WO2017091196A1 - Facteur de réplication c40 en tant que marqueur pronostique et cible dans le cancer du sein - Google Patents

Facteur de réplication c40 en tant que marqueur pronostique et cible dans le cancer du sein Download PDF

Info

Publication number
WO2017091196A1
WO2017091196A1 PCT/US2015/062118 US2015062118W WO2017091196A1 WO 2017091196 A1 WO2017091196 A1 WO 2017091196A1 US 2015062118 W US2015062118 W US 2015062118W WO 2017091196 A1 WO2017091196 A1 WO 2017091196A1
Authority
WO
WIPO (PCT)
Prior art keywords
rfc40
seq
cells
sequence
breast cancer
Prior art date
Application number
PCT/US2015/062118
Other languages
English (en)
Inventor
Rakhee S. GUPTE
Original Assignee
Raadysan Biotech, Inc.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Raadysan Biotech, Inc. filed Critical Raadysan Biotech, Inc.
Priority to PCT/US2015/062118 priority Critical patent/WO2017091196A1/fr
Publication of WO2017091196A1 publication Critical patent/WO2017091196A1/fr

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
    • G01N33/57407—Specifically defined cancers
    • G01N33/57415—Specifically defined cancers of breast
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/106—Pharmacogenomics, i.e. genetic variability in individual responses to drugs and drug metabolism
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/118—Prognosis of disease development
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the present invention extends to the diagnosis and/or treatment of breast cancer in mammals, particularly in humans, using replication factor C 40 (RFC40) and particularly to RFC40 protein and/or gene expression as a marker in breast cancer and to RFC40 as a novel and specific oncologic target for intervention in cancer, particularly in breast cancer, including estrogen sensitive, estrogen resistant and triple negative breast cancer (TNBC).
  • RFC40 replication factor C 40
  • TNBC triple negative breast cancer
  • identification of a human subject as a candidate for treatment with a composition disclosed herein is achieved using the same or similar criteria as for identifying an individual as a candidate for therapy with other anti-cancer agents, which is based on clinically established parameters and will be known to the skilled artisan.
  • the individual is suspected of having or is at risk for developing breast cancer.
  • the disclosure upon diagnosing an individual as having cancer, the disclosure further comprises administering to the individual a pharmaceutical composition of this disclosure.
  • methods for the treatment of breast cancer and/or the reduction of risk for breast cancer by modulating RFC40 are provided.
  • the treatment of breast cancer comprising modulating RFC40 expression or activity in cells, particularly and specifically in oncogenic or cancer cells versus normal or benign cells, particularly non-cancerous breast cells.
  • the method includes inhibitors of RCF40 expression and/or activity, and compositions comprising such inhibitors, wherein the inhibitor of the activity or expression of RFC40 is selected from the group consisting of a small interfering RNA (siRNA), microRNA (miRNA), an antisense polynucleotide, a ribozyme and a short-hairpin RNA (shRNA).
  • the inhibitor comprises or consists of a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence.
  • the nucleic acids can be approximately 17-30 contiguous nucleotides of a nucleic acid encoding RFC40 polypeptide, or the reverse complement of a nucleic acid encoding RFC40.
  • the disclosure includes an shRNA comprising the sequence ACUACGAACUGCCGUGGGUUG (SEQ ID NO: 20). In embodiments, the disclosure includes shRNAs comprising or consisting of the following sequences:
  • SEQ ID NO: 21 comprises the sequence
  • the NNNNNNNNN of SEQ ID NO: 24 comprises UUCAAGAGA (SEQ ID NO: 22) as the loop sequence.
  • the disclosure includes an shRNA comprising or consisting of the sequence:
  • SEQ ID NO: 25 This is also referred to herein as the hRFC2- shRNA# 2 sequence.
  • SEQ ID NO 25 may also include a transcription termination sequence, such as UUUUU, encoded by TTTTTT.
  • a method for prophylaxis and or therapy ofcancer, including breast cancer, in a mammal comprising administering to said mammal the above composition(s).
  • the method may further comprise administering an anticancer agent selected from an anti-mitotic agent, an immunomodulatory agent, and an agent targeting growth factor or estrogen receptors.
  • the disclosure includes a method for inhibiting growth of cancer cells.
  • the method comprises introducing into cancer cells at least one polynucleotide, wherein the polynucleotide comprises a sequence selected from any one or any combination of the sequences described herein.
  • the method comprises introducing into cancer cells at least one polynucleotide which comprises, consists of, and/or encodes any one or any combination of: SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, ACUACGAACUGCCGUGGGUUGAAAAAUAU (SEQ ID NO: 15), GUCCCGCUGUGCAGUCCUCCGGUACACAA (SEQ ID NO: 16), ACUACGAACUGCCGUGGGUUG (SEQ ID NO:20),
  • the diagnostic utility of the present invention includes in certain embodiments determining RFC40 protein amounts and/or gene amplification and/or expression in diagnosis and prognosis of cancer, particularly breast cancer.
  • the disclosure provides in various embodiments methods for determining or prognosing/prognosticating breast cancer in an individual comprising assessing of levels or activity of RFC40 protein, RFC40 mRNA or RFC40 gene amplification in breast tissue, whereby an individual having breast cancer or malignancy has elevated levels or activity of RFC40 protein, RFC40 mRNA or RFC40 gene amplification versus a normal or benign control.
