Cross-Reference to Related Application

This application claims benefit of U.S. provisional patent application number 61/253,532, filed Oct. 21, 2009, the entire contents of which are incorporated herein by reference.

Background of the Invention

Synthetic small interfering fragments of RNA (siRNA) are known to inhibit specific protein expression by suppressing target genes at the mRNA level by a mechanism called RNA interference (RNAi). 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. Many siRNA-delivery systems, including cationic polymers and lipids, possess inherent deficiencies associated with target cell-specific gene inhibition and biocompatibility. Pegylated liposomes have been used previously as vehicles for delivery of siRNA to a site of disease.

To further increase tumor selectivity of the siRNA drugs, control unloading of their cargo within tumors, and increase their therapeutic efficacy, the liposomal carriers of siRNA can be specifically targeted to tumor cells by conjugation with targeting ligands. For example, 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). Yao Xue Xue Bao 41, 142-48). It was shown that liposomes bearing a synthetic arginine-rich CPP are stable and can efficiently transfect lung tumor cells in vitro. Taken together, 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.

The integration of phage display technology with nanocarrier-based drug delivery platforms is emerging as one new approach to deliver RNAi products. 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). Initially, 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).

Later, a sequence encoding a foreign peptide was spliced in-frame into the gene pVIII encoding the major coat protein, leading to the "landscape" fashion of phage display allowing thousands copies of guest peptide fused to the phage coat protein to cover the whole virion surface

(Ilyichev et al. (1989) Doklady Biochemistry (Proc. Acad. Sci. USSR) - Engl. Tr. 307, 196-198). 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).

The use of landscape phage fusion protein has been proposed as targeting ligands for

drug-loaded pharmaceutical nanocarriers, such as liposomes (Brigati et al. (2008) Curr. Prot. Protein Sci. , chapter 18, units 18, 19; Jayanna et al. (2009) Nanomedicine 5, 83-89). To overcome the drawbacks of the chemical modification of nanocarriers with cancer-selective peptides, the use of intact preselected phage fused coat proteins for the integration with drug-loaded liposomes has been explored (US 2007 / 0077291). This new approach is justified by well-documented propensity of the phage major coat protein to reside in the bacterial internal membrane during phage infection, and its ability to spontaneously insert into bacterial membrane, without the help of the translocation machinery (Broome-Smith et al. (1994) Mol. Membr. Biol. 11, 3-8). Moreover, data are available indicating the ability of the protein to spontaneously integrate into the lipid bilayer of liposomes (Soekarjo et al. (1996) Biochemistry 35, 1232-41).

Based on these findings, it has been reasoned that 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). Thus, 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. Brief Summary of the Invention

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. As an example, 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.

Direct and fast screening for tumor-targeted peptides via phage display technology offers a promising approach to the targeting of various pharmaceutical agents. However, 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).

For example, for targeting of 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. Thus, 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.

As a result of 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. In an embodiment of the present invention, 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.

Brief Description of the Figures

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. B) Relative expression of the gene normalized against GAPDH using the Kodak ID image analysis software.

Figure 3. Optimization of RT-PCR A) temperature gradient of annealing temperature from 59°C to 66°C. B) Different concentration of RNA from 10 ng to 150 ng in the RT-PCR reaction, C) temperature gradient of extension temperature from 62°C to 72°C. Left lanes show DNA markers.

Figure 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) for 10-20 min at RT and then the mixture of 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.

Figure 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. Selectivity of VEEGGYIAA phage towards breast cancer cells MCF-7 in comparison with normal breast cells MCF-IOA and hepatocellular carcinoma HepG2. Selectivity is based on the interaction of phage guest peptides to receptors over-expressed on target cells in comparison with other cells (non-neoplastic breast epithelial cells, MCF-IOA and

hepatocellular carcinoma cells, HepG2) and serum. This was estimated as percentage phage recovery; output (cell-associated) phage to input phage. The unrelated phage bearing the peptide VPEGAFSS was the control. B. Mode of interaction of VEEGGYIAA 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 7. Chromatographic profile of DMPGTVLP-fusion major coat protein.

Figure 8. left, Mean size 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; and 6. siRNA-phage

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.

Figure 9A, B. left and right. RT-PCR analysis of PRDM14 gene transcription after 72 h transfection of cells, obtained from two different passages, with siRNA-liposome preparations. 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. A: 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. 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+S.D. (n = 2 or 3).

