(en)The invention provides methods for using the expression levels and subcellular localization of non-coding mitochondrial RNAs to select individuals or subpopulation of individuals for treatment with an anticancer therapy for multiple myeloma. Additional methods provided herein are useful for determining whether an individual in remission for multiple myeloma following successful treatment will be likely to suffer a relapse as well as to identify individuals who have suffered a relapse of multiple myeloma.

1.ApplicationNumber: US-201414776260-A
1.PublishNumber: US-2016138109-A1
2.Date Publish: 20160519
3.Inventor: VILLOTA ARCOS CLAUDIO E.
VILLEGAS OLAVARRIA JAIME E.
BURZIO MENENDEZ VERONICA A.
BURZIO ERIZ LUIS O.

4.Inventor Harmonized: VILLOTA ARCOS CLAUDIO E(CL)
VILLEGAS OLAVARRIA JAIME E(CL)
BURZIO MENENDEZ VERONICA A(CL)
BURZIO ERIZ LUIS O(CL)

5.Country: US
6.Claims:
(en)The invention provides methods for using the expression levels and subcellular localization of non-coding mitochondrial RNAs to select individuals or subpopulation of individuals for treatment with an anticancer therapy for multiple myeloma. Additional methods provided herein are useful for determining whether an individual in remission for multiple myeloma following successful treatment will be likely to suffer a relapse as well as to identify individuals who have suffered a relapse of multiple myeloma.

7.Description:
(en)CROSS REFERENCE TO RELATED APPLICATIONS
This application claims priority to U.S. Provisional Patent Application No. 61/785,269, filed Mar. 14, 2013 and U.S. Provisional Patent Application No. 61/790,072, filed Mar. 15, 2013, the disclosures of each of which are incorporated by reference herein in their entireties.


FIELD OF THE INVENTION
This invention provides methods for detecting and treating multiple myeloma based, in part, on the expression and subcellular localization of non-coding mitochondrial RNAs expressed in plasmocytes isolated from the bone marrow of individuals diagnosed with or thought to have multiple myeloma.
BACKGROUND OF THE INVENTION
Multiple myeloma is a neoplastic disease characterized by infiltration of bone and bone marrow by myeloma cells forming multiple tumor masses that lead to pathological fractures. The condition is usually progressive and fatal. Symptoms include anemia, renal damage and high globulin levels in blood and increased susceptibility to bacterial infections. The impaired abnormal immunoglobulin production observed in multiple myeloma may be due to the presence of a monocyte or macrophage that suppresses the maturation of normal B-lymphocytes into antibody secreting cells. Life expectancy is related to extent of the disease at diagnosis and response to treatment. The median life expectancy of responding patients is two years. High levels of myeloma protein in serum or urine, bone lesions, hypercalcemia, pancytopenia, and renal failure are unfavorable signs.
Although Multiple Myeloma remains incurable, the development of novel therapies has dramatically increased response rates and survival over recent years. Despite major advances in our understanding of this complex disease, a standard remission-induction therapeutic approach is taken to patients in similar categories of age and performance status in the great majority of treatment centers. High dose chemotherapy with autologous stem cell transplant remains the standard therapy for younger patients.
Monoclonal gammopathy of undetermined significance (MGUS) and multiple myeloma are the most frequent forms of monoclonal gammopathies. Monoclonal gammopathy of undetermined significance is the most common plasma cell dyscrasia with an incidence of up to 10% of population over age 75. The molecular basis of monoclonal gammopathy of undetermined significance and multiple myeloma are not very well understood and it is not easy to differentiate the two disorders. The diagnosis of multiple myeloma or monoclonal gammopathy of undetermined significance is identical in ⅔ of cases using classification systems that are based on a combination of clinical criteria such as the amount of bone marrow plasmocytosis, the concentration of monoclonal immunoglobulin in urine or serum, and the presence of bone lesions. Especially in early phases of multiple myeloma, the differential diagnosis is associated with a certain degree of uncertainty.
Furthermore, in the diagnosis of multiple myeloma, the clinician must exclude other disorders in which a plasma cell reaction may occur such as rheumatoid arthritis and connective tissue disorders, or metastatic carcinoma where the patient may have osteolytic lesions associated with bone metastases. Therefore, given that multiple myeloma is thought to have an extended latency and clinical features are recognized many years after the development of the malignancy, new molecular diagnostic techniques are needed in screening for the disease and providing differential diagnosis for multiple myeloma. Thus, there is a need for new and accurate methods for differentially diagnosing and identifying distinct and prognostically relevant clinical subgroups of multiple myeloma.
Complicating the search for such new methods is the fact that multiple myeloma cells are endowed with a multiplicity of anti-apoptotic signaling mechanisms that account for their resistance to current chemotherapy and thus the ultimately fatal outcome for most patients. While aneuploidy by interphase fluorescence in situ hybridization (FISH) and DNA flow cytometry are observed in >90% of cases, cytogenetic abnormalities in this typically hypoproliferative tumor are informative in only about 30% of cases and are typically complex, involving on average seven different chromosomes. It has therefore been difficult to establish correlations between genetic abnormalities and clinical outcomes.
Consequently, given the unreliability of individual gene expression within multiple myeloma tumor cells, there exists a need for a biomarker whose measurement can not only predict the likelihood that an individual has multiple myeloma and will likely benefit from anticancer therapies, but is also useful for determining whether individuals or subpopulations of individuals who are currently in remission following successful anticancer therapy for multiple myeloma are likely to suffer relapse. Such a marker would be helpful for guiding health care professionals involved in the treatment of an individual suffering from or thought to have multiple myeloma. Consequently, a marker of this sort would be useful not only for diagnostic purposes, but also to track prognosis following the initiation of treatment and establishment of remission.
This invention provides such a biomarker and uses the expression and subcellular localization of non-coding mitochondrial RNAs (ncmtRNAs) within plasmocytes isolated from bone marrow to select individuals or subpopulations of individuals who will benefit from an anti-multiple myeloma therapy as well as to identify individuals in remission who are likely to relapse or who have relapsed following remission.
Throughout this specification, various patents, patent applications and other types of publications (e.g., journal articles) are referenced. The disclosure of all patents, patent applications, and publications cited herein are hereby incorporated by reference in their entirety for all purposes.
SUMMARY OF THE INVENTION
The invention provided herein discloses, inter alia, methods for using the expression and subcellular localization of a non-coding mitochondrial RNAs (ncmtRNAs) to select individuals or subpopulations of individuals suspected of having or developing multiple myeloma that will benefit from anticancer therapies as well as methods for using ncmtRNA expression levels in plasmocytes isolated from the bone marrow of individuals to guide the treatment of individuals having or suspected of developing multiple myeloma.
Accordingly, in one aspect, the provided herein are methods for treating multiple myeloma in an individual in need thereof comprising: measuring the expression of a sense non-coding mitochondrial RNA (SncmtRNA) and an antisense non-coding mitochondrial RNA (ASncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein the expression of both a SncmtRNA and an ASncmtRNA indicates that the individual does not have multiple myeloma and wherein the expression of a SncmtRNA and the lack of expression of an ASncmtRNA indicates that the individual has multiple myeloma; and treating the individual with one or more anti-cancer therapeutics if the plasmocytes of the individual express only the SncmtRNA and do not express the ASncmtRNA. In another aspect, provided herein are methods for treating multiple myeloma in an individual in need thereof comprising: treating the individual with one or more anti-cancer therapeutics, wherein prior to treatment it has been determined that plasmocytes isolated from the bone barrow of the individual express a sense non-coding mitochondrial RNA (SncmtRNA) and do not express an antisense non-coding mitochondrial RNA (ASncmtRNA). In some embodiments of any of the embodiments provided herein, the anti-cancer therapeutics comprise one or more oligonucleotides sufficiently complementary to a human non-coding mitochondrial chimeric RNA molecule comprising (a) an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence or (b) a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence, wherein the oligonucleotides are able to hybridize with the mitochondrial chimeric RNA molecules to form a stable duplex. In some embodiments of any of the embodiments provided herein, wherein the anti-cancer therapeutics comprise remicade, docetaxel, celecoxib, melphalan, dexamethasone (Decadron®), steroids, gemcitabine, cisplatinum, temozolomide, etoposide, cyclophosphamide, temodar, carboplatin, procarbazine, gliadel, tamoxifen, topotecan, methotrexate, Arisa®, taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11, interferon alpha, pegylated interferon alpha (e.g., PEG INTRON-A), capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, liposomal daunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin, busulphan, prednisone, bortezomib (Velcade®), bisphosphonate, arsenic trioxide, vincristine, doxorubicin (Doxil®), paclitaxel, ganciclovir, adriamycin, estrainustine sodium phosphate (Emcyt®), sulindac, and etoposide. In some embodiments of any of the embodiments provided herein, the multiple myeloma relapsed after treatment with one or more of bortezomib (Velcade®), cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha, prednisone, thalidomide, and vincristine. In some embodiments of any of the embodiments provided herein, said anti-cancer therapy is administered as part of a salvage therapy in treating patients wherein the multiple myeloma has become refractory to other drugs. In some embodiments of any of the embodiments provided herein, the method further comprises administering one or more additional therapies. In some embodiments, the one or more additional therapies comprise allogenic stem cell transplant therapy. In some embodiments, the one or more additional therapies comprise autologous stem cell transplant therapy. In some embodiments, hematopoietic stem-cells and/or bone marrow for use in autologous stem cell transplantation are treated with an effective amount of one or more oligonucleotides sufficiently complementary to an ASncmtRNA or SncmtRNA molecule to form a stable duplex prior to transplantation into the affected individual. In some embodiments of any of the embodiments provided herein, the ASncmtRNA comprises a human mitochondrial chimeric RNA molecule comprising an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the ASncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments of any of the embodiments provided herein, the SncmtRNA comprises a human mitochondrial chimeric RNA molecule comprising a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the SncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments of any of the embodiments provided herein, expression of the SncmtRNA or ASncmtRNA is measured by RT-PCR or another PCR-based method, Northern Blot, in situ hybridization, or SAGE. In some embodiments, the SncmtRNA or ASncmtRNA is measured by quantitative RT-PCR (qRT-PCR). In some embodiments, the SncmtRNA or ASncmtRNA is measured by in situ hybridization. In some embodiments of any of the embodiments provided herein, the subcellular localization of the SncmtRNA or ASncmtRNA is measured by in situ hybridization.
In other aspects, provided herein are methods for preventing relapse of multiple myeloma in an individual in need thereof comprising: (a) measuring the subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein the cytoplasmic localization of the SncmtRNA indicates that the individual is in remission; and (b) treating the individual with one or more maintenance anti-cancer therapeutics if the SncmtRNA is localized to the cytoplasm of the plasmocytes. In another aspect, provided herein are methods for preventing relapse of multiple myeloma in an individual in need thereof comprising: treating the individual with one or more maintenance anti-cancer therapeutics, wherein prior to treatment it has been determined that plasmocytes isolated from the bone barrow of the individual exhibit subcellular cytoplasmic localization of a sense non-coding mitochondrial RNA (SncmtRNA). In some embodiments of any of the embodiments provided herein, the anti-cancer therapeutics comprise one or more oligonucleotides sufficiently complementary to a human non-coding mitochondrial chimeric RNA molecule comprising (a) an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence or (b) a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence, wherein the oligonucleotides are able to hybridize with the mitochondrial chimeric RNA molecules to form a stable duplex. In some embodiments of any of the embodiments provided herein, wherein the anti-cancer therapeutics comprise remicade, docetaxel, celecoxib, melphalan, dexamethasone (Decadron®), steroids, gemcitabine, cisplatinum, temozolomide, etoposide, cyclophosphamide, temodar, carboplatin, procarbazine, gliadel, tamoxifen, topotecan, methotrexate, Arisa®, taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11, interferon alpha, pegylated interferon alpha (e.g., PEG INTRON-A), capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, liposomal daunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin, busulphan, prednisone, bortezomib (Velcade®), bisphosphonate, arsenic trioxide, vincristine, doxorubicin (Doxil®), paclitaxel, ganciclovir, adriamycin, estrainustine sodium phosphate (Emcyt®), sulindac, and etoposide. In some embodiments of any of the embodiments provided herein, the multiple myeloma relapsed after treatment with one or more of bortezomib (Velcade®), cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha, prednisone, thalidomide, and vincristine. In some embodiments of any of the embodiments provided herein, said anti-cancer therapy is administered as part of a salvage therapy in treating patients wherein the multiple myeloma has become refractory to other drugs. In some embodiments of any of the embodiments provided herein, the method further comprises administering one or more additional therapies. In some embodiments, the one or more additional therapies comprise allogenic stem cell transplant therapy. In some embodiments, the one or more additional therapies comprise autologous stem cell transplant therapy. In some embodiments, hematopoietic stem-cells and/or bone marrow for use in autologous stem cell transplantation are treated with an effective amount of one or more oligonucleotides sufficiently complementary to an ASncmtRNA or SncmtRNA molecule to form a stable duplex prior to transplantation into the affected individual. In some embodiments of any of the embodiments provided herein, the ASncmtRNA comprises a human mitochondrial chimeric RNA molecule comprising an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the ASncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments of any of the embodiments provided herein, the SncmtRNA comprises a human mitochondrial chimeric RNA molecule comprising a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the SncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments of any of the embodiments provided herein, expression of the SncmtRNA or ASncmtRNA is measured by RT-PCR or another PCR-based method, Northern Blot, in situ hybridization, or SAGE. In some embodiments, the SncmtRNA or ASncmtRNA is measured by quantitative RT-PCR (qRT-PCR). In some embodiments, the SncmtRNA or ASncmtRNA is measured by in situ hybridization. In some embodiments of any of the embodiments provided herein, the subcellular localization of the SncmtRNA or ASncmtRNA is measured by in situ hybridization.
In still other aspects, provided herein are methods for treating relapsed multiple myeloma in an individual thereof comprising: (a) measuring the subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein the (i) nuclear or (ii) cytoplasmic and nuclear localization of the SncmtRNA indicates that the individual has relapsed; and (b) treating the individual with one or more anti-cancer therapeutics if the SncmtRNA is localized to (i) the nuclei or (ii) cytoplasm and nuclei of the plasmocytes. In another aspect, provided herein are methods for treating relapsed multiple myeloma in an individual thereof comprising: treating the individual with one or more anti-cancer therapeutics, wherein prior to treatment it has been determined that plasmocytes isolated from the bone barrow of the individual exhibit (i) nuclear or (ii) cytoplasmic and nuclear subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA). In some embodiments of any of the embodiments provided herein, the anti-cancer therapeutics comprise one or more oligonucleotides sufficiently complementary to a human non-coding mitochondrial chimeric RNA molecule comprising (a) an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence or (b) a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence, wherein the oligonucleotides are able to hybridize with the mitochondrial chimeric RNA molecules to form a stable duplex. In some embodiments of any of the embodiments provided herein, wherein the anti-cancer therapeutics comprise remicade, docetaxel, celecoxib, melphalan, dexamethasone (Decadron®), steroids, gemcitabine, cisplatinum, temozolomide, etoposide, cyclophosphamide, temodar, carboplatin, procarbazine, gliadel, tamoxifen, topotecan, methotrexate, Arisa®, taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11, interferon alpha, pegylated interferon alpha (e.g., PEG INTRON-A), capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, liposomal daunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin, busulphan, prednisone, bortezomib (Velcade®), bisphosphonate, arsenic trioxide, vincristine, doxorubicin (Doxil®), paclitaxel, ganciclovir, adriamycin, estrainustine sodium phosphate (Emcyt®), sulindac, and etoposide. In some embodiments of any of the embodiments provided herein, the multiple myeloma relapsed after treatment with one or more of bortezomib (Velcade®), cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha, prednisone, thalidomide, and vincristine. In some embodiments of any of the embodiments provided herein, said anti-cancer therapy is administered as part of a salvage therapy in treating patients wherein the multiple myeloma has become refractory to other drugs. In some embodiments of any of the embodiments provided herein, the method further comprises administering one or more additional therapies. In some embodiments, the one or more additional therapies comprise allogenic stem cell transplant therapy. In some embodiments, the one or more additional therapies comprise autologous stem cell transplant therapy. In some embodiments, hematopoietic stem-cells and/or bone marrow for use in autologous stem cell transplantation are treated with an effective amount of one or more oligonucleotides sufficiently complementary to an ASncmtRNA or SncmtRNA molecule to form a stable duplex prior to transplantation into the affected individual. In some embodiments of any of the embodiments provided herein, the ASncmtRNA comprises a human mitochondrial chimeric RNA molecule comprising an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the ASncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments of any of the embodiments provided herein, the SncmtRNA comprises a human mitochondrial chimeric RNA molecule comprising a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the SncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments of any of the embodiments provided herein, expression of the SncmtRNA or ASncmtRNA is measured by RT-PCR or another PCR-based method, Northern Blot, in situ hybridization, or SAGE. In some embodiments, the SncmtRNA or ASncmtRNA is measured by quantitative RT-PCR (qRT-PCR). In some embodiments, the SncmtRNA or ASncmtRNA is measured by in situ hybridization. In some embodiments of any of the embodiments provided herein, the subcellular localization of the SncmtRNA or ASncmtRNA is measured by in situ hybridization.
In other aspects, provided herein are methods for assessing a likelihood of a beneficial response to an anti-multiple myeloma therapy in an individual suspected of having or developing multiple myeloma, the method comprising: measuring the subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein the cytoplasmic subcellular localization of the SncmtRNA indicates a beneficial response to the anti-multiple myeloma therapy. In some embodiments of any of the embodiments provided herein, the SncmtRNA comprises a human mitochondrial chimeric RNA molecule comprising a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the SncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments of any of the embodiments provided herein, the subcellular localization of the SncmtRNA or ASncmtRNA is measured by in situ hybridization.
In another aspect, provided herein are methods for diagnosing multiple myeloma in an individual comprising: measuring the expression of a sense non-coding mitochondrial RNA (SncmtRNA) and an antisense non-coding mitochondrial RNA (ASncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein the expression of both a SncmtRNA and an ASncmtRNA indicates that the individual does not have multiple myeloma and wherein the expression of a SncmtRNA and the lack of expression of an ASncmtRNA indicates that the individual has multiple myeloma. In other aspects, provided herein are methods for determining whether an individual diagnosed with multiple myeloma that is in remission will relapse, the method comprising: measuring the subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein (a) cytoplasmic localization of the SncmtRNA indicates that the individual is in remission and (b) nuclear or cytoplasmic and nuclear localization of the SncmtRNA indicates that the individual will relapse. In some embodiments of any of the embodiments provided herein, the ASncmtRNA comprises a human mitochondrial chimeric RNA molecule comprising an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, wherein the ASncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. In some embodiments of any of the embodiments provided herein, the SncmtRNA comprises a human mitochondrial chimeric RNA molecule comprising a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the SncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments of any of the embodiments provided herein, expression of the SncmtRNA or ASncmtRNA is measured by RT-PCR or another PCR-based method, Northern Blot, in situ hybridization, or SAGE. In some embodiments, expression of the SncmtRNA or ASncmtRNA is measured by quantitative RT-PCR (qRT-PCR). In some embodiments, expression of the SncmtRNA or ASncmtRNA is measured by in situ hybridization. In some embodiments of any of the embodiments provided herein, the subcellular localization of the SncmtRNA or ASncmtRNA is measured by in situ hybridization.
In still other aspects, provided herein are kits for diagnosing multiple myeloma, the kit comprising one or more of: (a) one or more oligonucleotides sufficiently complementary to a human mitochondrial chimeric RNA molecule comprising (i) an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence or (ii) a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence, wherein the oligonucleotides are able to hybridize with the mitochondrial chimeric RNA molecules to form a stable duplex; (b) a conjugated antibody reagent for label detection; (c) hybridization and wash buffer; and/or (d) one or more slides of fixed plasmocytes (i) isolated from an individual diagnosed with multiple myeloma or a multiple myeloma cell line as a positive control and (ii) isolated from an individual without multiple myeloma or from a normally proliferating cell line as a negative control.
In another aspect, provided herein are kits for determining whether an individual diagnosed with multiple myeloma that is in remission will relapse, the kit comprising one or more of: (a) one or more oligonucleotides sufficiently complementary to a human mitochondrial chimeric RNA molecule comprising (i) an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence or (ii) a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence, wherein the oligonucleotides are able to hybridize with the mitochondrial chimeric RNA molecules to form a stable duplex; (b) a conjugated antibody reagent for label detection; (c) hybridization and wash buffer; (d) one or more slides of fixed plasmocytes (i) isolated from an individual diagnosed with multiple myeloma that has relapsed or isolated from a multiple myeloma cell line exhibiting nuclear or cytoplasmic and nuclear subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA) as a positive control and (ii) isolated from an individual without relapsed multiple myeloma or isolated from a multiple myeloma cell line exhibiting cytoplasmic subcellular localization of a SncmtRNA as a negative control.



BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 depicts hybridization signal to both SncmtRNA and ASncmtRNA probes in plasmocytes isolated from the bone marrow of healthy donors (upper panels). The lower panels show the same cells stained with DAPI (nuclear staining).
FIG. 2 depicts hybridization signal to both SncmtRNA and ASncmtRNA probes in plasmocytes isolated from the bone marrow of individuals who have been diagnosed with multiple myeloma prior to administration of any anticancer therapy. Myeloma cells are identified by their positive hybridization to the SncmtRNA probe and the lack of hybridization signal to the ASncmtRNA probe (upper panels). SncmtRNA is localized only in the cytoplasm. 18S rRNA serves as a positive control. The lower panels correspond to the phase image of the same cells ( FIGS. 2A-C ).
FIG. 3 depicts hybridization signal to both SncmtRNA and ASncmtRNA probes in plasmocytes isolated from the bone marrow of individuals who have been diagnosed with multiple myeloma and have subsequently relapsed following anticancer therapy. Myeloma cells are identified by their positive hybridization to the SncmtRNA probe and the lack of hybridization signal to the ASncmtRNA probe (upper panels). Additionally, SncmtRNA is localized wither primarily in the nucleus ( FIGS. 3C-D ) or in the cytoplasm and the nucleus ( FIGS. 3A-B and E). 18S rRNA serves as a positive control. The lower panels correspond to the phase image of the same cells.
FIG. 4 depicts hybridization signal to both SncmtRNA and ASncmtRNA probes in plasmocytes isolated from the bone marrow of an individual with monoclonal gammopathy of unknown significance (MGUS). These cells show SncmtRNA expression and localization consistent with individuals diagnosed with multiple myeloma but prior to relapse (cytoplasmic localization). ASncmtRNA is absent. The lower panels show the same cells stained with DAPI (nuclear staining).
FIG. 5 depicts survival of mice injected with untreated NSO2 cells, NSO2 cells treated with a control antisense oligonucleotide (ASO-C) or cells treated with the therapeutic antisense oligonucleotide ASO-1560S.