  • the disclosure includes a method for aiding in diagnosis of breast cancer in an individual.
  • a control quantity of RFC40 or antibodies thereto, or the like may be prepared and labeled with an enzyme, a specific binding partner and/or a radioactive element, and may then be introduced into a cellular sample.
  • the present invention includes an assay system which may be prepared in the form of a test kit for the quantitative analysis of the extent of the presence or activity of RFC40 protein or mRNA, or to identify drugs or other agents that may mimic or block their activity.
  • the system or test kit may comprise a labeled component prepared by one of the radioactive and/or enzymatic techniques discussed herein, coupling a label to the RFC40, their agonists and/or antagonists, and one or more additional immunochemical reagents, at least one of which is a free or immobilized ligand, capable either of binding with the labeled component, its binding partner, one of the components to be determined or their binding partner(s).
  • FIGURE 2 A and 2B Western blot analyses of RFC40 using MCF 10A (A and
  • FISH fluorescence in situ hybridization
  • MDA-MB231 cells used for implantation, were harvested during log phase growth and resuspended in cold PBS. Each mouse was injected subcutaneous ly in the right flank with 5 x 106 cells (0.1 mL cell suspension). Tumors were calipered in two dimensions to monitor growth as their mean volume approached the desired 90 to 130 mm3 range. Tumor weight was estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume.
  • Transcriptional and translational control sequences are DNA regulatory sequences, such as promoters, enhancers, polyadenylation signals, terminators, and the like, that provide for the expression of a coding sequence in a host cell.
  • a "promoter sequence” is a DNA regulatory region capable of binding RNA polymerase in a cell and initiating transcription of a downstream (3' direction) coding sequence.
  • the promoter sequence is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background.
  • a transcription initiation site (conveniently defined by mapping with nuclease SI), as well as protein binding domains (consensus sequences) responsible for the binding of RNA polymerase.
  • Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT” boxes.
  • Prokaryotic promoters contain Shine-Dalgarno sequences in addition to the -10 and -35 consensus sequences.
  • An "expression control sequence” is a DNA sequence that controls and regulates the transcription and translation of another DNA sequence.
  • a coding sequence is "under the control" of transcriptional and translational control sequences in a cell when RNA polymerase transcribes the coding sequence into mRNA, which is then translated into the protein encoded by the coding sequence.
  • oligonucleotide primer typically contains 15-25 or more nucleotides, although it may contain fewer nucleotides.
  • restriction endonucleases and “restriction enzymes” refer to bacterial enzymes, each of which cut double-stranded DNA at or near a specific nucleotide sequence.
  • a cell has been "transformed” by exogenous or heterologous DNA when such
  • Histidine Histidine (His or H) CAU or CAC
  • Lysine (Lys or K) AAA or AAG
  • Glycine GGU or GGC or GGA or GGG
  • Amino acids with nonpolar R groups Alanine, Valine, Leucine, Isoleucine, Proline, Phenylalanine, Tryptophan, Methionine
  • a DNA sequence is "operatively linked" to an expression control sequence when the expression control sequence controls and regulates the transcription and translation of that DNA sequence.
  • the term "operatively linked” includes having an appropriate start signal (e.g., ATG) in front of the DNA sequence to be expressed and maintaining the correct reading frame to permit expression of the DNA sequence under the control of the expression control sequence and production of the desired product encoded by the DNA sequence. If a gene that one desires to insert into a recombinant DNA molecule does not contain an appropriate start signal, such a start signal can be inserted in front of the gene.
  • RFC40 can be specifically inhibited in cancer cells, without effects in concomitant normal or benign cells, thereby providing RFC40 as a specific anti-cancer agent with onco-specificity in rapidly dividing and proliferating cancer cells, without toxic or unintended effects in normal cells.
  • the present invention contemplates pharmaceutical intervention in the expression, activity or necessary protein interactions of RFC40 in a cancer cell, to modulate the cell division and proliferation, including particularly the Gl to S phase transition.
  • Panels of monoclonal antibodies produced against RFC40 peptides can be screened for various properties; i.e., isotype, epitope, affinity, etc. Of particular interest are monoclonal antibodies that neutralize the activity of the RFC40 or its subunits. Such monoclonals can be readily identified in RFC40 activity assays.
  • the anti-RFC40 antibody used in the diagnostic methods of this invention is an affinity purified polyclonal antibody. More preferably, the antibody is a monoclonal antibody (mAb).
  • mAb monoclonal antibody
  • the anti-RFC40 antibody molecules used herein be in the form of Fab, Fab', F(ab')2 or F(v) portions of whole antibody molecules.
  • Monkey kidney cells e.g., COS 1, COS 7, BSC1, BSC40, and BMT10
  • insect cells e.g., COS 1, COS 7, BSC1, BSC40, and BMT10