Figure 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

(VEEGGYIAA or DMPGVTLP correspondingly)-targeted liposomes (40 nM) or scrambled siRNA-peptide 1 or 2 (VEEGGYIAA or DMPGTVLP)-targeted liposomes (40nM) and incubated for 48 and 72 h. A. 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. 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).

Figure 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. A. 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. B. Western blot band intensities were quantified using Image J software (NIH). All data represent the mean+SD (n = 2).

Figure 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.

Detailed Description of the Invention

In embodiments of the present invention, 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. As an example, 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

(Nishikawa et al. (2007) Cancer Res. 67, 9649-57) that 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

over-expression has been proposed as a biomarker of cancer in breast tissue.

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. GATA3 activation by

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. Among the other genes that are down-regulated is 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). Also inhibited is 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.

Direct and fast screening for tumor-targeted peptides via phage display technology provides a promising approach to advance the targeting of various pharmaceutical agents. However, the traditional phage display approach requires chemical synthesis of identified tumor- selective peptides and their conjugation with pharmaceuticals or pharmaceutical nanocarriers (such as liposomes). For example, for targeting of 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.

In embodiments of the present invention, we developed 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. The

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). Thus, 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.

That targeting of siRNA-loaded liposomes with cancer-specific phage proteins can increase their down-regulating activity towards an example target gene, PRDM14, has been demonstrated in the described experiments. 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. In contrast to the complex and poorly controllable conjugation procedures used for coupling of tumor-specific peptides and mAbs, 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). Furthermore, techniques for polyvalent phage display in the major coat protein pVIII for construction of large

(> 10 ⁹ clones) 8- and 9-mer landscape libraries have been developed. They rely on splicing of the corresponding degenerate coding sequence into the beginning of the pVIII coat-protein gene, replacing wild-type codons 2-4, or 2-5 (Kuzmicheva et al. (2009) Protein Eng. Des. Sel. 22, 9- 18; Petrenko et al. (1996) Protein Eng. 9, 797-801). Hundreds of targeted phage probes against prostate, glial and breast tumor cells were successfully selected from these libraries using advanced biopanning protocols (Brigati et al. (2008) Curr. Prot. Protein Sci., chapter 18, units 18, 19). As a result of siRNA-liposome targeting with the cancer cell-specific phage protein, we observed a much better uptake of doxorubicin into target cells upon their treatment with

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. 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, 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. In an embodiment of the present invention, 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.

Examples

Example 1. Materials and Methods

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) and phosphate saline buffer (lOx solution) was from Fisher Scientific (Fair Lawn, NJ). Dimethyl sulfoxide (DMSO), doxorubicin, and the three 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 NeutrAvidin™-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.

Cell lines were obtained from the American Type Culture Collection (ATCC, Manassas, VA). The MCF-7 human breast adenocarcinoma (HTB 22) cells were used as target cells. MCF-IOA (CRL-10317) cells, non-tumori genie human epithelial cells, and HepG2 (HB-8065) cells, human hepatocellular carcinoma cells, were used as controls. WI-38 (CCL-75) cells, normal human lung fibroblasts, were used for depletion of the phage libraries. All cells were grown in media as recommended by ATCC and incubated at 37°C, 5% C0 ₂ .

Example 2. Design and synthesis of siR As

For silencing of the PRDM 14 gene, we used two sets of siRNAs:

1) designed previously by Nishikawa et al. {Cancer Res. 67, 9649-57 (2007)):

5 -CCAG UGAAGUGAAGACCUAtt-3' (siPRDM14F) and

5 -UAGGUCUUCACUUCACUGG tt-3' (siPRDM14R),

5 ' -GGACAAGGGCGAUAGGAAAtt-3 ' (siPRDM14-2F) and

5'-UUUCCUAUCGCCCUUGUCCtt-3' (siPRDM14-2R),

5'-GGGAAAAUCUUCUCAGAUCtt-3' (siPRDM14-3F) and

5 ' -GAUCUG AGAAGAUUUUCCCtt-3 ' (siPRDM 14-3R).

