US 2010O261781A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2010/0261781 A1 Gmeiner (43) Pub. Date: Oct. 14, 2010

(54) MULTIVALENT APTAMER COMPLEXES Publication Classification (51) Int. Cl. (76) Inventor: William H. Gmeiner, Yadkinville, A63L/7088 (2006.01) NC (US) C7H 2L/04 (2006.01) C7H 2L/02 (2006.01) Correspondence Address: 44% 388: } MYERS BIGELSIBLEY & SAJOVEC (52) U.S. Cl...... 514/44 R; 536/23.1:435/325 POBOX37428 RALEIGH, NC 27627 (US) (57) ABSTRACT A compound of the formula A-B-C, is provided, wherein: A is (21) Appl. No.: 12/759,216 a first nucleic acid that specifically binds to an extracellular surface protein expressed by a cell of interest, B is an alkyl linker; and C is a second nucleic acid that hybridizes to a (22) Filed: Apr. 13, 2010 complementary nucleic acid. In some embodiments, the first nucleic acid is anaptamer. In some embodiments, the nucleic Related U.S. Application Data acid comprises an active compound, particularly cytotoxic nucleotides such as poly-FdUMP. Compositions and methods (60) Provisional application No. 61/169,058, filed on Apr. ofusing Such compounds for treating and/or detecting cancer 14, 2009. are also described. Patent Application Publication Oct. 14, 2010 Sheet 1 of 6 US 2010/0261781 A1

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FIGURES Aptamer Rotation

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Aptamer Rotation Aptamer 2 Aptamer 1

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FIGURE 8

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PC3-PSMA US 2010/0261781 A1 Oct. 14, 2010

MULTIVALENT APTAMER COMPLEXES 0008 A further aspect of the invention is the use of a composition as described above, and further herein, for treat RELATED APPLICATIONS ing and/or detecting cancer, or for the preparation of a medi 0001. This application claims the benefit of U.S. Provi cament for treating and/or detecting cancer. sional Patent Application Ser. No. 61/169,058, filed Apr. 14, 0009. A further aspect of the invention is a method of 2009, the disclosure of which is incorporated by reference introducing a nucleic acid of interest into a cell of interest, herein in its entirety. comprising contacting a composition as described above, and further herein, to the cell under conditions in which the GOVERNMENT SUPPORT nucleic acid of interest is internalized into the cell. In some embodiments the method is carried out in vitro; in other 0002 This invention was made with government support embodiments the method is carried out in vivo. For example, under grants from the Department of Defense and the the cell of interest may be a cancer cell in a subject afflicted National Institutes of Health. The government has certain with cancer (e.g., prostate cancer), and the contacting is car rights to this invention. ried out by administering the composition to the Subject in an amount effective to treat or detect the cancer. FIELD OF INVENTION 0003. The present invention concerns chemotherapeutic BRIEF DESCRIPTION OF THE DRAWINGS molecules and compositions thereof, and methods of use thereof for the treatment of cancer. 0010 FIG. 1. Fluorescence microscopy images of the PSMAO1 DNA aptamer binding to (A) C4-2 cells; (B) BACKGROUND OF THE INVENTION LNCaP cells; (C)PC3 cells. (D) Binding of the A10-3 RNA aptamerto C4-2 cells. Live cells were incubated in PBS with 0004 Cancer is the second-leading cause of death in the 1x10 M Rhodamine-conjugated aptamer for 2 hat room United States and is a serious public health concern. The temperature. Cells were fixed with 3.7% formaldehyde for 2 current generation of cytotoxic chemotherapeutic agents used min prior to visualization using an Olympus inverted micro for the treatment of cancer is not curative for a majority of Scope. patients. For many cancer patients, the use of chemotherapy 0011 FIG. 2. Depiction of dimeric aptamer complex for extends patient-life by only a few months and often results in mation through Watson-Crick base pair formation. PSMA01 serious side effects that reduce the quality of life. aptamers were synthesized with either a dA16 or dT16 tail 0005 Anticancer drugs that are utilized for cancer chemo and dimers were formed upon annealing the individual therapy include cytotoxic nucleoside analogs (Pratt et al., aptamer conjugates in a 1:1 ratio. Antimetabolites’ in The Anticancer Drugs, 2" ed. Oxford 0012 FIG. 3. Fluorescence microscopy images of (A) a University Press, New York. pp. 69-107 (1994)), such as monomeric aptamer conjugate of PSMA01 with a dT16 tail; analogs of the four nucleotides that are the principal compo (B) a dimeric aptamer complex consisting of 1:1 ratio of nents of DNA. Examples of cytotoxic analogs include the monomeric PSMA01 conjugates with dA16 and dT16 tails; fluoropyrimidines (FPS) such as 5FU and FdU, which are (C) the J591 mAb. All images were obtained using live C4-2 analogs of Ura and dU, the precursor for dT, the arabinosyl cells. For(A) and (B) cells were prepared as described in FIG. nucleotides AraC and AraA, which are analogs of dC and dA, 4. For (C), live cells were incubated with J591, fixed with respectively, dFdC (gemcitabine), which is an analog of dC. formalin, permeabilized with 0.5% Triton-X prior to addition and 6-mercaptopurine, which is an analog of dI, the precursor of secondary antibody (goat anti-mouse) and post-fixed with of dG. formalin prior to visualization. Yellow arrows point to inter nalized signal. SUMMARY OF THE INVENTION 0013 FIG. 4. Split x-y images using confocal microscopy 0006. A first aspect of the invention is a compound of the to detect two fluorescent dyes (Quasar 670 and Quasar 570). formula A-B-C, wherein: A is a first nucleic acid that specifi The PSMAO1 aptamer with the dA16 tail was labeled with cally binds to an extracellular Surface protein expressed by a Quasar 670 while the PSMA01 aptamer with the dT16 tail cell of interest, B is an alkyl linker; and C is a second nucleic was labeled with Quasar 570. The unconjugated PSMA01 acid that hybridizes to a complementary nucleic acid. In some aptamer was also labeled with Quasar 570. In each panel of embodiments, the first nucleic acid is an aptamer. In some four images is shown: (A) Quasar 670 image; (B) Quasar 570 embodiments, the first nucleic acid is from 30 to 150 nucle image; (C) overlay of (A) and (B); (D) Nomarski image. otides in length. In some embodiments, the alkyl linker com (Left) The PSMA01 monomeric aptamer is shown in the prises C2-C6 loweralkyl. In some embodiments, the second leftmost panel; (Center) Dimericaptamer complex consisting nucleic acid is from 8 to 100 nucleotides in length. In some of a 1:1 stoichiometry of PSMA01 aptamers with dA16 and embodiments, the cell of interest is a cancer cell, microbial dT16 tails; (Right) Dimeric Aptamer Complexes that also cell, or parasite cell. In some embodiments, the nucleic acid contain a flexible linker (see FIG. 5). The dimeric aptamer comprises an active compound, particularly cytotoxic nucle complex has greater internalized signal relative to the mono otides such as poly-FdUMP. meric aptamer. Inclusion of the alkyl spacer results in 0007. A second aspect of the present invention is a com enhanced cellular internalization relative to dimeric com position comprising a pair of compounds as described above plexes that do not have alkyl spacer (see FIG.9). Arrows point and further herein, each member of the pair having a second to internalized aptamer. nucleic acid that is complementary to and hybridized to the 0014 FIG. 5. Inclusion of flexible alkyl spacers (denoted second nucleic acid of the other member of the pair. The by “X”) in dimeric aptamer complexes. The flexible linkers composition may be provided in a pharmaceutically accept allow eachaptamer to rotate relative to the linker construct to able carrier. obtain maximal binding affinity. US 2010/0261781 A1 Oct. 14, 2010

