WO2009148552A2 - Méthodes et compositions pour le traitement de maladies - Google Patents

Méthodes et compositions pour le traitement de maladies Download PDF

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WO2009148552A2
WO2009148552A2 PCT/US2009/003303 US2009003303W WO2009148552A2 WO 2009148552 A2 WO2009148552 A2 WO 2009148552A2 US 2009003303 W US2009003303 W US 2009003303W WO 2009148552 A2 WO2009148552 A2 WO 2009148552A2
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sirna
cells
injection
ribonucleic acid
intra
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WO2009148552A3 (fr
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Dolores J. Takemoto
Thu A. Nguyen
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Kansas State University
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    • 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
    • C12N15/1138—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 against receptors or cell surface proteins
    • 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 nucleic acids [NA]

Definitions

  • This invention relates to the field of disease treatment, and in particular any disease which is associated with a hypoxic/ischemic component.
  • the invention relates to treating cancer, and in particular to a method of treating breast cancer in a human patient.
  • the invention relates to treating or preventing heart disease.
  • it relates to the treatment of human ocular retinoblastoma.
  • Breast cancer is the most common form of cancer and the second leading cause of mortality in women around the world. Over 182,500 (female) and 1,990 (male) estimated new cases are expected in United States in 2008. Breast cancer growth depends on the ratio of proliferating and dying cells. Estrogens are the main regulator of this ratio, and they act by both stimulating proliferation and inhibiting apoptosis. Despite the improvements in detection and treatments, about 40-50% of patients still succumb to the disease. The development of distant metastases is the major cause of these deaths, and breast cancer is no longer curable when metastases are detected.
  • This invention relates to hypoxic environments, and in particular any disease which is associated with a hypoxic/ischemic component.
  • the present invention contemplates a method of reducing the growth of cells in an hypoxic environment comprising inhibiting the expression of Connexin 46.
  • the invention relates to treating cancer, and in particular to a method of treating breast cancer in a human patient.
  • the invention relates to treating or preventing heart disease.
  • a third it relates to the treatment of ocular retinoblastoma.
  • the present invention provides a method of treating cancer in a subject.
  • the present invention contemplates a method of treating cancer in a subject, comprising administering to said subject an effective amount of a short interfering ribonucleic acid (siRNA) directed to Connexin 46 (CX46), a gap junction protein. Treating both humans and animals is contemplated.
  • the present invention contemplates administering anti-CX46 siRNA to control and potentially eliminate early breast cancers while at the hypoxic stage.
  • siRNA short interfering ribonucleic acid
  • the effective amount of the short interfering ribonucleic acid (siRNA) can range from about 1 nM to about 100 nM, or even greater (e.g. 1000 nM).
  • the present invention be limited by the cancer type or stage of the cancer.
  • a variety of cancers can be treating according to the present invention (e.g. prostate, ovarian, etc.), and in particular breast cancer (including metastatic breast cancer). It is not intended that the present invention be limited by the mode of administration.
  • the short interfering ribonucleic acid (siRNA) is administered in conjunction with a delivery reagent.
  • the delivery agent is selected from the group consisting of lipofectin, lipofectamine, cellfectin, polycations, nanoparticles and liposomes.
  • the siRNA target a sequence of Connexin 46. A variety of different target sequences are contemplated.
  • the siRNA is directed to the nucleic acid sequence CGCATGGAAGAGAAGAAGAAA (SEQ ID NO: 1).
  • the present invention be limited by the route of administration, hi one embodiment, the short interfering ribonucleic acid (siRNA) is administered by an enteral administration route, hi one embodiment, the enteral administration route is selected from the group consisting of oral, rectal, and intranasal, hi yet another embodiment, the short interfering ribonucleic acid (siRNA) is administered by a parenteral administration route.
  • enteral administration route is selected from the group consisting of oral, rectal, and intranasal
  • the short interfering ribonucleic acid (siRNA) is administered by a parenteral administration route.
