US008.956825B2

(12) United States Patent (10) Patent No.: US 8,956,825 B2 Weisbart (45) Date of Patent: Feb. 17, 2015

(54) INTRANUCLEAR PROTEIN TRANSDUCTION FOREIGN PATENT DOCUMENTS THROUGH ANUCLEOSDE SALVAGE PATHWAY WO WO 97.326O2 3, 1997 WO 98.16247 A1 4f1998 WO WO 98.29.437 A2 7, 1998 (75) Inventor: Richard H. Weisbart, Los Angeles, CA WO WO 2005,117562 A2 12/2005 (US) OTHER PUBLICATIONS (73) Assignee: The United States of America as represented by the Department of Avrameas et al., “Polyreactive anti-DNA monoclonal antibodies and Veterans Affairs, Washington, DC (US) a derived peptide as vectors for the intracytoplasmic and intranuclear translocation of macromolecules'. Proc. Natl. Acad. Sci., 95:5601 (*) Notice: Subject to any disclaimer, the term of this 5606 (1998). patent is extended or adjusted under 35 de Coupade et al., “Novel human-derived cell-penetrating peptides U.S.C. 154(b) by 705 days. for specific subcellular delivery of therapeutic biomolecules”. Biochem. J. 390:407-418 (2005). (21) Appl. No.: 12/126,810 Mackey et al., “Gemcitabine Transport in Xenopus oocytes Express ing Recombinant Plasma Membrane Mammalian Nucleoside Trans (22) Filed: May 23, 2008 porters”,Journal of the National Cancer Institute, 91(21): 1876-1881 (1999). (65) Prior Publication Data Hansen et al., “Intranuclear protein transduction through a nucleoside Salvage pathway”. J. Biol. Chem., 282(29):20790-3 US 2008/O292618 A1 Nov. 27, 2008 (2007). Nagaiet al., “Mouse equilibrative nucleoside transporter 2 (mENT2) Related U.S. Application Data transports nucleosides and purine nucleobases differing from human (60) Provisional application No. 60/931,855, filed on May and rat ENT2'. Biol. Pharm. Bull, 30(5):979-81 (2007). 24, 2007. Zhang et al., “The role of nucleoside transporters in cancer chemo therapy with nucleoside drugs'. Cancer Metastasis Rev. 26(1): 85 (51) Int. Cl. 110 (2007). GOIN 33/53 (2006.01) European Search Report from EP 08769716, Oct. 31, 2011. GOIN33/58 (2006.01) Kong et al., “Mammalian Nucleoside Transporters”. Current Drug GOIN 33/577 (2006.01) Metabolism, vol. 5: 63-84 (2004). C07K I6/44 (2006.01) C07K I6/46 (2006.01) * cited by examiner A6 IK 47/48 (2006.01) Primary Examiner — Phuong Huynh GOIN33/50 (2006.01) A61 K38/00 (2006.01) (74) Attorney, Agent, or Firm — DLA Piper LLP (US) (52) U.S. Cl. (57) ABSTRACT CPC ...... C07K 16/44 (2013.01); A61K 47/48538 Provided herein are conjugate molecules containing a Sub (2013.01); G0IN33/5035 (2013.01); A61 K strate for a nucleoside transport pathway linked to an active 38/00 (2013.01); C07K 2317/622 (2013.01); agent, wherein the conjugate can be transported into a cell or C07K 2317/77 (2013.01); C07K 2317/82 into the nucleus of a cell via a cellular nucleoside transport (2013.01) pathway. Further provided are methods of delivering a con USPC ...... 435/29: 435/7.21: 435/7.72: 514/1.1; jugate molecule to a target cell expressing a nucleoside trans 514/12:530/391.1 port pathway, wherein the conjugate contains a Substrate for (58) Field of Classification Search the nucleoside transport pathway linked to an active agent. None Also provided are methods for Screening for conjugates that See application file for complete search history. are transported by nucleoside transport pathways. Further (56) References Cited provided are methods of treating a patient having a disease or disorder affecting tissues expressing nucleoside transport U.S. PATENT DOCUMENTS pathways, in which a conjugate containing an agent effective in treating the disorder is administered to the patient. Also 4,812,397 A 3, 1989 Weisbart provided are methods of treating a patient having an autoim 6,130,065. A * 10/2000 Belt et al...... 435/69.1 6,232,444 B1 5, 2001 Weisbart mune disorder involving administering to the patient a com 7,381,799 B2 6/2008 Papathanassiu pound that inhibits a nucleoside transport pathway. 2005/OO74771 A1 4/2005 Cook et al...... 435/6 2006/0003362 A1* 1/2006 Zerangue ...... 435/6 15 Claims, 4 Drawing Sheets

