Title:

Method of treating a cancer

Document Type and Number:

United States Patent 7405287

Abstract:

The present invention provides isolated nucleic acids encoding delta DNA methyltransferase 3B molecules that are involved in the treatment and prevention of cancers such as, but not limited to, lung cancer. The delta DNA methyltransferase 3B molecules of the present invention are found to play a critical role in promoter-specific methylation of tumor suppressor genes. The DNA methyltransferase 3B molecules of the present invention are provided as therapeutic targets for identifying inhibitors of DNA methyltransferase. Such inhibitors are contemplated for the treatment and/or prevention of cancers. In particular embodiments, the present invention involves the treatment and prevention of a non-small cell lung cancer.

Inventors:

Mao, Li (Houston, TX, US)
Wang, Jie (Beijing, CN)
Wang, Luo (Pearland, TX, US)

Plaque It!

BACKGROUND OF THE INVENTION

The government owns rights in the present invention pursuant to grant number DAMD17-01-1-01689-1 from the Department of Defense and grant numbers CA 68437 and CA 91844 from the National Cancer Institute.

FIELD OF THE INVENTION

The present invention relates generally to the fields of molecular biology, cancer biology and cancer therapy. More particularly, it concerns identification of therapeutic targets of DNA methyltransferase 3B molecules, such as, for example, delta DNA methyltransferase 3B variants, for treating a cancer including, but not limited to, lung cancer, and/or selecting a patient for treatment based on expression of the molecules.

DESCRIPTION OF RELATED ART

In the United States, lung cancer leads all other cancers in both incidence and mortality rate (Khuri et al., 2001). Lung cancer is the primary cause of cancer death among both men and women in the United States and worldwide.

Non-small cell lung cancer (NSCLC) constitutes 80% of all primary lung cancers, which are the leading cause of cancer-related death in both men and women in the United States (Greenlee et al., 2001). Despite advances in the treatment of the disease over the past two decades, the prognosis of patients with NSCLC has improved only modestly, with the 5-year overall survival rate increasing from 11% in the 1970s to 15% in the late 1990s (Greenlee et al., 2000). Patients with early-stage NSCLC generally have a better survival than those with advanced-stage tumors. For example, patients with stage I NSCLC are expected to have an approximate 60% 5-year overall survival rate after surgical resection of their primary tumors, while those with stage IIIA disease have an estimated 25% 5-year overall survival rate after surgery followed by radiation with or without chemotherapy.

Biological features of NSCLC are determined by underlying molecular alterations of the tumors, including inactivation of tumor suppressor genes (Niklinksi et al., 2001; Fong et al., 2003; Hirsch et al., 2001). Besides mutations and deletions of genes, it is now clear that de novo promoter hypermethylation is a common mechanism to inactivate tumor suppressor genes (Zochbauer-Muller et al., 2002; Foracs et al., 2001; Merlo et al., 1995). The p16 ^INK4atumor suppressor gene located on 9p21 encodes a cyclin-dependent kinase inhibitor important for G1 cell cycle arrest (Zhang et al., 1999; Koh et al., 1995). Promoter hypermethylation of this gene has been frequently observed early in lung carcinogenesis, including in individuals exposed to tobacco carcinogens who do not exhibit evidence of cancer (Kim et al., 2001; Toyooka et al., 2001; Soria et al., 2002).

In contrast to p16 ^INK4a, which is inactivated early in lung carcinogenesis (Soria et al., 2002; Belinsky et al., 1998), hypermethylation of another tumor suppressor gene, RASSF1A, occurs relatively late (Belinsky et al., 2002; Dammann et al., 2000; Pfeifer et al., 2002; Burbee et al., 2001), suggesting RASSF1A might be important in NSCLC progression. The RASSF1A tumor suppressor gene is located at 3p21, a region frequently deleted in NSCLC (Brauch et al. 1987). RASSF1A has been shown to bind to the Ras-GTP binding protein Norel, consistent with its role as a negative effector of Ras oncoprotein (Ortiz-Vegas et al., 2002).

It is that believed that DNA methytransferases play a critical role in the hypermethylation status of these tumor suppressor genes. Thus, DNA methyltransferases provide novel therapeutic targets in the treatment of cancers.

SUMMARY OF THE INVENTION

The present invention regards a new class of DNMT3B isoforms, referred to herein as deltaDNMT3Bs, ΔDNMT3Bs or DDNMT3Bs, that play an important role in tumorigenesis. The expression of these isoforms is initiated through a novel promoter, in specific embodiments. In particular aspects of the invention, the abnormal expression of the isoforms correlates with promoter methylation of tumor suppressor genes, thereby leading to at least partial inhibition of their expression. In other aspects, inactivation of the isoforms restores expression of a tumor suppressor gene, such as the exemplary RASSF1A gene, through demethylation of relevant hypermethylated promoters. In specific embodiments of the invention, the isoforms provide therapeutic targets of cancer that comprise inactivation of tumor suppressor genes, such as RASSF1A, through inactivation of promoter hypermethylation. Thus, the present invention provides a novel mechanism for providing cancer therapy separate from other methylation preventing agents by utilizing deltaDNMT3B in a novel promoter-specific demethylation.

In one aspect of the invention, deltaDNMT3B2/4 participates in regulation of RASSF1A promoter-specific methylation. The rapid demethylation of the promoter, activation of the gene expression, and prolonged inheritable effect of a single siRNA or antisense treatment, for example, provide significant implications in cancer therapy.

As described herein, at least seven transcription variants of DDNMT3B were identified as the result of alternative pre-mRNA processing. DDNMT3B variants but not DNMT3Bs were the predominant transcripts in both non-small cell lung cancer (NSCLC) cell lines and primary tumors. A striking association was observed between expression of DDNMT3B4, for example, and promoter methylation of RASSF1A, but a weaker association was observed with p16INK4A promoter methylation. A specific knockout of DDNMT3B4/2 by RNA interference or antisense approach results in a rapid and prolonged demethylation of RASSF1A promoter and reactivation of RASSF1A gene expression but not p16INK4A in NSCLC cell lines. Therefore, in specific aspects of the invention, DDNMT3Bs, such as DDNMT3B4/2, play an important role in maintenance of promoter-specific methylation of RASSF1A and shed light in understanding mechanisms of tissue-specific methylation. In specific embodiments, the isoform may bind directly to a promoter with specific DNA structure to prevent methylated cytosine from being demethylated or alternatively to prevent the structure from being repaired by DNA repair mechanisms. In further specific embodiments, the isoform interacts with specific chromotin structures of the promoters and forms complexes to protect the modified DNA structure.

Although in particular aspects the present invention provides methods and compositions of cancer therapy for cancers involving inactivation of tumor suppressor promoters through hypermethylation, such as the exemplary RASSF1A, the invention may be useful for any cancer, including lung cancer, brain cancer, prostate cancer, colon cancer, breast cancer, ovarian cancer, pancreatic cancer, liver cancer, spleen cancer, cervical cancer, melanoma, leukemia, head and neck cancer, esophageal cancer, thyroid cancer, testicular cancer, and so on. In a specific embodiment of the invention, the present invention is particularly useful for non-small cell lung cancer.

In an embodiment of the present invention, there is an isolated DNA methyltransferase-3B variant nucleic acid sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. In a specific embodiment, the nucleic acid sequence is comprised in an expression vector, for example, a viral or plasmid vector. The viral vector may be an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes viral vector, polyoma viral vector or hepatitis B viral vector. The expression vector may be comprised in a non-viral delivery system. The non-viral delivery system may comprise one or more lipids. In a specific embodiment, the nucleic acid sequence is operatively linked to a promoter.

In an additional embodiment of the present invention, there is an isolated nucleic acid sequence encoding a DNA methyltransferase-3B variant having the amino acid sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.

In a further embodiment, there is a host cell comprising a nucleic acid sequence encoding a DNA methyltransferase-3B variant according to the present invention, wherein the nucleic acid sequence may be comprised in a vector.

In an additional embodiment of the present invention, there is an isolated DNA methyltransferase-3B variant having the amino acid sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, or SEQ ID NO:28.

In another embodiment of the present invention, there is a method of identifying an inhibitor of delta DNA methyltransferase 3B (dDNMT3B) activity, comprising: (a) providing in a cell or cell-free system a DNA methyltransferase 3B polypeptide corresponding to the sequence of SEQ. ID. NO:15, SEQ. ID. NO:16, SEQ. ID. NO:17, SEQ. ID. NO:18, SEQ. ID. NO:19, SEQ. ID. NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ. ID. NO:23, SEQ. ID. NO:24, SEQ. ID. NO:25, SEQ. ID. NO:26, SEQ. ID. NO:27, or SEQ ID NO:28; (b) contacting the DNA methyltransferase with a candidate substance (c) selecting an inhibitor of the DNA methyltransferase by assessing the effect of said candidate substance on DNA methyltransferase activity and (d) manufacturing the inhibitor.

In particular aspects of the invention, the candidate substance is a protein, a nucleic acid, a small molecule, an organo-pharmaceutical, or a combination thereof. The protein may be an antibody that binds immunologically to a dDNMT3B variant. The providing step may be further defined as providing a nucleic acid that encodes the DNA methyltransferase 3B polypeptide. In specific embodiments, the candidate substance is a nucleic acid, such as, for example, an antisense molecule or an siRNA molecule. In a particular embodiment, assessing comprises assaying for dDNMT3B activity, such as, for example, assessing the effect of the candidate substance on dDNMT3B activity comprises assaying for DNA methylation, which may be further defined as assaying for DNA methylation of a promoter. In a specific embodiment, the assessing step comprises polymerase chain reaction, a restriction endonuclease-based assay, or both.

In another embodiment of the present invention, there is a method of inhibiting the growth of a cancer cell comprising administering to the cell an effective amount of an inhibitor manufactured according to the present invention. In a specific embodiment, the administering of the inhibitor is further defined as inhibiting the enzymatic activity of DNA methyltransferase. The cell may be of any kind, but in specific embodiments the cancer cell is in a human. The cancer cell may be a cancer cell of the bladder, blood, bone, bone marrow, brain, breast, colon, esophagus, gastrointestine, gums, head, kidney, liver, lung, nasopharynx, neck, ovary, prostate, skin, stomach, testis, tongue, or uterus.