DETAILED DESCRIPTION OF THE INVENTION
This invention provides, inter alia, methods for diagnosing and treating individuals with or thought to have multiple myeloma. The inventors have observed that the subcellular localization and expression of particular species of non-coding mitochondrial RNAs (ncmtRNAs) in plasmocytes isolated from the bone marrow of individuals diagnosed with or thought to have multiple myeloma is associated with both the existence of the disease state itself and is indicative of whether an individual who is currently in remission for multiple myeloma will suffer a relapse or is currently suffering a relapse. Assessing individuals for the expression and subcellular localization of these ncmtRNAs is thus useful for selecting individuals or subpopulations of individuals for appropriate treatment and for determining the likelihood of relapse once an individual is in remission following successful initial therapy. This assessment is also useful as a component of a method for treating individuals diagnosed with or suspected of having multiple myeloma.
I. General Techniques
The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology, microbiology, cell biology, biochemistry, nucleic acid chemistry, and immunology, which are well known to those skilled in the art. Such techniques are explained fully in the literature, such as, Molecular Cloning: A Laboratory Manual , second edition (Sambrook et al., 1989) and Molecular Cloning: A Laboratory Manual , third edition (Sambrook and Russel, 2001), (jointly referred to herein as “Sambrook”); Current Protocols in Molecular Biology (F. M. Ausubel et al., eds., 1987, including supplements through 2001); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Harlow and Lane (1988) Antibodies, A Laboratory Manual , Cold Spring Harbor Publications, New York; Harlow and Lane (1999) Using Antibodies: A Laboratory Manual , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (jointly referred to herein as “Harlow and Lane”), Beaucage et al. eds., Current Protocols in Nucleic Acid Chemistry John Wiley & Sons, Inc., New York, 2000), Handbook of Experimental Immunology, 4th edition (D. M. Weir & C. C. Blackwell, eds., Blackwell Science Inc., 1987); and Gene Transfer Vectors for Mammalian Cells (J. M. Miller & M. P. Calos, eds., 1987). Other useful references include Harrison's Principles of Internal Medicine (McGraw Hill; J. Isseleacher et al., eds.), Dubois' Lupus Erythematosus (5th ed.; D. J. Wallace and B. H. Hahn, eds.
II. Definitions
As used herein, the singular form “a”, “an”, and “the” includes plural references unless indicated otherwise.
It is understood that aspects and embodiments of the invention described herein include “comprising,” “consisting,” and “consisting essentially of” aspects and embodiments.
An “individual” can be a vertebrate, a mammal, or a human. Mammals include, but are not limited to, farm animals, sport animals, pets, primates, mice and rats. Individuals also include companion animals including, but not limited to, dogs and cats. In one aspect, an individual is a human.
A “healthcare professional,” as used herein, can include, without limitation, doctors, nurses, physician assistants, lab technicians, research scientists, clerical workers employed by the same, or any person involved in determining, diagnosing, aiding in the diagnosis or influencing the course of treatment for the individual.
An “effective amount” or “therapeutically effective amount” refers to an amount of therapeutic compound, such as an oligonucleotide or other anticancer therapy, administered to an individual, either as a single dose or as part of a series of doses, which is effective to produce a desired therapeutic effect.
It is intended that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.
III. Methods for Using ncmtRNAs as a Biomarker for Diagnosis of Multiple Myeloma
The expression level and subcellular localization of non-coding mitochondrial RNAs (ncmtRNAs) in plasmocytes isolated from bone marrow can be used to determine whether an individual is suffering from multiple myeloma, whether an individual who is in remission for multiple myeloma following successful treatment with one or more anticancer therapies is likely to suffer a relapse, and/or whether an individual is currently suffering a relapse of multiple myeloma. Any of the methods described herein can be used by health care professionals to aid in a treatment plan or to make treatment decisions by choosing among the most appropriate treatment options for multiple myeloma for any individual, based on expression levels and subcellular localization of ncmtRNAs in plasmocytes isolated from the bone marrow of the individual. Similarly, any of the methods described herein can be used for aiding in the diagnosis of an individual or subpopulation of individuals who will benefit from an anticancer (such as an anti-multiple myeloma) therapy.
A. Human Chimeric Non-Coding Mitochondrial RNAs (ncmtRNAs)
Human cells express a number of unique chimeric mitochondrial RNA molecules. These molecules are non-coding (i.e., they are not known to serve as a template for the translation of a protein) and comprise the transcribed 16S mitochondrial ribosomal RNA gene covalently linked at the 5′ end to an inverted repeat sequence of the 16S mitochondrial ribosomal RNA gene. Chimeric mitochondrial RNA molecules are found in two forms: sense and antisense.
The sense chimeric non-coding mitochondrial RNA (SncmtRNA) molecule corresponds to the 16S mitochondrial ribosomal RNA gene transcribed from the “H-strand” of the circular mitochondrial genome. Covalently linked to the 5′ end of this RNA molecule is a nucleotide sequence corresponding to the inverted repeat sequence of the 16S mitochondrial ribosomal RNA gene, transcribed from the “L-strand” of the mitochondrial genome. The size of the inverted repeat sequence in the SncmtRNA can vary from about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, or 800 nucleotides or more to between about 100-200, 150-250, 200-300, 250-350, 400-500, 450-550, 500-600, 550-650, 600-700, 650-750, or 700-800 nucleotides or more, including any number in between these values. In one embodiment, the inverted repeat sequence in the SncmtRNA corresponds to a fragment of 815 nucleotides of the RNA transcribed from the L-strand of the 16S gene of the mitochondrial genome. In another embodiment, the inverted repeat sequence in the SncmtRNA corresponds to a fragment of 754 nucleotides of the RNA transcribed from the L-strand of the 16S gene of the mitochondrial genome. In still another embodiment, the inverted repeat sequence in the SncmtRNA corresponds to a fragment of 694 nucleotides of the RNA transcribed from the L-strand of the 16S gene of the mitochondrial genome. In another embodiment, the SncmtRNA corresponds to SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3.
The antisense chimeric non-coding mitochondrial RNA (ASncmtRNA) molecule corresponds to the 16S mitochondrial ribosomal RNA gene transcribed from the “L-strand” of the circular mitochondrial genome. Covalently linked to the 5′ end of this RNA molecule is a nucleotide sequence corresponding to the inverted repeat sequence of the 16S mitochondrial ribosomal RNA gene, transcribed from the “H-strand” of the mitochondrial genome. The size of the inverted repeat sequence in the ASncmtRNA can vary from about 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800 nucleotides or more to between about 100-200, 150-250, 200-300, 250-350, 400-500, 450-550, 500-600, 550-650, 600-700, 650-750, or 700-800 or more, including any number in between these values. In another embodiment, the SncmtRNA corresponds to SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6.
Further information related to chimeric mitochondrial RNA molecules can be found in U.S. Pat. No. 8,318,686, the disclosure of which is incorporated by reference herein in its entirety.
B. Detection of ncmtRNAs
The SncmtRNA or ASncmtRNA molecules disclosed herein can be used as biomarkers for the diagnosis of multiple myeloma, to determine the likelihood that an individual diagnosed with multiple myeloma will relapse, and/or to determine whether an individual diagnosed with multiple myeloma has relapsed following anticancer therapy by assessing the expression levels and subcellular localization of the ncmtRNA molecules in plasmocytes isolated from bone marrow.
The assessment of SncmtRNA or ASncmtRNA expression is at the level of the transcribed RNA. Assessment of RNA expression levels of gene transcripts is routine and well known in the art. For example, one flexible and sensitive quantitative method for assessing RNA expression levels derived from a biological sample (such as bone marrow or plasmocytes isolated from bone marrow) is by quantitative RT-PCR (qRT-PCR) or by any other comparable quantitative PCR-based method. Additional methods for assessing RNA expression include, but are not limited to, Northern blotting, microarrays, in situ hybridization, serial analysis of gene expression (SAGE), dot blot, oligonucleotide arrays for chimeric RNA and antisense chimeric RNAs, amplification of the RNA by in vitro transcription mediated amplification (TMA), or ribonuclease protection assays.
In one embodiment, expression and subcellular localization of SncmtRNA or ASncmtRNA expression is determined by fluorescent in situ hybridization (FISH). Bone marrow samples for FISH analysis can be obtained by any method known in the art. Once obtained, the bone marrow sample may be fixed, paraffin embedded, fresh, or frozen before expression levels and subcellular localization of ncmtRNAs are measured. In situ hybridization can be performed according to well-known methods in the art. For example, a hybridization solution comprising one or more labeled probes targeted to one or more of the sequences of SncmtRNA (for example, SEQ ID NO 1, SEQ ID NO 2, or SEQ ID NO 3) or ASncmtRNA (for example, SEQ ID NO 4, SEQ ID NO 5, or SEQ ID NO 6) within the plasmocyte is contacted with the cell under hybridization conditions. The hybridization signal is then compared with a predetermined hybridization pattern from normal or control plasmocytes isolated from healthy volunteers or individuals with multiple myeloma, respectively. Labeled probes for performing FISH can be RNA, DNA or synthetic nucleic acids and can be prepared by any method known in the art. Synthetic nucleic acids include riboprobes transcribed in vitro or PCR fragments. In one embodiment, synthetic complementary oligonucleotides can be used. In addition, the synthetic nucleic acid-based probes can have one or more alterations to the oligonucleotide phosphate backbone, sugar moieties, and/or nucleobase (such as any of those described herein) that increase resistance to degradation, such as by nuclease cleavage. The complementary oligonucleotide probes are at least about 10 (such as any of about 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50) nucleotides in length. In another embodiment, the oligonucleotide probes can be between about 10-15, 15-20, 20-25, 25-30, 30-35, 35-40, 40-45, or 45-50 oligonucleotides in length. In other embodiments, longer oligonucleotide probes can be used, such as 60, 70, 80, 90, or 100 nucleotides in length or more. In some embodiments, the probe is at least 90% (such as at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the sequences listed in SEQ ID NO:1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and/or SEQ ID NO: 6.
Oligonucleotide probes for use in in situ hybridization are labeled to detect the hybridization with SncmtRNA and/or ASncmtRNA. The probes can be labeled with a detectable marker by any method known in the art. Methods for labeling probes include random priming, end labeling, PCR, and nick translation. Enzymatic labeling is conducted in the presence of nucleic acid polymerase, three unlabeled nucleotides, and a fourth nucleotide which is either directly labeled, contains a linker arm for attaching a label, or is attached to a hapten or other molecule to which a labeled binding molecule (such as a secondary antibody) may bind. Suitable direct labels include radioactive labels such as 32 P, 33 P, 3 H, and 35 S and non-radioactive labels such as fluorescent markers. Fluorescent labels for use in FISH include 5(6)-carboxyfluorescein, 6-((7-amino-4-methylcoumarin.-3-acetyl)amino)hexanoic acid, 5(and 6)-carboxy-X-rhodamine, Cyanine 2 (Cy2) Dye, Cyanine 3 (Cy3) Dye, Cyanine 3.5 (Cy3.5) Dye, Cyanine 5 (Cy5) Dye, Cyanine 5.5 (Cy5.5) Dye Cyanine 7 (Cy7) Dye, Cyanine 9 (Cy9) Dye (Cyanine dyes 2, 3, 3.5, 5 and 5.5 are available as NHS esters from Amersham, Arlington Heights, Ill.) or the Alexa dyes comprising Alexa 488, Alexa 532, Alexa 556, Alexa 590, etc. (Molecular Probes, Eugene, Oreg.).
C. Methods for Diagnosing Multiple Myeloma
Provided herein are methods for diagnosing multiple myeloma based on the expression levels and subcellular localization of SncmtRNA and/or ASncmtRNA in plasmocytes isolated from the bone marrow of individuals having or suspected of having multiple myeloma. The expression of SncmtRNA and ASncmtRNA in plasmocytes isolated from the bone marrow of an individual suspected of having multiple myeloma indicates that the plasmocytes are normally proliferating. Further, if the bone marrow sample contains non-proliferating plasmocytes, assessment of ncmtRNA expression will show that neither SncmtRNA nor ASncmtRNA are expressed. However, if measurement of ncmtRNA levels shows expression of SncmtRNA and down regulation or the absence of the ASncmtRNA transcript, the individual has multiple myeloma.
Accordingly, in one aspect, provided herein are methods for diagnosing multiple myeloma in an individual comprising: measuring the expression of a sense non-coding mitochondrial RNA (SncmtRNA) and an antisense non-coding mitochondrial RNA (ASncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein the expression of both a SncmtRNA and an ASncmtRNA indicates that the individual does not have multiple myeloma and wherein the expression of a SncmtRNA and the lack of expression of an ASncmtRNA indicates that the individual has multiple myeloma. The expression of the ncmtRNAs can be assessed using any technique known in the art (such as, but not limited to, RT-PCR or another non-quantitative, semi-quantitative, or quantitative PCR-based method, Northern Blot, in situ hybridization (such as, fluorescence in situ hybridization (FISH), or SAGE).
The ASncmtRNA can be a human mitochondrial chimeric RNA molecule comprising an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the ASncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. The SncmtRNA can be a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the SncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
D. Methods for Determining Whether an Individual Diagnosed with Multiple Myeloma Will Relapse
The natural progression of multiple myeloma is of relapse following treatment. While new “maintenance” therapies have been effective in extending the duration of remission following initial treatment (e.g., lenalidomide (Revlimid) maintenance therapy), none have been effective in preventing it entirely. Depending on the individual's condition, prior treatment modalities used and the duration of remission, options for relapsed disease include re-treatment with the original therapeutic agent, use of other agents (such as melphalan, cyclophosphamide, thalidomide or dexamethasone, or the proteosome inhibitor bortezomib (Velcade®), careilzomib (Kyprolis®), pomalidomide (Pomalyst®), or additional autologous stem cell transplantation. Thus, the ability to predict or determine the likelihood that an individual diagnosed with multiple myeloma that is currently in remission following initial treatment will relapse would be of great value for assisting health care practitioners in the monitoring and treatment of the disease.
The expression levels and subcellular localization of SncmtRNA and ASncmtRNA in plasmocytes isolated from the bone marrow of individuals diagnosed with multiple myeloma can be used to determine whether the individual is in remission or is likely to relapse or has suffered a relapse. As described herein, if measurement of ncmtRNA levels shows expression of SncmtRNA and down regulation or the absence of the ASncmtRNA transcript, the individual has multiple myeloma. Further, the subcellular localization of the SncmtRNA within the plasmocyte is indicative of whether the individual will suffer a relapse or has already suffered relapse. If the SncmtRNA transcript is localized to the nucleus of the plasmocyte or is predominantly nuclear in subcellular localization or is localized to both the nucleus and cytoplasm of the plasmocyte, the individual will suffer relapse or is currently in relapse. However, solely cytoplasmic subcellular localization of the SncmtRNA transcript indicates that the individual is in remission.
Accordingly, provided herein are methods for determining whether an individual diagnosed with multiple myeloma that is in remission will relapse, the method comprising: measuring the subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein cytoplasmic localization of the SncmtRNA indicates that the individual is in remission and nuclear or cytoplasmic and nuclear localization of the SncmtRNA indicates that the individual will relapse. The expression and/or subcellular localization of the ncmtRNAs can be assessed using any technique known in the art (such as, but not limited to, RT-PCR or another non-quantitative, semi-quantitative, or quantitative PCR-based method, Northern Blot, in situ hybridization (such as, fluorescence in situ hybridization (FISH), or SAGE).
The ASncmtRNA can be a human mitochondrial chimeric RNA molecule comprising an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the ASncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. The SncmtRNA can be a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the SncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
E. Methods for Assessing a Likelihood of a Beneficial Response to an Anti-Multiple Myeloma Therapy
The expression levels and subcellular localization of SncmtRNA and ASncmtRNA in plasmocytes isolated from the bone marrow of individuals diagnosed with multiple myeloma can be used to determine the likelihood that the individual will respond to an anti-multiple myeloma therapy (such as any of the therapies or treatments for multiple myeloma disclosed herein). As previously described herein, if measurement of ncmtRNA levels shows expression of SncmtRNA and down regulation or the absence of the ASncmtRNA transcript, the individual has multiple myeloma. Further, the subcellular localization of the SncmtRNA within the plasmocyte is indicative of whether the individual will respond to an anti-multiple myeloma therapy. If the SncmtRNA transcript is localized solely to the cytoplasm of the plasmocyte, the individual will likely exhibit a beneficial response to an anti-multiple myeloma therapy. Thus, cytoplasmic localization of the SncmtRNA transcript in the plasmocyte isolated from the bone marrow of individuals diagnosed with multiple myeloma is indicative of a good prognosis with respect to responding to one or more anticancer therapeutics (such as any of those described herein).
Accordingly, provided herein are methods for assessing a likelihood of a beneficial response to an anti-multiple myeloma therapy in an individual suspected of having or developing multiple myeloma, the method comprising: measuring the subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein the cytoplasmic subcellular localization of the SncmtRNA indicates a beneficial response to the anti-multiple myeloma therapy. The expression and/or subcellular localization of the ncmtRNAs can be assessed using any technique known in the art (such as, but not limited to, RT-PCR or another non-quantitative, semi-quantitative, or quantitative PCR-based method, Northern Blot, in situ hybridization (such as, fluorescence in situ hybridization (FISH), or SAGE).
The ASncmtRNA can be a human mitochondrial chimeric RNA molecule comprising an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the ASncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6. The SncmtRNA can be a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence. In some embodiments, the SncmtRNA comprises one or more nucleotide sequences selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3.
F. Fixed or Data-Storage Media
The diagnosis of multiple myeloma, the determination that an individual diagnosed with multiple myeloma will relapse, or the assessment of the likelihood of a beneficial response to an anticancer therapy in an individual suspected of having or developing multiple myeloma may be provided to a healthcare professional by being recorded on a fixed or data storage medium and/or being accessible via a system for reading the storage medium. For example, a system for reading a data storage medium may include a computer including a central processing unit (“CPU”), a working memory which may be, e.g., RAM (random access memory) or “core” memory, mass storage memory (such as one or more disk drives or CD-ROM drives), one or more display devices (e.g., cathode-ray tube (“CRT”) displays, light emitting diode (“LED”) displays, liquid crystal displays (“LCDs”), electroluminescent displays, vacuum fluorescent displays, field emission displays (“FEDs”), plasma displays, projection panels, etc.), one or more user input devices (e.g., keyboards, microphones, mice, touch screens, etc.), one or more input lines, and one or more output lines, all of which are interconnected by a conventional bidirectional system bus. The system may be a stand-alone computer, or may be networked (e.g., through local area networks, wide area networks, intranets, extranets, or the internet) to other systems (e.g., computers, hosts, servers, etc.). The system may also include additional computer controlled devices such as consumer electronics and appliances.
Input hardware may be coupled to the computer by input lines and may be implemented in a variety of ways. Machine-readable data of this invention may be inputted via the use of a modem or modems connected by a telephone line or dedicated data line. Alternatively or additionally, the input hardware may include CD-ROM drives or disk drives. In conjunction with a display terminal, a keyboard may also be used as an input device.
Output hardware may be coupled to the computer by output lines and may similarly be implemented by conventional devices. By way of example, the output hardware may include a display device for displaying a graphical representation of an active site of this invention using a program such as QUANTA. Output hardware might also include a printer, so that hard copy output may be produced, or a disk drive, to store system output for later use.
Machine-readable storage devices useful in the present invention include, but are not limited to, magnetic devices, electrical devices, optical devices, and combinations thereof. Examples of such data storage devices include, but are not limited to, hard disk devices, CD devices, digital video disk devices, floppy disk devices, removable hard disk devices, magneto-optic disk devices, magnetic tape devices, flash memory devices, bubble memory devices, holographic storage devices, and any other mass storage peripheral device. It should be understood that these storage devices include necessary hardware (e.g., drives, controllers, power supplies, etc.) as well as any necessary media (e.g., disks, flash cards, etc.) to enable the storage of data.
A person having skill in the art will appreciate that any other method or technique to communicate or store data is may also be contemplated for communication of the diagnosis of multiple myeloma, the determination that an individual diagnosed with multiple myeloma will relapse, or the assessment of the likelihood of a beneficial response to an anticancer therapy in an individual suspected of having or developing multiple myeloma to a healthcare professional.
IV. Methods for Treating Multiple Myeloma
The methods disclosed herein for using the expression and subcellular localization of ncmtRNAs for diagnosing multiple myeloma as well as for identifying individuals who are likely to suffer a relapse of multiple myeloma or who have relapsed can additionally be used in conjunction with methods for the treatment and/or prevention of a multiple myeloma.
The present invention is accordingly directed to methods for inhibiting the symptoms or conditions (disabilities, impairments) associated with multiple myeloma as described in detail below. As such, it is not required that all effects of the condition be entirely prevented or reversed, although the effects of the presently disclosed methods likely extend to a significant therapeutic benefit for the patient. As such, a therapeutic benefit is not necessarily a complete prevention or cure for the condition, but rather, can encompass a result which includes reducing or preventing the symptoms that result from multiple myeloma, reducing or preventing the occurrence of such symptoms (either quantitatively or qualitatively), reducing the severity of such symptoms or physiological effects thereof, and/or enhancing the recovery of the individual after experiencing multiple myeloma symptoms.
Specifically, the therapies of the present invention, when administered to an individual, can treat or prevent one or more of the symptoms or conditions associated with multiple myeloma and/or reduce or alleviate symptoms of or conditions associated with this disorder. As such, protecting an individual from the effects or symptoms resulting from multiple myeloma includes both preventing or reducing the occurrence and/or severity of the effects of the disorder and treating a patient in which the effects of the disorder are already occurring or beginning to occur. A beneficial effect can easily be assessed by one of ordinary skill in the art and/or by a trained clinician who is treating the patient. Preferably, there is a positive or beneficial difference in the severity or occurrence of at least one clinical or biological score, value, or measure used to evaluate such patients in those who have been treated with the methods of the present invention as compared to those that have not.
The methods can be practiced in an adjuvant setting. “Adjuvant setting” refers to a clinical setting in which an individual has had a history of multiple myeloma, and generally (but not necessarily) been responsive to therapy, which includes, but is not limited to, surgery (such as surgical resection), radiotherapy, and chemotherapy. However, because of their history of the proliferative disease (such as multiple myeloma), these individuals are considered at risk of development of the disease. Treatment or administration in the “adjuvant setting” refers to a subsequent mode of treatment. The degree of risk (i.e., when an individual in the adjuvant setting is considered as “high risk” or “low risk”) depends upon several factors, most usually the extent of disease when first treated.
The methods provided herein can also be practiced in a “neoadjuvant setting,” i.e., the method can be carried out before the primary/definitive therapy. In some embodiments, the individual has previously been treated. In some embodiments, the individual has not previously been treated. In some embodiments, the treatment is a first line therapy.
A. Anticancer Therapies
Provided herein are methods for treating individuals who have been diagnosed with multiple myeloma according to any of the diagnostic methods disclosed herein. Additionally, the invention also provides methods for preventing relapse of multiple myeloma or for treating an individual who has suffered a relapse as determined by the expression and/or subcellular localization of ncmtRNAs within plasmocytes isolated from the bone marrow of individuals diagnosed with or thought to have multiple myeloma according to any of the methods disclosed herein.