0015 FIG. 6. Flow cytometry evaluation of the binding of 0020. The disclosures of all Patent references cited herein fluorescently labeled aptamers and dimeric aptamer com are incorporated herein by reference in their entirety. plexes with and without flexible linkers to C4-2 cells. Aptamer labeling with Quasar 570 and Quasar 670 was done 1. DEFINITIONS as described in FIG. 8. The mean fluorescence intensity for 0021 Aptamer(s) or “aptamer sequence(s) as used the monomeric aptamer is right-shifted relative to back herein are meant to refer to single stranded nucleic acids ground. Monitoring of Quasar 670 fluorescence clearly (RNA or DNA) whose distinct nucleotide sequence deter shows the dimeric complex shifted relative to background. mines the folding of the molecule into a unique three dimen Introduction of flexible linkers further increases the mean sional structure. Aptamers comprising 15 to 120 nucleotides fluorescence intensity. The results are consistent with dimeric can be selected in vitro from a randomized pool of oligonucle aptamer complexes having enhanced cellular binding relative otides (10.sup. 14-10.sup.15 molecules). The “aptamers or to monomeric aptamers and with flexible linkers further aptamer sequences’ comprise a sequence (sometimes a enhancing cell binding. degenerate or random sequence), and can further comprise fixed sequences flanking that sequence. The term “aptamer 0016 FIG. 7. Plot of tumor size versus time for PC3 as used herein further contemplates the use of both native and Xenografts in nude mice. Data are shown for four treatment modified DNA and RNA bases, e.g. beta-D-Glucosyl-Hy groups (n=8 in each group): 1) control 1: 2) Fall JMP 10:3) droxymethyluracil. See, e.g., U.S. Pat. No. 7,329.742. control 2; 4) 5-FU. The FdUMP10 and 5FU studies were 0022 "Detectable compounds' as used herein include, but done consecutively (rather than concurrently) so that two are not limited to, radiolabels (e.g., S, 'I, ‘P, H, C, control groups were required. Tumors from animals treated ''I), enzyme labels (e.g., horseradish peroxidase, alkaline with FdUMP10 displayed statistically significant reduced phosphatase), gold beads, chemiluminescence labels, ligands growth throughout the study while 5FU did not reduce tumor (e.g., biotin, digoxin) and/or fluorescence labels (e.g., growth rates relative to control. rhodamine, phycoerythrin, fluorescein), a fluorescent protein 0017 FIG. 8. (Top) Western blot demonstrating expres including, but not limited to, green fluorescent protein or one Sion of PSMA in PSMA-transduced PC3 cells but not in of its many modified forms, a nucleic acid segment in accor mock-transfected PC3 cells or PC3 cells obtained from dance with known techniques, and energy absorbing and ATCC. (Bottom)—Confocal microscopy images demonstrat energy emitting agents. ing a lack of binding by both the J591 mAb and the dimeric 0023 Active compound as used herein includes, but is aptamer complex to PSMA-PC3 cells (top panels). Signifi not limited to, cytotoxic nucleosides or nucleotides, antisense cant surface binding is observed for both the dimericaptamer oligonucleotides, radionuclides, energy absorbing and complex and the mab to the PSMA+PC3 cells (bottom pan energy emitting agents, and other cytotoxic agents. Other cytotoxic agents include, but are not limited to, ricin (or more els). particularly the ricin A chain), aclacinomycin, diphtheria toxin, Monensin, Verrucarin A, Abrin, Tricothecenes, and DETAILED DESCRIPTION OF THE PREFERRED Pseudomonas exotoxin A, taxol, cytochalasin B, gramicidin EMBODIMENTS D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, anti-mitotic agents such as the Vinca alkaloids 0018. The present invention is explained in greater detail (e.g., Vincristine and vinblastine), colchicin, anthracyclines below. This description is not intended to be a detailed catalog Such as doxorubicin and daunorubicin, dihydroxy anthracin of all the different ways in which the invention may be imple dione, mitoxantrone, mithramycin, actinomycin D, 1-dehy mented, or all of the features that may be added to the instant drotestosterone, glucocorticoids, procaine, tetracaine, invention. For example, features illustrated with respect to lidocaine, propranolol, and puromycin and analogs or one embodiment may be incorporated into other embodi homologs thereof, antimetabolites (e.g., methotrexate, ments, and features illustrated with respect to a particular 6-mercaptopurine, 6-thioguanine, cytarabine, and 5-fluorou embodiment may be deleted from that embodiment. In addi racil decarbazine), alkylating agents (e.g., mechlorethamine, tion, numerous variations and additions to the various thioepa chlorambucil, melphalan, carmustine (BSNU), embodiments Suggested herein will be apparent to those lomustine (CCNU), cyclothosphamide, busulfan, dibromo skilled in the artin light of the instant disclosure which do not mannitol, Streptozotocin, mitomycin C, and cis-dichlorodi depart from the instant invention. Hence, the following speci amine platinum(II) (DDP)), and antibiotics, including but not fication is intended to illustrate Some particular embodiments limited to, dactinomycin (formerly actinomycin), bleomycin, of the invention, and not to exhaustively specify all permuta mithramycin, calicheamicin, and anthramycin (AMC). tions, combinations and variations thereof. 0024 “Cytotoxic nucleoside or nucleotide' as used herein 0019. As used in the description of the invention and the includes, but is not limited to, 2,2'-difluorodeoxycytidine, appended claims, the singular forms “a”, “an and “the are (dFdC, gemcitabine), 5-fluorouracil (5-FU), 5-fluoro-2'- intended to include the plural forms as well, unless the con deoxyuridine-5'-O-monophosphate (FdUMP), 5-fluoro-2'- text clearly indicates otherwise. Furthermore, the term deoxyuridine (FdU), arabinosylcytosine (Ara-C), arabinosyl “about as used herein when referring to a measurable value adenosine (Ara-A), fluorouracil arabinoside, mercaptopurine Such as an amount of a compound, dose, time, temperature, riboside, 5-aza-2'-deoxycytidine, arabinosyl 5-azacytosine, and the like, is meant to encompass variations of 20%, 10%, 6-azauridine, azaribine, 6-azacytidine, trifluoro-methyl-2'- 5%, 1%, 0.5%, or even 0.1% of the specified amount. Also, as deoxyuridine, thymidine, thioguanosine, 3-deaZautidine, used herein, “and/or refers to and encompasses any and all 2-Chloro-2'-deoxyadenosine (2-CdA), AZT (azidothymi possible combinations of one or more of the associated listed dine), 2',3'-dideoxyinosine (ddI), cytotoxic nucleoside-corti items, as well as the lack of combinations when interpreted in costeroid phosphodiester, 5-bromodeoxyuridine 5'-meth the alternative (“or”). ylphosphonate, 5-fluorodeoxyuridine (FdUrd), fludarabine US 2010/0261781 A1 Oct. 14, 2010

