WO2011050178A2 - Agents thérapeutiques de modulation ciblée de gènes ou protéines prdm - Google Patents

Agents thérapeutiques de modulation ciblée de gènes ou protéines prdm Download PDF

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WO2011050178A2
WO2011050178A2 PCT/US2010/053575 US2010053575W WO2011050178A2 WO 2011050178 A2 WO2011050178 A2 WO 2011050178A2 US 2010053575 W US2010053575 W US 2010053575W WO 2011050178 A2 WO2011050178 A2 WO 2011050178A2
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phage
sirna
protein
liposomes
cells
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WO2011050178A3 (fr
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Lonnie Bookbinder
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Lonnie Bookbinder
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/10—Dispersions; Emulsions
    • A61K9/127—Liposomes
    • A61K9/1271—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers
    • A61K9/1272—Non-conventional liposomes, e.g. PEGylated liposomes, liposomes coated with polymers with substantial amounts of non-phosphatidyl, i.e. non-acylglycerophosphate, surfactants as bilayer-forming substances, e.g. cationic lipids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7088—Compounds having three or more nucleosides or nucleotides
    • A61K31/713—Double-stranded nucleic acids or oligonucleotides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/62—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being a protein, peptide or polyamino acid
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/69—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit
    • A61K47/6905—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion
    • A61K47/6911—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the conjugate being characterised by physical or galenical forms, e.g. emulsion, particle, inclusion complex, stent or kit the form being a colloid or an emulsion the form being a liposome
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents

Definitions

  • siRNA Synthetic small interfering fragments of RNA
  • RNAi RNA interference
  • siRNAs have been studied extensively to treat various cancers and two major factors have been revealed that are important for their practical applications as therapeutics: the development of appropriate delivery systems and the discovery of cancer- related mRNA targets.
  • Pegylated liposomes have been used previously as vehicles for delivery of siRNA to a site of disease.
  • the liposomal carriers of siRNA can be specifically targeted to tumor cells by conjugation with targeting ligands.
  • transferrin-targeted liposomal siRNA demonstrated specific inhibition of Her-2 expression in breast cancer animal model and tumor growth inhibition in pancreatic cancer animal model (Pirollo & Chang (2008) Cancer Res. 68, 1247-50; Pirollo et al. (2006) Hum. Gene Ther. 17, 117-24).
  • Attachment of cell-penetrating peptides (CPP), a family of peptides able to translocate across the cell membrane, has also been used to deliver siRNA into cells (Wang et al. (2006).
  • liposomes bearing a synthetic arginine-rich CPP are stable and can efficiently transfect lung tumor cells in vitro.
  • targeted liposomes containing siRNA and anticancer drugs, such as doxorubicin represent a promising treatment avenue for breast cancer.
  • a new challenge, within the framework of this concept is development of selective, stable, active and physiologically acceptable ligands and integrated targeted nanoparticulate formulations to deliver RNAi products safely and effectively.
  • Phage techniques evolved as a result of advances in combinatorial chemistry and phage display that has opened a door to identifying tumor-specific peptides in a high-throughput fashion (Aina et al. (2007). Mol. Pharm. 4, 631- 651 ; Mori (2004) Curr. Pharm. Des. 10, 2335-43; Sergeeva et al. (2006) Adv. Drug Deliv. Rev. 58, 1622-54).
  • a foreign protein was fused to the N-terminus of the minor coat protein pill of filamentous phage yielding a chimeric "fusion phage," in which up to five copies of the foreign antigen were displayed on the tip of a virion (Smith (1985) Science 228, 1315-17).
  • Tumor-specific phage can be affinity-selected from multi-billion clone libraries (Petrenko (2008) Expert Opin. Drug Deliv. 5, 825-36) by their ability to interact specifically with cancer cell surface receptors and/or penetrate cells ⁇ see US 2007 / 0077291).
  • hybrid phage pVIII coat proteins fused to the tumor-specific peptides could serve a dual function in liposome targeting: the water-exposed N-terminal binding segment can serve as a targeting moiety, while the C-terminal hydrophobic segment can serve as an anchor within the liposome membrane (Fig. 1).
  • the major coat fusion proteins isolated from the tumor- specific landscape phages can be directly inserted into liposomes, leaving the targeting peptide on their surface (US 2007 / 0077291). Accordingly, the chemical conjugation procedure can be avoided and the targeting ligand, the tumor-specific peptide fused to the major coat protein, can be exposed on the surface of the drug-loaded vesicle.
  • Embodiments of the present invention comprise new chemical entities comprising targeted nanocarriers containing a PRDM gene (e.g., PRDM14) or protein- modulating agent.
  • the invention provides a therapeutic agent comprised of a nanocarrier labeled with one or more cell- or tissue-targeting moiety, which can deliver a therapeutic payload that modulates the activity of the PRDM genes or proteins or associated genes or proteins.
  • Embodiments of the present invention comprise phage protein-liposome fusions for the construction of siRNA- (e.g., siRNA to PRDM14) and drug (e.g., doxorubicin)-loaded liposomes that specifically interact with cancer cells. It has been found that, for example,
  • siRNA-doxorubicin-loaded PEGylated liposomes modified with proteins specific for, e.g., MCF-7 breast cancer cells demonstrate (phage-inherited) strong, specific binding, specific silencing of the target gene (e.g., PRDM14), and increased peptide-directed cytotoxicity towards the target cells in vitro.
  • the PRDM14 gene was selected as a target for siRNA-mediated silencing because it may play an important role in carcinogenesis. It has been shown that PRDM14 mRNA is over-expressed and expression of PRDM14 protein is up-regulated in about two-thirds of breast cancer cells tested (Nishikawa et al. (2007) Cancer Res. 67, 9649-57). Introduction of PRDM14 into cancer cells enhanced cell growth and reduced their sensitivity to
  • chemotherapeutic drugs Knockdown of the PRDM14 gene, for example, using siRNA, can increase their sensitivity to chemotherapeutic drugs, suggesting that up-regulated expression of PRDM14 gene may play an important role in the proliferation of breast cancer cells. Further, little or no expression of PRDM14 is seen in non-cancerous tissues.
  • phage display technology offers a promising approach to the targeting of various pharmaceutical agents.
  • the traditional phage display approach requires chemical synthesis of identified tumor-selective peptides and their conjugation with pharmaceuticals or pharmaceutical nanocarriers (such as, e.g., liposomes).
  • a synthetic peptide is first coupled to a lipid anchor to form a lipopeptide, and this derivative is then integrated into a liposome.
  • the chemical conjugation step not only complicates the use of peptides to target liposomes, but can also compromise the targeting ability of the peptides, due to the grafting of the phage-displayed peptides and because of the modification.
  • Embodiments of the present invention comprise novel, siRNA-based nanopharmaceuticals targeted to breast cancer cells via their association with phage fusion proteins, "substitute antibodies” that have high selectivity, affinity, and stability.
  • Tumor-specific peptides were genetically fused to all the copies of the phage's major coat protein pVIII.
  • the peptides are affinity- selected from multi-billion clone libraries by their ability to bind specifically to breast cancer cells and/or to penetrate the cells.
  • the selected tumor-specific phage was converted into drug-loaded liposomal vesicles in which the fusion phage proteins span the lipid bilayer, displaying the tumor-binding peptides on the surface of the vesicles.
