US008O84017B2

(12) United States Patent (10) Patent No.: US 8,084,017 B2 Ahrens (45) Date of Patent: Dec. 27, 2011

(54) CONTRASTAGENTS FOR MAGNETIC Cohen, B., et al., “ as an Endogenous MRI Reporter for RESONANCE MAGING AND METHODS Noninvasive Imaging of Expression in C6 Glioma Tumors.” RELATED THERETO Neoplasia, 7(2):109-117 (2005). Genove, G., “A new transgene reporter for in vivo magnetic reso (75) Inventor: Eric Ahrens, Pittsburgh, PA (US) nance imaging.” Nature Medicine, 11(4):450-454 (2005). Ichikawa, T., et al., “MRI of Transgene Expression: Correlation of (73) Assignee: Carnegie Mellon University, Pittsburgh, Therapeutic .” Neoplasia, 4(6):523-530 (2002). PA (US) Moore, A., et al., “Measuring transferrin receptor gene expression by NMR imaging.” Biochimica et Biophysica Acta, 14 02:239-249 (*) Notice: Subject to any disclaimer, the term of this (1998). patent is extended or adjusted under 35 Walter, G., et al., “Noninvasive measurement of gene expression in U.S.C. 154(b) by 738 days. skeletal muscle.” PNAS,97(10):5151-5155 (2000). Weissleder, R., et al., “MR Imaging and Scintigraphy of Gene (21) Appl. No.: 10/384,496 Expression through Melanin Induction.” Radiology, 204:425-429 (1997). (22) Filed: Mar. 7, 2003 Ahrens, et al., “A model for MRI contrast enhancement using T. agents”. Proc. Acad. Sci. USA. vol. 95, pp. 8443-8448, Applied (65) Prior Publication Data Biological Sciences (1998). Ahrens, et al., “Peripheral somatosensory fMRI in mouse at 11.7T". US 2003/0219385 A1 Nov. 27, 2003 NMR in Biomedicine, NMR Biomed. 14:318-324 (2001). Ahrens, et al., “Receptor-Mediated Endocytosis of Iron-Oxide Par Related U.S. Application Data ticles Provides Efficient Labeling of Dendritic Cells for In Vivo MR (60) Provisional application No. 60/363,163, filed on Mar. Imaging, Magnetic Resonance in Medicine 49:1006-1013 (2003). 7, 2002. Beckmann, et al., “From Anatomy to the Target. Contributions of Magnetic Resonance Imaging to Preclinical Pharmaceutical (51) Int. Cl. Research”. The Anatomical Record (New Anat.) 265:85-100, (2001). A6B 5/55 (2006.01) Corsi, et al., “Transient overexpression of human H- and L-ferritin CI2N IS/00 (2006.01) chains in COS cells”, Biochem. J. (1998) 330, 315-320. CI2N 15/82 (2006.01) Cozzi, et al., “Overexpression of Wild Tjipe and Mutated Human Ferritin H-chain in HeLa Cells—In Vivo Role of Ferritin Ferroxidase (52) U.S. Cl...... 424/9.3: 435/455; 435/468 Activity”. The Journal of Biological Chemistry, vol. 275, No. 33, pp. (58) Field of Classification Search ...... None 25122-25129, (2000). See application file for complete search history. Donlin, et al., “Analysis of Iron in Ferritin, the Iron-Storage , A General Chemistry Experiment”, Journal of Chemical Education, (56) References Cited vol. 75 No. 4, (1998). Donlin, et al., “Ferritin Molecular-Graphics Tutorial. Iron in Biol U.S. PATENT DOCUMENTS ogy. Study of the Iron Content in Ferritin, The Iron-storage Protein'. 5,707,605 A 1/1998 Meade et al...... 424,935 J. Chem. Edu. 75, 437, (1998). 6,232,295 B1 5/2001 Kayyem et al. Engelstad, et al. “Contrast Agents”. 6,315,981 B1 1 1/2001 Unger Epsztejn, et al., “H-Ferritin Subunit Overexpression in Erythroid 6,316,692 B1 1 1/2001 Readhead et al...... 800/14 6,495.355 B1 12/2002 Contaget al. Cells Reduces the Oxidative Stress Response and Induces Multidrug 6,511,967 B1 1/2003 Weissleder et al. Resistance Properties”. Blood, vol. 94, No. 10, (1999):pp. 3593 6,812,339 B1 1 1/2004 Venter et al. 3603. 7,097.841 B2 8, 2006 Carter et al. Fernandez, et al., “Gene Expression Systems. using nature for the art 2002fOO25296 A1 2, 2002 Knaus et al. of expression.”. San Diego: Academic Press, 1999. 2003/0219385 A1 11/2003 Ahrens Fleming, et al., “Transferrin receptor 2. Continued expression in 2004/OOO6001 A1 1/2004 Carter et al. mouse liver in the face of iron overload and in hereditary FOREIGN PATENT DOCUMENTS hemochromatosis”, PNAS, vol.97, No. 5. pp. 2214-2219 (2000). WO WO98,295.35 7, 1998 Fleming, et al., “Targeted mutagenesis of the murine transferrin WO WO-98.33809 8, 1998 receptor-2 gene produces hemochromatosis”, PNAS, vol.99, No. 16, WO WO-O3,O75747 A2 9, 2003 pp. 10653-10658 (2002). OTHER PUBLICATIONS (Continued) Weissleder et al., Nature, 2000, 6:351-354.* Primary Examiner — Ileana Popa Gottesfeld et al., Magn Reson Med, 1996, 35: 514-520.* Weissleder et al., Radiology, 1997, 204: 425-429.* (74) Attorney, Agent, or Firm — Ropes & Gray LLP Vymazal et al., Cell Mol Biol, 2000, 46: 835-842.* Rucker et al., Protein Engineering, 1997, 10:967-973.* (57) ABSTRACT Santambrogio et al., J Biol Chem, 1993, 268, 12744-12748.* In certain aspects the present invention provides methods and Ponka et al., Semin Hematol, 1998, 35:35-54, Abstract.* compositions related to contrast agents for magnetic reso Cozzi et al., J. Biol. Chem., 2000, 275: 25122-25129.* Database accession No. UNIPROT:PO2794 retrieved from UniProt nance imaging. In certain variations, contrast agents provided database; Ferritin heavy chain (created Jul. 21, 1986; last sequence herein are generated in situ via genetic instructions and update Aug. 13, 1987; last annotation update Sep. 13, 2005). become potent upon sequestering available metal atoms. Database accession No. EM-PRO: B.CO16354 retrieved from EMBL Exemplary contrast agents include metal-binding . database; Homo sapiens ferritin, light polypeptide (created Nov. 13, 2001; last updated Apr. 17, 2005). 13 Claims, 15 Drawing Sheets US 8,084.017 B2 Page 2

OTHER PUBLICATIONS Trinder, et al., “Transferrin receptor 2. a new molecule in iron metabolism'. The International Journal of Biochemistry & Cell Biol Hamlin, Nadia. “New Use for MRI. The Scientist 14(7):28 (2000). ogy 35 (2003) p. 292-296. Harrison, et al. “The : molecular properties, iron storage Weissleder, et al., “Molecular Imaging'. Molecular Imaging Radi fianction and cellular regulation'. Biochimica et Biophysica Acta ology, 2001, vol. 219, No. 2. pp. 316-333. Yamamuro, et al. "Structure of Self Assembled Fe and FeFt 1275 (1996) 161-203. Nanoparticle Arrays', Mat. Res. Soc. Symp. Proc. vol. 636. D10.8. Holbrook, et al. “In Vivo Observation of Cavitation and Embolism 1-D10.8.6. Medical Sciences. Repair Using Magnetic Resonance Imaging”. Plant Physiology, Zhou, et al., “HFE gene knockout produces mouse model of heredi vol. 126, pp. 27-31 (2001). tary hemochromatosis”. Proc. Natl. Acad. Sci. USA. vol. 95, pp. Guo, et al., “Expression and Loading of Recombinant Heavy and 2492-2497, 1998. Light Chain Homopolymers of Rat Liver Ferritin'. Archives of Bio Strausberg, R., Direct Submission, Database GeneEmbl. Acession chemistry and Biophysics vol. 335, No. 1, pp. 197-204 (1996). No. BC016009, Homo sapiens (human), Oct. 29, 2001, p. 1 bridging Jacobs, et al., “Looking deeper into vertebrate development, trends p. 2. in 9:73-76 (1999). BartZokis and Tishler, 2000, MRI Evaluation of Basal Ganglia Fer Jacobs, et al., “Towards a microMRI atlas of mouse development”. ritin Iron and Neurotoxicity in Alzheimer's and Huntington's Dis Computerized Medical Imaging and Graphics 23 (1999) 15-24. ease, Cell. Mol. Biol. 46:821-833. LaVaute, et al., “Targeted deletion of the gene encoding iron regula Cherry, 2004. In vivo molecular and genomic imaging: new chal tory protein-2 causes misregulation of iron metabolism and lenges for imaging physics, Phys. Med. Biol. 49:R13-R48. neurodegenerative disease in mice', nature genetices, 27:209-214 Fielden and Zacharewski, 2001, Challenges and limitations of gene (2001). expression profiling in mechanistic and predictive toxicology, Tox. Lois, et al. “Germline Transmission and Tissue-Specific Expression Sci. 60:6-10. of Transgenes Delivered by Lentiviral Vectors'. Science, 295:868 Goncalves, 2005. A concise peer into the background, initial thoughts 872 (2002). and practices of human gene therapy, BioEssays 27:506-517. Lok, Corie...'Medical imaging techniques are being adapted to study Lee et al., 2002, Active human ferritin HVL-hybrid and sequence gene expression and other cellular activities in living animals. Corie effection folding efficiency in Escherichia coli, Biochem. & Biophys. Loktalks to the pioneers who are watching cells at work in the natural ReS. Comm. 298:225-229. habitat”. Nature, 412:372-374 (2001). Makrides, 1999, Components of vectors for genetransfer and expres Louie, et al., “In vivo visualization of gene expression using magnetic sion in mammalian cells, Protein Exp. and Pur. 17:183-202. resonance imaging”. Nature Biotechnology, 18:321-325 (2000). Stic Sequence Search on SEQID No. 2 (pp. 1-3). Mathur-de, et al., “Invited review. Biophysical properties and clinical Koretsky, Nuclear Magnetic Resonance Detection of the Conse applications of magnetic resonance imaging contrast agents. The quences of Transgene Expression, News Physiol. Sci. 9:197-202 British Journal of Radiology, 1995, vol. 68, No. 807, pp. 225-247. (1994). Miklos, et al., “Integrating molecular medicine with functional Koretsky etal, Genetic Control of MRI Contrast by Expression of the proteomics. Realities and expectations”. Proteomics 2001. 1, 30-41. Transferrin Receptor, Int. Soc. Magn. Res. Med., New York, p. 5471 Moats, et al., “A 'Smart" Magnetic Resonance Imaging Agent That (1996). Reports on Specific Enzymatic Activity”. Angew. Chem. Int. Ed. Engl. Pereira et al., Rapid and parallel formation of Fe3+ multimers, 1997, 36, No. 7. pp. 726-728. including a trimer, during H-type subunit ferritin mineralization, Mulvey, et al., “Induction of Ferritin Synthesis in Cells Infected with Biochemistry 36:7917-7927 (1977). Mego Virus'. The Journal of Biological Chemistry, vol. 271. Boydet al., “Structural and functional relationships of human ferritin 16:9851-9857, (1996). H and L chains deduced from cDNA clones.' J. Biol. Chem. Narasimhan, et al., “Neuroanatomical Micromagnetic Resonance 260(21): 11755-11761 (1985). Imaging', Brain Mapping: The Methods, pp. 147-166, Academic Dhar et al., “Sequence of a cDNA encoding the ferritin H-chain from Press (1996). an 11-week-old human fetal brain.” Gene 126(2):275-278 (1993). Picard, et al., “Overexpression of the Ferritin HSubunit in Cultured Dorner et al., “Structure of human ferritin light subunit messenger Erythroid Cells Changes the Intracellular Iron Distributon”. Blood, RNA: comparison with heavy Subunit message and functional impli vol. 87, No. 5, 1996: pp. 2057-2064. cations.” PNAS, 82:3139-3143 (1985). Picard, et al., “Role of Ferritin in the Control of the Labile Iron Pool Kreiss et al., Plasmid DNA size does not affect the physiochemical in Murine Erythroleukemia Cells'. The Journal of Biological Chem properties of lipoplexes but modulates gene transfer efficiency, Nucl. istry, vol. 273, No. 25, pp. 15382-15386, 1998. Acids Res. 27(19):3792-3798 (1999). Rudin, Mark. “Target watching with a beady eye'. Nature McGregor et al., Rational Approaches to the Design of Cationic Biotechnology, vol. 18, p. 383 (2000). Gemini Surfactants for Gene Delivery, J. Amer. Chem. Soc. Sadun, et al., “First Application of Extremely High-resolution Mag 123(26):6215-6220 (2001). netic Resonance Imaging to Study Microscopic Features of Normal Vymazal et al., “Magnetic resonance imaging of brain iron in health and LHON Human Optic Nerve”. Ophthalmology vol. 109, No. 6, and disease.” J. Neurol. Sci. 134(Suppl.):19-26 (1995). 2002. pp. 1085-1091. Wong et al., Electrostatically Mediate Interactions between Cationic Scraba, et al., “The Mechanism of Ferritin Induction in Cells Infected Lipid-DNA Particles and an Anionic Surface, Arch. Biochem with Mengo Virus'. Protein Synthesis: Regulation (969–974). Streicher, et al., “3D modelling of gene expression patterns'. Biophy, 366(1):31-39 (1999). TRENDS in Biotechnology vol. 19, No. 4, (2001), p. 145-148. * cited by examiner U.S. Patent Dec. 27, 2011 Sheet 1 of 15 US 8,084,017 B2