  • the parenteral administration route is selected from the group consisting of intravascular administration, peri- and intra-tissue injection, subcutaneous injection or deposition, subcutaneous infusion, and direct application at or near the site of the tumor
  • the intravascular administration is selected from the group consisting of intravenous bolus injection, intravenous infusion, intra- arterial bolus injection, intra-arterial infusion and catheter instillation into the vasculature
  • the peri- and intra-tissue injection is selected from the group consisting of peri-tumoral injection, and intra- tumoral injection.
  • the present invention contemplates a method of inhibiting expression of Connexin 46 in a tumor comprising: administering to a subject having a tumor an effective amount of a short interfering ribonucleic acid (siRNA) directed to a sequence of Connexin 46.
  • a short interfering ribonucleic acid (siRNA) directed to a sequence of Connexin 46.
  • the cancer is breast cancer (e.g. metastatic breast cancer)
  • said siRNA is directed to the SEQ ID NO:1.
  • compositions are also contemplated.
  • the present invention contemplates a short interfering ribonucleic acid (siRNA) directed to a sequence of Connexin 46.
  • said sequence of Connexin 46 is SEQ ED NO:1.
  • the siRNA would be modified for a longer half-life.
  • the present invention also provides a method of treating other diseases in a subject, hi one embodiment, the present invention contemplates down-regulating Cx46 or inhibiting (e.g. reducing) the expression of Cx46 in the context of any condition or disease which is associated with a hypoxic/ischemic component.
  • the present invention contemplates a method of treating or preventing heart disease (or simply treating or preventing damage to the heart) in a subject, comprising administering to said subject an effective amount of a short interfering ribonucleic acid (siRNA) directed to Connexin 46. Treating both humans and animals is contemplated.
  • siRNA short interfering ribonucleic acid
  • Cx46 hypoxia-specific gap junction protein
  • Cx46 loss is directly related to breast tumor growth
  • the hypoxia-directed increase in Cx46 may provide the growing and hypoxic tumor with a survival advantage.
  • Cx46 is a hypoxia-specific gap junction protein which provides survival benefits for the hypoxic breast cancer tumor. This occurs through a proteasomal/ubiqui tin-driven degradation of Cx43. If this is the case, then, directed down-regulation of Cx46 could provide a novel treatment for breast cancer.
  • Figure Ia is a Western Blot showing upregulation of Cx46 in HLEC.
  • Figure Ib is a Western Blot showing siRNA knockdown of Cx46 in HLEC.
  • Figure Ic is a bar graph showing the magnitude of the knockdown.
  • Figure Id is a Western Blot showing the lack of knockdown with a non-silencing siRNA control.
  • Figure Ie is a bar graph demonstrating the impact of siRNA on cell viability.
  • Figure 2 is a graph showing that overexpression of Cx46 confers survival of neuronal cells in hypoxia.
  • Figure 3a is a Western Blot showing expression of Cx46 in human breast cancer tumors.
  • Fig. 3b is a gel showing RT-PCT results which agree with the results of Fig 3b.
  • Figure 3c are immunohistochemistry results showing that pre-metastatic breast tumors express Cx46.
  • Figure 3d is a Western Blot showing that Cx46 is upregulated in breast tumors.
  • Figure 3e is a Western Blot showing siRNA knockdown of Cx46 in breast cancer cells.
  • Figure 3f is a Western Blot showing the lack of siRNA knockdown with a non-silencing siRNA control.
  • Figure 3g is a bar graph showing the impact on breast cancer cell viability.
  • Figures 4a, 4b and 4c are Western blots showing knockdown by anti-Cx46 siRNA (with controls) of tumors grown and treated in vivo.
  • hypoxia gene regulator HIFl alpha and the estrogen receptor.
  • This hypoxia gene regulator directly correlates with the degree of metastasis of breast cancer tumors.
  • hypoxia gene regulator directly correlates with the degree of metastasis of breast cancer tumors.
  • hypoxic tumors one other tissue in the human body thrives under hypoxia, the lens. Normal tissues cannot live under hypoxic conditions and this is one of the survival mechanisms for a tumor. The human lens cells also survive in 1% oxygen throughout life. The lens has the normal and ubiquitous gap junction proteins, Cx43 and Cx50.
  • Cx46 the gap junction protein inside the lens.