  • a nucleic acid sequence provided or of use in accordance with the present invention can be prepared synthetically using methods known and available in the art, including commercially available synthesizers, laboratory bench methods and in vitro methods.
  • Such an inhibitor of the activity or expression of RFC40 includes and may be a small interfering RNA (siRNA), microRNA (miRNA), an antisense polynucleotide, a ribozyme ora short-hairpin RNA (shRNA), particularly wherein said inhibitor comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence of about 17 to about 30 contiguous nucleotides of a nucleic acid encoding RFC40 polypeptide.
  • siRNA small interfering RNA
  • miRNA microRNA
  • antisense polynucleotide a ribozyme or a short-hairpin RNA (shRNA)
  • shRNA short-hairpin RNA
  • MicroRNAs regulate gene expression in a sequence specific manner by hybridization and recruitment of multi-protein complexes to complementary messenger RNA (mRNA) target sequences.
  • miRNA function can transiently be antagonized by antagomirs— chemically modified oligonucleotides complementary to individual miRNAs.
  • oligonucleotides and nucleic acids which specifically inhibit or block the expression and activity of miR A(s).
  • Antagomirs of miR A(s) are provided and assessed herein, with demonstrated anti-oncogenic and anti-proliferative activity.
  • siRNA according to the present invention comprises a sense strand of 15-30, particularly 17-30, most particularly 17-25 nucleotides complementary or homologous to a contiguous 17-25 nucleotide sequence selected from the group of exemplary siRNA sequences described in FIGURE 18, and an antisense strand of 15-30, particularly 17-30, most particularly 17-25, more specifically 19-21 nucleotides complementary to the sense strand of encoding RNA, particularly complementary to encoding nucleic acid as set out in FIGURE 18.
  • siRNA comprises sense and anti-sense strands that are 100 per cent complementary to each other and the TARGET polynucleotide sequence.
  • the siRNA further comprises a loop region linking the sense and the antisense strand.
  • the siRNA can be modified to confirm resistance to nucleolytic degradation, or to enhance activity, or to enhance cellular distribution, or to enhance cellular uptake, such modifications may consist of modified internucleoside linkages, modified nucleic acid bases, modified sugars and/or chemical linkage the siRNA to one or more moieties or conjugates.
  • the nucleotide sequences are selected according to siRNA designing rules that give an improved reduction of the TARGET sequences compared to nucleotide sequences that do not comply with these siRNA designing rules (For a discussion of these rules and examples of the preparation of siRNA, WO 2004/094636 and US 2003/0198627, are hereby incorporated by reference).
  • Ribozymes are RNA molecules possessing the ability to specifically cleave other single stranded RNA molecules in a manner somewhat analogous to DNA restriction endonucleases. Because they are sequence-specific, only mRNAs with particular sequences are inactivated. Ribozymes are catalytic RNA molecules (RNA enzymes) that have separate catalytic and substrate binding domains. The substrate binding sequence combines by nucleotide complementarity and, possibly, non-hydrogen bond interactions with its target sequence. The catalytic portion cleaves the target RNA at a specific site. The substrate domain of a ribozyme can be engineered to direct it to a specified mRNA sequence.
  • the ribozyme recognizes and then binds a target mRNA through complementary base pairing. Once it is bound to the correct target site, the ribozyme acts enzymatically to cut the target mRNA. Cleavage of the mRNA by a ribozyme destroys its ability to direct synthesis of the corresponding polypeptide. Once the ribozyme has cleaved its target sequence, it is released and can repeatedly bind and cleave at other mRNAs.
  • Exemplary ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) motif or Neurospora VS RNA motif.
  • Ribozymes possessing a hammerhead or hairpin structure are readily prepared since these catalytic RNA molecules can be expressed within cells from eukaryotic promoters (Chen, et al. (1992) Nucleic Acids Res. 20:4581-9).
  • a ribozyme of the present invention can be expressed in eukaryotic cells from the appropriate DNA vector. If desired, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ventura, et al. (1993) Nucleic Acids Res.
  • Ribozymes may be chemically synthesized by combining an
  • oligodeoxyribonucleotide with a ribozyme catalytic domain (20 nucleotides) flanked by sequences that hybridize to the TARGET mRNA after transcription.
  • oligodeoxyribonucleotide is amplified by using the substrate binding sequences as primers.
  • the amplification product is cloned into a eukaryotic expression vector.
  • Ribozymes are expressed from transcription units inserted into DNA, RNA, or viral vectors. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol (I), RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters will be expressed at high levels in all cells; the levels of a given pol II promoter in a given cell type will depend on nearby gene regulatory sequences. Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate cells (Gao and Huang, (1993) Nucleic Acids Res. 21 :2867-72). It has been demonstrated that ribozymes expressed from these promoters can function in mammalian cells (Kashani-Sabet, et al.
  • antisense nucleic acids, siRNAs and miRs are particularly preferred.
  • oligonucleotides and may consist entirely of ribonucleotides, modified ribonucleotides, deoxyribo-nucleotides, modified deoxyribonucleotides, or some combination of both.