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US 8,084,017 B2 1. 2 CONTRASTAGENTS FOR MAGNETIC cally inert. The exogenous contrast agents are used to high RESONANCE MAGING AND METHODS light anatomy with poor intrinsic contrast, as well as to visu RELATED THERETO alize various pathologies that disrupt normal vascular flow or cause a break in the blood-brain-barrier. None of these agents RELATED APPLICATIONS cross cellular membranes easily and therefore the existing technology is difficult to adapt for the analysis of intracellular This application claims the benefit of priority to U.S. Pro eVentS. visional Patent Application No. 60/363,163, filed on Mar. 7, A new generation of MRI contrast agents is required to 2002, which application is hereby incorporated by reference adapt this powerful imaging technology to the needs of in its entirety. 10 molecular medicine and biological research. INTRODUCTION SUMMARY OF THE INVENTION Tools that enable one to visualize gene expression in vivo In certain aspects, the invention relates to contrast agents are of fundamental importance to the future of medicine and 15 the biological Sciences. The emerging field of genetic medi for magnetic resonance imaging that are synthesized in a cine requires non-invasive imaging methods that can indicate Subject material as directed by a nucleic acid sequence. The where, when and if therapeutic have been delivered and contrast agents are made potent by sequestering available whether the desired protein has been expressed. In the realm metal atoms, typically iron atoms. In certain aspects, the of basic biological research, the ability to image the timing nucleic acid sequence encodes a metal binding protein that and location of gene expression in vivo is a fundamental need. acts, directly or indirectly, to impart a contrast effect on the Scientists typically monitor gene expression by incorpo cell in which it is produced. The invention further relates to rating a marker gene that is expressed along with the gene of methods of generating and employing the Subject contrast interest, often as either a transcriptional or translational agents. fusion. Detection of the marker gene products is most often 25 In certain embodiments, the invention relates to methods of achieved using histological preparations (e.g. using a B-ga generating an image of a Subject material by imaging a Sub lactosidase assay), or by using fluorescence microscopy (e.g. ject material comprising a plurality of cells wherein a Subset using green fluorescent protein, or GFP). Neither of these of the cells contain an MRI-detectable amount of contrast methods permit non-invasive imaging of tissues or other mac protein. In preferred embodiments, the amount of contrast roscopic assemblies of cells. Markers that require histological 30 protein present in different cells is distinguishable, and preparation cannot be detected without sacrificing the Subject optionally, cells comprising measurable amounts of contrast material. Fluorescent markers can be imaged in living cells, protein are distinguishable from cells or other components of but even with the most Sophisticated optical technologies the material that do not comprise the measurable amount of available, it is not possible to image at tissue depths exceeding contrast protein. approximately 500 um. Other methods such as PET (positron 35 In another embodiment, methods of the invention comprise emission tomography), gamma cameras, and SPECT (single detecting gene expression by imaging a cell comprising a photon emission computed tomography) have been used to recombinant nucleic acid encoding a contrast agent. Prefer detect gene expression in vivo, but all of these suffer from ably, detection of the contrast protein by magnetic resonance limited spatial resolution, which is on the order of cubic imaging indicates that the nucleic acid encoding the contrast millimeters or larger. 40 protein is and/or has been expressed. Optionally, the contrast MRI is a widely used clinical diagnostic tool that allows agent is a protein, preferably a metal-binding protein. Exem non-invasive imaging of optically opaque Subjects and pro plary classes of metal binding proteins include ferritin pro vides contrast among Soft tissues at high spatial resolution. In teins; transferrin receptor proteins; iron regulatory proteins; the majority of clinical applications, the MRI signal is derived and iron Scavenger proteins. Exemplary metal binding pro from protons of the water molecules present in the materials 45 teins of the invention include metal binding proteins that are being imaged. The image intensity of tissues is determined by at least 60%, optionally at least 70%, 80%, 90%, 95%,99% or a number of factors. The physical properties of a specific 100% identical to a sequence as shown in any of SEQID Nos: tissue, such as the proton density, spin lattice relaxation time 2, 4, 6, 8, 10, 12, and 14. Alternatively, the protein is at least (T1), and the spin-spin relaxation time (T2) often determine 60%, optionally at least 70%, 80%, 90%. 95%, 99% or 100% the amount of signal available. 50 identical to a sequence as shown in any of SEQID Nos: 16, A number of compositions termed “contrast agents' have 18, 20 or 22. been developed to provide enhanced contrast between differ Methods described herein may be used with essentially any ent tissues. Contrast agents commonly affect T1, T2 or both. material capable of generating the contrast agent in situ. For In general, contrast agents are made potent by incorporating example, the Subject material may be a cell, optionally a cell metals with unpaired dorf electrons. For example, T1 con 55 that is part of a cell culture, part of an in vitro tissue or part of trast agents often include a lanthanide metal ion, usually a multicellular organism, such as, for example, a fungus, a Gd", that is chelated to a low molecular-weight molecule in plant, or an animal. In preferred embodiments, the Subject order to limit toxicity. T2-agents often consist of Small par material is a living mammal such as a mouse or a human. ticles of magnetite (FeC) FeO) that are coated with dext In further aspects, the invention provides vectors for trans ran. Both types of agents interact with mobile water in tissue 60 fection of a multicellular organism comprising a recombinant to produce contrast; the details of this microscopic interaction nucleic acid encoding a contrast agent. In certain embodi differ depending on the agent type. ments, the contrast agent is a metal-binding protein. Option Most widely used contrast agents are exogenous, meaning ally, the vector is a viral vector derived from a virus selected that the contrast agent is produced externally and then deliv from the group: an adenovirus, an adenovirus-associated ered to the tissue or cells to be imaged. Exogenous contrast 65 virus, a herpes simplex virus, a retrovirus, an alphavirus, a agents are generally delivered through the vascular system, poxvirus, an arena virus, a vaccinia virus, an influenza virus, typically have a nonselective distribution, and are physiologi a polio virus and a hybrid of any of the foregoing. US 8,084,017 B2 3 4 In additional aspects, the invention includes delivery sys value of the control pellet, which is 1.5 mg/ml of ferritin; the tems for introducing nucleic acids of the invention into Sub experimental samples were incubated with various concen ject material. In certain embodiments, the invention provides tration of ferric ammonium citrate (FAC) and the control viral particles Suitable for transfecting a mammalian cell, samples were incubated in the absence of FAC. The error bars comprising a nucleic acid comprising a coding sequence for a represent the standard deviation for N=4 experimental runs. contrast agent, such as a contrast agent described above. Optionally, the viral particle is derived from one or more of FIG. 2. Data showing the percent of the total number cells the following: an adenovirus, an adenovirus-associated virus, remaining after the 16 hour period of ferritin loading. For aherpes simplex virus, a retrovirus, an alphavirus, a poxvirus, each FAC concentration (and control), cells before and after an arena virus, a vaccinia virus, an influenza virus and a polio 10 the incubation period were counted 3-times using a hemocy virus. In additional embodiments, the invention provides col tometer and the results were averaged. The error bars repre loidal Suspensions suitable for transfecting a mammalian cell sent the standard deviation for the separate (N=4) incubation comprising a nucleic acid comprising a coding sequence for a experiments. contrast agent, such as a contrast agent described above. FIG. 3. MRI image of three simulated tumor samples. Optional types of colloidal Suspensions include one or more 15 Here, (a) is the control and (b) and (c) are the samples con of the following: a macromolecule complex, a nanocapsule, a taining a ferritin increase of 2.7 and 4, respectively. Contrast microsphere, a bead, an oil-in-water emulsions, a micelle, a among these samples is readily apparent in this T2-weighted mixed micelle, and a liposomes. image. Images were acquired simultaneously using a Bruker In yet further aspects, the invention provides cells, cell cultures, organized cell cultures, tissues, organs and non 7-Tesla MRI system with TE/TR-45/2000 ms, 128x128 human organisms comprising a recombinant nucleic acid image points, and a 1 mm-thick slice. The pellet size was comprising a coding sequence for a contrast agent, such as a approximately 4 mm in diameter. contrast agent described above. In certain embodiments, the FIG. 4: MRI image through pelleted 9L glioma cells trans organism is selected from the group consisting of a mouse, a fected with contrast proteins light (LF) and heavy (HF) chain rat, a dog, a monkey, a pig, a fruit fly, a nematode worm and 25 ferritin. The sample on the left is the control (no DNA added a fish, or alternatively a plant or fungus. In further embodi during incubation). Image contrast is readily apparent ments, the cells, cell cultures, organized cell cultures, tissues, between the two pellets. Expression of the reporter turns cells organs and non-human organisms may comprise a vector as dark in the MR image. This image was acquired using an 11.7 described above. Tesla MRI system with a standard T-weighted 2DFT pulse The practice of the present invention may employ, unless 30 sequence. This image was acquired at 4°C. otherwise indicated, conventional techniques of cell biology, FIG. 5: MRI image through pelleted 9L cells infected with cell culture, , transgenic biology, microbi contrast proteins light (LF) and heavy (HF) chain ferritin via ology, recombinant DNA, and immunology, which are within an adenovirus. The sample on the left is the control (unin the skill of the art. Such techniques are explained fully in the fected cells). Image contrast is readily apparent between the literature. See, for example, Molecular Cloning A Laboratory 35 two pellets. (Note that the intense dark spots in the pellets are Manual, 2nd Ed., ed. by Sambrook, Fritsch and Maniatis bubble artifacts.) This image was acquired using an 11.7 Tesla (Cold Spring Harbor Laboratory Press: 1989); DNA Cloning, MRI system and a standard T-weighted 2DFT pulse Volumes I and II (D. N. Glover ed., 1985); Oligonucleotide sequence. This image was acquired at 4°C. Synthesis (M. J. Gait ed., 1984); Mullis et al. U.S. Pat. No. 4,683, 195; Nucleic Acid Hybridization (B. D. Hames & S.J. 40 FIG. 6: Human ferritin heavy chain cDNA sequence Higgins eds. 1984); Transcription And Translation (B. D. (BC016009) (SEQ ID NO:1). The coding region is under Hames & S. J. Higgins eds. 1984); Culture Of Animal Cells lined. (R. I. Freshney, Alan R. Liss, Inc., 1987); Immobilized Cells FIG. 7: Human ferritin light chain cDNA sequence (XM And Enzymes (IRL Press, 1986); B. Perbal, A Practical Guide 050469) (SEQ ID NO:3) The coding region is underlined. To Molecular Cloning (1984); the treatise, Methods. In Enzy 45 FIG. 8: Mus musculus ferritin heavy chain cDNA sequence mology (Academic Press, Inc., N.Y.); Gene Transfer Vectors (NM 010239.1) (SEQ ID NO:5). The coding region is For Mammalian Cells (J. H. Miller and M. P. Calos eds., underlined. 1987, Cold Spring Harbor Laboratory); Methods In Enzymol FIG.9: Mus musculus ferritin light chain 1 cDNA sequence ogy, Vols. 154 and 155 (Wu et al. eds.), Immunochemical (NM 010240.1) (SEQ ID NO:7). The coding region is Methods. In Cell And Molecular Biology (Mayer and Walker, 50 underlined. eds. Academic Press, London, 1987); Handbook Of Experi mental Immunology, Volumes I-IV (D. M. Weir and C. C. FIG. 10: Mus musculus ferritin light chain 2 cl DNA Blackwell, eds., 1986); Manipulating the Mouse Embryo, sequence (NM 008049.1) (SEQ ID NO:9). The coding (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, region is underlined. N.Y., 1986). 55 FIG. 11: Rattus norvegicus ferritin subunit H clNA Other features and advantages of the invention will be sequence (NM 012848.1) (SEQ ID NO:11). The coding apparent from the following detailed description, and from region is underlined. the claims. FIG. 12: Homo sapiens transferrin receptor cDNA The claims provided below are hereby incorporated into sequence (NM 003234) (SEQ ID NO:15). The coding this section by reference. 60 region is underlined. BRIEF DESCRIPTION OF THE DRAWINGS FIG. 13: Homo sapiens transferrin receptor 2 cl DNA sequence (NM 003227) (SEQ ID NO:17). The coding FIG. 1. Correlation between ferritin increase and 1/T (a) region is underlined. and 1/T (b) in simulated tumors. The solid line is least 65 FIG. 14: Mus musculus transferrin receptor 2 nucleic acid squares fit through the data (guide for the eye). The values are sequence (NM 015799) (SEQ ID NO:21). The coding normalized to give the ferritin increase over the meanbaseline region is underlined. US 8,084,017 B2 5 6 DETAILED DESCRIPTION OF THE INVENTION tions such as temperature, pH, oxidation state etc. that are readily controlled external to the site of transcription. 