complement of the contiguous nucleotide sequence. According to certain embodiments of the invention a probe or primer further comprises a label, for example by incorporating a radioisotope, fluorescent compound, enzyme, or enzyme co-factor.

[0133] Oligonucleotides that exhibit differential or selective binding to polymorphic sites can readily be designed by one of ordinary skill in the art. For example, an oligonucleotide that is perfectly complementary to a sequence that encompasses a polymorphic site (i.e., a sequence that includes the polymorphic site within it or at one or the other end) will generally hybridize preferentially to a nucleic acid comprising that sequence as opposed to a nucleic acid comprising an alternate polymorphic variant.

[0134] In order to detect polymorphisms and/or polymorphic variants, it will frequently be desirable to amplify a portion of DNA encompassing the polymorphic site. Such regions can be amplified and isolated by PCR using oligonucleotide primers designed based on genomic and/or cDNA sequences that flank the site. See e.g., PCR Primer: A Laboratory Manual, Dieffenbach, C. W. and Dveksler, G. S. (eds.); PCR Basics: From Background to Bench, Springer Verlag, 2000; McPherson et al; Mattila et al., Nucleic Acids Res. 19: 4967, 1991; Eckert et al, PCR Methods and Applications 1: 17, 1991; PCR (eds. McPherson et al., IRL Press, Oxford); and U.S. Pat. No. 4,683,202. Other amplification methods that can be employed include the ligase chain reaction (LCR) (Wu and Wallace, Genomics 4: 560, 1989; Landegren et al., Science 241: 1077, 1988) transcription amplification (Kwoh et al., Proc. Natl. Acad. ScL USA 86: 1173, 1989), self- sustained sequence replication (Guatelli et al., Proc. Nat. Acad. ScL USA 87: 1874, 1990), and nucleic acid based sequence amplification (NASBA). Guidelines for selecting primers for PCR amplification are well known in the art. See, e.g., McPherson et al., PCR 2000, cited supra. A variety of computer programs for designing primers are available, e.g., "Oligo" (National Biosciences, Inc, Plymouth MN), Mac Vector (Kodak/IBI), and the GCG suite of sequence analysis programs (Genetics Computer Group, Madison, WI 53711)

[0135] According to certain methods for diagnosing early age-onset lung cancer or susceptibility to early age-onset lung cancer, hybridization methods, such as Southern analysis, Northern analysis, or in situ hybridizations, can be used {see Ausubel et al., supra). For example, a sample (e.g., a sample comprising genomic DNA, RNA, or cDNA), is obtained from a subject suspected of being susceptible to or having lung cancer disease, e.g., early age-onset lung cancer. The DNA, RNA, or cDNA sample is then examined to determine whether a polymorphic variant in a coding or noncoding portion of a gene set forth in Table 4, or a polymorphic variant in a genomic region linked to a coding or noncoding portion of a gene encoding as set forth in Table 4 is present. The presence of the polymorphic variant can be indicated by hybridization of the gene in the genomic DNA, RNA, or cDNA to a nucleic acid probe, e.g., a DNA probe (which includes cDNA and oligonucleotide probes) or an RNA probe. The nucleic acid probe can be designed to specifically or preferentially hybridize with a particular polymorphic variant, e.g., a polymorphic variant indicative of susceptibility to early age-onset lung cancer. [0136] In order to diagnose susceptibility to early age-onset lung cancer, a hybridization sample is formed by contacting the sample with at least one nucleic acid probe. The probe is typically a nucleic acid probe (which can be labeled, e.g., with a radioactive, fluorescent, or enzymatic label or tag) capable of hybridizing to mRNA, genomic DNA, and/or cDNA sequences encompassing detecting a polymorphic variant in a coding or noncoding portion of a gene encoding LELAl, or a polymorphic variant in a genomic region linked to a coding or noncoding portion of a gene wherein the gene has at least one single nucleotide polymorphism including, but not limited to, a polymorphism as disclosed in Table 4. The nucleic acid probe can be, for example, a full-length nucleic acid molecule, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to appropriate mRNA, cDNA, or genomic DNA.

[0137] The hybridization sample is maintained under conditions selected to allow specific hybridization of the nucleic acid probe to a region encompassing the polymorphic site. Specific hybridization can be performed under high stringency conditions or moderate stringency conditions, for example, as described above. In a particularly preferred embodiment, the hybridization conditions for specific hybridization are high stringency. In general, the probe can be perfectly complementary to the region to which it hybridizes, i.e., perfectly complementary to a region encompassing the polymorphic site when the site contains any particular polymorphic sequence. Multiple nucleic acid probes {e.g., multiple probes differing only at the polymorphic site, or multiple probes designed to detect polymorphic variants at multiple polymorphic sites) can be used concurrently in this method. Specific hybridization of any one of the nucleic acid probes is indicative of a polymorphic variant in a genomic region linked to a coding or noncoding portion of LELAl or fragment thereof, or detecting a polymorphic variant or a polymorphism in a genomic region linked to such a gene, wherein the at least one single nucleotide polymorphism is, for example, a single nucleotide polymorphism (SNP) in the LELAl gene and is thus diagnostic of susceptibility to early age-onset lung cancer.

[0138] Northern analysis can be performed using similar nucleic acid probes in order to detect a polymorphic variant or a polymorphism in a coding or noncoding portion of a gene encoding LELAl, or detecting a polymorphic variant or a polymorphism in a genomic region linked to such a gene, for example, wherein the at least one single nucleotide polymorphism isin the LELAl gene. See, e.g., Ausubel et al, supra.

[0139] According to certain embodiments of the invention, a peptide nucleic acid (PNA) probe can be used instead of a nucleic acid probe in the hybridization methods described above. PNA is a DNA mimetic with a peptide-like, inorganic backbone, e.g., N-(2- aminoethyl)glycine units, with an organic base (A, G, C, T or U) attached to the glycine nitrogen via a methylene carbonyl linker (see, for example, Nielsen et al., 1994, Bioconjugate Chemistry 5 American Chemical Society, p. 1 (1994). The PNA probe can be designed to specifically hybridize to a nucleic acid comprising a polymorphic variant conferring susceptibility to or indicative of the presence of early age-onset lung cancer.

[0140] According to another method, restriction digest analysis can be used to detect the existence of a polymorphic variant or a polymorphism, if alternate polymorphic variants of the polymorphism result in the creation or elimination of a restriction site. A sample containing genomic DNA is obtained from the individual. Polymerase chain reaction (PCR) can be used to amplify a region comprising the polymorphic site, and restriction fragment length polymorphism analysis is conducted (see, e.g., Ausubel et al., supra). The digestion pattern of the relevant DNA fragment indicates the presence or absence of a particular polymorphic variant of the polymorphism and is therefore indicative of the presence or absence of susceptibility to early age-onset lung cancer.

[0141] Sequence analysis can also be used to detect specific polymorphic variants. A sample comprising DNA or RNA is obtained from the subject. PCR or other appropriate methods can be used to amplify a portion encompassing the polymorphic site, if desired. The sequence is then ascertained, using any standard method, and the presence of a polymorphic variant is determined.

[0142] Allele- specific oligonucleotides can also be used to detect the presence of a polymorphic variant, e.g., through the use of dot-blot hybridization of amplified oligonucleotides with allele- specific oligonucleotide (ASO) probes (see, for example, Saiki et al., Nature 324: 163-166, 1986). An "allele- specific oligonucleotide" (also referred to herein as an "allele- specific oligonucleotide probe") is typically an oligonucleotide of approximately 10-50 base pairs, preferably approximately 15-30 base pairs, that specifically hybridizes to a nucleic acid region that contains a polymorphism, e.g., a polymorphism associated with a susceptibility to early age-onset lung cancer. An allele-specific oligonucleotide probe that is specific for particular a polymorphism can be prepared, using standard methods (see Ausubel et al., supra).

[0143] To determine which of multiple polymorphic variants is present in a subject, a sample comprising DNA is obtained from the individual. PCR can be used to amplify a portion encompassing the polymorphic site. DNA containing the amplified portion can be dot-blotted, using standard methods, and the blot contacted with the oligonucleotide probe. The presence of specific hybridization of the probe to the DNA is then detected. Specific hybridization of an allele- specific oligonucleotide probe (specific for a polymorphic variant indicative of susceptibility to early age-onset lung cancer) to DNA from the subject is indicative of susceptibility to early age-onset lung cancer.

