USOO8492.133B2

(12) United States Patent (10) Patent No.: US 8.492,133 B2 Rosin-Arbesfeld et al. (45) Date of Patent: Jul. 23, 2013

(54) MIR-21 PROMOTER DRIVEN TARGETED Loffler et al., 2007, Blood, 110: 1330-1333.* CANCERTHERAPY Fujita et al., 2008, J. Mol. Biol. 378, 492-504.* KumarSwamy et al., 2001, RNA Biology, 8: 706-713.* (75) Inventors: Rina Rosin-Arbesfeld, Herzliya (IL); Amaral, Fernando Colbari et al., (2008) differentially Ella Sklan, Raanana (IL); Alona expressed in ACTH-secreting pituitary tumors. J Clin Endocrinol Zilberberg, Tel Aviv (IL); Naama Metab 94(1):320-323. David, Givatayim (IL) Barnes, Dwight et al., (2008) Harnessing Endogenous miRNAs to (73) Assignee: Ramot at Tel Aviv University, Ltd., Tel Control Virus Tissue Tropism as a Strategy for Developing Attenu Aviv (IL) ated Virus Vaccines. Cell Host Microbe 4(3):239-248. Bartel, David P. (2004) MicroRNAs: genomics, biogenesis, mecha (*) Notice: Subject to any disclaimer, the term of this nism, and function. Cell 116(2):281-297. patent is extended or adjusted under 35 Betel, D. et al., (2008) The microRNA.org resource: targets and U.S.C. 154(b) by 0 days. expression. Nucleic Acids Res 36(Database issue): D149-153. Cai, Xuezhong et al., (2004) Human microRNAs are processed from (21) Appl. No.: 13/145,332 capped, polyadenylated transcripts that can also function as mRNAs. RNA 10(12): 1957-1966. (22) PCT Filed: Jan. 19, 2010 Chan, Jennifer A. et al., (2005) MicroRNA-21 is an antiapoptotic factor in human glioblastoma cells. Res 65 (14):6029-6033. (86). PCT No.: PCT/L2O1O/OOOO49 Chan, Shin-Hsuan et al., (2008) miR-21 microRNA expression in human gastric carcinomas and its clinical association. Anticancer Res S371 (c)(1), 28(2A):907-911. (2), (4) Date: Nov. 17, 2011 Chang, Steven S. et al., (2008) MicroRNA alterations in head and neck squamous cell carcinoma. IntJ Cancer 123 (12):2791-2797. (87) PCT Pub. No.: WO2010/084488 Connolly, Erin et al., (2008) Elevated expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-associated hepatocellular PCT Pub. Date: Jul. 29, 2010 carcinoma contributes to the malignant phenotype. Am J Pathol 173(3):856-864. (65) Prior Publication Data Dillhoff, Mary et al., (2008) MicroRNA-21 is Overexpressed in US 2012/OO65251A1 Mar. 15, 2012 and a Potential Predictor of Survival. J Gastrointest Surg 12(12):2171-2176. Related U.S. Application Data Feber, Andrew et al., (2008) MicroRNA expression profiles of esophageal cancer. J Thorac Cardiovasc Surg 135 (2):255-260; dis (60) Provisional application No. 61/145,751, filed on Jan. cussion 260. 20, 2009. Fujita, Shuji et al., (2008) miR-21 Expression Triggered by AP-1 Is Sustained through a Double-Negative Feedback Mechanism. (51) Int. Cl. J Mol Biol 378(3):492-504. CI2N 15/85 Fujita, Shuji and IBA, Hideo (2008) Putative promoter regions of (2006.01) miRNA involved in evolutionarily conserved regulatory sys (52) U.S. Cl. tems among vertebrates. Bioinformatics 24(3):303-308. USPC ...... 435/252.3 Fulci, Valerio et al., (2007) Quantitative technologies establish a (58) Field of Classification Search novel microRNA profile of chronic lymphocytic leukemia. Blood None 109(11):4944-4951. See application file for complete search history. Gabriely, Galina et al., (2008) MicroRNA 21 promotes glioma inva sion by targeting matrix metalloproteinase regulators. Mol Cell Biol (56) References Cited 28(17):5369-5380. Gao, Jie et al., (2007) Cloning and identification of microRNA from U.S. PATENT DOCUMENTS human osteosarcoma cell line SOSP-9607. Chinese Journal of Can cer (Ai Zheng) 26(6):561-565 article in Chinese with English 5,998.205 A * 12/1999 Hallenbeck et al...... 435/325 7,041,654 B2 5/2006 Hochberg abstract. 2006, O105360 A1 5, 2006 Croce Giladi, Nis et al., (2007) Gene therapy approach in 2008/0050744 A1 2/2008 Brown cells using an active Wnt signal. Biomedicine & Pharmacotherapy 2008.0171715 A1 7/2008 Brown 61(9):527-530. 2008. O199961 A1 8, 2008 Rasko 2008/0261908 A1 10, 2008 Croce (Continued) 2008/0306006 A1 12, 2008 Croce 2008/0306017 A1 12, 2008 Croce Primary Examiner — Deborah Crouch 2008/0306018 A1 12, 2008 Croce Assistant Examiner — Titilayo Moloye 2009/0192101 A1* 7/2009 Hung et al...... 514,44 (74) Attorney, Agent, or Firm — Greenberg Traurig, LLP FOREIGN PATENT DOCUMENTS WO 99, 18195 4f1999 (57) ABSTRACT WO 03/093441 11, 2003 WO WO 2005/052129 * 6/2005 The invention provides a nucleic acid construct comprising a WO 2006/065938 6, 2006 promoter sequence derived from microRNA-21 (miR-21) WO 2007/034487 3, 2007 linked to a nucleic acid sequence encoding an anti-cancer WO 2008/073303 6, 2008 agent, an example of which is a toxin. The constructs of the WO 2008/087642 T 2008 invention are particularly useful for treating tumors express OTHER PUBLICATIONS ing miR-21. Esquela-Kerscher et al., 2006, Nature Reviews: Cancer, 6: 259-269.* 5 Claims, 5 Drawing Sheets US 8.492.133 B2 Page 2

OTHER PUBLICATIONS Pichiorri, Flavia et al., (2008) MicroRNAs regulate critical genes Iorio, Marilena V. et al., (2005) MicroRNA deregu associated with multiple myeloma pathogenesis. Proc Natl Acad Sci lation in human breast cancer. Cancer Res 65 (16):7065-7070. USA 105(35): 12885-12890. Iorio, Marilena V. et al., (2007) MicroRNA signatures in human Qian, Biyun et al., (2008) High miR-21 expression in breast cancer . Cancer Res 67(18):8699-8707. Jiang, Jiami et al., (2008) Schmittgen, Association of MicroRNA associated with poor disease-free Survival in early stage disease and expression in hepatocellular carcinomas with hepatitis infection, cir high TGF-beta1. Breast Cancer Res Treat 117(1): 131-140. rhosis, and patient survival. Clin Cancer Res 14(2):419-427. Roldo, Claudia et al., (2006) MicroRNA expression abnormalities in Kutay, Huban et al., (2006) Downregulation of miR-122 in the rodent pancreatic endocrine and acinar tumors are associated with distinc and human hepatocellular carcinomas. J Cell Biochem 99(3):671 tive pathologic features and clinical behavior. J. Clin Oncol 678. 24(29):4677-4684. Ladeiro, Yannicket al., (2008) MicroRNA profiling in hepatocellular Schetter, Aaron J. et al., (2008) MicroRNA expression profiles asso tumors is associated with clinical features and oncogenetumor Sup ciated with prognosis and therapeutic outcome in colon pressor gene mutations. Hepatology 47(6):1955-1963. adenocarcinoma. JAMA 299(4):425-436. Lawrie, Charles H. et al., (2007) MicroRNA expression distinguishes Sempere, Lorenzo F. et al., (2007) Altered MicroRNA expression between germinal center B cell-like and activated B cell-like sub confined to specific epithelial cell Subpopulations in breast cancer. types of diffuse large B cell lymphoma. Int J Cancer 121(5): 1156 Cancer Res 67(24): 11612-11620. 1161. Si, M.-L. et al., (2007) miR-21-mediated tumor growth. Lawrie, Charles H. et al., (2008) Detection of elevated levels of 26(19):2799-2803. tumour-associated microRNAS in serum of patients with diffuse large Slaby, O. et al., (2007) Altered expression of miR-21, miR-31, miR B-cell lymphoma. BrJ Haematol 141(5):672-675. 143 and miR-145 is related to clinicopathologic features of colorectal Lee, Eun Joo et al., (2006) Expression profiling identifies microRNA cancer. Oncology 72(5-6):397-402. signature in pancreatic cancer. IntJ Cancer 120 (5):1046-1054. Tran, Nham et al. (2007) MicroRNA expression profiles in head and Loffler, Dennis et al., (2007) Interleukin-6 dependent survival of neck cancer cell lines. Biochem Biophys Res Commun358(1):12-17. multiple myeloma cells involves the Stat3-mediated induction of Varda-Bloom, N. et al., (2008) Specific Induction of Tumor microRNA-21 through a highly conserved enhancer. Blood Neovasculature Death by Modified Murine PPE-1 Promoter Armed 110(4): 1330-1333. with HSV-TK. Pathobiology 75(6):346-355. Lui, Weng-Onn et al., (2007) Patterns of known and novel Small Volinia, Stefano et al., (2006) A microRNA expression signature of in human . Cancer Res 67 (13):6031-6043. human solid tumors defines cancer gene targets. Proc Natl Acad Sci Markou, Athina et al., (2008) Prognostic value of mature microRNA USA 103(7): 2257-2261. 21 and microRNA-205 overexpression in non-Small cell Wang, Tongsheng et al., (2007) A micro-RNA signature associated by quantitative real-time RT-PCR. Clin Chem 54(10): 1696-1704. with race, tumor size, and target gene activity in human uterine Meng, Fanyin et al., (2006) Involvement of human micro-RNA in leiomyomas. Genes Cancer 46(4):336-347. growth and response to chemotherapy inhuman cholangiocarcinoma Yan, Li-Xu et al., (2008) MicroRNA miR-21 overexpression in cell lines. Gastroenterology 130(7):2113-2129. human breast cancer is associated with advanced clinical stage, Nam, Eun Ji et al., (2008) MicroRNA expression profiles in serous lymph node and patient poor prognosis. RNA ovarian carcinoma. Clin Cancer Res 14 (9):2690-2695. 14(11):2348-23.60. Neely, Lori A. et al., (2008) microRNA expression ratio defining the Zhang, Zhiyu et al., (2008) miR-21 plays a pivotal role in gastric invasive phenotype in bladder tumors. Urol Oncol 28(1):39-48. cancer pathogenesis and progression. Lab Invest 88 (12): 1358-1366. Ohana, Patricia et al., (2005) Regulatory sequences of H19 and IGF2 ISR of PCT/IL2010/000049 mailed May 18, 2010. genes in DNA-based therapy of colorectal ratliver metastases. J Gene Med 7(3):366-374. * cited by examiner U.S. Patent Jul. 23, 2013 Sheet 1 of 5 US 8.492,133 B2

U.S. Patent Jul. 23, 2013 Sheet 2 of 5 US 8.492,133 B2

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Figure 2 U.S. Patent Jul. 23, 2013 Sheet 3 of 5 US 8.492,133 B2

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U.S. Patent Jul. 23, 2013 Sheet 5 of 5 US 8.492,133 B2

