Patent Grant
7638497
The invention relates to anti sense IAP nucleobase oligomers and methods of using them to induce apoptosis.
One way by which cells die is referred to as apoptosis, or programmed cell death. Apoptosis often occurs as a normal part of the development and maintenance of healthy tissues. The process may occur so rapidly that it is difficult to detect.
The apoptosis pathway is now known to play a critical role in embryonic development, viral pathogenesis, cancer, autoimmune disorders, and neurodegenerative diseases, as well as other events. The failure of an apoptotic response has been implicated in the development of cancer, autoimmune disorders, such as lupus erythematosis and multiple sclerosis, and in viral infections, including those associated with herpes virus, poxvirus, and adenovirus.
The importance of apoptosis in cancer has become clear in recent years. The identification of growth promoting oncogenes in the late 1970's gave rise to an almost universal focus on cellular proliferation that dominated research in cancer biology for many years. Long-standing dogma held that anti-cancer therapies preferentially targeted rapidly dividing cancer cells relative to “normal” cells. This explanation was not entirely satisfactory, since some slow growing tumors are easily treated, while many rapidly dividing tumor types are extremely resistant to anti-cancer therapies. Progress in the cancer field has now led to a new paradigm in cancer biology wherein neoplasia is viewed as a failure to execute normal pathways of programmed cell death. Normal cells receive continuous feedback from their neighbors through various growth factors, and commit “suicide” if removed from this context. Cancer cells somehow bypass these commands and continue inappropriate proliferation. It is now believed that many cancer therapies, including radiation and many chemotherapies, previously thought to act by causing cellular injury, actually work by triggering apoptosis.
Both normal cell types and cancer cell types display a wide range of susceptibility to apoptotic triggers, although the determinants of this resistance are only now under investigation. Many normal cell types undergo temporary growth arrest in response to a sub-lethal dose of radiation or cytotoxic chemical, while cancer cells in the vicinity undergo apoptosis. This differential effect at a given dose provides the crucial treatment window that allows successful anti-cancer therapy. It is therefore not surprising that resistance of tumor cells to apoptosis is emerging as a major category of cancer treatment failure.
Several potent endogenous proteins that inhibit apoptosis have been identified, including Bcl-2, and IAP (inhibitor-of apoptosis) families in mammalian cells. Certain members of the latter family directly inhibit terminal effector caspases, i.e. casp-3 and casp-7, engaged in the execution of cell death, as well as the key mitochondrial initiator caspase, casp-9, important to the mediation of cancer chemotherapy induced cell death. The IAPs are the only known endogenous caspase inhibitors, and thus play a central role in the regulation of apoptosis.
The IAPs have been postulated to contribute to the development of some cancers, and a postulated causal chromosomal translocation involving one particular IAP (cIAP2/HIAP1) has been identified in MALT lymphoma. A recent correlation between elevated XIAP, poor prognosis, and short survival has been demonstrated in patients with acute myelogenous leukemia. XIAP was highly over-expressed in many tumor cell lines of the NCI panel.
There exists a need for improved cancer therapeutics and, in particular, therapeutics that can induce cancer cells to undergo apoptosis and override anti-apoptotic signals provided in such cells.
In general, the invention features methods and reagents useful for inducing apoptosis in a cell. The methods and reagents of the invention are useful in treating cancers, and other proliferative diseases.
The present invention features nucleobase oligomers, particularly oligonucleotides, for use in modulating the function of a polynucleotide encoding an IAP. These oligomers reduce the amount of an IAP produced, allowing a cell normally expressing the IAP to undergo apoptosis. This is accomplished by providing nucleobase oligomers that specifically hybridize with one or more polynucleotides encoding an IAP. The specific hybridization of the nucleobase oligomer with an IAP polynucleotide (e.g., RNA, DNA) interferes with the normal function of that IAP polynucleotide, reducing the amount of IAP protein produced. This modulation of function of a target nucleic acid by compounds that specifically hybridize to the target is generally referred to as “antisense.”
In one aspect, the invention features a nucleobase oligomer of up to 30 nucleobases in length, the oligomer including at least eight consecutive nucleobases of a sequence selected from SEQ ID NOs: 1-99, 143, 147, 151, 163-260, 287, 289, and 300-460. Desirably, when administered to a cell, the oligomer inhibits expression of an IAP.
In certain embodiments, the nucleobase oligomer includes a sequence selected from SEQ ID NOs: 1-99, 143, 147, 151, 163-260, 287, 289, and 300-460. It is desirable that the nucleobase oligomer consists of (or essentially of) one or more of the foregoing SEQ ID NOs. For example, the nucleobase oligomer may be a XIAP antisense nucleic acid that includes a sequence chosen from SEQ ID NOs 97, 98, 99, 143, 147, 151, 287, and 289, a HIAP1 antisense nucleic acid that includes a sequence chosen from SEQ ID NOs 300-389, or a HIAP2 antisense nucleic acid includes a sequence chosen from SEQ ID NOs 390-460. In a particularly desirable embodiment, the invention features a nucleobase oligomer having eleven DNA residues flanked on each side by four 2′-O-methyl RNA residues, and consists of one of the following sequences: 5′-AUUGGTTC CAATGTGUUCU-3′ (SEQ ID NO: 155); 5′-ACACGACCGCTAAGAAACA-3′ (SEQ ID NO: 16); 5′-ACAGGACTACCACTTGGAA-3′ (SEQ ID NO: 157); 5′-UGCCAGTGTTGATGCUGAA-3′ (SEQ ID NO: 27); 5′-GCUGAGTCTCCATATUGCC-3′ (SEQ ID NO: 141); 5′-UCGGGTATATGGTGTCUGA-3′ (SEQ. ID NO: 41); 5′-AAGCACTGCACTTGGUCAC-3′ (SEQ ID NO: 47); 5′-CCGGCCCAAAACAAAGAAG-3′ (SEQ ID NO: 51); 5′-ACCCTGGATACCATTUAGC-3′ (SEQ ID NO: 63); 5′-UGUCAGTACATGTTGGCUC-3′ (SEQ ID NO: 161); and 5′-UGCACCCTGGATA CCAUUU-3′ (SEQ ID NO: 151).
A nucleobase oligomer of the present invention may include at least one modified linkage (e.g., a phosphorothioate, a methylphosphonate, a phosphotriester, a phosphorodithioate, or a phosphoselenate linkage), modified nucleobase (e.g., a 5-methyl cytosine), and/or a modified sugar moiety (e.g., a 2′-O-methoxyethyl group or a 2′-O-methyl group). In one embodiment, the oligomer is a chimeric oligomer (e.g., an oligonucleotide that includes DNA residues linked together by phosphorothioate or phosphodiester linkages, flanked on each side by at least one, two, three, or four 2′-O-methyl RNA residue linked together by a phosphorothioate linkage).
In another aspect, the invention features a method of enhancing apoptosis in a cell. This method includes the step of administering to the cell a nucleobase oligomer of the present invention so that expression of an IAP (e.g., XIAP, HIAP1, or HIAP2) is inhibited. The nucleobase oligomer may be, e.g., a component of an antisense compound, a double-stranded RNA, or a ribozyme. This administering step may be performed alone, or in combination with a second step (e.g., administration of a chemotherapeutic agent, a biological response modifying agent, and/or a chemosensitizer). The cell can be in vitro or in vivo. In one embodiment, the cell is a cancer cell (e.g., a human cancer cell) or a cell of lymphoid or myeloid origin.
In a related aspect, the invention features a method for treating an animal (e.g., a human) having a proliferative disease (e.g., a cancer, lymphoproliferative disorder, or myelodysplastic syndrome) or preventing the development of such a disease, by administering to the animal an effective amount of a nucleobase oligomer of the present invention.
The cancer may be, for example, acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, myelodysplastic syndrome, chronic lymphocytic leukemia, polycythemia vera, lymphoma, Hodgkin's disease, Waldenstrom's macroglobulinemia, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, or retinoblastoma. When treating a cancer, it may be desirable to also administer one or more chemotherapeutic agents, biological response modifying agents, and/or chemosensitizers. Desirably, the administration of one or more of these agents is within five days of the administration of the nucleobase oligomer. Exemplary chemotherapeutic agents are adriamycin (doxorubicin), vinorelbine, etoposide, taxol, and cisplatin. While any route of administration that results in an effective amount at the desired site may be used, particularly desirable routes are by intravenous and intratumoral administration.
In another aspect, the invention features a pharmaceutical composition that includes a nucleobase oligomer of the present invention and a pharmaceutically acceptable carrier. If desirable, the pharmaceutical composition may further include additional components (e.g., a colloidal dispersion system or a chemotherapeutic agent).
The invention also features a catalytic RNA molecule capable of cleaving XIAP, HIAP1, or HIAP2 mRNA. In desirable embodiments, the catalytic RNA molecule includes, in its binding arms, at least eight consecutive nucleobases corresponding to a nucleobase oligomer of the invention (e.g., a nucleobase sequence of any one of Tables 1, 2, 6, and 7). The RNA molecule is desirably in a hammerhead motif, but may also be in a hairpin, hepatitis delta virus, group 1 intron, VS RNA or RNAseP RNA motif.
The invention also features an expression vector including a nucleic acid encoding one or more catalytic RNA molecules of the invention positioned for expression in a mammalian cell.
The invention also features a method of treating an animal having a cancer or lymphoproliferative disorder by administering to the animal an effective amount of a catalytic RNA molecule described above, or an expression vector encoding such a catalytic RNA molecule.
In still another aspect, the invention features a double-stranded RNA molecule having between 21 and 29 nucleobases, wherein at least eight consecutive nucleobases corresponding to a sequence of any one of Tables 1, 2, 6, and 7 are present.
In a related aspect, the invention also features a double-stranded RNA molecule having between 50 and 70 nucleobases, the RNA molecule having a first domain of between 21 and 29 nucleobases that include least eight consecutive nucleobases corresponding to a sequence of any one of Tables 1, 2, 6, and 7; a second domain complementary to the first domain, and a loop domain situated between the first and second domains such that the first and second domains are capable of duplexing to form a double-stranded RNA molecule. The invention also features an expression vector (e.g., an adenoviral vector or a retroviral vector) encoding such a double stranded RNA.
The invention also features a method of treating an animal having a cancer or lymphoproliferative disorder by administering to the animal an effective amount of a double-stranded RNA molecule described above
By a “nucleobase oligomer” is meant a compound that includes a chain of at least eight nucleobases joined together by linkage groups. Included in this definition are natural and non-natural oligonucleotides, both modified and unmodified, as well as oligonucleotide mimetics such as Protein Nucleic Acids, locked nucleic acids, and arabinonucleic acids. Numerous nucleobases and linkage groups may be employed in the nucleobase oligomers of the invention, including those described in detail herein in the section entitled “Oligonucleotides and other nucleobase oligomers,” infra.