[0144] According to another embodiment of the invention, arrays of oligonucleotide probes that are complementary to nucleic acid portions from a subject can be used to identify polymorphisms. Biochips as described herein can be used.

[0145] The array typically includes oligonucleotide probes capable of specifically hybridizing to different polymorphic variants. According to the method, a nucleic acid of interest, e.g., a nucleic acid encompassing a polymorphic site, (which is typically amplified) is hybridized with the array and scanned. Hybridization and scanning are generally carried out according to standard methods. See, e.g., Published PCT Application Nos. WO 92/10092 and WO 95/11995, and U.S. Pat. No. 5,424,186. After hybridization and washing, the array is scanned to determine the position on the array to which the nucleic acid hybridizes. The hybridization data obtained from the scan is typically in the form of fluorescence intensities as a function of location on the array.

[0146] Arrays can include multiple detection blocks (i.e., multiple groups of probes designed for detection of particular polymorphisms). Such arrays can be used to analyze multiple different polymorphisms. Detection blocks can be grouped within a single array or in multiple, separate arrays so that varying conditions (e.g., conditions optimized for particular polymorphisms) can be used during the hybridization. For example, it can be desirable to provide for the detection of those polymorphisms that fall within G-C rich stretches of a genomic sequence, separately from those falling in A-T rich segments.

[0147] Additional description of use of oligonucleotide arrays for detection of polymorphisms can be found, for example, in U.S. Pat. Nos. 5,858,659 and 5,837,832. In addition, to oligonucleotide arrays, cDNA arrays can be used similarly in certain embodiments of the invention.

[0148] Other methods of nucleic acid analysis can be used to detect polymorphisms and/or polymorphic variants. Such methods include, e.g., direct manual sequencing (Church and Gilbert, Proc. Natl. Acad. ScL USA 81: 1991-1995, 1988; Sanger et al, Proc. Natl. Acad. ScL USA 74: 5463-5467, 1977; Beavis et al, U.S. Pat. No. 5,288,644); automated fluorescent sequencing; single- stranded conformation polymorphism assays (SSCP); clamped denaturing gel electrophoresis (CDGE); denaturing gradient gel electrophoresis (DGGE) (Sheffield et al., Proc. Natl. Acad. ScL USA 86: 232-236, 1991), mobility shift analysis (Orita et al., Proc. Natl. Acad. ScL USA 86: 2766-2770, 1989), restriction enzyme analysis (Flavell et al., Cell 15: 25, 1978; Geever et al, Proc. Natl. Acad. ScL USA 78: 5081, 1981); heteroduplex analysis; chemical mismatch cleavage (CMC) (Cotton et al, Proc. Natl. Acad. ScL USA 85: 4397-4401, 1985; RNase protection assays (Myers et al., Science 230: 1242, 1985); use of polypeptides that recognize nucleotide mismatches, e.g., E. coli mutS protein; allele- specific PCR.

[0149] In certain embodiments of the invention fluorescence polarization template- directed dye-terminator incorporation (FP-TDI) is used to determine which of multiple polymorphic variants of a polymorphism is present in a subject. This method is based on template-directed primer extension and detection by fluorescence polarization. According to this method, amplified genomic DNA containing a polymorphic site is incubated with oligonucleotide primers (designed to hybridize to the DNA template adjacent to the polymorphic site) in the presence of allele- specific dye-labeled dideoxyribonucleoside triphosphates and a commercially available modified Taq DNA polymerase. The primer is extended by the dye- terminator specific for the allele present on the template, increasing 10-fold the molecular weight of the fluorophore. At the end of the reaction, the fluorescence polarization of the two dye- terminators in the reaction mixture are analyzed directly without separation or purification. This homogeneous DNA diagnostic method has been shown to be highly sensitive and specific and is suitable for automated genotyping of large number of samples. (Chen et al., Genome Research 9: 492-498, 1999). Note that rather than involving use of allele- specific probes or primers, this method employs primers that terminate adjacent to a polymorphic site, so that extension of the primer by a single nucleotide results in incorporation of a nucleotide complementary to the polymorphic variant at the polymorphic site.

[0150] Real-time pyrophosphate DNA sequencing is yet another approach to detection of polymorphisms and polymorphic variants (Alderborn et al., Genome Research 10: 1249-1258, 2000). Additional methods include, for example, PCR amplification in combination with denaturing high performance liquid chromatography (dHPLC) (Underhill et al. , Genome Research 7: 996-1005, 1997).

[0151] In general, it will be of interest to determine the genotype of a subject with respect to both copies of the polymorphic site present in the genome. For example, the complete genotype can be characterized as -/-, as -/+, or as +/+, where a minus sign indicates the presence of the reference or wild type sequence at the polymorphic site, and the plus sign indicates the presence of a polymorphic variant other than the reference sequence. If multiple polymorphic variants exist at a site, this can be appropriately indicated by specifying which ones are present in the subject. Any of the detection means above can be used to determine the genotype of a subject with respect to one or both copies of the polymorphism present in the subject's genome. [0152] According to certain embodiments of the invention it is preferable to employ methods that can detect the presence of multiple polymorphic variants (e.g., polymorphic variants at a plurality of polymorphic sites) in parallel or substantially simultaneously. Oligonucleotide arrays represent one suitable means for doing so. Other methods, including methods in which reactions (e.g., amplification, hybridization) are performed in individual vessels, e.g., within individual wells of a multi-well plate or other vessel can also be performed so as to detect the presence of multiple polymorphic variants (e.g., polymorphic variants at a plurality of polymorphic sites) in parallel or substantially simultaneously according to certain embodiments of the invention.

[0153] The invention provides a database comprising a list of polymorphic sequences stored on a computer-readable medium, wherein the polymorphic sequences occur in a coding or noncoding portion of a gene set forth in Table 4 or fragment thereof, or in a genomic region linked to such a gene, or in a genomic region linked to such a gene, and wherein the list is largely or entirely limited to polymorphisms have been identified as useful in performing genetic diagnosis or prognosis of early age-onset lung cancer or susceptibility to early age-onset lung cancer, or for performing genetic studies of early age-onset lung cancer or susceptibility to early age-onset lung cancer.

METHODS AND REAGENTS FOR IDENTIFICATION OF EARLY AGE-ONSET LUNG CANCER SUSCEPTIBILITY LOCI AND FUNCTIONAL MUTATIONS

[0154] A systematic approach is provided to identifying additional early age-onset lung cancer susceptibility loci, polymorphisms useful in diagnosis of early age-onset lung cancer or susceptibility to early age-onset lung cancer, and to identifying functional mutations that cause or contribute to early age-onset lung cancer. The invention provides a variety of methods for the diagnosis or prognosis of early age-onset lung cancer. In particular, a method of detecting an increased susceptibility to early age-onset lung cancer in an individual is provided comprising analyzing a DNA sample from the individual for the presence of at least one single nucleotide polymorphism of a LELAl gene sequence, wherein the presence of the at least one single nucleotide polymorphism is indicative of an increased susceptibility to early age-onset lung cancer. In some methods, the at least one single nucleotide polymorphism is in the LELAl gene. If linkage or association exists, the polymorphism is useful in diagnosis or prognosis of early age-onset lung cancer or susceptibility to early age-onset lung cancer. Such polymorphisms can thus be located in or define early age-onset lung cancer susceptibility locus. The set of samples can comprise samples obtained from one or more families affected with lung cancer disease (e.g., early age-onset lung cancer) and can comprise both related and unrelated individuals. In other embodiments, the at least one single nucleotide polymorphism is, for example, in the LELAl gene. See Table 4.

[0155] The invention further provides a method of screening drug candidates to treat early age-onset lung cancer in a mammalian subject comprising: providing a cell or tissue from a patient with early age-onset lung cancer; treating the cell or tissue with a compound that increase LELAl gene expression or LELAl gene product, determining at least one single nucleotide polymorphism including, but not limited to, a single nucleotide polymorphism (SNP) in the LELAl gene in the cell or tissue; adding a drug candidate to the cell; and determining an effect of the drug candidate on an increased LELAl gene expression or LELAl gene product.

[0156] The methods can further comprise analyzing expression of the gene in normal subjects and in subjects affected with lung cancer, e.g., early age-onset lung cancer, which includes examining the mRNA abundance, size, and tissue expression pattern, examining the abundance, size, tissue expression pattern and/or activity of the encoded protein, and the like.