  • the nucleic acids can be synthetic oligonucleotides.
  • the nucleic acids and oligonucleotides may be chemically modified, if desired, to improve stability and/or selectivity.
  • oligonucleotides envisioned for this invention include those containing modified backbones, for example, phosphorothioates, phosphotriesters, methyl phosphonates, short chain alkyl or cycloalkyl intersugar linkages or short chain heteroatomic or heterocyclic intersugar linkages. Since oligonucleotides are susceptible to degradation by intracellular nucleases, the modifications can include, for example, the use of a sulfur group to replace the free oxygen of the phosphodiester bond. This modification is called a phosphorothioate linkage. Phosphorothioate antisense oligonucleotides are water soluble, polyanionic, and resistant to endogenous nucleases.
  • RNA-DNA duplex activates the endogenous enzyme ribonuclease (RNase) H, which cleaves the mRNA component of the hybrid molecule.
  • RNase ribonuclease
  • Oligonucleotides may also contain one or more substituted sugar moieties.
  • oligonucleotides comprise one of the following at the 2' position: OH, SH, SCH3, F, OCN, heterocycloalkyl; heterocycloalkaryl; aminoalkylamino; polyalkylamino; substituted silyl; an RNA cleaving group; a reporter group; an intercalator; a group for improving the pharmacokinetic properties of an oligonucleotide; or a group for improving the pharmacodynamic properties of an oligonucleotide and other substituents having similar properties. Similar modifications may also be made at other positions on the oligonucleotide, particularly the 3' position of the sugar on the 3' terminal nucleotide and the 5' position of 5' terminal nucleotide.
  • antisense oligonucleotides with phosphoramidite and polyamide [0153] In addition, antisense oligonucleotides with phosphoramidite and polyamide
  • (peptide) linkages can be synthesized. These molecules should be very resistant to nuclease degradation. Furthermore, chemical groups can be added to the 2' carbon of the sugar moiety and the 5 carbon (C-5) of pyrimidines to enhance stability and facilitate the binding of the antisense oligonucleotide to its TARGET site. Modifications may include 2'-deoxy, O- pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy phosphorothioates, modified bases, as well as other modifications known to those of skill in the art.
  • DNA sequences described herein may thus be used to prepare antisense molecules against, and ribozymes that cleave mRNAs for RFC40 and their ligands.
  • the inhibitory agent particularly comprises antisense polynucleotide, a ribozyme, and a small interfering RNA (siRNA), wherein said agent comprises a nucleic acid sequence complementary to, or engineered from, a naturally-occurring polynucleotide sequence of RFC40, including as set out in FIGURE 18 (SEQ ID NO:2).
  • Another embodiment of the present invention relates to a method for identifying a compound for treatment of cancer, particularly breast cancer, wherein said compound is an expression-inhibiting agent such as a nucleic acid expressing the antisense RNA, antisense oligodeoxynucleotide (ODN), a ribozyme that cleaves the polyribonucleotide coding for RFC40, including as set out in FIGURE 18 (SEQ ID NO:2), a small interfering RNA (siRNA, particularly shRNA,) that is sufficiently complementary to a portion of the polyribonucleotide coding for RFC40, including as set out in FIGURE 18 (SEQ ID NO:2), such that the antisense RNA, ODN, ribozyme, particularly siRNA, particularly shRNA, interferes with the translation of the target RFC40 polyribonucleotide to the target RFC40 polypeptide.
  • the expression-inhibiting agent is an antisense RNA, ribo
  • Such shRNA sequences may also comprise a suitable transcription termination signal, such as a poly T sequence (which can be poly U, such as UUUUU in shRNA). Additional sequences may also be part of, or contiguous with, the shRNA sequence, such as expression-vector derived sequences, or other sequences that will be apparent to those skilled in the art given the benefit of the present disclosure, provided the sequences retain their intended function.
  • a suitable transcription termination signal such as a poly T sequence (which can be poly U, such as UUUUU in shRNA).
  • Additional sequences may also be part of, or contiguous with, the shRNA sequence, such as expression-vector derived sequences, or other sequences that will be apparent to those skilled in the art given the benefit of the present disclosure, provided the sequences retain their intended function.
  • Antisense nucleic acids of the invention are particularly nucleic acid fragments capable of specifically hybridizing with all or part of a nucleic acid encoding a target RFC40 polypeptide or the corresponding messenger RNA.
  • antisense nucleic acids may be designed which decrease expression of the nucleic acid sequence capable of encoding a target RFC40 polypeptide by inhibiting splicing of its primary transcript. Any length of antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of a nucleic acid coding for target RFC40.
  • a number of fluorescent materials are known and can be utilized as detectable labels. These include, for example, fluorescein, rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow.
  • a particular detecting material is anti-rabbit antibody prepared in goats and conjugated with fluorescein through an isothiocyanate.
  • the RFC40 or its binding partner(s) can also be labeled with a radioactive element or with an enzyme. The radioactive label can be detected by any of the currently available counting procedures.