  • a major principle of the present invention is that synergistic combinations of appropriate siRNA and anticancer drugs, such as doxorubicin, encapsulated into a targeted liposome, can be delivered to the cancer cells of interest that were used for the selection of the targeting peptide. That targeting of siRNA-loaded liposomes with cancer-specific phage proteins can increase their down-regulating activity towards an example target gene, PRDM14, is demonstrated in the Examples described here. Targeted siRNA preparations were shown to enhance the cytotoxic effects of doxorubicin towards breast cancer cells. These effects can be further studied, for example, in murine models harboring xenografts of, for example, MCF-7 breast cancer cells.
  • siRNA-liposome targeting with the cancer cell-specific peptide-phage protein fusions we observed much better uptake of doxorubicin into target cells upon their treatment with siRNA-phage-liposomes, which also confirmed the preservation of the specific activity of the peptide fragments fused to the coat protein associated with liposomes and the fact that part of the binding peptides is exposed beyond the liposome surface and able to interact with the target cancer cell.
  • Our results demonstrated that the phage pVIII coat protein displaying cancer cell-targeting peptides can serve as an anchor for the integration of these peptides with siRNA-loaded liposomes without significant effects on liposome integrity.
  • Targeting peptide moieties become exposed on the liposome surface and allow the specific targeting of siRNA-loaded phage-liposomes to the cancer cells against which the specific phage was selected, enhancing the silencing effect of siRNA towards the target gene PRDM14.
  • Embodiments of the present invention comprise siRNA-containing pharmaceutical nanocarriers, such as liposomes, targeted with breast cancer-specific phage proteins.
  • siRNA encapsulated in phage protein-targeted PEGylated liposomes, was demonstrated to be active against cancer cells and may be useful as an anti-breast cancer medicine, optionally in combination with traditional anti-cancer chemotherapeutics, such as doxorubicin.
  • Figure 1 siRNA-loaded liposome targeted by the fusion pVIII protein.
  • the hydrophobic helix of the pVIII protein spans the lipid bilayer and binding peptide is displayed on the surface of the vesicle. Some phospholipid domains are conjugated with PEG.
  • Figure 2. A) RT-PCR analysis of PRDM14 gene expression in cancer cells HepG2 , ZR-75-1, MCF-7 and normal breast cells MCF-IOA.
  • P Positive control using RNA and primers included in the RT-PCR kit.
  • Nl Negative control with PRDM14 primers but without any RNA.
  • N2 Negative control, with GAPDH (housekeeping gene) primers, without RNA.
  • T represents the target gene
  • PRDM14 and C represents the housekeeping gene, GAPDH.
  • FIG. 3 Optimization of RT-PCR
  • FIG. 4A RT-PCR analysis of PRDM14 gene after 72 h transfection with siRNA or dsRNA.
  • PRDM14 gene siRNA or dsiRNA (40 nM) or scrambled siRNA (40 nM) were mixed with
  • Lipofectamine RNAiMAX transfection reagent
  • siRNA-Lipofectamine was incubated with MCF-7 cells for a period of 72 h.
  • PRDM14 gene expression was analyzed by RT-PCR B. levels of PRDM14 gene expression were normalized to GAPDH using Kodak ID image analysis software, and the range was calibrated to the value of the negative control.
  • FIG 5A Selectivity of DMPGTVLP phage towards breast cancer cells MCF-7 in comparison with normal breast cells MCF-IOA and hepatocellular carcinoma HepG2.
  • the unrelated phage bearing the peptide VPEGAFSS was the control.
  • B Mode of interaction of DMPGTVLP phage with cells MCF-7 under three different conditions. Selectivity and mode of interaction were estimated as percentage phage recovery: output (cell-associated) phage to input phage, rtp-sf represents room temperature-serum free, whereas sf and s depict serum free and serum, respectively.
  • Figure 6A illustrates output (cell-associated) phage to input phage, rtp-sf represents room temperature-serum free, whereas sf and s depict serum free and serum, respectively.
  • DMPGTVLP protein right, Zeta potential and standard deviation of liposome formulations: 1. modified with phage VEEGGYIAA protein; 2. modified with phage DMPGTVLP protein; 3. plain phage protein-free; 4. plain siRNA-free; 5. siRNA-phage VEEGGYIAA protein; 6.
  • siRNA-phage DMPGTVLP protein siRNA-phage DMPGTVLP protein.
  • MCF-7 cells were treated with PRDM14 gene-specific siRNA-peptide 1 or 2 (VEEGGYIAA or DMPGVTLP correspondingly)-liposomes (40 nM), control siRNA-liposomes (nontargeted), and control scrambled siRNA-liposomes (nontargeted) (40 nM), for 72 h.
  • PRDM14 gene expression was analyzed by RT-PCR. The bands show relative transcription level of the target gene in cells treated with: 1. siRNA-VEEGGYIAA-liposomes, 2.
  • siRNA-DMPGTVLP- liposomes 3. siRNA-liposomes (non-targeted), 4. scrambled siRNA-liposome, B.
  • FIG. 9C D. RT-PCR analysis of PRDM14 gene transcription after 72 h (left) and 48 h (right) transfection of the MCF-7 cells with siRNA-liposome preparations. Two plates with the same passage of MCF-7 cells were treated with PRDM14 gene-specific siRNA-peptide 1 or 2
  • siRNA-VEEGGYIAA-liposomes 2. Scrambled siRNA-VEEGGYIAA-liposomes, 3.
  • siRNA-DMPGTVLP-liposomes 4. Scrambled siRNA-DMPGTVLP-liposome, 5. Control non- treated MCF-7 cells.
  • B. The relative Quantification was normalized against GAPDH using Kodak ID image analysis software, and the range was calibrated to the value of the negative control. All data represent the mean+SD (n 2 or 3).
  • FIG. 9E Western blot analysis of PRDM14 protein after 48 hrs transfection of MCF-7 cells with siRNA.
  • MCF-7 cells from the same passage as described in Figure 9C, D were treated with PRDM14 gene-specific siRNA-peptide (VEEGGYIAA or DMPGVTLP)-liposomes (40 nM), scrambled siRNA-peptide (1 and 2)-liposomes (40 nM), or siRNA-lipofectamine mix (40 nM) and scrambled siRNA-lipofectamine mix (40 nM) for 48 h.
  • PRDM14 protein expression was analyzed by Western blotting. 1. siRNA-VEEGGYIAA-liposomes, 2. scrambled siRNA-VEEGGYIAA-liposomes, 3. siRNA- DMPGTVLP-liposomes, 4. scrambled
  • siRNA-DMPGVTLP-liposomes 5. control (non-treated MCF-7 cells), 6. siRNA-Lipofectamine, 7. scrambled siRNA-Lipofectamine.
  • FIG. 10 Cell viability percentage.
  • the phage-DMPGTVLP-siRNA-DOXO (doxorubicin) liposome formulation shows a higher cytotoxicity efficiency compared to phage- VEEGGYIAA- siRNA-DOXO for same concentration of protein, siRNA and DOXO. The efficacy becomes much higher when compared to phage-free/siRNA-free/DOXO for same DOXO concentration.
  • P value for comparison of phage-DMPGTVLP-siRNA-DOXO and phage-free/siRNA- free/DOXO 0.001
  • P value for comparison of phage-VEEGGYIAA-siRNA-DOXO and phage- free/siRNA-free/DOXO 0.018. Both were calculated as two tailed t-test by Excel. The corresponding plain formulations only made of lipids did not show any cytotoxic effect.