“Protein” or “polypeptide” or “polypeptide fragment” means any chain of more than two amino acids, regardless of post-translational modification (e.g., glycosylation or phosphorylation), constituting all or part of a naturally occurring polypeptide or peptide, or constituting a non-naturally occurring polypeptide or peptide.
“Apoptosis” means the process of cell death wherein a dying cell displays a set of well-characterized biochemical hallmarks that include cell membrane blebbing, cell soma shrinkage, chromatin condensation, and DNA laddering. Cells that die by apoptosis include neurons (e.g., during the course of neurodegenerative diseases such as stroke, Parkinson's disease, and Alzheimer's disease), cardiomyocytes (e.g., after myocardial infarction or over the course of congestive heart failure), and cancer cells (e.g., after exposure to radiation or chemotherapeutic agents). Environmental stress (e.g., hypoxic stress) that is not alleviated may cause a cell to enter the early phase of the apoptotic pathway, which is reversible (i.e., cells at the early stage of the apoptotic pathway can be rescued). At a later phase of apoptosis (the commitment phase), cells cannot be rescued, and, as a result, are committed to die.
Proteins and compounds known to stimulate and inhibit apoptosis in a diverse variety of cells are well known in the art. For example, intracellular expression and activation of the caspase (ICE) family induces or stimulates apoptotic cell death, whereas expression of the IAPs or some members of the Bcl-2 family inhibit apopiotic cell death. In addition, there are survival factors that inhibit cell death in specific cell types. For example, neurotrophic factors, such as NGF inhibit neuronal apoptosis.
By “IAP gene” is meant a gene encoding a polypeptide having at least one BIR domain and that is capable of modulating (inhibiting or enhancing) apoptosis in a cell or tissue when provided by other intracellular or extracellular delivery methods (see, e.g., U.S. Pat. No. 5,919,912). In preferred embodiments, the IAP gene is a gene having about 50% or greater nucleotide sequence identity (e.g., at least 85%, 90%, or 95%) to at least one of human or murine XIAP, HIAP1, or HIAP2 (each of which is described in U.S. Pat. No. 6,156,535). Preferably the region of sequence over which identity is measured is a region encoding at least one BIR domain and a ring zinc finger domain. Mammalian IAP genes include nucleotide sequences isolated from any mammalian source. Preferably the mammal is a human.
By “IAP protein” or “IAP polypeptide”is meant a polypeptide, or fragment thereof, encoded by an IAP gene.
By “IAP biological activity” is meant any activity known to be caused in vivo or in vitro by an IAP polypeptide.
By “enhancing apoptosis” is meant increasing the number of cells that apoptose in a given cell population (e.g., cancer cells, lymphocytes, fibroblasts, or any other cells). It will be appreciated that the degree of apoptosis enhancement provided by an apoptosis-enhancing compound in a given assay will vary, but that one skilled in the art can determine the statistically significant change in the level of apoptosis that identifies a nucleobase oligomer that enhances apoptosis otherwise limited by an IAP. Preferably, “enhancing apoptosis” means that the increase in the number of cells undergoing apoptosis is at least 10%, more preferably the increase is 25% or even 50%, and most preferably the increase is at least one-fold, relative to cells not administered a nucleobase oligomer of the invention but otherwise treated in a substantially similar manner. Preferably the sample monitored is a sample of cells that normally undergo insufficient apoptosis (i.e., cancer cells). Methods for detecting changes in the level of apoptosis (i.e., enhancement or reduction) are described herein.
By a nucleobase oligomer that “inhibits the expression” of a target gene (e.g., an IAP) is meant one that reduces the amount of a target mRNA, or protein encoded by such mRNA, by at least about 5%, more desirable by at least about 10%, 25%, or even 50%, relative to an untreated control. Methods for measuring both mRNA and protein levels are well-known in the art; exemplary methods are described herein.
“Hybridization” means hydrogen bonding, which may be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding, between complementary nucleobases. For example, adenine and thymine are complementary nucleobases that pair through the formation of hydrogen bonds.
By “proliferative disease” is meant a disease that is caused by or results in inappropriately high levels of cell division, inappropriately low levels of apoptosis, or both. For example, cancer is an example of a proliferative disease. Examples of cancers include, without limitation, leukemias (e.g., acute leukemia, acute lymphocytic leukemia, acute myelocytic leukemia, acute myeloblastic leukemia, acute promyelocytic leukemia, acute myelomonocytic leukemia, acute monocytic leukemia, acute erythroleukemia, chronic leukemia, chronic myelocytic leukemia, chronic lymphocytic leukemia), polycythemia vera, lymphoma (Hodgkin's disease, non-Hodgkin's disease), Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors such as sarcomas and carcinomas (e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatoma, nile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical cancer, uterine cancer, testicular cancer, lung carcinoma, small cell lung carcinoma, bladder carcinoma, epithelial carcinoma, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodenroglioma, schwannoma, meningioma, melanoma, neuroblastoma, and retinoblastoma). Lymphoproliferative disorders are also considered to be proliferative diseases.
Preferably, a nucleobase oligomer of the invention is capable of enhancing apoptosis and/or decreasing IAP mRNA or protein levels when present in a cell that normally does not undergo sufficient apoptosis. Preferably the increase is by at least 10%, relative to a control, more preferably 25%, and most preferably 1-fold or more. Preferably a nucleobase oligomer of the invention includes from about 8 to 30 nucleobases, wherein at least eight consecutive nucleobases are from a sequence selected from SEQ ID NOs: 1-99, 143, 147, 151, 163-260, 287, 289, and 300-460. A nucleobase oligomer of the invention may also contain, e.g., an additional 20, 40, 60, 85, 120, or more consecutive nucleobases that are complementary to an IAP polynucleotide. The nucleobase oligomer (or a portion thereof) may contain a modified backbone. Phosphorothioate, phosphorodithioate, and other modified backbones are known in the art. The nucleobase oligomer may also contain one or more non-natural linkages.
By “chemotherapeutic agent” is meant an agent that is used to kill cancer cells or to slow their growth. Accordingly, both cytotoxic and cytostatic agents are considered to be chemotherapeutic agents.
By “biological response modifying agent” is meant an agent that stimulates or restores the ability of the immune system to fight disease. Some, but not all, biological response modifying agents may slow the growth of cancer cells and thus are also considered to be chemotherapeutic agents.” Examples of biological response modifying agents are interferons (alpha, beta, gamma), interleukin-2, rituximab, and trastuzumab.
By “chemosensitizer” is meant an agent that makes tumor cells more sensitive to the effects of chemotherapy.
By “an effective amount” is meant the amount of a compound (e.g., a nucleobase oligomer) required to ameliorate the symptoms of a disease, inhibit the growth of the target cells, reduce the size or number of tumors, inhibit the expression of an IAP, or enhance apoptosis of target cells, relative to an untreated patient. The effective amount of active compound(s) used to practice the present invention for therapeutic treatment of abnormal proliferation (i.e., cancer) varies depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician or veterinarian will decide the appropriate amount and dosage regimen. Such amount is referred to as an “effective” amount.
By “lymphoproliferative disorder” is meant a disorder in which there is abnormal proliferation of cells of the lymphatic system (e.g., T-cells and B-cells), and includes multiple sclerosis, Crohn's disease, lupus erythematosus, rheumatoid arthritis, and osteoarthritis.
By “ribozyme” is meant an RNA that has enzymatic activity, possessing site specificity and cleavage capability for a target RNA molecule. Ribozymes can be used to decrease expression of a polypeptide. Methods for using ribozymes to decrease polypeptide expression are described, for example, by Turner et al., (Adv. Exp. Med. Biol. 465:303-318, 2000) and Norris et al., (Adv. Exp. Med. Biol. 465:293-301, 2000).
By “reporter gene” is meant a gene encoding a polypeptide whose expression may be assayed; such polypeptides include, without limitation, glucuronidase (GUS), luciferase, chloramphenicol transacetylase (CAT), and beta-galactosidase.
By “promoter” is meant a polynucleotide sufficient to direct transcription.
By “operably linked” is meant that a first polynucleotide is positioned adjacent to a second polynucleotide that directs transcription of the first polynucleotide when appropriate molecules (e.g., transcriptional activator proteins) are bound to the second polynucleotide.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims.
The present invention provides nucleobase oligomers that inhibit expression of an LAP, and methods for using them to induce apoptosis in a cell. The nucleobase oligomers of the present invention may also be used to form pharmaceutical compositions. The invention also features methods for enhancing apoptosis in a cell by administering an oligonucleotide of the invention in combination with a chemotherapeutic agent such as a cytotoxic agent, cytostatic agent, or biological response modifying agent (e.g., adriamycin, vinorelbine, etoposide, taxol, cisplatin, interferon, interleukin-2, monoclonal antibodies). The chemotherapeutic agent may be, for example. If desirable, a chemosensitizer (i.e., an agent that makes the proliferating cells more sensitive to the chemotherapy) may also be administered. Any combination of the foregoing agents may also be used to form a pharmaceutical composition. These pharmaceutical compositions may be used to treat a proliferative disease, for example, cancer or a lymphoproliferative disorder, or a symptom of a proliferative disease. The nucleobase oligomer of the invention may also be used in combination with radiotherapy for the treatment of cancer or other proliferative disease.
Activation of apoptosis in cancer cells offers novel, and potentially useful approaches to improve patient responses to conventional chemotherapy or radiotherapy. XIAP is the most potent member of the IAP gene family in terms of its ability to directly inhibit caspases and to suppress apoptosis. We investigated the effect of XIAP down-regulation by antisense (AS) oligonucleotides on human non-small cell lung cancer (NIH-H460) growth in vitro and in vivo. In cultured H460 human lung cancer cells, oligonucleotide G4 AS was identified as the most potent compound, effectively down-regulated XIAP mRNA by 55% and protein levels up to 60%, as determined by real-time RT-PCR and western blotting, respectively, and induced 60% cell death at 1.2 μM concentrations. In contrast, the scrambled control G4 oligonucleotide caused little XIAP loss and less than 10% cell death. Treatment with G4 AS induced apoptosis, as revealed by degradation of pro-caspase-3 and PARP proteins, with significant nuclear DNA condensation and fragmentation at 1.2 μM concentrations. Moreover, XIAP AS oligonucleotides sensitized H460 cells to the cytotoxic effects of doxorubicin, taxol, vinorelbine, and etoposide. In animal models, we demonstrate that G4 AS at 15 mg/kg had significant sequence-specific growth inhibitory effects on human H460 tumors in xenograft models of SCID/RAG2-immunodeficient mice by systemic intraperitoneal administration. Systemic AS ODN administration was associated with an 85% down-regulation of XIAP protein in tumor xenografts. The combination of 15 mg/kg G4 AS with 5 mg/kg vinorelbine significantly inhibited tumor growth, more than either agent alone. These studies indicate that down-regulation of XIAP is a potent death signal in lung carcinoma cells and is able to induce apoptosis in vitro as well as inhibit tumor growth in vivo. These studies support the contention that IAPs are suitable targets for cancer therapy in human non-small cell lung cancer, as well as other solid tumors.
