Methods and compositions for treating cancer

Abstract

Described herein are methods and compositions relating to the treatment of cancer (e.g., breast cancer) using, for example, aptamer-siRNA chimera molecules.

Description

[0001] This application is a divisional application of patent application No. 201580059104.2, filed on August 28, 2015, with the invention name “Methods and compositions for treating cancer”.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS

[0003] This application claims the benefit under 35 USC §119(e) of U.S. Provisional Application No. 62 / 043,803, filed August 29, 2014, the contents of which are incorporated herein by reference in their entirety.

[0004] Government support

[0005] This invention was made with Federal funds awarded by the U.S. Department of Defense under Grant No. W81 XWH-09-1-0058. The U.S. Government has certain rights in this invention.

[0006] Sequence Listing

[0007] This application includes a sequence listing, which has been submitted electronically in ASCII format and is incorporated herein by reference in its entirety. The ASCII copy was created on August 28, 2015, is named 701039-082401-PCT_SL.txt, and is 8,984 bytes in size. Technical Field

[0008] The technology described herein relates to chimeric molecules comprising an EpCAM binding molecule and an inhibitory nucleic acid, and methods of using such combinations to treat cancer (eg, epithelial cancer). Background Art

[0009] RNA interference (RNAi) has been explored for its medical use in reducing gene expression in the liver. However, the liver is uniquely susceptible to transfection with RNAi molecules. In other tissues, small RNA delivery and the resulting gene knockdown are consistently inefficient and ultimately ineffective. In particular, delivery barriers are a major obstacle to using RNAi to treat cancer. Summary of the Invention

[0010] As described herein, the inventors have developed novel chimeric aptamer-siRNA molecules (AsiCs) that target cancer cell markers to direct siRNA specifically to cancer cells, increasing delivery potency and therapeutic effectiveness while reducing the potential for side effects.

[0011] In one aspect, described herein are chimeric molecules comprising an aptamer domain that binds a cancer marker and an inhibitory nucleic acid domain. In some embodiments, the cancer marker is EpCAM or EphA2. In some embodiments, the inhibitory nucleic acid specifically binds to a gene product that is upregulated in cancer cells. In some embodiments, the inhibitory nucleic acid inhibits the expression of a gene selected from the group consisting of: Plk1, MCL1, EphA2, PsmA2, MSI1, BMI1, XBP1, PRPF8, PFPF38A, RBM22, USP39, RAN, NUP205, and NDC80. In some embodiments, the cancer marker is EpCAM and the inhibitory nucleic acid domain inhibits the expression of Plk1.

[0012] In some embodiments, the molecule is an aptamer-siRNA chimera (AsiC). In some embodiments, the aptamer domain that binds a cancer marker comprises the sequence of SEQ ID NO: 33. In some embodiments, the aptamer domain that binds a cancer marker consists essentially of the sequence of SEQ ID NO: 33. In some embodiments, the inhibitory nucleic acid domain comprises the sequence of SEQ ID NO: 2. In some embodiments, the inhibitory nucleic acid domain consists essentially of the sequence of SEQ ID NO: 2. In some embodiments, the molecule comprises the sequence of one of SEQ ID NO: 1 to SEQ ID NO: 3. In some embodiments, the molecule consists essentially of the sequence of one of SEQ ID NO: 1 to SEQ ID NO: 3.

[0013] In some embodiments, the 3' end of the molecule comprises dTdT. In some embodiments, the molecule comprises at least one 2'-F pyrimidine.

[0014] In one aspect, described herein are pharmaceutical compositions comprising a chimeric molecule as described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition comprises at least two chimeric molecules as described herein, wherein the chimeric molecules have different aptamer domains and / or inhibitory nucleic acid domains. In some embodiments, the different aptamers or inhibitory nucleic acid domains recognize different targets. In some embodiments, the different aptamers or inhibitory nucleic acid domains have multiple sequences and recognize the same target.

[0015] In one aspect, described herein are methods of treating cancer comprising administering a chimeric molecule and / or composition as described herein. In some embodiments, the cancer is an epithelial cancer or a breast cancer. In some embodiments, the breast cancer is a triple-negative breast cancer. In some embodiments, the administration is subcutaneous. In some embodiments, an additional cancer therapy is further administered to the subject. In some embodiments, the cancer therapy is paclitaxel. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figures 1A to 1H The EpCAM aptamer was shown to specifically target basal A (BasalA) breast cancer cells. Design of EpCAM-AsiC comprising the EpCAM aptamer and PLK1 siRNA (sense strand disclosed as SEQ ID NO: 1 and antisense strand disclosed as SEQ ID NO: 2) ( Figure 1C Compared with normal breast epithelial cell line (BPE), epithelial breast cancer cell line (BPLER) overexpresses EpCAM protein ( Figure 1A to Figure 1B EpCAM-AsiC targeting GFP was labeled with Cy3 or Alexa647 at the 3' end of the antisense siRNA strand and incubated with BPLER and BPE cells. Uptake was assessed by flow cytometry after 24 hours ( Figure 1D ). Data are representative of 3 independent experiments. Cy3- and Alexa647-labeled EpCAM-AsiC were taken up by MB468 and BPLER (EpCAM+ cells), respectively, but not by BPE (EpCAM-). The MFI of each peak is shown. To test gene silencing, BPLER and BPE were treated with EpCAM-AsiC targeting GFP (4 μM) and compared with a transfection control using Dharmafect and GFP-siRNA (100 nM). Knockdown was assessed by flow cytometry after 72 hours of incubation. The control was a mock and only Dharmafect treated (lipid) (n=4) ( Figure 1D EpCAM-AsiC targeting AKT1 selectively knocked down AKT1 mRNA in basal A and luminal breast cancer cell lines ( Figure 1E ) and protein ( Figure 1F) expression, without knocking down AKT1 mRNA and protein expression in basal B cells or human fibroblasts (hFb). Transfection with siRNA targeting AKT1 induced gene knockdown in all cell lines, while treatment with EpCAM-AsiC targeting GFP did not affect AKT1 mRNA and protein levels (*p<0.05, p<0.01). Figure 3 shows AKT1 protein and gene knockdown compared to the effect of EpCAM-AsiC on siRNA transfection. EpCAM-AsiC-induced knockdown correlated with EpCAM expression ( Figures 1E to 1H (n=3, normalized to the mean ± SEM of the mock, *P<0.05, **P<0.01, two-tailed t test).

[0017] Figures 2A to 2E The results showed that EpCAM-AsiC targeting PLK1 specifically inhibited cell proliferation in basal A breast cancer cells. Using the cell-titer-glo assay (CTG), the effect of EpCAM-AsiC targeting PLK1 on cell proliferation was tested in 10 breast cancer cell lines representing basal A, basal B, and luminal cell lines. EpCAM-AsiC targeting PLK1 reduced cell proliferation in both basal A and luminal cell lines, but had no effect on basal B cells ( Figure 2A 、 Figure 2C A correlation was observed between EpCAM expression levels and cell viability ( Figure 2B Basal A (EpCAM+GFP-) cells were co-cultured with BPE (EpCAM-GFP+) cells and treated with or without EpCAM-AsiC targeting PLK1. Untreated co-cultures showed similar cell ratios as follows: after treatment with EpCAM-AsiC targeting PLK1, EpCAM+ cells decreased and EpCAM- cells increased. Representative flow cytometry images ( Figure 2D ), the experimental quantitative analysis of the ratio of GFP+ / GFP- cells in four different cell lines ( Figure 2E ). (n=4, *p<0.05, p<0.01).

[0018] Figures 3A to 3D The results showed that human TNBC tissues specifically take up Cy3-EpCAM aptamers. Experimental design: Cy3-EpCAM-AsiC targeting GFP, Alexa647-siRNA-GFP, or Alexa647-chol-siRNA-GFP (2 μM each) were added to breast cancer and control explants and incubated for 24 hours. The tissues were then digested with collagenase to single-cell suspensions and analyzed by flow cytometry. Figure 3A Tumor biopsies overexpressed EpCAM and cytokeratin, epithelial cell markers ( Figure 3B Representative histogram from one of three independent experiments showing that siRNA and chol-siRNA penetrate tumor and healthy tissue with similar efficacy, whereas EpCAM-AsiC is selectively taken up by tumor tissue biopsies but not by healthy control tissue samples ( Figure 3C The uptake experiments were repeated in tumors from three different patients, and each biopsy received was tested three times for each treatment group. Figure 3D (n=3, mock, gray EpCAM, red, *P<0.05, **P<0.005, t-test CD4-AsiC vs. mock treatment).

[0019] Figures 4A to 4C The results showed that EpCAM-AsiC targeting PLK1 specifically inhibited tumor initiation in basal A breast cancer cells. Colony assays of breast cancer cell lines were treated with EpCAM-AsiC targeting PLK1 or GFP (4 μM) or paclitaxel (100 nM) for 24 hours and cultured in drug-free medium for 8 days. Treatment with paclitaxel reduced colony formation in all cell lines, while treatment with EpCAM-AsiC targeting PLK1 abolished colony formation only in luminal (MCF7) and basal A (HCC1954) cells, and treatment with EpCAM-AsiC targeting GFP had no effect ( Figure 4A The experiment was repeated in 3 more cell lines and the results were reproducible ( Figure 4B Mammosphere formation assays showed similar results, with EpCAM-AsiC targeting PLK1 reducing sphere numbers only in basal A and luminal cells, but not in basal B cells ( Figure 4C MB468-luc cells were treated with either EpCAM-AsiC targeting GFP or PLK1 for 24 hours and injected subcutaneously into the flanks of nude mice. Mice were imaged every five days for 20 days. Untreated mice and mice treated with EpCAM-AsiC targeting GFP showed an increase in tumor initiation, while mice injected with cells pretreated with EpCAM-AsiC targeting PLK1 showed no tumor initiation.

[0020] Figures 5A to 5C This indicates that Alexa750-EpCAM-AsiC is selectively taken up into EpCAM+ tumors. Figure 5AThe experimental setup is depicted; nude mice were injected with MB468-luc (left flank) and MB231-luc-mCherry (right flank) cells and 5 days after injection, Alexa750 labeled GFP-targeted EpCAM-AsiC (0.5 mg / kg) was injected subcutaneously into the neck. Mice were imaged immediately after injection and again after 24 hours, 48 ​​hours and 5 days. Alexa750 labeled GFP-targeted EpCAM-AsiC co-localized with luciferase tumors in MB468-luc tumors (EpCAM+) but not in MB231-luc-mCherry (EpCAM-) tumors. Analysis of 7 mice demonstrated a significant increase in Alexa750 in MB468 (EpCAM+) tumors ( Figure 5B ). Figure 5C Graph depicting the Alexa750 uptake rate.

[0021] Figures 6A to 6B showed that EpCAM-AsiC targeting PLK1 specifically inhibited tumor growth in basal A breast cancer cells. Figure 6A MB231-luc-mCherry cells (5×10 5 ) or MB468-luc cells (5×10 6 ) nude mice were treated or not treated. Figure 6B MB468-luc tumors treated with EpCAM-AsiC targeting PLK1 decreased in size as early as 6 days after treatment and completely disappeared in many mice by day 14. Untreated EpCAM+ and EpCAM- tumors increased in size over 14 days.

[0022] Figure 7 EpCAM-AsiC was shown to be stable in human and mouse serum. eGFPEpCAM-AsiC synthesized using 2'-fluoro-pyrimidine, chemically stable cholesterol-conjugated eGFP siRNA (chol-siRNA), or unmodified eGFP siRNA were incubated with equal volumes of human or mouse serum. Aliquots were removed at regular intervals, resuspended in a gel containing loading buffer, and stored at -80°C before electrophoresis on a denaturing PAGE gel. The average intensity (± SEM) of the bands from two independent experiments quantified by densitometry after staining is shown.

[0023] Figures 8A to 8BInjection of EpCAM-AsiC showed that it did not stimulate innate immunity in mice.Mice were injected subcutaneously with eGFPEpCAM-AsiC (5 mg / kg, n=3) or intraperitoneally with poly(I:C) (5 mg / kg or 50 mg / kg, n=2 / dose). Figure 8A : IFNβ, IL-6, and IP-10 were assessed by multiplex immunoassay in serum samples collected at baseline and 6 and 16 hours after treatment. *p<0.05, **p<0.01, ***p<0.001 compared to baseline. Figure 8B : mRNA expression of IFN-induced genes and cytokines was assessed by qRT-PCR relative to gapdh in total splenocytes harvested 16 hours after treatment. **p<0.01 compared to untreated (NT, n=3).

[0024] Figure 9 A table of sequences is depicted (SEQ ID NO: 1 to SEQ ID NO: 2 and SEQ ID NO: 23 to SEQ ID NO: 32, respectively, in order of appearance).

[0025] FIG. 10A to FIG. 10B Aptamer-siRNA chimera (AsiC) is depicted. Figure 10A Depicts an image of AsiC (an aptamer covalently linked to one strand of siRNA) that specifically recognizes a cancer cell surface receptor, is internalized, and subsequently released into the cytosol, where it is processed like an endogenous pre-miRNA to knock down the target gene. Bars indicate two delivery hurdles: cellular uptake at the localization of Dicer and the RNA-induced silencing complex (RISC), and release from endosomes into the cytosol. Figure 10B Depicted is the design of EpCAM-AsiC targeting PLK1 (sense strand disclosed as SEQ ID NO: 1 and antisense strand disclosed as SEQ ID NO: 2).

[0026] 11A to 11D These results indicate that EpCAM-AsiC knockdown and antitumor effects are associated with EpCAM levels and inhibit epithelial breast tumor T-IC. Figures 11A to 11B :Representative experiments ( Figure 11A ) and AKT1 knockdown compared with lipid siRNA transfection and EpCAM-AsiC ( Figure 11B ). Figure 11C : The anti-proliferative effect of PLK1 knockdown by EpCAM-AsiC only occurs in EpCAM+ cell lines. Figure 11DPLK1 EpCAM-AsiC inhibited colony formation in luminal MCF and basal-A TNBC HCC1143 cells, but not in mesenchymal basal-B MB231 cells.

[0027] FIG. 12A to FIG. 12B Demonstrating identification of functional EphA2 aptamers. Figure 12A : Incubation of EphA2+ basal-B MB231 cells with EphA2 aptamer (EphA2apt) caused EphA2 degradation and transient reduction of active Akt (pAkt). Figure 12B : EphA2+ breast cancer cells incubated for 2 hours with EphA2apt (0 to 100 nM) but not a control non-binding aptamer (ctl), show reduced EphA2. Ephrin A was added as a positive control for EphA2 degradation.

[0028] 13A to 13C The results showed that EpCAM-AsiC knocked down GFP protein only in EpCAM+ cell lines, but not in immortalized mammary epithelial cell lines (BPE) or mesenchymal basal cells B TNBC or human fibroblasts. Figure 13A ) and AKT1mRNA( Figures 13B to 13C ). The transfected siRNA was nonspecific in its knockdown. *P<0.05.

[0029] Figure 14 Normal breast tissue and basal cell-A TNBC tumor biopsies from the same subjects were incubated with Cy3-labeled EpCAM-AsiC, and single-cell suspensions were analyzed for uptake by flow cytometry three days later. Naked siRNA was not taken up, while cholesterol-conjugated siRNA was equally taken up. However, EpCAM-AsiC was taken up specifically by tumors. Representative tissues are shown on the left.

[0030] Figures 15A to 15C In an in vitro assay of T-IC function, treatment of EpCAM+ (but not EpCAM-) breast cancer lines with PLK1 EpCAM-AsiC inhibited colony formation ( Figure 15A 、 Figure 15B ) and mammospheres ( Figure 15C )Function.

[0031] Figure 16 It was shown that ex vivo treatment of MB468 cells with PLK1 EpCAM-AsiC abolished their ability to form tumors in nude mice. Equal numbers of viable cells were implanted the day after treatment.

[0032] 17A to 17BThese results indicate that EpCAM-AsiC is selectively taken up into EpCAM+ tumors, but not EpCAM- and TNBC tumors. Figure 17A Depicts the experimental protocol. Figure 17B Depicted are the concentrations of EpCAM-AsiC in resected tumors at the time of sacrifice.

[0033] 18A to 18B PLK1 EpCAM-AsiC caused complete tumor regression of EpCAM+ TNBC xenografts but had no effect on EpCAM-basal-B xenografts. Figure 18A The experimental design is depicted. Luciferase activity imaging was performed sequentially in left and right flank tumors over a 2-week period. Figure 18B Graph depicting tumor size as a function of luciferase activity. In mice treated with PLK1 AsiC, all EpCAM+ tumors rapidly regressed, while the others continued to grow.

[0034] 19A to 19C The results showed that basal cell-dependent genes included four tri-snRNP spliceosome complex genes (PFPF8, PRPF38A, RBM22, USP39), two nuclear export genes (NUP205, RAN) and a centromere gene (NDC80). Figure 19A Depicted are cell viability normalized to control siRNA after 3 days of knockdown. Figure 19B Depicted are colony formation assessed by plating live cells 2 days after knockdown. Figure 19C Depicted is that caspase activation after 2 days of knockdown is specific to MB468 and does not occur in BPE cells.

[0035] Figure 20 Some possible designs for multimerizing EpCAM-AsiC to improve endocytosis are depicted. In these designs, the sense and antisense strands can be interchanged and the linker can be changed.

[0036] 21A to 21D It was demonstrated that the EpCAM aptamer specifically targets basal A breast cancer cells. Figure 21A Depicted is the design of EpCAM-AsiC containing the EpCAM aptamer and PLK1 siRNA (sense strand disclosed as SEQ ID NO: 1 and antisense strand disclosed as SEQ ID NO: 2). Figure 21BFigure depicts an epithelial breast cancer cell line (BPLER) that overexpresses EpCAM protein compared to a normal breast epithelial cell line (BPE). EpCAM-AsiC targeting GFP was labeled with Alexa647 or Cy3 at the 3' end of the antisense siRNA strand and incubated with BPLER and BPE cells. Uptake was assessed by flow cytometry after 24 hours ( Figure 21C Data are representative of three independent experiments. Cy3- and Alexa647-labeled EpCAM-AsiC were taken up by MB468 and BPLER (EpCAM+ cells), respectively, but not by BPE (EpCAM-). The MFI of each peak is shown (mock, gray). Figure 21D Depicts an experiment in which BPLER and BPE were treated with EpCAM-AsiC targeting GFP (4 μM) to measure gene silencing and compared to a control transfection using Dharmafect and GFP-siRNA (100 nM). Knockdown was assessed by flow cytometry 72 hours after incubation. Controls were mock and Dharmafect-only (lipid) treatment. (n=4).

[0037] Figure 22 Figure 2 shows that the EpCAM aptamer does not bind to mouse EpCAM. Mouse ESA (EpCAM) levels were determined using flow cytometry with the mCD326 antibody. 4T1 cells (epithelial mouse breast cancer cell line) showed high expression levels of EpCAM. RAW (mouse monocytic cell line) and MB468 (human basal A cell line) both showed an increase in EpCAM expression, but much smaller than that of 4T1 cells. The mouse mesenchymal carcinoma cell line (67NR) showed the smallest increase in EpCAM expression. Uptake experiments showed that the EpCAM-aptamer was not taken up by either 4T1 or 67NR cells.

[0038] Figure 23 Depicted is a graph demonstrating that EpCAM is overexpressed in basal A and luminal breast cancer cell lines, but not in basal B breast cancer cell lines. Representative FACS plots for eight different breast cancer cell lines tested for EpCAM expression levels by flow cytometry using hEpCAM antibodies. EpCAM is overexpressed in all basal A and luminal cell lines, but not in basal B cell lines (mock, shaded gray, EpCAM, black).

[0039] 24A to 24FEpCAM-AsiC specifically silences gene expression in basal A breast cancer cells. EpCAM-AsiC targeting AKT1 selectively knocks down AKT1 mRNA in basal A and luminal breast cancer cell lines, but not in basal B or human fibroblasts (hFb). Figure 24A ) and protein ( Figure 24B 、 Figure 24C ) expression. Transfection with siRNA targeting AKT1 induced gene knockdown in all cell lines, whereas treatment with EpCAM-AsiC targeting GFP did not affect AKT1 mRNA and protein levels (*p<0.05, p<0.01). Figure 3 compares the effect of EpCAM-AsiC on siRNA transfection and AKT1 protein and gene knockdown. EpCAM-AsiC-induced knockdown correlated with EpCAM expression ( Figure 24D 、 Figure 24E (n=3, normalized to the mean ± SEM of the mock; *P<0.05, **P<0.01, two-tailed t-test). Figure 24F The results of flow cytometry analysis are depicted.

[0040] Figures 25A to 25E These results indicate that human TNBC tissues specifically take up the Cy3-EpCAM aptamer. Figure 25A The experimental design is depicted; breast cancer and control explants were added with Cy3-EpCAM-AsiC targeting GFP, Alexa647-siRNA-GFP, or Alexa647-chol-siRNA-GFP (2 μM each) and incubated for 24 hours, after which the tissues were digested with collagenase into single-cell suspensions and analyzed by flow cytometry. Figure 25B Depicted are graphs demonstrating that tumor biopsies overexpress EpCAM and cytokeratin, epithelial cell markers. Figure 25C Depicted is a representative histogram from one of three independent experiments, showing that both siRNA and chol-siRNA penetrate tumor and healthy tissue with similar potency, while EpCAM-AsiC is selectively taken up by tumor tissue biopsies but not by healthy control tissue samples. The uptake experiment was repeated in tumors from three different patients, and each biopsy obtained was assayed three times for each treatment. Figure 25D Representative tumors are depicted. An overview of all three patients is given in Figure 25E (n=3, *P<0.05, **P<0.005, t-test CD4-AsiC vs. mock treatment).

[0041] Figure 26Depicted are graphs showing EpCAM-AsiC uptake by healthy and colon cancer biopsies. GFP-targeted Cy3-EpCAM-AsiC, Alexa647-siRNA-GFP, or Alexa647-chol-siRNA-GFP (2 μM each) were added to colon cancer and control explants and incubated for 24 hours. The tissues were then digested with collagenase to single-cell suspensions and analyzed by flow cytometry. Representative histograms show that EpCAM-AsiC, siRNA, and chol-siRNA penetrated tumor and healthy tissue with similar potency.

[0042] 27A to 27D The results showed that EpCAM-AsiC targeting PLK1 specifically inhibited cell proliferation in basal A breast cancer cells. Using a cell titer assay (CTG), the effect of EpCAM-AsiC targeting PLK1 on cell proliferation was tested in 10 breast cancer cell lines representing basal A, basal B, and luminal cell lines. EpCAM-AsiC targeting PLK1 reduced cell proliferation in basal A and luminal cell lines, but had no effect on basal B cells ( Figure 27A A correlation was observed between EpCAM expression levels and cell viability ( Figure 27B Basal A (EpCAM+GFP-) cells were co-cultured with BPE (EpCAM-GFP+) cells and treated or left untreated with EpCAM-AsiC targeting PLK1. Following treatment with EpCAM-AsiC targeting PLK1, the untreated co-cultures showed similar cell ratios, with a decrease in the ratio of EpCAM+ cells and an increase in the ratio of EpCAM- cells. Figure 27C Representative flow cytometry plots are depicted. Figure 27D Depicted is a quantitative graph of an experiment analyzing the ratio of GFP+ / GFP- cells in four different cell lines (n=4, *p<0.05, p<0.01).

[0043] Figure 28 Depicted is a graph demonstrating that specific reduction in cell viability in basal A breast cancer cell lines is PLK1-dependent. Ten different breast cancer cell lines representing basal A, basal B, and luminal cell types were treated with EpCAM-AsiC targeting PLK1 or with the EpCAM-aptamer alone and compared to untreated controls. None of the cell lines treated with the EpCAM-aptamer showed a reduction in cell viability, while the basal A and luminal cell lines showed a reduction in cell viability after treatment with the EpCAM-AsiC targeting PLK1.

[0044] Figures 29A to 29CThe results showed that EpCAM-AsiC targeting PLK1 specifically inhibited tumor initiation in basal A breast cancer cells. Colony assays of breast cancer cell lines were performed with EpCAM-AsiC targeting PLK1 or GFP (4 μM) or paclitaxel (100 nM) for 24 hours and cultured in drug-free medium for 8 days. Paclitaxel treatment reduced colony formation in all cell lines, while treatment with EpCAM-AsiC targeting PLK1 abolished colony formation only in luminal cells (MCF7) and basal A cells (HCC1954). Treatment with EpCAM-AsiC targeting GFP had no effect. Figure 29A The test results are depicted in the graph. Figure 29B As shown in the depicted figures, the experiment was repeated in three more cell lines and the results were reproducible. Figure 29C Figure 1 shows similar results in the sphere formation assay, with EpCAM-AsiC targeting PLK1 reducing the number of spheres only in basal A and luminal cells, but not in basal B cells. MB468-luc cells were treated with EpCAM-AsiC targeting GFP or PLK1 for 24 hours and injected subcutaneously into the flanks of nude mice. Mice were imaged every 5 days for 20 days. Untreated mice and mice treated with EpCAM-AsiC targeting GFP showed an increase in tumor initiation, while mice injected with cells pretreated with EpCAM-AsiC targeting PLK1 showed no tumor initiation.

[0045] FIG. 30A to FIG. 30B EpCAM AsiC was shown to be stable in human and mouse serum for 36 hours. EpCAM-AsiC targeting GFP, synthesized using 2'-fluoro-pyrimidine, chemically stabilized 21-mer cholesterol-conjugated GFP-siRNA (chol-siRNA), and unmodified 21-mer GFP-siRNA were added to 100 μl of human or mouse serum, each in 100 μl of PBS. 20 μL was removed at intervals and resuspended in gel loading buffer and frozen at -80°C before electrophoresis on a denaturing PAGE gel. Figure 30A A representative PAGE gel is depicted. Figure 30B Depicted are graphs showing the mean intensity (± SEM) of bands from two independent experiments analyzed by densitometry.Both stabilized cholesterol-conjugated siRNA and EpCAM-AsiC were stable over the 36 hours of the experiment.

[0046] Figures 31A to 31B This indicates that Alexa750-EpCAM-AsiC is selectively taken up into EpCAM+ tumors. Figure 31ADescribe the experimental setup; Nude mice were injected with MB468-luc (left flank) and MB231-luc-mCherry (right flank) cells, and 5 days after injection, Alexa750-labeled GFP-targeted EpCAM-AsiC (0.5 mg / kg) was injected subcutaneously into the neck. Mice were imaged immediately after injection and imaged again 24 hours, 48 ​​hours, and 5 days after injection. Alexa750-labeled GFP-targeted EpCAM-AsiC was colocalized with the luciferase tumor in the MB468-luc tumor (EpCAM+) rather than the MB231-luc-mCherry (EpCAM-) tumor. Figure 31B Depicted is an analysis of seven mice, demonstrating a significant increase in Alexa 750 in MB468 (EpCAM+) tumors. On day 5, tumors were removed and visualized to verify that Alexa 750-labeled GFP-targeted EpCAM-AsiC had indeed entered the tumor. Increased Alexa 750 levels were negatively correlated with mCherry levels (n=8, *P<0.05, t-test, EpCAM+ vs. EpCAM- cells).

[0047] FIG. 32A to FIG. 32B showed that EpCAM AsiC targeting PLK1 specifically inhibited tumor growth in basal A breast cancer cells. Figure 32A The experimental setup is depicted. MB231-luc-mCherry cells (5×10 5 ) or MB468-luc cells (5×10 6 Mice were imaged every 72 h for 14 days using an IVIS Spectra imaging system. Figure 32B Depicted is a graph showing that MB468-luc tumors treated with EpCAM-AsiC targeting PLK1 decreased in size as early as 6 days after treatment and completely disappeared in many mice by day 14. Untreated tumors, EpCAM+ and EpCAM-, increased in size over 14 days.

[0048] Figure 33 A graph of tumor growth is depicted, demonstrating that MB468 tumors regressed only after treatment with PLK1 EpCAM-AsiC. Mice bearing subcutaneous (sc) MB468 tumors were treated with 5 mg / kg RNA 2x / wk starting when tumors became palpable. Samples treated with PLK1 EpCAM-AsiC, GFP SpCAM-AsiC, EpCAM aptamer, PLK1 siRNA, and mock were analyzed as indicated.

[0049] Figure 34 PLK1 siRNA associates with Argonaute (AGO) in cells treated with PLK1 EpCAM-AsiC. MB-468 cells treated with PLK1 EpCAM-AsiC or siRNA for 2 days were lysed, and cell lysates were immunoprecipitated with a pan-AGO antibody or an IgG isotype control. The amount of PLK1 siRNA in the immunoprecipitates was quantified by Taqman qRT-PCR and expressed as mean ± SEM relative to the log2 of miR-16. **P < 0.01 compared to siRNA-treated cells by Student's t-test. ND, not detectable. PLK1 siRNA was found in RISC after treatment with PLK1 EpCAM-AsiCs. However, AGO immunoprecipitation did not significantly deplete PLK1 siRNA from the supernatant. This may be because most RNA taken up by cells is not released from endosomes to the cytosol (A. Wittrup et al., Visualizing lipid-formulated siRNA release from endosomes and target gene knockdown. Nature Biotechnology 2015, in press).

[0050] Figure 35 PLK EpCAM AsiC suppresses MCF10CA1a (CA1a) tumor growth. The top panel depicts the experimental protocol. In this experiment, AsiC was injected subcutaneously into the flank near the tumor, but not into the tumor itself. The bottom panel depicts a plot of the Log2 total luminescence photon flux of the tumor (N=4). *P<0.05 by Student's t-test. DETAILED DESCRIPTION

[0051] The present inventors have demonstrated the unexpected efficacy of AsiC (aptamer-siRNA chimeric molecules) in treating cancer. AsiC described herein utilizes aptamers that specifically target the chimeric molecule to cancer cells, providing effective on-target repression of genes targeted by siRNA.