  • phage protein-liposome fusion was used for the construction of siRNA- and doxorubicin-siRNA-loaded liposomes that specifically interacted with target cancer cells. It was found that siRNA-doxorubicin-loaded PEGylated liposomes modified with proteins specific towards MCF-7 breast cancer cells demonstrate (phage-inherited) strong, specific binding, specific silencing of the target gene, and increased peptide-directed cytotoxicity towards the target cells in vitro.
  • the PRDM14 gene was selected as a target for siRNA-mediated silencing because it may play an important role in carcinogenesis.
  • PRDM14 is a member of a family of genes encoding proline-rich domain proteins (PRDM). In particular, it has been shown
  • PRDM14 mRNA is over-expressed and expression of PRDM14 protein is up-regulated in about two-thirds of the breast cancer cells examined
  • PRDM 14 is a target of gene amplification on chromosome 8ql3, a region where gene amplification has frequently been detected in various human tumors
  • introduction of PRDM14 into cancer cells enhanced cell growth and reduced their sensitivity to chemotherapeutic drugs
  • knockdown of the PRDM14 gene, by siRNA can increase their sensitivity to chemotherapeutic drugs, suggesting that up-regulated expression of PRDM14 gene may play an important role in the proliferation of breast cancer cells, and little or no expression of PRDM14 is seen in non-cancerous tissues. As little or no expression is seen in normal tissues, PRDM14
  • PRDM 14 over-expression is associated with up-regulation of 116 genes and down-regulation of 110 genes (Nishikawa et al. (2007) Cancer Res. 67, 9649-57).
  • CEACAM6 is up-regulated and is associated with increased cell surface expression of this adhesion protein, as well as oncogenesis, migration, adhesion, and invasion.
  • Oncogene activation is a result of PRDM14 over-expression, including c-MYC and PEG- 10 activation. Cell survival is enhanced by the increased protection of telomere gene, inducing radio-resistance to breast cancer cells.
  • PRDM14 is oncogenic and increases the number of estrogen receptors and estrogen (Nishikawa et al. (2007) Cancer Res. 67, 9649-57). This may increase the resistance of breast cancer cells to monoclonal antibodies, chemotherapy, and anti-estrogens.
  • a tumor suppressor that inhibits the oncogenic pathway initiated by PI3K/Akt.
  • PI3K/Akt activation increases the resistance of breast cancer to monoclonal antibodies, chemotherapy and anti-estrogens (Clark et al. (2002) Mol. Cancer Ther. 1, 707-17).
  • PI3K/Akt PI3K/Akt activation increases the resistance of breast cancer to monoclonal antibodies, chemotherapy and anti-estrogens (Clark et al. (2002) Mol. Cancer Ther. 1, 707-17).
  • a key gene involved in apoptosis insulin-like growth factor binding protein-7.
  • This tumor suppressor is anti-angiogenic, anti-migratory, and anti-invasive.
  • Over-expression of PRDM14 is thought to be an early event resulting in a poor prognosis in breast cancer and present in three-quarters of advanced breast cancers. These data suggest that PRDM14 could be a therapeutic target for the treatment of breast cancer.
  • phage display technology provides a promising approach to advance the targeting of various pharmaceutical agents.
  • the traditional phage display approach requires chemical synthesis of identified tumor- selective peptides and their conjugation with pharmaceuticals or pharmaceutical nanocarriers (such as liposomes).
  • a synthetic peptide is first coupled to a lipid anchor to form a lipopeptide, and this derivative is then integrated into a liposome (Lee et al. (2007) Cancer Res. 67, 10958-65).
  • the chemical conjugation step not only complicates the application of the discovered peptides to target liposomes, but can also compromise the targeting ability of the peptides, due to the grafting of the phage-displayed peptides and because of the modification.
  • nanopharmaceuticals targeted to breast cancer cells via their association with phage fusion proteins “substitute antibodies” that have high selectivity, affinity, and stability.
  • tumor-specific peptides genetically fused to all 4,000 copies of the phage's major coat protein pVIII were affinity-selected from multi-billion clone libraries by their ability to bind specifically to breast cancer cells and/or penetrate the cells.
  • the selected tumor-specific phage was converted into drug-loaded liposomal vesicles in which the fusion phage proteins span the lipid bilayer, displaying the tumor-binding peptides on the surface of the vesicles (Fig. 1).
  • a major principle of the present invention is that synergistic combinations of appropriate siRNA and anticancer drugs, such as doxorubicin, encapsulated into a targeted liposome can be delivered to cancer cells that have been used for selection of the targeting phage.
  • siRNA-loaded liposomes with cancer-specific phage proteins can increase their down-regulating activity towards an example target gene, PRDM14.
  • the targeted siRNA preparations were shown to enhance the cytotoxic effects of doxorubicin towards breast cancer cells. These effects can be further studied, for example, in murine models harboring xenografts of MCF-7 breast cancer cells.
  • the landscape phage-based approach used here relies on the powerful and precise mechanisms of selection, biosynthesis, and self assembly.
  • a culture of cells secreting filamentous phage is an efficient and convenient protein production system, yielding up to 300 mg/liter of pure phage, with the major coat protein constituting 98% of the total protein mass of the virion; such purity is hardly reachable in normal synthetic and bioengineering procedures.
  • the phage itself and its components are not toxic and have been already tested for safety in preclinical trials (Krag et al. (2006) Cancer Res. 66, 7724-33).
  • siRNA-phage-liposomes which additionally confirmed the preservation of specific activity by peptide fragments of the coat fused protein associated with liposomes and the fact that a good part of the binding peptides is exposed and fit for the interaction on the liposome surface.
  • Targeting peptide moieties become exposed on the liposome surface and allow the specific targeting of siRNA-loaded phage-liposomes to the cancer cells against which the specific phage was selected, dramatically increasing the silencing effect of siRNA towards the target gene PRDM14.
  • Embodiments of the present invention comprise siRNA-containing pharmaceutical nanocarriers, such as liposomes, targeted with breast tumor- specific phage proteins.
  • siRNA encapsulated in phage protein-targeted PEGylated liposomes, was demonstrated to be active against cancer cells and may be useful as an anti-breast cancer medicine, optionally in combination with traditional anti-cancer chemotherapeutics, such as doxorubicin.
  • Egg phosphatidylcholine (ePC), cholesterol (CHOL), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl trimethylammonium propane (DOTAP), and polyethylene glycol (2000) distearoyl phosphoetanolamine (PEG2k-PE) were purchased from Avanti Polar Lipids (Alabaster, Al).
  • Sodium cholate hydrate was from Sigma (St. Louis, MO)
  • RNase/DNase-free water was obtained from MP Biomedicals (Solon, OH)
  • phosphate saline buffer (lOx solution) was from Fisher Scientific (Fair Lawn, NJ).
  • DMSO Dimethyl sulfoxide
  • doxorubicin Dimethyl sulfoxide
  • doxorubicin Trimethyl sulfoxide
  • 3 siRNA sequences that the target PRDM-14 gene used for encapsulation into the liposomes were from by Sigma, sodium cholate was from Sigma, 16% non-gradient Tris-Tricine gels were from Jule Inc., the Immobilon-P PVDF membrane from Millipore; the NeutrAvidinTM-HRP and BCA protein assay kits were from PIERCE, and the Cell Titer Blue assay kit was from Invitrogen.
  • the RNA STAT reagent was from Tel Test.