And

2)

5 ' - CCAGUGAAGUGAAGACCUACGGAga-3 ' (DsiPRDM14F) and

5 ' -UCUCCGUAGGUCUUCACUUCACUGGAC-3 ' (DsiPRDM 14R),

5 -GGACAAGGGCGAUAGGAAAUUUCcc-3 ' (DsiPRDM14-2F) and

5 ' -GGGAAAUUUCCUAUCGCCCUUGUCC AC-3 ' (DsiPRDM 14-2R)

5 -GGGAAAAUCUUCUC AGAUC AAGAaa-3 ' (DsiPRDM14-3F) and 5 ' -UUUCUUGAUCUGAGAAGAUUUUCCCCA-3 ' (DsiPRDM14-3R).

Lower case ga and tt depict deoxyribonucleotides and bolded letters show common nucleotide structures in two sets of siRNAs nucleotides. siRNAs were from IDT (for silencing

experiments), or Sigma (for preparation of targeted liposomes).

Example 3. Primers

In preliminary experiments (Fig. 2) we explored primers for RT-PCR of the PRDM14 gene with following structures:

Upper Primer (sense): GTGCGGTCCCGGGATGGCTCTAC (Tm 68°C)

Lower Primer (antisense): GGGGCGGTGGAATTAAAGTGTCAG (Tm 73°C)

However, using these primers we did not obtain consistent results: the bands in the agarose gels were very faint and sometimes not visible. We believe the problem was a significant difference in the melting temperatures (> 5°C) of the sense and antisense primers. Thus, using Vector-NTI software, we designed a new set of primers with very close melting temperatures:

Upper Primer (sense): TGGATGTCCTATGTCAACTGTGCC (Tm 65°C)

Lower Primer (antisense): GAGGCCGGTGCTTCTCATGA (Tm 66°C)

After optimization of RNA concentration, annealing and extension temperatures the results of the RT-PCR experiments were consistent (Fig 4) and these conditions were used in subsequent experiments.

Example 4. RNA isolation

RNA was isolated from MCF-7 cells using RNASTAT, followed by ethanol/chloroform precipitation. Briefly, 250 \L of RNA STAT-60 was added to cells grown in 6-well culture plates (Corning). Chloroform (40 was added to the lysates, and they were transferred to 1.5-mL microfuge tubes and vortexed vigorously. The homogenate was centrifuged (14,000 rpm, 15 min, 4°C). The top aqueous phase was transferred to new 1.5-mL microfuge tubes.

Isopropanol (250 was added to the tubes, the mixture was mixed gently, and kept at 20°C for 5-10 min. The mixture was centrifuged (14,000 rpm, 10 min, 4°C). The supernatant was carefully removed. Then, 250 \L of 75% ethanol was added to the remaining pellet. The tube was centrifuged (14,000 rpm, 10 min) and the supernatant was carefully removed. The pellet was air-dried for 10 min and redissolved in 25 μΤ of DEPC-water. RNA was characterized by UV spectroscopy and stored at -80°C.

Example 5. RT-PCR analysis

Reverse transcription of total RNA and cDNA amplification by PCR was carried out using 25 ng of total RNA in a 25-μΤ 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 μΜ. 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.

Example 6. Knockdown of PRDM 14

MCF-7 cells grown in a 75-cm flask were collected by routine trypsinization. For PRDM14 gene knockdown studies, 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. Three siRNA that target PRDM14 gene are described in Example 2, above. Briefly, 4.8 μΕ of each siRNA (106 nM) was mixed with 250 μΕ of Opti-MEM I medium in a 1.5-mL microfuge tube. Lipofectamine RNAiMAX was mixed gently before use by inverting and 6 μΐ _^ of the homogenized preparation was added to 250 μL· of Opti-MEM I medium and the composition was mixed gently by inverting. 250 μΐ _^ of Lipofectamine was added to the diluted 250 μL 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. The medium was changed every 24 h. After a 72-h incubation, RNA was isolated for RT-PCR analysis. For knockdown of the PRDM14 gene by siRNA-phage fusion protein-liposomes, 1.6 μL of siRNA-VEEGGYIAA-liposome (50 μΜ), 1.6 μΐ, of siRNA-DMPGTVLP-liposomes (50 μΜ) or 0.53 μΐ, of siRNA-liposomes (150 μΜ) 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. The plates were processed and RNA was isolated as described above.