Therapy
Therapy may be provided wherever cancer therapy is performed: at home, the doctor's office, a clinic, a hospital's outpatient department, or a hospital. Treatment generally begins at a hospital so that the doctor can observe the therapy's effects closely and make any adjustments that are needed. The duration of the therapy depends on the kind of cancer being treated, the age and condition of the patient, the stage and type of the patient's disease, and how the patient's body responds to the treatment. Drug administration may be performed at different intervals (e.g., daily, weekly, or monthly). Therapy may be given in on-and-off cycles that include rest periods so that the patient's body has a chance to build healthy new cells and regain its strength.
Depending on the type of cancer and its stage of development, the therapy can be used to slow the spreading of the cancer, to slow the cancer's growth, to kill or arrest cancer cells that may have spread to other parts of the body from the original tumor, to relieve symptoms caused by the cancer, or to prevent cancer in the first place.
As used herein, the terms “cancer” or “neoplasm” or “neoplastic cells” is meant a collection of cells multiplying in an abnormal manner. Cancer growth is uncontrolled and progressive, and occurs under conditions that would not elicit, or would cause cessation of, multiplication of normal cells.
A nucleobase oligomer of the invention, or other negative regulator of the IAP anti-apoptotic pathway, may be administered within a pharmaceutically-acceptable diluent, carrier, or excipient, in unit dosage form. Conventional pharmaceutical practice may be employed to provide suitable formulations or compositions to administer the compounds to patients suffering from a disease that is caused by excessive cell proliferation. Administration may begin before the patient is symptomatic. Any appropriate route of administration may be employed, for example, administration may be parenteral, intravenous, intraarterial, subcutaneous, intratumoral, intramuscular, intracranial, intraorbital, ophthalmic, intraventricular, intrahepatic, intracapsular, intrathecal, intracisternal, intraperitoneal, intranasal, aerosol, suppository, or oral administration. For example, therapeutic formulations may be in the form of liquid solutions or suspensions; for oral administration, formulations may be in the form of tablets or capsules; and for intranasal formulations, in the form of powders, nasal drops, or aerosols.
Methods well known in the art for making formulations are found, for example, in “Remington: The Science and Practice of Pharmacy” Ed. A. R. Gennaro, Lippincourt Williams & Wilkins, Philadelphia, Pa., 2000. Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for IAP modulatory compounds include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, for example, polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.
The formulations can be administered to human patients in therapeutically effective amounts (e.g., amounts which prevent, eliminate, or reduce a pathological condition) to provide therapy for a disease or condition. The preferred dosage of a nucleobase oligomer of the invention is likely to depend on such variables as the type and extent of the disorder, the overall health status of the particular patient, the formulation of the compound excipients, and its route of administration.
As described above, if desired, treatment with a nucleobase oligomer of the invention may be combined with therapies for the treatment of proliferative disease (e.g., radiotherapy, surgery, or chemotherapy).
For any of the methods of application described above, a nucleobase oligomer of the invention is desirably administered intravenously or is applied to the site of the needed apoptosis event (e.g., by injection).
Oligonucleotides and Other Nucleobase Oligomers
At least two types of oligonucleotides induce the cleavage of RNA by RNase H: polydeoxynucleotides with phosphodiester (PO) or phosphorothioate (PS) linkages. Although 2′-OMe-RNA sequences exhibit a high affinity for RNA targets, these sequences are not substrates for RNase H. A desirable oligonucleotide is one based on 2′-modified oligonucleotides containing oligodeoxynucleotide gaps with some or all internucleotide linkages modified to phosphorothioates for nuclease resistance. The presence of methylphosphonate modifications increases the affinity of the oligonucleotide for its target RNA and thus reduces the IC50. This modification also increases the nuclease resistance of the modified oligonucleotide. It is understood that the methods and reagents of the present invention may be used in conjunction with any technologies that may be developed, including covalently-closed multiple antisense (CMAS) oligonucleotides (Moon et al., Biochem J. 346:295-303, 2000; PCT Publication No. WO 00/61595), ribbon-type antisense (RiAS) oligonucleotides (Moon et al., J. Biol. Chem. 275:4647-4653, 2000; PCT Publication No. WO 00/61595), and large circular antisense oligonucleotides (U.S. Patent Application Publication No. US 2002/0168631 A1).
As is known in the art, a nucleoside is a nucleobase-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. For those nucleosides that include a pentofuranosyl sugar, the phosphate group can be linked to either the 2′, 3′ or 5′ hydroxyl moiety of the sugar. In forming oligonucleotides, the phosphate groups covalently link adjacent nucleosides to one another to form a linear polymeric compound. In turn, the respective ends of this linear polymeric structure can be further joined to form a circular structure; open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.
Specific examples of preferred nucleobase oligomers useful in this invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, nucleobase oligomers having modified backbones include those that retain a phosphorus atom in the backbone and those that do not have a phosphorus atom in the backbone. For the purposes of this specification, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone are also considered to be nucleobase oligomers.
Nucleobase oligomers that have modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl-phosphotriesters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity, wherein the adjacent pairs of nucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and free acid forms are also included. Representative United States patents that teach the preparation of the above phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.
Nucleobase oligomers having modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts. Representative United States patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439, each of which is herein incorporated by reference.
In other nucleobase oligomers, both the sugar and the internucleoside linkage, i.e., the backbone, are replaced with novel groups. The nucleobase units are maintained for hybridization with an IAP. One such nucleobase oligomer, is referred to as a Peptide Nucleic Acid (PNA). In PNA compounds, the sugar-backbone of an oligonucleotide is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The nucleobases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. Methods for making and using these nucleobase oligomers are described, for example, in “Peptide Nucleic Acids: Protocols and Applications” Ed. P. E. Nielsen, Horizon Press, Norfolk, United Kingdom, 1999. Representative United States patents that teach the preparation of PNAs include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262, each of which is herein incorporated by reference. Further teaching of PNA compounds can be found in Nielsen et al., Science, 1991, 254, 1497-1500.
In particular embodiments of the invention, the nucleobase oligomers have phosphorothioate backbones and nucleosides with heteroatom backbones, and in particular —CH2—NH—O—CH2—, —CH2—N(CH3)—O—CH2— (known as a methylene (methylimino) or MMI backbone), —CH2—O—N(CH3)—CH2—, —CH2—N(CH3)—N(CH3)—CH2—, and —O—N(CH3)—CH2—CH2—. In other embodiments, the oligonucleotides have morpholino backbone structures described in U.S. Pat. No. 5,034,506.
Nucleobase oligomers may also contain one or more substituted sugar moieties. Nucleobase oligomers comprise one of the following at the 2′ position: OH; F; O—, S—, or N-alkyl; O—, S—, or N-alkenyl; O—, S— or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl, and alkynyl may be substituted or unsubstituted C1 to C10 alkyl or C2 to C10 alkenyl and alkynyl. Particularly preferred are O[(CH2)nO]mCH3, O(CH2)nOCH3, O(CH2)nNH2, O(CH2)nCH3, O(CH2)nONH2, and O(CH2)nON[(CH2)nCH3)]2, where n and m are from 1 to about 10. Other preferred nucleobase oligomers include one of the following at the 2′ position: C1 to C10 lower alkyl, substituted lower alkyl, alkaryl, aralkyl, O-alkaryl, or O-aralkyl, SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, NH2, heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleaving group, a reporter group, an intercalator, a group for improving the pharmacokinetic properties of a nucleobase oligomer, or a group for improving the pharmacodynamic properties of an nucleobase oligomer, and other substituents having similar properties. Preferred modifications are 2′-O-methyl and 2′-methoxyethoxy (2′-O—CH2CH2OCH3, also known as 2′-O-(2-methoxyethyl) or 2′-MOE). Another desirable modification is 2′-dimethylaminooxyethoxy (i.e., O(CH2)2ON(CH3)2), also known as 2′-DMAOE. Other modifications include, 2′-aminopropoxy (2′-OCH2CH2CH2NH2) and 2′-fluoro (2′-F). Similar modifications may also be made at other positions on an oligonucleotide or other nucleobase oligomer, particularly the 3′ position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linked oligonucleotides and the 5′ position of 5′ terminal nucleotide. Nucleobase oligomers may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative United States patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, each of which is herein incorporated by reference in its entirety.
Nucleobase oligomers may also include nucleobase modifications or substitutions. As used herein, “unmodified” or “natural” nucleobases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine; 2-propyl and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine and 2-thiocytosine; 5-halouracil and cytosine; 5-propynyl uracil and cytosine; 6-azo uracil, cytosine and thymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines; 5-halo (e.g., 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-deazaadenine; and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in U.S. Pat. No. 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. I., ed. John Wiley & Sons, 1990, those disclosed by Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613, and those disclosed by Sanghvi, Y. S., Chapter 15, Antisense Research and Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993. Certain of these nucleobases are particularly useful for increasing the binding affinity of an antisense oligonucleotide of the invention. These include 5-substituted pyrimidines, 6-azapyrimidines, and N-2, N-6 and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine. 5-methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2.degree. C. (Sanghvi, Y. S., Crooke, S. T. and Lebleu, B., eds., Antisense Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are desirable base substitutions, even more particularly when combined with 2′-O-methoxyethyl or 2′-O-methyl sugar modifications. Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066; 5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908; 5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091; 5,614,617; 5,681,941; and 5,750,692, each of which is herein incorporated by reference.
Another modification of a nucleobase oligomer of the invention involves chemically linking to the nucleobase oligomer one or more moieties or conjugates that enhance the activity, cellular distribution, or cellular uptake of the oligonucleotide. Such moieties include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 86:6553-6556, 1989), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let, 4:1053-1060, 1994), a thioether, e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 660:306-309, 1992; Manoharan et al., Bioorg. Med. Chem. Let., 3:2765-2770, 1993), a thiocholesterol (Oberhauser et al., Nucl. Acids Res., 20:533-538: 1992), an aliphatic chain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J., 10:1111-1118, 1991; Kabanov et al., FEBS Lett., 259:327-330, 1990; Svinarchuk et al., Biochimie, 75:49-54, 1993), a phospholipid, e.g., di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3—H-phosphonate (Manoharan et al., Tetrahedron Lett., 36:3651-3654, 1995; Shea et al., Nucl. Acids Res., 18:3777-3783, 1990), a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 14:969-973, 1995), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 36:3651-3654, 1995), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1264:229-237, 1995), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 277:923-937, 1996. Representative United States patents that teach the preparation of such nucleobase oligomer conjugates include U.S. Pat. Nos. 4,587,044; 4,605,735; 4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,828,979; 4,835,263; 4,876,335; 4,904,582; 4,948,882; 4,958,013; 5,082,830; 5,109,124; 5,112,963; 5,118,802; 5,138,045; 5,214,136; 5,218,105; 5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,414,077; 5,416,203, 5,451,463; 5,486,603; 5,510,475; 5,512,439; 5,512,667; 5,514,785; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,565,552; 5,567,810; 5,574,142; 5,578,717; 5,578,718; 5,580,731; 5,585,481; 5,587,371; 5,591,584; 5,595,726; 5,597,696; 5,599,923; 5,599,928; 5,608,046; and 5,688,941, each of which is herein incorporated by reference.