1. Definitions The term “Ferritin protein' is intended to include any of a "Coding sequence' is used herein to refer to the portion of group of diron-carboxylate proteins characterized by the a nucleic acid that encodes a particular protein. A coding 5 tendency to form a multimeric structure with bound iron and region may be interrupted by introns and other non-coding having a helix-bundle structure comprising an iron-coordi sequences that are ultimately removed prior to translation. nating Glu residue in a first helix and a Glu-X-X-His motif in “Colloidal suspension' is used herein to refer to a colloidal a second. Certain ferritins maintain bound iron in a primarily Suspension that comprises one or more nucleic acids for Fe(III) state. Bacterioferritins tend to be haem proteins. Ver 10 tebrate ferritins tend to be assembled from two or more sub delivery to cells. The material in a colloidal suspension is units, and mammalian ferritins are often assembled from a generally designed so as to protect nucleic acids and facilitate heavy chain and a light chain. Many ferritins form hollow the delivery of nucleic acids across cell membranes. Exem structures with an iron-rich aggregate in the interior. Exem plary colloidal Suspensions include, but are not limited to, plary ferritins are presented in Table 1 below. lipid micelles, tubes, rafts, sandwiches and other lipid struc 15 “Homology” or “identity” or “similarity” refers to tures, often comprising cationic lipids. Other colloidal Sus sequence similarity between two polypeptides or between pensions include nanocapsules, microbeads and Small, two nucleic acid molecules. Homology and identity can each nucleic acid-binding polymeric structures, etc. be determined by comparing a position in each sequence The term “contrast agent' is used herein to refer to a mol which may be aligned for purposes of comparison. When an ecule that generates a contrasting effect in Vivo, whether the equivalent position in the compared sequences is occupied by effect is director indirector both. In exemplary embodiments, the same base or amino acid, then the molecules are identical “contrast agent' is used interchangeably with “contrast pro at that position; when the equivalent site occupied by the same tein' or “contrast polypeptide.” In the case of a direct effector, or a similar amino acid residue (e.g., similar in steric and/or the contrast protein will typically form a complex that affects electronic nature), then the molecules can be referred to as the relaxation times T1, T2 or T2*. Often direct contrast 25 homologous (similar) at that position. Expression as a per proteins form metalloprotein complexes. Exemplary catego centage of homology/similarity oridentity refers to a function ries of contrast proteins include, for example, metal binding of the number of identical or similar amino acids at positions proteins and/or agents that stimulate production of one or shared by the compared sequences. A sequence which is more metal-binding protein, etc. Indirect effectors include “unrelated' or “non-homologous' shares less than 40% iden molecules that cause a cell to produce a direct contrast protein 30 tity, though preferably less than 25% identity with a sequence and/or modulate a functional, biochemical, and/or biophysi of the present invention. cal characteristic of a direct contrast protein, thereby creating The term “homology describes a mathematically based a contrast effect. Exemplary categories of indirect effectors comparison of sequence similarities which is used to identify include, for example, proteins and/or nucleic acids that affect genes or proteins with similar functions or motifs. The expression of a direct contrast protein, modulate the activity 35 nucleic acid and protein sequences of the present invention of a direct contrast protein, modulate metal binding to a may be used as a “query sequence' to perform a search metal-binding protein, modulate expression of an iron regu against public databases to, for example, identify other family latory protein, and/or modulate the activity of an iron regula members, related sequences or homologs. Such searches can tory protein, etc. be performed using the NBLAST and XBLAST programs The term “contrast effect”, as used herein with respect to 40 (version 2.0) of Altschul, et al. (1990).J Mol. Biol. 215:403 MRI, includes any alteration in the MRI signal that renders 10. BLAST nucleotide searches can be performed with the one cell or tissue detectably different from another. A contrast NBLAST program, score=100, wordlength=12 to obtain effect may involve effects on T1, T2 and/or T2*. In MRI, a nucleotide sequences homologous to nucleic acid molecules Subject containing mobile water is generally placed in a large of the invention. BLAST protein searches can be performed static magnetic field. The field tends to align some of the 45 with the XBLAST program, score=50, wordlength=3 to magnetic moments (spins) of the hydrogen nuclei in the water obtain amino acid sequences homologous to protein mol along the field direction. The spin lattice relaxation time (T1) ecules of the invention. To obtain gapped alignments for is the time constant for a population of nuclei placed in a comparison purposes, Gapped BLAST can be utilized as magnetic field to equilibrate along the magnetic field direc described in Altschulet al., (1997)Nucleic Acids Res. 25(17): tion. T1 is the time constant for the transfer of energy from the 50 3389-3402. When utilizing BLAST and Gapped BLAST pro spin system to the environment (the lattice). The spin-spin grams, the default parameters of the respective programs relaxation time (T2) is the time constant for nuclei precessing (e.g., XBLAST and BLAST) can be used. See http://www. at the Larmor frequency to remain in phase with each other. incbi.nlm.nih.gov. Alternatively. T2 is called the spin-phase memory time. This As used herein, “identity” means the percentage of identi loss of phase coherence is attributed to low-frequency fluc 55 cal nucleotide or amino acid residues at corresponding posi tuations of the magnetic field that are commonly due to inter tions in two or more sequences when the sequences are actions among spins. The relaxation time T2 is defined as aligned to maximize sequence matching, i.e., taking into 1/T2*=1/T2+YAB, where Y is the nuclear gyromagnetic ratio accountgaps and insertions. Identity can be readily calculated and AB is the static external magnetic field inhomogeneity. by known methods, including but not limited to those The terms “contrast gene' or “contrast nucleic acid are 60 described in (Computational Molecular Biology, Lesk, A.M., used interchangeably herein to refer to a nucleic acid com ed., Oxford University Press, New York, 1988: Biocomput prising a coding sequence for a contrast protein. ing: Informatics and Genome Projects, Smith, D. W., ed., An “externally regulated promoter is a nucleic acid that Academic Press, New York, 1993: Computer Analysis of affects transcription in response to conditions that may be Sequence Data, Part I, Griffin, A.M., and Griffin, H. G., eds., provided in a controlled manner by one of skill in the art. 65 Humana Press, New Jersey, 1994; Sequence Analysis in Externally regulated promoters may be regulated by specific Molecular Biology, von Heinje, G., Academic Press, 1987: chemicals, such as tetracycline or IPTG, or by other condi and Sequence Analysis Primer, Gribskov, M. and Devereux, US 8,084,017 B2 7 8 J., eds., M. Stockton Press, New York, 1991; and Carillo, H., in one embodiment, the helper plasmid encodes the AAV and Lipman, D., SIAM J. Applied Math., 48: 1073 (1988). genome, with the exception of the AAV ITR regions, which Methods to determine identity are designed to give the largest are replaced with adenovirus ITR sequences. This permits match between the sequences tested. Moreover, methods to replication and encapsidation of the AAV replication defec determine identity are codified in publicly available computer 5 tive recombinant vector, while preventing generation of wild programs. Computer program methods to determine identity type AAV virus, e.g., by recombination. between two sequences include, but are not limited to, the A “regulatory nucleic acid' or “regulatory sequence' GCG program package (Devereux, J., et al., Nucleic Acids includes any nucleic acid that can exert an effect on the Research 12(1): 387 (1984)), BLASTP, BLASTN, and transcription of an operably linked open reading frame. A FASTA (Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 10 regulatory nucleic acid may be a core promoter, an enhancer (1990) and Altschul et al. Nuc. Acids Res. 25: 3389-3402 or repressor element, a complete transcriptional regulatory (1997)). The BLAST X program is publicly available from region or a functional portion of any of the preceding. Mutant NCBI and other sources (BLAST Manual, Altschul, S., et al., versions of the preceding may also be considered regulatory NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., et al., J. nucleic acids. Mol. Biol. 215: 403-410 (1990). 15 The term “iron binding protein’ as used herein is intended A “transcriptional fusion' is a nucleic acid construct that to include proteins that bind to iron under physiologically causes the expression of an mRNA comprising at least two relevant conditions. Certain iron binding proteins interact coding regions. In other words, two or more open reading with iron through a cofactor Such as heme. Many other exem frames may be organized into a transcriptional fusion Such plary cofactors are also described herein. Other iron binding that both open reading frames will be expressed as part of a proteins form an iron binding site with the appropriate amino single mRNA and then give rise, as specified by the host cell, acids, including but not limited to, histidine, aspartate, to separate polypeptides. The open reading frames in a tran glutamate, asparagine and glutamine. Although iron binding Scriptional fusion tend to be subject to the same transcrip proteins of the invention bind iron, they are also likely to bind tional regulation, but the encoded polypeptides may be Sub to other metals. Accordingly, “iron binding protein’ as used 25 ject to distinct post-translational fates (eg. degradation, etc.). herein is not meant to indicate that the protein binds iron A “transcriptional fusion may be contrasted with a “transla exclusively, or even that the protein binds iron more tightly tional fusion' in which two or more open reading frames are than other metals. connected so as to give rise to a single polypeptide. The fused An “iron regulatory protein’ refers to a protein that is polypeptides in a “translational fusion' tend to experience involved in iron utilization, processing, and/or accumulation 30 similar transcriptional, translational and post-translational in a cell. Iron regulatory proteins include, for example, pro regulation. teins that regulate iron homeostasis, proteins that regulate As used herein, the term “transfection' means the intro iron trafficking into or out of a cell, proteins involved in duction of a nucleic acid, e.g., an expression vector, into a regulating the production of iron related elements, such as, for recipient cell, and is intended to include commonly used example, ferritin and transferring, etc. Iron regulatory pro 35 terms such as “infect” with respect to a virus or viral vector. teins may or may not bind iron directly. The term “transduction' is generally used herein when the As used herein, the term “nucleic acid refers to polynucle transfection with a nucleic acid is by viral delivery of the otides such as deoxyribonucleic acid (DNA), and, where nucleic acid. “Transformation', as used herein, refers to a appropriate, ribonucleic acid (RNA). The term should also be process in which a cell's genotype is changed as a result of the understood to include analogs of either RNA or DNA made 40 cellular uptake of exogenous DNA or RNA, and, for example, from nucleotide analogs (including analogs with respect to the transformed cell expresses a recombinant form of a the base and/or the backbone, for example, peptide nucleic polypeptide or, in the case of anti-sense expression from the acids, locked nucleic acids, mannitol nucleic acids etc.), and, transferred gene, the expression of a naturally-occurring form as applicable to the embodiment being described, single of the recombinant protein is disrupted. Stranded (Such as sense or antisense), double-stranded or 45 As used herein, the term “transgene' refers to a nucleic higher order polynucleotides. acid sequence which has been introduced into a cell. Daugh The term “operably linked' is used herein to refer to the ter cells deriving from a cell in which a transgene has been relationship between a regulatory sequence and a gene. If the introduced are also said to contain the transgene (unless it has regulatory sequence is positioned relative to the gene Such been deleted). A transgene can encode, e.g., a polypeptide, that the regulatory sequence is able to exert a measurable 50 partly or entirely heterologous, i.e., foreign, to the transgenic effect on the amount of gene product produced, then the animal or cell into which it is introduced. Optionally, a trans regulatory sequence is operably linked to the gene. gene-encoded polypeptide may be homologous to an endog A "polylinker is a nucleic acid comprising at least two, enous gene of the transgenic animal or cell into which it is and preferably three, four or more restriction sites for cleav introduced, but may be designed to be inserted, or is inserted, age by one or more restriction enzymes. The restriction sites 55 into the genome in Such a way as to alter the genome of the may be overlapping. Each restriction sites is preferably five, cell into which it is inserted (e.g., it is inserted at a location six, seven, eight or more nucleotides in length. which differs from that of the natural gene). Alternatively, a A “recombinant helper nucleic acid' or more simply transgene can also be present in an episome. A transgene can “helper nucleic acid' is a nucleic acid which encodes func include one or more transcriptional regulatory sequences and tional components that allow a second nucleic acid to be 60 any other nucleic acid, (e.g. intron), that may be necessary for encapsidated in a capsid. Typically, in the context of the optimal expression of a selected coding sequence. A trans present invention, the helper plasmid, or other nucleic acid, gene may also contain no polypeptide coding region, but in encodes viral functions and structural proteins which allow a Such cases will generally direct expression of a functionally recombinant viral vector to be encapsidated into a capsid. In active RNA, such as an rRNA, tRNA, ribozyme, etc. A “thera one preferred embodiment, a recombinant adeno-associated 65 peutic transgene' is a transgene that is introduced into a cell, virus (AAV) helper nucleic acid is a plasmid encoding AAV tissue and/or organism for the purpose of altering a biological polypeptides, and lacking the AAVITR regions. For example, function in a manner that is beneficial to a subject. US 8,084,017 B2 10 “Transient transfection” refers to cases where exogenous latory sequence (or is operably linked to a weak promoter). nucleic acid is retained for a relatively short period of time, This type of “promoterless’ construct may be used to identify often when the nucleic acid does not integrate into the endogenous sequences that Supply regulatory activity in a genome of a transfected cell, e.g., where episomal DNA is manner analogous to an "enhancer trap'. transcribed into mRNA and translated into protein. A cell has In certain exemplary embodiments, methods and compo been “stably transfected with a nucleic acid construct com sitions of the invention are used to monitor the expression of prising viral coding regions when the nucleic acid construct a transgene of interest, Such as a therapeutic transgene. Sub has been introduced inside the cell membrane and the viral ject material is contacted with both a transgene of interest, coding regions are capable of being inherited by daughter Such as a therapeutic transgene, and a nucleic acid construct cells. 