In particular embodiments, the cancer cell is a lung cancer cell, such as a malignant cancer cell or a metastatic lung cancer cell. In another specific embodiment, the lung cancer is a non-small cell lung cancer, a small cell lung cancer or a rare lung cancer cell. In another specific embodiment, the non-small cell lung cancer is a squamous cell carcinoma, an adenocarcinoma or a large cell carcinoma. The rare lung cancer cell may be an adenoid cystic carcinoma, a mesothelioma, a hamartoma, a lymphoma or a sarcoma. The lung cancer may be a carcinoid tumor. In a specific embodiment, the method further comprises inducing apoptosis in a cancer cell.

In another embodiment of the present invention, there is a nucleic acid sequence that is antisense to at least a portion of a DNA methyltransferase 3B nucleic acid of SEQ. ID. NO:1, SEQ. ID. NO:2, SEQ. ID. NO:3, SEQ. ID. NO:4, SEQ. ID. NO:5, SEQ. ID. NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, or SEQ ID NO:14. The nucleic acid sequence may be further defined as an siRNA sequence. The nucleic acid sequence may be further defined as being antisense to an exon/intron junction of a DNA methyltransferase 3B nucleic acid. In a specific embodiment, there is a DNA sequence encoding the antisense nucleic acid sequence.

In a specific embodiment, there is a pharmaceutical composition comprising a nucleic acid sequence of the present invention, in a pharmaceutically acceptable vehicle.

In an additional embodiment of the present invention, there is a method of inhibiting the growth of a cancer cell comprising providing to the cell an effective amount of a pharmaceutical composition in accordance with the present invention. In specific embodiments, the cancer cell is in a human cancer patient.

In specific aspects of the invention, there is selecting of a patient for treatment based on the expression of molecules of the invention. This is because in certain embodiments only tumors with abnormal expression of these molecules will respond to a therapy, such as an inhibitor, and therefore selection of patients based on the expression of the molecule is needed and may be considered part of treatment.

In certain aspects of the invention, small molecules are developed to target, for example exon-exon junctions and/or structures formed by these junctions to inhibitor the molecules.

Embodiments discussed with respect to one embodiment or example of the invention may be employed or implemented with respect to any other embodiment of the invention.

The use of the word “a” or “an” when used in conjunction with the term “comprising” in the claims and/or the specification may mean “one,” but it is also consistent with the meaning of “one or more,” “at least one,” and “one or more than one.” Some embodiments of the invention may consist of or consist essentially of one or more elements, method steps, and/or methods of the invention. It is contemplated that any method or composition described herein can be implemented with respect to any other method or composition described herein.

Other objects, features and advantages of the present invention will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating specific embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and are included to further demonstrate certain aspects of the present invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of specific embodiments presented herein.

FIGS. 1A-1B. Examples of promoter methylation status measured using MSP. FIG. 1 A—PCR products of methylated or unmethylated p16INK4a promoter from primary NSCLC and corresponding normal lung tissues. FIG. 1 B—PCR products of methylated or unmethylated RASSF1A promoter from primary NSCLC and corresponding normal lung tissues. Molecular weight markers are listed on left side Neg indicates negative controls using unmethylated DNA Pos indicates positive controls using methylated DNA and methylation-specific primer sets Ts indicate primary tumors Ns indicate corresponding normal lung tissues; U indicates unmethylated promoter and M indicates methylated promoter.

FIGS. 2A-2F. Association between the p16INK4a promoter methylation status (FIGS. 2A, 2 B, and 2 C) or RASSF1A promoter methylation status (FIGS. 2D, 2 E, and 2 F) and overall, disease-specific, and disease-free survival. 0 indicates groups without methylation of promoter 1 indicates groups with methylation of promoter. E/N indicates number of events/total number in each group.

FIG. 3. Analysis of the effect of p16 ^INK4aand RASSF1A promoter methylation on patients' survival in patients with stage IIIA tumors.

FIG. 4. Association between the p16INK4a promoter methylation status (A, B, and C) or RASSF1A promoter methylation status (D, E, and F) and overall, disease-specific, and disease-free survival. - indicates groups without promoter methylation (number of events/total in group [E/N]: 12/28 for A 9/28 for B, 12/28 for C 6/17 for D 5/17 for E and 9/17 for F) ••• indicates groups with methylation of the p16INK4a or the RASSF1A promoter (E/N: 16/23 for A 13/23 for B 16/23 for C 14/21 for D 13/21 for E 14/21 for F) - - - indicates groups with methylation of both promoters (E/N: 13/19 for A 13/19 for B 14/19 for C 11/11 for D-F).

FIGS. 5A-5B. Expression of DNMT3B6. FIG. 5 A—Expression of DNMT3B by RT-PCR using different 5′ primers located at exon 2 (E1), exon 4 (E3), and exon 6 (E5) of DNMT3B. FIG. 5 B—Primer extension assay showing expression initiation sites of DNMT3B6.

FIGS. 6A-6B. Promoter activity of DNMT3B6 detected by luciferase assay. FIG. 6 A—Schematic representation of reagents utilized in the assay. FIG. 6 B—Effect of C/T transition polymorphism (C46359T) on the DNMT3B6 promoter activity. FIG. 6 B—Effect of T-C transition polymorphism on promoter activity of DNMT3B6.

FIGS. 7A-7B. Identification of ΔDNMT3B variants highly expressed in lung cancer. FIG. 7 A—Relative expression levels and pattern in NSCLC cell lines and paired primary lung cancers. FIG. 7 B—Structural scheme of novel ΔDNMT3B isoforms.

FIGS. 8A-8D. Alternative or aberrant splicing variants of ΔDNMT3B subfamily. FIG. 8 A—Location of the primers used to amplify individual ΔDNMT3B variants in this study. FIG. 8 B—Expression patterns of ΔDNMT3B variants in NSCLC cell lines 1-7 represent ΔDNMT3B1-7, respectively. FIG. 8 C—Expression patterns of DNMT3Bs, with more proximal exons corresponding to ΔDNMT3B1-4 and ΔDNMT3B6. FIG. 8 D—Multiplex PCR using primer sets for DNMT3B1 and ΔDNMT3B1 with different ratios in concentration (concentration of DNMT3B1 primer set was serially diluted from 1-9 and serially increased from 1-9 1, DNMT3B1 primer set alone 9, ΔDNMT3B1 primer set alone 5, equal concentrations for both primer sets). The upper band represents the DNMT3B1 product and the lower band represents the ΔDNMT3B1 product. The lower panel shows relative intensity of the product bands.

FIGS. 9A-9E. Effects of ΔDNMT3B4/2 knockout on H1299 cells. FIG. 9 A—Expression of ΔDNMT3B1, ΔDNMT3B2, and ΔDNMT3B4 at different time points after treatment measured by RT-PCR. FIG. 9 B—Promoter methylation status of p16INK4a and RASSF1A at different time points after treatment measured by MSP. FIG. 9 C—RASSF1A gene expression status at different time points after treatment measured by RT-PCR. For A, B, and C, M indicates size marker 1, treated with medium alone 2, treated with lipofectamine alone 3, treated with lipofectamine plus 40 nM GAPDH-specific siRNA 4, treated with lipofectamine plus 40 nM scramble siRNA 5, treated with lipofectamine plus 10 nM ΔDNMT3B4/2-specific siRNA 6, treated with 20 nM ΔDNMT3B4/2-specific siRNA 7, treated with 40 nM ΔDNMT3B4/2-specific siRNA 8, treated with 40 nM ΔDNMT3B4/2 antisense RNA −, negative control +, positive control. FIG. 9 D—DNMT1 protein level in cells treated with or without ΔDNMT3B4/2-specific siRNA at the 48-h time point measured by Western blot analysis. The open circles indicate unmethylated cytosine residuals, and the solid circles indicate methylated cytosine residuals in the CpG sites. Each line represents DNA from a single clone. FIG. 9 E—Methylation status of individual CpG sites in a RASSF1A promoter region from cells treated with or without ΔDNMT3B4/2-specific siRNA.

FIG. 10A-10B. Growth inhibition by siRNA to ΔDNMT3B4/2. FIG. 10 A—Cell indexes (measured every 30 min) reflecting the cell number and the area of cell attachment to the plastic surface using ACEA RT-CES System. FIG. 10 B—Cell cycle distribution measured by flow cytometry. (A) treated with medium (B) treated with lipofectamine alone (C) treated with lipofectamine plus 40 nM GAPDH-specific siRNA (D) Treated with lipofectamine plus 40 nM scramble siRNA (E) treated with lipofectamine plus 10 nM ΔDNMT3B4/2-specific siRNA; (F) treated with 20 nM ΔDNMT3B4/2-specific siRNA (G) treated with 40 nM ΔDNMT3B4/2-specific siRNA (H), treated with 40 nM ΔDNMT3B4/2 antisense RNA.

FIG. 11. Expression of Rare ΔDNMT3B5 (E), ΔDNMT3B6 (F), and ΔDNMT3B7 (G) and clinical outcome in NSCLC.

FIG. 12. Expresion of ΔDNMT3B4 in bronchial brush cell.

FIG. 13. Antibody recognition of ΔDNMT3B variant proteins in lysate of H460 NSCLC cell line.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

I. The Present Invention

The present invention overcomes the deficiencies of the current cancer therapies involving demethylating agents. Currently tested demethylating agents in cancer therapy are non-specific (therefore toxic), have low efficiency (only partial demethylation can be achieved), and are transient (DNA is methylated quickly after drug withdrawal), for example. Thus, studies to understand functions of individual DNA methyltransferases and variants thereof in the control of promoter specific methylation may lead to development of novel strategies for cancer therapy.

In specific aspects, the present invention relates to the identification of a novel DNA methyltransferase 3B (DNMT3B) subfamily, delta DNA methyltransferase 3B (ΔDNMT3B, DDNMT3B, or dDNMT3B), whose expression is initiated through a novel promoter. The present invention provides seven transcription variants of ΔDNMT3B that were identified as the result of alternative pre-mRNA processing.

The association between the promoter methylation status and tumorigenesis was examined, and it was determined that expression of ΔDNMT3B is a critical factor in promoter methylation of tumor suppressor genes, such as RASSF1A, in primary tumors. This indicates that inhibitors targeting delta DNA methyltransferase variants of the present invention may be useful to remove promoter demethylation, subsequently allowing expression of tumor suppressors.