  • the overexpression of Cx46 causes the ubiquitin-proteasomal driven degradation of Cx43. This is not due to a change in transcript, is reversible by using a proteasome inhibitor, and causes an increase in ubiquitination of Cx43.
  • breast cancer cell lines which have elevated estrogen receptor also have a high level of Cx46.
  • human breast cancer tumors strongly express Cx46. While not intended to limit the present invention to any particular mechanism, we believe that Cx46 plays a role in the breast tumor to protect from hypoxia and to enhance metastasis. Further, as in the lens, it is likely that the Cx46 will enhance the degradation of Cx43, thus, playing a role in the well documented decrease in Cx43 in breast cancer.
  • compositions and methods of the present invention will have high impact as regulation of the hypoxia stage of a breast cancer tumor would provide an entirely new line of drugs to treat (and perhaps eradicate) breast cancer, through a directed down-regulation of Cx46.
  • Targeted down-regulation of Cx46 can be used therapeutically to cause hypoxic tissues such as tumors to die. It is expected that the side effects of down-regulation of Cx46 would be very minimal. Indeed, side effects on the lens or eye would be non-existent since very little gets across the blood/ocular barrier.
  • RNA interference is a method of post-transcriptional gene regulation that is conserved throughout many eukaryotic organisms. RNAi is induced by short (i.e. ⁇ 30 nucleotide) double stranded RNA (“dsRNA”) molecules which are present in the cell (Fire A et al. (1998), Nature 391: 806-811).
  • dsRNA double stranded RNA
  • siRNAs messenger RNAs
  • RISC RNA-induced silencing complex
  • siRNA-mediated RNAi degradation of an mRNA is therefore more effective than currently available technologies for inhibiting expression of a target gene.
  • siRNA-induced RNAi degradation has been demonstrated in several recent in vitro studies, including the siRNA -directed inhibition of HIV-I infection (Novina C D et al. (2002), Nat. Med. 8: 681-686) and reduction of neurotoxic polyglutamine disease protein expression (Xia H et al. (2002), supra).
  • Gap junctions, especially Cx43, are abundant in myocardium where they localize at regions connecting the cardiomyocytes. Cx43 is essential for fast propagation of the action potential necessary for coordinated contraction. Cx43 has been proven to be a key in the process of ischemic reperfusion injury and in cardiac protection from ischemia. More recently this process has been found to include the translocation of Cx43 to the mitochondria. Sold, G., Willecke, K. Cardiovasc Res 62:228-232.(2004). The phosphor- ylation of Cx43 by PKC ⁇ is essential for control of Cx43 in response to ischemia, and some anti-arrhythmic drugs are directed at this target.
  • HIF-I Hypoxia inducible factor- 1
  • HEF-I is a heterodimer composed of an Ch-sensing HIF- l ⁇ subunit and a constitutively expressed HEF-I ⁇ subunit, or aryl hydrocarbon receptor nuclear translocator (ARNT).
  • HIF-I primarily functions as a response element to protect cells from accumulated reactive oxygen species (ROS) or hypoxic insult which causes cell death. Under normoxic conditions, HIF-l ⁇ is continuously synthesized and degraded. conserveed proline residues are hydroxylated allowing the E3 ubiquitin ligase complex to bind and target the HIF- l ⁇ for proteasomal degradation.
  • Hypoxic conditions decrease the essential rate-limiting oxygen levels for proline hydroxylation which, in turn, decreases the rate of ubiquitination. This allows the HIF- l ⁇ to dimerize with the HIF-I ⁇ , translocate to the nucleus, and subsequent expression of genes with HIF-I response elements occurs.
  • One of these is the gap junction protein Cx46.
  • Cx46 is usually only found in the lens, a tissue which thrives naturally under hypoxic conditions. Normal tissues, including the heart, cannot live under hypoxic conditions and this is one of the survival mechanisms which is required for the lens. The human lens cells survive in 1% oxygen throughout life. The lens has the normal and ubiquitous gap junction proteins, Cx43 and Cx50.
  • Cx46 the gap junction protein inside the lens.