(2-F-ara-AMP), 6-mercaptopurine and 6-thioguanine, rial encoding contrast agents include, but are not limited to, 2-chlorodeoxyadenosine (CdA), 2'-deoxycoformycin (pen paramagnetic reporter genes such as ferredoxin; paramag to statin), 4'-thio-beta-D-arabinofuranosylcytosine, and any netic tag(s) on liposomal lipids such as paramagnetic chelat other cytotoxic dA, dC, dT, dG, dU, or homologs thereof. ing groups added to PEG, detectable probes; and luciferin/ 0025 Antisense oligonucleotide.” as used herein, refers luciferase reporter system. to a nucleic acid that is complementary to and specifically 0028 “Nucleic acid' as used herein refers to single- or hybridizes to a specified DNA or RNA sequence. Antisense double-stranded molecules which may be deoxyribonucleic oligonucleotide includes, but is not limited to, ribozymes, acid (DNA), ribonucleic acid (RNA), or homologs thereof small interfering RNAs, short hairpin RNAs, micro RNAs, such as peptide nucleic acid (PNA), which is comprised of triplex-forming oligonucleotides, and/or PNAS. Antisense stretches of nucleic acid polymers linked together by peptide oligonucleotides and nucleic acids that encode the same can linkers, or a combination thereof. The nucleic acid may rep be made in accordance with conventional techniques. See, resent a coding strand or its complement. The nucleic acids of e.g., U.S. Pat. No. 5,023.243 to Tullis; U.S. Pat. No. 5,149, this invention may be comprised of any combination of natu 797 to Pederson et al. Those skilled in the art will appreciate rally-occurring nucleosides (A, G, C, TU), and/or the nucleic that it is not necessary that the antisense oligonucleotide be acids may comprise nucleoside or nucleotide analogs and/or fully complementary to a target sequence, as long as the derivatives as are well known in the art, including cytotoxic, degree of sequence similarity is sufficient for the antisense synthetic, rare, non-natural bases or altered nucleotide bases. nucleotide sequence to specifically hybridize to its target and A nucleic acid molecule in the form of a polymer of DNA may reduce production of the polypeptide (e.g., by at least about be comprised of one or more segments of cDNA, genomic 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more). DNA or synthetic DNA. In addition, a modification can be 0026 “Radionuclide' as described herein may be any incorporated to reduce exonucleolytic degradation, such as a radionuclide Suitable for delivering a therapeutic dosage of reverse (3'->5') linkage at the 3'-terminus. radiation to a tumor or cancer cell, including, but not limited 0029. “Cell of interest” as used herein may be any suitable to,225Ac, 227Ac, 21. At 13 Ba.77Br 10°Cd, 5 Cr,67Cu, 165Dy, cell, including but not limited to cancer cells, tissue cells 155Eu, 153Gd, Au, 16Ho, 113In, 115min, 123, 125I, 13 II, generally (e.g., muscle, bone, nerve, liver, lung, etc.), patho 189Ir, 191 Ir, 192Ir, 194Ir, 52Fe, 55 Fe, Fe, 77Lu, 109Pd, 32p, logical and non-pathological microbial cells (e.g., bacterial, 226Ra, 186Re, 188Re, SSm, 46Sc, *7Sc, 72Se, 7Se, Ag, mycobacterial, spirochetal rickettsial, chlamydial, mycoplas 89Sr. 35S, 177Ta, '''mSn, 12'Sn, 166Yb, 169Yb. 90Y, 212Bi, mal, and fungal, etc.), parasitic cells (e.g., protozoal, helm 213Bi, 19Sb, 7Hg, 7Ru, 100Pd, 10 Rh. and 212Pb. inth, etc.), and plant cells, etc. 0027 “Energy absorbing and energy emitting agent as 0030 "Cancer cell as used herein may be any cancer cell, used herein includes, but is not limited to, diagnostic agents, including, but not limited to, lung, colon, ovarian, prostate, contrast agents, iodinated agents, radiopharmaceuticals, fluo bone, nerve, liver, leukemia, and lymphoma cells. rescent compounds and fluorescent compounds coencapsu 0031 “Bacterial cell as used herein may be any bacterial lated with a quencher, agents containing MRS/MRI sensitive cell including, but not limited to, Gram-negative bacteria, nuclides, genetic material encoding contrast agents, and Gram-positive bacteria and other bacteria. energy absorbing and heat emitting nanomaterials including, 0032 Examples of Gram-negative bacteria include, but but not limited to, single-walled nanotubes and gold nano are not limited to, bacteria of the genera, Salmonella, Escheri cages. Some examples of contrast agents include, but are not chia, Klebsiella, Haemophilus, Pseudomonas, Proteus, Neis limited to, metal chelates, polychelates, multinuclear cluster seria, Vibro, Helicobacter, Brucella, Bordetella, Legionella, complexes (U.S. Pat. No. 5,804,161), halogenated Xanthene Campylobacter, Francisella, Pasteurella, Yersinia, Bar or a functional derivative of a halogenated Xanthene (U.S. Pat. tonella, Bacteroides, Streptobacillus, Spirillum, Moraxella No. 6,986,740), gadolinium-diethylenetriaminepentaacetic and Shigella. Furthermore, bacterial cell of interest includes acid (gadopentetate dimeglumine, GdDTPA; Magnavist), Gram-negative bacteria including, but not limited to, Escheri gadoteridol (ProHance), gadodiamide, gadoterate meglu chia coli, Pseudomonas aeruginosa, Neisseria meningitides, mine (Gd-DOTA), gadobenate dimeglumine (Gd-BOPTA/ Neisseria gonorrhoeae, Salmonella typhimurium, Salmo Dimeg; MultiHance), mangafodipir trisodium (Mn-DPDP), nella entertidis, Klebsiella pneumoniae, Haemophilus influ ferumoxides, paramagnetic analogue of doxorubicin, and enzae, Haemophilus ducreyi, Proteus mirabilis, Vibro chol ruboxyl (Rb). Some examples of iodinated agents include, but era, Helicobacter pylori, Brucella abortis, Brucella are not limited to, diatrizoate (3,5-di(acetamido)-2,4,6-tri melitensis, Brucella suis, Bordetella pertussis, Bordetella iodobenzoic acid), iodipamide (3.3'-adipoyl-diimino-di(2.4. parapertussis, Legionella pneumophila, Campylobacter 6-triiodobenzoic acid), acetrizoate 3-acetylamino-2,4,6-tri fetus, Campylobacter jejuni, Francisella tularensis, Pas iodobenzoic acid. aminotrizoate 3-amino-2,4,6- teurella multocida, Yersinia pestis, Bartonella bacilliformis, triiodobenzoic acid), and iomeprol. Examples of Bacteroides fragilis, Bartonella henselae, Streptobacillus radiopharmaceuticals include, but are not limited to, fluorine moniliformis, Spirillum minus, Moraxella catarrhalis (Bra 18 fluorodeoxyglucose (18FFDG), Tc-99m Depreotide, inhamella catarrhalis), and Shigella dysenteriae. carbon-11 hydroxyephedrine (HED), 18Fsetoperone, me 0033 Examples of Gram-positive bacteria include, but are thyl-11C thymidine, 99 mTc-hexamethyl propyleneamine not limited to, bacteria of the genera Listeria, Staphylococ oxime (HMPAO), 99mTc-L,L-ethylcysteinate dimer (ECD), cus, Streptococcus, Bacillus, Corynebacterium, Peptostrep 99mTc-sestamibi, thallium 201, I-131 metaiodobenzylguani tococcus, and Clostridium. Furthermore, bacterial cell of dine (MIBG), 123I N-isopropyl-p-iodoamphetamine interest includes Gram-positive bacteria including, but not (IMP), 99 mTc-hexakis-2-methoxyisobutylisonitrile 99 limited to, Listeria monocytogenes, Staphylococcus aureus, mTc-tetrofosmin. Examples of agents containing MRS/MRI Streptococcus pyogenes, Streptococcus pneumoniae, Bacil sensitive nuclides include, but are not limited to, perfluoro lus cereus, Bacillus anthracis, Clostridium botulinum, carbons and fluorodeoxyglucose. Examples of genetic mate Clostridium perfingens, Clostridium difficile, Clostridium US 2010/0261781 A1 Oct. 14, 2010 tetani, Corynebacterium diphtherias, Corynebacterium Trypanosoma, Babesia, Naegleria, Acanthamoeba, Bala ulcerans, and Peptostreptococcus anaerobius. muthia, Enterobius, Strongyloides, Ascaradia, Trichuris, 0034 Additional bacteria include bacterial genera includ Necator; Ancylostoma, Uncinaria, Onchocerca, Mesoces ing, but not limited to, Actinomyces, Propionibacterium, toides, Echinococcus, Taenia, Diphylobothrium, Hymenolep Nocardia and Streptomyces. Furthermore, bacterial cell of sis, Moniezia, Dicytocaulus, Dirofilaria, Wuchereria, Brugia, interest of the present invention includes, but is not limited to, Toxocara, Rhabditida, Spirurida, Dicrocoelium, Clonorchis, Actinomyces israeli, Actinomyces gerencseriae, Actinomyces Echinostoma, Fasciola, Fascioloides, Opisthorchis, Para viscosus, Actinomyces naeslundii, Propionibacterium propi gonimus, and Schistosoma. Additionally, parasitic cell of the onicus, Nocardia asteroides, Nocardia brasiliensis, Nocardia present invention includes, but is not limited to, Entamoeba Otitidiscaviarum and Streptomyces somaliensis. histolytica, Dientamoeba fragilis, Giardia lamblia, Balan 0035 “Mycobacterial cell” as used herein may be any tidium coli, Trichomonas vaginalis, Cryptosporidium par mycobacterial cell, including but not limited to mycobacteria vum, Isospora belli, Plasmodium malariae, Plasmodium belonging to the mycobacteria families including, but not ovale, Plasmodium falciparum, Plasmodium vivax, Leishma limited to, Mycobacteriaceae. Additionally, mycobacterial nia braziliensis, Leishmania donovani, Leishmania tropica, cell of the present invention includes, but is not limited to, Trypanosoma Cruzi, Trypanosoma brucei, Babesia divergens, Mycobacterium tuberculosis, Mycobacterium leprae, Myco Babesia microti, Naegleria fowleri, Acanthamoeba culbert bacterium avium-intracellulare, Mycobacterium kansasii, soni, Acanthamoeba polyphaga, Acanthamoeba castellanii, and Mycobacterium ulcerans. Acanthamoeba astronyxis, Acanthamoeba hatchetti, Acan 0036 “Spirochetal cell as used herein may be any spiro thamoeba rhysodes, Balamuthia mandrillaris, Enterobius chetal cell, including but not limited to spirochetes belonging vermicularis, Strongyloides Stercoralis, Strongyloides fille to the genera including, but not limited to, Treponema, Lep borni, Ascaris lumbricoides, Trichuris trichiura, Necator to spira, and Borrelia. Additionally, spirochetal cell of the americanus, Ancylostoma duodenale, Ancylostoma ceylani present invention includes, but is not limited to, Treponema cum, Ancylostoma braziliense, Ancylostoma Caninum, Unci palladium, Treponema pertenue, Treponema carateum, Lep naria Stenocephala, Onchocerca volvulus, Mesocestoides to spira interrogans, Borrelia burgdorferi, and Borrelia variabilis, Echinococcus granulosus, Taenia solium, Diphy recurrentis. lobothrium latum, Hymenolepis nana, Hymenolepis 0037 "Rickettsial cell as used herein may be any rickett diminuta, Moniezia expansa, Moniezia benedeni, Dicytocau sial cell, including but not limited to rickettsia belonging to lus viviparous, Dicytocaulus filarial, Dicytocaulus arnfieldi, the genera including, but not limited to, Rickettsia, Ehrlichia, Dirofilaria repens, Dirofilaria immitis, Wuchereria bancrofti, Orienta, Bartonella and Coxiella. Furthermore, rickettsial Brugia malayi, Toxocara Canis, Toxocara Cati, Dicrocoelium cell includes, but is not limited to, Rickettsia rickettsii, Rick dendriticum, Clonorchis sinensis, Echinostoma, Echinos ettsia akari, Rickettsia prowazekii, Rickettsia typhi, Rickett toma illocanum, Echinostoma jassyenese, Echinostoma sia Conorii, Rickettsia Sibirica, Rickettsia australis, Rickettsia malayanum, Echinostoma caproni, Fasciola hepatica, Fas japonica, Ehrlichia chafeensis, Orienta tsutsugamushi, Bar ciola gigantica, Fascioloides magna, Opisthorchis viverrini, tonella quintana, and Coxiella burni. Opisthorchis felineus, Opisthorchis sinensis, Paragonimus 0038 “Chlamydial cell as used herein may be any westermani, Schistosoma japonicum, Schistosoma mansoni, chlamydial cell belonging to the genera including, but not Schistosoma haematobium and Schistosoma haematobium. limited to, Chlamydia. Furthermore, chlamydial cell of the 0042 “Extracellular surface protein’ as used herein may present invention includes, but is not limited to, Chlamydia be any extracellular Surface protein including, but not limited trachomatis, Chlamydia caviae, Chlamydia pneumoniae, to, growth factor receptors, receptor tyrosine kinases, folate Chlamydia muridarum, Chlamydia psittaci, and Chlamydia hydrolases, GPI-anchored cell Surface antigens, pumps, and pecorum. cell Surface receptors including, but not limited to, G-protein 0039. “Mycoplasmal cell as used herein may be any coupled receptors, ion channel-linked receptors, and enzyme mycoplasmal cell belonging to the genera including, but not linked receptors. Extracellular surface proteins of interest limited to, Mycoplasma and Ureaplasma. In addition, myco may be those “differentially expressed by a targeted cell of plasmal cell includes but is not limited to, Mycoplasma pneu interest, in comparison to a cell that is not to be targeted by a moniae, Mycoplasma hominis, Mycoplasma genitalium, and cytotoxic nucleotide. For example, the cancer cells differ Ureaplasma urealyticum. from normal cells in many respects, including the up- or 0040 “Fungal cell as used herein may be any fungal cell down-regulation of numerous genes. Among the genes that belonging to the genera including, but not limited to, are differentially regulated in cancer cells are genes that Aspergillus, Candida, Cryptococcus, Coccidioides, Tinea, encode proteins that are expressed on the extracellular Sur Sporothrix, Blastomyces, Histoplasma, Pneumocystis and face. As an example, specific proteins are expressed on the Saccharomyces. Additionally, fungal cell of the present extracellular surface of prostate cancer (PC) cells that are not invention includes, but is not limited to, Aspergillus filmiga expressed (or are expressed at very low levels) by normal tus, Aspergillus flavus, Aspergillus niger; Aspergillus terreus, prostatic epithelial cells and cells from other normal tissues. Aspergillus nidulans, Candida albicans, Coccidioides immi Extracellular proteins that are expressed exclusively by PC tis, Cryptococcus neoformans, Tinea unguium, Tinea cor cells are excellent candidates for specific targeting of malig poris, Tinea cruris, Sporothrix schenckii, Blastomyces der nant cells with anticancer drugs. Cytotoxic oligodeoxynucle matitidis, Histoplasma capsulatum, Histoplasma duboisii, otides (ODNs) may be internalized by malignant cells that and Saccharomyces cerevisiae. express specific ODN receptor proteins (Corrias et al., Bio 0041. “Parasitic cell as used herein may include any para chem. Pharmacol. 55: 1221-1227 (1998)). The expression of sitic cell belonging to the genera including, but not limited to, prostate specific membrane antigen (PSMA) is limited to PC Entamoeba, Dientamoeba, Giardia, Balantidium, Trichomo cells and cells of the tumor neovasculature (Schulke et al., nas, Cryptosporidium, Isospora, Plasmodium, Leishmania, Proc. Natl. Acad. Sci. USA 100: 12590-12595 (2003)). A US 2010/0261781 A1 Oct. 14, 2010