The strong correlation between expression of a particular DNA methyltransferase variant and methylation of a specific gene promoter in primary tumors has never been reported previously. Knockout of an exemplary DDNMT3B variant resulted in demethylation of a specific promoter and activation of its gene expression, and this supports the critical role of the variant in control of the gene-specific promoter methylation. The complete demethylation of the promoter, such as in a 12 hour period, for example, indicates a novel, replication-independent mechanism regulating methylation in CpG sites, which provides information concerning gene-specific promoter methylation and replication-independent DNA demethylation.

The present invention provides at least nucleic acid and polypeptide or peptide compositions of the dDNMT3B subfamily and novel DNA methyltransferase 3B inhibitors, specifically delta DNA methyltransferase 3B (dDNMT3B) inhibitors, as therapeutic agents for treating or preventing cancers. Inhibition of DNMT3B may be of any suitable kind, including agents that inhibit at least partially one or more of the following: transcription, post-transcriptional process, translation, post-translational process, and/or protein activity and/or half-life, for example.

II. DNA Methyltransferase 3B (DNMT3B)

DNA methylation plays an essential role in normal development of a mammalian embryo by regulating gene transcription through genomic imprinting, X chromosome inactivation, and genomic stability (Jaenisch, 1997; Jones and Gonzalgo, 1997; Robertson and Wolfee, 2000, Surani, 1998). It is believed that DNA methylation patterns in somatic cells are established during gametogenesis and early embryonic development via consecutive waves of demethylation and de novo methylation (Monk et al., 1987).

DNMT3 consists of DNMT3A and DNMT3B and has been shown to be the major de novo DNA methyltransferases that preferentially methylate the cytosine in CpG sites (Okano et al., 1998; Li and Jaenisch, 2000). Human DNMT3B is highly homologous to the mouse gene and comprises 24 exons spanning about 47-kb of genomic DNA (GeneID: 1789 and 13436; GenBank Accession No. AL035071 (SEQ ID NO:64)). Two alternative 5′ exons of DNMT3B have been reported. However, both of these are believed to result in the same full-length DNMT3B protein (DNMT3B1 and DNMT3B2) (Robertson et al., 1999). Three additional transcriptional variants (DNMT3B3-6) resulting from alternative splicing have also been reported (Robertson et al., 1999). Some of the variants lacking the DNA methyltransferase activity compete with variants with the enzyme activity resulting in DNA hypomethylation (Saito et al., 2002), suggesting a complex role of the DNMT3B variants.

Increased expression of DNMT3B has been frequently observed in human cancer cell lines and primary tumors compared to most of the normal tissues except testis, pancreas, thyroid, and bone marrow (Robertson et al., 1999; Saito et al., 2002; Oue et al., 2001). Although the expression level of DNMT3B was found to be higher in cancer cell lines and primary tumor tissues, most of the studies did not find a strong association between the expression level of DNMT3B and promoter methylation status of tumor suppressor genes (Oue et al., 2001; Yakushiji et al., 2003; Sato et al., 2002), suggesting the presence of a more complex mechanism in regulating methylation of these promoters. In fact, only a small number of CpG-rich promoters are methylated in normal adult tissues or tumor tissues and these methylated promoters are different in different tissues, a phenomenon termed as “tissue-specific methylation” (Reik et al., 2001; Cedar, 1988). The methylation in CpG-rich promoter regions results in transcriptional silencing of corresponding genes, a major mechanism to inactivate tumor suppressor genes in tumorigenesis (Baylin et al., 2002).

Because the expression of DNMT3B might be highly regulated in the cell cycle, it was believed that the increased expression observed in tumors might be merely a reflection of an increased proliferation status (Robertson et al., 2000). Several recent studies further underscore this notion by demonstrating that the maintenance of methylated promoters of tumor suppressor genes could only be effectively disrupted when both DNMT3B and DNMT1 genes were knocked out while a single knockout of either DNMT3B or DNMT1 had minimal effects (Rhee et al., 2002; Rhee et al., 2000; Leu et al., 2003). However, these studies did not address potential effects of individual variants of DNMT3B. A dominant-negative effect of DNMT3b4, which lacks methyltransferase enzymatic motifs, in competing with DNMT3b3 has been suggested and resulted in DNA hypomethylation on pericentromeric satellite regions (Saito et al., 2002).

During tumorigenesis, de novo DNA methylation occurs in certain promoters, particularly tumor suppressor genes (Jones and Gonzalgo, 1997; Robertson and Wolffe, 2000). Global analysis of promoter methylation in different tumors indicates a number of promoters, including genes unlikely critical in tumorigenesis, are methylated. However, the patterns in terms of which genes and the total number of genes vary depending on the tumors (Esteller et al., 2003), suggesting that de novo promoter methylation occurring in tumorigenesis is a complex biologic operation. Weisenberger et al. (2004) recently studied several DNMT3B variants for their role in methylation of selected sequences and found that certain DNMT3B variants, despite the lack of a catalytically active domain, may still be biologically important in controlling methylation of certain sequence structures, although such variants alone may be not sufficient for the control.

The nucleic acid sequence of the seven exemplary ΔDNMT3B variants of the invention is provided respectively in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. The corresponding encoded amino acid sequences for SEQ ID NOS:1-7 are provided respectively in SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, and SEQ ID NO:21. In specific embodiments, exemplary ΔDNMT3B variants lacking the C-terminal enzymatic domains are provided respectively in SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14. The corresponding encoded amino acid sequences for SEQ ID NOS:8-14 are provided respectively in SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, SEQ ID NO:26, SEQ ID NO:27, and SEQ ID NO:28.

III. Nucleic Acids Encoding DNA Methyltransferase 3B Molecules

In particular embodiments, the present invention provides isolated nucleic acid sequences encoding DNA methyltransferase 3B variants, and more particularly, delta DNA methyltransferase 3B (dDNMT3B) inhibitors such as antisense or siRNA molecules, for treating or preventing a cancer. In further particular embodiments, the present invention provides isolated nucleic acid sequences of DNA methyltransferase-3B variants comprising SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14, for example. The term “comprises SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14” means that the nucleic acid sequence substantially corresponds to a portion of SEQ ID NO:1 to SEQ ID NO:7. In some embodiments, the present invention employs a nucleic acid sequence that is antisense to at least a portion of the coding sequence of a DNA methyltransferase 3B polypeptide, and these nucleic acids correspond to the sequence of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

The term “nucleic acid” generally refers to at least one molecule or strand of DNA, RNA or a derivative or mimic thereof, comprising at least one nucleotide base, such as, for example, a naturally occurring purine or pyrimidine base found in DNA (e.g., adenine “A,” guanine “G,” thymine “T,” and cytosine “C”) or RNA (e.g., A, G, uracil “U,” and C). The term “nucleic acid” encompasses the terms “oligonucleotide” and “polynucleotide.” These definitions generally refer to at least one single-stranded molecule, but in specific embodiments will also encompass at least one additional strand that is partially, substantially or fully complementary to the at least one single-stranded molecule. Thus, a nucleic acid may encompass at least one double-stranded molecule or at least one triple-stranded molecule that comprises one or more complementary strand(s) or “complement(s)” of a particular sequence comprising a strand of the molecule. An “isolated nucleic acid” as contemplated in the present invention may comprise transcribed nucleic acid(s), regulatory sequences, coding sequences, or the like, isolated substantially away from other such sequences, such as other naturally occurring nucleic acid molecules, regulatory sequences, polypeptide or peptide encoding sequences, etc.

Nucleic acids according to the present invention may comprise an entire DNA methyltransferase 3B polynucleotide, or any fragment or variant of DNA methyltransferase 3B as set forth herein. A nucleic acid of the present invention may be derived from genomic DNA, i.e., cloned directly from the genome of a particular organism. It is contemplated that the nucleic acids of the present invention may comprise complementary DNA (cDNA). The term “cDNA” is intended to refer to DNA prepared using messenger RNA (mRNA) as template. The advantage of using a cDNA, as opposed to genomic DNA or DNA polymerized from a genomic, non- or partially-processed RNA template, is that the cDNA primarily contains coding sequences of the corresponding protein. There may be times when the full or partial genomic sequence is preferred, such as where the non-coding regions are required for optimal expression or where non-coding regions such as introns are to be targeted in an antisense strategy.

It also is contemplated that a polynucleotide of a given DNA methyltransferase 3B variant may be represented by natural or synthetic variants that have slightly different nucleic acid sequences but, nonetheless, encode the same or homologous protein (Table 1). As used in this application, the term “polynucleotide” refers to a nucleic acid molecule that has been isolated free of total cellular nucleic acid. In exemplary embodiments, the invention concerns a nucleic acid sequence essentially as set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14.

TABLE 1

Amino Acids	Codons


Alanine	Ala	A	GCA	GCC	GCG	GCU
Cysteine	Cys	C	UGC	UGU
Aspartic acid	Asp	D	GAC	GAU
Glutamic acid	Glu	E	GAA	GAG
Phenylalanine	Phe	F	UUC	UUU
Glycine	Gly	G	GGA	GGC	GGG	GGU
Histidine	His	H	CAC	CAU
Isoleucine	Ile	I	AUA	AUC	AUU
Lysine	Lys	K	AAA	AAG
Leucine	Leu	L	UUA	UUG	CUA	CUC	CUG	CUU
Methionine	Met	M	AUG
Asparagine	Asn	N	AAC	AAU
Proline	Pro	P	CCA	CCC	CCG	CCU
Glutamine	Gln	Q	CAA	CAG
Arginine	Arg	R	AGA	AGG	CGA	CGC	CGG	CGU
Serine	Ser	S	AGC	AGU	UCA	UCC	UCG	UCU
Threonine	Thr	T	ACA	ACC	ACG	ACU
Valine	Val	V	GUA	GUC	GUG	GUU
Tryptophan	Trp	W	UGG
Tyrosine	Tyr	Y	UAC	UAU

Allowing for the degeneracy of the genetic code, sequences that have at least about 80%, preferably at least about 90% and most preferably about 95% of nucleotides that are identical to the nucleotides of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 are contemplated. Sequences that are essentially the same as those set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 may also be functionally defined as sequences that are capable of hybridizing to a nucleic acid sequence containing the complement of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 under standard conditions. The term “functionally equivalent codon” is used herein to refer to codons that encode the same amino acid, such as the six codons for arginine or serine (Table 1), and also refers to codons that encode biologically equivalent amino acids, as discussed herein.