1. Oligonucleotides Complementary to ncmtRNAs
In some aspects, the anticancer therapy for use in any of the methods described herein is administration of oligonucleotides complementary to a sense or antisense chimeric non-coding mitochondrial RNA (ncmtRNA) molecule, such as any of these disclosed herein, to individuals diagnosed with multiple myeloma according to any method disclosed herein, for preventing relapse of multiple myeloma, or for treating an individual who has relapsed as determined by any of the methods disclosed herein.
The oligonucleotides for use in any of the methods described herein can be complementary to the SncmtRNA and/or to the ASncmtRNA molecules disclosed herein. Without being bound to theory, it is believed that the complementary oligonucleotides bind to the ncmtRNAs and interfere with their cellular functions. As used herein, an oligonucleotide sequence is “complementary” to a portion of an ncmtRNA, as referred to herein, if the oligonucleotide possesses a sequence having sufficient complementarity to be able to hybridize with the ncmtRNA to form a stable duplex. The ability to hybridize will depend on both the degree of complementarity and the length of the oligonucleotide. Generally, the longer the hybridizing oligonucleotide, the more base mismatches with an ncmtRNA it may contain and still form a stable duplex. In some aspects, the oligonucleotides used as an anticancer therapy according to the methods disclosed herein are at least 8 (such as at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50 or more) base pairs in length. Those skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. In some embodiments, the oligonucleotides are at least 85% (such as at least 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) complementary to the SncmtRNA and/or to the ASncmtRNA molecule. In some embodiments, the complementary oligonucleotide is an antisense oligonucleotide. In one embodiment, the oligonucleotides are complementary to the ncmtRNAs encoded by one or more of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6. In another embodiments, the oligonucleotides comprise one or more of SEQ ID NOs:7-196.
a. Oligonucleotide Modifications
The naturally occurring internucleoside linkage of RNA and DNA is a 3′ to 5 phosphodiester linkage. The oligonucleotides (for example, an antisense oligonucleotides) used for treating multiple myeloma according to any of the methods disclosed herein can have one or more modified, i.e. non-naturally occurring, internucleoside linkages. With respect to therapeutics, modified internucleoside linkages are often selected over oligonucleotides having naturally occurring internucleoside linkages because of desirable properties such as, for example, enhanced cellular uptake, enhanced affinity for target nucleic acids, and increased stability in the presence of nucleases.
Oligonucleotides (such as an antisense oligonucleotide) having modified internucleoside linkages include internucleoside linkages that retain a phosphorus atom as well as internucleoside linkages that do not have a phosphorus atom. Representative phosphorus containing internucleoside linkages include, but are not limited to, phosphodiesters, phosphotriesters, methylphosphonates, phosphoramidate, and phosphorothioates. Methods of preparation of phosphorous-containing and non-phosphorous-containing linkages are well known.
In one embodiment, oligonucleotides (such as antisense oligonucleotides) targeted to the SncmtRNA and/or to the ASncmtRNA molecules disclosed herein comprise one or more modified internucleoside linkages. In some embodiments, the modified internucleoside linkages are phosphorothioate linkages. In other embodiments, each internucleoside linkage of an oligonucleotide compound is a phosphorothioate internucleoside linkage.
As is known in the art, a nucleoside is a base-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, however, open linear structures are generally preferred. Within the oligonucleotide structure, the phosphate groups are commonly referred to as forming the internucleoside backbone of the oligonucleotide. The normal linkage or backbone of RNA and DNA is a 3′ to 5′ phosphodiester linkage.
Specific though nonlimiting examples of oligonucleotides (such as antisense oligonucleotides) useful in the methods of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. As defined in this specification, oligonucleotides 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, and as sometimes referenced in the art, modified oligonucleotides that do not have a phosphorus atom in their internucleoside backbone can also be considered to be oligonucleosides.
In some embodiments, modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotri-esters, methyl and other alkyl phosphonates including 3′-alkylene phosphonates, 5′-alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3′-amino phosphoramidate and aminoalkylphosphoramidates, thiono-phosphoramidates, thionoalkylphosphonates, thionoalkylphospho-triesters, selenophosphates and boranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs of these, and those having inverted polarity wherein one or more internucleotide linkages is a 3′ to 3′, 5′ to 5′ or 2′ to 2′ linkage. Oligonucleotides having inverted polarity comprise a single 3′ to 3′ linkage at the 3′-most internucleotide linkage i.e. a single inverted nucleoside residue which may be abasic (the nucleobase is missing or has a hydroxyl group in place thereof) can also be employed. Various salts, mixed salts and free acid forms are also included. 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; riboacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH 2 component parts.
In other embodiments, both the sugar and the internucleoside linkage, i.e., the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for hybridization with an appropriate nucleic acid target compound. One such oligomeric compound, an oligonucleotide mimetic 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. Representative United States patents that teach the preparation of PNA compounds 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.
Representative United States patents that teach the preparation of the above phosphorus-containing and non-phosphorus-containing linkages include, but are not limited to, U.S. Pat. Nos. 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; 5,194,599; 5,565,555; 5,527,899; 5,721,218; 5,672,697 and 5,625,050, 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; 5,792,608; 5,646,269 and 5,677,439, each of which is herein incorporated by reference.
Modified oligonucleotides (such as antisense oligonucleotides) complementary to SncmtRNA and/or ASncmtRNA used as anticancer therapies in conjunction with any of the methods disclosed herein may also contain one or more substituted sugar moieties. For example, the furanosyl sugar ring can be modified in a number of ways including substitution with a substituent group, bridging to form a bicyclic nucleic acid “BNA” and substitution of the 4′-O with a heteroatom such as S or N(R) as described in U.S. Pat. No. 7,399,845, hereby incorporated by reference herein in its entirety. Other examples of BNAs are described in published International Patent Application No. WO 2007/146511, hereby incorporated by reference herein in its entirety.
The oligonucleotides (such as antisense oligonucleotides) for use in the methods for treating multiple myeloma as disclosed herein can optionally contain one or more nucleotides having modified sugar moieties. Sugar modifications may impart nuclease stability, binding affinity or some other beneficial biological property to the antisense compounds. The furanosyl sugar ring of a nucleoside can be modified in a number of ways including, but not limited to: addition of a substituent group, particularly at the 2′ position; bridging of two non-geminal ring atoms to form a bicyclic nucleic acid (BNA); and substitution of an atom or group such as —S—, —N(R)— or —C(R1)(R2) for the ring oxygen at the 4′-position. Modified sugars include, but are not limited to: substituted sugars, especially 2′-substituted sugars having a 2′-F, 2′-OCH2 (2′-OMe) or a 2′-O (CH 2 ) 2 —OCH 3 (2′-O-methoxyethyl or 2′-MOE) substituent group; and bicyclic modified sugars (BNAs), having a 4′-(CH 2 )n-O-2′ bridge, where n=1 or n=2. Methods for the preparations of modified sugars are well known to those skilled in the art.
In certain embodiments, a 2′-modified nucleoside has a bicyclic sugar moiety. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is a D sugar in the beta configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the alpha configuration. In certain such embodiments, the bicyclic sugar moiety is an L sugar in the beta configuration.
In other embodiments, the bicyclic sugar moiety comprises a bridge group between the 2′ and the 4′-carbon atoms. In certain such embodiments, the bridge group comprises from 1 to linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises from 1 to 4 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises 2 or 3 linked biradical groups. In certain embodiments, the bicyclic sugar moiety comprises 2 linked biradical groups. In certain embodiments, a linked biradical group is selected from —O—, —S—, —N(R1)-, —C(R1)(R2)-, —C(R1)=C(R1)-, —C(R1)=N—, —C(═NR1)-, —Si(R1)(R2)-, —S(═O)2-, —S(O)—, —C(═O)— and —C(═S)—; where each R1 and R2 is, independently, H, hydroxyl, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, C5-C20 aryl, substituted C5-C20 aryl, a heterocycle radical, a substituted hetero-cycle radical, heteroaryl, substituted heteroaryl, C5-C7 alicyclic radical, substituted C5-C7 alicyclic radical, halogen, substituted oxy (—O—), amino, substituted amino, azido, carboxyl, substituted carboxyl, acyl, substituted acyl, CN, thiol, substituted thiol, sulfonyl (S(═O)2-H), substituted sulfonyl, sulfoxyl (S(═O)—H) or substituted sulfoxyl; and each substituent group is, independently, halogen, C1-C12 alkyl, substituted C1-C12 alkyl, C2-C12 alkenyl, substituted C2-C12 alkenyl, C2-C12 alkynyl, substituted C2-C12 alkynyl, amino, substituted amino, acyl, substituted acyl, C1-C12 amino alkyl, C1-C12 aminoalkoxy, substituted C1-C12 aminoalkyl, substituted C1-C12 aminoalkoxy or a protecting group.
Oligonucleotides (such as antisense oligonucleotides) for use in any of the methods disclosed herein may also include nucleobase (often referred to in the art simply as “base”) modifications or substitutions. Nucleobase modifications or substitutions are structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic unmodified nucleobases. Both natural and modified nucleobases are capable of participating in hydrogen bonding. Such nucleobase modifications may impart nuclease stability, binding affinity or some other beneficial biological property to oligonucleotide compounds. Modified nucleobases include synthetic and natural nucleobases such as, for example, 5-methylcytosine (5-me-C). Certain nucleobase substitutions, including 5-methylcytosine substitutions, are particularly useful for increasing the binding affinity of an oligonucleotide compound (such as an antisense oligonucleotide compound) for a target nucleic acid (such as an ncmtRNA).
Additional unmodified nucleobases include 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 (—C≡C—CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 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 particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.
Heterocyclic base moieties may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. Nucleobases that are particularly useful for increasing the binding affinity of antisense compounds include 5-substituted pyrimidines, 6-azapyrimidines and N-2, N-6 and 0-6 substituted purines, including 2 aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.
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 (—C≡C—CH 3 ) uracil and cytosine and other alkynyl derivatives of pyrimidine bases, 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 particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 2-F-adenine, 2-amino-adenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further modified nucleobases include tricyclic pyrimidines such as phenoxazine cytidine (1H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), phenothiazine cytidine (1H-pyrimido[5,4-b][1,4]benzothiazin-2(3H)-one), 0-clamps such as a substituted phenoxazine cytidine (e.g. 9-(2-aminoethoxy)-H-pyrimido[5,4-b][1,4]benzoxazin-2(3H)-one), carbazole cytidine (2H-pyrimido[4,5-b]indol-2-one), pyridoindole cytidine (H-pyrido[3′,2′:4,5]pyrrolo[2,3-d]pyrimidin-2-one). Modified nucleobases may also include those in which the purine or pyrimidine base is replaced with other heterocycles, for example 7-deaza-adenine, 7-deazaguanosine, 2-aminopyridine and 2-pyridone. 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.
Representative United States patents that teach the preparation of certain of the above noted modified nucleobases as well as other modified nucleobases include, but are not limited to, U.S. Pat. Nos. 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; 5,645,985; 5,830,653; 5,763,588; 6,005,096; and 5,681,941, each of which is herein incorporated by reference.
b. Ribozymes
In another embodiment of the invention, ribozymes can be used to interfere with the ncmtRNA molecules described herein to induce cell death in proliferative cells associated with multiple myeloma. The sequence of the ribozyme can be designed according to the sequence of the ASncmtRNA (for example, SEQ ID NO 4, SEQ ID NO 5, SEQ ID NO 6) or the SncmtRNA (for example, SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3) to cleave specific regions of the transcript. Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA (Rossi, Curr. Biology 4:469-471, 1994). The mechanism of ribozyme action involves sequence specific hybridization of the ribozyme molecule to complementary target RNA, followed by an endonucleolytic cleavage. The composition of ribozyme molecules must include one or more sequences complementary to the RNA, and must include the well-known catalytic sequence responsible for RNA cleavage, and described in U.S. Pat. No. 5,093,246, the disclosure of which is incorporated by reference herein in its entirety. As such, within the scope of the invention hammerhead ribozyme molecules can be engineered that specifically and efficiently catalyze endonucleolytic cleavage of the ASncmtRNA or SncmtRNA molecules disclosed herein. The construction and production of hammerhead ribozymes is well known in the art and it was described (Haseloff et al., Gene, 82:43-52, 1989). Ribozymes of the present invention can also include RNA endoribonucleases (Zaug et al., Science, 224:574-578, 1984).
c. RNA Interference
In another aspect, interference with the function of the ASncmtRNA and/or SncmtRNA molecules disclosed herein for the treatment of multiple myeloma in any of the methods disclosed herein can be achieved by RNA interference or RNA silencing. RNA interference (RNAi) has emerged as a novel and promising approach for gene silencing in mammalian cells (Elbashir et al., Nature 411:494-498, 2001; McManus et al., Nature Rev. Genet. 3:737-747, 2002). Synthetically synthesized double stranded RNA molecules of about 8 to 40 (such as about 10 to 36, 14 to 32, 18-28, or 22-24) base pairs (bp) or at least about 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 bp in length hybridize specifically to their complementary target RNA, leading to degradation of the RNA. Several different genes have been silenced successfully by small interfering RNA or siRNA (Lu et al., Curr. Opin. Mol. Ther. 5:225-234, 2003; Wacheck et al., Oligonucleotides 13:393-400, 2003). Therefore, synthetic double stranded RNA targeted to the ASncmtRNA and/or SncmtRNA molecules disclosed herein can be used to degrade these transcripts and induce tumor cell death. Those familiar in the art will understand that the sequence of the siRNA has to be complementary to any region of the ASncmtRNA and/or SncmtRNA molecules (such as complementary to any of SEQ ID NO 1, SEQ ID NO 2, SEQ ID NO 3, SEQ ID NO 4, SEQ ID NO 5, and/or SEQ ID NO 6).
d. Gene Therapy
In one embodiment, a recombinant vector can be used for delivering one or more oligonucleotides (such as any of the oligonucleotides disclosed herein) complementary to a sense and/or antisense chimeric non-coding mitochondrial RNA molecule to the individual. This can include both systemic delivery and delivery localized to a particular region of the body (such as, the bone marrow). Any vector capable of enabling recombinant production of one or more oligonucleotides complementary to a sense or antisense chimeric ncmtRNA molecule and/or which can deliver one or more oligonucleotides complementary to a sense or antisense chimeric ncmtRNA molecule into a host cell. The vector can be either RNA or DNA, either prokaryotic or eukaryotic, and typically is a virus or a plasmid. The vector can be part of a DNA vaccine or used as part of any other method for delivering a heterologous gene for expression in a host cell that is known to one having skill in the art. Recombinant vectors are capable of replicating when transformed into a suitable host cell. Viral vectors infect a wide range of non-dividing human cells and have been used extensively in live vaccines without adverse side effects. A viral vector (such as, but not limited to, an adenoviral vector or an adeno-associated viral (AAV) vector (e.g. AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, etc. or hybrid AAV vectors comprising the same) is an example of a vector for use in the present methods for delivering one or more oligonucleotides complementary to a sense or antisense chimeric ncmtRNA molecule to multiple myeloma cancer cells (such as a plasmocyte; see, e.g. U.S. Patent Application Publication No. 2004/0224389, the disclosure of which is incorporated by reference herein).
2. Other Anticancer Therapies
In some aspects, any of the methods of treatment described herein can comprise administering one or more additional anticancer therapies to the individual. Various classes of anticancer agents can be used. Non-limiting examples include: alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, podophyllotoxin, antibodies (e.g., monoclonal or polyclonal), tyrosine kinase inhibitors (e.g., imatinib mesylate (Gleevec® or Glivec®)), hormone treatments, soluble receptors and other antineoplastics.
Topoisomerase inhibitors are also another class of anticancer agents that can be used. Topoisomerases are essential enzymes that maintain the topology of DNA. Inhibition of type I or type II topoisomerases interferes with both transcription and replication of DNA by upsetting proper DNA supercoiling. Some type I topoisomerase inhibitors include camptothecins: irinotecan and topotecan. Examples of type II inhibitors include amsacrine, etoposide, etoposide phosphate, and teniposide. These are semisynthetic derivatives of epipodophyllotoxins, alkaloids naturally occurring in the root of American Mayapple ( Podophyllum peltatum ).
Antineoplastics include the immunosuppressant dactinomycin, doxorubicin, epirubicin, bleomycin, mechlorethamine, cyclophosphamide, chlorambucil, ifosfamide. The antineoplastic compounds generally work by chemically modifying a cell's DNA.
Alkylating agents can alkylate many nucleophilic functional groups under conditions present in cells. Cisplatin and carboplatin, and oxaliplatin are alkylating agents. They impair cell function by forming covalent bonds with the amino, carboxyl, sulfhydryl, and phosphate groups in biologically important molecules.
Vinca alkaloids bind to specific sites on tubulin, inhibiting the assembly of tubulin into microtubules (M phase of the cell cycle). The vinca alkaloids include: vincristine, vinblastine, vinorelbine, and vindesine.
Anti-metabolites resemble purines (azathioprine, mercaptopurine) or pyrimidine and prevent these substances from becoming incorporated in to DNA during the “S” phase of the cell cycle, stopping normal development and division. Anti-metabolites also affect RNA synthesis.
Plant alkaloids and terpenoids are derived from plants and block cell division by preventing microtubule function. Since microtubules are vital for cell division, without them, cell division cannot occur. The main examples are vinca alkaloids and taxanes.
Podophyllotoxin is a plant-derived compound which has been reported to help with digestion as well as used to produce two other cytostatic drugs, etoposide and teniposide. They prevent the cell from entering the G1 phase (the start of DNA replication) and the replication of DNA (the S phase).
Taxanes as a group includes paclitaxel and docetaxel. Paclitaxel is a natural product, originally known as Taxol and first derived from the bark of the Pacific Yew tree. Docetaxel is a semi-synthetic analogue of paclitaxel. Taxanes enhance stability of microtubules, preventing the separation of chromosomes during anaphase.
In some aspects, the anticancer therapeutics can be selected from remicade, docetaxel, celecoxib, melphalan, dexamethasone (Decadron®), steroids, gemcitabine, cisplatinum, temozolomide, etoposide, cyclophosphamide, temodar, carboplatin, procarbazine, gliadel, tamoxifen, topotecan, methotrexate, Arisa®, taxol, taxotere, fluorouracil, leucovorin, irinotecan, xeloda, CPT-11, interferon alpha, pegylated interferon alpha (e.g., PEG INTRON-A), capecitabine, cisplatin, thiotepa, fludarabine, carboplatin, liposomal daunorubicin, cytarabine, doxetaxol, pacilitaxel, vinblastine, IL-2, GM-CSF, dacarbazine, vinorelbine, zoledronic acid, palmitronate, biaxin, busulphan, prednisone, bortezomib (Velcade®), bisphosphonate, arsenic trioxide, vincristine, doxorubicin (Doxil®), paclitaxel, ganciclovir, adriamycin, estrainustine sodium phosphate (Emcyt®), sulindac, or etoposide.
In other embodiments, the anticancer therapeutics can be selected from bortezomib, cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha, prednisone, thalidomide, or vincristine.
3. Stem Cell Transplantation and Ex Vivo Treatment of Autologous Hematopoietic Stem Cells
In other aspects, any of the methods of treatment described herein can include either autologous or allogenic stem cell transplantation therapy. In recent years, high-dose chemotherapy with autologous hematopoietic stem-cell transplantation has become the preferred treatment for multiple myeloma patients under the age of 65. While not curative, this procedure does prolong overall survival and complete remission. Prior to stem-cell transplantation, these patients receive an initial course of induction chemotherapy. The most common induction regimens used today are thalidomide-dexamethasone, bortezomib based regimens, and lenalidomide-dexamethasone (Kyle & Rajkumar, 2008, Blood. 111 (6): 2962 72). Autologous peripheral stem cell transplantation is useful for up to 50% of multiple myeloma patients. Despite a low mortality rate, problems with such transplant therapy include the inability to eradicate the tumor and the difficulty in the removal of myeloma cells and their precursors from the stem cell collection used for transplantation. Autologous stem cell transplantation is typically used for individuals under the age of 65 years who do not have substantial heart, lung, renal or liver dysfunction.
Allogenic transplant (the transplantation of a healthy person's stem cells into the affected individual), is another therapy option for treating multiple myeloma, but is less frequently used since the mortality rate at 100 days is 25-30% and it does not provide a cure. Only 5-10% of patients with multiple myeloma are eligible for allogeneic bone marrow transplantation because of their age and the paucity of a human leukocyte antigen (HLA)-matched sibling donor. Use of allogenic transplant for the treatment of relapsed myeloma also remains a treatment strategy with limited clinical benefit. Most studies evaluating its use in this setting demonstrate long-term disease-free survival of 10-20%, with a significant fraction of patients developing relapse.
When included as a treatment for multiple myeloma according to any of the methods disclosed herein, autologous stem cell transplantation can also include the step of treating the hematopoietic stem-cells and/or bone marrow to be transplanted into the affected individual with any of the anticancer therapeutics disclosed herein, prior to transplantation into the affected individual. In one embodiment, hematopoietic stem-cells and/or bone marrow for use in autologous stem cell transplantation can be treated with an effective amount of one or more oligonucleotides (such as antisense oligonucleotides) sufficiently complementary to an ASncmtRNA or SncmtRNA molecule (such as any of the ASncmtRNA and/or SncmtRNA molecules disclosed herein) to form a stable duplex prior to transplantation into the affected individual. In another embodiment, the oligonucleotides are sufficiently complementary to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, to form a stable duplex. In other embodiments, the oligonucleotides comprise one or more of SEQ ID Nos:7-196.
It has been shown that autologous transplantation of bone marrow or hematological stem cells can also be used to treat other forms of hematological cancers (such as, but not limited to, leukemia and lymphoma). Accordingly, in some aspects, when included as a treatment for a hematological cancer, provided herein is a method of performing autologous stem cell transplantation which includes the step of treating the hematopoietic stem-cells and/or bone marrow to be transplanted into the affected individual with any of the anticancer therapeutics disclosed herein, prior to transplantation into the affected individual. In one embodiment, hematopoietic stem-cells and/or bone marrow for use in autologous stem cell transplantation in an individual with a hematological cancer can be treated with an effective amount of one or more oligonucleotides (such as antisense oligonucleotides) sufficiently complementary to an ASncmtRNA or SncmtRNA molecule (such as any of the ASncmtRNA and/or SncmtRNA molecules disclosed herein) to form a stable duplex prior to transplantation into the affected individual. In another embodiment, the oligonucleotides are sufficiently complementary to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6, to form a stable duplex. In other embodiments, the oligonucleotides comprise one or more of SEQ ID Nos:7-196.
4. Pharmaceutical Compositions
Any of the anticancer therapies (such as oligonucleotide-based therapies) disclosed herein can be administered in the form of pharmaceutical compositions. These compounds can be administered by a variety of routes including oral, rectal, transdermal, subcutaneous, intravenous, intramuscular, and intranasal. These compounds are effective as both injectable and oral compositions. Such compositions are prepared in a manner well known in the pharmaceutical art and comprise at least one active compound. When employed as oral compositions, the oligonucleotides and another disclosed herein are protected from acid digestion in the stomach by a pharmaceutically acceptable protectant.
This invention also includes pharmaceutical compositions which contain, as the active ingredient, one or more of the anticancer therapies disclosed herein associated with one or more pharmaceutically acceptable excipients or carriers. In making the compositions of this invention, the active ingredient is usually mixed with an excipient or carrier, diluted by an excipient or carrier or enclosed within such an excipient or carrier which can be in the form of a capsule, sachet, paper or other container. When the excipient or carrier serves as a diluent, it can be a solid, semi-solid, or liquid material, which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), ointments containing, for example, up to 10% by weight of the active compound, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders.