US 8,956,825 B2 1. 2 INTRANUCLEAR PROTEIN TRANSDUCTION SUMMARY OF THE INVENTION THROUGH ANUCLEOSDE SALVAGE PATHWAY The present invention is based on the discovery that a DNA-binding antibody that can penetrate cells and localize RELATED APPLICATIONS into the nucleus is transported by a nucleoside transport path way. This application is a utility application and claims priority According to one embodiment of the invention, there are under 35 U.S.C. S 119(e) of U.S. Provisional application Ser. provided conjugates including a substrate that is capable of No. 60/931,855 filed May 24, 2007. being transported by a nucleoside transport pathway, and an 10 active agent linked to the Substrate, wherein the conjugate is GRANT INFORMATION transported by the nucleoside transport pathway. In particular embodiments in which the substrate is an antibody, the anti This invention was made with government Support by the body is not the monoclonal antibody 3E10 or a fragment U.S. Department of Veterans Affairs. The government has thereof. In certain embodiments, the nucleoside transport certain rights in the invention. 15 pathway includes an equilibrative nucleoside transporter or a FIELD OF THE INVENTION concentrative nucleoside transporter. In embodiments in which the nucleoside transport pathway involves an equili The present invention relates generally to conjugate mol brative nucleoside transporter, such a transporter may be ecules and more specifically, to conjugate molecules and their insensitive to inhibition by low concentrations of nitroben use in the delivery of active agents into cells using endog Zylmercaptopurine riboside (NBMBR). enous cellular transport pathways. According to another embodiment of the invention, there are provided methods for delivering a conjugate to a target BACKGROUND OF THE INVENTION cell expressing a nucleoside transportpathway. Such methods 25 involve contacting the target cell expressing the nucleoside Transporter proteins are involved in the cellular uptake of transport pathway with a conjugate including a substrate that various molecules into and/or through cells. Carrier-mediated is capable of being transported by a nucleoside transport transport systems use proteins that are anchored to the cell pathway, and an active agent linked to the Substrate, wherein membrane, typically by a plurality of membrane-spanning the conjugate is transported by the nucleoside transport path domains and function by transporting their Substrates via 30 way. In particular embodiments in which the Substrate is an active or passive mechanisms. Carrier-mediated transport antibody, the antibody is not the monoclonal antibody 3E10 systems are involved in the active or non-active, facilitated or a fragment thereof. In certain embodiments, the nucleoside transport of many important nutrients such as vitamins, Sug transport pathway includes an equilibrative nucleoside trans ars, and amino acids. Carrier-mediated transporters are also porter or a concentrative nucleoside transporter. In embodi present in organs such as the liver and kidney, in which the 35 ments in which the nucleoside transport pathway involves an proteins are involved in the excretion or re-absorption of equilibrative nucleoside transporter, such a transporter may circulating compounds. Polar or hydrophilic compounds be insensitive to inhibition by low concentrations of nitroben typically diffuse poorly across the lipid bilayers that consti Zylmercaptopurine riboside (NBMBR). tute cellular membranes. For many Small molecules (e.g., According to a further embodiment of the invention, there amino acids, di- and tripeptides, monosaccharides, nucleo 40 are provided methods of Screening a conjugate for transport sides and water-soluble vitamins) there exist specific carrier by a nucleoside transport pathway. Such methods include, mediated transporters for active transport of the solute mol contacting a cell expressing the nucleoside transportpathway, ecules across biological membranes. with a conjugate under Suitable conditions for transport to The uptake or release physiological nucleosides and many occur, and determining whether the conjugate is transported of their synthetic analogs by mammalian cells occurs prima 45 into the cell by the nucleoside transport pathway. In certain rily by means of specific carrier-mediated transporters known embodiments, the determining step includes comparing the as nucleoside transporters. Nucleoside transporters have been amount of conjugate transported into a cell expressing the classified into two categories: (i) equilibrative (facilitated nucleoside transport system to the amount of conjugate trans diffusion) and (ii) concentrative (secondary active) sodium ported into a control cell not expressing the nucleoside trans dependent. Two equilibrative transport systems with similar 50 port system. In these embodiments, an increase in transport of broad substrate specificities have been identified and desig conjugate of the cell expressing the nucleoside transport path nated as the es (equilibrative sensitive) and ei (equilibrative way as compared to the control cell indicates transport is by insensitive) transporters, on the basis of their sensitivity or that nucleoside transport pathway. insensitivity to inhibition by nitrobenzylthioinosine (NB According to yet another embodiment of the invention, MPR, 1), respectively. As many as six sodium ion-coupled 55 there are provided methods for treating a disease or disorder (concentrative) nucleoside transport systems designated cif in a cell or tissue expressing a nucleoside transport pathway. NI, cit/N2, cib/N3, cit/N4, cs/N5 and csg/N6 have also been The method includes administering to a patient having the functionally identified in mammalian tissues. disease or disorder a conjugate including a substrate that is The anti-DNA antibody fragment 3E10 Fv has been dem capable of being transported by the nucleoside transport path onstrated to be a novel molecular delivery vehicle due to its 60 way expressed in the affected cell or tissue and an active agent penetration into living cells with specific nuclear localization, for treating disease or disorder, wherein the conjugate is also absence of toxicity, and successful delivery of therapeutic transported by the nucleoside transport pathway. In this way, cargo proteins in vitro and in vivo. Elucidation of the pathway the conjugate is transported into the affected cells or tissue, that allows 3E10 Fv to cross cell membranes is critical to the thereby delivering the active agent. In certain embodiments development of new molecular therapies, which rely on the 65 the disease or disorder involves skeletal muscle and the con regulation of gene expression by intranuclear transduction of jugate is transported into the skeletal muscle cells, thereby macromolecules. delivering the active agent. US 8,956,825 B2 3 4 According to yet another embodiment, there are provided FIG. 4 shows the nucleotide and amino acid sequences of methods for treating a genetic disorder wherein the method mAb3E10 Vk light chains, 3E1 OVkIII (GenBank Accession includes, administering to a patient having a genetic disorder No. L34051; SEQID NOS:7 and 8, for nucleotide and amino a conjugate including: a Substrate that is capable of being acid sequences, respectively) and 3E10VkSER (GenBank transported by an equilibrative nucleoside transporter, and an Accession No. L16981; SEQID NOs:9 and 10, for nucleotide active agent for treating the genetic disorder, wherein the and amino acid sequences, respectively). active agent is linked to the substrate, and further wherein the conjugate is transported by the equilibrative nucleoside trans DETAILED DESCRIPTION OF THE INVENTION porter, whereby the conjugate is transported into cells, 10 Before the present methods are described, it is to be under thereby delivering the active agent. In certain embodiments, stood that this invention is not limited to particular composi the active agent is a gene or protein that is deficient in patients tions, methods, and experimental conditions described, as having the genetic disease. Such compositions, methods, and conditions may vary. It is In other aspects, this disclosure provides a method of treat also to be understood that the terminology used herein is for ing a cancer, wherein the method includes, administering to a 15 purposes of describing particular embodiments only, and is patient having cancera conjugate including: a Substrate that is not intended to be limiting, since the scope of the present capable of being transported by an equilibrative nucleoside invention will be limited only in the appended claims. transporter, and an active agent for treating cancer, wherein As used in this specification and the appended claims, the the active agent is linked to the substrate, and further wherein singular forms “a”, “an', and “the include plural references the conjugate is transported by the equilibrative nucleoside unless the context clearly dictates otherwise. Thus, for transporter, whereby the conjugate is transported into cancer example, references to “the method’ includes one or more ous cells, thereby delivering the active agent. In certain methods, and/or steps of the type described herein which will embodiments, the active agent is a tumor Suppressor gene or become apparent to those persons skilled in the art upon tumor Suppressor protein. reading this disclosure and so forth. According to still another embodiment of the invention, 25 Unless defined otherwise, all technical and scientific terms there are provided methods of treating an autoimmune disor used herein have the same meaning as commonly understood der including administering to a patient having the autoim by one of ordinary skill in the art to which this invention mune disorder a compound that inhibits transport by a belongs. nucleoside transport pathway. In accordance with the present invention, there are pro The present disclosure also provides a pharmaceutical 30 vided conjugates containing a Substrate that is capable of composition including a conjugate described herein and an being transported by a nucleoside transport pathway, and an agent that promotes ENT2 expression in a tissue. In some active agent linked to the substrate, wherein the conjugate is aspects, the agent that promotes ENT2 expression in a tissue transported by the nucleoside transport pathway. In this way, is an agent that treats or inhibits hypoxia or an agent that the conjugate, and thus, the active agent, is transported into inhibits HIF-1. The tissue may be a hypoxic tissue, such as a 35 the target cell. In particular embodiments in which the sub hypoxic tumor, a tissue with insufficient vasculature, an ulcer, strate is an antibody, the antibody is not the monoclonal a diabetic ulcer, a poorly-healing wound, an ischemic area, an antibody 3E10 or a fragment thereof. In certain embodiments, ischemic area resulting from stroke, or an ischemic area the nucleoside transport pathway includes an equilibrative resulting from cardiovascular disease. In certain embodi nucleoside transporter or a concentrative nucleoside trans ments, the agent that inhibits HIF-1C. is a siRNA, an RNAi 40 porter. In embodiments in which the nucleoside transport construct, a hairpin RNA, or a miRNA that reduces HIF-1C. pathway involves an equilibrative nucleoside transporter, expression. In some embodiments, the HIF-1C. inhibitor is a such a transporter may be insensitive to inhibition by low chemotherapeutic drug, topotecan, NSC 644221, PX-478, concentrations of nitrobenzylmercaptopurine riboside (NB YC-1, 17-AAG, or bevacizumab. In certain embodiments, the MBR) agent that treats or inhibits hypoxia is an agent that normal 45 “Nucleoside transport pathways' refer to systems of one or izes tumor vasculature, or an agent that alters the redox state more transport proteins that effect the transport of a substrate of a tissue. The agent that treats or inhibits hypoxia may be across one or more biological membranes. For example, a excess oxygen, TSC, or almitrine. nucleoside transport pathway may mediate the step-wise Furthermore, herein is provided a method of treating an transport of a Substrate across the plasma membrane followed ENT-2 deficient tissue, wherein the method includes: a) 50 by the transport of the substrate across the membrane of an administering an agent that promotes ENT2 expression and/ intracellular organelle. The transport proteins or nucleoside or activity, and b) administering one of the conjugates dis transporters responsible for Such a step-wise translocation of closed herein. a Substrate across two biological membranes may be the same type of nucleoside transporter or may be of different types. In DESCRIPTION OF THE FIGURES 55 certain embodiments, the nucleoside transporter may be an equilibrative nucleoside transporter. In other embodiments, FIGS. 1A-B show the nucleotide sequence (SEQID NO:1; the nucleoside transporter may be a concentrative nucleoside GenBank Accession No. U81375) and amino acid sequence transporter. (SEQ ID NO:2: GenBank Accession No. AAC51 103.1) of A “transport protein’ or “transporter is a protein that has human ENT1, respectively. 60 a direct or indirect role in transporting a molecule across a FIGS. 2A-B show the nucleotide sequence (SEQID NO:3: membrane. The term includes, for example, membrane GenBank Accession No. AF02.9358) and amino acid bound proteins that recognize a Substrate and effects its entry sequence (SEQ ID NO:4: GenBank Accession No. into, or exit from a cell by a carrier-mediated transporter or by AAC39526.1) of human ENT2, respectively. receptor-mediated transport. Transporters may be present on FIG. 3 shows the nucleotide sequence (SEQ ID NO:5; 65 plasma membranes or the membranes of intracellular GenBank Accession No. L16982) and amino acid sequence organelles. Thus, transporters facilitate the transport of mol (SEQ ID NO:6) of mAb 3E10 V. ecules into the cytoplasm or into an intracellular organelle. US 8,956,825 B2 5 6 Two different families of nucleoside transporters (NTs) employ sodium gradients resulting from a difference in intra have been characterized: equilibrative nucleoside transport cellular versus extracellular sodium concentration. This con ers and concentrative nucleoside transporters. “Equilibrative centration gradientallows an uphill or concentrative transport nucleoside transporters' or “ENTs’ refer to transporters that of Substrate across biological membranes. In general, the translocate Substrate down the Substrate's concentration gra Sodium concentration gradient across mammalian cell mem dient via passive transport or facilitated diffusion. ENT activ branes favors movement of Sodium and nucleoside into the ity does not require a sodium ion (or other ion) gradient and cell. CNTs are therefore considered “Na'-dependent' trans are therefore termed “Na'-independent' transporters. ENTs porters. There are currently three cloned members of the CNT are categorized into one of two Subtypes based on sensitivity family and differ from each other with respect to substrate to inhibition by nitrobenzylmercaptopurine riboside (NB 10 selectivity and Substrate to sodium ratio. MBR). One subtype of ENT (equilibrative, sensitive or “es”), CNT1 is known to transport pyrimidine nucleosides as well is inhibited by s1 nM NBMPR, whereas the other subtype as adenosine, the latter in a high-affinity, low-capacity man (equilibrative, insensitive or “ei'), is unaffected by low con ner. Transport via CNT1 occurs at a ratio of 1:1 sodium-to centrations (e.g., <1 uM) of NBMPR. nucleoside ratio. Exemplary polynucleotide sequences Four members of the ENT family have been cloned and are 15 encoding human CNT1 include GenBank Accession No. termed ENT1, ENT2, ENT3, and ENT4. All 4 transport U62968 and GenBank Accession No. AAB53839.1 repre adenosine but differ from each other with respect to their sents the corresponding amino acid sequence. CNT1 is pri ability to transport other nucleosides or nucleobases. ENT1 is marily expressed in epithelial cells of tissues, such as Small an es Subtype transporter. Exemplary polynucleotide intestine, kidney, and liver, as well as in many regions of the sequences encoding human ENT1 include GenBank Acces brain. Sion No. U81375 and GenBank Accession No. AAC51 103.1 CNT2 is known to transport purine nucleosides as well as represents the corresponding amino acid sequence. ENT1 is uridine. Transport via CNT2 occurs at a ratio of 1:1 sodium ubiquitously expressed in human and rodent tissues, although to-nucleoside ratio. Exemplary polynucleotide sequences expression levels vary between tissues. ENT1 is known to encoding human CNT2 include GenBank Accession No. transport a wide range of purine and pyrimidine nucleosides. 25 AF036109 and GenBank Accession No. AAB88539 repre ENT2 is an ei subtype transporter. Exemplary polynucle sents the corresponding amino acid sequence. CNT2 is otide sequences encoding human ENT2 include GenBank expressed in a wide range of human tissues such as the heart, Accession No. AF02.9358 and GenBank Accession No. liver, kidney, brain, placenta, pancreas, skeletal muscle, AAC39526 represents the corresponding amino acid colon, and the Small intestine. sequence. ENT2 is expressed in a wide range of human and 30 CNT3 is known to be broadly selective, transporting purine rodent tissues, including vascular endothelium, heart, brain, and pyrimidine nucleosides, as well as various nucleoside placenta, thymus, pancreas, prostate, kidney, and muscle, analogs. Transport via CNT3 occurs at a ratio of 2:1 sodium skeletal muscle, cardiac muscle, blood, skin, and ENT2-ex to-nucleoside ratio. Exemplary polynucleotide sequences pressing cancer cells. ENT2-expressing cancer cells include, encoding human CNT3 include GenBank Accession No. for example, certain renal tumor cells, breast tumor cells, 35 AF305210 and GenBank Accession No. AAG.22551 repre prostate cancer cells, colon cancer cells, stomach cancer cells, sents the corresponding amino acid sequence. CNT3 is leukemia cells, lung cancer cells, and ovarian cancer cells. expressed in tissues such as the trachea, pancreas, bone mar Other types of ENT-2 expressing cancer cells are known in row, and mammary gland, as well as in low levels in the the art; for example see Lu X et al., Journal of Experimental intestine, lung, placenta, prostate, testis, and liver. Therapeutics and Oncology 2:200-212, 2002, and Penny 40 A “conjugate' as used herein generally refers to a molecule cooke Met al., Biochemical and Biophysical Research Com which contains a Substrate that is capable of being transported munications 208,951-959, 2001. ENT2 exhibits high expres by a nucleoside transport pathway linked to an active agent. sion levels in skeletal muscle. ENT2 is also expressed in the The conjugate is also capable of being transported by a membrane of organelles such as the nucleus. ENT2 is known nucleoside transporter. to transport a wide range of purine and pyrimidine nucleo 45 A “substrate of a transport protein, as used generally sides and nucleobases. herein, is a compound whose uptake into a cell or organelle is ENT3 is an ei subtype transporter. Exemplary polynucle facilitated by the transport protein. Substrates have charac otide sequences encoding human ENT3 include GenBank teristic kinetic parameters (e.g., V, and K.) for a particular Accession No. NM 018344 and GenBank Accession No. transporter. V. refers to the number of molecules of Sub NP 060814 represents the corresponding amino acid 50 strate transported per unit time at Saturating concentration of sequence. ENT3 is widely expressed in different tissues and is the substrate. Krefers to the concentration of the substrate at abundant in placenta. ENT3 appears to be predominantly an which the Substrate is transported at half of V. In general, intracellular protein and co-localizes with lysosomal markers a high value of V is desirable for a Substrate of a trans in cultured cells. ENT3 is known to transporta wide range of porter. A low value of K is desirable for transport of low purine and pyrimidine nucleosides. 55 concentrations of a compound, and a high value of K is ENT4 is weakly inhibited by NBMPR. Exemplary poly desirable for transport of high concentrations of a compound. nucleotide sequences encoding human ENT4 include Gen V is affected both by the intrinsic turnover rate of a trans Bank Accession No. BC047592 and GenBank Accession No. porter (molecules/transporter protein) and transporter density AAH47592 represents the corresponding amino acid in plasma membrane that depends on expression level. For sequence. ENT4 is fairly ubiquitously expressed and is abun 60 these reasons, the intrinsic capacity of a compound to be dant in brain, skeletal muscle, and heart. ENT4 is also sub transported by a particular transporter is usually expressed as stantially expressed in intestine, pancreas, kidney, liver, bone the ratio V of the compound/V of a control compound marrow, and lymph node. ENT4 is known to transport a wide known to be a substrate for the transporter. range of purine and pyrimidine nucleosides and serotonin. A “substrate that is capable of being transported by a “Concentrative nucleoside transporters' or “CNTs’ refer 65 nucleoside transport pathway' refers to a molecule com to a group of nucleoside transporters that transport nucleo pound whose uptake into a cell or organelle is facilitated by a sides and nucleoside analogs by active transport. CNTs nucleoside transport protein or nucleoside transporter. Sub US 8,956,825 B2 7 8 strates used in the invention conjugates may be known Sub ZNF9, and PABPN1, Glycogen synthase, Glucose-6-phos strates of nucleoside transporters or may be identified using phatase, Debranching enzyme, Transglucosidase, Myophos methods known in the art and provided herein. Substrates phorylase, Phosphorylase, Phosphofructokinase, Acid Mal may include a nucleoside, a nucleobase, a nucleotide, a tase Deficiency, Carnitine Palmityl Transferase, nucleoside analog, an oligonucleotide, a peptide, a polypep Phosphoglycerate Kinase, or Phosphoglycerate Mutase, or a tide, an antibody, an antibody fragment. nucleic acid encoding any of said proteins. The term “nucleobase' refers to purine or pyrimidine In certain embodiments, the Substrate is an antibody or bases. Examples include adenine, cytosine, guanine, uracil, fragment thereof. For example, the antibody or fragment and thymine. Nucleobases include modified bases, such as thereof may bind nucleosides, nucleotides, nucleobases, oli pseudouridine, dihydrouridine, inosine, ribothymidine, 10 gonucleotides, polynucleotides, or nucleic acid. In certain 7-methylguanosine (m7G), hypoxanthine, and Xanthine. embodiments in which the substrate is an antibody, it is not The term “nucleoside' refers to a purine orpyrimidine base the 3E10 antibody or the antibody produced by the hybri that is covalently linked to a 5-carbon Sugar (i.e., pentose). doma having the ATCC accession number PTA 2439. When the sugar is ribose, the nucleoside is a ribonucleoside: In certain embodiments the Substrate portion of the conju when it is 2-deoxyribose, the nucleoside is a deoxyribo 15 gate may be a DNA-binding autoantibody. Examples of such nucleoside. Exemplary nucleosides include cytidine, uridine, DNA-binding autoantibodies include an antibody having the adenosine, guanosine, and thymidine, and the corresponding binding specificity of the antibody as produced by the hybri deoxyribonucleosides, which form the basis of the nucle doma having ATCC accession number PTA 2439, antibody otides that form DNA and RNA. mAb3E10, and variants and/or functional fragments thereof. The term “nucleoside analog as used herein refers to a The nucleotide and amino acid sequences for the variable nucleoside in which the base moiety, the Sugar moiety or both region of the heavy chain of mAb 3E10 are provided in FIG. has been modified. Such analogs are generally synthetic and 3. The nucleotide and amino acid sequences for the variable mimic natural nucleosides so that they may take the place of region of the light chains of mAb 3EI 0 are provided in FIG. a nucleoside in cellular functions. For example, nucleosides 4. In particular, the light chain designated VkIII contains the may be incorporated into DNA or RNA in place of the natural 25 DNA binding capability for mAb 3E10. Thus, VkIII is the corresponding nucleoside. Certain nucleoside analogs so preferred light chain for 3E10 to be used in the methods of the incorporated can, for example, prevent further elongation of present invention. the nucleic acid chain during synthesis. Many nucleoside Although antibodies that penetrate living cells are fre analogs have anti-viral oranti-cancer properties. Examples of quently toxic or injurious and may explain some of the patho nucleoside analogs include inosine, deoxyadenosine analogs 30 logic manifestations of the autoimmune diseases in which such as didanosine (2',3'-dideoxyinosine, ddI) and vidarabine they are found, antibody mAb 3E10, in contrast, shows no (9-O-D-ribofuranosyladenine), deoxycytidine analogs such harm to cells that it penetrates in tissue culture. Moreover, as cytarabine (cytosine arabinoside, emitricitabine, lamivu studies in vitro have shown that mAb 3E10 and schv frag dine (2',3'-dideoxy-3'-thiacytidine, 3TC), and Zalcitabine (2'- ments of mAb 3E10 can transport relatively large proteins, 3'-dideoxycytidine, ddC), deoxyguanosine analogs such as 35 Such as catalase, into the nucleus of cells in tissue culture. abacavir, (deoxy-)thymidine analogs such as stavudine (2'-3'- Moreover, mab 3E10 or fragments thereof (e.g., FV) should didehydro-2'-3'-dideoxythymidine, d4T) and zidovudine not generate significant inflammation in vivo which could (azidothymidine, or AZT), and deoxyuridine analogs such as hinder therapeutic efficacy of a biologically active molecule idoxuridine and trifluridine. conjugated thereto. Monoclonal antibody 3E10 is produced As used herein, the phrase “active agent” refers to a mol 40 by the hybridoma 3E10 placed permanently on deposit with ecule that has a biological effect in a cell. In certain embodi the American Type Culture Collection, 10801 University ments the active agent may be a nucleic acid, an inorganic Blvd., Manassas, Va. 20110-2209, USA, on Aug. 31, 2000, molecule, an organic molecule, a small organic molecule, a according to the terms of the Budapest Treaty under ATCC drug compound, a peptide, a polypeptide, an antibody, an accession number PTA-2439 and are thus maintained and antibody fragment, a peptidomrnimetic, a lipid, DNA, RNA, 45 made available according to the terms of the Budapest Treaty. a ribozyme, hairpin RNA, siRNA (small interfering RNAs) of Availability of Such strains is not to be construed as a license varying chemistries, miRNA, an antagomir, a PNA (peptide to practice the invention in contravention of the rights granted nucleic acid), an LNA (locked nucleic acids), or a mor under the authority of any government in accordance with its pholino. In certain embodiments, the active agent is a patent laws. polypeptide. 