The present invention also includes nucleobase oligomers that are chimeric compounds. “Chimeric” nucleobase oligomers are nucleobase oligomers, particularly oligonucleotides, that contain two or more chemically distinct regions, each made up of at least one monomer unit, i.e., a nucleotide in the case of an oligonucleotide. These nucleobase oligomers typically contain at least one region where the nucleobase oligomer is modified to confer, upon the nucleobase oligomer, increased resistance to nuclease degradation, increased cellular uptake, and/or increased binding affinity for the target nucleic acid. An additional region of the nucleobase oligomer may serve as a substrate for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNase H is a cellular endonuclease which cleaves the RNA strand of an RNA:DNA duplex. Activation of RNase H, therefore, results in cleavage of the RNA target, thereby greatly enhancing the efficiency of nucleobase oligomer inhibition of gene expression. Consequently, comparable results can often be obtained with shorter nucleobase oligomers when chimeric nucleobase oligomers are used, compared to phosphorothioate deoxyoligonucleotides hybridizing to the same target region.
Chimeric nucleobase oligomers of the invention may be formed as composite structures of two or more nucleobase oligomers as described above. Such nucleobase oligomers, when oligonucleotides, have also been referred to in the art as hybrids or gapmers. Representative United States patents that teach the preparation of such hybrid structures include U.S. Pat. Nos. 5,013,830; 5,149,797; 5,220,007; 5,256,775; 5,366,878; 5,403,711; 5,491,133; 5,565,350; 5,623,065; 5,652,355; 5,652,356; and 5,700,922, each of which is herein incorporated by reference in its entirety.
The nucleobase oligomers used in accordance with this invention may be conveniently and routinely made through the well-known technique of solid phase synthesis. Equipment for such synthesis is sold by several vendors including, for example, Applied Biosystems (Foster City, Calif.). Any other means for such synthesis known in the art may additionally or alternatively be employed. It is well known to use similar techniques to prepare oligonucleotides such as the phosphorothioates and alkylated derivatives.
The nucleobase oligomers of the invention may also be admixed, encapsulated, conjugated or otherwise associated with other molecules, molecule structures or mixtures of compounds, as for example, liposomes, receptor targeted molecules, oral, rectal, topical or other formulations, for assisting in uptake, distribution and/or absorption. Representative United States patents that teach the preparation of such uptake, distribution and/or absorption assisting formulations include U.S. Pat. Nos. 5,108,921; 5,354,844; 5,416,016; 5,459,127; 5,521,291; 5,543,158; 5,547,932; 5,583,020; 5,591,721; 4,426,330; 4,534,899; 5,013,556; 5,108,921; 5,213,804; 5,227,170; 5,264,221; 5,356,633; 5,395,619; 5,416,016; 5,417,978; 5,462,854; 5,469,854; 5,512,295; 5,527,528; 5,534,259; 5,543,152; 5,556,948; 5,580,575; and 5,595,756, each of which is herein incorporated by reference.
The nucleobase oligomers of the invention encompass any pharmaceutically acceptable salts, esters, or salts of such esters, or any other compound that, upon administration to an animal, is capable of providing (directly or indirectly) the biologically active metabolite or residue thereof. Accordingly, for example, the disclosure is also drawn to prodrugs and pharmaceutically acceptable salts of the compounds of the invention, pharmaceutically acceptable salts of such prodrugs, and other bioequivalents.
The term “prodrug” indicates a therapeutic agent that is prepared in an inactive form that is converted to an active form (i.e., drug) within the body or cells thereof by the action of endogenous enzymes or other chemicals and/or conditions. In particular, prodrug versions of the oligonucleotides of the invention can be prepared as SATE [(S-acetyl-2-thioethyl)phosphate] derivatives according to the methods disclosed in PCT publication Nos. WO 93/24510 or WO 94/26764.
The term “pharmaceutically acceptable salts” refers to salts that retain the desired biological activity of the parent compound and do not impart undesired toxicological effects thereto. Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Examples of metals used as cations are sodium, potassium, magnesium, calcium, and the like. Examples of suitable amines are N,N′-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, dicyclohexylamine, ethylenediamine, N-methylglucamine, and procaine (see, for example, Berge et al., J. Pharma Sci., 66:1-19, 1977). The base addition salts of acidic compounds are prepared by contacting the free acid form with a sufficient amount of the desired base to produce the salt in the conventional manner. The free acid form may be regenerated by contacting the salt form with an acid and isolating the free acid in the conventional manner. The free acid forms differ from their respective salt forms somewhat in certain physical properties such as solubility in polar solvents, but otherwise the salts are equivalent to their respective free acid for purposes of the present invention. As used herein, a “pharmaceutical addition salt” includes a pharmaceutically acceptable salt of an acid form of one of the components of the compositions of the invention. These include organic or inorganic acid salts of the amines. Preferred acid salts are the hydrochlorides, acetates, salicylates, nitrates and phosphates. Other suitable pharmaceutically acceptable salts are well known to those skilled in the art and include basic salts of a variety of inorganic and organic acids, such as, for example, with inorganic acids, such as for example hydrochloric acid, hydrobromic acid, sulfuric acid or phosphoric acid; with organic carboxylic, sulfonic, sulfo or phospho acids or N-substituted sulfamic acids, for example acetic acid, propionic acid, glycolic acid, succinic acid, maleic acid, hydroxymaleic acid, methylmaleic acid, fumaric acid, malic acid, tartaric acid, lactic acid, oxalic acid, gluconic acid, glucaric acid, glucuronic acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, salicylic acid, 4-aminosalicylic acid, 2-phenoxybenzoic acid, 2-acetoxybenzoic acid, embonic acid, nicotinic acid or isonicotinic acid; and with amino acids, such as the 20 alpha-amino acids involved in the synthesis of proteins in nature, for example glutamic acid or aspartic acid, and also with phenylacetic acid, methanesulfonic acid, ethanesulfonic acid, 2-hydroxyethanesulfonic acid, ethane-1,2-disulfonic acid, benzenesulfonic acid, 4-methylbenzenesulfonic acid, naphthalene-2-sulfonic acid, naphthalene-1,5-disulfonic acid, 2- or 3-phosphoglycerate, glucose-6-phosphate, N-cyclohexylsulfamic acid (with the formation of cyclamates), or with other acid organic compounds, such as ascorbic acid. Pharmaceutically acceptable salts of compounds may also be prepared with a pharmaceutically acceptable cation. Suitable pharmaceutically acceptable cations are well known to those skilled in the art and include alkaline, alkaline earth, ammonium and quaternary ammonium cations. Carbonates or hydrogen carbonates are also possible.
For oligonucleotides and other nucleobase oligomers, suitable pharmaceutically acceptable salts include (i) salts formed with cations such as sodium, potassium, ammonium, magnesium, calcium, polyamines such as spermine and spermidine, etc.; (ii) acid addition salts formed with inorganic acids, for example hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid and the like; (iii) salts formed with organic acids such as, for example, acetic acid, oxalic acid, tartaric acid, succinic acid, maleic acid, fumaric acid, gluconic acid, citric acid, malic acid, ascorbic acid, benzoic acid, tannic acid, palmitic acid, alginic acid, polyglutamic acid, naphthalenesulfonic acid, methanesulfonic acid, p-toluenesulfonic acid, naphthalenedisulfonic acid, polygalacturonic acid, and the like; and (iv) salts formed from elemental anions such as chlorine, bromine, and iodine.
The present invention also includes pharmaceutical compositions and formulations that include the nucleobase oligomers of the invention. The pharmaceutical compositions of the present invention may be administered in a number of ways depending upon whether local or systemic treatment is desired and upon the area to be treated. Administration may be topical (including ophthalmic and to mucous membranes including vaginal and rectal delivery), pulmonary, e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), oral, or parenteral. Parenteral administration includes intravenous, intraarterial, subcutaneous, intraperitoneal, or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular, administration.
Locked Nucleic Acids
Locked nucleic acids (LNAs) are nucleobase oligomers that can be employed in the present invention. LNAs contain a 2′O, 4′-C methylene bridge that restrict the flexibility of the ribofuranose ring of the nucleotide analog and locks it into the rigid bicyclic N-type conformation. LNAs show improved resistance to certain exo- and endonucleases and activate RNAse H, and can be incorporated into almost any nucleobase oligomer. Moreover, LNA-containing nucleobase oligomers can be prepared using standard phosphoramidite synthesis protocols. Additional details regarding LNAs can be found in PCT publication No. WO 99/14226 and U.S. Patent Application Publication No. US 2002/0094555 A1, each of which is hereby incorporated by reference.
Arabinonucleic Acids
Arabinonucleic acids (ANAs) can also be employed in methods and reagents of the present invention. ANAs are nucleobase oligomers based on D-arabinose sugars instead of the natural D-2′-deoxyribose sugars. Underivatized ANA analogs have similar binding affinity for RNA as do phosphorothioates. When the arabinose sugar is derivatized with fluorine (2′ F-ANA), an enhancement in binding affinity results, and selective hydrolysis of bound RNA occurs efficiently in the resulting ANA/RNA and F-ANA/RNA duplexes. These analogs can be made stable in cellular media by a derivatization at their termini with simple L sugars. The use of ANAs in therapy is discussed, for example, in Damha et al., Nucleosides Nucleotides & Nucleic Acids 20: 429-440, 2001.
Delivery of Nucleobase Oligomers
We demonstrate herein that naked oligonucleotides are capable on entering tumor cells and inhibiting IAP expression. Nonetheless, it may be desirable to utilize a formulation that aids in the delivery of oligonucleotides or other nucleobase oligomers to cells (see, e.g., U.S. Pat. Nos. 5,656,611, 5,753,613, 5,785,992, 6,120,798, 6,221,959, 6,346,613, and 6,353,055, each of which is hereby incorporated by reference).
Ribozymes
Catalytic RNA molecules or ribozymes that include an antisense IAP sequence of the present invention can be used to inhibit expression of an IAP polynucleotide in vivo. The inclusion of ribozyme sequences within antisense RNAs confers RNA-cleaving activity upon them, thereby increasing the activity of the constructs. The design and use of target RNA-specific ribozymes is described in Haseloff et al., Nature 334:585-591. 1988, and U.S. Patent Application Publication No. 2003/0003469 A1, each of which is incorporated by reference.