[0052] In particular, the aptamers described herein (e.g., those targeting EpCAM and EphA2) allow for the targeting of tumor-initiating cells (also known as cancer stem cells) for therapeutic purposes. These cells are not only responsible for tumor initiation, recurrence, and metastasis, but are also relatively resistant to conventional cytotoxic therapies. Thus, the compositions and methods described herein allow for the effective treatment of underlying pathologies in a manner that existing therapies have failed to achieve. The success of the AsiCs described herein is particularly surprising given that direct targeting of EpCAM with antibodies has previously been investigated and found to lack effectiveness.

[0053] In addition, AsiC described herein has been shown to be unexpectedly effective in treating epithelial cancers such as breast cancer, for example, triple-negative breast cancer (TNBC). There is currently no targeted therapy for TNBC, and available treatments typically cause metastasis within 3 years, leading to death. Compared to healthy cells, AsiC described herein specifically exhibits effective gene knockdown in luminal and basal-A TNBC cells, suppresses colony and mammosphere formation in vitro, and eliminates tumor initiation ex vivo. In vitro treatment with AsiC results in therapeutic targeted delivery and rapid tumor regression.

[0054] In one aspect, described herein are chimeric molecules comprising a domain that binds a cancer marker and an inhibitory nucleic acid domain. As used herein, a "domain that binds a cancer marker" refers to a domain and / or molecule that can specifically bind to a molecule (cancer marker) that is more highly expressed on the surface of cancer cells compared to healthy cells of the same type. In some embodiments, the cancer marker can be a protein and / or polypeptide. In some embodiments, the cancer marker can be selected from EpCAM or EphA2. In some embodiments, the domain that binds a cancer marker can be an aptamer.

[0055] As used herein, "EpCAM" or "epithelial cell adhesion molecule" refers to a molecule that mediates Ca 2+ Transmembrane glycoprotein for independent homotypic cell-cell adhesion. The sequence of EpCAM is known for various species, such as human EpCAM (see, for example, NCBI Gene ID: 4072; protein sequence: NCBI Ref Seq: NP_002345.2).

[0056] As used herein, "EphA2" or "EPH receptor A2" refers to an ephirin-type protein-tyrosine kinase receptor. EphA2 binds to the ephrin-A ligand and allows Kaposi's sarcoma-associated herpesvirus to enter host cells. The sequence of EphA2 is known for various species, such as human EphA2 (see, e.g., NCBI Gene ID: 1969; protein sequence: NCBI Ref Seq: NP_004422.2).

[0057] As used herein, an "inhibitory nucleic acid domain" refers to a domain comprising an inhibitory nucleic acid. In some embodiments, the inhibitory nucleic acid can be siRNA.

[0058] Inhibitory nucleic acid domains can inhibit (e.g., can target) the expression of gene products that are upregulated in cancer cells and / or the expression of genes required for cell growth and / or survival. In some embodiments, the inhibitory nucleic acid domain can inhibit the expression of genes selected from the group consisting of: Plk1 (e.g., "polo-like kinase 1," NCBI Gene ID: 5347), MCL1 (e.g., myeloid cell leukemia 1, NCBI Gene ID: 4170), EphA2 (NCBI Gene ID: 1969), PsmA2 (e.g., proteasome subunit α2, NCBI Gene ID: 5683), MSI1 (e.g., musashi RNA binding protein 1, NCBI Gene ID: 4440), BMI1 (e.g., B lymphoma Mo-MLV insert 1, NCBI Gene ID: 648), XBP1 (X-box binding protein 1, NCBI Gene ID: 7494), PRPF8 (e.g., pre-mRNA processing factor 8, NCBI Gene ID: 1167), PRPF9 (e.g., proteomic factor 1, NCBI Gene ID: 1169), PRPF10 (e.g., proteomic factor 2, NCBI Gene ID: 1171), PRPF11 (e.g., proteomic factor 3, NCBI Gene ID: 1167), PRPF12 (e.g., proteomic factor 4, NCBI Gene ID: 1169), PRPF13 (e.g., proteomic factor 5, NCBI Gene ID: 1172), PRPF14 (e.g., proteomic factor 6, NCBI Gene ID: 1169), PRPF15 (e.g., proteomic factor 7, NCBI Gene ID: 11 ID: 10594), PFPF38A (e.g., pre-mRNA processing factor 38A, NCBI Gene ID: 84950), RBM22 (e.g., RNA binding motif protein 22, NCBI Gene ID: 55696), USP39 (e.g., ubiquitin-specific peptidase 39, NCBI Gene ID: 10713), RAN (e.g., ras-related nuclear protein, NCBI Gene ID: 5901), NUP205 (e.g., nucleoporin 205 kDa, NCBI Gene ID: 23165), and NDC80 (e.g., NDC80 centromere complex component, NCBI Gene ID: 10403). The sequences of these genes (e.g., human mRNA) are readily available from the NCBI database and can be used by those skilled in the art to design inhibitory nucleic acids. In addition, exemplary inhibitory nucleic acid domains are provided herein, for example, nucleic acids having the sequence of SEQ ID NO: 2.

[0059] In some embodiments, the compositions described herein may comprise a cancer marker binding domain comprising an aptamer and an inhibitory nucleic acid domain comprising an siRNA, for example, the composition may comprise an aptamer-siRNA chimera (AsiC).

[0060] In some embodiments, the methods described herein relate to treating a subject suffering from or diagnosed with cancer with the compositions described herein. Subjects suffering from cancer can be identified by a doctor using current methods for diagnosing cancer. Symptoms and / or complications of cancer that characterize these conditions and aid in diagnosis are well known in the art and include, but are not limited to, for example, in the case of breast cancer: a lump or mass in breast tissue, swelling of the entire breast or part of the breast, skin irritation, breast inversion, breast or nipple pain, nipple inversion, redness, scaly, or irritation of the breast or nipple and nipple discharge. Tests that may help diagnose, for example, breast cancer include, but are not limited to, mammograms, x-rays, MRIs, ultrasounds, breast ductography, biopsies, and ductal lavages. A family history of cancer or exposure to risk factors for cancer (e.g., smoke, radiation, pollutants, BRCA1 mutations, etc.) can also help determine whether a subject may have cancer or aid in cancer diagnosis.

[0061] As used herein, the terms "malignancy," "malignant condition," "cancer," or "tumor" refer to the uncontrolled growth of cells that interferes with the normal function of the body's organs and systems.

[0062] As used herein, the term "cancer" generally refers to a class of diseases or conditions in which abnormal cells divide uncontrollably and can invade nearby tissues. Cancer cells can also spread to other parts of the body through the blood and lymphatic system.

[0063] "Cancer cell" or "tumor cell" refers to a single cell or tissue that is a cancerous growth. A tumor generally refers to a swelling or lesion formed by the abnormal growth of cells, which can be benign, pre-malignant, or malignant. Most cancer cells form tumors, but some (such as leukemias) do not necessarily form tumors. For those cancer cells that form tumors, the terms cancer (cell) and tumor (cell) are used interchangeably.

[0064] The subject that suffers from cancer or tumor is the subject that objectively measurable cancer cell exists in the subject's body.Include malignant, active proliferative cancer in this definition, and potential dormant tumor or micrometastasis.Cancer that migrates from its original position and is seeded to other vital organs can eventually cause the death of the subject by the functional degradation of the affected organ.Hematopoietic cancer (such as leukemia) can compete with the normal hematopoietic compartment in the subject, thereby causes hematopoietic failure (being in the form of anemia, thrombocytopenia and neutropenia), eventually causes death.

[0065] Examples of cancer include, but are not limited to, carcinoma; lymphoma; blastoma; sarcoma; leukemia; basal cell carcinoma; bile duct cancer; bladder cancer; bone cancer; brain and CNS cancer; breast cancer; peritoneal cancer; cervical cancer; choriocarcinoma; colon and rectal cancer; connective tissue cancer; digestive system cancer; endometrial cancer; esophageal cancer; eye cancer; head and neck cancer; gastric cancer (including gastrointestinal cancer); glioblastoma (GBM); hepatic carcinoma; hepatoma; intraepithelial neoplasia; kidney cancer (renal cancer); laryngeal cancer; leukemia; liver cancer. cancer); lung cancer (e.g., small cell lung cancer, non-small cell lung cancer, lung adenocarcinoma, and lung squamous cell carcinoma); lymphoma, including Hodgkin and non-Hodgkin lymphomas; melanoma; myeloma; neuroblastoma; oral cancer (e.g., lip, tongue, mouth, and pharynx); ovarian cancer; pancreatic cancer; prostate cancer; retinoblastoma; rhabdomyosarcoma; rectal cancer; respiratory cancer; salivary gland cancer; sarcoma; skin cancer; squamous cell carcinoma; stomach cancer; testicular cancer; thyroid cancer; uterine or endometrial cancer; urinary tract cancer; vulvar cancer, as well as other malignancies and sarcomas; and B-cell lymphomas (including low-grade / follicular non-Hodgkin lymphoma ( In some embodiments, the cancer may be an epithelial cancer. In some embodiments, the cancer may be a breast cancer. In some embodiments, the cancer may be a triple-negative breast cancer.

[0066] "Cancer cell" is a cancerous, precancerous or transformed cell in vivo, in vitro or in tissue culture, which has a spontaneous or induced phenotypic change that is not necessarily related to the uptake of new genetic material. Although conversion can be derived from the infection of a transforming virus and the integration of new genomic nucleic acids or the uptake of exogenous nucleic acids, it can also occur spontaneously or after being exposed to a carcinogen, thereby mutating endogenous genes. Conversion / cancer is related to: for example, morphological changes, cell immortalization, abnormal growth control, focus formation, non-anchorage dependence, malignant tumors, contact inhibition loss and growth density restriction, growth factor or serum independence, tumor-specific markers, invasiveness or metastasis, tumor growth in a suitable animal host (for example, nude mice). See, for example, Freshney, CULTURE ANIMAL CELLS: MANUAL BASIC TECH. (3rd edition, 1994).

[0067] The compositions and methods described herein can be administered to a subject suffering from or diagnosed with cancer. In some embodiments, the methods described herein comprise administering to a subject an effective amount of a composition described herein to alleviate the symptoms of cancer. As used herein, "alleviating the symptoms of cancer" means improving any condition or symptom associated with cancer. Such alleviation is a reduction of at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, 99% or more, as measured by any standard technique, compared to an equivalent untreated control. A variety of means for administering the compositions described herein to a subject are known to those skilled in the art. Such methods may include, but are not limited to, oral administration, parenteral administration, intravenous administration, intramuscular administration, subcutaneous administration, transdermal administration, airway administration (aerosol), pulmonary administration, cutaneous administration, topical administration, injection, or intratumoral administration. Administration can be local or systemic. In some embodiments, administration is subcutaneous. In some embodiments, administration of the AsiC described herein is subcutaneous.

[0068] The term "effective amount" as used herein refers to the amount of the composition required for alleviating at least one or more symptoms of a disease or disorder, and relates to the amount of a pharmaceutical composition sufficient to provide a desired effect. Therefore, the term "therapeutically effective amount" refers to an amount sufficient to provide a specific anti-cancer effect when administered to a typical subject. In various contexts, the effective amount used herein also includes an amount sufficient to postpone the progression of disease symptoms, an amount sufficient to change the course of disease symptoms (such as, but not limited to, the progression of disease symptoms), or an amount sufficient to reverse disease symptoms. Therefore, it is generally impractical to specify an exact "effective amount". However, for any given situation, an appropriate "effective amount" can be determined by those of ordinary skill in the art using only routine experiments.

[0069] Effective amounts, toxicity, and therapeutic efficacy can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, such as those used to determine the LD50 (the dose lethal to 50% of the population) and the ED50 (the dose therapeutically effective in 50% of the population). The dosage can vary depending on the dosage form employed and the route of administration utilized. The dose ratio between toxic effects and therapeutic effects is the therapeutic index and can be expressed as the ratio LD50 / ED50. Compositions and methods that exhibit large therapeutic indices are preferred. The therapeutically effective dose can be initially estimated from cell culture assays. Alternatively, the dose can be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the composition that achieves half-maximal inhibition of symptoms) determined in cell culture or in a suitable animal model. Levels in plasma can be measured, for example, by high performance liquid chromatography. The effects of any particular dose can be monitored by suitable bioassays, such as those for, among others, tumor size. The dosage can be determined by a physician and adjusted, if necessary, to suit the observed therapeutic effect.

[0070] In some embodiments, the technology described herein relates to pharmaceutical compositions described herein and, optionally, pharmaceutically acceptable carriers. Pharmaceutically acceptable carriers and diluents include saline, aqueous buffer solutions, solvents, and / or dispersion vehicles. The use of such carriers and diluents is well known in the art. Some non-limiting examples of materials that can serve as pharmaceutically acceptable carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, methylcellulose, ethylcellulose, microcrystalline cellulose, and cellulose acetate; (4) tragacanth; (5) malt; (6) gelatin; (7) lubricants, such as magnesium stearate, sodium lauryl sulfate, and talc; (8) excipients, such as cocoa butter and suppositories; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) diols, such as propylene glycol; (11) polyols, such as glycerol, sorbitol, mannitol, and polyethylene glycol (PEG); (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethanol; (20) pH buffered solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) bulking agents, such as polypeptides and amino acids; (23) serum components, such as serum albumin, HDL, and LDL; (24) C2-C 12Alcohols, such as ethanol; and (25) other non-toxic compatible substances used in pharmaceutical formulations. Wetting agents, colorants, release agents, coating agents, sweeteners, flavorings, fragrances, preservatives, and antioxidants may also be present in the formulation. Terms such as "excipient," "carrier," "pharmaceutically acceptable carrier," and the like are used interchangeably herein. In some embodiments, for example, as described herein, the carrier inhibits degradation of the active agent.

[0071] In some embodiments, the pharmaceutical compositions described herein may be in parenteral dosage form. Because administration of parenteral dosage forms typically bypasses a patient's natural defenses against contaminants, parenteral dosage forms are preferably sterile or capable of being sterilized prior to administration to a patient. Examples of parenteral dosage forms include, but are not limited to, solutions ready for injection, dry products to be dissolved or suspended in a pharmaceutically acceptable carrier for injection, suspensions ready for injection, and emulsions. In addition, controlled-release parenteral dosage forms may be prepared for administration to a patient, including but not limited to dosage form and dose-dumping.

[0072] Suitable carriers disclosed herein that can be used to provide parenteral dosage forms are well known to those skilled in the art. Examples include, but are not limited to, sterile water; USP water for injection; saline solution; dextrose solution; aqueous carriers such as, but not limited to, sodium chloride injection, Ringer's injection, dextrose injection, dextrose and sodium chloride injection, and lactated Ringer's injection; water-miscible carriers such as, but not limited to, ethanol, polyethylene glycol, and propylene glycol; and non-aqueous carriers such as, but not limited to, corn oil, cottonseed oil, peanut oil, sesame oil, ethyl oleate, isopropyl myristate, and benzyl benzoate. Compounds that alter or modify the solubility of the pharmaceutically acceptable salts of the compositions disclosed herein can also be incorporated into the parenteral dosage forms of the present disclosure, including conventional and controlled-release parenteral dosage forms.

[0073] Pharmaceutical compositions can also be formulated for oral administration, for example, as discrete dosage forms such as, but not limited to, tablets (including but not limited to scored or coated tablets), pills, caplets, capsules, chewable tablets, powder packets, cachets, lozenges, wafers, aerosol sprays, or liquids such as, but not limited to, syrups, elixirs, solutions or suspensions in aqueous liquids, non-aqueous liquids, oil-in-water emulsions, or water-in-oil emulsions. Such compositions contain a predetermined amount of a pharmaceutically acceptable salt of a disclosed compound and can be prepared using pharmaceutical methods well known to those skilled in the art. See generally, Remington: The Science and Practice of Pharmacy, 21st ed., Lippincott, Williams and Wilkins, Philadelphia, PA (2005).

[0074] Conventional dosage forms generally provide rapid or immediate release of the drug from the formulation. Depending on the pharmacology and pharmacokinetics of the drug, the use of conventional dosage forms can result in significant fluctuations in the drug concentration in the patient's blood and other tissues. These fluctuations may affect many parameters, such as dosage frequency, onset time, duration of efficacy, maintenance of therapeutic blood levels, toxicity, side effects, etc. Advantageously, controlled-release formulations can be used to control the onset time, duration of action, plasma levels within the therapeutic window, and peak blood levels of the drug. In particular, controlled-release or extended-release dosage forms or formulations can be used to ensure the maximum effect of the drug while minimizing potential side effects and potential safety hazards, which may occur when the drug is administered in insufficient amounts (i.e., below the minimum therapeutic level) and when exceeding the toxicity level of the drug. In some embodiments, the composition can be given in a sustained-release formulation.

[0075] Controlled-release pharmaceutical products have the common goal of improving drug therapy over that achieved by their non-controlled-release counterparts. Ideally, the characteristic of using an optimally designed controlled-release formulation in medical treatment is that the condition is cured or controlled in the shortest possible time using the least amount of drug substance. Advantages of controlled-release formulations include: 1) prolonged drug activity; 2) reduced dosage frequency; 3) improved patient compliance; 4) less total amount of drug used; 5) reduced local or systemic side effects; 6) minimized drug accumulation; 7) reduced fluctuations in blood levels; 8) improved therapeutic efficacy; 9) reduced potentiation or loss of drug activity; and 10) improved speed of control of the disease or condition (Kim, Cherng-ju, Controlled Release Dosage Form Design, 2 (Technomic Publishing, Lancaster, Pa.: 2000)).

[0076] Most controlled-release formulations are designed to initially release a certain amount of drug (active ingredient) to immediately produce the desired therapeutic effect, and gradually and continuously release additional amounts of drug to maintain this level of therapeutic or preventive effect over a long period of time. In order to maintain this constant level of drug in the body, the drug must be released from the dosage form at a certain rate to replace the amount of drug metabolized and excreted from the body. The controlled release of the active ingredient can be stimulated by a variety of conditions, including but not limited to: pH, ionic strength, osmotic pressure, temperature, enzymes, water and other physiological conditions or compounds.

[0077] Various known controlled-release or sustained-release dosage forms, formulations, and devices may be suitable for use with the salts and compositions described herein. Examples include, but are not limited to, those described in U.S. Patent Nos. 3,845,770, 3,916,899, 3,536,809, 3,598,123, 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, 5,733,566, and 6,365,185B1; each of which is incorporated herein by reference. These dosage forms may be used using, for example, hydroxypropyl methylcellulose, other polymer matrices, gels, permeable membranes, osmotic systems (e.g., (Alza Corporation, Mountain View, Calif. USA)) or a combination thereof provides slow or controlled release of one or more active ingredients, thereby providing a desired release profile in varying proportions.

[0078] The methods described herein may further comprise administering a second agent and / or treatment to the subject, for example as part of a combination therapy. Non-limiting examples of second agents and / or treatments may include radiation therapy, surgery, gemcitabine, cisplastin, paclitaxel, carboplatin, bortezomib, AMG479, vorinostat, rituximab, temozolomide, rapamycin, ABT-737, PI-103; alkylating agents such as thiotepa and Cyclophosphamide; alkyl sulfonates, such as busulfan, improsulfan, and piposulfan; aziridines, such as benzodopa, carboquinone, meturedopa, and uredopa; ethyleneimines and methylamelamines, including hexamethylmelamine, triethylmelamine, triethylenephosphoramide, triethylenethiophosphoramide, and trimethylolmelamine; polyacetyl groups (particularly bratacin and bratacinone); camptothecins (including the synthetic analogue topotecan); bryostatin; callystatin; CC-1065 (including its synthetic analogues adolesine, carzelesin, and biszelesin); cryptophycins (particularly cryptophycin 1 and cryptophycin 2); 8); dolastatin; duocarmycin (including synthetic analogs KW-2189 and CB1-TM1); eleutherobin; pancratistatin; sarcodictyin; spongistatin; nitrogen mustards, such as chlorambucil, naphthyl mustard, cholophosphamide, estramustine, ifosfamide, dichloromethyldiethylamine, oxychloride, melphalan, new mustard, phenesterine ), chlorambucil, cyclophosphamide, uracil nitrogen mustard; nitrosureas, such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimnustine; antibiotics, such as enediyne antibiotics (e.g., calicheamicins, particularly calicheamicin γll and calicheamicin ωll (see, e.g., Agnew, Chem. Intl. Ed. Engl., 33:183-186 (1994)); dynemicins, including dynemicin A; bisphosphonates such as clodronate; esperamicin; and neocarcinogen chromophores and related chromoprotein enediyne antibiotic chromophores), aclacinomycins, actinomycins, anthramycins, azaserine, bleomycin, actinomycin C, carabicin, caminomycin, carcinoglossin, chromomycinis, actinomycin D, daunorubicin, detoximum iodide, 6-diazo-5-oxo-L-norleucine, Doxorubicin (including morpholinodoxorubicin, cyanomorpholinodoxorubicin, 2-pyrrolineodoxorubicin and deoxydoxorubicin), epirubicin, esorubicin, desmethoxydaunorubicin, masiromycin, mitomycins such as mitomycin C, mycophenolic acid, nogamycin, olivomycin, peplomycin, potfiromycin, puromycin, triferric doxorubicin, rhodorubicin, streptozotocin, streptozotocin, tuberculin, ubenimex, zoloft, daunorubicin; antimetabolites such as methotrexate and 5-fluorouracil (5-F U); folic acid analogs, such as dimethylfolate, methotrexate, pteropterin, trimesate; purine analogs, such as fludarabine, 6-mercaptopurine, thiopurine, thioguanine; pyrimidine analogs, such as cyclocytidine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens, such as calosterone, methylandrostanolone propionate, cyclothioandrostol, melastane, testolactone; antiadrenal drugs, such as aminoglutethimide, mitotane, trilostane; folic acid supplements, such as folinic acid (frolinic acid); acid); acetylglucuronolactone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demeclocycline; diacrylamide; elformithine; elformithine; epothilone; ethidium; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids, such as maytansine and ansamitocin; mitoxantrone; mitoxantrone; mopidanmol; nitraerine; pentostatin; methamidine; pirarubicin; losoxantrone; ethylhydrazine; procarbazine; Polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofuran; spirogermanium; tenuisporic acid; triimidoquinone; 2,2',2"-trichlorotriethylamine; trichothecenes (particularly T-2 toxin, verracurin A, baculozolin A, and serpentin); carbamates; vindesine; dacarbazine; mannitol mustard; dibromomannitol; dibromodulcitol; pipobroman; gacytosine; cytarabine ("Ara-C"); cyclophosphamide; thiotepa; taxanes, such as Paclitaxel (Bristol-Myers Squibb Oncology, Princeton, NJ), Cremophor-free, albumin-engineered nanoparticle formulation of paclitaxel (American Pharmaceutical Partners, Schaumberg, 111.) and doxetaxel (Rhone-Poulenc Rorer, Antony, France); chloranbucil; Gemcitabine; 6-thioguanine; mercaptopurine; methotrexate; platinum analogs such as cisplatin, oxaliplatin, and carboplatin; vinblastine; platinum; etoposide (VP-16); ifosfamide; mitoxantrone; vincristine; navelbine TM Vinorelbine; novocycline; teniposide; edatrexate; daunorubicin; aminopterin; xeloda; ibandronate; irinotecan (Camptosar, CPT-11) (including regimens of irinotecan with 5-FU and leucovorin); the topoisomerase inhibitor RFS2000; difluoromethylornithine (DMFO); retinoids, such as retinoic acid; capecitabine; combretastatin; leucovorin (LV); oxaliplatin, including oxaliplatin regimens (FOLFOX); lapatinib (Tykerb TM ); PKC-α, Raf, H-Ras, EGFR (e.g., erlotinib) that reduce cell proliferation ) and VEGF-A inhibitors, and pharmaceutically acceptable salts, acids or derivatives of any of the above substances.

[0079] Treatment methods may also include the use of radiation or radiotherapy. Treatment methods may also include the use of surgery.

[0080] In some embodiments of any aspect described herein, the chimeric molecules described herein can be administered in combination with a taxane (e.g., docetaxel or paclitaxel). In some embodiments of any aspect described herein, the chimeric molecules described herein can be administered in combination with paclitaxel. In some embodiments of any aspect described herein, AsiC as described herein can be administered in combination with a taxane. In some embodiments of any aspect described herein, AsiC as described herein can be administered in combination with paclitaxel.

[0081] In certain embodiments, the effective dose of the compositions described herein can be administered to the patient once. In certain embodiments, the effective dose of the compositions can be administered to the patient repeatedly. For systemic administration, a therapeutic amount of the compositions can be administered to the patient, including, for example, 0.1 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 5 mg / kg, 10 mg / kg, 15 mg / kg, 20 mg / kg, 25 mg / kg, 30 mg / kg, 40 mg / kg, 50 mg / kg or more.

[0082] In some embodiments, after the initial treatment regimen, treatment can be given on a less frequent basis. For example, after three months of biweekly treatment, treatment can be repeated monthly for up to six months or a year or longer. Treatment according to the methods described herein can reduce the level of a marker or a symptom of a condition by, for example, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% or more.

[0083] The dosage of compositions as herein described can be determined by clinicians and adjusted if necessary, to adapt to the observed therapeutic effect. As for the treatment duration and the frequency of treatment, generally by skilled clinicians monitoring subjects to determine when treatment provides a therapeutic effect, and determine whether to increase or decrease dosage, increase or reduce the frequency of administration, do not continue treatment, resume treatment or make other changes to the treatment plan. The dosing schedule can change from once a week to once a day, and this depends on many clinical factors, such as the sensitivity of the subject to the component. The desired dosage or amount of the active effect can be given once or divided into sub-doses, such as 2-4 sub-doses and given over a period of time (for example, with the appropriate time intervals in a day or other appropriate plans). In some embodiments, giving can be long-term, such as once or multiple administration and / or treatment in the time period of several weeks or months. The example of administration and / or treatment plan is in 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months or 6 months or longer time period, giving every day, twice a day, three times a day or four times a day or more. The composition can be administered over a period of time, for example, over 5 minutes, 10 minutes, 15 minutes, 20 minutes, or 25 minutes.

[0084] For convenience, the meanings of some terms and phrases used in the specification, examples and appended claims are provided below. Unless otherwise indicated or implied from the context, the following terms and phrases include the meanings provided below. These definitions are provided to help describe specific embodiments and are not intended to limit the claimed invention, as the scope of the invention is limited only by the claims. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those generally understood by those of ordinary skill in the art to which the invention belongs. If there is a clear difference between the use of a term in the art and the definition provided herein, the definition provided in this specification shall prevail.

[0085] For convenience, some terms used herein in the specification, examples, and appended claims are collected here.

[0086] As used herein, the terms "reduce", "lowered", "reduce" or "inhibit" all refer to a statistically significant amount of reduction. In some embodiments, "decline", "reduce", "reduce" or "inhibit" generally refer to a reduction of at least 10% compared to a reference level (e.g., lacking a given treatment), and can include, for example, a reduction of at least about 10%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, at least about 98%, at least about 99% or more. "Reduce" or "inhibit" as used herein does not encompass complete inhibition or reduction compared to a reference level. "Complete inhibition" is 100% inhibition compared to a reference level. Reduction can preferably drop to a level accepted within the normal range for individuals without a given disorder.

[0087] As used herein, the terms "increased / elevated", "increase / elevate", "enhance" or "activate / activate" all refer to an increase in a statistically significant amount. In some embodiments, the terms "increased / elevated", "increase / elevate", "enhance" or "activate / activate" can mean an increase of at least 10% compared to a reference level, such as an increase of at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90%, or up to and including a 100% increase, or any increase between 10-100% compared to a reference level, or at least about 2-fold, or at least about 3-fold, or at least about 4-fold, or at least about 5-fold or at least about 10-fold, or any increase between 2-fold and 10-fold compared to a reference level or more. In the context of a marker or symptom, "increase / elevate" is a statistically significant increase / elevation of such levels.

[0088] As used herein, a "subject" refers to a human or an animal. Typically, the animal is a vertebrate, such as a primate, a rodent, a domesticated animal, or a game animal. Primates include chimpanzees, crab-eating macaques, spider monkeys, and macaques such as rhesus macaques. Rodents include mice, rats, woodchucks, ferrets, rabbits, and hamsters. Domesticated and game animals include cows; horses; pigs; deer; bison; buffalo; felines, such as house cats; canines, such as dogs, foxes, wolves; birds, such as chickens, emus, ostriches; and fish, such as trout, catfish, and salmon. In some embodiments, the subject is a mammal, such as a primate, such as a human. The terms "individual," "patient," and "subject" are used interchangeably herein.

[0089] Preferably, the subject is a mammal. The mammal may be a human, non-human primate, mouse, rat, dog, cat, horse, or cow, but is not limited to these examples. Mammals other than humans can advantageously be used as subjects representing animal models of cancer. The subject may be male or female.

[0090] The subject can be a subject who has previously been diagnosed or confirmed to have or have a condition (e.g., cancer) that needs treatment or who has or has one or more complications related to such conditions, and has optionally received treatment for cancer or the treatment of one or more complications related to cancer. Alternatively, the subject can also be a subject who has not previously been diagnosed as having cancer or one or more complications related to cancer. For example, the subject can be a subject who shows one or more risk factors for cancer or shows one or more risk factors for one or more complications related to cancer, or a subject who does not show risk factors.

[0091] A "subject in need thereof" for treatment of a particular disorder may be a subject having the disorder, diagnosed as having the disorder, or diagnosed as being at risk of developing the disorder.

[0092] As used herein, the terms "protein" and "polypeptide" are used interchangeably herein to designate a series of amino acid residues that are interconnected by peptide bonds between the α-amino and carboxyl groups of adjacent residues. Regardless of their size or function, the terms "protein" and "polypeptide" refer to polymers of amino acids, including modified amino acids (e.g., phosphorylation, saccharification, glycosylation, etc.) and amino acid analogs. "Protein" and "polypeptide" are typically used to refer to larger polypeptides, while the term "peptide" is typically used to refer to small polypeptides, but the usage of these terms overlaps in the art. When referring to gene products and fragments thereof, the terms "protein" and "polypeptide" are used interchangeably herein. Thus, exemplary polypeptides or proteins include gene products, naturally occurring proteins, homologs, orthologs, paralogs, fragments, and other equivalents, variants, fragments, and analogs of the above substances.