THERAPEUTIC APPLICATIONS

[0157] The LELAl polypeptide, or mimetic, analog or derivative thereof, identified by the methods as described herein can be used in a variety of methods of treatment of cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma. Activators of LELAl gene expression or LELAl polypeptide activity can be used in a variety of methods of treatment of cancer. Thus, the present invention provides compositions and methods for treating cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma. The composition includes a LELAl polypeptide, or mimetic, analog or derivative thereof, and a pharmaceutically acceptable carrier. The LELAl composition can be administered alone or in combination with other compositions. The composition further includes an activator of LELAl gene expression or LELAl polypeptide activity, and a pharmaceutically acceptable carrier. The LELAl activator composition can be administered alone or in combination with other therapeutic compositions.

[0158] A LELAl polypeptide, or mimetic, analog or derivative thereof, as described herein, can be used in methods for treating lung cancer or early age onset lung adenocarcinoma in a mammalian subject. This nature of LELAl polypeptide is of particular importance for the potential clinical usage of LELAl polypeptide as a factor inhibiting telomerase activity. LELAl polypeptide or mimetic, analog or derivative thereof, or activators of LELAl, thus has an unusual safety profile with minimum side effect as a survival molecule. It may therefore be used to treat a broad array of diseases related to neoplatic disease, including, but not limited to, cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma. LELAl polypeptide, or activators of LELAl, therefore offers a new and better therapeutic option for the treatment of disease.

[0159] Preferably, treatment using LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl gene expression or LELAl polypeptide activity, in an aspect of the present invention could either be by administering an effective amount of the LELAl polypeptide or mimetic, analog or derivative thereof, to the patient. Moreover, the polypeptide or peptidomimetic as provided herein can be used to reduce or eliminate lung cancer or early age onset lung adenocarcinoma.

PHARMACEUTICAL COMPOSITIONS

[0160] A LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl, useful in the present compositions and methods can be administered to a human patient per se, in the form of a stereoisomer, prodrug, pharmaceutically acceptable salt, hydrate, solvate, acid salt hydrate, N-oxide or isomorphic crystalline form thereof, or in the form of a pharmaceutical composition where the compound is mixed with suitable carriers or excipient(s) in a therapeutically effective amount, for example, to treat cancer, renal cell carcinoma, lung carcinoma, or early age onset lung adenocarcinoma.

[0161] "Therapeutically effective amount" refers to that amount of the therapeutic agent, LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl gene expression or LELAl polypeptide activity, sufficient to result in the amelioration of one or more symptoms of a disorder, e.g., renal cancer, lung cancer, or early age onset lung adenocarcinoma or prevent advancement of a disorder, cause regression of the disorder, or to enhance or improve the therapeutic effect(s) of another therapeutic agent. With respect to the treatment of lung cancer or early age onset lung adenocarcinoma, a therapeutically effective amount refers to the amount of a therapeutic agent sufficient to reduce or eliminate lung cancer or early age onset lung adenocarcinoma. Preferably, a therapeutically effective amount of a therapeutic agent reduces or eliminates lung cancer or early age onset lung adenocarcinoma, by at least 5%, preferably at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or at least 100%. "Therapeutic protocol" refers to a regimen for dosing and timing the administration of one or more therapeutic agents, such as a LELAl polypeptide, or mimetic, analog or derivative thereof or activators of LELAl.

[0162] Pharmaceutically acceptable carriers are determined in part by the particular composition being administered, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable formulations of pharmaceutical compositions for administering the antibody compositions (see, e.g., latest edition of Remington' s Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, incorporated herein by reference). The pharmaceutical compositions generally comprise a differentially expressed protein, LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl, in a form suitable for administration to a patient. The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.

TREATMENT REGIMES

[0163] Aspects of the invention provide pharmaceutical compositions comprising one or a combination of LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl, formulated together with a pharmaceutically acceptable carrier. Some compositions include a combination of multiple {e.g., two or more) LELAl polypeptide, or mimetic, analog or derivative thereof.

[0164] In prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition (i.e., lung cancer or early age onset lung adenocarcinoma) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. In therapeutic applications, compositions or medicants are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient immune response has been achieved. Typically, the immune response is monitored and repeated dosages are given if the immune response starts to wane.

EFFECTIVE DOSAGES

[0165] Effective doses of the LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl gene expression or LELAl polypeptide activity, for the treatment of lung cancer or early age onset lung adenocarcinoma, as described herein vary depending upon many different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Usually, the patient is a human but nonhuman mammals including transgenic mammals can also be treated. Treatment dosages need to be titrated to optimize safety and efficacy.

[0166] For administration with a LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl gene expression or LELAl polypeptide activity, the dosage ranges from about 0.0001 to 100 mg/kg, and more usually 0.01 to 5 mg/kg, of the host body weight. For example dosages can be 1 mg/kg body weight or 10 mg/kg body weight or within the range of 1-10 mg/kg. An exemplary treatment regime entails administration once per every two weeks or once a month or once every 3 to 6 months. In some methods, two or more LELAl polypeptides, or mimetic, analog or derivative thereof, or activators of LELAl, with different binding specificities are administered simultaneously, in which case the dosage of each LELAl polypeptide is usually administered on multiple occasions. Intervals between single dosages can be weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of LELAl polypeptide in the patient. In some methods, dosage is adjusted to achieve an concentration of 1-1000 μg/ml LELAl polypeptide and in some methods 25-300 μg/ml. Alternatively, LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl, can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the compound in the patient. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of lung cancer or early age onset lung adenocarcinoma. Thereafter, the patent can be administered a prophylactic regime.

[0167] Doses for a nucleic acid vector encoding LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl, range from about 10 ng to 1 g, 100 ng to 100 mg, 1 μg to 10 mg, or 30-300 μg DNA per patient. Doses for infectious viral vectors vary from 10-100, or more, virions per dose.

PRODRUGS

[0168] The present invention is also related to prodrugs of the agents obtained by the methods disclosed herein. Prodrugs are agents which are converted in vivo to active forms (see, e.g., R.B. Silverman, 1992, The Organic Chemistry of Drug Design and Drug Action, Academic Press, Chp. 8). Prodrugs can be used to alter the biodistribution {e.g., to allow agents which would not typically enter the reactive site of the protease) or the pharmacokinetics for a particular agent. For example, a carboxylic acid group, can be esterified, e.g., with a methyl group or an ethyl group to yield an ester. When the ester is administered to a subject, the ester is cleaved, enzymatically or non-enzymatically, reductively, oxidatively, or hydrolytically, to reveal the anionic group. An anionic group can be esterified with moieties (e.g., acyloxymethyl esters) which are cleaved to reveal an intermediate agent which subsequently decomposes to yield the active agent. The prodrug moieties may be metabolized in vivo by esterases or by other mechanisms to carboxylic acids.

[0169] Examples of prodrugs and their uses are well known in the art (see, e.g., Berge et al., "Pharmaceutical Salts", /. Pharm. ScL 66: 1-19, 1977). The prodrugs can be prepared in situ during the final isolation and purification of the agents, or by separately reacting the purified agent in its free acid form with a suitable derivatizing agent. Carboxylic acids can be converted into esters via treatment with an alcohol in the presence of a catalyst.

[0170] Examples of cleavable carboxylic acid prodrug moieties include substituted and unsubstituted, branched or unbranched lower alkyl ester moieties, (e.g., ethyl esters, propyl esters, butyl esters, pentyl esters, cyclopentyl esters, hexyl esters, cyclohexyl esters), lower alkenyl esters, dilower alkyl-amino lower-alkyl esters (e.g., dimethylaminoethyl ester), acylamino lower alkyl esters, acyloxy lower alkyl esters (e.g., pivaloyloxymethyl ester), aryl esters (phenyl ester), aryl-lower alkyl esters (e.g., benzyl ester), substituted (e.g., with methyl, halo, or methoxy substituents) aryl and aryl-lower alkyl esters, amides, lower-alkyl amides, dilower alkyl amides, and hydroxy amides.

ROUTES OF ADMINISTRATION

[0171] A LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl gene expression or LELAl polypeptide activity, for treatment or amelioration of lung cancer or early age onset lung adenocarcinoma can be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic as inhalants for LELAl polypeptide, or mimetic, analog or derivative thereof, preparations targeting lung cancer or early age onset lung adenocarcinoma in a variety of tissues, and/or therapeutic treatment. The most typical route of administration of an immunogenic agent is subcutaneous although other routes can be equally effective. The next most common route is intramuscular injection. This type of injection is most typically performed in the arm or leg muscles. Intramuscular injection or intravenous infusion are preferred for administration of antibody. In some methods, antibodies are administered as a sustained release composition or device, such as a Medipad™ device.