second protein that displays characteristics suitable for devel virus, JC virus, B19 virus, Adeno-associated virus, Adenovi oping targeted therapeutics for PC is prostate stem cell anti rus, serotypes 3.7.14.21, Adenovirus, serotypes 11.21, Aden gen (PSCA: Saffran et al., Proc. Natl. Acad. Sci. USA 98: ovirus, Hepatitis B virus, Coronavirus, Human T-cell lym 2658-2663 (2001)). photrophic virus, Human immunodeficiency virus, Human 0043. In one embodiment, extracellular proteins that form foamy virus, Influenza viruses, types A, B, C, and Thogotovi dimers are preferred. Examples include, but are not limited to, U.S. PSMA and transferrin receptor. 0046 “Treat or “treatment as used herein refers to an 0044. In another embodiment, extracellular proteins that action resulting in a reduction in the severity of the Subject's are associated with the development of tumor neovasculature condition, wherein the condition is at least partially improved are preferred. PSMA is a non-limiting example thereof. orameliorated, and/or there is some alleviation, mitigation or decrease in at least one clinical symptom (or agricultural 0.045 “Viral disease' as used herein includes, but is not index for plants), and/or there is a delay in the progression of limited to, those caused by viruses belonging to the viral the condition, and/or prevention or delay of the onset of the families including, but not limited to, Flaviviridae, Arenavi condition. Thus, the term “treat” refers to both prophylactic radae, Bunyaviridae, Filoviridae, Poxyiridae. Togaviridae, and therapeutic treatment regimes. Compounds generated by Paramyxoviridae, Herpesviridae, Picornaviridae, Caliciviri the methods of the present invention may be used for the dae, Reoviridae, Rhabdoviridae, Papovaviridae, Parvoviri diagnosis and/or treatment of human Subjects, or animal Sub dae, Adenoviridae, Hepadnaviridae, Coronaviridae, Retro jects for veterinary or drug development purposes. Examples viridae, and Orthomyxoviridae. Furthermore, viral diseases of animal Subjects include mammalian (e.g., dog, cat, mouse, that can be treated using the compounds of the present inven rat, horse, cow, pig, sheep, etc.), reptile, amphibian, and avian tion can be caused by the viruses including, but not limited to, (e.g., parrot, budgie, chicken, turkey, duck, geese, quail, Yellow fever virus, St. Louis encephalitis virus, Dengue pheasant) Subjects. virus, Hepatitis G. virus, Hepatitis C virus, Bovine diarrhea virus, West Nile virus, Japanese Bencephalitis virus, Murray 2. ACTIVE COMPOUNDS Valley encephalitis virus, Central European tick-borne encephalitis virus, Far eastern tick-born encephalitis virus, 0047. In general, compounds (sometimes also referred to Kyasanur forest virus, Louping ill virus, Powassan virus, as “active compounds' herein) are compounds of the general Omsk hemorrhagic fever virus, Kumilinge virus, Absetarov formula A-B-C, wherein: anzalova hypr virus, Ilheus virus, Rocio encephalitis virus, 0048 A is a first nucleic acid that specifically binds to an Langat virus, Lymphocytic choriomeningitis virus, Junin extracellular surface protein expressed by a cell of interest, virus, Bolivian hemorrhagic fever virus, Lassa fever virus, 0049 B is an alkyl linker; and California encephalitis virus, Hantaan virus, Nairobi sheep 0050 C is a second nucleic acid that hybridizes to a disease virus, Bunyamwera virus, Sandfly fever virus, Rift complementary nucleic acid. Each of these is discussed in Valley fever virus, Crimean-Congo hemorrhagic fever virus, further detail below. Marburg virus, Ebola virus, Variola virus, Monkeypox virus, 0051 A. First nucleic acid. The first nucleic acid is, in Vaccinia virus, Cowpox virus, Orf virus, Pseudocowpox general, selected to specifically bind to an extracellular Sur virus, Molluscum contagiosum virus, Yaba monkey tumor face protein expressed by a cell of interest. The first nucleic virus, Tanapox virus, Raccoonpox virus, Camelpox virus, acid optionally but preferably contains cytotoxic nucleotides. Mousepox virus, Tanterapox virus, Volepox virus, Buffa Numerous such nucleic acids, including those sometimes lopox virus, Rabbitpox virus, Uasingishu disease virus, referred to as “aptamers', are known or can be identified in Sealpox virus, Bovine papular stomatitis virus, Camel conta accordance with known techniques, such as described in Wil gious eethyma virus, Chamios contagious eethyma virus, Red liam H. Gmeiner, Cytotoxic nucleotides for targeted thera squirrel parapox virus, Juncopox virus, Pigeonpox virus, peutics, US Patent Application 20080026947 (published Jan. Psittacinepox virus, Quailpox virus, Sparrowpox virus, Star 31, 2008). Other methods of identifying nucleic acids that can lingpox virus, Peacockpox virus, Penguinpox virus, Mynah be used as the first nucleic acid herein include but are not pox virus, Sheeppox virus, Goatpox virus, Lumpy skin dis limited to those described in U.S. Pat. Nos. 7,329,742; 7,312, ease virus, Myxoma virus, Hare fibroma virus, Fibromavirus, 325; 6,867,289; 6,858,390; and 6,369,208, or variations Squirrel fibroma virus, Malignant rabbit fibroma virus, thereof that will be apparent to those skilled in the art given Swinepox virus, Yaba-like disease virus, Albatrosspox virus, the present disclosure. Cotia virus, Embu virus, Marmosetpox virus, Marsupialpox 0052. In general, an aptamer would be selected as a spe virus, Mule deer poxvirus virus, Volepox virus, Skunkpox cies of a nucleic acid pool, and then synthetically resynthe virus, Rubella virus, Eastern equine encephalitis virus, West sized in whole or in part to provide active compounds. As ern equine encephalitis virus, Venezuelan equine encephalitis such, the elements of the aptamer will depend upon the fea virus, Sindbis virus, Semliki forest virus, Chikungunyavirus, tures of the nucleic acid with the pool from which the aptamer Onyong-nyong virus, Ross river virus, Parainfluenza virus, is produced. In some embodiments, the size of the nucleic Mumps virus, Measles virus (rubeola virus), Respiratory syn acids species within the pool can be in a range of about 30 cytial virus, Herpes simplex virus type 1, Herpes simplex nucleotides to about 150 nucleotides. In preferred embodi virus type 2, Varicella-Zoster virus, Epstein-Barr virus, ments, the nucleic acid species of the present invention com Cytomegalovirus, Human b-lymphotrophic virus, Human prises three regions: a “random” region flanked by two “con herpesvirus 7, Human herpesvirus 8, Poliovirus, Coxsackie A stant” regions. It should be noted while the sequence in this virus, Coxsackie B virus, ECHOvirus, Rhinovirus, Hepatitis region is random during the course of SELEX or other pro A virus, Mengovirus, ME virus, Encephalomyocarditis cedure to identify sequences that have the desired character (EMC) virus, MM virus, Columbia SK virus, Norwalk agent, istics (e.g. binding to extracellular protein) these sequences Hepatitis E virus, Colorado tick fever virus, Rotavirus, are not random, but selected for in the course of practicing the Vesicular stomatitis virus, Rabies virus, Papilloma virus, BK invention. The two "constant regions need not be identical to US 2010/0261781 A1 Oct. 14, 2010 each other, but comprise known nucleotide sequences. These synthesized onto the growing polymer molecule in a continu “constant regions are used for the annealing of PCR primers ous automated fashion, or synthesized separately and then during PCR amplification. The lengths of the “constant” covalently coupled to other portions of the molecule. Flexible regions can be in a range of about 8 nucleotides to about 35 linkers as described above can optionally be included in the nucleotides. In some embodiments the lengths of the “con second nucleic acid. stant regions are in a range of about 12 nucleotides to about 0057 D. Additional features. To aid in detection, the at 22 nucleotides. The length of the constant regions need not be least one nucleic acid from the first Subpopulation may be the same as one another, and indeed each region may be labeled with a detectable label using methods standard in the modified in length and/or sequence based on folding predic art, wherein the detectable label can include, but is not limited tions or results following the identification of optimal “ran to, fluorescent dyes, fluorophores, chromophores, affinity dom” regions. The “random” region of the nucleic acids labels, metal chelates, chemically reactive groups, enzymes, species within the pool consists of random arrangements of radionuclides, electrochemically detectable moieties, and nucleotide sequences. The length of the "random” region is energy absorbing or energy emitting compounds. not critical, but in general can be from 10 or 20 nucleotides in 0.058 Fluorescent dyes that can be used with the present length up to 80 or 100 nucleotides in length, or more. invention are any capable of binding to nucleic acids as 0053 Synthesizing a nucleic acid having a sequence cor defined herein and include, but are not limited to, the cou responding to a selected nucleic acid and incorporating a marin dyes, acetyl azide, fluorescein isothiocyanate, 1,2-di compound of interest may done according to any method hexadecanoyl-sn-glycero-3-phosphoethanolamine, 8-(6- standard in the art including, but not limited to, de novo aminohexyl)amino adenosine 3',5'-cyclicmonophosphate, chemical synthesis of polynucleotides, such as by presently bis(triethylammonium) salt, rhodamine dyes, Sulfonyl chlo available automated DNA synthesizers, and standard phos ride, CyDyeTM flors, and carboxynaphtofluorescein. The hap phoramidite chemistry. De novo chemical synthesis of a poly tenes that may be used for labeling include, but are not limited nucleotide can be conducted using any Suitable method, to, biotin, digoxigenin, and 2,4-dinitrophenyl. The haptenes including, but not limited to, the phosphotriester or phos require fluorescently-labeled antibodies or specific proteins phodiester methods. See Naranget al., Meth. Enzymol., 68:90 for visualization/detection. (1979); U.S. Pat. No. 4,356.270; Itakura et al., Ann. Rev. 0059 Labeling of nucleic acids with electrophore mass Biochem., 53:323-56 (1989); Brown et al., Meth. Enzymol., labels is described, for example, in Xu et al., J. Chromatog 68:109 (1979); and U.S. Pat. No. 6,911,310 issued to Heller. raphy 764:95-102 (1997). Electrophores are compounds that In one embodiment of the present invention, automated can be detected with high sensitivity by electron capture mass nucleic acid synthesis is conducted using an Applied Biosys spectrometry (EC-MS). Electrophore mass labels can be tem 394TM automated DNA/RNA synthesizer (Applied Bio attached to a probe using chemistry that is well known in the systems, Foster City, Calif.). art for reversibly modifying a nucleotide (e.g., well-known 0054 B. Alkyl linkers. Alkyl linkers (also referred to as nucleotide synthesis chemistry teaches a variety of methods alkyl spacers) may for example be partially Saturated or fully for attaching molecules to nucleotides as protecting groups). saturated C2-C6 or C10 alkyl groups, which can be linear or Electrophore mass labels are detected using a variety of well branched and may optionally contain one or more hetero known electron capture mass spectrometry devices. Further, atoms (e.g., one, two, three or four heteroatoms selected from techniques that may be used in the detection of electrophore N, O, and S), as long as their is at least one alkyl bond, mass labels include, for example, fast atomic bombardment —CH2—CH2—, in the main chain between the two linked mass spectrometry (See Koster et al., Biomedical Environ. groups (though in Some preferred embodiments, heteroatoms Mass Spec. 14:111-116 (1987)); plasma desorption mass are excluded therefrom). Alkyl linkers include groups of the spectrometry; electrospray/ionspray (See Fennet al., J. Phys. formula —X Y—Z—, where X and Y may be present or Chem. 88:4451-59 (1984), PCT Applin. No. WO 90/14148, absent; at least one of X, Y, and Z is an alkyl group of the Smith et al., Anal. Chem. 62:882-89 (1990)); and matrix formula—CH2— where n is an integer of from 1, 2 or 3 up assisted laser desorption/ionization (Hillenkamp et al. Bio to 5 or 10; and otherwise each of X, Y, and Z can be selected logical Mass Spectrometry (Burlingame and McCloskey, from the group consisting of alkenyl, alkynyl, alkoxy, etc. The eds.), Elsevier Science Pub., Amsterdam, pp. 49-60, 1990); linker may be of any suitable length, for example, 10, 20 or 50 Huth-Fehre et al., Rapid Communications in Mass Spectrom Angstroms in length, up to 100, 200 or 500 Angstroms in etry, 6:209-13 (1992)). (See also U.S. Pat. No. 6,979,548 length, or more. issued to Ford et al.) 0055 Alkyl linkers suitable for phosphoramidite synthe 0060 Methods for conjugation of detectable labels to sis of nucleic acids are known and available. A currently nucleic acids are well known in the art, for example, Schubert preferred example is 3-(4,4'-Dimethoxytrityloxy)propyl-1- et al., Nucleic Acids Research 18:3427 (1990) Smith et al., (2-cyanoethyl)-(N,N-diisopropyl)-phosphoramidite, com Nature, 321:674-679 (1986); Agarawal et al., Nucleic Acids mercially available as Spacer Phosphoramidite C3 (Product Research, 14:6227-6245 (1986); Chu et al., Nucleic Acids No. 10-1913-XX) from Glen Research Corporation, 22825 Research, 16:3671-3691 (1988). Davis Drive, Sterling, Va. 20164 USA. 0061. In some embodiments, modified oligonucleotides 0056 C. Second nucleic acid. The second nucleic acid incorporate activated anticancer drugs into three-dimensional (which optionally but preferably contains cytotoxic nucle nucleic acid structures that selectively bind to and are inter otides) can be of any suitable length to provide hybridization nalized by cancer cells. Modified oligonucleotides are com to another nucleic acid, according to the well-known prin prised, in part, of relatively low molecular weight activated ciples of Watson-Crick pairing. In general, the second nucleic drugs. Thus, the three-dimensional structures of modified acid can be of from 5 or 10 to 40 or 60 nucleotides in length, oligonucleotides that facilitate selective binding to and pen or more. Depending upon how the alkyl linker is synthesized etration of targeted cells are formed based upon the chemical in the molecule, the second nucleic acid can be continuously and structural properties of the component drugs or cytotoxic US 2010/0261781 A1 Oct. 14, 2010