Accordingly, the invention also features a catalytic RNA molecule that includes, in the binding arm, an antisense RNA having between eight and nineteen consecutive nucleobases corresponding to a sequence of any one of Tables 1, 2, 6, and 7. In preferred embodiments of this invention, the catalytic nucleic acid molecule is formed in a hammerhead or hairpin motif, but may also be formed in the motif of a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) or Neurospora VS RNA. Examples of such hammerhead motifs are described by Rossi et al., Aids Research and Human Retroviruses, 8:183, 1992. Example of hairpin motifs are described by Hampel et al., “RNA Catalyst for Cleaving Specific RNA Sequences,” filed Sep. 20, 1989, which is a continuation-in-part of U.S. Ser. No. 07/247,100 filed Sep. 20, 1988, Hampel and Tritz, Biochemistry, 28:4929, 1989, and Hampel et al., Nucleic Acids Research, 18: 299, 1990. An example of the hepatitis delta virus motif is described by Perrotta and Been, Biochemistry, 31:16, 1992. The RNaseP motif is described by Guerrier-Takada et al., Cell, 35:849, 1983. The Neurospora VS RNA ribozyme motif is described by Collins et al. (Saville and Collins, Cell 61:685-696, 1990; Saville and Collins, Proc. Natl. Acad. Sci. USA 88:8826-8830, 1991; Collins and Olive, Biochemistry 32:2795-2799, 1993). These specific motifs are not limiting in the invention and those skilled in the art will recognize that all that is important in an enzymatic nucleic acid molecule of this invention is that it has a specific substrate binding site which is complementary to one or more of the target gene RNA regions, and that it have nucleotide sequences within or surrounding that substrate binding site which impart an RNA cleaving activity to the molecule.
RNA interference
The nucleobase oligomers of the present invention may be employed in double-stranded RNAs for RNA interference (RNAi)-mediated knock-down of IAP expression. RNAi is a method for decreasing the cellular expression of specific proteins of interest (reviewed in Tuschl, Chembiochem 2:239-245, 2001; Sharp, Genes & Devel. 15:485-490, 2000; Hutvagner and Zamore, Curr. Opin. Genet. Devel. 12:225-232, 2002; and Hannon, Nature 418:244-251, 2002). In RNAi, gene silencing is typically triggered post-transcriptionally by the presence of double-stranded RNA (dsRNA) in a cell. This dsRNA is processed intracellularly into shorter pieces called small interfering RNAs (siRNAs). The introduction of siRNAs into cells either by transfection of dsRNAs or through expression of siRNAs using a plasmid-based expression system is increasingly being used to create loss-of-function phenotypes in mammalian cells.
In one embodiment of the invention, double-stranded RNA (dsRNA) molecule is made that includes between eight and nineteen consecutive nucleobases of a nucleobase oligomer of the invention. The dsRNA can be two distinct strands of RNA that have duplexed, or a single RNA strand that has self-duplexed (small hairpin (sh)RNA). Typically, dsRNAs are about 21 or 22 base pairs, but may be shorter or longer (up to about 29 nucleobases) if desired. dsRNA can be made using standard techniques (e.g., chemical synthesis or in vitro transcription). Kits are available, for example, from Ambion (Austin, Tex.) and Epicentre (Madison, Wis.). Methods for expressing dsRNA in mammalian cells are described in Brummelkamp et al. Science 296:550-553, 2002; Paddison et al. Genes & Devel. 16:948-958, 2002. Paul et al. Nature Biotechnol. 20:505-508, 2002; Sui et al. Proc. Natl. Acad. Sci. USA 99:5515-5520, 2002; Yu et al. Proc. Natl. Acad. Sci. USA 99:6047-6052, 2002; Miyagishi et al. Nature Biotechnol. 20:497-500, 2002; and Lee et al. Nature Biotechnol. 20:500-505 2002, each of which is hereby incorporated by reference.
Small hairpin RNAs consist of a stem-loop structure with optional 3′ UU-overhangs. While there may be variation, stems can range from 21 to 31 bp (desirably 25 to 29 bp), and the loops can range from 4 to 30 bp (desirably 4 to 23 bp). For expression of shRNAs within cells, plasmid vectors containing either the polymerase III H1-RNA or U6 promoter, a cloning site for the stem-looped RNA insert, and a 4-5-thymidine transcription termination signal can be employed. The Polymerase III promoters generally have well-defined initiation and stop sites and their transcripts lack poly(A) tails. The termination signal for these promoters is defined by the polythymidine tract, and the transcript is typically cleaved after the second uridine. Cleavage at this position generates a 3′ UU overhang in the expressed shRNA, which is similar to the 3′ overhangs of synthetic siRNAs. Additional methods for expressing the shRNA in mammalian cells are described in the references cited above.
The following examples are to illustrate the invention. They are not meant to limit the invention in any way.
We selected 96 and 98, mostly non-overlapping, 19-mer nucleobase sequences for human XIAP and HIAP1, respectively, based on the selection criteria listed below. In the case of XIAP, we selected 96 sequences (each being 19 nucleobases in length) (SEQ ID NOs: 1 through 96; Table 1), from a region approximately 1 kb upstream of the start codon to approximately 1 kb downstream of the stop codon of the cDNA sequence. This blanketed approximately 50% of the coding region, and immediate 5′ and 3′ UTR sequences (i.e., 96 19-mers span 1.8 kb of sequence, while the targeted region is approximately 3.5 kb in length, comprised of a coding region of 1.5 kb plus 1 kb at either side of UTR sequences).
Note that in any of the foregoing nucleobase oligomers, or any other nucleobase oligomers described herein, each nucleobase may independently be a DNA residue or RNA residue, such as a 2′-O-methyl or 2′-O-methoxyelthyl RNA residue. For example, the nucleobase sequence of SEQ ID NO: 3 may be, for example, 5′-CAGATATATATG TAACACT-3′,5′-CAGATATATATGTAACACU-3′, or 5′-mCmAGATATATATGTA ACAmCmU-3′ (wherein mX represents a 2′-O-methyl X residue). Additional modified nucleobases are known in the art. The linkages may be phosphodiester (PO), phosphorothioate (PS), or methylphosphonate (MP) linkages, or may have a mixed backbone (MB). The backbone may be any suitable backbone that allows hybridization of the nucleobase oligomer to the target IAP polynucleotide. Exemplary backbones are described herein. In other embodiments, the nucleobase oligomers include acridine-protected linkages, cholesteryl or psoralen components, C5-propynyl pyrimidines, or C5-methyl pyrimidines. Suitable modifications to the nucleobase oligomers of the invention include those described above, as well as those in U.S. Patent Application Publication No. US 2002/0128216 A1, hereby incorporated by reference.
Examples of nucleobase oligomers are provided in Table 2, below (wherein mX represents a 2′-O-methyl X RNA residue).
A similar identification approach was taken for designing nucleobase oligomers against HIAP1. Initially, 98 19-mer nucleobase oligomers were chosen (SEQ ID NOs: 163-260; Table 3). Of these 98 nucleobase oligomers targeted to the HIAP1 sequence, fifteen (SEQ ID NOs: 163-170, 173, 179, 202, 222, 223, 247, and 259) were selected for further evaluation. These fifteen candidate nucleobase oligomers included four nucleobase oligomers targeting the coding region (SEQ ID NOs: 202, 222, 223, and 247), one nucleobase oligomer targeting the 3′ UTR (SEQ ID NO: 259), seven nucleobase oligomers targeting the 5′ UTR (SEQ ID NOs: 166-170, 173, and 179; one of the seven nucleobase oligomers overlapped the start codon), and three other oligonucleotides (SEQ ID NOs: 163-165) that were designed to target an intronic segment of the 5′ UTR.
Nucleobase Oligomer Selection Criteria
The computer program OLIGO (previously distributed by National Biosciences Inc.) was used to select candidate nucleobase oligomers based on the following criteria:
The ability of nucleobase oligomers to inhibit IAP expression was tested using oligonucleotides as exemplary nucleobase oligomers. The oligonucleotides were synthesized by IDT (Integrated DNA Technologies, USA) as chimeric, second-generation oligonucleotides, consisting of a core of phosphodiester DNA residues flanked on either side by two 2′-O-methyl RNA residues with a phosphorothioate linkage between the flanking RNA residues. The oligonucleotides were provided in a 96-well plate, as well as matching tubes, with a minimum of 12 ODs of nucleobase oligomer, which provided ample material for transfections (greater than a hundred assays in the 96-well format) when the detection method is a sensitive method, such as TaqMan quantitative PCR, or an ELISA. Once the positive hits were identified (see below), oligonucleotides were re-synthesized with three, instead of two, flanking RNA residues to further increase stability/nuclease resistance. In addition, for validation purposes, appropriate controls (such as scrambled, 4-base mismatch, and reverse polarity oligonucleotides) were synthesized for some of the targets that yielded the highest activity.
Our approach to identifying nucleobase oligomers capable of inhibiting expression of an IAP was to screen the above-described oligonucleotide libraries for specific decreases (knock-down) of the RNA and/or protein for the specific IAP gene targeted. Any number of standard assays may be used to detect RNA and protein levels in cells. For example, RNA levels can be measured using standard northern blot analysis or RT-PCR techniques. Protein levels can be measured, for example, by standard western blot analyses or immunoprecipitation techniques. Alternatively, cells administered an antisense IAP nucleic acid may be examined for cell viability, according to methods described, for example, in U.S. Pat. Nos. 5,919,912, 6,156,535, and 6,133,437, incorporated herein by reference.
We used TaqMan quantitative PCR (described below) to assay for changes in mRNA levels after oligonucleotide treatment. We employed ELISA for determining XIAP protein levels and western blotting for determining HIAP1 protein levels. Transfection conditions were optimized with Lipofectamine plus or Lipofectamine 2000 (Life Technologies, Canada) on T24 bladder carcinoma cells or H460 non-small cell lung carcinoma cells, or lipofectin on SF-295 glioblastoma cells, using a fluorescein-tagged sense oligonucleotide (5′-mGmAGAAGATGACTGGTAAmCmA-3′; SEQ ID NO: 261) from XIAP spanning the start codon as a control. The results were visualized and gauged by epi-fluorescence microscopy. In the case of T24 cells, transfections were further optimized based on the ability of a published oligonucleotide to downregulate survivin expression (Li et al., Nat. Cell Biol. 1:461-466, 1999) (5′-U/TGTGCTATTCTGTGAA U/TU/T-3′ SEQ ID NO: 262). We optimized the transfection conditions based on the TaqMan results of survivin RNA knock-down detected with PCR primers and fluorescent probe, described in detail below. Optimal conditions for oligonucleotide uptake by the cells were found to be 940 nM oligonucleotide and 4 μL PLUS reagent and 0.8 μL Lipofectamine in a total of 70 μL for three hours. We then applied these conditions to screen for XIAP protein knock-down using the oligo library against T24 cells.