[0172] Agents of the invention can optionally be administered in combination with other agents that are at least partly effective in treating lung cancer or early age onset lung adenocarcinoma.

FORMULATION

[0173] A LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl, for the treatment of cancer, e.g., lung carcinoma or early age onset lung adenocarcinoma, can be administered as pharmaceutical compositions comprising an active therapeutic agent, i.e., and a variety of other pharmaceutically acceptable components. See latest edition of Remington's Pharmaceutical Science (Mack Publishing Company, Easton, Pa.). The preferred form depends on the intended mode of administration and therapeutic application. The compositions can also include, depending on the formulation desired, pharmaceutically- acceptable, non-toxic carriers or diluents, which are defined as vehicles commonly used to formulate pharmaceutical compositions for animal or human administration. The diluent is selected so as not to affect the biological activity of the combination. Examples of such diluents are distilled water, physiological phosphate-buffered saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, nonimmunogenic stabilizers and the like.

[0174] Pharmaceutical compositions can also include large, slowly metabolized macromolecules such as proteins, polysaccharides such as chitosan, polylactic acids, polyglycolic acids and copolymers (such as latex functionalized Sepharose™, agarose, cellulose, and the like), polymeric amino acids, amino acid copolymers, and lipid aggregates (such as oil droplets or liposomes). Additionally, these carriers can function as immuno stimulating agents (i.e., adjuvants).

[0175] For parenteral administration, compositions of aspects of the invention can be administered as injectable dosages of a solution or suspension of the substance in a physiologically acceptable diluent with a pharmaceutical carrier that can be a sterile liquid such as water oils, saline, glycerol, or ethanol. Additionally, auxiliary substances, such as wetting or emulsifying agents, surfactants, pH buffering substances and the like can be present in compositions. Other components of pharmaceutical compositions are those of petroleum, animal, vegetable, or synthetic origin, for example, peanut oil, soybean oil, and mineral oil. In general, glycols such as propylene glycol or polyethylene glycol are preferred liquid carriers, particularly for injectable solutions. Antibodies can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained release of the active ingredient. An exemplary composition comprises monoclonal antibody at 5 mg/mL, formulated in aqueous buffer consisting of 50 mM L-histidine, 150 mM NaCl, adjusted to pH 6.0 with HCl.

[0176] Typically, compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared. The preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997. The agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.

[0177] Additional formulations suitable for other modes of administration include oral, intranasal, and pulmonary formulations, suppositories, and transdermal applications.

[0178] For suppositories, binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%. Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%- 95% of active ingredient, preferably 25%-70%.

[0179] Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins. Glenn et al., Nature 391: 851, 1998. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.

[0180] Alternatively, transdermal delivery can be achieved using a skin patch or using transferosomes. Paul et al., Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al., Biochem. Biophys. Acta 1368: 201-15, 1998.

[0181] The pharmaceutical compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration. TOXICITY

[0182] Preferably, a therapeutically effective dose of LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl gene expression or LELAl polypeptide activity, described herein will provide therapeutic benefit without causing substantial toxicity.

[0183] Toxicity of the proteins described herein can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., by determining the LD50 (the dose lethal to 50% of the population) or the LD₁Oo (the dose lethal to 100% of the population). The dose ratio between toxic and therapeutic effect is the therapeutic index. The data obtained from these cell culture assays and animal studies can be used in formulating a dosage range that is not toxic for use in human. The dosage of the proteins described herein lies preferably within a range of circulating concentrations that include the effective dose with little or no toxicity. The dosage can vary within this range depending upon the dosage form employed and the route of administration utilized. 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., 1975, In: The Pharmacological Basis of Therapeutics, Ch. 1,

KITS

[0184] Also within the scope of the invention are kits comprising a LELAl polypeptide, or mimetic, analog or derivative thereof, or activators of LELAl gene expression or LELAl polypeptide activity, of aspects of the invention and instructions for use. The kit can further contain a least one additional reagent, or one or more additional human antibodies of aspects of the invention (e.g., a human antibody having a complementary activity which binds to an epitope in the antigen distinct from the first human antibody). Kits typically include a label indicating the intended use of the contents of the kit. The term label includes any writing, or recorded material supplied on or with the kit, or which otherwise accompanies the kit.

[0185] The invention will be further described with reference to the following examples; however, it is to be understood that the invention is not limited to such examples.

[0186] The following examples of specific aspects for carrying out the present invention are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way. EXEMPLARY ASPECTS

EXAMPLE 1

Loss of Heterozygosity (LOH) at Two Adjacent Microsatellite Markers on Chromosome

3p21 Is Associated with Early Age Onset Lung Cancer

[0187] Lung cancer is one of the most common cancers and the leading cause of cancer death worldwide. In general, most patients develop lung cancer between the ages 60-70 but about 10 percent are diagnosed at extreme ages, either before age 50 or after age 80. Cigarette smoking is a major risk factor contributing to lung cancer. However, only a small portion of smokers ever develop the disease and some affected patients are non-smokers. These facts indicate that genetic background of the individual plays a role in lung cancer development. Indeed, studies have suggested the presence of a genetic factor contributing to lung cancer onset.

[0188] In an effort to identify the genetic component contributing to lung cancer, we examined five commonly altered chromosome regions for genetic changes in lung adenocarcinomas from patients who were diagnosed of the disease before age 50 or at age 80 or older. See Table 1, microsatellite markers used for initial LOH study. We observed that loss of heterozygosity (LOH) at two adjacent microsatellite markers on chromosome 3p21 occurred much more frequently in tumors from patients with early age onset lung cancer compared to those with late age onset (Tablel). One of the two microsatellite markers, D3S1581 lies within the minimal telomerase (hTERT) suppressor locus previously identified in a renal cell carcinoma cell (RCC23) line having a somatically transferred chromosome 3 (RCC23 +Ch.3). Since telomerase activation is a critical event associated with neoplastic transformation, we hypothesized that the gene associated with early age lung cancer onset might normally function to suppress hTERT expression. Overlapping the early onset associated region in primary lung adenocarcinoma with the telomerase suppression locus identified in RCC cells with or without chromosome 3 narrowed the candidate region to approximately 2Mb encoding approximately 70 known or predicted genes on chromosome 3p21.31.

Table 1. 38 microsatellite markers on chromosome 3p used for the initial LOH study

D17S1828 D17S1876 D17S796 D17S1791

D19S814 D19S883 D19S565 D19S424 D19S894 D19S884

D3S2338 D3S3659 D3S1266 D3S1609 D3S1277 D3S3521 D3S3685 D3S1581 D3S1289 D3S1300 D3S1600 D3S3697 D3S1566

D3S1279 D3S3668 D3S3715 D3S3592 D3S1580 D3S1601 D3S2748 D3S1265

D9S1751

D9S1748

D9S157

D9S1684

D9S169

D9S161

D9S1817

[0189] Table 2 shows genetic loss of chromosome 3p21 region in primary lung cancers. Markers used are as indicated. The frequency of allelic loss were calculated for each age group and tested for significance of difference in frequencies using T test.

[0190] To identify the candidate gene, we designed 65 quantitative RT-PCR probe sets to examine the gene expression changes for 58 analyzable genes within this region. We searched for candidate genes that had reduced expression in lung tumors with LOH in this region, absent or lowly expressed in the telomerase positive parental RCC23 cells but high in the telomerase negative RCC23 cells transferred with the wild type Chromosome 3. See list of genes and complete expression results in Table 3. This approach led us to identify two hypothetical proteins MGC35097 and LOC339834 (Figure IA). MGC35097 mRNA was about 60% on average in primary lung tumors having LOH in the region compared to MGC35097 mRNA levels in the tumors without LOH. In RCC lines, MGC35097 expression was reduced by 9.5 fold in telomerase positive RCC23 cells compared to the telomerase negative RCC23+Ch. 3 cells. The message for LOC339834 (CCDC36 gene) was about 40% in lung tumors with LOH compared to those without LOH in the region. In RCC lines, CCDC36 was undetectable in telomerase positive RCC23 parental cells but easily detected in telomerase negative RCC23Ch. 3 cells. To determine the potential relevance of these two candidate genes, we used in situ hybridization to evaluate their expression in normal lung epithelium (Figure IB). Hybridization using antisense rRNA probes for each gene showed that only CCDC36, but not MGC35097, is present in normal lung epithelium cells even though they are both expressed in the muscle fibroblast cells underlining the vessels in the lung. We named CCDC36 (LOC339834) gene LELAl for "loss in early onset lung adenocarcinoma 1."