50 As used herein, “specific binding refers to antibody bind In other embodiments, the active agent is selected from the ing to a predetermined antigen. Typically, the antibody binds group consisting of C-glucosidase, C-L-iduronidase, C-ga with an affinity corresponding to aK, of about 10 Morless, lactosidase A, arylsulfatase, N-acetylgalactosamine-6-sulfa and binds to the predetermined antigen with an affinity (as tase or 3-galactosidase, iduronate 2-sulfatase, ceramidase, expressed by K) that is at least 10 fold less, and preferably at galactocerebrosidase, B-glucuronidase, Heparan N-sulfatase, 55 least 100 fold less than its affinity for binding to a non-specific N-Acetyl-O-glucosaminidase, Acetyl CoA-O-glucosaminide antigen (e.g., BSA, casein) other than the predetermined anti N-acetyl transferase, N-acetyl-glucosamine-6 Sulfatase, gen or a closely-related antigen. Alternatively, the antibody Galactose 6-sulfatase, Arylsulfatase A, B, or C, Arylsulfatase can bind with an affinity corresponding to a K of about 10 A Cerebroside, Ganglioside, Acid Bgalactosidase G. M', or about 107 M', or about 10 M', or 10 M' or Gaiglioside, Acid B-galactosidase, Hexosaminidase A, Hex 60 higher, and binds to the predetermined antigen with an affin osaminidase B, CX-fucosidase, Cl-N-Acetyl galactosamini ity (as expressed by K) that is at least 10 fold higher, and dase, Glycoprotein Neuraminidase, Aspartylglucosamine preferably at least 100 fold higher than its affinity for binding amidase, Acid Lipase, Acid Ceramidase, Lysosomal Sphin to a non-specific antigen (e.g., BSA, casein) other than the gomyelinase and other Sphingomyelinase. In certain embodi predetermined antigen or a closely-related antigen. In some ments, the active agent is dystrophin, components of dystro 65 embodiments the antibody variant or functional fragment will phin-glycoprotein complex, the laminin-C2 chain, fukutin have the same K or K, as an antibody produced by the related protein, LARGE, fukutin, EMD, LMNA, DMPK, hybridoma having ATCC accession number PTA 2439. In US 8,956,825 B2 10 certain embodiments, the antibody variant or functional frag Harlow and Lane, Antibodies. A Laboratory Manual (Cold ment will have the same K or K, as mAb3E10. Spring Harbor Laboratory 1988), which is incorporated The term "k" (sec'), as used herein, is intended to refer to herein by reference). Both anti-peptide and anti-conjugate the dissociation rate constant of a particular antibody-antigen antibodies can be used (see, for example, Bahouth et al., interaction. This value is also referred to as the off value. 5 Trends Pharmacol. Sci. 12:338 (1991); Ausubel et al., Cur The term "k,” (M'sec'), as used herein, is intended to rent Protocols in Molecular Biology (John Wiley and Sons, refer to the association rate constant of a particular antibody NY 1989) which are incorporated herein by reference). See in antigen interaction. The term “K (M), as used herein, is particular, FIGS. 2 and 3 for specific nucleotide and amino intended to refer to the association equilibrium constant of a acid sequences of the illustrative antibody of the invention particular antibody-antigen interaction. 10 designated mAb 3E10. The term “K,” (M'), as used herein, is intended to refer to For example, antibodies may be humanized by replacing the dissociation equilibrium constant of aparticular antibody sequences of the Fv variable region which are not directly antigen interaction. involved in antigen binding with equivalent sequences from Naturally occurring antibodies are generally tetramers human Fv variable regions. General reviews of humanized containing two light chains and two heavy chains. Experi 15 chimericantibodies are provided by Morrison et al., (Science mentally, antibodies can be cleaved with the proteolytic 229:1202-1207, 1985) and by Oietal. (BioTechniques 4:214, enzyme papain, which causes each of the heavy chains to 1986). Those methods include isolating, manipulating, and break, producing three separate subunits. The two units that expressing the nucleic acid sequences that encode all or part consist of a light chain and a fragment of the heavy chain of immunoglobulin Fv variable regions from at least one of a approximately equal in mass to the light chain are called the heavy or light chain. Sources of Such nucleic acid are well Fab fragments (i.e., the “antigen binding fragments). The known to those skilled in the art and, for example, may be third unit, consisting of two equal segments of the heavy obtained from for example, an antibody producing hybri chain, is called the Fc fragment. The Fc fragment is typically doma. The recombinant DNA encoding the humanized or not involved in antigen-antibody binding, but is important in chimeric antibody, or fragment thereof, can then be cloned later processes involved in ridding the body of the antigen. 25 into an appropriate expression vector. Humanized antibodies As used herein, the phrase “functional fragments of an can alternatively be produced by CDR substitution U.S. Pat. antibody having the binding specificity of the antibody as No.5.225,539; Jones (1986) Nature 321:552-525; Verhoeyan produced by the hybridoma having ATCC accession number etal. 1988 Science 239:1534; and Beidler (1988).J. Immunol. PTA 2439” refers to a fragment that retains the same cell 141:4053-4060. Thus, in certain embodiments, the antibody penetration characteristics and binding specificity as mAb 30 used in the conjugate is a humanized or CDR-grafted form of 3E10. Thus, in certain embodiments, a functional fragment of an antibody produced by the hybridoma having ATCC acces an antibody having the binding specificity of the antibody as sion number PTA 2439. In other embodiments the antibody is produced by the hybridoma having ATCC accession number a humanized or CDR-grafted form of antibody mAb 3E10. PTA 2439 or antibody mAb3E10 is used in the conjugate. In For example, the CDR regions of the illustrative antibody of Some embodiments, the functional fragment used in the con 35 the invention, as shown in FIGS. 2 and 3, can include amino jugate is selected from the group consisting of Fab., F(ab'), acid substitutions such as 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino Fv, and single chain FV (ScFV) fragments. In certain embodi acid differences from those shown in the figures. In some ments the functional fragment is an Fv fragments oran Schv instances, there are anywhere from 1-5 amino acid differ fragment. In one example, the functional fragment includes at CCCS, least the antigen-binding portion of mAb 3E10. In another 40 As used herein, reference to variants of an antibody having example, the functional fragments is an ScFV fragment the binding specificity of an antibody as produced by the including the variable region of the heavy chain (VH) and hybridoma having ATCC accession number PTA 2439" variable region of the kappa light chain (VK) of mAb 3E10. includes variants retaining the same cell penetration charac For increased expression in the polynucleotide from which teristics and binding specificity as mab 3E10, as well as the ScFv is expressed, the nucleic acids encoding the chains of 45 variants modified by mutation to improve the utility thereof mAb E310 are placed in reverse order with the VKc cDNA (e.g., improved ability to target specific cell types, improved being placed 5' of VH. In addition, one or more tags known in ability to penetrate the cell membrane, improved ability to the art, preferably peptide (e.g., myc or His), may be incor localize to the cellular DNA, and the like). Such variants porated into a conjugate to facilitate in vitro purification or include those wherein one or more conservative substitutions histological localization of the conjugate. In some embodi 50 are introduced into the heavy chain, the light chain and/or the ments, the a myc tag and a His tag are added to the C-termi constant region(s) of the antibody. In some embodiments the nus of VH. variant has a light chain having an amino acid sequence at As readily recognized by those of skill in the art, altered least 80% or at least 90% or at least 95% identical to the amino antibodies (e.g., chimeric, humanized, CDR-grafted, bifunc acid sequence set forth in SEQ ID NO:8. In other embodi tional, antibody polypeptide dimers (i.e., an association of 55 ments, the variant has a heavy chain having an amino acid two polypeptide chain components of an antibody, e.g., one sequence at least 80% or at least 90% or at least 95% identical arm of an antibody including a heavy chain and a light chain, to the amino acid sequence set forthin SEQID NO:6. Further, or an Fab fragment including V, V, C, and C1 antibody the invention includes antibodies that are encoded by nucleic domains, oran Fv fragment comprising a V, domain and a V acid sequences that hybridize under stringent conditions to domain), single chain antibodies (e.g., an Schv (i.e., single 60 the 3E10 variable region coding sequence (e.g., SEQID NO:5 chain FV) fragment including a V, domain linked to a V and/or SEQID NO:7) or encode amino acid sequences at least domain by a linker, and the like) can also be produced by 80% or at least 90% or at least 95% identical to the amino acid methods well known in the art. Such antibodies can also be sequence set forth in SEQID NO:6 or SEQID NO:8. produced by hybridoma, chemical synthesis or recombinant Such variants include those wherein one or more substitu methods described, for example, in (Sambrooketal. Molecu 65 tions are introduced into the heavy chain nucleotide lar Cloning: A Laboratory Manual 2d Ed. (Cold Spring Har sequence, the light chain nucleotide sequence and/or the con bor Laboratory, 1989); incorporated herein by reference and stant region(s) of the antibody. In some embodiments the US 8,956,825 B2 11 12 variant has a light chain having a nucleotide sequence at least S2, Spodoptera SJ9, CHO, COS (e.g. COS-7), or Bowes 80% or at least 90% or at least 95% identical to the nucleotide melanoma cells are all suitable host cells for use in practice of sequence set forth in SEQ ID NO:7. In other embodiments, the invention methods. the variant has a heavy chain having a nucleotide sequence at Conjugates in which the Substrate is a polypeptide and the least 80% or at least 90% or at least 95% identical to the active agent is a small molecule or drug compound may be nucleotide sequence set forth in SEQID NO:5. generated using methods known in the art. For example, One exemplary variant contemplated for use in the practice methods for attaching a drug or other Small molecule phar of the present invention is anmAb3E10 VH variant involving maceutical to protein include bifunctional chemical linkers a single change of the aspartic acid residue at position 31 to Such as N-Succinimidyl (4-iodoacetyl)-aminobenzoate; Sul 10 foSuccinimidyl(4-iodoacetyl)-aminobenzoate, 4-succinim asparagine (i.e., mAb 3E10-31). The preparation of this vari idyl-oxycarbonyl-O-(2-pyridyldithio) toluene; SulfoSuccin ant and further variants and a demonstration of its cell pen imidyl-6-O-methyl-O-(pyridyldithiol)-toluamido etration ability is described in U.S. Pat. No. 7,189,396. This hexanoate; N-Succinimidyl-3-(2-pyridyldithio)-proprionate; particular mAb 3E10 variant is especially well suited for Succinimidyl-6-3 (-(-2-pyridyldithio)-proprionamidohex delivery of biological molecules to kidney and brain cells. 15 anoate; SulfoSuccinimidyl-6-3(-(-2-pyridyldithio)-propi Other 3E10 variants and/or functional fragments thereof may onamidohexanoate; 3-(2-pyridyldithio)-propionyl be used to provide targeting of biologically active molecules. hydrazide, Ellman's reagent, dichlorotriazinic acid, S-(2-thi A wide variety of variants and/or functional fragments opyridyl)-L-cysteine, and the like. Further bifunctional link thereof are possible provided that they exhibit substantially ing molecules are disclosed in U.S. Pat. Nos. 5,349.066: the same cell penetration characteristics as mab 3E10 or 5,618,528; 4,569,789: 4,952,394; and 5,137,877, each of mAb 3E10-31 after conjugation to a selected biologically which is incorporated herein by reference in its entirety. active molecule. According to a further embodiment of the invention, there In other embodiments, novel Substrates can be generated are provided methods of Screening a conjugate for transport that target one or more specific nucleoside transporter. Such by a nucleoside transport pathway in which the method novel Substrates could be generated using, for example 25 includes, contacting a cell expressing the nucleoside transport molecular modeling and protein mimetic methodologies pathway, with a conjugate under Suitable conditions for trans based on structures of known Substrates. port to occur; and determining whether the conjugate is trans Conjugates in which the Substrate and active agent are ported into the cell by the nucleoside transport pathway. In polypeptides (i.e., protein conjugates) can be designed to certain embodiments, the determining step includes compar place the active agent at the amino or carboxy terminus the 30 ing the amount of conjugate transported into a cell expressing substrate using well-known recombinant DNA methodolo the nucleoside transport system to the amount of conjugate gies. Such conjugates can be expressed in a host cell as a transported into a control cell not expressing the nucleoside fusion protein. Alternatively, the Substrate and active agent transport system, wherein an increase in transport of conju can be chemically linked by a peptide bond or by a chemical gate of the cell expressing the nucleoside transport pathway or peptide linker molecule of the type well known in the art. 35 as compared to the control cell indicates transport is by that The linker may be one or more tags (e.g., myc or His (SEQ nucleoside transport pathway. IDNO:12)) or may be one or more repeats of the known linker In particular embodiments of the above screening method, sequence GGGGS (SEQID NO:11). Additional peptide link the nucleoside transport pathway includes an equilibrative ers are known in the art. The skilled artisan will recognize that nucleoside transporter or a concentrative nucleoside trans the linker sequence may be varied depending on the polypep 40 porter. In some embodiments, the equilibrative nucleoside tide to be linked to the antibody. transporter is selected from the group consisting of ENT1, Vectors suitable for use in preparation of protein conju ENT2, ENT3, and ENT4. In certain embodiments, the equili gates include those selected from baculovirus, phage, plas brative nucleoside transporter is insensitive to low concentra mid, phagemid, cosmid, fosmid, bacterial artificial chromo tions of nitrobenzylmercaptopurine riboside (NBMBR). In some, viral DNA, Pl-based artificial chromosome, yeast 45 particular embodiments, the cell is transfected with DNA plasmid, and yeast artificial chromosome. For example, the encoding the nucleoside transporter. Such embodiments may viral DNA vector can be selected from vaccinia, adenovirus, further include a step in which the amount of conjugate trans foul pox virus, pseudorabies and a derivative of SV40. Suit ported into the cell transfected with DNA encoding the able bacterial vectors for use in practice of the invention nucleoside transporter is compared to the amount of conju methods include pGE70, pGE60, pGE-9, pBLUESCRIPT 50 gate transported into a control cell not transfected with the SK, pBLUESCRIPT KS, pTRC99a, pKK223-3, plDR540, nucleoside transporter, wherein an increase in transport of PAC and pRIT2T. Suitable eukaryotic vectors for use in prac conjugate of the transfected cell as compared to the control tice of the invention methods include pWLNEO, pXTI, cell indicates transport is by the nucleoside transporter. pSG5, pSVK3, pBPV, pMSG, and pSVLSV40. Suitable Screening methods may further include compounds that eukaryotic vectors for use in practice of the invention meth 55 inhibit nucleoside transport activity. For example, NBMPR, ods include pWLNEO, pXTI, pSG5, pSVK3, pBPV, pMSG, dilaZep, dipyridamole, and draflazine are inhibitors of certain and pSVLSV40. es nucleoside transporters (e.g., ENT1). Thus, in certain Those of skill in the art can select a suitable regulatory embodiments low concentrations of NBMPR may be region to be included in Such a vector, for example from lacI. included. lacz, T3, T7, apt, lambda PR, PL, trp, CMV immediate early, 60 In certain embodiments, the conjugate may further contain HSV thymidine kinase, early and late SV40, retroviral LTR, a detectable label. Such labels are known in the art and and mouse metallothionein-I regulatory regions. include radio-isotopes and fluorescent labels. Host cells in which the vectors containing the polynucle Conjugates can be screened directly for their capacity to be otides encoding the protein conjugates can be expressed transported by nucleoside transport pathways. The screening include a bacterial cell, a eukaryotic cell, a yeast cell, an insect 65 is typically performed on cells expressing the nucleoside cell, or a plant cell. For example, E. coli, Bacillus, Strepto transport pathway. In some methods, the cells are transfected myces, Pichia pastoris, Salmonella typhimurium, Drosophila with DNA encoding the a particular nucleoside transporter US 8,956,825 B2 13 14 (NT). In other methods, cells expressing an endogenous NT complementary mRNA and inhibit translation or induce are used. Cells may express endogenous CNTs and/or an RNaseH-mediated degradation of the transcript; siRNA oli ENTs. In some methods, an ENT is the only NT expressed. In gonucleotides, which are short, double-stranded RNAs that other methods, cells expressing both ENT1 and ENT2 are activate the RNA interference (RNAi) pathway leading to used. mRNA degradation: ribozymes, which are oligonucleotide Internalization of a compound evidencing passage through based endonucleases that are designed to cleave specific transporters can be detected by detecting a signal from within mRNA transcripts; and nucleic acid aptamers and decoys, a cell from any of a variety of reporters. The reporter can be as which are non-naturally occurring oligonucleotides that bind simple as a label Such as a fluorophore, a chromophore, a to and block protein targets in a manner analogous to Small radioisotope. Confocal imagining can also be used to detect 10 internalization of a label as it provides sufficient spatial reso molecule drugs. lution to distinguish between fluorescence on a cell Surface As used herein, the term “nucleic acid refers to polynucle and fluorescence within a cell; alternatively, confocal imag otides such as deoxyribonucleic acid (DNA), and, where ing can be used to track the movement of compounds over appropriate, ribonucleic acid (RNA). The term should also be time. In another approach, internalization of a compound is 15 understood to include, as appropriate to the context or as detected using a reporter that is a Substrate for an enzyme applicable to the embodiment being described, both single expressed within a cell. Once the complex is internalized, the Stranded polynucleotides (such as antisense) and double Substrate is metabolized by the enzyme and generates an stranded polynucleotides (such as siRNAs). The term optical signal or radioactive decay that is indicative of uptake. “nucleic acid encompasses, for example, DNA molecules, Light emission can be monitored by commercial PMT-based RNA molecules, RNAi molecules and siRNA molecules, instruments or by CCD-based imaging systems. In addition, microRNA molecules, native RNA molecules, ribozyme assay methods utilizing LCMS detection of the transported RNA molecules, aptamers, plasmids, cDNA molecules, anti compounds or electrophysiological signals indicative of sense DNA strands, and oligonucleotides. It further encom transport activity are also employed. passes DNA molecules (in the form of plasmids, cDNA, In some methods, multiple conjugates are screened simul 25 linear DNA, oligos or anti-sense DNA stands) RNA mol taneously and the identity of each agent or conjugate moiety ecules (in the form of siRNA, mRNA, shRNA, ribozymes, is tracked using labels linked to the conjugates. In some RNAi.) aptamers, proteins (antibodies, polypeptides, pep methods, the screening can be performed in a competition tides or fragment of proteins), nucleic acids conjugated to format in which an a conjugate under test and a known Sub other compounds (such as fluorescent dyes, Small molecular strate of the nucleoside transporter are applied to the same 30 inhibitors of specific proteins). There are a number of nucleic cells. Typically, the conjugate and known Substrate are dif acid-based therapeutic agents in various stages of develop ferentially labeled in such assays. Alternatively, the known ment at this time. Among them are antisense agents, aptam Substrate may be labeled and parallel measurements of uptake ers, ribozymes, and small interfering RNAs (siRNAs). M. of labeled Substrate in the presence and absence test conjugate Faria, H. Ulrich, Curr. Cancer Drug Targets 2002, 2:355-368. may be compared. 35 Antisense agents may be the most advanced class of these In such comparative assays, the Vmax of a conjugate can be agents, with one product (fomivirsen) on the market for the compared with that of known Substrate. If a conjugate has a treatment of CMV retinitis, another (alicaforsen) in advanced Vmax of at least 1%, preferably at least 5%, more preferably clinical trials for treatment of Crohn's disease, and Gena at least 10%, even more preferably at least 20%, and most senseTM (oblimersen sodium), AffinitacTM, and Oncomyc preferably at least 50% of known substrate for the transporter 40 NGTM in clinical trials for treatment of cancer. Antisense then the conjugate can be considered to be a substrate for that agents are typically short, chemically-modified oligonucle NT. otide chains that hybridize to a specific complementary area Nucleic Acid Therapeutics of a targeted mRNA. The resulting mRNA duplex is recog In certain embodiments, the compositions herein may be nized and degraded by RNAse H, thereby destroying the used to deliver nucleic acids, or analogs thereof, to a targeted 45 mRNA. Because the mRNA instructions fail to reach the tissue or cell type. For example, protein expression can be ribosome, production of the protein encoded by the targeted specifically down-regulated using oligonucleotides such, for mRNA is prevented. By inhibiting the production of proteins example, as antisense, locked nucleic acids (LNA), peptide involved indisease, antisense drugs can produce atherapeutic nucleic acids (PNA), morpholino nucleic acids (Morpholi benefit. nos) and small interfering RNAs (siRNA) of various chem 50 An aptamer is a DNA or RNA molecule that has been istries. Alternatively, expression constructs may be delivered selected from a random or biased pool of oligonucleic acids, to cells, to induce expression of a desired gene product. based on its ability to bind to a target molecule. Aptamers can Nucleic acids which modulate the expression of a certain be selected which bind nucleic acids, proteins, Small organic gene or gene product may be administered. As used herein, “a compounds and specific cell Surfaces, and several have been nucleic acid that modulates expression of . . . . encompasses 55 developed which bind to proteins which are associated with nucleic acids that up-regulate and down-regulate the expres disease states. Aptamers are in general more easily manufac sion of the given gene or gene product. For example, an tured and are more amenable to chemical modification than expression construct can expresses the gene of interest and are antibodies, and they can be "evolved for tighter binding cause up-regulation. Alternatively, a nucleic acid that causes to the target by an iterative process of random modification down-regulation can be, for example, a siRNA, a construct 60 and affinity-based selection. The evolved aptamers often have that expresses an antisense RNA (such as a short hairpin antibody-like specificities, and are therefore expected to have RNA), or a ribozyme. utility in those applications, such as therapeutics and in vitro Nucleic acid therapeutics, such as oligonucleotides and in vivo diagnostics, where antibodies have already proved directed against intracellular targets (mRNA or protein), are useful. At least one product, MacugenTM (pegaptainib Sodium, powerful therapeutic agents. Examples of oligonucleotide 65 a PEGylated aptamer with high affinity for VEGF), is in therapeutic agents include: antisense oligonucleotides, which advanced clinical trials for the treatment of age-related macu are short, single-stranded DNAs and RNAs that bind to lar degeneration. US 8,956,825 B2 15 16 Ribozymes, or RNA enzymes, are RNA molecules that can acids, and nucleic acid oligomers that have otherwise been catalyze a chemical reaction. All ribozymes found naturally chemically modified as described above. Again, any of these so far catalyze the cleavage of RNA. They range in size from species may also feature chemically modified purines and the large “hammerhead ribozymes to the so-called pyrimidines or may be lipophilically modified and/or PEGy “minizymes' which are synthetic constructs containing the 5 lated. See M. Rimmele, Chembiochem. 