Aptamer Compositions and Methods of Use Thereof
An aptamer platform capable of efficiently delivering and silencing one, two or more genes in vivo or in vitro is provided. Methods of using the aptamer compositions for selectively targeting cells to down-regulate the expression of multiple genes are also provided.
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This application is a continuation of U.S. patent application Ser. No. 16/234,710 filed on Dec. 28, 2018, which claims benefit of and priority to U.S. Provisional Application No. 62/611,292 filed on Dec. 28, 2017 and 62/738,236 filed on Sep. 28, 2018, both of which are incorporated by reference in their entirety herein.
STATEMENT REGARDING FEDERALLY SPONSORED RESEARCHThis invention was made with government support under W81XWH-15-1-0333 awarded by the US Army Medical Research and Material Command. The government has certain rights in the invention.
REFERENCE TO SEQUENCE LISTINGThe Sequence Listing submitted on Dec. 28, 2018, as a text file named “064466.082_seqlisting_ST25.txt” created on Dec. 27, 2018 and having a size of 6.57 kilobytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).
FIELD OF THE INVENTIONThe invention is generally directed to nucleic acid compositions for inhibiting gene expression in targeted cells.
BACKGROUND OF THE INVENTIONTwenty to thirty percent of breast cancer is characterized with HER2 overexpression that correlates with poor prognosis, high aggressiveness, and extensive drug resistance (Slamon, et al., Science, 235:177-182 (1987)). Trastuzumab (Herceptin®), a humanized antibody, is the first line treatment for patients with HER2 positive breast cancer. However, the majority of patients eventually develop resistance.
The resistance to HER2 targeted therapies is associated with the increased levels of other HER family receptors (Narayan, et al., Cancer Res, 69: 2191-2194 (2009)). The HER family consists of EGFR (HER1), HER2, HER3 and HER4 (Arteaga, et al., Cancer Cell, 25: 282-303 (2014)). It is well known that the HER family is interdependent and displays functional redundancy in that the blockade of one HER receptor can often be compensated by another HER family member (Olayioye, et al., EMBO, 19: 3159-3167 (2000)). EGFR, HER2 and HER3 contribute to the initiation and progression of many cancers, and are well recognized therapeutic targets.
Ligand binding to HER receptors results in the formation of homo- and hetero-dimers. EGFR is one of the most studied HER-family receptors and a key oncogenic driver in many epithelial cancers (Nicholson, et al., Eur J Cancer, 37: S9-S15 (2001)). Accumulating evidence shows that HER3 is a key node in many cancers and involved in the resistance against EGFR- and HER2-targeted therapies through activating a compensatory PI3K-AKT survival pathway (Lee-Hoeflich, et al., Cancer Res, 68: 5878-5887 (2008); Holbro, et al., Proc Natl Acad Sci USA, 100: 8933-8938 (2003)). Resistance to trastuzumab in breast cancer cell lines is associated with upregulation of EGFR and HER3 (Narayan, et al., Cancer Res, 69: 2191-2194. (2009); Ritter, et al., Clin Cancer Res, 13: 4909-4919 (2007)).
HER2/HER3 heterodimer has been identified to function like an oncogene to support the proliferation of HER2-overexpressing tumor cells (Holbro, et al., Proc Natl Acad Sci USA, 100: 8933-8938 (2003)). The cross-talk and compensatory functionalities of HER family receptors provide strong rationales for co-targeting of EGFR, HER2 and HER3 in HER-dependent cancer treatment. In line with the notion of combining treatment, bi-specific antibodies and pan-HER antibodies have been developed and show more efficacious than single receptor targeting antibodies. Bispecific MM11 antibody targeting both HER2 and HER3 is able to suppress the proliferation of HER2-expressing tumor cells (Kontermann, et al., Drug Discov Today, 20:838-847 (2015)). Pan-HER with six antibody mixture targeting EGFR, HER2 and HER3 has been developed and displayed superior potency compared to agents targeting single receptors in preclinical studies (Jacobsen, et al., Clin Cancer Res, 21: 4110-4122 (2015)).
Since HER3 lacks intrinsic kinase activity, TKIs (tyrosine-kinase inhibitors) will be not effective in inhibiting HER3. Antibody combinations are the current strategies to inhibit HER family signaling network (Hu, et al., Cancer Res, 75: 159-170 (2015); Iida, et al., Mol Cancer Ther, 15: 2175-2186 (2016)). However, most antibodies are expensive to produce and have high immunogenicity. To address immunogenicity and high cost of antibody, a nucleic acid-based multiple function molecule with a siRNA and two RNA aptamers was engineered. The new siRNA-aptamer chimera is able to target EGFR/HER2/HER3 simultaneously with low toxicity. Recently, aptamer-siRNA chimera, employing only RNA molecules, is attractive for cell type-specific gene silencing. Aptamers are ssDNA or RNA and selected through in vitro enrichment process (Ellington, et al., Nature, 346: 818-822 (1990)). Like antibodies, aptamers can bind to target with high affinity and specificity. Due to small and oligonucleotide properties, aptamers offer many advantages over antibodies including non-immunogenicity, high tissue penetration, thermostability, low cost, and ease of synthesis and modification (Keefe, et al., Nat Rev Drug Discov, 9:537-550 (2010); Zhou et al., Nat Drug Rev Discov, 16: 181-202 (2017)). Current cell type-specific RNA aptamers have been used for targeting delivery of siRNA and drugs (Wheeler, et al., J Clin Inves, 121: 2401-2412 (2011); Neff, et al., Sci Transl Med.)
HER3 aptamer has been identified and is able to specifically bind to the extracellular domains of HER3 (Chen, et al., Proc Natl Acad Sci USA, 100: 9226-9231 (2003)). HER3 aptamer inhibits HRG-dependent tyrosine phosphorylation of HER2 (Takahashi, et al., Sci Rep, 6: 33697 (2016)). With a similar generation approach, HER2 aptamer has been identified and synthesized. HER2 aptamer shows high specificity to HER2 positive but not negative cancer cell lines (Kim, et al., Nucleic Acid Ther, 21:173-178 (2011)).
Therefore, it is an object of the invention to provide immunotherapies that have reduced off-target effects for the treatment of cancer including but not limited to, ovarian cancer, non-small-cell lung cancer, cervical cancer, prostate cancer, esophageal and stomach cancers, preferably breast cancer,
It is also an object of the invention to provide methods of treating cancer using non-antibody therapies.
SUMMARY OF THE INVENTIONAn aptamer platform capable of efficiently delivering and silencing two or more genes in vivo or in vitro is provided. Methods of using the aptamers compositions for selectively targeting cells to down-regulate the expression of multiple genes are also provided.
Currently, most chimeras are designed as the fusion of one aptamer with one siRNA (Dassie, et al., Nat Biotechnol, 27: 839-849 (2009); Herrmann, et al., J Clin Invest, 124: 2977-2987 (2014); Zhou, et al., Mol Ther, 16: 1481-1489 (2008); Hussain, et al., Nucleic Acid Ther, 23: 203-212 (2013); Wheeler, et al., J Clin Invest, 121: 2401-2412 (2011)). Importantly, simultaneous delivery of multiple siRNAs has not been reported. In one embodiment, the disclosed aptamers delivers at least one siRNAs against one or more different genes to cells expressing a specific cell surface protein or secreting a specific protein into the microenvironment of the cell. The genes to be down-regulated include, but are not limited to oncogenes, proto-oncogenes, tumor specific antigens, and viruses.
One embodiment provides an aptamer composition that contains an siRNA that down-regulates expression of EGFR. The aptamer composition also contains two aptamers on either end of the chimera that specifically bind to a cell surface protein, for example a tumor specific antigen or a viral antigen. Using multiple aptamers specific to a cell surface protein increases efficiency of delivering the siRNAs to the targeted cell. The disclosed aptamer compositions are processed by cellular RNA interference machinery (Siomi, et al., Nature, 457: 396-404 (2009)) to produce separate and active siRNAs that inhibit expression of three different genes. In one embodiment, the aptamer composition can simultaneously silence EGFR, HER2, and HER3 in vitro and in vivo. The data in the Examples demonstrates a profound efficacy against breast tumor growth through cell cycle arrest and the induction of apoptosis. Delivery of an siRNA by a tumor targeting aptamer-siRNA chimera represents a new approach for combination therapy of using siRNA molecules.
In one embodiment, the aptamer composition specifically binds to HER2 and HER3. Thus, a method for treating HER2 or HER3 expressing breast cancer is provided which includes administering to a subject in need thereof an effective amount of aptamer composition having aptamers that specifically bind to HER2 and HER3 and siRNA constructs that are processed to produce siRNA that inhibits expression of EGFR.
Another embodiment provides a pharmaceutical composition containing one or more aptamer compositions in an amount effective to down-regulate at least two different genes in a target cell.
Another embodiment provides a method for treating a viral infection by administering to a subject in need thereof an effective amount of an aptamer-composition that targets virally infected cells and down regulates two or more genes of the virus infecting the subject.
The use of the terms “a,” “an,” “the,” and similar referents in the context of describing the presently claimed invention (especially in the context of the claims) are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context.
Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein.
Use of the term “about” is intended to describe values either above or below the stated value in a range of approx. +/−10%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/−5%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/−2%; in other embodiments the values may range in value either above or below the stated value in a range of approx. +/−1%. The preceding ranges are intended to be made clear by context, and no further limitation is implied. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.
As used herein, the term “effective amount” in the context of the administration of a therapy to a subject refers to the amount of a therapy that achieves a desired prophylactic or therapeutic effect.
As used herein, the term “in combination” refers to the use of more than one therapy (e.g., one or more prophylactic and/or therapeutic agents). The use of the term “in combination” does not restrict the order in which therapies are administered to a subject with a disease or disorder, or the route of administration. A first therapy (e.g., a prophylactic or therapeutic agent) can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second therapy (e.g., a prophylactic or therapeutic agent) to a subject with a disease or disorder or a symptom thereof.
As used herein, the terms “subject” and patient” are used interchangeably and refer to an animal. In a specific embodiment, such terms refer to a mammal such as a non-primate (e.g., cows, pigs, horses, cats, dogs, rats etc.) and a primate (e.g., monkey and human), most preferably a human. In certain embodiments, such terms refer to a non-human animal (e.g., a non-human animal such as a pig, horse, cow, cat, or dog). In some embodiments, such terms refer to a pet or farm animal. In specific embodiments, such terms refer to a human.
As used herein, the terms “treat”, “treating” and “treatment” in the context of the administration of a disclosed aptamer compositions to a subject refer to the beneficial effects that a subject derives from the therapy. In certain embodiments, treatment of a subject with the aptamer composition achieves one, two, three, four, or more of the following effects: (i) reduction or amelioration the severity of disease or symptom associated therewith; (ii) reduction in the duration of a symptom associated with a disease; (iii) prevention of the progression of a disease or symptom associated therewith; (iv) regression of a disease or symptom associated therewith; (v) prevention of the development or onset of a symptom associated with a disease; (vi) prevention of the recurrence of a symptom associated with a disease; (vii) reduction in the hospitalization of a subject; (viii) reduction in the hospitalization length; (ix) an increase in the survival of a subject with a disease; (x) a reduction in the number of symptoms associated with a disease; (xi) an enhancement, improvement, supplementation, complementation, or augmentation of the prophylactic or therapeutic effect(s) of another therapy.
The term “oncogene” refers to a gene that can in some circumstances transform a cell into a cancerous cell or a gene that promotes the survival of a cancer cell.
II. Aptamer-siRNA ChimerasAn aptamer platform capable of efficiently delivering and silencing two or more genes in vivo or in vitro is provided. Methods of using the aptamer compositions for treating infections or cancer, for example breast cancer, are also provided.
A. Aptamer Platform Design
In one embodiment, the aptamer composition contains at least one siRNA that inhibits or reduces expression of one or more oncogenes (
In one embodiment, the aptamer and siRNAs are separated by a linker. The linker can be about 2-10, 3-9, or preferably about 4 nucleotides. An exemplary linker is four Adenine (“A”). The linker helps to warrant the cleavage with dicer but not mixing two genes, since dicer is able to measure and cut 21-25-nt RNA duplex. The size of the exemplary HER2 aptamer-EGFR siRNA-HER3 aptamer is about 55.4 Kd, which is larger than each single aptamer, and larger than renal glomerulus cutoff mass (30-50 Kd), but smaller than antibody (150 Kd). Chimera with two aptamers are expected to have a longer circulation time than single aptamer alone.
In one embodiment, the RNA chimera incorporates 2′ F into the entire RNA chimera by T7 RNA polymerase-driven transcription. Previously reported aptamer-siRNA chimeras contain one strand unmodified siRNA (Xiao, et al., Mol Cancer Ther, 15: 689-701 (2016); Dassie, et al., Nat Biotechnol, 27: 839-849 (2009)). 2′ F completely modified RNA offers more serum stability than partial modified chimeras. The efficacy in tumor targeting and gene knockdown of EGFR, HER 2, and HER3 was confirmed, and the profound reduction of tumor size and induction of apoptosis have been achieved, suggesting the efficacy of targeting on multiple proliferation pathways.