nucleotides. In preferred embodiments, the activated drug is 0066. In some embodiments, ODNs may be synthesized to 5-fluoro-2'-deoxyuridine-5'-O-monophosphate (FdUMP). incorporate compounds of interest Such as cytotoxic nucleo 0062 Incorporation of a compound of interest into a side analogs, either before or after the enrichment selections selected nucleic acid sequence requires that the selected of candidate ODN sequences. In a preferred embodiment, ODNs selected in the first and second pools do not comprise nucleic acid sequence retains its original three-dimensional a compound of interest. The selected ODNs are sequenced structure of the native sequence following the incorporation. and analyzed to determine whether the incorporation of a In some embodiments, folding calculations are performed to compound on interest will affect their activity towards a bio compare the predicted folding patterns of the chemical struc logical target of interest. Cytotoxic ODNs are then subse ture of the native nucleic acid sequence with that of a nucleic quently synthesized consistent with analysis predictions (e.g. acid sequence incorporating one or more compound of inter predicted folding). However, synthesis of ODNs containing a est. Calculations can be performed with, e.g., folding pro compound on interest Such as a cytotoxic nucleoside analog grams such as mFOLD (Michael Zuker, Burnet Institute). may also be performed prior to the enrichment steps. Such calculations apply an algorithm to the native sequence 0067 Modified ODNs of the present invention can be of the nucleic acid to determine folding patterns that yield the optimized, e.g., for treatment of PC and other malignancies. most stable secondary structures. This approach provides In some embodiments of the present invention, the modified insight into the likely location of double helical regions that ODNs target XPSM using FdUMP as the active drug. In other occur within the three-dimensional structure of the nucleic embodiments, the modified ODNs target extracellular surface acid. The structural characteristics of the native and modified proteins that are differentially expressed specifically on the nucleic acids can also be determined using circular dichroism Surface of certain PC cells (e.g. prostate stem cell antigen). In (CD) spectroscopy and ultraviolet (UV) hyperchromicity further embodiments the modified ODNs administered to a measurements. Other methods of comparison will be appar particular patient may be customized to reflect the protein ent to those skilled in the art. Preferred nucleic acids of profile expressed by a specific patient. Additionally, the interest are those that incorporate compounds of interest in choice of drugs for inclusion into the modified ODN structure Such a way as to not significantly alter the folding character may be expanded to reflect the drug-profile that provides the istics of the native sequences. maximum response for aparticular malignancy. The ODNs of 0063. In some embodiments, modified nucleic acids are the present invention are compatible with a wide-range of further evaluated for the extent to which they selectively kill cytotoxic compounds, including, but not limited to, nucleo cells of interest, e.g., through the release of cytotoxic nucle side analogs, cytotoxic drugs, radionuclides, modifiers of otides by 3'-O-exonucleolytic degradation. Cell viability can gene expression and nanoparticles. be evaluated, e.g., using 3-(4,5-dimethylhiazole-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, 3. COMPOSITIONS AND FORMULATIONS inner salt (MTS) assays. Preferred nucleic acids are those that 0068. Initially, a pair of active compounds of the present are cytotoxic towards cells of interest and not cytotoxic to invention, as described above, are hybridized to one another non-targeted cells. in accordance with well-known techniques (e.g., simply mix 0064. In some embodiments of the present invention, a ing together in an aqueous Solution, or annealing by gentle synthetic nucleic acid may comprise one compound of inter heating and cooling in accordance with known techniques) to est. In other embodiments, a synthetic nucleic acid may incor produce a hybridized composition. They hybridized compo porate more than one compound of interest. In some embodi sition is, for convenience. Sometimes also referred to as an ments, one of the compounds of interest incorporated into the “active compound herein. synthetic nucleic acid may be a detectable compound, and/or 0069. The active compounds described above may be for an active compound. mulated for administration in a pharmaceutical carrier in 0065. In preferred embodiments, cytotoxic oligodeoxy accordance with known techniques. See, e.g., Remington, nucleotides are oligodeoxynucleotides (ODNs) that contain The Science And Practice of Pharmacy (9' Ed. 1995). In the one or more cytotoxic nucleoside analogs. Once incorporated manufacture of a pharmaceutical formulation according to into an ODN, the 5'-O-monophosphate form of the nucleo the invention, the active compound (including the physiologi side is present as an intact unit that is embedded in the ODN cally acceptable salts thereof) is typically admixed with, inter polymer. The cytotoxic nucleoside analogs may be incorpo alia, an acceptable carrier. The carrier must, of course, be rated as a stretch of several ODNs, or may be incorporated at acceptable in the sense of being compatible with any other various places in the nucleotide species. In some embodi ingredients in the formulation and must not be deleterious to ments, cytotoxic ODNs are arranged as a stretch of 2, 3, 4, or the patient. The carrier may be a solid or a liquid, or both, and 5 to 20, 25, 30, or 40 ODNs. A preferred example of a is preferably formulated with the compound as a unit-dose cytotoxic ODN is FdUMPI 10, a linear homopolymer of formulation, for example, a tablet, which may contain from FdUMP, the thymidylate synthase inhibitory metabolite of 0.01 or 0.5% to 95% or 99% by weight of the active com the anticancer drug 5-fluorouracil (5FU). (Gmeiner, Curr. pound. One or more active compounds may be incorporated Med. Chem. 12: 1345-1359 (2005); Gmeiner et al., Nucl. in the formulations of the invention, which may be prepared Nuct. Nucl. Acids 23: 401-410 (2004)). Another preferred by any of the well known techniques of pharmacy comprising example is FdUMP5). The cytotoxic ODNs may be included admixing the components, optionally including one or more in the synthesis of a desired nucleotide species, or may be accessory ingredients. appended to a desired nucleotide species. Synthesis and tox 0070 The formulations of the invention include those suit icity of FdUMP are found in U.S. Pat. Nos. 5,457,187 able for oral, rectal, topical, buccal (e.g., Sub-lingual), vagi (Gmeiner et al.); 5.614,505 (Gmeiner et al.); 5,663.321 nal, parenteral (e.g., Subcutaneous, intramuscular, intrader (Gmeiner et al.); 5,741,900 (Gmeiner et al.); and 6,342,485 mal, or intravenous), topical (i.e., both skin and mucosal (Gmeiner). Surfaces, including airway Surfaces) and transdermal admin US 2010/0261781 A1 Oct. 14, 2010 istration, although the most Suitable route in any given case prepared by admixing the active compound with one or more will depend on the nature and severity of the condition being conventional Solid carriers, for example, cocoa butter, and treated and on the nature of the particular active compound then shaping the resulting mixture. which is being used. 0074 Formulations suitable for topical application to the 0071. Formulations suitable for oral administration may skin preferably take the form of an ointment, cream, lotion, be presented in discrete units. Such as capsules, cachets, loz paste, gel, spray, aerosol, or oil. Carriers which may be used enges, or tablets, each containing a predetermined amount of include petroleum jelly, lanoline, polyethylene glycols, alco the active compound; as a powder or granules; as a solution or hols, transdermal enhancers, and combinations of two or a suspension in an aqueous or non-aqueous liquid; or as an more thereof. oil-in-water or water-in-oil emulsion. Such formulations may be prepared by any suitable method of pharmacy which 0075 Formulations suitable for transdermal administra includes the step of bringing into association the active com tion may be presented as discrete patches adapted to remain in pound and a suitable carrier (which may contain one or more intimate contact with the epidermis of the recipient for a accessory ingredients as noted above). In general, the formu prolonged period of time. Formulations suitable for transder lations of the invention are prepared by uniformly and inti mal administration may also be delivered by iontophoresis mately admixing the active compound with a liquid or finely (see, for example, Pharmaceutical Research 3 (6):318 divided solid carrier, or both, and then, if necessary, shaping (1986)) and typically take the form of an optionally buffered the resulting mixture. For example, a tablet may be prepared aqueous solution of the active compound. Suitable formula by compressing or molding a powder or granules containing tions comprise citrate or bis\tris buffer (pH 6) or ethanol/ the active compound, optionally with one or more accessory water and contain from 0.1 to 0.2M active ingredient. ingredients. Compressed tablets may be prepared by com 0076 Further, the present invention provides liposomal pressing, in a suitable machine, the compound in a free formulations of the compounds disclosed herein and salts flowing form, Such as a powder or granules optionally mixed thereof. The technology for forming liposomal Suspensions is with a binder, lubricant, inert diluent, and/or surface active/ dispersing agent(s). Molded tablets may be made by molding, well known in the art. When the compound or salt thereofisan in a suitable machine, the powdered compound moistened aqueous-soluble salt, using conventional liposome technol with an inert liquid binder. ogy, the same may be incorporated into lipid vesicles. In Such 0072 Formulations suitable for buccal (sub-lingual) an instance, due to the water solubility of the compound or administration include lozenges comprising the active com salt, the compound or salt will be substantially entrained pound in a flavoured base, usually Sucrose and acacia or within the hydrophilic center or core of the liposomes. The tragacanth; and pastilles comprising the compound in an inert lipidlayer employed may be of any conventional composition base Such as gelatin and glycerin or Sucrose and acacia. and may either contain cholesterol or may be cholesterol-free. 0073 Formulations of the present invention suitable for When the compound or salt of interest is water-insoluble, parenteral administration comprise Sterile aqueous and non again employing conventional liposome formation technol aqueous injection solutions of the active compound(s), which ogy, the salt may be substantially entrained within the hydro preparations are preferably isotonic with the blood of the phobic lipid bilayer which forms the structure of the lipo intended recipient. These preparations may contain anti-oxi some. In either instance, the liposomes which are produced dants, buffers, bacteriostats and solutes which render the for may be reduced in size, as through the use of standard Soni mulation isotonic with the blood of the intended recipient. cation and homogenization techniques. Aqueous and non-aqueous sterile Suspensions may include 0077. Of course, the liposomal formulations containing Suspending agents and thickening agents. The formulations the compounds disclosed herein or salts thereof, may be may be presented in unit\dose or multi-dose containers, for lyophilized to produce a lyophilizate which may be reconsti example sealed ampoules and vials, and may be stored in a tuted with a pharmaceutically acceptable carrier, such as freeze-dried (lyophilized) condition requiring only the addi water, to regenerate a liposomal Suspension. tion of the sterile liquid carrier, for example, Saline or water for-injection immediately prior to use. Extemporaneous 0078. Other pharmaceutical compositions may be pre injection Solutions and Suspensions may be prepared from pared from the water-insoluble compounds disclosed herein, sterile powders, granules and tablets of the kind previously or salts thereof. Such as aqueous base emulsions. In such an described. For example, in one aspect of the present inven instance, the composition will contain a sufficient amount of tion, there is provided an injectable, stable, sterile composi pharmaceutically acceptable emulsifying agent to emulsify tion comprising an active compound(s), or a salt thereof, in a the desired amount of the compound or salt thereof. Particu unit dosage form in a sealed container. The compound or salt larly useful emulsifying agents include phosphatidyl cho is provided in the form of a lyophilizate which is capable of lines, and lecithin. being reconstituted with a suitable pharmaceutically accept 0079. In addition to active compound(s), the pharmaceu able carrier to form a liquid composition Suitable for injection tical compositions may contain other additives, such as pH thereof into a Subject. The unit dosage form typically com adjusting additives. In particular, useful pH-adjusting agents prises from about 10 mg to about 10 grams of the compound include acids, such as hydrochloric acid, bases or buffers, or salt. When the compound or salt is substantially water Such as sodium lactate, sodium acetate, Sodium phosphate, insoluble, a Sufficient amount of emulsifying agent which is Sodium citrate, Sodium borate, or Sodium gluconate. Further, physiologically acceptable may be employed in Sufficient the compositions may contain microbial preservatives. Use quantity to emulsify the compound or salt in an aqueous ful microbial preservatives include methylparaben, propylpa carrier. One Such useful emulsifying agent is phosphatidyl raben, and benzyl alcohol. The microbial preservative is typi choline. Formulations suitable for rectal administration are cally employed when the formulation is placed in a vial preferably presented as unit dose Suppositories. These may be designed for multidose use. Of course, as indicated, the phar US 2010/0261781 A1 Oct. 14, 2010