  • the overexpression of Cx46 causes the ubiquitin- proteasomal driven degradation of Cx43. This is not due to a change in transcript, is reversible by using a proteasome inhibitor, and causes an increase in ubiquitination of Cx43.
  • Cx46 is a hypoxia-specific gap junction protein which causes the loss of Cx43. This likely occurs through a proteasomal/ubiquitin-driven degradation of Cx43. If this is the case, then directed down-regulation of Cx46 could provide a novel treatment for heart disease.
  • the present invention contemplates down-regulation of CX46 using siRNA, in order to restore CX43 levels and protect cardiac cells from transient hypoxia.
  • Rabbit lens epithelial NNl 003 A cells, human lens epithelial cells (HLEC) and murine neuronal N2A cells were cultured in Dulbecco's modified Eagle's medium (DMEM, low glucose; Invitrogen, San Diego, CA) supplemented with 10% fetal bovine serum and 50 ⁇ g/mL gentamicin, 0.05 U/mL penicillin, and 50 ⁇ g/mL streptomycin (pH 7.4) at 37 °C in 5% CO 2 and 21% O 2 (normoxic conditions).
  • DMEM Dulbecco's modified Eagle's medium
  • DMEM low glucose
  • Invitrogen San Diego, CA
  • penicillin 50 ⁇ g/mL
  • streptomycin 50 ⁇ g/mL streptomycin
  • MCF-7 cells were grown in Minimal Essential Medium (MEM) supplemented with Earl's salts, 10% FBS, glutamine, ImM sodium pyruvate, 0.01 mM nonessential amino acids, antibiotic/antimycotic, and 1.5 g/L sodium bicarbonate at 37 0 C under normoxic conditions (21% O 2 , 5% CO 2 ).
  • MEM Minimal Essential Medium
  • FBS FBS
  • glutamine ImM sodium pyruvate
  • 0.01 mM nonessential amino acids antibiotic/antimycotic
  • 1.5 g/L sodium bicarbonate 1.5 g/L sodium bicarbonate at 37 0 C under normoxic conditions (21% O 2 , 5% CO 2 .
  • To generate a stable line of neuronal N2A cells expressing Cx46 or Cx43 the cDNA of rat Cx46 or rat Cx43 was cloned in pEGFP- N3 vector (BD Biosciences; cat. no. 6080-1) and transfection was carried out using
  • Antibodies Rabbit polyclonal anti-Cx46 was purchased from US Biological, Massachusetts, MA (cat. no. C7858-07A). Anti-Cx50 (cat. no. 33-4300), anti-Cx26 (cat. no. 71-0500) and mouse anti- ⁇ -tubulin (cat. no. 32-2500) were purchased from Zymed- Invitrogen (San Francisco, CA). Anti- ⁇ -actin was purchased from Sigma (cat. no. A5441). Hypoxia. Hypoxia conditions were considered as 1% O 2 , 5% CO 2 at 37°C and 100% relative humidity. Hypoxia conditions were created by a Proox C21 hypoxic chamber (BioSpherix, NY) using nitrogen and CO 2 as displacement gases.
  • Normoxia conditions were considered as 21% O 2 , 5% CO 2 at 37 0 C and 100% relative humidity.
  • 6 ⁇ l ⁇ 5 rabbit NNl 003 A cells were pre-incubated with DMEM low glucose media (supplemented with 10% FBS) at 21% O 2 , 5% CO 2 (normoxic conditions) for 12 h. Following this incubation, the media was replaced with DMEM low glucose complete media that was pre-equilibrated to 1% O 2 . Then the cells were incubated under hypoxic conditions (1% O 2 , 5% CO 2 ) in Proox C21 hypoxic chamber (Biospherix, NY) and harvested after 1-7 days. Whole cell lysates were run on 8% SDS-PAGE followed by western blot to check the expression levels of different proteins.
  • siRNA transfection Anti-Cx46 siRNA (cat. no. SI00131670, Target: CGC ATG GAA GAG AAG AAG AAA) and negative non-silencing control siRNA (cat. no. 1023076) were purchased from Qiagen (Valencia,CA).
  • HLEC or MCF-7 cells were cultured in their respective medium under normoxic conditions (5% CO 2 , 21% O 2 ) on 60 mm dishes.