[0191] Figure IA shows expression of LELAl gene in lung cancers with LOH and RCC +Ch. 3 cells with no tTERT expression. Left side represents the average fold difference in gene expression between lung cancer samples with LOH and without LOH. The right side shows the fold difference in gene expression between RCC23 cell line ( with no hTERT ) and RCC23+3 cell line ( with hTERT expression). The gene expression was measured by RT-PCR. A total of 36 tumor and 18 non-tumor lung samples were included in the gene expression analysis.

[0192] Figure IB shows in situ hybridization demonstrating the expression of genes, vWF (positive control), LELAl, and MGC35097 (adjacent to LELAl and serving as negative control). Tissues used are indicated at the bottom of the figures.

[0193] Table 4 shows unique changes in LELAl gene associated with early age onset lung cancer cases.

EXAMPLE 2

Methylation State of CpG Island in LELAl Gene

[0194] Structurally, LELAl is a highly conserved gene predicted to encode a 583 amino acid protein based on the human genome sequencing database. It contains a coil-coiled domain and shares structure homology to the yeast myosin like protein (MLPl) known to participate in telomerase maintenance (Figure 2A). A 1024bp long CpG island is located 5' of the protein translation initiation codon (ATG) and is completely methylated in 12 of 15 lung cancer cell lines that do not have detectable LELAl but invariably carried an unmethylated allele in the 3 cell lines expressing the gene. See Figure 2B and Table 5. In primary tissues, LELAl is methylated in two third of the lung tumors tested but rarely methylated or at much lower levels in adjacent normal lung tissues (Figure 2C). Of the primary tumors that have been tested, 44/65 (68%) of all tumors tested were methylated. In patients who were 50 or younger, 15/22 were methylated at LELAl promoter while 7/12 were methylated at the same region in those who had lung cancer after age 79 (p = 0.05).

[0195] Figure 2 shows frequent LELAl promoter methylation in primary lung tumors and lung cancer cell lines. A. Genomic structure of LELAl; translated exons are shown in pink, untranslatwd exons in black, coil-coil domain in green and CpG island in red; B. Fold difference between methylated and unmethylated alleles (Methylation) in different cell lines by MBSE method. C. Comparison of methylations between normal lung and primary lung tumors in all ages, younger than 50 years and age at diagnosis more then 79 years as indicated on the x-axis. Numbers indicate the number of samples in each group, p-value was assigned by t-test.

[0196] To assess whether the methylation state of the CpG island directly affects the expression of LELAl, we treated lung cancer cells lines with or without endogenous LELAl expression with the demethylating agent, 5'-d-azacytosine (Figure 3). This treatment resulted in demethylation of the CpG sites 5' of LELAl and re-expression of the gene in all cell lines that are normally methylated at LELA promoter site but only moderately (H 1299) or not at all (H2023 and H2122) in cell lines already expressing the gene. We also tested hTERT message level in cells treated with 5'azacytosine and observed that hTERT message levels were reduced in five of six cell lines re-expressed LELAl. In contrast, hTERT levels increased or remain essentially unchanged in the three cell lines already expressing LELAl prior to demethylation treatment. Since 5'-azacytosine affects the methylation state of many genes in the cells; it is possible that the reduction of hTERT expression was not directly related to LELAl activation. To exclude this possibility, we transfected two independent constructs carrying the full length LELAl cDNA into lung cancer cell lines H1299, A549 and RCC23 cells. Gene expression changes in LELAl and hTERT were monitored by quantitative RT-PCR at 24hr post transfection or at confluence after selection under the selective marker. In all three cell lines, exogenous expression of LELAl resulted in a consistent reduction of hTERT in the cells and the reduced hTERT activity (Figure 4). These results jointly establishes LELAl as a gene whose expression leads to the reduction of hTERT and supports our hypothesis that it contributes to lung cancer early onset via dysregulation of hTERT.

[0197] Figure 3 show demethylation of LELAl led to re-expression LELAl and reduced expression of hTERT in lung cancer cell lines. LELAl and hTERT expression were determined after 5-azacytosine (demethylation) treatment, by RT-PCR. The status of LELAl expression and LELAl promoter methylation in the untreated cells are shown in the lower panels underneath each line. [0198] Figure 4 shows LELAl overexpression suppresses hTERT. A and B. Gene expression of LELAl and hTERT in H 1299 and RCC23 cell lines transfected with LELAl (Pl or P2) and MOCK plasmids at 24hr. after transfection (dl) and at confluence (dc). Gene expression was assigned by RT-PCR and shown as fold difference between the gene expression in the sample the those measured in cells transfectionted with the plasmid containing GFP and collected at day 1 (dl). C and D. TRAP5 assay for H1299 and RCC23 cell lines transfection samples as in A and B. Images show the serial dilution ( 1:2, 1:4 or both) of the cell lysates for the cells collected at day of confluency (dc) after selection with G418. See Example 4 for methods for TRAP Assay to assess the function of hTERT in cells expressing LELAl.

EXAMPLE 3

Genetic Basis Associated With LELAl Gene in Lung Cancer Onset

[0199] To assess the genetic changes associated with LELAl in lung cancer onset, we sequenced each of the 11 exons for LELAl in 60 lung adenocarcinomas from patients with different age of cancer onset. This screen identified seven unique somatic and germline changes and five common single nucleotide changes. See Table 4. Based on these tested samples involving 120 chromosomes, the distribution of the five common SNPs were not statistically different among the young and old age group. However, five of the seven unique changes have only been found in tumors of patients who had lung adenocarcinoma before the age of 50.

[0200] Loss of chromosome 3p21 and its surrounding regions is one of the most common and early event in non-small cell lung cancers. It is also involved in many other cancers such as kidney cancers. Many of the genes in this region have been extensively sequence analyzed and some shown to have tumor suppressive effect when overexpressed. However, the identification of a tumor suppressor gene whose inactivation directly contributes to lung cancer development remains elusive. Our results suggest that instead of directly affecting cell growth, LELAl affects the likelihood of cancer via modulation of hTERT activity.

Table 2. LOH at 3p21 with age of lung cancer onset (19 pairs <50, 20 pairs >79)

LOH % p-val

Marker on3p21 Position Mb Age<50 Age>79 <50 vs.>70 d3s1478 46.4 53% 33% 0.24 d3s1581 48.5 59% 20% 0.03 d3s3615 50.5 82% 30% 0.02 d3s3688 51.8 33% 10% 0.19 d3s1578 53.6 50% 24% 0.11 d3s1289 54.4 41% 20% 0.20 d3s3719 55.4 67% 45% 0.28 d3s1295 57.9 69% 38% 0.12