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Figure 5 US 8,492,133 B2 1. 2 MR-21 PROMOTER DRIVEN TARGETED lications which Suggest elevating or inhibiting the expression CANCERTHERAPY of miR-21 include U.S. Patent Application Publication Nos. 2008/0171715, 2008/01999.61 and 2008/0050744. Certain FIELD OF THE INVENTION attempts to use miRNAs to control tissue tropism have also been reported (see for example, Barnes et al., Cell Host The present invention is directed to the field of cancer Microbe, 2008. 4(3):239-248). treatment, and provides a nucleic acid construct particularly Selective targeting of a specific cancer using differentially useful for treating tumors expressing miR-21. expressed genes has been previously demonstrated Success fully where regulatory sequences of the H19 gene differen BACKGROUND OF THE INVENTION 10 tially expressed in bladder cancer were used to control the MicroRNAs (miRNAs) are an abundant class of short (20 expression of diphtheria toxin (Ohana et al., J Gene Med, 24 nt) endogenous noncoding RNAS that act as post-tran 2005.7(3):366-374). Scriptional regulators of gene expression by base-pairing with WO99/18195 and U.S. Pat. No. 7,041,654 teach the spe cific expression of heterologous sequences, particularly their target mRNAs (for review see for example, Bartel, Cell 15 2004, 116:281-297). In general, genes encoding miRNAs are genes encoding cytotoxic products (e.g. diphtheria toxin), in transcribed by RNA polymerase II as part of capped and tumor cells under the control of a cancer specific promoter polyadenylated primary transcripts (pri-miRNAs) that can be (e.g., H19 and IGF promoters). either protein-coding or non-coding. The primary transcript is WO 2007/034487 discloses a nucleic acid construct com cleaved by the ribonuclease III enzyme to produce an prising: (i) a first nucleic acid sequence encoding TNF alpha; approximately 70 nt stem-loop precursor miRNA (pre (ii) a second nucleic acid sequence encoding a Diphtheria miRNA), which is further cleaved by the cytoplasmic toxin; and (iii) at least one additional nucleic acid sequence ribonuclease to generate the mature miRNA and antisense comprising a cancer specific promoter (e.g. H19 and IGF miRNA star (miRNA) products. The mature miRNA is promoters); the TNF alpha and Diphtheria toxin encoding incorporated into a RNA-induced silencing complex (RISC), 25 sequences being under an expression control of the cancer which recognizes target mRNAS through imperfect specific promoter. Also disclosed are construct systems and ing with the miRNA and most commonly results in transla methods and uses thereof. tional inhibition or destabilization of the target mRNA. WO 2008/087642 discloses compositions and methods for To date, several hundred miRNAs have been described in the treatment of cancer and other conditions that are associ humans and a large number of them have been implicated in 30 ated with elevated expression of the H19 gene, utilizing con cancer. miRNAs that have been experimentally shown structs encoding H19-silencing nucleic acid agents such as directly to induce tumor phenotypes (i.e., formation) have inhibitory RNA. been termed “. MiR-21 (also known as MIRN21, WO 2006/065938 discloses, interalia, a method for treat miRNA21, hsa-mir-21 and MIR21) is such an shown ing cancer in a mammal comprising administering to the to target multiple tumor/metastasis Suppressor genes and to 35 mammal an effective amount of a nucleic acid that encodes an have a role in tumor growth, invasion and metastasis. Various anticancer agent operably linked to a SPANX-N1 promoter. studies show that miR-21 is exclusively expressed in cancer WO 2003/093441 discloses a method of inhibiting expres ous cell lines and solid human tumors, but not in non-trans sion of a gene in a cell comprising introducing into said cella formed cell lines or in the adjacent non cancerous tissue (see DNA construct comprising a promoter functional in said cell for example, Iorio et al., Cancer Res, 2005. 65(16): 7065 40 operably linked to a nucleic acid sequence encoding an 7070; Si et al., Oncogene, 2007. 26(19): 2799-2803; Volinia miRNA precursor having a stem loop structure and compris et al., Proc Natl Acad Sci USA, 2006. 103(7): 2257-2261). ing in said stem a sequence complementary to a portion of an Thus, evaluation of miR-21 expression has been suggested in RNA transcript of said gene, wherein, following introduction cancer diagnosis (see, for example, U.S. Patent Application of said construct into said cell, said nucleic acid sequence is Publication No. 2006/0105360). 45 transcribed and processed so that said miRNA precursor is U.S. Patent Application Publication No. 2008/0306.018 produced. Further disclosed is a plasmid construct compris discloses methods for diagnosing pancreatic cancer compris ing precursor miRNA-21 linked to a CMV promoter. ing measuring the level of at least one miRNA gene product, Caietal. disclose a plasmid containing the firefly luciferase including miR-21, in a test sample from the Subject. This gene linked to an ~1 kb DNA fragment of the pri-miR-21 publication further discloses a method of treating pancreatic 50 transcription unit corresponding to the region from ~959 to cancer in a Subject in which at least one miRNA gene product +49 relative to the T1 transcription start site (Cai et al., RNA is downregulated or upregulated in the cancer cells relative to (2004) 10:1957-1966). According to this disclosure, firefly control cells, inter alia when miR-21 is upregulated in the luciferase activity was detected in 293T cells transfected with cancer cells, the method comprising administering to the Such a plasmid, leading the authors to conclude that Subject an effective amount of at least one compound for 55 sequences located 5' to the pri-miR-21 transcription unit can inhibiting expression of the at least one miRNA gene product, function as an mRNA promoter. such that proliferation of cancer cells in the subject is inhib Fujita and Iba disclose a putative promoter region of miR ited. Also disclosed are pharmaceutical compositions for 21 462 nucleotides in length (Fujita and Iba Bioinformatics treating pancreatic cancer, comprising at least one isolated 2008, 24(3):303-308), and Fujita et al disclose the miR-21 miRNA gene product or an inhibitor thereof. 60 promoter including binding sites for activation protein 1 and Similar methods and compositions have been disclosed for PU. 1 (Fujita et al., J Mol Biol 2008 May 2: 378(3):492-504). the treatment of lung cancer (U.S. Patent Application Publi While several therapeutic approaches utilizing gene cation No. 2008/0306017), breast cancer (U.S. Patent Appli therapy in cancer patients have been Suggested, there exists a cation Publication No. 2008/0261908) and solid such need for additional efficacious anti-cancer agents and vectors. as prostate cancer, Stomach cancer, pancreatic cancer, lung 65 As tumors are known to exhibit significant genomic instabil cancer, breast cancer and colon cancer (U.S. Patent Applica ity and heterogeneity, the use of hitherto known gene therapy tion Publication No. 2008/0306006). Other exemplary pub vectors is likely to fail in a substantial number of the patients. US 8,492,133 B2 3 4 Nowhere in the prior art is it taught or Suggested that a gal toxin is selected from the group consisting of C.-sarcin, miR-21 promoter can be used in recombinant nucleic acid restrictocin, mitogillin, enomycin, RNase T1 and phenomy constructs for expressing selectively cytotoxic agents in can C1. cer cells. Nor does the art demonstrate the use of such vectors In particular embodiments, the toxin comprises a cytotoxic as an effective and safe anti-cancer treatment in vivo. There fragment of a naturally occurring toxin. In particular embodi remains an unmet medical need for developing gene therapy, ments, the cytotoxic fragment of a toxin is fragment A of vectors having enhanced therapeutic activity, minimized tox diphtheria toxin (DTA). In particular embodiments, DT-A icity and abroad target range for treating neoplastic disorders. has the amino acid sequence of SEQID NO: 19. In particular embodiments, the nucleic acid sequence encoding DT-A SUMMARY OF THE INVENTION 10 comprises SEQID NO: 18. In particular embodiments, the drug-metabolizing enzyme The invention is directed to nucleic acid constructs and is a kinase. In particular embodiments, the kinase is a thymi methods for providing targeted cancer therapy. Specifically, dine kinase. In particular embodiments, the prodrug target of the constructs of the invention comprise at least one nucleic the thymidine kinase is ganciclovir. In particular embodi 15 ments, the kinase is a viral thymidine kinase. In particular acid sequence encoding a cytotoxic or cytostatic anti-cancer embodiments, the inducer of is selected from the molecule. Such as a toxin, the nucleic acid sequence being group consisting of PUMA; BAX: BAK; Bcl-XS: BAD: operably linked to an miR-21 transcription-regulating BIM; BIK; BID; HRK; Ad ElB: an ICE-CED3 protease: sequence. Vectors comprising these constructs, pharmaceuti TRAIL: SARP-2; and apoptin. cal compositions comprising them and use thereof for treat In a particular embodiment, the construct comprises SEQ ing cancers and solid tumors are also provided. ID NO: 1 as the miR-21 promoter sequence and further com The present invention discloses for the first time novel prises SEQID NO: 18 as the nucleic acid sequence encoding nucleic acid constructs and vectors in which expression of an an anti-cancer agent. In particular embodiments, the nucleic anti-cancer agent is placed under the transcriptional control acid sequence comprising an miR-21 promoter sequence is ofan miR-21 promoter sequence. The inventors of the present 25 Substantially devoid of a nucleic acid sequence corresponding invention have Surprisingly demonstrated that Such con to or complementary to an miR-21 transcription or processing structs have activity in depressing de novo protein synthesis product. In particular embodiments, the nucleic acid con in cancer cells in vitro, and moreover inhibit tumor growth struct is Substantially devoid of a nucleic acid sequence cor and metastasis in vivo. responding to or complementary to a form of miR-21 selected The invention is based, in part, on the finding that direct 30 from the group consisting of a primary transcript of miR-21 injection into tumors induced in mice, of a nucleic acid con (pri-miR-21); a precursor of miR-21 (pre-miR-21); an RNA struct having an miR-21 promoter operatively linked to a duplex of miR-21, and a mature miR-21. In particular nucleic acid sequence encoding diphtheria toxin, results embodiments, the nucleic acid construct is Substantially either in complete eradication of the tumor or substantial devoid of a nucleic acid sequence corresponding to or 35 complementary to a nucleic acid sequence selected from the inhibition of tumor growth. group consisting of: SEQID NO: 2: SEQID NO:3: SEQID. Thus, in a first aspect the present invention provides a NO: 4: SEQID NO: 5 and SEQID NO: 26. nucleic acid construct comprising a miR-21 promoter In a particular embodiment, the nucleic acid construct sequence and at least one nucleic acid sequence encoding an comprises a sequence selected from the group consisting of anti-cancer agent, wherein the nucleic acid sequence encod 40 SEQID NO: 7 and SEQID NO: 8. ing the anti-cancer agent is operably linked to the miR-21 In a particular embodiment, the miR-21 promoter promoter sequence. sequence is a variant of a native miR-21 promoter, such as a In particular embodiments, the nucleic acid construct is the homolog, analog or fragment of a native miR-21 promoter. product of recombinant methods, a chemical synthesis or a In another aspect, the invention provides a vector compris combination thereof. 45 ing the recombinant nucleic acid construct of the invention. In particular embodiments, the miR-21 promoter sequence In another aspect, the invention provides an isolated host has at least 80% identity with SEQ ID NO: 1. In particular cell comprising the vector of the invention. embodiments, the miR-21 promoter sequence has at least In a particular embodiment, the vector is selected from the 85%, or at least 90% identity, or at least 95% identity with group consisting of SEQID NO:9 and SEQID NO: 10. SEQID NO: 1. In particular embodiments, the miR-21 pro 50 In a particular embodiment, the construct encodes a fusion moter sequence comprises SEQID NO: 1. protein comprising the anti-cancer agent. In a particular In particular embodiments, the anti-cancer agent is embodiment, the construct further comprises a nucleic acid selected from cytotoxic and cytostatic proteins and peptides sequence that encodes an antibody or a fragment thereof capable of killing a target cell or inhibiting its growth or comprising at least the antigen binding portion of the anti proliferation. In particular embodiments, the anti-cancer 55 body, wherein the antibody or antibody fragment is specific agent is selected from a toxin, a drug-metabolizing enzyme for a cancer-related protein. In a particular embodiment, the which converts a prodrug to a drug having cytotoxic activity, construct comprises a nucleic acid sequence encoding a an inducer of apoptosis, and a combination thereof. In par fusion protein wherein the fusion protein comprises segments ticular embodiments, the toxin is selected from the group corresponding to the anti-cancer agent and to the antibody or consisting of a bacterial toxin, a plant toxin, a fungal toxin, 60 antibody fragment. and a combination thereof. In particular embodiments, the In another aspect, the invention provides a pharmaceutical bacterial toxin is selected from the group consisting of diph composition comprising as an active ingredient at least one theria toxin, Pseudomonas exotoxin, cholera toxin, anthrax nucleic acid construct of the invention and a pharmaceutically toxin, botulinum toxin, pertussis toxin, E. coli enterotoxin, acceptable carrier, excipient or diluent. and Shiga toxin. In particular embodiments, the plant toxin is 65 In another embodiment, the present invention provides a selected from the group consisting of ricin, modeccin, abrin, method for treating cancer in a human Subject, the method Volkensin and Viscumin. In particular embodiments, the fun comprising administering to a human Subject in need thereof US 8,492,133 B2 5 6 a therapeutically effective amount of a nucleic acid construct a particular embodiment, the cancer is selected from the of the present invention, thereby treating cancer in the human group consisting of a sarcoma, a carcinoma, an adenocarci Subject. noma, a lymphoma, and a leukemia. In a particular embodi In another embodiment, the present invention provides a ment, the cancer is selected from the group consisting of method for inhibiting tumor progression in a human Subject, breast cancer, colon cancer, , cervi the method comprising administering to a human Subject in cal cancer, cholangiocarcinoma, endometrioid ovarian carci need thereof a therapeutically effective amount of a nucleic noma, esophageal cancer, glioblastoma, head and neck can acid construct of the present invention, thereby inhibiting cer, leukemia, lymphoma, lung cancer, multiple myeloma, tumor progression in the human Subject. pancreatic cancer, osteosarcoma, pituitary tumor, prostate In another embodiment, the present invention provides a 10 method for inhibiting tumor metastasis in a human Subject, cancer, stomach cancer, and uterine leiomyoma. In a particu the method comprising administering to a human Subject in lar embodiment, the cancer is selected from the group con need thereof a therapeutically effective amount of a nucleic sisting of breast cancer, colon cancer and hepatocellular car acid construct of the present invention, thereby inhibiting cinoma. tumor metastasis in the human Subject. 15 Other objects, features and advantages of the present In another embodiment, the present invention provides a invention will become clear from the following description method for reducing oralleviating a symptom associated with and drawings. a neoplastic disorder in a human Subject, the method com prising administering to a human Subject in need thereof a BRIEF DESCRIPTION OF THE DRAWINGS therapeutically effective amount of a nucleic acid construct of the present invention, thereby reducing or alleviating a symp FIG. 1 provides a schematic representation of the nucleic tom associated with a neoplastic disorder in the human Sub acid construct of the invention, in which a nucleic acid ject. sequence encoding an anti-cancer agent ("killer gene') is In the methods of the invention, said subject is afflicted, in operably linked to a miR-21 promoter sequence. one embodiment, with a cancer, tumor or a neoplastic disor 25 FIG. 2 demonstrates miR-21 promoter-driven expression der characterized by endogenous expression of miR-21 in at of a reporter luciferase gene in different cell lines. A vector least a portion of the cells thereof. containing the firefly luciferase gene (Luc) under the control In particular embodiments, the administering is carried out of the miR-21 promoter (miR-21-Luc, SEQID NO: 11: black by a route selected from the group consisting of injection, bars), and a control vector containing the firefly luciferase infusion and direct injection into the tumor. 30 gene under the control of the TATA box sequence of the In particular embodiments, the administering comprises miR-21 promoter (TATA-Luc, SEQID NO:20; white bars) administering a single dose or multiple doses of the nucleic were each transfected into different cell lines. The cell lines acid construct. tested included Chinese hamster ovary (CHO, right bars) and In particular embodiments, the methods further comprise a the human cell lines HCT 116 (left bars) and step of determining the level of miR-21 transcriptional activ 35 ity in a biological sample e.g. cells or tissue, from the Subject. COLO320 (middle bars). Y axis-relative luciferase activity. In particular embodiments, the methods are carried out in FIG. 3 demonstrates inhibition of Renilla luciferase conjunction with at least one additional cancer therapy expression in a model of reduction of de novo protein Syn modality. In particular embodiments, the additional cancer thesis induced by constructs encoding a fragment of diphthe therapy modality is selected from the group consisting of 40 ria toxin (DTA). Cells were co-transfected with a plasmid bone marrow transplant, cord blood cell transplant, Surgery, encoding Renilla luciferase under the control of an SV-40 chemotherapy, radiation therapy, immunotherapy and a com promoter (SEQ ID NO: 28) and either a plasmid encoding bination thereof. DTA under the control of the miR-21 promoter (miR-21 Pr In another aspect, the present invention provides use of a DTA: SEQID NO:9), or a plasmid encoding DT-A under the nucleic acid construct of the invention for the preparation of 45 control of the TATA-box sequence of the miR-21 promoter a medicament for treating cancer in a human Subject. (miR-21 TATA box DT-A (SEQ ID NO: 13). Cells were In another aspect, the present invention provides use of a harvested 72 hours post-transfection and subjected to nucleic acid construct of the invention for the preparation of luciferase assay. a medicament for inhibiting tumor progression in a human FIG. 4 demonstrates inhibition of expression of Renilla Subject. 50 luciferase or green fluorescent protein (GFP) in models of In another aspect, the present invention provides use of a reduction of de novo protein synthesis. nucleic acid construct of the invention for the preparation of FIG. 4A (white bars) shows the results of co-transfection a medicament for inhibiting tumor metastasis in a human experiments in which cells were co-transfected with miR-21 Subject. Pr DT-A (SEQ ID NO: 9) or miR-21 prPUMA (a vector In another aspect, the present invention provides use of a 55 encoding the p53 up-regulated modulator of apoptosis nucleic acid construct of the invention for the preparation of (PUMA) under the control of the miR-21 promoter: SEQID a medicament for reducing or alleviating a symptom associ NO: 10), together with a reporter plasmid encoding Renilla ated with a neoplastic disorder. luciferase. FIG. 4A (black bars) shows the results of co In another aspect, the present invention provides a kit con transfection experiments in control systems in which cells taining i) one or more dosage units of a nucleic acid construct 60 were co-transfected with miR-21 TATA box DTA (SEQ ID of the invention sufficient for one or more courses of treat NO: 13) or TATAPUMA (a vector encoding PUMA under the ment for a cancer, tumor or neoplasm expressing miR-21; and control of the TATA-box sequence of the miR-21 promoter; ii) instructions for administering said nucleic acid construct SEQID NO: 29), together with the plasmid encoding Renilla to a subject in need thereof. luciferase. The compositions, methods and kits of the present inven 65 FIG. 4B shows the results of co-transfection experiments tion are useful in the treatment of a variety of cancers and performed as for those depicted in FIG. 4A, except that a neoplastic disorders associated with expression of miR-21. In plasmid encoding GFP was used as the reporter plasmid. US 8,492,133 B2 7 8 Cells were harvested 72 hours post-transfection and sub to mature miR-21, including a primary transcript of miR-21 jected to luciferase assay (FIG. 4A) or Western blot analysis (pri-miR-21). Such as those having GenBank accession num (FIG. 4B). bers BC053563 and AY699265;an intermediate precursor of FIG. 5 demonstrates that miR-21 Pr DTA expression miR-21 (pre-miR-21). Such as those having GenBank acces inhibits tumor formation in vivo. Nude mice (n=3 per group) sion numbers NR 029493, AF 48.0546 and AF 480557: were injected with 1x107 HCT116 cells. Two weeks follow and mature miR-21. Such as that having GenBank accession ing tumor cell injection, mice were administered endotoxin number NR 029493. free miR-21 Pr DT-A (SEQID NO: 9) by intra-tumor injec As used herein, the term “nucleic acid sequence corre tion (dose of 25ug). An additional injection was administered sponding to or complementary to an miR-21 transcription or 7 days later. Tumor size was recorded at days 0, 7 and 14 10 processing product” refers to a DNA or RNA sequence that is following tumor cell injection using a manual caliper. Con either identical to i.e. “corresponds to the sequence of an trol-treated mice were administered miR-21-Luc (SEQ ID miR-21 transcription or processing product, or could demon NO: 11) by intra-tumor injection. strate complete base-pairing i.e. is "complementary to” to the miR-21 transcription or processing product. For example, a DETAILED DESCRIPTION OF THE INVENTION 15 subject DNA would “correspond to a cDNA produced from an miR-21 RNA transcript, provided that the sequences of the The present invention is directed to nucleic acid constructs subject DNA and the cDNA were identical, whereas the same and methods for providing targeted cancer therapy. Specifi subject DNA would be “complementary to the aforemen cally, the constructs of the invention comprise at least one tioned miR-21 transcript. nucleic acid sequence encoding a heterologous anti-cancer As used herein, the term “nucleic acid construct” refers to gene product, the nucleic acid sequence being operably a continuous polynucleotide molecule comprising at least linked to a miR-21 transcription-regulating sequence, in par two heterologous nucleic acid sequences that are joined ticular, an miR-21 promoter sequence. Vectors comprising together to form a single unit. The nucleic acid sequences these constructs, pharmaceutical compositions comprising comprising the construct may include polypeptide coding them and methods of use thereof are also provided. 25 sequences and non-coding sequences. The efficacy of the present invention is unexpected over The terms “polynucleotide'. “polynucleotide sequence'. prior art disclosures which teach suppression of miR-21 “nucleic acid”, “nucleic acid sequence' and "oligonucle driven processes for treatment of cancers in which miR-21 is otide' are used interchangeably herein to refer to polymeric highly expressed. Such as by administration of agents which forms of nucleotides of any length, Such as deoxyribonucle inhibit miR-21 activity, or use of miR-21 for inactivating 30 otides, ribonucleotides, or modified forms thereof in the form genes involved in oncogenesis. That is, the harnessing of the of an individual fragment or as a component of a larger miR-21 promoter for driving expression of tumor killing construct, in a single strand or in a double strand or multi agents, as taught by the present invention, provides a reversal strand form. The polynucleotides to be used in the invention of prior art approaches to therapy of miR-21-associated can include sense and antisense sequences of DNA or RNA, CS. 35 genomic DNA, cDNA, DNA-RNA hybrids, or a polymer Definitions comprising purine and pyrimidine bases or other natural, As used herein, the terms “microRNA and “miRNA chemically or biochemically modified, non-natural, or interchangeably refer to a single-stranded non-coding regu derivatized nucleotide bases. Further included are mRNA or latory RNA of approximately 22-25 nucleotides in length, cDNA that comprise intronic sequences The backbone of the generated by the action of RNase-III-type enzymes on an 40 polynucleotide can comprise Sugars and phosphate groups (as endogenous primary transcript (pri-miRNA). typically be found in RNA or DNA), or modified or substi As used herein, the terms “miRNA biogenesis”, “miRNA tuted Sugar orphosphate groups. Alternatively, the backbone pathway' and “miRNA processing pathway interchange of the polynucleotide can comprise a polymer of synthetic ably refer to the RNA metabolic process leading to miRNA Subunits such as phosphoramidites and thus can be an oli formation that includes transcription of the primary miRNA 45 godeoxynucleoside phosphoramidate or a mixed phosphora transcript (pri-miRNA), cleavage of pri-miRNA to create an midate-phosphodiester oligomer (see, e.g., Peyrottes et al. intermediate precursor miRNA (pre-miRNA) and subsequent (1996) Nucl. Acids Res. 24:1841-1848; Chaturvedi et al processing of pre-miRNA to create the mature miRNA. (1996) Nucl. Acids Res. 24:2318-2323). A polynucleotide As used herein, the term “miR-21’ refers to the mature may comprise modified nucleotides, such as methylated processed form of the miRNA having the sequence of SEQID 50 nucleotides and nucleotide analogs, uracyl, other Sugars, and NO: 5. linking groups such as fluororibose and thioate, and nucle As used herein, the term “miR-21 promoter refers to the otide branches. The sequence of nucleotides may be inter native nucleic acid sequence which is located upstream of an rupted by non-nucleotide components. A polynucleotide may miR-21 gene and is required for the transcription thereof, so be further modified after polymerization, such as by conju as to produce a primary transcript of miR-21 (pri-miR-21). 