  • 20 X 10 5 HLEC or MCF-7 cells were transfected with 512 ng of Cx46 siRNA (Final cone.1OnM) or 512 ng negative control siRNA and 20 uL of HiPerFect transfectant reagent (Qiagen, cat. no. 301704).
  • Cell Viability Assay was performed using CellTiter-Blue ® Cell Viability Assay kit (Promega Madison, WI). The kit applies the fiuorometric detection of resorufin converted from resazurin by viable cells. The amount of fluorescence measured is directly proportional to the number of viable cells.
  • 3O x 10 3 HLEC or MCF-7cells in 100 uL of DMEM low glucose media or MEM media respectively, were seeded into each well in 96 well micro-titer plates and cultured for 14 h at 37°C under normoxic conditions (21% O 2 , 5% CO 2 ).
  • the cells were then transfected with IOng (per well, final cone.1OnM) of Cx46 siRNA or 10 ng negative non-silencing siRNA along with .75ul (per well) of HiPerFect transfectant reagent and incubated for 24 h under normoxic conditions (21% O 2 , 5% CO 2 ). Cells with no siRNA treatment were considered as controls. Following this incubation, the media for HLEC or MCF-7 cells were changed with lOOuL (per well) of their respective media that was equilibrated to 1% O 2 .
  • PCR initial activation was done at 95 0 C for 15 min, denaturation at 95 0 C for 30 sec, annealing at 60 0 C for 30 sec. PCR was performed at 35 cycles with final extension at 70 0 C for 10 min.
  • the primer set for Cx46 cDNA were 5'-CTG GCC CTG CTG GCC TTG-3' and 5'-CCA CCA CCT GCT GAT GAC-3'.
  • the primers for ⁇ -actin were 5'-GAA ATC GTG CGT GAC ATT AAG-3' and 5'-CTA GAA GCA TTT GCG GTG GAC GAT-3'.
  • the PCR products were run on 1% agarose gel at 90 V for 80 min. Gels were photographed using Fotodyne software. PCR products were sequenced for confirmation.
  • Immunohistochemical study Immunohistochemsitry was performed on paraffin- embedded human breast tumor tissue (Histologic type: Infiltrating ductal carcinoma;
  • Tissue slides were baked for 1 h at 56°C. Paraffin was washed off the tissue by rinsing three times in xylene for 5 min each, followed by graded series of ethanol washes. Slides were washed with distilled water twice before incubating with antigen retrieval in a steamer for 20 min. Slides were incubated in 3% hydrogen peroxide and washed twice in PBS-T. Slides were blocked in horse serum for 1 h and then rabbit
  • Human breast tumor and normal tissues The human breast tissues were purchased as tissue lysates from Protein Biotechnologies (Ramona, CA). The characterisations of human breast tumors tissues are as follows; Tumor breast tissue 1 : Infiltrating Ductal Carcinoma, grade 2, stage EtA. T2N0M0, source: female, 42 years; Tumor breast tissue 2: Invasive Ductal Carcinoma, grade 2, stage HA. T2N0M0, source: female,42 years.
  • the Human adult normal tissue lysate was purchased from Novus Biologicals (Littleton, CO). 20 ug of total protein of each lysate was loaded and run in 8% agarose gel. The blot was probed with rabbit polyclonal anti-Cx46 antibody (1 :500; US Biological) and developed as described previously 22 .
  • Xenograft tumors of MCF-7 cells in Nu/Nu Mice were purchased from Charles River Laboratory. Mice were implanted with 17 ⁇ -estradiol (1.7 mg/pellet) one week before the injection of 1 x 10 MCF-7 breast cancer cells subcutaneously into the inguinal region of mammary fat pad. Cell viability of MCF-7 cells was performed prior to the injection. Mice were observed for any change in behavior, appearance or weight. Tumors were injected with 7.5 ug of anti-Cx46 siRNA or negative non-silencing siRNA or no siRNA (control) every 40 h for minimum of 10 days to maximum of 20 days at two-three different locations of each tumor.
  • the tumor size was measured in two dimensions by a caliper every other day before injection.