Dysregulation of cell cycle progression and apoptosis are hallmarks of cancer formation and progression. Cell cycle genes include but are not limited to cyclins and cyclin-dependent kinases (CDK), P53, and Ki67. Apoptotic genes include but are not limited to Fas, Fas ligands, caspases, P21, PARP, and Bcl-2 family members. The data show that H2EH3 enables down-regulation of the expression of HER2, HER3, and EGFR, thereby triggering cell cycle arrest and apoptosis. HER2+HER3+ cells lines treated with H2EH3 showed G2/M arrest and an increase in Annexin V positive cells.
The data show that siRNAs can be delivered to targeted cells by the aptamer compositions. Delivery of siRNA to a cell targeted by two different aptamers in one chimera provides a new and efficient approach for combination therapy. Since the system is highly modular, it can be applied to many targeting co-delivery designs by using siRNA and aptamers. The data also demonstrated that repeated administration is well tolerated and did not elicit an innate immune response.
Small interfering RNA (siRNA) has great potential for sequence-specific silencing of any genes and has emerged as a promising new therapeutic paradigm for “undruggable” targets (Hannon, et al., Nature, 418: 244-251 (2002); Jensen, et al., Sci Transl Med, 5: 209ra152. doi: 10.1126/scitranslmed.3006839 (2013); Wu, et al., Sci Transl Med, 6: 240ps7, doi:10.1126/scitranslmed.3008362 (2014)). However, the use of siRNA as a therapeutic has been hampered by the difficulty of delivery (Whitehead, et al., Nat Rev Drug Discov, 8: 129-138 (2009)). Recently, aptamers (synthetic DNA/RNA ligands) have proven to be a promising platform for delivering siRNA into cells. Selected in a process known as SELEX (systematic evolution of ligands by exponential enrichment) (Ellington, et al., Nature, 346: 818-822 (1990); Tuerk, et al., Science, 249: 505-510 (1990)), aptamers can specifically bind to various targets including organics, peptides, proteins and cells (Zhou, et al., Front Genet, 3: 234, doi:10.3389/fgene.2012.00234 (2012)). Particularly, cell-based SELEX allows the selection of internalized aptamers, which can induce the intracellular delivery of cargo through receptor-mediated endocytosis (Zhou, et al., J Vis Exp, 52: e2954, doi: 10.3791/2954 (2011)). Aptamers have specific 3-dimensional structures for target binding with high affinity, which can be maintained in vivo. Aptamer-siRNA chimera (AsiC), employing only RNA molecules, is a new targeting therapeutic (Dassie, et al., Ther Deliv, 4: 1527-1546 (2013); Wang, et al., Theranostics, 5: 1456-1472 (2015)) and has shown the promise of minimizing off-target effects that are usually associated with small molecule drugs and immunogenicity of antibody-based therapeutic. As a single-component entity, AsiC also has advantages in ease of synthesis and high tissue penetrability. Importantly, AsiC-based drugs can utilize endogenous enzymes (e.g., dicer, argonaute) and enable cell type- and mRNA sequence-specific gene silencing, which can provide selective and effective inhibition of protein targets regardless their cellular localization. For examples, CD4 aptamer-tat/rev siRNA chimera has shown the efficacy in inhibition of HIV transmission (Neff, et al., Sci Transl Med, 3: 66ra6 (2011)), PSMA aptamer-PLK1siRNA enables the regression of prostate cancer (Dassie, et al., Nat Biotechnol, 27: 839-849 (2009)). CTLA4 aptamer-STAT3 siRNA inhibits tumor-associated Tregs and reduces tumor burden in multiple mouse tumor models (Herrmann, et al., J Clin Invest, 124: 2977-2987 (2014)). EpCAM aptamer-survivin siRNA enables reversal of doxorubicin resistance and prolongs survival in mice bearing chemoresistant tumors (Wang, et al., Theranostics, 5: 1456-1472 (2015)).
B. Aptamers
In one embodiment, the aptamer composition contains two aptamers. The aptamers can specifically bind the same target, or in some embodiments, the aptamers can specifically bind to different targets. In a preferred embodiment, the aptamers bind to the same target, for example HER 2 or HER3.
Aptamers are molecules that interact with a target molecule, preferably in a specific way. Typically aptamers are small nucleic acids ranging from 15-200 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets. Aptamers can bind to protein, cells, small organic, peptide. Aptamers can bind small molecules, such as ATP and theophiline, as well as large molecules, such as reverse transcriptase and thrombin. Aptamers can bind very tightly with Kd's from the target molecule of less than 10−12 M. It is preferred that the aptamers bind the target molecule with a Kd less than 10−6, 10−8, 10−10, or 10−12. Aptamers can bind the target molecule with a very high degree of specificity. For example, aptamers have been isolated that have greater than a 10,000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule. It is preferred that the aptamer have a Kd with the target molecule at least 10, 100, 1000, 10,000, or 100,000 fold lower than the Kd with a background binding molecule. It is preferred when doing the comparison for a molecule such as a polypeptide, that the background molecule be a different polypeptide.
III. Targeted Cell Surface ProteinsIn one embodiment, the aptamers in the aptamer compositions can specifically bind to a cell surface protein or a protein or peptide secreted into the microenvironment of cell to be treated, for example a cancer cell, tumor cell, or virally infected cell. In some embodiments, the protein or peptide that is specifically recognized by the aptamers of the aptamer composition can be cell surface proteins involved in signal transduction, tumor specific antigens, tumor neovasculature antigens, viral proteins or viral peptides displayed in the surface of cells, cytokines, and cytokine receptors. These targeted proteins or peptides may be substances produced by a cell or may be substances which accumulate at a cell microenvironment site, or on cell surfaces.
In one embodiment, the aptamers specifically bind to PDGF, nucleolin, P-selectin, EpCAM, CD44, Mucin, AXL, PSMA, ICAM-1, VCAM-1, transferrin receptor, ErbB2, VEGFR, HIV-1 Tat protein, HIV Nuceocapsid, integrin, Her3, IL-10, anti-NF-KB, Kanamycin A, catenin, ERK2, C-reactive protein, L-tryptophan, SARS Coronavirus, influenza B, thrombin Hemagglutinin, tumor necrosis factor-alpha, VEGF, streptavidin, Kit-129, HIV Reverse transcriptase, insulin, PSA, RNase H1, Swine influenza A virus, Human neutrophil elastase, anti-IgE, L-selectin, 4-1BB, Tenascin-C, Protein Kinase C, RBP4, Enterotoxin B, her2, Hepatocyte growth factor receptor, Hepatitis C, Fibrogen, HGF, IgG, EGFR, survivin, Osteopontin, P-selectin, neurotrophin receptor, interferon-γ, Human matrix metalloprotease 9, Keratinocyte growth factor, MCP-1, von-Willebrand factor, Plasminogen activator inhibitor-1, OX40, CD4, CD3, CD8, Tenascin-C, androgen receptor (AR), androgen receptor splicing variants (ARV7 (AR3), ARV12, ARV3, ARV1, ARV9, ARV2, ARV5/6, ARV8, ARV9, ARV10, ARV11).
In one embodiment, the aptamers of the aptamer composition specifically bind to HER2 or HER3.
A. Tumor Specific Antigens
In some embodiments disclosed aptamers may be specific to or selective for a variety of cell surface or disease-associated antigens. In certain embodiments, such as treating tumors, the aptamers of the disclosed compositions specifically bind tumor-associated antigens. These antigenic markers may be substances produced by a tumor or may be substances which accumulate at a tumor site, or on tumor cell surfaces.
In some embodiments, the targeting domains bind to antigens, ligands or receptors that are specific to tumor cells or tumor-associated neovasculature, or are upregulated in tumor cells or tumor-associated neovasculature compared to normal tissue.
1. Oncogenes
Tumor-associated antigens that are targeted by the disclosed compositions may include, for example, cellular oncogene-encoded products or aberrantly expressed proto-oncogene-encoded products (e.g., products encoded by the neu, ras, trk, and kit genes), or mutated forms of growth factor receptor or receptor-like cell surface molecules (e.g., surface receptor encoded by the c-erb B gene). Other tumor-associated antigens include molecules that may be directly involved in transformation events, or molecules that may not be directly involved in oncogenic transformation events but are expressed by tumor cells (e.g., carcinoembryonic antigen, CA-125, melanoma associated antigens, etc.).
Genes that encode cellular tumor associated antigens include cellular oncogenes and proto-oncogenes that are aberrantly expressed. In general, cellular oncogenes encode products that are directly relevant to the transformation of the cell, and because of this, these antigens are particularly preferred targets for immunotherapy. An example is the tumorigenic neu gene that encodes a cell surface molecule involved in oncogenic transformation. Other examples include the ras, kit, and trk genes. The products of proto-oncogenes (the normal genes which are mutated to form oncogenes) may be aberrantly expressed (e.g., overexpressed), and this aberrant expression can be related to cellular transformation. Thus, the product encoded by proto-oncogenes can be targeted. Some oncogenes encode growth factor receptor molecules or growth factor receptor-like molecules that are expressed on the tumor cell surface. An example is the cell surface receptor encoded by the c-erbB gene. Other tumor-associated antigens may or may not be directly involved in malignant transformation. These antigens, however, are expressed by certain tumor cells and may therefore provide effective targets. Some examples are carcinoembryonic antigen (CEA), CA 125 (associated with ovarian carcinoma), and melanoma specific antigens.
Exemplary oncogenes that can be targeted to direct the disclosed compositions to tumors, tumor cells, or tumor microenvironments include, but are not limited to ABL1, ABL2, AKT1, AKT2, ATF1, BCL11A, BCL2, BCL3, BCL6, BCR, BRAF, CARD11, CBLB, CBLC, CCND1, CCND2, CCND3, CDX2, CTNNB1, DDB2, DDIT3, DDX6, DEK, EGFR, ELK4, ERBB2, ETV4, ETV6, EVI1, EWSR1, FEV, FGFR1, FGFR1OP, FGFR2, FUS, GOLGA5, GOPC, HMGA1, HMGA2, HRAS, IRF4, JUN, KIT, KRAS, LCK, LMO2, MAF, MAFB, MAML2, MDM2, MET, MITF, MPL, MYB, MYC, MYCL1, MYCN, NCOA4, NFKB2, NRAS, NTRK1, NUP214, PAX8, PDGFB, PIK3CA, PLAG1, PPARG, PTPN11, RAF1, REL, RET, ROS1, SMO, SS18, TCL1A, TET2, TFG, MLL, TLX1, TPR, and USP6.
In ovarian and other carcinomas, for example, tumor associated antigens are detectable in samples of readily obtained biological fluids such as serum or mucosal secretions. One such marker is CA125, a carcinoma associated antigen that is also shed into the bloodstream, where it is detectable in serum (e.g., Bast, et al., N. Eng. J. Med., 309:883 (1983); Lloyd, et al., Int. J. Canc., 71:842 (1997). CA125 levels in serum and other biological fluids have been measured along with levels of other markers, for example, carcinoembryonic antigen (CEA), squamous cell carcinoma antigen (SCC), tissue polypeptide specific antigen (TPS), sialyl TN mucin (STN), and placental alkaline phosphatase (PLAP), in efforts to provide diagnostic and/or prognostic profiles of ovarian and other carcinomas (e.g., Sarandakou, et al., Acta Oncol., 36:755 (1997); Sarandakou, et al., Eur. J. Gynaecol. Oncol., 19:73 (1998); Meier, et al., Anticancer Res., 17(4B):2945 (1997); Kudoh, et al., Gynecol. Obstet. Invest., 47:52 (1999)). Elevated serum CA125 may also accompany neuroblastoma (e.g., Hirokawa, et al., Surg. Today, 28:349 (1998), while elevated CEA and SCC, among others, may accompany colorectal cancer (Gebauer, et al., Anticancer Res., 17(4B):2939 (1997)).