55 gation with a labeling component, capping, Substitution of Exemplary human pri-miR-21 sequences include: GenBank one or more of naturally occurring nucleotides with an ana accession number BC053563 (disclosed herein as SEQ ID log, and introduction of means for attaching the polynucle NO: 2); GenBank accession number AY.699265 (disclosed otide to proteins, metal ions, labeling components, other herein as SEQID NO:3), and that disclosed in Fujita et al., J polynucleotides, or a solid Support. Mol Biol 2008 May 2: 378(3):492-504 (disclosed herein as 60 A polynucleotide can comprise a nucleotide sequence dis SEQID NO: 26). As used herein, the term “miR-21 promoter closed herein wherein thymidine (T) can also be uracil (U); sequence” refers to a nucleic acid sequence derived from an this definition pertains to the differences between the chemi miR-21 promoter and having at least about 80% identity to cal structures of DNA and RNA, in particular the observation SEQID NO:1. that one of the four major bases in RNA is uracil (U) instead As used herein, the term “an miR-21 transcription or pro 65 of thymidine (T). cessing product” refers to a nucleic acid transcription or pro The term “recombinant nucleic acid construct” as used cessing product derived from an miR-21 gene in the pathway herein refers to a nucleic acid construct that has been sub US 8,492,133 B2 10 jected to molecular manipulation in vitro, Such as for example less cytotoxic to tumor cells compared to the parent drug and joining together nucleic acid sequences by ligation. is capable of being enzymatically activated or converted into As used herein, the term “operably linked refers to the the more active parent form. structural and functional relationship between two or more As used herein, the term "heterologous' when used in nucleic acid sequences joined together as part of the same reference to distinct nucleic acid sequences or distinct protein polynucleotide, generally a regulatory sequence and a protein sequences, means that the sequences differ with respect to coding sequence. For example, a nucleic acid sequence their sequence, structure and/or the organism from they are encoding a protein is operably linked to a promoter if tran derived or produced. Scription is initiated from the promoter, or a nucleic acid As used herein, the term “homolog” refers to a molecule, 10 generally a polypeptide or nucleic acid sequence, which sequence encoding a protein is operably linked to a ribosome exhibits homology to another molecule, by having sequences binding site if of the corresponding mRNA is of chemical residues that are identical or similar at corre initiated from the ribosome binding site. In the present inven sponding positions. tion, a nucleic acid encoding a protein having anti-cancer Sequence similarity and sequence identity are calculated activity is operably linked to an miR-21 transcription control 15 based on a reference sequence, which may be a Subset of a sequence, preferably an miR-21 promotersequence, Such that larger sequence, such as a conserved motif coding region, transcription of the nucleic acid is effected or enhanced. Gen flanking region, etc. A reference sequence will usually be at erally, “operably linked' means that the nucleic acid least about 18 nt long, more usually at least about 30 nt long, sequences being linked are contiguous, although enhancers and may extend to the complete sequence that is being com do not have to be contiguous. Linking is accomplished by pared. In general, percent sequence identity is calculated by ligation at convenient restriction sites, or if such sites do not counting the number of residue matches (e.g., nucleotide exist, synthetic oligonucleotide adaptors or linkers may be residue or amino acid residue) between the query and test used in accordance with conventional practice. sequence and dividing total number of matches by the number Transcription control sequences are sequences, which con of residues of the individual sequences found in the region of trol the initiation, elongation, and termination of transcrip 25 strongest alignment, or along a complete region following the tion. Particularly important transcription control sequences introduction of gaps, as is known in the art. Algorithms for are those which control transcription initiation, such as pro computer-based alignment and sequence analysis are known moter, enhancer, operator and repressor sequences. in the art (see, e.g., Altschul et al., (1990) J. Mol. Biol. As used herein, the term “vector” refers to a nucleic acid 215:403-10), and publicly available software tools such as construct, comprising a regulatory sequence linked to a het 30 BLAST, BLAST-2, ALIGN, ALIGN-2 or Megalign (DNAS erologous polynucleotide that is inserted to target cells for TAR), are well known in the art. replication and/or expression therein. The vector can be a Accordingly, "percent (%) nucleic acid sequence identity” viral expression vector, a plasmid or a construct of naked with respect to a miR-21 promoter nucleic acid sequence DNA, and, optionally, can include additional sequences identified herein is defined as the percentage of nucleotides in required for construction, selection, stability, penetration, etc. 35 a candidate sequence that are identical with a disclosed miR As used herein, the terms “protein’ and “polypeptide' refer 21 promoter nucleic acid sequence, after aligning the interchangeably to a polymer of at least about 10amino acids sequences and introducing gaps, if necessary, to achieve the joined together through peptide bonds. Throughout the speci maximum percent sequence identity. Alignment for purposes fication, standard three letter or single letter designations for of determining percent nucleic acid sequence identity can be amino acids are used. 40 achieved using the aforementioned computer Software tools. As used herein, the terms “recombinant protein’ and As used herein, the term “variant” refers to substantially “recombinant polypeptide' refer interchangeably to a protein similar sequences possessing common qualitative biological produced using cells that do not have, in their native state, an activities. An oligonucleotide variant includes a pharmaceu endogenous copy of the nucleic acid (DNA or RNA) capable tically acceptable salt, homolog, analog, extension or frag of expressing the protein. The cells produce the recombinant 45 ment of a nucleotide sequence useful for the invention, Such protein because they have been genetically altered by the as an miR-21 promoter sequence. Encompassed within the introduction of the appropriate isolated nucleic acid term “variant” are chemically modified natural and synthetic sequence, for example as a component of an expression vec nucleotide molecules. Also encompassed within the term tOr. “variant” are substitutions (conservative or non-conserva As used herein, the term “fusion protein’ as used herein 50 tive), additions or deletions within the nucleotide sequence of refers to a protein sequence composed of segments i.e. amino the molecule, as long as the required function is sufficiently acid sequences, which correspond to heterologous protein maintained. Oligonucleotide and polynucleotides variants sequences. Such as proteins of different function and/or pro may share at least 50%, 55%, 60%. 65%, 70%, 75%, 80%, teins from different organisms. The segments are joined 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity. either directly or indirectly to each other via peptide bonds, 55 As used herein in the context of polynucleotides, the terms and may correspond to either full-length proteins or frag “hybridize”, “hybridizing, “hybridizes” and the like, refer to ments thereof. By indirect joining it is meant that an inter conventional hybridization conditions. vening amino acid sequence, such as a peptide linker is jux “Stringency' of hybridization reactions is readily deter taposed between segments forming the fusion protein. Fusion minable by one of ordinary skill in the art, and generally is an proteins may be produced by recombinant technology from a 60 empirical calculation dependent upon probe length, washing polynucleotide comprising the nucleic acid sequences encod temperature, and salt concentration. In general, longer probes ing the various segments of the fusion protein. require higher temperatures for proper annealing, while As used herein, the term “drug-metabolizing enzyme’ shorter probes need lower temperatures. Hybridization gen refers to an enzyme which converts a prodrug into a cytotoxic erally depends on the ability of denatured DNA to reanneal product. 65 when complementary strands are present in an environment As used herein, the term “prodrug” refers to a precursor or below their melting temperature. The higher the degree of derivative form of a pharmaceutically active substance that is desired homology between the probe and hybridizable US 8,492,133 B2 11 12 sequence, the higher the relative temperature which can be The term "sarcoma is recognized by those skilled in the art used. As a result, it follows that higher relative temperatures and refers to malignant tumors of connective tissue such as would tend to make the reaction conditions more stringent, bone or cartilage. while lower temperatures less so. For additional details and As used herein, the term “leukemia' and “leukemic can explanation of stringency of hybridization reactions, see cer” refer to all cancers of the hematopoietic and immune Ausubel et al., Current Protocols in Molecular Biology, John systems (blood and lymphatic system). These terms refer to a Wiley and Sons, Baltimore, Md., 1988. progressive, malignant disease of the blood-forming organs, "Stringent conditions' or “high Stringency conditions', as marked by distorted proliferation and development of leuko defined herein, may be identified by those that: (1) employ cytes and their precursors in the blood and bone marrow. low ionic strength and high temperature for washing, for 10 Myelomas refer to other types of tumors of the blood, bone example 0.015M sodium chloride/0.0015M sodium citrate/ marrow cells. Lymphomas refer to tumors of the lymph tis 0.1% sodium dodecyl sulfate at 50° C.; (2) employ during SC. hybridization a denaturing agent, such as formamide, for As used herein, the term “pharmaceutical composition' example, 50% (v/v) formamide with 0.1% bovine serum refers to a preparation comprising one or more pharmaceuti albumin/0.1% Ficoll/0.1% polyvinylpyrrollidone/50 mM 15 cally active ingredients e.g. a nucleic acid construct encoding sodium phosphate buffer at pH 6.5 with 750 mM sodium an anti-cancer agent, and generally further comprising at least chloride, 75 mM sodium citrate at 42°C.; or (3) employ 50% one pharmaceutically acceptable diluent, carrier or excipient. formamide, 5xSSC (0.75 M NaCl, 0.075 M sodium citrate), The purpose of a pharmaceutical composition is to facilitate 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophos administration of a pharmaceutically active ingredient to a phate, 5xDenhardt’s solution, sonicated salmon sperm DNA Subject. (50 g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C., with As used herein, the term “active ingredient” refers to a washes at 42°C. in 0.2xSSC (sodium chloride/sodium cit component of a pharmaceutical composition that provides the rate) and 50% formamide at 55° C., followed by a high primary pharmaceutical benefit, as opposed to an “inactive stringency wash consisting of 0.1 xSSC containing EDTA at ingredient' which is generally recognized as providing no 550 C. 25 pharmaceutical benefit. As used herein, the term "isolated in reference to a nucleic As used herein, the term “carrier, excipient or diluent acid molecule means that the nucleic acid molecule is initially refers to an inactive ingredient, for example atonicity adjust separated from different (in terms of sequence or structure) ing agent, wetting agent or preservative, which facilitates and unwanted nucleic acid molecules Such that a population formulation and/or administration of an active pharmaceuti of isolated nucleic acids is at least about 90% homogenous, 30 cal ingredient. and may be at least about 95, 96, 97.98.99, or 100% homog As used herein, the term “pharmaceutically acceptable' enous with respect to other polynucleotide molecules. In refers to a non-toxic and inert substance that is physiologi many embodiments, a nucleic acid is isolated by virtue of it cally compatible with humans or other mammals. having been synthesized in vitro separate from endogenous As used herein, the term “subject” refers to any mammal of nucleic acids in a cell. It will be understood, however, that 35 any age having a cancer, in particular a human Subject, but isolated nucleic acids may be Subsequently mixed or pooled also including non-human mammals, such as primate, canine, together. feline, bovine, equine and murine species. As used herein, the term “anti-cancer agent” refers to a The term “antibody' (also referred to as an “immunoglo protein or peptide that exerts an inhibitory or cytotoxic effect bulin') is used in the broadest sense, and refers to polypep on a cancer cell, either directly or indirectly. For example, a 40 tides which exhibit binding specificity to a specific antigen. cytotoxic drug that kills a cancer cell exerts a direct effect on The term encompasses polyclonal and monoclonal antibod the cancer cell, while an enzyme which converts a prodrug to ies, including both full length antibodies and antibody frag its active cytotoxic form which then kills the cancer cell, ments so long as they exhibit the desired biological activity. exerts an indirect effect. The terms “native' and “full length' antibodies' as used The terms “cancer and “neoplasm' are used herein inter 45 herein interchangeably refer to heterotetrameric glycopro changeably to refer to a disease state characterized by cells in teins of about 150,000 daltons, composed of two identical an abnormal state or condition characterized by rapid prolif light (L) chains and two identical heavy (H) chains. Each light eration. A “tumor containing Such cells may be either chain is linked to a heavy chain by covalent disulfide bond(s), benign, premalignant or malignant. The terms include disease while the number of disulfide linkages varies between the states characterized by all types of hyperproliferative growth, 50 heavy chains of different immunoglobulin isotypes. Each hyperplastic growth, cancerous growths, oncogenic pro heavy and light chain also has regularly spaced intrachain cesses, metastatic tissues, malignantly transformed cells, tis disulfide bridges. Each heavy chain has, at one end, a variable Sues, or organs, irrespective of histopathologic type or stage domain (V) followed by a number of constant domains. of invasiveness. Each light chain has a variable domain at one end (V) and a The term “carcinoma is recognized by those skilled in the 55 constant domain at its other end; the constant domain of the art and refers to malignancies of epithelial or endocrine tis light chain is aligned with the first constant domain of the Sues including respiratory system carcinomas, gastrointesti heavy chain, and the light chain variable domain is aligned nal system carcinomas, genitourinary system carcinomas, with the variable domain of the heavy chain. testicular carcinomas, breast carcinomas, prostatic carcino Depending on the amino acid sequence of the constant mas, endocrine system carcinomas, and melanomas. Exem 60 domain of their heavy chains, antibodies can be assigned to plary carcinomas include those forming from tissue of the different classes. There are five major classes of antibodies cervix, lung, prostate, breast, head and neck, colon and ovary. IgA, Ig|D, IgE, IgG, and IgM, and several of these may be The term also includes carcinosarcomas, e.g., which include further divided into Subclasses (isotypes), e.g., IgG1, IgG2. malignant tumors composed of carcinomatous and sarcoma IgG3, IgG4. tous tissues. An "adenocarcinoma’ refers to a carcinoma 65 Antibody fragments’ comprise a portion of a full length derived from glandular tissue or in which the tumor cells form antibody, generally the antigen binding or variable region recognizable glandular structures. thereof. Examples of antibody fragments include Fab, Fab', US 8,492,133 B2 13 14 F(ab'), and Fv fragments: diabodies; linear antibodies: nemestrina); AY865962 (from Pongo pygmaeus); AY865961 single-chain antibody molecules; and multispecific antibod (from Gorilla gorilla); AY865960 (from Pan troglodytes), and ies formed from antibody fragments. AY865959 (from Macaca mulata). The terms “polyclonal antibody' and “polyclonal antise The maturation of miRNA from pri-miRNAs involves rum' as used herein refer to a population of antibody mol trimming of pri-miRNAs into hairpin intermediates called ecules synthesized by a population of B cells. Polyclonal precursor miRNAs (pre-miRNAs), that are subsequently antibodies encompass a population of different antibody mol cleaved into mature miRNAs. The stem-loop structure of ecules which specifically bind to aparticular antigen, wherein pri-miRNA molecules are cleaved by the nuclear RNase III various antibodies in the mixture recognize different epitopes enzyme Drosha to release the pre-miRNA molecules. Drosha (also termed antigenic determinants). 10 is a large protein of approximately 160 kDa, and, in humans, The term “monoclonal antibody” as used herein refers to forms an even larger complex of approximately 650 kDa an antibody obtained from a population of Substantially known as the Microprocessor complex. The enzyme is a Class homogeneous antibodies, i.e., the individual antibodies com II RNAse III enzyme having a double-stranded RNA binding prising the population are identical except for possible natu domain (dsRBD). rally occurring mutations that may be present in minor 15 Human sequences of pre-miR-21 include those available amounts. Monoclonal antibodies are highly specific, being under GenBank accession numbers NR 29493: AF48.0546, directed against a single antigenic site. Furthermore, in con and AF480557. Highly homologous non-human pre-miR-21 trast to polyclonal antibody preparations, each monoclonal sequences include those available under GenBank accession antibody is directed against a single epitope on the antigen. numbers NR 032094 (Equus cabalus): NR 030880 (Bos The modifier "monoclonal indicates the character of the taurus): NR 029738 (Mus musculus); and NR 032151 antibody as being obtained from a Substantially homoge (Monodelphis domestica). neous population of antibodies, and is not to be construed as Following export of pre-miRNA molecules to the cyto requiring production of the antibody by any particular plasm, another RNase III enzyme called “Dicer cleaves the method. For example, monoclonal antibodies may be made pre-miRNA to produce the mature miRNA. Mature miRNAs by the hybridoma method first described by Kohler et al., 25 are incorporated into an effector complex known as the Nature 256:495 (1975), or may be made by recombinant miRNA-containing RNA-induced silencing complex or DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The miRISC. "monoclonal antibodies' may also be isolated from phage Mature human miR-21 is available under GenBank acces antibody libraries, as is known in the art, for example using sion number NR 29493 and is disclosed herein as SEQ ID techniques such as those described in Clackson et al., Nature 30 NO:5. Identical or nearly identical non-human miR-21 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581 sequences include those available under GenBank accession 597 (1991). numbers NR 032094 (Equus cabalus): NR 030880 (Bos The terms “specifically interacts”, “specifically binds” and taurus): NR 029738 (Mus musculus); and NR 032151 “having specificity for are used herein interchangeably to (Monodelphis domestica). refer to high avidity and/or high affinity binding of an anti 35 Additional information concerning miRNAS and associ body to an antigen or epitope thereof, e.g., an epitope on a ated pri-miRNA and pre-miRNA sequences is available in cancer cell. Antibody binding to its epitope is stronger than miRNA databases such as miRBase (Griffiths-Jones et al. binding of the same antibody to any other epitope. Antibodies 2008 Nucl Acids Res 36, (Database Issue:D154-D158) and which bind specifically to a polypeptide of interest may be the NCBI database. capable of binding other polypeptides at a weak, yet detect 40 It is to be noted that the sequences referred to and disclosed able, level (e.g., 10% or less of the binding shown to the herein are expressed with DNA thymine bases (T) rather than polypeptide of interest). Such weak binding, or background the corresponding RNAuracil (U) bases, even in cases where binding, is readily discernible from the specific antibody the native sequences are RNA, such as for example mature binding to the compound or polypeptide of interest, e.g., by miR-21. use of appropriate controls. 45 Nucleic Acid Constructs, Vectors and Host Cells The singular forms “a”, “an', and “the include plural The nucleic acid construct of the invention comprises a referents unless the context clearly dictates otherwise. Thus, nucleic acid sequence encoding an anti-cancer agent operably for example, reference to “a construct” includes one or more linked to an miR-21 promoter sequence. of Such constructs, and so forth. The miR-21 promoter sequence corresponds to a region miR-21 Processing Pathway 50 positioned upstream of an miR-21 gene that functions in Transcription of miRNA genes, including that of miR-21. transcription of the corresponding pri-miR-21 transcript. The is mediated by RNA polymerase II (pol II) to produce long miR-21 promoter sequence used for the present invention primary transcripts (pri-miRNAs) that are often several kilo may be the native sequence or a fragment thereof, as long as bases long. Pri-miRNA transcripts contain both a 5' terminal it retains the capability to drive transcription of the nucleic cap structure and a 3' terminal poly(A)tail. Several poly(A)- 55 acid sequence to which it is operably linked. containing transcripts containing both miRNA sequences and The miR-21 promoter for use in the invention may be that regions of adjacent mRNAs have been characterized. A which drives transcription of any of the known forms of human pri-miR-21, 3463 nucleotides in length, is disclosed pri-miR-21. Such as the pri-miR-21 transcripts disclosed under GenBank accession number AY.699265, and a highly under GenBank accession numbers AY699265 and similar sequence from a human cDNA clone, 3404 nucle 60 BC053563 and in Fujita et al., J Mol Biol 2008 May 2: otides in length, is disclosed under GenBank accession num 378(3):492-504. Transcripts of miR-21 may be characterized ber BC053563. A human pri-miR-21 is disclosed by Fujita et by methods known in the art, for example rapid amplification al., J Mol Biol 2008 May 2: 378(3):492-504. of cDNA ends (RACE), polymerase chain reaction (PCR) and Homologous miR-21 genes of non-human species are also primer extension, as described for example in Caiet al., RNA known, including for example those available under Gen 65 (2004) 10:1957-1966. Bank accession numbers AY865965 (from Pan paniscus); Putative promoter sequences located upstream i.e. 5' of AY865964 (from Ateles geoffroyi); AY865963 (from Macaca known or identified pri-miR-21 transcription units may be US 8,492,133 B2 15 16 PCR amplified from genomic DNA using appropriate prim In a particular embodiment, the nucleic acid construct ers, and isolated and purified using standard methods known comprises a sequence selected from the group consisting of in the art. Such purified fragments may then be ligated into SEQID NO: 7 and SEQID NO: 8. vectors comprising heterologous nucleic acid sequences The constructs, vectors and nucleic acid sequences of the encoding reporter proteins, for example firefly luciferase. 5 present invention may be produced using standard recombi Such vectors are then transfected into cells and analyzed for nant, enzymatic and chemical synthetic methods well known the presence of the reporter protein, thereby indicating in the art. A combination of such techniques may be used. An whether the test sequence functions as an mRNA promoter isolated nucleic acid sequence can be obtained from its natu capable of driving expression of the reporter sequence. In ral Source, either as an entire (i.e., complete) gene or a portion 10 thereof. The desired nucleic acid molecule may be produced parallel systems, one or more shorter subfragments may be by polymerase chain reaction (PCR) amplification from a separately cloned into the same vector and evaluated for genomic template using suitable primer sequences. The reporter sequence expression, so as to identify the minimal amplified fragment may be manipulated by addition of Syn region with promoteractivity. A short Subfragment, such as a thetic restriction endonuclease sites, linkers etc for cloning putative TATA box, as well as antisense orientations of the 15 into a particular vector enabling propagation and/or further various test sequences, may be similarly processed to serve as manipulation and/or expression of a particular nucleic acid negative controls. sequence. Such procedures enable construction of a single Exemplary experiments of this type are disclosed in molecule e.g. construct or vector, characterized by the juxta Example 2 herein, which shows that an miR-21 promoter position of specific heterologous nucleic acid sequences and sequence of SEQID NO: 1 drives expression of a luciferase 20 including Suitable flanking sequences which enable the reporter gene, while a short fragment of the miR-21 promoter desired functionalities of the construct. In addition, at least corresponding only to the TATA box (SEQ ID NO: 6), is Some of the desired nucleic acid molecules may be produced incapable of driving expression of the same reporter by assembly of chemically synthesized. oligonucleotides (see Sequence. e.g. Sambrook et al., 2001, Molecular Cloning: A Laboratory Accordingly, an exemplary miR-21 promoter sequence for 25 Manual; Ausubel et al., Supra). For example, a nucleic acid use in the invention is SEQ ID NO: 1. SEQID NO 1 corre sequence produced by oligonucleotide assembly may be sponds to nucleotides -1326 to -608 relative to the pri-mir-21 ligated to a heterologous sequence that was produced by PCR T1 transcription start site disclosed in Fujita et al., J Mol Biol amplification, and the resultant molecule may be manipulated 2008 May 2: 378(3):492-504. In other embodiments, the by addition of synthetic restriction endonuclease sites, linkers 30 etc for cloning into a particular expression vector, as is well miR-21 promoter sequence has at least 80% identity with known in the art. SEQID NO: 1, such as at least 85%, or at least 90% identity, Nucleic acid sequences useful for the invention include or at least 95% identity with SEQ ID NO: 1. In particular natural nucleic acid sequences and homologs thereof, includ embodiments, the miR-21 promoter sequence comprises ing, but not limited to, natural allelic variants and modified SEQID NO: 1. 35 nucleic acid sequences in which nucleotides have been In particular embodiments, the miR-21 promoter sequence inserted, deleted, Substituted, and/or inverted in Such a man comprises recognition sequences typically present in eukary ner that such modifications do not substantially interfere with otic promoters, in particular, the TATA box. Other promoter the intended function of the nucleic acid such as encoding an elements which may be present in the miR-21 promoter anti-cancer agent or acting as a promoter for transcription sequence include initiator, CCAAT box and GC box sites. 