  • the Access RT-PCR kit was from Promega. Oligos for PRDM 14 and GAPDH were from Invitrogen and siRNA specific for PRDM 14 were from IDT DNA. Negative scrambled siRNA was from Applied Biosystems.
  • MCF-7 human breast adenocarcinoma (HTB 22) cells were used as target cells.
  • siRNAs were from IDT (for silencing
  • Reverse transcription of total RNA and cDNA amplification by PCR was carried out using 25 ng of total RNA in a 25- ⁇ reaction mixture, using the one-step Access RT-PCR kit, according to the manufacturer's protocol.
  • the primers for PRDM14 and GAPDH genes were used at final concentrations of 0.1 ⁇ .
  • One cycle of reverse transcription of isolated RNA at 48°C (45 min) and 94°C (2 min) was followed by 35 cycles of PCR at 62°C (30 s), 68°C (1 min), and 68°C (7 min). Relative levels of gene expression were quantified using A Kodak imager.
  • MCF-7 cells grown in a 75-cm flask were collected by routine trypsinization.
  • 100,000 MCF-7 cells in 6-well culture plates were transfected with PRDM14-specific siRNA (40 nM) or scrambled siRNA (40 nM) mixed with the Lipofectamine RNAimax reagent.
  • PRDM14-specific siRNA 40 nM
  • scrambled siRNA 40 nM
  • RNAimax reagent Three siRNA that target PRDM14 gene are described in Example 2, above. Briefly, 4.8 ⁇ of each siRNA (106 nM) was mixed with 250 ⁇ of Opti-MEM I medium in a 1.5-mL microfuge tube.
  • Lipofectamine RNAiMAX was mixed gently before use by inverting and 6 ⁇ ⁇ of the homogenized preparation was added to 250 ⁇ L ⁇ of Opti-MEM I medium and the composition was mixed gently by inverting. 250 ⁇ ⁇ of Lipofectamine was added to the diluted 250 ⁇ L of siRNA in Opti-MEM I medium, mixed gently and incubated for 10-20 min at room temperature.
  • the siRNA-Lipofectamine preparation was mixed with 100,000 MCF-7 cells in a 6-well culture plate and adjusted to 2 mL with L-15 media (with 10% FBS, without antibiotics) resulting in a 40 nM total concentration of siRNA. The plate was gently rocked back and forth at room temperature and incubated at 37 °C for 72 h.
  • siRNA-VEEGGYIAA-liposome 50 ⁇
  • siRNA-DMPGTVLP-liposomes 50 ⁇
  • 0.53 ⁇ of siRNA-liposomes (150 ⁇ ) were mixed with 100,000 MCF-7 cells in a 6-well culture plate and adjusted to 2 mL with L-15 media (with 10% FBS, without antibiotics), resulting in a 40-nM total concentration of siRNA.
  • Example 7 Analysis of PRDM14 protein expression in MCF-7 cells by Western blotting
  • MCF-7 cells were treated with PRDM i4-specific siRNA-phage fusion protein (VEEGGYIAA or DMPGTVLP), scrambled siRNA-phage fusion protein (VEEGGYIAA or DMPGTVLP;
  • siRNA-lipofectamine 40 nM
  • siRNA-lipofectamine 40 nM
  • scrambled siRNA-lipofectamine 40 nM
  • cells were lysed with 70 ⁇ of RIPA buffer (Sigma, #0278) containing protease inhibitor cocktail (7 ⁇ ) and PMSF (2 mM final concentration).
  • the protein concentration in whole cell lysate was measured by the Biorad DC protein assay. 15 ⁇ g of whole cell extract was separated on Tris-HCl gels (4-20%; Biorad, #161-1159) by electrophoresis and transferred to PVDF membrane.
  • the membrane was blocked in wash buffer (lx PBS) with 5% non-fat dry milk and incubated at room temperature for 1 h and incubated overnight at 4°C with polyclonal anti-PRDM14 antibody (1:500 dilution; Genway, 180003-42347).
  • the membrane was washed with PBS/0.5% Tween-20 four times and incubated with peroxidase-conjugated Affinipure goat anti-rabbit IgG (1:5000; Jackson Immunoresearch, #111-035-003) at room temperature for 1 h.
  • the membrane was washed again with PBS/0.5% Tween-20 four times and incubated with 5 mL of West Pico Luminol/Enhancer Solution and 5 mL West Pico Stable Peroxide Solution (Pierce Super Signal West Pico Biotin Detection Kit, prod. no. 34085) for 10 min.
  • the membrane was loaded into a cassette and was exposed to radiographic film for 1-2 min. Images were scanned and quantified using the NIH Image J software.
  • Example 8 Preparation of phage proteins specifically binding to MCF-7 breast cancer cells Selection of the breast cancer cell-binding clones from the f8/8 and f8/9 libraries was conducted in parallel. An aliquot of each phage library containing 100 billion phage particles in blocking buffer (0.5% BSA in serum-free medium) was added to an empty cell culture flask and incubated for 1 h at room temperature to deplete phage particles that bound to the cell culture flask. At the same time, confluent MCF-7 cells were incubated for 1 h at room temperature in serum-free medium that was removed immediately before application of phages.
  • Unbound phages recovered from the depletion flask were transferred to confluent MCF-7 cells already incubated with serum-free medium, and incubated for 1 h at room temperature. Thereafter, unbound phage particles were removed and cells were washed 10 times with washing buffer (0.1% BSA, 0.1% Tween 20 in serum-free medium). Unbound phage and washings were stored for titering in host E. coli (K91 BlueKan) cells. Cell-surface bound phages were eluted with 2 mL elution buffer (0.1 M glycine-HCl) for 10 min on ice and neutralized with 376 iL 1 M Tris (pH 9.1).
  • Phages in eluate were concentrated using centrifugal concentrators (Centricone 100 kDa, Fisher Scientific, Pittsburgh, PA) to an approximate volume of 80 iL.
  • the concentrated eluted phages were titered and amplified in host E. coli bacteria and used as an input in further rounds of selection, which were similar to the procedure described above, except for the lack of depletion with the cell culture flask.
  • Four rounds of selection were performed altogether and clones selected in different rounds were randomly picked, isolated as individual clones, sequenced, and propagated for further characterization.
  • the enrichment of phages binding to the cells was determined by titering of input and output phages. The ratio of output to input phage increased from one round to the next, indicating successful selection of phage clones that bound to the target MCF-7 cells.
  • Binding specificity and selectivity of the phage was determined in a phage capture assay (Brigati et al. (2008) Curr. Prot. Protein Sci., chapter 18, units 18, 19) adapted to the 96-well culture plate format. Briefly, target cells (MCF-7, MCF-IOA, HepG2 cells) were cultivated in triplicate to confluence in separate wells of 96-well cell culture plates. Cell culture growth medium was incubated in separate wells in triplicates as controls. The experiment commenced by aspirating the cell culture growth media from wells containing confluent cells and control wells. Cells were washed and incubated with serum-free medium at room temperature for 1 h. Phages
  • DMPGTVLP VEEGGYIAA or control
  • unrelated phage VPEGAFSS streptavidin binder (Petrenko and Smith (2000) Protein Eng. 13, 589-92; 10 6 cfu) in 100 ⁇ blocking buffer were added to the corresponding well and incubated for 1 h at room temperature or 37°C. Unbound phage were carefully removed and cells were washed with 100 ⁇ washing buffer for 5 min, eight times. Then, cells were treated with 25 ⁇ lysis buffer (2.5% CHAPS: 3-[(3- cholamidopropyl)dimethylammonio]-l-propanesulfonate; Sigma-Aldrich) for 10 min on a shaker. The concentration of the phage was measured by titering it in E. coli (K91 BlueKan) host bacterial cells; phage recovery was calculated as the ratio of input phage to output phage. The test was performed in triplicate.