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;

40 nM), siRNA-lipofectamine, or scrambled siRNA-lipofectamine (40 nM) for 48 h. At 48 h, cells were lysed with 70 μΕ of RIPA buffer (Sigma, #0278) containing protease inhibitor cocktail (7 μΕ) and PMSF (2 mM final concentration). The protein concentration in whole cell lysate was measured by the Biorad DC protein assay. 15 μg 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. In each round, 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.

Example 9. Phage specificity and selectivity

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 μΤ 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 μΤ washing buffer for 5 min, eight times. Then, cells were treated with 25 μΤ 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. To determine a role of cell metabolism in the association of the phage with live cells, 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. In the "cool" experiment, the cells were incubated with 100 μΤ serum-free medium at room temperature for 1 h and phage clone (~10 ⁶ cfu in 100 μΤ 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 μΤ 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 μΤ neutralizing buffer (1 M Tris, pH 9.1). Wells were additionally washed twice with 25 μΤ washing buffer for 5 min per wash and post-elution washings were collected (fractions PEW-1 and PEW-2). To retrieve internalizing phages, wells were treated with 25 μΤ lysis buffer (2.5% CHAPS) for 10 min on a rocker. The eluate, PEW-1, PEW-2, and lysate fractions were titered in E. coli (K91 BlueKan) and phage recovery was calculated as the ratio of input phage to output phage. The procedure was modified by carrying out the incubation of cells with phage at 37 °C instead of room temperature to study the effect of temperature on the binding and internalization of phages. In another experiment, the incubation was carried out at 37 °C in medium supplemented with serum. The blocking buffer was also prepared from serum-containing medium. Example 11. Preparation and purification of the pVIII coat fusion protein

Landscape phage harboring breast cancer cell-specific peptide DMPGTVLP was selected from the 8-mer landscape library f8/8 (Petrenko et al. (1996) Protein Eng. 9, 797-801 ; Petrenko and Smith (2000) Protein Eng. 13, 589-92) using biopanning against human mammary

adenocarcinoma cells MCF-7 (Fagbohun et al. (2008) Nanotech. 2, 461-464). 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 μΐ _^ 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). The concentration of the protein was determined spectrophotometrically, using the formula: absorbance unit, ΑΙ½ο = 0.7 mg/mL, using the PROTEAN program (DNA STAR Inc., Madison, Wisconsin).

Example 12. Liposomal formulations: Phage-liposome formulation

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.

Example 13. siRNA-liposome formulation

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.

Example 14. siRNA-phage-liposome formulation

To make siRNA -phage-liposomes, the siRNA-liposomes and phage-liposomes were incubated in a 1 :2 volume ratio (50 μΕ siRNA-liposomes in 100 μΕ phage-liposomes) overnight at 4°C.

Example 15. Doxorubicin-loaded siRNA-liposome formulation

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. Then, 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.

Example 16. Empty and doxorubicin-loaded siRNA-phage-liposome formulations

To make empty and doxorubicin-loaded siRNA-phage-liposomes, the siRNA-liposomes and phage-liposomes were incubated at a 1:2 volume ratio (50 μΤ siRNA-liposomes in 100 μΤ phage-liposomes) overnight at 4°C.

Example 17. Doxorubicin loading determination

Doxorubicin (DOXO) 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 μg/mL).

Example 18. Size distribution and zeta potential (ζ) analysis

All formulations were characterized by size, size distribution, and zeta potential (ζ) using dynamic light scattering (DLS) on a Zeta Plus instrument (Brookhaven Instrument Corporation, Holtsville, NY) and Zeta Phase Analysis Light Scattering (PALS) with an ultrasensitive zeta (ζ) potential analyzer instrument (Brookhaven Instruments). Then, 5 μΐ _^ of each liposome suspension was diluted to 1 mL and then analyzed for size distribution; for the zeta potential, each sample was diluted in KC1 1 mM (5 μυΐ.5 mL). Example 19. PicoGreen fluorescent assay

To check the amount of free siRNA in solution, a fluorescent assay was used, based on the interaction between the PicoGreen probe and siRNA. The fluorescent probe PicoGreen is an intercalating agent that reacts with siRNA. Fluorescence intensity is proportional to the amount of the siRNA. Only free siRNA is available and can interact with the probe. The higher the amount of siRNA reacting with the intercalating agent, the higher is the resulting fluorescence. 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. 1 μΤ of siRNA-Phage EL-7-liposomes and siRNA-Phage 28-4-liposomes diluted in 10 μΕ nuclease-free water was incubated with 990 μΕ 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. As controls, 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

fluorescence. The siRNA associated with liposomes is shielded and not accessible to the probe. The free siRNA was calculated according to the following formula: % siRNA in solution = (Picogreen fluorescence liposomes/Picogreen fluorescence free).