Naturally, the present invention also encompasses nucleic acid sequences that are complementary, or essentially complementary, to the sequences set forth herein, for example, in SEQ ID NO:1. Nucleic acid sequences that are “complementary” are those that are capable of base-pairing according to the standard Watson-Crick complementarity rules. As used herein, the terms “complementary sequences” and “essentially complementary sequences” means nucleic acid sequences that are substantially complementary to, as may be assessed by the same nucleotide comparison set forth above, or are able to hybridize to a nucleic acid segment of one or more sequences set forth herein, for example SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:1, SEQ ID NO:12, SEQ ID NO:13, and SEQ ID NO:14 under relatively stringent conditions such as those described herein. Such sequences may encode an entire DNA methyltransferase 3B molecule or functional or non-functional fragments thereof.

The hybridizing sequences may be short oligonucleotides. Sequences of 17 bases long should occur only once in the human genome and, therefore, suffice to specify a unique target sequence. Although shorter oligomers are easier to make and increase in vivo accessibility, numerous other factors are involved in determining the specificity of hybridization. Both binding affinity and sequence specificity of an oligonucleotide to its complementary target increases with increasing length. It is contemplated that exemplary oligonucleotides of about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 70, 80 or more base pairs will be used, although longer polynucleotides are contemplated. Such oligonucleotides will find use, for example, as probes in Southern and Northern blots and as primers in amplification reactions.

Suitable hybridization conditions will be well known to those of skill in the art. In certain applications, for example, substitution of amino acids by site-directed mutagenesis, it is appreciated that lower stringency conditions are required. Under these conditions, hybridization may occur even though the sequences of the probe and the target strand are not perfectly complementary, but are mismatched at one or more positions. Conditions may be rendered less stringent by increasing salt concentration and decreasing temperature. For example, a medium stringency condition could be provided by about 0.1 to 0.25 M NaCl at temperatures of about 37° C. to about 55° C., while a low stringency condition could be provided by about 0.15 M to about 0.9 M salt, at temperatures ranging from about 20° C. to about 55° C. Thus, hybridization conditions can be readily manipulated, and thus will generally be a method of choice depending on the desired results.

In other instances, hybridization may be achieved under conditions of, for example, 50 mM Tris-HCl (pH 8.3), 75 mm KCl, 3 mM MgCl ₂, 10 mM dithiothreitol, at temperatures between approximately 20° C. to about 37° C. Other hybridization conditions utilized could include approximately 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 1.5 μM MgCl ₂, at temperatures ranging from approximately 40° C. to about 72° C. Formamide and SDS also may be used to alter the hybridization conditions.

IV. Vectors Comprising Nucleic Acid Encoding DDNMT3B Molecules

Within certain embodiments of the present invention, an isolated nucleic acid sequence comprising a delta DNA methyltansferase 3B variant may be comprised in an expression vector. Expression requires that appropriate signals be provided in the vectors, which include various regulatory elements, such as enhancers/promoters from both viral and mammalian sources that drive expression of the genes of interest in host cells. Expression vectors utilized in the present invention may be a viral or plasmid vector.

The term “vector” is used to refer to a carrier nucleic acid molecule into which a nucleic acid sequence can be inserted for introduction into a cell where it can be replicated. A nucleic acid sequence can be “exogenous,” which means that it is foreign to the cell into which the vector is being introduced or that the sequence is homologous to a sequence in the cell but in a position within the host cell nucleic acid in which the sequence is ordinarily not found. Vectors include plasmids, cosmids, viruses (bacteriophage, animal viruses, etc.), and artificial chromosomes (e.g., YACs). One of skill in the art would be well equipped to construct a vector through standard recombinant techniques (see, for example, Maniatis et al., 1989 and Ausubel et al., 1994, both incorporated herein by reference).

The term “expression vector” refers to any type of genetic construct comprising a nucleic acid coding for a DNA methyltransferase molecule. In some cases, DNA molecules are then translated into a protein, polypeptide, or peptide. In other cases, these sequences are not translated, for example, in the production of antisense molecules or ribozymes. Expression vectors can contain a variety of “control sequences,” which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operably linked coding sequence in a particular host cell. In addition to control sequences that govern transcription and translation, vectors and expression vectors may contain nucleic acid sequences that serve other functions as well and are described infra. It is contemplated in the present invention, that virtually any type of vector may be employed in any known or later discovered method to deliver nucleic acids encoding a DNA methyltransferase molecule. Where incorporation into an expression vector is desired, the nucleic acid encoding a DNA methyltransferase molecule may also comprise a natural intron or an intron derived from another gene. Such vectors may be viral or non-viral vectors as described herein, and as known to those skilled in the art. An expression vector comprising a nucleic acid encoding a DNA methyltransferase molecule may comprise a virus or engineered construct derived from a viral genome.

The ability of certain viruses to enter cells via receptor-mediated endocytosis and to integrate into the host cell genome and express viral genes stably and efficiently have made them attractive candidates for the transfer of foreign genes into mammalian cells (Ridgeway, 1988; Nicolas and Rubenstein, 1988; Baichwal and Sugden, 1986; Temin, 1986). The first viruses used as vectors were DNA viruses including the papovaviruses (simian virus 40, bovine papilloma virus, and polyoma) (Ridgeway, 1988; Baichwal and Sugden, 1986) adenoviruses (Ridgeway, 1988; Baichwal and Sugden, 1986) and adeno-associated viruses. Retroviruses also are attractive gene transfer vehicles (Nicolas and Rubenstein, 1988; Temin, 1986) as are vaccina virus (Ridgeway, 1988) and adeno-associated virus (Ridgeway, 1988). Such vectors may be used to (i) transform cell lines in vitro for the purpose of expressing the DNA methyltransferase molecules or inhibitors thereof, such as antisense or siRNA molecules of the present invention or (ii) to transform cells in vitro or in vivo to provide therapeutic molecules for gene therapy. Thus, the present invention contemplates viral vectors such as, but not limited to, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a lentiviral vector, a herpes viral vector, polyoma viral vector or hepatitis B viral vector.

In particular embodiments of the invention, a plasmid vector is contemplated for use to transform a host cell. In general, plasmid vectors containing replicon and control sequences that are derived from species compatible with the host cell are used in connection with these hosts. The vector ordinarily carries a replication site, as well as marking sequences that are capable of providing phenotypic selection in transformed cells. Plasmid vectors are well known and are commercially available. Such vectors include, but are not limited to, the commercially available pSupervector (OligoEngine, Seattle, Wash.) and pSilencer™ siRNA expression vectors (Ambion, Austin Tex.). Other vectors that may be employed in the present invention include, but are not limited to, the following eukaryotic vectors: pWLNEO, pSV2CAT, pOG44, PXT1, pSG (Stratagene) pSVK3, pBSK, pBR322, pUC vectors, vectors that contain markers that can be selected in mammalian cells, such as pcDNA3.1, episomally replicating vectors, such as the pREP series of vectors, pBPV, pMSG, pSVL (Pharmacia), adenovirus vector (AAV pCWRSV, Chatterjee et al. (1992)) retroviral vectors, such as the pBABE vector series, a retroviral vector derived from MoMuLV (pG1Na, Zhou et al., (1994)); and pTZ18U (BioRad, Hercules, Calif.).

Regulatory Elements. Throughout this application, the term “expression construct” is meant to include any type of genetic construct containing a nucleic acid coding for a DNA methyltransferase 3B molecule of the present invention in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript may be translated into a protein, but it need not be. Expression includes both transcription of a gene and translation of mRNA into a gene product. In other instances, expression only includes transcription of the nucleic acid encoding a gene of interest.

In preferred embodiments, the nucleic acid encoding a delta DNA methyltansferase is under transcriptional control of a promoter. A “promoter” refers to a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrases “operatively positioned,” “operatively linked,” “under control,” and “under transcriptional control” means that the promoter is in the correct location and orientation in relation to the nucleic acid to control transcriptional initiation and/or expression of that sequence.

The term promoter will be used here to refer to a group of transcriptional control modules that are clustered around the initiation site for RNA polymerase II. Much of the thinking about how promoters are organized derives from analyses of several viral promoters, including those for the HSV thymidine kinase (tk) and SV40 early transcription units. These studies, augmented by more recent work, have shown that promoters are composed of discrete functional modules, each consisting of approximately 7-20 bp of DNA, and containing one or more recognition sites for transcriptional activator or repressor proteins.

At least one module in each promoter functions to position the start site for RNA synthesis. The best-known example of this is the TATA box, but in some promoters lacking a TATA box, such as the promoter for the mammalian terminal deoxynucleotidyl transferase gene and the promoter for the SV40 late genes, a discrete element overlying the start site itself helps to fix the place of initiation.

Additional promoter elements regulate the frequency of transcriptional initiation. Typically, these are located in the region 30-110 bp upstream of the start site, although a number of promoters have recently been shown to contain functional elements downstream of the start site as well. The spacing between promoter elements frequently is flexible, so that promoter function is preserved when elements are inverted or moved relative to one another. In the tk promoter, the spacing between promoter elements can be increased to 50 bp apart before activity begins to decline. Depending on the promoter, it appears that individual elements can function either co-operatively or independently to activate transcription.

Promoters such as the human cytomegalovirus (CMV) immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular or bacterial phage promoters which are well-known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose.

Enhancers are genetic elements that increase transcription from a promoter located at a distant position on the same molecule of DNA. Enhancers are organized much like promoters. That is, they are composed of many individual elements, each of which binds to one or more transcriptional proteins. The basic distinction between enhancers and promoters is operational. An enhancer region as a whole must be able to stimulate transcription at a distance; this need not be true of a promoter region or its component elements. On the other hand, a promoter must have one or more elements that direct initiation of RNA synthesis at a particular site and in a particular orientation, whereas enhancers lack these specificities. Promoters and enhancers are often overlapping and contiguous, often seeming to have a very similar modular organization.