[0093] As used herein, the terms "nucleic acid" or "nucleic acid sequence" refer to any molecule, preferably a polymeric molecule, that incorporates units of ribonucleic acid, deoxyribonucleic acid, or their analogs. A nucleic acid can be single-stranded or double-stranded. A single-stranded nucleic acid can be one strand of denatured double-stranded DNA. Alternatively, a single-stranded nucleic acid can be a single-stranded nucleic acid that is not derived from any double-stranded DNA. In one aspect, the nucleic acid can be DNA. In another aspect, the nucleic acid can be RNA. Suitable nucleic acid molecules are DNA, including genomic DNA or cDNA. Other suitable nucleic acid molecules are RNA, including mRNA.

[0094] The inhibitor of the expression of a given gene can be an inhibitory nucleic acid or an inhibitory oligonucleotide. In some embodiments, the inhibitory nucleic acid is an inhibitory RNA (iRNA). In some embodiments, the inhibitory nucleic acid is an inhibitory DNA (iDNA). Double-stranded RNA molecules (dsRNA) have been shown to block gene expression using a highly conservative regulatory mechanism known as RNA interference (RNAi). Inhibitory nucleic acids as described herein can include an RNA chain or DNA chain (antisense strand) having a length of 30 or less nucleotides (i.e., a length of 8-30 nucleotides, a length generally of 19-24 nucleotides), which is substantially complementary to at least a portion of a precursor or mature form of the transcript of the target gene. Using these inhibitory oligonucleotides allows for targeted degradation of the target gene, causing the expression and / or activity of the target gene to decrease.

[0095] As used herein, the terms "inhibitory oligonucleotide," "inhibitory nucleic acid," or "antisense oligonucleotide" (ASO) refer to agents containing oligonucleotides, e.g., DNA or RNA molecules that mediate targeted cleavage of RNA transcripts. In one embodiment, the inhibitory oligonucleotides described herein affect inhibition of expression and / or activity of a target gene. Inhibitory nucleic acids useful in the methods and compositions of the present invention include antisense oligonucleotides, ribozymes, exogenous guide sequence (EGS) oligonucleotides, siRNA compounds, single-stranded or double-stranded RNA interference (RNAi) compounds (e.g., siRNA compounds), modified bases / locked nucleic acids (LNA), antagomirs, peptide nucleic acids (PNA), and other oligomeric compounds or oligonucleotide mimetics that hybridize to at least a portion of a target nucleic acid and modulate its function. In some embodiments, the inhibitory nucleic acid comprises antisense RNA, antisense DNA, chimeric antisense oligonucleotides, antisense oligonucleotides comprising modified linkages, interfering RNA (RNAi), short interfering RNA (siRNA); microinterfering RNA (miRNA); temporal regulatory small RNA (stRNA); or short hairpin RNA (shRNA); small RNA-induced gene activation (RNAa); small activating RNA (saRNA), or a combination thereof. For further disclosure of inhibitory nucleic acids, see US2010 / 0317718 (antisense oligonucleotides), US2010 / 0249052 (double-stranded ribonucleic acid (dsRNA)), US2009 / 0181914 and US2010 / 0234451 (LNA), US2007 / 0191294 (siRNA analogs), US2008 / 0249039 (modified siRNA), and WO2010 / 129746 and WO2010 / 040112 (inhibitory nucleic acids).

[0096] In certain embodiments, a cell is contacted with an inhibitor (e.g., an inhibitory oligonucleotide) such that the level of target mRNA in the cell is reduced to at least about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, up to and including 100% of the level of target mRNA found in the cell in the absence of the inhibitory oligonucleotide.

[0097] As used herein, the term "iRNA" refers to an agent containing RNA as defined herein, and mediates the targeted cleavage of RNA transcripts through an RNA-induced silencing complex (RISC) pathway. In one embodiment, the iRNA described herein affects the expression and / or inhibition of the activity of a target gene. In one aspect, RNA interference agents include single-stranded RNA that interacts with a target RNA sequence to guide the cleavage of the target RNA. Without wishing to be bound by theory, long double-stranded RNA introduced into plant and invertebrate cells is broken down into siRNA by a type III endonuclease called Dicer (Sharp et al., Genes Dev., 2001, 15: 485). Dicer (ribonuclease-III-like enzyme) processes dsRNA into short interfering RNAs of 19-23 base pairs with characteristic two-base 3' overhangs (Bernstein et al., (2001), Nature, 409: 363). The siRNA is then incorporated into an RNA-induced silencing complex (RISC), where one or more helicases unwind the siRNA duplex, allowing the complementary antisense strand to guide target recognition (Nykanen et al., (2001), Cell, 107: 309). Upon binding to the appropriate target mRNA, one or more endonucleases within the RISC cleave the target to induce silencing (Elbashir et al., (2001) Genes Dev., 15: 188). Thus, in one aspect, RNA interference agents involve double-stranded RNA that promotes the formation of a RISC complex containing single-stranded RNA, which directs the complex to cleave at a target region of the target transcript to affect silencing of the target gene.

[0098] In some embodiments, inhibitory oligonucleotide can be a double-stranded nucleic acid (such as dsRNA). Double-stranded nucleic acid comprises two nucleic acid chains, and two nucleic acid chains are fully complementary to form a duplex structure when using double-stranded nucleic acid. One chain (antisense strand) of double-stranded nucleic acid includes a complementary region, which is substantially complementary to the target sequence and is generally fully complementary. The target sequence can be derived from the sequence of the mRNA and / or mature miRNA formed during the expression of the target gene. The other chain (sense strand) includes a region complementary to the antisense strand, so that when combined under appropriate conditions, the two chains hybridize and form a duplex structure. Generally, the length of the duplex structure is between 8 and 30 base pairs, including end values; more generally between 18 and 25 base pairs, including end values; more generally between 19 and 24 base pairs, including end values; and most generally between 19 and 21 base pairs, including end values. Similarly, the length of the region complementary to the target sequence is between 8 and 30 nucleotides, including the end values; more generally between 18 and 25 nucleotides, including the end values; more generally between 19 and 24 nucleotides, including the end values; and most generally between 19 and 21 nucleotides, including the end values. In some embodiments, the length of the dsRNA is between 15 and 20 nucleotides, including the end values; and in other embodiments, the length of the dsRNA is between 25 and 30 nucleotides, including the end values. As will be appreciated by those of ordinary skill, the targeting region of the RNA targeted for cutting is most generally a part of a larger RNA molecule (typically an mRNA molecule). Relatedly, the "part" of an mRNA and / or miRNA target is a continuous sequence of an mRNA target having a length sufficient to become a substrate for antisense-guided cleavage (i.e., cleavage by RISC pathway). In some cases, a double-stranded nucleic acid with a duplex as short as 8 base pairs can mediate antisense-guided RNA cleavage. Most commonly targets will be at least 15 nucleotides in length, preferably 15-30 nucleotides in length.

[0099] Those skilled in the art will also recognize that the duplex region is the primary functional portion of a double-stranded inhibitory nucleic acid, such as a duplex region of 8 to 36 (e.g., 15-30) base pairs. Thus, in one embodiment, an inhibitory nucleic acid molecule or a complex of inhibitory nucleic acid molecules having a duplex region of more than 30 base pairs is a double-stranded nucleic acid to the extent that it is processed into a functional duplex of, for example, 15-30 base pairs (targeting the desired RNA for cleavage). Thus, those of ordinary skill will recognize that, in one embodiment, the miRNA is a dsRNA. In another embodiment, the dsRNA is not a naturally occurring miRNA. In another embodiment, an inhibitory nucleic acid agent for targeting target gene expression is not produced in the target cell by cutting a larger double-stranded nucleic acid molecule.

[0100] Although target sequences are typically 15-30 nucleotides in length, there is wide variation in the suitability of specific sequences within this range for directing the cleavage of any given target RNA. When targeting miRNA, the target sequence can be as short as 8 nucleotides, including a "seed" region (e.g., nucleotides 2-8). Various software packages and guides presented herein provide guidance for identifying the optimal target sequence for any given gene target, but an empirical approach can also be employed in which a "window" or "mask" of a given size (non-limiting example, 21 nucleotides) is literally or figuratively (including, for example, in silico) placed on the target RNA sequence to identify sequences within a size range that can be used as target sequences. By gradually moving the sequence "window" to one nucleotide upstream or downstream of the initial target sequence position, the next potential target sequence can be identified until a complete set of possible sequences is identified for any given target size selected. This process, together with the systematic synthesis and testing (using assays described herein or known in the art) of identified sequences to identify those that perform best when targeted with inhibitory nucleic acid agents, can identify those RNA sequences that mediate optimal inhibition of target gene expression when targeted with inhibitory nucleic acid agents.

[0101] The double-stranded inhibitory nucleic acids described herein may further include one or more single-stranded nucleotide overhangs. The double-stranded inhibitory nucleic acids can be synthesized by standard methods known in the art as further discussed below, for example, by using an automated DNA synthesizer commercially available, for example, from Biosearch, Applied Biosystems, Inc. In one embodiment, the antisense strand of the double-stranded inhibitory nucleic acid has 1-10 nucleotide overhangs at the 3' end and / or the 5' end. In one embodiment, the sense strand of the double-stranded inhibitory nucleic acid has 1-10 nucleotide overhangs at the 3' end and / or the 5' end. In one embodiment, at least one end of the double-stranded inhibitory nucleic acid has a single-stranded nucleotide overhang of 1 to 4, typically 1 or 2 nucleotides. Double-stranded inhibitory nucleic acids with at least one nucleotide overhang have unexpectedly superior inhibitory properties relative to their blunt-ended counterparts.

[0102] In another embodiment, one or more nucleotides in the overhang are substituted with nucleoside phosphorothioates.

[0103] As used herein, the term "nucleotide overhang" refers to at least one unpaired nucleotide that protrudes from the duplex structure of an inhibitory nucleic acid (e.g., dsRNA). For example, a nucleotide overhang exists when the 3'-end of one strand of a double-stranded inhibitory nucleic acid extends beyond the 5'-end of the other strand, and vice versa. A double-stranded inhibitory nucleic acid may comprise an overhang of at least one nucleotide; alternatively, the overhang may comprise at least two nucleotides, at least three nucleotides, at least four nucleotides, at least five nucleotides, or more. The nucleotide overhang may comprise or consist of nucleotide / nucleoside analogs, including deoxynucleotides / nucleosides. The overhang may be on the sense strand, the antisense strand, or any combination thereof. In addition, the nucleotides of the overhang may be present at the 5' end, the 3' end, or both ends of the antisense strand or the sense strand of the double-stranded inhibitory nucleic acid.

[0104] As used herein with respect to double-stranded inhibitory nucleic acids, the term "blunt" or "blunt-ended" refers to the absence of unpaired nucleotides or nucleotide analogs at a given end of the dsRNA, i.e., the absence of nucleotide overhangs. One or both ends of a double-stranded inhibitory nucleic acid may be blunt-ended. A double-stranded inhibitory nucleic acid is said to be blunt-ended when both ends are blunt-ended. It will be appreciated that a "blunt-ended" double-stranded inhibitory nucleic acid is a double-stranded inhibitory nucleic acid that is blunt-ended at both ends, i.e., has no nucleotide overhangs at either end of the molecule. Most commonly, such molecules are double-stranded throughout their entire length.

[0105] In this regard, one of the two strands is complementary to the other of the two strands, wherein one strand is substantially complementary to the sequence of the target gene precursor or mature miRNA. Thus, in this regard, a double-stranded inhibitory nucleic acid will comprise two oligonucleotides, wherein one oligonucleotide is described as the sense strand and the second oligonucleotide is described as the corresponding antisense strand of the sense strand. As described elsewhere herein and known in the art, the complementary sequence of a double-stranded inhibitory nucleic acid can also be contained as a self-complementary region of a single nucleic acid molecule, as opposed to being on separate oligonucleotides.

[0106] The skilled artisan will be well aware that inhibitory nucleic acids having a duplex structure of 20 to 23, particularly 21, base pairs are believed to be particularly effective in inducing antisense-mediated inhibition (Elbashir et al., EMBO 2001, 20: 6877-6888). However, others have found that shorter or longer inhibitory nucleic acids may also be effective.

[0107] In addition, it is contemplated that for any sequence identification, further optimization can be achieved by systematically adding or removing nucleotides to generate longer or shorter sequences and assaying those sequences as well as sequences generated by shifting the window of longer or shorter size up or down the target RNA from that point. Furthermore, coupling this method of generating new candidate targets to testing for the effectiveness of inhibitory nucleic acids based on those target sequences in inhibition assays known in the art or as described herein can further enhance the inhibitory efficacy. Furthermore, such optimized sequences can be adjusted, for example, by introducing modified nucleotides as described herein or known in the art, adding or changing overhangs, or other modifications known in the art and / or discussed herein (e.g., increasing serum stability or circulation half-life, increasing thermal stability, enhancing transmembrane delivery, targeting to specific locations or cell types, increasing interaction with silencing pathway enzymes, increasing release from endosomes, etc.) to further optimize molecules as expression inhibitors.

[0108] Inhibitory nucleic acids as described herein may contain one or more mismatches with the target sequence. In one embodiment, inhibitory nucleic acids as described herein contain no more than 3 mismatches. If the antisense strand of the inhibitory nucleic acid contains a mismatch with the target sequence, then preferably the mismatch region is not located at the center of the complementary region. If the antisense strand of the inhibitory nucleic acid contains a mismatch with the target sequence, then preferably the mismatch is limited to the last 5 nucleotides at the 5' end or 3' end of the complementary region. For example, for an inhibitory nucleic acid reagent chain of 23 nucleotides complementary to the region of the target gene or its precursor, the chain generally does not contain any mismatch within the central 13 nucleotides. Methods described herein or methods known in the art can be used to determine whether an inhibitory nucleic acid containing a mismatch with the target sequence can effectively inhibit the expression of the target gene. It is important to consider the effectiveness of inhibitory nucleic acids with mismatches in inhibiting target gene expression, especially when the specific region of complementation in the known target gene has a polymorphic sequence variation within the population.

[0109] In another embodiment, the nucleic acid of the inhibitory nucleic acid (e.g., dsRNA) is chemically modified to enhance stability or other beneficial properties. The nucleic acids featured in the present invention can be modified and / or synthesized by methods well established in the art, such as those described in "Current protocols in nucleic acid chemistry," Beaucage, SL et al. (eds.), John Wiley & Sons, Inc., New York, NY, USA, which is incorporated herein by reference. Modifications include, for example, (a) terminal modifications, such as 5' terminal modifications (phosphorylation, conjugation, reverse ligation, etc.), 3' terminal modifications (conjugation, DNA nucleotides, reverse ligation, etc.); (b) base modifications, such as substitutions with stable bases, unstable bases, or bases that pair with a partner that expands the repertoire, removal of bases (absic nucleotides), or conjugation of bases; (c) sugar modifications (e.g., at the 2' or 4' position) or sugar substitutions; and (d) backbone modifications, including modifications or substitutions of phosphodiester linkages. Specific examples of nucleic acid compounds used in the embodiments described herein include, but are not limited to, nucleic acids containing modified backbones or non-natural internucleoside linkages. Among them, nucleic acids with modified backbones include nucleic acids that do not have a phosphorus atom in the backbone. For the purposes of this specification, and as sometimes cited in the art, modified nucleic acids that do not have a phosphorus atom in their internucleoside backbones can also be considered oligonucleotides. In specific embodiments, modified nucleic acids will have a phosphorus atom in their internucleoside backbones.

[0110] Modified backbones can include, for example, boranophosphates, phosphothioates, chiral phosphothioates, phosphorodithioates, phosphotriesters, aminoalkylphosphotriesters, methyl and other alkylphosphonates (including 3'-alkylenephosphonates and chiral phosphonates), phosphinates, phosphoramidates (including 3'-phosphoamidoaminoesters and aminoalkylphosphoamidates), thionylphosphoramidates, thionylphosphoramidates, and thionylphosphotriesters with normal 3'-5' connections; analogs thereof with 2'-5' connections; and substances with reverse polarity (wherein adjacent nucleoside unit pairs are connected with 3'-5' to 5'-3' or 2'-5' to 5'-2' connections). Various salts, mixed salts, and free acid forms are also included.

[0111] Representative U.S. patents that teach the preparation of the above-mentioned phosphorus-containing linkages include, but are not limited to, U.S. Patent Nos. 3,687,808, 4,469,863, 4,476,301, 5,023,243, 5,177,195, 5,188,897, 5,264,423, 5,276,019, 5,278,302, 5,286,717, 5,321,131, 5,399,676, 5,405,939, 5,453,496, 5,455,233, 5,466,677, 5,476,925, 5,519,126, 5,536,821, 5,541,316, 5,550,111, 5,563,253, 5,571,7 99, 5,587,361, 5,625,050, 6,028,188, 6,124,445, 6,160,109, 6,169,170, 6,172,209, 6,239,265, 6,277,603, 6,326,199, 6,346,614, 6,444,423, 6,531,5 90, 6,534,639, 6,608,035, 6,683,167, 6,858,715, 6,867,294, 6,878,805, 7,015,315, 7,041,816, 7,273,933, 7,321,029, and U.S. Pat. No. RE39464, each of which is incorporated herein by reference.

[0112] The modified backbones that do not contain a phosphorus atom have backbones formed by: short-chain alkyl or cycloalkyl nucleoside linkages, mixed heteroatom and alkyl or cycloalkyl nucleoside linkages, or one or more short-chain heteroatom or heterocyclic nucleoside linkages. These backbones include: backbones with morpholino linkages (partially formed by the sugar portion of the nucleoside); siloxane backbones; sulfide backbones, sulfoxide backbones, or sulfone backbones; formacetyl backbones and thioformacetyl backbones; methylene formacetyl backbones and methylene thioformacetyl backbones; olefin-containing backbones; sulfamate backbones; methyleneimino backbones and methylenehydrazine backbones; sulfonate backbones and sulfonamide backbones; amide backbones; and other backbones with mixed N, O, S, and CH2 components.

[0113] Representative U.S. patents that teach the preparation of the above oligonucleotides include, but are not limited to, U.S. Patent Nos. 5,034,506, 5,166,315, 5,185,444, 5,214,134, 5,216,141, 5,235,033, 5,64,562, 5,264,564, 5,405,938, 5,434,257, 5,466,677, 5,470, 967, 5,489,677, 5,541,307, 5,561,225, 5,596,086, 5,602,240, 5,608,046, 5,610,289, 5,618,704, 5,623,070, 5,663,312, 5,633,360, 5,677,437, and 5,677,439, each of which is incorporated herein by reference.

[0114] In other nucleic acid mimics suitable for or considered for use in inhibitory nucleic acids, the sugar and internucleoside connections (i.e., backbones) of the nucleotide units are replaced with new groups. The base units are maintained for hybridization with appropriate nucleic acid target compounds. One such oligomeric compound (a nucleic acid mimic that has been shown to have good hybridization properties) is referred to as peptide nucleic acid (PNA). In PNA compounds, the sugar backbone of the nucleic acid is replaced by an amide-containing backbone (particularly an aminoethylglycine backbone). The core base is retained and directly or indirectly bound to the nitrogen-nitrogen atom of the amide portion of the backbone. Representative U.S. patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Patent Nos. 5,539,082, 5,714,331, and 5,719,262, each of which is incorporated herein by reference. Further teachings of PNA compounds can be found in, for example, Nielsen et al., Science, 1991, 254, 1497-1500.

[0115] Some embodiments of the present invention include nucleic acids having a phosphorothioate backbone and oligonucleosides having a heteroatom backbone, and in particular, --CH2--NH--CH2--, --CH2--N(CH3)--O--CH2-- [referred to as a methylene(methylimino) backbone or MMI backbone], --CH2--O--N(CH3)--CH2--, --CH2--N(CH3)--N(CH3)--CH2--, and --N(CH3)--CH2--CH2-- [wherein the natural phosphodiester backbone is represented as --O--P--O--CH2--] in the aforementioned cited U.S. Patent No. 5,489,677; and the amide backbone in the aforementioned cited U.S. Patent No. 5,602,240. In some embodiments, the inhibitory nucleic acids characterized herein have the morpholino backbone structure of the aforementioned cited U.S. Patent No. 5,034,506.

[0116] The modified nucleic acid may also contain one or more substituted sugar moieties. The inhibitory nucleic acids characterized herein may contain one of the following at the 2' position: OH; F; O-alkyl, S-alkyl, or N-alkyl; O-alkenyl, S-alkenyl, or N-alkenyl; O-alkynyl, S-alkynyl, or N-alkynyl; or O-alkyl-O-alkyl, wherein alkyl, alkenyl, and alkynyl groups may be substituted or unsubstituted C1-C 10 Alkyl or C2-C 10 Alkenyl or C2-C 10 Alkynyl. Exemplary suitable modifications include: O[(CH2) n O] m CH3, O(CH2) n OCH3, O(CH2) n NH2, O(CH2) n CH3, O(CH2) n ONH2 and O(CH2) n ON[(CH2) n CH3)]2, wherein n and m are from 1 to about 10. In another embodiment, the dsRNA comprises one of the following at the 2' position: C1-C 10 Lower alkyl; substituted lower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl; SH, SCH3, OCN, Cl, Br, CN, CF3, OCF3, SOCH3, SO2CH3, ONO2, NO2, N3, NH2; heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino, substituted silyl, RNA cleavage group, reporter group, intercalator, group for improving the pharmacokinetic properties of inhibitory nucleic acid, or group for improving the pharmacodynamic properties of inhibitory nucleic acid, and other substituents with similar properties. In some embodiments, the modification includes 2'-methoxyethoxy (2'-O--CH2CH2OCH3, also known as 2'-O-(2-methoxyethyl) or 2'-MOE) (Martin et al., Helv. Chim. Acta, 1995, 78: 486-504), i.e., an alkoxy-alkoxy group. Another exemplary modification is 2'-dimethylaminooxyethoxy, i.e., O(CH2)2ON(CH3)2 group, also known as 2'-DMAOE, as described in the Examples below; and 2'-dimethylaminoethoxyethoxy (also known in the art as 2'-O-dimethylaminoethoxyethyl or 2'-DMAEOE), i.e., 2'-O--CH2--O--CH2--N(CH2)2, as described in the Examples below.

[0117] Other modifications include 2'-methoxy (2'-OCH3), 2'-aminopropoxy (2'-OCH2CH2CH2NH2), and 2'-fluoro (2'-F). Similar modifications can also be made at other positions on the nucleotides of the modified nucleic acid, particularly the 3' position of the sugar in a 2'-5' linked dsRNA or on the 3' terminal nucleotide, and the 5' position of the 5' terminal nucleotide. Inhibitory nucleic acids can also have sugar mimetics, such as cyclobutyl moieties in place of the pentofuranosyl sugar. Representative U.S. patents that teach the preparation of such modified sugar structures include, but are not limited to, U.S. Patent Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265; 5,658,873; 5,670,633; and 5,700,920, some of which are commonly owned as provisional applications and are each incorporated herein by reference.

[0118] Inhibitory nucleic acids may also include modifications or substitutions of nucleobases (often referred to in the art as "bases"). As used herein, "unmodified" or "natural" nucleobases include: the purine bases adenine (A) and guanine (G); and the pyrimidine bases thymine (T), cytosine (C), and uracil (U). Modified nucleobases include other synthetic and natural nucleobases, such as: 5-methylcytosine (5-me-C); 5-hydroxymethylcytosine; xanthine; hypoxanthine; 2-aminoadenine; 6-methyl derivatives and other alkyl derivatives of adenine and guanine; 2-propyl derivatives and other alkyl derivatives of adenine and guanine; 2-thiouracil, 2-thiothymine, and 2-thiocytosine; 5-halouracil and 5-halocytosine; 5-propynyluracil and 5-propynylcytosine; 6-azouracil, 6-methyl- ...6-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl-2-thiouracil, 6-methyl-2-thiouracil -azocytosine and 6-azothymine; 5-uracil (pseudouracil); 4-thiouracil; 8-halogen, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxy and other 8-substituted adenines and guanines; 5-halogen (especially 5-bromo), 5-trifluoromethyl and other 5-substituted uracils and cytosines; 7-methylguanine and 7-methyladenine; 8-azaguanine and 8-azaadenine; 7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine. Further nucleobases include those disclosed in: U.S. Patent No. 3,687,808; Modified Nucleosides in Biochemistry, Biotechnology and Medicine, Herdewijn, P., ed., Wiley-VCH, 2008; The Concise Encyclopedia Of Polymer Science And Engineering, pp. 858-859, Kroschwitz, JL, ed., John Wiley & Sons, 1990; Englisch et al., Angewandte Chemie, International Edition, 1991, 30, 613; and Sanghvi, Y S., dsRNA Research and Applications, Chapter 15, pp. 289-302, Crooke, ST and Lebleu, B., eds., CRC Press, 1993. Some of these nucleobases are particularly useful for increasing the binding affinity of the oligomeric compounds featured in the present invention. These nucleobases include: 5-substituted pyrimidines; 6-azapyrimidines; and N-2, N-6, and O-6 substituted purines, including 2-aminopropyladenine, 5-propynyluracil, and 5-propynylcytosine.5-Methylcytosine substitutions have been shown to increase nucleic acid duplex stability by 0.6-1.2°C (Sanghvi, YS, Crooke, ST and Lebleu, B., eds., dsRNA Research and Applications, CRC Press, Boca Raton, 1993, pp. 276-278) and are exemplary base substitutions, even more particularly when combined with 2'-O-methoxyethyl sugar modifications.

[0119] Representative U.S. patents that teach the preparation of some of the aforementioned modified nucleobases, as well as other modified nucleobases, include, but are not limited to, the aforementioned U.S. Patent No. 3,687,808, and U.S. Patent Nos. 4,845,205, 5,130,30, 5,134,066, 5,175,273, 5,367,066, 5,432,272, 5,457,187, 5,459,255, 5,484,908, 5,502,177, 5,525,711, 5,552,540, 5,587,469, 5,5 7,427,672, and 7,495,088, each of which is incorporated herein by reference; and U.S. Patent No. 5,750,692, which is also incorporated herein by reference.

[0120] The nucleic acid of the inhibitory nucleic acid can also be modified to include one or more locked nucleic acids (LNA). Locked nucleic acids are nucleotides with a modified ribose moiety, wherein the ribose moiety includes an additional bridge connecting the 2' and 4' carbons. This structure effectively "locks" the ribose in a 3'-inward structural conformation. It has been shown that adding locked nucleic acids to siRNA increases the stability of siRNA in serum and reduces off-target effects (Elmen, J. et al., (2005) Nucleic Acids Research 33(1):439-447; Mook, OR. et al., (2007) Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic Acids Research 31(12):3185-3193).

[0121] Representative U.S. patents that teach the preparation of locked nucleic acid nucleotides include, but are not limited to, the following: U.S. Patent Nos. 6,268,490, 6,670,461, 6,794,499, 6,998,484, 7,053,207, 7,084,125, and 7,399,845, each of which is incorporated herein by reference in its entirety.

[0122] Another modification of the nucleic acid of the inhibitory nucleic acids featured in the invention involves chemically linking to the inhibitory nucleic acid one or more ligands, moieties, or conjugates that enhance the cellular uptake, activity, cellular distribution, or pharmacokinetic properties of the inhibitory nucleic acid. Such moieties include, but are not limited to, lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acid. Sci. USA, 1989, 86:6553-6556); cholic acid (Manoharan et al., Biorg. Med. Chem. Let., 1994, 4:1053-1060); thioethers, such as beryl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993, 3:2765-2770); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533-538); aliphatic chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanov et al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie, 1993, 75:49-54); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al., Nucl. Acids Res., 1990, 18:3777-3783); polyamines or polyethylene glycol chains (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969-973); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654); a palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229-237); or an octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923-937).

[0123] In one embodiment, the ligand alters the distribution, targeting, or lifetime of the inhibitory nucleic acid agent into which it is incorporated. In preferred embodiments, the ligand provides enhanced affinity for a selected target (e.g., a molecule, a cell or cell type, a compartment (e.g., a cell or organ compartment of the body), a tissue, an organ, or a region) compared to a species in which such a ligand does not exist. Preferred ligands will not participate in duplex pairing in duplex nucleic acids.

[0124] The ligand can include naturally occurring substances, such as proteins (e.g., human serum albumin (HSA), low-density lipoprotein (LDL), or globulins); carbohydrates (e.g., dextrose, pullulan, chitin, chitosan, inulin, cyclodextrin, or hyaluronic acid); or lipids. The ligand can also be a recombinant or synthetic molecule, such as a synthetic polymer, such as a synthetic polyamino acid. Examples of polyamino acids include polylysine (PLL), poly-L-aspartic acid, poly-L-glutamic acid, styrene-maleic anhydride copolymer, poly(L-lactide-co-glycolylated) copolymer, divinyl ether-maleic anhydride copolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA), polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane, poly(2-ethyl acrylic acid), N-isopropylacrylamide polymer, or polyphosphazene. Examples of polyamines include polyethyleneimine, polylysine (PLL), spermine, spermidine, polyamines, pseudopeptide-polyamines, peptidomimetic polyamines, dendrimer polyamines, arginine, amidine, protamine, cationic lipids, cationic porphyrins, quaternary salts of polyamines, or α-helical peptides.

[0125] The ligand can also include a targeting group, such as a cell or tissue targeting agent, such as a lectin, glycoprotein, lipid or protein (e.g., an antibody) that binds to a specific cell type (e.g., hepatocytes or macrophages). The targeting group can be thyrotropin, melanocyte stimulating hormone, a lectin, a glycoprotein, surfactant protein A, a mucin carbohydrate, multivalent lactose, multivalent galactose, N-acetylgalactosamine, N-acetyl glucosamine multivalent mannose, multivalent fucose, a glycosylated polyamino acid, multivalent galactose, transferrin, a bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, a steroid, bile acid, folic acid, vitamin B12, vitamin A, biotin, or an RGD peptide or RGD peptide mimetic.