In preparing a formulation, it may be necessary to mill the active lyophilized compound to provide the appropriate particle size prior to combining with the other ingredients. If the active compound is substantially insoluble, it ordinarily is milled to a particle size of less than 200 mesh. If the active compound is substantially water soluble, the particle size is normally adjusted by milling to provide a substantially uniform distribution in the formulation, e.g. about 40 mesh.
Some examples of suitable excipients or carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starches, gum acacia, calcium phosphate, alginates, tragacanth, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, sterile water, syrup, and methyl cellulose. The formulations can additionally include: lubricating agents such as talc, magnesium stearate, and mineral oil; wetting agents; emulsifying and suspending agents; preserving agents such as methyl- and propylhydroxy-benzoates; sweetening agents; and flavoring agents. The compositions of the invention can be formulated so as to provide quick, sustained or delayed release of the active ingredient after administration to the patient by employing procedures known in the art.
The compositions can be formulated in a unit dosage form, each dosage containing from about 5 mg to about 100 mg or more, such as any of about 1 mg to about 5 mg, 1 mg to about 10 mg, about 1 mg to about 20 mg, about 1 mg to about 30 mg, about 1 mg to about 40 mg, about 1 mg to about 50 mg, about 1 mg to about 60 mg, about 1 mg to about 70 mg, about 1 mg to about 80 mg, or about 1 mg to about 90 mg, inclusive, including any range in between these values, of the active ingredient. The term “unit dosage forms” refers to physically discrete units suitable as unitary dosages for individuals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient or carrier.
The anticancer therapies disclosed herein are effective over a wide dosage range and are generally administered in a therapeutically effective amount. It will be understood, however, that the amount of the anticancer therapies actually administered will be determined by a physician, in the light of the relevant circumstances, including the condition to be treated, the chosen route of administration, the actual compound administered, the age, weight, and response of the individual patient, the severity of the patient's symptoms, and the like.
For preparing solid compositions such as tablets, the principal active ingredient anticancer therapy is mixed with a pharmaceutical excipient or carrier to form a solid preformulation composition containing a homogeneous mixture of a compound of the present invention. When referring to these preformulation compositions as homogeneous, it is meant that the active ingredient is dispersed evenly throughout the composition so that the composition can be readily subdivided into equally effective unit dosage forms such as tablets, pills and capsules.
The tablets or pills of the present invention can be coated or otherwise compounded to provide a dosage form affording the advantage of prolonged action and to protect the anticancer therapies (such as an oligonucleotide) from acid hydrolysis in the stomach. For example, the tablet or pill can comprise an inner dosage and an outer dosage component, the latter being in the form of an envelope over the former. The two components can be separated by an enteric layer which serves to resist disintegration in the stomach and permit the inner component to pass intact into the duodenum or to be delayed in release. A variety of materials can be used for such enteric layers or coatings, such materials including a number of polymeric acids and mixtures of polymeric acids with such materials as shellac, cetyl alcohol, and cellulose acetate.
The liquid forms in which the novel compositions of the present invention can be incorporated for administration orally or by injection include aqueous solutions, suitably flavored syrups, aqueous or oil suspensions, and flavored emulsions with edible oils such as corn oil, cottonseed oil, sesame oil, coconut oil, or peanut oil, as well as elixirs and similar pharmaceutical vehicles.
Compositions for inhalation or insufflation include solutions and suspensions in pharmaceutically acceptable, aqueous or organic solvents, or mixtures thereof, and powders. The liquid or solid compositions can contain suitable pharmaceutically acceptable excipients as described herein. The compositions can be administered by the oral or nasal respiratory route for local or systemic effect. Compositions in pharmaceutically acceptable solvents can be nebulized by use of inert gases. Nebulized solutions can be inhaled directly from the nebulizing device or the nebulizing device can be attached to a face mask tent, or intermittent positive pressure breathing machine. Solution, suspension, or powder compositions can also be administered, orally or nasally, from devices which deliver the formulation in an appropriate manner.
B. Methods for Treating Multiple Myeloma
Provided herein are method for treating multiple myeloma in an individual in need thereof comprising: measuring the expression of a sense non-coding mitochondrial RNA (SncmtRNA) and an antisense non-coding mitochondrial RNA (ASncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein the expression of both a SncmtRNA and an ASncmtRNA indicates that the individual does not have multiple myeloma and wherein the expression of a SncmtRNA and the lack of expression of an ASncmtRNA indicates that the individual has multiple myeloma; and treating the individual with an effective amount of one or more anticancer therapeutics (such as any of the anticancer therapeutics disclosed herein) if the plasmocytes of the individual express only the SncmtRNA and do not express the ASncmtRNA.
Also provided herein is a method for treating multiple myeloma in an individual in need thereof comprising: treating the individual with an effective amount of one or more anticancer therapeutics, wherein prior to treatment it has been determined that plasmocytes isolated from the bone barrow of the individual express a sense non-coding mitochondrial RNA (SncmtRNA) and do not express an antisense non-coding mitochondrial RNA (ASncmtRNA).
In some embodiments, the anticancer therapeutics comprise one or more oligonucleotides (such as antisense oligonucleotides) sufficiently complementary to a human non-coding mitochondrial chimeric RNA molecule comprising an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence or a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence, wherein the oligonucleotides are able to hybridize with the mitochondrial chimeric RNA molecules to form a stable duplex. In some embodiments, the anticancer therapeutic is an RNAi oligonucleotide, an antisense oligonucleotide, or a ribozyme. In yet other embodiments, the oligonucleotide contains one or more modifications to a phosphate backbone linkage, a sugar, or a base, such as any of the modifications disclosed herein.
In another embodiment, the anticancer therapeutics can be selected from bortezomib, cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha, prednisone, thalidomide, or vincristine or any other compound disclosed herein for the treatment of multiple myeloma.
In a further embodiment, the individual is treated with a combination of one or more oligonucleotides (such as antisense oligonucleotides) sufficiently complementary to any of the SncmtRNA and/or ASncmtRNA molecules disclosed herein and an anticancer therapeutic selected from bortezomib, cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha, prednisone, thalidomide, or vincristine or any other compound disclosed herein for the treatment of multiple myeloma. In yet another embodiment, the individual is treated with stem cell transplant therapy, either autologous or allogenic.
As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of a compound of the present invention in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
In another embodiment, the method further comprises administering one or more additional therapies. These additional therapies can include, without limitation, allogenic or autologous stem cell transplant therapy. In embodiments where the additional therapy includes autologous stem cell transplant therapy, the method can further comprise the step of treating hematopoietic stem-cells and/or bone marrow for use in autologous stem cell transplantation with an effective amount of one or more oligonucleotides (such as antisense oligonucleotides) sufficiently complementary to an ASncmtRNA or SncmtRNA molecule (such as any of the ASncmtRNA and/or SncmtRNA molecules disclosed herein) to form a stable duplex prior to transplantation into the affected individual.
C. Methods for Preventing Relapse of Multiple Myeloma
In other aspects, provided herein are methods for preventing relapse of multiple myeloma in an individual who has responded to initial treatment and is in remission. A number of randomized studies have shown a benefit from maintenance therapy with novel agents at least in terms of response rate and progression-free survival (PFS) following initial successful treatment (Harousseau, 2009, Hematol. Rep., 1(2)).
Accordingly, provided herein are methods for preventing relapse of multiple myeloma in an individual in need thereof comprising measuring the subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein the cytoplasmic localization of the SncmtRNA indicates that the individual is in remission; and treating the individual with an effective amount of one or more maintenance anticancer therapeutics if the SncmtRNA is localized to the cytoplasm of the plasmocytes.
Also provided herein are methods for preventing relapse in multiple myeloma in an individual in need thereof comprising: treating the individual with an effective amount of one or more maintenance anticancer therapeutics, wherein prior to treatment it has been determined that plasmocytes isolated from the bone barrow of the individual exhibit subcellular cytoplasmic localization of a sense non-coding mitochondrial RNA (SncmtRNA).
In some embodiments, the maintenance anticancer therapeutics comprise any of the oligonucleotide-based (such as antisense-based) therapies disclosed herein and/or any other therapy for the treatment of multiple myeloma disclosed herein.
In other embodiments, a “maintenance schedule” may be used in which one or more maintenance anticancer therapies are administered less frequency than in the original treatment administered prior to remission, such as once per week or once every two weeks. The maintenance schedule can be continued either for a fixed period of time, generally 1-2 years, or indefinitely as long as the patient is continuing to show no signs of progressive disease and is tolerating the treatment without significant toxicity.
D. Methods for Treating Relapsed Multiple Myeloma
In yet other aspects, provided herein are methods for treating relapsed multiple myeloma in an individual thereof. “Relapsed and/or refractory multiple myeloma” refers to multiple myeloma that has become unresponsive to a drug or a therapy. For example and without limitation, relapsed and/or refractory multiple myeloma includes multiple myeloma in patients whose first progression occurs in the absence of any treatment following successful treatment with a drug or a therapy; multiple myeloma in patients who progress on a treatment, or within 60 days of the treatment; and multiple myeloma in patients who progress while receiving treatment.
Accordingly, in some aspects, provided herein are methods for treating relapsed multiple myeloma in an individual thereof comprising measuring the subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA) in plasmocytes isolated from the bone barrow of the individual, wherein the (i) nuclear or (ii) cytoplasmic and nuclear localization of the SncmtRNA indicates that the individual has relapsed; and treating the individual with one or more anti-cancer therapeutics if the SncmtRNA is localized to (i) the nuclei or (ii) cytoplasm and nuclei of the plasmocytes.
Also provided herein, in other aspects, are methods for treating relapsed multiple myeloma in an individual thereof comprising treating the individual with one or more anti-cancer therapeutics, wherein prior to treatment it has been determined that plasmocytes isolated from the bone barrow of the individual exhibit (i) nuclear or (ii) cytoplasmic and nuclear subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA).
In another embodiment, the therapeutically effective amount of said composition is administered as part of a salvage therapy in treating patients wherein the multiple myeloma has become refractory to other drugs for treating multiple myeloma. In some embodiments, the drug for treating multiple myeloma to which the multiple myeloma is refractory, includes, without limitation, bortezomib, cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha, prednisone, thalidomide, and vincristine.
In some embodiments, the anticancer therapeutics for treating relapsed multiple myeloma comprise one or more oligonucleotides (such as antisense oligonucleotides) sufficiently complementary to a human non-coding mitochondrial chimeric RNA molecule comprising an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence or a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence, wherein the oligonucleotides are able to hybridize with the mitochondrial chimeric RNA molecules to form a stable duplex. In some embodiments, the anticancer therapeutic is an RNAi oligonucleotide, an antisense oligonucleotide, or a ribozyme. In yet other embodiments, the oligonucleotide contains one or more modifications to a phosphate backbone linkage, a sugar, or a base, such as any of the modifications disclosed herein.
In another embodiment, the anticancer therapeutics can be selected from bortezomib, cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha, prednisone, thalidomide, or vincristine or any other compound disclosed herein for the treatment of multiple myeloma.
In a further embodiment, the individual is treated with a combination of one or more oligonucleotides (such as antisense oligonucleotides) sufficiently complementary to any of the SncmtRNA and/or ASncmtRNA molecules disclosed herein and an anticancer therapeutic selected from bortezomib, cyclophosphamide, dexamethasone, doxorubicin, interferon-alpha, lenalidomide, melphalan, pegylated interferon-alpha, prednisone, thalidomide, or vincristine or any other compound disclosed herein for the treatment of multiple myeloma. In yet another embodiment, the individual is treated with stem cell transplant therapy, either autologous or allogenic.
As non-limiting examples, treatment according to the present invention may be provided as a daily dosage of a compound of the present invention in an amount of about 0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 after initiation of treatment, or any combination thereof, using single or divided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.
In another embodiment, the method for treating relapsed multiple myeloma further comprises administering one or more additional therapies. These additional therapies can include, without limitation, allogenic or autologous stem cell transplant therapy. In embodiments where the additional therapy includes autologous stem cell transplant therapy, the method can further comprise the step of treating hematopoietic stem-cells and/or bone marrow for use in autologous stem cell transplantation with an effective amount of one or more oligonucleotides (such as antisense oligonucleotides) sufficiently complementary to an ASncmtRNA or SncmtRNA molecule (such as any of the ASncmtRNA and/or SncmtRNA molecules disclosed herein) to form a stable duplex prior to transplantation into the affected individual.
V. Kits
In other aspects, provided herein are kits suitable for performing an assay which detect the expression and subcellular localization of one or more SncmtRNA and/or ASncmtRNA molecules (such as any of those disclosed herein) in plasmocytes isolated from the bone marrow of individuals diagnosed with or thought to have multiple myeloma.
In one embodiment, the kit is for diagnosing multiple myeloma. This kit can comprise one or more of one or more oligonucleotides sufficiently complementary to a human mitochondrial chimeric RNA molecule comprising (i) an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence or (ii) a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence, wherein the oligonucleotides are able to hybridize with the mitochondrial chimeric RNA molecules to form a stable duplex, one or more conjugated antibody reagents for label detection, one or more hybridization and/or wash buffers; and/or one or more slides of fixed plasmocytes (i) isolated from an individual diagnosed with multiple myeloma or a multiple myeloma cell line as a positive control and (ii) isolated from an individual without multiple myeloma or from a normally proliferating cell line as a negative control.
In another embodiment, the kit is for determining whether an individual diagnosed with multiple myeloma that is in remission will relapse. This kit can comprise one or more of one or more oligonucleotides sufficiently complementary to a human mitochondrial chimeric RNA molecule comprising (i) an antisense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence or (ii) a sense 16S mitochondrial ribosomal RNA covalently linked at its 5′ end to the 3′ end of a polynucleotide with an inverted repeat sequence, wherein the oligonucleotides are able to hybridize with the mitochondrial chimeric RNA molecules to form a stable duplex, one or more conjugated antibody reagents for label detection, one or more hybridization and/or wash buffers; and/or one or more slides of fixed plasmocytes (i) isolated from an individual diagnosed with multiple myeloma that has relapsed or isolated from a multiple myeloma cell line exhibiting nuclear or cytoplasmic and nuclear subcellular localization of a sense non-coding mitochondrial RNA (SncmtRNA) as a positive control and (ii) isolated from an individual without relapsed multiple myeloma or isolated from a multiple myeloma cell line exhibiting cytoplasmic subcellular localization of a SncmtRNA as a negative control.
EXAMPLES
Example 1
Determination of Expression and Subcellular Localization of ncmtRNAs in Plasmocytes Isolated from Healthy Individuals
In this study, differential expression of antisense non-coding mitochondrial RNA (ASncmtRNA) and sense non-coding mitochondrial RNA (SncmtRNA) was determined in plasmocytes isolated from the bone marrow of healthy volunteers.
Materials and Methods
Isolation of plasmocytes from bone marrow: To obtain plasmocytes, a bone marrow sample of 1-2 ml was obtained from the patient by inserting a needle into the breastbone under local anesthesia. A small sample of bone marrow (1-2 ml) was removed and the mononuclear cells separated by centrifugation in a histopaque gradient. The layer of cells was recovered, washed with PBS (50 mM sodium phosphate, 150 mM NaCl and 2 mM EDTA, pH 9.0) and incubated with magnetic beads (Miltenyi Biotech) loaded with anti-CD 138 antibodies. The plasmocytes (CD138 positive) were purified with a magnetic column and suspended in PBS. Samples containing between 200 to 500 cells in 100-200 μl were cytospinned on positively charged slides.
Fluorescence in situ hybridization (FISH) to detect ncmtRNAs: Isolated plasmocytes fixed on slides were incubated with 100 μl 0.2N HCl for 5 min at room temperature. The acid was discarded and the cells washed twice with PBS. The cells were then incubated with 100 μl hybridization buffer (50% formamide, 150 μg/ml herring sperm DNA, 4×SSC, 2 mM EDTA) containing 0.5 μM Texas Red Alexa labeled probe P1 (5′ GTTCTTGGGTGGGTGTGGG 3′), complementary to the SncmtRNA. Another slide was hybridized with 0.05 μM each of two 5′ Texas Red-labeled probes P2 (5′ GATAACAGCGCAATCCTATT 3′) and P3 (5′ ACCGTGCAAAGGTAGCATAATCA 3′), complementary to the ASncmtRNAs. A third slide was hybridized with the positive hybridization control corresponding to a 5′-Texas Red-labeled probe complementary to 18S rRNA (P4: 5′ AGTGGACTCATTCCAATTACA 3′). The cells were finally stained with DAPI (1 mg/ml) and analyzed by fluorescence microscopy on an Olympus BX-51 micro scope.
Results
The results of the analysis of plasmocytes from healthy donors (LC and NF) are depicted in FIG. 1 . Normal cells are identified by their hybridization signal to both SncmtRNA and ASncmtRNA probes. Additionally, the transcripts for both species of ncmtRNAs display cytoplasmic subcellular localization.
Example 2
Determination of Expression and Subcellular Localization of ncmtRNAs in Plasmocytes Isolated from Individuals Diagnosed with Multiple Myeloma
In this example, plasmocytes were obtained from the bone marrow of individuals who had previously been diagnosed with multiple myeloma. Plasmocytes were isolated from individuals prior to treatment with any anticancer therapy.
Methods
Plasmocytes were isolated from the bone marrow of individuals positively diagnosed with multiple myeloma and FISH performed to detect the expression and subcellular localization of SncmtRNA and ASncmtRNA as described above. A probe to the 18S rRNA subunit was used as a positive control.
Results
Results are depicted in FIG. 2 . FISH staining of plasmocytes reveals positive hybridization to the SncmtRNA probe and the lack of hybridization signal to the ASncmtRNA probe. Additionally, for plasmocytes isolated from individuals who have been diagnosed with multiple myeloma, SncmtRNA is localized only in the cytoplasm of the plasmocytes ( FIGS. 2A-C ). This pattern of SncmtRNA expression and subcellular localization was generally associated with a more favorable prognosis.
Example 3
Determination of Expression and Subcellular Localization of ncmtRNAs in Plasmocytes Isolated from Individuals Diagnosed with Multiple Myeloma Who have Relapsed Following Treatment
In this example, plasmocytes were obtained from the bone marrow of individuals who had previously been diagnosed with multiple myeloma. Plasmocytes were isolated from individuals following treatment with bortezomib (Velcade®) who had suffered subsequent relapse of symptoms.
Methods
Plasmocytes were isolated from the bone marrow of individuals positively diagnosed with multiple myeloma and FISH performed to detect the expression and subcellular localization of SncmtRNA and ASncmtRNA as described above. A probe to the 18S rRNA subunit was used as a positive control.
Results
Results are depicted in FIG. 3 . FISH staining of plasmocytes reveals positive hybridization to the SncmtRNA probe and the lack of hybridization signal to the ASncmtRNA probe. Additionally, for plasmocytes isolated from individuals who have been diagnosed with multiple myeloma, treated with bortezomib, and suffered a subsequent relapse of multiple myeloma symptoms, SncmtRNA was localized either mainly in the nucleus of the plasmocytes ( FIGS. 3C-D ) or both in the cytoplasm and the nucleus ( FIGS. 3A-B and E). A probe to the 18S rRNA subunit was used as a positive control.
Example 4
Determination of Expression and Subcellular Localization of ncmtRNAs in Plasmocytes Isolated from Individuals with MGUS
In this example, plasmocytes were obtained from the bone marrow of individuals who had previously been diagnosed with monoclonal gammopathy of unknown significance (MGUS).
Methods Plasmocytes were isolated from the bone marrow of individuals with MGUS and FISH performed to detect the expression and subcellular localization of SncmtRNA and ASncmtRNA as described above. A probe to the 18S rRNA subunit was used as a positive control.
Results
Results are depicted in FIG. 4 . FISH staining of plasmocytes reveals positive hybridization to the SncmtRNA probe and the lack of hybridization signal to the ASncmtRNA probe. Significantly, SncmtRNA is localized only in the cytoplasm of the plasmocytes, consistent with the subcellular localization of SncmtRNA in the plasmocytes of individuals diagnosed with multiple myeloma but prior to relapse.
Example 5
Treatment of a Murine Myeloma Cell Line with Antisense Oligonucleotides to ncmtRNAs Followed by Transplantation into Mice
Autologous bone marrow transplantation has become a major treatment option for an increasing number of patients with hematologic cancer such as multiple myeloma. Autologous transplantation (AT) has several potential advantages over allogeneic transplantation. However, the major obstacle to use AT is that the infusion of bone marrow occults tumor cells harbored within the harvested marrow would result in more rapid relapse of disease. To minimize relapse, many authors have taken a new approach: treatment or purge the collected bone marrow with drugs to eliminate the malignant cells or their precursors (cancer stem cells). Post-treatment, the bone marrow will eventually be cleaned of malignant cells.
As a cell model for the purging of bone marrow, we work with the murine myeloma cell line NSO2. Treatment of these cells in vitro with antisense oligonucleotides complementary to the mouse ASncmtRNA induces massive cell death by apoptosis without affecting normal mouse cells. Knocking down the ASncmtRNA induces activation of caspases, DNA fragmentation and causes cells to become annexin V positive, which are all hallmarks of apoptoses. Accordingly, based on these results, we employed these cells to determine if treatment of NSO2 cells with antisense oligonucleotide complementary to the mouse ASncmtRNA prior to transplantation into mice can prevent or decrease symptoms associated with multiple myeloma.
Materials and Methods
NSO2 cells were transfected with 150 nM antisense oligonucleotide control (ASO-C) or 150 nM of an oligonucleotide complementary to the mouse ASncmtRNA (Therapeutic ASO or ASO-1560S) or left untreated (no treatment) for 48 hours. Transfection was carried out with Lipofectamin 2000 according to Invitrogene Instructions. At 48 hours post-transfection, cells were harvested and counted.
Post-treatment, 200,000 cells (controls or treated with the therapeutic ASO or ASO-1560S) were injected intraperitoneally into balb/c mice. Mice survival was follow for 90 days post-cell injection.
Results
As shown in FIG. 5 , around day 40 post-cell injection (day 0 on the graph), mice injected with untreated cells or cells treated with ASO-C died. In contrast, mice injected with cells treated with the therapeutic ASO or ASO-1560S exhibit 100% survival after 90 days. At this time, mice were sacrificed for histopathological studies. Compared with untreated mice, no histopathological alteration of liver, spleen, kidney, intestine, heart and lung was observed.
The examples, which are intended to be purely exemplary of the invention and should therefore not be considered to limit the invention in any way, also describe and detail aspects and embodiments of the invention discussed above. The foregoing examples and detailed description are offered by way of illustration and not by way of limitation. All publications, patent applications, and patents cited in this specification are herein incorporated by reference as if each individual publication, patent application, or patent were specifically and individually indicated to be incorporated by reference. In particular, all publications cited herein are expressly incorporated herein by reference for the purpose of describing and disclosing compositions and methodologies which might be used in connection with the invention. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.