  • a test kit may be prepared for the determination and quantitation of RFC40 protein in cells or a cellular or biopsy sample, comprising:
  • test kit may be prepared and used for the purposes stated above, and comprises:
  • RFC40 is a Molecular Marker for Breast Cancer
  • RFC40a a non-receptor based molecular marker for breast cancers specifically, estrogen positive (ER positive), estrogen negative (ER negative), and progesterone, estrogen and human epidermal growth factor receptor 2-HER2 negative or triple negative breast cancers (TNBC).
  • TNBC triple negative breast cancers
  • BTMAs patient breast tissue microarray
  • FIG. 1 To determine the expression of RFC40 protein in patient breast tissues, we used a 96-cores patient breast tissue microarray (BTMAs; Pantomics, Inc., CA, USA) containing 36 cases of breast cancers (20-ER positive and 15-ER negative) and 12 cases of normal, reactive and benign tumor tissues of the breast, in duplicates (FIGURE 3).
  • BTMAs have been extensively used and well characterized in several studies (Moreira JM et al (2010) Mol Oncol4(6):539-61 ; Unger K et al (2010) Endocr Relat Cancer 17(l):87-98).
  • the tissues in the microarrays are fixed in 10% neutral buffered formalin for 24 to 48 hours. Tissue sections were cut fresh upon receiving an order.
  • RNA targeting endogenous RFC40 by blocking translation using siRNA, miRNA, as well as via antisense or ribozyme approaches provides a new and directed approach to breast cancer, whether estrogen sensitive, estrogen resistant or TNBC, and a means to inhibit cell division and growth of cancer cells or tumors, via inhibiting or blocking DNA replication specifically in cancer or tumor cells.
  • glucose-6-phosphate-dehydrogenase (LAC; 100 nM; FIGURE 19B & C), glucose-6-phosphate-dehydrogenase (G6PD; 100 nM;
  • FIGURE 19A and RFC40 siRNA -smartpool (100 nM; cocktail of four different sequences;
  • FIGURE 19A-D as indicated in the figure for 72 hr using 2.5 ⁇ of Dharmafect Reagent 1 (Dharmacon, Inc., TX, USA).
  • Cells lysates were subjected to Western blot analysis using anti-RFC40, anti-RIa (Pharmingen, Inc., CA, USA) and anti-G6PD (Santa Cruz, CA, USA) antibodies, respectively.
  • ⁇ -Actin was used as loading control.
  • siRNA sequences against RFC40 normal and breast cancer cell lines were transfected with individual siRNAs from the On-Targetplus-Smartpool selection, particularly with a single siRNA and particularly either of RFC40-siRNA-Sl, RFC40-siRNA-S2, RFC40-siRNA-S3, or RFC40-siRNA-S4, using the transfection protocol as described above.
  • miRDB site provides 295 predicted target mRNAs and RFC40 (RFC2) is not among the predicted targets.
  • RFC40 RFC40
  • the miR-hsa-125a-3p sequence is
  • RFC40 protein levels was determined by Western blot analysis using anti-RFC40 antibody. ⁇ -Actin was used as a loading control. RFC40 protein was reduced after transfection of miR-hsa-125a-3p, but not affected in untreated or miRNA negative control#l treated MDA-MB-231 cells (FIGURE 25). No change or difference was observed in MCFIOA cells.
  • MDA-MB231 human breast carcinoma xenograft in female NCr nu/nu mice.
  • CR Discovery Services specifically complies with the recommendations of the Guide for Care and Use of Laboratory Animals with respect to restraint, husbandry, surgical procedures, feed and fluid regulation, and veterinary care.
  • the animal care and use program at CR Discovery is accredited by the Association for Assessment and Accreditation of
  • the MDA-MB231 cells used for implantation were harvested during log phase growth and resuspended in cold PBS containing. Each mouse was injected subcutaneously in the right flank with 5 x 106 cells (0.1 mL cell suspension). Tumors were calipered in two dimensions to monitor growth as their mean volume approached the desired 90 to 130 mm3 range. Tumor size, in mm3, was calculated from:
  • Tumor weight can be estimated with the assumption that 1 mg is equivalent to 1 mm3 of tumor volume.
  • AD-GFP (1.2 x 10 11 PFU /mL), Ad-GFP-U6-hRFC2-shRNA#l (1.0 xlO 11
  • Tumors were measured using calipers twice per week.
  • the study endpoint was defined as a mean tumor volume of 1500 mm3 in the control group or 30 days, whichever came first.
  • Treatment efficacy was determined using data from the final day (D28).
  • the MTV (n) the median tumor volume for the number of animals, n, on the final day, was determined for each group.
  • Percent tumor growth inhibition (%TGI) was defined as the difference between the MTV of the designated control group (Group 1) and the MTV of the drug treated group, expressed as a percentage of the MTV of the control group:
  • the data set for TGI analysis includes all animals in a group, except those that die due to treatment-related (TR) or non-treatment-related (NTR) causes.
  • TR treatment-related
  • NTR non-treatment-related
  • CR Discovery Services considers an agent that produces at least 60% TGI in this assay to be potentially therapeutically active.
  • a death was to be classified as TR if it was attributable to treatment side effects as evidenced by clinicalsigns and/or necropsy, or may also be classified as TR if due to unknown causes during the dosing period or within 14 days of the last dose.
  • a death was classified as NTR if there was evidence that the death was related to the tumor model, rather than treatment related. NTR deaths are further categorized as NTRa (due to accident or human error), NTRm (due to necropsy- confirmed tumor dissemination by invasion or metastasis), and NTRu (due to unknown causes).