Table 3

Order on RCC23+3/RCC23 LOH/noLOH

3p21 ABI_Probe Neg. Control Gene Fold Fold

Hs00264218_s1 Undetermined UCN2 3.521315514 2.77272354

HsOOI 64310_m1 Undetermined COL7A1 2.0851 15788 1.19365067

HsOOI 63415_m1 Undetermined UQCRC1 1.197446749 1.02933284

Hs00224747_m1 Undetermined SLC26A6 1.962500471 1.00576211

Hs00370470_m1 Undetermined SLC26A6 1.717649889 1.1 1819475

Hs00609783_m1 Undetermined CELSR3 1.814322599 1.67143938

Hs00356575_m1 Undetermined NCKIPSD 0.851817338 1.32681356

HsOOI 79614_m1 Undetermined IHPK2 2.045969304 1.17527608

Hs00383065_m1 Undetermined NULL 1.468679907 1.978062610 HsOOI 77760_m1 Undetermined PRKAR2A 2.15727037 0.92075011 Hs00386383_m1 Undetermined SLC25A20 1.634167086 0.696133462 HsOOI 71838_m1 Undetermined ARIH2 1.944181623 1.129166063 Hs00214665_m1 Undetermined PH 0.721332084 1.504395424 Hs00250592_g1 Undetermined WDR6 1.289744684 1.001634435 Hs00382925_g1 Undetermined DKFZP564J0123 1.793865843 1.009542596 Hs00404252_g1 Undetermined DKFZP564J0123 2.498597473 1.145439687 HsOOI 68418_m1 Undetermined IMPDH2 0.419792996 1.01625618 Hs00214646_m1 Undetermined FLJ20259 1.905104981 0.923262539 HsOOI 92530_m1 Undetermined QARS 0.823723755 1.085271020 HsOOI 58642_m1 Undetermined LAMB2 2.764974164 1.064978111 Hs00293902_m1 Undetermined MGC35097 9.464658814 0.605368712 HS0069831 1_m1 Undetermined LOC339834 2896.309376 0.444676953 Hs00737144_m1 Undetermined FLJ43654 1.150366445 0.478333424 Hs00234300_m1 Undetermined USP4 1.414094466 0.947163955 Hs00608203_m1 Undetermined USP4 1.757030823 0.827337976 Hs00357608_m1 Undetermined RHOA 1.85454079 1.313083667 HsOOI 85328_m1 Undetermined TCTA 1.359303395 1.792033518 HsOOI 66628_m1 Undetermined AMT 1.431167469 1.186787499 Hs00276884_m1 Undetermined NICN1 1.044466702 0.875707080 HsOOI 89308_m1 Undetermined DAG 1 1.771198002 0.8399911 HsOOI 85720_m1 Undetermined BSN 1.203873662 3.522841762 HsOOI 55756_m1 Undetermined APEH 0.992954145 1.048797263 Hs00360684_m1 Undetermined MST1 0.644588341 1.443851874 Hs00222902_m1 Undetermined RNF123 0.989238298 1.274473825 Hs00362722_g1 Undetermined GMPPB 2.417032714 0.868727596 Hs00384812_m1 Undetermined IHPK1 1.767072828 1.023063457 Hs00293736_m1 Undetermined LOC3891 19 2.900871721 0.750279868 HsOOI 63295_m1 Undetermined UBE1 L 1.186025476 0.7851 14239 HsOOI 83394_m1 Undetermined TRIP 3.067355752 1.856591980 Hs00225234_m1 Undetermined MGC8407 0.207673063 10.95563331 Hs00234013_m1 Undetermined MST1 R 0.476060225 0.962178132 Hs00260615_m1 Undetermined MGC13272 4.144802905 1.422015093 HsOOI 72915_m1 Undetermined RBM6 2.0132551 17 1.178907414 HsOOI 72952_m1 Undetermined RBM5 1.94092181 1.074422245 HsOOI 88273_m1 Undetermined SEMA3F 0.680599502 0.784996526 HsOOI 81 100_m1 Undetermined GNAT1 ND ND 7 HsOOI 99177_m1 Undetermined SLC38A3 0.8684857 4.72113688 HsOOI 79998_m1 Undetermined GNAI2 3.218976401 0.804502029 HsOOI 90328_m1 Undetermined SEMA3B 1.440502336 1.042786640 Hs00414920_m1 Undetermined FLJ38608 ND 0.43273163 HsOOI 98888_m1 Undetermined IFRD2 1.51 1619988 1.12894433

Hs00273329_s1 Undetermined NAT6 1.950954998 1.45816292

Hs00201046_m1 Undetermined HYAL1 0.331996745 0.47798385

Hs00537920_g1 Undetermined HYAL1 0.201706223 0.80449082

Hs00738390_m1 Undetermined HYAL1 0.253839678 1.01263068

HsOOI 86839_m1 Undetermined HYAL2 ND 0.88013839

HsOOI 86841_m1 Undetermined HYAL2 2.297061527 0.80524266

Hs00200725_m1 Undetermined TUSC2 1.689931 185 1.22757565

Hs00200394_m1 Undetermined RASSF1 2.889348048 1.04827416

Hs00210720_m1 Undetermined ZMYND10 1.30282156 1.30177722

HsOOI 98012_m1 Undetermined TUSC4 1.477889085 1.03596345

HsOOI 99737_m1 Undetermined CYB561 D2 1.944999791 1.23896834

HsOOI 83656_m1 Undetermined PL6 1.364404577 1.01 171759

HsOOI 95772_m1 Undetermined CACNA2D2 ND 1.18866933

Hs002751 11_m1 Undetermined NULL ND ND

Hs00737806_m1 Undetermined LOC51 161 6.684093896 0.92357771

Hs00275076_m1 Undetermined HEMK1 1.710627929 1.15314593

S18 4342379 1

Table 5

MSB E Assay Summary

Ceil lines tested; M/U ratio

H358 4.316268487

A549 3.031048623

H460 5.922546012

H522 2.949394939

H 1299 0.255208333

RCC23 8.234782609

RCC23#3 0.568485915

Rev 10.8072445

Primary c&

Paϊrwfe© comparison; 28 paired samples (Mayo)

T/N

M/U ratio 1 ratio

Tumor 21/ 28 pairs

Normal 2/ 28 pairs

Non-pairwϊse

Tumor (Mayo) 29/ 41 samples 70% Tumor (Harris) 4/ 7 samples 57% Normal (mayo) 2/ 28 samples 7%

LELA expression and promoter methylation status in lung cancer cell lines

Supplementary Table3

Gene expressioon change after demethylation treatment.

LELA-1 hTERT expression after expression

LELA1 CpCa Mediation ■5-d-Aza after §-d*A&a

2023 1 allele unmeth no change no change

2122 1 allele unmeth no change no change

H 1299 1 allele unmeth no change Increased

H358 methylated

H322 methylated increased decreased

H522 methylated increased decreased

HOP92 methylated increased no change Table 5 (Cont.)

HOP62 methylated

EKVX methylated increased Increased

H23 methylated increased decfgased

H226 methylated increased cteereasee!

A549 methylated increased cteefeased

H520 methylated

H460 methylated

H2170 methylated

1568 methylated increased decreased

EXAMPLE 4 Materials and Methods

[0201] Samples: Sixty Lung Adenocarcinoma tissue samples and paired normal lung samples were obtained from Mayo Clinic. They consisted of three age groups: < 50, 57-79, and > 79. Samples were selected to have similar clinical features (similar in stage, grade, and survival) and smoking history (similar intensity and year smoked) between the age groups.

[0202] LOH study: We performed initial LOΗ study for chromosomes 3p, 9p, 17p and 19p using 38 polymorphic microsatellite markers. We further used 8 markers around chromosome region 3p21 for more precise LOΗ mapping. All LOΗ studies were performed on ABI 3730x1 DNA Analyser using standard ABI protocol and ABI markers (See the list of 8 markers in Supplemental materials).

[0203] RT-PCR of3p21 region: We performed quantitative PCR study using custom TaqMan® Low Density Array (micro fluidic card) with 68 probes for 61 genes belonging to the 2 Mb region of interest, probe for TERT and control probe for S18 via standard protocol (See the list of the probes in Supplemental materials).

[0204] Sequencing: We sequenced all exons of LELAl (CCDC36) on ABI 3730x1 DNA Analyser using standard ABI protocol and ABI designed primer ( See the list of the primers in Supplemental materials).

[0205] Methylation study: The methylation status of LELAl CpG island was determined using methylation single base extension (MSBE) method. Specifically, 1 ug of genomic DNA was used for the sodium bisulfite treatment. Modified genomic DNA was amplified with LLCl gene-specific primers corresponding to the sodium bisulfite-treated sequences. The amplified products were then purified with a QIAquickTM PCR Purification Kit (Qiagen, Valencia, CA). For the SBE reaction, SNaPshotTM Ready Reaction Mix (Applied Biosystems) was used. The single base-extended samples were treated with 1 unit of shrimp alkaline phosphatase (Roche) and resulting dephosphorylated samples were analyzed on an ABI Prism® (Applied Biosystems) automatic sequencer, to determine the peak heights for the extended products corresponding to either methylated or unmethylated DNA.

[0206] The degree of methylation was analyzed based on the ratio of the methylated and unmethylated (M/U) peaks between the paired tumor and normal tissues or the average of MIU ratios between the 2 groups using r-test.

[0207] To determine whether CpG island methylation played a role in LELAl expression, lung cancer cell lines (A549, Η1299 Hop92, 2023 2122 1568) were treated with culture medium containing a demethylating agent, 5-aza-deoxycytidine (final 2 mM; Sigma, St. Louis, MO), for 4 days with a media change at day two. The expression and DNA promoter methylation status of LELAi gene was determined by Q-PCR and MSBE, respectively.