  • Example 10 Analysis of the mode of phage binding to breast cancer cells
  • Target MCF-7 cells were cultivated in triplicate per phage clone to confluence in wells of a 96-well cell culture plate until confluence.
  • Cell culture growth medium was aspirated and wells containing the confluent cells were washed with serum-free growth media.
  • the incubation of phage with cells was carried out at room temperature without serum, at 37°C without serum, and at 37°C with serum.
  • the cells were incubated with 100 ⁇ serum-free medium at room temperature for 1 h and phage clone ( ⁇ 10 6 cfu in 100 ⁇ blocking buffer) was added to the corresponding well and incubated for 1 h at room temperature. Unbound phage were carefully removed and the cells were washed with 100 ⁇ washing buffer for 5 min, eight times, to remove any remaining unbound phage. Surface-bound phage were recovered by treating wells with acid elution buffer (pH 2.2). The eluate was neutralized with 4.7 ⁇ neutralizing buffer (1 M Tris, pH 9.1).
  • Phage fusion 55-mer coat proteins ADMPGTVLPDPAKAAFDSLQASATEYIGYAWAMVVVIVGAT- IGIKLFKKFTSKAS and AVEEGGYIAAPAKAAFDSLQASATEYIGYAWAMVVVIV- GATIGIKLFKKFTSKAS (foreign peptides shown in bold) were prepared by stripping the phage in cholate buffer (Jayanna et al. (2009) Nanomedicine 5, 83-89; Spruijt et al., (1989)
  • Biochemistry 28, 9158-65 Briefly, a mixture of 350 ⁇ ⁇ phage in TBS buffer ( ⁇ 1 mg/mL) and 700 of 120 mM cholate in 10 mM Tris-HCl, 0.2 mM EDTA, pH 8.0, was incubated at 37°C for 1 h.
  • the fusion protein was purified from the viral DNA and traces of bacterial proteins by size-exclusion chromatography using a Sepharose 6B-CL (Amersham Biosciences) column (1x45 cm), which was eluted with 10 mM cholate in 10 mM Tris-HCl, 0.2 mM EDTA, pH 8.0.
  • the chromatographic profile was monitored using an Econo UV monitor (Bio-Rad, CA); 5-mL fractions were collected and stored at 4°C.
  • the protein was isolated as an aggregate with molecular weight -46 kDa (8-mer), determined by chromatography on a column calibrated with standard molecular weight markers (aprotinin, 6.5 kDa, cytochrome C, 12.4 kDa, carbonic anhydrase, 29 kDa, and bovine serum albumin, 66 kDa; Sigma), as described by Spruijt et al. (1989).
  • a lipid film composed of ePC:CHOL:DPPG:DOTAP:PEG2k-PE (45:30:20:2:3 molar ratio) was prepared in a round-bottomed flask by removing the organic solvent. The film was further dried for 4 h under high vacuum, then it was rehydrated in sterile PBS buffer, pH 7.4 (made in nuclease-free water to avoid contamination in subsequent steps) to a final liposome concentration of 40 mg/niL. To obtain "plain" liposomes (liposome formulation with no phage), hydrated lipids were bath-sonicated for 10-15 min and finally extruded through a 200-nm polycarbonate membrane.
  • Each phage-peptide was incorporated into the lipid formulation by an overnight incubation at 37°C (1 :200 wt phage-protein:liposomes) at a final sodium cholate concentration of 15 mM and up to a final lipid concentration of 10.3 mg/niL.
  • the formulation was dialyzed overnight (dialysis bag cutoff size 2000 Da) against sterile PBS buffer, pH 7.4 (in nuclease-free water) to remove excess sodium cholate.
  • siRNA-liposomes A similar preparation was used to prepare siRNA-liposomes. Briefly, a lipid film composed of ePC:CHOL:DOTAP:PEG2k-PE (60:30:10:2 molar ratio) was made in a round-bottomed flask, removing the chloroform. The film was further dried for 4 h under high vacuum, then it was rehydrated in sterile PBS buffer, pH 7.4 (in nuclease-free water), a final liposome concentration of 10.3 mg/niL. The hydrated lipids were bath-sonicated for 10-15 min and finally extruded through a 200-nm polycarbonate membrane. Then, "plain" liposomes (liposome formulation with no siRNA) were incubated at room temperature for 3.5 h with a mixture of the three siRNA together at a molar ratio DOTAP: siRNA of 10: 1.
  • siRNA-liposomes and phage-liposomes were incubated in a 1 :2 volume ratio (50 ⁇ siRNA-liposomes in 100 ⁇ phage-liposomes) overnight at 4°C.
  • a lipid film composed of ePC:CHOL:DOTAP:PEG 2k -PE (60:30: 10:2 molar ratio) was made in a round-bottomed flask, removing the chloroform.
  • a methanol solution of doxorubicin at 1 % wt ratio on the total lipids was added to the solution.
  • the film was further dried for 4 h under high vacuum, then it was rehydrated in sterile PBS buffer, pH 7.4 (in nuclease-free water) to a final liposome concentration of 10.3 mg/niL.
  • the hydrated lipids were bath-sonicated for 10-15 min and finally extruded through a 200-nm polycarbonate membrane.
  • the siRNA-free liposomes (liposome formulation with no siRNA) were incubated at room temperature for 3.5 h with a mixture of the three siRNA together at a molar ratio DOT AP: siRNA of 10: 1.
  • siRNA-liposomes and phage-liposomes were incubated at a 1:2 volume ratio (50 ⁇ siRNA-liposomes in 100 ⁇ phage-liposomes) overnight at 4°C.
  • Doxorubicin possesses fluorescent properties.
  • a known amount of liposomes was dissolved in methanol and the doxorubicin loading was assessed by fluorescence spectrometry (Hitachi F-2000 fluorescence spectrometer (Hitachi, Japan) at Ex 480 nm and Em 550 nm.
  • the DOXO loading was determined using a calibration curve obtained using standard concentrations of drug in methanol (ranging from 12.2 ng/mL to 6.25 ⁇ g/mL).
  • PicoGreen-siRNA fluorescence intensity was detected at an excitation wavelength of 480 nm and emission of 520 nm. A 1/200 dilution of the probe in TBE buffer was prepared.
  • siRNA-Phage EL-7-liposomes and siRNA-Phage 28-4-liposomes diluted in 10 ⁇ nuclease-free water was incubated with 990 ⁇ of PicoGreen solution.
  • the same amount of free siRNA in PicoGreen solution was used as a reference to determine to amount of siRNA not associated with lipids.
  • the same dilution of phage EL-7-liposomes, phage 28-4-liposomes, and plain PicoGreen solution were used and subtracted from the final sample fluorescence. Each sample was incubated at 37 °C for 10 min. Then, the fluorescence was detected with a Hitachi F-2000 fluorescence spectrometer. Only free siRNA is able to react with the probe and emit
  • % siRNA in solution (Picogreen fluorescence liposomes/Picogreen fluorescence free).