A tumor antigen may include a cell surface molecule. Tumor antigens of known structure and having a known or described function, include the following cell surface receptors: HER1 (GenBank Accession No. U48722), HER2 (GenBank Acc. Nos. X03363 and M17730), HER3 (GenBank Acc. Nos. U29339 and M34309), HER4 (GenBank Acc. Nos. L07868 and T64105), epidermal growth factor receptor (EGFR) (GenBank Acc. Nos. U48722, and KO3193), vascular endothelial cell growth factor (GenBank No. M32977), vascular endothelial cell growth factor receptor (GenBank Acc. Nos. AF022375, 1680143, U48801 and X62568), insulin-like growth factor-I (GenBank Acc. Nos. X00173, X56774, X56773, X06043), insulin-like growth factor-II (GenBank Acc. Nos. X03562, X00910, M17863 and M17862), transferrin receptor (Trowbridge and Omary, Proc. Nat. Acad. USA, 78:3039 (1981); GenBank Acc. Nos. X01060 and M11507), estrogen receptor (GenBank Acc. Nos. M38651, X03635, X99101, U47678 and M12674), progesterone receptor (GenBank Acc. Nos. X51730, X69068 and M15716), follicle stimulating hormone receptor (FSH-R) (GenBank Acc. Nos. Z34260 and M65085), retinoic acid receptor (GenBank Acc. Nos. L12060, M60909, X77664, X57280, X07282 and X06538), MUC-1 (Barnes, et al., Proc. Nat. Acad. Sci. USA, 86:7159 (1989); GenBank Acc. Nos. M65132 and M64928) NY-ESO-1 (GenBank Acc. Nos. AJ003149 and U87459), NA 17-A, Melan-A/MART-1 GenBank Acc. Nos. U06654 and U06452), tyrosinase (GenBank Acc. No. M26729), Gp-100 (GenBank Acc. No. S73003), MAGE (GenBank Acc. Nos. U93163, AF064589, U66083, D32077, D32076, D32075, U10694, U10693, U10691, U10690, U10689, U10688, U10687, U10686, U10685, L18877, U10340, U10339, L18920, U03735 and M77481), BAGE (GenBank Acc. No. U19180), GAGE (GenBank Acc. Nos. AF055475, AF055474, AF055473, U19147, U19146, U19145, U19144, U19143 and U19142), any of the CTA class of receptors including in particular HOM-MEL-40 antigen encoded by the SSX2 gene (GenBank Acc. Nos. X86175, U90842, U90841 and X86174), carcinoembryonic antigen (CEA; GenBank Acc. Nos. M59710, M59255 and M29540), and PyLT (GenBank Acc. Nos. J02289 and J02038); p97 (melanotransferrin),
Additional tumor associated antigens include prostate surface antigen (PSA); β-human chorionic gonadotropin (β-HCG); glycosyltransferase β-1,4-N-acetylgalactosaminyltransferases (GalNAc); NUC18; melanoma antigen gp75 (GenBank Accession No. X51455); human cytokeratin 8; high molecular weight melanoma antigen.
Tumor antigens of interest include antigens regarded in the art as “cancer/testis” (CT) antigens that are immunogenic in subjects having a malignant condition. CT antigens include at least 19 different families of antigens that contain one or more members and that are capable of inducing an immune response, including but not limited to MAGEA (CT1); BAGE (CT2); MAGEB (CT3); GAGE (CT4); SSX (CT5); NY-ESO-1 (CT6); MAGEC (CT7); SYCP1 (C8); SPANXB1 (CT11.2); NA88 (CT18); CTAGE (CT21); SPA17 (CT22); OY-TES-1 (CT23); CAGE (CT26); HOM-TES-85 (CT28); HCA661 (CT30); NY-SAR-35 (CT38); FATE (CT43); and TPTE (CT44).
Additional tumor antigens that can be targeted, including a tumor-associated or tumor-specific antigen, include, but not limited to, alpha-actinin-4, Bcr-Abl fusion protein, Casp-8, beta-catenin, cdc27, cdk4, cdkn2a, coa-1, dek-can fusion protein, EF2, ETV6-AML1 fusion protein, LDLR-fucosyltransferaseAS fusion protein, HLA-A2, HLA-A11, hsp70-2, KIAAO205, Mart2, Mum-1, 2, and 3, neo-PAP, myosin class I, OS-9, pml-RARα fusion protein, PTPRK, K-ras, N-ras, Triosephosphate isomeras, Bage-1, Gage 3,4,5,6,7, GnTV, Herv-K-mel, Lage-1, Mage-A1,2,3,4,6,10,12, Mage-C2, NA-88, NY-Eso-1/Lage-2, SP17, SSX-2, and TRP2-Int2, MelanA (MART-I), gp100 (Pmel 17), tyrosinase, TRP-1, TRP-2, MAGE-1, MAGE-3, BAGE, GAGE-1, GAGE-2, p15(58), CEA, RAGE, NY-ESO (LAGE), SCP-1, Hom/Mel-40, PRAME, p53, H-Ras, HER-2/neu, BCR-ABL, E2A-PRL, H4-RET, IGH-IGK, MYL-RAR, Epstein Barr virus antigens, EBNA, human papillomavirus (HPV) antigens E6 and E7, TSP-180, MAGE-4, MAGE-5, MAGE-6, p185erbB2, p180erbB-3, c-met, nm-23H1, PSA, TAG-72-4, CA 19-9, CA 72-4, CAM 17.1, NuMa, K-ras, β-Catenin, CDK4, Mum-1, p16, TAGE, PSMA, PSCA, CT7, telomerase, 43-9F, 5T4, 791Tgp72, α-fetoprotein, 13HCG, BCA225, BTAA, CA 125, CA 15-3 (CA 27.29\BCAA), CA 195, CA 242, CA-50, CAM43, CD68\KP1, CO-029, FGF-5, G250, Ga733 (EpCAM), HTgp-175, M344, MA-50, MG7-Ag, MOV18, NB\70K, NY-CO-1, RCAS1, SDCCAG16, TA-90 (Mac-2 binding protein\cyclophilin C-associated protein), TAAL6, TAG72, TLP, and TPS. Other tumor-associated and tumor-specific antigens are known to those of skill in the art and are suitable for targeting by the disclosed fusion proteins.
2. Tumor Neovasculature Antigens
The targeted antigen may be specific to tumor neovasculature or may be expressed at a higher level in tumor neovasculature when compared to normal vasculature. Exemplary antigens that are over-expressed by tumor-associated neovasculature as compared to normal vasculature include, but are not limited to, VEGF/KDR, Tie2, vascular cell adhesion molecule (VCAM), endoglin and α5β3 integrin/vitronectin. Other antigens that are over-expressed by tumor-associated neovasculature as compared to normal vasculature are known to those of skill in the art and are suitable for targeting by the disclosed fusion proteins.
B. Chemokine or Chemokine Receptors
In another embodiment, the aptamer on the composition specifically binds to a chemokine or a chemokine receptor. Chemokines are soluble, small molecular weight (8-14 kDa) proteins that bind to their cognate G-protein coupled receptors (GPCRs) to elicit a cellular response, usually directional migration or chemotaxis. Tumor cells secrete and respond to chemokines, which facilitate growth that is achieved by increased endothelial cell recruitment and angiogenesis, subversion of immunological surveillance and maneuvering of the tumoral leukocyte profile to skew it such that the chemokine release enables the tumor growth and metastasis to distant sites. Thus, chemokines are important for tumor progression.
Based on the positioning of the conserved two N-terminal cysteine residues of the chemokines, they are classified into four groups namely CXC, CC, CX3C and C chemokines. The CXC chemokines can be further classified into ELR+ and ELR− chemokines based on the presence or absence of the motif ‘glu-leu-arg (ELR motif)’ preceding the CXC sequence. The CXC chemokines bind to and activate their cognate chemokine receptors on neutrophils, lymphocytes, endothelial and epithelial cells. The CC chemokines act on several subsets of dendritic cells, lymphocytes, macrophages, eosinophils, natural killer cells but do not stimulate neutrophils as they lack CC chemokine receptors except murine neutrophils. There are approximately 50 chemokines and only 20 chemokine receptors, thus there is considerable redundancy in this system of ligand/receptor interaction.
Chemokines elaborated from the tumor and the stromal cells bind to the chemokine receptors present on the tumor and the stromal cells. The autocrine loop of the tumor cells and the paracrine stimulatory loop between the tumor and the stromal cells facilitate the progression of the tumor. Notably, CXCR2, CXCR4, CCR2 and CCR7 play major roles in tumorigenesis and metastasis. CXCR2 plays a vital role in angiogenesis and CCR2 plays a role in the recruitment of macrophages into the tumor microenvironment. CCR7 is involved in metastasis of the tumor cells into the sentinel lymph nodes as the lymph nodes have the ligand for CCR7, CCL21. CXCR4 is mainly involved in the metastatic spread of a wide variety of tumors.
In one embodiment, tumor or tumor-associated neovasculature targeting domains are ligands that bind to cell surface antigens or receptors that are specifically expressed on tumor cells or tumor-associated neovasculature or are overexpressed on tumor cells or tumor-associated neovasculature as compared to normal tissue. Tumors also secrete a large number of ligands into the tumor microenvironment that affect tumor growth and development. Receptors that bind to ligands secreted by tumors, including, but not limited to growth factors, cytokines and chemokines, including the chemokines provided above, are suitable for use in the disclosed fusion proteins. Ligands secreted by tumors can be targeted using soluble fragments of receptors that bind to the secreted ligands. Soluble receptor fragments are fragments polypeptides that may be shed, secreted or otherwise extracted from the producing cells and include the entire extracellular domain, or fragments thereof.
In another embodiment the aptamers of the disclosed compositions specifically bind to target antigens selected from the group consisting of carbonic anhydrase IX, CCL19, CCL21, CSAp, CD1, CD1a, CD2, CD3, CD4, CD S, CD8, CD11A, CD14, CD15, CD16, CD18, CD19, IGF-1R, CD20, CD21, CD22, CD23, CD25, CD29, CD30, CD32b, CD33, CD37, CD38, CD40, CD40L, CD45, CD46, CD52, CD54, CD55, CD59, CD64, CD66a-e, CD67, CD70, CD74, CD79a, CD80, CD83, CD95, CD126, CD133, CD138, CD147, CD154, CXCR4, CXCR7, CXCL12, HIF-1α, AFP, PSMA, CEACAM5, CEACAM6, c-met, B7, ED-B of fibronectin, Factor H, FHL-1, Flt-3, folate receptor, GROB, HMGB-1, hypoxia inducible factor (HIF), HM1.24, insulin-like growth factor-1 (ILGF-1), IFN-γ, IFN-α, IL-2, IL-4R, IL-6R, IL-13R, IL-15R, IL-17R, IL-18R, IL-6, IL-8, IL-12, IL-15, IL-17, IL-18, IL-25, IP-10, MAGE, mCRP, MCP-1, MIP-1A, MIP-1B, MIF, MUC1, MUC2, MUC3, MUC4, MUC5, NCA-95, NCA-90, Ia, HM1.24, EGP-1, EGP-2, HLA-DR, tenascin, Le(y), RANTES, T101, TAC, Tn antigen, Thomson-Friedenreich antigens, tumor necrosis antigens, TNF-α, TRAIL receptor (R1 and R2), VEGFR, EGFR, P1GF, complement factors C3, C3a, C3b, C5a, C5, PLAGL2, and an oncogene product. A particularly preferred target antigen is CEACAM5 (CEA).
C. Viral Antigens
In some embodiments, the protein that is bound by the aptamer is a viral protein selected from the group consisting of a pox virus, smallpox virus, ebola virus, marburg virus, dengue fever virus, influenza virus, parainfluenza virus, respiratory syncytial virus, rubeola virus, human immunodeficiency virus, human papillomavirus, varicella-zoster virus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovirus, rotavirus, rhinovirus, adenovirus, papillomavirus, parvovirus, picornavirus, poliovirus, virus that causes mumps, virus that causes rabies, reovirus, rubella virus, togavirus, orthomyxovirus, retrovirus, hepadnavirus, coxsackievirus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus.
D. Genes to be Down-Regulated
The disclosed aptamer composition contains one or more siRNAs that specifically inhibit or down-regulate expression of one or more different genes. In some embodiments, the one or more different genes are oncogenes. In a preferred embodiment, at least one siRNA inhibits or reduces the expression of EGFR.
1. Oncogenes
Exemplary oncogenes or proto-oncogenes that can be inhibited by the siRNA include, but are not limited to the oncogenes and proto-oncogenes discussed above is Section II.A.1. Representative oncogenes that can be down-regulated by the siRNA in the chimeras include, for example ABL1, ABL2, AKT1, AKT2, ATF1, BCL11A, BCL2, BCL3, BCL6, BCR, BRAF, BIRC5, CARD11, CBLB, CBLC, CCND1, CCND2, CCND3, CCNE1, CDX2, CTNNB1, DDB2, DDIT3, DDX6, DEK, EGFR, ELK4, ERBB2, ETV4, ETV6, EVIL EWSR1, FEV, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, FGFR1OP, FUS, GOLGA5, GOPC, HMGA1, HMGA2, HRAS, IRF4, JUN, KIT, KRAS, LCK, LMO2, MAF, MAFB, MAML2, MDM2, MET, MITF, MPL, MYB, MYC, MYCL1, MYCN, NCOA4, NFKB2, NRAS, NTRK1, NUP214, PAX8, PDGFB, PIK3CA, PIM1, PLAG1, PPARG, PTPN11, RAF1, REL, RET, ROS1, SMO, SS18, TCL1A, TET2, TFG, MLL, TLX1, TPR, and USP6.
Sequence information for these oncogenes are known in the art, and one of skill in the art could readily make siRNA constructs to specifically inhibit oncogene expression.
2. Virus Expression to be Inhibited.
Genes encoding viruses or virus components can be targeted for siRNA inhibition using the aptamer compositions Exemplary viruses to be targeted for siRNA inhibition include, but are not limited to pox virus, smallpox virus, ebola virus, marburg virus, dengue fever virus, influenza virus, parainfluenza virus, respiratory syncytial virus, rubeola virus, human immunodeficiency virus, human papillomavirus, varicella-zoster virus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovirus, rotavirus, rhinovirus, adenovirus, papillomavirus, parvovirus, picornavirus, poliovirus, virus that causes mumps, virus that causes rabies, reovirus, rubella virus, togavirus, orthomyxovirus, retrovirus, hepadnavirus, coxsackievirus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus.