HIAP1 knock-down was studied in SF-295 cells because these cells had easily detectable and discernable 70 kDa HIAP1 protein, while many cell lines do not express high levels of the protein, or are not distinguishable from the large amounts of the similarly sized 68 kDa HIAP2 protein.
Real-Time PCR
RNA was extracted from cells lysed in RLT buffer (QIAGEN, Valencia, Calif.), and purified using QIAGEN RNeasy columns/kits. Real-time quantitative PCR was performed on a Perkin-Elmer ABI 7700 Prism PCR machine. RNA was reverse transcribed and amplified according to the TaqMan Universal PCR Master Mix protocol of PE Biosystems, using primers and probes designed to specifically recognize XIAP, HIAP1, survivin, or GAPDH. For human survivin, the forward primer was 5′-TCTGCTTCAAGGAGCTGGAA-3′ (SEQ ID NO: 263), the reverse primer was 5′-GAAAGGAAAGCGCAACCG-3′ (SEQ ID NO: 264), and the probe was 5′-(FAM)AGCCAGATGACGACCCCATAGAGGAACATA(TAMRA)-3′ (SEQ ID NO: 265). For human HIAP1, the forward primer was 5′-TGGAGATGATCCATGGGTTCA-3′ (SEQ ID NO: 266), the reverse primer was 5′-GAACTCCTGTCCTTTAATTCTTATCAAGT-3′ (SEQ ID NO: 267), and the probe was 5′-(FAM)CTCACACCTTGGAAACCACTTGGCATG (TAMRA)-3′ (SEQ ID NO: 268). For human XIAP, the forward primer was 5′-GGTGATAAAGTAAAGTGCTTTCACTGT-3′ (SEQ ID NO: 269), the reverse primer was 5′-TCAGTAGTTCTTACCAGACACTCCTCAA-3′ (SEQ ID NO: 270), and the probe was 5′-(FAM)CAACATGCTAAATGGTATCCAGGGTGCAAATATC(TAMRA)-3′ (SEQ ID NO: 271). For human GAPDH, the forward primer was 5′-GAAGGTGAAGGTCGGAGTC-3′ (SEQ ID NO: 272), the reverse primer was 5′-GAAGATGGTGATGGGATTC-3′ (SEQ ID NO: 273), and the probe was 5′-(JOE)CAAGCTTCCCGTTCTCA GCC(TAMRA)-3′ (SEQ ID NO: 274). FAM is 6-carboxyfluoroscein, JOE is 6-carboxy-4,5-dichloro-2,7-dimethoxyfluoroscein, and TAMRA is 6-carboxy-N,N,N′,N′-tetramethylrhodamine. FAM and JOE are 5′ reporter dyes, while TAMRA is a 3′ quencher dye.
Relative quantification of gene expression was performed as described in the PE Biosystems manual using GAPDH as an internal standard. The comparative Ct (cycle threshold) method was used for relative quantitation of IAP mRNA levels compared to GAPDH mRNA levels. Briefly, real-time fluorescence measurements were taken at each PCR cycle and the threshold cycle (Ct) value for each sample was calculated by determining the point at which fluorescence exceeded a threshold limit of 30 times the baseline standard deviation. The average baseline value and the baseline SD are calculated starting from the third cycle baseline value and stopping at the baseline value three cycles before the signal starts to exponentially rise. The PCR primers and/or probes for the specific IAPs were designed to span at least one exon-intron boundary separated by 1 kb or more of genomic DNA, to reduce the possibility of amplifying and detecting genomic DNA contamination. The specificity of the signal, and possible contamination from DNA, were verified by treating some RNA samples with either DNase or RNase, prior to performing the reverse transcription and PCR reaction steps.
XIAP ELISA and HIAP1 Western Immunoblots
A standard colorimetric XIAP ELISA assay was performed using an affinity-purified rabbit polyclonal antibody to XIAP as a capture antibody, and was detected with a XIAP monoclonal antibody (MBL, Japan) and a biotinylated anti-mouse Ig antibody and horseradish peroxidase-conjugated streptavidin and TMB substrate. Alternatively, a polyclonal XIAP or HIAP1 antibody may be used to measure XIAP or HIAP1 protein levels, respectively.
HIAP1 was detected on a western immunoblot using an affinity-purified anti-rat HIAP1 rabbit polyclonal antibody as a primary antibody and was detected by ECL (Amersham) on X-ray film with a secondary horseradish-peroxidase-conjugated anti-rabbit Ig antibody and a chemiluminescent substrate. The anti-HIAP1 polyclonal antibody is raised against a GST-fusion of the rat HIAP1. This antibody cross-reacts with both human and murine HIAP1 and HIAP2.
The XIAP synthetic library of 96 antisense oligonucleotides was first screened for decreases in XIAP protein levels. Specifically, T24 cells (1.5×104 cells/well) were seeded in wells of a 96-well plate on day 1, and were cultured in antibiotic-free McCoy's medium for 24 hours. On day 2, the cells were transfected with XIAP antisense oligonucleotides as described above (oligonucleotides are labeled according to their plated position, i.e., A1 to H12, and include two repeats, A13 and B13 that contain lyophilized DNA pellets that stuck to the sealing membrane). Briefly, the nucleobase oligomers were diluted in 10 μl/well of serum-free, antibiotic-free McCoy's medium and then PLUS reagent was added. Lipofectamine was diluted in 10 μl/well of serum-free, antibiotic-free McCoy's medium, and both mixes were incubated for 15 minutes at room temperature. The mixes were then combined and incubated for 15 minutes at room temperature.
In the meantime, the complete medium was removed from the cells and 50 μl/well of serum-free, antibiotic-free medium was added to the cells. The transfection mixes were added to the well, and the cells were incubated for three hours. Then 30 μl/well of serum-free, antibiotic-free medium and 100 μl/well of antibiotic-free complete medium, containing 20% fetal bovine serum were added to each well.
At day 3, XIAP RNA levels were measured using quantitative real-time PCR techniques, as described above. At day 4, XIAP protein levels were measured by ELISA (
The oligonucleotide library was also screened for decreases in RNA levels, using TaqMan-specific PCR primers and fluorescent probes at the appropriate optimal time, using the primers and probes described above. Time course experiments determined mRNA to be optimally decreased at 6 to 9 hours. These results agree well with the protein results.
The first screen (although performed at a sub-optimal time point when XIAP levels are returning to normal, possibly due to an outgrowth of non-transfected cells) identified 16 antisense oligonucleotides (Table 1: C2, E2, E3, F3, C4, D4, E4, F4, G4, C5, D5, B6, F6, D7, D8, F8) out of the 96 nucleobase oligomers tested that showed some decrease in XIAP protein levels relative to total protein, compared to mock (no nucleobase oligomer) transfection levels (
The 16 antisense nucleobase oligomers that showed some decrease in relative XIAP protein levels compared to mock transfection, were re-tested alone or in combination, with one control nucleobase oligomer (D2) included, for their ability to knock-down XIAP protein at a more optimal time point (12 hours) based on the above described time course studies (
There was a good correlation between the ability of a nucleobase oligomer to decrease XIAP protein levels (
A portion of the 96 nucleobase oligomers depicted in Table 1 were rescreened for their ability to knock-down XIAP mRNA in NCI-H460 cells at 9 hours post-transfection. The data are summarized in Table 4, below.
We also determined whether 4×4 MBOs (all PS, DNA residues flanked on both sides by four 2′-O-methyl RNA residues) were capable of knocking-down XIAP protein in H460 cells. As shown in
We investigated if XIAP antisense nucleobase oligomers could chemosensitize the highly drug resistant T24 cells to traditional chemotherapeutic agents, such as adriamycin or cisplatin. Antisense oligonucleotides were chosen to represent some of the different XIAP target regions and tested for their cytotoxic effects, alone or in combination with other oligonucleotides or drugs. Five XIAP antisense oligonucleotides were tested for their ability to kill or chemosensitize T24 bladder carcinoma cells, and were compared to the effects of three corresponding scrambled control oligonucleotides.
T24 cells were transfected with XIAP antisense oligonucleotides, scrambled oligonucleotides, no oligonucleotides (mock transfected), or were left untreated. The cells were tested for viability 20 hours after transfection (with the exception of the untreated control) using the WST-1 tetrazolium dye assay in which WST-1 tetrazolium dye is reduced to a colored formazan product in metabolically active cells (
The occurrence of cytoxicity induced by oligonucleotide E4 was examined by visually inspecting T24 cells that were left untreated, mock transfected, or transfected with E4, E4 scrambled, E4 reverse polarity, or E4 mismatched oligonucleotides. Twenty hours after transfection, the cells were examined for morphology (
To examine the effects of the nucleobase oligomers on the chemosensitization of the T24 cells to cisplatin or adriamycin, oligonucleotides were tested for their ability to further kill T24 cells in the presence of a fixed dose of adriamycin (0.5 μg/ml). Cells were first transfected with a oligonucleotide, then adriamycin was added for another 20 hours. Viability was measured by WST-1 at the end of the 20-hour drug treatment (
All five nucleobase oligomers tested (F3, E4, G4, C5, D7) as well as combinations of E4+C5 and G4+C5, killed the T24 cells, leaving only 10-15% surviving cells after 24 hours, as compared to the mock (no oligonucleotide) transfected cells, or to cells transfected with three corresponding scrambled controls to F3 (5′-mCmAmGAGATTTCATTTAAmCmGmU-3′; SEQ ID NO: 275), E4 (5′-mCmUmACGCTCGCCATCGTm UmCmA-3′; SEQ ID NO: 276) and C5 (5′-mUmGmCCCAAGAATACTAGmUmC mA-3′; SEQ ID NO: 277)(
The addition of a fixed dose of adriamycin or cisplatin at the end of the three hour transfection period resulted in a further decrease in survival for some of the tested oligonucleotides, a further 40% drop in survival after 20 hours for nucleobase oligomers F3, D7 and G4+C5 combination (
By using real-time PCR, antisense oligonucleotides (2×2 MBO, composed of two flanking 2′-O-methyl RNA residues at either end with phosphorothioate linkages, and a central core of 15 phosphodiester DNA residues) were examined for their effects on XIAP mRNA in H460 cells. In this configuration, nucleobase oligomers F3, G4, C5, AB6 and DE4 reduced the mRNA level by 50-70%, compared to untreated control, while D7 AS nucleobase oligomers reduced the mRNA level by 30% (
We characterized the potency of nucleobase oligomers F3, G4 and C5 in oligonucleotide configuration on the XIAP protein expression by western blot analysis (
Having demonstrated that XIAP AS nucleobase oligomers were capable of reducing viability of H460 cells and T24 bladder carcinoma cells after, we determined whether the observed cell death was due to the induction of apoptosis. As shown in
To analyze biological effects of nucleobase oligomers associated with down-regulation of XIAP expression and apoptosis, the growth of H460 cells treated with G4 AS oligonucleotides was investigated by MTT assay. Forty-eight hours after the transfection, G4 AS oligonucleotides had reduced H460 cell growth in a dose-dependent manner, exhibiting a 55% reduction relative to untreated control levels at 1.2 μM (
To investigate whether down-regulation of XIAP expression has the potential to sensitize H460 cells to chemotherapy, combination treatments using G4 AS oligonucleotides and one of the following anticancer drugs: doxorubicin (DOX), taxol, vinorelbine (VNB) and etoposide (Etop) were performed.