  • MCF-7 human breast cancer cell line In vitro cell viability tests were performed on the MCF-7 human breast cancer cell line. Assay results were evaluated using the MTT test according to a routine protocol. MCF-7 cells were grown in 75 -cm 2 flasks to 80% confluence. Then, they were seeded (7.7x103 J cells/well) in 96- well plates and maintained for 24 h at 37°C and 5% C0 2 . The cells were washed with
  • MTT is reduced to the purple formazan in the mitochondria of living cells. This reduction takes place only when mitochondrial reductase enzymes are active, and therefore conversion can be directly related to the number of viable (living) cells.
  • Cell viability was determined by measuring the absorbance of the produced formazan with a microplate reader (Bioteck Synergy HT) at 540 nm and expressed as the percentage of live cells of the total untreated cells. P values were calculated with Excel (two tailed t-test) and were deemed to be statistically significant when ⁇ 0.05.
  • Example 21 Profiling candidate breast cancer cells for activity of the PRDM14 gene
  • RNASTAT Human mammary cell lines, breast carcinoma MCF-7, breast ductal carcinoma ZR-75-1, and normal breast cells MCF-10, were used.
  • RNASTAT total RNA was isolated from the cells using RNASTAT and ethanol precipitation. RNA quantity and purity was controlled by UV spectroscopy.
  • PRDM14 gene expression was analyzed by semi-quantitative RT-PCR using Access RT-PCR kit (Promega) and primers described below, followed by gel electrophoresis. The relative expression of the gene was normalized against the housekeeping glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene (Fig. 2).
  • GPDH housekeeping glyceraldehyde-3-phosphate dehydrogenase
  • RNA isolated from breast cancer cells using RNASTAT kits and ethanol precipitation was spectrally pure and contained no contaminating proteins (260/280 > 1.8) or impurities inhibiting RT-PCR.
  • RT-PCR analysis of RNA isolated from breast cancer cells ZR-75-1 and MCF-7, hepatocellular carcinoma HepG2, and normal breast MCF-IOA cells showed that while the PRDM14 gene was highly expressed in MCF-7 and moderately so in ZR-75-1 and HepG2 cells, it was not active in normal breast cells.
  • the production of the PRDM14 protein itself in the MCF-7 cells was demonstrated by Western blotting (Fig. 9E).
  • Example 22 RT-PCR experiments
  • RT-PCR reaction conditions were assessed, including the following parameters: a) structure of primers, b) temperature of annealing of the primers with the template, c) RNA concentration in the RT-PCR reaction mixture, and d) temperature of extension of the polymerase reaction.
  • the product of the RT-PCR reaction (412 bp DNA fragment) was analyzed by electrophoresis in agarose gel followed by SYBR Green staining (Fig. 3). An annealing temperature of 62°C, an RNA concentration of 1 ⁇ g/mL, and an extension temperature 68°C were determined to be optimal and were used in the subsequent experiments.
  • siRNAs reported by Nishikawa et al. Based on preliminary experiments (Fig. 4), 40 nM siRNAs was chosen; this concentration showed visible effects of target gene silencing induced by gene-specific siRNA in comparison with negative control siRNAs and control unrelated siRNA.
  • dsRNA "dicer substrate siRNA”
  • IDT siRNA substrate siRNA
  • Direct and fast screening for tumor-targeted peptides via the phage display technology provides a promising approach to advance the targeting of various pharmaceutical agents.
  • the traditional phage display approach requires the chemical synthesis of the identified tumor- selective peptides and their conjugation with pharmaceuticals or pharmaceutical nanocarriers (such as liposomes).
  • a synthetic peptide is first coupled to a lipid anchor to form a lipopeptide, and this derivative is then integrated into a liposome.
  • the chemical conjugation step not only complicates the application of the discovered peptides to target liposomes, but can also compromise the targeting ability of the peptides, due to the grafting of the phage-displayed peptide(s) into a new environment and because of their modification.
  • liposome targeting of intact hybrid fusion coat proteins has been suggested. They can be isolated from cancer cell-specific phages selected from "landscape libraries," multi-billion collections of phage with all 4000 copies of the major coat proteins pVIII fused to foreign, random peptides. Almost 90%, by mass, of a landscape phage is a hybrid coat protein, a chimeric polypeptide 55-56 amino acids long, composed of a foreign targeting peptide segment fused to the N-terminus of the phage protein. The propensity of the major coat protein pVIII to integrate into liposomes is a result of its intrinsic function as membrane protein, judged by its biological, chemical, and structural properties.
  • the protein is synthesized as a water- soluble cytoplasmic precursor, which contains an additional leader sequence of 23 residues at its N-terminus.
  • the leader sequence is cleaved off by a leader peptidase.
  • the newly synthesized proteins are transferred from the membrane into the coat of the emerging phage.
  • the major coat protein can change its conformation to accommodate the various distinctly different forms during phage infection and assembly.
  • the major coat protein is determined by its unique architecture.
  • the 50 amino acid-long pVIII protein is very hydrophobic and largely insoluble in water when separated from virus particles or membranes. In virus particles, it forms a single, somewhat distorted a-helix with only the first four or five residues mobile and unstructured.
  • the membrane nature of the pVIII major coat protein is retained even when a short foreign target peptide is fused to its N-terminal because the "membranophilicity" of the fusion proteins and their ability to proceed normally during the phage infection and morphogenesis has evolved during the propagation of phage libraries in the host bacteria and is ensured by the observed viability of the selected phage.
  • the pVIII major coat protein segment can serve as a membrane anchor within liposome bilayer for the targeting peptide, which should be exposed on/over the liposome surface.
  • Phages DMPGTVLP and VEEGGYIAA (designated by the structure of the borne foreign peptide) demonstrated high selectivity and specificity towards the target cells versus control unrelated cells, as discussed below.
  • amino acids 45-55 They have a positively charged C-terminus (amino acids 45-55), which can navigate the protein through the liposome membrane, a highly hydrophobic "membranophilic" segment (amino acids 27-40), which allows the proteins to integrate readily in the membrane, and an amphiphilic N-terminus (amino acids 1-26), which is soluble in water, but can also readily interact with PEG residues on the surface of the "stealth" Doxil-like liposomes and display the N-terminal cancer cell-binding octamers and nonamers on the liposome shell.
  • the proteins were obtained in the form of cholate- stabilized octadomains by stripping of phages DMPGTVLP and VEEGGYIAA, selected by biopanning against MCF-7 breast cancer cells.
  • Phage fusion coat protein-targeted liposomes (“phage-liposomes")
  • phage-liposomes To prepare phage fusion coat protein-targeted liposomes ("phage-liposomes”), we modified previously developed procedures for the insertion of membrane proteins into liposomes during their reconstitution, to ensure the integrity of preformed PEGylated liposomes.
  • the highly hydrophobic pVIII coat fusion protein was solubilized using the detergent sodium cholate at its CMC concentration, then inserted the coat fusion protein into the liposome membrane by incubating mixed micelles of sodium cholate and coat fusion protein with liposomes, and then removed sodium cholate by dialysis, yielding phage-liposomes.
  • the presence of uniform population of liposomal nanoparticles in the protein- modified preparation was confirmed by the FFEM and size distribution analysis.
  • Western blotting also demonstrated that pVIII major coat fusion protein was associated with liposomes.
  • Previously selected breast cancer cell-binding and cell-penetrating phages were used. Unique phage clones were identified by sequencing of their DNA. Selected clones, represented by 136 unique variants that belonged to 32 peptide families, were tested for their selectivity towards target breast cancer cells in comparison with control "normal" breast cells MCF-IOA and other cancer cells. A novel high-throughput screening method in 96-well plates with three different cell cultures was developed to accomplish this analysis. The most selective phages were also characterized for their distribution in different parts of the cells and characterized as "binding" or "penetrating" phages.