[0126] Other examples of ligands include dyes, intercalating agents (e.g., acridine), cross-linking agents (e.g., psoralen, mitomycin C), porphyrins (TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g., phenazine, dihydrophenazine), artificial endonucleases (e.g., EDTA), lipophilic molecules (e.g., cholesterol, cholic acid, adamantaneacetic acid, 1-pyrenebutyric acid, dihydrotestosterone, 1,3-bis-O-(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol, borneol, menthol, 1,3-propylene glycol, heptadecyl group, palmitic acid, myristic acid, O3-(oleoyl)lithocholic acid, O3-(oleoyl) ) cholenic acid, dimethoxytrityl or phenoxazine) and peptide conjugates (e.g., antennapedia peptides, Tat peptides), alkylating agents, phosphates, amino groups, sulfhydryl groups, PEG (e.g., PEG-40K), MPEG, [MPEG]2, polyamino, alkyl, substituted alkyl, radiolabeled markers, enzymes, haptens (e.g., biotin), transport / absorption enhancers (e.g., aspirin, vitamin E, folic acid), synthetic ribonucleases (e.g., imidazole, bisimidazole, histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+ complexes of tetraazamacrocycles), dinitrophenyl, HRP or AP.

[0127] The ligand can be a protein, such as a glycoprotein or a peptide, for example a molecule with a specific affinity for a co-ligand, or an antibody (e.g., an antibody that binds to a specified cell type, such as a hepatocyte or a macrophage). The ligand can also include hormones and hormone receptors. They can also include non-peptide substances, such as lipids, lectins, carbohydrates, vitamins, cofactors, multivalent lactose, multivalent galactose, N-acetylgalactosamine, N-acetylglucosamine multivalent mannose or multivalent fucose.

[0128] The ligand can be, for example, a drug that increases the uptake of the inhibitory nucleic acid agent into the cell by, for example, disrupting the cell's cytoskeleton (e.g., by disrupting the cell's microtubules, microfilaments, and / or intermediate filaments). The drug can be, for example, a taxon, vincristine, vinblastine, cytochalasin, nocodazole, japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, or myoservin.

[0129] In some embodiments, the ligand connected to the inhibitory nucleic acid as described herein serves as a pharmacokinetic (PK) regulator. "PK regulator" as used herein refers to a pharmacokinetic regulator. PK regulators include lipophiles, bile acids, steroids, phospholipid analogs, peptides, protein binders, PEG, vitamins, etc. Examples of PK regulators include, but are not limited to, cholesterol, fatty acids, cholic acid, lithocholic acid, dialkyl glycerides, diacylglyceride phospholipids, sphingolipids, naproxen, ibuprofen, vitamin E, biotin, etc. It is also known that the oligonucleotides comprising many phosphorothioate connections are bound to serum proteins. Therefore, short oligonucleotides (such as oligonucleotides of about 5 bases, 10 bases, 15 bases or 20 bases) comprising multiple phosphorothioate connections in the backbone can be used as ligands (such as PK regulators) in the present invention. In addition, the aptamers in conjunction with serum components (such as serum proteins) are also suitable for use as PK regulators in the embodiments described herein.

[0130] For macromolecular drugs and hydrophilic drug molecules that can not easily pass through double membrane, the retention in the endosome / lysosome compartment of cell is considered to be the biggest obstacle of being effectively delivered to its site of action. Many methods and strategies have been designed to solve this problem. For liposome preparations, the use of fusion lipids in preparations is modal method (Singh, RS, Goncalves, C. etc. (2004), On the Gene Delivery Efficacies of pH-Sensitive Cationic Lipids via Endosomal Protonation.A Chemical Biology Investigation, Chem.Biol., 11,713-723). Other components of the endosomal lysis activity that show pH sensitivity by protonation and / or pH-induced conformational change include charged polymer and peptide. Examples can be found in: Hoffman, AS, Stayton, PS et al. (2002), Design of “smart” polymers that can direct intracellular drug delivery. Polymers Adv. Technol., 13, 992-999; Kakudo, Chaki, T., S. et al. (2004), Transferrin-Modified Liposomes Equipped with a pH-Sensitive FusogenicPeptide: An Artificial Viral-like Delivery System. Biochemistry 436, 5618-5628; Yessine, MA and Leroux, JC (2004), Membrane-destabilizing polyanions: interaction with lipid bilayers and endosomal escape of biomacromolecules., Adv.DrugDeliv.Rev., 56, 999-1021; Oliveira, S., van Rooy, I. et al. (2007), Fusogenic peptidesenhance endosomal escape improving inhibitory nucleic acid-induced silencing of oncogenes., Int. J. Pharm., 331, 211-4.They are commonly used in the context of drug delivery systems, such as liposomes or lipid complexes. For the delivery of folate receptor-mediated delivery using liposome formulations, for example, pH-sensitive fusogenic peptides have been incorporated into liposomes and demonstrated enhanced activity by improving the unloading of drugs during the uptake process (Turk, MJ, Reddy, JA et al. (2002), Biochim.Biophys.Acta, 1559, 56-68. Characterization of a novel pH-sensitive peptide that enhances drug release from folate-targeted liposomes at endosomal pHs).

[0131] In some embodiments, the endosomal dissolution component (endosmolytic component) can be a polyanionic peptide or peptide mimetic that shows pH-dependent membrane activity and / or fusogenicity. Peptide mimetic can be a small protein-like chain designed to mimic peptides. Peptide mimetic can be produced from the modification of existing peptides to change the properties of the molecule, or use non-natural amino acids or their analogs to synthesize peptide-like molecules. In some embodiments, when compared with peptides, they have improved stability and / or biological activity. In some embodiments, the endosomal dissolution component presents its active conformation under endosomal pH (e.g., pH 5-6). "Active" conformation is a conformation in which the endosomal dissolution component promotes the endosomal cleavage of the modular composition of the present invention (modular composition) or any of its components (e.g., nucleic acids) and / or is transported from the endosomal to the cytoplasm of the cell.

[0132] Exemplary endosomolytic components include GALA peptide (Subbarao et al., Biochemistry, 1987, 26: 2964-2972), EALA peptide (Vogel et al., J. Am. Chem. Soc., 1996, 118: 1581-1586) and derivatives thereof (Turk et al., Biochem. Biophys. Acta, 2002, 1559: 56-68). In certain embodiments, the endosomolytic component may contain a chemical group (e.g., an amino acid) that undergoes a charge change or protonation in response to a change in pH. The endosomolytic component may be linear or branched. Exemplary primary sequences of endosomolytic components include H2N-(AALEALAEALEALAEALEALAEAAAAGGC)-CO2H (SEQ ID NO: 16), H2N-(AALAEALAEALAEALAEALAEALAEALAAAAGGC)-CO2H (SEQ ID NO: 17), and H2N-(ALEALAEALEALAEA)-CONH2 (SEQ ID NO: 18).

[0133] In certain embodiments, more than one endosomolytic component can be incorporated into the inhibitory nucleic acid agents of the present invention. In some embodiments, this requires incorporating more than one of the same endosomolytic component into the inhibitory nucleic acid agent. In other embodiments, this requires incorporating two or more different endosomolytic components into the inhibitory nucleic acid agent.

[0134] These endosomal lytic components can mediate endosomal escape by, for example, changing the conformation at endosomal pH. In certain embodiments, the endosomal lytic component can exist in a random coil conformation at neutral pH and rearrange to an amphipathic helix at endosomal pH. As a result of this conformational transition, these peptides may be inserted into the lipid membrane of the endosome, causing the endosomal contents to leak into the cytoplasm. Since the conformational transition is pH-dependent, the endosomal lytic component can show little or no fusogenic activity (fusogenic activity) when circulating in the blood (pH ~ 7.4). "Fusogenic activity" as used herein is defined as the activity that causes the endosomal lytic component to destroy the lipid membrane. An example of fusogenic activity is that the endosomal lytic component destroys the endosomal membrane, causing endosomal lysis or leakage, and transporting one or more components (e.g., nucleic acids) of the modular composition of the present invention from the endosome to the cytoplasm.

[0135] Suitable endosomal dissolution components can be tested and identified by those skilled in the art. For example, the ability of a compound to respond to, for example, a change in charge according to a pH environment can be determined by conventional methods, such as cell assays. In certain embodiments, the test compound is combined with a cell or contacted with a cell, and the cell is allowed to internalize the test compound, for example, by endocytosis. Then, an endosomal preparation is made from the contacted cell, and the endosomal preparation is compared with an endosomal preparation from a control cell. Relative to the endosomal portion from the control cell, a change (e.g., a decrease) in the endosomal portion from the contacted cell indicates that the test compound can act as a fusogenic agent. Alternatively, the contacted cells and control cells can be evaluated, for example, by microscopy (e.g., by optical microscopy or electron microscopy) to determine the difference in the endosomal population in the cell. The test compound and / or endosome can be labeled, for example, to quantify endosomal leakage.

[0136] In another test type, use one or more tests or the fusogenic agent of inference to build inhibitory nucleic acid reagent as herein described.Inhibitory nucleic acid reagent can be marked so that observation.In case inhibitory nucleic acid reagent is taken up by cells, can for example, by preparing endosome preparation or by microscopy (this makes the inhibitory nucleic acid acidic agent through marking in the Cytoplasm of cell be visualized) come the ability that endosome dissolving component is promoted to escape from endosomal body and is assessed.In some other embodiments, the inhibition of gene expression or any other physiological parameter can be used as the surrogate marker of endosome escape.

[0137] In other embodiments, circular dichroism spectroscopy can be used to identify compounds that exhibit pH-dependent structural transitions. A two-step assay can also be performed, wherein a first assay evaluates the ability of a test compound alone to respond to a change in pH, and a second assay evaluates the ability of a modular composition comprising the test compound to respond to a change in pH.

[0138] In one embodiment of the aspects described herein, the ligand or conjugate is a lipid or lipid-based molecule. Such lipid or lipid-based molecules preferably bind to serum proteins, such as human serum albumin (HSA). HSA binding ligands allow the conjugate to be distributed to target tissues, such as non-renal target tissues of the body. Other molecules that can bind to HSA can also be used as ligands. For example, neproxin or aspirin can be used. The lipid or lipid-based ligand can (a) increase resistance to degradation of the conjugate, (b) increase targeting or transport to target cells or cell membranes, and / or (c) be used to modulate binding to serum proteins (e.g., HSA).

[0139] On the other hand, the ligand is a cell permeating agent, preferably a helical cell permeating agent. Preferably, such agents are amphipathic. Exemplary agents are peptides, such as tat or antennapedia peptides. If the agent is a peptide, it may be modified, including peptide mimetics, reverse mimics, non-peptide or pseudopeptide linkages, and the use of D-amino acids. The helical agent is preferably an α-helical agent, which preferably has a lipophilic phase and a lipophobic phase.

[0140] Peptides suitable for use in the present invention can be natural peptides (e.g., tat or antennapedia peptides), synthetic peptides, or peptide mimetics. In addition, the peptide can be a modified peptide, for example, the peptide can contain non-peptide or pseudopeptide connections and D-amino acids. Peptide mimetics (also referred to herein as oligopeptide mimetics) are molecules that can fold into a limited three-dimensional structure similar to a natural peptide. Peptides and peptide mimetics attached to inhibitory nucleic acid agents can, for example, affect the pharmacokinetic distribution of inhibitory nucleic acids by enhancing cell recognition and absorption. The length of the peptide or peptide mimetic portion can be about 5-50 amino acids, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids.

[0141] The peptide or peptide mimetic can be, for example, a cell-penetrating peptide, a cationic peptide, an amphipathic peptide, or a hydrophobic peptide (e.g., composed primarily of Tyr, Trp, or Phe). The peptide portion can be a dendrimer peptide, a contrained peptide, or a cross-linked peptide. In another alternative, the peptide portion can include a hydrophobic membrane translocation sequence (MTS). An exemplary hydrophobic MTS-containing peptide is RFGF having the amino acid sequence AAVALLPAVLLALLAP (SEQ ID NO: 19). An RFGF analog containing a hydrophobic MTS (e.g., the amino acid sequence AALLPVLLAAP (SEQ ID NO: 20)) can also be a targeting moiety. The peptide portion can be a "delivery" peptide that can carry large polar molecules including peptides, oligonucleotides, and transcellular membrane proteins. For example, sequences from the HIVTat protein (GRKKRRQRRRPPQ (SEQ ID NO: 21)) and the Drosophila antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 22)) have been found to be effective as delivery peptides. The peptide or peptide mimetic can be encoded by a random sequence of DNA, such as a peptide identified from a phage display library or a "one bead one compound" (OBOC) combinatorial library (Lam et al., Nature, 354:82-84, 1991). Preferably, the peptide or peptide mimetic linked to the dsRNA agent via an incorporated monomer unit is a cell targeting peptide, such as an arginine-glycine-aspartic acid (RGD)-peptide or RGD mimetic. The peptide portion can be from about 5 amino acids to about 40 amino acids in length. The peptide portion can have structural modifications, such as to increase stability or direct conformational properties. Any of the structural modifications described below can be used.

[0142] "Cell penetrating peptides" are capable of penetrating cells (e.g., microbial cells such as bacterial or fungal cells, or mammalian cells such as human cells). Microbial cell penetrating peptides can be, for example, α-helical linear peptides (e.g., LL-37 or CeropinP1), disulfide bond-containing peptides (e.g., α-defensin, β-defensin, or bovine antimicrobial peptide), or peptides containing only one or two dominant amino acids (e.g., PR-39 or indolicidin). Cell penetrating peptides can also include a nuclear localization signal (NLS). For example, a cell penetrating peptide can be a bidirectional amphipathic peptide, such as MPG (Simeoni et al., Nucl. Acids Res, 31: 2717-2724, 2003), which is derived from the fusion peptide domain of HIV-1 gp41 and the NLS of the SV40 large T antigen.

[0143] In some embodiments, the inhibitory nucleic acid oligonucleotides described herein further comprise carbohydrate conjugates. Carbohydrate conjugates are beneficial for the in vivo delivery of nucleic acids, and compositions described herein for in vivo therapeutic applications. As used herein, "carbohydrate" refers to a compound that is itself a carbohydrate consisting of one or more monosaccharide units with at least 6 carbon atoms (which may be linear, branched, or cyclic) and oxygen atoms, nitrogen atoms, or sulfur atoms bonded to each carbon atom, or a compound having a carbohydrate moiety consisting of one or more monosaccharide units as part thereof, each of which has at least six carbon atoms (which may be linear, branched, or cyclic), wherein oxygen atoms, nitrogen atoms, or sulfur atoms are bonded to each carbon atom. Representative carbohydrates include sugars (monosaccharides, disaccharides, trisaccharides, and oligosaccharides containing about 4-9 monosaccharide units) and polysaccharides (e.g., starch, glycogen, cellulose, and polysaccharide gums). Specific monosaccharides include sugars with C5 or more (preferably C5-C8); disaccharides and trisaccharides (including sugars with two or three monosaccharide units (preferably C5-C8)). In some embodiments, the carbohydrate conjugate further comprises other ligands, such as, but not limited to, PK modulators, endosomolytic ligands, and cell penetrating peptides.

[0144] In some embodiments, the conjugates described herein can be linked to inhibitory nucleic acid oligonucleotides using various linkers, cleavable or non-cleavable.The term "linker" or "linking group" refers to an organic moiety that connects two parts of a compound. The linker typically comprises a direct bond or an atom such as oxygen or sulfur, a unit such as NR8, C(O), C(O)NH, SO, SO2, SO2NH, or a chain of atoms such as, but not limited to, substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, arylalkyl, arylalkenyl, arylalkynyl, heteroarylalkyl, heteroarylalkenyl, heteroarylalkynyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, cycloalkenyl, alkylarylalkyl, alkylarylalkenyl, alkylarylalkenyl, alkenylarylalkyl, alkenylarylalkenyl, alkenylarylalkynyl, alkynylarylalkyl, alkynylarylalkenyl, alkynylarylalkynyl, alkylheteroarylalkyl, alkylheteroarylalkenyl, alkyl heteroarylalkynyl, alkenylheteroarylalkyl, alkenylheteroarylalkenyl, alkenylheteroarylalkynyl, alkynylheteroarylalkyl, alkynylheteroarylalkenyl, alkynylheteroarylalkynyl, alkylheterocyclylalkyl, alkylheterocyclylalkenyl, alkylheterocyclylalkynyl, alkenylheterocyclylalkyl, alkenylheterocyclylalkenyl, alkenylheterocyclylalkynyl, alkylaryl, alkenylaryl, alkynylaryl, alkylheteroaryl, alkenylheteroaryl, alkynylheteroaryl, wherein one or more methylene groups may be interrupted by O, S, S(O), SO2, N(R8), C(O), substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, wherein R8 is hydrogen, acyl, aliphatic or substituted aliphatic. In one embodiment, the linker is 1-24 atoms, preferably 4-24 atoms, preferably 6-18 atoms, more preferably 8-18 atoms and most preferably 8-16 atoms.

[0145] A cleavable linking group is a linking group that is sufficiently stable outside the cell but is cleaved upon entry into the target cell to release the two moieties held together by the linker. In a preferred embodiment, the cleavable linking group is cleaved at least 10 times faster, preferably at least 100 times faster, in the target cell or under a first reference condition (which can, for example, be selected to simulate or represent conditions within the cell) than in the blood of a subject or under a second reference condition (which can, for example, be selected to simulate or represent conditions found in blood or serum).

[0146] The cleavable linking group is sensitive to a cleavage agent (e.g., pH, redox potential, or the presence of a degrading molecule). Typically, the cleavage agent is more prevalent or found at higher levels or activity within cells than in serum or blood. Examples of such degrading agents include: redox agents that are selected for specific substrates or that do not have substrate specificity, including, for example, oxidizing or reductive enzymes or reducing agents (e.g., thiols) present in cells that can degrade the redox-cleavable linking group by: reduction; esterases; endosomal or acidic environment-generating agents (e.g., those that result in a pH below 5); enzymes that can hydrolyze or degrade acid-cleavable linking groups by acting as general acids, peptidases (which can be substrate-specific), and phosphatases.

[0147] The cleavable linking group (e.g., disulfide bond) can be sensitive to pH. The pH of human serum is 7.4, while the average intracellular pH is slightly lower, ranging from about 7.1-7.3. Endosomes have a more acidic pH, ranging from 5.5-6.0, and lysosomes have an even more acidic pH of about 5.0. Some linkers will have a cleavable linking group that is cleaved at a preferred pH, thereby releasing the cationic lipid from the ligand inside the cell, or releasing it into the desired compartment of the cell.

[0148] The linker may comprise a cleavable linking group that can be cleaved by a specific enzyme. The type of cleavable linking group incorporated into the linker may depend on the cell to be targeted. Further examples of cleavable linking groups include, but are not limited to, redox cleavable linking groups (e.g., disulfide linking groups (-SS-)), phosphate-based cleavable linking groups, ester-based cleavable linking groups, and peptide-based cleavable linking groups. Representative U.S. patents for teaching the preparation of RNA conjugates include, but are not limited to, U.S. Patent Nos. 4,828,979, 4,948,882, 5,218,105, 5,525,465, 5,541,313, 5,545,730, 5,552,538, 5,578,717, 5,580,731, 5,591,584, 5,109,124, 5,118,802, 5,138,045, 5,414,07 7、5,486,603、5,512,439、5,578,718、5,608,046、4,587,044、4,605,735、4,667,025、4,762,779、4,789,737、4,824,941、4,835,263、4,876,335、4,904,582、4,958,013、5,082,830、5,112,963、5,214,136、 5,082,830、5,112,963、5,214,136、5,245,022、5,254,469、5,258,506、5,262,536、5,272,250、5,292,873、5,317,098、5,371,241、5,391,723、5,416,203、5,451,463、5,510,475、5,512,667、5,514,785、5, 7,037,646, each of which is incorporated herein by reference.

[0149] Typically, the suitability of a candidate's cleavable linking group can be assessed by testing the ability of a degradation agent (or condition) to cut a candidate's linking group. It is also desirable that the ability of the candidate's cleavable linking group to resist cutting in blood or when contacted with other non-targeted tissues is tested. Therefore, the relative susceptibility of the cutting between the first condition and the second condition can be determined, wherein the first condition is selected to select the cutting shown in the target cell, and the second condition is selected to illustrate the cutting in other tissues or biofluids (such as blood or serum). Assessment can be carried out in a cell-free system, cell, cell culture, organ or tissue culture or whole animal. Under cell-free conditions or under culture conditions, preliminary assessment is carried out and confirmed by further assessment in whole animal and may be useful. In a preferred embodiment, useful candidate compounds in cells (or under in vitro conditions selected for simulating intracellular conditions) are faster than cutting at least 2 times, 4 times, 10 times or 100 times in blood or serum (or under conditions selected for simulating extracellular conditions).

[0150] It is not necessary to uniformly modify all positions in a given compound; in fact, more than one of the above modifications may be incorporated into a single compound or even into a single nucleoside within an inhibitory nucleic acid. The present invention also includes inhibitory nucleic acid compounds that are chimeric compounds. In the context of the present invention, "chimeric" inhibitory nucleic acid compounds or "chimeras" are inhibitory nucleic acid compounds, such as dsRNA, that contain two or more chemically distinct regions, each region consisting of at least one monomeric unit (i.e., nucleotides in the case of dsRNA compounds). These inhibitory nucleic acids typically contain at least one region in which the nucleic acid is modified to impart increased resistance to nuclease degradation, increased cellular uptake, and / or increased binding affinity to the target nucleic acid to the inhibitory nucleic acid. Additional regions of the inhibitory nucleic acid can serve as substrates for enzymes capable of cleaving RNA:DNA or RNA:RNA hybrids. For example, RNase H is a cellular endonuclease that cleaves the RNA strand of an RNA:DNA duplex. Thus, activation of RNase H results in cleavage of the RNA target, thereby greatly increasing the effectiveness of the inhibitory nucleic acid in inhibiting gene expression. Thus, when using chimeric inhibitory nucleic acids, comparable results can generally be achieved using shorter inhibitory nucleic acids than, for example, phosphothioate deoxy dsRNAs that hybridize to the same target region. Cleavage of the RNA target can be routinely detected by gel electrophoresis and, if necessary, by relevant nucleic acid hybridization techniques known in the art.

[0151] In some cases, the nucleic acid of the inhibitory nucleic acid can be modified with a non-ligand group. Many non-ligand molecules have been conjugated to inhibitory nucleic acids to enhance the activity, cellular distribution or cellular uptake of the inhibitory nucleic acid, and procedures for performing such conjugations are available in the scientific literature. Such non-ligand moieties include lipid moieties, such as cholesterol (Kubo, T. et al., Biochem. Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl. Acids. Sci. USA, 1989, 86:6553); cholic acid (Manoharan et al., Bioorg. Med. Chem. Lett., 1994, 4:1053); thioethers, such as hexyl-S-tritylthiol (Manoharan et al., Ann. NY Acad. Sci., 1992, 660:306; Manoharan et al., Bioorg. Med. Chem. Lett., 1993, 3:2765); thiocholesterol (Oberhauser et al., Nucl. Acids Res., 1992, 20:533); fatty chains, such as dodecanediol or undecyl residues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk et al., Biochimie, 1993, 75:49); phospholipids, such as di-hexadecyl-rac-glycerol or triethylammonium 1,2-di-O-hexadecyl-rac-glycero-3-H-phosphate (Manoharan et al., Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990, 18:3777); polyamine or polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14:969); or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36:3651); palmitoyl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229); or octadecylamine or hexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative U.S. patents teaching the preparation of such nucleic acid conjugates are listed above. A typical conjugation protocol involves synthesizing a nucleic acid bearing an amino linker at one or more positions in the sequence. The amino group is then reacted with the molecule to be conjugated using a suitable coupling agent or activator.The conjugation reaction can be performed with the nucleic acid still bound to the solid support or after cleavage of the nucleic acid in solution phase. Purification of the nucleic acid conjugate by HPLC generally provides a pure conjugate.

[0152] The term "aptamer" refers to a nucleic acid molecule that is capable of binding to a target molecule (e.g., a polypeptide). For example, the aptamers of the present invention can specifically bind to a target molecule or to a molecule in a signaling pathway that regulates the expression and / or activity of the target molecule. The production and therapeutic use of aptamers are well known in the art. See, for example, U.S. Patent No. 5,475,096.

[0153] As used herein, the term "specific binding" refers to a chemical interaction between two molecules, compounds, cells, and / or particles wherein a first entity binds to a second target entity with greater specificity and affinity than the first entity binds to a third, non-target entity. In some embodiments, specific binding can mean that the first entity has an affinity for the second target entity that is at least 10-fold, at least 50-fold, at least 100-fold, at least 500-fold, at least 1000-fold, or greater than the affinity of the first entity for a third, non-target entity. An agent specific for a given target is one that exhibits specific binding to that target under the assay conditions used.

[0154] As used herein, the terms "treat / treatment / treating" or "alleviate" refer to treatment with a therapeutic agent, wherein the purpose is to reverse, alleviate, reduce, inhibit, slow down or stop the progression or severity of a condition associated with a disease or disorder (e.g., cancer). The term "treat" includes reducing or alleviating at least one adverse effect or symptom of a condition, disease or disorder associated with cancer. A treatment is generally "effective" if one or more symptoms or clinical markers are reduced. Alternatively, a treatment is "effective" if the progression of the disease is reduced or stopped. That is, "treatment" includes not only improvement of symptoms or markers, but also cessation or at least slowing of the progression or worsening of symptoms compared to what would be expected without treatment. Beneficial or desired clinical results (whether measurable or unmeasurable) include, but are not limited to: alleviating one or more symptoms, reducing the extent of the disease, stabilizing the disease state (i.e., not worsening), retarding or slowing the progression of the disease, alleviating or slowing the disease state, palliating (partially or completely), and / or reducing mortality. The term "treating" a disease also includes providing relief from the symptoms or side effects of the disease (including palliative care).

[0155] As used herein, the term "pharmaceutical composition" refers to an active agent in combination with a pharmaceutically acceptable carrier (e.g., a carrier commonly used in the pharmaceutical industry). As used herein, the phrase "pharmaceutically acceptable" refers to compounds, materials, compositions, and / or dosage forms that are, within the scope of sound medical judgment, suitable for use in contact with the tissues of humans and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio.

[0156] As used herein, the term "administer" refers to administering a compound disclosed herein to a subject by a method or route such that at least a portion of the agent is delivered to the desired site. Pharmaceutical compositions containing the compounds disclosed herein can be administered by any appropriate route to produce effective treatment in the subject.

[0157] The term "statistically significant" or "significantly" refers to statistical significance, and typically refers to a difference of 2 standard deviations (2SD) or greater.

[0158] Other than in the operating examples, or where otherwise indicated, all numbers expressing quantities of ingredients or reaction conditions used herein are to be understood as modified in all instances by the term “about.” The term “about” when used in connection with a percentage can mean ±1%.

[0159] As used herein, the terms "comprising" or "including" refer to components that are essential to a method or composition, method, and their respective ingredients, and are open to the inclusion of unspecified elements, whether essential or non-essential.

[0160] The term "consisting of" means that the compositions, methods, and their respective ingredients described herein exclude any elements not recited in the description of the embodiment.

[0161] As used herein, the term "consisting essentially of refers to the elements required for a given embodiment. The term permits the presence of elements that do not materially affect the basic and novel or functional characteristics of the embodiment.

[0162] Unless the context clearly indicates otherwise, the singular terms "a / an / the" include plural referents. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "eg" is derived from the Latin exempli gratia and is used herein to indicate a non-limiting example. Therefore, the abbreviation "eg" is synonymous with the term "for example."

[0163] Definitions of commonly used terms in cell biology and molecular biology can be found in the following references: "The Merck Manual of Diagnosis and Therapy", 19th edition, published by Merck Research Laboratories, 2006 (ISBN 0-911910-19-0); Robert S. Porter et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9); Benjamin Lewin, Genes X, published by Jones & Bartlett Publishing, 2009 (ISBN-10: 0763766321); Kendrew et al. (eds.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8); and Current Protocols in Protein Sciences 2009, Wiley Intersciences, Coligan et al., eds.

[0164] Unless otherwise indicated, the present invention was performed using standard procedures described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual (4th Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA (2012); Davis et al., Basic Methods in Molecular Biology, Elsevier Science Publishing Inc., New York, USA (1995); or Methods in Enzymology: Guide to Molecular Cloning Techniques, Vol. 152, ed. S. L. Berger and A. R. Kimmel, Academic Press Inc., San Diego, USA (1987); Current Protocols in Protein Science (CPPS) (ed. John E. Coligan et al., John Wiley and Sons, Inc.); Current Protocols in Cell Biology (CPCB) (ed. Juan S. Bonifacino et al., John Wiley and Sons, Inc.); and Culture of Animal Cells: A Manual of Basic Technique, R. Ian Freshney, published by Wiley-Liss, 5th edition (2005); Animal Cell Culture Methods (Methods in Cell Biology, Vol. 57, Jennie P. Mather and David Barnes, eds., Academic Press, 1st edition, 1998), which are incorporated herein by reference in their entirety.

[0165] Additional terms are defined herein in the description of various aspects of the invention.

[0166] For the purpose of description and disclosure, all patents and other publications (including text publications, issued patents, published patent applications and simultaneously pending patent applications) cited in this application are expressly incorporated herein by reference, for example, the methodology described in such publications that can be used in conjunction with the technology described herein. These publications are provided only because they are disclosed earlier than the filing date of this application. In this regard, it should not be regarded as an admission that the inventors have no right to advance such disclosures by means of previous inventions or for any other reason. All statements about the dates of these documents or the representations about the contents of these documents are based on information available to the applicant and do not constitute any admission about the correctness of the dates or contents of these documents.