We first determined whether intra-tumoral injection of XIAP antisense 2×2-MBOs into SCID-RAG2 mice carrying sub-cutaneous H460 human lung carcinoma xenografts reduced the amount of tumor growth. Treatment started 14 days after tumor cell inoculation (s.c. shoulder injection of 106 cells) by injecting MBOs (50 μg 2′-O-methyl RNA oligonucleotides per g tumor) into the palpable tumor mass three times per week for two weeks. Vinorelbine (VNB; also referred to as navelbine (NVB) (15 mg/kg i.p.) was injected on days 17 and 24. Tumor size was measured with calipers three times per week. At the end of the treatment period (day 24), the mean relative tumor growth of mice treated with a combination of C5+G4 AS MBOs and VNB was ˜70% reduced compared to those treated with scrambled control MBOs and VNB. Treatments with C5 AS MBO and VNB resulted in a ˜60% reduction of tumor size, compared to scrambled control (
Initial systemic PS-oligonucleotide studies were designed without any chemotherapeutic agents. SCID-RAG2 mice were inoculated with H460 human lung carcinoma cells (s.c. shoulder injection of 106 cells) and treatments with G4 and F3 AS PS-oligonucleotides, as well as a scrambled control, were initiated three days after tumor inoculation. Nucleobase oligomer injections were administered i.p. at 12.5 mg/kg three times a week for three weeks. At the end of the treatment period, mean tumor sizes in the groups treated with either G4 or F3 AS oligonucleotides were ˜50% smaller than in the group treated with a scrambled control oligonucleotides (
We conducted additional examination of the antitumor effects of G4 AS oligonucleotides in SCID-RAG2 mice bearing xenografts of H460 human non-small-cell lung tumors implanted subcutaneously. Saline-treated control tumors grew reproducibly to a size of 0.75 cm3 within approximately 24 days (
To correlate the tumor growth inhibitory effects of G4 AS oligonucleotides with XIAP protein expression, we examined the changes in XIAP expression at the end of the in vivo treatment with 15 mg/kg of G4 AS and SC oligonucleotides. At day 21 or 24 post-tumor inoculation when tumors reached 1 cm3 in size (
To evaluate whether XIAP AS oligonucleotide administration results in direct tumor cell kill, we examined the histology of tumors after treatment both for morphology and ubiquitin immunostaining (
The degradation of proteins is largely ubiquitin-proteasome-dependent; increased ubiquitin expression has been observed during apoptosis. Thus, we examined the ubiquitin expression in the tumors sections used for hematoxylin and eosin staining. As shown in
To evaluate whether combined treatments of G4 AS nucleobase oligomers and vinorelbine (VNB), a chemotherapeutic agent used for lung cancer treatment, may result in any cooperative effects, we compared the therapeutic efficacy of VNB in the presence and absence of G4 AS oligonucleotides or G4 SC oligonucleotides. Treatment regimens were initiated on day 3 after tumor inoculation.
Methods
The results obtained in Examples 5-13 were obtained using the following methods.
Oligonucleotide Synthesis
A library of over 96 non-overlapping chimeric, or mixed-backbone (MBO), 19-mer antisense oligonucleotides was synthesized as 2×2 MBO oligonucleotides, composed of two flanking 2′-O-methyl RNA residues at either end with phosphorothioate linkages, and a central core of 15 phosphodiester DNA residues. Each final product was desalted by Sephadex G-25 chromatography (IDT Inc., Coralville, Iowa). This chimeric wingmer configuration, and mix of phosphorothioate and phosphodiester linkages (referred to as 2×2 PS/PO), provided adequate stability while also reducing non-specific toxicity associated with phosphorothioate residues. Fully phosphorothioated non-chimeric (DNA) antisense oligonucleotides for in vivo and in vitro studies were synthesized by Trilink Biotech and purified by RP-HPLC.
Antisense Oligonucleotide Screening
T24 bladder carcinoma cells, transfected with 1-1.2 μM oligonucleotide-lipofectin complexes for 24-48 hours, were assessed to determine the ability of each oligonucleotide to knock-down XIAP protein. Positive hits were reconfirmed for their ability to knock-down (i) XIAP protein levels at 12-18 hours of transfection by western analysis, and (ii) XIAP mRNA levels at 6-9 hours of transfection by quantitative RT-PCR (see below) in T24 bladder carcinoma cells and H460 lung carcinoma cells. Candidate oligonucleotides were identified and tested further. Identified 2×2 PS/PO chimeric oligonucleotides showed a dose-dependent ability to decrease XIAP mRNA levels at 6-9 hours in the range of 400-1200 nM concentrations. Exemplary oligonucleotides are shown in Table 5.
ATCTTCTCTTGAAAATAGG
AU
CTTCTCTTGAAAATAGG
GGATAAAAGTTCTCTTCTA
GCTGAGTCTCCATATTGCC
GC
TGAGTCTCCATATTGCC
GGCTCTTTGCCCACTGAAT
ACCATTCTGGATACCAGAA
AC
CATTCTGGATACCAGAA
AAGACCATAGGTCTTACCA
GGGTTCCTCGGGTATATGG
GGTATATGGCGTCCTTGGG
GGTATCTCCTTCACCAGTA
ATGACCACTTCCTCTATGG
AATTATAACGTTCACTTAG
Tumor Cell Line and Animal Xenografts Model
The human non-small cell lung cancer cell line (large cell type) NCI-H460 (H460) was obtained from ATCC and maintained in RPMI 1640 supplemented with 10% FCS at 37° C. in a humidified atmosphere containing 5% CO2. Cells were used in exponential growth phase, up to a maximum of 25 in vitro passages. Male SCID-RAG2 mice (7-9 weeks old, 23-26 g) were obtained from British Columbia Cancer Agency Joint Animal Facility breeding colony and kept in aseptic environments. A tumor model of NCI-H460 cells in SCID-RAG2 mice was established by subcutaneous implantation of 1×106 NCI-H460 cells on the back of mice.
Treatment of Cells with Antisense and Anticancer Drugs
One day prior to transfection, H460 cells were plated in 6- or 96-well tissue culture plates. Phosphorothioate antisense oligonucleotides were delivered into cells with Lipofectamine 2000 (Life Technologies) in the form of liposome-oligonucleotide complexes. Following a 4.5 or 6 h transfection, the transfection medium was replaced with RPMI medium containing 10% FBS, and the cells incubated for another 24 or 48 h.
Real-Time Quantitative RT-PCR
Total RNA from H460 cells treated with liposome-oligonucleotide complexes for 6 hours was immediately isolated using RNeasy mini spin columns and DNase treatment (QIAGEN, Valencia, Calif.). Specific XIAP mRNA was measured using a real-time quantitative RT-PCR method. XIAP forward and reverse primers (600 nM) and probe (200 nM) (5′-GGTGATAAAGTAAAGTGCTTTCACTGT-3′ (SEQ ID NO 293), 6FAM-CAACATGCTAAATGGTTCCAGGGTGCAAATATC-TAMRA (SEQ ID NO: 294), and 5′-TCAGTAGTTCTTACCAGACACTCCTCAA-3′ (SEQ ID NO: 295) were designed to span exon 3-4 and 4-5 junctions. One of the primers, as well as the probe, was designed to overlap an intron-exon boundary to block detection of any possible genomic DNA contamination. The RNA was reverse-transcribed and PCR amplified using the TaqMan EZ RT-PCR kit (PE/ABI, Foster City, Calif.) in the ABI prism 7700 Sequence Detection System (PE/ABI). The thermal cycling condition for the RT step were 50° C. for 2 min, 60° C. for 30 min, and 95° C. for 5 min, followed by 45 cycles of PCR (at 94° C. for 20 s and 60° C. for 1 min per cycle). The XIAP mRNA level of each sample was calculated relative to untreated control cells. XIAP mRNA levels were determined by the cycle threshold method (Ct) using a threshold of 30× the baseline SD, and XIAP levels were normalized for GAPDH content, using PE/ABI supplied primers and probe.
Western Blot Analysis
The cells or tumor tissue samples were lysed with ice-cold lysis buffer (50 mM Tris, 150 mM NaCl, 2.5 mM EDTA, 0.1% SDS, 0.5% sodium deoxycholate, 1% NP-40, 0.02% sodium azide) containing protease inhibitors (Complete-Mini protease inhibitor tablets; Boehringer Mannheim GmBH, Mannheim, Germany). After incubation for 30 min on ice, samples were centrifuged at 10,000 rpm for 15 min, and stored at −20° C. Protein content in the lysed extracts was determined using a detergent-compatible Bio-Rad assay (Bio-Rad Labs, Hercules, Calif.). Equal amounts of protein (40 μg/lane) were separated on 12% SDS-polyacrylamide gels or 4-15% gradient SDS-polyacrylamide pre-made gels (Bio-Rad) and transferred to nitrocellulose membranes. Primary antibodies against XIAP, Bcl-2 (DAKO, Glostrup, Denmark), Bax (Sigma, St. Louis, Mo.), β-actin (Sigma), caspase-3 (BD PharMingen, San Diego, Calif.), and PARP (BD PharMingen) were used. The secondary antibody was the appropriate horseradish-conjugated anti-mouse or anti-rabbit IgG (Promega, Madison, Wis.). Proteins were detected by enhanced chemiluminescence (ECL; Amersham Pharmacia Biotech, Buckinghamshire, England) and visualized after exposure to Kodak autoradiography film. Scanning densitometry (Molecular Dynamics, Sunnyvale, Calif.) was performed to quantify band intensities by volume/area integration. The amount of XIAP, caspase-3, Bcl-2 and Bax in cells was normalized to their respective lane β-actin levels, upon stripping and reprobing.
Measurement of Cell Growth and Viability or Death
Growth inhibition of H640 cells was determined by the colorimetric MTT cell viability/proliferation assay. In brief, cells were treated with liposome-oligonucleotide complexes for 4.5 h, then incubated for another 48 h at 37° C. in medium without transfection reagent or oligonucleotides in the presence or absence of anticancer drugs. MTT (25 μg/well) was added to each well, and the plates incubated for 3 h at 37° C. Following the incubation step, the colored formazan product was dissolved by the addition of 200 μl DMSO. Plates were read using the microtiter plate reader (Dynex Technologies Inc., Chantilly, Va.) at a wavelength of 570 nm. The percentage of surviving cells in wells treated with oligonucleotides was normalized to untreated controls. All assays were performed in triplicate.