2003, 4:963-71 and minimal structures needed for activity. DNA-based enzymes A. Vater and S. Klussmann, Curr. Opin. Drug Discov. Devel. (deoxyribozymes, or DNAZymes) having similar properties 2003, 6: 253-61 for recent reviews of aptamer technology. It have also been prepared. The ability of ribozymes to recog will be appreciated that many members of this second class nize and cut specific mRNA molecules gives them consider will, in addition to their structure-specific affinity for the able potential as therapeutic agents. A ribozyme designed to 10 target molecule, have sequence-specific affinity for a putative catalyze the cleavage of a specific mRNA would be useful as DNA or RNA sequence. a therapeutic agent in the same way that a complimentary The third class of nucleic acid-based therapeutic agents, antisense nucleic acid would be, but with the advantage that a referred to herein as “nucleic acid enzymes, comprises single ribozyme molecule can destroy many copies of the nucleic acids that are capable of recognizing and catalyzing mRNA. A synthetic ribozyme (AngiozymeTM) that cleaves 15 the cleavage of target RNA molecules, in a sequence-specific the mRNA encoding a VEGF receptor subtype is currently in manner. The class includes hammerhead ribozymes, mini clinical trials for treatment of cancer. mized hammerheads (“minizymes'), 10-23 deoxyri RNA interference (RNAi) is the phenomenon of gene bozymes (“DNAZymes'), and the like. As with antisense and specific post-transcriptional silencing by double-stranded aptamer molecules, the class includes catalytic species that RNA oligomers (Elbashir et al. Nature 2001, 411: 494-498: have been chemically modified. Caplen et al., Proc. Natl. Acad. Sci. U.S.A. 2001, 98: 9742 The term “pharmaceutically acceptable salts' refers to 9747). Small inhibitory RNAs (siRNAs), like antisense oli physiologically and pharmaceutically acceptable salts of the gonucleic acids and ribozymes, have the potential to serve as compounds of the invention, e.g., salts that retain the desired therapeutic agents by reducing the expression of harmful biological activity of the parent compound and do not impart proteins. The double-stranded siRNA is recognized by a pro 25 undesired toxicological effects thereto. tein complex (the RNA induced silencing complex), which A "protein coding sequence' or a sequence that “encodes' strips away one of the strands, facilitates hybridization of the a particular polypeptide or peptide, is a nucleic acid sequence remaining strand to the target mRNA, and then cleaves the that is transcribed (in the case of DNA) and is translated (in target Strand. DNA-based vectors capable of generating the case of mRNA) into a polypeptide in vitro or in vivo when siRNA within cells are also of interest for the same reason, as 30 placed under the control of appropriate regulatory sequences. are short hairpin RNAs that are efficiently processed to form The boundaries of the coding sequence are determined by a siRNAs within cells. siRNAs capable of specifically targeting start codon at the 5' (amino) terminus and a translation stop endogenously and exogenously expressed genes have been codon at the 3' (carboxyl) terminus. A coding sequence can described; see for example Paddison et al., Proc. Natl. Acad. include, but is not limited to, cDNA from prokaryotic or Sci. U.S.A., 2002, 99: 1443-1448; Paddison et al., Genes & 35 eukaryotic mRNA, genomic DNA sequences from prokary Dev. 2002, 16:948-958; Sui et al. Proc. Natl. Acad. Sci. otic or eukaryotic DNA, and even synthetic DNA sequences. U.S.A. 2002, 8: 5515-5520; and Brummelkamp et al., Sci A transcription termination sequence will usually be located ence 2002, 296: 550-553. 3' to the coding sequence. The term “nucleic acid-based therapeutic agent” as used As used herein, the term “RNAi construct” is a generic herein refers to three classes of compounds. The term also 40 term including siRNA, hairpin RNA, and other RNA species includes pharmaceutically acceptable salts, esters, prodrugs, which can be cleaved in vivo to form siRNAs. RNAi con codrugs, and protected forms of the compounds, analogs and structs herein also include expression vectors (also referred to derivatives described below. The first class, referred to herein as RNAi expression vectors) capable of giving rise to tran collectively as “antisense nucleic acids, comprises nucleic scripts which form dsRNAs or hairpin RNAs in cells, and/or acids, preferably oligomers of about 50 monomer units or 45 transcripts which can be converted into siRNAs in vivo. fewer, which have the ability to hybridize in a sequence As used herein, the term “vector” refers to a nucleic acid specific manner to a targeted single-stranded RNA or DNA molecule capable of transporting another nucleic acid to molecule. Members of this class include ordinary DNA and which it has been linked. One type of vector is a genomic RNA oligomers, DNA and RNA having modified backbones, integrated vector, or “integrated vector, which can become including but not limited to phosphorothioates, phospho 50 integrated into the chromosomal DNA of the host cell. rodithioates, methylphosphonates, and peptide nucleic acids, Another type of vector is an episomal vector, e.g., a nucleic 2'-deoxy derivatives, and nucleic acid oligomers that feature acid capable of extra-chromosomal replication. Vectors chemically modified purine and pyrimidine bases, or have capable of directing the expression of genes to which they are been lipophilically modified and/or PEGylated to modify operatively linked are referred to herein as “expression vec their pharmacodynamics. Oligomers that serve as precursors 55 tors.” In the present specification, "plasmid' and “vector' are for Such agents, such as hairpin RNAS that are converted to used interchangeably unless otherwise clear from the context. siRNAs within cells, are also considered to be within this In the expression vectors, regulatory elements controlling class. transcription can be generally derived from mammalian, The second class of nucleic acid-based therapeutic agents microbial, viral or insect genes. The ability to replicate in a is aptamers. Aptamers comprises nucleic acids, preferably 60 host, usually conferred by an origin of replication, and a oligomers of about 50 monomer units or fewer, which have selection gene to facilitate recognition of transformants may the ability to bind with structural specificity to a non-oligo additionally be incorporated. Vectors derived from viruses, nucleotide target molecule, or to an oligonucleotide in a man Such as retroviruses, adenoviruses, and the like, may be ner other than through sequence-specific hybridization. employed. Members of this class include DNA and RNA aptamers, and 65 In one embodiment, the present disclosure relates to the use modifications thereof including but not limited to mirror of antisense nucleic acid to decrease expression of a targeted image DNA and RNA (“Spiegelmers'), peptide nucleic disease-related protein. Such an antisense nucleic acid can be US 8,956,825 B2 17 18 delivered, for example, as an expression plasmid which, when cell) of double-stranded material may yield more effective transcribed in the cell, produces RNA which is complemen inhibition, while lower doses may also be useful for specific tary to at least a unique portion of the cellular mRNA which applications. encodes the targeted disease-related protein. Alternatively, The subject RNAi constructs can be “small interfering the construct is an oligonucleotide which is generated ex vivo 5 RNAs' or “siRNAs.” These nucleic acids are less than about and which, when introduced into the cell causes inhibition of 50, and preferably around 19-30 nucleotides in length, more expression by hybridizing with the mRNA and/or genomic preferably 21-23 nucleotides in length. The siRNAs are sequences encoding the targeted disease-related protein. thought to recruit nuclease complexes and guide the com Such oligonucleotides are optionally modified so as to be plexes to the target mRNA by pairing to the specific 10 sequences. As a result, the target mRNA is degraded by the resistant to endogenous exonucleases and/or endonucleases. nucleases in the protein complex. In a particular embodiment, Exemplary nucleic acid molecules for use as antisense oligo the 21-23 nucleotides siRNA molecules comprise a 3 nucleotides are phosphoramidate, phosphothioate and meth hydroxyl group. In certain embodiments, the siRNA con ylphosphonate analogs of DNA (see for example U.S. Pat. structs can be generated by processing of longer double Nos. 5,176,996; 5,264,564; and 5.256,775). General 15 stranded RNAs, for example, in the presence of the enzyme approaches to constructing oligomers useful in nucleic acid DICER. In one embodiment, the Drosophila in vitro system is therapy have been reviewed, for example, by van der Krolet used. In this embodiment, dsRNA is combined with a soluble al., (1988) Biotechniques 6:958-976; and Stein et al., (1988) extract derived from Drosophila embryo, thereby producing a Cancer Res 48: 2659-2668. combination. The combination is maintained under condi In other embodiments, this application relates to the use of tions in which the dsRNA is processed to RNA molecules of RNA interference (RNAi) to effect knockdown of the targeted about 21 to about 23 nucleotides. The siRNA molecules can gene. RNAi constructs comprise double stranded RNA that be purified using a number of techniques known to those of can specifically block expression of a target gene. RNAi skill in the art, such as gel electrophoresis. Alternatively, constructs can comprise either long stretches of dsRNA iden non-denaturing methods, such as column chromatography, tical or Substantially identical to the target nucleic acid 25 size exclusion chromatography, glycerol gradient centrifuga sequence, or short stretches of dsRNA identical or substan tion, and affinity purification can be used to purify siRNAs. tially identical to only a region of the target nucleic acid Production of RNAi constructs can be carried out by Sequence. chemical synthetic methods or by recombinant nucleic acid Optionally, the RNAi constructs may contain a nucleotide techniques. Endogenous RNA polymerase of the treated cell sequence that hybridizes under physiologic conditions of the 30 may mediate transcription in vivo, or cloned RNA poly cell to the nucleotide sequence of at least a portion of the merase can be used for transcription in vitro. The RNAi mRNA transcript for the gene to be inhibited (the “target” constructs may include modifications to either the phosphate gene). The double-stranded RNA need only be sufficiently Sugar backbone or the nucleoside, e.g., to reduce Susceptibil similar to natural RNA that it has the ability to induce RNAi. ity to cellular nucleases, improve bioavailability, improve Thus, the invention contemplates embodiments that are tol 35 formulation characteristics, and/or change other pharmaco erant of sequence variations that might be expected due to kinetic properties. For example, the phosphodiester linkages genetic mutation, polymorphic sites, or evolutionary diver of natural RNA may be modified to include at least one gence in a targeted sequence. The number of tolerated nucle nitrogen or sulfur heteroatom. Modifications in RNA struc otide mismatches between the target sequence and the RNAi ture may be tailored to allow specific genetic inhibition while construct sequence may be as high as 1 in 5 base pairs, but is 40 avoiding a general response to dsRNA. Likewise, bases may preferably no higher than 1 in 10 base pairs. Mismatches in be modified to block the activity of adenosine deaminase. The the center of the siRNA duplex are most critical and may RNAi construct may be produced enzymatically or by partial/ essentially abolish cleavage of the target RNA. In contrast, total organic synthesis, any modified ribonucleotide can be nucleotides at the 3' end of the siRNA strand that is comple introduced by in vitro enzymatic or organic synthesis. Meth mentary to the target RNA do not significantly contribute to 45 ods of chemically modifying RNA molecules can be adapted specificity of the target recognition. Sequence identity may be for modifying RNAi constructs (see, e.g., Heidenreich et al. optimized by sequence comparison and alignment algorithms (1997) Nucleic Acids Res. 25: 776-780: Wilson et al. (1994) known in the art (see Gribskov and Devereux, Sequence J. Mol. Recog. 7: 89-98: Chen et al. (1995) Nucleic Acids Analysis Primer, Stockton Press, 1991, and references cited Res. 23: 2661-2668; Hirschbein et al. (1997) Antisense therein) and calculating the percent difference between the 50 Nucleic Acid Drug, Dev. 7: 55-61). For example, the back nucleotide sequences by, for example, the Smith-Waterman bone of an RNAi construct can be modified with phospho algorithm as implemented in the BESTFIT software program rothioates, phosphoramidate, phosphodithioates, chimeric using default parameters (e.g., University of Wisconsin methylphosphonate-phosphodie-sters, peptide nucleic acids, Genetic Computing Group). Between 90% and 100% 5-propynyl-pyrimidine containing oligomers or Sugar modi sequence identity between the inhibitory RNA and the por 55 fications (e.g., 2-substituted or 2'-deoxy ribonucleosides, tion of the target gene is preferred. Alternatively, the duplex ..alpha.-configurations, etc.) region of the RNA may be defined functionally as a nucle In some embodiments, at least one strand of the siRNA otide sequence that is capable of detectably hybridizing with molecules may have a 3' overhang from about 1 to about 6 the target genetranscript after hybridization for 12 to 16 hours nucleotides in length. Preferably, the 3' overhangs are 1-3 at 50...degree. C. to 70.degree. C. in 400 mM. NaCl, 40 mM 60 nucleotides in length. In certain embodiments, one Strand has PIPES pH 6.4, and 1.0 mM EDTA, followed by washing. a3' overhang and the other strand is blunt-ended or also has an The double-stranded structure may be formed by a single overhang. The length of the overhangs may be the same or self-complementary RNA strand or two complementary different for each strand. In order to further enhance the RNA strands. Formation of the dsRNA may be initiated stability of the siRNA, the 3' overhangs can be stabilized inside or outside of the cell. The RNA may be introduced in an 65 against degradation. In one embodiment, the RNA is stabi amount which allows delivery of at least one copy per cell. lized by including purine nucleotides, such as adenosine or Higher doses (e.g., at least 5, 10, 100, 500 or 1000 copies per guanosine nucleotides. Alternatively, Substitution of pyrimi US 8,956,825 B2 19 20 dine nucleotides by modified analogues, e.g., Substitution of (or nearly unique) target sequence. Preferably, the sequence is uridine nucleotide 3' overhangs by 2'-deoxythymidine, may a G/C rich stretch of approximately 18 to 22 nucleotides. be tolerated without reducing the effectiveness of the RNAi. High G/C content helps insure a stronger interaction between The absence of a 2 hydroxyl significantly enhances the the DNA enzyme and the target sequence. When synthesizing nuclease resistance of the overhang in tissue culture medium, the DNA enzyme, the specificantisense recognition sequence and may be also beneficial in vivo. that will target the enzyme to the message is divided so that it The RNAi construct can also be in the form of a long comprises the two arms of the DNA enzyme, and the DNA double-stranded RNA, which is digested intracellularly to enzyme loop is placed between the two specific arms. Meth produce a siRNA sequence within the cell. Alternatively, the ods of making and administering DNA enzymes can be RNAi construct may be in the form of a hairpin RNA. It is 10 found, for example, in U.S. Pat. No. 6,110,462. known in the art that siRNAs can be produced by processing The methods described herein may be used to deliver a hairpin RNAs in the cell. Hairpin RNAs can be synthesized variety of molecules, including but not limited to Small mol exogenously or can be formed by transcribing from RNA ecules (including Small molecules that do not have optimal polymerase III promoters in vivo. Examples of making and cell-permeability), lipids, nucleosides, nucleotides, nucleic using hairpin RNAS for gene silencing in mammalian cells 15 acids, polynucleotides, oligonucleotides, antibodies, toxins, are described in, for example, Paddison et al., Genes Dev, negatively charged polymers and other polymers, for 2002, 16:948-58; McCaffrey et al., Nature, 2002, 418:38-9: example proteins, peptides, hormones, carbohydrates, or McManus et al., RNA, 2002, 8: 842-50; Yu et al., Proc. Natl. polyamines, across cellular membranes. Non-limiting Acad. Sci. USA, 2002,99: 6047-52). Preferably, such hairpin examples of polynucleotides that can be delivered across RNAS are engineered in cells or in an animal to ensure con cellular membranes using the compounds and methods of the tinuous and stable Suppression of a desired gene. invention include short interfering nucleic acid (siNA), anti PCT application WO 01/77350 describes an exemplary sense, enzymatic nucleic acid molecules, 2',5'-oligoadeny vector for bi-directional transcription of a transgene to yield late, triplex forming oligonucleotides, aptamers, and decoys. both sense and antisense RNA transcripts of the same trans Biologically active molecules that may be delivered include gene in a eukaryotic cell. Accordingly, in certain embodi 25 antibodies (e.g., monoclonal, chimeric, humanized etc.), cho ments, the present invention provides a recombinant vector lesterol, hormones, antivirals, peptides, proteins, chemo having the following unique characteristics: it comprises a therapeutics, Small molecules, vitamins, co-factors, nucleo viral replicon having two overlapping transcription units sides, nucleotides, oligonucleotides, enzymatic nucleic acids, arranged in an opposing orientation and flanking a transgene antisense nucleic acids, triplex forming oligonucleotides, for an RNAi construct of interest, wherein the two overlap 30 2.5-A chimeras, allozymes, aptamers, decoys and analogs ping transcription units yield both sense and antisense RNA thereof, and Small nucleic acid molecules. Such as short inter transcripts from the same transgene fragment in a host cell. fering nucleic acid (siNA), short interfering RNA (siRNA), In another embodiment, the present disclosure relates to double-stranded RNA (dsRNA), micro-RNA (miRNA), the use of ribozyme molecules designed to catalytically antagomirs, and short hairpin RNA (shRNA) molecules, to cleave an mRNA transcript to prevent translation of the 35 relevant cells and/or tissues, such as in a subject or organism. mRNA (see, e.g., PCT International Publication WO90/ The compounds, compositions, and methods of the inven 11364, published Oct. 4, 1990; Sarver et al., 1990, Science tion can increase delivery or availability of biologically active 247: 1222-1225; and U.S. Pat. No. 5,093,246). While any molecules (e.g., siNAs, siRNAs, miRNAs, siRNA and ribozyme that cleaves the target mRNA at a site-specific miRNA inhibitors, nucleic acids, polynucleotides, oligo recognition sequence can be used to destroy that particular 40 nucleotides, peptides, polypeptides, proteins, hormones, anti mRNA, the use of hammerhead ribozymes is preferred. Ham bodies, and Small molecules) to cells or tissues compared to merhead ribozymes cleave mRNAs at locations dictated by delivery of the molecules in the absence of the compounds, flanking regions that form complementary base pairs with the compositions, and methods of the invention. As such, the target mRNA. The sole requirement is that the target mRNA level of a biologically active molecule inside a cell, tissue, or have the following sequence of two bases: 5'-UG-3'. The 45 organism is increased in the presence of the compounds and construction and production of hammerhead ribozymes is compositions of the invention compared to when the com well known in the art and is described more fully in Haseloff pounds and compositions of the invention are absent. and Gerlach, 1988, Nature, 334: 585-591. The ribozymes of The term “ligand” refers to any compound or molecule, the present invention also include RNA endoribonucleases Such as a drug, peptide, hormone, or neurotransmitter that is (“Cech-type ribozymes') such as the one which occurs natu 50 capable of interacting with another compound, such as a rally in Tetrahymena thermophila (known as the IVS or L-19 receptor, either directly or indirectly. The receptor that inter IVS RNA) and which has been extensively described (see, acts with a ligand can be present on the Surface of a cell or can e.g., Zaug, et al., 1984, Science, 224: 574-578; Zaug and alternately be an intercellular receptor. Interaction of the Cech, 1986, Science, 231: 470-475; Zaug, et al., 1986, ligand with the receptor can result in a biochemical reaction, Nature, 324: 429–433; published International patent appli 55 or can simply be a physical interaction or association. Non cation No. WO88/04300 by University Patents Inc.; Been and limiting examples of ligands include Sugars and carbohy Cech, 1986, Cell, 47: 207-216). drates such as galactose, galactosamine, and N-acetylgalac In a further embodiment, the invention relates to the use of tosamine; hormones such as estrogen, testosterone, DNA enzymes to inhibit expression of a targeted gene. DNA progesterone, glucocortisone, adrenaline, insulin, glucagon, enzymes incorporate some of the mechanistic features of both 60 cortisol, Vitamin D, thyroid hormone, retinoic acid, and antisense and ribozyme technologies. DNA enzymes are growth hormones; growth factors such as VEGF, EGF, NGF, designed so that they recognize aparticular target nucleic acid and PDGF; cholesterol; bile acids; neurotransmitters such as sequence, much like an antisense oligonucleotide; however, GABA, Glutamate, acetylcholine; NOGO; inostitol triphos much like a ribozyme, they are catalytic and specifically phate; diacylglycerol; epinephrine; norepinephrine; Nitric cleave the target nucleic acid. Briefly, to design an ideal DNA 65 Oxide, peptides, vitamins such as folate and pyridoxine, enzyme that specifically recognizes and cleaves a target drugs, antibodies and any other molecule that can interact nucleic acid, one of skill in the art must first identify a unique with a receptor in vivo or in vitro. The ligand can be attached US 8,956,825 B2 21 22 to a compound of the invention using a linker molecule. Such Examples of linking reagents which contain cleavable dis as an amide, amido, carbonyl, ester, peptide, disulphide, ulfide bonds (reducable bonds) include, but are not limited to silane, nucleoside, abasic nucleoside, polyether, polyamine, “DPDPB, 1,4-di-3'-(2-pyridyldithio)propionamidobu polyamide, peptide, carbohydrate, lipid, polyhydrocarbon, tane: “SADP, (N-succinimidyl (4-azidophenyl) 1,3'-dithio phosphate ester, phosphoramidate, thiophosphate, alkylphos propionate); "Sulfo-SADP' (Sulfosuccinimidyl (4-azi phate, or photolabile linker. In one embodiment, the linker is dophenyldithio) propionate; DSP-Dithic bis a biodegradable linker. (Succinimidylproprionate); “DTSSP-33'-Dithio bis Linkers. (sulfoSuccinimidylpropionate); “DTBP'-dimethyl A variety of linkers may be used to link the substrate 3.3dithiobispropionimidate-2HCI, all available from Pierce capable of being transported to the active agent. For example, 10 Chemicals (Rockford, Ill.). degradable and cleavable linkers may be used. Examples of linking reagents cleavable by oxidation are The term “degradable linker as used herein, refers to “DST'-disuccinimidyl tartarate; and “Sulfo-DST'-disuccin linker moieties that are capable of cleavage under various imidyl tartarate. Again, these linkers are available from conditions. Conditions Suitable for cleavage can include but 15 PierceChemicals. are not limited to pH, UV irradiation, enzymatic activity, Examples of non-cleavable linkers are "Sulfo-LC-SMPT'- temperature, hydrolysis, elimination, and Substitution reac (sulfosuccinimidyl 6-alphamethyl-alpha-(2-pyridylthio) tions, and thermodynamic properties of the linkage. The term toluamidohexanoate: “SMPT: “ABH-Azidobenzoyl “photolabile linker” as used herein, refers to linker moieties hydrazide; “NHS-ASA-N-Hydroxysuccinimidyl-4-azi as are known in the art that are selectively cleaved under dosalicyclic acid; “SASD-Sulfosuccinimidyl 2-(pazidosali particular UV wavelengths. Compounds of the invention con cylamido)ethyl-1,3-dithiopropionate: “APDP-N-(4-(p-azi taining photolabile linkers can be used to deliver compounds dosalicylamido) buthy-3" (2-pyidyldithio)propionamide: to a target cell or tissue of interest, and can be Subsequently “BASED'-Bis-beta-(4-azidosalicylamido)ethyl disulfide: released in the presence of a UV source. “HSAB'-N-hydroxysuccinimidyl-4 azidobenzoate: “APG”- The term “linker as used herein is any bond, small mol 25 p-Azidophenyl glyoxal monohydrate: "SANPAH'-N-Suc ecule, or other vehicle which allows the substrate and the ciminidyl-6 (4-azido-2'-mitrophenyl-amimo) hexanoate; active agent to be targeted to the same area, tissue, or cell. In “Sulfo-SANPAH'-Sulfosuccinimidyl6-(4'-azido-2'-nitro certain embodiments, the linker is cleavable. phenylamino) hexanoate: “ANB-NOS N-5-Azido-2-ni In one embodiment the linker is a chemical bond between trobenzyoyloxysuccinimide: "SAND'-Sulfosuccinimidyl-2- one or more Substrates and one or more therapeutic moieties. 