[0167] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the disclosure to the specific forms disclosed. Although specific embodiments and examples of the present disclosure are described herein for illustrative purposes, as will be appreciated by those skilled in the relevant art, various equivalent modifications may be made within the scope of the present disclosure. For example, when method steps or functions are given in a given order, alternative embodiments may perform the functions in a different order or may perform the functions substantially simultaneously. The teachings of the present disclosure provided herein may be applied to other appropriate procedures or methods. The various embodiments described herein may be combined to provide further embodiments. If necessary, aspects of the present disclosure may be modified to adopt combinations, functions, and concepts of the above-mentioned references and applications to provide further embodiments of the present disclosure. In addition, due to considerations of biological functional equivalence, some changes may be made to the protein structure in kind or quantity that do not affect the biological or chemical effects. These and other changes may be made to the present disclosure based on the inspiration of the detailed specification. All of these modifications are intended to be included within the scope of the appended claims.

[0168] Specific elements in any of the above embodiments may be combined or replaced with elements in other embodiments. In addition, although advantages associated with some embodiments of the present disclosure have been described in the context of these embodiments, other embodiments may also exhibit such advantages, and not all embodiments must exhibit such advantages to fall within the scope of the present disclosure.

[0169] The technology described herein is further illustrated by the following examples, which should not be construed as further limiting.

[0170] Some implementations of the technology described herein may be defined according to any of the following numbered paragraphs:

[0171] 1. A chimeric molecule comprising an aptamer domain that binds to a cancer marker and an inhibitory nucleic acid domain.

[0172] 2. The molecule of paragraph 1, wherein the cancer marker is EpCAM or EphA2.

[0173] 3. The molecule of any of paragraphs 1-2, wherein the molecule is an aptamer-siRNA chimera (AsiC).

[0174] 4. The molecule of any of paragraphs 1-3, wherein the inhibitory nucleic acid specifically binds to a gene product that is upregulated in cancer cells.

[0175] 5. The molecule of any of paragraphs 1-4, wherein the inhibitory nucleic acid inhibits expression of a gene selected from the group consisting of:

[0176] Plk1, MCL1, EphA2, PsmA2, MSI1, BMI1, XBP1, PRPF8, PFPF38A, RBM22, USP39, RAN, NUP205, and NDC80.

[0177] 6. The molecule of any of paragraphs 1-5, wherein the cancer marker is EpCAM and the inhibitory nucleic acid domain inhibits expression of Plk1.

[0178] 7. The molecule of any of paragraphs 1-6, wherein the cancer marker binding aptamer domain comprises the sequence of SEQ ID NO: 33.

[0179] 8. The molecule of any of paragraphs 1-6, wherein the cancer marker binding aptamer domain consists essentially of the sequence of SEQ ID NO: 33.

[0180] 9. The molecule of any of paragraphs 1-8, wherein the inhibitory nucleic acid domain comprises the sequence of SEQ ID NO: 2.

[0181] 10. The molecule of any of paragraphs 1-8, wherein the inhibitory nucleic acid domain consists essentially of the sequence of SEQ ID NO: 2.

[0182] 11. The molecule of any of paragraphs 1-10, comprising a sequence of one of SEQ ID NO: 1 to SEQ ID NO: 3.

[0183] 12. The molecule of any of paragraphs 1-11, consisting essentially of the sequence of one of SEQ ID NO: 1 to SEQ ID NO: 3.

[0184] 13. The molecule of any of paragraphs 1-12, wherein the 3' end of the molecule comprises dTdT.

[0185] 14. The molecule of any of paragraphs 1-13, wherein the molecule comprises at least one 2'-F pyrimidine.

[0186] 15. A pharmaceutical composition comprising the molecule of any of paragraphs 1-14 and a pharmaceutically acceptable carrier.

[0187] 16. The composition of paragraph 15, comprising at least two chimeric molecules of any of paragraphs 1-14, wherein the chimeric molecules have different aptamer domains or inhibitory nucleic acid domains.

[0188] 17. The composition of paragraph 16, wherein different aptamers or inhibitory nucleic acid domains recognize different targets.

[0189] 18. The composition of paragraph 16, wherein the different aptamers or inhibitory nucleic acid domains have multiple sequences and recognize the same target.

[0190] 19. A method of treating cancer comprising administering a molecule or composition as described in any one of paragraphs 1-18.

[0191] 20. The method of paragraph 19, wherein the cancer is epithelial cancer or breast cancer.

[0192] 21. The method of paragraph 20, wherein the breast cancer is triple-negative breast cancer.

[0193] 22. The method of any of paragraphs 19-21, wherein the administration is subcutaneous.

[0194] 23. The method of any of paragraphs 19-22, wherein the subject is further administered an additional cancer treatment.

[0195] 24. The method of paragraph 23, wherein the cancer therapy is paclitaxel.

[0196] Example

[0197] Example 1: Gene knockdown by EpCAM aptamer-siRNA chimera inhibits basal-like triple-negative breast cancer and its tumor-initiating cells

[0198] It is necessary to use RNA interference to treat cancer by delivering siRNA in vivo to knock down genes in cells outside the liver. EpCAM is a tumor-associated antigen that is highly expressed in common epithelial cancers and their tumor-initiating cells (T-IC, also referred to as cancer stem cells). This paper demonstrates that aptamer-siRNA chimeras (AsiC, EpCAM aptamers connected to siRNA sense strands and annealed to siRNA antisense strands) are selectively absorbed and knock down gene expression in human cancer biopsies and in vitro EpCAM+ cancer cells. PLK1 EpCAM-AsiC inhibits colony and mammosphere formation (in vitro T-IC assay) in nude mice, as well as tumor initiation caused by EpCAM+luminal and basal cell-A triple-negative breast cancer (TNBC) cell lines and not EpCAM-mesenchymal basal cells-B TNBCs. Subcutaneously administered EpCAM-AsiC concentrates and inhibits growth in EpCAM+Her2+ and TNBC tumors. Therefore, EpCAM-AsiC provides an attractive strategy for treating epithelial cancers.

[0199] introduce

[0200] RNA interference (RNAi) provides an opportunity to treat diseases by knocking down disease-causing genes 1 Recent early-stage clinical trials have demonstrated robust (75-95%), sustained (lasting weeks or up to months), and safe knockdown of a small number of gene targets in the liver using lipid nanoparticle-encapsulated siRNA or GalNAc-conjugated siRNA. 2-5 The liver is the body's primary filtration organ, trapping particles and therefore relatively easy to transfect. However, the major obstacles to using RNAi to treat most diseases (i.e., gene knockdown in cells other than the liver and effective delivery of small RNAs) have not yet been overcome. In particular, delivery barriers are a major obstacle to using RNAi to treat cancer. 6 .

[0201] Triple-negative breast cancer (TNBC) is a heterogeneous group of poorly differentiated cancers defined by the lack of expression of estrogen, progesterone, and Her2 receptors, which have the worst prognosis of any breast cancer subtype. 7-9 . Although a sizable minority is mesenchymal (basal-B subtype), the majority of TNBCs are epithelial in nature and are classified as basal-like or belonging to the basal-A subtype. TNBC affects young women and is a subtype associated with BRCA1 gene mutations. No targeted therapies are available. Although most TNBC patients respond to chemotherapy, about one-third develop metastases and ultimately die within 3 years. Therefore, new strategies are needed.

[0202] Described here is a flexible target platform for gene knockdown and treatment of basal-like TNBC, which may also be applicable to the treatment of most common (epithelial) cancers. We delivered small interfering RNAs (siRNAs) into epithelial cancer cells by linking them to RNA aptamers that bind to EpCAM, the first described tumor antigen and a cell surface receptor overexpressed in epithelial cancers including basal-like TNBC. Aptamer-linked siRNAs, termed aptamer-siRNA chimeras (AsiC), have been used in small animal models to treat prostate cancer and prevent HIV infection. 10-18 We chose EpCAM for targeting basal-like TNBC because EpCAM is highly expressed in epithelial cancers. High-affinity EpCAM aptamers were previously identified. {Shigdar, 2011#17903} EpCAM also marks tumor-initiating cells (T-ICs, also known as cancer stem cells). 20-27 These cells are not only thought to be responsible for initiating tumors, but are also relatively resistant to traditional cytotoxic therapies and are thought to be responsible for tumor recurrence and metastasis. Designing therapies to eliminate T-ICs is an important but unmet goal in cancer research. 28 .

[0203] In normal epithelial cells, EpCAM is only weakly expressed at basolateral gap junctions that may be inaccessible to drugs. 29 In epithelial cancers, it is not only more abundant (by orders of magnitude) but also distributed along the cell membrane. Ligation of EpCAM promotes adhesion and enhances cell proliferation and invasiveness. Proteolytic cleavage of EpCAM releases intracellular fragments that increase stem cell factor transcription. 30、31 The oncogenic properties of EpCAM may make it difficult for tumor cells to develop resistance by downregulating EpCAM. In one study, approximately two-thirds of TNBC (estimated to be the basal-A subtype) strongly stained for EpCAM. 25 The number of EpCAM+ circulating cells is associated with poor prognosis in breast cancer 32-36 EpCAM antibodies have been clinically evaluated for epithelial cancers but have limited efficacy on their own. 37-39 EpCAM expression identifies circulating tumor cells in an FDA-approved test for monitoring treatment of metastatic breast, colon, and prostate cancers. 32-36 Furthermore, approximately 97% of human breast cancers and nearly 100% of other common epithelial cancers (including lung, colon, pancreatic, and prostate cancers) brightly stain EpCAM, indicating that the platform developed here is adaptable to RNAi-based therapy of common cancers.

[0204] It is demonstrated herein that all epithelial breast cancer cell lines tested stained brightly for EpCAM, while immortalized normal mammary epithelial cells, fibroblasts, and mesenchymal tumor cell lines did not stain. EpCAM-AsiC caused targeted gene knockdown in luminal and basal cell-A TNBC cancer cells and human breast cancer tissue in vitro, but did not cause targeted gene knockdown in normal epithelial cells, mesenchymal tumor cells, or normal human breast tissue. Knockdown was proportional to EpCAM expression. In addition, knockdown of PLK1 (a gene required for mitosis) mediated by EpCAM-AsiC inhibited in vitro T-IC functional assays (colony and mammosphere formation) of epithelial breast cancer cell lines. Ex vivo treatment specifically eliminated the initiation of tumors. Subcutaneously injected PLK1 EpCAM-AsiC was specifically taken up by EpCAM+ basal cell-A triple-negative breast cancer (TNBC) orthotopic xenografts of poor prognosis basal cell-A and Her2 breast cancer, and caused rapid tumor regression.

[0205] EpCAM is highly expressed in epithelial breast cancer cell lines

[0206] First, EpCAM expression was examined in breast cancer cell lines. Based on gene expression data from the Cancer Cell Line Encyclopedia40, EpCAM mRNA was highly expressed in basal-A TNBC and luminal breast cancer cell lines, but poorly expressed in basal-B (mesenchymal) TNBC ( Figure 1A Surface EpCAM staining assessed by flow cytometry was 2-3 times higher in all tested luminal and basal-like cell lines compared to that in hTERT(BPE) cells. 41 Brighter in normal epithelial cells, fibroblasts or mesenchymal TNBC immortalized ( Figure 1B ). Thus, EpCAM is highly expressed in epithelial breast cancer cell lines compared to normal cells or mesenchymal tumors.

[0207] EpCAM-AsiC selectively knocks down gene expression in EpCAM+ breast cancer cells

[0208] Identified by SELEX that binds to human EpCAM with an affinity of 12 nM 19 19 nucleotide (nt) aptamer 42、43. It does not bind to mouse EpCAM (data not shown). A small amount of EpCAM-AsiC was designed to connect the sense or antisense strand of the siRNA to the 3' end of the aptamer through several linkers and was synthesized using 2'-fluoropyrimidine substitutions and 3'-dTdT overhangs to improve in vivo stability, avoid off-target knockdown of partially complementary genes with similar sequences, and limit innate immune receptor stimulation. In order to test RNA delivery, gene knockdown and anti-tumor effects, siRNA was incorporated into knocked-down eGFP (as a useful marker gene); AKT1 (an endogenous gene expressed in all studied cell lines, whose knockdown is not lethal) and PLK1 (a kinase required for mitosis, whose knockdown is lethal) ( Figure 9 The AsiC that performed best in the dose-response study of gene knockdown linked the 19nt EpCAM aptamer to the sense (inactive) strand of the siRNA via a UUU linker ( Figure 1C EpCAM-AsiC was prepared by annealing a chemically synthesized chain of approximately 42-44 nt (19 nt aptamer + linker + 20-22 nt siRNA sense strand) to a 20-22 nt antisense siRNA strand. It was commercially synthesized using 2'-fluoropyrimidines (Jackson, 2003#11353; Scacheri, 2004#11912; Jackson, 2006#13758; Wheeler, 2011#17906), which are RNase-resistant and very stable in human serum (T 1 / 2 >>36h, Figure 7 ) and did not trigger innate immunity when injected into tumor-bearing mice in vivo ( Figure 7 ).

[0209] To verify the selective uptake of EpCAM+ tumor cells, confocal fluorescence microscopy was used to compare EpCAM+ MDA-MB-468TNBC cells with BPE and EpEAM. dim Internalization of EpCAM aptamers fluorescently labeled with Cy3 at the 5'-end in immortalized mammary epithelial cells (data not shown). Without wishing to be bound by theory, since AsiCs contain only one aptamer, they do not cross-link with the receptors they recognize. Therefore, cellular internalization is slow because it likely occurs through receptor recycling rather than the more rapid activation-induced endocytosis process.

[0210] Only MDA-MB-468 cells took up the aptamer. Uptake was clearly detected at 22 hours, but greatly increased after 43 hours. To test whether EpCAM-AsiC is specifically taken up by EpCAM bright cell lines, the 3' end of the antisense strand of AsiC was fluorescently labeled. EpCAM+BPLER is a basal cell-A TNBC cell line transformed by BPE by transfection with human TERT, SV40 early region and H-RASV12. When analyzed after 24 hours of incubation, EpCAM+BPLER took up Alexa-647 EpCAM-AsiC, but BPE cells did not take up ( Figure 1D Previous studies have shown that AsicC is processed intracellularly by Dicer to release siRNA from aptamers (10, 12, 15). To verify whether the released siRNA is taken up by the RNA-induced silencing complex (RISC), qRT-PCR was used to amplify PLK1 siRNA immunoprecipitated with Ago when MDA-MB-468 cells were incubated with PLK1EpCAM-AsiC ( Figure 33 ). When the same cells were incubated with PLK1 siRNA, no PLK1 siRNA bound to Ago.

[0211] Alexa-467 EpCAM-AsiC was taken up by TNBC cells, but no uptake was detected in BPE cells ( Figure 1E ). Next, to assess whether gene knockdown was specific to EpCAM+ tumors, we compared eGFP knockdown in these same cell lines (which stably express eGFP) by lipofection of eGFP siRNA and eGFPEpCAM-AsiC ( Figure 1D While transfection of eGFP siRNA knocked down gene expression equally in BPE and BPLER, incubation of EpCAM-AsiC in the absence of any transfection lipid selectively knocked down expression only in BPLER. AsiC knockdown was uniform and comparable to that achieved with lipid transfection. Next, we compared endogenous AKT1 gene knockdown specifically by AKT1 AsiC and transfected AKT1 siRNA in six breast cancer cell lines compared to normal human fibroblasts ( Figure 1E ). AKT1 is only expressed on EpCAM brightEpCAM-AsiC targeting AKT1 selectively knocked down luminal and basal-A TNBCs, but not mesenchymal basal-B TNBCs, fibroblasts, or BPE (data not shown). As expected, AsiC targeting eGFP had no effect on AKT1 levels, and transfection of AKT1 siRNA knocked down expression equally in all cell lines studied. Furthermore, EpCAM-AsiC knockdown of AKT1 strongly correlated with EpCAM expression ( Figure 1G Similar results were obtained when AKT1 protein was analyzed by flow cytometry in stained transfected cells ( Figure 1G 、 Figure 1H ). Therefore, in vitro knockdown of EpCAM-AsiC has an effect on EpCAM bright It is effective and specific for tumor cells.

[0212] PLK1 EpCAM-AsiC selectively kills EpCAM in vitro bright tumor cells

[0213] To explore whether EpCAM-AsiC could be used as an antitumor agent in breast cancer, we examined the effects of AsiC targeting PLK1 (a kinase required for mitosis) on the survival of 10 breast cancer cell lines, including 5 basal-A TNBCs, 2 luminal-type cell lines, and 3 basal-B TNBCs, using the CellTiterGlo assay. EpCAM-AsiC targeting PLK1, but not the control AsiC targeting eGFP, reduced cell proliferation in basal-A and luminal-type cell lines, but did not inhibit basal-B cells ( Figure 2A PLK1 siRNA lipofection inhibited the growth of all cell lines. The antiproliferative effect was closely related to EpCAM expression ( Figure 2B The reduction in viable EpCAM+ cells after knockdown was attributed to the induction of apoptosis, assessed by Annexin V-propidium iodide staining and caspase activation (data not shown). To determine whether ligation of the EpCAM aptamer contributes to the anti-proliferative effects of EpCAM-AsiC, we compared the survival of cells treated with the PLK1 EpCAM-AsiC to that of cells treated with the aptamer on its own ( Figure 2C The aptamer itself had no reproducible effect on the survival of any breast cancer cell line, likely because, as a monomeric reagent, it does not crosslink EpCAM receptors and alter EpCAM signaling. Therefore, PLK1 EpCAM-AsiC demonstrated its specific antitumor effect against EpCAM+ breast cancer cells by genetic knockdown.

[0214] To determine when EpCAM dim To determine whether EpCAM-AsiCs specifically target EpCAM+ cells when mixed with non-transformed epithelial cells, we incubated co-cultures of GFP-TNBC cells and GFP+BPE cells with PLK1 EpCAM-AsiCs or culture medium and determined their relative viability by flow cytometry using GFP fluorescence after 3 days ( Figure 2D 、 Figure 2E EpCAM-AsiC targeting PLK1 significantly reduced the proportion of viable EpCAM+ Basal-A tumor cells but had no effect on the survival of the EpCAM-Basal-B cell line. Therefore, PLK1 EpCAM-AsiC is selectively cytotoxic to EpCAM+ tumor cells when mixed with normal cells.

[0215] EpCAM-AsiC is concentrated in EpCAM+ breast tumor biopsy specimens

[0216] Next, we examined whether EpCAM-AsiC is concentrated in human breast tumors relative to normal breast samples within intact tissue. Paired normal tissue and breast tumor biopsies from three breast cancer patients were cut into cubes with approximately 3 mm edges and placed in culture dishes. The tumor sample cells were all EpCAM-AsiC-positive. bright , normal tissue cells are EpCAM dim ( Figure 3A Fluorescently labeled Alexa647-siRNA (not expected to be taken up by normal tissue or tumor), Alexa647-cholesterol conjugated siRNA (chol-siRNA, expected to be taken up by both), or Cy3-EpCAM-AsiC were added to the culture medium and the tissues were incubated for 24 hours before harvesting. The Cy3 signal of AsiC, which can be visualized by the naked eye, was concentrated only in tumor specimens and was not detected in normal tissues ( Figure 3B To quantify RNA uptake, tissue specimens (representative tumor-normal tissue pairs ( Figure 3C ), from 3 EpCAM bright Mean ± SD of triplicate biopsies of paired breast tumor-normal tissue samples ( Figure 3D )) were subjected to flow cytometry analysis of the rinsed single cell suspension. EpCAM-AsiC was significantly taken up by tumors but not normal tissues, while unconjugated siRNA was not taken up. Both took up chol-siRNA to some extent. Therefore, in intact tissues, EpCAM-AsiC was selectively delivered to EpCAM relative to normal tissues. bright tumor.

[0217] PLK1 EpCAM-AsiC inhibits T-IC in EpCAM+ tumors

[0218] EpCAM was chosen for targeting in part because it marks T-ICs and metastasis-initiating cells (M-ICs). 20、22、26、27、31 To investigate whether EpCAM-AsiC inhibits T-IC, we compared colony formation and mammosphere formation after mock treatment, treatment with paclitaxel, or EpCAM-AsiC against eGFP or PLK1 (T-IC functional replacement assay). PLK1 EpCAM-AsiC inhibited colony and mammosphere formation more strongly than paclitaxel in EpCAM+ basal-A TNBC and luminal-type cell lines, but had no activity against EpCAM- basal-B TNBC ( Figures 4A-4C T-IC inhibition was specific, as eGFP AsiC had no effect. Incubation with PLK1EpCAM-AsiC, but not eGFP AsiC, also reduced the proportion of cells with a T-IC phenotype, specifically CD44 in basal-A and luminal breast cancer cell lines. + CD24 low / - and ALDH + To evaluate the effect of EpCAM-AsiC on tumor initiation, EpCAM+MB468 cells stably expressing luciferase were treated overnight with culture medium or PLK1 or eGFP EpCAM-AsiC, and then an equal number of viable cells were subcutaneously implanted into nude mice. PLK1 EpCAM-AsiC completely blocked tumor formation as assessed by in vivo tumor cell luminescence (data not shown). In contrast, similar treatment of basal-B MB436 cells had no effect on tumor initiation (data not shown). Thus, PLK1 EpCAM-AsiC selectively inhibits in vitro T-IC assays and tumor initiation in EpCAM+ breast cancer.

[0219] Subcutaneously administered EpCAM-AsiC is selectively taken up by distant EpCAM+ TNBC

[0220] To be clinically useful, EpCAM-AsiC needs to be taken up by disseminated tumor cells. Intravenous injection of fluorescent EpCAM-AsiC in the tail vein of mice did not cause significant accumulation of AsiC in subcutaneous tumors implanted in the flanks of nude mice (data not shown), probably because their size (approximately 25 kDa) is below the threshold of renal filtration and they are rapidly excreted. Linkage to polyethylene glycol greatly enhanced the circulation half-life, tumor accumulation and antitumor therapeutic effect of PSMA-AsiC in a mouse xenograft model of prostate cancer11. However, to see whether this modification could be circumvented, we examined whether subcutaneous injection of Alexa750-labeled eGFP EpCAM-AsiC in the scruff of the neck of 7 mice caused accumulation in distant EpCAM+MB468 and EpCAM-MB231 TNBCs implanted subcutaneously in each flank by live animal epifluorescence imaging ( Figure 5A 、 Figure 5B EpCAM-AsiC was concentrated exclusively in EpCAM+ tumors on the day of injection and persisted for at least 4 days. EpCAM-AsiC was detected around the injection site on day 2, but was found exclusively within EpCAM+ tumors on day 4.

[0221] PLK1 EpCAM AsiC causes regression of basal-A TNBC and Her2 breast cancer xenografts

[0222] Because subcutaneously injected EpCAM-AsiC concentrates in distant EpCAM+ tumors, we next investigated whether subcutaneous injection of PLK1EpCAM-AsiC could selectively inhibit the growth of EpCAM+ TNBC xenograft tumors. EpCAM+MB468-luc cells were implanted in Matrigel in one flank of nude mice, and EpCAM-MB231-luc-mCherry cells were implanted in the opposite flank. Once the luciferase signal of both tumors was clearly detected above background, groups of 5-6 mice were mock treated or injected subcutaneously with 5 mg / kg of EpCAM-AsiC targeting PLK1 or eGFP every 3 days for 2 weeks. Tumors grew and then glowed. All EpCAM+ tumors rapidly and completely regressed only in mice that received AsiC targeting PLK1 ( Figure 6A 、 Figure 6B ). EpCAM+ tumors and all EpCAM tumors in mice treated with eGFP-targeting AsiC continued to grow. This experiment was repeated with similar results after injection of PLK1AsiC. Tumors continued to grow without significant changes except for the control group of mice treated with only EpCAM aptamer or PLK1 siRNA (data not shown) and the group of mice bearing Her2+MCF10A-CA1a ( Figure 34 Thus, subcutaneously injected PLK1EpCAM-AsiC showed specific antitumor activity against basal-A TNBC and EpCAM+ human xenografts.

[0223] discuss

[0224] Current targeted therapies rely on the use of tumor-specific antibodies or oncogenic kinase inhibitors. Previously, no one has demonstrated that unconjugated AsiC can have effective antitumor effects or that AsiC can be administered subcutaneously. Currently, there are no targeted therapies for TNBC or T-IC. Developing targeted therapies for TNBC and developing methods to eliminate T-IC are important unmet goals in cancer research.

[0225] This paper demonstrates that EpCAM-AsiC can be used to selectively knock down genes in epithelial breast cancer cells and their stem cells, while sparing normal epithelial cells and stroma, to induce tumor regression and inhibit tumor initiation. In a very aggressive TNBC xenograft model, EpCAM-AsiC caused complete tumor regression after only three injections. This may be an effective and flexible targeted therapy platform for all common epithelial cancers (which uniformly express high levels of EpCAM).

[0226] Although EpCAM-AsiC targeting PLK1 was used herein, siRNAs can be tailored to knockdown any tumor-dependent gene to the molecular signature of an individual patient's tumor or tumor subtype. A cocktail of AsiCs targeting more than one gene would be ideal for cancer therapeutics to reduce the chance of developing drug resistance. Targeted cancer therapies have so far relied on the use of tumor-specific antibodies or small molecule inhibitors of oncogenic kinases. The use of EpCAM as an AsiC ligand and the development of RNAi therapies targeting cancer stem cells are novel. No one has previously demonstrated that unconjugated AsiCs can have effective antitumor effects or that AsiCs can be administered subcutaneously. Furthermore, preliminary studies of subcutaneously administered CD4-AsiCs in humanized mice showed robust knockdown of CD4 cells in the spleen and distal lymph nodes, suggesting that AsiCs targeting receptors on cells elsewhere in the body can also be administered subcutaneously. Currently, no targeted therapies are available for TNBC or T-IC. Targeted delivery offers the advantages of reduced dose and reduced toxicity to bystander cells.

[0227] A major obstacle to using RNAi for cancer is delivering small RNAs to disseminated cells. Described herein is the use of AsiCs to overcome this obstacle. Described herein is a new class of effective anticancer drugs. AsiCs are a flexible platform capable of targeting diverse cell surface receptors and knocking down any gene or combination of genes. {Burnett, 2012#18447; Zhou, 2011#18448; Thiel, 2010#18445}. By modifying the aptamer, the AsiC platform can overcome the delivery barriers that have hindered the application of RNAi-based therapies to most diseases. This approach is ideal for personalized cancer treatment because the choice of target genes can be tailored to the molecular characteristics of the tumor. Furthermore, RNA mixtures can knock down multiple genes at once to predict and overcome drug resistance. AsiCs are the most attractive approach for gene knockdown outside the liver. They are superior to complex liposome, nanoparticle, or conjugated methods for delivering RNA because they are single chemical entities that are stable in the blood, easy to manufacture, non-immunogenic, can easily penetrate tissues, and are not trapped in filtering organs.

[0228] An important cancer research goal is to eliminate T-ICs (cancer stem cells). T-ICs are relatively resistant to chemotherapy and are thought to be responsible for tumor recurrence and metastasis. {Federici, 2011#19371}. The AsiCs described herein target (epithelial) T-ICs with high potency. Thus, they can eliminate this aggressive subpopulation within tumors that are at risk for progressive disease (see Figure 6A 、 Figure 6B ).

[0229] The small size of the EpCAM aptamer used here is ideal for AsiC drugs because RNA <60 nt can be efficiently synthesized.

[0230] In addition to their potential therapeutic uses, EpCAM-AsiC is also a powerful in vivo research tool for identifying T-ICs and tumor dependency genes to define new drug targets. In principle, aptamer chimeras can be designed to deliver not only siRNA, but also miRNA mimics or antagomirs, antisense oligonucleotides that act through mechanisms other than RNAi, and even longer mRNAs or noncoding RNAs (50, 51). They can also be designed to incorporate more than one aptamer, multiple siRNAs, or even toxins or small molecule anticancer drugs.

[0231] Its small size is ideal for AsiC drugs because RNAs ≤ 60 nt can be efficiently synthesized. Not only can siRNAs be targeted to tumors, but drug targets can also be selected to attack the tumor's Achilles' heel by knocking down tumor-dependent genes. This flexibility can be used to personalize cancer treatments by targeting the molecular vulnerabilities of individual patients' cancers.

[0232] Materials and methods

[0233] Cell culture. Human BPE and BPLER cells were grown in WIT medium (Stemgent). MB468 was transduced with a luciferase reporter. Unless otherwise noted, all other human cell lines were obtained from ATCC and grown in DMEM (MB231, BT549, MB436), MEM (MCF7, BT474), McCoy's 5A (SKBR3), or RPMI1640 (HCC1806, HCC1143, HCC1937, HCC1954, HCC1187, MB468, T47D) supplemented with 10% FBS, 1 mM L-glutamine, and penicillin / streptomycin (Gibco). 4T1 mouse breast cancer cells were grown in 10% FBSDMEM. For in vivo imaging, MB468 cells stably expressing firefly luciferase (MB468-luc) were used, and MB231 cells stably expressing firefly luciferase and mCherry (MB231-luc-mCherry) were selected after infection with pLV-Fluc-mCherry-Puro lentivirus (provided by Andrew Kung, Columbia University). MB231 cells were selected with puromycin.

[0234] For uptake and silencing, cells were seeded at low density (10,000 cells / well in a 96-well plate) and immediately treated. All AsiC and siRNA treatments were performed in OptiMEM or WIT medium. Cell viability was assessed in 96-well plates by CellTiter-Glo (Promega) or trypan blue staining.