Example 20. Viability of human breast cancer cell MCF-7

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

100 μΕ/well of fresh DMEM complete medium and incubated with different dilutions of DOXO in phage EL-V-siRNA-liposomes, phage 28-4-siRNA-liposomes, or phage-free/siRNA-free liposomes. As a positive control, untreated cells and phage-free/siRNA-free/empty-liposomes were used. The concentrations incubated are shown in Table 1. After an additional 72 h at 37 °C and 5% C0 ₂ , the cells were washed with DMEM, and incubated at 37°C with 20 μΐ. of MTT, diluted in 100 μΤ of medium for about 3 h. 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

Human mammary cell lines, breast carcinoma MCF-7, breast ductal carcinoma ZR-75-1, and normal breast cells MCF-10, were used. To analyze activity of the PRDM-14 gene in these cells, 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). In a parallel experiment, the activity of the PRDM14 gene was studied using Western blotting of cellular proteins (Fig. 9E).

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 μg/mL, and an extension temperature 68°C were determined to be optimal and were used in the subsequent experiments.

Example 23. siRNA concentration

In some experiments we used the mixture of 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.

The silencing effect of "dicer substrate siRNA" (dsRNA; IDT) in comparison with the siRNAs suggested by Nishikawa et al. was also evaluated. dsRNA has been shown to be more potent than siRNA in studies by Kim and colleagues (FEBS Lett. (2005) 579, 5974-81). In these experiments, siRNA was more or equally potent as dsRNA (Fig. 4). To our knowledge, activity of dsRNA towards PRDM14 gene has not been studied previously.

Example 24. Time points for siRNA treatment

Knockdown of PRDM-14 gene was analyzed at 48 and 72 h after treatment of the cells with preparation of siRNA. The concentration of lipofectamine was optimized for siRNA delivery to avoid its toxic effects. As a result of analyzing PRDM14 gene expression and its silencing, conditions were established that allowed the study of the effects of different targeted liposomal siRNA preparations on the silencing of the PRDM14 gene. Example 25. Selection of phages specific for breast tumor cells MCF-7

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. However, 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). For example, for targeting of 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.

To simplify the procedure and exclude any such chemical modifications, the use in 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. Thus, during infection of the host Escherichia coli, the positively charged C-terminus of the protein spans the bacterial cytoplasmic membrane, leaving the negatively charged N-terminus in the periplasm. In an infected cell, the protein is synthesized as a water- soluble cytoplasmic precursor, which contains an additional leader sequence of 23 residues at its N-terminus. When this protein is inserted into the membrane, the leader sequence is cleaved off by a leader peptidase. Later, during phage assembly, the newly synthesized proteins are transferred from the membrane into the coat of the emerging phage. Thus, the major coat protein can change its conformation to accommodate the various distinctly different forms during phage infection and assembly. This structural flexibility of 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. Thus, 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.

Example 26. Phages DMPGTVLP and VEEGGYIAA

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. The fusion protein carrying tumor-cell binding peptides DMPGTVLP and VEEGGYIAA, used in this work for targeting of siRNA-loaded liposomes, inherited the major structural features of the wild-type major coat protein. 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.

Example 27. Phage fusion coat protein-targeted 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. To explore the spontaneous insertion of MCF-7-specific pVIII coat fusion proteins into preformed liposomes, the following procedure was used: 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.

Example 28. Phage characterization

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. To determine the localization of the phage in the 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. It was found that 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.

Binding to the cells increased with temperature from 20 to 37°C in the presence of growth factors in serum, suggesting an active role of cellular metabolism in the binding of the phage. Bound phage was located on the surface of the target cells and was not seen in internal cellular compartments (Fig. 5, right).

Another 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).

Two phage clones, VEEGGYIAA and DMPGTVLP, for the preparation of breast

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 (ζ, 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

DMPGTVLP liposomes, it was confirmed that the majority of the siRNA was shielded in liposomes and protected from the nucleases. Free siRNA constituted about 4% and 9% of the total amount in siRNA-phage VEEGGYIAA-liposomes and siRNA-phage

DMPGTVLP-liposomes, respectively.