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Biotechnology (AREA)
  • Analytical Chemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Pathology (AREA)
  • General Health & Medical Sciences (AREA)
  • Biochemistry (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Hospice & Palliative Care (AREA)
  • Oncology (AREA)
  • Biophysics (AREA)
  • Hematology (AREA)
  • Urology & Nephrology (AREA)
  • Cell Biology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • General Physics & Mathematics (AREA)
  • Plant Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Abstract

La présente divulgation concerne, de manière générale, le cancer et, en particulier, le cancer du sein, comprenant le cancer du sein sensible aux oestrogènes, résistant aux oestrogènes et triple négatif (TN) et des procédés de diagnostic et de pronostic correspondants et une intervention thérapeutique impliquant le facteur de réplication C 40 (FRC40). L'invention concerne des procédés et des essais pour évaluer le cancer du sein. La divulgation concerne également l'inhibition ou la modulation du FRC40 dans le traitement ou l'atténuation d'un cancer, notamment le cancer du sein. Des inhibiteurs du FRC40, comprenant des ARNsi, des miARN et des ARNsh, qui touchent spécifiquement les cellules cancéreuses, en particulier de cellules de cancer du sein sont décrits.
PCT/US2015/062118 2015-11-23 2015-11-23 Facteur de réplication c40 en tant que marqueur pronostique et cible dans le cancer du sein WO2017091196A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/US2015/062118 WO2017091196A1 (fr) 2015-11-23 2015-11-23 Facteur de réplication c40 en tant que marqueur pronostique et cible dans le cancer du sein