Where a cDNA insert is employed, one will typically desire to include a polyadenylation signal to effect proper polyadenylation of the gene transcript. The nature of the polyadenylation signal is not believed to be crucial to the successful practice of the invention, and any such sequence may be employed such as human growth hormone and SV40 polyadenylation signals. Also contemplated as an element of the expression cassette is a terminator. These elements can serve to enhance message levels and to minimize read through from the cassette into other sequences.

Selectable Markers. In certain embodiments of the invention, cells containing a nucleic acid constructs of the present invention may be identified in vitro or in vivo by including a marker in the expression construct. Such markers would confer an identifiable change to the cell permitting easy identification of cells containing the expression construct. Usually the inclusion of a drug selection marker aids in cloning and in the selection of transformants, for example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers. Alternatively, enzymes such as herpes simplex virus thymidine kinase (tk) or chloramphenicol acetyltransferase (CAT) may be employed. Immunologic markers also can be employed. The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a gene product. Further examples of selectable markers are well known to one of skill in the art.

Multigene Constructs and IRES. In certain embodiments of the invention, the use of internal ribosome binding sites (IRES) elements are used to create multigene, or polycistronic, messages. IRES elements are able to bypass the ribosome scanning model of 5′ methylated Cap dependent translation and begin translation at internal sites (Pelletier and Sonenberg, 1988). IRES elements from two members of the picanovirus family (polio and encephalomyocarditis) have been described (Pelletier and Sonenberg, 1988), as well an IRES from a mammalian message (Macejak and Sarnow, 1991). IRES elements can be linked to heterologous open reading frames. Multiple open reading frames can be transcribed together, each separated by an IRES, creating polycistronic messages. By virtue of the IRES element, each open reading frame is accessible to ribosomes for efficient translation. Multiple genes can be efficiently expressed using a single promoter/enhancer to transcribe a single message.

Any heterologous open reading frame can be linked to IRES elements. This includes genes for secreted proteins, multi-subunit proteins, encoded by independent genes, intracellular or membrane-bound proteins and selectable markers. In this way, expression of several proteins can be simultaneously engineered into a cell with a single construct and a single selectable marker.

Host Cells. As used herein, the terms “cell,” “cell line,” and “cell culture” may be used interchangeably. All of these terms also include their progeny, which is any and all subsequent generations formed by cell division. It is understood that all progeny may not be identical due to deliberate or inadvertent mutations. A host cell may be “transfected” or “transformed,” which refers to a process by which exogenous nucleic acid is transferred or introduced into the host cell. A transformed cell includes the primary subject cell and its progeny. As used herein, the terms “engineered” and “recombinant” cells or host cells are intended to refer to a cell into which an exogenous nucleic acid sequence, such as, for example, a DNA methyltransferase 3B molecule or antisense or siRNA or a construct thereof. Therefore, recombinant cells are distinguishable from naturally occurring cells that do not contain a recombinantly introduced nucleic acid.

In certain embodiments, it is contemplated that nucleic acid or proteinaceous sequences may be co-expressed with other selected nucleic acid or proteinaceous sequences in the same host cell. Co-expression may be achieved by co-transfecting the host cell with two or more distinct recombinant vectors. Alternatively, a single recombinant vector may be constructed to include multiple distinct coding regions for nucleic acids, which could then be expressed in host cells transfected with the single vector.

V. Methods for Identifying Inhibitors DNA Methyltransferase 3B Activity

A. Inhibitors of Delta DNA Methyltransferase 3B

The present invention further comprises methods for identifying, making, generating, providing, manufacturing or obtaining inhibitors of delta DNA methyltransferase 3B activity. Delta DNA methyltransferase 3B nucleic acid or polypeptide may be used as a target in identifying compounds that inhibit, decrease or down-regulate its expression or activity in cancer cells, such as lung cancer cells. In other embodiments, compounds screened for would demethylate a hypermethylated promoter, such as a tumor suppressor gene promoter in a cancer cell. These assays may comprise random screening of large libraries of candidate substances; alternatively, the assays may be used to focus on particular classes of compounds selected with an eye towards structural attributes that are believed to make them more likely to inhibit the function of delta DNA methyltransferase 3B molecules. By function, it is meant that one may assay for inhibition of activity of delta DNA methyltransferase 3B in cancer cells, for demethylation of a methylated promoter or inhibition of the ability of the delta DNA methyltransferase 3B to methylate a promoter, and/or for the ability to increase apoptosism, for example.

To identify, make, generate, provide, manufacture or obtain a delta DNA methyltransferase 3B inhibitor, one generally will determine the activity of the delta DNA methyltransferase 3B molecule in the presence, absence, or both of the candidate substance, wherein an inhibitor is defined as any substance that down-regulates, reduces, inhibits or decreases delta DNA methyltransferase 3B expression or activity. For example, a method may generally comprise:

a) providing in a cell or cell free-system a DNA methyltransferase 3B polypeptide corresponding to the sequence of SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20 or SEQ ID NO:21;

b) contacting the DNA methyltransferase with a candidate substance; and

c) selecting an inhibitor of the DNA methyltransferase by assessing the effect of the candidate substance on DNA methyltransferase 3B activity. Upon identification of the inhibitor, the method may further provide manufacturing of the inhibitor.

Assays may be employed to assess the effect of the candidate substance on DNA methyltransferase activity, such as the following exemplary assays: (1) methylation specific PCR (MSP) or bisulfide sequencing analysis may be used to determine methylation status of specific promoters (2) RT-PCR may be used to determine reactivation of gene expression as a result of promoter demethylation of specific genes (3) global gene expression status measured by using DNA microarrays can be used to determine promoter methylation/demethylation status following manipulation of individual DNA methyltransferases; and/or (4) vectors containing specific promoter sequences with or without methylation may be co-transfected with vectors carrying individual DNA methyltransferase sequence into cells to determine at least one in vivo role of each isoform in controlling promoter methylation.

DNA methylation is a major determinant in the epigenetic silencing of genes. It is a complex process wherein three DNA methyltransferases catalyze the addition of a methyl group from S-adenosyl-L-methionine to the 5-carbon position of cytosine. A number of methods known to one of ordinary skill in the art may be used to detect DNA methylation. For example, such methods may include an enzyme based methodology or by chemical modificaton. In particular, such assays may include restriction endonuclease-based assays, restriction-enzyme based techniques, or developing methods based on polymerase chain reaction of sodium bisulfite-modified DNA, but is not limited to such. DNA array based techniques such as a differential methylation hybridization (Huang et al., 1999) assay may also be employed to perform a screen for hypermethylated promoter in a variety of cancer cell samples. PCR based methodologies may include methylation-sensitive restriction fingerprinting (Huang et al., 1997) to screen for changes in DNA methylation in tumors.

1. Inhibitors

As used herein the term “candidate substance” or “candidate compound” refers to any molecule that may potentially inhibit the activity of a delta DNA methyltransferase 3B molecule, that negatively affects its expression, or both. A delta DNA methyltransferase 3B inhibitor may be a compound that overall affects delta DNA methyltransferase 3B activity, which may be accomplished by inhibiting delta DNA methyltransferase 3B expression, function, or more directly by preventing its activity. Any compound or molecule described in the methods and compositions herein may be an inhibitor of delta DNA methyltransferase 3B activity.

The candidate substance may be a protein or fragment thereof, a small molecule, or even a nucleic acid molecule. It may prove to be the case that the most useful pharmacological compounds will be compounds that are structurally related to delta DNA methyltransferase 3B or other DNA methyltransferases, or that binds delta DNA methyltransferase 3B. In specific embodiments, the inhibitors may be dominant negative forms of the known DNMT3B forms or of the inventive DDNMT3B variants. Using lead compounds to help develop improved compounds is known as “rational drug design” and includes not only comparisons with known inhibitors, but predictions relating to the structure of target molecules.

Candidate compounds or inhibitors of the present invention will likely function to inhibit, decrease or down-regulate the expression or activity of delta DNA methyltransferase 3B in a cancer cell such as a lung cancer cell. Such candidate compounds may be inhibitors or regulators of DNA methyltransferases may have the ability to demethylate a methylated promoter or may likely be involved in controlling cellular proliferation in a cancer or tumor cell, such as lung cancer cells. These candidate compounds may be antisense molecules, ribozymes, interfering RNAs or siRNAs, antibodies (including single chain antibodies), small molecules, and/or organopharmaceuticals, but are not limited to such.

2. Rational Drug Design

The present invention also provides methods for developing drugs that inhibit delta DNA methyltransferase 3B activity that may be used to treat a cancer, such as lung cancer. One such method involves the prediction of the three dimensional structure of a validated DNA methyltransferase target using molecular modeling and computer stimulations. The resulting structure may then be used in docking studies to identify potential small molecule inhibitors. Inhibitors identified may then be tested in biochemical assays to further identify delta DNA methyltransferase 3B drug targets for cancer treatment, e.g., lung cancer treatment.

Rational drug design is therefore used to produce structural analogs of substrates for delta DNA methyltransferase 3B. By creating such analogs, it is possible to fashion drugs which are more active or stable than the natural molecules, which have different susceptibility to alteration or which may affect the function of various other molecules. In one approach, one would generate a three-dimensional structure for the delta DNA methyltransferase 3B targets of the invention or a fragment thereof. This could be accomplished by X-ray crystallography, computer modeling or by a combination of both approaches.

It is also possible to use antibodies to ascertain the structure of a target compound inhibitor. In principle, this approach yields a pharmacore upon which subsequent drug design can be based. It is possible to bypass protein crystallography altogether by generating anti-idiotypic antibodies to a functional, pharmacologically active antibody. As a mirror image of a mirror image, the binding site of anti-idiotype would be expected to be an analog of the original antigen. The anti-idiotype could then be used to identify and isolate peptides from banks of chemically- or biologically-produced peptides. Selected peptides would then serve as the pharmacore. Anti-idiotypes may be generated using the methods described herein for producing antibodies, using an antibody as the antigen.

On the other hand, one may simply acquire, from various commercial sources, small molecule libraries that are believed to meet the basic criteria for useful drugs in an effort to “brute force” the identification of useful compounds. Such libraries, including combinatorially generated libraries (e.g., peptide libraries), provide a rapid and efficient way to screen a large number of related (and unrelated) compounds for activity. Combinatorial approaches also lend themselves to rapid evolution of potential drugs by the creation of second, third and fourth generation compounds model of active, but otherwise undesirable compounds.