SEQUENCES







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gggtaataat gacttgttgg ttgattgtag atattgggct gttaattgtc agttcagtgt
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tttaatctga cgcaggctta tgcggaggag aatgttttca tgttacttat actaacatta
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gttcttctat agggtgatag attggtccaa ttgggtgtga ggagttcagt tatatgtttg
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gttatttcta gttaattcat tatgcagaag gtataggggt tagtccttgc tatattatgc
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tctatcgcct atactttatt tgggtaaatg gtttggctaa acctagcccc aaacccactc
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caccttacta ccagacaacc ttagccaaac catttaccca aataaagtat aggcgataga
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gcataatata gcaaggacta acccctatac cttctgcata atgaattaac tagaaataac
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tttgcaagga gagccaaagc taagaccccc gaaaccagac gagctaccta agaacagcta
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aaagagcaca cccgtctatg tagcaaaata gtgggaagat ttataggtag aggcgacaaa
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cctaccgagc ctggtgatag ctggttgtcc aagatagaat cttagttcaa ctttaaattt
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gcccacagaa ccctctaaat ccccttgtaa atttaactgt tagtccaaag aggaacagct
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ctaaaagcag ccaccaatta agaaagcgtt caagctcaac acccactacc taaaaaatcc
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caaacatata actgaactcc tcacacccaa ttggaccaat ctatcaccct atagaagaac
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taatgttagt ataagtaaca tgaaaacatt ctcctccgca taagcctgcg tcagattaaa
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acactgaact gacaattaac agcccaatat ctacaatcaa ccaacaagtc attattaccc
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tcactgtcaa cccaacacag gcatgctcat aaggaaaggt taaaaaaagt aaaaggaact
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cggcaaatct taccccgcct gtttaccaaa aacatcacct ctagcatcac cagtattaga
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ggcaccgcct gcccagtgac acatgtttaa cggccgcggt accctaaccg tgcaaaggta
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gcataatcac ttgttcctta aatagggacc tgtatgaatg gctccacgag ggttcagctg
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tctcttactt ttaaccagtg aaattgacct gcccgtgaag aggcgggcat aacacagcaa
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gacgagaaga ccctatggag ctttaattta ttaatgcaaa cagtacctaa caaacccaca
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ggtcctaaac taccaaacct gcattaaaaa tttcggttgg ggcgacctcg gagcagaacc
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caacctccga gcagtacatg ctaagacttc accagtcaaa gcgaactact atactcaatt
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gatccaataa cttgaccaac ggaacaagtt accctaggga taacagcgca atcctattct
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gcagccgcta ttaaaggttc gtttgttcaa cgattaaagt cctacgtgat ctgagttcag
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accggagtaa tccaggtcgg tttctatcta ccttcaaatt cctccctgta cgaaaggaca
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agagaaataa ggcctacttc acaaagcgcc ttcccccgta aatgatatca tctcaactta
2340

gtattatacc cacacccacc caagaacagg gttt
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ccagacgagc tacctaagaa cagctaaaag agcacacccg tctatgtagc aaaatagtgg
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tagaatctta gttcaacttt aaatttgccc acagaaccct ctaaatcccc ttgtaaattt
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aactgttagt ccaaagagga acagctcttt ggacactagg aaaaaacctt gtagagagag
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taaaaaattt aacacccata gtaggcctaa aagcagccac caattaagaa agcgttcaag
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ctcaacaccc actacctaaa aaatcccaaa catataactg aactcctcac acccaattgg
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accaatctat caccctatag aagaactaat gttagtataa gtaacatgaa aacattctcc
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tccgcataag cctgcgtcag attaaaacac tgaactgaca attaacagcc caatatctac
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aatcaaccaa caagtcatta ttaccctcac tgtcaaccca acacaggcat gctcataagg
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aaaggttaaa aaaagtaaaa ggaactcggc aaatcttacc ccgcctgttt accaaaaaca
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tcacctctag catcaccagt attagaggca ccgcctgccc agtgacacat gtttaacggc
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cgcggtaccc taaccgtgca aaggtagcat aatcacttgt tccttaaata gggacctgta
1080