10 comprising the coding sequence for a contrast protein that is “Viral particle' is an assemblage of at least one nucleic acid operably linked to a regulatory sequence. In one variation, and a coat comprising at least one viral protein. In general, production of the transgene of interest and production of the viral particles for use in delivering nucleic acids to cells will contrast protein are both modulated by functionally similar retain the ability to insert the nucleic acid into a cell, but may (optionally identical) regulatory sequences. For example, if be defective for many other functions, such as self-replica 15 Subject material has been contacted with a transgene under tion. direction of a strong constitutive promoter, Such as certain 2. Exemplary Methods viral terminal repeat promoters, then expression of the gene In some aspects, the invention relates to methods for per encoding the contrast protein should also be under direction forming MRI using an intracellular contrast agent that is of the same promoter or a promoter designed to have a similar generated in situ via genetic instructions and made potent by expression pattern. In some variations, the transgene of inter the sequestering of metal atoms. The sequestered metal atoms estis introduced first, and then at a later time the nucleic acid are preferably endogenous metal atoms Such as, for example, encoding the contrast protein is introduced. In other varia ironatoms. In certain embodiments, methods of the invention tions, the nucleic acid encoding the contrast protein is intro comprise contacting Subject material with a nucleic acid duced at the same time as the transgene of interest, and encoding instructions for the synthesis of an intracellular 25 optionally the contrast nucleic acid and the transgene of inter contrast agent, such as a metal binding protein. In Such an est are located on the same vector. In certain embodiments, embodiment, upon internalization by an appropriate cell, the the contrast nucleic acid is expressed as a transcriptional nucleic acid directs production of the metal binding protein fusion with the transgene of interest. In further embodiments, which becomes potent as a contrast agent by binding to avail the contrast gene and the transgene of interest (or a second able metal atoms. In another embodiment, the methods of the 30 copy thereof) may be expressed as a fusion protein. The invention comprise contacting Subject material with a protein fusion protein approach may be desirable where it is thought or nucleic acid that indirectly affects contrast, for example, by that the effectiveness of the therapeutic transgene is influ increasing the amount of metal in the cell or by affecting the enced by post-transcriptional regulation. Subject material expression and/or activity of a metal binding protein. Intrac may be imaged by MRI, and cells having the contrast protein ellular contrast agents described herein may be employed in 35 may be detected and distinguished from cells that do not have the imaging of essentially any biological material that is the contrast protein. In preferred embodiments, the level of capable of producing Such an agent, including but not limited contrast detected by MRI will correlate with, or be indicative to: cultured cells, tissues, and living organisms ranging from of the level of expression of the transgene of interest. unicellular organisms to multicellular organisms (e.g. In further exemplary embodiments, methods and compo humans, non-human mammals, other vertebrates, higher 40 sitions of the invention may be used to investigate the in situ plants, insects, nematodes, fungi etc.). While most biological regulatory activity of a regulatory sequence of interest. Sub systems contain a variety of metals that have potent contrast ject material is contacted with a nucleic acid encoding a effects, it is understood that iron is generally the only Such contrast protein, where the nucleic acid is operably linked to metal that is sufficiently concentrated to be useful in render the regulatory sequence of interest. Once internalized within ing an intracellular contrast agent potent. However, if desired, 45 an appropriate cell, the contrast gene is expressed at a level material to be imaged may be Supplemented with exogenous that is regulated by the regulatory sequence of interest. In metal atoms, and such protocols will preferably be optimized preferred embodiments, the level of contrast detected by MRI to reduce deleterious effects caused by the exogenous metal will be correlated with the level of activity of the regulatory atOmS. sequence of interest. The regulatory sequence of interest may In certain embodiments, the novel contrast technology 50 be essentially any regulatory sequence, including but not described herein may be employed to investigate the regula limited to a promoter, an enhancer, an entire promoter/en tion of gene expression in situ. For example, a nucleic acid hancer region, a mutated or altered form of the preceding, or encoding a contrast protein may be introduced into a cell, one or more portions of the preceding. tissue, and/or Subject of interest. Those cells having appro In further exemplary embodiments, the methods described priate intracellular conditions for expression of the contrast 55 herein may be used to determine whether a physiologically protein may be distinguished by MRI from cells that do not important regulatory sequence is active in situ. For example, produce the contrast protein. In certain embodiments, the the p53 protein is a widely recognized regulator of cell pro nucleic acid encoding the contrast protein is operably linked liferation and apoptosis that exerts its regulatory influences to a constitutively active regulatory sequence. In further partly in response to DNA damage. Therefore, a construct embodiments, the contrast protein is operably linked to a 60 comprising a p53-responsive regulatory sequence operably regulatory sequence so that production of the contrast protein linked to a nucleic acid encoding a contrast protein would may be regulated by application of one or more exogenously permit detection of cells, in situ, in which the p53 regulatory controlled conditions, such as temperature changes, concen pathway has been activated. Similarly, methods of the inven tration of an inducer or repressor, etc. In yet another embodi tion may be employed to investigate, for example, the status ment, the activity of the regulatory sequence is at least par 65 of pro-proliferative signaling pathways (e.g. to identify can tially unknown. In a further embodiment, the nucleic acid cerous or pre-cancerous cells), or to assess the status of encoding a contrast protein is not operably linked to a regu inflammatory pathways (e.g. in host and/or donor tissues in or US 8,084,017 B2 11 12 near transplanted organs), or to non-invasively image pro ing the Subject material with a composition that elicits pro moter activation during the course of development, etc. In duction of the contrast agent. For example, cells may be view of this disclosure, one of skill in the art will be able contacted with an agent, such as an iron Source, that causes develop myriad related methods. cells to produce ferritin, which is an effective contrast agent. An analogy may be drawn between the traditional reporter Accordingly, it is understood that the invention encompasses gene assays routinely performed by biologists, such as assays agents that are not direct contrast agents and may be neither employing B-galactosidase (B-Gal) or green fluorescent pro nucleic acid nor protein but which nonetheless are useful for tein (GFP), and certain embodiments of the present invention. inducing in situ production of an intracellular contrast agent. Accordingly, certain methods of the invention may be used as In certain aspects, nucleic acids of the invention may be an alternative for other commonly used cell-screening meth 10 introduced into biological material by using any of a variety ods. For example, a method for assessing candidate pharma of vectors, whether general or organism/tissue/cell-type spe ceuticals may traditionally involve contacting the candidate cific, and in combination with any of a variety of delivery pharmaceutical with a cell carrying an informative reporter systems, such as for example, liposomes, viral particles, elec gene construct. Now, the standard reporter gene may be troporation, etc. In additional aspects, proteins of the inven replaced with a contrast gene, and the standard detection 15 tion may also be administered directly to cells in a variety of system may be replaced with an MRI system. While certain ways, such as liposome fusion, electroporation, attachment to embodiments of the present invention may be used to substi a moiety that is internalized by cells, etc. tute for traditional reporter gene assays, these traditional In certain embodiments where a nucleic acid encoding a assays are far more limited in their utility. For example, tra contrast protein is introduced into cells, it may be desirable to ditional assays use optically-based readout technologies that have that gene active or present in the cells for only a short are ineffective in visualizing gene expression deep within period of time, or optionally for a regulated period of time. If intact tissue, and often require histological processing of the desired, a transient transfection system may be used, and biological materials. By contrast, certain embodiments of the preferably a vector that permits expression for, on average, present invention employ an MRI contrast agent as a reporter fewer than one or two days. Alternatively, or in conjunction, gene, allowing signal readout deep within optically opaque 25 gene expression may be controlled by using an externally tissues by MRI and, if desired, readouts may be obtained with regulated promoter, or as a further example, the contrast gene little or no disruption of the biological function of the subject or a portion thereof may be situated with respect to one or material. more recombination sites Such that activation of a recombi In yet another exemplary embodiment, methods and com nase causes inactivation (or, if preferred, activation) of the positions of the invention may be used to assess the distribu 30 nucleic acid encoding the contrast protein. tion of a vector that has been administered to subject material. Many embodiments of the invention involve the use of For example, a vector designed to transfect an organism may nucleic acids encoding multiple contrast proteins, such as, for include a nucleic acid encoding a contrast protein operably example, nucleic acids encoding heavy and light chains of a linked to a suitable promoter. Optionally, a promoter will be mammalian ferritin, or nucleic acids encoding a ferritin and a selected to provide detectable levels of expression in a wide 35 transferrin receptor. range of tissue types. For example, a strong constitutive pro In certain embodiments, the intracellular contrast agent moter might be selected. The transfected biological material will be chosen for safety in the subject material, and where the is imaged by MRI to identify cells that have been transfected Subject is a human Subject, the intracellular contrast agent is with the vector. This exemplary method may be coupled with preferably safe for use in humans. numerous different methods of administering the vector (e.g. 40 3. Contrast Agents introduction into an anatomical region or organ of particular In many aspects, as described above, methods of