Sequence information for the genes of these viruses are known in the art, and one of skill in the art could readily make siRNA constructs to specifically inhibit viral gene expression.
IV. siRNAIn one embodiment, the aptamer compositions contain aptamers and siRNA that specifically inhibit expression of two or more genes. The siRNA in the chimera is processed using cellular siRNA machinery to produce siRNA in active form.
Gene expression can be effectively silenced in a highly specific manner through RNA interference (RNAi). This silencing was originally observed with the addition of double stranded RNA (dsRNA) (Fire, et al. (1998) Nature, 391:806-11; Napoli, et al. (1990) Plant Cell 2:279-89; Hannon, (2002) Nature, 418:244-51). Once dsRNA enters a cell, it is cleaved by an RNase III-like enzyme, Dicer, into double stranded small interfering RNAs (siRNA) 21-23 nucleotides in length that contains 2 nucleotide overhangs on the 3′ ends (Elbashir, et al. (2001) Genes Dev., 15:188-200; Bernstein, et al. (2001) Nature, 409:363-6; Hammond, et al. (2000) Nature, 404:293-6). In an ATP dependent step, the siRNAs become integrated into a multi-subunit protein complex, commonly known as the RNAi induced silencing complex (RISC), which guides the siRNAs to the target RNA sequence (Nykanen, et al. (2001) Cell, 107:309-21). At some point the siRNA duplex unwinds, and it appears that the antisense strand remains bound to RISC and directs degradation of the complementary mRNA sequence by a combination of endo and exonucleases (Martinez, et al. (2002) Cell, 110:563-74). However, the effect of iRNA or siRNA or their use is not limited to any type of mechanism.
Short Interfering RNA (siRNA) is a double-stranded RNA that can induce sequence-specific post-transcriptional gene silencing, thereby decreasing or even inhibiting gene expression. In one example, an siRNA triggers the specific degradation of homologous RNA molecules, such as mRNAs, within the region of sequence identity between both the siRNA and the target RNA. For example, WO 02/44321 discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with 3′ overhanging ends, herein incorporated by reference for the method of making these siRNAs. Sequence specific gene silencing can be achieved in mammalian cells using synthetic, short double-stranded RNAs that mimic the siRNAs produced by the enzyme dicer (Elbashir, et al. (2001) Nature, 411:494 498) (Ui-Tei, et al. (2000) FEBS Lett 479:79-82). siRNA can be chemically or in vitro-synthesized or can be the result of short double-stranded hairpin-like RNAs (shRNAs) that are processed into siRNAs inside the cell. Synthetic siRNAs are generally designed using algorithms and a conventional DNA/RNA synthesizer. Suppliers include Ambion (Austin, Tex.), ChemGenes (Ashland, Mass.), Dharmacon (Lafayette, Colo.), Glen Research (Sterling, Va.), MWB Biotech (Esbersberg, Germany), Proligo (Boulder, Colo.), and Qiagen (Vento, The Netherlands). siRNA can also be synthesized in vitro using kits such as Ambion's SILENCER® siRNA Construction Kit.
The production of siRNA from a vector is more commonly done through the transcription of a short hairpin RNAse (shRNAs). Kits for the production of vectors comprising shRNA are available, such as, for example, Imgenex's GENESUPPRESSOR™ Construction Kits and Invitrogen's BLOCK-IT™ inducible RNAi plasmid and lentivirus vectors.
V. Methods of UseThe disclosed aptamer compositions can be used to down-regulate specific genes in targeted cells. In some embodiments, the chimeras are used to down-regulated two or more genes in a targeted cell through siRNA inhibition. The aptamers in the chimeras can be designed to target a protein or peptide that is expressed by the cell.
A. Cancer
In one embodiment, the aptamer compositions are administered to a subject having or suspected of having cancer in an amount effective to inhibit expression of two or more genes in the cancer survival pathway. For example, the aptamer composition can have two aptamers that specifically bind to a cancer antigen or a tumor specific antigen.
A preferred tumor specific antigen is HER2. The genes to be down-regulated in the cancer or tumor cell are typically oncogenes or proto-oncogenes, for example EGFR. In another embodiment, EGFR is down-regulated and an oncogene selected from the group consisting of ABL1, ABL2, AKT1, AKT2, ATF1, BCL11A, BCL2, BCL3, BCL6, BCR, BRAF, CARD11, CBLB, CBLC, CCND1, CCND2, CCND3, CDX2, CTNNB1, DDB2, DDIT3, DDX6, DEK, ELK4, ERBB2, ETV4, ETV6, EVI1, EWSR1, FEV, FGFR1, FGFR1OP, FGFR2, FUS, GOLGA5, GOPC, HMGA1, HMGA2, HRAS, IRF4, JUN, KIT, KRAS, LCK, LMO2, MAF, MAFB, MAML2, MDM2, MET, MITF, MPL, MYB, MYC, MYCL1, MYCN, NCOA4, NFKB2, NRAS, NTRK1, NUP214, PAX8, PDGFB, PIK3CA, PIM1, PLAG1, PPARG, PTPN11, RAF1, REL, RET, ROS1, SMO, SS18, TCL1A, TET2, TFG, MLL, TLX1, TPR, and USP6 is also down-regulated.
Thus, methods for treating breast cancer are provided. Other cancers that can be treated include, but are not limited to bone sarcoma, osteosarcoma, chondrosarcoma, Ewing's sarcoma, malignant giant cell tumor, fibrosarcoma of bone, chordoma, periosteal sarcoma, soft-tissue sarcomas, angiosarcoma (hemangiosarcoma), fibrosarcoma, Kaposi's sarcoma, leiomyosarcoma, liposarcoma, lymphangiosarcoma, neurilemmoma, rhabdomyosarcoma, synovial sarcoma; brain tumors including, but not limited to, glioma, astrocytoma, brain stem glioma, ependymoma, oligodendroglioma, nonglial tumor, acoustic neurinoma, craniopharyngioma, medulloblastoma, meningioma, pineocytoma, pineoblastoma, primary brain lymphoma; breast cancer including, but not limited to, adenocarcinoma, lobular (small cell) carcinoma, intraductal carcinoma, medullary breast cancer, mucinous breast cancer, tubular breast cancer, papillary breast cancer, Paget's disease, and inflammatory breast cancer; adrenal cancer, including, but not limited to, pheochromocytom and adrenocortical carcinoma; thyroid cancer such as but not limited to papillary or follicular thyroid cancer, medullary thyroid cancer and anaplastic thyroid cancer; pancreatic cancer, including, but not limited to, insulinoma, gastrinoma, glucagonoma, vipoma, somatostatin-secreting tumor, and carcinoid or islet cell tumor; pituitary cancers including, but not limited to, Cushing's disease, prolactin-secreting tumor, acromegaly, and diabetes insipius; eye cancers including, but not limited to, ocular melanoma such as iris melanoma, choroidal melanoma, and ciliary body melanoma, and retinoblastoma; vaginal cancers, including, but not limited to, squamous cell carcinoma, adenocarcinoma, and melanoma; vulvar cancer, including, but not limited to, squamous cell carcinoma, melanoma, adenocarcinoma, basal cell carcinoma, sarcoma, and Paget's disease; cervical cancers including, but not limited to, squamous cell carcinoma, and adenocarcinoma; uterine cancers including, but not limited to, endometrial carcinoma and uterine sarcoma; ovarian cancers including, but not limited to, ovarian epithelial carcinoma, borderline tumor, germ cell tumor, and stromal tumor; esophageal cancers including, but not limited to, squamous cancer, adenocarcinoma, adenoid cyctic carcinoma, mucoepidermoid carcinoma, adenosquamous carcinoma, sarcoma, melanoma, plasmacytoma, verrucous carcinoma, and oat cell (small cell) carcinoma; stomach cancers including, but not limited to, adenocarcinoma, fungating (polypoid), ulcerating, superficial spreading, diffusely spreading, malignant lymphoma, liposarcoma, fibrosarcoma, and carcinosarcoma; colon cancers; rectal cancers; liver cancers including, but not limited to, hepatocellular carcinoma and hepatoblastoma, gallbladder cancers including, but not limited to, adenocarcinoma; cholangiocarcinomas including, but not limited to, papillary, nodular, and diffuse; lung cancers including, but not limited to, non-small cell lung cancer, squamous cell carcinoma (epidermoid carcinoma), adenocarcinoma, large-cell carcinoma and small-cell lung cancer; testicular cancers including, but not limited to, germinal tumor, seminoma, anaplastic, classic (typical), spermatocytic, nonseminoma, embryonal carcinoma, teratoma carcinoma, choriocarcinoma (yolk-sac tumor), prostate cancers including, but not limited to, adenocarcinoma, leiomyosarcoma, and rhabdomyosarcoma; penal cancers; oral cancers including, but not limited to, squamous cell carcinoma; basal cancers; salivary gland cancers including, but not limited to, adenocarcinoma, mucoepidermoid carcinoma, and adenoidcystic carcinoma; pharynx cancers including, but not limited to, squamous cell cancer, and verrucous; skin cancers including, but not limited to, basal cell carcinoma, squamous cell carcinoma and melanoma, superficial spreading melanoma, nodular melanoma, lentigo malignant melanoma, acral lentiginous melanoma; kidney cancers including, but not limited to, renal cell cancer, adenocarcinoma, hypernephroma, fibrosarcoma, transitional cell cancer (renal pelvis and/or ureter); Wilms' tumor; bladder cancers including, but not limited to, transitional cell carcinoma, squamous cell cancer, adenocarcinoma, carcinosarcoma. Cancers that can be prevented, treated or otherwise diminished by the MDNPs include myxosarcoma, osteogenic sarcoma, endotheliosarcoma, lymphangioendotheliosarcoma, mesothelioma, synovioma, hemangioblastoma, epithelial carcinoma, cystadenocarcinoma, bronchogenic carcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, and gastric cancer.
B. Tumor Burden
Another embodiment provides a method for reducing the tumor burden of a subject by administering an effective amount of an aptamer composition that specifically binds a tumor specific antigen produced by the tumor and is processed by cellular iRNA machinery to produce two or more siRNAs that inhibit the expression of at least two genes in the tumor to promote apoptosis of tumor cells.
C. Viral Infections
Another embodiment provides a method for treating a viral infection in a subject in need thereof by administering an effective amount of aptamer composition that contain aptamers that specifically bind to proteins on the virus expressed on the surface of virally infected cells and wherein the chimera is internalized by a virally infected celled and processed to produce two or more siRNAs that inhibit viral genes.
Exemplary viruses that can be treated include, but are not limited to pox virus, smallpox virus, ebola virus, marburg virus, dengue fever virus, influenza virus, parainfluenza virus, respiratory syncytial virus, rubeola virus, human immunodeficiency virus, human papillomavirus, varicella-zoster virus, herpes simplex virus, cytomegalovirus, Epstein-Barr virus, JC virus, rhabdovirus, rotavirus, rhinovirus, adenovirus, papillomavirus, parvovirus, picornavirus, poliovirus, virus that causes mumps, virus that causes rabies, reovirus, rubella virus, togavirus, orthomyxovirus, retrovirus, hepadnavirus, coxsackievirus, equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, hepatitis A virus, hepatitis B virus, hepatitis C virus, hepatitis D virus, and hepatitis E virus.
D. Co-Therapies
The aptamer compositions can be used in combination or alternation with a second therapeutic.
1. Cancer Co-Therapies
Non-limiting examples of one or more other therapies that can be used in combination with the aptamer compositions include immunomodulatory agents, such as but not limited to, chemotherapeutic agents and non-chemotherapeutic immunomodulatory agents. Non-limiting examples of chemotherapeutic agents include cyclophosphamide, methotrexate, cyclosporin A, leflunomide, cisplatin, ifosfamide, taxanes such as taxol and paclitaxol, topoisomerase I inhibitors (e.g., CPT-11, topotecan, 9-AC, and GG-211), gemcitabine, vinorelbine, oxaliplatin, 5-fluorouracil (5-FU), leucovorin, vinorelbine, temodal, cytochalasin B, gramicidin D, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin homologs, and cytoxan. Examples of non-chemotherapeutic immunomodulatory agents include, but are not limited to, anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1 (DEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (DEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH 1H (Ilex)), anti-CD2 antibodies (e.g., MEDI-507 (MedImmune, Inc., International Publication Nos. WO 02/098370 and WO 02/069904), anti-CD11a antibodies (e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC)); anti-cytokine receptor antibodies (e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, and anti-IL-12 receptor antibodies), anti-cytokine antibodies (e.g., anti-IFN antibodies, anti-TNF-alpha antibodies, anti-IL-1alpha antibodies, anti-IL-6 antibodies, anti-IL-8 antibodies (e.g., ABX-TL-8 (Abgenix)), anti-IL-12 antibodies and anti-IL-23 antibodies)); CTLA4-immunoglobulin; LFA-3TIP (Biogen, International Publication No. WO 93/08656 and U.S. Pat. No. 6,162,432); soluble cytokine receptors (e.g., the extracellular domain of a TNF-alpha receptor or a fragment thereof, the extracellular domain of an IL-1 alpha receptor or a fragment thereof, and the extracellular domain of an IL-6 receptor or a fragment thereof); cytokines or fragments thereof (e.g., interleukin (IL)-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-15, IL-23, INF-alpha, INF-beta, interferon (IFN)-alpha, IFN-beta, IFN-gamma, and GM-CSF); and anti-cytokine antibodies (e.g., anti-IL-2 antibodies, anti-IL-4 antibodies, anti-IL-6 antibodies, anti-IL-10 antibodies, anti-IL-12 antibodies, anti-IL-15 antibodies, anti-TNF-alpha antibodies, and anti-IFN-gamma antibodies), and antibodies that immunospecifically bind to tumor-associated antigens (e.g., Herceptin®). In certain embodiments, an immunomodulatory agent is an immunomodulatory agent other than a chemotherapeutic agent. In other embodiments an immunomodulatory agent is an immunomodulatory agent other than a cytokine or hemapoietic such as IL-1, IL-2, IL-4, IL-12, IL-15, TNF, IFN-alpha, IFN-beta, IFN-gamma, M-CSF, G-CSF, IL-3 or erythropoietin. In yet other embodiments, an immunomodulatory agent is an agent other than a chemotherapeutic agent and a cytokine or hemapoietic factor.