40 thereof. The TATA box, typically located 25-30 base pairs upstream of Any of the miR-21 promoter sequences used for the inven the transcription initiation site, is thought to be involved in tion can be altered by additions, substitutions or deletions and directing RNA polymerase to begin RNA synthesis. The assayed for the level of expression of sequences operably other upstream promoter elements determine the rate at linked thereto, in cancer cells. Similarly, sequences encoding which transcription is initiated. Preferably, the miR-21 pro- 45 anti-cancer agents may be altered in order to manipulate any moter sequence utilized in the nucleic acid construct of the of a number of properties, such as for example the level of present invention is active in the specific target cell popula their toxicity, targeting capacity, in vivo stability, etc. tion. In the constructs of the invention, it may be preferable to Sequence alterations can be generated using a variety of position the miR-21 promoter sequence relative to the chemical and enzymatic methods known to those skilled in sequence encoding the anti-cancer agent, such that the TATA 50 the art. For example, regions of the sequences defined by box is the same distance from the transcription initiation site restriction sites can be deleted. Oligonucleotide-directed as occurs in the native gene encoding the anti-cancer agent. mutagenesis can be employed to alter the sequence in a The nucleic acid sequence comprising an miR-21 promoter defined way and/or to introduce restriction sites in specific sequence may, in some embodiments, be substantially devoid regions within the sequence. Additionally, deletion mutants of a nucleic acid sequence corresponding to or complemen- 55 can be generated using DNA nucleases such as Bal31 or tary to an miR-21 transcription or processing product. fur ExoII and S1 nuclease. Progressively larger deletions in the thermore, the nucleic acid construct may be substantially regulatory sequences are generated by incubating the DNA devoid of a nucleic acid sequence corresponding to or with nucleases for longer periods of time (See, e.g., Ausubel complementary to a precursor or intermediate form of miR et al., Supra for a review of mutagenesis techniques). 21, Such as a primary transcript of miR-21 (pri-miR-21); a 60 Sequence diversity can be introduced by a variety of precursor of miR-21 (pre-miR-21); an RNA duplex of miR mutagenesis schemes known in the art. Random, non-specific 21, and a mature miR-21. In particular embodiments, the methods of introducing mutations include classical in vivo nucleic acid construct of the invention is substantially devoid mutagenesis techniques. Such as exposure of entire organisms of a nucleic acid sequence corresponding to or complemen to radiation or chemical mutagens, or use of transposons. tary to a nucleic acid sequence selected from the group con- 65 More localized mutations in microorganisms in vivo can be sisting of SEQID NO: 2: SEQID NO:3: SEQID NO: 4; and performed by using mutator Strains of bacteria, transducing SEQID NO: 5. phages, episomes, or homologous recombination. US 8,492,133 B2 17 18 Mutations can be induced in vitro by many other methods meret al., 1995, Gene 164:49-53). The method relies on DNA in addition to homologous recombination and random polymerase, rather than DNA ligase, to build increasingly mutagenesis, which include deletion analysis, linker muta longer DNA fragments during the assembly process (see e.g., tions, reporter gene fusion, restriction/ligation assembly, oli U.S. Pat. Nos. 5,605,793 and 5,830,696). gonucleotide-directed mutagenesis, and cassette mutagen- 5 In sexual PCR, also called DNA shuffling, related but not esis. PCR-based methods that can introduce sequence identical DNA sequences are randomly fragmented, after diversity are also available such as error-prone PCR and which the fragments are reassembled by assembly PCR under assembly PCR (also known as parallel PCR). Furthermore, conditions that permit homologous recombination (Stemmer, various combinations of the above techniques have led to 1994, Nature 370:389 391). Repeated cycles of point specialized methods which include sexual PCR (also known 10 mutagenesis, recombination, and selection produce in vitro as DNA shuffling), recursive ensemble mutagenesis and molecular evolution. This process is repeated for as many exponential ensemble mutagenesis. cycles as necessary to obtain a desired property or function Homologous recombination occurs naturally in living (Stemmer, 1994, Proc Natl Acad. Sci. 91:10747 10751). cells. In eucaryotes, homologous recombination occurs at Recursive Ensemble Mutagenesis (“REM) is a protein meiosis, and is thought to be one of the inherent evolutionary 15 engineering method that employs multiple cycles of cassette mechanisms. A number of patents describe the use of in vivo mutagenesis to identify "optimal' amino acids at targeted homologous recombination to manipulate DNA sequences positions in a given protein. REM uses information gained (see, e.g., U.S. Pat. Nos. 5,093,257; 5,413,923; 5,521,077; from previous iterations of combinatorial cassette mutagen 5,202,238; 5,763,240; and 6,015,708). esis to search sequence space more efficiently. Through mul Means of performing random, or localized random, in vitro 20 tiple rounds of optimized point mutation and recombination, mutagenesis are essentially similar to those that can be used rapid evolution of DNA sequences is achieved (Arkin and for in vivo mutagenesis, e.g., irradiation, chemical mutagens, Youvan, 1992, Proc Natl Acad. Sci. 89:781 17815; Delagrave transposon mutagenesis. Aside from random mutagenesis et al., 1993, Protein Engineering 6:32733 1). methods, any of various techniques by which specific muta In exponential ensemble mutagenesis, several nucleotides tions can be made in vitro can be considered to be a form of 25 are randomized in parallel to identify amino acids, at each site-directed mutagenesis (Kendrew, J., 1994, in The Ency altered position, that lead to functional proteins. The method clopedia of Molecular Biology, Blackwell Science Inc., Lon thereby generates combinatorial libraries with a high percent don). Non-PCR-based in vitro approaches to site-directed age of optimized proteins. Exponential ensemble mutagen mutagenesis can be grouped generally into the categories of esis can be advantageous when it is desirable to change many oligonucleotide-directed mutagenesis, methods that restruc- 30 residues simultaneously. With the greater frequency of func ture fragments of DNA (e.g., cassette mutagenesis, gene tional mutants which is obtained by this method, entire pro assembly), and localized random mutagenesis (Botstein and teins can be mutagenized combinatorially (Delagrave and Shortle, 1985, Science 229, 1193 1201). Youvan, 1993, Biotechnology 11:1548 1552; U.S. Pat. No. Oligonucleotide-directed mutagenesis is based on the prin 5,521,077). ciple that an oligonucleotide encoding the desired mutation 35 The altered sequences created by use of any technique (s) is annealed to one strand of the DNA of interest and serves known in the art to introduce sequence diversity, including as a primer for initiation of DNA synthesis to produce a strand those disclosed herein, are evaluated for their ability to direct containing the mutation. Various forms of the basic tech expression of heterologous polynucleotides in appropriate nique, including single or multiple Substitutions, insertions or host cells, particularly miR-21-expressing cancer derived deletions, are described by Kramer et al., 1982, Nucleic Acids 40 cells e.g., pancreatic carcinoma cells. It is within the scope of Res. 10:64756485; Zoller and Smith, 1982, Nucleic Acids the present invention to use any altered miR-21 promoter Res. 10:6487 6500; Smith et al., 1982, “Site-Directed sequence that can direct tumor-specific expression to create a Mutagenesis'. Trends in Biochem. Sci., 7:440442; and Nor recombinant expression vector. ris et al., 1983, Nucleic Acids Res. 11:5103 5112. Synthetic methods suitable for preparing nucleic acid mol Cassette mutagenesis is characterized by replacement, 45 ecules include for example, assembly of oligonucleotides typically by restriction/ligation, of a portion of the endog (see e.g. U.S. Pat. No. 5,583,013), or in vitro chemical syn enous gene with a "cassette', which often is a synthetic oli thesis using phosphotriester, phosphite or phosphoramidite gonucleotide (see, e.g., Estell et al., 1985, J. Biol. Chem. chemistry and Solid phase techniques such as those described 260(11):6518 6521; Wells et al., 1985, Gene 34:315 323; in EP 266.032, or via deoxynucleoside H-phosphonate inter Beck von Bodman et al., 1986, Proc Natl Acad. Sci. 83:9443 50 mediates (see e.g. U.S. Pat. No. 5,705,629). In the present 9447: Reidhaar-Olson and Sauer, 1988, Science 241:53 57; invention, one or more oligonucleotides may be used to pre U.S. Pat. Nos. 4,894,331; 5,155,033; and 5,182,204). pare the various nucleic acid molecules. Various different Gene assembly entails the use of oligonucleotides, which mechanisms of oligonucleotide synthesis have been dis can be a mixture of synthetic oligomers and fragmented closed in for example, U.S. Pat. Nos. 4,659,774; 4.816,571; native sequences. Stochastic polymerization of the oligo- 55 5,141,813: 5,264,566; 4,959,463; 5,428,148; 5,554,744: nucleotide pool by treatment with a DNA ligase results in the 5,574,146, and 5,602,244. assembly of novel polynucleotide sequences (see, e.g. U.S. The nucleotide sequence encoding an anti-cancer agent Pat. No. 5,723,323). can be selected from those encoding a wide variety of proteins Error-prone PCR is a means of randomly introducing sev Such as, but not limited to, genes encoding toxic gene prod eral point mutations in a PCR product as a result of using a 60 ucts, cytostatic gene products, gene products essential for DNA polymerase that demonstrates low fidelity (see, e.g., viral replication, genes encoding drug metabolizing enzymes Leung et al., 1989, Technique 1:11 15, 1989; U.S. Pat. No. and genes encoding inducers of apoptosis, as further 5,223,408). described below. Assembly PCR describes a process whereby, using a pool The constructs of the invention may further comprise addi of oligonucleotides, many different PCR reactions occur in 65 tional heterologous protein-encoding sequences in addition parallel in the same reaction mixture, with the products of one to those encoding the anti-cancer agent. These sequences may PCR reaction priming the products of another reaction (Stem encode markers useful for selection ordetection of cells trans US 8,492,133 B2 19 20 formed with the desired constructs in the course of cloning, integrate into the mammalian for long-term sta propagation, manipulation, cell transfer, etc., such as, but not bility using a linked selectable marker gene. Alternatively, limited to, an enzyme oran antigenic marker. However, it is to derivatives of viruses such as the bovine papillomavirus be explicitly understood that the present invention specifi (BPV-1) or Epstein-Barr virus can be used for transient cally excludes constructs comprising an miR-21 promoter expression sequence operably linked to a nucleic acid sequence encoding The term “expression vector” refers to a vector containing a reporter protein, Such as luciferase, as are known in the art. a nucleic acid sequence coding for at least part of a gene Although the present invention relates to cell-specific product capable of being transcribed. Expression vectors can expression of heterologous polynucleotides to target cancer contain a variety of “control sequences.” which refer to cells, greater specificity of treatment can be achieved by using 10 nucleic acid sequences necessary for the transcription and cancer-specific delivery of the vectors of the invention. For translation of an operably linked coding sequence in a par example, antibodies that recognize cell Surface antigens ticular host organism. In addition to control sequences that unique to cancer cells, or that are more prevalent on cancer govern transcription and translation, vectors and expression cells, compared to normal cells are known in the art, and can vectors may contain nucleic acid sequences that serve other be used together with a vector of the invention to specifically 15 functions as well. target and kill tumor cells (See, e.g., Dillman, “Antibody Control sequences typically found in expression vectors Therapy: Principles of Cancer Oldham (ed.), Raven Press, include transcription and translation initiation sequences, Ltd., New York, 1987). Accordingly, constructs according to transcription and translation terminators, and a polyadenyla the invention may include nucleic acid sequences encoding tion signal. antibodies orantigenbinding fragments thereofthat can serve Enhancer elements can augment transcription up to 1,000 to target the anti-cancer agent to the target cancer cell. In fold from linked homologous or heterologous promoters. particular embodiments, the construct encodes a fusion pro Enhancers are active when placed downstream or upstream tein, i.e. a single protein comprising heterologous protein from the transcription initiation site. Many enhancer elements segments, in which one segment is the anti-cancer agent, and derived from viruses have a broad host range and are active in the other segment is an antibody or antibody fragment which 25 a variety of tissues. For example, the SV40 early gene is specific for a cancer-specific marker or a cancer-related enhancer is suitable for many cell types. Other enhancer/ protein. promoter combinations that are suitable for the present inven The nucleic acid constructs of the invention may be com tion include those derived from polyoma virus, human or prised in a vector, in particulara eukaryotic expression vector. murine cytomegalovirus (CMV), the long term repeat from In particular embodiments, a vector useful for the invention 30 various retroviruses Such as murine leukemia virus, murine or may have the nucleic acid sequence of SEQID NO:9 or SEQ Rous sarcoma virus and HIV. See, Enhancers and Eukaryotic ID NO: 10. Expression, Cold Spring Harbor Press, Cold Spring Harbor, The term “vector” is used to refer to a carrier nucleic acid N.Y. 1983. molecule into which a nucleic acid sequence or construct can In the construction of the expression vector, the promoteris be inserted for introduction into a cell where it can be repli 35 preferably positioned approximately the same distance from cated. A nucleic acid sequence can be “exogenous,” which the heterologous transcription start site as it is from the tran means that it is foreign to the cell into which the vector is Scription start site in its natural setting. As is known in the art, being introduced or that the sequence is homologous to a however, some variation in this distance can be accommo sequence in the cell but in a position within the host cell dated without loss of promoter function. nucleic acid in which the sequence is ordinarily not found. 40 Polyadenylation sequences can also be added to the expres Vectors include plasmids, cosmids, viruses (bacteriophage, sion vector in order to increase RNA stability. Two distinct animal viruses, and plant viruses), and artificial chromo sequence elements are required for accurate and efficient Somes (e.g.,YACs). The vectors of the present invention may polyadenylation: GU or U rich sequences located down include sequences which render the vector suitable for repli stream from the polyadenylation site and a highly conserved cation and integration in a particular type of host cells, for 45 sequence of six nucleotides, AAUAAA, located 11-30 nucle example, prokaryotes or eukaryotes, or they may be capable otides upstream. Exemplary termination and polyadenylation of propagation in more than one type of host e.g. shuttle signals that are suitable for the present invention include vectors which may be propagated in both prokaryotes and those derived from SV40. eukaryotes. It is to be understood that while the miR-21 promoter Vectors suitable for cultivation of the subject polynucle 50 sequence according to the invention may comprise at least otides in bacterial cells (e.g., E. coli), include, but are not Some of the aforementioned elements, the construct or vector limited to, plasmids of the types: p3R322-derived plasmids, which comprises the miR-21 promotersequence may contain pEMBL-derived plasmids, pEX-derived plasmids, pBTac additional types of such elements. In addition to the elements derived plasmids, and puC-derived plasmids. For replication already described, the expression vector of the present inven in yeast, cloning and expression vehicles useful for introduc 55 tion may typically contain other specialized elements ing genetic constructs in S. cerevisiae include, but are not intended to increase the level of expression of cloned nucleic limited to, the YEP24, YIP5, YEP51, pYES2 and YRP17 acids or to facilitate the identification of cells that carry the plasmids (See, e.g., Broach et al., 1993, in Experimental recombinant DNA. For example, a number of animal viruses Manipulation of Gene Expression, ed. M. Inouye, Academic contain DNA sequences that promote the extra chromosomal Press, p. 83). These vectors can replicate in E. coli due to the 60 replication of the viral genome in permissive cell types. Plas presence of the pBR322 ori, and in yeast due to the replication mids bearing these viral replicons are replicated episomally determinant of the yeast 2 um circle plasmid. In addition, as long as the appropriate factors are provided by genes either drug-resistant markers such as amplicillin can be used. carried on the plasmid or with the genome of the host cell. Similarly, mammalian vectors for the polynucleotides of In certain embodiments of the invention, the expression the invention may comprise prokaryotic sequences to facili 65 vector comprises a virus or an engineered vector derived from tate the propagation of the vector in bacteria. Such vectors, a viral genome. The ability of certain viruses to enter cells via when transfected into mammalian cells, can be designed to receptor-mediated endocytosis, to integrate into host cell US 8,492,133 B2 21 22 genome and express viral genes stably and efficiently have ods of the invention may have all or part of the viral E1 and E3 made them attractive candidates for the transfer of foreign genes deleted, but retains as much as 80% of the adenoviral genes into mammalian cells (see e.g. Nicolas and Rubenstein, genetic material (see, e.g., Jones et al., 1979, Cell 16:683; Biotechnology. 1988: 10:493-513; Baichwal and Sugden, Berkner et al., supra; Graham et al. in Methods in Molecular Oncogene. 1988 May; 2(5):461-7: Gélinas and Temin Proc Biology, Vol. 7, E. J. Murray (ed.), Humana, Clifton N.J. Natl AcadSci USA. 1986 December, 83 (23):921 1-5; Coupar (1991) pp. 109-127). et al., Gene. 1988 Aug. 15: 68(1): 1-10). Nucleic acid con Adeno-associated virus (AAV) can also be used in accor structs can be inserted into a viral vector, such as, but not dance with the present invention (see, e.g., Walsh et al., 1993, limited to, a papovavirus, a retrovirus, an adenovirus, an Proc. Soc. Exp. Biol. Med. 204:289-300; U.S. Pat. No. 5,436, adeno-associated virus, a vaccinia virus or a herpesvirus (for 10 146). Adeno-associated virus is a naturally occurring defec review, see for example Viral Vectors. Gene Therapy and tive virus that requires another virus, Such as an adenovirus or Neuroscience Applications, Kaplitt and Loewy (eds.), Aca a herpesvirus, as a helper virus for efficient replication and a demic Press, San Diego (1995)). productive life cycle (see, e.g., Muzyczka et al., 1992, Curr The infection spectrum of viruses and viral-based vectors Top Microbiol Immunol. 158:97-129). Also, AAV is one of can be limited by modifying the viral packaging proteins on 15 the few viruses that can integrate its DNA into non-dividing the surface of the viral particle (See, e.g., PCT publications cells, and exhibits a high frequency of stable integration (see, WO93/25234 and WO94/06920). Strategies for modifying e.g., Flotte et al., 1992, Am J Respir Cell Mol. Biol. 7:349 the infection spectrum of retroviral vectors include, but are 354; Samulski et al., 1989, J. Virol. 63:38223828; McLaugh not limited to, coupling antibodies specific for cell Surface linet al., 1989, J. Virol. 63:1963 1973). antigens to the viral env protein (Roux et al., 1989, Proc. Nat. Vectors containing as few as 300 base pairs of AAV can be Acad. Sci. USA 86:9079 9083; Julan et al., 1992, J. Gen. packaged and can integrate. The capacity for the insertion of Virol. u3:3251 3255; Goud et al., 1983, Virology 163:251 exogenous DNA is limited to about 4.5 kb. An AAV vector 254) or coupling cell Surface receptor ligands to the viral env such as that described in Tratschin et al. (1985, Mol. Cell. proteins (Neda et al., 1991, J. Biol. Chem. 266:14143 14146). Biol. 5:3251-3260) can be used to introduce DNA into cells. Coupling can be in the form of chemical crosslinking with a 25 A variety of nucleic acids have been introduced into different protein or other compound (e.g., lactose to convert the env cell types using AAV vectors (see, e.g. Hermonat et al., 1984, protein to an asialogycoprotein), or in the form of fusion Proc Natl Acad. Sci. 81:6466-6470; Tratschin et al., 1985, proteins (e.g., single-chain antibody/env fusion proteins). Mol. Cell. Biol. 4:2072-2081; Wondisford et al., 1988, Mol. Accordingly, cancer cells can be targeted to the Surface of a Endocrinol. 2:32 39: Tratschin et al., 1984, J. Virol. 51: 611 recombinant virus by using, for example, coupling antibodies 30 619; Flotte et al., 1993, J. Biol. Chem. 268:3781-3790). that are directed against tumor-associated molecules, or by Mammalian expression vectors which may serve as vector using cancer cell surface proteins. Such techniques, while backbone for the constructs of the present invention include, useful for limiting, or otherwise directing, the infection to but are not limited to, pcDNA3, pcDNA3.1 (+/-), pGL3, certain tissue types, can also be used to convert an ecotropic pZeoSV2(+/-), pSecTag2, poisplay, pFF/myc/cyto, pCMV/ vector into an amphotropic vector. 35 myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, In a particular embodiment, an adenovirus-derived vector pNMT41, and pNMT81, available from Invitrogen R; pCI may be used in the present invention. The genome of an available from Promega(R), pMbac, pPbac, pFBK-RSV and adenovirus can be manipulated Such that it encodes and pBK-CMV, available from StrategeneR); and pTRES, avail expresses a gene product of interest, but is inactivated interms able from Clontech(R). of its ability to replicate in a normal lytic viral life cycle (see, 40 Other exemplary vectors include pSVT7 and pMT2 (de e.g., Berkner et al., 1988, BioTechniques 6:616; Rosenfeld et rived from SV40): pBV-1MTHA (derived from bovine pap al., 1991, Science 252:431 434; Rosenfeld et al., 1992, Cell illoma virus); pHEBO and p2O5; (derived from Epstein-Barr 68:143 155). Suitable adenoviral vectors derived from the virus); pMSG; pAV009/A"; pMTO10/A"; pMAMneo-5, and adenovirus strain AD type 5 d1324 or other strains of aden baculovirus p)SVE. ovirus (e.g., Ad2. Ad3, Ad7, etc.), are well known to those 45 Host cells transfected with vectors of the invention can be skilled in the art. any prokaryotic or eukaryotic cell. Transforming or transfect Recombinant adenoviruses can be advantageous in certain ing the vector into host cells, either eukaryotic (e.g., yeast, circumstances in that they can be used to infect a wide variety avian, insect or mammalian) or prokaryotic (e.g., bacterial of cell types, including airway epithelium (Rosenfeld et al., cells) are standard procedures used widely in the microbial or 1992, Cell 68:143 155), endothelial cells (Lemarchand et al., 50 tissue-culture technologies. 1992, Proc Natl Acad. Sci. 89:6482 6486), hepatocytes (Herz Methods suitable for nucleic acid delivery to effect expres and Gerard, 1993, Proc Natl Acad Sci. 90:2812 2816), and sion of anti-cancer agents according to the present invention muscle cells (Quantin et al., 1992, Proc Natl Acad Sci. include virtually any method by which a nucleic acid (e.g., 89:2581 2584). Furthermore, the virus particle is relatively DNA, including viral and nonviral vectors) can be introduced stable, amenable to purification and concentration, and can be 55 into an organelle, a cell, a tissue oran organism, as described modified so as to affect the spectrum of infectivity. herein or as would be known to one of ordinary skill in the art. Additionally, introduced adenoviral DNA (and foreign Such methods include, but are not limited to, direct delivery DNA contained therein) is generally not integrated into the of DNA such as by injection (see e.g. U.S. Pat. Nos. 5,994, genome of a host cell but remains episomal, thereby avoiding 624, 5,981,274, 5,945,100, 5,780,448, 5,736,524, 5,702,932, potential problems (e.g., insertional mutagenesis) that can 60 5,656,610, 5,589,466 and 5,580,859), including microinjec occur when introduced DNA (e.g., retroviral DNA) becomes tion (U.S. Pat. No. 5,789.215); by electroporation (U.S. Pat. integrated into the host genome. No. 5,384.253); by calcium phosphate precipitation (Chen Moreover, the carrying capacity of the adenoviral genome and Okayama, Mol. Cell. Biol, 7(8):2745-2752, 1987); by for foreign DNA is large (up to 8 kilobases) relative to other using DEAE-dextran followed by polyethylene glycol (Go gene delivery vectors (Berkner et al., 1988, BioTechniques 65 pal, Mol. Cell. Biol. 5:1188-1190, 1985); by direct sonic 6:616; Haj-Ahmand and Graham, 1986, J. Virol. 57:267). A loading (Fechheimer et al., Proc. Natl. Acad. Sci. USA, replication-defective adenoviral vector useful for the meth 84:8463-8467, 1987); by liposome mediated transfection US 8,492,133 B2 23 24 (Nicolau and Sene, Biochim. Biophys. Acta, 721:185-190, Biol. Chem. 264: 14256-14261, 1989). Other forms of PE 1982; Kaneda et al., Science, 243:375-378, 1989: Kato et al, which may be encoded and expressed by the constructs of the J. Biol. Chem., 266:3361-3364, 1991); by microprojectile invention include for example, PE38 (see e.g., U.S. Pat. No. bombardment (PCT Application Nos. WO 94/09699 and 5,608,039, and Pastan et al., 1997, Biochim. Biophys. Acta 95/06128: U.S. Pat. Nos. 5,610,042; 5,322,783 5,563,055, 1333:C1-C6), disclosed herein as SEQ ID NO: 21; PE38 5,550,318, 5.538,877 and 5,538,880; by agitation with silicon KDEL (see e.g., Brinkmann et al 1991, Proc Nat Acad Sci carbide fibers (U.S. Pat. Nos. 5,302,523 and 5,464,765); by USA 88:8616-8621), disclosed herein as SEQ ID NO: 22: Agrobacterium-mediated transformation (U.S. Pat. Nos. PE37 (see e.g., U.S. Pat. No. 5,602,095) disclosed herein as 5,591,616 and 5.563,055); or by PEG-mediated transforma SEQID NO:23; and PE40 (see e.g., U.S. Pat. No. 6,051.405) tion of protoplasts (U.S. Pat. Nos. 4,684,611 and 4,952.500; 10 disclosed herein, as SEQID NO: 24. by desiccation/inhibition-mediated DNA uptake (Potrykus et The invention further encompasses use of a nucleic acid al., Mol. Gen. Genet., 199(2):169-77, 1985). Through the encoding a drug-metabolizing enzyme which converts a pro application of techniques such as these, organelle(s), cell(s), drug into a cytotoxic product. tissue(s) or organism(s) may be stably or transiently trans In a particular embodiment, the drug-converting enzyme is formed. 