Candidate compounds may include fragments or parts of naturally-occurring compounds, or may be found as active combinations of known compounds, which are otherwise inactive. It is proposed that compounds isolated from natural sources, such as animals, bacteria, fungi, plant sources, including leaves and bark, and marine samples may be assayed as candidates for the presence of potentially useful pharmaceutical agents. It will be understood that the pharmaceutical agents to be screened could also be derived or synthesized from chemical compositions or man-made compounds. Thus, it is understood that the candidate substance identified by the present invention may be a peptide, polypeptide, polynucleotide, small molecule inhibitors or any other compounds that may be designed through rational drug design starting from known inhibitors or stimulators.

Other suitable compounds include antisense molecules, ribozymes, and antibodies (including single chain antibodies), each of which would be specific for the target molecule. Such compounds are described in greater detail elsewhere in this document. For example, an antisense molecule that bound to a translational or transcriptional start site, or splice junctions, would be ideal candidate inhibitors.

In addition, it is also contemplate that sterically similar compounds may be formulated to mimic the key portions of the structure of the inhibitors. Such compounds, may include peptidomimetics of peptide inhibitors. Regardless of the type of inhibitor identified by the present screening methods, the effect of the inhibition by such a compound results in the regulation of delta DNA methyltransferase 3B activity as compared to that observed in the absence of the added candidate substance.

The term “drug” as contemplated herein is intended to refer to a chemical entity, whether in the solid, liquid, or gaseous phase which is capable of providing a desired therapeutic effect when administered to a subject. The term “drug” should be read to include synthetic compounds, natural products and macromolecular entities such as polypeptides, polynucleotides, or lipids and also small entities such as neurotransmitters, ligands, hormones or elemental compounds. The term “drug” is meant to refer to that compound whether it is in a crude mixture or purified and isolated.

3. In Vitro Assays

A quick, inexpensive and easy assay to run is an in vitro assay. Such assays generally use isolated molecules, and can be run quickly and in large numbers, thereby increasing the amount of information obtainable in a short period of time. A variety of vessels may be used to run the assays, including test tubes, plates, dishes and other surfaces such as dipsticks or beads.

One example of a cell-free assay is a binding assay. While not directly addressing function, the ability of a compound to bind or contact to a target molecule, such as DNA methyltransferase of the present invention, in a specific fashion is strong evidence of a related biological effect, which can be assessed in a screening assay. For example, binding of a molecule to a delta DNA methyltransferase 3B molecule of the present invention may, in and of itself, be inhibitory, due to steric, allosteric or charge-charge interactions. The delta DNA methyltransferase 3B molecule may be either free in solution, fixed to a support, and/or expressed in or on the surface of a cell. Either the delta DNA methyltransferase 3B molecule or the compound may be labeled, thereby permitting measuring of the binding. Competitive binding formats can be performed in which one of the agents is labeled, and one may measure the amount of free label versus bound label to determine the effect on binding.

A technique for high throughput screening of compounds is described in WO 84/03564. Large numbers of small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface. Bound polypeptide is detected by various methods.

4. In Cyto Assays

The present invention also contemplates identifying compounds for their ability to inhibit a delta DNA methyltransferase 3B variant disclosed herein, in cells. Various cell lines can be utilized for such screening assays, including cells specifically engineered for this purpose. The present invention particularly contemplates the use of cancer cells, such as lung cancer cells. Depending on the assay, culture may be required. The cell is examined using any of a number of different physiologic assays. Alternatively, molecular analysis may be performed, for example, looking at protein expression, mRNA expression (including differential display of whole cell or polyA RNA) by methods as described herein and that are well known to those of skill in the art.

5. In Vivo Assays

In vivo assays involve the use of various animal models, including transgenic animals that have been engineered to have specific defects such as overexpression of a delta DNA methyltransferase 3B molecule, or that carry markers that can be used to measure the ability of a candidate substance to reach and effect different cells within the organism. Due to their size, ease of handling, and information on their physiology and genetic make-up, mice are a preferred embodiment, especially for transgenics. However, other animals are suitable as well, including rats, rabbits, hamsters, guinea pigs, gerbils, woodchucks, cats, dogs, sheep, goats, pigs, cows, horses and monkeys (including chimps, gibbons and baboons). Assays for inhibitors may be conducted using an animal model derived from any of these species.

In such assays, one or more candidate substances are administered to an animal, and the ability of the candidate substance(s) to alter one or more characteristics, as compared to a similar animal not treated with the candidate substance(s), identifies an inhibitor. The characteristics may be any of those discussed above with regard to delta DNA methyltransferase 3B activity, or it may be broader in the sense of “treating” the condition present in the animal.

Treatment of these animals with test compounds will involve the administration of the compound, in an appropriate form, to the animal. Administration will be by any route that could be utilized for clinical or non-clinical purposes, including but not limited to oral, nasal, buccal, topical or by a nebulizer or atomizer. Alternatively, administration may be by intratracheal instillation, bronchial instillation, intradermal, subcutaneous, intramuscular, intraperitoneal or intravenous injection. Specifically contemplated routes are systemic intravenous injection, regional administration via blood or lymph supply, or directly to an affected site.

Determining the effectiveness of a compound in vivo may involve measuring toxicity and dose response which can be performed in animals in a more meaningful fashion than in in vitro or in cyto assays.

VI. DNA Methyltransferase Inhibitors for Cancer Therapy

The present invention embodies a method of treating cancer such as lung cancer, by the delivery of a delta DNA methyltransferase 3B inhibitor to a cancer cell. Such a cell may be located in a patient having a cancer. Examples of cancers contemplated for treatment include leukemia, ovarian cancer, breast cancer, lung cancer, colon cancer, liver cancer, prostate cancer, testicular cancer, stomach cancer, brain cancer, bladder cancer, head and neck cancer, melanoma, and any other cancer that may be treated by inhibiting or decreasing the enzymatic activity of delta DNA methyltransferase 3B. Such inhibitors may include antisense molecules, RNA interference or siRNA methodology, or ribozymes.

A. Antisense Methodology

As discussed above, the present invention may also employ an antisense molecule in inhibiting the activity of a DNA methyltransferase, such as delta DNA methyltransferase. Antisense methodology takes advantage of the fact that nucleic acids tend to pair with “complementary” sequences. By complementary, it is meant that the polynucleotides are those capable of base-pairing according to the standard Watson-Crick complementary rules. That is, the larger purines will base pair with the smaller pyrimidines to form combinations of guanine paired with cytosine (G:C) and adenine paired with either thymine (A:T) in the case of DNA, or adenine paired with uracil (A:U) in the case of RNA. Inclusion of less common bases such as inosine, 5-methylcytosine, 6-methyladenine, hypoxanthine and others in hybridizing sequences does not interfere with pairing.

Targeting double-stranded (ds) DNA with polynucleotides leads to triple-helix formation targeting RNA leads to double-helix formation. Antisense polynucleotides, when introduced into a target cell, specifically bind to their target polynucleotide and interfere with transcription, RNA processing, transport, translation and/or stability. Antisense molecules, or DNA encoding such antisense molecules, may be employed to inhibit gene transcription or translation or both within a host cell, either in vitro or in vivo, such as within a host animal, including a human subject.

Antisense molecules may be designed to bind to the promoter and/or other control regions, exons, introns or even exon-intron boundaries of a gene. It is contemplated that the most effective antisense constructs will include regions complementary to intron/exon splice junctions. Thus, it is proposed that a preferred embodiment includes one or more antisense molecules with complementarity to regions within about 50 of an intron-exon splice junction. It has been observed that some exon sequences can be included in the construct without seriously affecting the target selectivity thereof. The amount of exonic material included will vary depending on the particular exon and intron sequences used. One can readily test whether too much exon DNA is included simply by testing the constructs in vitro to determine whether normal cellular function is affected or whether the expression of related genes having complementary sequences is affected.

As stated above, “complementary” or “antisense” means polynucleotide sequences that are substantially complementary over their entire length and have very few base mismatches. For example, sequences of fifteen bases in length may be termed complementary when they have complementary nucleotides at thirteen or fourteen positions. Naturally, sequences which are completely complementary will be sequences which are entirely complementary throughout their entire length and have no base mismatches. Other sequences with lower degrees of homology also are contemplated. For example, an antisense construct which has limited regions of high homology, but also contains a non-homologous region (e.g., ribozyme see below) could be designed. These molecules, though having less than 50% homology, would bind to target sequences under appropriate conditions. It may be advantageous to combine portions of genomic DNA with cDNA or synthetic sequences to generate specific constructs. For example, where an intron is desired in the ultimate construct, a genomic clone will need to be used. The cDNA or a synthesized polynucleotide may provide more convenient restriction sites for the remaining portion of the construct and, therefore, would be used for the rest of the sequence.

In particular aspects of the invention, siRNA compositions may target an exon-exon junctions, such as of particular variants, including the exemplary one comprising sense strand provided in SEQ ID NO:61 and the antisense strand provided in SEQ ID NO:62, which targets the exemplary sequence of SEQ ID NO:63.

B. RNA Interference (RNAi)

The present invention also contemplates the use of RNA interference in inhibiting, reducing or downregulating the activity of delta DNA methyltransferase 3B molecules of the present invention. RNA interference (also referred to as “RNA-mediated interference” or RNAi) is a mechanism by which gene expression can be reduced or eliminated. Double stranded RNA (dsRNA) has been observed to mediate the reduction, which is a multi-step process. dsRNA activates post-transcriptional gene expression surveillance mechanisms that appear to function to defend cells from virus infection and transposon activity (Fire et al., 1998; Grishok et al., 2000; Ketting et al., 1999; Lin and Avery, 1999; Montgomery et al., 1998; Sharp, 1999; Sharp and Zamore, 2000; Tabara et al., 1999; Hutvagner et al., 2001; Tuschl, 2001; Waterhouse et al., 2001; Zamore, 2001). Activation of these mechanisms target mature, dsRNA-complementary mRNA for destruction. Moreover, dsRNA has been shown to silence genes in a wide range of systems, including plants, protozoans, fungi, C. elegans, Trypanasoma and Drosophila (Grishok et al., 2000; Sharp, 1999; Sharp and Zamore 2000).