30 (m-azido-o-mitrobenzamido)-ethyl-1,3'-dithiopropionate; Thus, the bond may be covalent or ionic. An example of a “PNP-DTP'-p-nitrophenyl-2-diazo-3,3,3-trifluoropropi therapeutic complex where the linker is a chemical bond onate: “SMCC'-Succinimidyl-4-(N-maleimidomethyl)cy would be a fusion protein. In one embodiment, the chemical clohexane-1-carboxylate: “Sulfo-SMCC'-Sulfosuccinim bond is acid sensitive and the pH sensitive bond is cleaved idyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate: upon going from the blood stream (pH 7.5) to the transcytotic 35 vesicle or the interior of the cell (pH about 6.0). Alternatively, “MBS’ m-Maleimidobenzoyl-N-hydroxysuccinimide ester; the bond may not be acid sensitive, but may be cleavable by a “sulfo-MBS'-m-Maleimidobenzoyl-N-hydroxysulfosuccin specific enzyme or chemical which is Subsequently added or imide ester: “SIAB'-N-Succinimidyl (4-iodoacetyl)ami naturally found in the microenvironment of the targeted site. nobenzoate: "SulfSIAB'-N-Sulfosuccinimidyl (4-io Alternatively, the bond may be a bond that is cleaved under 40 doacetyl)aminobenzoate: “SMPB-Succinimidyl reducing conditions, for example a disulfide bond. 4-(pmalenimidophenyl) butyrate; "Sulfo-SMPB'-Sulfosuc Alternatively, the bond may not be cleavable. cinimidyl 4-(p-malenimidophenyl) butyrate: “DSS-Disuc Any kind of acid cleavable or acid sensitive linker may be cinimidyl suberate; “BSSS'-bis(sulfosuccinimidyl) suber used. Examples of acid cleavable bonds include, but are not ate: “BMH-Bis maleimidohexane: “DFDNB'-1,5-difluoro limited to: a class of organic acids known as cipolycarboxylic 45 2,4-dinitrobenzene: “DMA'-dimethyl adipimidate 2HCI; alkenes. This class of molecule contains at least three car “DMP'-Dimethyl pimelimidate-2HCI: “DMS'-dimethyl boxylic acid groups (COOH) attached to a carbon chain that suberimidate-2-HCl, “SPDPN-succinimidyl-3-(2-py contains at least one double bond. These molecules as well as ridylthio) propionate: "Sulfo-HSAB'-Sulfosuccinimidyl how they are made and used is disclosed in Shen, et al. U.S. 4-(pazidophenyl) butyrate: "Sulfo-SAPB-Sulfosuccinim Pat. No. 4,631,190. 50 idyl 4-(p-azidophenylbutyrate); ASIB'-1-9p-azidosalicyla Alternatively, molecules Such asamino-sulfhydryl cross mido)-4-(iodoacetamido) butane: 'ASBA’-4-(p-Azidosali linking reagents which are cleavable under mildly acidic con cylamido) butylamine. All of these linkers are available from ditions may be used. These molecules are disclosed in Blattler Pierce Chemicals. et al., U.S. Pat. No. 4,569,789. In another embodiment the linker is a small molecule such Alternatively, the acid cleavable linker may be a time 55 as a peptide linker. In one embodiment the peptide linker is release bond, such as a biodegradable, hydrolyzable bond. not cleavable. In a further embodiment the peptide linker is Typical biodegradable carrier bonds include esters, amides or cleavable by base, under reducing conditions, or by a specific urethane bonds, so that typical carriers are polyesters, polya enzyme. In one embodiment, the enzyme is indigenous. mides, polyurethanes and other condensation polymers hav Alternatively, the small peptide may be cleavable by an non ing a molecular weight between about 5,000 and 1,000,000. 60 indigenous enzyme which is administered after or in addition Examples of these carriers/bonds are shown in Peterson, et to the therapeutic complex. Alternatively, the small peptide al., U.S. Pat. No. 4,356,166. Other acid cleavable linkers may may be cleaved under reducing conditions, for example, be found in U.S. Pat. Nos. 4,569,789 and 4,631,190 or Blatt when the peptide contains a disulfide bond. Alternatively, the ner et al. in Biochemistry 24: 1517-1524 (1984). The linkers Small peptide may be pH sensitive. Examples of peptide are cleaved by natural acidic conditions, or alternatively, acid 65 linkers include: poly(L-Gly), (Poly L-Glycine linkers); poly conditions can be induced at a target site as explained in (L-Glu), (PolyL-Glutamine linkers); poly(L-Lys), (Poly Abrams et al., U.S. Pat. No. 4,171,563. L-Lysine linkers). In one embodiment, the peptide linker has US 8,956,825 B2 23 24 the formula (amino acid) n, where n is an integer between 2 large unilamellar vesicles (LUV), giant unilamellar vesicles and 100, preferably wherein the peptide comprises a polymer (GUV), or multivesicular vesicles (MVV). Each of these of one or more amino acids. liposome structures are well known in the art (see Storm, et al. In a further embodiment, the peptide linker is cleavable by PSTT 1: 19-31 (1998)). proteinase such as one having the sequence Gly-(D) Phe-Pro In one embodiment, the liposome is a “micromachine' that Arg-Gly-Phe-Pro-Ala-Gly-Gly (SEQ ID NO:13) (Suzuki, et evulses pharmaceuticals for example by the application of al. 1998, J. Biomed. Mater. Res. October: 42 (1): 112-6). This specific frequency radio waves. In another embodiment, the embodiment has been shown to be advantageous for the treat liposomes can be degraded such that they will release the ment of bacterial infections, particularly Pseudomonas active agent in the targeted cell, for example, the liposomes aeruginosa. Gentamicin or an alternate antibiotic is cleaved 10 may be acid or alkaline. Sensitive, or degraded in the presence only when the wounds are infected by Pseudomonas aerugi of a low or high pH, such that the active agent is released nosa because there is significantly higher activity of throm within the cell. Alternatively, the liposomes may be bin-like proteinase enzymes then in noninfected tissue. uncharged so that they will be taken up by the targeted cell. In a further embodiment the linker is a cleavable linker The liposomes may also be pH sensitive or sensitive to reduc including, poly(ethylene glycol) (PEG) and a dipeptide.L- 15 ing conditions. alanyl-L-valine (Ala-Val), cleavable by the enzyme thermol One type of liposome which may be advantageously used ysin. This linker is advantageous because thermolysin-like in the present invention is that identified in Langer et al., U.S. enzyme has been reported to be expressed at the site of many Pat. No. 6,004,534, issued Dec. 21, 1999. In this application tumors. Alternatively, a 12 residue spacer Thr-Arg-His-Arg a method of producing modified liposomes which are pre Gln-Pro-Arg-Gly-Trp-Glu-Gln-Leu (SEQ ID NO: 14) may pared by polymerization of double and triple bond-containing be used which contains the recognition site for the protease monomeric phospholipids is disclosed. These liposomes have furin (Goyal, et al. Biochem. J. 2000 Jan. 15: 345 Pt 2: Surprisingly enhanced Stability against the harsh environment 247-254). of the gastrointestinal tract. Thus, they have utility for oral The chemical and peptide linkers can be bonded between and/or mucosal delivery of the active agent. It has also been the Substrate and the active agent by techniques known in the 25 shown that the liposomes may be absorbed into the systemic art for conjugate Synthesis, i.e. using genetic engineering, or circulation and lymphatic circulation. The liposomes are gen chemically. The conjugate synthesis can be accomplished erally prepared by polymerization (i.e., radical initiation or chemically via the appropriate antibody by classical coupling radiation) of double and triple bond-containing monomeric reactions of proteins to other moieties at appropriate func phospholipids. tional groups. 30 In other embodiments of the present invention, the linker Examples of the functional groups present in proteins and can also be a liposome having a long blood circulation time. utilized normally for chemical coupling reactions are out Such liposomes are well known in the art, (see U.S. Pat. Nos. lined as follows. The carbohydrate structures may be oxi 5,013,556; 5,225,212:5,213,804: 5,356,633; and 5,843,473). dized to aldehyde groups that in turn are reacted with a com Liposomes having long blood circulation time are character pound containing the groupH2NNH R (wherein R is the 35 ized by having a portion of their phosphoslipids derivatized compound) to the formation of a C=NH NH R group. with polyethylene glycol (PEG) or other similar polymer. In The thiol group (cysteines in proteins) may be reacted with a some embodiments, the end of the PEG molecule distal to the compound containing a thiol-reactive group to the formation phospholipid may be activated so a to be chemically reactive. of a thioether group or disulfide group. The free amino group Such a reactive PEG molecule can be used to linka substrate (at the amino terminus of a protein or on a lysine) in amino 40 to the liposome. One example of a reactive PEG molecule is acid residues may be reacted with a compound containing an the maleimide derivative of PEG described in U.S. Pat. No. electrophilic group, Such as an activated carboxy group, to the 5,527,528). formation of an amide group. Free carboxy groups in amino Alternatively, the linker may be a microcapsule, a nano acid residues may be transformed to a reactive carboxy group particle, a magnetic particle, and the like (Kumar, J. Pharm. and then reacted with a compound containing an amino group 45 Sci., May-August 3 (2) 234-258, 2000; and Gill et al., Trends to the formation of an amide group. Biotechnol. November; 18(11): 469-79, 2000), with the lipo The linker may alternatively be a liposome. Many methods philic active agent on or in the container, and the container for the preparation of liposomes are well known in the art. For functioning as the linker in the therapeutic complex. example, the reverse phase evaporation method, freezethaw Alternatively, the linker may be a photocleavable linker. methods, extrusion methods, and dehydration-rehydration 50 For example, a 1-2-(nitrophenyl)ethyl moiety can be cleaved methods. (see Storm, et al. PSTT 1:19-31 (1998).). using 300 to 360 nm light (see Pierce catalog no. 21332ZZ). The liposomes may be produced in a solution containing It can be envisioned that the photocleavable linker would the active agent so that the Substance is encapsulated during allow activation and action of the drug in an even more spe polymerization. Alternatively, the liposomes can be polymer cific area, for example a particular part of the organ. The light ized first, and the biologically active substance can be added 55 could be localized using a catheter into the vessel. Alterna later by resuspending the polymerized liposomes in a solution tively, light may be used to localize treatment to a specific part ofa biologically active Substance and treating with Sonication of the digestive tract and the light may be manipulated to affect encapsulation of the active agent. The liposomes can through a natural orifice to the area. be polymerized in the presence of the substrate such that the Alternatively, the light can be Surgically manipulated to the substrate becomes a part of the phospholipid bilayer. In one 60 aca. embodiment, the liposome contains the active agent on the Alternatively, the linker may not be cleavable, but the inside and the substrate on the outside. active agent or Substrate is. An example of this is when the The liposomes contemplated in the present invention can active agent is a prodrug and the enzyme which cleaves the comprise a variety of structures. For example, the liposomes prodrug is administered with the therapeutic complex. Alter can be multilamellar large vesicles (MLV), oligolamellar 65 natively, the enzyme is part of the therapeutic complex or vesicles (OLV), unilamellar vesicles (UV), small unilamellar indigenous and the prodrug is administered separately. Pref vesicles (SUV), medium sized unilamellar vesicles (MUV), erably, the enzyme or prodrug which is administered sepa US 8,956,825 B2 25 26 rately is administered within about 48 hours of the first muscles include Phosphorylase Deficiency, Acid Maltase administration. Alternatively, the prodrug or enzyme which is Deficiency, Mitochondrial Myopathy, Carnitine Palmityl administered separately may be administered between about Transferase Deficiency, Phosphoglycerate Kinase Defi 1 min and 24 hours, alternatively between about 2 min and 8 ciency, and Phosphoglycerate Mutase Deficiency. hours. In particular embodiments, the active agent effective in the The prodrug or enzyme which is administered separately, treatment of a skeletal muscle disorder may be, for example, may be readministered at a later date and may continue to be an enzyme that is lacking in a patient with the muscle disor administered until the effect of the drug is not longer needed. der. For example, the following skeletal muscle diseases and According to yet another embodiment of the invention, disorders may be treated with the following enzymes, or a there are provided methods for treating a disease or disorder 10 nucleic acid that modulates the expression of said enzymes, in in a cell or tissue expressing a nucleoside transport pathway. accordance with the methods herein: C-glucosidase (Pompe The method includes administering to a patient having the Disease), C-L-iduronidase (Hurler Syndrome), C-galactosi disease or disorder a conjugate including a substrate that is dase A (Fabry Disease), arylsulfatase (Maroteaux-Lamy Syn capable of being transported by the nucleoside transport path drome), N-acetylgalactosamine-6-sulfatase or B-galactosi way expressed in the affected cell or tissue and an active agent 15 dase (Morquio Syndrome), iduronate 2-sulfatase (Hunter for treating disease or disorder, wherein the conjugate is also Syndrome), ceramidase (Farber Disease), galactocerebrosi transported by the nucleoside transport pathway. In this way, dase (Krabbe Disease), B-glucuronidase (Sly Syndrome), the conjugate is transported into the affected cells or tissue, Heparan N-sulfatase (Sanfilippo A), N-Acetyl-O-glu thereby delivering the active agent. cosaminidase (Sanfilippo B), Acetyl CoA-O-glucosaminide Diseases or disorders which may be treated using a conju N-acetyltransferase, N-acetyl-glucosamine-6 Sulfatase (San gate of the invention include diseases or disorders involving filippo D), Galactose 6-sulfatase (Morquio A), Arylsulfatase tissues such as muscle (including skeletal muscle and cardiac A, B, and C (Multiple Sulfatase Deficiency), Arylsulfatase A muscle), glycogen-storing cells, vascular endothelium, heart, Cerebroside (Metachromatic Leukodystrophy), Ganglioside brain, placenta, thymus, pancreas, prostate, kidney, blood, (Mucolipidosis IV), Acid B-galactosidase G. Sub.MI Gaiglio skin, and ENT2-expressing cancer cells. 25 side (G. Sub.MI Gangliosidosis), Acid B-galactosidase (Ga In certain embodiments the disease or disorder involves lactosialidosis), Hexosaminidase A (Tay-Sachs and Variants), muscle Such as skeletal or cardiac muscle and the conjugate is Hexosaminidase B (Sandhoff), C.-fucosidase (Fucsidosis), transported into the muscle cells (such as skeletal muscle cells Cl-N-Acetylgalactosaminidase (Schindler Disease). Glyco or cardiac muscle cells), thereby delivering the active agent. protein Neuraminidase (Sialidosis), Aspartylglucosamine In certain embodiments, the muscle disorder is selected from 30 amidase (Aspartylglucosaminuria), Acid Lipase (Wolman the group consisting of cachexia, muscle dystrophies, lyso Disease), Acid Ceramidase (Farber Lipogranulomatosis), somal muscle disorders, skeletal muscle disorders, smooth Lysosomal Sphingomyelinase and other Sphingomyelinase muscle disorders, and cardiac muscle disorders. In certain (Nieman-Pick). In certain embodiments, the active agent is embodiments, these designations may overlap. Muscle dys dystrophin, components of dystrophin-glycoprotein com trophies include Becker's muscular dystrophy (BMD), Con 35 plex, the laminin-C2 chain, fukutin-related protein, LARGE. genital muscular dystrophy, Duchenne muscular dystrophy fukutin, EMD, LMNA, DMPK, ZNF9, and PABPN1, or a (DMD), Distal muscular dystrophy, Emery-Dreifuss muscu nucleic acid that modulates the expression of said proteins. lar dystrophy, Facioscapulohumeral muscular dystrophy In certain embodiments the disease or disorder involves (FSHD), Limb-girdle muscular dystrophy (LGMD), Myo glycogen-storing cells and the conjugate is transported into tonic muscular dystrophy, and Oculopharyngeal muscular 40 the glycogen-storing cells, thereby delivering the active dystrophy. In certain embodiments, a lysosomal disorder is agent. Glycogen-storing cells include muscle cells, liver Pompe Disease, Hurler Syndrome, Fabry Disease, Maro cells, and also kidney and intestinal cells. In certain embodi teaux-Lamy Syndrome, Morquio Syndrome, Hunter Syn ments, the glycogen-storage disorder is selected from the drome, Farber Disease, Krabbe Disease, Sly Syndrome, San group consisting of Glycogen synthase deficiency, Glucose filippo (including A, B, and D), Morquio A. Multiple 45 6-phosphatase deficiency (von Gierke disease), Debranching Sulfatase Deficiency, Metachromatic Leukodystrophy, enzyme deficiency (Forbes-Cori disease), Transglucosidase Mucolipidosis IV. G. sub.MI Gangliosidosis, Galactosialido deficiency, (Andersen disease, amylopectinosis), Myophos sis, Tay-Sachs and Tay-Sachs Variants, Sandhoff, Fucsidosis, phorylase deficiency (McArdle disease), Phosphorylase defi Schindler Disease, Sialidosis, Aspartylglucosaminuria, Wol ciency (Hers disease), and Phosphofructokinase deficiency man Disease, Farber Lipogranulomatosis, and Nieman-Pick 50 (Tauri disease). In particular embodiments, the active agent disease. In certain embodiments, the cardiac muscle disorder effective in the treatment of a glycogen-storage disorder is is cardiomyopathy, cardiac ischemia, congestive heart fail Glycogen synthase, Glucose-6-phosphatase, Debranching ure, ischemia-reperfusion injury, Coronary heart disease, enzyme, Transglucosidase, Myophosphorylase, Phosphory Cardiovascular disease, Ischaemic heart disease, Heart fail lase, Phosphofructokinase, Acid Maltase Deficiency, Car ure, Hypertensive heart disease, Inflammatory heart disease, 55 nitine Palmityl Transferase, Phosphoglycerate Kinase, or and Valvular heart disease. The muscle disorder may be sar Phosphoglycerate Mutase, or a nucleic acid that up-regulates copenia. In some embodiments, the muscle disorder is muscle the expression of the deficient proteins. wasting caused by another disease, such as AIDS or cancer. In certain embodiments the disease or disorder involves Other muscle disorders include diseases of the neuromuscu vascular endothelium and the conjugate is transported into the lar junction, such as myasthenia gravis, Lambert-Eaton Syn 60 vascular endothelium, thereby delivering the active agent. In drome, and Congenital Myasthenic Syndrome, motor neuron certain embodiments, the vascular endothelium disorder is diseases (such as ALS, spinal muscular atrophy, Charcot selected from the group consisting of inappropriate angiogen Maria-Tooth disease, and Freidrich's Ataxia), inflammatory esis (for example, Surrounding a tumor), deficient angiogen myopathies (such as dermatomyositis, polymyositis, and esis (for example, in a slowly-healing wound or ulcer), rest inclusion body myositis), endocrine abnormalities (such as 65 enosis, atherosclerosis, Scarring after Surgery or injury, and hyperthyroid myopathy), myotonia, nemaline myopathy, and vasculitis. Examples of diseases associated with uncontrolled myotubular myopathy. Enzyme deficiency disorders of the angiogenesis that may be treated with the compositions and US 8,956,825 B2 27 28 methods herein include, but are not limited to retinal/choroi ments, the placental disorder is selected from the group con dal neovascularization and corneal neovascularization. sisting of Placenta accreta, Placenta praevia, and Placental Examples of retinal/choroidal neovascularization include, abruption. but are not limited to, Bests diseases, myopia, optic pits, In certain embodiments the disease or disorder involves the Stargarts diseases, Pagets disease, vein occlusion, artery thymus and the conjugate is transported into cells of the occlusion, sickle cell anemia, sarcoid, syphilis, pseudoxan thymus, thereby delivering the active agent. In certain thoma elasticum carotid apo structive diseases, chronic uvei embodiments, the thymus disorder is selected from the group tis/vitritis, mycobacterial infections, Lyme’s disease, sys consisting of an autoimmune disease, a disease resulting from temic lupus erythematosus, retinopathy of prematurity, Eales faulty positive selection or faulty negative selection of T cells, disease, diabetic retinopathy, macular degeneration, Bechets 10 and cancer of the thymus. Some examples of autoimmune diseases, infections causing a retinitis or chroiditis, presumed diseases include Alopecia Areata, Ankylosing Spondylitis, ocular histoplasmosis, pars planitis, chronic retinal detach Antiphospholipid Syndrome, Autoimmune Addison's Dis ment, hyperviscosity syndromes, toxoplasmosis, trauma and ease, Autoimmune Hemolytic Anemia, Autoimmune Hepati post-laser complications, diseases associated with rubesis 15 tis, Behcet’s Disease, Bullous Pemphigoid, Cardiomyopathy, (neovascularization of the angle) and diseases caused by the Celiac Sprue-Dermatitis, Chronic Fatigue Immune Dysfunc abnormal proliferation of fibrovascular or fibrous tissue tion Syndrome (CFIDS), Chronic Inflammatory Demyclinat including all forms of proliferative vitreoretinopathy. ing Polyneuropathy, Churg-Strauss Syndrome, Cicatricial Examples of corneal neovascularization include, but are not Pemphigoid, CREST Syndrome, Cold Agglutinin Disease, limited to, epidemic keratoconjunctivitis, Vitamin A defi Crohn's Disease, Discoid Lupus, Essential Mixed Cryoglo ciency, contact lens overwear, atopic keratitis, Superior limbic bulinemia, Fibromyalgia-Fibromyositis, Graves’ Disease, keratitis, pterygium keratitis sicca, Sjogrens, acne rosacea, Guillain-Barr, Hashimoto's Thyroiditis, Hypothyroidism, phylectenulosis, diabetic retinopathy, retinopathy of prema Idiopathic Pulmonary Fibrosis, Idiopathic Thrombocytope turity, corneal graft rejection, Mooren ulcer, Terrien’s mar nia Purpura (ITP), IgA Nephropathy, Insulin dependent Dia ginal degeneration, marginal keratolysis, polyarteritis, Wege 25 betes, Juvenile Arthritis, Lichen Planus, Lupus, Meniere's ner sarcoidosis, Scleritis, periphigoid radial keratotomy, Disease, Mixed Connective Tissue Disease, Multiple Sclero neovascular glaucoma and retrolental fibroplasia, syphilis, sis, Myasthenia Gravis, Pemphigus Vulgaris, Pernicious Ane Mycobacteria infections, lipid degeneration, chemical burns, mia, Polyarteritis Nodosa, Polychondritis, Polyglandular bacterial ulcers, fungal ulcers, Herpes simplex infections, Syndromes, Polymyalgia Rheumatica, Polymyositis and Herpes Zoster infections, protozoan infections and Kaposi 30 Dermatomyositis, Primary Agammaglobulinemia, Primary Biliary Cirrhosis, Psoriasis, Raynaud's Phenomenon, Reit sarcoma. Example of deficient angiogenesis include ulcers er's Syndrome, Rheumatic Fever, Rheumatoid Arthritis, Sar Such as skin ulcers and diabetic ulcers. In one embodiment, coidosis, Scleroderma, Sjogren's Syndrome, Stiff-Man Syn the active agent effective in the treatment of a disease of the drome, Takayasu Arteritis, Temporal Arteritis/Giant Cell vascular endothelium is serum amyloid P (SAP), or a nucleic 35 Arteritis, Ulcerative Colitis, Uveitis, Vasculitis, Vitiligo, acid that increases SAP expression. SAP inhibits fibrocytes Wegener's Granulomatosis, and myasthenia gravis. In par from causing pathological scarring lesions. In another ticular embodiments, the active agent effective in the treat embodiment, the disease of the vascular endothelium is ath ment of a disease of the thymus is immunosuppressive or erosclerosis, which may be treated using statins, niacin, intes antiinflammatory. The agent may be, for example, an anti tinal cholesterol absorption-inhibiting Supplements such as 40 body including muromab, basiliximab, and daclizumab, or a eZetimibe and fibrates, aspirin, human Apo-Al Milano HDL, nucleic acid encoding one of those antibodies. Examples of or a nucleic acid that increases Apo-Al Milano HDL expres immunosuppressive and antiinflammatory drugs that may be Sion. One may also administer nucleic acids that reduce Syn used as the active agent