15 a thymidine kinase (e.g. from herpes simplex virus or vari Nucleic Acid Sequences Encoding Anti-Cancer Agents cella Zoster virus), which has the capacity to convert the The nucleotide sequence encoding an anti-cancer agent prodrug form of ganciclovir (2-amino-9-(1,3-dihydrox can be selected from those encoding a wide variety of proteins ypropan-2-yl)oxymethyl-6,9-dihydro-3H-purin-6-one) Such as, but not limited to, cytotoxic gene products, cytostatic into its active phosphorylated form, which is a deoxygua gene products, viral replication proteins, drug metabolizing nosine triphosphate (dGTP) analog. This analog competi enzymes and inducers of apoptosis. tively inhibits the incorporation of dGTP into cellular DNA, The anti-cancer agent may be a toxin, Such as a bacterial resulting in cell death. toxin, a plant toxin, or a fungal toxin, either the full-length Drug metabolizing enzymes that convert a prodrug into a protein, but more preferably a cytotoxic fragment of a natu cytotoxic product include, but are not limited to, thymidine rally occurring toxin. Exemplary bacterial toxins include, but 25 kinase (e.g. from herpes simplex virus or varicella Zoster are not limited to, diphtheria toxin, Such as diphtheria toxin virus), cytosine deaminase, nitoreductase, cytochrome p. 450 fragment A (DTA), Pseudomonas exotoxin (PE), cholera 2B1, thymidine phosphorylase, E. coli guanine phosphoribo toxin, anthrax toxin, botulinum toxin, pertussis toxin, E. coli Syl transferase, purine nucleoside phosphorylase, alkaline enterotoxin, and shiga toxin. Exemplary plant toxins include, phosphatase, carboxypeptidases A and G2, linamarase, beta but are not limited to, ricin, modeccin, abrin, Volkensin and 30 lactamase, Xanthine oxidase, and variants thereof (see, e.g., Viscumin. Examples of fungal toxins include O.-sarcin (see for Pandha et al., 1999, J. Clin Oncol. 17:2180 2189; Rigg and example, Nagano et al., J Antibiot (Tokyo). 1996 January: Sikora, 1997, Mol Med. Today. 3:359366). 49(1): 81-5); restrictocin (see e.g., Rathore et al FEBS Lett 2 The anti-cancer agent may further be an inducer of apop Sep.1996, 392(3):259-262); mitogillin (see e.g., Better et al., tosis, such as PUMA (see, e.g. GenBank accession No. J Biol Chem August 15: 1992 267, 16712-16718); enomycin 35 AF354654); BAX (see, e.g. GenBank accession No. (see e.g., Takeuchi et al., J Antibiot (Tokyo). 1997 January; AY217036); BAK (see, e.g. GenBank accession No. 50(1):27-31); a fungal ribotoxin, for example RNase T1 (for NR 027882); Bcl-XS (see, e.g. GenBank accession No. review see e.g., Lacadena et al., FEMS Microbiol Rev. 2007 NM 001191); BAD (see, e.g. GenBank accession No. March;31(2):212-37) and phenomycin (see e.g., Sakata et al., BCO01901); BIM (see, e.g. GenBank accession No. J Antibiot (Tokyo). 1994 March; 47(3):370-1). 40 BC033694); BIK (see, e.g. GenBank accession No. U34584): The toxin for use in the invention may be the A chain of a BID (see, e.g. GenBank accession No. BC036364); HRK ricin-like protein, as disclosed for example in U.S. Pat. No. (see, e.g. GenBank accession No. HSU76376): Ad ElB; ICE 7,375,186. CED3 proteases (see, e.g. GenBank accession Nos. A diphtheria toxin for use in the invention may correspond NM 001080 124 and NP 001073593); TRAIL (see, e.g. to the full-length 535 amino acid polypeptide secreted by 45 GenBank accession Nos. NM 003810 and NP 003801); Corynbacterium diphtheria or it may be a cytotoxic fragment SARP-2 (see, e.g. GenBank accession Nos. NM 003012 and thereof, in particular fragment A comprising the N-terminal NP 003003); and apoptin (see, e.g. GenBank accession Nos. catalytic domain (see, e.g., Genbank Accession Nos. A04646 NM 012234 and NP 036366). and AY820132; Greenfield et al., 1983 Proc. Natl. Acad. Sci. Oligonucleotide sequences useful for preparing nucleic USA 80:6853-6857: Tweten et al., 1983 J. Bacteriol. 156: 50 acid sequences encoding Such apoptosis inducers are dis 680-685; Kaczoreket al., 1983 Science 221:855-858: Leong closed for example in U.S. Patent Application publication No. et al., 1983 Science 220:515-517). 2009/O298071. Examples 3 and 4 herein disclose a construct comprising a Examples 3 and 4 herein discloses construction and use of nucleic acid sequence encoding diphtheria toxin fragment A a construct and vector comprising a sequence encoding operably linked to a miR-21 promoter sequence and its cyto 55 PUMA for use in anti-cancer therapy. toxic effect on cancer cells in vitro and in vivo. Polynucleotides encoding cytostatic agents that Suppress In a particular embodiment, a nucleic acid sequence encod cell growth and multiplication are useful for the invention. ing a diphtheria toxin comprises SEQ ID NO: 18. In a par These include, but are not limited to, gene products derived ticular embodiment, the construct encodes a diphtheria toxin from p21 (Waf1), p27(Kip1), p53, p53175P. p57 (Kip2), p15 comprising an amino acid sequence of SEQID NO: 19. 60 (INK4b), p16 (INK4a), p 18(INK4c), p19(Arf), p73, In other embodiments, a nucleic acid sequence encoding a GADD45, APC1, p73RB1, WT1, NF1, VHL, and variants Pseudomonas exotoxin (PE) may be used in the invention, for thereof (see e.g., Koga et al., Hepatology. 33:1087 1097; example that corresponding to the full-length protein of 613 Huanget al., 2001, Cancer Res.61:3373.3381; Liet al., 2001, amino acids secreted by Pseudomonas aeruginosa, or that Cancer Res.61:1493 1499; MaZur et al., 1998, Biotechnol encoding a cytotoxic fragment thereof. Such as a cytotoxic 65 Prog. 14:705713: Tsugu et al., 2000, Am J Pathol. 157:919 fragment corresponding to domain III i.e. amino acid residues 932: Dyer et al., 2001, J. Neurosci. 21:425.94571; Kovalev et 400-613 (see, e.g., U.S. Pat. No. 5,602,095; Siegallet al., J. al., 2001, J. Immunol. 167:3285 3292; Modesitt et al., 2001, US 8,492,133 B2 25 26 Clin Cancer Res. 7:1765 1772; Wong et al., 2001, J Pathol. hydroxypropylmethyl-cellulose, sodium carboxymethylcel 194:35 42: Shapiro et al., 2000, Cell Biochem Biophys. lulose, and/or polyvinylpyrrolidone (PVP); granulating 33:189 197: Fuxe et al., 2000, Cell Growth Differ. 11:373 agents; and binding agents. If desired, disintegrating agents 384; Latres et al., 2000, EMBO.J. 19:3496.3506; Weberet al., may be added. Such as the cross-linked polyvinylpyrrolidone, 2000, Genes Dev. 14:23.58 2365; Sasaki et al., 2001, Gene 5 agar, oralginic acid or a salt thereof Such as sodium alginate. Ther. 8:1401 1408; Zhu et al., 1998, Cancer Res. 58:5061 If desired, Solid dosage forms may be sugar-coated or enteric 5065; Mullanet al., 2001, Oncogene. 20:6123 6131; Velasco coated using standard techniques. For oral liquid preparations Miguel et al., 1999. Oncogene. 18:127 137: Jorgensen et al., Such as, for example, Suspensions, elixirs and solutions, Suit 2001, Gene. 262:51 59; Kurasawa and Todokoro, 1999, able carriers, excipients or diluents include water, glycols, Oncogene 18:5131 5137; Basu et al., 1999, Int J Oncol. 10 oils, alcohols, etc. Additionally, flavoring agents, preserva 15:701 708; Yamagami et al., 1998, Leuk Res. 22:383 384; Murata et al., 1997, FEBS Lett. 409:41 45; Uhlmann et al., tives, coloring agents and the like may be added. For buccal 2001, Cell Biochem Biophys. 34:61 78; Zhanget al., 1998, J administration, the molecules may take the form of tablets, Exp Med. 187: 1893 1902: Norton et al., 1996, Neuroreport. lozenges, etc. formulated in conventional manner. 7:601604; Baba et al., 2001, Oncogene. 20:2727 2736: Davi 15 The nucleic acid molecules may also be formulated as a dowitz et al., 2001, Mol Cell Biol. 21:865 874). depot preparation. Such long acting formulations may be Pharmaceutical Compositions, Formulations and Modes of administered by implantation (for example Subcutaneously or Administration intramuscularly) or by intramuscular injection. Thus, for The nucleic acid constructs and vectors of the invention example, the molecules may be formulated with suitable may be administered to a Subject alone or in the form of a polymeric or hydrophobic materials (for example as an emul pharmaceutical composition. Pharmaceutical compositions sion in an acceptable oil) or ion exchange resins, or as spar may beformulated in conventional manner using one or more ingly soluble derivatives, for example, as a sparingly soluble physiologically acceptable carriers, diluents, excipients or salt. auxiliaries which facilitate processing of the nucleic acids A nucleic acid of the invention may be administered in into preparations which can be used pharmaceutically. Proper 25 combination with a carrier or lipid to increase cellular uptake. formulation is dependent upon the route of administration For example, nucleic acids may be administered in combina chosen. tion with a cationic lipid. Examples of cationic lipids include, Systemic formulations include those designed for admin but are not limited to, Lipofectin R, DOTMA, DOPE, and istration by injection, e.g. Subcutaneous, intravenous, intra DOTAP, and T-shaped cholesterol ester derivative (see e.g. muscular, intrathecal or intraperitoneal injection, as well as 30 Lee etal Biorg MedChem Lett 14 (2004):2637-2641. Various those designed for transdermal, transmucosal, inhalation, nucleic acid formulations and methods of administration are oral or pulmonary administration. For injection, the nucleic disclosed for example in U.S. Pat. Nos. 7,470,675; 5,844,107: acids of the invention may be formulated in aqueous solu 5,877,302: 6,008,336; 6,077,835; 6,200,801, and 5,972,900, tions, preferably in physiologically compatible buffers such and in U.S. Patent Application Publication Nos. 2003/ as Hanks Solution, Ringer's Solution, or physiological saline 35 0203865; 2002/0150626; 2003/0032615, and 2004/0048787. buffer. The solution may contain formulatory agents such as In addition, liposomes and emulsions are well-known Suspending, stabilizing and/or dispersing agents. Alterna examples of delivery vehicles that may be used to deliver tively, the nucleic acid molecules may be in powder form for nucleic acids of the invention. Suitable liposomes can be reconstitution with a suitable vehicle, e.g., sterile pyrogen formed from standard vesicle-forming lipids, which gener free water, before use. For transmucosal administration, pen 40 ally include neutral or negatively charged phospholipids and etrants appropriate to the barrier to be permeated are used in a sterol. Such as cholesterol. The selection of lipids is gener the formulation. For administration by inhalation, the mol ally guided by consideration of factors, such as the desired ecules for use according to the present invention are conve liposome size and half-life of the liposomes in the blood niently delivered in the form of an aerosol spray from pres stream. A variety of methods are known for preparing lipo surized packs or a nebulizer, with the use of a suitable 45 somes, described example, in Szoka et al. (1980), Ann. Rev. propellant, e.g., dichlorodifluoromethane, trichlorofluo Biophys. Bioeng.9:467; and U.S. Pat. Nos. 4,235,871, 4,501, romethane, dichlorotetrafluoroethane, carbon dioxide or 728, 4,837,028, and 5,019,369. other suitable gas. In the case of a pressurized aerosol the Liposomes for use in the present methods can comprise a dosage unit may be determined by providing a valve to deliver ligand molecule that targets the liposome to cancer cells, for a metered amount. Capsules and cartridges of gelatin for use 50 example a monoclonal antibody that binds a tumor cell anti in an inhaler or insufflator may be formulated containing a gen. powder mix of the nucleic acids and a suitable powder base can also be modified so as to avoid clearance by the mono Such as lactose or starch. The nucleic acid molecules may also nuclear macrophage system (“MMS) and reticuloendothe be formulated in rectal or vaginal compositions such as Sup lial system (“RES). Such modified liposomes have positories or retention enemas, e.g., containing conventional 55 opSonization-inhibition moieties on the Surface or incorpo Suppository bases such as cocoa butter or other glycerides. rated into the liposome structure. In a particular embodiment, For oral administration, the nucleic acids can be readily for a liposome of the invention can comprise both an opSoniza mulated by combining the molecules with pharmaceutically tion-inhibition moiety and a ligand. acceptable carriers well known in the art. Such carriers enable Opsonization-inhibiting moieties for use in preparing the the nucleic acids of the invention to be formulated as tablets, 60 liposomes of the invention are typically large hydrophilic pills, dragees, capsules, liquids, gels, syrups, slurries, Suspen polymers that are bound to the liposome membrane. As used sions and the like, for oral ingestion by a patient to be treated. herein, an opSonization-inhibiting moiety is “bound to a For oral Solid formulations such as, for example, powders, liposome membrane when it is chemically or physically capsules and tablets, Suitable excipients include fillers such as attached to the membrane, e.g., by the intercalation of a Sugars, e.g. lactose, Sucrose, mannitol and Sorbitol; cellulose 65 lipid-soluble anchor into the membrane itself, or by binding preparations such as maize starch, wheat starch, rice starch, directly to active groups of membrane lipids. These opsoniza potato starch, gelatin, gum tragacanth, methyl cellulose, tion-inhibiting hydrophilic polymers form a protective Sur US 8,492,133 B2 27 28 face layer that significantly decreases the uptake of the lipo capsules may, depending on their chemical nature, release the somes by the MMS and RES: e.g., as described in U.S. Pat. molecules for a few weeks up to over 100 days. Depending on No. 4,920,016. the chemical nature and the biological stability of the chi Examples of opsonization-inhibiting moieties Suitable for meric molecules, additional strategies for molecule stabiliza modifying liposomes are water-soluble polymers with a num tion may be employed. ber-average molecular weight from about 500 to about 40,000 Nucleic acids may be included in any of the above-de daltons, such as about 2,000 to about 20,000 daltons. Such scribed formulations as the free acids or bases or as pharma polymers include polyethylene glycol (PEG) or polypropy ceutically acceptable salts. Pharmaceutically acceptable salts lene glycol (PPG) derivatives; e.g., methoxy PEG or PPG, are those salts that substantially retain the biologic activity of and PEG or PPG stearate; synthetic polymers, such as poly 10 acrylamide or poly N-vinyl pyrrolidone; linear, branched, or the free bases and which are prepared by reaction with inor dendrimeric polyamidoamines; polyacrylic acids; polyalco ganic acids. Pharmaceutical salts tend to be more soluble in hols, e.g., polyvinylalcohol and polyxylitol to which car aqueous and other protic solvents than are the corresponding boxylic or amino groups are chemically linked, as well as free base forms. gangliosides, such as ganglioside GM1. Copolymers of PEG, 15 Pharmaceutical compositions of the present invention methoxy PEG, or methoxy PPG, or derivatives thereof, are comprise an effective amount of one or more nucleic acid also Suitable. In addition, the opsonization-inhibiting poly molecules or vectors dissolved or dispersed in a pharmaceu mercan be a block copolymer of PEG and either a polyamino tically acceptable carrier. The phrases “pharmaceutically acid, polysaccharide, polyamidoamine, polyethyleneamine, acceptable' refers to molecular entities and compositions that or polynucleotide. The opsonization-inhibiting polymers can do not produce an adverse, allergic or other untoward reaction also be natural polysaccharides containing amino acids or when administered to an animal. Such as, for example, a carboxylic acids, e.g., galacturonic acid, glucuronic acid, human, as appropriate. The preparation of a pharmaceutical mannuronic acid, hyaluronic acid, pectic acid, neuraminic composition that contains at least one nucleic acid ingredient acid, alginic acid, carrageenan; aminated polysaccharides or will be known to those of skill in the art in light of the present oligosaccharides (linear or branched); or carboxylated 25 disclosure, as exemplified by Remington’s Pharmaceutical polysaccharides or oligosaccharides, e.g., reacted with Sciences, 18th Ed. Mack Printing Company, 1990. Moreover, derivatives of carbonic acids with resultant linking of car for animal (e.g., human) administration, it will be understood boxylic groups. Preferably, the opSonization-inhibiting moi that preparations should meet Sterility, pyrogenicity, general ety is a PEG, PPG, or a derivative thereof. Liposomes modi safety and purity standards as required by health regulatory fied with PEG or PEG-derivatives are sometimes called 30 agencies such as the FDA Office of Biological Standards. “PEGylated liposomes.” As used herein, "pharmaceutically acceptable carrier, dilu An opsonization-inhibiting moiety can be bound to the ent or excipient” includes any and all solvents, dispersion liposome membrane by any one of numerous well-known media, coatings, Surfactants, antioxidants, preservatives (e.g., techniques. For example, an N-hydroxySuccinimide ester of antibacterial agents, antifungal agents), isotonic agents, PEG can be bound to a phosphatidyl-ethanolamine lipid 35 absorption delaying agents, salts, preservatives, drugs, drug soluble anchor, and then bound to a membrane. Similarly, a stabilizers, gels, binders, excipients, disintegration agents, dextran polymer can be derivatized with a stearylamine lipid lubricants, Sweetening agents, flavoring agents, dyes, such soluble anchor via reductive amination using Na(CN) BH like materials and combinations thereof, as would be known and a solvent mixture, Such as tetrahydrofuran and water in a to one of ordinary skill in the art. Except insofar as any 30:12 ratio at 60° C. 40 conventional carrier is incompatible with the active ingredi Liposomes modified with opSonization-inhibition moi ent, its use in the therapeutic or pharmaceutical compositions eties remain in the circulation much longer than unmodified is contemplated. liposomes. For this reason, such liposomes are sometimes The pharmaceutical compositions comprising nucleic called “stealth' liposomes. Stealth liposomes are known to molecules may comprise different types of carriers depend accumulate in tissues fed by porous or “leaky' microvascu 45 ing on whether it is to be administered in Solid, liquid or lature. Thus, tissue characterized by Such microvasculature aerosol form, and in accordance with the general need for defects, for example, Solid tumors, will efficiently accumu sterility for such routes of administration as injection. The late these liposomes; see Gabizon, et al. (1988), Proc. Natl. present invention can be administered intravenously, intrad Acad. Sci., U.S.A., 18:6949-53. In addition, the reduced ermally, intraarterially, intraperitoneally, intralesionally, uptake by the RES lowers the toxicity of stealth liposomes by 50 intracranially, intraarticularly, intraprostatically, intrapleu preventing significant accumulation of the liposomes in the rally, intratracheally, intranasally, intravitreally, intravagi liver and spleen. Thus, liposomes that are modified with nally, intrarectally, topically, intratumorally, intramuscularly, opSonization-inhibition moieties are particularly Suited to intraperitoneally, Subcutaneously, Subconjunctival, intrave deliver the or nucleic acids of the invention to tumor cells. Sicularly, mucosally, intrapericardially, intraumbilically, The nucleic acids may also be administered in combination 55 intraocularally, orally, topically, locally, inhalation (e.g. aero with a cationic amine Such as poly (L-lysine). Nucleic acids Sol inhalation), injection, infusion, continuous infusion, may also be conjugated to a chemical moiety, Such as trans localized perfusion bathing target cells directly, via a catheter, ferrin and cholesteryls. In addition, oligonucleotides may be via a lavage, in lipid compositions (e.g., liposomes), or by targeted to certain organelles by linking specific chemical other method or any combination of the forgoing as would be groups to the oligonucleotide. For example, linking the oli 60 known to one of ordinary skill in the art. gonucleotide to a suitable array of mannose residues will The actual dosage amount of a composition of the present target the oligonucleotide to the liver. invention administered to the Subject, such as a human Additionally, the molecules may be delivered using a sus patient, can be determined by physical and physiological tained-release system, such as semipermeable matrices of factors such as body weight, severity of condition, the type of Solid polymers containing the therapeutic agent. Various of 65 disease being treated, previous or concurrent therapeutic Sustained-release materials have been established and are interventions, idiopathy of the patient and on the route of well known by those skilled in the art. Sustained-release administration. The practitioner responsible for administra US 8,492,133 B2 29 30 tion will, in any event, determine the concentration of active cancer, colon cancer, hepatocellular carcinoma, cervical can ingredient(s) in a composition and appropriate dose(s) for the cer, cholangiocarcinoma, endometrioid ovarian carcinoma, individual subject. esophageal cancer, glioblastoma, head and neck cancer, leu In certain embodiments, pharmaceutical compositions kemia (e.g. chronic lymphocytic leukemia), lymphoma (e.g. may comprise, for example, at least about 0.1% of an active diffuse large B cell lymphoma, activated B cell-like lym compound. In other embodiments, the an active compound phoma), lung cancer, multiple myeloma, pancreatic (e.g. may comprise between about 2% to about 75% of the weight endocrine and acinar) cancer, osteosarcoma, pituitary tumor, of the unit, or between about 25% to about 60%, for example, prostate cancer, stomach cancer, and uterine leiomyoma. and any range derivable therein. In other non-limiting The constructs and vectors of the invention treat cancer by examples, a dose may also comprise from at least about 1 10 inhibiting cancer cell proliferation, and/or by arresting or microgram/kg/body weight, including about 5, about 10, slowing the growth of the cancer cells and/or by inhibiting about 50, about 100, about 200, about 350 weight, about 500 tumor progression, and/or by inhibiting tumor metastasis. microgram/kg/body weight; or at least about 1 milligram Any of the aforementioned effects may be permanent or tem (mg)/kg/body weight, such as about 5, about 10, about 50, porary. Inhibition of cancer cell proliferation can be inferred about 100, about 200, about 350, about 500, to about 1000 15 if the number of such cells in the subject remains constant or mg/kg/body weight or more per administration, and any decreases after administration of the constructs and vectors. range derivable therein. An inhibition of cancer cell proliferation can also be inferred The composition may comprise various antioxidants to if the absolute number of such cells increases, but the rate of retard oxidation of one or more component. Additionally, the tumor growth decreases. composition may comprise preservatives such as various The number of cancer cells in the body of a subject can be antibacterial and antifungal agents, including but not limited determined by direct measurement, or by estimation from the to parabens (e.g., methylparabens, propylparabens), chlo size of primary or metastatic tumor masses. For example, the robutanol, phenol, Sorbic acid, thimerosal or combinations number of cancer cells in a subject can be measured by thereof. immunohistological methods, flow cytometry, or other tech In embodiments where the composition is in a liquid form, 25 niques designed to detect characteristic Surface markers of a carrier can be a solvent or dispersion medium comprising cancer cells. but not limited to, water, ethanol, polyol (e.g., glycerol, pro The size of a tumor mass can be ascertained by direct visual pylene glycol, liquid polyethylene glycol, etc.), lipids (e.g., observation, or by diagnostic imaging methods, such as triglycerides, vegetable oils, liposomes) and combinations X-ray, magnetic resonance imaging, ultrasound, and Scintig thereof. The proper fluidity can be maintained, for example, 30 raphy. Diagnostic imaging methods used to ascertain size of by the use of a coating, such as lecithin; by the maintenance of the tumor mass can be employed with or without contrast the required particle size by dispersion in carriers such as, for agents, as is known in the art. The size of a tumor mass can example liquid polyol or lipids; by the use of surfactants such also be ascertained by physical means, such as palpation of as, for example hydroxypropylcellulose; or combinations the tissue mass or measurement of the tissue mass with a thereof such methods. In many cases, it will be preferable to 35 measuring instrument, such as a caliper. include isotonic agents. Such as, for example, Sugars, sodium Any mode of administration of the constructs and vectors chloride or combinations thereof. can be used so long as it results in the expression of the In other embodiments, one may use nasal Solutions or anti-cancer agent in the desired tissue, in an amount Sufficient sprays, aerosols or inhalants in the present invention. Such to be therapeutically and/or prophylactically effective. The compositions are generally designed to be compatible with 40 miR-21 promoter containing constructs and vectors can be the target tissue type. In a non-limiting example, nasal solu administered to a subject by any suitable enteral or parenteral tions are usually aqueous solutions designed to be adminis administration route. Suitable enteral administration routes tered to the nasal passages in drops or sprays. Nasal Solutions for the present methods include, e.g., oral, rectal, or intranasal are prepared so that they are similar in many respects to nasal delivery. Suitable parenteral administration routes include, secretions, so that normal ciliary action is maintained. Thus, 45 e.g., intravascular administration (e.g., intravenous bolus in preferred embodiments the aqueous nasal solutions usually injection, intravenous infusion, intra-arterial bolus injection, are isotonic or slightly buffered to maintain a pH of about 5.5 intra-arterial infusion and catheter instillation into the vascu to about 6.5. lature); peri- and intra-tissue injection (e.g., peri-tumoral and The composition must be stable under the conditions of intra-tumoral injection, intra-retinal injection, or Subretinal manufacture and storage, and preserved against the contami 50 injection); Subcutaneous injection or deposition, including nating action of microorganisms, such as bacteria and fungi. Subcutaneous infusion (such as by osmotic pumps); direct It will be appreciated that endotoxin contamination should be application to the tissue of interest, for example by a catheter kept minimally at a safe level, for example, less that 0.5 ng/mg or other placement device (e.g., a retinal pellet or a Supposi protein. tory oran implant comprisingaporous, non-porous, or gelati Therapeutic Uses and Methods of Treatment 55 nous material e.g. gelfoam sponge depots; hydrogels) or topi The nucleic acid constructs and vectors of the invention cal applications during Surgery; and inhalation. Particularly may be used for treating a subject having cancer, Such as for Suitable administration routes are injection, infusion and inhibiting tumor progression, for inhibiting tumor metastasis, direct injection into the tumor. or for reducing or alleviating a symptom associated with a Means of injection include biolistic injectors and particle neoplastic disorder. 