  • Selectivity of the phage was determined by measuring its binding to breast cancer cells, in comparison with serum, normal breast epithelial MCF-IOA cells, and hepatocellular carcinoma HepG2 cells.
  • different ways of recovering the cell-associated phage were used: with acid buffer for elution of surface-bound phage, followed by post-elution washing with neutral buffer and finally with CHAPS buffer for recovery of cell-integrated and -penetrated phage particles.
  • the amount of phage in different fractions was determined by titration.
  • phage DMPGTVLP demonstrated high selectivity towards the target breast cancer MCF-7 cells, binding them at a level 26-fold higher than normal breast MCF-IOA cells, and 12 times higher then liver cancer HepG2 cells (Fig. 5, left).
  • the control phage VPEGAFSS bound the same cells at a level about 70 times lower than the selected phage DMPGTVLP.
  • Bound phage was located on the surface of the target cells and was not seen in internal cellular compartments (Fig. 5, right).
  • phage was chosen for targeting of the siRNA-liposomes, VEEGGYIAA; it also bound selectively to MCF-7 cells (Fig. 6, left), but in contrast to phage DMPGTVLP, it was able to penetrate into the cells at 37°C (Fig. 6, right).
  • cancer-targeted liposomes were selected from f8/9 and f8/8 landscape phage libraries, respectively, using biased selection and exhibited high selectivity towards MCF-7 cells.
  • Example 29 Isolation of phage proteins specifically binding breast cancer cells
  • the phage VEEGGYIAA and DMPGTVLP were stripped in cholate/chloroform buffer and purified by size-exclusion chromatography.
  • the proteins were stabilized in cholate buffer and characterized by Western blotting and chromatography on the mass-calibrated column (Fig. 7) and used for preparation of the targeted siRNA-liposomes.
  • Example 30 Synthesis of liposomal siRNA complementary to the PRDM14 gene and decorated with cell-targeted phage proteins
  • siRNA-phage-liposomes and doxorubicin-siRNA-phage-liposomes were prepared using two different liposome formulations fused together in the last step, by coincubation overnight at 4°C. All liposome formulations were PEGylated. Both intermediate preparations (phage-liposomes and siRNA-liposomes) were made with PEG-PE. Liposome formulations were characterized by measuring their size, size distribution, and surface charge ( ⁇ , zeta). To check the amount of free siRNA in solution, a fluorescent assay based on the interaction between the PicoGreen probe and siRNA was used. Only free siRNA is available and can interact with the probe. The higher is the amount of siRNA reacting with the intercalating agent, the higher is the resulting fluorescence. By this analysis, with the siRNA-phage VEEGGYIAA liposomes and siRNA-phage
  • Both phage-liposome formulations showed a mean size comparable to their starting plain formulation (that did not contain phage; Fig. 8, left), but a quite different ⁇ because of the incorporated proteins: from a positive ⁇ for plain liposomes (+49.3 mV) to negative (-46.6 mV and -42.3 mV for phages VEEGGYIAA and DMPGTVLP, respectively; Fig. 8, right).
  • siRNA-liposomes This inversion of ⁇ highlights the incorporation of the proteins in the lipid structure.
  • the size and ⁇ of the plain formulation used to make siRNA-liposomes was also compared with the final siRNA-phage formulations. Although phage-free siRNA-liposomes demonstrated a bigger size than phage-liposomes after an overnight incubation, "fused" formulations demonstrated sizes closer to that of initial phage-liposomes. The ⁇ does not change strongly, compared with the phage-liposomes. This may be due to the high level of shielding of siRNA in lipoplexes, where siRNA is hidden and more stable. PicoGreen analysis of the siRNA-phage
  • VEEGGYIAA-liposomes and siRNA-phage DMPGTVLP-liposomes confirmed that the majority of the siRNA was shielded in liposomes and protected from nucleases. Phage-siRNA-liposomes were sufficiently stable: in buffer solution, size and size distribution did not change over one week; in 10% serum, no change in size or size distribution occurred upon overnight incubation.
  • Doxorubicin-loaded phage-siRNA-liposome formulations (with phage DMPGTVLP and phage VEEGGYIAA) and phage-free/siRNA-free liposomes were shown to be comparable in size distribution and their mean size was about 150 nm, with a different polydispersity index, below 0.2, for each of them. The ⁇ did not change strongly among the doxorubicin-containing formulations.
  • Example 31 Gene-silencing activity of siRNA-phage fusion peptide-liposomes preparation in vitro towards MCF-7 breast cancer cells
  • MCF-7 cells were treated with 40 nM siRNA-phage protein (VEEGGYIAA or
  • DMPGTVLP DMPGTVLP-encapsulated liposomes for 48 and 72 h.
  • Total RNA was isolated from treated cells and analyzed by RT-PCR with primers specific for PRDM14 gene. Relative expression of the gene was normalized to the GAPDH gene, as suggested by Nishikawa et al. (2007). Gene expression was analyzed by semi-quantitative PCR that has been successfully used previously by other researchers and confirmed by Western blot analysis of the expressed PRDM14 protein.
  • PRDM14 gene expression by gene-specific siRNA targeted by peptide 2 (DMPGTVLP), compared with non-targeted siRNA, non-specific siRNA, and gene-specific siRNA targeted by peptide 1 (VEEGGYIAA).
  • siRNA-lipofectamine preparation as a positive control. Consistent with the semi-quantitative RT-PCR analysis, we observed a significant silencing effect of siRNA targeted by phage proteins.
  • the phage fusion protein-targeted PEGylated liposomal siRNA down-regulated the PRDM14 gene in breast cancer cells at a much higher level than non-targeted siRNA-liposomes.
  • the targeted siRNA liposomes demonstrated silencing activity comparable to the known, but pharmaceutically unacceptable, Lipofectamine preparations. These results demonstrate the superiority of the selected peptides and targeted delivery versus non-targeting. These results demonstrate that siRNA, encapsulated in phage protein-targeted PEGylated liposomes, may be useful as a potential therapeutic for the modulation of activity of cancer-related genes, such as the PRDM14 gene.
  • Doxorubicin is known to have a proapoptotic effect on MCF-7 breast cancer cells.
  • the siRNA used was specifically targeted and effective in gene silencing in this cell line.
  • Table 1 Summary of the composition for each liposome formulations. These amounts are associated to the different DOXO-liposome formulations.
  • the phage-free/siRNA-free/DOXO liposomes did not contain any phage-protein and/or siRNA.
  • phage-DMPGTVLP-siRNA-DOXO liposome formulation was also demonstrated to be more efficacious than phage- VEEGGYIAA-siRNA-DOXO for the same concentration of protein, siRNA, and DOXO (Fig. 10).
  • the higher targeted activity of the phage DMPGTVLP protein correlated well with the higher silencing activity of the siRNA liposomes targeted with this protein (Fig. 10).
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein the carrier particle is a liposome.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein one of the drug molecules is a therapeutically active polynucleotide.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein one of the drugs is an siRNA.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein one of the drugs is an siRNA targeting the PRDM14 gene.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein one of the drugs is a dicer substrate (ds) siRNA targeting the PRDM14 gene.