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/US2015/062118 WO2017091196A1 (fr) 2015-11-23 2015-11-23 Facteur de réplication c40 en tant que marqueur pronostique et cible dans le cancer du sein

Publications (1)

Publication Number Publication Date
WO2017091196A1 true WO2017091196A1 (fr) 2017-06-01

Family

ID=58763674

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2015/062118 WO2017091196A1 (fr) 2015-11-23 2015-11-23 Facteur de réplication c40 en tant que marqueur pronostique et cible dans le cancer du sein

Country Status (1)

Country Link
WO (1) WO2017091196A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11236337B2 (en) 2016-11-01 2022-02-01 The Research Foundation For The State University Of New York 5-halouracil-modified microRNAs and their use in the treatment of cancer

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001071042A2 (fr) * 2000-03-23 2001-09-27 Pe Corporation (Ny) Necessaires de detection, tels que des jeux ordonnes d'echantillons d'acide nucleique, servant a detecter l'expression d'au moins 10.000 genes de drosophila et leur utilisation
US20040265849A1 (en) * 2002-11-22 2004-12-30 Applera Corporation Genetic polymorphisms associated with Alzheimer's disease, methods of detection and uses thereof
US20120197540A1 (en) * 2011-02-01 2012-08-02 Consiglio Nazionale Delle Ricerche Markers to predict survival of breast cancer patients and uses thereof
US9193970B1 (en) * 2013-03-14 2015-11-24 Raadysan Biotech, Inc. Replication factor C-40 (RFC40/RFC2) as a prognostic marker and target in estrogen positive and negative and triple negative breast cancer