60 accelerators (e.g., "gene guns' or pneumatic “needleless' The invention is particularly useful for treating types of injectors), such as those marketed as Med-E-JetTM (Vahlsing, cancer, tumors or neoplastic disorders characterized interalia H., et al., J. Immunol. Methods 171, 11-22 (1994)), Biojec by endogenous expression of miR-21 in at least a portion of torTM (Davis, H., et al., Vaccine 12, 1503-1509 (1994): the cells thereof. Gramzinski, R., et al., Mol. Med. 4, 109-118 (1998)), Advan The cancer, tumor or neoplastic disorder may be a sarcoma, 65 taletTM (Linmayer, I., et al., Diabetes Care 9:294-297 (1986)), a carcinoma, an adenocarcinoma, a lymphoma, and a leuke and Medi-JectorTM (Martins, J., and Roedl, E. J. Occup. Med. mia. Included within the scope of the invention are breast 21:821-824 (1979)). US 8,492,133 B2 31 32 The nucleic acid constructs and vectors may be adminis sample from the subject, so that the levels of miR-21 gene tered as a single dose or multiple doses, administered at product produced from a miR-21 gene in cells from the sub suitable time intervals. jects sample can be compared to the corresponding miR-21 The term “therapeutically effective amount” refers to an gene product levels from cells of the control sample. amount of a construct or vector of the invention that is effec Some cancers are characterized by an alteration, particu tive to achieve the desired result following administration. larly an increase, in the level of an miR-21 gene product in a The constructs and vectors may be administered in con sample obtained from the subject, relative to the level of a junction with one or more additional modalities of cancer corresponding miR-21 gene product in a control sample. therapy including, but not limited to bone marrow transplant, When the level of at least one miR-21 gene product in the test cord blood cell transplant, Surgery, chemotherapy, radiation 10 sample is greater than the level of the corresponding miR-21 therapy, and immunotherapy. The polynucleotide construct gene product in the control sample, the expression of the or pharmaceutical composition of the present invention can miR-21 gene product is "up-regulated'. be administered prior to the commencement of one or more of The relative miR-21 gene expression in the control and the additional cancer therapies, during the practice of one or normal samples can be determined with respect to one or more of the additional modalities of cancer therapy, and after 15 more RNA expression standards. The standards can com the end of one or more of the additional modalities of cancer prise, for example, a Zero miR-21 gene expression level, the therapy. miR-21 gene expression level in a standard cell line, or the Types of bone marrow transplant include, but are not lim average level of miR-21 gene expression previously obtained ited to autologous bone marrow transplant and heterologous for a population of normal human controls. (i.e., from a donor) bone marrow transplant. The level of a miR-21 gene product in a sample can be Chemotherapeutic agents include, but are not limited to measured using any technique that is Suitable for detecting alkylating agents, including mechlorethamine, cyclophos RNA expression levels in a biological sample. Suitable tech phamide, ifosfamide, melphalan, chlorambucil, dicarbazine, niques for determining RNA expression levels in cells from a streptazocine, carmustine, lomustine, Semustine, chlorozoto biological sample (e.g., Northern blot analysis, RT-PCR, in cin, buSulfan, triethylenemelamine, thiotepa, hexameth 25 situ hybridization) are well known to those of skill in the art. ylmelamine; antimetabolites, including methotrexate; pyri In a particular embodiment, the level of at least one miR-21 midine analogs, including fluorouracil, 5-fluorouracil, gene product is detected using Northern blot analysis. For floxuridine (5'-fluoro-2'-deoxyuridine), idoxuridine, cytara example, total cellular RNA can be purified from cells by bine, N-phosphonoacetyl-L-aspartate, 5-azacytidine, azarib homogenization in the presence of nucleic acid extraction ine, 6-azauridine, pyrazofuran, 3-deaZauridine, acivicin; 30 buffer, followed by centrifugation. Nucleic acids are precipi purine analogs, including thioguanine, mercaptopurine, aza tated, and DNA is removed by treatment with DNase and thioprine, pentostatin, erythrohydroxynonyladenine; Vinca precipitation. The RNA molecules are then separated by gel alkaloids, including Vincristine and vinblastine; epipodo electrophoresis on agarose gels according to standard tech phyllotoxins, including etoposide and teniposide; antibiotics, niques, and transferred to nitrocellulose filters. The RNA is including dactinomycin, daunorubicin, doxorubicin, bleo 35 then immobilized on the filters by heating. Detection and mycin Sulfate, plicamycin, mitomycin; enzymes, including quantification of specific RNA is accomplished using appro L-asparaginase; platinum coordination complexes, including priately labeled DNA or RNA probes complementary to the cisplatin, carboplatin; hydroxyurea, procarbazine, mitotane; RNA in question. See, for example, Sambrook et al., supra and hormones or related agents, including adrenocorticoster Chapter 7. oids such as prednisone and prednisolone; aminoglutethim 40 Suitable probes for Northern blot hybridization of a given ide; progestins such as hydroxyprogesterone caproate, miR-21 gene product can be produced from their known medroxyprogesterone acetate, megesterol acetate, estrogens nucleic acid sequences, as disclosed herein and in publicly and androgens such as diethylstilbestrol, fluoxymesterone, available databases. Methods for preparation of labeled DNA ethynyl estradiol, antiestrogens such as tamoxifen, and gona and RNA probes, and the conditions for hybridization thereof dotropin-releasing hormone analogs such as leuprolide. 45 to target nucleotide sequences, are described in Sambrook et The methods of the invention may further comprise a step al., Supra. of determining the level of miR-21 transcriptional activity in Probes can be labeled for example with a radionuclide, a a biological sample e.g. cells or tissue, from the Subject. The heavy metal; a ligand capable of functioning as a specific presence or level of at least one miR-21 gene product in the binding pair member for a labeled ligand (e.g., biotin, avidin sample may be indicative that the cancer or tumor or neo 50 or an antibody), a fluorescent molecule, a chemiluminescent plasm is treatable by the methods of the invention. molecule, an enzyme or the like, to high specific activity The level of a miR-21 gene product can be measured in a using methods well known in the art. Autoradiographic biological sample obtained from the Subject. For example, a Detection of hybridization is performed by a means appro tissue sample can be removed from a subject Suspected of priate for the labeling entity e.g. autoradiography for radio having breast cancer by conventional biopsy techniques. In 55 labeled probes; reacting biotinylated probes with biotin-bind another example, a blood sample can be removed from the ing proteins that produce color reactions. subject, and white blood cells can be isolated for DNA extrac In situ hybridization involves depositing whole cells onto a tion by standard techniques. The blood or tissue sample is microscope cover slip and probing the nucleic acid content of preferably obtained from the subject prior to initiation of the cell with a solution containing radioactive or otherwise treatment according to the invention or any other type of 60 labeled nucleic acid probes. This technique is particularly intervention Such as radiotherapy, chemotherapy or other well-suited for analyzing tissue biopsy samples from Subjects therapeutic treatment. A corresponding control tissue or (see e.g. U.S. Pat. No. 5,427,916). blood sample can be obtained from unaffected tissues of the The relative number of miR-21 gene transcripts in cells can Subject, from a normal human individual or population of also be determined by reverse transcription of miR-21 gene normal individuals, or from cultured cells corresponding to 65 transcripts, followed by amplification of the reverse-tran the majority of cells in the subjects sample. The control scribed transcripts by polymerase chain reaction (RT-PCR). tissue or blood sample is then processed along with the The levels of miR-21 gene transcripts can be quantified in US 8,492,133 B2 33 34 comparison with an internal standard, for example, the level The following examples are presented in order to more of mRNA from a “housekeeping gene present in the same fully illustrate some embodiments of the invention. They sample. A Suitable “housekeeping gene for use as an internal should, in no way be construed, however, as limiting the standard includes, e.g., myosin or glyceraldehyde-3-phos broad scope of the invention. phate dehydrogenase (G3PDH). The methods for quantitative 5 RT-PCR and variations thereof are within the skill in the art. In some instances, it may be desirable to simultaneously EXAMPLES determine the expression level of a plurality of different miRNA gene products in a sample. In other instances, it may Example 1 be desirable to determine the expression level of the tran 10 scripts of all known miRNA genes correlated with a cancer. Suitable microarray techniques are known in the art, as Differential Expression of Endogenous miR-21 in described for example in U.S. Pat. Application Publication Cancer Cells and Tissues No. 2008/0306O18. 15 Kits Table 1 summarizes the differential miR-21 expression in The present invention also provides kits containing com cancerous vs. non-cancerous tissues and cells (NS not speci positions of the invention or compositions to implement fied). methods of the invention. In some embodiments, the kits are for therapeutic use and contain multiple dosage units of the TABLE 1 constructs or vectors and instructions for use for administer Fold over ing to a Subject having cancer. Tumoricancer expression In some embodiments, the kits are for prognostic use, for type Method (Mean) Reference example to evaluate the susceptibility of one or more miR-21 Glioblastoma Microarray, Northern 5 expressing target cells to the cytotoxic effects of the construct 25 blots of the invention. real-time PCR 1O-40 6 Individual components of the kit may be provided in con Breast cancer Microarray 1.6 7 In situ hybridization NS 8 centrated amounts; in Some embodiments, a component is Real-time PCR 5 9 provided individually in the same concentration as it would Real-time PCR NS O be in a solution with other components. 30 Microarray 2 1 Negative and/or positive control vectors are included in Hepatocellular Microarray -2 2 carcinomas Small RNA cloning and 4.5 3 some kit embodiments. The control molecules can be used to Sequencing verify transfection efficiency and/or control for transfection real-time PCR >2 4 induced changes in cells. The kit may also include one or Microarray, real-time 1O-45 5 35 PCR more transfection reagent(s) to facilitate delivery of the con Cholangiocarcinoma Northern blots and real- 6-12 6 struct or vector to cells. time polymerase chain The components of the kits may be packaged either in reaction aqueous media or in lyophilized form. The container means Pancreatic endocrine Midroarray NS 7 and acinar tumors of the kits will generally include at least one vial, test tube, Pancreatic cancer Microarray 15.7 8 flask, bottle, Syringe or other container means, into which a 40 In situ hybridization NS 9 component may be placed, and preferably, Suitably aliquoted. Lung, breast, stomach, Microarray NS 2O Where there is more than one component in the kit, the kit also prostate, colon, and pancreatic tumors will generally contain a second, third or other additional Chronic lymphocytic miRNA cloning and -3 21 container into which the additional components may be sepa eukemia real-time-PCR rately placed. However, various combinations of components 45 Osteosarcoma miRNA cloning and NS 22 may be comprised in a vial. The kits of the present invention real-time-PCR, also will typically include a means for containing the nucleic Northern blots Endometrioid ovarian Microarray, Northern 4 23 acids, and any other reagent containers in close confinement carcinoma blots for commercial sale. Such containers may include injection or Diffuse large B cell Microarray 2O 24 blow-molded plastic containers into which the desired vials 50 ymphoma (activated B 25 are retained. cell-like ) Cervical cancer Direct sequencing 5 26 When the components of the kit are provided in one and/or Multiple myeloma NS NS 27 more liquid solutions, the liquid solution is an aqueous solu Microarray, Real-time- 28 tion, with a sterile aqueous solution being particularly pre PCR ferred. 55 Colon cancer Real-time-PCR 5 29 Microarray NS 30 However, the components of the kit may be provided as Microarray NS 31 dried powder(s). When reagents and/or components are pro Real-time-PCR 10-70 32 vided as a dry powder, the powder can be reconstituted by the Head and neck cancer Microarray, Northern NS 33 blots addition of a Suitable solvent, provided in a separate container Esophageal cancer Microarray 7.2 34 CaS. 60 Microarray 3-5 35 The kits may also include components that preserve or Uterine leiomyomas Microarray 1.2-5 36 maintain the constructs and vectors or that protect against its Pituitary tumors Real-time-PCR 2.4 37 degradation. Lung cancer Real time RT-PCR NS 38 Ovarian carcinoma Microarray, Northern 3 39 A kit will also include instructions for employing the kit blots components as well the use of any other reagent not included 65 Bladder cancer Microarray 10 40 in the kit. Instructions may include variations that can be implemented. US 8,492,133 B2 35 36 Example 2 The DNA sequence encoding DT-A, and the corresponding amino acid sequence are provided herein as SEQID NO: 18 Expression of Heterologous Genes Under miR-21 and SEQID NO: 19, respectively. The constructed plasmids Promoter Control containing the miR-21 promoter sequence of SEQID NO:1 or its TATA box of SEQ ID NO: 6 were respectively desig A recombinant vector that expresses the firefly luciferase nated miR-21 Pr-DTA (SEQ ID NO: 9) and miR-21 TATA gene (Luc) under the control of a miR-21 promoter sequence box DT-A (SEQID NO: 13). (miR-21-Luc, SEQ ID NO: 11) was produced by standard Apoptosis levels and protein synthesis rates were deter recombinant procedures, as detailed herein. As a negative mined to confirm the miR-21 promoter-driven expression of control, a vector containing the TATA box of the miR-21 10 DTA. To determine the reduction in de novo protein synthe promoter (TATA-Luc, SEQID NO:20), rather than the miR sis, miR-21 Pr DT-A and a expression vector containing 21 promoter, was prepared. Renilla luciferase under the control of an SV-40 promoter The human hepatocellular carcinoma cell line Huh? was were cotransfected into cells. As demonstrated in FIG. 3, used as a genomic DNA template for amplification of an 15 transfectants of three different cancer cell types (Huh7. miR-21 promoter sequence of SEQ ID NO: 1, using the human hepatocellular carcinoma; MCF7, human breast can following primers: fiv 5'-GGGGTACCGAAGGAGCTC cer; HCT 116, human colon carcinoma) containing miR-21 Pr CGAGTA CATAAAT-3 (SEQ ID NO: 14) and rev DT-A showed strong reduction in luciferase activity, com 5'-CCCAAGCTTCTACTCTGGTATGGCACAAAG 3' pared to the corresponding transfectants containing miR-21 (SEQ ID NO: 15). The PCR product was cloned into the TATA box DT-A, indicating the efficiency of the miR-21 pGL3-Basic (Promega, Madison, Wis.) vector, which carries promoter-driven approach. the firefly luciferase gene. The next set of experiments was performed using a plasmid A DNA sequence corresponding to the TATA box of the in which the nucleic acid sequence encoding the p53 up miR-21 promoter (SEQID NO: 6) was constructed by anneal regulated modulator of apoptosis (PUMA) replaced the Luc ing the following oligonucleotides: fiv 5'-CCTAGTGGT 25 sequence in the plasmids described in Example 2. Thus, the GATAAATGTGGGACTTCTGAGAAGTCAT. plasmid miR-21 prPUMA (SEQ ID NO: 10), having the TCATTTTATTCTTTGTGCC ATACCAGAGTACAA-3' PUMA sequence under the control of the miR-21 promoter (SEQ ID NO: 16) and 5'-TCGAAACATGAGACCATACC sequence of SEQID NO:1 was produced. miR-21 prPUMA GTGTTTCTTATTTTACTTACTGAA and miR-21 Pr DT-A were co-transfected into cells together GAGTCTTCAGGGTGTAAATAGTGGTGATCCCAT G-3 30 with a luciferase expressing plasmid. Forty eight hours post (SEQID NO:17). The resultant fragment was directly cloned transfection the cells were harvested and luciferase activity into pGL3-Basic to yield the vector TATA-Luc (SEQID NO: was determined. Reduction in de novo protein synthesis by 20) DTA and induction of apoptosis by PUMA were observed in To examine the selective expression of heterologous gene cells transfected with the miR-21 promoter driven plasmids, products under miR-21 promoter control in cancerous cells, 35 but not in cells transfected with the TATA box control plas miR-21-Luc and its control TATA-Luc were transfected into mids (FIG. 4A). different transformed and untransformed cells and the A similar experiment was carried out in which a plasmid luciferase expression levels of the transfected cell lines were encoding green fluorescent protein (GFP, SEQ ID NO: 25 compared. was co-transfected together with the miR-21 promoter driven FIG. 2 demonstrates miR-21 promoter-driven expression 40 plasmids (miR-21 PrDT-A or miR-21 prPUMA) or the TATA of the luciferase gene in Chinese hamster ovary cells (CHO) box control plasmids (miR-21 TATA box DTA or miR-21 and the human colorectal cancer cell lines HCT116 and TATA box PUMA). After 48 hours the cells were lysed and COLO320. As can be seen from FIG. 2, the human cancer cell the lysates subjected to Western blot analysis with an anti lines transfected with miR-21-Luc exhibited significantly GFP antibody (FIG. 4B). As seen in FIG. 4B, the GFP signal higher expression of luciferase, as compared to the cancer 45 was completely eliminated in the cotransfection systems with cells transfected with the control vector TATA-Luc. In addi miR-21 DT-A and with miR-21 PUMA, but not in the systems tion, the CHO cells transfected with either vector showed low with the TATA box driven controls. levels of expression of luciferase, similar to the levels exhib ited by the cancer cells transfected with the control vector Example 4 TATA-Luc. These results indicate that heterologous gene 50 products may be expressed at high levels in miR-21-express miR-21 Promoter Driven DT-A Expression Inhibits ing cells, such as certain cancers, using constructs encoding Tumor Formation in Vivo the heterologous gene under the control of an mix-21 pro moter sequence. Nude mice (n-3 in each group) were injected with 1x107 55 HCT116 cells. Two weeks post injection tumor starting size Example 3 was determined. Two weeks following tumor cell injection, endotoxin-free miR-21 PrDTA (25 ug) complexed with the miR-21 Promoter Driven “Killer Gene Expression transfection reagent JETPEITM (Polyplus, France) was injected intratumorly. An additional injection was adminis To determine the ability of regulatory promoter sequences 60 tered 7 days later. Tumor size was recorded at days 0, 7 and 14 of miR-21 to specifically drive the expression of a “killer using a manual caliper. Injection of miR-21-Luc (described in gene', the luciferase coding regions of the reporter and con Example 2) served as a control. In one mouse injected with trol vectors described in Example 2 were replaced with a miR-21 Pr DT-A, the tumor completely disappeared. In nucleic acid sequence encoding the cytotoxic fragment A of another mouse similarly treated with miR-21 Pr DT-A, the diphtheria toxin (DTA). DTA inhibits protein synthesis, 65 tumor growth was inhibited but its size was not reduced. triggers apoptosis and detachment of cells, and has been Overall, the mean fold change for the treated mice was 1 previously used as a "killer gene' for targeting cancer cells. compared to 1.6 in the control mice, as shown in FIG. 5. US 8,492,133 B2 37 38 In additional experiments, the expression level of miR-21 miR-122 in the rodent and human hepatocellular carcinomas. may be examined in cancer cells, such as cell lines of human JCell Biochem, 2006.99(3): p. 671-678. hepatocellular carcinoma, colorectal cancer, and breast can 13. Connolly, E., M. Melegari, P. Landgraf, T. Tchaik cer. Particularly preferred are cancer cells which have shown ovskaya, B. C. Tennant, B. L. Slagle, L. E. Rogler, M. to be susceptible to the cytotoxic effects of the constructs and Zavolan, T. Tuschl, and C. E. Rogler, Elevated expression of vectors of the invention upon transfection and expression of the miR-17-92 polycistron and miR-21 in hepadnavirus-as the anti-cancer agent encoded therein. Determination of Sociated hepatocellular carcinoma contributes to the malig expression levels of miR-21 may be carried out using for nant phenotype. Am J Pathol, 2008. 173(3): p. 856-864. example, real-time RT-PCR. 14. Jiang, J. Y. Gusev, I. 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Ai Zheng, cancer associated with poor disease-free Survival in early 2007. 26(6): p. 561-565. stage disease and high TGF-beta1. Breast Cancer Res Treat, 23. Iorio, M. V., R. Visone, G. Di Leva, V. Donati, F. 2008. 60 Petrocca, P. Casalini, C. Taccioli, S. Volinia, C. G. Liu, H. 11. Yan, L. X., X. F. Huang, Q. Shao, M.Y. Huang, L. Deng, Alder, G. A. Calin, S. Menard, and C. M. Croce, MicroRNA Q. L. Wu, Y. X. Zeng, and J. Y. Shao, MicroRNA miR-21 signatures in human ovarian cancer. Cancer Res, 2007. overexpression in human breast cancer is associated with 67(18): p.8699-8707. advanced clinical stage, lymph node metastasis and patient 24. Lawrie, C. H. S. Soneji, T. Marafioti, C. D. Cooper, S. poor prognosis. RNA, 2008. 14(11): p. 2348-23.60. 65 Palazzo, J. C. Paterson, H. Cattan, T. Enver, R. Mager, J. 12. Kutay, H., S. Bai, J. Datta, T. Motiwala, I. Pogribny, W. Boultwood, J. S. Wainscoat, and C. S. Hatton, MicroRNA Frankel, S. T. Jacob, and K. 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Br J Thorac Cardiovasc Surg, 2008. 135(2): p. 255-260; discus Haematol, 2008. 141(5): p. 672-675. sion 260. 26. Lui, W. O., N. Pourmand, B. K. Patterson, and A. Fire, 36. Wang, T., X. Zhang, L. Obijuru, J. Laser, V. Axis, P. Lee, Patterns of known and novel Small RNAs in human cervical 10 K. Mittal, P. Soteropoulos, and J. J. Wei. A micro-RNA sig cancer. Cancer Res, 2007. 67(13): p. 6031-6043. nature associated with race, tumor size, and target gene activ 27. Loffler, D., K. Brocke-Heidrich, G. Pfeifer, C. Stocsits, ity in human uterine leiomyomas. Genes Chromosomes Can J. Hackermuller, A. K. Kretzschmar, R. Burger, M. Gra cer, 2007. 46(4): p. 336-347. matzki, C. Blumert, K. Bauer, H. Cvijic, A. K. Ullmann, P. F. 37. Amaral, F. C., N. Torres, F. Saggioro, L. Neder, H. R. Stadler, and F. Horn, Interleukin-6 dependent survival of mul 15 Machado, W. A. Silva, Jr., A. C. Moreira, and M. Castro, tiple myeloma cells involves the Stat3-mediated induction of MicroRNAs differentially expressed in ACTH-secreting microRNA-21 through a highly conserved enhancer. Blood, pituitary tumors. J Clin Endocrinol Metab, 2008. 2007. 110(4): p. 1330-1333. 38. Markou, A., E. G. Tsaroucha, L. Kaklamanis, M. Foti 28. Pichiorri, F. S. S. Suh, M. Ladetto, M. Kuehl, T. nou, V. Georgoulias, and E. S. Lianidou, Prognostic value of Palumbo, D. Drandi, C. Taccioli, N. Zanesi, H. Alder, J. P. mature microRNA-21 and microRNA-205 overexpression in Hagan, R. Munker, S. Volinia, M. Boccadoro, R. Garzon, A. non-small cell lung cancer by quantitative real-time RT-PCR. Palumbo, R. I. Aqeilan, and C. M. Croce, MicroRNAs regu Clin Chem, 2008. 54(10): p. 1696-1704. late critical genes associated with multiple myeloma patho 39. Nam, E. J., H. Yoon, S.W. Kim, H. Kim, Y.T. Kim, J. genesis. Proc Natl Acad Sci USA, 2008. 105(35): p. 12885 H. Kim, J. W. Kim, and S. Kim, MicroRNA expression pro 12890. 25 files in serous ovarian carcinoma. Clin Cancer Res, 2008. 29. Slaby, O., M. Svoboda, P. Fabian, T. Smerdova, D. 14(9): p. 2690-2695. Knoflickova, M. Bednarikova, R. Nenutil, and R. VyZula, 40. Neely, L.A., K. M. Rieger-Christ, B.S. Neto, A. Erosh Altered expression of miR-21, miR-31, miR-143 and miR kin, J. Garver, S. Patel, N. A. Phung, S. McLaughlin, J. A. 145 is related to clinicopathologic features of colorectal can Libertino, D. Whitney, and I. C. Summerhayes. A microRNA cer. Oncology, 2007. 72(5-6): p. 397-402. 30 expression ratio defining the invasive phenotype in bladder 30. Chan, S. H., C. W. Wu, A. F. Li, C. W. Chi, and W. C. tumors. Urol Oncol, 2008. Lin, miR-21 microRNA expression in human gastric carci 41. Betel, D., M. Wilson, A. Gabow, D. S. Marks, and C. nomas and its clinical association. Anticancer Res, 2008. Sander, The microRNA.org resource: targets and expression. 28(2A): p. 907-911. Nucl. Acids Res., 2008. 36(suppl. 1): p. D149-153. 31. Schetter, A.J., S.Y. Leung, J. J. Sohn, K. A. Zanetti, E. 35 The foregoing description of the specific embodiments will D. Bowman, N. Yanaihara, S. T. Yuen, T. L. Chan, D. L. so fully reveal the general nature of the invention that others Kwong, G. K. Au, C. G. Liu, G. A. Calin, C. M. Croce, and C. can, by applying current knowledge, readily modify and/or C. Harris, MicroRNA expression profiles associated with adapt for various applications such specific embodiments prognosis and therapeutic outcome in colonadenocarcinoma. without undue experimentation and without departing from JAMA, 2008. 299(4): p. 425-436. 40 the generic concept, and, therefore, such adaptations and 32. Zhang, Z., Z. Li, C. Gao, P. Chen, J. Chen, W. Liu, S. modifications should and are intended to be comprehended Xiao, and H. Lu, miR-21 plays a pivotal role in gastric cancer within the meaning and range of equivalents of the disclosed pathogenesis and progression. Lab Invest, 2008. embodiments. It is to be understood that the phraseology or 33. Tran, N. T. McLean, X. Zhang, C. J. Zhao, J. M. terminology employed herein is for the purpose of descrip Thomson, C. O’Brien, and B. Rose, MicroRNA expression 45 tion and not of limitation. The means, materials, and steps for profiles in head and neck cancer cell lines. Biochem Biophys carrying out various disclosed functions may take a variety of Res Commun, 2007. 358(1): p. 12-17. alternative forms without departing from the invention.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 3 O