include corticosteroids, rolipram, thesis of cholesterol, such as siRNA constructs designed to calphostin, CSAIDs; interleukin-10, glucocorticoids, salicy reduce expression of cholesterol synthetic enzymes. Choles 45 lates, nitric oxide; nuclear translocation inhibitors, such as terol synthetic enzymes include HMG-CoA synthase, HMG deoxyspergualin (DSG); non-steroidal antiinflammatory CoA reductase, mevalonate kinase, phosphomevalonate drugs (NSAIDs) such as ibuprofen, celecoxib and rofecoxib; kinase, and lanosterol synthase. steroids such as prednisone or dexamethasone; antiviral In certain embodiments the disease or disorder involves the agents such as abacavir, antiproliferative agents such as brain and the conjugate is transported into the brain cells, 50 methotrexate, leflunomide, FK506 (tacrolimus, Prograf): thereby delivering the active agent. In certain embodiments, cytotoxic drugs such as azathioprine and cyclophosphamide; the brain disorder is selected from the group consisting of TNF-alpha. inhibitors such as tenidap, anti-TNF antibodies neurodegenerative diseases (such as Alzheimer's disease, or soluble TNF receptor, and rapamycin (sirolimus or Rapa Parkinson's disease, motor neuron disease, and Huntington's mune) or derivatives thereof. When the disease is cancer of disease), mental illnesses, such as clinical depression, Schizo 55 the thymus, the active agent may be a chemotherapeutic drug phrenia, bipolar disorder, and post-traumatic stress disorder; or other type of anti-cancer therapeutic. infectious diseases including meningitis, viral, bacterial, and In certain embodiments the disease or disorder involves the prion diseases, inherited disorders such as Tay-Sachs disease, pancreas and the conjugate is transported into cells of the Fragile X syndrome, and Down syndrome, and lysosomal pancreas, thereby delivering the active agent. In certain storage disorders. In particular embodiments, the active agent 60 embodiments, the pancreas disorder is selected from the effective in the treatment of a disease of the brain is an group consisting of Pancreatitis, Diabetes mellitus, Exocrine enzyme absent (or present at reduced levels) in a patient with pancreatic insufficiency, complications of Cystic fibrosis, a lysosomal storage disorder; examples of lysosomal disor Pseudocysts, or pancreatic cancer. In particular embodi ders, and compositions for treating them, are listed above. ments, the active agent effective in the treatment of a disease In certain embodiments the disease or disorder involves the 65 of the pancreas is insulin, a Pancreatic Enzyme Product (PEP) placenta and the conjugate is transported into the placental Such as pancrelipase, or a nucleic acid that up-regulates cells, thereby delivering the active agent. In certain embodi expression of the same. US 8,956,825 B2 29 30 In certain embodiments the disease or disorder involves the dermatitis, bullous dermatoses, pigmented dermatoses, pho prostate and the conjugate is transported into cells of the tosensitive dermatoses, dermatoses caused by collagen dis prostate, thereby delivering the active agent. In certain eases, dermatoses due to internal diseases, Xerosis, urticaria, embodiments, the prostate disorder is selected from the group atopic dermatitis, eczyma, lichen simplex chronicus, psoria consisting of Prostatitis, Benign prostatic hyperplasia, or 5 sis, Scabies, wound, Sun burn, cold Sores, acne, insect bite, Prostate cancer. In particular embodiments, the active agent radiotherapy or chemotherapy-induced dermatitis, paraneo effective in the treatment of a disease of the prostate is an plastic syndrome, malignancy, melanoma, primary skin can anti-cancer agent; examples of Such agents are listed else cer, and metastatic skin cancer. In particular embodiments, where in this application. the active agent effective in the treatment of a disease of the In certain embodiments the disease or disorder involves the 10 skin is anthralin, calpotriene, coal tar, diclofenac, T4 endo kidney and the conjugate is transported into cells of the kid nuclease, isotretinoin, acitretin, cidofoir, a corticosteroid, an ney, thereby delivering the active agent. In certain embodi antibiotic, an analgesic, an immunomodulator, including oral ments, the kidney disorder is selected from the group consist immunomodulator Such as tacrolimus and pimecrolimus, and ing of Diabetic nephropathy, Glomerulonephritis, topical immunomodulators; an immunosuppressant, an anti Hydronephrosis, Kidney Stones, Kidney tumors (such as 15 angiogenic, including anti-VEGF, anti-FGF, anti-EGF and Wilms tumor and Renal cell carcinoma), Lupus nephritis, anti-HGF; a leukotriene modifier, an aminosalicylate, an Minimal change disease, Pyelonephritis, nephrotic Syn anesthetic, a non-steroidal antiinflammatory, a modifier of a drome, and Renal failure (such as Acute renal failure and solubilized interleukin receptor, an inhibitor of a tyrosine Stage 5 Chronic Kidney Disease). In particular embodiments, kinase receptor, a protein kinase C inhibitor, methotrexate, the active agent effective in the treatment of a disease of the cyclosporine, and methylprednisolone acetate. kidney is an agent that treats autoimmune disease, or an In certain embodiments the disease or disorder is a type of anti-cancer therapeutic, both of which are listed elsewhere in cancer and the conjugate is transported into cancer cells, the present application. thereby delivering the active agent. In certain embodiments, In certain embodiments the disease or disorder involves the the type of cancer is selected from the group consisting of blood and the conjugate is transported into cells of the blood, 25 rhabdomyosarcoma, ovarian cancer, colon cancer, and breast thereby delivering the active agent. In certain embodiments, cancer. In other embodiments, the cancer is selected from the blood disorder is selected from the group consisting of leukemia, lymphomas, melanomas, squamous cell carcino primary immunodeficiency (including SCID, hemophilia A, mas, breast cancer, prostrate cancer, bladder cancer, lung and hemophilia B), reduced hematopoietic function, reduced cancer including non Small-cell lung cancer and Small-cell immune function, reduced neutrophil count, reduced neutro 30 lung cancer, ovarian cancer, colon cancer, Squamous cell phil mobilization, mobilization of peripheral blood progeni carcinoma, astrocytoma, Kaposi's sarcoma, glioblastoma, tor cells, sepsis, severe chronic neutropenia, bone marrow bladder cancer, head and neck cancer, glioma, colorectal can transplants, infectious diseases, leucopenia, thrombocytope cer, genitourinary cancer and gastrointestinal cancer. In cer nia, anemia, bone marrow disorders caused by radiation, tain embodiments, the active agent is a chemotherapeutic chemical or chemotherapeutic induced bone marrow aplasia 35 drug. Chemotherapeutic drugs are well-known in the art and or myelosuppression, acquired immune deficiency syn include alkylating agents such as cisplatin, anti-metabolites drome, and polycythemia rubra Vera. In particular embodi Such as mercaptopurine, taxanes such as paclitaxel, topoi ments, the active agent effective in the treatment of a disease Somerase inhibitors such as topotecan, and antitumor antibi of the blood is selected from the group consisting of corticos otics such as doxorubicin. Anti-tumor active agents also teroids, anti-leukemic agents, growth factors, and clotting 40 include antibodies such as Herceptin. In particular embodi factors. In certain embodiments, the clotting factor is Factor ments, the active agent effective in the treatment of cancer is VIII or IX. In certain aspects, SCID is caused by a recessive a protein (or nucleic acid encoding the same) selected from: a mutation and may be treated by administering a wild-type bispecific antibody that binds Pax-FKHR fusion protein or a copy of the missing protein (or a nucleic acid encoding that tumor suppressor such as p53, pRb, PTEN, APC, and CD95, protein). For example, X-linked SCID may be treated with 45 BRCA1, BRCA2, DNA repair enzymes, proapoptotic genes, IL2RG, Jak3 gene mutations may be treated with JAK3, ADA p16', WT1, NF1 (neurofibromin 1), NF2 (merlin or neu gene mutations may be treated with ADA, IL-7R alpha-chain rofibromin 2), TSC1 (hamartin), TSC2 (tuberin), DPC4, mutations may be treated with IL7R alpha, CD3 delta or SMAD4, DCC, LKB1, STK11, MSH2, MLH1, CDH1 epsilon mutations may be treated with CD3 delta or epsilon, (E-cadherin), VHL, PTCH, (patched), MENI, BLM, NBS1, RAG1/RAG2 mutations may be treated with RAG1/RAG2. 50 MRE 11A, ATM, hRad50, NER enzymes (such as XPA, XPB, Artemis gene mutations may be treated with ARTEMIS, and XPC, XPD, DDB2, ERCC4, RAD2, and POLH), ERCC6, CD45 gene mutations may be treated with CD45. Other types ERCC8, RECQL2, FANCA, FANCC, FANCD2, FANCE, of primary immunodeficiency are deficiencies in the follow FANCF, FANCG, FANCI, FANCJ, FANCL, FANCM, ing proteins: DNA ligase type I, CD40 ligand, CD40. Purine MLH1, MSH2, MSH6, PMS, and PMS2. nucleoside phosphorylase (PNP), MHC class II, CD3y, CD8, 55 In certain embodiments the compositions and methods ZAP-70, TAP-/2, Winged helix protein, CD19, TACI, BAFF herein may be used to treat a disease or disorder involving receptor, AICDA, uracil-DNA glycosylase, perforin, dysfunction of nuclear receptors, and the conjugate is trans MUNC13D, syntaxin 11, CD95, Fas ligand, CASP8, and ported into cells in which altered nuclear receptor function is CASP10. These deficiencies may be treated by administra desired. In certain embodiments, the nuclear receptors are tion of the deficient protein or a nucleic acid encoding it. 60 steroid, thyroid, retinoid, or orphan nuclear receptors. In cer In certain embodiments the disease or disorder involves the tain embodiments, the orphan nuclear receptor is a SAR skin and the conjugate is transported into cells of the skin, (selective androgen receptor), PPAR, PPARB, PPAR, NUC1, thereby delivering the active agent. In certain embodiments, FAAR, PPAR, RevErbA, EAR-1, RVR, RevErbAB, BD73, the skin disorder is selected from the group consisting of HZF2, ROR, RZR, RORB, RZRf8, ROR, TOR, LXR, RLD1, dermatomyositis, papulosquamous dermatoses, bacterial 65 LXRB, UR, NER, RIP15, OR1, FXR, RIP14, HRR1, PXR. 1, dermatoses, viral dermatoses, mycolic skin infections, granu PXR.2, SXR, ONR1, xOR6, BXR, hCAR1, MB67, mCAR1, lomatous dermatoses, parasitic skin dermatoses, exfoliative HNF4, HNF4 B, HNF4, RXR, RXRB, H2RIIBP, RXR, TR2, US 8,956,825 B2 31 32 TR2-11, xDOR2, alOR1, TR4, TAK1, TR2R1, T1x, TLL, NOR-1) is thought to be involved in obesity, energy balance, xTLL COUP-TFI, COUPTFA, EAR3, SVP44, COUP-TFII, homeostasis, lipid utilization, and lipid and carbohydrate COUPTFB ARPI, SVP40, XCOUP-TFIII, COUP-TF, homeostasis. Based on the disclosures herein in combination SVP46, EAR2, ERR, ERR1, ERRB, ERR2, ERR, NGFI-B, with the state of the art, one of skill in the art will recognize NUR77, N10, TR3, NAKI, TISI, NURR1, NOT, RNR1, which orphan nuclear receptor protein, orphan nuclear recep HZF-3, TINUR TR3 B, NOR-1, MINOR, TEC, CHN, FTZ tor-modulating nucleic acid, or orphan nuclear receptor ago F1, SF1, ELP, AD4BP, FTF, LRH1, PHR1, CPF, FFLR, nist or antagonist, may be administered in conjunction with FF1r A, GCNF, RTR, DAX1, AHCH, or SHP. Depending on the methods and compositions herein, in order to effectively the disease to be treated, one of skill in the art will recognize treat an orphan nuclear receptor-mediated disease. whether the disease should be treated by increasing the levels 10 of a nuclear receptor or decreasing the levels or activity of a In certain embodiments the compositions and methods nuclear receptor. Levels of the nuclear receptor may be herein may be used to treat a disease or disorder involving increased, for example, by administering a nucleic acid dysfunction of factors controlling chromatin modification, encoding the nuclear receptor. Activity of the nuclear receptor and the conjugate is transported into cells in which altered chromatin modification state is desired. In certain embodi may be decreased, by example, by administering an inhibi 15 tory antibody. Nuclear receptors, and the diseases caused by ments, the chromatin modification factors are histone mutations in them, are as follows: Androgen receptor (CAIS/ deacetylases, histone methyltransferases, histone kinases, PAIS, complete/partial androgen insensitivity syndrome; histone phosphatases, histone ubiquitinylating enzymes, or Gynecomastia; interfility; SBMA; Kennedy's disease; Pros histone poly-ADP-ribosylases. Also, chromatin assembly tate Cancer; perineal hypospadias), DAX-1 (adrenal hypopla factors and nucleosome remodeling factors (and nucleic acids sia congenita, Adrenal insufficiency, delayed-onset, and modulating their expression) may be administered. In certain hypogonadotropic hypogonadism), Vitamin 3D receptor (Vi embodiments, DNA methyltransferases (such as DNMT1, tamin D Resistant-rickets type IIA); HNF4 alpha (Maturity DNMT2, DNMT3) or nucleic acids modulating their expres onset diabetes of the young); Mineralocoricoid receptor sion may be administered in order to treat diseases associated (Pseudohypoaldosteronism, type 1; autosomal dominant; 25 with altered DNA methyltransferase function, such as ICF Hypertension, early-onset, autosomal dominant, with exac Syndrome. erbation in pregnancy), Thyroid hormone beta-1 (thyroid hor In certain embodiments, the disease or disorder involves mone resistance), Glucocorticoid receptor (Primary cortisol incorrect hormone levels. The hormone levels may be inap resistance familial Glucocorticoid resistance), PPARgamma propriately high or low. If the disease is associated with low (Diabetes Mellitus, insulin-resistant, with acanthosis nigri 30 hormone levels, one may administer a gene (or gene product) cans and hypertension; colon cancer, Inflammatory bowel in the synthesis pathway for that hormone. For example, to disease), HNF4 alpha (Type II Diabetes), and ERa (Os treat low testosterone levels, a gene or gene product of 17B teoporosis, Breast cancer). In particular embodiments, the hydroxysteroid dehydrogenase, 3B hydroxysteroid dehydro active agent effective in the treatment of a nuclear receptor genase, or 17.20 lyase may be administered. Alternatively, if mediated disorder is the protein deficient in the above-men 35 the disease is associated with elevated hormone levels, a tioned diseases, or a nucleic acid that up-regulates its expres nucleic acid (such as a siRNA) designed to reduce levels of sion. Those of the above diseases that are caused by the hormone synthesis pathway components may be admin inappropriately high expression of the mutant gene may be istered. Hormones include testosterone, estrogen, estradiol. treated by administering a nucleic acid that down-regulates its and progesterone. expression. 40 In certain embodiments, the methods herein may be used to In some aspects, a skeletal muscle disorder may be treated target therapeutic antibodies, or nucleic acids encoding them, by altering the activity and/or levels of an orphan nuclear to particular target cells. The antibodies may be, for example, receptor. Orphan nuclear receptors and diseases associated monoclonal antibodies, polyclonal antibodies, single-chain with them are known in the art, for example in Smith et al., antibodies, or bi-specific antibodies. Suitable therapeutic “Orphan Nuclear Receptors: therapeutic opportunities in 45 antibodies include, but are not limited to, Anti EGFrantibod skeletal muscle” Am J Physiol Cell Physiol 291:203-217, ies (e.g., panitumamab, ErbituX (cetuximab), matuZumab, 2006. For example, dysfunction of LXR-C. LXR-B, farnesoid IMC-IIF 8. TheraCIM hR3), denosumab, Avastin (bevaci X receptor (FXR), PPAR-C, -?3/ö, and -y, liver receptor Zumab), Anti-HGF antibodies, Humira (adalimumab), Anti homolog-1, and the Small heterodimeric partner can cause Ang-2 antibodies, Herceptin (trastuzumab), Remicade (in dyslipidemia, diabetes, obesity, inflammation, and cardiovas 50 fliximab), Anti-CD20 antibodies, rituximab, Synagis cular disease. In addition, ERR-O, ROR-O, Rev-erb-C. and -3. (palivizumab), Mylotarg (gemtuzumab oxogamicin), Raptiva and Nur77 control several processes including lipid absorpo (efalizumab), TySabri (natalizumab), Zenapax (dacliximab), tion, lipolysis, inflammation, and myokine expression. Spe NeutroSpec (Technetium ("Tc) fanolesomab), tocili cifically, PPAR-8 coordinates glucose tolerance, fatty acid Zumab, ProstaScint (Indium-Ill labeled Capromab Pen oxidation, and energy expenditure in skeletal muscle as well 55 detide), Bexxar (to situmomab), Zevalin (ibritumomab tiux as in adipose tissue. PPAR-A regulates fatty acid oxidation, etan (IDEC-Y2B8) conjugated to yttrium 90), Xolair and stimulates mitochondrial B-Oxidation and thermogenesis (omalizumab), MabThera (Rituximab), ReoPro (abciximab), in the muscles. In addition, LXR-O. and -3 regulate lipid MabCampath (alemtuzumab), Simulect (basiliximab), Leu metabolism in skeletal muscle. ERR-C. -B, and -y are koScan (sulesomab), CEA-Scan (arcitumomab), Verluma involved in obesity, lipid metabolism, and oxidative phospho 60 (nofetumomab), Panorex (Edirecolomab), alemtuzumab, rylation, and mitochondrial respiration in skeletal and cardiac CDP870, and natalizumab. muscle. Also, ROR nuclear receptors (including ROR-O1, In certain embodiments, the therapeutic agent is a protea -C2, C3, and -CO) are involved in muscle dysfunction Such as some inhibitor. Proteasome inhibitors may be used, for ataxia, as well as dyslipidemia, atherosclerosis, and hyper example, in the treatment of infectious diseases like HIV/ sensitive inflammatory response. Rev-erbs (including Rev 65 AIDS and Hepatitis C, and for cancer therapy. In certain erbC. and Rev-erbB) are also involved in dyslipidemia. In embodiments, the protease inhibitor is an antibody that binds addition, the NR4A family (including Nurr1, Nur11, and a protease. US 8,956,825 B2 33 34 In certain embodiments, the Subject conjugates can be used mal expression of the nucleic acids in the cell(s). Sufficient to deliver an expression construct to cells, such as muscle expression, however, may sometimes be obtained without cells, that encodes a therapeutic protein. For instance, the Such additional elements. expression construct can encode a therapeutic protein that is Vectors also can include other elements. For example, a secreted by the transduced cell. For example, the expression 5 vector can include a nucleic acid that encodes a signal peptide construct acid can encode an angiogenic growth factor Such Such that the encoded polypeptide is directed to a particular as VEGF, a fibroblast growth factor such as basic FGF or cellular location (e.g., a signal secretion sequence to cause the FGF-4, placental growth factor, hepatocyte growth factor, protein to be secreted by the cell) or a nucleic acid that angiogenin, angiopoietin-1, pleiotrophin, transforming encodes a selectable marker. Non-limiting examples of 10 selectable markers include puromycin, adenosine deaminase growth factor (C. or 3), or tumor necrosis factor C. The expres (ADA), aminoglycoside phosphotransferase (neo, G418, sion construct also can encode a natiuretic peptide Such as an APH), dihydrofolate reductase (DHFR), hygromycin-B- atrial natiuretic peptide (ANP) or a brain natriuretic peptide phosphtransferase, thymidine kinase (TK), and Xanthin-gua (BNP), prostacyclin synthase, nitric oxide synthase, nine phosphoribosyltransferase (XGPRT). Such markers are angiostatin, endostatin, erythropoietin (EPO), blood factors 15 useful for selecting stable transformants in culture. (such as coagulation factors like Factor I, II, III, IV, V, VII, Viral vectors can be used to form the conjugates, and VIII, IV, X, XI, XII and XIII), GM-CSF, or an interleukin include adenovirus, adeno-associated virus (AAV), retrovi such as IL-1,2,3,4,5,6,7,8,9, 10, 11, 12, 13, 14, 15, 16, 17, ruses, lentiviruses, vaccinia virus, measles viruses, herpes or 18. The expression construct can encode an adhesion mol viruses, and bovine papilloma virus vectors. See, Kay et al. ecule Such as a selectin (e.g., E. L., or Pselectin), an extracel (1997) Proc. Natl. Acad. Sci. USA 94:12744-12746 for a lular matrix protein (e.g., collagen type I, III, or IV: fibronec review of viral and non-viral vectors. Viral vectors are modi tin, laminin; or vitronectin), an integrin (e.g., C.s.f3), or an fied so the native tropism and pathogenicity of the virus has intracellular adhesion molecule such as ICAM or a vascular been altered or removed. The genome of a virus also can be cell adhesion molecule (VCAM). modified to increase its infectivity and to accommodate pack In either case, the expression construct that is delivered 25 aging of the nucleic acid encoding the polypeptide of interest. typically is part of a vector in which a regulatory element Such Non-viral vectors can also be used in the Subject conju as a promoter is operably linked to the nucleic acid of interest. gates. The promoter can be constitutive or inducible. Non-limiting To further illustrate, in one embodiment, the mammalian examples of constitutive promoters include cytomegalovirus serum protein that is encoded by the vector is selected from 30 the group consisting of a tissue-type plasminogen activator, a (CMV) promoter and the Rous sarcoma virus promoter. As receptor of a tissue-type plasminogen activator, a streptoki used herein, “inducible” refers to both up-regulation and nase, a staphylokinase, a urokinase, and coagulation factors. down regulation. An inducible promoter is a promoter that is The invention also provides a method for treating associated capable of directly or indirectly activating transcription of with the formation of clots in its circulation, including the one or more DNA sequences or genes in response to an 35 step of administering to the mammal a conjugate that causes inducer. In the absence of an inducer, the DNA sequences or the recombinant expression and secretion into the blood, Such genes will not be transcribed. The inducer can be a chemical as from transduced muscle cells, ofatherapeutically effective agent such as a protein, metabolite, growth regulator, phe amount of Such a mammalian serum protein. nolic compound, or a physiological stress imposed directly In another embodiment, the mammalian serum protein is by, for example heat, or indirectly through the action of a 40 glucocerebrosidase. The invention also provides a method of pathogen or disease agent Such as a virus. The inducer also treating a patient having Gaucher disease, including the step can be an illumination agent Such as light and lights various of administering to the patient a conjugate that causes the aspects, which include wavelength, intensity, fluorescence, recombinant expression and Secretion into the blood, such as direction, and duration. from transduced muscle cells, of a therapeutically effective An example of an inducible promoter is the tetracycline 45 amount of glucocerebrosidase. (tet)-on promoter system, which can be used to regulate tran In still another embodiment, the mammalian serum protein Scription of the nucleic acid. In this system, a mutated Tet is C.