maceutical compositions of the present invention may be aptamers to PSMA that we are aware of. We contracted with lyophilized using techniques well known in the art. Kinakeet Biotechnology (Richmond, Va.) to express the 706 amino acids comprising the extracellular domain of PSMA 4. USE from Sf9 cells using baculovirus. RNA was extracted from 0080. As noted above, compounds of the present invention pelleted LNCaP cells and primers were designed for cloning may be used to detect and/or selectively kill or inhibit the of the extracellular domain of PSMA from the corresponding growth of cells of interest, including but not limited to cancer cDNA. The accuracy of all cloning steps was verified by DNA cells, tissue cells generally (e.g., muscle, bone, nerve, liver, sequencing. The recombinant protein was purified by affinity lung, etc.), pathological and non-pathological microbial cells chromatography. Protein purity was determined by gel elec (e.g., bacterial, mycobacterial, spirochetal rickettsial, trophoresis and the identity of the protein was confirmed by chlamydial, mycoplasmal, and fungal, etc.), parasitic cells Western blotting and mass spectrometry. The recombinant (e.g., protozoal, helminth, etc.), and plant cells, etc. Such PSMA includes a His-tag that was used for attachment of the methods may be carried out in vitro or in vivo (e.g., by recombinant protein to Dynabeads Talon (Dynal Biotech) for administering the compound to a plant or animal host carry use as an affinity matrix for aptamer selection. The Suitability ing or harboring undesired cells of interest, such as cancer of the affinity matrix for DNA aptamer selection was verified cells, pathological microbial cells, parasitic cells, etc.) by demonstrating that the A10-3 RNA aptamer to PSMA 0081 Compounds of the present invention may also be bound the affinity matrix. SELEX methodology was used to used to control, e.g., kill or inhibit the growth of microbes identify DNAaptamers to PSMA. The DNA library used for that may otherwise contaminate an industrial fermentation. SELEX included a 45 nucleotide random sequence flanked 0082. Additionally, compounds of the present invention by two 21 nucleotide fixed regions. The ssDNA was con may be used as an herbicide. The compounds of the present verted to dsDNA using a series of “fill-in reactions with T7 invention may be applied to the Surface of the plant including, DNA polymerase. These “fill-in reactions were each run on but not limited to, leaves, stems, flowers, fruits, roots, cells or a 2 ug scale. The resulting dsDNA was amplified using mul callus tissue. Alternatively, the compounds of the present tiplex PCR to create 20-30 ug of dsDNA. PCR was done using invention may be introduced into the plant via methods stan a primer to the original ssDNA that was 5'-phosphorylated. dard in the art including, but not limited to, microinjection, Following amplification, the dsDNA was converted to electroporation, particle bombardment, and Agrobacterium ssDNA using exonuclease w. The ssDNA product was ana mediated transformation. lyzed by gel electrophoresis and quantified by UV absorption. 0083. Further, compounds of the present invention may Typically 5-10 ug ssDNA were used for binding reactions for also be used for treatment of infection of plants and plant cells SELEX. The ssDNA was incubated with the PSMA affinity by plant pathogens, the plant pathogens including, but not matrix for 60 minat37°C. The supernatant was removed, and limited to, bacteria, fungi, oomycetes, viruses, and nema bound material was eluted from the affinity matrix by heating todes. For the purpose of treatment of plant pathogenic infec to 90° C. ssDNA was converted to dsDNA, PCR amplified, tions, the compounds of the present invention may be applied converted back to ssDNA and repeated rounds of forward and to the Surface of a plant including, but not limited to, leaves, counter SELEX were performed to create a DNA pool stems, flowers, fruits, roots, cells or callus tissue. Alterna enriched in sequences that bound with high affinity to PSMA. tively, the compounds of the present invention may be intro The resulting DNA was cloned into the pGEMT-Easy vector duced into the plant via methods standard in the art including, (Promega) which was then used to transform competent but not limited to, microinjection, electroporation, particle BL21 E. coli cells. DNA sequences were determined from bombardment, and Agrobacterium-mediated transformation. individual clones at the core DNA sequencing facility of the 0084. The therapeutically effective dosage of any specific CCCWFU. The initial SELEX procedure resulted in identi compound, the use of which is in the scope of present inven fication of 10 DNA sequences—several of which were tion, will vary somewhat from compound to compound, and chemically synthesized and shown to bind selectively to patient to patient, and will depend upon the condition of the recombinant PSMA (BSA as the negative control) using fluo patient and the route of delivery. As a general proposition, a rescence anisotropy. These sequences were also shown using dosage from about 0.1 to about 50 mg/kg will have therapeu fluorescence microscopy to bind selectively to PSMA-ex tic efficacy, with all weights being calculated based upon the pressing prostate cancer cells (LNCaP. C4-2) relative to PC3 weight of the active compound, including the cases where a cells. While we had identified DNA aptamers that were suit salt is employed. In some embodiments, a dosage from about able for the next stage of Cytotoxamer development, a few of 10 mg/kg to about 50 mg/kg may be employed for oral admin our experiments using scrambled sequences as negative con istration. In some embodiments, a dosage from about 0.5 trols had ambiguous results. Further, while we consistently mg/kg to 5 mg/kg may be employed for intramuscular injec observed surface binding to PSMA-expressing cells by fluo tion. For agricultural use, the compounds may be applied to rescence microscopy, we observed little evidence for cellular plants or crops by any suitable technique, such as by spraying. internalization. Somewhat Surprisingly, only Surface binding 0085. The present invention is explained in greater detail was also observed with the A10 RNA aptamer that was in the following non-limiting Examples. reported to be internalized into PSMA-expressing cells by other research groups (Lupold et al., 2002; Chu et al., 2006). EXAMPLES We thus decided to repeat the SELEX procedure. The second SELEX procedure used different primer sequences, but was I0086) Developing DNAAptamers to PSMA. We have has otherwise undertaken using identical methodology. Sequenc formed an affinity matrix using recombinant PSMA ing of 10 clones from this procedure resulted in nine of the expressed from baculovirus. This affinity matrix has been clones having a single sequence indicating the final DNA used in completion of two SELEX procedures to identify pool was highly enriched in this sequence which we termed novel DNA aptamers to PSMA. These are the first DNA PSMAO1. The sequence of PSMAO1 is: US 2010/0261781 A1 Oct. 14, 2010 10