Non-limiting examples of anti-cancer agents that can be used as therapies in combination with the aptamer compositions, include, but are not limited to: acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; ametantrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; bleomycin sulfate; brequinar sodium; bropirimine; busulfan; cactinomycin; calusterone; caracemide; carbetimer; carboplatin; carmustine; carubicin hydrochloride; carzelesin; cedefingol; chlorambucil; cirolemycin; cisplatin; cladribine; crisnatol mesylate; cyclophosphamide; cytarabine; dacarbazine; dactinomycin; daunorubicin hydrochloride; decitabine; dexormaplatin; dezaguanine; dezaguanine mesylate; diaziquone; docetaxel; doxorubicin; doxorubicin hydrochloride; droloxifene; droloxifene citrate; dromostanolone propionate; duazomycin; edatrexate; eflornithine hydrochloride; elsamitrucin; enloplatin; enpromate; epipropidine; epirubicin hydrochloride; erbulozole; esorubicin hydrochloride; estramustine; estramustine phosphate sodium; etanidazole; etoposide; etoposide phosphate; etoprine; fadrozole hydrochloride; fazarabine; fenretinide; floxuridine; fludarabine phosphate; fluorouracil; flurocitabine; fosquidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-I a; interferon gamma-I b; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride; lometrexol sodium; lomustine; losoxantrone hydrochloride; masoprocol; maytansine; mechlorethamine hydrochloride; megestrol acetate; melengestrol acetate; melphalan; menogaril; mercaptopurine; methotrexate; methotrexate sodium; metoprine; meturedepa; mitindomide; mitocarcin; mitocromin; mitogillin; mitomalcin; mitomycin; mitosper; mitotane; mitoxantrone hydrochloride; mycophenolic acid; nocodazole; nogalamycin; ormaplatin; oxisuran; paclitaxel; pegaspargase; peliomycin; pentamustine; peplomycin sulfate; perfosfamide; pipobroman; piposulfan; piroxantrone hydrochloride; plicamycin; plomestane; porfimer sodium; porfiromycin; prednimus tine; procarbazine hydrochloride; puromycin; puromycin hydrochloride; pyrazofurin; riboprine; rogletimide; safingol; safingol hydrochloride; semustine; simtrazene; sparfosate sodium; sparsomycin; spirogermanium hydrochloride; spiromustine; spiroplatin; streptonigrin; streptozocin; sulofenur; talisomycin; tecogalan sodium; tegafur; teloxantrone hydrochloride; temoporfin; teniposide; teroxirone; testolactone; thiamiprine; thioguanine; thiotepa; tiazofurin; tirapazamine; toremifene citrate; trestolone acetate; triciribine phosphate; trimetrexate; trimetrexate glucuronate; triptorelin; tubulozole hydrochloride; uracil mustard; uredepa; vapreotide; verteporfin; vinblastine sulfate; vincristine sulfate; vindesine; vindesine sulfate; vinepidine sulfate; vinglycinate sulfate; vinleurosine sulfate; vinorelbine tartrate; vinrosidine sulfate; vinzolidine sulfate; vorozole; zeniplatin; zinostatin; zorubicin hydrochloride. Other anti-cancer drugs include, but are not limited to: 20-epi-1,25 dihydroxyvitamin D3; 5-ethynyluracil; abiraterone; aclarubicin; acylfulvene; adecypenol; adozelesin; aldesleukin; ALL-TK antagonists; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsalizing morphogenetic protein-1; antiandrogen, prostatic carcinoma; antiestrogen; antineoplaston; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulacrine; atamestane; atrimustine; axinastatin 1; axinastatin 2; axinastatin 3; azasetron; azatoxin; azatyrosine; baccatin III derivatives; balanol; batimastat; BCR/ABL antagonists; benzochlorins; benzoylstaurosporine; beta lactam derivatives; beta-alethine; betaclamycin B; betulinic acid; bFGF inhibitor; bicalutamide; bisantrene; bisaziridinylspermine; bisnafide; bistratene A; bizelesin; breflate; bropirimine; budotitane; buthionine sulfoximine; calcipotriol; calphostin C; camptothecin derivatives; canarypox IL-2; capecitabine; carboxamide-amino-triazole; carboxyamidotriazole; CaRest M3; CARN 700; cartilage derived inhibitor; carzelesin; casein kinase inhibitors (ICOS); castanospermine; cecropin B; cetrorelix; chlorins; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomifene analogues; clotrimazole; collismycin A; collismycin B; combretastatin A4; combretastatin analogue; conagenin; crambescidin 816; crisnatol; cryptophycin 8; cryptophycin A derivatives; curacin A; cyclopentanthraquinones; cycloplatam; cypemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodidemnin B; deslorelin; dexamethasone; dexifosfamide; dexrazoxane; dexverapamil; diaziquone; didemnin B; didox; diethylnorspermine; dihydro-5-azacytidine; dihydrotaxol, 9-; dioxamycin; diphenyl spiromustine; docetaxel; docosanol; dolasetron; doxifluridine; droloxifene; dronabinol; duocarmycin SA; ebselen; ecomustine; edelfosine; edrecolomab; eflornithine; elemene; emitefur; epirubicin; epristeride; estramustine analogue; estrogen agonists; estrogen antagonists; etanidazole; etoposide phosphate; exemestane; fadrozole; fazarabine; fenretinide; filgrastim; finasteride; flavopiridol; flezelastine; fluasterone; fludarabine; fluorodaunorunicin hydrochloride; forfenimex; formestane; fostriecin; fotemustine; gadolinium texaphyrin; gallium nitrate; galocitabine; ganirelix; gelatinase inhibitors; gemcitabine; glutathione inhibitors; hepsulfam; heregulin; hexamethylene bisacetamide; hypericin; ibandronic acid; idarubicin; idoxifene; idramantone; ilmofosine; ilomastat; imidazoacridones; imiquimod; immunostimulant peptides; insulin-like growth factor-1 receptor inhibitor; interferon agonists; interferons; interleukins; iobenguane; iododoxorubicin; ipomeanol, 4-; iroplact; irsogladine; isobengazole; isohomohalicondrin B; itasetron; jasplakinolide; kahalalide F; lamellarin-N triacetate; lanreotide; leinamycin; lenograstim; lentinan sulfate; leptolstatin; letrozole; leukemia inhibiting factor; leukocyte alpha interferon; leuprolide+estrogen+progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogue; lipophilic disaccharide peptide; lipophilic platinum compounds; lissoclinamide 7; lobaplatin; lombricine; lometrexol; lonidamine; losoxantrone; HMG-CoA reductase inhibitor (such as but not limited to, Lovastatin, Pravastatin, Fluvastatin, Statin, Simvastatin, and Atorvastatin); loxoribine; lurtotecan; lutetium texaphyrin; lysofylline; lytic peptides; maitansine; mannostatin A; marimastat; masoprocol; maspin; matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; merbarone; meterelin; methioninase; metoclopramide; MIF inhibitor; mifepristone; miltefosine; mirimostim; mismatched double stranded RNA; mitoguazone; mitolactol; mitomycin analogues; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofarotene; molgramostim; monoclonal antibody, human chorionic gonadotrophin; monophosphoryl lipid A+myobacterium cell wall sk; mopidamol; multiple drug resistance gene inhibitor; multiple tumor suppressor 1-based therapy; mustard anticancer agent; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagrestip; naloxone+pentazocine; napavin; naphterpin; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidasc; nilutamide; nisamycin; nitric oxide modulators; nitroxide antioxidant; nitrullyn; O6-benzylguanine; octreotide; okicenone; oligonucleotides; onapristone; ondansetron; ondansetron; oracin; oral cytokine inducer; ormaplatin; osaterone; oxaliplatin; oxaunomycin; paclitaxel; paclitaxel analogues; paclitaxel derivatives; palauamine; palmitoylrhizoxin; pamidronic acid; panaxytriol; panomifene; parabactin; pazelliptine; pegaspargase; peldesine; pentosan polysulfate sodium; pentostatin; pentrozole; perflubron; perfosfamide; perillyl alcohol; phenazinomycin; phenylacetate; phosphatase inhibitors; picibanil; pilocarpine hydrochloride; pirarubicin; piritrexim; placetin A; placetin B; plasminogen activator inhibitor; platinum complex; platinum compounds; platinum-triamine complex; porfimer sodium; porfiromycin; prednisone; propyl bis-acridone; prostaglandin J2; proteasome inhibitors; protein A-based immune modulator; protein kinase C inhibitor; protein kinase C inhibitors, microalgal; protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridine; pyridoxylated hemoglobin polyoxyethylene conjugate; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ras-GAP inhibitor; retelliptine demethylated; rhenium Re 186 etidronate; rhizoxin; ribozymes; RH retinamide; rogletimide; rohitukine; romurtide; roquinimex; rubiginone B1; ruboxyl; safingol; saintopin; SarCNU; sarcophytol A; sargramostim; Sdi 1 mimetics; semustine; senescence derived inhibitor 1; sense oligonucleotides; signal transduction inhibitors; signal transduction modulators; single chain antigen binding protein; sizofiran; sobuzoxane; sodium borocaptate; sodium phenylacetate; solverol; somatomedin binding protein; sonermin; sparfosic acid; spicamycin D; spiromustine; splenopentin; spongistatin 1; squalamine; stern cell inhibitor; stem-cell division inhibitors; stipiamide; stromelysin inhibitors; sulfinosinc; superactive vasoactive intestinal peptide antagonist; suradista; suramin; swainsonine; synthetic glycosaminoglycans; tallimustine; tamoxifen methiodide; tauromustine; tazarotene; tecogalan sodium; tegafur; tellurapyrylium; telomerase inhibitors; temoporfin; temozolomide; teniposide; tetrachlorodecaoxide; tetrazomine; thaliblastine; thiocoraline; thrombopoietin; thrombopoietin mimetic; thymalfasin; thymopoietin receptor agonist; thymotrinan; thyroid stimulating hormone; tin ethyl etiopurpurin; tirapazamine; titanocene bichloride; topsentin; toremifene; totipotent stern cell factor; translation inhibitors; tretinoin; triacetyluridine; triciribinc; trimetrexate; triptorelin; tropisetron; turosteridc; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector system, erythrocyte gene therapy; velaresol; veramine; verdins; verteporfin; vinorelbine; vinxaltine; Vitaxin®; vorozole; zanotcrone; zeniplatin; zilascorb; and zinostatin stimalamer. Additional anti-cancer drugs are 5-fluorouracil and leucovorin. These two agents are particularly useful when used in methods employing thalidomide and a topoisomerase inhibitor. In specific embodiments, the anti-cancer agent is not a chemotherapeutic agent.
2. Antiviral Co-Therapies
Antiviral agents that can be used in combination with aptamer compositions include, but are not limited to, non-nucleoside reverse transcriptase inhibitors, nucleoside reverse transcriptase inhibitors, protease inhibitors, and fusion inhibitors. In one embodiment, the antiviral agent is selected from the group consisting of amantadine, oseltamivir phosphate, rimantadine, and zanamivir. In another embodiment, the antiviral agent is a non-nucleoside reverse transcriptase inhibitor selected from the group consisting of delavirdine, efavirenz, and nevirapine. In another embodiment, the antiviral agent is a nucleoside reverse transcriptase inhibitor selected from the group consisting of abacavir, didanosine, emtricitabine, emtricitabine, lamivudine, stavudine, tenofovir DF, zalcitabine, and zidovudine. In another embodiment, the antiviral agent is a protease inhibitor selected from the group consisting of amprenavir, atazanavir, fosamprenav, indinavir, lopinavir, nelfinavir, ritonavir, and saquinavir. In another embodiment, the antiviral agent is a fusion inhibitor such as enfuvirtide.