[0235] For colony formation assays, 1,000 viable cells were plated in a round-bottom 96-well plate for 6 hours and then transferred to a 10 cm plate containing serum-containing culture medium. The culture medium was changed every 3 days. After 8-14 days, the cells were fixed in methanol (-20°C) and stained with crystal violet. For mammosphere formation assays, 1,000 viable cells / ml were plated in a round-bottom 96-well plate for 6 hours and then suspended in serum-free DMEM / F12 1:1 (Invitrogen) supplemented with EGF (20 ng / ml, BD Biosciences), B27 (1:50, Invitrogen), 0.4% bovine serum albumin (Sigma), and 4 μg / ml insulin (Sigma). Mammospheres were counted after 1 or 2 weeks.

[0236] siRNA transfection. Cells were transfected with Dharmafect I according to the manufacturer's protocol. All siRNA sequences are available at Figure 9 .

[0237] Flow cytometry. For flow cytometry, cells were stained as previously described (Yu, F. et al. (2007), let-7 Regulates Self Renewal and Tumorigenicity of Breast Cancer Cells, Cell 131, 1109-1123). Briefly, EpCAM and AKT1 were directly immunostained using a 1:50 dilution of hAb at 4°C for 30-60 minutes (BioLegend / BD). Cells were stained in PBS containing 0.5% FCS, 1 mM EDTA, and 25 mM HEPES. Samples were washed twice in the same buffer. Data were acquired using a FACS-Canto II (BD Biosciences). Three replicates were analyzed, and 10,000 gated events / sample were counted. All data analyses were performed using FlowJo (Treestar Inc.).

[0238] RNA analysis. qRT-PCR analysis was performed as described in (Petrocca, F. et al. (2008). E2F1-regulated microRNAs impair TGFbeta-dependent cell-cycle arrest and apoptosis in gastric cancer. Cancer Cell 13, 272-286). Briefly, total RNA was extracted with Trizol (Invitrogen) using the manufacturer's SYBR Green MasterMix (Applied Biosystems) and a BioRad C1000 Thermal Cycler (Biorad), and cDNA was prepared from 1000 ng of total RNA using the Thermoscript RT kit (Invitrogen). Relative CT values ​​were normalized to GAPDH and converted to a linear scale.

[0239] Collagenase digestion of human breast tissue. Fresh breast or colon cancer and control biopsies were received from the UMASS tissue bank and cut into 3 × 3 × 3 mm samples and placed in a 96-well plate with 100 μl RPMI. The samples were treated with Alexa647-siRNA-GFP, Alexa647-chol-siRNA-GFP or Cy3-AsiC-GFP for 24 hours. The samples were photographed and digested. Three samples from each treatment were merged and placed in 10 ml RPMI containing 1 mg / ml collagenase II (Sigma-Aldrich) and shaken at 37°C for 30 minutes. Before and after collagenase digestion, the spleen program was used to break the sample in a gentle MACS dissociator (Miltenyi) at 37°C for 30 minutes. The cell suspension was passed through a 70 μm cell strainer (BD Falcon), washed with 30 ml RPMI, and stained for flow cytometry.

[0240] Animal Experimentation. All animal procedures were approved by the Animal Care and Use Committees of Harvard Medical School and Boston Children's Hospital. Nude mice were purchased from the Jackson Laboratory.

[0241] In vivo experiments. For tumor initiation studies, 8-week-old female Nu / J mice (Stock#002019, Jackson Laboratory) were subcutaneously injected with MB468-luc (5×10 6) cells. Cells were trypsinized using Tryple Express (Invitrogen), resuspended in WIT medium, and injected subcutaneously into the flank. Following intraperitoneal injection of 150 mg / kg D-luciferin (Caliper Life Sciences), whole-body luminescence images were captured every 5 days for a total of 20 days using an IVIS Spectra system (Caliper Life Sciences).

[0242] For AsiC uptake experiments, MB468-luc (5 × 10 6 ) and MB231-luc-mCherry (5×10 5 ) cells were resuspended in a 1:1 WIT-Matrigel solution and injected subcutaneously into the flank of 8-week-old female Nu / J mice (Stock #002019, Jackson Laboratory). Tumor size was analyzed daily using an IVIS Spectra system (Caliper Life Sciences). After 5 days, tumors were clearly visible, and mice were subcutaneously injected with Alexa750-EpCAM-AsiC-GFP (0.5 mg / kg) in the neck region. AsiC localization relative to the tumor was measured every 48 hours for 7 days.

[0243] For tumor inhibition studies, MB468-luc (5 × 10 6 ) and MB231-luc-mCherry (5×10 5 ) cells were resuspended in a 1:1 WIT-Matrigel solution and injected subcutaneously into 8-week-old female Nu / J mice (Stock #002019, Jackson Laboratory). Tumor size was analyzed daily using an IVIS Spectra, and tumors were clearly visible after 5 days. Mice bearing tumors of similar size were randomly divided into 5 groups and treated with 5 mg / kg of EpCAM-AsiC-PLK1, EpCAM-AsiC-GFP, EpCAM-aptamer, or siRNA-PLK1, or left untreated. Mice were treated every 72 hours for 14 days.

[0244] Using Living All images were analyzed using Caliper software (Caliper Life Sciences).

[0245] Statistical analysis. The significance between treated samples and controls was analyzed using a Student's t-test calculated with Microsoft Excel, with the test type set to one-tailed distribution and two-sample equal variance. To assess innate immune stimulation, a one-way analysis of variance (ANOVA) with Bonferroni's multiple comparison test was performed using GraphPad Prizm 4 software (GraphPad Software, San Diego, CA). P < 0.05 was considered significant.

[0246] Measurement of innate immune stimulation. Mice were injected subcutaneously with eGFP EpCAM-AsiCs (5 mg / kg) or intraperitoneally with poly (I: C) (5 mg / kg or 50 mg / kg). Serum samples collected at baseline and 6 hours and 16 hours after treatment were stored at -80 ° C and then measured using ProcartaPlex Multiplex Immunoassay (Affymetrix / eBioscience, San Diego, CA) for IFNβ, IL-6, and IP-10. Before RNA was extracted using TRIZOL (Invitrogen) with a gentle MACS dissociator (MACS Miltenyi Biotec, San Diego, CA), spleens obtained after 16 hours of treatment were stored in RNAlater (Qiagen). cDNA was synthesized using Superscript III and random hexamers (Invitrogen), and PCR was performed using the following primers with SsoFast EvaGreen Supermix and a Bio-Rad CFX96 Real-Time PCR System (Bio-Rad Laboratories, Hercules, CA).

[0247] Gapdh forward primer: 5′-TTCACCACCATGGAGAAGGC-3′ (SEQ ID NO: 4);

[0248] Gapdh reverse primer: 5′-GGCATGGACTGTGGTCATGA-3′ (SEQ ID NO: 5);

[0249] ifnb forward primer: 5′-CTGGAGCAGCTGAATGGAAAG-3′ (SEQ ID NO: 6);

[0250] ifnb reverse primer: 5′-CTTGAAGTCCGCCCTGTAGGT-3′ (SEQ ID NO: 7);

[0251] il-6 forward primer: 5′-TGCCTTCATTTATCCCTTGAA-3′ (SEQ ID NO: 8);

[0252] il-6 reverse primer: 5′-TTACTACATTCAGCCAAAAAGCAC-3′ (SEQ ID NO: 9);

[0253] ip-10 forward primer: 5′-GCTGCCGTCATTTTCTGC-3′ (SEQ ID NO: 10);

[0254] ip-10 reverse primer: 5′-TCTCACTGGCCCGTCATC-3′ (SEQ ID NO: 11);

[0255] oas-1 forward primer: 5′-GGAGGTTGCAGTGCCAACGAAG-3′ (SEQ ID NO: 12);

[0256] oas-1 reverse primer: 5′-TGGAAGGGAGGCAGGGCATAAC-3′ (SEQ ID NO: 13);

[0257] stat1 forward primer: 5′-TTTGCCCAGACTCGAGCTCCTG-3′ (SEQ ID NO: 14);

[0258] stat1 reverse primer: 5'-GGGTGCAGGTTCGGGATTCAAC-3' (SEQ ID NO: 15).

[0259]

[0260] EpCAM is overexpressed in basal-A and luminal breast cancer cell lines, but not in basal-B breast cancer cell lines (data not shown). Eight different breast cancer cell lines were subjected to FACS, and EpCAM expression levels were measured by flow cytometry using hEpCAM antibodies. EpCAM was overexpressed in all basal-A and luminal cell lines, but not in basal-B cells.

[0261] Specific reduction in cell viability in basal-A breast cancer cell lines is PLK1-dependent. Ten different breast cancer cell lines representing basal-A, basal-B, and luminal cell types were treated with EpCAM-AsiC targeting PLK1 or with the EpCAM aptamer alone and compared to untreated controls. None of the cell lines treated with the EpCAM-aptamer showed a reduction in cell viability, while the basal-A and luminal cell lines showed a reduction in cell viability after treatment with EpCAM-AsiC targeting PLK1 (data not shown).

[0262] EpCAM-AsiC is taken up by healthy and colon cancer biopsies. Cy3-EpCAM-AsiC, Alexa647-siRNA-GFP or Alexa647-chol-siRNA-GFP targeting GFP (2 μM each) were added to colon cancer and control explants and incubated for 24 hours, after which the tissues were digested with collagenase to single cell suspensions and analyzed by flow cytometry. EpCAM-AsiC, siRNA and chol-siRNA penetrated tumors and healthy tissues with similar efficacy. On day 5, the tumors were removed and visualized to confirm that Alexa750-labeled EpCAM-AsiC targeting GFP had indeed entered the tumor. Elevated levels of Alexa750 were negatively correlated with mCherry levels (n=8, *P<0.05, t-test EpCAM+ versus EpCAM- cells) (data not shown).

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[0315] Example 2

[0316] This article describes the development of targeted siRNA delivery (aptamer-siRNA chimeras (AsiCs)) using chimeric RNA composed of structured RNA (called aptamers), which are selected for high affinity binding to cell surface proteins covalently linked to siRNA. These AsiCs are taken up by cells expressing receptors, which are recognized by the aptamers and processed inside the cells to release active siRNA. This is a flexible platform that can be modified to target different cells by targeting specific cell surface receptors and can be designed to knock down any gene or combination of genes.

[0317] This aptamer was selected for high affinity binding to human EpCAM (CD326 or ESA), which is expressed on all epithelial cells but is more highly expressed in poorly differentiated breast cancers such as basal-like TNBC. All common cancers (lung, pancreas, prostate, breast, and colon) have high EpCAM expression and can be effectively targeted.

[0318] Here, we demonstrate that epithelial breast cancer cells, but not mesenchymal or normal epithelial cells, selectively take up EpCAM-AsiC and undergo gene knockdown in vitro. Furthermore, the extent of knockdown correlates closely with EpCAM levels. Knockdown of PLK1, a gene required for mitosis, using EpCAM-AsiC abolished cancer cell line growth and stem cell properties, including tumor initiation in xenografts and colony and mammosphere formation. This platform can be used to eliminate both cancer cells and malignant cancer stem cells within epithelial tumors.

[0319] EpCAM AsiCs can be delivered specifically to basal-like tumors and inhibit tumor growth. These AsiCs can also serve as powerful research tools to identify genes that T cells depend on, which may be good targets for conventional drugs or RNAi-based drugs.

[0320] Example 3

[0321] The ubiquitous mechanism for regulating gene expression is called RNA interference. It uses small RNAs loaded with short complementary sequences to block the conversion of genetic information into proteins. Exploiting this endogenous process offers exciting possibilities for treating diseases by knocking down the expression of pathogenic genes. The main obstacle is delivering small RNAs into cells where the RNA interference machinery is located. In the past year, preliminary clinical studies have shown very promising results, with no significant toxicity in a few diseases caused by abnormal gene expression in the liver. However, delivery to the liver (an organ that captures particles in the blood) is easier to achieve than delivering drugs to metastatic tumor cells. Described herein is a strategy for targeting RNA into epithelial cancer cells, which is particularly suitable for targeting the most aggressive breast cancer, triple-negative breast cancer (TNBC). In addition, it also targets the most malignant subpopulation in most breast cancers, called cancer stem cells. These cells are resistant to chemotherapy drugs and are believed to be the cause of tumor recurrence and metastasis. An important goal of current cancer research is to replace cytotoxic chemotherapy drugs that are toxic to both cancer cells and normal dividing cells (such as blood-forming cells and intestinal cells) with agents that have selective activity against tumors, particularly against cancer stem cells within tumors.

[0322] Targeted therapies for one type of breast cancer (Her2+) have dramatically changed treatment and improved survival rates. Currently, there are no targeted therapies for TNBC or breast cancer stem cells.

[0323] Described herein are data demonstrating that RNA interfering with RNA attached to structured RNAs (aptamers) that recognize cell surface proteins can knock down gene expression in aggressive breast cancer cells. Aptamers that bind to proteins highly expressed on breast cancer stem cells and most TNBC cells can specifically knock down proteins required for cancer cell division or survival in the most common subtypes of TNBC. These RNAs can be tested in tissue culture and mouse models of TNBC. This paper describes a platform for treating breast cancer with poor prognosis using RNA-based drugs and demonstrates its effectiveness in mouse models.

[0324] Ultimate applicability of the treatment for breast cancer (which patients, how it will help them, clinical application / benefits / risks, expected time to patient-relevant outcomes). The protein that the treatment can target is expressed on all epithelial cancer cells, but is more strongly expressed on the least differentiated (and therefore the most malignant) cancer cells. This approach can be used to treat not only the majority of epithelial breast cancers (and most breast cancer cells are epithelial cells), but also has the potential to treat common cancers (including colon cancer, lung cancer, pancreatic cancer and prostate cancer). Our focus is on the most aggressive and poorest prognosis breast cancer, TNBC, which preferentially invades young women and women in a small population. This approach allows for a new breast cancer treatment platform. Any gene or combination of genes that causes or promotes cancer can be knocked down, making this strategy suitable for the future era of personalized cancer treatment, in which each patient's treatment will be customized according to the molecular characteristics of their individual tumor.

[0325] In addition, if the tumor is unresponsive or becomes resistant, the mixture of target genes can be adjusted flexibly. Because normal epithelial cells express low levels of the protein used for targeting, normal epithelial cells may take up some and have some toxicity to normal epithelial cells, which was evaluated in this article. However, the platform is flexible, making it possible to choose therapeutic siRNA cargo to kill tumor cells with minimal toxicity to normal cells.

[0326] This article describes the design and testing of multiple molecules capable of causing tumor-specific gene knockdown and tumor suppression in a mouse TNBC model.

[0327] There are no targeted therapies for TNBC, or the highly malignant tumor-initiating cell subset in breast cancer.

[0328] Triple-negative breast cancer (TNBC) has the worst prognosis among breast cancers 1-4There is no targeted therapy, and TNBC often relapses. This article describes the development of small RNA-based drugs that knock down tumor-dependent genes in basal-like (or basal-A) TNBC. In principle, RNA interference (RNAi) can be used to knock down disease-causing genes to treat any disease. 5-9 However, translating small RNAs into drugs is challenging. Recent phase I and II clinical trials have demonstrated dramatic and durable gene knockdown in the liver (approximately 80%-95%, lasting nearly a month after a single injection) without significant toxicity. 10-16 However, realizing the potential of gene knockdown for cancer therapy requires robust methods to deliver RNA to diffuse cancer cells, which cannot be achieved with liver-targeted RNA.7 An ideal treatment would selectively knock down genes in cancer cells, minimizing toxicity to normal cells. 17 .

[0329] AsiC consists of an RNA aptamer (structured RNA with high affinity for a receptor) covalently linked to the siRNA 18、19 constitute( Figures 10A-10B ).

[0330] This article describes the use of AsiC, an EpCAM aptamer, for gene knockdown in epithelial cancers. 37 EpCAM (the first tumor antigen described) is highly expressed in all common epithelial cancers 38-45 In epithelial breast cancer, EpCAM is approximately 400 times more abundant than in normal breast tissue. 46 EpCAM 39、45、47-53 It is also highly expressed on most epithelial cancer tumor-initiating cells (T-ICs, also known as cancer stem cells). 39、45、47-53 .

[0331] EpCAM aptamers have high affinity (12 nM) and are short (19 nt), which is ideal for AsiC drugs because RNA <60 nt can be synthesized cheaply and efficiently. EpCAM-AsiC (19 nt aptamer + 3 nt linker + 20-22 nt siRNA sense strand) consisting of a 42-44 nt strand is annealed to a 20-22 nt antisense (active) siRNA strand ( Figure 10B They are commercially synthesized with 2'-fluoropyrimidine, which improves serum stability (T1 / 2 > 3d) and blocks innate immune recognition 28、54-56 .

[0332] EpCAM targeting can cause selective gene knockdown in basal-like TNBC relative to normal epithelial cells. Selective knockdown will reduce drug dose and normal tissue toxicity. In normal epithelial cells, EpCAM is only expressed at basolateral gap junctions, which may be inaccessible. In epithelial cancers, it is more abundant and distributed along the entire cell membrane. EpCAM promotes adhesion and therefore enhances proliferation and invasiveness. Proteolytic cleavage of EpCAM releases intracellular fragments that increase stem cell factor transcription. The oncogenic properties of EpCAM may make it difficult for tumor cells to develop resistance by downregulating EpCAM. The number of EpCAM+ circulating cells is associated with poor prognosis in breast cancer. In fact, enumeration of circulating EpCAM+ cells is the basis of FDA-approved methods for monitoring treatment of metastatic breast cancer, colon cancer, and prostate cancer. In our study, 9 out of 9 basal-ATNBC and luminal-type breast cancer cell lines were strongly EpCAM+, while normal breast cancer epithelial cell lines and mesenchymal TNBC were close to background levels ( Figure 1B ). Therefore, most basal-like TNBC and luminal breast cancers will likely be targeted by EpCAM-AsiC. In preliminary data, EpCAM-AsiC selectively knocked down expression in EpCAM+ breast and colon cancer cell lines, but not in normal epithelial or mesenchymal tumor cells; the knockdown was uniform and comparable to lipofection, which uniformly knocked down gene expression in all cell lines ( Figure 3A-3C ).

[0333] AKT1 knockdown and cell proliferation inhibition by EpCAM-AsiC targeting PLK1 (a kinase required for mitosis) correlated with EpCAM levels. When normal transformed epithelial cells (BPE) 57 were mixed with epithelial TNBC cell lines, EpCAM-AsiC caused PLK1-sensitive cell death only in tumor cells (but not in BPE cells) (not shown). In addition, when tumor biopsies and normal tissue biopsies were co-incubated with fluorescent AsiC, only tumors took up AsiC and fluoresced (not shown). These results indicate that EpCAM-AsiC is specific for epithelial tumor cells compared to normal epithelial cells.

[0334] EpCAM also marks T-IC 40、45、58 An important goal in cancer research is to develop methods to target T-ICs. Although the stem cell hypothesis is controversial and may not apply to all cancers, there is good evidence that breast cancer includes a subpopulation of T-ICs. 59-82. T-ICs are relatively resistant to chemotherapy and are also thought to be responsible for tumor recurrence and metastasis. The AsiCs described herein are designed to target (epithelial) T-ICs with high efficacy. Therefore, they may be suitable for eliminating this aggressive subpopulation in patients at risk of recurrence. To investigate whether EpCAM-AsiC inhibits TNBC T-ICs, we compared mammosphere and colony formation (in vitro surrogates for T-IC function) of breast tumor cells that were mocked or treated with EpCAM-AsiC targeting eGFP or PLK1. PLK1 EpCAM-AsiC (but not control GFP AsiC) eliminated mammosphere and colony formation of breast luminal and basal-like TNBC cell lines. Figure 11D PLK1EpCAM-AsiC also reduced CD44+CD24low and Aldefluor+ cells (not shown). Importantly, treatment with PLK1EpCAM-AsiC abolished tumor initiation in basal-like TNBC, but, as expected, had no effect on basal-B TNBC tumor initiation (data not shown). Luciferase-expressing cell lines were treated with AsiC overnight or mock-treated prior to orthotopic implantation in the mammary fat pad.

[0335] AsiC targets EphA2, which is important in EGF receptor signaling 83-92 Also considered herein. EphA2 is expressed on epithelial and mesenchymal (basal-A and basal-B, respectively) TNBC cell lines (including its T-IC), but is less expressed than EpCAM and is only weakly expressed on other breast cancers. Inhibition of EphA2 reduces tumor growth and angiogenesis in multiple cancer models. Furthermore, EphA2 can be selectively accessible to cancer cells but not normal cells.

[0336] Also considered in this paper are mouse-human cross-reactive AsiCs, which are valuable for future drug development as they will allow us to assess toxicity and efficacy in spontaneous mouse tumor models.

[0337] AsiC targeting EphA2 can generate dual-functional RNA that inhibits EphA2 signaling and cell proliferation and knocks down genes.

[0338] AsiC is ideal for personalized cancer therapy because the genes targeted for knockdown can be tailored to the molecular signature of the tumor. In addition, mixtures of RNA can be assembled to knockdown multiple genes at once for anticipated combination therapies and to overcome drug resistance. AsiC not only targets drugs to tumors, but also allows the selection of siRNAs to attack specific tumor Achilles' heel. siRNAs also provide a unique opportunity to target "undruggable" genes. AsiC that knockdown tumor-dependent genes (required by the tumor but not by normal cells for survival) should have reduced toxicity. To identify genetic dependencies of basal-like TNBC that we could knockdown, we performed a genome-wide siRNA lethality screen comparing two TNBC cell lines (human 10 breast epithelial cells transformed with the same oncogene in different culture media): basal-like BPLER and myoepithelial HMLER cells. 57、93 .

[0339] Although essentially isogenic, BPLER is highly malignant and enriched in T-ICs, with only 50 cells forming tumors in nude mice, whereas HMLER requires >105 cells to initiate tumors. The screen identified 154 genes that are dependent on BPLER but not HMLER. Proteasome genes were highly enriched (P<10-14). BPLER-dependent gene expression was associated with poor prognosis in breast cancer (but not lung or colon cancer). Because TNBC is heterogeneous, 1、3、4、94To identify shared dependencies in basal-like TNBC, we performed another screen to test whether 17 breast cancer cell lines depended on 154 BPLER-dependent genes (unpublished). Although many BPLER dependencies were shared only with a subset of basal-like TNBC cell lines, proteasome, MCL1, some splicing genes, and several other novel genes stood out, as nearly all (at least 8 of 9) basal-like TNBC cell lines depended on these genes, while normal cells did not. As predicted by the screen, the proteasome inhibitor bortezomib killed basal-A TNBC and also blocked T-IC function (assessed by colony and mammosphere formation), and was most selective in basal-like TNBC. Brief exposure to bortezomib also inhibited colony formation and tumor suppression in mouse epithelial TNBC cell lines. Bortezomib strongly inhibited tumor growth in multiple human basal-A lines and primary TNBCs that spontaneously arose in Tp53+ / - mice, but not in basal-B or luminal cell lines. Bortezomib also blocked metastatic lung colonization of IV-injected TNBC cells. However, bortezomib does not penetrate well into solid tumors. A maximum tolerated dose is required to inhibit proteasome activity and suppress tumors. Although proteasome inhibitors can improve tumor penetration during development, proteasome gene knockdown may provide more durable and more effective proteasome inhibition.

[0340] EpCAM- and EphA2-AsiCs can be used for targeted gene knockdown to treat basal-like TNBC cancers, sparing normal cells and eliminating T-ICs within them. Some uptake may be present in normal epithelial cells that weakly express EpCAM or EphA2, but gene knockdown will be concentrated in tumor cells that are highlighted by the aptamer ligand.

[0341] It is possible to determine which breast cancer subtypes are targeted by EpCAM- and EphA2-AsiC and to determine how aptamer ligand levels affect gene silencing. Uptake / knockdown in cancerous tissue versus normal epithelium can also be assessed. The effectiveness of EpCAM-AsiC in inducing knockdown in basal-like TNBC can be compared to that of EphA2-AsiC. It is possible to determine whether EpCAM-AsiC and EphA2-AsiC can target T-ICs to inhibit tumor initiation.

[0342] Pharmacokinetic (PK) / pharmacodynamic (PD) studies of EpCAM- and EphA2-AsiC can be performed using in vivo imaging of mice bearing orthotopic TNBC xenografts. Toxicity and innate immune activation assays can be performed on treated tissue samples and animals, and AsiC can be chemically modified if needed to improve PK / PD or reduce toxicity. As a proof of principle, the anti-tumor effect of PLK1 knockdown will be evaluated. The methods described herein can be used to suppress recently identified basal cell-A TNBC-dependent genes, such as MCL1 and proteasome genes.

[0343] Among those considered in this article are:

[0344] Cross-species reactive aptamers that recognize EpCAM and EphA2 and are selectively internalized into basal cells, TNBCs, and normal epithelial cells;

[0345] Validate the in vitro selective uptake, gene silencing, and cytotoxicity of TNBC-targeted AsiCs in breast cancer cell lines versus normal epithelial cells, identify the subtypes of breast cancer cell lines they transfect, and evaluate their potential to transfect and eliminate breast T-IC cells;

[0346] To assess in vivo systemic delivery and tumor concentrations, define the PK and PD and maximum tolerated dose of TNBC-targeted AsiC, and evaluate the antitumor effects of optimized TNBC-targeted AsiC that knocks down PLK1 and basal-like TNBC-dependent genes in mouse primary and metastatic cancer models of human TNBC cell lines.

[0347] Selection of aptamers targeting TNBC. Aptamers that bind to selected targets are identified by iterative screening of combinatorial nucleic acid sequence libraries of broad complexity (typically 1012-1014 different sequences) through a process called SELEX (Systematic Evolution of Ligands by Exponential enrichment). 95、96 In the classic approach, an RNA pool is incubated with a protein target, and the bound RNA is isolated and amplified to produce a bound RNA pool. These are then applied again in multiple cycles to produce increasing, enriched pools of high-affinity RNA. Identification of sequences that emerge after multiple rounds of SELEX is achieved in advance by cloning and sequencing <100 individual sequences.

[0348] Although this generally provides a sufficient number of winning sequences to identify aptamers, the number of sequences analyzed is quite small compared to the sequence complexity of the evolving oligonucleotide pool. With multiple selection cycles, some winning aptamer sequences that are not effectively amplified may be exhausted and lost. Next-generation deep sequencing methods and bioinformatics can allow the evaluation of more sequences in the early-cycle SELEX sequence pool to identify winning aptamer sequences in early selection rounds, thereby reducing the time and resources required to complete the identification of high-affinity aptamers. 30、97-104 .

[0349] An important property of aptamers for incorporation into AsiC is efficient internalization into cells. Some ligands of cell surface proteins are efficiently internalized after binding to their cell surface protein targets, while other ligands cannot. Another strategy ("toggle SELEX") selects cross-reactive aptamers that recognize the same ligand from different species, which is a useful property for preclinical development. By switching cycles between the selection of orthologous protein ligands (e.g., mouse and human forms), cross-species reactive aptamers can be enriched. 105 .

[0350] These SELEX techniques enable the identification of high affinity, cross-species reactive aptamers to EpCAM and EphA2 that are internalized in human (and mouse) basal-like TNBCs but not in normal immortalized epithelial cell lines. To select additional EpCAM and EphA2 aptamers with antagonistic activity and / or cross-recognition of the corresponding mouse antigens (published EpCAM aptamers do not recognize mouse EpCAM (data not shown)), we can start with a library of 1012 RNA sequences containing 2'-fluoropyrimidines, switching between commercially available mouse and human purified recombinant target proteins. This library of 51 nt oligonucleotides was designed with a random region of 20 nucleotides flanked by constant regions of known sequence used for PCR amplification in each selection round. The previously described method will be used to select high affinity RNAs that bind to immobilized C-terminally tagged proteins. 37 (This exposes the N-terminal region to aid in the selection of aptamers that recognize the extracellular domain.) Tagged control proteins can be used to pre-clean the RNA aptamer library to remove non-specific binders. 7-10 iterative rounds of SELEX can be performed to enrich for specific aptamers. Concentration after each round can be monitored by surface plasmon resonance. Enriched pools that show specific binding can be sequenced using high-throughput sequencing. Sequences for experimental validation can be selected using bioinformatics analysis of the enriched library sequences as described. 97、98、106 .

[0351] The top 10-15 sequences from high-throughput sequencing and bioinformatics analysis can be evaluated by surface plasmon resonance to assess for example using previously characterized human aptamers for comparison. 99、106 The relative binding affinity.

[0352] An alternative strategy to identify high-affinity cross-reactive aptamers is cell-internalized SELEX, with positive selection on 293T cells transfected to express human or mouse EpCAM and pre-depleted of cells expressing a control protein. 37 The ability of the five highest affinity aptamers to be internalized into EpCAM / EphA2+ cells will be compared with previously selected aptamers by qRT-PCR and flow cytometry (using fluorescently tagged aptamers).

[0353] These aptamers can also be evaluated for their ability to specifically inhibit the proliferation of tumor cell lines. Aptamers with this property can be receptor antagonists and will be validated by examining their effects on cell signaling. Given the high homology between the extracellular domains of human and mouse EphA2 (>90% identity; >90% structural homology), identifying aptamers that cross-react with human and mouse EphA2 can be as simple as testing selected aptamers for cross-reactivity against mouse. Therefore, the ability of the existing panel of 20 human EphA2 aptamers to bind to mouse EphA2 can be evaluated first. Alternatively, the above strategy can be followed. For a small number of top aptamers, truncated sequences (lacking one or two library adapter sequences) can be synthesized to define the minimum sequence required for binding.

[0354] Aptamers of approximately 20-35 nt in length can be identified for each ligand, which can be designed into AsiCs suitable for chemical synthesis.

[0355] In vitro evaluation of TNBC-targeted AsiC and its activity against T-ICs. It will be possible to define which breast cancer subtypes can be efficiently transfected with TNBC-targeted AsiC and assess whether tumor knockdown is specific relative to normal tissue cells, first in a cell line and then in 10 tumor specimens to validate the translation of the cell line to 10 tissues. We will also assess the efficacy of transfection of TNBC-targeted AsiC and targeting breast T-ICs.