[0208] Cell lines: H1299 cell line was purchased from ATCC and propagated in RPMI1640 media with 10%FBS and 1% P/S. RCC23 cell line was a courtesy of Dr Izumi Horikawa (NIH/NCI) and were propagated in RPMI1640 media with 10%FBS, 4mm L- Glutamine and 1% P/S.

[0209] Plasmids: LELAl plasmid 1 was purchased from GeneCopoeia (Germantown, MD), that produced it by cloning the PCR product into the EX_T7584-M03 vector. LELAl plasmid 2 was produced in the lab by cloning the PCR product into pcDNA3 vector from Invitrogen ( Carlbad, CA)using the standard manufacture protocol. The empty pcDNA3 vector was used as MOCK control. GFP containing pMaxGFP plasmid from AMAXA (Gaithersburg, MD) was used as day 1 control.

[0210] Transfection: The day before transfection the cells were seeded in T25 flasks to be 60-80 % confluent the next day. The next day the cell were transfected with lμg of plasmid using Effectine Transfection Reagent from Qiagen ( Valencia, CA) and the standard manufacture's protocol. Cells were collected at two time points - dayl after transfection and at confluency after G418 selection. H1299 was selected for 1 week with 1.2 mg/ml G418, RCC23 was selected for 3 weeks with 0.35 mg/ml G418.

[0211] RT-PCR for transfected cells: Total RNA was extracted from collected cells using Trizol reagent, DNase trated by RNase-Free DNase Set (Quigen) and purified with Quigen RNeasy Mini Kit. lμg of total RNA was converted to cDNA usind High Capacity cDNA Reverse Transcription Kit from Applied Biosystems ( Foster City, CA). RT-PCR primers for LELAl (Hs00698311_ml), hTERT (Hs00162669_ml) and control GUSB (Hu GUSB 2Ox) were purchased from Applied Biosystems. Quantitative RT-PCR was performed in duplicates or triplicates using ABI 7500.

[0212] Telomeric repeat amplification protocol with fluorescent probe (TRAP5): Cells were trysinized, counted and collected by centrifugation. TRAP5 assay was performed as described in Greider & Blackburn. Cell 43: 405, 1985; Kim at el. Science 266: 2011, 1994; Kim and Wu Nuc. Acid. Rec. 25: 2595, 1997. See the protocol in Supplementary Materials. Telomeric repeat amplification protocol with fluorescent probe (TRAP5)

• Telomerase extraction:

1. Wash cells with PBS or Hanks once in an Eppendorf tube (1.5 ml), and spin at 6,000 rpm for 5 min. at 4 ⁰C to pellet cells.

2. Re-suspend cells with ice-cold CHAPS lysis buffer (50-200 μl per 10⁶ cells), incubate on ice for 30 min., and centrifuge at 14,000 RPM for 20 min. at 4 ⁰C.

3. Collect the supernatant, take 6 μl (1 and 5 μl each) for telomerase assay, freeze the rest on dry ice immediately, and store tubes in a -80 ⁰C freezer (stable for 6-12 month).

• Telomere synthesis by telomerase: (For 10 μl reaction)

4. In a 0.5 ml Eppendorf tube or PCR tube, add 5 μl CHAPS cell lysate, 2.5 μl DEPC treated water, 2.5 μl reaction mix 1, and incubate at 30 ⁰C (Room temperature) for 40 min. Take 2 μl for amplification and store the rest in a -20 ⁰C freezer (stable for 1-2 weeks)

Reaction mix 1 contains following reagents: (Using premixed 2X)

1.0 μl 1O X Telomerase reaction buffer

1.0 μl 20 mM dATP, dGTP, and dTTP each (GE Bioscience)

0.5 μl 20 pmole/μl Ts-TAMRA (Operon, PAGE purified)

• Amplification of telomere repeats by PCR: (For 20 μl reaction)

5. Add the following reagents (using 20 μl as example) into PCR tube (make a master mix of the following reagents add 18 μl of mix to each well)

2 μl 10 X PCR reaction buffer,

1.2 μl 25 mM MgCl₂

2 μl 2 mM dNTP,

0.2 μl 20 μM of Ts- TAMRA,

0.1 μl 20 μM of ACX and NT,

0.2 μl Taq polymerase,

1 μl 0.1-5 amole/μl TSNT (need readjust for every new series of experiment),

11.2 μl pure water.

THEN, add 2 μl of telomere products (from step 4), CHAPS lysis buffer as negative and R8 as positive controls. Mix them well before put in the PCR.

6. PCR condition and cycle number: 94 ⁰C for 30 seconds, then 94 ⁰C for 20 seconds and 60 ⁰C for 45 seconds for 30-33 cycles (approx. 35 minutes).

• PAGE analysis:

7. Make 12% polyacrylamide non-denaturing gel: For 10 gels, mix 24 ml 40% acrylamide solution stock, 8 ml 10 X TBE, 1 ml 10% Ammonium persulfate, and 47 ml distal water, filter through a 0.45 μm filter, degas under vacuum for 5 minutes. Add 20 μl of TEMED, and pour the gel (polymerize at RT for 15-30 minutes). 8. Add 4 μl of 6 X loading buffer (without dye or minimum dye) to each sample, mix and take 8 μl for loading onto the acrylamide gel. Set the electrophoresis condition with constant volts at 300 for 35 min or at 200 volts for 60 min. Stop the running when the second dye (Xylene) reaches 5 mm to the bottom of the gel.

9. Collecting the image using Typhoon:

Fluorescent with Emission filter at 580 BP Cy3 TAMRA... , PMT at 600, Laser at Green 532, Sensitivity at Normal.

• Controls:

10. Use a tumor cell line (293) as a positive control, and R8 as other control. RNase A treatment of cell extract or single primer as the negative controls. Incubate cell extract in 50 ng/μl of RNase A at 23 ⁰C for 20 min. before step 5.

Reagents:

1. CHAPS Lysis buffer (TRAP 2) 50 ml. Stored at -20 ⁰C.

Stock Amount

10 mM Tris-HCl (pH 7.5) 1 M 500 μl

1 mM Mg Cl₂ 1 M 50 μl

1 mM EGTA 0.5 M 100 μl

5 μM β-mercaptoethanol 14.4 M 17.4 μl

0.5% CHAPS 0.25 g

10% Glycerol 100 % 5 ml

Water 44 ml

* Add 1 μl of 0.1 M PMSF (Phenylmethylsulfonyl Fluoride) per 1 ml CHAPS lysis buffer right before use.

2. Telomerase Reaction Buffer (10X): 10 ml (stored at -20 ⁰C)

10 X concentration Stock Amount

50O mM Tris-OAc (pH 8.5) I M 5 ml

1O mM MgCl₂ I M 100 μl

50O mM KOAc 5 M I mI

5O mM β-mercaptoethanol 14.4 M 35 μl

1O mM Spermidine I M 100 μl

DEPC treated water 3765 μl

3. Oligos: (PAGE or HPLC purified, Genosys)

Ts-TAMRA 5'- AAT CCG TCG AGC AGA GTT-S' (18 mer), 20 pmole/μl

ACX 5'- GCG CGG CTT ACC CTT ACC CTT ACC CTA ACC-3' (30 mer), 20 pmole/μl.

NT 5'-ATC GCT TCT CGG CCT TTT-S' (18 mer), 20 pmole/μl TSNT 5'-AAT CCG TCG AGC AGA GTT AAA AGG CCG AGA AGC GAT-3' (36 mer), 0.1 amole/μl.

R8 5'-AAT CCG TCG AGC AGA GTT [GGT TAG]₈-3' (66 mer), 0.1-1 amole/μl. 4. Chemicals:

CHAPS (Cholamidopropyl dimeththylammonio-propanesulfonate, Calbiochem, Cat #220201).

EGTA (Sigma, E-3889). β-mercaptoethanol (Sigma, M 3148)

Phenylmethylsulfonyl Fluoride (Sigma, P-7626)

Spermidine (Sigma, S-0266).

40% acrylamide solution (BioRad 161-0154)

SYBR green I (Molecular Probes: Cat S7563)

See, for example, Greider & Blackburn. Cell 43: 405, 1985; Kim at el. Science 266: 2011, 1994; Kim and Wu Nuc. Acid. Rec. 25: 2595, 1997.

[0213] All publications and patent applications cited in this specification are herein incorporated by reference in their entirety for all purposes as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference for all purposes.