the inven interest, introduction into the circulatory system, the lymph tion will employ one or more contrast proteins that generate system, etc.), and may be used to compare vector distribution MRI contrast in vivo. The contrast protein will impart MRI and transcription levels obtained with each of these contrast directly, or indirectly, by causing the cell to produce approaches. In the case of delivery systems that are targeted to 45 a secondary protein(s) that imparts MRI contrast. In the case a particular tissue, the exemplary methodology may be used of the direct effector, the contrast protein will typically form to confirm or optimize tissue specificity. As another illustra a complex that creates a change in at least one of relaxation tion, the present methods may be employed to optimize or times T1, T2, and/or T2*, where the change leads to a contrast develop a genetherapy protocol by allowing an investigator to effect during MRI. Often direct contrast proteins form met determine the location and optionally the level of gene 50 alloprotein complexes. In the case of indirect effectors, the expression obtained after administration of a particular gene contrast agent may be, for example, a protein or nucleic acid therapy system. that regulates iron homeostasis, regulates expression of an Many embodiments of the invention pertain to the genera endogenous gene coding for a direct contrast agent, and/or tion of an artificially induced intracellular contrast agent. In regulates the activity of an endogenous protein that may act as many of the preceding embodiments, production of the intra 55 a direct contrast agent, thereby producing a contrast effect. In cellular contrast agent is achieved by introducing a nucleic certain embodiments, the methods described herein may acid encoding a direct contrast protein. Generally, production involve both direct and indirect contrast agents. In an exem of the contrast agent may be achieved by alternative methods. plary embodiment, the methods and/or compositions For example, in situ production of an intracellular contrast described herein comprises an indirect contrast agent that agent may be stimulated by introducing a nucleic acid encod 60 affects iron homeostasis and a direct contrast agent, Such as a ing an indirect contrast agent. An indirect contrast agent may metal binding protein. be, for example, a protein or nucleic acid that regulates iron In aspects of the invention employing a metal-binding homeostasis, regulates expression of an endogenous gene polypeptide as a direct contrast agent, the metal-binding pro coding for a direct contrastagent, and/or regulates the activity tein will preferably bind to one or more metals that provide of an endogenous protein that may act as a direct contrast 65 effective contrasting. A variety of metals are effective as agent, such as, for example, ferritin. As another example, elements of a contrasting agent, particularly those with production of the contrast agent may be provoked by contact unpaired electrons in the dor forbitals. Such as, for example, US 8,084,017 B2 13 14 iron (Fe), cobalt (Co), manganese (Mn), nickel (Ni), gado structure with bound iron and having a helix-bundle structure linium (Gd), etc. As noted above, iron is of particular interest comprising an iron-coordinating Glu residue in a first helix because it is present at relatively high levels in mammals and and a Glu-X-X-His motif in a second. Certain ferritins main most other organisms, and therefore, detectable accumula tain bound iron in a primarily Fe(III) form. A list of exemplary tions of iron may be generated without the aid of exogenous ferritins is provided in Table 1. This list is intended to provide iron Supplementation. Accordingly, preferred metal-binding examples and is not intended to be comprehensive. Many proteins of the invention are iron-binding proteins. In those known ferritins are not included, and it is understood that embodiments employing a T2 contrast agent, the geometry of most vertebrate species will have a form, offerritin that can be metal binding is not important, but the contrast will tend to be used as a contrast agent. In view of this specification, one of greater when larger amounts of metal are concentrated 10 ordinary skill in the art will be able to identify additional together. In certain preferred embodiments, the effective ferritin homologs. In certain embodiments, a ferritin for use metal should be bound into a metal-rich aggregate, optionally as a contrasting agent should have at least 50% identity with a crystal-like aggregate, greater than 10 picometers in diam the amino acid sequence of SEQ ID NO:2 and/or SEQ ID eter, optionally greater than 100 picometers, greater than 1 NO:4, and optionally at least 60%, 70%, 80%, 90%. 95%, nanometer, greater than 10 nanometers or greater than 100 15 98%, 99% or 100% identity with the amino acid sequence of nanometers in diameter. Alternatively the metal-rich aggre SEQID NO:2 and/or SEQID NO:4. gate should be in the range of 1-100 nanometers in diameter In many embodiments, methodologies of the invention will within the polypeptide complex. In a particularly preferred employ a vertebrate ferritin as a contrast agent. Vertebrate embodiment, the metal-rich aggregate exhibits properties of ferritins typically form a large complex that assembles in a Superparamagnetism. When an iron-binding polypeptide is shell to delimit a cavity where iron is accumulated in a min used, it is preferable if the polypeptide retains the iron in the eral and compact form. Most mammalian ferritins are com nontoxic Fe(III) oxidation state. Fe(II) is also an effective posed of two subunit types, the H- and L-chains. Typically the contrasting agent, but Fe(II) may participate in the iron-cata endogenous mRNAs for the two chains have nearly identical lyzed HaberWeiss reaction that yields potentially damaging iron-responsive elements (IREs) close to the 5' termini that hydroxyl radicals. 25 regulate ferritin translation by binding to iron-regulatory pro In a preferred embodiment, a direct contrast protein of the teins (IRPs). When designing nucleic acid constructs for the invention has the following properties: rapid intracellular ectopic expression of ferritins, it will often be desirable to protein assembly and metal loading, the tendency to promote omit or otherwise disrupt the IRE sequences. Contacting formation of a metal-rich aggregate that has a large paramag cultured cells with an elevated iron concentration typically netic susceptibility, and the ability to retain the metal in a 30 causes a strong up-regulation of both the L- and H-chains, relatively non-toxic form (e.g. in the case of iron, the Fe(III) whereas treatment with iron chelating agents. Such as desfer state). rioxamine, suppresses ferritin production. Preferred ferritins In certain aspects, metal-binding polypeptides may also of the invention catalyze both an iron oxidation step from the change the contrast properties of a cell by perturbing metal Fe(II) form to the Fe(III) form and also catalyze the nucle metabolism and stimulating the expression of endogenous 35 ation and growth of an iron mineral core. In the case of metal-binding polypeptides that have contrast effects. This ferritins composed of multiple subunits, it will typically be may also lead to an accumulation or depletion of a particular desirable to express all Subunits at a stoichiometry approxi metal in the cell. For example, transient expression of high mating that found in the native complexes. However, it is affinity iron-binding proteins may create a temporary notable that a wide range of subunit ratios will typically be decrease in the intracellular labile iron pool and stimulate 40 effective. For example, human H chain is capable of forming production of transferrin receptor, thereby increasing the net a homopolymer that binds iron. Excess ferritin resulting from iron uptake into the cell. overexpression is typically degraded inside the cell, and the Although the exact binding affinity of a metal-binding primary decay product is hemosiderin deposits; these are also protein for different metals is not critical, it is generally effective as contrast agents. expected that polypeptides with a sub-nanomolar affinity for 45 one or more effective metals may be useful, and optionally the TABLE 1 polypeptide will have a dissociation constant less than 10 M, 10' M, or less for one or more effective metals. It is Exemplary Ferritin Proteins and Nucleic Acids understood that many metal binding proteins will bind to Amino Acid Nucleic Acid more than one type of metal. For example, lactoferrin will 50 Sequence Sequence form complexes with metals such as manganese and zinc. Name (Acc. No.) (Acc. No.) Ferritin-iron complexes are generally expected to contain erritin, heavy polypeptide 1 AAH16009.1 BCO16009.1 Some Small (perhaps infinitesimal) amounts of other metals. Homo sapiens In general, iron binding proteins are likely to bind to metals erritin, light polypeptide XP 050469.1 XM 050469.1 Such as manganese, cobalt, Zinc and chromium, although in 55 Homo sapiens Vivo the concentration and abundance of iron is so much erritin heavy chain Mus NP 034369.1 NM 010239.1 miscuits higher than these other metals that an iron binding protein will erritin light chain 1 Mus NP 034370.1 NM 01024.0.1 be primarily associated with iron. miscuits Several exemplary metal-binding polypeptides of the erritin light chain 2 Mus NP 032075.1 NM 008049.1 miscuits invention are provided. This is in no way intended to be an 60 erritin subunit H Rattus NP 036980.1 NM O12848.1 exhaustive list, and, in view of the teachings herein, one of norvegicus skill in the art will be able to identify or design other useful erritin light chain 1 Rattus NP 071945.1 NM O22500.1 metal-binding polypeptides. norvegicus erritin heavy chain Cavia BAB7O615.1 ABO73371.1 In certain exemplary embodiments, one or more ferritins porcellus may be used as a contrast protein. Ferritins of the invention 65 erritin light chain Cavia AAF36408.1 AF233445 1 include any of the group of diiron-carboxylate proteins char porcellus acterized by the tendency to form a dimeric or multimeric US 8,084,017 B2 15 16 TABLE 1-continued are encoded by mRNAs that include one or more stem-loop motifs, termed an Iron Responsive Element (IRE). Under low Exemplary Ferritin Proteins and Nucleic Acids iron conditions, IRPs bind to IREs and modulate the stability Amino Acid Nucleic Acid or translation of the affected mRNA. In general, when an IRE Sequence Sequence is positioned in the 5' UTR region of an mRNA (e.g. the Name (Acc. No.) (Acc. No.) ferritins), the IRP blocks translation, causing decreased pro erritin heavy chain rabbit P25915 tein production in low iron conditions. When an IRE is posi erritin light chain rabbit SO1239 tioned in the 3' UTR (e.g. transferrin receptor), the IRP typi erritin H subunit Bos BAA248.18.1 ABOO3093.1 cally stabilizes the mRNA, thereby increasing production of taurus 10 the gene product in response to low iron conditions. Mice erritin L subunit Bos BAA248.19.1 ABOO3094.1 taurus having a targeted deletion of the gene encoding IRP2 show erritin heavy chain Gallus A26886 significant accumulations of iron in neural tissues (LaVaute et gallus al., 2001, Nat. Genet. 27(2):209-14). Accordingly, manipula erritin Canis familiaris AAK82992.1 AF285177.1 tion of IRPs by, for example, antisense or RNAi methodolo erritin H chain Macaca AAF98711.1 AF162481. 1 15 mitiatta gies may provide contrast effects. IRPs of the invention will erritin heavy chain FRXL typically have the ability to bind to IREs in an iron-regulated Xenopus laevis manner. Preferred IRPS of the invention will be vertebrate erritin heavy chain Danio AAG378.37.1 AF295373 1 IRPs such as: human IRP1 (Ace. Nos. P21399 and Z11559), rerio yolk ferritin Paragonimus AAG17056.1 AF18872O 1 human IRP2 (Ace. Nos. AAA69901 and M58511), rat IRE westernani BP1 (Ace. Nos. Q63270 and L23874), mouse IRE-BP1 (Ace. erritin Taenia Saginata CAA6SO97.1 Nos. P28271 and X61147), chicken IRE-BP (Ace. No. 26 kDa ferritin subunit AAG41120.1 AF14234.0.1 Q90875 and D16150), etc. In general, it will be desirable to Galleria mellonella nonheme iron-containing NP 207447.1 NC OOO915.1 employ an IRP that binds to the IREs of the subject biological erritin (pfr) Helicobacter material, and in certain embodiments, this may be accom pylori 26695 25 plished by using an IRP that is derived from the subject erritin Glycine max AALO9920.1 AYO49920.1 species. In certain aspects of the invention, a contrast protein comprises an amino acid sequence at least 60% identical to In a further embodiment, a metal binding protein of the that of human IRP1 and/or IRP2, and optionally at least 70%, invention is a metal scavenger, defined as a protein that binds 80%, 90%, 95%, 98%, 99% or 100% identical. metal with very high affinity through a siderophore. Such 30 In further aspects, a contrast protein of the invention may be one that perturbs cellular iron homeostasis. For example, a proteins may be used as contrast agents. While not wishing to transferrin receptor protein, and/or a molecule that regulates be bound to a mechanism, it is expected that Such proteins will the expression and/or function of a transferrin receptor pro act primarily as indirect contrast agents. For example, iron tein, may be used as a contrast agent. Transferrin receptor Scavenging proteins expressed in a cell may scavenge and 35 mediates the receptor mediated endocytosis of the iron-car tightly bind iron from the labile iron pool within the intrac rying protein transferrin and thereby mediates cellular iron ellular space. Thus MRI contrast may be enhanced by a com uptake. Therefore, in one embodiment of the invention, the bination of the iron-bound chelate itself and the additional level and/or activity of a transferrin receptor in targeted cells iron that is sequestered and stored as a result of the cell's own may be modulated so as to produce an increase in cellular iron iron regulation mechanisms. Exemplary siderophores that 40 uptake thereby causing the cell to produce ferritin. The end may be present in metal scavenging proteins include hemo result will be an accumulation of excess ferritin that will yield globin, and any other agent that provides an octahederal coor MRI contrast. Exemplary transferrin receptors include SEQ dination sphere for the iron, usually formed-by six oxygen ID Nos: 16, 18, 20 and 22. In certain aspects of the invention, atoms. In general these fall into two categories: (a) catechols a contrast protein comprises an amino acid sequence at least Such as enterobactin which comprises a cyclic structure com 45 60% identical to that of human transferrin receptor 1 and/or posed of three molecules of 2,3-dihydroxy-N-benzoyl serine. human transferrin receptor 2, and optionally at least 70%, Further examples include agents wherein the serine is Substi 80%,90%. 