  • the tumor size measured prior to first siRNA injection is considered day 0 measurement.
  • two anti-Cx46 siRNA treated, one negative non-silencing siRNA treated and one control (no siRNA) tumors were dissected out.
  • Three anti-Cx46 siRNA treated tumors were dissected out at day 16.
  • the rest of the anti-Cx46 siRNA treated tumors were isolated from euthanised mice at day 20. Tumors dissected out were homogenised and lysed in RIPA buffer. The lysates were sonicated for 10 sec for three times.
  • Whole tumor tissue lysate were quantitated by Bio-Rad Protein Assay and analysed by western blot.
  • Cx46 protein is upregulated in response to hypoxia (1% O 2 ), in vitro, in rabbit lens epithelial NNl 003 A cells (Fig. Ia).
  • Cells were subjected to hypoxia or normoxia and harvested at 1-7 days.
  • Cx46 protein level was analysed by western blot using anti-Cx46 antibody.
  • Tubulin acted as a loading control.
  • Normoxia treatment (21% O 2 , 5% CO 2 ) had no effect on Cx46 proteins level.
  • HLEC human lens epithelial cells
  • HLEC with Cx46 knocked-down showed significant reduction in cell viability under hypoxic conditions (Fig. Ie).
  • Anti-Cx46 siRNA treated HLEC viability was reduced to 23% at 6 h, 38% at 12 h and 42% at 18 h compared to untreated (control) or non- silencing siRNA treated cells under hypoxia.
  • Cx46 downregulation had no effect on HLEC viability under normoxic conditions which suggested that Cx46 provides protections to lens cells only against hypoxia induced death.
  • Cx46 was stably overexpressed as a GFP tagged fusion protein in murine neuronal N2A cells.
  • a stable line of N2A cells overexpressing Cx43-GFP fusion protein was also generated to test the effect of another gap junction protein, Cx43, on hypoxia induced death.
  • N2A cells do not express endogenous Cx46 or Cx43 proteins.
  • N2A cells transfected with Cx43-GFP or Cx46-GFP expressed fusion proteins of predicted molecular weight of ⁇ 71kDa and 73kDa, respectively as determined by western blot using antibody against GFP (data not shown).
  • N2A cells overexpressing Cx46-GFP or Cx43-GFP were incubated under hypoxic conditions (1% O 2 , 5% CO 2 ) or at normoxic conditions (21% O 2 , 5% CO 2 ) and cell viability was assessed by fluorometric resazurin reduction method. More specifically, 30 ⁇ l0 3 wild type N2A cells or N2A cells overexpressing Cx46 or Cx43 were incubated under hypoxic conditions (1% O 2 , 5% CO 2 ). The viability was assessed every 4 h for up to 24 h by fluorometric resazurin reduction assay. The data are plotted as mean ⁇ s.e.m of three independent experiments.
  • Asterisk indicates the statistical significance (PO.01) between indicated data and control (N2AWT).
  • Wild type N2A cells were hypoxia sensitive as they began to die after 4 h under hypoxic conditions and cell viability was reduced 40% at 12 h and almost 90% at 24 h (Fig. 2).
  • the cell viability of N2A cells overexpressing Cx43-GFP showed the same pattern as wild type N2A cells with cell viability reducing significantly during 4-24 h time period at 1% O 2 (Fig. 2b).
  • N2A cells overexpressing Cx46-GFP remained viable to a considerable extent even after 12 h at 1% O 2 .
  • the cell viability of these cells was only reduced 3% at 12 h and 51% at 24 h at 1% O 2 .
  • Cx46 protein was upregulated in pre-metastatic breast tumor tissue.
  • Tumor breast tissue 1 Infiltrating Ductal Carcinoma, grade 2, stage HA.
  • T2N0M0 source: female, 42 years
  • Tumor breast tissue 2 Invasive Ductal Carcinoma, grade 2, stage DA.
  • T2N0M0, source female, 54 years.
  • Cx46 protein was present in pre- metastatic breast tumor and the protein was upregulated in tumor tissues as compared to normal breast tissue (Fig. 3d).