[0356] AsiC Design and Initial Testing The most attractive aptamers identified above (optimized based on selectivity for binding and expression in poor prognosis cancers versus normal cells, affinity, truncation to shorter lengths, importance of ligands in tumor formation and stem cell behavior, receptor antagonism, and cross-species reactivity) can be designed into AsiCs by ligation to siRNAs targeting eGFP, AKT1, and PLK1 (relative to control scrambled siRNAs) used for the initial EpCAM-AsiC described herein above.

[0357] A basal-like NBC cell line that stably expresses unstable (d1) EGFP (protein T1 / 2 of approximately 1 hour) was previously generated using lentivirus. GFP expression can be easily quantified by flow cytometry and imaging, and its knockdown has no biological consequences. The short T1 / 2 allows for rapid and sensitive detection of knockdown. AKT1, expressed in all cells, is a good endogenous gene for research because its knockdown has little effect on cell viability.

[0358] PLK1 is used for its anti-tumor effects because its knockdown is cytotoxic to cell division. Described herein is a robust and reproducible gene knockdown using EpCAM-AsiC targeting each of these genes. AsiC will be chemically synthesized with 2'-fluoropyrimidine to stabilize and inhibit innate immune recognition and a dT residue at their 3' end to prevent exonuclease digestion. The two strands are annealed to produce the final RNA ( Figures 10A-10B ). These AsiCs can be evaluated and compared with original EpCAM-AsiC (as positive control) and CD4- or PSMA-AsiC (as negative control) in in vitro dose response experiments with respect to AsiC uptake (using, for example, a fluorophore (such as AF-647) conjugated to the 3' end of the short chain that does not affect AsiC activity), gene knockdown, and reduced tumor cell line growth and survival. Selective uptake, gene knockdown, and anti-tumor effects can be quantified in several human basal cell-ATNBC cell lines (MB468, HCC1937, BPLER vs. immortalized epithelial cells) by flow cytometry, flow cytometry and qRT-PCR, and Cell-TiterGlo and Annexin-PI staining, respectively. These experiments can enable the selection of a few best-performing AsiCs that recognize EpCAM and EphA2.

[0359] Types of breast cancer that respond to AsiCs targeting TNBC. It can be determined which types of breast cancer can be transfected with the selected AsiCs and how specific gene knockdown is in tumors relative to normal epithelial cells. In vitro knockdown by selected AsiCs can be evaluated in 20 human breast cancer cell lines representing common breast cancer subtypes but with a bias towards TNBC (14 TNBC cell lines, plus liminal and Her2+ cell line samples). 93 . Aptamer ligand expression, uptake and gene silencing of fluorescently labeled AsiC can be compared with BPE57 and fibroblast cell lines as negative controls. This large panel of cell lines allows assessment of how cell surface EpCAM and EphA2 levels affect RNA uptake and gene silencing, and whether there is an expression threshold required for effective knockdown. Dose response experiments can allow verification of the high affinity of the aptamers retained in AsiC. The specificity of uptake (relative to non-specific "adhesion") will be verified by using acid wash to remove loosely adhering aptamers and showing that binding is competed by unlabeled aptamers and eliminated when cells are trypsinized before treatment. AsiC-mediated transfection is compared with lipid transfection as a positive control and naked siRNA as a negative control. Knockdown is assessed by flow cytometry and qRT-PCR after 5 days, and 5 days is the optimal time for AsiC-mediated knockdown. It is expected that uptake and gene silencing will be related to aptamer ligand levels. To verify that tumor cell specificity was maintained in mixtures of ligand+ and liganddim / - untransformed mammary epithelial cells, we compared fluorescent AsiC uptake, gene knockdown, and survival when PLK1 was the gene target in mixtures of tumor cells expressing varying levels of the aptamer ligand and varying numbers of GFP+ BPE cells.

[0360] Do epithelial primary breast cancer cells preferentially take up TNBC-targeted AsiC and show knockdown relative to normal epithelial cells in tissue explants? To assess primary tumor uptake and knockdown and anticipate potential toxicity to normal tissue cells, we next evaluated in situ transfection and gene knockdown in explants of 10 luminal, Her2+, and TNBC breast cancers and surrounding normal tissue. We can analyze samples from approximately 25 tumors to provide a comprehensive understanding of common tumor subtypes. Tumor subtypes can be confirmed by histology and immunohistochemistry (IHC) staining for ER, PR, Her2, and E-cadherin. If the aptamer recognizes a mouse ligand, we can also use mouse tumor / normal tissue to assess potential toxicity to normal epithelium. We can compare normal tissue, which lacks a significant source of competing tumor cells, with tissue containing tumor cells. This may be important for predicting toxicity in cases where AsiC is administered to patients with low / undetectable tumor burden after treatment or surgery. These experiments will also allow assessment of whether knockdown in the 10 tumors is matched to the cell line, whether tissue architecture affects uptake / knockdown in tumor cells, and how different tumor subtypes transfect. Among the considerations here is that epithelial breast cancers will undergo efficient gene knockdown, but normal epithelial cells will not.

[0361] Can be cut into about 3×3×3mm 3 Biopsies of 100 microtiter plates were transfected in microtiter wells, which should mimic in vivo uptake after SQ or IV infusion. Both lipofectamine-encapsulated siRNA and cholesterol-conjugated siRNA were effective in gene knockdown in normal epithelial cells in polarized columnar and squamous genital mucosa. 108、109 , while naked siRNA is not taken up. Similar results are expected with these controls in normal mammary epithelial tissue. We can analyze the knockdown of 10 cells digested with collagenase in parallel to compare the knockdown with the results achieved in tissues and cancer cell lines. We can first confirm these controls using siRNA targeting epithelial genes that we have previously knocked down (e.g., E-cadherin, cytokeratin (CK)-5 (a good marker for basal cells) and 14, and integrin-1). 93、108、109, their expression can be easily followed by IHC, fluorescence microscopy (FM) or flow cytometry of isolated cells. Staining of target genes can be correlated with staining of phenotypic markers and fluorescently labeled siRNA to determine which cell types are targeted. Pan-CK antibodies can distinguish epithelial cells (normal and tumor) from stroma. Of particular interest is the delivery and CK5 knockdown in rare basal cell tissue stem cells, because EpCAM-AsiC can target these cells and effectively cause depletion of normal tissue stem cells. Tissue toxicity and inflammation will be assessed by H&E staining of tissue sections and qRT-PCR assays of type I interferons and inflammatory cytokines (IL-1, IL-6, TNF-α). If detected, additional chemical modifications of the RNA sequence (in addition to 2'-fluoropyrimidine) will also be introduced to eliminate potential harmful inflammation.

[0362] Can AsiC targeting TNBC target breast tumor-initiating cells? We chose EpCAM and EphA2 as aptamer targets, in part because of their potential to transfect T-ICs. Mammary T-ICs are not uniquely defined by phenotypic markers (and they may in fact be heterogeneous). 93、 110-113), which makes experiments challenging because T-ICs are functionally defined by their ability to initiate small tumors that can be serially transplanted. Staining for CD44, CD24, EpCAM, CD133, CD49f, or ALDH1 in different combinations enriched T-ICs. 59、72、78、114-121 Different approaches define overlapping but distinct subsets of potential T-ICs. Without wishing to be bound by theory, it is contemplated herein that EpCAM- and EphA2-AsiCs will be taken up by T-ICs and induce gene silencing in T-ICs, and may be used for targeted therapy to eliminate or impair T-IC capacity within tumors.

[0363] To analyze AsiC uptake and gene silencing in T-IC subsets, multicolor flow cytometry of EpCAM, EphA2, CD44, and CD24 in a panel of breast cancer cell lines (luminal, Her2+, basal-A, and basal-B TNBC) can be used to identify which breast cell lines have putative T-IC populations containing cells that stain brightly for EpCAM and / or EphA2. We can also examine EpCAM / EphA2 staining of mammospheres and Aldefluor+ cells generated from these cell lines. 112、123-125. We can select approximately 4-5 of the brightest / most uniform EpCAM / EphA2 expressing lines in T-IC as the most attractive cell lines to study in this sub-target and can generate stable (d1)GFP expressing variants. These cell lines and their mammospheres and Aldefluor+ subpopulations can be incubated with AF647-labeled AsiC carrying GFP siRNA (and non-targeted PSMA-AsiC as a negative control). AsiC uptake is assessed by AF-647 fluorescence together with EpCAM or EphA2, CD44 and CD24 and Aldefluor staining. AsiC can be taken up by EpCAM+ or EphA2+CD44+CD24- / dim Aldefluor+ cells. To assess gene knockdown in T-IC phenotype cells, we can monitor GFP expression and remaining cells in the T-IC population by flow cytometry and qRT-PCR after treatment with eGFP or AsiC loaded with control siRNA. We can also assess knockdown of endogenous PLK1 and AKT1.

[0364] These experiments could indicate whether T-ICs in different subtypes of breast cancer are targeted by EpCAM / EphA2-AsiC. In subsequent experiments, we will focus on cell lines in which we have >80% knockdown in T-IC-enriched populations. If knockdown is inefficient, we can modify transfection conditions (amount of AsiC, cell number, volume, etc.). Next, we can assess whether AsiCs inhibit mammosphere and colony formation, reduce subpopulations expressing CD44 and ALDH1, and the size of side population cells. In addition to knockdown of PLK1, we can design and evaluate AsiCs targeting a small number of additional genes on which mammary T-ICs depend for self-renewal or maintenance of pluripotency. Basal-like TNBC T-ICs are selectively sensitive to proteasome inhibition93.

[0365] Therefore, we can evaluate the expression of proteasome components (PSMA2) and potentially other selective T-IC dependent genes such as MSI1 (Musashi), which regulate Wnt and Notch signaling in mammary T-ICs. 126-130 RNA-binding protein or BMI1, a polycomb component required for stem cell self-renewal 131-134) knockdown. After verifying that these genes are expressed and knocked down in mammosphere cells, we can treat adherent cells and mammospheres with AsiC targeting PLK1, MSI1, BMI1, or PSMA2 or AsiC targeting eGFP as a negative control and measure the size of the T-IC subpopulation after 5-7 days by staining with CD44, CD24, EpCAM, CD133, CD49f, and ALDH1. We can also measure the proportion of cells that efflux small molecule dyes ("side population cells"). These experiments can be supplemented by functional assays that quantify the frequency of colony-forming cells and mammospheres. Serial replating can assess whether the ability of continuously proliferating T-ICs as mammospheres is inhibited. It is considered in this article that knocking down PLK1, MSI1, BMI1, or PSMA2 can reduce the number, proliferation, and function of T-ICs in T-ICs from some cell lines, but different genes may be more active in different breast cell lines. For example, proteasome inhibition inhibits T-IC elimination in basal-like TNBC, but only in 1 of 3 mesenchymal TNBC cell lines and not in more differentiated non-TNBC tumors. 93 .

[0366] Knockdown strategies to inhibit T-ICs can be further investigated through experiments using available chemical inhibitors such as bortezomib, or by examining whether knockdown of other genes in the same pathway (e.g., NOTCH1, β-catenin, or WNT1, for MSI1) also has anti-T-IC activity. Next, using AsiCs, which have shown promise in vitro, we determined whether short-term ex vivo exposure of basal-like TNBC cell lines to AsiCs inhibits TNBC tumor initiation, as a final measure of their ability to inhibit T-ICs. Viability was assessed in cell lines treated overnight with selected AsiCs (as well as AsiCs containing PSMA aptamers or eGFP siRNA as negative controls). After verifying that short-term siRNA exposure did not affect viability, ex vivo treated cells were injected orthotopically into NOD / scid / "c- / - (NSG) mice (these mice have the highest tumor implant uptake) at a range of cell numbers. Bortezomib treatment for 24 hours (at which point approximately 40% of cells remain viable) served as a positive control.

[0367] In vivo evaluation of AsiC targeting TNBC

[0368] The best performing few AsiCs can then be evaluated in vivo in nude mice carrying mammary fat pad xenografts of aptamer ligands + basal-A TNBC cell lines (e.g., MB468 or HCC1187), compared to ligand-breast cancer cell lines (e.g., basal-B MB231), with approximately 5-8 mice / gp to obtain reproducible statistics based on our experience with these models. For in vivo imaging, we have generated stable luminescent / fluorescent cell lines by infection with lentivirus expressing mCherry and luciferase.

[0369] Systemic delivery and knockdown in tumor cells. Because unmodified AsiCs are small (approximately 30 kDa), they are rapidly eliminated by renal filtration when injected IV or IP. 20 kDa polyethylene glycol (PEG) can be attached to the 5' end of the inactive strand (passenger strand) of the siRNA. 21 . IV-injected PEGylated PSMA-AsiC concentrated in subcutaneous tumors; PEGylation extended the circulating T1 / 2 of IP-injected AsiC from <35 minutes to >>30 hours, increased the durability of gene silencing to approximately 5 days, and reduced the effective tumor-suppressive dose 8-fold to 250 pmol×5 injections. We also found (not shown) that SQ injection of 5 mg / kg unmodified CD4-AsiC caused systemic specific knockdown of CD4+ cells in the spleen and proximal and distal lymph nodes of humanized mice. Therefore, by using AF-790-conjugated AsiC and IVIS Spectrum in vivo imaging and by Taqman assay of active chains in blood, urine, liver, and tumor samples, we can compare AsiC levels after IV and SQ administration of the original AsiC construct and PEG-AsiC. Samples were collected frequently on the first day and samples could be analyzed within 5 days. Tissue damage can be assessed on tissue sections, and blood can be analyzed for hematologic, liver, and kidney toxicity by blood cell count and serum chemistry. Toxicity related to induction of innate immunity or inflammation can be assessed by ELISA assays for serum interferon and inflammatory cytokines. Circulating T1 / 2 and the proportion of injected drug confined to EpCAM+ tumors can be calculated. Based on our preliminary experiments with SQ and IV administration of CD4-AsiC and PSMA-AsiC 9、21、25 Based on our in vivo experience, without wishing to be bound by theory, it is contemplated herein that unPEGylated AsiC will be rapidly excreted following IV administration, but SQ EpCAM-AsiC and IV PEG-AsiC will more favorably localize to tumor xenografts.

[0370] Knockdown of mCherry and PLK1 following a single AsiC injection over a range of concentrations can be assessed by in vivo imaging and by qRT-PCR, FM, and flow cytometry of tumor samples harvested 4, 7, and 12 days after treatment. These experiments can provide estimates of the effective dose required for 50%, 75%, and 90% (ED50, ED75, ED90) of peak tumor gene knockdown, as well as the durability of knockdown in the tumor (quantified as T-KD50 = the time it takes for tumor expression to return to control midway from peak knockdown). These parameters can be determined for each construct. We can also determine the maximum tolerated dose (MTD) of the PLK1 construct. Signs of inappropriate PK / PD or innate immune stimulation will lead us to adjust chemical modifications (addition of 2'-OMe ribose to certain residues) or add longer PEG polymers to improve these parameters using simple methods.

[0371] Antitumor effect. We can test how the best AsiC targeting TNBC works against basal cell-A tumors injected IV in nude mice (as a metastasis model) or implanted in the mammary fat pad by in vivo imaging. We can start by targeting PLK1 as a proof of principle. 21、107 . PLK1-AsiC can be injected SQ and / or IV in groups of 8 mice bearing basal cell-ATNBC fat pad tumors (group size selected for statistical significance based on previous experiments) using a dosing regimen selected based on PK / PD results. Mice can be treated once tumors become palpable. The effects on representative ligand+ and ligand- tumors will be compared. Control mice can be treated with PBS or naked siRNA, AsiC carrying scrambled siRNA, and PLK1PSMA-AsiC. Tumor size can be quantified by imaging and calipers. If the antitumor effect is unsatisfactory, the dosing regimen can be adjusted to the maximum tolerated regimen.

[0372] We can also compare the effects of PLK1 knockdown and standard-of-care chemotherapy, both on their own and in combination, in anticipation of potential clinical studies. If complete tumor regression is achieved, we can evaluate dose reductions. Effective regimens can also be evaluated in mice implanted with a few other basal-A TNBC cell lines to confirm the generalizability of the anti-tumor response. We can also evaluate AsiC treatment after IV injection of tumor cells to determine effectiveness against distant metastases. At sacrifice, mice can be sacrificed, and residual microscopic or macroscopic tumors in the mammary fat pad can be examined by FM, H&E, and IHC. Residual tumor cells can also be assessed for EpCAM / EphA2 expression to determine whether tumor resistance may develop as a result of downregulating the aptamer ligand. Treated mice can also be observed for clinical signs of toxicity, and intestinal and myelotoxicity can be carefully examined at sacrifice by blood cell counts and pathological examination of the intestine, bone marrow, and spleen. AsiC designed with cross-reactive aptamers can be used to assess toxicity to normal epithelium. Using our best AsiC designs, we can next begin to compare PLK1 knockdown with knockdown of TNBC-dependent genes identified in our siRNA screen 93 (such as PSMA2 or MCL1), tested alone or in combination with PLK1.

[0373] References:

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[0508] Example 4

[0509] RNA interference (RNAi) provides an exciting opportunity for treating diseases by knocking down pathogenic genes. Recent early clinical trials have shown promising and sustained gene knockdown and / or clinical benefits in a few diseases caused by abnormal gene expression in the liver. The main obstacle to using RNAi for cancer treatment is the delivery of small RNAs to disseminated cancer cells. Most epithelial cancer cells and tumor-initiating cells (T-ICs) therein highly express EpCAM, the first described tumor antigen. All epithelial breast cancer cell lines we tested showed bright staining of EpCAM, while immortalized normal mammary epithelial cells and fibroblasts did not. Chimeric RNAs composed of structured RNA (called aptamers) can be used to achieve targeted gene knockdown in epithelial cancer cells in vivo, which is covalently linked to siRNA and selected for binding to EpCAM with high affinity. These EpCAM aptamer-siRNA chimeras (AsiC) are taken up by EpCAM+ cells and selectively cause gene knockdown in epithelial breast cancer cells rather than normal epithelial cells. Furthermore, knockdown of PLK1 using EpCAM-AsiC suppressed colony and mammosphere formation in epithelial breast cancer cell lines in in vitro assays of tumor-initiating potential and tumor initiation.

[0510] Subcutaneously injected PLK1 EpCAM-AsiC is specifically taken up by EpCAM+ basal-A triple-negative breast cancer (TNBC) orthotopic xenografts and causes rapid tumor regression. TNBC has the worst prognosis of any breast cancer, and there are no targeted therapies for TNBC. Specifically contemplated herein is the use of EpCAM-AsiC for targeted gene knockdown to treat epithelial (basal-like) TNBC cancers while sparing normal cells and eliminating the T-ICs within them. It will be possible to define which breast cancer subtypes can be targeted by EpCAM-AsiC and determine how EpCAM levels affect uptake and gene silencing. The relative uptake / knockdown in EpCAM-expressing cancer cells and normal epithelial cells can be assessed in human breast cancer tissue explants. It will also be determined whether EpCAM-AsiC can target breast T-ICs to disrupt tumor initiation.

[0511] The drug-like properties of EpCAM-AsiC can be optimized. Cellular uptake, endosomal release, systemic delivery, and in vivo gene knockdown of EpCAM-AsiC can be optimized. The pharmacokinetics (PK) and pharmacodynamics (PD) of EpCAM-AsiC uptake and gene silencing, as well as tumor suppression, can be evaluated using in vivo animal imaging in TNBC cell line xenograft models. As a proof of principle, the anti-tumor effect of knockdown of PLK1, which is required for cell proliferation, can be evaluated. In addition, knockdown of new gene targets identified in a genome-wide siRNA screen for TNBC genetic dependencies was evaluated in a mouse xenograft model. The optimized EpCAM-AsiC and its understanding of PK, PD, and possible toxicity can be used in experiments for further toxicity and other preclinical studies.

[0512] Described herein is the development of EpCAM aptamer-siRNA chimeras as a method for targeted gene knockdown in tumor-initiating cells in basal cell-like triple-negative breast cancer and other epithelial cancers and within them. Currently, there are no targeted therapies for triple-negative breast cancer, which frequently recurs, or for highly malignant tumor-initiating cell subpopulations in breast cancer, which may be responsible for drug resistance and recurrence in some cases. These RNA pairs provide a versatile and flexible platform for RNA-based drugs to treat breast cancer with a poor prognosis.

[0513] Example 5

[0514] We demonstrate that: (1) the EpCAM aptamer itself does not affect cell growth or viability of EpCAM+ breast tumor cell lines (not shown); (2) when normal breast biopsies are mixed with EpCAM+ TNBC human breast tumor tissue in vitro, fluorescent EpCAM-AsiC is concentrated only in the tumor ( Figure 14 ); (3) Treatment of EpCAM+luminal and basal-A TNBC cells, but not mesenchymal TNBC cells, with PLK1 EpCAM-AsiC blocked in vitro assays of tumor-initiating cells (T-IC, colony and mammosphere formation) and in vivo tumor initiation ( Figures 15A-15C and Figure 16 ); (4) Subcutaneously (sc) injected EpCAM-AsiC was concentrated in EpCAM+ tumors on both sides away from the injection site in mice bearing EpCAM+ and EpCAM-TNBC ( Figures 17A-18B ); and (5) Most importantly, subcutaneous injection of PLK1 EpCAM-AsiC caused complete regression of palpable basal-A TNBC xenografts ( Figures 18A-18B). In addition, (6) a novel siRNA screen identified a novel shared genetic dependency of basal-A TNBCs with EpCAM-AsiC knockdown ( FIG19 ).

[0515] Without wishing to be bound by theory, T-ICs are heterogeneous and plastic in epithelial / mesenchymal gene expression. Although mesenchymal properties may contribute to initial tissue invasion, the formation (colonization) of clinically significant metastases may require epithelial properties. EpCAM-mediated siRNA delivery effectively blocked tumor initiation, but only in epithelial (basal cell-A TNBC, luminal) breast cancer.

[0516] The high affinity of the EpCAM aptamer and our uptake, gene knockdown, and proliferation experiments in homogeneous and mixed cell populations showed specific targeting to EpCAM+ cells. Normal epithelial cells and fibroblasts were not targeted. The new data showing that EpCAM-AsiC is not taken up by normal human breast biopsies is convincing.

[0517] Triple negative breast cancer (TNBC) is a diverse group of highly malignant cancers that do not express estrogen, progesterone and Her2 receptors, with the worst breast cancer prognosis. There is no targeted therapy for TNBC, and TNBC often recurs after cytotoxic therapy. Described herein is a platform for gene knockdown therapy using specifically targeted RNA interference (RNAi) for basal cell-like TNBC. RNAi can selectively knock down pathogenic genes. However, it is recognized that the therapeutic potential of gene knockdown for treating cancer requires an effective method to deliver RNA to diffuse cancer cells. There are two bottlenecks: RNA is made to cross the cell membrane, and from endosomes, it is spanned to the target cell cytoplasm at the location of RNAi mechanism. An ideal therapeutic method is to selectively knock down the gene in the cancer cell while retaining most normal cells to minimize toxicity.

[0518] Described herein is gene knockdown in basal cell-like TNBC (most TNBC) with chimeric RNA using aptamers, which are structured nucleic acids selected for high affinity binding to a target molecule for EpCAM (also known as CD326 or ESA), the first tumor antigen described. EpCAM is highly expressed in epithelial breast cancers (including basal cell-like TNBC), averaging 400-fold higher than normal breast tissue. It is also highly expressed in other epithelial cancers and is a marker for "cancer stem cells," also known as tumor initiating cells (T-ICs). Aptamer-siRNA chimeras (AsiC) covalently link a targeting aptamer to a siRNA ( Figure 10B ). Dicer cleaves siRNA from internal cellular aptamers.

[0519] Epithelial breast cancer cells, but not mesenchymal or normal epithelial cells, selectively took up EpCAM-AsiC and underwent gene knockdown in vitro. Furthermore, knockdown correlated closely with EpCAM levels. Knockdown of PLK1, a gene required for mitosis, using EpCAM-AsiC abolished colony and mammosphere formation (in in vitro assays relevant to self-renewal and tumor initiation) as well as tumor initiation in vivo, suggesting that EpCAM-AsiC may be used to target T-ICs. Subcutaneous injection of PLK1EpCAM-AsiC caused complete regression of EpCAM+ TNBC xenografts but had no effect on EpCAM- mesenchymal TNBCs.

[0520] Here we describe the use of EpCAM-AsiC for targeted gene knockdown to treat basal-like TNBC cancers, while sparing normal cells and eliminating T-ICs within them. In addition to selective delivery to target cells, AsiCs have important advantages for cancer therapy compared to RNA delivery via nanoparticles, liposomes, or RNA-binding proteins: (1) they bypass capture in the liver and lungs and concentrate in tumors; (2) as single RNA molecules, they are simpler and cheaper to manufacture than multicomponent drugs; (3) they have little toxicity and do not stimulate innate immunity or inflammation or produce significant off-target effects; (4) they can be used repeatedly because they do not elicit antibodies; and (5) they are stable in serum and other body fluids.

[0521] It is possible to define which breast cancer subtypes can be targeted by EpCAM-AsiC and determine how EpCAM levels affect uptake and gene silencing. The relative uptake / knockdown of cancerous tissue relative to normal epithelium can be assessed. It is also possible to determine whether EpCAM-AsiC can target breast T-ICs to inhibit tumor initiation. Important goals for optimizing EpCAM-AsiC are uptake, endosomal release, systemic delivery, and in vivo knockdown. The pharmacokinetics (PK) and pharmacodynamics (PD) of EpCAM-AsiC uptake, gene silencing, and tumor suppression were assessed by in vivo animal imaging in TNBC orthotopic xenografts. As a proof of principle, the antitumor effect of PLK1, which is required for cell proliferation, can be evaluated. Knockdown of other genes identified as genetic dependencies of basal-like TNBC in our genome-wide RNAi screen can be assessed. Described herein is the development of optimized EpCAM-AsiC, the knowledge of its PK, PD, and potential toxicity, and the identification of new basal-like TNBC-dependent genes for targeting.

[0522] Described herein are: the demonstration of selective EpCAM-AsiC activity in epithelial breast cancer compared to normal epithelium, and the evaluation of the potential of EpCAM-AsiC to transfect and eliminate mammary T-ICs (i.e., cancer stem cells); the optimization of EpCAM-AsiC for gene transfection and knockdown in epithelial TNBC cells in vitro and for systemic delivery and tumor localization in vivo, as well as the definition of PK and PD and the maximum tolerated dose; and the evaluation of the antitumor effects of an optimized EpCAM-AsiC targeting PLK1 and a novel dependency gene of basal-like TNBC in mouse models of primary and metastatic cancer in human epithelial TNBC.

[0523] Although most TNBC patients respond to chemotherapy, approximately one-third develop metastases and eventually die within 3 years. Therefore, new strategies are needed. TNBC is a heterogeneous, poorly differentiated tumor that may require subtype-based or personalized therapy. 1、3、4、72 Most TNBCs are basal-like or belong to the basal-A subtype. This paper describes a flexible targeting platform for the treatment of basal-like TNBCs suitable for personalized therapy. Drugs can not only be targeted to the tumor, but also selectively target the drug to attack the tumor's Achilles' heel by knocking down tumor-dependent genes. This strategy delivers small interfering RNA (siRNA) to epithelial cancer cells ( Figure 10B ), EpCAM is a cell surface receptor overexpressed on epithelial cancers (including basal-like TNBC). EpCAM is highly expressed on epithelial cancers and their T-ICs.

[0524] EpCAM targeting resulted in selective gene knockdown in basal-like TNBC but not in normal epithelial cells, which could reduce both drug dose and tissue toxicity.

[0525] As described herein, 9 of 9 basal-A TNBC and luminal breast cancer cell lines were strongly EpCAM+, whereas normal breast epithelial cell lines, fibroblasts, and mesenchymal TNBCs were close to background EpCAM ( Figure 1B ). Thus, almost all basal-like TNBCs (and possibly luminal breast cancers) will be targeted by EpCAM-AsiC. In addition, since approximately 100% of epithelial cancers (including lung, colon, pancreatic, and prostate cancers) stain brightly for EpCAM, this platform can also be used for RNAi-based treatment of common cancers.

[0526] When RNAi was discovered in mammals, small RNAs were hailed as the next new class of drugs. Researchers soon realized that getting RNAi to work as a drug was not simple. However, after solving the main obstacle to RNA therapy (cellular uptake), there is now optimism for RNAi-based drugs. Recent Phase I / II studies have shown 80%-95% gene knockdown in hypercholesterolemia, transthyretin-associated amyloidosis, hepatitis C, hemophilia, and liver metastases caused by abnormal liver gene expression. However, applying RNAi to cancer treatment remains a dream. The main obstacle to using RNAi for cancer is the delivery of small RNAs to diffuse cells. Methods and compositions (e.g., by using AsiC) to overcome this problem are described herein.

[0527] AsiC is a flexible platform that can target diverse cell surface receptors and knockdown any gene or combination of genes. By modifying the aptamer, the AsiC platform can overcome the delivery barriers that have hindered the application of RNAi-based therapeutics to most diseases. This strategy is ideal for personalized cancer therapy because the choice of target genes can be tailored to the molecular characteristics of the tumor. Furthermore, RNA cocktails can be used to knockdown multiple genes at once to predict and overcome drug resistance.

[0528] This article describes the development of an optimized EpCAM-AsiC with well-defined PK / PD.

[0529] An important cancer research goal is to eliminate T-ICs (cancer stem cells). T-ICs are relatively resistant to chemotherapy and are thought to be responsible for tumor recurrence and metastasis. The AsiCs described herein are designed to target (epithelial) T-ICs with high efficiency. They can therefore eliminate this aggressive subpopulation within tumors that are at risk for progressive disease (see Figure 16 ).

[0530] In addition to its potential therapeutic uses, EpCAM-AsiC can also be a powerful in vivo research tool for identifying tumor and T-IC dependency genes to define new drug targets.