Additional, non-limiting examples of antiviral agents for use in combination with aptamer composition include the following: rifampicin, nucleoside reverse transcriptase inhibitors (e.g., AZT, ddI, ddC, 3TC, d4T), non-nucleoside reverse transcriptase inhibitors (e.g., delavirdine efavirenz, nevirapine), protease inhibitors (e.g., aprenavir, indinavir, ritonavir, and saquinavir), idoxuridine, cidofovir, acyclovir, ganciclovir, zanamivir, amantadine, and palivizumab. Other examples of anti-viral agents include but are not limited to acemannan; acyclovir; acyclovir sodium; adefovir; alovudine; alvircept sudotox; amantadine hydrochloride (SYMMETREL™); aranotin; arildone; atevirdine mesylate; avridine; cidofovir; cipamfylline; cytarabine hydrochloride; delavirdine mesylate; desciclovir; didanosine; disoxaril; edoxudine; enviradene; enviroxime; famciclovir; famotine hydrochloride; fiacitabine; fialuridine; fosarilate; foscamet sodium; fosfonet sodium; ganciclovir; ganciclovir sodium; idoxuridine; kethoxal; lamivudine; lobucavir; memotine hydrochloride; methisazone; nevirapine; oscltamivir phosphate (TAMIFLU™); penciclovir; pirodavir; ribavirin; rimantadine hydrochloride (FLUMADINE™); saquinavir mesylate; somantadine hydrochloride; sorivudine; statolon; stavudine; tilorone hydrochloride; trifluridine; valacyclovir hydrochloride; vidarabine; vidarabine phosphate; vidarabine sodium phosphate; viroxime; zalcitabine; zanamivir (RELENZA™); zidovudine; and zinviroxime.
3. Genetic Disorder Co-Therapies
Genetic disorders including: Down's Syndrome, muscular dystrophy, Huntington's Disease, asthma, heart disease, diabetes, obesity, hypertension, X-linked dominant genetic diseases, and autosomal dominant genetic diseases can be treated using the disclosed aptamer composition. The genes known to be involved in the genetic disease or syndrome can be targeted for down-regulation using the disclosed aptamer platform.
E. Administration and Formulations
The disclosed aptamers can be formulated as pharmaceutical compositions for parenteral administration. The formulations can contain one or more pharmaceutically acceptable excipients.
EXAMPLES Example 1: Engineering of an Aptamer-siRNA Chimera. Bivalent HER2 Aptamer-EGFR siRNA Aptamer Chimera (HEH)Materials and Methods
Chemicals and Cell culture. Vendors for specific chemicals are listed below. Cell culture products were purchased from Life Technologies (Carlsbad, Calif.). Antibodies were from Cell Signaling Technology (Danvers, Mass.). Single stranded DNAs were synthesized by Integrated DNA Technologies (IDT, Coralville, Iowa). TranscriptAid T7 High Yield Transcription Kits were purchased from Thermo Fisher Scientific. HER2 and HER3 siRNAs were purchased from Life Technologies Corporation. LysoTracker Green DND-26 and Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kits were from Invitrogen. ELISA kits for detection of IFNα and IL-6 were obtained from RayBiotech (Norcross, Ga.). TUNEL assay kit was purchased from R&D systems (Minneapolis, Minn.). 2′Fluoro-2′-deoxycytidine-5′triphosphate and 2′-Fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5 labeled 2′fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, Calif.). Cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Va.). Human serum (Normal Pool) was obtained from Thermo Fisher Scientific.
Aptamer-siRNA Chimera synthesis. The ssDNA templates and primers were synthesized from IDT. For HEH chimera synthesis, two RNAs (RNA1 and RNA2) were generated separately.
RNA1: HER2 aptamer-EGFR sense siRNA.
RNA1 and RNA2 were generated by in vitro transcription with PCR products as templates. The PCR products were put into T-A cloning pCR2.1 vector (Invitrogen) and sequenced. Transcription was performed with Transcript Aid T7 High Yield Transcription Kits. 2′ F-modified pyrimidines were incorporated into RNAs to replace CTP and UTP. The transcribed RNAs were purified with phenol/chloroform/isoamyl alcohol (25:24:1) (Sigma-Aldrich), precipitated with isopropanol (Sigma-Aldrich) followed by cold 70% ethanol wash. The RNA pellets were dissolved in nuclease free water (IDT). The purification procedures were used for all transcribed RNAs. RNA1 and RNA2 were mixed at a molar ratio of 1:1 and annealed to form one entity by heating at 94° C. for 3 min, followed by slowly cooling to room temperature.
For HER2 aptamer (RNA3) synthesis, RNA1 PCR template and RNA1 5′-primer will be used as the above sequences, and RNA3 3′-primer is 5′-AGCCGCGCCCTACCCTATCCCT-3′ (SEQ ID NO:6) (P4).
For mutant HEH3 synthesis, RNA4 and RNA5 will be separately synthesized and annealed together.
RNA4: mutant HER2 aptamer-EGFR sense siRNA.
RNA 5: mutant HER2 aptamer-EGFR antisense siRNA.
For HER2 aptamer-scrambled siRNA synthesis, RNA6 and RNA7 will be separately synthesized and annealed together.
RNA 6: HER2-scrambled sense siRNA.
Western Blot Analysis. Whole-cell protein was extracted with RIPA lysis buffer containing 1× Halt Protease Inhibitor Cocktails and quantitated with Bio-Rad Protein Assay. Protein (100 μg per sample) was resolved on 10% SDS-PAGE and transferred to PVDF membrane. After blocking for 2 h at room temperature in 5% milk in TBS/0.1% Tween™-20, membrane were incubated overnight at 4° C. with the indicated primary antibodies (HER2, EGFR, Cleaved Caspase-3, GAPDH, 1:1000 dilution, Cell signaling), followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. After ECL Western Blotting Substrate (Pierce) was added onto membrane, the signals were captured by the exposure to X-ray film.
Cytotoxicity Assay. Cellular cytotoxicity was quantified by measuring WST-8 formazan using Cell Counting. Cells in RPMI 1640 containing 5% fetal bovine serum were seeded into 96-well plate at a density 5×103 per well for 24 h at 37° C., and then cells were treated with HEH (1 μM), HER2 aptamer (2 μM), EGFR siRNA (1 μM), HScH (1 μM), and muHEH (1 μM) for 72 h at quadruplicate wells without transfection reagents (e.g., Lipofectamine). CCK-8 solution (10 μl) (Dojindo, Japan) was added to each well and incubated at 37° C. for 4 h. Absorbance at 450 nm was measured using a plate reader.
HEH Stability in Cell Culture Medium. 2′ F-modified HEH (0.2 nmoles) were put into 40 μL of RPMI 1640 containing 5% fetal bovine serum, which was the condition for chimera treatment, for different time periods. RNA integrity was detected with 3% agarose gel electrophoresis. HEH intensity was measured with ImageJ.
qRT-PCR Assay. Total RNA from BT474 and MDA-MB-231 cells was extracted with RNAeasy plus kits (Qiagen). The quantity of RNAs was determined by NanoDrop. cDNA was generated with iScript cDNA synthesis kits (Bio-Rad). qRT-PCR analyses were performed using SYBR Green Master Mix™ (Bio-Rad) and further carried out on a CFX96™ Real-Time System (Bio-Rad).
Detection of Apoptosis by Flow Cytometry. SKBR3 and BT474 cells were treated with HEH (1 μM) or HER2 aptamer (2 μM) or muHEH for 48 h and 72 h. The cells were harvested and washed in cold PBS. Cells were stained with Alexa Fluor 488 annexin V-PI solution for 1 h at room temperature. Cells were acquired by BD FACSCalibur and analyzed using BD FACStation software.
Cellular Uptake Assay with Laser Scanning Confocal Microscopy. Cells were seeded into 35 mm glass-bottom Petri dishes for 24 h in RPMI 1640 supplemented with 5% fetal bovine serum. Cy5-labeled HEH (1 μM), HER2 aptamer (2 μM), or EGFR siRNA (1 μM) was added into culture for 12 h at 37° C. At the same time, LysoTracker Green DND-26 (80 nM) and DAPI (10 μg/mL) were added to the culture medium for imaging. Images were captured using confocal laser scanning microscope (Zeiss 780 inverted). The internalization of treatments was captured by “Z” stacking using an oil-immersion lens (63× magnification). Data were analyzed with 249 Zeiss LSM image Browser.
Cell-type Binding Specificity. Cells including BT474, SKBR3, MDA-MB-231, MCF7, and Hs578T were grown and harvested. After washing, cells were incubated with Cy5-labeled HEH (2 μM) or Cy3-labeled muHEH (2 μM) in the presence of yeast tRNA (300 μg/mL) and sperm DNA (500 μg/mL) for 1 h at 37° C. Cell binding was detected using BD FACSCalibur flow cytometry.
Biodistribution Assay. Athymic nude female mice were implanted with 2×106 BT474 cells. After 4 weeks of implantation, tumor-bearing mice (n=3 per group) were intravenously administered Cy5-labeled HEH (20 nmoles, 200 mL) or an equal mole amount of Cy5-labeled muHEH. The whole-body images were obtained at 0.5 h, 3 h, 12 h, and 24 h using the Xenogen IVIS100 imaging system by setting the wavelength at an excitation of 640 nm and emission at 710 nm.
Mouse Xenograft Models and Drug Administration. Athymic nude female mice (4-6-week old) were obtained from Envigo. BT474 cells (2×106) were injected into the flank of the mouse. Once tumor reached 100 mm3, mice were randomly divided into three groups (n=4). Mice were i.p. injected with PBS (200 μL), HEH (10 nmoles, 200 μL), HER2 aptamer (20 nmoles, 200 μL) three times per week for 4 weeks. Tumor sizes were measured weekly and calculated with the formula V=(L×W2)/2 (W, width; L, length; V, volume). The animals were euthanized 2 days after the last treatment. The tumors and organs were removed.
Histology Assay. Tumor tissue samples and major organs were collected from xenografts and fixed in 10% neutral-buffered formalin and paraffin-embedded. Sections (6 μm) were cut and mounted on the slides and then deparaffinized in xylene and ethyl alcohol. Each block has a section for HE staining. For immunohistochemistry assay, antigen retrieval was performed in 10 mmol/L citrate buffer (pH6) at 95° C. for 30 min, and then sections were incubated in 3% normal goat serum for 2 h and incubated with primary antibodies: caspase-3 (1:200), HER2 (1:800), and EGFR (1:50). After washing, the sections were incubated with biotinylated secondary antibody (1:200) (Vector Laboratories, Burlingame, Calif.) for 1 h. Following washing, the sections were incubated with VECTASTAIN ABC reagents for 30 min. The images were captured with a Nuance fluorescence microscope with a bright field imaging system.
Statistical Analysis. GraphPad Prism™ software was used to perform all statistical analyses. The results were expressed as a mean±SE. All data were analyzed using two-tailed Student's t test by comparing with the control group, and P<0.05 was considered statistically significant.