5'-GCGTTTTCGCTTTTGCGTTTTGGGTCATCTGCTTACGATAGCAATCGT (SEQ ID NO: 1)

I0087 Studies with the PSMA01 DNA aptamer verified The images indicated cellular internalization and retention of that this sequence selectively binds to PSMA-expressing cells the dimeric complexes within endocytic vesicles. The two and has no affinity for cells that do not express PSMA (FIG. dyes co-localized in endocytic vesicles, as well, consistent 1). In these studies, live (non-fixed cells) cells were incubated with the aptamer complex remaining in dimeric form follow in the presence of fluorescently-labeled aptamer for 2 hat ing cellular internalization. room temperature. In these studies, C4-2 and LNCaP cells (0090. Increased Flexibility Enhances Cellular Uptake of were used to evaluate binding to PSMA+ cells while PC3 Dimeric Aptamer Complexes: While the linker domain con cells were used as a PSMA-negative control. The PSMA01 sisting of 16 Watson-Crick base pairs was stable at 37°C. and DNA aptamer that we identified in our laboratory displayed conferred an advantage in terms of cellular binding and inter selective binding to PSMA+C4-2 and LNCaP cells and dis nalization relative to the monomericaptamer conjugates, we played no binding to PSMA-PC3 cells. The binding of reasoned that additional binding avidity of the dimeric PSMA-01 was similar to the A10-3 RNA aptamer that had aptamer complex for PSMA could be conferred by including been previously described. Neither our PSMA-01 DNA one or more alkyl linkers into the structure. Alkyl linkers are aptamer nor the A10-3 aptamer were internalized into highly flexible, with flexibility that surpasses the deoxyribo PSMA+ cells to any significant extent under any of the con nucleotide components of the dsDNA that composes the ditions analyzed (incubation at room temperature or 37° C.). linker sequence (FIG. 5). We reasoned that this increased 0088 Dimeric Aptamer Complexes Show Enhanced Cel flexibility would allow each componentaptamer to adopt an lular Internalization: At this time, we obtained a sample of the optimal conformation for binding to PSMA with minimal J591 mAb to PSMA (kindly provided by Dr. Neil Bander, penalty in terms of free energy associated with structural Cornell-Weill Medical School). During the course of studies distortion. Preliminary studies are promising, with dimeric with J591, it became clear that the mab not only had some aptamer complexes including one alkyl linker between the what greater surface binding to PSMA-expressing cells rela PSMA01 aptamer and the tail (either dA16 or dT16) showing tive to PSMA01 (and other aptamers, both DNA aptamers enhanced cellular binding and internalization relative to developed in our laboratory and RNA aptamers that had been dimericaptamer complexes that do not include an alkyl linker previously described), but J591 was also more efficiently (FIG. 4). internalized into PSMA-expressing cells. In contemplating (0091. The relative binding of the PSMAO1 DNA aptamer the physical basis for the increased binding and internaliza in monomeric form as well as in dimeric form with and tion of the J591 antibody relative to the monomericaptamer without flexible linkers was also evaluated towards C4-2 cells conjugates, we focused on the bivalent structure of the mAb using flow cytometry. The results are summarized in FIG. 6. as likely contributing significantly to the observed more Cells were treated in the same manner as described for the favorable binding and cellular internalization characteristics confocal microscopy experiments and were detached using of the mAb.We designed bivalent dimericaptamer complexes an enzyme-free cell-dissociation buffer (Invitrogen). Cells that were formed through Watson-Crick base pairing (FIG. were analyzed using a BD FACSCanto flow cytometry sys 2). Initial constructs used homopolymeric dA and dT tails tem and the data were analyzed using BDFACSDiva software since this strategy readily permits rendering these dimeric (BD Biosciences, San Jose, Calif.). aptamer complexes potentially cytotoxic through T->FdU 0092 Safety and Efficacy of FdUMPI 10 Towards Pros substitution. The tails were designed to form a 16 base pair tate Cancer Xenografts: The principal strategy that will be linker sequence that would be stable at 37° C. but not be so used to render the dimericaptamer complexes we have devel stable as to inhibit nuclease degradation following cellular oped selectively cytotoxic towards targeted cancer cells is to internalization. Although this linker is probably not of opti include cytotoxic nucleotides, such as FdU, in the structure. mal geometry, the dimeric aptamer complex displayed While other modalities may also be included to render these greater Surface binding and fluorescence microscopy images modified aptamers cytotoxic to cancer cells (e.g. siRNA, were consistent with increased cellular internalization rela toxins), there is considerable merit to the concept that dimeric tive to the monomeric aptamer conjugates (FIG. 3). aptamer complexes are the best method for delivery of FdU 0089 Confocal Microcopy Demonstrates Enhanced Cell and other cytotoxic nucleotide analogs in the future. In prin Internalization of Dimeric Aptamer Complexes: The cellular ciple, delivery of cytotoxic nucleotide analogs in aptamers internalization of dimeric aptamer complexes was further increases the selectivity and potency of these drugs while investigated using confocal microscopy. For these studies, minimizing systemic toxicities. In this regard, it is important both component monomeric aptamer conjugates were fluo to consider the advantages obtained by inclusion of FdU in rescently labeled. PSMA01-da16 was labeled with Quasar single-stranded DNA (e.g. FauMP10) relative to delivery 670 and PSMAO1-dT16 was labeled with Quasar 570. These as the nucleobase (5FU). We have shown that not only does long wavelength dyes are resistant to bleaching and have delivery of fluoropyrimidine (FP) as FdUMPI 10 increase minimal spectral overlap permitting simultaneous scanning efficacy and decrease toxicity, it also changes the spectrum of of these two wavelengths to demonstrate through co-local malignancies that are responsive to FP treatment. Recent ization studies to what extent the dimer structure was formed unpublished data from our laboratory has demonstrated that and whether the aptamer remained in dimeric form while FdUMPI 10 is efficacious towards PC3 xenografts, a model bound to the cell surface and following cellular internaliza of hormone-refractory prostate cancer (FIG. 7). PSMA, the tion. Representative images are shown in FIG. 4. The data target for the dimericaptamer complexes being developed by confirmed co-localization of the Quasar 570 and Quasar 670 us, is frequently expressed in advanced prostate cancer. dyes indicating that the aptamer complexes bound as dimers. PSMA is also frequently expressed in tumor neovasculature. US 2010/0261781 A1 Oct. 14, 2010