RNAi offers major experimental advantages for the study of gene function. These advantages include a very high specificity, ease of movement across cell membranes, and prolonged down-regulation of the targeted gene. RNAi may be used to identify genes that are essential for a particular biological pathway, identify disease-causing genes, study structure function relationships, and implement therapeutics and diagnostics. As with other types of gene inhibitory compounds, such as antisense and triplex forming oligonucleotides, tracking these potential drugs in vivo and in vitro is important for drug development, pharmacokinetics, biodistribution, macro and microimaging metabolism and for gaining a basic understanding of how these compounds behave and function.

In RNAi the dsRNA is typically directed to an exon, although some exceptions to this have been shown (see Plasterk and Ketting, 2000). Also, a homology threshold (probably about 80-85% over 200 bases) is required. Most tested sequences are 500 base pairs or greater, though sequences of 30 nucleotides or fewer evade the antiviral response in mammalian cells (Baglioni et al., 1983; Williams, 1997). The targeted mRNA is lost after RNAi. The effect of RNAi is non-stoichiometric, and thus incredibly potent. In fact, it has been estimated that only a few copies of dsRNA are required to knock down >95% of targeted gene expression in a cell (Fire et al., 1998).

Due to a potent antiviral response pathway in mammalian cells that induces global changes in gene expression when the cells are challenged with long (>30 nucleotides) dsRNA molecules, RNAi was used in non-mammalian cells. This limitation in the art was overcome by the discovery of a method to bypass the antiviral response and induce gene specific silencing in mammalian cells (Elbashir et al., 2001). Several nucleotide (nt) dsRNAs with 2 nt 3′ overhangs were transfected into mammalian cells without inducing the antiviral response. These small dsRNAs, referred to as small interfering RNAs (siRNAs) proved capable of inducing the specific suppression of target genes. In addition, it was demonstrated that siRNAs could reduce the expression of several endogenous genes in human cells. The use of siRNAs to modulate gene expression in mammalian cells has since been demonstrated (Caplen et al., 2001; Hutvagner et al., 2001).

Thus, small interfering RNA (siRNA), which are generally 12-15 or 21-23 nucleotides in length and which possess the ability to mediate RNA interference are also contemplated in the present invention. For example, such siRNA may be of at least 21 nucleotides. siRNAs of the present invention may be synthesized chemically or may be produced recombinantly. They may be subsequently isolated and/or purified.

When made in vitro, siRNA is formed from one or more strands of polymerized ribonucleotide. When formed of only one strand, it takes the form of a self-complementary hairpin-type or stem and loop structure that doubles back on itself to form a partial duplex. The self-duplexed portion of the RNA molecule may be referred to as the “stem” and the remaining, connecting single stranded portion referred to as the “loop” of the stem and loop structure. When made of two strands, they are substantially complementary. siRNAs of the present invention may be synthesized chemically or may be produced recombinantly. They may be subsequently isolated and/or purified. dsRNA for use as siRNA may also be enzymatically synthesized through the use of RNA dependent RNA polymerases such as Q beta replicase, Tobacco mosaic virus replicase, brome mosaic virus replicase, potato virus replicase, etc. Methods for synthesizing dsRNA are well-described (Fire et al., 1998). Briefly, sense and antisense RNA are synthesized from DNA templates using T7 polymerase (MEGAscript, Ambion). After the synthesis is complete, the DNA template is digested with DNaseI and RNA purified by phenol/chloroform extraction and isopropanol precipitation. RNA size, purity and integrity are assayed on denaturing agarose gels. Sense and antisense RNA are diluted in potassium citrate buffer and annealed at 80° C. for 3 min to form dsRNA. As with the construction of DNA template libraries, a procedure may be employed to aid this time intensive procedure. The sum of the individual dsRNA species is designated as a “dsRNA library.”

Reaction conditions for use of these RNA polymerases are well known in the art (U.S. Pat. RE 35,443, and U.S. Pat. No. 4,786,600, each incorporated herein by reference). The result of contacting the appropriate template with an appropriate polymerase is the synthesis of an RNA product, which is typically double-stranded. In some instances a single stranded RNA or single stranded DNA template may be utilized. If utilizing a single stranded DNA template, the enzymatic synthesis results in a hybrid RNA/DNA duplex that is also contemplated as useful as siRNA.

The templates for enzymatic synthesis of siRNA are nucleic acids, typically, though not exclusively DNA. A nucleic acid may be made by any technique known to one of ordinary skill in the art. Non-limiting examples of synthetic nucleic acid, particularly a synthetic oligonucleotide, include a nucleic acid made by in vitro chemical synthesis using phosphotriester, phosphite or phosphoramidite chemistry and solid phase techniques such as described in EP 266,032, incorporated herein by reference, or via deoxynucleoside H-phosphonate intermediates as described by Froehler et al. (1986), and U.S. Pat. No. 5,705,629, each incorporated herein by reference. A non-limiting example of enzymatically produced nucleic acid include one produced by enzymes in amplification reactions such as PCR™ (see for example, U.S. Pat. Nos. 4,683,202 and 4,682,195, each incorporated herein by reference), or the synthesis of oligonucleotides described in U.S. Pat. No. 5,645,897, incorporated herein by reference. A non-limiting example of a biologically produced nucleic acid includes recombinant nucleic acid production in living cells (see for example, Sambrook, 2001; incorporated herein by reference). Methods for the production of siRNA to induce gene silencing can be found in United States Patent Application 20030166282, incorporated herein by reference.

The nucleic acid(s) of the present invention, regardless of the length of the sequence itself, may be combined with other nucleic acid sequences, including but not limited to, promoters, enhancers, polyadenylation signals, restriction enzyme sites, multiple cloning sites, coding segments, and the like, to create one or more nucleic acid construct(s). The overall length may vary considerably between nucleic acid constructs. Thus, a nucleic acid segment of almost any length may be employed, with the total length preferably being limited by the ease of preparation or use in the intended protocol.

C. Ribozymes

The present invention also contemplates the use of DNA methyltransferase 3B specific ribozymes to down-regulate or inhibit delta DNA methyltransferase 3B enzymatic activity. Ribozymes are RNA-protein complexes that cleave nucleic acids in a site-specific fashion. Ribozymes comprise of eight classes including seven that modify the nucleic acid backbone which include hammerhead, hairpin, HDV (hepatitis delta virus), ribonuclease P, group I intron, group II intron, and VS ribozyme. The eighth type, the ribosome's peptidyl transferase center, builds peptide bonds.

Ribozymes have specific catalytic domains that possess endonuclease activity (Kim and Cook, 1987;; Gerlach et al., 1987; Forster and Symons, 1987). For example, a large number of ribozymes accelerate phosphoester transfer reactions with a high degree of specificity, often cleaving only one of several phosphoesters in an oligonucleotide substrate (Michel and Westhof, 1990; Reinhold-Hurek and Shub, 1992). This specificity has been attributed to the requirement that the substrate binds via specific base-pairing interactions to the internal guide sequence (“IGS”) of the ribozyme prior to chemical reaction.

Ribozyme catalysis has primarily been observed as part of sequence-specific cleavage/ligation reactions involving nucleic acids (Joyce, 1989). For example, U.S. Pat. No. 5,354,855 reports that certain ribozymes can act as endonucleases with a sequence specificity greater than that of known ribonucleases and approaching that of the DNA restriction enzymes. Thus, sequence-specific ribozyme-mediated inhibition of gene expression may be particularly suited to therapeutic applications (Scanlon et al., 1991; Sarver et al., 1990).

VII. Generating Antibodies Reactive with DNA Methyltransferase 3B Molecules

In a particular embodiment, the present invention contemplates antibodies that are immunoreactive with a DNA methyltransferase 3B or variants thereof. Such an antibody can be a polyclonal or a monoclonal antibody, although in a preferred embodiment the antibody is a monoclonal antibody. Means for preparing and characterizing antibodies are well known in the art (see, e.g., Harlow and Lane, 1988). Briefly, a polyclonal antibody is prepared by immunizing an animal with an immunogen comprising a polypeptide of the present invention and collecting antisera from that immunized animal. A wide range of animal species can be used for the production of antisera. Typically an animal used for production of anti-antisera is a non-human animal including rabbits, mice, rats, hamsters, pigs or horses. Because of the relatively large blood volume of rabbits, a rabbit is a preferred choice for production of polyclonal antibodies.

Antibodies, both polyclonal and monoclonal, specific for an antigen may be prepared using conventional immunization techniques, as are generally known to those of skill in the art. A composition containing antigenic epitopes of the compounds of the present invention can be used to immunize one or more experimental animals, such as a rabbit or mouse, which will then proceed to produce specific antibodies against the compounds of the present invention. Polyclonal antisera may be obtained, after allowing time for antibody generation, simply by bleeding the animal and preparing serum samples from the whole blood.

It is proposed that the antibodies of the present invention will find useful application in standard immunochemical procedures, such as ELISA and Western blot methods and in immunohistochemical procedures such as tissue staining, as well as in other procedures which may utilize antibodies specific to DNA methyltransferase 3B variants.

In general, both polyclonal and monoclonal antibodies against delta DNA methyltransferase 3B variants of the present invention may be used in a variety of embodiments. For example, they may be employed in antibody cloning protocols to obtain cDNAs or genes encoding other DNA methyltransferase 3B molecules. They may also be used in inhibition studies to analyze the effects of delta DNA methyltransferase 3B in cells or animals. Antibodies comprising DNA methyltransferase 3B variants will also be useful in immunolocalization studies to analyze the distribution of these molecules during various cellular events, for example, to determine the cellular or tissue-specific distribution of DNA methyltransferase 3B polypeptides at different points in the cell cycle. A particularly useful application of such antibodies is in purifying native or recombinant DNA methyltransferase 3B molecules, for example, using an antibody affinity column. The operation of such immunological techniques are well known to those of skill in the art.

As is well known in the art, a given composition may vary in its immunogenicity. It is often necessary therefore to boost the host immune system, as may be achieved by coupling a peptide or polypeptide immunogen to a carrier. Exemplary and preferred carriers are keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA). Other albumins such as ovalbumin, mouse serum albumin or rabbit serum albumin can also be used as carriers. Means for conjugating a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, m-maleimidobencoyl-N-hydroxysuccinimide ester, carbodiimide and bis-biazotized benzidine.