-galactosidase A. The invention also provides a method of repressor (TetR) is fused to the activation domain of herpes treating a mammal having Fabry disease, including the step of simplex VP16 (transactivator protein) to create a tetracy administering to the patient a conjugate that causes the cline-controlled transcriptional activator (tTA), which is 50 recombinant expression and Secretion into the blood, such as regulated by tet or doxycycline (dox). In the absence of anti from transduced muscle cells, of a therapeutically effective biotic, transcription is minimal, while in the presence oftet or amount of C-galactosidase A. dox, transcription is induced. Alternative inducible systems In still another embodiment, the mammalian serum protein include the ecdysone or rapamycin Systems. Ecdysone is an is a cytokine. The cytokine can be selected from the group insect molting hormone whose production is controlled by a 55 consisting of IFN-O, IFN-B, IFN-y, IL-2, IL-3, IL-4, IL-5, heterodimer of the ecdysone receptor and the product of the IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 and IL-15. The invention ultraspiracle gene (USP). Expression is induced by treatment also provides a method of treating a mammal having cancer or with ecdysone or an analog of ecdysone such as muristerone a bacterial or viral infection, including the step of adminis A. tering to the mammala conjugate that causes the recombinant Additional regulatory elements that may be useful in vec 60 expression and secretion into the blood, such as from trans tors, include, but are not limited to, polyadenylation duced muscle cells, of a therapeutically effective amount of sequences, translation control sequences (e.g., an internal Such a cytokine. ribosome entry segment, IRES), enhancers, or introns. Such In still another embodiment, the mammalian serum protein elements may not be necessary, although they may increase is a peptide hormone. The peptide hormone can be selected expression by affecting transcription, stability of the mRNA, 65 from the group consisting of antimullerian hormone (AMH), translational efficiency, or the like. Such elements can be adiponectin, adrenocorticotropic hormone (ACTH), angio included in a nucleic acid construct as desired to obtain opti tensinogen and angiotensin, antidiuretic hormone (ADH), US 8,956,825 B2 35 36 atrial-natriuretic peptide (ANP), calcitonin, cholecystokinin mode of administration, and the like. Therefore, it is difficult (CCK), corticotropin-releasing hormone (CRH), erythropoi to generalize an exact 'effective amount, yet, a Suitable etin (EPO), follicle stimulating hormone (FSH), gastrin, glu effective amount may be determined by one of ordinary skill cagon, gonadotropin-releasing hormone (GnRH), growth in the art. hormone-releasing hormone (GHRH), human chorionic The term “pharmaceutically acceptable” refers to the fact gonadotropin (hCG), growth hormone (GH), insulin, insulin that the carrier, diluent or excipient must be compatible with like growth factor (IGF), leptin, luteinizing hormone (LH), the other ingredients of the formulation and not deleterious to melanocyte stimulating hormone (MSH or C-MSH), neu the recipient thereof. For example, the carrier, diluent, or ropeptide Y, oxytocin, parathyroid hormone (PTH), prolactin excipient or composition thereof may be administered to a (PRL), relaxin, renin, secretin, Somatostatin, thrombopoietin, 10 Subject along with a conjugate of the invention without caus thyroid-stimulating hormone (TSH), and thyrotropin-releas ing any undesirable biological effects or interacting in an ing hormone (TRH). The invention also provides a method undesirable manner with any of the other components of the for hormone replacement therapy in a mammal, including the pharmaceutical composition in which it is contained. step of administering to the mammal a conjugate that causes Pharmaceutical compositions including the conjugate may the recombinant expression and secretion into the blood, Such 15 be administered by any suitable means, for example, as from transduced muscle cells, ofatherapeutically effective parenterally, such as by Subcutaneous, intravenous, intramus amount of Such a peptide hormone. cular, intrathecal, or intracisternal injection or infusion tech In still other embodiments, the Subject conjugate can be niques (e.g., as sterile injectable aqueous or non-aqueous selected to include an expression vector that causes the Solutions or Suspensions) in dosage formulations containing recombinant expression and Secretion into the blood, such as non-toxic, pharmaceutically acceptable vehicles or diluents. from transduced muscle cells, of an enzyme selected from the In certain embodiments the conjugate is administered group consisting of L-asparagine, L-glutaminase-L-aspara parenterally, or more preferably, intravenously. ginase, L-methioninase, L-phenylalanine, ammonialyase, The mode of delivery chosen for administration of conju L-arginase, L-tyrosinase, L-serine dehydratase, L-threonine gates according to the present invention to a Subject, such as deaminase, indolyl-3-alkane hydroxylase, neuraminidase, 25 a human patient or mammalian animal, will depend in large ribonuclease, a protease, pepsin, and a carboxypeptidase. part on the particular active agent present in the conjugate and Such constructs can be used as part of a treatment program for the target cells. In general, the same dosages and administra CaCC. tion routes used to administer the active agent alone will also In another embodiment, the Subject conjugate can be be used as the starting point for the conjugate. However, it is selected to include an expression vector that causes the 30 preferred that smaller doses be used initially due to the recombinant expression and secretion into the blood of lyso expected increase in cellular penetration of the active agent. staphin. The invention also provides a method of treating a The actual final dosage for a given route of administration is mammal having a bacterial infection, including the step of easily determined by routine experimentation. In general the administering Such a conjugate. same procedures and protocols that have been previously In certain embodiments, the Subject methods and compo 35 used for other antibody-based targeting conjugates (e.g., sitions are used to deliver a prodrug of any of the drugs listed parenterally, intravenous, intrathecal, and the like) are also herein. Suitable for the conjugates of the present invention. Pharmaceutical compositions including a disclosed conju The pharmaceutical compositions of the conjugate can be gate may be used in the methods described herein. Thus, in administered either alone or in combination with other thera one embodiment, a pharmaceutical composition including a 40 peutic agents, may conveniently be presented in unit dose conjugate present in an amount effective to treat a disease or form and may be prepared by any of the methods well known disorder affecting a tissue expressing a nucleoside transport in the art of pharmacy. All methods include bringing the pathway in a subject is used in methods described herein. In conjugate into association with the carrier, which constitutes another embodiment, a pharmaceutical composition includ one or more accessory ingredients. In general, the pharma ing a conjugate present in an amount effective to treat a 45 ceutical compositions are prepared by uniformly and inti disease or disorder of skeletal muscle in a subject is used in mately bringing the active ingredient into association with a methods described herein. In addition to the conjugate, the liquid carrier. In a pharmaceutical composition, the conjugate pharmaceutical composition may also contain other thera is included in an amount Sufficient to produce the desired peutic agents, and may be formulated, for example, by effect upon the process or condition of disease. employing conventional vehicles or diluents, as well as phar 50 Depending on the condition being treated, these pharma maceutical additives of a type appropriate to the mode of ceutical compositions may be formulated and administered desired administration (for example, excipients, preserva systemically or locally. Techniques for formulation and tives, etc.) according to techniques known in the art of phar administration may be found in the latest edition of “Rem maceutical formulation. ington's Pharmaceutical Sciences” (Mack Publishing Co. In certain embodiments, the compositions disclosed herein 55 Easton Pa.). Suitable routes may, for example, parenteral are formulated with additional agents that promote entry into delivery, including intramuscular, Subcutaneous, intramedul the desired cell or tissue. Such additional agents include lary, intrathecal, intraventricular, intravenous, or intraperito micelles, liposomes, and dendrimers. neal. For injection, the pharmaceutical compositions of the The term “effective amount of an active agent refers an invention may beformulated in aqueous solutions, preferably amount that is non-toxic to a subject or a majority or normal 60 in physiologically compatible buffers such as Hanks solu cells, but is an amount of the active agent that is sufficient to tion, Ringer's solution, or physiologically buffered saline. provide a desired effect (e.g., treatment of a skeletal muscle The present disclosure also provides a pharmaceutical disorder, metabolic disorder, blood disorder, or cancer). This composition including a conjugate described herein and an amount may vary from Subject to Subject, depending on the agent that promotes ENT2 expression in a tissue. In some species, age, and physical condition of the Subject, the sever 65 aspects, the agent that promotes ENT2 expression in a tissue ity of the disease that is being treated, the particular conju is an agent that inhibits hypoxia or an agent that inhibits gate, or more specifically, the particular active agent used, its HIF-1. The tissue may be a hypoxic tissue. Such as a hypoxic US 8,956,825 B2 37 38 tumor, a tissue with insufficient vasculature, an ulcer, a dia Therapeutic antibodies may be used to normalize tumor betic ulcer, a poorly-healing wound, an ischemic area, an vasculature. For example, a neutralizing antibody (A4.6.1) ischemic area resulting from stroke, or an ischemic area against VEGF/VPF is described in Yuan F et al. (Proc Natl resulting from cardiovascular disease. In certain embodi AcadSci USA. 1996 Dec. 10; 93(25): 14765-70.) Permeabo ments, the agent that inhibits HIF-1C. is a siRNA, an RNAi lization of the tumor vasculature was observed a few hours construct, a hairpin RNA, or a miRNA that reduces HIF-1C. after injection and lasted about 5 days. Also, the (VEGFR)-2 expression. In some embodiments, the HIF-1C. inhibitor is a neutralizing antibody DC101 may be used to normalize chemotherapeutic drug, topotecan, NSC 644221, PX-478, tumor vasculature as described in Kadambi et al., (Cancer YC-1, 17-AAG, or bevacizumab. In certain embodiments, the Res. 2001 Mar. 15: 61(6):2404-8). Humanized versions of agent that inhibits hypoxia is an agent that normalizes tumor 10 these antibodies, and antibody variants such as single-chain vasculature, or an agent that alters the redox state of a tissue. antibodies, may be used in accordance with the methods The agent that inhibits hypoxia may be excess oxygen, TSC, disclosed herein. or almitrine. Excess oxygen may be delivered, for example, The invention will now be described in greater detail by by intubation, an oxygen mask, or a hyperbaric chamber. In reference to the following non-limiting examples. certain embodiments, the agent that promotes ENT2 expres 15 sion is an inhibitor of a gene that downregulates ENT2, such EXAMPLE 1. as HIF-1C. In other embodiments, the agent that promotes ENT2 expression is a nucleic acid encoding ENT2, for The anti-DNA antibody fragment 3E10 Fv has received example an expression construct that drives expression of attention as a novel molecular delivery vehicle due to its ENT2 or any fragment thereof having essentially the same penetration into living cells with specific nuclear localization, therapeutic transport activity as full-length ENT2. absence of toxicity, and successful delivery of therapeutic Furthermore, herein is provided a method of treating an cargo proteins in vitro and in vivo. In the present study, the ENT-2 deficient tissue, wherein the method includes: a) pathway that allows 3E10 Fv to cross cell membranes was administering an agent that promotes ENT2 expression and/ elucidated. In particular, the present study demonstrates that or activity, and b) administering one of the conjugates dis 25 3E10 Fv penetrates cells through a nucleoside salvage trans closed herein. porter. The results showed that 3E10 Fv is unable to penetrate In certain aspects, a conjugate as described herein may be into cells deficient in the equilibrative nucleoside transporter, adminstered together with ATP or an ATP-generating agent. ENT2, and reconstitution of ENT2 into ENT2-deficient cells These agents may be used to inhibit hypoxia and/or ischemia restores 3E10 Fv transport into cell nuclei. These results In certain aspects, ATP is specifically delivered to the target 30 represented the first demonstration of protein transport tissue, for example, using liposomes. Methods of delivering through a nucleoside Salvage pathway. ATP to ischemic tissue are known in the art, and are described Cell Lines. in U.S. Pat. No. 7,056,529 and Verma D et al., “ATP-loaded COS-7, K562, and CEM/ENT1 cells were purchased from Liposomes Effectively Protect Mechanical Functions of the the American Type Culture Collection (Rockville, Md.). A Myocardium from Global Ischemia in an Isolated Rat Heart 35 nucleoside transport-deficient porcine kidney tubular epithe Model”, JControl Release. 2005 Nov. 28; 108(2-3): 460-471. lial cells (PKNTD) were generated and transfected with plas In certain embodiments, a patient is treated with a hypoxia mids containing nucleic acid encoding hENT1 or hENT2 to inhibiting agent and a conjugate herein prior to Surgery, as a form PKNTD/ENT1 and PKNTD/ENT2 cells, respectively, prophylactic treatment for ischemia caused by Surgery. as previously described (Wardet al., J. Biol. Chem. 275:8375 A number of drugs affect tumor vasculature. While the 40 81, 2000). mechanism of such drugs is not fully understood, there appear Plasmids. to be three broad classes of vasculature-targeting agents. A construct for expression of 3E10 Fv in the X-33 strain of First, an agent may be anti-angiogenic. Such agents prevent Pichia pastoris, pPICZOA-Fv, was generated by ligating the growth of new blood vessels, starving the tumor of blood cDNA encoding the single-chain Fv fragment of mAb 3E10 and oxygen. Such agents make a tumor more hypoxic. Sec 45 into pPICZOA, as previously described (Weisbartet al., Can ond, an agent may collapse pre-existing tumor vasculature, cer Lett. 195:211-9, 2003; and Weisbart et al., Int J Oncol also increasing the hypoxia of the tumor. Third, vasculature 2004; 25:1113-8). normalizing agents reduce the abnormalities of the tumor Purification of 3E10 Fv. 3E10 Fv was purified from the vasculature. For example, they may reduce the number of supernatant of P. pastoris transfected with pPICZOA-Fv as excess epithelial cells in the tumor vasculature. These agents 50 previously described (Weisbartet al., Cancer Lett. 195:211-9, improve blood flow to the tumor and reduce hypoxia. Para 2003). doxically, vasculature-normalizing agents may be used to Cellular Penetration Assays. impede tumor growth, by allowing other therapeutic mol Purified 3E10 Fv was exchange-dialyzed into PBS prior to ecules (such as chemotherapeutic drugs) better access to the application to cells. After dialysis, 10% fetal calf serum was tumor. 55 added to the buffer containing the 3E10 Fv. Control buffer Some therapies previously thought to be anti-angiogenic was PBS with 10% fetal calf serum. For adherent cell lines may instead produce vasculature normalization. For example, (COS-7, PKNTD/ENT1, and PKNTD/ENT2), 50 uL of con one may block vascular endothelial growth factor (VEGF) or trol buffer or 3E10 Fv in PBS+10% fetal calf serum was its receptor (VEGFR2), causing apoptosis of endothelial added to cells on 96-well plates for one hour. After incubation cells. Consequently there is a decrease in blood vessel diam 60 with 3E10 Fv, the antibody fragment was removed and cells eter, density and permeability. There is also a decrease in were washed, fixed in chilled 100% ethanol, and stained with interstitial fluid pressure and, at least in Some instances, the 9E10 C-myc antibody as previously described (Weisbart elevated oxygen tension (reviewed in Jain R et al., Nature et al., Cancer Lett. 195:211-9, 2003). For non-adherent cells Medicine 7,987-989 (2001)). Various other therapeutics also (K562 and CEM/ENT1) cell pellets composed of -200,000 contribute to vasculature normalization, including ST 1571, 65 cells were re-suspended in 100 uL control buffer or 10 LM C225, and Herceptin, which block PDGFR, HER1 and HER2 3E10 Fv and allowed to incubate with intermittent shaking at signaling, respectively. 37° C. for one hour. Cells were then centrifuged at 100 g for US 8,956,825 B2 39 40 2 minutes and washed three times with PBS. Next, cells were Western blot analysis of cell lysates or immunocytochemical spread on glass slides and allowed to dry overnight. Cells staining of cells (FIG. 1C, top panels). Western blot analysis were then fixed in chilled 100% ethanol for ten minutes, of cell lysates demonstrated the presence of a ~30 kDa myc washed three times in PBS, and stained with the 9E10 C-myc tagged protein inside cells treated with 3E10 Fv, which indi antibody. cated penetration of the full-length antibody fragment. Fur Nucleoside Transporter Inhibition Assay. thermore, immunocytochemical staining confirmed nuclear Nitrobenzylmercaptopurine riboside (NBMPR) was pur localization by 3E10 Fv (FIG. 1C, top right panel), consistent chased from Sigma (St. Louis, Mo.), and a stock Solution of with previous confocal microscopy and immunocytochemi 100 mM NBMPR in DMSO was prepared. To control for the cal studies on the antibody (Hansen et al., Brain Res. 1088(1), effects of DMSO in cell culture, DMSO was added to control 10 187-96, 2006; and Weisbartet al., J. Immunol. 164(11), 6020 buffers not containing NBMPR. The concentration of DMSO 6, 2000). Next, COS-7 cells were pre-treated for 30 minutes in all control and experimental buffers was 0.1%. COS-7 cells with control buffer or buffer containing 100 uM NBMPR were pre-treated for 30 minutes with control buffer (PBS+ prior to a one hour incubation with 10 uM 3E10 Fv in the 10% fetal calf serum) or buffer containing 10 uM or 100 uM presence or absence of NBMPR. Subsequent immunocy NBMPR. Buffers were then replaced with control buffer or 10 15 tochemical staining of the cells demonstrated that 100 uM uM3E10 Fv in the presence or absence of 10 uM or 100 uM NBMPR suppressed nuclear penetration by 3E10 Fv (FIG. NBMPR for one hour. Cells were then washed, fixed, and 1C, bottom left panel), which suggested that ENT1 or ENT2 stained with the 9E10 C-myc antibody. is involved in 3E10 Fv transport. Microscopic Images. To resolve which of the ENTS was linked to 3E10 Fv Images of cells were acquired with an Olympus IX70 transport, 3E10 Fv penetration into COS-7 cell nuclei was inverted microscope with RC reflected light fluorescent tested in the presence of a lower dose of NBMPR to take attachment and MagnaFire SP Digital Imaging System advantage of the different K, of NBMPR for ENT1 and ENT2 (Olympus, Melville, N.Y.) as described previously (Weisbart (0.4 nM versus 2.8 LM, respectively) (Ward et al., J. Biol. et al., J. Immunol 164.6020-6, 2000). Scale bar in cell Chem. 275(12):8375-81, 2000). Thus, at 10 uM NBMPR images=5um. 25 ENT1 activity is completely inhibited while ENT2 retains The single chain Fv fragment of the 3E10 anti-DNA moderate activity. In contrast to the distinct inhibition of autoantibody (3E10 Fv) has recently been harnessed as a 3E10 Fv transport provided by 100 uM NBMPR (FIG. 1C, novel molecular delivery vehicle due to its specific nuclear bottom left panel), 3E10 Fv successfully penetrated COS-7 localization and apparent lack of toxicity (Weisbart et al., J. cell nuclei in the presence of 10 uM NBMPR (FIG. 1C, Autoimmun. 11(5), 539-46, 1998). 3E10 Fv and Fv-fusion 30 bottom right panel). This result suggested that ENT2, not proteins readily transduce across cell membranes and pen ENT1, mediated transport of 3E10 Fv. The decreased nuclear etrate into cell nuclei, and 3E10 Fv has successfully delivered staining intensity in cells treated with 3EI 0 Fv4-10 uM biologically active proteins such as Hsp70 (Hansen et al., NBMPR compared to cells treated with 3E10 Fv alone likely Brain Res. 1088(1), 187-96, 2006) and p53 (Weisbartet al., reflects partial inhibition of 3E10 Fv transport due to the Int. J. Oncol. 25(6), 1867-1873, 2004) into living cells in 35 expected >50% suppression of ENT2 activity by 10 uM vitro. Moreover, 3E10 Fv mediated full-length p53 protein NBMPR. therapy in vivo (Hansen et al., Cancer Res. 67(4), 1769-74, As an additional approach to identifying the equilibrative 2007). The pathway that carries 3E10 Fv across cell mem nucleoside transporter(s) involved in 3E10 Fv transduction, branes and into cell nuclei, however, has not been identified 3E10 Fv transduction into the CEMAENT1 cell line that previously. 40 expresses ENT1 but lacks ENT2 (Crawford et al., J. Biol. Previous studies implicated DNA binding as important in Chem. 273(9), 5288-93, 1998) was examined. K562 leuke 3E10 Fv transduction into cell nuclei. Specifically, mutations mia cells, which express both ENT1 and ENT2 (Huang et al., that abrogate DNA binding by the antibody render it inca Nucleosides Nucleotides Nucleic Acids 23(8-9), 1445-50, pable of cellular penetration (Zacket al., J. Immunol. 157(5), 2004), were used as a positive control. CEM/ENT1 cells 2082-8, 1996). The association between cellular penetration 45 (which express ENT1 but not ENT2) and K562 cells were and DNA binding distinguished 3E10 Fv from other protein incubated with control buffer or 10 uM3E10 Fv for one hour. transduction domains and Suggested the potential involve Cells were then washed, fixed, and stained with the C-myc ment of nucleoside salvage pathways in 3E10 Fv transport. antibody. Control K562 cells and CEM/ENT1 cells showed Both concentrative (CNT) and equilibrative (ENT) nucleo no staining. Consistent with the studies in COS-7 cells, side transporters mediate the uptake of nucleosides and 50 immunocytochemical staining of the 3E10 Fv-treated K562 nucleobases by mammalian cells (Kong et al., Curr: Drug cells demonstrated penetration of the antibody fragment into Metab. 5(1):63-84, 2004). Other studies demonstrating 3E10 100% of the cells. CEM/ENT1 cells treated with 3E10 Fv, Fv penetration into COS-7 cells that lack endogenous CNTs, however, showed no staining. This result demonstrated that suggested that CNTs do not play a major role in 3E10 Fv absence of ENT2 significantly impaired transduction by transport (Hansen et al., Brain Res. 1088(1), 187-96, 2006: 55 3E10 Fv into cells. Taken together, the inhibition of 3E10 Fv and Toan et al., Pflugers Arch. 447(2), 195-204, 2003). Thus, transport by high concentrations of NBMPR and failure of the role of ENTs in 3E10 Fv transport was examined in the 3E10 Fv to penetrate a cell line lacking ENT2 strongly sup present study. port a role of ENT2 in 3E10 Fv transport. ENT1 and ENT2, which each mediate equilibrative To verify that ENT2 facilitates 3E10 Fv intranuclear pro nucleoside transport in mammalian cells, are inhibited by 60 tein transduction, experiments were performed on nucleoside high concentrations of NBMPR (Ward et al., J. Biol. Chem. transporter-deficient PK15 cells (PKNTD) with either ENT1 275(12):8375-81, 2000). NBMPR was tested for inhibition of or ENT2 reconstituted through stable transfection and 3E10 Fv transport. Purified 3E10 Fv (constructed with His expression of ENT1 or ENT2 cDNA (Ward et al., J. Biol. tag for purification and myc tag for identification) migrated as Chem. 275(12):8375-81, 2000). PKNTD/ENT1 and a single -30 kDa protein on SDS-PAGE. Transduction of 65 PKNTD/ENT2 cells were treated with control buffer (i.e., 3E10 Fv into COS-7 cells was confirmed by incubating cells control cells) or 10 uM3E10 Fv for one hour. Cells were then with 10 uM 3E10 Fv for one hour at 37° C., followed by washed, fixed, and stained with the C-myc antibody. Control US 8,956,825 B2 41 42 PKNTD/ENT1 and PKNTD/ENT2 cells showed an absence across both cellular and nuclear membranes or if another of staining. Similarly, PKNTD/ENT1 cells treated for one pathway is involved in nuclear penetration (Mani et al., J. hour with 10 uM 3E10 Fv showed an absence of staining, Biol. Chem. 273(46), 30818-325, 1998). exhibiting no evidence of nuclear penetration, which indi With regard to molecular therapy, the linkage between cated a failure of the antibody fragment to penetrate the ENT2 and nuclear penetration by 3E10 Fv reported herein ENT2-deficient cells. This result was consistent with the further establishes 3E10 Fv as a novel molecular delivery results obtained with the CEM/ENT1 cells. In contrast, 3E10 vehicle that is distinct from other protein transduction Fv-treated PKNTD/ENT2 cells exhibited distinct nuclear domains previously described. EndoSomal localization by staining, which indicated that restoration of ENT2 to the cell-penetrating peptides limits their role in molecular nucleoside transporter deficient cells significantly augmented 10 therapy (Jones et al., Br. J. Pharmacol. 145 (8), 1093-102, nuclear penetration by 3E10 Fv. This result confirmed that the 2005; and Kaplanet al., J. Control Release 102(1):247-53, presence of ENT2 facilitates nuclear penetration by 3E10 Fv 2005), but the identification of 3E10 Fv transport through and verified protein transduction of the antibody fragment ENT2 provides a rationale for future studies on the use of through the ENT2-mediated nucleoside salvage pathway. 3E10 Fv in delivering molecules such as siRNAs, antisense The nucleoside Salvage pathways have been studied in 15 oligonucleotides, and transcription factors to cell nuclei. detail, but protein transport through or related to nucleoside Moreover, it is possible that toxic cell-penetrating antibod salvage has not been previously described. While not wishing ies utilize a nucleoside Salvage pathway in cellular penetra to be bound by any particular theory, 3E10 Fv may be carried tion. Thus, inhibition of nucleoside transporters may be a into cells by virtue of its binding to nucleosides or nucleo- means of limiting tissue damage by cytotoxic autoantibodies bases that are subsequently transported into cells by ENT2. 20 in certain autoimmune diseases. The discovery of intra Alternatively, 3E10 Fv may mimic the structure of a nucleo- nuclear protein transduction by 3E10 Fv through the ENT2 side or nucleobase that is recognized and transported into mediated nucleoside Salvage pathway has profound implica cells by ENT2. Elucidation of the specific mechanism by tions for cell biology, pharmacology, and medicine. which ENT2 facilitates 3E10 Fv transport should yield fur- Although the invention has been described with reference ther insights into both protein transduction and nucleoside 25 to the above example, it will be understood that modifications salvage pathways. Furthermore, since ENT2 is located in and variations are encompassed within the spirit and scope of both plasma and nuclear membranes, it will be important to the invention. Accordingly, the invention is limited only by ascertain whether ENT2 facilitates transport of 3E10 Fv the following claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS : 14