  • Cx46 in breast tumors led us to hypothesise that breast cancer cells and breast tumors also use Cx46, as an adaptation to hypoxia, to survive and grow.
  • Cx46 protein was downregulated in MCF-7 cells using anti-Cx46 siRNA.
  • 20 X 10 5 MCF-7 cells were transfected with 512 ng anti-Cx46 siRNA (final cone. 1OnM) or negative non-silencing siRNA.
  • the level of knockdown was determined by western blot at 12, 24 and 48 h after transfection. Maximum knockdown using anti-Cx46 siRNA was achieved between 24-48hr time period (Fig 3e).
  • a Cx26 blot was done to show the absence of any non-specific action of anti-Cx46 siRNA in MCF-7 cells.
  • the blot associated with the negative non-silencing siRNA (Fig. 3f) showed no significant knockdown.
  • MCF-7 Downregulation of Cx46 remarkably reduced the MCF-7 cell viability (Fig. 3g). Briefly, 30> ⁇ 10 3 MCF-7 cells (per well of 96 well micro-titer plate) were pre-incubated with IOng (per well, final cone. 1OnM) negative control siRNA or anti-Cx46 siRNA for 24 h at normoxic conditions (21% O 2 , 5% CO 2 ) and then cells were kept at hypoxic conditions (1% O 2 , 5% CO 2 ). Cell viability was assessed every 12 h intervals for up to 24 h. The data are represented as mean ⁇ s.e.m of three independent experiments.
  • the asterisk indicates significant statistical difference (PO.01) between indicated data and control to approximately 34% at 12 h and 40% at 24 h under hypoxic conditions (1% O 2 , 5% CO 2 ) as compared to control (no siRNA) or negative non-silencing siRNA treated cells (Fig.3g).
  • PO.01 significant statistical difference between indicated data and control to approximately 34% at 12 h and 40% at 24 h under hypoxic conditions (1% O 2 , 5% CO 2 ) as compared to control (no siRNA) or negative non-silencing siRNA treated cells (Fig.3g).
  • the downregulation of Cx46 had no effect on MCF-7 cell viability under normoxia (data not shown). This data clearly demonstrated that human breast cancer cells MCF-7 utilize Cx46 to survive against death caused by hypoxia.
  • Tumor size was measured perpendicularly by a caliper every alternate day prior to siRNA injection and hence the tumor size measured prior to first injection was considered as day 0 (measurement).
  • Cx43 The gap junction protein, Cx43, is known to have a turnover rate of 1.5-5 h. Degradation of Cx43 is known to be through both the lysosomal and proteasomal pathways and ubiquitination is essential for both. Cx43 ubiquitination is controlled by a MAPK pathway which directs binding of the E3 ubiquitin ligase Nedd4, a process which is controlled by Cx43 phosphorylation.
  • the interaction of the Nedd4 is at a proline-rich region conforming to the consensus PY sequence (XPPXY) which is on the C-terminus of Cx43 at residues 282-286.
  • Ubiquitination on lysines occurs at many sites on Cx43 but one K is at residue 287.
  • Cx46 also has a XPPXY site at residues 274-279 but lacks the nearby K site.
  • Gap junction plaques are known to exist in eaveolin-containing lipid rafts which contain both Cx43 and Cx46. Overexpression of Cx46 should, in a myocardiac cell, cause the ubiquitination and degradation of Cx43. This should not occur in similar cells which over-express a Cx46 which lacks the correct Nedd4 site.
  • Myocardiac cells in culture will be transfected with plasmid for Cx46 or Nedd4- site mutated Cx46. Following overexpression, cells will be examined for growth properties using a cell viability dye assay, presence of Cx43 protein and transcript, ubiquitination of Cx43, using both confocal co-localization and Western blot, and reversal by a proteasomal inhibitor. Over-expression of cells with Cx50 will be used as a control. For mutations the Cx46 will be altered as: P275L and Y277F/Y278F using standard site-directed mutagenesis procedures. In these studies we will also examine how Cx46 overexpression can occur in these normal myocardiac cells.