  • ds dicer substrate
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein one of the drug molecules is an anti-cancer drug molecule.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein one of the drugs is doxorubicin.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein the binding peptide specifically binds to a cancer cell.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein the binding peptide specifically binds to a breast cancer cell.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein the binding peptide specifically binds to a MCF-7 breast cancer cell.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein the landscape phage protein assembly is a filamentous bacteriophage protein assembly.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein the landscape phage protein assembly is a filamentous bacteriophage protein assembly and wherein the filamentous bacteriophage protein assembly displays the binding peptide in a pVIII major coat protein.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein the zeta potential of the targeted drug delivery nanocarrier is between about -25 mV and about -50 mV.
  • a targeted drug delivery nanocarrier comprising a plurality of amphipathic molecules; a targeting landscape phage protein assembly; and one or more drug molecules; wherein the amphipathic molecules form a carrier particle having one or more drug molecules contained therein and the targeting landscape phage protein assembly is complexed to the carrier particle and wherein the targeting landscape phage protein assembly displays a binding peptide previously selected to specifically and selectively bind to a selected cancer cell, wherein the zeta potential of the targeted drug delivery nanocarrier is between about -26 mV and about -47 mV.

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Abstract

Une nouvelle entité chimique médicale est créée si un nanocarrier est combiné avec un agent de modulation de gènes ou de protéines PRDM. La présente invention concerne un agent thérapeutique comprenant un nanocarrier étiqueté avec un ou plusieurs fragments ciblant des cellules ou tissus, pouvant fournir une charge thérapeutique qui module l'activité d'un ou de plusieurs gènes ou protéines ou de gènes ou protéines associés (par exemple le gène ou la protéine PRDM14). Les nanocarriers incorporant la charge (par exemple un agent de modulation de gènes ou de protéines PRDM) et les fragments ciblants sont assemblés. Le composé est ensuite administré à un animal (par exemple un être humain) atteint d'une affection ou d'une maladie associée au gène ou à la protéine ou à une autre substance endogène contribuant à l'évolution de la maladie (par exemple cancer, maladie proliférante ou autre trouble génétique) de manière à entraîner un effet thérapeutique. AA Peptide ciblant BB Double couche lipidique SiRNA siARN
PCT/US2010/053575 2009-10-21 2010-10-21 Agents thérapeutiques de modulation ciblée de gènes ou protéines prdm WO2011050178A2 (fr)

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WO2011140365A1 (fr) * 2010-05-05 2011-11-10 Auburn University Particules cibles comprenant des protéines de fusion de phage d'environnement et un acide nucléique hétérologue
WO2016084979A1 (fr) * 2014-11-28 2016-06-02 株式会社クオリーメン Dendrimère de carbosilane et support et pouvant être agrégé obtenu en utilisant ledit dendrimère pour système de distribution de médicament
WO2017094792A1 (fr) * 2015-11-30 2017-06-08 株式会社Ｑｕａｒｒｙｍｅｎ＆Ｃｏ． Enveloppe ciblée pour utilisation dans un système d'administration de médicament utilisant un dendrimère de carbosilane
WO2021007465A3 (fr) * 2019-07-09 2021-02-18 Lonnie Bookbinder Traitement du cancer faisant appel à des formulations pharmaceutiques d'parni ciblées pour réguler à la baisse l'expression de la protéine prdm14
CN113616593A (zh) * 2021-08-23 2021-11-09 嘉兴学院 一种纳米靶向聚合物胶束在制备靶向给药系统中的应用
US11510987B2 (en) * 2016-12-01 2022-11-29 Saitama University Endocytosis enhancer for drug delivery system
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EP1940355B1 (fr) * 2005-09-30 2012-12-19 Auburn University Nanovecteurs à libération d'un principe actif contenant un ensemble des proteines cibles de bacteriophages d'environnement

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WO2011140365A1 (fr) * 2010-05-05 2011-11-10 Auburn University Particules cibles comprenant des protéines de fusion de phage d'environnement et un acide nucléique hétérologue
US9226972B2 (en) 2010-05-05 2016-01-05 Auburn University Targeted particles comprising landscape phage fusion proteins and heterologous nucleic acid
WO2016084979A1 (fr) * 2014-11-28 2016-06-02 株式会社クオリーメン Dendrimère de carbosilane et support et pouvant être agrégé obtenu en utilisant ledit dendrimère pour système de distribution de médicament
CN107206096B (zh) * 2014-11-28 2024-04-02 株式会社Cysay 碳硅烷树枝状体及用于药物递送系统的使用该树枝状体获得的可聚集载体
CN107206096A (zh) * 2014-11-28 2017-09-26 石匠株式会社 碳硅烷树枝状体及用于药物递送系统的使用该树枝状体获得的可聚集载体
JPWO2016084979A1 (ja) * 2014-11-28 2017-11-09 株式会社Ｑｕａｒｒｙｍｅｎ＆Ｃｏ． カルボシランデンドリマー、そのデンドリマーを用いた薬物送達システム用凝集性担体
US10918726B2 (en) 2014-11-28 2021-02-16 Quarrymen & Co. Inc. Carbosilane dendrimer and aggregatable carrier obtained using said dendrimer for drug delivery system
US10772973B2 (en) 2015-11-30 2020-09-15 Quarrymen & Co. Inc. Targeted shell for use in drug delivery system utilizing carbosilane dendrimer
WO2017094792A1 (fr) * 2015-11-30 2017-06-08 株式会社Ｑｕａｒｒｙｍｅｎ＆Ｃｏ． Enveloppe ciblée pour utilisation dans un système d'administration de médicament utilisant un dendrimère de carbosilane
JPWO2017204337A1 (ja) * 2016-05-27 2019-04-04 国立大学法人埼玉大学 カルボシランデンドリマーを用いた標的組織特異的送達型ドラッグデリバリーシステム用カプセル
WO2017204337A1 (fr) * 2016-05-27 2017-11-30 国立大学法人埼玉大学 Capsule pour systèmes d'administration de médicament du type à administration spécifique au tissu cible utilisant un dendrimère de carbosilane
US11160877B2 (en) 2016-05-27 2021-11-02 Saitama University Capsule for drug delivery systems of targeted tissue-specific delivery type using carbosilane dendrimer
US11510987B2 (en) * 2016-12-01 2022-11-29 Saitama University Endocytosis enhancer for drug delivery system
WO2021007465A3 (fr) * 2019-07-09 2021-02-18 Lonnie Bookbinder Traitement du cancer faisant appel à des formulations pharmaceutiques d'parni ciblées pour réguler à la baisse l'expression de la protéine prdm14
CN114727958A (zh) * 2019-07-09 2022-07-08 阿莱兹精准医疗公司 使用靶向的sirna药物制剂下调prdm14蛋白质的表达的癌症治疗
CN114727958B (zh) * 2019-07-09 2024-02-09 阿莱兹精准医疗公司 使用靶向的sirna药物制剂下调prdm14蛋白质的表达的癌症治疗
EP3996684A4 (fr) * 2019-07-09 2024-06-19 Ariz Prec Medicine Inc Traitement du cancer faisant appel à des formulations pharmaceutiques d'parni ciblées pour réguler à la baisse l'expression de la protéine prdm14
US12012597B2 (en) 2020-05-14 2024-06-18 Ariz Precision Medicine, Inc. Cancer treatment using siRNA to modulate expression of PRDM2/RIZ protein
CN113616593A (zh) * 2021-08-23 2021-11-09 嘉兴学院 一种纳米靶向聚合物胶束在制备靶向给药系统中的应用

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