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001071042A2 (fr) * 2000-03-23 2001-09-27 Pe Corporation (Ny) Necessaires de detection, tels que des jeux ordonnes d'echantillons d'acide nucleique, servant a detecter l'expression d'au moins 10.000 genes de drosophila et leur utilisation
US20040265849A1 (en) * 2002-11-22 2004-12-30 Applera Corporation Genetic polymorphisms associated with Alzheimer's disease, methods of detection and uses thereof
US20120197540A1 (en) * 2011-02-01 2012-08-02 Consiglio Nazionale Delle Ricerche Markers to predict survival of breast cancer patients and uses thereof
US9193970B1 (en) * 2013-03-14 2015-11-24 Raadysan Biotech, Inc. Replication factor C-40 (RFC40/RFC2) as a prognostic marker and target in estrogen positive and negative and triple negative breast cancer

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DATABASE GenBank 29 March 2005 (2005-03-29), "Synthetic construct Homo sapiens clone FLH012715.01X replication factor C 2 (RFC2) mRNA", XP055385982, Database accession no. AY889681.1 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US11236337B2 (en) 2016-11-01 2022-02-01 The Research Foundation For The State University Of New York 5-halouracil-modified microRNAs and their use in the treatment of cancer

Similar Documents

Publication Publication Date Title
WO2015149056A1 (fr) Inhibition de tyrosine kinase de bruton (btk) dans le poumon pour traiter de l'inflammation pulmonaire grave et des blessures pulmonaires graves
WO2018048798A1 (fr) Tyrosine kinase de bruton utilisée comme cible médicamenteuse anticancéreuse
US20180251763A1 (en) Compositions and Methods for Inducing Senescence in Cancer Cells
US9453050B2 (en) Compositions for treating glioma
EP2838541A1 (fr) Thérapies à base d'interférence arn pour des myocardiopathies, des dystrophies musculaires et des laminopathies
KR20130107203A (ko) 섬유증의 검출 및 치료
US9822363B2 (en) Replication factor C-40 (RFC40/RFC2) as a prognostic marker and target in estrogen positive and negative and triple negative breast cancer
EP2454372B1 (fr) Gérer le traitement de troubles inflammatoires ou auto-immuns en utilisant l'expression de hom-1
JP2007517763A (ja) Ｈｅｒ２アンタゴニストを用いた治療への感受性を回復させる組成物および方法
US9193970B1 (en) Replication factor C-40 (RFC40/RFC2) as a prognostic marker and target in estrogen positive and negative and triple negative breast cancer
EP2963125B1 (fr) Agent therapeutique pour le traitement d'un cancer dans lequel le nrf2 est stabilise
US9970012B2 (en) Replication factor C-40 (RFC40/RFC2) as a prognostic marker and target in estrogen positive and negative and triple negative breast cancer
US8492356B2 (en) Methods of inhibiting tumor growth using TTK antagonists
JP2022532303A (ja) Ｆｍｒｐ及び癌治療
WO2013165320A1 (fr) Traitement du cancer par augmentation de l'expression de socs6
WO2017091196A1 (fr) Facteur de réplication c40 en tant que marqueur pronostique et cible dans le cancer du sein
ES2873377T3 (es) Procedimientos y composiciones farmacéuticas para el tratamiento de cáncer de pulmón
Park et al. Human plasminogen-derived N-acetyl-Arg-Leu-Tyr-Glu antagonizes VEGFR-2 to prevent blood-retinal barrier breakdown in diabetic mice
KR101913693B1 (ko) SS18-SSX 융합 유전자 특이적 siRNA 및 이를 포함하는 암 예방 또는 치료용 약학적 조성물
EP2502069B1 (fr) Composants d'inhibition de signalisation cd95 pour le traitement du cancer du pancréas
JP2011520781A (ja) 治療、診断及び検査のためのｍｓ４ａ１２を伴う方法、及びｍｓ４ａ１２を標的とする剤
EP4257146A1 (fr) Médicament de traitement pour lymphangiomes kystiques
US11788091B2 (en) Methods and compositions for diagnosing and treating prostate cancer based on long noncoding RNA overlapping the LCK gene that regulates prostate cancer cell growth
TW201825673A (zh) 修飾癌細胞及其應用
CN107723369B (zh) Setd1b蛋白及其编码基因在肝癌诊断治疗中的应用

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 15909393

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 15909393

Country of ref document: EP

Kind code of ref document: A1