<21 Os SEQ ID NO 1 &211s LENGTH: 2162 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens

<4 OOs SEQUENCE: 1 gggctg.cgct gtc.ca.gctgt ggctatggcc cc agcc.ccga gatgaggagg gagagalacta 60

ggggc.ccgca ggcctgggala titt.ccgt.ccc ccaccalagtic cqgatgctica ct coaaagtic 12O tdagcaggcc cctgagggag ggagctgtca gC cagggaala accgagaaca CC at Caccat 18O

gacaaccagt caccago: ct c aggacagata caaagctgtc. toggcttatct tctt catgct 24 O

gggtctggga acgctgctcc cqtggaattt ttt catgacg gccact cagt attt cacaaa 3 OO cc.gc.ctggac atgtc.ccaga atgtgtcCtt gg.tcactgct galactgagca aggacgcc.ca 360 ggcgt.ca.gcc gcc.cctgcag cacccttgcc tagcggaac totcticagtg cc at Cttcaa 42O

caatgtcatg accotatgtg ccatgctgcc cctogctgtta tt caccitacct caact cott 48O Cctgcatcag aggat.cccCC agtc.cgitacg gatcctgggc agcctggtgg cc at CCtgct 54 O

ggtgtttctg at cactgcca to Ctggtgaa ggtgcagctg gatgctctgc cctt Ctttgt 6 OO cat caccatg at caagat.cg togct cattaa tt catttggit gc catcctgc agggcagoct 660 gtttggtctg gctggccttic toctgc.cag ct acacggcc cc catcatga gtggcCaggg 72O Cctagdaggc titctttgcct cogtggc.cat gatctgcgct attgcc agtg gCtcggaact 78O

atcagaaagt gcctt cqgct acttitat cac agcc tigtgct gttat cattt tdac catcat 84 O

ctgttacct g ggcctg.cccc gcctggaatt ct accoctac taccagcagc ticaa.gcttga 9 OO

aggaccc.ggg gagcaggaga ccaagttgga cct cattagc aaaggagagg agccaagagc 96.O

aggcaaagag gaatctggag titt cagt ct c caact ct cag cccaccaatgaaagcc actic 102O

US 8,956,825 B2 45 46 - Continued

Lell Phe Gly Lieu Ala Gly Lieu. Lieu Pro Ala Ser Tyr Thir Ala Pro Ile 1.65 17O 17s

Met Ser Gly Glin Gly Lieu Ala Gly Phe Phe Ala Ser Wall Ala Met Ile 18O 185 19 O

Ala Ile Ala Ser G y Ser Glu Lieu. Ser Glu Ser Ala Phe Gly Tyr 195

Phe Ile Thir Ala Cys A a Wall Ile Ile Lieu. Thir Ile Ile Tyr Lieu. 21 O 215

Gly Luell Pro Arg Lieu G u Phe Tyr Arg Tyr Tyr Glin Glin Luell Llys Lieu. 225 235 24 O

Glu Gly Pro Gly Glu G in Glu. Thir Llys Lieu. Asp Lell Ile Ser Lys Gly 245 250 255

Glu Glu Pro Arg Ala G y Lys Glu Glu Ser Gly Wall Ser Wall Ser Asn 26 O 265 27 O

Ser Glin Pro Thir Asn. G u Ser His Ser Ile Llys Ala Ile Luell Lys Asn 28O 285

Ile Ser Wall Lieu Ala Phe Ser Wall Cys Phe Ile Phe Thir Ile Thir Ile 29 O 295 3 OO

Gly Met Phe Pro Ala Wall. Thir Wall Glu Val Lys Ser Ser Ile Ala Gly 3. OS 310 315 32O

Ser Ser Thir Trp. Glu Arg Tyr Phe Ile Pro Wall Ser Phe Lieu. Thir 3.25 330 335

Phe Asn Ile Phe Asp Trp Lieu. Gly Arg Ser Lieu. Thir Ala Wall Phe Met 34 O 345 35. O

Trp Pro Gly Lys Asp Ser Arg Trp Luel Pro Ser Lieu Wall Luell Ala Arg 355 360 365

Lell Wall Phe Wall Pro Lieu. Lieu. Lieu. Lieu. Cys Asn Ile Pro Arg Arg 37 O 375

Tyr Luell Thir Wall Wall Phe Glu. His Asp Ala Trp Phe Ile Phe Phe Met 385 390 395 4 OO

Ala Ala Phe Ala Phe Ser Asn Gly Tyr Lieu Ala Ser Lell Met Cys 4 OS 41O 415

Phe Gly Pro Llys Llys Val Llys Pro Ala Glu Ala Glu Thir Ala Gly Ala 425 43 O

Ile Met Ala Phe Phe Lieu. Cys Lieu Gly Lieu Ala Lell Gly Ala Wall Phe 435 44 O 445

Ser Phe Luell Phe Arg Ala Ile Val 450 45.5

SEQ ID NO 3 LENGTH: 1576 TYPE: DNA ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 3 gccatggCCC gaggagacgc cc.cgcgggac agctaccacc tgg togggat cagcttcttic 6 O atcCtggggg tgggcacc ct cct tcc.ctgg aactt Cttca t caccgc cat cc.cgtacttic 12 O

Caggcgcgac tgg.ccggggc cggcaa.ca.gc acagcCagga tcc tigagcac caaccacacg 18O ggtc.ccgagg atgcct tcaa Ctt Caacaat tgggtgacgc tgctgtcc.ca gctgc.ccctg 24 O ctgct citt.ca CCCtcCtcaa. citcct tcctg taccagtgcg tcc.cggagac ggtgcgcatt 3OO

Ctgggcagcc tgctggcc at actgctgctic tittgc cctoga Cagcagcgct ggit Caaggtg 360 gacatgagcc ccggacccitt citt ct coat c accatggcct cc.gtctgctt Cat Caactic C

US 8,956,825 B2 49 50 - Continued

1.65 17O 17s

Ala Met Luell Lieu. Ser Met Ala Ser Gly Val Asp Ala Glu Thir Ser Ala 18O 185 19 O

Lell Gly Tyr Phe Ile Thr Pro Tyr Val Gly Ile Lell Met Ser Ile Wall 195

Tyr Luell Ser Lieu. Pro His Lieu. Llys Phe Ala Arg Lieu Ala 21 O 215

Asn Ser Ser Glin Ala Glin Ala Glin Glu Lieu. Glu Thir Ala Glu 225 23 O 235 24 O

Lell Luell Glin Ser Asp Glu Asn Gly Ile Pro Ser Ser Pro Glin Llys Val 245 250 255

Ala Luell Thir Lieu. Asp Lieu. Asp Lieu Glu Lys Glu Pro Glu Ser Glu Pro 26 O 265 27 O

Asp Glu Pro Glin Llys Pro Gly Lys Pro Ser Wall Phe Thir Wall Phe Glin 27s 28O 285

Ile Trp Lieu. Thir Ala Lieu. Cys Lieu Wall Lieu Wall Phe Thir Wall. Thir 29 O 295 3 OO

Lell Ser Wall Phe Pro Ala Ile Thr Ala Met Wall Thir Ser Ser Thir Ser 3. OS 310 315 32O

Pro Gly Trp Ser Glin Phe Phe Asn. Pro Ile Phe Lieu. Luell 3.25 330 335

Phe Asn Ile Met Asp Trp Lieu. Gly Arg Ser Lieu. Thir Ser Tyr Phe Lieu. 34 O 345 35. O

Trp Pro Asp Glu Asp Ser Arg Lieu. Luell Pro Luell Lell Wall Lieu. Arg 355 360 365

Phe Luell Phe Wall Pro Leu Phe Met Lieu. Cys His Wall Pro Glin Arg Ser 37 O 375

Arg Luell Pro Ile Leu Phe Pro Glin Asp Ala Tyr Phe Ile Thir Phe Met 385 390 395 4 OO

Lell Luell Phe Ala Val Ser Asn Gly Tyr Lieu Val Ser Lell Thir Met Cys 4 OS 41O 415

Lell Ala Pro Arg Glin Val Lieu Pro His Glu Arg Glu Wall Ala Gly Ala 425 43 O

Lell Met Thir Phe Phe Lieu Ala Lieu. Gly Lieu. Ser Gly Ala Ser Luell 435 44 O 445

Ser Phe Luell Phe Lys Ala Lieu. Lieu 450 45.5

<210s, SEQ ID NO 5 &211s LENGTH: 348 &212s. TYPE: DNA &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 5 gaggtgcagc tiggtggagtic tgggggaggc ttagtgaagc CtggagggtC ccggaaactic 6 O t cct gtgcag cct ctoggatt cactitt cagt gactatggaa tgcactgggt cc.gtcaggct 12 O ccagagaagg ggctggagtg ggttgcatac attagtagtg gcagtag tac Cat Ctactat 18O gCagacacag tgaagggcc.g att Caccatc. tccagaga.ca atgccaagaa caccctgttc 24 O

Ctgcaaatga c cagtictaag gtctgaggac acago catgt attactgtgc aaggcggggg 3OO ttact acttg act actgggg cCaaggcacc act ct cacag t ct cott ca. 348

<210s, SEQ ID NO 6 &211s LENGTH: 116 US 8,956,825 B2 51 - Continued

212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 6

Glu Val Glin Lieu Val Glu Ser Gly Gly Gly Lieu Wall Pro Gly Gly 1. 5 1O 15

Ser Arg Llys Lieu. Ser Cys Ala Ala Ser Gly Phe Thir Phe Ser Asp Tyr 2O 25 3O

Gly Met His Trp Val Arg Glin Ala Pro Glu Lys Gly Lell Glu Trp Val 35 4 O 45

Ala Tyr Ile Ser Ser Gly Ser Ser Thr Ile Tyr Tyr Ala Asp Thir Wall SO 55 6 O

Lys Gly Arg Phe Thir Ile Ser Arg Asp Asn Ala Asn Thir Lieu. Phe 65 70 7s 8O

Lieu Gln Met Thr Ser Lieu. Arg Ser Glu Asp Thr Ala Met Tyr Cys 85 90 95

Ala Arg Arg Gly Lieu. Lieu. Lieu. Asp Tyr Trp Gly Glin Gly Thir Thir Lieu. 1OO 105 11 O

Thir Wal Ser Ser 115

<210s, SEQ ID NO 7 &211s LENGTH: 333 &212s. TYPE: DNA <213s ORGANISM: Mus musculus

<4 OO > SEQUENCE: 7 gacattgttgc tigacacagtic ticcitgct tcc ttagctgitat Ctctggggca gagggccacc 6 O atct Cotgca gggc.ca.gcaa aagtgtcagt a catctagot at agttacat gCactgg tac 12 O caacagaaac caggacagcc acccaaactic ct catcaagt atgcatccta cctagaatct 18O ggggttcCtg C caggttcag tigcagtggg totggga cag act t t cacct caa.catcCat 24 O

Cctgtggagg aggaggatgc tigcaa.cat at tactgtcagc acagtaggga gtttcc.gtgg 3OO acgttcggtg gaggcaccala gctggagttgaaa 333

<210s, SEQ ID NO 8 &211s LENGTH: 111 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 8

Asp Ile Val Lieu. Thr Glin Ser Pro Ala Ser Lieu. Ala Wall Ser Lieu. Gly 1. 5 1O 15

Glin Arg Ala Thir Ile Ser Cys Arg Ala Ser Lys Ser Wall Ser Thir Ser 2O 25 3O

Ser Tyr Ser Tyr Met His Trp Tyr Glin Glin Lys Pro Gly Glin Pro Pro 35 4 O 45

Llys Lieu. Lieu. Ile Llys Tyr Ala Ser Tyr Lieu. Glu Ser Gly Wall Pro Ala SO 55 6 O

Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Phe His Lell Asn Ile His 65 70 7s 8O

Pro Val Glu Glu Glu Asp Ala Ala Thr Tyr Tyr Glin His Ser Arg 85 90 95

Glu Phe Pro Trp Thr Phe Gly Gly Gly Thr Lys Lell Glu Luell 1OO 105 11 O US 8,956,825 B2 53 - Continued

<210s, SEQ ID NO 9 &211s LENGTH: 321 &212s. TYPE: DNA <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 9 agtattgttga tigacccagac toccaaattic ctdcctgitat Cagcaggaga Cagggitt acc 6 O atgacctgca aggc.cagt ca gagtgtgggit aataatgtag cctgg tacca acagaa.gc.ca 12 O ggacagt citc ctaaactgct gatatact at gcatccaatc gct acactgg agt ccct gat 18O cgct tcactg gcagtggat.c tdggacagat ttcactitt.ca c catcagcag tgtgcaggtt 24 O gaag acctgg cagtttattt citgtcagoag cattatagot Ctc.cgtggac gttcggtgga 3OO ggcaccalagc tiggaaatcaa a 321

<210s, SEQ ID NO 10 &211s LENGTH: 107 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 10

Ser Ile Val Met Thr Glin Thr Pro Llys Phe Leu Pro Wall Ser Ala Gly 1. 5 1O 15

Asp Arg Val Thr Met Thr Cys Lys Ala Ser Glin Ser Wall Gly Asn. Asn 2O 25

Val Ala Trp Tyr Glin Gln Lys Pro Gly Glin Ser Pro Lys Luell Lieu. Ile 35 4 O 45

Tyr Tyr Ala Ser ASn Arg Tyr Thr Gly Val Pro Asp Arg Phe Thr Gly SO 55 6 O

Ser Gly Ser Gly Thr Asp Phe Thr Phe Thr Ile Ser Ser Wall Glin Wall 65 70 7s 8O

Glu Asp Lieu Ala Val Tyr Phe Cys Glin Gln His Ser Ser Pro Trp 85 90 95 Thr Phe Gly Gly Gly Thr Lys Lieu. Glu Ile Llys 1OO 105

<210s, SEQ ID NO 11 &211s LENGTH: 5 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 11 Gly Gly Gly Gly Ser 1. 5

<210s, SEQ ID NO 12 &211s LENGTH: 6 212. TYPE: PRT <213> ORGANISM: Artificial sequence 22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct

<4 OOs, SEQUENCE: 12

His His His His His His 1. 5

<210s, SEQ ID NO 13 &211s LENGTH: 10 212. TYPE: PRT <213> ORGANISM: Artificial sequence US 8,956,825 B2 55 56 - Continued

22 Os. FEATURE: <223> OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 13 Gly Phe Pro Arg Gly Phe Pro Ala Gly Gly 1. 5 1O

SEQ ID NO 14 LENGTH: 12 TYPE PRT ORGANISM: Artificial sequence FEATURE: OTHER INFORMATION: Synthetic construct <4 OOs, SEQUENCE: 14 Thir Arg His Arg Glin Pro Arg Gly Trp Glu Gln Lieu. 1. 5 1O

I claim: (a) contacting a cell expressing human equilibrative 1. A method of screening a conjugate for transport by nucleoside transporter 2 (ENT2), with a conjugate under human equilibrative nucleoside transporter 2 (ENT2) com suitable conditions for transport to occur, wherein the prising, conjugate comprises (i) a Substrate comprising a 25 polypeptide, and (ii) an active agent linked to the Sub contacting a cell expressing human equilibrative nucleo strate; but does not include a substrate comprising a side transporter 2 (ENT2), with a conjugate under suit nucleoside or nucleoside analog; and able conditions for transport to occur, wherein the con (b) determining whether the conjugate is transported in the jugate includes (i) a Substrate comprising an antibody or cell by the human ENT2. antibody fragment, and (ii) an active agent linked to the 30 8. The method according to claim 7, wherein the cell is Substrate; and determining whether the conjugate is transfected with DNA encoding the human ENT2 transporter. transported into the cell by the human ENT2. 9. The method according to claim 8, wherein the determin 2. The method according to claim 1, wherein the equilibra ing step comprises comparing the amount of conjugate trans tive nucleoside transporter is insensitive to low concentra ported into the cell transfected with DNA encoding the tions of nitrobenzylmercaptopurine riboside (NBMBR). 35 human ENT2 to the amount of conjugate transported into a 3. The method according to claim 1, wherein the cell is control cell not transfected with the human ENT2, whereinan transfected with DNA encoding the human ENT2 transporter. increase in transport of conjugate of the transfected cell as 4. The method according to claim3, wherein the determin compared to the control cell indicates transport is by the ing step comprises comparing the amount of conjugate trans human ENT2 transporter. ported into the cell transfected with DNA encoding the 40 10. The method according to claim 7, wherein the conju human ENT2 to the amount of conjugate transported into a gate comprises a detectable label. control cell not transfected with the human ENT2, whereinan 11. The method according to claim 7, wherein the cell increase in transport of conjugate of the transfected cell as endogenously expresses the human ENT2 transporter. compared to the control cell indicates transport is by the 12. The method according to claim 1, wherein the active human ENT2 transporter. 45 agent is a peptide or a polypeptide. 5. The method according to claim 1, wherein the conjugate 13. The method according to claim 1, wherein the active comprises a detectable label. agent is an antibody or an antibody fragment. 6. The method according to claim 1, wherein the cell 14. The method according to claim 7, wherein the active endogenously expresses the human ENT2 transporter. agent is a peptide or a polypeptide. 7. A method of screening a conjugate for transport by 50 15. The method according to claim 7, wherein the active human equilibrative nucleoside transporter 2 (ENT2) com agent is an antibody or an antibody fragment. prising: k k k k k