Both phage-liposome formulations (with phage VEEGGYIAA and phage DMPGTVLP) showed a mean size comparable to their starting plain formulation (that did not contain phage; Fig. 8, left), but a quite different ζ because of the incorporated proteins: from a positive ζ for plain liposomes (+49.3 mV) to negative (-46.6 mV and -42.3 mV for phages VEEGGYIAA and DMPGTVLP, respectively; Fig. 8, right).

This inversion of ζ highlights the incorporation of the proteins in the lipid structure. The size and ζ 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 ζ 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 ζ did not change strongly among the doxorubicin-containing formulations. It was -34.6+10.3, -26.7+2.0, and -34.3+3.6 mV for phage-free / siRNA-free / DOXO, phage-DMPGTVLP-siRNA-DOXO, and phage- VEEGGYIAA-siRNA-DOXO liposomes, respectively, suggesting a contribution to the net negative charge also due to doxorubicin. The composition of the doxorubicin-loaded liposomes is shown in the Table 1.

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)-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.

In the first set of experiments (Fig. 9A, B), we compared the silencing effect of targeted and non-targeted siRNA-liposomes using two different passages of MCF-7 cell cultures after 72 h of treatment. In both experiments, we observed statistically significant down-regulation of

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).

In another set of experiments (Fig. 9C, D), we compared the silencing effect of siRNA preparations at different time points (48 vs. 72 h). In these experiments, in addition to previously used targeted gene-specific siRNA, we also tested targeted non-specific siRNA. We found that the silencing effect of the targeted specific siRNA could be observed after 48 h and persisted till 72 h. As in the previous set of experiments (Fig. 9A, B), a more profound effect was observed when gene-specific siRNA was targeted by peptide 2 (DMPGTVLP).

In a parallel experiment (Fig. 9E), using MCF-7 cells from the same passage that has been used in the experiment described above (Fig. 9C, D), we analyzed the expression of PRDM14 protein by Western blotting. In addition to the siRNA-liposome preparation, we used a

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.

Consistent with their high selectivity towards the breast cancer cell line, 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.

Example 32. Cytotoxicity of the preparations

Doxorubicin (DOXO) 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. We next examined whether the use of DOXO and siRNA together demonstrated a synergistic effect when used in the same formulation, so that the administered dose of DOXO could be reduced to a less toxic level.

An in vitro cell viability test was performed using MCF-7 human breast cancer cells. Assay results were evaluated using the MTT test, according to a routine protocol, using doxorubicin- loaded liposomes of the compositions (lipids, siRNA, phage proteins and doxorubicin) shown in Table 1. The table lists the amount of each component for each doxorubicin (DOXO) concentration. For example for a DOXO concentration of 64 ng/mL the other components in the same formulations are: 10.3 μg/mL of total lipid, 1330 ng/mL (100 nM) of siRNA, and

68.7 ng/mL of phage proteins. For comparison with the silencing experiments, molar concentrations of siRNA that correspond to the previously shown mass concentrations of siRNA in ng/mL are shown. For example, the concentration 665 ng/mL (50 nM) approximately corresponds to the concentration of 40 nM siRNA used in the silencing experiments (Fig. 9). The formulations used are similar to that of Doxil, but there are also additional lipid components such as DOTAP.

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.

After 72 h incubation, cell survival was analyzed. This showed that both phage-siRNA-DOXO formulations possessed higher cytotoxic efficacy than the phage-free/siRNA-free-DOXO liposomes. Moreover, the 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). The corresponding plain formulations (made only of lipids) did not show any cytotoxic effect. It was found also that at the maximum concentration of doxorubicin, control cells (normal and cancer cells: cardiomyocytes, fibroblasts, prostate cancer) die, as do MCF-7 cells (i.e., 60-70%), but the phage protein did not increase the level of death. siRNA itself (in phage-liposomes) caused very little cell death (within experimental error).

Embodiments of the present invention include the following:

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.

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.

All documents, books, manuals, papers, patents, published patent applications, guides, abstracts and other references cited herein are incorporated by reference in their entirety. Other embodiments of the invention will be apparent to those skilled in the art from consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and examples be considered as exemplary only, with the true scope and spirit of the invention being indicated by the following claims.