95%.98%,99% or 100% identical, and preferably tuted with either a glycine or a threonine. Also included retains transferrin receptor activity. herein are catechol siderophores having linear rather than In further embodiments, contrast proteins of the invention cyclic structures such as pseudobactin; (b) Hydroxamates 50 may be engineered, by for example, employing techniques of comprise a large and variable group having either cyclic or molecular biology. For example, it is possible to modify the linear peptides containing various types of hydroxamic acids. structure of the Subject contrast proteins for Such purposes as Common examples include ferrichrome, ferrioxamine, and enhancing contrast efficacy, stability (e.g., increased or aerobactin. Further examples include plant siderophores Such decreased resistance to proteolytic degradation in vivo), anti as phytosiderophore. Exemplary metal scavenging proteins 55 genicity, or safety, among other characteristics. Such modi include ferric binding proteins of the siderophilin family, fied proteins can be produced, for instance, by amino acid Such as mammalian transferring, ovotransferin, lactoferrins, Substitution, deletion, or addition. In addition, simple variants melanotransferrin, Sertoli transferrin, neurotransferrin, of any of the proteins discussed herein may be obtained by mucosal transferrin, and bacterial transferring, Such as those conservative Substitution. For instance, it is reasonable to found in Haemophilus influenzae, Neisseria gonorrhoeae, 60 expect that an isolated replacement of a leucine with an iso and Neisseria meningitidis. leucine or valine, an aspartate with a glutamate, a threonine In further embodiments, an iron regulatory protein (IRP) with a serine, or a similar replacement of an amino acid with may be used as a contrast protein. IRPs are iron-regulating a structurally related amino acid (i.e. conservative mutations) RNA binding proteins that modulate synthesis of proteins that will not have a major effect on the biological activity of the function in the uptake (e.g. transferrin receptor), utilization 65 resulting molecule. Conservative replacements are those that (e.g. erythroid 5-aminolevulinate synthase) or storage (e.g. take place within a family of amino acids that are related in H-ferritin and L-ferritin) of iron. Proteins regulated by IRPs their side chains. Genetically encoded amino acids are can be US 8,084,017 B2 17 18 divided into four families: (1) acidic aspartate, glutamate; but are not limited to, gene expression at a desired level, gene (2) basic-lysine, arginine, histidine; (3) nonpolar alanine, expression that is reflective of the expression of a different Valine, leucine, isoleucine, proline, phenylalanine, methion gene, easy clonability, transient or stable gene expression in ine, tryptophan; and (4) uncharged polar glycine, aspar Subject cells, etc. agine, glutamine, cysteine, serine, threonine, tyrosine. Phe 5 In certain aspects, it is desirable to use a vector that pro nylalanine, tryptophan, and tyrosine are sometimes classified vides transient expression of the contrast agent. Such vectors jointly as aromatic amino acids. In similar fashion, the amino will generally be unstable inside a cell, such that the nucleic acid repertoire can be grouped as (1) acidic aspartate, acids necessary for expression of the contrast agent are lost glutamate; (2) basic-lysine, arginine histidine, (3) after a relatively short period of time. Optionally, transient aliphatic glycine, alanine, Valine, leucine, isoleucine, serine, 10 expression may be effected by stable repression. Exemplary threonine, with serine and threonine optionally be grouped transient expression vectors may be designed to provide gene separately aS aliphatic-hydroxyl; (4) expression for an average time of hours, days, weeks, or aromatic phenylalanine, tyrosine, tryptophan; (5) perhaps months. Often transient expression vectors do not amide-asparagine, glutamine; and (6) Sulfur recombine to integrate with the stable genome of the host. containing cysteine and methionine. (see, for example, Bio 15 Exemplary transient expression vectors include: adenovirus chemistry, 2nd ed., Ed. by L. Stryer, W. H. Freeman and Co., derived vectors, adeno-associated viruses, herpes simplex 1981). derived vectors, hybrid adeno-associated/herpes simplex This invention further contemplates methods of generating viral vectors, influenza viral vectors, especially those based sets of combinatorial mutants of the Subject contrast proteins, on the influenza A virus, and alphaviruses, for example the as well as functional truncation mutants. The purpose of Sinbis and semliki forest viruses. screening Such combinatorial libraries is to generate, for In some aspects the invention provides a vector or construct example, engineered contrast proteins with any number of comprising a readily clonable nucleic acid encoding a con desirable qualities such as those mentioned above. trast protein. For example, the coding sequence may be There are many ways by which the library of potential flanked by a polylinker on one or both sides. Polylinkers are engineered contrast proteins can be generated. Chemical Syn 25 useful for allowing one of skill in the art to readily insert the thesis of a degenerate gene sequence can be carried out in an coding sequence in a variety of different vectors and con automatic DNA synthesizer, and the synthetic genes then be structs as required. In another example, the coding sequence ligated into an appropriate gene for expression. The purpose may be flanked by one or more recombination sites. A variety of a degenerate set of genes is to provide, in one mixture, all of commercially available cloning systems use recombina of the sequences encoding the desired set of potential contrast 30 tion sites to facilitate movement of the desired nucleic acid protein sequences. Such techniques have been employed in into different vectors. For example, the Invitrogen GatewayTM the directed evolution of other proteins (see, for example, technology utilizes a phage lambda recombinase enzyme to Scott et al., (1990) Science 249:386-390; Roberts et al., recombine target nucleic acids with a second nucleic acid. (1992) PNAS USA 89:2429-2433: Devlin et al., (1990) Sci Each nucleic acid is flanked with appropriate lambda recog ence 249: 404–406; Cwirla et al., (1990) PNAS USA 87: 35 nition sequence, Such as att, or attB. In other variations, a 6378-6382: as well as U.S. Pat. Nos. 5,223,409, 5,198,346, recombinase such as topoisomerase I may be used with and 5,096,815). nucleic acids flanked by the appropriate recognition sites. For Alternatively, otherforms of mutagenesis can be utilized to example, the Vaccinia virus topoisomerase I protein recog generate a combinatorial library. For example, engineered nizes a (C/T)CCTT sequence. These recombination systems contrast proteins can be generated and isolated from a library 40 permit rapid shuffling of flanked cassettes from one vector to by Screening using, for example, alanine Scanning mutagen another as needed. A construct or vector may include both esis and the like (see e.g. Ruf et al., (1994) Biochemistry flanking polylinkers and flanking recombination sites, as 33:1565-1572; Wang et al., (1994) J. Biol. Chem. 269:3095 desired. 3099: Balint et al., (1993), by linker scanning mutagenesis In certain aspects, the contrast gene is operably linked to a (Gustin et al., (1993) Virology 193:653-660; Brown et al., 45 promoter. The promoter may for example, be a strong or (1992) Mol. Cell Biol. 12:2644-2652; McKnight et al., constitutive promoter, such as the early and late promoters of (1982) Science 232:316); by saturation mutagenesis (Meyers SV40, or adenovirus or cytomegalovirus immediate early et al., (1986) Science 232:613); by PCR mutagenesis (Leung promoter. Optionally it may be desirable to use an externally et al., (1989) Method Cell Mol Biol 1:11-19); or by random regulated promoter, Such as a tet promoter, IPTG-regulated mutagenesis, including chemical mutagenesis, etc. (Miller et 50 promoters (GAL4, Plac), or the trp system. In view of this al., (1992) A Short Course in Bacterial Genetics, CSHL Press, specification, one of skill in the art will readily identify other Cold Spring Harbor, N.Y.; and Greener et al., (1994) Strate useful promoters depending on the downstream use. For gies in Mol Biol 7:32-34). example, the invention may utilize exemplary promoters such Whethera change in the amino acid sequence of a polypep as the T7 promoter whose expression is directed by T7 RNA tide results in a functional homologue can be readily deter 55 polymerase, the major operator and promoter regions of mined by assessing the ability of the variant polypeptide to, phage lambda, the control regions for fa coat protein, the for example, bind the desired metal, produce sufficient MRI promoter for 3-phosphoglycerate kinase or other glycolytic contrast in cells, and produce reduced cell toxicity. enzymes, the promoters of acid phosphatase, e.g., Pho5, the In further aspects, any combination of contrast proteins promoters of the yeast a-mating factors, the polyhedron pro may employed to obtain the desired contrast effects. 60 moter of the baculovirus system and other sequences known 4. Constructs and Vectors to control the expression of genes of prokaryotic or eukary In certain aspects, the invention provides vectors and otic cells or their viruses, and various combinations thereof. nucleic acid constructs comprising nucleic acids encoding In addition, as noted above, it may be desirable to have a one or more contrast agents. Other features of the vector or contrast gene operably linked to a promoter that provides construct will generally be designed to Supply desirable char 65 useful information about the condition of the cell in which it acteristics depending on how the contrast agent is to be gen is situated. In certain embodiments, it is anticipated that it will erated and used. Exemplary desirable characteristics include be desirable to achieve a concentration of contrast protein US 8,084,017 B2 19 20 within target cells that permits detection above background the host. See, for example, Dubensky et al. (1984) Proc. Natl. noise, and with certain detection systems this will translate Acad. Sci. USA 81,7529-7533; Kaneda et al., (1989) Science into a protein concentration of at least 1 nM or at least 10 nM. 243,375-378; Hiebert etal. (1989) Proc. Natl. Acad. Sci. USA Vectors of the invention may be essentially any nucleic acid 86, 3594-3598: Hatzoglu et al. (1990) J. Biol. Chem. 265, designed to introduce and/or maintain a contrast gene in a cell 5 17285-17293 and Ferry, et al. (1991) Proc. Natl. Acad. Sci. or virus. The pcDNAI/amp, pcDNAI/neo, pRc/CMV, USA 88, 8377-8381. The vector may be administered by pSV2gpt, pSV2neo, pSV2-dhfr, pTk2, pRSVneo, pMSG, injection, e.g. intravascularly or intramuscularly, inhalation, pSVT7... pko-neo and pHyg derived vectors are examples of or other parenteral mode. Non-viral delivery methods such as mammalian expression vectors suitable for transfection of administration of the DNA via complexes with liposomes or eukaryotic cells. Some of these vectors are modified with 10 by injection, catheter or biolistics may also be used. Gener sequences from bacterial plasmids, such as pBR322, to facili ally, in human subjects, it will be preferable to design the tate replication and drug resistance selection in both prokary nucleic acid and/or the delivery system to provide transient otic and eukaryotic cells. Alternatively, derivatives of viruses expression of the nucleic acid encoding the contrast agent. such as the bovine papilloma virus (BPV-1), or Epstein-Barr In general, the manner of introducing the nucleic acid will virus (pHEBo, pREP-derived and p205) may be used. Other 15 depend on the nature of the tissue, the efficiency of cellular vector