Example 2: Synthesis of Bivalent HER2 Aptamer-EGFR siRNA 298 Chimera and Comparison of CytotoxicityBivalent aptamer chimera was constructed with previously established methods. A HER2 aptamer with 34 nucleic acids was selected and was used in this investigation. This aptamer can specifically bind to HER2 expressing tumors in previous studies. Briefly, HER2 aptamer containing EGFR sense strand was fused with HER2 aptamer containing EGFR antisense strand to construct a HER2 aptamer-EGFR siRNA-HER2 aptamer chimera (HEH) as in
Next, the cytotoxicity of HEH was measured comparing with HER2 aptamer only. Prior to cytotoxicity detection, HER2 and EGFR expression among different breast cancer cell lines was evaluated. As shown in
Furthermore, cytotoxicity was measured after treated with HEH (1.0 μM), HER2 aptamer (2.0 μM), mutant HEH (muHEH) (1.0 μM), HER2 aptamer-scrambled siRNA-HER2 aptamer (HScH) (1.0 μM), and EGFR siRNA (1.0 μM) in the above five cell lines. Because one HEH has two copies of HER2 aptamer and one copy of EGFR siRNA, aptamer and siRNA was adjusted with the same amounts (i.e., moles) as HEH. After 72-h treatment, Cell Counting Kit-8 (CCK-8) reagents have been added into the cells. As shown in
Results:
To test if HEH induced cytotoxicity is through triggering apoptosis, HER2 positive SKBR3 and BT474 cells were treated with HER2 aptamer (2 μM), HScH (1 μM), or HEH (1 μM) for 48 h and 72 h. Cell apoptosis and death were measured with Annexin Vf2 Propidium Iodied (PI) staining. As shown in
Results:
EGFR siRNA between two HER2 aptamers is expected to silence EGFR gene. In previous studies, it was demonstrated that bivalent aptamer, like antibody, can cross-link cell surface receptors and active cells and thus induce significantly increased siRNA internalization compared with monovalent aptamer counter-parts. To validate if bivalent HER2 aptamer has driven EGFR siRNA internalization and enables endosomal escape, which is the prerequisite of siRNA silencing, confocal microscopy was performed to evaluate internalization of Cy5-labeled constructs. BT474 cells were treated with Cy5 labeled HEH, HER2 aptamer, muHEH, and EGFR siRNA. Nuclei were stained with DAPI and endosome/lysosomes were revealed by Lysotracker. After 12-h incubation, confocal laser scanning microscopy with Z-stack was performed to evaluate subcellular distribution of treatments. From Z-stack imaging shown in
HEH is capable of reducing expression levels of EGFR and HER2. Furthermore, the ability of HEH to induce EGFR silencing and reduce EGFR protein level was investigated. Western blot was performed to evaluate EGFR protein expression. As shown in
Next, the ability of HER2 aptamer to behave like antibody was evaluated. It has been well documented that antibody upon binding to its cell surface receptors can induce receptor-mediated endocytosis and initiate degradation of bound receptors. As shown in
Results:
To explore if HEH has different binding patterns on breast cancer cell lines, breast cancer cells were incubated with Cy5-HEH or Cy5-muHEH and detected with flow cytometry. As shown in
Results:
To explore tumor-targeting capability and biodistribution of HEH in vivo, tumor-bearing mice (BT474 cell derived xenografts) were i.v. injected with Cy5-HEH or Cy5-mHEH. Biodistribution was monitored with Xenogen IVIS 100 imaging system. As shown in
Results:
Because HEH can reduce protein expression of HER2 and EGFR and trigger cell apoptosis in vitro, treatment efficacy was evaluated to determine if it can be demonstrated in vivo. BT474 cells were implanted into one flank of athymic nude female mice, after tumor size reach about 100 mm, HEH (10 nmoles) was intraperitoneally injected into tumor bearing mice three times per week for 4 weeks. Tumor growth was measured weekly with digital caliper meter. As shown in
Results:
To confirm treatment efficacy, HE staining on excised tumors was performed. As shown in
As shown in
Results:
To evaluate potential systemic toxicity, after 4-week treatment, HE staining was performed on major organs including brain, heart, intestine, kidney, liver, lung, and spleen. There is no obvious histological difference between PBS- and HEH-treated mice (
Results:
To generate HEH, equal moles of HER2 aptamer-EGFR siRNA sense strand and HER2 aptamer-EGFR siRNA anti-sense strand were mixed together and heated to 95° C. for 3 min, followed by slowly cooling to room temperature. After annealing, in HEH lane, no free aptamer-EGFR siRNA sense strand or HER2 aptamer-EGFR siRNA anti-sense strand is detectable. Lane 1: Molecular weight marker; Lane 2: HER2 aptamer-EGFR siRNA sense strand; Lane 3: annealed HEH; Lane 4: HER2 aptamer-EGFR siRNA anti-sense strand (
HER2 positive BT474 cells and HER2 negative MDA-MB-231 cells were treated with HEH, muHEH or HER2 aptamer-scrambled siRNA at 2 μM for 48 h. RNA was extracted and reverse transcribed as described in material and methods. Gene copy numbers were normalized against GAPDH (
Cy5-labeled HEH, EGFRsiRNA, HER2 aptamer, or muH2EH3 was individually added into BT474 cells for 12 h at 37° C. LysoTracker Green was used to show lysosomes and endosomes. DAPI was used to display nucleus. Confocal laser scanning microscopy with z stack was performed to show cell binding and internalization. As a cell control, HER2 negative MDA-MB-231 cells were treated with Cy5-HEH for 12 h at 37° C. and the internalization was determined with Z-stack confocal microscopy.
Example 13: Engineering of a Three-in-One Aptamer-siRNA Chimera. HER2 Aptamer-EGFR siRNA-HER3 AptamerMaterials and Methods:
Chemicals and Cell culture. Vendors for specific chemicals are listed below. Cell culture products were purchased from Life Technologies (Carlsbad, Calif.). Antibodies were from Cell Signaling Technology (Danvers, Mass.). Single stranded DNAs were synthesized by Integrated DNA Technologies (IDT, Coralville, Iowa). TranscriptAid T7 High Yield Transcription Kits were purchased from Thermo Fisher Scientific. HER2 and HER3 siRNAs were purchased from Life Technologies Corporation. LysoTracker Green DND-26 and Alexa Fluor 488 Annexin V/Dead Cell Apoptosis kits were from Invitrogen. ELISA kits for detection of IFNα and IL-6 were obtained from RayBiotech (Norcross, Ga.). TUNEL assay kit was purchased from R&D systems (Minneapolis, Minn.). 2′Fluoro-2′-deoxycytidine-5′triphosphate and 2′-Fluoro-2′-deoxyuridine-5′-triphosphate, and Cy5 labeled 2′fluoro-labeled HER2 aptamer were purchased from TriLink Biotechnologies (San Diego, Calif.). Cell lines were purchased from the American Type Culture Collection (ATCC, Manassas, Va.). Human serum (Normal Pool) was obtained from Thermo Fisher Scientific.
Aptamer-siRNA Chimera synthesis. The ssDNA templates and primers were synthesized from IDT.
For H2EH3 chimera synthesis, RNA1 (HER3 aptamer-EGFR antisense siRNA):
The forward primer contains T7 RNA polymerase promoter site (bolded).
The forward primer contains T7 RNA polymerase promoter site (bolded).
Three RNAs were generated by in vitro transcription with PCR products as templates. The PCR products were put into T-A cloning pCR2.1 vector
(Invitrogen) and sequenced. Transcription was performed with Transcript Aid T7 High Yield Transcription Kit following manufacture's instruction. 2′ F-modified pyrimidines were incorporated into RNAs to replace CTP and UTP. The transcribed RNAs were purified following the methods described in Zheng, et al., Theranostics, 7: 1373-1388 (2017); Liu, et al., Sci Rep, 6:30346 (2016). Two RNAs were mixed at molar ratio 1:1 and annealed to form one entity by heating at 94° C. for 3 min followed by slowly cooling to room temperature within 1 h.
For HER3 aptamer (RNA 3) synthesis, RNA1 PCR template (SEQ ID NO:21) and RNA1 5′ primer (SEQ ID NO:22) were used as above, and RNA3 3′primer is 5′-GGAUCCC GAACGGACCGCCCA-3′. (SEQ ID NO:26)
For HER2 aptamer (RNA 4) synthesis, RNA2 PCR template (SEQ ID NO:25) and RNA 2 5′ primer (SEQ ID NO:2) were used as above, RNA4 3′primer is
The PCR products were transcribed into RNA3, RNA 4 and RNA 5, which were annealed into HER3, HER2 and MG aptamers individually.
For mutant H2EH3 synthesis. RNA 6 (mutant HER3 aptamer-EGFR antisense siRNA). PCR template (IDT):
RNA7 (mutant HER2 aptamer-EGFR sense siRNA). PCR template (IDT):
As above procedures, the PCR products were transcribed into RNAs. RNA6 and RNA7 were annealed into mutant H2EH3. Cy5-labeled aptamers were synthesized by TriLink Biotechnologies
Dicer assay. H2EH3 (4 μg) was digested using human recombinant dicer enzyme (2 units) at 37° C. for 12 h following manufacturer's instructions (Genlantis, San Diego, Calif.). Reaction was quenched by adding dicer stop solution. The digestion pattern was analyzed on 3.5% agarose gel electrophoresis.
Results
A three-in-one aptamer siRNA chimera was designed to target three important oncogenes: EGFR, HER2, and HER3, respectively. The 3′-terminus of HER3 aptamer (79 bases) (Chen, et al., Proc Natl Acad Sci USA, 100: 9226-9231 (2003)) was fused with the anti-sense strand of EGFR siRNA, and the 3′-terminus of HER2 aptamer (34 bases) (Kim, et al., Nucleic Acid Ther, 21: 173-178 (2011)) was fused with the sense strand of EGFR siRNA. Kd value of HER2 aptamer is 3.49 nM, and Kd value of HER3 aptamer is 45 nM, 2-4 unpaired “A” s were inserted between an aptamer and an siRNA to offer spatial flexibility to each aptamer (
H2EH3 possesses the activities of HER2 aptamer, EGFR siRNA and HER3 aptamer. The binding affinity of H2EH3 compared with HER2 aptamer and HER3 aptamer individually was investigated. H2EH3 showed strong binding affinity comparable with HER2 aptamer and HER3 aptamer (
Materials and Methods
Cytotoxicity assay. Cellular cytotoxicity was quantified by measuring WST-8 formazan using Cell Counting Kit-8 (CCK-8) (Dojindo, Japan). Cells were seeded in 96-well plate at a density of 5×103 in 5% CO2 incubator for 24 h at 37° C. Cell lines were incubated with the varying concentrations of H2EH3 or controls for 72 h without transfection reagents (e.g., Lipofectamine). CCK-8 solution (10 μl) was added to each well and incubated at 37° C. for 4 h. Absorbance at 450 nm was measured using a microplate reader.
Results
H2EH3 cytotoxicity on breast cancer cell lines was evaluated. HER2, HER3, and EGFR expression in breast cancer and ovarian cancer cell lines including BT474, BT20, ES2, OVCAR8, SKBR3, Hs587T, MDA-MB-231 and MCF7 were analyzed by Western blot. As shown in
The growth inhibitory effect of H2EH3 on these cell lines was examined using a CCK8 cell counting kit. Cell lines were treated with increasing concentrations of H2EH3 or controls including HER2 aptamer, HER3 aptamer, EGFR siRNA, mixture of HER2 aptamer, HER3 aptamer, and EGFR siRNA, non-targeting control aptamer MG (specific to Malachite Green), or mutant H2EH3. As shown in
Materials and Methods
Cell cycle analysis. SKBR3 and BT474 cells were treated with the different concentrations of H2EH3 for 24 h and 48 h. Collected cells were fixed in cold 70% ethanol. After fix for 30 min at 4° C., ethanol-fixed cells were centrifuged and washed once with PBS. The cells were treated with RNase A and stained with Propidium Iodide (PI) for 30 min at 37° C. Cellular DNA contents were measured by exciting PI at 488 nm and measuring the emission at 580 nm using a BD FACSCalibur flow cytometer.
Detection of apoptosis by flow cytometry. SKBR3 and BT474 cells were treated with the different concentrations of H2EH3 for 72 h. The cells were harvested and washed in cold phosphate-buffered saline (PBS). Cells were stained with Alexa Fluor 488 Annexin V-Propidium Iodide (PI) solution for 1 h at room temperature. Cells (1×104/sample) were acquired by BD FACSCalibur and analyzed using BD FACStation software.
Results
The decrease in cell viability may be attributed to reduced cell proliferation and/or increased cell death. To identify these effects, the effect of H2EH3 on cell cycle progression was examined. BT474 and SKBR3 cells were treated with H2EH3 at the different concentrations for 24 h and 48 h. As shown in
To further assess induction of apoptosis by H2EH3, SKBR3 cells and BT474 cells were treated with H2EH3 for 72 h and stained with Annexin V/PI (Propidium Iodide) for flow cytometry analyses. As shown in
Materials and Methods
Confocal Microscopy
BT474 cells were treated with Cy5-H2EH3 and control Cy5-muH2EH3 aptamer. Confocal microscopy with z-stack was performed to visualize the subcellular locations of H2EH3. Nuclei were stained with DAPI and endosome/lysosomes were revealed by Lysotracker.
Results
Because EGFR siRNA was inserted between HER2 and HER3 aptamers, EGFR gene knockdown was expected. It was hypothesized that H2EH3 binding to cell receptors of HER2 and HER3 will cross link two receptors and result in ligand-receptor complex internalization. The internalized H2EH3 may lead to EGFR silencing. First, the internalization of H2EH3 was validated, which is the prerequisite of siRNA silencing. As shown in
Materials and Methods
Western blot. Cells were lysed in lysis buffer (M-PER Mammalian Protein Extraction Reagent, Thermo Fisher Scientific) containing 1× Protease Inhibitor Cocktails (Sigma Aldrich). The cell lysates were centrifuged at 12,000 g for 10 min at 4° C. The supernatant was collected and the protein concentration was determined with Bio-Rad Protein Assay (Bio-Rad, Hercules, Calif.). Equal amount of proteins was mixed with 4× Laemmli sample buffer containing 5% β-mercaptoethanol and heated at 95° C. for 10 min. Denatured samples was separated on 8%-10% SDS-PAGE and transferred to PVDF membrane. The membranes were blocked with 5% non-fat milk overnight at 4° C., and then incubated with primary antibodies overnight at 4° C., followed by incubation with horseradish peroxidase-conjugated secondary antibodies for 2 h at room temperature. After ECL Western Blotting Substrate (Pierce) was added onto membrane, the signals were captured by the exposure to X-ray film.