As FPs are widely used for treatment of solid tumors, the (0095 Selective Uptake Into PC3 PSMA+/- cells. We have dimericaptamer complexes developed in these studies should evaluated the selective binding and uptake of our dimeric provide an improved mechanism for tumor-specific delivery aptamer complexes using in a matched pair of PC3 cells of FPS. 0093. We recently completed an in vivo xenograft study (PSMA+ and PSMA-). These cells were provided by Dr. W. evaluating the anti-tumor activity of FdUMP10 and 5FU D. Heston (Cleveland Clinic). Since these cells differ only in towards PC3 xenografts (FIG. 8). All groups had comparable PSMA expression, differences in surface binding and inter size tumors at baseline. Fall JMP 10 (150 mg/kg) was nalization can be attributed directly to PSMA-mediated pro injected i.v. 1x per week via jugular vein catheter. 5FU (100 cesses. Western blots confirmed PSMA expression selec mg/kg) was also injected 1xper week by the same route. The tively in the PSMA-transduced PC3 cells (FIG. 8). dose of 5FU administered was the maximum tolerated dose Representative confocal images demonstrating PSMA-spe and 5FU-treated animals lost weight compared to the con cific binding are also shown in FIG. 8. Neither the J591 mAb trols. FauMP10) was well-tolerated at the administered nor the dimeric aptamer complex displayed either Surface dose. A mixed effects model was fit to examine whether there binding or internalization into the PSMA-PC3 cells. Both the were differences between groups over the first 44 days after mAb and the dimeric aptamer complex, however, displayed administration of FdUMP 10 or 5FU. In this model, indi considerable Surface binding upon incubation with the vidual animals were considered as random effects and the PSMA+ PC3 cells at 4° C. and considerable internalized group and day variables were considered as fixed effects. The signal was evident upon incubation at 37° C. When incuba FdUMPI 10-treated mice displayed significantly reduced tion proceeds at 4°C., clustered signal, or Surface “patching tumor growth relative to control beginning on day 18 of is observed for both the J591 mAb and the dimeric aptamer treatment and persisting for all Subsequent time points (p<0. complex. This surface “patching' (Hopwood et al., 1982) is 0003 (day 30)). We expect that with dimeric aptamer com plexes including FdU, we will further increase the selectivity observed over the Golgi apparatus in PSMA+ PC3 cells. for malignant cells and further reduce systemic toxicities. Patching is characteristic of multivalent ligands and facili Thus, dimericaptamer complexes may ultimately become the tates endocytosis. Both the dimericaptamer complex and the preferred method for administering fluoropyrimidine chemo mAb are present in internalized vesicles in PSMA+ PC3 cells therapy in the era of molecularly targeted cancertherapeutics. following incubation at 37° C. 0094. We plan to conduct clonogenic assays with dimeric 0096. The foregoing is illustrative of the present invention, aptamer complexes containing FdU in a matched pair of PC3 and is not to be construed as limiting thereof. The invention is cells (PSMA+ and PSMA-). These cells were provided by defined by the following claims, with equivalents of the Dr. W. D. Heston (Cleveland Clinic). Since these cells differ claims to be included therein.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 1

<21 Os SEQ ID NO 1 &211s LENGTH: 48 &212s. TYPE: DNA <213> ORGANISM: Artificial 22 Os. FEATURE: <223> OTHER INFORMATION: PSMAO1 DNA aptamer sequence

<4 OOs SEQUENCE: 1

gcqttitt cqc ttittgcgttt togggt catct gcttacgata gcaatcgt. 48 only in PSMA expression, differences in cytotoxicity can be That which is claimed is: attributed directly to PSMA-mediated endocytosis of dimeric 1. A compound of the formula A-B-C, wherein: aptamer complexes. PC3 cells also have mutant p53 while A is a first nucleic acid that specifically binds to an extra C4-2 cells have wtp53. Ongoing studies from our laboratory cellular surface protein expressed by a cell of interest, show that PC3 cells are more sensitive to activated FPs (e.g. FdUMPI 10 than C4-2 cells and that the relative cytotoxicity B is an alkyl linker; and difference can be reduced either by shRNA knockdown of C is a second nucleic acid that hybridizes to a complemen p53 in C4-2 cells or viral transduction of wtp53 into PC3 tary nucleic acid. cells. As p53 mutations are among the most frequent mutation 2. The compound of claim 1, wherein said first nucleic acid in cancer and that mutated p53 generally makes tumors less is an aptamer. responsive to chemotherapy, these finding bode well for the 3. The compound of claim 1, wherein said first nucleic acid future clinical use of activated FPs, and especially dimeric is from 30 to 150 nucleotides in length. aptamer complexes containing FdU, for molecularly-targeted 4. The compound of claim 1, wherein said first nucleic acid chemotherapy. is selected from the group consisting of DNA and RNA. US 2010/0261781 A1 Oct. 14, 2010

5. The compound of claim 1, wherein said first nucleic acid is a DNA having the sequence of PSMA01:

5'-dgCGTTTTCGCTTTTGCGTTTTGGGTCATCTGCTTACGATAGCAATCGT. (SEQ ID NO: 1)

6. The compound of claim 1, wherein said alkyl linker 18. The compound of claim 17, wherein said extracellular comprises C2-C6 loweralkyl. Surface protein comprises an extracellular Surface portion of 7. The compound of claim 1, wherein said second nucleic prostate specific membrane antigen (PSMA). acid is from 8 to 100 nucleotides in length. 19. A composition comprising a pair of compounds of 8. The compound of claim 1, wherein said second nucleic claim 1, each member of said pair having a second nucleic acid is selected from the group consisting of DNA and RNA. acid that is complementary to and hybridized to the second 9. The compound of claim 1, wherein said cell of interest is nucleic acid of the other member of said pair. a cancer cell. 20. The composition of claim 19 in a pharmaceutically 10. The compound of claim 1, wherein said cell of interest acceptable carrier. is a microbial cell. 21. A method of introducing a nucleic acid of interest into 11. The compound of claim 1, wherein said cell of interest a cell of interest, comprising contacting a composition of is a parasitic cell. claim 19 to said cell under conditions in which said nucleic 12. The compound of claim 1, wherein said nucleic acid acid of interest is internalized into said cell. comprises an active compound. 22. The method of claim 21, wherein said method is carried 13. The compound of claim 1, wherein said nucleic acid out in vitro. comprises a detectable group. 23. The method of claim 21, wherein said method is carried 14. The compound of claim 1, where said nucleic acid out in vivo. comprises more than one active compound and/or detectable 24. The method of claim 21, wherein said cell of interest is group. a cancer cell in a subject afflicted with cancer, and said con 15. The compound of claim 12, wherein said active com tacting is carried out by administering said composition to pound comprises cytotoxic nucleotides. said Subject in an amount effective to treat or detect said 16. The compound of claim 15, wherein said cytotoxic CaCC. nucleotides comprise poly-FdUMP. 25. The method of claim 25, wherein said cancer is prostate 17. The compound of claim 1, wherein said extracellular CaCC. surface protein is an extracellular surface protein differen tially expressed by cancer cells.