systems suitable for gene therapy are described below. modification required, the number of opportunities to modify 5. Cells, Organized Cell Cultures, and Tissues the particular cells, the accessibility of the tissue to the In many aspects, the invention provides cells, organized nucleic acid composition to be introduced, and the like. The cell cultures, and tissues comprising a nucleic acid that DNA introduction need not result in integration. In fact, non encodes a contrast agent. Methods for generating trans integration often results in transient expression of the intro formed or transfected cells are widely known in the art, and it duced DNA, and transient expression is often sufficient or is anticipated that methods described herein may be used with even preferred. essentially any cell type of interest, including but not limited Any means for the introduction of polynucleotides into to bacterial, fungal, plant and animal cells. Preferred embodi mammals, human or non-human, may be adapted to the prac ments of the invention employ mammalian cells. Cells of 25 tice of this invention for the delivery of the various constructs particular interest may include transformed cells or other of the invention into the intended recipient. In one embodi cells that either are part of a tumor or are useful as a model for ment of the invention, the nucleic acid constructs are deliv cancer in vitro, stem or progenitor cells, and cells prepared for ered to cells by transfection, i.e., by delivery of “naked' a cell therapy for a patient. Cells of the invention may be nucleic acid or in a complex with a colloidal dispersion sys cultured cells, cell lines, cells situated in tissues and/or cells 30 tem. A colloidal system includes macromolecule complexes, that are part of an organism. nanocapsules, microspheres, beads, and lipid-based systems It is further anticipated that cells may be used to generate including oil-in-water emulsions, micelles, mixed micelles, organized cell cultures (i.e. cell cultures developing a non and liposomes. An exemplary colloidal system of this inven random structure) and to generate organs or organ-like struc tion is a lipid-complexed or liposome-formulated DNA. In tures for transplant into Subjects. It may be useful to non 35 the former approach, prior to formulation of DNA, e.g., with invasively monitor some aspect of gene expression in Such lipid, a plasmid containing a transgene bearing the desired cells, or to otherwise provide MRI contrast in such cells. For DNA constructs may first be experimentally optimized for example, muscle progenitor cells may be used to develop expression (e.g., inclusion of an intron in the 5' untranslated muscle-like organs for administration to injured muscle or for region and elimination of unnecessary sequences (Felgner, et administration as a packet of cells that produce a therapeutic 40 al., Ann NY AcadSci 126-139, 1995). Formulation of DNA, protein (see e.g. U.S. Pat. Nos. 5,399,346; 6.207,451; 5,538, e.g. with various lipid or liposome materials, may then be 722). Other cell culture methods have been used to produce effected using known methods and materials and delivered to neural, pancreatic, liver and many other organ types for trans the recipient mammal. See, e.g., Canonico et al. Am J Respir plant (see e.g. U.S. Pat. Nos. 6,146,889; 6,001,647; 5,888, Cell Mol Biol 10:24-29, 1994; Tsan et al. Am J Physiol 268; 705; 5,851,832 and PCT publication nos. WO 00/36091; WO 45 Alton et al., Nat Genet. 5:135-142, 1993 and U.S. Pat. No. 01/53461; WO 01/21767). Cells of this nature may be stably 5,679,647 by Carson et al. transfected with a contrast gene at an early stage of culture, or Optionally, liposomes or other colloidal dispersion sys the organized culture may be transiently or stably transfected tems are targeted. Targeting can be classified based on ana at a later point in culture to assess some aspect of cell func tomical and mechanistic factors. Anatomical classification is tion. Transfected cells may be administered to subjects in 50 based on the level of selectivity, for example, organ-specific, order to deliver a gene product, and this methodology is cell-specific, and organelle-specific. Mechanistic targeting effective as an ex vivo gene therapy or cell therapy method. A can be distinguished based upon whether it is passive or nucleic acid encoding a contrast protein may be introduced active. Passive targeting utilizes the natural tendency of lipo into Such cells and administered to a subject in order to somes to distribute to cells of the reticulo-endothelial system monitor gene expression or viability of the administered cells. 55 (RES) in organs, which contain sinusoidal capillaries. Active Cells transfected with the gene adenosine deaminase have targeting, on the other hand, involves alteration of the lipo been delivered to patients as an ex vivo gene therapy cure for Some by coupling the liposome to a specific ligand Such as a Severe Combined Immunodeficiency Syndrome (SCID) monoclonal antibody, Sugar, glycolipid, or protein, or by (Cavazzana-Calvo et al., 2000, Science 288(5466):669-72). changing the composition or size of the liposome in order to 6. Nucleic Acids for Delivery to Organisms and In vitro 60 achieve targeting to organs and cell types other than the Tissues naturally occurring sites of localization. Instead of ex vivo modification of cells, in many situations The surface of the targeted delivery system may be modi one may wish to modify cells in vivo. For this purpose, fied in a variety of ways. In the case of a liposomal targeted various techniques have been developed for modification of delivery system, lipid groups can be incorporated into the target tissue and cells in vivo. A number of viral vectors have 65 lipid bilayer of the liposome in order to maintain the targeting been developed, such as described above, which allow for ligand in stable association with the liposomal bilayer. Vari transfection and, in some cases, integration of the virus into ous linking groups can be used for joining the lipid chains to US 8,084,017 B2 21 22 the targeting ligand. A certain level of targeting may be useful for the treatment of cancers, killing the neoplastic cells achieved through the mode of administration selected. which proliferate faster than other cell types (Andreansky et In certain variants of the invention, the nucleic acid con al, 1996, 1997). A replication-restricted HSV-1 vector has structs are delivered to cells, and particularly cells in an been used to treat human malignant mesothelioma (Kuchari organism or a cultured tissue, using viral vectors. The trans Zuket al., 1997). In addition to neurons, wild type HSV-1 can gene may be incorporated into any of a variety of viral vectors infect other non-neuronal cell types, such as skin (Al-Saadi et useful in gene therapy, such as recombinant retroviruses, al, 1983), and HSV-derived vectors may be useful for deliv adenovirus, adeno-associated virus (AAV), herpes simplex ering transgenes to a wide array of cell types. Other examples derived vectors, hybrid adeno-associated/herpes simplex of herpes virus vectors are known in the art (U.S. Pat. No. viral vectors, influenza viral vectors, especially those based 10 5,631,236 and WO 00/08191). on the influenza A virus, and alphaviruses, for example the B. Adenoviral Vectors Sinbis and semliki forest viruses, or recombinant bacterial or A viral gene delivery system useful in the present invention eukaryotic plasmids. The following additional guidance on utilizes adenovirus-derived vectors. Knowledge of the the choice and use of viral vectors may be helpful to the genetic organization of adenovirus, a 36 kB, linear and practitioner. As described in greater detail below, such 15 double-stranded DNA virus, allows substitution of a large embodiments of the Subject expression constructs are specifi piece of adenoviral DNA with foreign sequences up to 8 kB. cally contemplated for use in various in vivo and ex vivo gene In contrast to retrovirus, the infection of adenoviral DNA into therapy protocols. host cells does not result in chromosomal integration because A. Herpes Virus Systems adenoviral DNA can replicate in an episomal manner without A variety of herpes virus-based vectors have been devel potential genotoxicity. Also, adenoviruses are structurally oped for introduction of genes into mammals. For example, stable, and no genome rearrangement has been detected after herpes simplex virus type 1 (HSV-1) is a human neurotropic extensive amplification. Adenovirus can infect virtually all virus of particular interest for the transfer of genes to the epithelial cells regardless of their cell cycle stage. In addition, nervous system. After infection of target cells, herpesviruses adenoviral vector-mediated transfection of cells is often a often follow either a lytic life cycle or a latent life cycle, 25 transient event. A combination of immune response and pro persisting as an intranuclear episome. In most cases, latently moter silencing appears to limit the time over which a trans infected cells are not rejected by the immune system. For gene introduced on an adenovirus vector is expressed. example, neurons latently infected with HSV-1 function nor Adenovirus is particularly Suitable for use as a gene trans mally and are not rejected. Some herpes viruses possess cell fer vector because of its mid-sized genome, ease of manipu type specific promoters that are expressed even when the 30 lation, high titer, wide target-cell range, and high infectivity. virus is in a latent form. The virus particle is relatively stable and amenable to purifi A typical herpes virus genome is a linear double stranded cation and concentration, and as above, can be modified so as DNA molecule ranging from 100 to 250kb. HSV-1 has a 152 to affect the spectrum of infectivity. Additionally, adenovirus kb genome. The genome may include long and short regions is easy to grow and manipulate and exhibits broad host range (termed UL and US, respectively) which are linked in either 35 in vitro and in vivo. This group of viruses can be obtained in orientation by internal repeat sequences (IRL and IRS). At the high titers, e.g., 10-10' plaque-forming unit (PFU)/ml, and non-linker end of the unique regions are terminal repeats they are highly infective. Moreover, the carrying capacity of (TRL and TRS). In HSV-1, roughly half of the 80-90 genes the adenoviral genome for foreign DNA is large (up to 8 are non-essential, and deletion of non-essential genes creates kilobases) relative to other gene delivery vectors (Berkner et space for roughly 40-50 kb of foreign DNA (Glorioso et al. 40 al., supra: Haj-Ahmand and Graham (1986) J. Virol. 57:267). 1995). Two latency active promoters which drive expression Most replication-defective adenoviral vectors currently in use of latency activated transcripts have been identified and may and therefore favored by the present invention are deleted for prove useful for vector transgene expression (Marconi et al. all or parts of the viral E1 and E3 genes but retain as much as 1996). 80% of the adenoviral genetic material (see, e.g., Jones et al., HSV-1 vectors are available in amplicons and recombinant 45 (1979) Cell 16:683; Berkner et al., supra; and Graham et al., HSV-1 virus forms. Amplicons are bacterially produced plas in Methods in Molecular Biology, E.J. Murray, Ed. (Humana, mids containing OriC, an Escherichia coli origin of replica Clifton, N.J., 1991) vol. 7. pp. 109-127). Expression of the tion, OriS (the HSV-1 origin of replication), HSV-1 packag inserted polynucleotide of the invention can be under control ing sequence, the transgene under control of an immediate of for example, the E1A promoter, the major late promoter early promoter & a selectable marker (Federoffet al., 1992). 