It is also well known in the art, that the immunogenicity of a particular immunogen composition can be enhanced by the use of non-specific stimulators of the immune response, known as adjuvants. Exemplary and preferred adjuvants include complete Freund's adjuvant (a non-specific stimulator of the immune response containing killed Mycobacterium tuberculosis ), incomplete Freund's adjuvants and aluminum hydroxide adjuvant.

The amount of immunogen composition used in the production of polyclonal antibodies varies upon the nature of the immunogen as well as the animal used for immunization. A variety of routes can be used to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies may be monitored by sampling blood of the immunized animal at various points following immunization. A second, booster, injection may also be given. The process of boosting and titering is repeated until a suitable titer is achieved. When a desired level of immunogenicity is obtained, the immunized animal can be bled and the serum isolated and stored, and/or the animal can be used to generate mAbs.

MAbs may be readily prepared through use of well-known techniques, such as those exemplified in U.S. Pat. No. 4,196,265, incorporated herein by reference. Typically, this technique involves immunizing a suitable animal with a selected immunogen composition, e.g., a purified or partially purified protein, polypeptide or peptide or cell expressing high levels. The immunizing composition is administered in a manner effective to stimulate antibody producing cells. Rodents such as mice and rats are preferred animals, however, the use of rabbit, sheep or frog cells is also possible. The use of rats may provide certain advantages (Goding, 1986), but mice are preferred, with the BALB/c mouse being most preferred as this is most routinely used and generally gives a higher percentage of stable fusions.

VIII. Delivery of a DNMT3B Inhibitor to a Cell

In some embodiments of the present invention delivery of a nucleic acid encoding a DNA methyltransferase inhibitor such as an antisense or siRNA or an expression construct thereof to a cell is contemplated. Virtually any method by which nucleic acids can be introduced into a cell, or an organism may be employed with the current invention, as described herein or as would be known to one of ordinary skill in the art.

Such methods include, but are not limited to direct delivery of a nucleic acid by: injection (U.S. Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each incorporated herein by reference) microinjection (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, incorporated herein by reference); electroporation (U.S. Pat. No. 5,384,253, incorporated herein by reference Tur-Kaspa et al., 1986; Potter et al., 1984) calcium phosphate precipitation (Graham and Van Der Eb, 1973; Chen and Okayama, 1987; Rippe et al., 1990) using DEAE-dextran followed by polyethylene glycol (Gopal, 1985) direct sonic loading (Fechheimer et al., 1987) liposome mediated transfection (Nicolau and Sene, 1982; Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; Kaneda et al., 1989; Kato et al., 1991) receptor-mediated transfection (Wu and Wu, 1987; Wu and Wu, 1988) microprojectile bombardment (PCT Application Nos. WO 94/09699 and 95/06128; U.S. Pat. Nos. 5,610,042 5,322,783 5,563,055, 5,550,318, 5,538,877 and 5,538,880, and each incorporated herein by reference) agitation with silicon carbide fibers (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464,765, each incorporated herein by reference) PEG-mediated transformation of protoplasts (Omirulleh et al., 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorporated herein by reference) desiccation/inhibition-mediated DNA uptake (Potrykus et al., 1985) or any combination of such methods.

In other embodiments, it is contemplated that a nucleic acid encoding an siRNA or an expression construct thereof may be delivered to a cell by hydrodynamic transfection/injection, or by liposomes.

It is also contemplated in the present invention that a siRNA may be delivered directly to a cell. In other embodiments, the siRNA may be delivered to a cell indirectly by introducing one or more vectors that encode both single strands of a siRNA (or, in the case of a self-complementary RNA, the single self-complementary strand) into the cell. The vectors of these embodiments contain elements of the templates described above such that the RNA is transcribed inside the cell, annealed to form siRNA and effects attenuation of the target gene expression. See WO 99/32619, WO 00/44914, WO 01/68836 (each of which is expressly incorporated herein by reference) and references therein for further examples of methods known in the art for introducing siRNA into cells. In some embodiments, an siRNA of the present invention may be delivered along with components that enhance RNA uptake by the cell, stabilize the annealed strands, or otherwise increase inhibition of DNA methyltransferase 3B activity.

Wherein the inhibitor is not a nucleic acid, such as an antibody, for example, standard means in the art may be utilized to deliver the inhibitor, such as by liposomes.

IX. Therapeutic/Pharmaceutical Compositions

In some embodiments, the present invention provides a method of treating or preventing a cancer by providing or administering to a patient a therapeutically effective amount of DNA methyltransferase 3B inhibitor, such as a delta DNA methytransferase 3B inhibitor which includes but is not limited to an antisense or siRNA molecule.

“Therapeutically effective amounts” are those amounts effective to produce beneficial results, particularly with respect to cancer treatment, in the recipient animal or patient. Such amounts may be initially determined by reviewing the published literature, by conducting in vitro tests or by conducting metabolic studies in healthy experimental animals. Before use in a clinical setting, it may be beneficial to conduct confirmatory studies in an animal model, preferably a widely accepted animal model of the particular disease to be treated. Preferred animal models for use in certain embodiments are rodent models, which are preferred because they are economical to use and, particularly, because the results gained are widely accepted as predictive of clinical value.

Diseases contemplated for treatment with the DNA methyltransferase 3B inhibitors of the present invention include, but are not limited to, cancers. Examples of cancers contemplated for treatment with a delta DNA methyltransferase 3B inhibitor may include breast cancer, lung cancer, head and neck cancer, bladder cancer, bone cancer, bone marrow cancer, brain cancer, colon cancer, esophageal cancer, gastrointestinal cancer, gum cancer, kidney cancer, liver cancer, nasopharynx cancer, ovarian cancer, prostate cancer, skin cancer, stomach cancer, testis cancer, tongue cancer, or uterine cancer. In some instances the cancer to be treated using a delta DNA methyltransferase 3B inhibitor as disclosed herein, may be a malignant or metastatic cancer but not limited to such.

To inhibit DNA methylation, kill cells, induce cell cycle arrest, inhibit cell growth, inhibit metastasis, inhibit angiogenesis or otherwise reverse or reduce the malignant phenotype of cancer cells, using the methods and compositions of the present invention, one would generally contact a cell with the DNA methyltransferase 3B inhibitor, in particular a delta DNA methyltransferase inhibitor. The terms “contacted” and “exposed,” when applied to a cell, tissue or organism, are used herein to describe the process by which the therapeutic compositions of the invention is delivered to a target cell, tissue or organism or are placed in direct juxtaposition with the target cell, tissue or organism. To achieve cell killing or stasis, an amount effective of the therapeutic composition is delivered to one or more cells to kill the cell(s) or prevent them from dividing.

Pharmaceutical aqueous compositions of the present invention comprise the delta DNA methyltransferase 3B inhibitor and/or an additional agent(s) dissolved or dispersed in a pharmaceutically acceptable carrier or aqueous medium. The phrases “pharmaceutically or pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to a human. As used herein, “pharmaceutically acceptable carrier” includes any and all solvents, dispersion media, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the active ingredient, its use in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

The actual dosage amount of a delta DNA methyltransferase 3B inhibitory composition of the present invention (and/or an additional agent) for administration to a patient can be determined by physical and physiological factors such as body weight, severity of condition, idiopathy of the patient and on the route of administration. With these considerations in mind, the dosage of a lipid composition for a particular subject and/or course of treatment can readily be determined.

Treatment may vary depending upon the host treated and the particular mode of administration. For example, in the invention the dose range of a DNA methyltransferase 3B inhibitor may be about 0.5 mg/kg body weight to about 500 mg/kg body weight. The term “body weight” is applicable when an animal is being treated. When isolated cells are being treated, “body weight” as used herein should read to mean “total cell weight”. The term “total weight may be used to apply to both isolated cell and animal treatment. All concentrations and treatment levels are expressed as “body weight” or simply “kg” in this application are also considered to cover the analogous “total cell weight” and “total weight” concentrations. However, those of skill will recognize the utility of a variety of dosage range, for example, 1 mg/kg body weight to 450 mg/kg body weight, 2 mg/kg body weight to 400 mg/kg body weight, 3 mg/kg body weight to 350 mg/kg body weight, 4 mg/kg body weight to 300 mg/kg body weight, 5 mg/kg body weight to 250 mg/kg body weighty, 6 mg/kg body weight to 200 mg/kg body weight, 7 mg/kg body weight to 150 mg/kg body weight, 8 mg/kg body weight to 100 mg/kg body weight, or 9 mg/kg body weight to 50 mg/kg body weight. Further, those of skill will recognize that a variety of different dosage levels will be of use, for example, 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg, 7.5 mg/kg, 10 mg/kg, 12.5 mg/kg, 15 mg/kg, 17.5 mg/kg, 20 mg/kg, 25 mg/kg, 30 mg/kg, 35 mg/kg, 40 mg/kg, 45 mg/kg, 50 mg/kg, 60 mg/kg, 70 mg/kg, 80 mg/kg, 90 mg/kg, 100 mg/kg, 120 mg/kg, 140 mg/kg, 150 mg/kg, 160 mg/kg, 180 mg/kg, 200 mg/kg, 225 mg/kg, 250 mg/kg, 275 mg/kg, 300 mg/kg, 325 mg/kg, 350 mg/kg, 375 mg/kg, 400 mg/kg, 450 mg/kg, 500 mg/kg, 550 mg/kg, 600 mg/kg, 700 mg/kg, 750 mg/kg, 800 mg/kg, 900 mg/kg, 1000 mg/kg, 1250 mg/kg, 1500 mg/kg, 1750 mg/kg, 2000 mg/kg, 2500 mg/kg, and/or 3000 mg/kg. Of course, all of these dosages are exemplary, and any dosage in-between these points is also expected to be of use in the invention. Any of the above dosage ranges or dosage levels may be employed for a DNA methyltransferase 3B inhibitor of the present invention.

In certain embodiments, pharmaceutical compositions may comprise, for example, at least about 0.1% of an active compound. In other embodiments, the active compound may comprise

WO/1999/067397	December, 1999	$i(DE NOVO) DNA CYTOSINE METHYLTRANSFERASE GENES, POLYPEPTIDES AND USES THEREOF
WO/2002/009126	November, 2002	SPIN VALVE STRUCTURE
WO/2004/030615	April, 2004	COMPOSITIONS AND METHODS FOR THE DIAGNOSIS AND TREATMENT OF TUMOR