SEO ID NO 1 LENGTH: SOf TYPE: DNA ORGANISM: Artificial Sequence FEATURE; OTHER INFORMATION: Synthetic <4 OOs SEQUENCE: 1

tgt attctgg gtaagaagga gotcc.gagta catalaattta toaaagat.ca citat cocaat 60

catct cagaa caagctgtta ctaatgtact ggagtttctg togcaaactgt ctaccataaa 12O

ccatgaaagg attcaaagtt catagttcct tctttgttcc tttgttaatc actgacttct 18O

US 8,492,133 B2 47 48 - Continued gcattcc.caa aatgct citat tittagataga ttaac attaa cca acataat tttttittaga 288O tcqagt cago ataaatttct aagt cagoct c tagt cqtgg ttcatct citt to acctgcat 294 O tittatttggt gtttgtctga agaaaggaaa gaggaaagca aatacgaatt gtact atttg 3 OOO taccaaatct ttgggattica ttggcaaata attt cagtgt gigtigt attat taaatagaaa 3 O 6 O aaaaaattitt gtttcc tagg ttgaaggit ct aattgatacg tttgactitat gatgaccatt 312 O tatgcactitt caaatgaatt tgctttcaaa ataaatgaag agcagctgtc. cittctitt cot 318O cittittaagtg ttcagotgtg gcatgcticag aggttcCtgc tiggattic cag Ctggagcggit 324 O gtgataccct tctttitt cag Ctgttcgtgc citt cotttct tdtgtccacc aaagtggaga 33 OO caaatacatg atctoaaaga tacacagtac c tact taatt coagctgat g g gagaccaaa 3360 gaatttgcaa gtggatggitt tggitat cact gtaaataaaa agagggcct g g gaattcttg 342O cgattic catc tictaaaaaaa aaaaaaaaaa. aaaaaaaaaa aaa. 3463

<210s, SEQ ID NO 4 &211s LENGTH: 72 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic

<4 OOs, SEQUENCE: 4 tgtcgggtag Cttatcagac tatgttgac tttgaatct catggcaa.ca C cagt catg 6 O ggctgtctga Ca 72

<210s, SEQ ID NO 5 &211s LENGTH: 22 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic

<4 OOs, SEQUENCE: 5 tagctitat ca gactgatgtt ga 22

<210s, SEQ ID NO 6 &211s LENGTH: 68 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic

<4 OOs, SEQUENCE: 6 tagtggtgat aaatgtggga cittctgagaa gtcatt catt ttatt ctittg togc catacca 6 O gagtacag 68

<210s, SEQ ID NO 7 &211s LENGTH: 1052 &212s. TYPE: DNA <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: 223 OTHER INFORMATION: Synthetic

<4 OO > SEQUENCE: 7 accgaaggag Ctcc.gagtac atalaattitat caaagat cac tat cocaatc atcto agaac 6 O aagctgttac taatgtactg gagtttctgt gcaaactgtc taccataaac catgaaagga 12 O ttcaaagttc at agttcc tt citttgttcct ttgttaatca citgacttctg act agtggga 18O ggtgcctic cc aagtttgcta atgcattctt tittggataag gatgacgcac agattgtc.ct 24 O

US 8,492,133 B2 65 66 - Continued cc caagctitc tactctggta togg cacaaag 3 O

SEQ ID NO 16 LENGTH: 70 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic SEQUENCE: 16 cctagtggtg ataaatgtgg gacttctgag aagt cattca ttittatt citt tdtgc catac 6 O cagagtacaa 70

SEO ID NO 17 LENGTH: 78 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic

SEQUENCE: 17 tcqaaacatg agaccatacc gtgtttctta ttt tact tac tdaagagtct t cagggtgta 6 O aatagtggtg atcc catg 78

SEQ ID NO 18 LENGTH: 558 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic SEQUENCE: 18 atggg.cgctg atgatgttgt tdatt cittct aaatcttittg tgatggaaaa cittitt citt.cg 6 O taccacggga ctaaacct gg titatgtag at tcc attcaaa alaggtataca aaa.gc.caaaa 12 O tctggtacac aaggaaatta tacgatgat taaagggit tittatag tac cqacaataaa 18O tacgacgctg. c.gggat actic titagataat gaaaaccc.gc t ct ctggaaa agctggaggc 24 O gtggtcaaag tacgt at Co aggactgacg aaggttct cq Cactaaaagt ggataatgcc 3OO gaaact atta agaaagagtt aggtttaagt ct cactgaac cgttgatgga gcaagttctgga 360 acggaagagt ttatcaaaag gttcggtgat ggtgctt.cgc gtgtagt gct cagcc titcc c 42O titcgctgagg ggagttctag cqttgaat at attaataact gggaac aggc gaaag.cgitta 48O agcgtagaac ttgagattaa ttittgaalacc C9tggaaaac gtggccalaga tigcgatgitat 54 O gagtatatgg Ctcaa.gc.c 558

SEQ ID NO 19 LENGTH: 186 TYPE PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthetic SEQUENCE: 19

Met Gly Ala Asp Asp Val Val Asp Ser Ser Lys Ser Phe Wal Met Glu 1. 15 Asn Phe Ser Ser Tyr His Gly Thr Llys Pro Gly Tyr Val Asp Ser Ile 2O 25 3O Gln Lys Gly Ile Gln Lys Pro Llys Ser Gly Thr Glin Gly Asn Tyr Asp 35 4 O 45

Asp Asp Trp Llys Gly Phe Tyr Ser Thr Asp Asn Llys Tyr Asp Ala Ala US 8,492,133 B2 67 68 - Continued

SO 55 6 O

Gly Tyr Ser Val Asp Asn. Glu Asn Pro Leu Ser Gly Ala Gly Gly 65 70 7s 8O

Wall Wall Val Thr Tyr Pro Gly Lieu. Thir Lys Wall Lell Ala Lieu Lys 85 90 95

Wall Asp Asn Ala Glu Thir Ile Llys Lys Glu Lieu. Gly Lell Ser Lieu. Thir 105 11 O

Glu Pro Luell Met Glu Glin Val Gly Thr Glu Glu Phe Ile Arg Phe 115 12 O 125

Gly Asp Gly Ala Ser Arg Val Val Luell Ser Luell Pro Phe Ala Glu Gly 13 O 135 14 O

Ser Ser Ser Val Glu Tyr Ile Asn Asn Trp Glu Glin Ala Ala Lieu 145 150 155 160

Ser Wall Glu Lieu. Glu Ile ASn Phe Glu Thir Arg Gly Arg Gly Glin 1.65 17O 17s

Asp Ala Met Tyr Glu Tyr Met Ala Glin Ala 18O 185

SEQ ID NO 2 O LENGTH: 1752 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthet ic SEQUENCE: 2O