[0214] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

Claims

What is Claimed:

1. A method for treating cancer in a mammalian subject which comprises administering a LELAl polypeptide or a mimetic, analog, or derivative thereof, in an amount effective to reduce or eliminate cancer in the mammalian subject or to prevent its occurrence or recurrence.

2. The method of claim 1 wherein the subject has a loss-of-function or reduced function of a LELAl gene or LELAl polypeptide.

3. The method of claim 2 wherein the subject has a loss-of-function or reduced function mutation in the LELAl gene.

4. The method of claim 1 wherein the LELAl polypeptide, or a mimetic, analog, or derivative thereof, is a peptide, peptidomimetic, or a small chemical molecule.

5. The method of claim 1 wherein administering the LELAl polypeptide or a mimetic, analog, or derivative thereof modulates LELAl gene activity or LELAl polypeptide activity in the mammalian subject.

6. The method of claim 5 wherein administering the LELAl polypeptide or a mimetic, analog, or derivative thereof activates LELAl gene activity or LELAl polypeptide activity in the mammalian subject.

7. The method of claim 1, wherein the cancer is lung carcinoma or renal cell carcinoma.

8. The method of claim 7 wherein the cancer is an early age onset lung adenocarcinoma.

9. The method of claim 1 wherein the mammalian subject is a human subject.

10. A method for treating cancer in a mammalian subject which comprises administering a recombinant nucleic acid molecule encoding a LELAl polypeptide or a mimetic, analog, or derivative thereof, in an amount effective to reduce or eliminate cancer in the mammalian subject or to prevent its occurrence or recurrence.

11. The method of claim 10 wherein the subject has a loss-of- function or reduced function of a LELAl gene or LELAl polypeptide.

12. The method of claim 11 wherein the subject has a loss-of-function or reduced function mutation in the LELAl gene.

13. The method of claim 10 wherein administering the nucleic acid molecule modulates LELAl gene activity or LELAl polypeptide activity in the mammalian subject.

14. The method of claim 13 wherein administering the nucleic acid molecule activates LELAl gene activity or LELAl polypeptide activity in the mammalian subject.

15. The method of claim 10, wherein the cancer is lung carcinoma or renal cell carcinoma.

16. The method of claim 15, wherein the cancer is an early age onset lung adenocarcinoma.

17. The method of claim 10 wherein the mammalian subject is a human subject.

18. The method of claim 10 wherein the nucleic acid is in a recombinant viral vector.

19. The method of claim 18 wherein the viral vector is an adenoviral vector, an adeno-associated viral vector, a lentiviral vector, an Epstein Barr viral vector, a papovaviral vector, a vaccinia viral vector, a herpes simplex viral vector, a baculoviral vector, or a bacterial plasmid vector.

20. The method of claim 10 wherein the nucleic acid is administered to the mammalian subject as naked nucleic acid, or nucleic acid complexed with polycations, lipids, charged liposomes, liposome-polycation-peptide complex, liposome-polycation complex, liposome-ligands, viral protein microparticles or viral protein nanoparticles.

21. The method of claim 10 wherein the nucleic acid is transfected into a cell ex vivo and the transfected cell is administered to the mammalian subject.

22. A method for identifying a compound which reduces proliferation of lung cancer cells which comprises contacting the test compound with a cell-based assay system comprising a cell capable of expressing LELAl and telomerase, activating telomerase gene expression or telomerase activity in the cell-based assay system, and detecting an effect of the test compound on activation of LELAl gene expression or LELAl polypeptide activity and on inhibition of telomerase activity, effectiveness of the test compound in the assay being indicative of reduced proliferation of lung cancer cells.

23. A method for diagnosing a risk factor for early age onset lung cancer in a mammalian subject which comprises removing cells or tissue from the subject, measuring production of LELAl gene product or LELAl polypeptide activity in the cells or tissue, and detecting reduced function or loss-of-function of the LELAl gene product or LELAl polypeptide activity in the cell or tissue of the mammalian subject indicating an increased risk for early age onset lung cancer.

24. The method of claim 23 which further comprises measuring production of telomerase gene product or telomerase activity in the cells or tissue, and detecting an increase in telomerase gene product or telomerase activity in the cell or tissue.

25. The method of claim 23 which further comprises detecting a single nucleotide polymorphism in the LELAl gene of the mammalian subject.

26. A method for diagnosing and treating early age onset lung cancer in a mammalian subject which comprises removing cells or tissue from the subject, identifying one or more risk alleles related to LELAl gene or LELAl polypeptide activity, and correlating the one or more risk alleles with reduced function or loss-of-function of the LELAl gene activity or LELAl polypeptide activity in the cell or tissue of the subject indicating an increased risk for early age onset lung cancer, and modulating a gene activity or a gene product activity in the subject in an amount effective to reduce or eliminate early age onset lung cancer in the subject.

27. The method of claim 26 wherein the gene activity or gene product activity is a gene of the risk allele.

28. The method of claim 27 wherein the gene activity or gene product activity is from a LELAl gene.

29. The method of claim 28 further comprising activating a LELAl gene activity or LELAl polypeptide activity in an amount effective to reduce or eliminate early age onset lung cancer in the subject.

30. The method of claim 29 further comprising administering the LELAl polypeptide or a mimetic, analog, or derivative thereof activates LELAl gene activity or LELAl polypeptide activity in the subject.

31. The method of claim 26 wherein the risk allele a single nucleotide polymorphism in the LELAl gene of the mammalian subject.

32. A method for diagnosing early age onset lung cancer in a mammalian subject which comprises removing cells or tissue from the subject, identifying one or more risk alleles related to LELAl gene or LELAl polypeptide activity, and correlating the one or more risk alleles with reduced function or loss-of-function of the LELAl gene activity or LELAl polypeptide activity in the cell or tissue of the subject indicating an increased risk for early age onset lung cancer.

33. The method of claim 32 wherein the gene activity or gene product activity is a gene of the risk allele.

34. The method of claim 33 wherein the gene activity or gene product activity is from a LELAl gene.

35. The method of claim 32 wherein the risk allele a single nucleotide polymorphism in the LELAl gene of the mammalian subject.

36. A method for diagnosing a LELAl gene loss-of-function-induced disorder or a genetic predisposition therefore in a mammalian subject, which comprises determining the presence of a mutated LELAl protein or a nucleic acid encoding a mutated LELAl protein in a cell sample, protein sample or nucleic acid sample obtained from the mammalian subject, wherein the presence of the mutated LELAl protein or nucleic acid is indicative of a LELAl gene loss-of-function-induced disorder or a genetic predisposition therefore.

37. The method of claim 36 wherein the LELAl gene loss-of-function-induced disorder is cancer.

38. The method of claim 37 wherein the cancer is renal cell carcinoma, lung carcinoma or early age onset lung carcinoma.

39. The method of claim 36 wherein the nucleic acid is a single nucleotide polymorphism in the LELAl gene of the mammalian subject.

40. A transgenic non-human animal comprising a heterologous nucleic acid, wherein the nucleic acid comprises a loss -of -function allele of a LELAl gene, and the animal exhibits a phenotype, relative to a wild-type phenotype, comprising susceptibility to early age onset lung cancer.

41. A method for associating a lung cancer with early age onset lung cancer, which comprises determining an amount or activity of a LELAl gene product in a lung cancer sample, and comparing the amount or activity of the LELAl gene product to an amount of a LELAl gene product determined in a non-pathological lung tissue sample, wherein a decreased amount of the LELAl gene product or activity in the lung cancer sample relative to the non-pathological lung tissue sample associates with the early onset lung cancer.

42. The method of claim 41 further comprising the step of using the determined comparative gene product information to formulate a diagnosis.

43. The method of claim 41 further comprising the step of using the determined comparative gene product information to formulate a prognosis.

44. The method of claim 41 further comprising the step of using the determined comparative gene product information to formulate a treatment plan.

45. The method of claim 41 wherein the gene product is mRNA.

46. The method of claim 41 wherein the gene product is protein.

47. The method of claim 41 wherein the lung tissue sample which is non-pathological comprises normal lung small airway epithelial cells.

48. The method of claim 41 wherein the lung tissue sample which is non-pathological comprises normal bronchial/tracheal epithelial cells.

49. The method of claim 41 wherein the amount of the gene product is determined using a microarray.

50. The method of claim 49 wherein cRNA is hybridized to probes on the microarray to determine the amount of the gene product.

51. The method of claim 41 which further comprises detecting a single nucleotide polymorphism in the LELAl gene of the mammalian subject.