Results
EGFR siRNA triggered gene silencing. EGFR expression was evaluated with Western blot. As shown in
The effect of H2EH3 on protein levels of HER2 and HER3 was examined next. SKBR3 and BT474 cells were treated with the varying concentrations of H2EH3 for 72 h, HER2 and HER3 levels were quantified by Western blot. As shown in
Next, the effect of H2EH3 on downstream apoptotic signaling was examined. Upon treatment with H2EH3, BT474 and SKBR3 cells were probed for the expression of p21 and Cleaved Caspase-3 (CC3) by Western blot. CC3 is regarded as a signature marker of apoptosis, and p21 upregulation is closely related to cell cycle arrest and apoptosis. As shown in
Materials and Methods
Knockdown of HER2 and/or HER3. BT474 cells were plated in 6-well plates at a density of 5×105 cells/well for 24 h. HER2 siRNA and/or HER3 siRNA or control siRNA were transfected into cells using Lipofectamine RNAi MAX (Life Technologies) according to the manufacturer's instruction. Cells were harvested 72 h post-transfection and Western blot was performed to confirm gene knockdown. HER2 and/or HER3 silenced cells were subjected to aptamer binding assay by BD flow cytometry.
Results
To investigate whether H2EH3 indeed binds to HER2 and HER3 receptors, siRNAs were used to silence each receptor to assess binding specificity. HER2 and/or HER3 siRNAs were transfected into BT474 cells via lipofectamine, then binding pattern was detected after receptor knockdown. HER2 and/or HER3 knockdown was confirmed with Western blot (
Materials and Methods:
Bio-distribution assay. To evaluate the bio-distribution properties of H2EH3, tumor bearing mice were intravenously administrated with Cy5-labeled H2EH3 (20 nmoles, 200 μl) or equal mole amount of Cy5-labeled MG aptamer. The whole-body images were obtained at 0.5 h and 6 h using Xenogen IVIS100 imaging system by setting wavelength at excitation 640 nm and emission 710 nm. After 6-h injection of Cy5-labeled aptamers, mice were euthanized with CO2 following whole-body imaging, and organs (heart, lung, liver, spleen, kidney, muscle, brain, stomach, and intestine) were removed. The ex vivo imaging of Cy5 signal were performed using Xenogen IVIS100.
In vivo xenograft treatment study. 4 to 5-week-old female athymic nu/nu mice were injected with tumor cells (BT474 or MDA-MB-231) (2×106) mixed with Matrigel (v/v 1:1) (Corning, N.Y.) subcutaneously at the left flank of mice, or orthotopically at the mammary fat pads of mice. Following the establishment of tumors (100 mm3), mice were randomly divided into three groups in subcutaneous xenografts, H2EH3 (8 nmoles, 200 μl), or mixture of HER2 aptamer &EGFR siRNA & HER3 aptamer (each 8 nmoles, total 200 μl), or PBS (200 μl) was intra-tumorally injected to mice at every other day for 5 weeks. In the orthotopic xenografts, upon tumor reaching 50 mm3, mice were administrated with H2EH3 (8 nmoles, 200 μl) or PBS every three days through tail-vein intravenous injection for 4 weeks. Tumor sizes and body weights were measured twice a week. The animals were euthanized two days after the last treatment. The tumors and organs (liver, spleen, kidney, brain, heart, muscle, blood and intestine) were removed and fixed in 10% formalin buffer. The sections of tissues were analyzed by immunohistochemistry.
Results:
To explore tumor targeting capability and bio-distribution in vivo, BT474 tumor bearing mice were tail-vein injected with Cy5-H2EH3 or non-targeting control aptamer. Thirty minutes after injection with control aptamer or H2EH3, the tumors in H2EH3 treated mice but not tumors in non-targeting control aptamer treated mice show high fluorescence intensity that is clearly delineated from the surrounding background tissues with Xenogen IVIS100 imaging system (
Materials and Methods
In vivo xenograft treatment study. 4 to 5-week-old female athymic nu/nu mice were injected with tumor cells (BT474 or MDA-MB-231) (2×106) mixed with Matrigel (v/v 1:1) (Corning, N.Y.) subcutaneously at the left flank of mice, or orthotopically at the mammary fat pads of mice. Following the establishment of tumors (100 mm3), mice were randomly divided into three groups in subcutaneous xenografts, H2EH3 (8 nmoles, 200 μl), or mixture of HER2 aptamer, EGFR siRNA, and HER3 aptamer (each 8 nmoles, total 200 μl), or PBS (200 μl) was intra-tumorally injected to mice at every other day for 5 weeks. In the orthotopic xenografts, upon tumor reaching 50 mm3, mice were administrated with H2EH3 (8 nmoles, 200 μl) or PBS every three days through tail-vein intravenous injection for 4 weeks. Tumor sizes and body weights were measured twice a week. The animals were euthanized two days after the last treatment. The tumors and organs (liver, spleen, kidney, brain, heart, muscle, blood and intestine) were removed and fixed in 10% formalin buffer. The sections of tissues were analyzed by immunohistochemistry.
Results
To investigate whether the in vitro anti-proliferative effect of H2EH3 could translate into in vivo efficacy, H2EH3 treatment was tested in subcutaneous and orthotopic breast cancer xenografts. BT474 cells were subcutaneously injected into 5 to 6 week-old female athymic nu/nu mice at the left flank of mice. Upon tumor reaching 100 mm3, mice were treated with PBS, mixture (HER2 aptamer, HER3 aptamer, and EGFR siRNA) (8 nmoles of each component) or H2EH3 (8 nmoles) by intra-tumor injection every other day for 5 weeks. As shown in
Next, mammary orthotopic xenografts were set up by injection of BT474 cells in mammary fat pads. Tumor (50 mm3) bearing mice were intravenously (via tail-vein) treated with H2EH3 (8 nmoles), mixture (HER2 aptamer, HER3 aptamer, and EGFR siRNA) or PBS every three days for 4 weeks. As shown in
Materials and Methods
Histology assay. Tumors and organs (spleen, lung, kidney, intestine, heart, liver and muscle) were collected from xenografts, and fixed with 4% paraformaldehyde. Sections (6 μm) were cut and mounted on the slides, and deparaffinized in xylene and ethyl alcohol. For immunohistochemistry assay, sections were incubated in 3% normal goat serum for 2 h and incubated with primary antibodies: caspase-3 (1:200), HER2 (1:800), HER3 (1:250), p21 (1:50), and EGFR (1:50). After washing, the sections were incubated with biotinylated secondary antibody (1:200) (VECTOR, Burlingame, Calif.) for 1 h. Following washing, the sections were incubated with VECTASTAIN ABC reagents for 30 min. The images were captured with Nuance fluorescence microscope with bright field imaging system.
Results
To determine the target specificity of H2EH3, immunohistochemistry (IHC) assay was performed to validate the gene knockdown and induction of apoptosis in vivo. Tumor tissues from subcutaneous and orthotopic xenografts were collected and examined for the expression of HER2, HER3, EGFR, p21 and CC3. As shown in
Materials and Methods
Histology assay. Tumors and organs (spleen, lung, kidney, intestine, heart, liver and muscle) were collected from xenografts, and fixed with 4% paraformaldehyde. Sections (6 μm) were cut and mounted on the slides, and deparaffinized in xylene and ethyl alcohol. For immunohistochemistry assay, sections were incubated in 3% normal goat serum for 2 h and incubated with primary antibodies: caspase-3 (1:200), HER2 (1:800), HER3 (1:250), p21 (1:50), and EGFR (1:50). After washing, the sections were incubated with biotinylated secondary antibody (1:200) (VECTOR, Burlingame, Calif.) for 1 h. Following washing, the sections were incubated with VECTASTAIN ABC reagents for 30 min. The images were captured with Nuance fluorescence microscope with bright field imaging system.
ELISA assay. Normal human peripheral blood mononuclear cells (PBMCs) were separated with Ficoll-Plaque Plus (Stem Cell Technologies). Cells were seeded into 24-well plates at 2×106/well for 24 h in RPMI-1640 medium containing 10% fetal bovine serum. H2EH3 with the varying concentrations was added into cells, and cells were incubated for 48 h in 5% CO2 incubator at 37° C. The cell culture supernatant was detected with human IFNα and IL-6 ELISA kits following the manufacture's instruction.
Results:
During the treatment, the body weights of mice were measured. There was no significant difference of body weights between PBS- and H2EH3-treated groups (
While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been put forth for the purpose of illustration, it will be apparent to those skilled in the art that the invention is susceptible to additional embodiments and that certain of the details described herein can be varied considerably without departing from the basic principles of the invention.
All references cited herein are incorporated by reference in their entirety. The present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof and, accordingly, reference should be made to the appended claims, rather than to the foregoing specification, as indicating the scope of the invention.
Claims
1. An aptamer-siRNA chimera comprising:
- first and second ends, wherein the first and second ends comprise an aptamer that specifically binds a target protein; and
- an siRNA construct between the first and second ends, wherein the siRNA construct is processed by cellular RNAi machinery to produce at least two different siRNAs that specifically inhibit expression of two or more different genes in a cell expressing the target protein.
2. The chimera of claim 1, wherein at least one of the two or more different genes are survivin and EGFR and the target protein is HER2, HER3 or PSMA.
3. The chimera of claim 1, wherein the two or more different genes are oncogenes selected from the group consisting of ABL1, ABL2, AKT1, AKT2, ATF1, BCL11A, BCL2, BCL3, BCL6, BCR, BIRC5, BRAF, CARD11, CBLB, CBLC, CCND1, CCND2, CCND3, CCNE1, CDX2, CTNNB1, DDB2, DDIT3, DDX6, DEK, EGFR, ELK4, ERBB2, ETV4, ETV6, EVI1, EWSR1, FEV, FGFR1, FGFR2, FGFR3, FGFR4, FGFR6, EGFR, FGFR1OP, FUS, GOLGA5, GOPC, HMGA1, HMGA2, HRAS, IRF4, JUN, KIT, KRAS, LCK, LMO2, MAF, MAFB, MAML2, MDM2, MET, MITF, MPL, MYB, MYC, MYCL1, MYCN, NCOA4, NFKB2, NRAS, NTRK1, NUP214, PAX8, PDGFB, PIK3CA, PIM1, PLAG1, PPARG, PTPN11, RAF1, REL, RET, ROS1, SMO, SS18, TCL1A, TET2, TFG, MLL, TLX1, TPR, and USP6.
4. The chimera of claim 1, wherein the target protein is selected from the group consisting of CLPP, CEA, Her-2/neu, Bladder Tumor Antigen, Thyroglobulin, Alpha-fetoprotein, PSA, CA 125, CA19.9, CA 15.3, leptin, prolactin, osteopontin, IGF-II, CD98, fascin, sPIgR, EpCAM, transferrin receptor, CD44, AXL, Human matrix metalloprotease 9, VEGFR, EGFR, Her3, ICAM-1, VCAM-1, Chemokine receptors, CD3, CD4, CD8, TNFR, L (P,E) selectin, and 14-3-3.
5. The chimera of claim 3, wherein the target protein is HER2 or HER3.
6. The chimera of claim 1, wherein the target protein is a tumor neovascular antigen.
7. A method for treating breast cancer in a subject in need thereof, comprising:
- administering to the subject an effective amount of the chimera of claim 2.
8. A method for reducing tumor burden in a subject in need thereof comprising:
- administering to the subject an effective amount of an aptamer to induce or promote apoptosis of the tumor cells, wherein the aptamer comprises first and second ends, wherein the first and second ends comprise an aptamer that specifically binds a cell surface protein expressed by the tumor cells; and an siRNA construct between the first and second ends, wherein the siRNA construct is processed by cellular RNAi machinery of the cancer cells to produce at least one or two different siRNAs that specifically inhibit expression of one or two or more different genes in the tumor cells to promote apoptosis of the tumor cells and thereby reduce tumor burden in the subject.
9. The method of claim 8, wherein the two or more different genes comprise EGFR and survivin.
10. The method of claim 9, wherein the two or more different genes comprise an oncogene expressed by the cancer cell.
11. A method of reducing tumor associated angiogenesis in a subject in need thereof comprising:
- administering to the subject an effective amount of the aptamer of claim 1 to reduce tumor associated angiogenesis, wherein the first and second ends comprise an aptamer that specifically binds a cell surface protein expressed by the tumor; and wherein the siRNA construct is processed by cellular RNAi machinery of tumor to produce at least one or two different siRNAs that specifically inhibit expression of two or more different genes in the tumor, wherein the two or more different genes comprise EGFR and survivin.
12. A method for treating a viral infection in a subject in need thereof comprising:
- administering to the subject an effective amount of the aptamer of claim 1 to inhibit expression genes of the virus infecting the subject, wherein the first and second ends comprise an aptamer that specifically binds a cell surface viral protein expressed by virally infected cells; and
- wherein the siRNA construct is processed by cellular RNAi machinery of the cells to produce at least two different siRNAs that specifically inhibit expression of two or more different genes of the infecting virus.
13. A pharmaceutical composition comprising an effective amount of the aptamer of claim 1.
14. The pharmaceutical composition of claim 14, wherein the aptamer comprises SEQ ID NOs: 31 and 33.
15. A nucleic acid composition according to FIG. 11A.
Type: Application
Filed: Feb 9, 2021
Publication Date: Jun 3, 2021
Applicant: Augusta University Research Institute, Inc. (Augusta, GA)
Inventor: Hong Yan LIU (Martinez, GA)
Application Number: 17/171,050