Ctagtggtga taalatgttggg act tctgaga agt catt cat titt at tott t gtgcc at acc 6 O agagtacaag cittggcattc cgg tactgtt ggtaaagcca c catggalaga cgc.caaaaac 12 O ataaagaaag attictato cq Ctggaagatg galacc.gctgg agagcaactg 18O

Catalaggcta tgaagagata cgc.cctggitt Cctggaacaa ttgcttttac agatgcacat 24 O atcgaggtgg a catcactta cgctgagtac titcgaaatgt cc.gttcggitt ggcagaa.gct 3OO atgaaacgat atgggctgaa tacaaatcac agaatcgt.cg tatgcagtga aaact. Ct. Ctt 360

Caattctitta ggg.cgc.gtta tittatcggag ttgcagttgc gcc.cgcgaac gacatttata atgaacgtga attgct caac agtatgggca titt cqcago c taccgtggtg titcgtttcca aaaaggggtt gcaaaaaatt ttgaacgtgc aaaaaaagct CCCaatcatC 54 O

Caaaaaatta titat catgga ttctaaaacg gattacCagg gattt cagtic gatgtacacg titcgtcacat CtcatCtaCC tcc.cggttitt aatgaatacg attttgttgcc agagt ccttic 660 gatagggaca agacaattgc actgat catg aactic ct citg gat ct actgg tctgcctaaa 72 O ggtgtc.gctic tgcct catag aactgcctgc gtgagattct agatcc tatt tittggcaatc aaatcatt CC ggatactg.cg attittaagtg ttgttccatt c catcacggit 84 O tittggaatgt ttact acact cggat atttg atatgtggat titcgagt cqt cittaatgitat 9 OO agatttgaag aagagctgtt tctgaggagc citt caggatt acaagattica aagtgcgctg 96.O

Ctggtgccaa coct attctg cittctitcgcc aaaag cactic tgattgacaa atacgattta totaatttac acgaaattgc ttctggtggc gct coccitct Ctalaggaagt cggggaa.gc.g 108 O gttgccalaga ggttccatct gcc aggtatic aggcaaggat atgggct cac tgagact aca 114 O t cagct attc tgattacacc cgagggggat gataalaccgg taaagttgtt 12 OO c catttitttg aag.cga-aggt tgtggatctg gat accggga aaacgctggg cgittaat caa 126 O agaggcgaac tgttgttgtgag aggtoctatg attatgtc.cg gttatgtaaa Caatc.cggaa 132O US 8,492,133 B2 69 - Continued gcgacCaacg ccttgattga Caaggatgga tggctacatt Ctggaga cat agctt actgg 1380 gacgalagacg alacact tott catcgttgac cgc.ctgaagt citctgattaa gtacalaaggc 144 O tatic aggtgg ctic cc.gctga attggaatcc atc.ttgcticc acac Co. Cala catctitcgac 15OO gCaggtgtc.g caggtott co cgacgatgac gcc.ggtgaac titc.ccgc.cgc cgttgttgtt 1560 ttggagcacg gaaagacgat gacggaaaaa gagat.cgtgg attacgt.cgc Cagt caagta 162O acaa.ccgcga aaaagttgcg cggaggagtt gtgtttgttgg acgaagtacc gaaaggit ctt 168O accggaaaac tcgacgcaag aaaaatcaga gagat cotca taaaggccala galagggcgga 1740 aagat.cgc.cg td 1752

SEQ ID NO 21 LENGTH: 613 TYPE : PRT ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthet ic

<4 OOs, SEQUENCE: 21 Ala Glu Glu Ala Phe Asp Lieu. Trp Asin Glu. Cys Ala Ala Cys Val 1. 5 1O 15

Lell Asp Luell Lys Asp Gly Val Arg Ser Ser Arg Met Ser Wall Asp Pro 25

Ala Ile Ala Asp Thr Asn Gly Glin Gly Val Lieu. His Tyr Ser Met Wall 35 4 O 45

Lell Glu Gly Gly Asn Asp Ala Lieu. Llys Lieu Ala Ile Asp Asn Ala Lieu SO 55 6 O

Ser Ile Thir Ser Asp Gly Lieu. Thr Ile Arg Lieu. Glu Gly Gly Wall Glu 65 70 7s 8O

Pro Asn Pro Val Arg Tyr Ser Tyr Thr Arg Glin Ala Arg Gly Ser 85 90 95

Trp Ser Luell Asn Trp Leu Val Pro Ile Gly His Glu Pro Ser Asn 105 11 O

Ile Wall Phe Ile His Glu Lieu. Asn Ala Gly Asn Glin Luell Ser His 115 12 O 125

Met Ser Pro Ile Tyr Thr Ile Glu Met Gly Asp Glu Lell Luell Ala Lys 13 O 135 14 O

Lell Ala Arg Asp Ala Thr Phe Phe Val Arg Ala His Glu Ser Asn. Glu 145 150 155 160

Met Glin Pro Thir Lieu. Ala Ile Ser His Ala Gly Wall Ser Wall Wal Met 1.65 17O 17s

Ala Glin Thir Glin Pro Arg Arg Glu Lys Arg Trp Ser Glu Trp Ala Ser 18O 185 19 O

Gly Wall Lieu. Cys Lieu. Lieu. Asp Pro Lieu. Asp Gly Wall Asn Tyr 195

Lell Ala Glin Glin Arg Cys Asn Lieu. Asp Asp Thr Trp Glu Gly Lys Ile 21 O 215

Tyr Arg Wall Lieu Ala Gly Asn Pro Ala Lys His Asp Lell Asp Ile Llys 225 23 O 235 24 O

Pro Thir Wall Ile Ser His Arg Lieu. His Phe Pro Glu Gly Gly Ser Luell 245 250 255

Ala Ala Luell Thir Ala His Glin Ala Cys His Lieu. Pro Lell Glu Thir Phe 26 O 265 27 O

Thir Arg His Arg Glin Pro Arg Gly Trp. Glu Gln Lell Glu Glin 27s 285 US 8,492,133 B2 71 - Continued Tyr Pro Val Glin Arg Lieu Val Ala Lieu. Tyr Lieu Ala Ala Arg Lieu. Ser 29 O 295 3 OO Trp Asn Glin Val Asp Glin Val Ile Arg Asn Ala Lieu Ala Ser Pro Gly 3. OS 310 315 32O Ser Gly Gly Asp Lieu. Gly Glu Ala Ile Arg Glu Glin Pro Glu Glin Ala 3.25 330 335 Arg Lieu Ala Lieu. Thir Lieu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg 34 O 345 35. O Glin Gly. Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn Ala Asp Val Val 355 360 365 Ser Lieu. Thir Cys Pro Val Ala Ala Gly Glu. Cys Ala Gly Pro Ala Asp 37 O 375 38O Ser Gly Asp Ala Lieu. Lieu. Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe 385 390 395 4 OO Lieu. Gly Asp Gly Gly Asp Wal Ser Phe Ser Thr Arg Gly. Thr Glin Asn 4 OS 41O 415 Trp Thr Val Glu Arg Lieu. Lieu. Glin Ala His Arg Glin Lieu. Glu Glu Arg 42O 425 43 O Gly Tyr Val Phe Val Gly Tyr His Gly Thr Phe Lieu. Glu Ala Ala Glin 435 44 O 445 Ser Ile Val Phe Gly Gly Val Arg Ala Arg Ser Glin Asp Lieu. Asp Ala 450 45.5 460 Ile Trp Arg Gly Phe Tyr Ile Ala Gly Asp Pro Ala Lieu Ala Tyr Gly 465 470 47s 48O Tyr Ala Glin Asp Gln Glu Pro Asp Ala Arg Gly Arg Ile Arg ASn Gly 485 490 495 Ala Lieu. Leu Arg Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr SOO 505 51O Arg Thir Ser Lieu. Thir Lieu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu 515 52O 525 Arg Lieu. Ile Gly His Pro Lieu Pro Lieu. Arg Lieu. Asp Ala Ile Thr Gly 53 O 535 54 O Pro Glu Glu Glu Gly Gly Arg Lieu. Glu Thir Ile Lieu. Gly Trp Pro Lieu. 5.45 550 555 560 Ala Glu Arg Thr Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg 565 st O sts Asn Val Gly Gly Asp Lieu. Asp Pro Ser Ser Ile Pro Asp Llys Glu Glin 58O 585 59 O Ala Ile Ser Ala Lieu Pro Asp Tyr Ala Ser Glin Pro Gly Llys Pro Pro 595 6OO 605 Arg Glu Asp Lieu Lys 610

<210s, SEQ ID NO 22 &211s LENGTH: 353 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OOs, SEQUENCE: 22 Ala Ala Ala Ser Gly Gly Pro Glu Gly Gly Ser Lieu Ala Ala Lieu. Thr 1. 5 1O 15 Ala His Glin Ala Cys His Leu Pro Leu Glu Thir Phe Thr Arg His Arg 2O 25 3O

Gln Pro Arg Gly Trp Glu Gln Leu Glu Glin Cys Gly Tyr Pro Val Glin US 8,492,133 B2 73 - Continued

35 4 O 45 Arg Lieu Val Ala Lieu. Tyr Lieu Ala Ala Arg Lieu. Ser Trp Asn Glin Val SO 55 6 O Asp Glin Val Ile Arg Asn Ala Lieu Ala Ser Pro Gly Ser Gly Gly Asp 65 70 7s 8O Lieu. Gly Glu Ala Ile Arg Glu Glin Pro Glu Glin Ala Arg Lieu Ala Lieu. 85 90 95 Thir Lieu Ala Ala Ala Glu Ser Glu Arg Phe Val Arg Glin Gly Thr Gly 1OO 105 11 O Asn Asp Glu Ala Gly Ala Ala Asn Gly Pro Ala Asp Ser Gly Asp Ala 115 12 O 125 Lieu. Lieu. Glu Arg Asn Tyr Pro Thr Gly Ala Glu Phe Lieu. Gly Asp Gly 13 O 135 14 O Gly Asp Val Ser Phe Ser Thr Arg Gly Thr Glin Asn Trp Thr Val Glu 145 150 155 160 Arg Lieu. Lieu. Glin Ala His Arg Glin Lieu. Glu Glu Arg Gly Tyr Val Phe 1.65 17O 17s Val Gly Tyr His Gly Thr Phe Leu Glu Ala Ala Glin Ser Ile Val Phe 18O 185 19 O Gly Gly Val Arg Ala Arg Ser Glin Asp Lieu. Asp Ala Ile Trp Arg Gly 195 2OO 2O5 Phe Tyr Ile Ala Gly Asp Pro Ala Lieu Ala Tyr Gly Tyr Ala Glin Asp 21 O 215 22O Glin Glu Pro Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Lieu. Lieu. Arg 225 23 O 235 24 O Val Tyr Val Pro Arg Ser Ser Leu Pro Gly Phe Tyr Arg Thr Ser Leu 245 250 255 Thir Lieu Ala Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Lieu. Ile Gly 26 O 265 27 O His Pro Lieu Pro Lieu. Arg Lieu. Asp Ala Ile Thr Gly Pro Glu Glu Glu 27s 28O 285 Gly Gly Arg Lieu. Glu Thir Ile Lieu. Gly Trp Pro Lieu Ala Glu Arg Thr 29 O 295 3 OO Val Val Ile Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly 3. OS 310 315 32O Asp Lieu. Asp Pro Ser Ser Ile Pro Asp Llys Glu Glin Ala Ile Ser Ala 3.25 330 335 Lieu Pro Asp Tyr Ala Ser Glin Pro Gly Llys Pro Pro Arg Lys Asp Glu 34 O 345 35. O

Lell

<210s, SEQ ID NO 23 &211s LENGTH: 334 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic <4 OOs, SEQUENCE: 23 Met Trp Glu Gln Lieu. Glu Gln Ser Gly Tyr Pro Val Glin Arg Lieu Val 1. 5 1O 15 Ala Lieu. Tyr Lieu Ala Ala Arg Lieu. Ser Trp Asn Glin Val Asp Glin Val 2O 25 3O Ile Arg Asn Ala Lieu Ala Ser Pro Gly Ser Gly Gly Asp Lieu. Gly Glu 35 4 O 45 US 8,492,133 B2 75 - Continued Ala Ile Arg Glu Gln Pro Glu Glin Ala Arg Lieu Ala Lieu. Thir Lieu Ala SO 55 6 O Ala Ala Glu Ser Glu Arg Phe Val Arg Glin Gly Thr Gly Asn Asp Glu 65 70 7s 8O Ala Gly Ala Ala Asn Ala Asp Val Val Ser Lieu. Thir Cys Pro Val Ala 85 90 95 Ala Gly Glu. Cys Ala Gly Pro Ala Asp Ser Gly Asp Ala Lieu. Lieu. Glu 1OO 105 11 O Arg Asn Tyr Pro Thr Gly Ala Glu Phe Lieu. Gly Asp Gly Gly Asp Wall 115 12 O 125 Ser Phe Ser Thr Arg Gly Thr Glin Asn Trp Thr Val Glu Arg Lieu. Leu 13 O 135 14 O Glin Ala His Arg Glin Lieu. Glu Glu Arg Gly Tyr Val Phe Val Gly Tyr 145 150 155 160 His Gly Thr Phe Lieu. Glu Ala Ala Glin Ser Ile Val Phe Gly Gly Val 1.65 17O 17s Arg Ala Arg Ser Glin Asp Lieu. Asp Ala Ile Trp Arg Gly Phe Tyr Ile 18O 185 19 O Ala Gly Asp Pro Ala Lieu Ala Tyr Gly Tyr Ala Glin Asp Glin Glu Pro 195 2OO 2O5 Asp Ala Arg Gly Arg Ile Arg Asn Gly Ala Lieu. Lieu. Arg Val Tyr Val 21 O 215 22O Pro Arg Ser Ser Lieu Pro Gly Phe Tyr Arg Thr Ser Lieu. Thir Lieu Ala 225 23 O 235 24 O Ala Pro Glu Ala Ala Gly Glu Val Glu Arg Lieu. Ile Gly His Pro Lieu. 245 250 255 Pro Lieu. Arg Lieu. Asp Ala Ile Thr Gly Pro Glu Glu Glu Gly Gly Arg 26 O 265 27 O Lieu. Glu Thir Ile Lieu. Gly Trp Pro Lieu Ala Glu Arg Thr Val Val Ile 27s 28O 285 Pro Ser Ala Ile Pro Thr Asp Pro Arg Asn Val Gly Gly Asp Lieu. Asp 29 O 295 3 OO Pro Ser Ser Ile Pro Asp Llys Glu Glin Ala Ile Ser Ala Lieu Pro Asp 3. OS 310 315 32O Tyr Ala Ser Glin Pro Gly Llys Pro Pro Arg Glu Asp Lieu Lys 3.25 330

<210s, SEQ ID NO 24 &211s LENGTH: 361 212. TYPE: PRT <213> ORGANISM: Artificial Sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic

<4 OOs, SEQUENCE: 24 Gly Gly Ser Lieu Ala Ala Lieu. Thir Ala His Glin Ala Cys His Lieu Pro 1. 5 1O 15 Lieu. Glu Thr Phe Thr Arg His Arg Gln Pro Arg Gly Trp Glu Gln Leu 2O 25 3O Glu Gln Cys Gly Tyr Pro Val Glin Arg Lieu Val Ala Lieu. Tyr Lieu Ala 35 4 O 45 Ala Arg Lieu. Ser Trp Asn Glin Val Asp Glin Val Ile Arg Asn Ala Lieu SO 55 6 O Ala Ser Pro Gly Ser Gly Gly Asp Lieu. Gly Glu Ala Ile Arg Glu Glin 65 70 7s 8O

Pro Glu Glin Ala Arg Lieu Ala Lieu. Thir Lieu Ala Ala Ala Glu Ser Glu US 8,492,133 B2 77 78 - Continued

85 90 95

Arg Phe Wall Arg Glin Gly Thr Gly Asn Asp Glu Ala Gly Ala Ala Asn 105 11 O

Ala Asp Wall Val Ser Lieu. Thr Cys Pro Wall Ala Ala Gly Glu Cys Ala 115 12 O 125

Gly Pro Ala Asp Ser Gly Asp Ala Luell Luell Glu Arg Asn Pro Thir 13 O 135 14 O

Gly Ala Glu Phe Lieu. Gly Asp Gly Gly Asp Wall Ser Phe Ser Thr Arg 145 150 155 160

Gly Thir Glin Asn Trp Thr Val Glu Arg Luell Luell Glin Ala His Arg Glin 1.65 17O 17s

Lell Glu Glu Arg Gly Tyr Val Phe Wall Gly His Gly Thir Phe Lieu. 18O 185 19 O

Glu Ala Ala Glin Ser Ile Wall Phe Gly Gly Wall Arg Ala Arg Ser Glin 195

Asp Luell Asp Ala Ile Trp Arg Gly Phe Ile Ala Gly Asp Pro Ala 21 O 215

Lell Ala Gly Tyr Ala Glin Asp Glin Glu Pro Asp Ala Arg Gly Arg 225 23 O 235 24 O

Ile Arg Asn Gly Ala Lieu. Lieu. Arg Wall Tyr Wall Pro Arg Ser Ser Luell 245 250 255

Pro Gly Phe Tyr Arg Thr Ser Leu Thir Luell Ala Ala Pro Glu Ala Ala 26 O 265 27 O

Gly Glu Wall Glu Arg Lieu. Ile Gly His Pro Luell Pro Lell Arg Lieu. Asp 285

Ala Ile Thir Gly Pro Glu Glu Glu Gly Gly Arg Lell Glu Thir Ile Lieu. 29 O 295 3 OO

Gly Trp Pro Lieu Ala Glu Arg Thr Wall Wall Ile Pro Ser Ala Ile Pro 3. OS 310 315 32O

Thir Asp Pro Arg Asn Val Gly Gly Asp Luell Asp Pro Ser Ser Ile Pro 3.25 330 335

Asp Glu Glin Ala Ile Ser Ala Luell Pro Asp Ala Ser Gln Pro 34 O 345 35. O

Gly Pro Pro Arg Glu Asp Lieu 355 360

SEO ID NO 25 LENGTH: 720 TYPE: DNA ORGANISM: Artificial Sequence FEATURE: OTHER INFORMATION: Synthet ic <4 OOs, SEQUENCE: 25 atggctagda aaggagaaga act t t t cact ggagttgtc.c caattcttgt tgaattagat 6 O ggtgatgtta atgggcacaa attittctgtc agtggagagg gtgaaggtga tgctacatac 12 O ggaaagctta CCCttalaatt tatttgcact actggaaaac tacctgttcc atggccaa.ca 18O cittgtcacta citt tot citta tggtgttcaa tgcttitt coc gttatcc.gga t catatgaaa 24 O cggcatgact ttittcaagag tgc catgc cc gaaggittatg tacaggaacg Cactatatot 3OO ttcaaagatg acgggalacta Caagacgcgt. gctgaagtica agtttgaagg tgatacc citt 360 gttaatcgta tcq agttaaa agg tattgat tittaaagaag atggaaacat t ct cqgacac aaacticgagt acalactataa. Ctcacacaat gtata catca cggcagacaa acaaaagaat 48O ggaatcaaag Ctalactitcaia aatticgc.cac aac attgaag atggat.ccgt. t caactagoa 54 O

US 8,492,133 B2 91 92 - Continued a catctgtgt gttggtttitt tdtgtgaatc gatag tacta acatacgctic ticcatcaaaa 366 O caaaacga aa caaaacaaac tagcaaaata ggctgtc.ccc agtgcaa.gtg Caggtgc.ca.g 372 O aacatttctic tat cqata 3.738

The invention claimed is: 10 3. An isolated host cell comprising the vector of claim 2. 1. A nucleic acid construct comprising a miR-21 promoter 4. A pharmaceutical composition comprising as an active sequence and at least one nucleic acid sequence encoding an ingredient the construct of claim 1, and at least one pharma anti-cancer agent, wherein the nucleic acid sequence encod ceutically acceptable carrier, excipient or diluent. ing the anti-cancer agent is operably linked to the miR-21. said construct comprising the sequence as set forth in SEQID 5. Akit comprisingi) one or more dosage units of the vector NO: 7. of claim 2; and ii) instructions for administering said nucleic 2. A vector comprising the nucleic acid construct of claim acid construct to a Subject in need thereof. 1, wherein the vector has the nucleic acid sequence of SEQID NO: 9.