Apoptotic Flow Cytometric Assays
Cells were treated with liposome-oligonucleotide complexes for 4.5 h, and incubated for another 48 h in the medium without transfection reagent at 37° C. Following incubation, cells were harvested, washed twice with sample buffer (0.5% glucose in PBS without Ca++ and Mg++), and fixed in cold 70% ethanol at 4° C. for at least 18 hrs. Samples were centrifuged at 3000 rpm for 10 min, then resuspended in sample buffer containing 50 μg/ml propidium iodide (PI) and 400 U/ml RNase A. Samples were incubated for 30 min at room temperature and 30 min on ice, followed by flow cytometry analysis. EXPO Software (Applied Cytometry Systems, Sacramento, Calif.) was used to generate histograms, which were used to determine the cell cycle phase distribution after debris exclusion. The Sub G1/G0 cell fraction was considered as representative for apoptotic cells.
Nuclear Morphology
Cells were treated with liposome-oligonucleotide complexes for 4.5 h, and incubated for another 48 h at 37° C. in the medium without transfection reagent or oligonucleotides. Cells were harvested and stained with 0.10 μg/ml DAPI (4′,6-diamidino-2-2-phenylindole) for 30 min at room temperature. Cells were placed on a glass slide, cytospun, and viewed with a Leica microscope and 40× objective lens under UV fluorescent illumination. Digital images were captured using Imagedatabase V. 4.01 Software (Leica, Germany).
In Vivo Antitumor Activity
Efficacy experiments were conducted in male RAG2 immunodeficient mice bearing NCI-H460 tumours or female RAG2 mice bearing LCC6 tumors. Treatments were commenced on day 3 after tumor inoculation. Saline (controls), G4 AS oligonucleotides (5 or 15 mg/kg), or G4 SC oligonucleotides (5 or 15 mg/kg) were administered i.p. daily for five doses a week over a three week regimen. Vinorelbine (VNB, 5 or 10 mg/kg) was administered i.v. via the tail vein, either alone or in combination with oligonucleotides, at day 3, 7, 11 and 17 after tumor inoculation. When oligonucleotides were administered in combination with VNB, the drug treatment was performed four hours after ODN treatment.
Mice were observed daily. Body weight measurements and signs of stress (e.g., lethargy, ruffled coat, ataxia) were used to detect possible toxicities. Animals with ulcerated tumor, or tumor volumes of 1 cm3 or greater were killed. Digital caliper measurements of tumors were converted into mean tumor size (cm3) using the formula: ½[length (cm)]×[width (cm)]2. An average tumor size per mouse was used to calculate the group mean tumor size±SE (n=6 mice) from at least two independent experiments per group.
Tumor and Tissue Processing
Mouse tumors were collected on day 21 or 24 post-tumor inoculation and treatment. One portion of the tumor tissue was fixed in formalin. Paraffin-embedded tissues were sectioned and subjected to gross histopathology using hematoxylin and eosin staining and immunohistochemistry for ubiquitin expression. The other portion of the tumor was homogenized in lysis buffer for western blot analysis (see above).
Statistical Analyses
Student's t test was used to measure statistical significance between two treatment groups. Multiple comparisons were done using one-way ANOVA and a post-hoc test that compared different treatment groups by the Shellé test criteria (Statistica release 4.5, StatSoft Inc., Tulsa, Okla.). Data were considered significant for a P-value of <0.05.
A library of 15 HIAP1 antisense nucleobase oligomers as oligonucleotides was screened for protein knock-down by western blot analysis and for RNA knock-down by TaqMan, using the primers and probes described in Example 3, above, under two different conditions. HIAP1 RNA levels may be detected using standard Northern blot analyses or RT-PCR techniques. The antisense oligonucleotides were administered to cells under basal conditions or under cycloheximide-induction conditions (24 hour treatment with sub-toxic doses). Cycloheximide (CHX) can lead to a 10- to 200-fold induction of HIAP1 mRNA depending on the cell line treated. This in turn leads to an increase in HIAP1 protein, as seen on a Western blot (70 kDa band). This effect of CHX is via two distinct mechanisms of action. First, CHX activates NFkB, a known transcriptional inducer of HIAP1, by blocking the de novo synthesis of a labile protein, IkB, which is an inhibitor of NFkB. This effect is mimicked by puromycin, another protein synthesis inhibitor, and by TNF-alpha, which induces a signaling cascade leading to the phosphorylation, ubiquination, and degradation of IkB. Only CHX leads to a further stabilization of the HIAP1 mRNA, as seen by the decreased rate of disappearance of HIAP1 message in the presence of actinomycin D, to block de novo transcription, and CHX, as opposed to actinomycin D and puromycin or TNF-alpha combined.
SF295 glioblastoma cells were transfected with lipofectin and oligonucleotide (scrambled survivin, no oligonucleotide, antisense APO 1 to APO 15) or left untreated. RNA was isolated from the cells six hours after transfection and the level of HIAP1 mRNA was measured by quantitative PCR (TaqMan analysis), normalized for GAPDH mRNA, with the value for the scrambled survivin oligonucleotide transfection set as 1.0. The results of this experiment, a compilation of three separate experiments, are shown in
The effect of HIAP1 antisense nucleobase oligomers on HIAP1 protein and mRNA expression was also examined in cells induced to express HIAP1. SF295 cells were transfected with oligonucleotides, or were mock transfected. The transfected cells were then treated with 10 μg/ml cycloheximide for 24 hours to induce 70 kDa HIAP1 mRNA and protein. Protein levels were measured by western blot analysis with an anti-HIAP1 polyclonal antibody, and normalized against actin protein in a re-probing of the same blots. Scans of the western blot results are shown in
Of the 15 tested nucleobase oligomers, six of them (APO 1, 2, 7, 8, 12, and 15) showed high activity, or had significant activity in both the protein and mRNA assays, and did not cause a stress-induced increase in HIAP1 mRNA, such as that seen with APO 4, 6, 11, 13, 14 (
To further investigate the efficacy of HIAP1 antisense oligonucleotides under cycloheximide induction conditions, changes in HIAP1 mRNA were measured by TaqMan real time PCR 6 hours after transfection with APO 2, which targets an Alu repeat within an intron of HIAP1 and results in the greatest block of CHX-induced upregulation of HIAP1 mRNA and protein. Controls for this experiment were three oligonucleotides for APO 2: one scrambled sequence (same base composition but random order, 5′-AAGGGCGGCGGAGTGAGAC-3′; SEQ ID NO: 297), one reverse polarity (same base composition, same sequential order but in the opposite direction, 5′-AGAGGACGGAGTCGGAGGC-3′; SEQ ID NO: 298), and one mismatch sequence (containing four base mismatches out of 19 bases, 5′-CGGAGCGTGAGGATGGAGA-3′; SEQ ID NO: 299).
Transfection of the APO 2 antisense into cells resulted in a 50% decrease in mRNA compared to a scrambled survivin control and matched perfectly with the protein results, while the scrambled control for APO 2 (H1 sc apo 2 in
The six antisense HIAP1 nucleobase oligomers include two very effective oligonucleotides against an intronic sequence (APO 1, and APO 2, with APO 2 demonstrating the better activity). These oligonucleotides could be used therapeutically for treatment of cancer or autoimmune disorders. The oligonucleotides against an intronic sequence would likely only target pre-mRNA (very short-lived target) and not the mature, processed form of HIAP1. Typically, introns are not targeted for antisense except when one wants to alter splicing by targeting the intron-exon boundaries or the branching point. These usually result in the skipping of an exon rather than RNase-mediated degradation of the message. Both mechanisms would likely be favorable for the enhancement of apoptosis, as the skipping would result in the loss of the exon encoding the first two important BIR domains of HIAP1. The APO 2 antisense ODN also targets an intron of survivin for 18 consecutive bases out of 19, but we did not see any loss of survivin protein; only HIAP1 was decreased after the oligo treatment, demonstrating the specificity of the HIAP1 antisense oligonucleotide. These antisense oligonucleotides hit Alu sequences in the HIAP1 intron and potentially in many other genes, and induce the cancer cells to die (see below), which may be as a result of down regulating HIAP1 and some other critical genes, and thus of therapeutic value if it is not too toxic to normal cells.
Cancer cells have reportedly more Alu-containing transcripts and may therefore be more sensitive to apoptosis induction with an Alu targeting nucleobase oligomer. Furthermore, this killing effect of nucleobase oligomers APO 1 and APO 2 may be due to the combined effect of both targeting Alu sequences and HIAP1 simultaneously. This dual effect would result in an effective way to prevent the normal stress response of HIAP1 induction through the NFkB pathway, when the cell is exposed to certain toxic agents. This stress response is most likely part of the cancer cell's anti-apoptotic program. By blocking HIAP1 expression, we counter this anti-apoptotic stress response and precipitate the cancer cell's demise.
The effect of HIAP1 antisense nucleobase oligomers on the chemosentization of SF295 cells was also evaluated. Cells were transfected with one of three different antisense oligonucleotides (APO 7, APO 15, and SC APO 2 (control)). Twenty-four hours after transfection with the oligonucleotides, the cells were incubated with adriamycin for an additional 24 hours before assaying by for cell survival by assaying WST-1.
The WST-1 survival curves for SF295 cells transfected with the above-described HIAP1 oligonucleotides and then treated with increasing concentrations of adriamycin are shown in
An additional 89 HIAP1 antisense sequences that can be employed in the methods of the invention are shown in Table 6. Sequences that are 100% identical between human HIAP1 and human HIAP2, or have one or two mismatches, are in bold.
GACTCCTTTCTGAGACAGG
CAGAAGCATTTGACCTTGT
CCAGCATCAGGCCACAACA
CTGCATGTGTCTGCATGCT
CATAATAAAAACCCGCACT
CTCCAGATTCCCAACACCT
GGAAACCACTTGGCATGTT
TAAAGCCCATTTCCACGGC
CTATAATTCTCTCCAGTTG
AGTTCTCTTGCTTGTAAAG
TCGTATCAATCAGTTCTCT
GCCACAACAGAAGCATTTG
We also analyzed human HIAP2 for sequences suitable for use as antisense nucleobase oligomers. Identified sequences are shown in Table 7.
All publications and patent applications mentioned in this specification, including U.S. Pat. Nos. 5,919,912, 6,156,535, and 6,133,437, are herein incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
While the invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications and this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known or customary practice within the art to which the invention pertains and may be applied to the essential features hereinbefore set forth.
This application is a continuation of U.S. patent application Ser. No. 10/400,382 filed Mar. 27, 2003, now U.S. Pat. No. 7,091,333, which claims benefit from U.S. provisional application 60/367,853 filed Mar. 27, 2002.
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