[0531] This article describes a novel targeted therapy for epithelial cancers and T-ICs thereof by targeting EpCAM, a tumor antigen widely overexpressed in epithelial cancers and their T-ICs. Current targeted therapies rely on the use of tumor-specific antibodies or oncogenic kinase inhibitors. No one has previously demonstrated that unconjugated AsiC can have effective anti-tumor effects or that AsiC can be administered subcutaneously. Currently, there are no targeted therapies for TNBC or T-ICs. Developing targeted therapies for TNBC and developing methods to eliminate T-ICs are important unmet goals in cancer research.

[0532] The method described here targets tumors in two ways: aptamers specifically deliver therapeutic RNA to tumor cells, while genes for knockdown can be selected based on their specific molecular dependencies on the target tumor. In vivo knockdown testing demonstrated that basal-like TNBC and its T-ICs selectively depend on the proteasome, MCL1, and the U4 / U6-U5 tri-snRNP splicing complex. This work could identify a new set of drug targets amenable to conventional and RNAi-based therapies.

[0533] The trafficking of siRNAs in transfected cells can be examined, and individual steps of RNA processing in cells can be systematically optimized to improve the drug properties of siRNAs.

[0534] CD4-AsiC can durably knock down gene expression in CD4+ T lymphocytes and macrophages and inhibit HIV transmission to humanized mice. CD4-AsiC specifically suppresses gene expression in polarized human cervicovaginal tissue explants and macrophages and CD4+ T cells in the female reproductive tract of humanized mice. Because they are monomeric and do not cross-link with receptors, CD4-AsiC do not activate target cells. They also do not stimulate innate immunity. Intravaginal administration of only 80 pmol of CD4-AsiC targeting HIV genes and / or CCR5 to humanized mice completely blocks sexual transmission of HIV. RNAi-mediated gene knockdown persists in vivo for several weeks. Transmission is blocked by CCR5 CD4-AsiC administered 2 days before challenge. Significant, but incomplete, protection also occurs when exposure is delayed by 4 or 6 days. CD4-AsiC targeting gag / vif provides protection when administered post-exposure. Therefore, CD4-AsiC has promise as an HIV microbicide.

[0535] Protection against HIV transmission requires local knockdown in the reproductive tract. However, systemic delivery is more challenging and is required for cancer. Because AsiCs are small enough to be filtered by the kidneys, they are rapidly eliminated and cannot effectively cause gene silencing. In some embodiments, polyethylene glycol (PEG) can be attached to the 5'-end of the inactive (passenger) chain of the siRNA. Intravenously injected PEG-AsiC concentrates in subcutaneous tumors. PEGylation extends the circulating T1 / 2 of ip-injected AsiC from <35 minutes to >>30 hours, improves the durability of gene silencing to about 5 days, and reduces the required dose by 8-fold. Subcutaneous injection of unmodified CD4-AsiC specifically causes approximately 80% gene knockdown in CD4+ cells in the spleen, proximal and distal lymph nodes of humanized mice (not shown). Subcutaneous injection of EpCAM-AsiC similarly causes specific concentration / knockdown in EpCAM+ tumors (see below).

[0536] EpCAM-AsiC selectively knocks down gene expression in EpCAM+ cancer cells. EpCAM-AsiC has a 42-44 nt chain (19 nt aptamer + linker + 20-22 nt siRNA chain) annealed to a 20-22 nt complementary siRNA chain ( Figure 10B ). Commercially synthesized with 2'-fluoropyrimidines, they are RNase-resistant (T1 / 2 in serum > 3 days, data not shown) and do not trigger innate immunity 37、91-93 .

[0537] Surface EpCAM was high in all luminal and basal-like cell lines tested, but not in cells expressing hTERT (BPE). 94 , fibroblasts and mesenchymal TNBC immortalized normal epithelial cells close to the background ( Figure 1B ). Several of the designs tested (sense and antisense strand swapping, and several linkers) specifically knocked down gene expression in EpCAM+ cell lines, but the most effective design was Figure 10B As shown. Knockdown of eGFP and AKT1 by EpCAM-AsiC was uniform and selective for EpCAM+ cells and was as effective as nonselective siRNA lipid transfection ( Figures 13A-13C In eight breast cancer cell lines, AKT1 knockdown and cell proliferation inhibition by PLK1 EpCAM-AsiC were closely associated with EpCAM levels ( Figure 11B-11C ). The EpCAM aptamer itself had no effect on cell proliferation (not shown). When EpCAM-BPE cells were mixed with an epithelial TNBC cell line, EpCAM-AsiC knocked down AKT1 and caused PLK1-sensitive cell death only in tumor cells, not in normal epithelial cells (not shown). The proportion of viable tumor cells decreased 7-fold after 3 days. When we added fluorescent AsiC, cholesterol-conjugated siRNA (chol-siRNA taken up by normal epithelial cells), or naked siRNA to normal breast and tumor biopsies, EpCAM-AsiC concentrated only in tumors ( Figure 14 ). Therefore, EpCAM-AsiC is specific for epithelial tumor cells.

[0538] EpCAM-AsiC inhibits T-ICs of EpCAM+ tumors. EpCAM was chosen for partial targeting because EpCAM marks T-ICs and metastasis-initiating cells (M-ICs). To investigate whether EpCAM-AsiC inhibits T-ICs, we compared colony and mammosphere formation following mock treatment, paclitaxel, or EpCAM-AsiC directed against eGFP or PLK1 (functional surrogates for T-ICs). PLK1 EpCAM-AsiC inhibited colony and mammosphere formation in multiple EpCAM+ basal-like TNBC and luminal cell lines more strongly than paclitaxel but had no activity against EpCAM-basal-B TNBCs ( Figures 15A-15C To evaluate the effect of EpCAM-AsiC on tumor initiation, viable luc+EpCAM+MB468 and EpCAM-MB231 cells treated overnight with medium or PLK1 or GFP EpCAM-AsiC were subcutaneously implanted into nude mice. PLK1 EpCAM-AsiC blocked tumor formation, but only in EpCAM+ tumors ( Figure 16 , data not shown). Thus, EpCAM-AsiC inhibits tumor initiation in EpCAM+ breast cancer.

[0539] EpCAM-AsiC was selectively taken up by EpCAM+ TNBC and caused tumor regression. To investigate the potential clinical usefulness of EpCAM-AsiC, we first examined the effect of Alexa750-labeled EpCAM-AsiC (EpCAM-AsiC) injected subcutaneously into the scruff of the neck of mice bearing EpCAM+ and EpCAM- TNBC in both flanks. Figures 17A-17B ) delivery. EpCAM-AsiC was concentrated only in EpCAM+ tumors. Mice bearing bilateral tumors were mock treated or injected every two weeks with PLK1 or GFP EpCAM-AsiC, and the tumors grew and subsequently glowed. EpCAM+ tumors rapidly and completely regressed only in mice that received AsiC targeting PLK1 ( Figures 18A-18B The experiment was repeated with additional control groups (EpCAM aptamer itself or PLK1 siRNA), both of which showed no antitumor activity (data not shown). Therefore, subcutaneously injected EpCAM-AsiC showed specific antitumor activity against basal-A TNBC cells.

[0540] Live cell imaging of siRNA uptake, endosomal release, and gene silencing. An optimized spinning disk confocal microscope capable of single-molecule detection was used to detect weak cytosolic signals of fluorescent RNA that had previously been impossible to release. HeLa cells incubated with Alexa647-siRNA lipoplexes were imaged every 3 seconds. Late endosomes containing RNA released a small amount of cargo RNA, which rapidly diffused to fill the cytosol (data not shown). Release occurred in a short period of time, about 15-20 minutes after endocytosis. About 104 siRNAs were released in a typical event. In HeLa cells stably expressing eGFP-d1, GFP siRNA caused a rapid decrease in GFP expression after endosomal release with a T1 / 2 of about 2.5 hours. Effective gene silencing only required about 1000 cytosolic siRNAs. Release triggered autophagy, which isolated the RNA-containing endosomes within the double autophagic membrane. No release was subsequently achieved.

[0541] We applied this approach to study the uptake / release of Cy3-labeled EpCAM-AsiC, comparing EpCAM+MB468 TNBC and EpCAM-BPE cells. Uptake and release were negligible in BPE but significantly reduced in MB468. This imaging approach and our understanding of siRNA trafficking can be used to optimize EpCAM-AsiC design for improved endosomal release and knockdown.

[0542] Identification of basal-like TNBC-dependent genes (BDGs). To identify the genetic dependencies of basal-like TNBCs targetable by EpCAM-AsiC, a genome-wide siRNA lethal screen was performed comparing human primary mammary epithelial cells, basal-like BPLER, and myoepithelial HMLER cells transformed with the same oncogene in different culture media. Although essentially isogenic, BPLER is highly malignant and enriched for T-ICs, with only 50 cells forming tumors in nude mice, while HMLER requires >105 cells to initiate tumors. The screen identified 154 genes that BPLER, but not HMLER, depends on. Proteasome genes were highly enriched (P<10-14). Expression of BPLER-dependent genes was associated with poor prognosis in breast cancer, but not lung or colon cancer. Proteasome inhibitor sensitivity is a common feature of basal-like TNBCs and is associated with MCL1 dependency. Normal mammary epithelial cells, luminal breast cancer cell lines, and mesenchymal TNBC cell lines do not depend on the proteasome or MCL1. Proteasome inhibition not only killed basal-A TNBCs but also blocked T-IC function in colony and mammosphere assays and was again selective primarily in basal-like TNBCs. Brief exposure to bortezomib also inhibited tumor initiation in a mouse basal-like TNBC cell line.

[0543] Next, we tested whether proteasome inhibition inhibits the growth of basal-like TNBC tumors in mice. Bortezomib does not penetrate well into solid tumors, which limits its clinical application. A maximum tolerated intravenous dose (MTD) is required to inhibit proteasome activity in subcutaneous tumors. Treatment with the MTD strongly inhibited tumor growth of three human basal-A TNBC cell lines and one mouse basal-A TNBC cell line, as well as 10 TNBCs, that spontaneously arose in Tp53+ / - mice, but was ineffective against basal-B or luminal cell lines. Similar results were obtained with carfilzomib. Bortezomib also blocked lung colonization of intravenously injected mouse TNBC cells. Therefore, epithelial TNBC growth, tumor initiation, and metastasis selectively require the proteasome. Although tumor penetration and PD may improve with newer proteasome inhibitors, proteasome gene knockdown may provide more effective proteasome inhibition.

[0544] Because TNBC is heterogeneous 1、3、4、72 We rescreened 154 BPLER-dependent genes in four basal-A TNBC and three luminal human cancer cell lines. Our goal was to identify additional shared dependencies of basal-like TNBC cell lines that serve as potential EpCAM-AsiC targets. Only 21 of the 154 BPLER-dependent genes reduced activity by at least 2-fold in three of the four basal-A cell lines tested. These putative BDGs clustered in four functional groups: four proteasome genes and MCL1 (previously validated), 10 genes involved in RNA splicing, two genes involved in mitosis, and two genes required for nuclear export. Twenty of the 21 BDG genes were retested using a new set of siRNAs, and 14 genes were reconfirmed (the additional "hits" may be secondary to off-target effects, or their knockdown may not be sufficient to cause lethality). Notably, nine of the 10 splicing genes were reconfirmed. They include four members of the U4 / U6-U5 tri-snRNP complex, PRPF8, PFPF38A, RBM22, and USP39. Other shared hits of interest are the RAN nuclear export G protein and the nucleoporin NUP205, as well as NDC80, a centromere component that anchors the centromere to the mitotic spindle. USP39 is also required for the mitotic spindle checkpoint.

[0545] TNBC is known to be particularly sensitive to anti-mitotic agents. USP39 is overexpressed in breast cancer cells and normal breast tissue, and USP39 knockdown inhibits the proliferation and colony formation of luminal MCF7 cells. In addition, in zebrafish, USP39 mutations lead to splicing defects in tumor suppressor genes such as rb1 and p21. To explore the therapeutic effect of inhibiting splicing in basal-like TNBC, we silenced four spliceosomal tri-snRNP complexes BDG (PRPF8, PRPF38A, RBM22, USP39) in six basal-like cell lines and luminal MCF7 cells (Figure 19). Knockdown of PRPF8, PRPF38A or RBM22, but not MCF7, activated caspase-3 and caused lethality in six of the six basal-like cell lines; USP39 knockdown killed three of the six basal-like cell lines. Spliceosomal proteins are frequently upregulated in breast cancer cell lines of all subtypes. The viability of all six basal-like cell lines, but not MCF7 cells, was reduced at least 2-fold by knockdown of the mitotic centromere gene NDC80 or the nuclear export genes RAN or NUP205. Furthermore, in three of the three basal-like TNBC cell lines, knockdown of each of the tri-snRNP complex genes, RAN, NUP205, or NDC80, blocked colony formation (a potential T-IC surrogate).

[0546] EpCAM-AsiC can cause targeted gene knockdown in EpCAM+ tumors and T-ICs within them. Although there may be some uptake in normal epithelial cells that weakly express EpCAM, gene knockdown will be concentrated in EpCAM. bright As described herein, EpCAM-AsiC can be optimized for favorable PK / PD, thereby suppressing tumor growth and metastasis of basal-like TNBC with acceptable toxicity in a mouse model.

[0547] EpCAM-AsiCs targeting eGFP, AKT1, and PLK1 were used herein as models for evaluating gene knockdown and optimizing AsiC design. Lentiviruses can generate cell lines that stably express unstable (d1) EGFP with a protein T1 / 2 of approximately 1 hour. GFP expression can be easily quantified by flow cytometry and imaging, and its knockdown has no biological consequences. The short T1 / 2 allows for rapid and sensitive detection of knockdown. AKT1, expressed in all cells tested, is an excellent endogenous gene to study, as its knockdown in TNBC does not affect cell viability. PLK1 was used as proof of principle for its antitumor effect, as its knockdown is cytotoxic to all dividing cells. We previously showed that PLK1 knockdown using different delivery strategies significantly suppressed Her2+ breast cancer in mice. In a recent screen, PLK1 was unique among kinase genes in that its knockdown eliminated mammary T-ICs. We have achieved reliable and reproducible gene knockdown using EpCAM-AsiCs targeting each of these genes.

[0548] EpCAM-AsiC can be purchased, for example, as non-GMP RNA from TriLink or NITTO Avecia. Each strand of EpCAM-AsiC is synthesized with 2'-fluoropyrimidine, protected at their 3'-ends with dT residues to prevent exonuclease digestion, and then annealed to produce the final RNA ( Figure 10B As we optimize AsiC, other chemical modifications can be substituted and tested to determine if they confer improved activity. Aptamer alone and AsiC carrying non-targeting siRNA can serve as controls. Some eGFP EpCAM-AsiC can also be annealed to an antisense strand modified with a fluorophore at the 3' end (which does not affect AsiC activity (not shown)) to quantify AsiC uptake and transport within cells and in vivo.

[0549] Specific EpCAM-AsiC knockdown in epithelial breast cancer and breast T-ICs versus normal epithelial cells. It will be possible to determine which breast cancer subtypes are transfected with EpCAM-AsiC and assess whether tumor knockdown is specific to cancer cells, first in cell lines and then in 10 tumor tissues to confirm the results of orthotopic transfer of cell lines to tissues. Because EpCAM-AsiC may also transfect normal tissue stem cells, knockdown and toxicity in these rare basal cells will be assessed in tissue experiments. The potential of EpCAM-AsiC to transduce and target breast T-ICs will also be evaluated.

[0550] Types of breast cancer that respond to EpCAM-AsiC. We first needed to know which types of breast cancer could be transfected with EpCAM-AsiC and how specific the gene knockdown was in tumors relative to normal epithelial cells. We performed this by transducing EpCAM-AsiC into a panel of 14 breast cancer cell lines representing common breast cancer subtypes, but focusing on TNBC (14 TNBC cell lines, plus luminal and Her2+ cell line samples). 95 We evaluated in vitro knockdown in a panel of 20 human breast cancer cell lines, extending our initial studies ( Figures 13A-13C and Figure 11B-11C ). EpCAM expression, uptake of Cy3 labeled AsiC and gene silencing in tumor cell lines can be compared with BPE94 and fibroblasts. This large tumor panel will enable us to assess how cell surface EpCAM levels affect gene silencing and whether there is an EpCAM expression threshold for effective knockdown. We can also use a variety of EpCAM+ cell lines (the reported high binding affinity of EpCAM aptamers is retained in AsiC) for verification in dose response experiments. The specificity of uptake (relative to nonspecific "adhesion") can be verified by using acid washes to remove loosely adhered aptamers and showing that binding is competed by unlabeled aptamers and eliminated when cells are trypsinized before treatment. EpCAM-AsiC mediated transfection can be compared with lipid transfection and naked siRNA as controls. Knockdown was assessed by flow cytometry and qRT-PCR after 5 days, which is the optimal time for AsiC mediated knockdown. We expect uptake and gene silencing to be correlated with EpCAM levels. In order to verify that the uptake and gene silencing are correlated with EpCAM levels in EpCAM+ and EpCAM dim Specificity for EpCAM+ cells was maintained in mixtures of untransfected mammary epithelial cells, allowing us to compare fluorescent EpCAM-AsiC uptake, gene knockdown, and viability when PLK1 was gene-targeted in mixtures of tumor cells expressing varying levels of EpCAM (MFI range 100-1000) and varying numbers of GFP+ BPE cells.

[0551] Do epithelial breast cancer cells preferentially take up EpCAM-AsiC and show knockdown relative to normal epithelial cells in tissue explants? To assess knockdown in primary tumors and anticipate potential toxicity to normal tissue cells, we will next evaluate orthotopic transfection and gene knockdown in surrounding normal tissue from mastectomy specimens and explants of 10 luminal, Her2+, and TNBC breast cancers. Samples from approximately 25 tumors can be analyzed to provide a comprehensive view of tumor subtypes. Tumor subtype can be confirmed by histology and IHC staining for ER, PR, Her2, and E-cadherin. Normal tissue, which lacks a significant source of competing EpCAM+ cells, can be compared with tissue containing tumor cells. This may be important for predicting toxicity when administering AsiC to patients with low / undetectable tumor burden following treatment or surgery. These experiments will allow assessment of whether knockdown in the 10 tumors is compatible with the cell line, whether tissue architecture influences uptake / knockdown in tumor cells, and how well different tumor subtypes transfect.

[0552] Based on this article (e.g. Figure 14 ) provided data, it is considered here that epithelial breast cancer cells, but not normal epithelial cells, can undergo effective gene knockdown. 3The tissue of the sample is transfected in Optimem solution. Lipid-complexed siRNA and chol-siRNA both knock down genes in normal columnar and squamous reproductive tract epithelial cells, while naked siRNA is not taken in. We can first use siRNA to target epithelial genes (such as E-cadherin, claudin3, cytokeratin (CK) -5 (a good marker for basal cells) and integrins -1) that we have previously knocked down, and these controls are verified, and their expression can be easily processed by IHC, fluorescence microscopy (FM) or flow cytometry of isolated cells. The staining of the target gene product may be related to the staining of fluorescent siRNA and phenotypic markers to determine which cell type in the tissue is targeted. Pan-CK antibodies can be used to distinguish epithelial cells (normal and tumor) from matrix. We can also compare the knockdown of 10 cells digested with collagenase with the knockdown of tissue. Without wishing to be bound by theory, since EpCAM-AsiC may target these cells and potentially cause toxicity, the delivery and CK5 knockdown in rare basal tissue stem cells can be evaluated. Because toxicity to the GI tract is often dose-limiting for cancer drugs, we can repeat these studies using colon tumor specimens to determine whether colon cancer cells, normal intestinal epithelial cells, and crypt stem cells are transfected. These experiments can provide useful data on clinical toxicity and the selection of genes to be knocked down, i.e., if tissue stem cells are effectively transfected, we may be able to knock down cancer-dependent genes that are not essential for normal stem cells. Hematopoietic cells do not express EpCAM, so no hematologic toxicity is expected.

[0553] Can EpCAM-AsiC be used to target breast tumor-initiating cells? One reason we chose EpCAM as an aptamer target was its potential to transfect T-ICs ("cancer stem cells"). T-ICs are drug-resistant and are thought to be responsible for tumor initiation, recurrence, and metastasis. Breast T-ICs are not uniquely defined by phenotype, which makes experiments challenging because T-ICs are functionally defined by their ability to initiate tumors that can be serially transplanted. Staining for CD44, CD24, EpCAM, CD133, CD49f, or ALDH1 in different combinations enriched T-ICs. 49、61、67、107-111 .

[0554] Different schemes define overlapping but distinct subsets of potential T-ICs. T-ICs are heterogeneous and display plasticity in their epithelial versus mesenchymal properties (and may actually possess some features of both states simultaneously). 28、95、112-118Some breast T-ICs are mesenchymal and do not express EpCAM. However, increasing evidence suggests that the ability of basal-like TNBCs to colonize distant tissues and form macrometastases, arguably the most clinically important function of T-ICs, depends on their epithelial nature. In addition, our new data on the effect of EpCAM-AsiC on T-IC function and tumor initiation ( Figures 15A-15C and Figure 16 ) showed that EpCAM-AsiC has anti-T-IC activity against basal-like TNBC. We speculate that EpCAM-AsiC is taken up by basal-like TNBC T-ICs and could be used for targeted therapy to impair T-IC function.

[0555] To analyze EpCAM-AsiC uptake and gene silencing in T-ICs, we can first stain a panel of breast cancer cell lines with EpCAM, CD44, and CD24 to identify breast cell lines whose putative T-IC populations contain cells that stain brightly for EpCAM. We can also examine mammospheres and Aldefluor+ cells generated from these cell lines. 111、123、124 EpCAM staining. We can select approximately 4-5 lines with the most uniform EpCAM expression in T-ICs as the most attractive cell lines to study in this subpopulation (as well as 1-2 basal-B cell lines whose T-ICs may lack EpCAM staining as controls) and generate stable eGFP-expressing variants. These cell lines and their mammosphere and Aldefluor+ subpopulations can be incubated with fluorescent eGFP EpCAM-AsiC (as well as non-targeted PSMA-AsiC as a control). AsiC uptake can be assessed along with EpCAM, CD44, and CD24, as well as Aldefluor staining. AsiC should be taken up by EpCAM+CD44+CD24- / dim Aldefluor+ cells. To assess gene knockdown in cells with the T-IC phenotype, we can monitor GFP and the remaining cells in the T-IC population by flow cytometry and qRT-PCR (Aldefluor+ or mammosphere populations). We can also assess knockdown of endogenous PLK1 and AKT1. These experiments could inform us whether T-ICs in different breast cancer subtypes are targeted by EpCAM-AsiC. Next, we assessed whether AsiC inhibited mammosphere and colony formation and reduced phenotypic T-IC subpopulations or side populations.

[0556] We can also design and evaluate AsiCs targeting additional genes required for self-renewal or pluripotency. Since basal-like TNBC T-ICs are sensitive to proteasome inhibition, we can evaluate knockdown of a proteasome component (PSMA2). Other potential T-IC-dependent genes we will evaluate are MSI1, a gene highly expressed in mammary T-ICs that regulates Wnt and Notch signaling. 125-129 ), BMI1 (polycomb component required for self-renewal 130-133 ) and possibly several novel BDGs identified in our recent siRNA screen ( FIG19 ). MSI1 knockdown reduces mammosphere formation and stem cell markers in MCF7 and T47D cells 129 .

[0557] After verifying that these genes are expressed and knocked down in mammosphere cells, we can treat adherent cells and mammospheres with AsiC targeting these genes or eGFP as a negative control and measure the size of the T-IC subpopulation 5-7 days later by staining for CD44, CD24, EpCAM, CD133, CD49f, and ALDH1. We can also measure the proportion of cells that efflux small molecule dyes ("side population cells"). These experiments can be supplemented by functional assays that quantify colony-forming cells and mammospheres. Serial repetitions can investigate whether the dissemination of T-ICs as mammospheres is inhibited.

[0558] Knockdown of PLK1, MSI1, BMI1, or PSMA2 reduces T-IC number, proliferation, and function in some breast cancer subtypes, but different genes may be more active in different breast cell lines (i.e., proteasome inhibition eliminates T-ICs in basal-like TNBC but not in non-TNBC tumors and only one of three basal-B TNBCs). 95 Knockdown strategies to suppress T-IC can be further investigated by experiments using available chemical inhibitors and / or by knocking down other genes in the same pathway (e.g., NOTCH1, β-catenin, or WNT1 for MSI1). The effect of EpCAM-AsiC on T-IC can be compared with the effect of the EpCAM aptamer itself and an EpCAM antibody (adecatumumab, Amgen).

[0559] Next, we determined whether short-term ex vivo exposure of basal-like TNBC cell lines to EpCAM-AsiC inhibits tumor initiation as a final measure of T-IC inhibition. The most promising AsiCs can be tested in vivo. Viability can be assessed for cell lines treated overnight with AsiC (as well as AsiCs containing PSMA aptamers or eGFP siRNA as negative controls). After verifying that short-term siRNA exposure does not affect viability, ex vivo treated cells are injected orally into NOD / scid / !c- / - (NSG) mice (these mice have the highest tumor implantation uptake) at a range of cell numbers. Bortezomib, which reduces tumor initiation in basal-like TNBC, is used for pretreatment, or adecatumumab is used as a control.

[0560] Optimizing EpCAM-AsiC. To improve the drug properties of EpCAM-AsiC, we can optimize the various steps of in vitro gene knockdown and in vivo delivery. We can also modify the chemical composition of EpCAM-AsiC (if necessary) to minimize off-target effects.

[0561] In preliminary studies and published work, the AsiC concentration required for optimal knockdown in vitro was approximately 1 μM-4 μM, which is many times higher than the approximately 100 nM (or lower) concentration used for lipofection. For knockdown, EpCAM-AsiC follows the following steps: (1) cell receptor binding; (2) endocytosis; (3) endosomal release; (4) Dicer processing; (5) incorporation into the RNA-induced silencing complex (RISC); and (6) target mRNA cleavage. We can systematically optimize each step, focusing on steps (2) and (3), where we expect to obtain the greatest improvement in efficacy. AsiC design variables are the EpCAM aptamer, whose affinity affects steps 1 and 2; the linker sequence between the aptamer and siRNA, which controls step 4; and the siRNA sequence, which controls step 6. In addition, each residue used for chemical synthesis from the phosphoramidite building block can be chemically modified to reduce nuclease digestion, off-target inhibition of partially complementary sequences, binding and stimulation of innate immune RNA sensors, and improve cellular uptake and in vivo PK. The most common chemical modifications are replacing O with S in the phosphate backbone to create RNase-resistant phosphorothioate (PS) linkages, and replacing the 2'-OH group in the ribose sugar with 2'-F, 2'-O-methyl (2'OMe), or 2'-O-methoxyethyl (2'MOE). PS, 2'-F, and 2'-OMe modifications have been well tolerated in clinical trials, so we focused on them. 2'-OMe is naturally present in rRNA and tRNA and is therefore safe, and 2'-F is also well tolerated. Heavily Ps-modified nucleotides are sticky (and induce binding to serum proteins, which can improve circulating T1 / 2) and can cause undesirable side effects. Mildly modified PS-RNA is nontoxic. Chemical modifications can simultaneously inhibit and enhance gene silencing in steps 5 and 6. This can be an iterative process; by leveraging lessons learned from previous candidates, modifications in one step can be made to optimize the most attractive modified candidates for the other steps. We can verify that modified AsiCs selected for further development do not stimulate innate immunity or produce cytotoxicity. If they stimulate innate immunity or produce cytotoxicity, we can further modify our design to avoid these issues.

[0562] Optimizing in vitro knockdown

[0563] (1) EpCAM binding. The EpCAM aptamer has an affinity of 12 nM. It can be confirmed that this affinity is retained in EpCAM-AsiC. If AsiC has a lower affinity than the aptamer, we can use biolayer interferometry (OctetRED System, ICCB-Longwood Core) to recombinant EpCAM to compare the affinity of the aptamer and AsiC. If AsiC has a lower binding affinity, it may not fold properly. To enhance folding into the desired conformation, we can try to change the type and length of the linker between the aptamer and the AsiC sense chain (i.e., we can incorporate more 3C linkers or triethylene or hexaethylene glycol spacers).

[0564] ...

Claims

1. A chimeric molecule comprising an aptamer domain that binds to a cancer marker and an inhibitory nucleic acid domain that inhibits the expression of the MCL1 gene, wherein: The cancer marker is EpCAM or EphA2.

2. The molecule according to claim 1, wherein The molecule is an aptamer-siRNA chimera (AsiC).

3. The molecule according to claim 1 or 2, wherein The cancer marker-binding aptamer domain comprises the sequence of SEQ ID NO:

33.

4. The molecule according to any one of claims 1 to 3, wherein The cancer marker binding aptamer domain consists essentially of the sequence of SEQ ID NO:

33.

5. The molecule according to any one of claims 1 to 4, wherein The 3' end of the molecule contains dTdT.

6. The molecule according to any one of claims 1 to 5, wherein The molecule comprises at least one 2'-F pyrimidine.

7. A pharmaceutical composition comprising the molecule of any one of claims 1 to 6 and a pharmaceutically acceptable carrier.

8. The composition of claim 7, comprising at least two chimeric molecules according to any one of claims 1 to 6, wherein The chimeric molecules have different aptamer domains or inhibitory nucleic acid domains.

9. The composition according to claim 8, wherein Different aptamers or inhibitory nucleic acid domains recognize different targets.

10. The composition according to claim 8, wherein Different aptamers or inhibitory nucleic acid domains have different sequences and recognize the same target.

11. Use of a molecule according to any one of claims 1 to 6 or a composition according to any one of claims 7 to 10 in the preparation of a medicament for treating cancer.

12. The use according to claim 11, wherein The cancer is epithelial cancer or breast cancer.

13. The use according to claim 12, wherein The breast cancer is triple-negative breast cancer.

14. The use according to any one of claims 11 to 13, wherein The drug is for subcutaneous administration.

15. The use according to any one of claims 11 to 14, wherein The drug further comprises paclitaxel.

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