tgaatggctc cacgagggtt cagctgtctc ttacttttaa ccagtgaaat tgacctgccc
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gtgaagaggc gggcataaca cagcaagacg agaagaccct atggagcttt aatttattaa
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tgcaaacagt acctaacaaa cccacaggtc ctaaactacc aaacctgcat taaaaatttc
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ggttggggcg acctcggagc agaacccaac ctccgagcag tacatgctaa gacttcacca
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gtcaaagcga actactatac tcaattgatc caataacttg accaacggaa caagttaccc
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aaattcctcc ctgtacgaaa ggacaagaga aataaggcct acttcacaaa gcgccttccc
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ccgtaaatga tatcatctca acttagtatt atacccacac ccacccaaga acagggttt
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tataggggtt agtccttgct aaacctagcc ccaaacccac tccaccttac taccagacaa
120

ccttagccaa accatttacc caaataaagt ataggcgata gaaattgaaa cctggcgcaa
180

tagatatagt accgcaaggg aaagatgaaa aattataacc aagcataata tagcaaggac
240

taacccctat accttctgca taatgaatta actagaaata actttgcaag gagagccaaa
300

gctaagaccc ccgaaaccag acgagctacc taagaacagc taaaagagca cacccgtcta
360

tgtagcaaaa tagtgggaag atttataggt agaggcgaca aacctaccga gcctggtgat
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agctggttgt ccaagataga atcttagttc aactttaaat ttgcccacag aaccctctaa
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atccccttgt aaatttaact gttagtccaa agaggaacag ctctttggac actaggaaaa
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aaccttgtag agagagtaaa aaatttaaca cccatagtag gcctaaaagc agccaccaat
600

taagaaagcg ttcaagctca acacccacta cctaaaaaat cccaaacata taactgaact
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cctcacaccc aattggacca atctatcacc ctatagaaga actaatgtta gtataagtaa
720

catgaaaaca ttctcctccg cataagcctg cgtcagatta aaacactgaa ctgacaatta
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acagcccaat atctacaatc aaccaacaag tcattattac cctcactgtc aacccaacac
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aggcatgctc ataaggaaag gttaaaaaaa gtaaaaggaa ctcggcaaat cttaccccgc
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ctgtttacca aaaacatcac ctctagcatc accagtatta gaggcaccgc ctgcccagtg
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acacatgttt aacggccgcg gtaccctaac cgtgcaaagg tagcataatc acttgttcct
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taaataggga cctgtatgaa tggctccacg agggttcagc tgtctcttac ttttaaccag
1080

tgaaattgac ctgcccgtga agaggcgggc ataacacagc aagacgagaa gaccctatgg
1140

agctttaatt tattaatgca aacagtacct aacaaaccca caggtcctaa actaccaaac
1200

ctgcattaaa aatttcggtt ggggcgacct cggagcagaa cccaacctcc gagcagtaca
1260

tgctaagact tcaccagtca aagcgaacta ctatactcaa ttgatccaat aacttgacca
1320

acggaacaag ttaccctagg gataacagcg caatcctatt ctagagtcca tatcaacaat
1380

agggtttacg acctcgatgt tggatcagga catcccaatg gtgcagccgc tattaaaggt
1440

tcgtttgttc aacgattaaa gtcctacgtg atctgagttc agaccggagt aatccaggtc
1500

ggtttctatc tacttcaaat tcctccctgt acgaaaggac aagagaaata aggcctactt
1560

cacaaagcgc cttcccccgt aaatgatatc atctcaactt agtattatac ccacacccac
1620

ccaagaacag ggttt
1635



<210> SEQ ID NO 4


<211> LENGTH: 1921


<212> TYPE: DNA


<213> ORGANISM: Homo sapiens


<400> SEQUENCE: 4


aacctccgag cagtacatgc taagacttca ccagtcaaag cgaactacta tactcaattg
60

atccaataac ttgaccaacg gaacaagtta ccctagggat aacagcgcaa tcctattcta
120

gagtccatat caacaatagg gtttacgacc tcgatgttgg atcaggacat cccaatggtg
180

cagccgctat taaaggttcg tttgttcaac gattaaagtc ctacgtgatc tgagttcaga
240

ccggagtaat ccaggtcggt ttctatctac ttcaaattcc tccctgtacg aaaggacaag
300

agaaataagg cctacttcac aaagcgcctt cccccgtaaa tgatatcatc tcaacttagt
360

attataccct gttcttgggt gggtgtgggt ataatactaa gttgagatga tatcatttac
420

gggggaaggc gctttgtgaa gtaggcctta tttctcttgt cctttcgtac agggaggaat
480

ttgaagtaga tagaaaccga cctggattac tccggtctga actcagatca cgtaggactt
540

taatcgttga acaaacgaac ctttaatagc ggctgcacca tcgggatgtc ctgatccaac
600

atcgaggtcg taaaccctat tgttgatatg gactctagaa taggattgcg ctgttatccc
660

tagggtaact tgttccgttg gtcaagttat tggatcaatt gagtatagta gttcgctttg
720

actggtgaag tcttagcatg tactgctcgg aggttgggtt ctgctccgag gtcgccccaa
780

ccgaaatttt taatgcaggt ttggtagttt aggacctgtg ggtttgttag gtactgtttg
840

cattaataaa ttaaagctcc atagggtctt ctcgtcttgc tgtgttatgc ccgcctcttc
960

acgggcaggt caatttcact ggttaaaagt aagagacagc tgaaccctcg tggagccatt
900

catacaggtc cctatttaag gaacaagtga ttatgctacc tttgcacggt tagggtaccg
1020

cggccgttaa acatgtgtca ctgggcaggc ggtgcctcta atactggtga tgctagaggt
1080

gatgtttttg gtaaacaggc ggggtaagat ttgccgagtt ccttttactt tttttaacct
1140

ttccttatga gcatgcctgt gttgggttga cagtgagggt aataatgact tgttggttga
1200

ttgtagatat tgggctgtta attgtcagtt cagtgtttta atctgacgca ggcttatgcg
1260

gaggagaatg ttttcatgtt acttatacta acattagttc ttctataggg tgatagattg
1320

gtccaattgg gtgtgaggag ttcagttata tgtttgggat tttttaggta gtgggtgttg
1380

agcttgaacg ctttcttaat tggtggctgc ttttaggcct actatgggtg ttaaattttt
1440

tactctctct acaaggtttt ttcctagtgt ccaaagagct gttcctcttt ggactaacag
1500

ttaaatttac aaggggattt agagggttct gtgggcaaat ttaaagttga actaagattc
1560

tatcttggac aaccagctat caccaggctc ggtaggtttg tcgcctctac ctataaatct
1620

tcccactatt ttgctacata gacgggtgtg ctcttttagc tgttcttagg tagctcgtct
1680

ggtttcgggg gtcttagctt tggctctcct tgcaaagtta tttctagtta attcattatg
1740

cagaaggtat aggggttagt ccttgctata ttatgcttgg ttataatttt tcatctttcc
1800

cttgcggtac tatatctatt gcgccaggtt tcaatttcta tcgcctatac tttatttggg
1860

taaatggttt ggctaaggtt gtctggtagt aaggtggagt gggtttgggg ctaggtttag
1920

c
1921



<210> SEQ ID NO 5


<211> LENGTH: 1744


<212> TYPE: DNA


<213> ORGANISM: Homo sapiens


<400> SEQUENCE: 5


tagggataac agcgcaatcc tattctagag tccatatcaa caatagggtt tacgacctcg
60

atgttggatc aggacatccc gatggtgcag ccgctattaa aggttcgttt gttcaacgat
120

taaagtccta cgtgatctga gttcagaccg gagtaatcca ggtcggtttc tatctacctt
180

caaattcctc cctgttcttg ggtgggtgtg ggtataatac taagttgaga tgatatcatt
240

tacgggggaa ggcgctttgt gaagtaggcc ttatttctct tgtcctttcg tacagggagg
300

aatttgaagt agatagaaac cgacctggat tactccggtc tgaactcaga tcacgtagga
360

ctttaatcgt tgaacaaacg aacctttaat agcggctgca ccatcgggat gtcctgatcc
420

aacatcgagg tcgtaaaccc tattgttgat atggactcta gaataggatt gcgctgttat
480

ccctagggta acttgttccg ttggtcaagt tattggatca attgagtata gtagttcgct
540

ttgactggtg aagtcttagc atgtactgct cggaggttgg gttctgctcc gaggtcgccc
600

caaccgaaat ttttaatgca ggtttggtag tttaggacct gtgggtttgt taggtactgt
660

ttgcattaat aaattaaagc tccatagggt cttctcgtct tgctgtgtta tgcccgcctc
720

ttcacgggca ggtcaatttc actggttaaa agtaagagac agctgaaccc tcgtggagcc
780

attcatacag gtccctattt aaggaacaag tgattatgct acctttgcac ggttagggta
840

ccgcggccgt taaacatgtg tcactgggca ggcggtgcct ctaatactgg tgatgctaga
900

ggtgatgttt ttggtaaaca ggcggggtaa gatttgccga gttcctttta ctttttttaa
960

cctttcctta tgagcatgcc tgtgttgggt tgacagtgag ggtaataatg acttgttggt
1020

tgattgtaga tattgggctg ttaattgtca gttcagtgtt ttaatctgac gcaggcttat
1080

gcggaggaga atgttttcat gttacttata ctaacattag ttcttctata gggtgataga
1140

ttggtccaat tgggtgtgag gagttcagtt atatgtttgg gattttttag gtagtgggtg
1200

ttgagcttga acgctttctt aattggtggc tgcttttagg cctactatgg gtgttaaatt
1260

ttttactctc tctacaaggt tttttcctag tgtccaaaga gctgttcctc tttggactaa
1320

cagttaaatt tacaagggga tttagagggt tctgtgggca aatttaaagt tgaactaaga
1380

ttctatcttg gacaaccagc tatcaccagg ctcggtaggt ttgtcgcctc tacctataaa
1440

tcttcccact attttgctac atagacgggt gtgctctttt agctgttctt aggtagctcg
1500

tctggtttcg ggggtcttag ctttggctct ccttgcaaag ttatttctag ttaattcatt
1560

atgcagaagg tataggggtt agtccttgct atattatgct tggttataat ttttcatctt
1620

tcccttgcgg tactatatct attgcgccag gtttcaattt ctatcgccta tactttattt
1680

gggtaaatgg tttggctaag gttgtctggt agtaaggtgg agtgggtttg gggctaggtt
1740

tagc
1744



<210> SEQ ID NO 6


<211> LENGTH: 1854


<212> TYPE: DNA


<213> ORGANISM: Homo sapiens


<400> SEQUENCE: 6


gaactcggca aatcttaccc cgcctgttta ccaaaaacat cacctctagc atcaccagta
60

ttagaggcac cgcctgccca gtgacacatg tttaacggcc gcggtaccct aaccgtgcaa
120

aggtagcata atcacttgtt ccttaaatag ggacctgtat gaatggctcc acgagggttc
180

agctgtctct tacttttaac cagtgaaatt gacctgcccg tgaagaggcg ggcatgacac
240

agcaagacga gaagacccta tggagcttta atttattaat gcaaacagta cctaacaaac
300

cctgttcttg ggtgggtgtg ggtataatac taagttgaga tgatatcatt tacgggggaa
360

ggcgctttgt gaagtaggcc ttatttctct tgtcctttcg tacagggagg aatttgaagt
420

agatagaaac cgacctggat tactccggtc tgaactcaga tcacgtagga ctttaatcgt
480

tgaacaaacg aacctttaat agcggctgca ccatcgggat gtcctgatcc aacatcgagg
540

tcgtaaaccc tattgttgat atggactcta gaataggatt gcgctgttat ccctagggta
600

acttgttccg ttggtcaagt tattggatca attgagtata gtagttcgct ttgactggtg
660

aagtcttagc atgtactgct cggaggttgg gttctgctcc gaggtcgccc caaccgaaat
720

ttttaatgca ggtttggtag tttaggacct gtgggtttgt taggtactgt ttgcattaat
780

aaattaaagc tccatagggt cttctcgtct tgctgtgtta tgcccgcctc ttcacgggca
840

ggtcaatttc actggttaaa agtaagagac agctgaaccc tcgtggagcc attcatacag
900

gtccctattt aaggaacaag tgattatgct acctttgcac ggttagggta ccgcggccgt
960

taaacatgtg tcactgggca ggcggtgcct ctaatactgg tgatgctaga ggtgatgttt
1020

ttggtaaaca ggcggggtaa gatttgccga gttcctttta ctttttttaa cctttcctta
1080

tgagcatgcc tgtgttgggt tgacagtgag ggtaataatg acttgttggt tgattgtaga
1140

tattgggctg ttaattgtca gttcagtgtt ttaatctgac gcaggcttat gcggaggaga
1200

atgttttcat gttacttata ctaacattag ttcttctata gggtgataga ttggtccaat
1260

tgggtgtgag gagttcagtt atatgtttgg gattttttag gtagtgggtg ttgagcttga
1320

acgctttctt aattggtggc tgcttttagg cctactatgg gtgttaaatt ttttactctc
1380

tctacaaggt tttttcctag tgtccaaaga gctgttcctc tttggactaa cagttaaatt
1440

tacaagggga tttagagggt tctgtgggca aatttaaagt tgaactaaga ttctatcttg
1500

gacaaccagc tatcaccagg ctcggtaggt ttgtcgcctc tacctataaa tcttcccact
1560

attttgctac atagacgggt gtgctctttt agctgttctt aggtagctcg tctggtttcg
1620

ggggtcttag ctttggctct ccttgcaaag ttatttctag ttaattcatt atgcagaagg
1680

tataggggtt agtccttgct atattatgct tggttataat ttttcatctt tcccttgcgg
1740

tactatatct attgcgccag gtttcaattt ctatcgccta tactttattt gggtaaatgg
1800

tttggctaag gttgtctggt agtaaggtgg agtgggtttg gggctaggtt tagc
1854



<210> SEQ ID NO 7


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 7




taggtttagc accgcaaggg
20

<210> SEQ ID NO 8


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 8


taggtttagc aaggactaac
20



<210> SEQ ID NO 9


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 9


ggggtaagat ttgccgag
18



<210> SEQ ID NO 10


<211> LENGTH: 22


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 10


atgctagagg tgatgttttt gg
22



<210> SEQ ID NO 11


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 11


cggtgcctct aatactgg
18



<210> SEQ ID NO 12


<211> LENGTH:
20

<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 12


gttaaacatg tgtcactggg
20



<210> SEQ ID NO 13


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 13


ttgcacggtt agggtacc
18



<210> SEQ ID NO 14


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 14


ggaacaagtg attatgctac c
21



<210> SEQ ID NO 15


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 15


ggagccattc atacaggtcc c
21



<210> SEQ ID NO 16


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 16


agtaagagac agctgaaccc
20



<210> SEQ ID NO 17


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 17


ggcaggtcaa tttcactgg
19



<210> SEQ ID NO 18


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 18


gctgtgttat gcccgcctc
19



<210> SEQ ID NO 19


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 19


agctccatag ggtcttctc
19



<210> SEQ ID NO 20


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 20


gttaggtact gtttgcatta
20



<210> SEQ ID NO 21


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 21


aagtcttagc atgtactg
18



<210> SEQ ID NO 22


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 22


tagtagttcg ctttgactg
19



<210> SEQ ID NO 23


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: OAntisense Oligonucleotide