  • the Cx46 gene contains a hypoxia response element. Following exposure of the myocardiac cells to transient hypoxia (1-20% oxygen for 1 hr, at 4 hr intervals for up to 4 days) the ceils will be examined for changes in Cx46 and Cx43 protein and transcript levels. Long term exposure of epithelial cells to hypoxia causes a strong up-regulation of Cx46 protein. Oxygen levels will be varied to reflect different ischemic conditions (1,5, 10 and the normoxic 20% oxygen).
  • the cells will be transfected using Lipofectamine 2000 (invitrogen).
  • Lipofectamine 2000 invitrogen
  • Four ⁇ g of the DNA plasmid E.g. GFP-CX46 plasmid cloned into a Clontech pEGFP-N3 vector
  • 0.1 mg of Lipofectamine in 250 ⁇ l of serum and antibiotic free media will be used for each transfection.
  • the cells will be incubated with the DNA plasmid and Lipofectamine for 24 h at 37 0 C. After incubation, media containing twice the amount of fetal calf serum (20%) will be added for 24 h.
  • transfected cells will be selected with 1 mg/ml G418 (Research Products International) for 6 weeks and grown in half that concentration of G418 thereafter. Transfection will be monitored by confocal microscopy and Western blot. Standard protocols will be used for site directed mutants of Cx46. For hypoxia studies cells will be cultured in a O 2 /CO 2 dual controlled chamber (BioSpherix, ProOx model C21).
  • Connexin Proteins will be done as follows: Degradation of 43 caused by overexpression of Cx46 or by hypoxia, will be measured by standard PCR and by Western blot analyses.
  • the H9c2 heart-derived cardiomyoblast cells will be cultured in DMEM. Cells will be harvested and 5 x 10 cells in a 75 cm culture flask will be lysed with ice cold extraction buffer: 2OmM Tris, pH 7.5, 0.5 mM EDTA, 0.5 mM EGTA, 0.5% Triton X-100, 25 ktg/ml aprotinin and leupeptin. Equal amount of protein, determined by Pierce Protein assay, will be used.
  • Proteins will be separated on SDS- PAGE and transferred to nitrocellulose membranes and probed with Cx43 or Cx46 antisera then visualized by ECL. This will identify which connexin proteins are present and/or the effect of overexpressed Cx46 on Cx43 protein levels. Effects on transcript and reversal by proteasome inhibitors will also be done using treatments with (ALLN at lOO ⁇ M and clasto-Lactacystin ⁇ - lactone at 10 ⁇ M, both for 4 hrs) proteasomal inhibitors. Binding of Cx43 or Cx46 to Nedd4 will be determined using co-immunoprecipitation studies and by confocal co-localization. EXAMPLE VI
  • siRNA transfectant reagent Cat. No 301605
  • siRNA Cat. No. S 100131670 against Connexin 46(Gene ID 2700) will be purchased from Qiagen (Valencia, CA).
  • Qiagen Valencia, CA.
  • the target sequence for this siRNA is GCATGGAAGAGAAGAAGAAA.

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Abstract

La présente invention concerne des méthodes et des compositions utiles pour le traitement de maladies et notamment toute condition ou maladie qui est associée à une composante hypoxique/ischémique. Dans un mode de réalisation, cette invention concerne le traitement du cancer et notamment une méthode de traitement du cancer du sein et du rétinoblastome oculaire chez un patient humain. Dans un autre mode de réalisation, cette invention concerne le traitement ou la prévention des maladies cardiaques.
PCT/US2009/003303 2008-06-02 2009-05-29 Méthodes et compositions pour le traitement de maladies Ceased WO2009148552A2 (fr)

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US20050119211A1 (en) * 2001-05-18 2005-06-02 Sirna Therapeutics, Inc. RNA mediated inhibition connexin gene expression using short interfering nucleic acid (siNA)
EP2314689A3 (fr) * 2003-12-03 2012-12-19 Coda Therapeutics (NZ) Ltd Composés anti-sens ciblés contre les connexines et procédés d'utilisation associés
RU2438696C2 (ru) * 2004-12-21 2012-01-10 Маск Фаундейшн Фор Рисерч Дивелопмент Композиции и способы, используемые для ускорения заживления ран и регенерации тканей
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