<400> SEQUENCE: 23


caagttattg gatcaattg
19



<210> SEQ ID NO 24


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 24


gggtaacttg ttccgttg
18



<210> SEQ ID NO 25


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 25


aataggattg cgctgtta
18



<210> SEQ ID NO 26


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 26


cctattgttg atatggac
18



<210> SEQ ID NO 27


<211> LENGTH: 17


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 27


ctgatccaac atcgagg
17



<210> SEQ ID NO 28


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 28


tagcggctgc accattgg
18



<210> SEQ ID NO 29


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 29


gttgaacaaa cgaaccttt
19



<210> SEQ ID NO 30


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 30


aactcagatc acgtaggac
19



<210> SEQ ID NO 31


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 31


cgacctggat tactccgg
18



<210> SEQ ID NO 32


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 32


ggaatttgaa gtagatag
18



<210> SEQ ID NO 33


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 33


ctcttgtcct ttcgtacag
19



<210> SEQ ID NO 34


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 34


ggcgctttgt gaagtagg
18



<210> SEQ ID NO 35


<211> LENGTH: 22


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 35


gttgagatga tatcatttac gg
22



<210> SEQ ID NO 36


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 36


cacccaccca agaacagg
18



<210> SEQ ID NO 37


<211> LENGTH: 25


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 37


caacttagta ttatacccac accca
25



<210> SEQ ID NO 38


<211> LENGTH: 22


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 38


tcccccgtaa atgattacat ct
22



<210> SEQ ID NO 39


<211> LENGTH: 25


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 39


gagaaataag gcctacttca caaag
25



<210> SEQ ID NO 40


<211> LENGTH: 22


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 40


caaattcctc cctgtacgaa ag
22



<210> SEQ ID NO 41


<211> LENGTH: 24


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 41


agtaatccag gtcggtttct atct
24



<210> SEQ ID NO 42


<211> LENGTH: 24


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 42


aagtcctagc tgatctgagt tcag
24



<210> SEQ ID NO 43


<211> LENGTH: 25


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 43


gctattaaag gttcgtttgt tcaac
25



<210> SEQ ID NO 44


<211> LENGTH: 16


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 44


tcccgatggt gcagcc
16



<210> SEQ ID NO 45


<211> LENGTH: 22


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 45


ttacgacctc gatgttggat ca
22



<210> SEQ ID NO 46


<211> LENGTH: 25


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 46


atcctattct agagtccata tcaac
25



<210> SEQ ID NO 47


<211> LENGTH: 24


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 47


aataggattg cgctgttatc ccta
24



<210> SEQ ID NO 48


<211> LENGTH: 24


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 48


tagggataac agcgcatacc tatt
24



<210> SEQ ID NO 49


<211> LENGTH: 23


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 49


ggaacaagtt accctaggga taa
23



<210> SEQ ID NO 50


<211> LENGTH: 23


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 50


ttgatccaat aacttgacca acg
23



<210> SEQ ID NO 51


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 51


acttcaccag tcaaagcgaa c
21



<210> SEQ ID NO 52


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 52


aacccaacct ccgagcag
18



<210> SEQ ID NO 53


<211> LENGTH: 16


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 53


gttggggcga cctcgg
16



<210> SEQ ID NO 54


<211> LENGTH: 22


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 54


aaactaccaa acctgcttaa aa
22



<210> SEQ ID NO 55


<211> LENGTH: 24


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 55


aaacagtacc taacaaaccc acag
24



<210> SEQ ID NO 56


<211> LENGTH: 25


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 56


gaccctatgg agctttaatt tatta
25



<210> SEQ ID NO 57


<211> LENGTH: 23


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 57


cataacacag caagacgaga aga
23



<210> SEQ ID NO 58


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 58


tgacctgccc gtgaagag
18



<210> SEQ ID NO 59


<211> LENGTH: 26


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 59


cagctgtctc ttacttttaa ccagtg
26



<210> SEQ ID NO 60


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 60


ctgtatgaat ggctccacga
20



<210> SEQ ID NO 61


<211> LENGTH: 26


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 61


agcataatca cttgttcctt aaatag
26



<210> SEQ ID NO 62


<211> LENGTH: 23


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 62


accgtgcaaa ggtagcataa tca
23



<210> SEQ ID NO 63


<211> LENGTH: 23


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 63


tgattatgct acctttgcac ggt
23



<210> SEQ ID NO 64


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 64


gtaccctaac cgtgcaaag
19



<210> SEQ ID NO 65


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 65


cctgcccagtgacacatgtt t
21



<210> SEQ ID NO 66


<211> LENGTH: 25


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 66


cacctctagc atcaccagta ttaga
25



<210> SEQ ID NO 67


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 67


cttaccccgc ctgtttacca
20



<210> SEQ ID NO 68


<211> LENGTH: 26


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 68


aggttaaaaa aagtaaaagg aactcg
26



<210> SEQ ID NO 69


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 69


cccaacacag gcatgctca
19



<210> SEQ ID NO 70


<211> LENGTH: 24


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 70


accaacaagt cattattacc ctca
24



<210> SEQ ID NO 71


<211> LENGTH: 24


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 71


tgacaattaa cagcccaata tcta
24



<210> SEQ ID NO 72


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 72


gcctgcgtca gattaaaaca c
21



<210> SEQ ID NO 73


<211> LENGTH: 24


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 73


gtaacatgaa aacattctcc tccg
24



<210> SEQ ID NO 74


<211> LENGTH: 28


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 74


tatcacccta tagaagaact aatgttag
28



<210> SEQ ID NO 75


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 75


ctgaactcct cacacccaat t
21



<210> SEQ ID NO 76


<211> LENGTH: 23


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 76


cactacctaa aaaatcccaa aca
23



<210> SEQ ID NO 77


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 77


ttaagaaagc gttcaagctc a
21



<210> SEQ ID NO 78


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 78


catagtaggc ctaaaagcag c
21



<210> SEQ ID NO 79


<211> LENGTH: 26


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 79


aaaccttgta gagagagtaa aaaatt
26

<210> SEQ ID NO 80




<211> LENGTH: 24


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 80


aaagaggaac agctctttgg acac
24



<210> SEQ ID NO 81


<211> LENGTH: 24


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 81


aatccccttg taaatttaac tgtt
24



<210> SEQ ID NO 82


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 82


ctttaaattt gcccacagaa c
21



<210> SEQ ID NO 83


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 83


ggttgtccaa gatagaatct
20



<210> SEQ ID NO 84


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 84


acaaacctac cgagcctgg
19



<210> SEQ ID NO 85


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 85


aagatttata ggtagaggcg
20



<210> SEQ ID NO 86


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 86


cccgtctatg tagcaaaata
20



<210> SEQ ID NO 87


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 87


acctaagaac agctaaaaga
20



<210> SEQ ID NO 88


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 88


taagaccccc gaaaccagac
20



<210> SEQ ID NO 89


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 89


ataactttgc aaggagagcc
20



<210> SEQ ID NO 90


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 90


cttctgcata atgaattaac
20



<210> SEQ ID NO 91


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 91


atatagcaag gactaacccc
20



<210> SEQ ID NO 92


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 92


agatgaaaaa ttataaccaa
20



<210> SEQ ID NO 93


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 93


caatagatat agtaccgcaa
20



<210> SEQ ID NO 94


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 94


aggcgataga aattgaaacc
20



<210> SEQ ID NO 95


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 95


tagccaaacc atttacccaa
20



<210> SEQ ID NO 96


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 96


caccttacta ccagacaacc
20



<210> SEQ ID NO 97


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 97


ctaaacctag ccccaaacc
19



<210> SEQ ID NO 98


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 98


ctagcatcac cagtattaga
20



<210> SEQ ID NO 99


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 99


ttaccaaaaa catcacctct
20



<210> SEQ ID NO 100


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 100


gaactcggca aatcttaccc
20



<210> SEQ ID NO 101


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Sense Oligonucleotide


<400> SEQUENCE: 101


gggtaagatt tgccgagttc
20



<210> SEQ ID NO 102


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 102


gctcataagg aaaggttaaa a
21



<210> SEQ ID NO 103


<211> LENGTH: 17


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 103


gtcaacccaa cacaggc
17



<210> SEQ ID NO 104


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 104


accaacaagt cattattacc c
21



<210> SEQ ID NO 105


<211> LENGTH: 22


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Sense Oligonucleotide


<400> SEQUENCE: 105


ggttgattgt agatattggg ct
22



<210> SEQ ID NO 106


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 106


attaacagcc caatatctac
20



<210> SEQ ID NO 107


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 107


tgcgtcagat taaaacactg
20



<210> SEQ ID NO 108


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 108


aaaacattct cctccgcata
20



<210> SEQ ID NO 109


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 109


gttagtataa gtaacatg
18



<210> SEQ ID NO 110


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 110


tggaccaatc tatcaccct
19



<210> SEQ ID NO 111


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 111


acatataact gaactcctca
20



<210> SEQ ID NO 112


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 112


cacccactac ctaaaaaatc
20



<210> SEQ ID NO 113


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 113


caccaattaa gaaagcgttg
20



<210> SEQ ID NO 114


<211> LENGTH: 22


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 114


taggcctaaa agcagccacc aa
22



<210> SEQ ID NO 115


<211> LENGTH: 22


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Sense Oligonucleotide


<400> SEQUENCE: 115


ttggtggctg cttttaggcc to
22



<210> SEQ ID NO 116


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 116


taacacccat agtaggcct
19



<210> SEQ ID NO 117


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 117


aaccttgtag agagagtaaa
20



<210> SEQ ID NO 118


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 118


aacagctctt tggacactag
20



<210> SEQ ID NO 119


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 119


aactgttagt ccaaagag
18



<210> SEQ ID NO 120


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 120


ctctaaatcc ccttgtaaa
19



<210> SEQ ID NO 121


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 121


actttaaatt tgcccacag
19



<210> SEQ ID NO 122


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 122


ggttgtccaa gatagaatc
19



<210> SEQ ID NO 123


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 123


acaaacctac cgagcctcc
19



<210> SEQ ID NO 124


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 124


atttataggt tagaggcg
18



<210> SEQ ID NO 125


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 125


atgtagcaaa atagtgggaa
20



<210> SEQ ID NO 126


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 126


taagaacagc taaaagagca c
21



<210> SEQ ID NO 127


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 127


cgaaaccaga cgagctac
18



<210> SEQ ID NO 128


<211> LENGTH: 22


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Sense Oligonucleotide


<400> SEQUENCE: 128


ggggtcttag ctttggctct cc
22



<210> SEQ ID NO 129


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 129


taactttgca aggagagcca
20



<210> SEQ ID NO 130


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 130


accttctgca taatgaat
18



<210> SEQ ID NO 131


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 131


atatagcaag gactaaccc
19



<210> SEQ ID NO 132


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 132


gatgaaaaat tataaccaag
20



<210> SEQ ID NO 133


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 133


aatagatata gtaccgcaag
20



<210> SEQ ID NO 134


<211> LENGTH: 17


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 134


cgatagaaat tgaaacc
17



<210> SEQ ID NO 135


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Sense Oligonucleotide


<400> SEQUENCE: 135


tactttattt gggtaaatgg
20



<210> SEQ ID NO 136


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 136


ccatttaccc aaataaagta
20



<210> SEQ ID NO 137


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 137


ttagccaaac catttaccca
20



210> SEQ ID NO 138


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 138


aaggtggagt gggtttgggg c
21



<210> SEQ ID NO 139


<211> LENGTH: 17


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 139


gctaaggttg tctggta
17



<210> SEQ ID NO 140


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 140


atcgcctata ctttatttgg
20



<210> SEQ ID NO 141


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 141


atctattgcg ccaggtttca
20



<210> SEQ ID NO 142


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 142


ttttcatctt tcccttgcg
19



<210> SEQ ID NO 143


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 143


tccttgctat attatgcttg
20



<210> SEQ ID NO 144


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 144


cattatgcag aaggtatagg
20



<210> SEQ ID NO 145


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 145


tctccttgca aagttatt
18



<210> SEQ ID NO 146


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 146


tttcgggggt cttagctttg
20



<210> SEQ ID NO 147


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 147


ctgttcttag gtagctcg
18



<210> SEQ ID NO 148


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 148


tgctacatag acgggtgtg
19



<210> SEQ ID NO 149


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 149


cctctaccta taaatcttcc
20



<210> SEQ ID NO 150


<211> LENGTH: 17


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 150


gctatcacca ggctcgg
17



<210> SEQ ID NO 151


<211> LENGTH: 17


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 151


aagttgaact aagattc
17



<210> SEQ ID NO 152


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 152


gagggttctg tgggcaaatt
20



<210> SEQ ID NO 153


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 153


acagttaaat ttacaaggg
19



<210> SEQ ID NO 154


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 154


gtgtccaaag agctgttcc
19



<210> SEQ ID NO 155


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 155


tactctctct acaaggtttt
20



<210> SEQ ID NO 156


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 156


taggcctact atgggtgtta
20



<210> SEQ ID NO 157


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 157


aacgctttct taattggtgg c
21



<210> SEQ ID NO 158


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 158


ttttaggtag tgggtgttga
20



<210> SEQ ID NO 159


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 159


ggagttcagt tatatgtttg
20



<210> SEQ ID NO 160


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 160


tgatagattg gtccaattgg
20



<210> SEQ ID NO 161


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 161


ctaacattag ttcttctata g
21



<210> SEQ ID NO 162


<211> LENGTH: 18


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 162


atgcggagga gaatgttt
18



<210> SEQ ID NO 163


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 163


tcagtgtttt aatctgacg
19



<210> SEQ ID NO 164


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Antisense Oligonucleotide


<400> SEQUENCE: 164


gtagatattg ggctgttaatt
21



<210> SEQ ID NO 165


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 165


gtgagggtaa taatgacttg
20



<210> SEQ ID NO 166


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 166


atgagcatgc ctgtgttggt
20



<210> SEQ ID NO 167


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 167


ggtaagattt gccgagttc
19



<210> SEQ ID NO 168


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 168


tggtgatgct agaggtgatg
20



<210> SEQ ID NO 169


<211> LENGTH: 15


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 169


gcggtgcctc taata
15



<210> SEQ ID NO 170


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 170


ggccgttaaa catgtgtcac
20



<210> SEQ ID NO 171


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 171


tgattatgct acctttgcac
20



<210> SEQ ID NO 172


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 172


ttaaggaaca agtgattatg
20



<210> SEQ ID NO 173


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 173


tggagccatt catacaggtc
20



<210> SEQ ID NO 174


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 174


aaaagtaaga gacagctgaa
20



<210> SEQ ID NO 175


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 175


cacgggcagg tcaatttcac
20



<210> SEQ ID NO 176


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 176


gtcttgctgt gttatgcccg
20



<210> SEQ ID NO 177


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 177


aattaaagct ccatagggt
19



<210> SEQ ID NO 178


<211> LENGTH: 21


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 178


gtttgttagg tactgtttgc a
21



<210> SEQ ID NO 179


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 179


aggtttggta gtttaggac
19



<210> SEQ ID NO 180


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 180


gccccaaccg aaatttttaa
20



<210> SEQ ID NO 181


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 181


ctcggaggtt gggttctgct
20



<210> SEQ ID NO 182


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 182


ctggtgaagt cttagcatgt
20



<210> SEQ ID NO 183


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 183


caattgagta tagtagttcg
20



<210> SEQ ID NO 184


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 184


tgttccgttg gtcaagtta
19



<210> SEQ ID NO 185


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 185


aataggattg cgctgttatc
20



<210> SEQ ID NO 186


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 186


attgttgata tggactctag
20



<210> SEQ ID NO 187


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 187


atccaacatc gaggtcgtaa
20



<210> SEQ ID NO 188


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 188


gcggctgcac catcgggat
19



<210> SEQ ID NO 189


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 189


ttgaacaaac gaaccttta
19



<210> SEQ ID NO 190


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 190


aactcagatc acgtaggact
20



<210> SEQ ID NO 191


<211> LENGTH: 19


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 191


aaaccgacct ggattactc
19



<210> SEQ ID NO 192


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 192


agggaggaat ttgaaggtag
20



<210> SEQ ID NO 193


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 193


ggccttattt ctcttgtcct
20



<210> SEQ ID NO 194


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 194


ggaaggcgct ttgtgaagta
20



<210> SEQ ID NO 195


<211> LENGTH: 20


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 195


aagttgagat gatatcattt
20



<210> SEQ ID NO 196


<211> LENGTH: 17


<212> TYPE: DNA


<213> ORGANISM: Artificial Sequence


<220> FEATURE:


<223> OTHER INFORMATION: Oligonucleotide


<400> SEQUENCE: 196


cctgttcttg ggtgggt
17