50 (MLP) and associated leader sequences, the viral E3 pro The ampliconistransfected into a cell line containing a helper moter, or exogenously added promoter sequences. virus (a temperature sensitive mutant) which provides all the The genome of an adenovirus can be manipulated Such that missing structural and regulatory genes in trans. More recent it encodes a gene product of interest, but is inactivated in amplicons include an Epstein-Barr virus derived sequence for terms of its ability to replicate in a normal lytic viral life cycle plasmid episomal maintenance (Wang & Vos, 1996). Recom 55 (see, for example, Berkner et al., (1988) BioTechniques binant viruses are made replication deficient by deletion of 6:616; Rosenfeld et al., (1991) Science 252:431-434; and one the immediate-early genes e.g. ICP4, which is provided Rosenfeld et al., (1992) Cell 68:143-155). Suitable adenovi in trans. Deletion of a number of immediate-early genes ral vectors derived from the adenovirus strain Adtype 5 d1324 Substantially reduces cytotoxicity and allows expression or other Strains of adenovirus (e.g., Ad2, Ad3, Ad7 etc.) are from promoters that would be silenced in the wild type latent 60 well known to those skilled in the art. virus. These promoters may be of use in directing long term Adenoviruses can be cell type specific, i.e., infect only gene expression. Replication-conditional mutants replicate in restricted types of cells and/or express a transgene only in permissive cell lines. Permissive cell lines supply a cellular restricted types of cells. For example, the viruses may be enzyme to complement for a viral deficiency. Mutants include engineered to comprise a gene under the transcriptional con thymidine kinase (During et al., 1994), ribonuclease reductase 65 trol of a transcription initiation region specifically regulated (Kramm et al., 1997), UTPase, or the neurovirulence factor by target host cells, as described e.g., in U.S. Pat. No. 5,698, g34.5 (Kesari et al., 1995). These mutants are particularly 443, by Henderson and Schuur, issued Dec. 16, 1997. Thus, US 8,084,017 B2 23 24 replication competent adenoviruses can be restricted to cer AAV stocks can be produced as described in Hermonatand tain cells by, e.g., inserting a cell specific response element to Muzyczka (1984) PNAS 81:6466, modified by using the regulate a synthesis of a protein necessary for replication, pAAV/Ad described by Samulski et al. (1989) J. Virol. e.g., E1A or E1B. 63:3822. Concentration and purification of the virus can be DNA sequences of a number of adenovirus types are avail achieved by reported methods such as banding in cesium able from Genbank. For example, human adenovirus type 5 chloride gradients, as was used for the initial report of AAV has GenBank Accession No.M73260. The adenovirus DNA vector expression in vivo (Flotte, et al. J. Biol. Chem. 268: sequences may be obtained from any of the 42 human aden 3781-3790, 1993) or chromatographic purification, as ovirus types currently identified. Various adenovirus strains described in O'Riordan et al., WO97/08298. Methods for in 10 vitro packaging AAV vectors are also available and have the are available from the American Type Culture Collection, advantage that there is no size limitation of the DNA pack Rockville, Md., or by request from a number of commercial aged into the particles (see, U.S. Pat. No. 5,688,676, by Zhou and academic sources. A transgene as described herein may et al., issued Nov. 18, 1997). This procedure involves the be incorporated into any adenoviral vector and delivery pro preparation of cell free packaging extracts. tocol, by restriction digest, linkerligation or filling in of ends, 15 D. Hybrid Adenovirus-AAV Vectors and ligation. Hybrid Adenovirus-AAV vectors have been generated and Adenovirus producer cell lines can include one or more of are typically represented by an adenovirus capsid containing the adenoviral genes E1, E2a, and E4 DNA sequence, for a nucleic acid comprising a portion of an adenovirus, and 5' packaging adenovirus vectors in which one or more of these and 3' inverted terminal repeat sequences from an AAV which genes have been mutated or deleted are described, e.g., in flanka selected transgene under the control of a promoter. See PCT/US95/15947 (WO 96/18418) by Kadan et al.: PCT/ e.g. Wilson et al. International Patent Application Publication US95/07341 (WO95/346671) by Kovesdiet al.; PCT/FR94/ No. WO 96/13598. This hybrid vector is characterized by 00624 (WO94/28152) by Imleret al.:PCT/FR94/00851 (WO high titer transgene delivery to a host cell and the ability to 95/02697) by Perrocaudet et al., PCT/US95/14793 (WO96/ stably integrate the transgene into the host cell 14061) by Wang et al. 25 in the presence of the rep gene. This virus is capable of C. AAV Vectors infecting virtually all cell types (conferred by its adenovirus Yet another viral vector system useful for delivery of the sequences) and stable longterm transgene integration into the Subject polynucleotides is the adeno-associated virus (AAV). host cell genome (conferred by its AAV sequences). Adeno-associated virus is a naturally occurring defective The adenovirus nucleic acid sequences employed in this virus that requires another virus, Such as an adenovirus or a 30 vector can range from a minimum sequence amount, which herpes virus, as a helper virus for efficient replication and a requires the use of a helper virus to produce the hybrid virus productive life cycle. (For a review, see Muzyczka et al., Curr. particle, to only selected deletions of adenovirus genes, Topics in Micro. and Immunol. (1992) 158:97-129). which deleted gene products can be supplied in the hybrid AAV has not been associated with the cause of any disease. viral process by a packaging cell. For example, a hybrid virus AAV is not a transforming or oncogenic virus. AAV integra 35 can comprise the 5' and 3' inverted terminal repeat (ITR) tion into of human cell lines does not cause any sequences of an adenovirus (which function as origins of significant alteration in the growth properties or morphologi replication). The left terminal sequence (5') sequence of the cal characteristics of the cells. These properties of AAV also Ad5 genome that can be used spans bp 1 to about 360 of the recommend it as a potentially useful human gene therapy conventional adenovirus genome (also referred to as map Vector. 40 units 0-1) and includes the 5' ITR and the packaging/enhancer AAV is also one of the few viruses that may integrate its domain. The 3' adenovirus sequences of the hybrid virus DNA into non-dividing cells, e.g., pulmonary epithelial cells, include the right terminal 3' ITR sequence which is about 580 and exhibits...a high frequency of stable integration (see for nucleotides (about bp 35,353-end of the adenovirus, referred example Flotte et al., (1992) Am. J. Respir. Cell. Mol. Biol. to as about map units 98.4-100). 7:349-356: Samulski et al., (1989) J. Virol. 63:3822-3828: 45 For additional detailed guidance on adenovirus and hybrid and McLaughlin et al., (1989) J. Virol. 62: 1963-1973). Vec adenovirus-AAV technology which may be useful in the prac tors containing as little as 300 base pairs of AAV can be tice of the Subject invention, including methods and materials packaged and can integrate. Space for exogenous DNA is for the incorporation of a transgene, the propagation and limited to about 4.5 kb. An AAV vector such as that described purification of recombinant virus containing the transgene, in Tratschinet al., (1985) Mol. Cell. Biol. 5:3251-3260 can be 50 and its use in transfecting cells and mammals, see also Wilson used to introduce DNA into cells. A variety of nucleic acids et al., WO 94/28938, WO 96/13597 and WO 96/26285, and have been introduced into different cell types using AAV references cited therein. vectors (see for example Hermonat et al., (1984) PNAS USA E. Retroviruses 81:6466-6470; Tratschin et al., (1985) Mol. Cell. Biol. In order to construct a retroviral vector, a nucleic acid of 4:2072-2081; Wondisford et al., (1988) Mol. Endocrinol. 55 interest is inserted into the viral genome in the place of certain 2:32-39: Tratschin et al., (1984) J. Virol. 51:611-619; and viral sequences to produce a virus that is replication-defec Flotte et al., (1993) J. Biol. Chem. 268:3781-3790). tive. In order to produce virions, a packaging cell line con The AAV-based expression vector to be used typically taining the gag, pol, and env genes but without the LTR and includes the 145 nucleotide AAV inverted terminal repeats psi components is constructed (Mann et al. (1983) Cell (ITRs) flanking a restriction site that can be used for subclon 60 33: 153). When a recombinant plasmid containing a human ing of the transgene, either directly using the restriction site cDNA, together with the retroviral LTR and psi sequences is available, or by excision of the transgene with restriction introduced into this cell line (by calcium phosphate precipi enzymes followed by blunting of the ends, ligation of appro tation for example), the psi sequence allows the RNA tran priate DNA linkers, restriction digestion, and ligation into the Script of the recombinant plasmid to be packaged into viral site between the ITRs. The capacity of AAV vectors is usually 65 particles, which are then secreted into the culture media about 4.4 kb (Kotin, R. M., Human Gene Therapy 5:793-801, (Nicolas and Rubenstein (1988) “Retroviral Vectors”, In: 1994 and Flotte, et al. J. Biol. Chem. 268:3781-3790, 1993). Rodriguez, and Denhardt ed. Vectors: A Survey of Molecular US 8,084,017 B2 25 26 Cloning Vectors and their Uses. Stoneham:Butterworth; Furthermore, it has been shown that it is possible to limit Temin, (1986) “Retrovirus Vectors for Gene Transfer: Effi the infection spectrum of retroviruses and consequently of cient Integration into and Expression of Exogenous DNA in retroviral-based vectors, by modifying the viral packaging Vertebrate Cell Genome'. In: Kucherlapatied. Gene Transfer. proteins on the surface of the viral particle (see, for example New York: Plenum Press: Mann et al., 1983, supra). The PCT publications WO93/25234, WO94/06920, and WO94/ media containing the recombinant retroviruses is then col 11524). For instance, strategies for the modification of the lected, optionally concentrated, and used for gene transfer. infection spectrum of retroviral vectors include: coupling Retroviral vectors are able to infect a broad variety of cell antibodies specific for cell surface antigens to the viral env types. Integration and stable expression require the division protein (Roux et al., (1989) PNAS USA 86:9079-9083; Julan 10 et al., (1992) J. Gen Virol 73:3251-3255; and Goud et al., of host cells (Paskind et al. (1975) Virology 67:242). This (1983) Virology 163:251-254); or coupling cell surface aspect is particularly relevant for the treatment of PVR, since ligands to the viral env proteins (Neda et al., (1991) J. Biol. these vectors allow selective targeting of cells which prolif Chem. 266:14143-14146). Coupling can be in the form of the erate, i.e., selective targeting of the cells in the epiretinal chemical cross-linking with a protein or other variety (e.g. membrane, since these are the only ones proliferating in eyes 15 lactose to convert the env protein to anasialoglycoprotein), as of PVR subjects. well as by generating fusion proteins (e.g. single-chain anti A major prerequisite for the use of retroviruses is to ensure body/env fusion proteins). This technique, while useful to the safety of their use, particularly with regard to the possi limit or otherwise direct the infection to certain tissue types, bility of the spread of wild-type virus in the cell population. and can also be used to convert an ecotropic vector in to an The development of specialized cell lines (termed “packaging amphotropic vector. cells') which produce only replication-defective retroviruses F. Other Viral Systems has increased the utility of retroviruses for gene therapy, and Other viral vector systems that can be used to deliver a defective retroviruses are well characterized for use in gene polynucleotide of the invention have been derived from vac transfer for gene therapy purposes (for a review see Miller, cinia virus, alphavirus, poxvirus, arena virus, polio virus, and A.D. (1990) Blood 76:271). Thus, recombinant retrovirus can 25 the like. Such vectors offer several attractive features for be constructed in which part of the retroviral coding sequence various mammalian cells. (Ridgeway (1988) In: Rodriguez R (gag, pol, env) has been replaced by nucleic acid encoding a L., Denhardt D.T. ed. Vectors: A Survey of molecular cloning protein of the present invention, e.g., a transcriptional activa vectors and their uses. Stoneham: Butterworth; Baichwal and tor, rendering the retrovirus replication defective. The repli Sugden (1986) In: Kucherlapati R, ed. Gene transfer. New cation defective retrovirus is then packaged into virions 30 York: Plenum Press; Coupar et al. (1988) Gene, 68:1-10; which can be used to infect a target cell through the use of a Walther and Stein (2000) Drugs 60:249-71; Timiryasova et al. helper virus by standard techniques. Protocols for producing (2001) J Gene Med 3:468-77; Schlesinger (2001) Expert recombinant retroviruses and for infecting cells in vitro or in Opin Biol Ther 1:177-91: Khromykh (2000) Curr Opin Mol vivo with such viruses can be found in Current Protocols in Ther 2:555-69: Friedmann (1989) Science, 244:1275-1281; Molecular Biology, Ausubel, F. M. et al., (eds.) Greene Pub 35 Ridgeway, 1988, supra; Baichwal and Sugden, 1986, supra; lishing Associates, (1989), Sections 9.10-9.14 and other stan Coupar et al., 1988; Horwich et al. (1990) J. Virol. 64:642 dard laboratory manuals. Examples of suitable retroviruses 650). include pl.), pZIP pWE and pEM which are well known to 7. Transgenic Animals those skilled in the art. A preferred retroviral vector is a pSR While the techniques described herein may be used to MSVtkNeo (Muller et al. (1991) Mol. Cell Biol. 11:1785 and 40 deliver nucleic acids to human or animal Subjects, other meth pSR MSVCXbal) (Sawyers et al. (1995) J. Exp. Med. 181: ods are available to generate non-human transgenic animals 307) and derivatives thereof. For example, the unique BamHI incorporating a recombinant nucleic acid encoding a contrast sites in both of these vectors can be removed by digesting the protein. vectors with BamHI, filling in with Klenow and religating to In an exemplary embodiment, the “transgenic non-human produce pSMTN2 and pSMTX2, respectively, as described in 45 animals' of the invention are produced by introducing trans PCT/US96/09948 by Clackson et al. Examples of suitable genes into the germline of the non-human animal. Embryonal packaging virus lines for preparing both ecotropic and target cells at various developmental stages can be used to amphotropic retroviral systems include Crip, Cre, 2 and Am. introduce transgenes. Different methods are used depending Retroviruses, including lentiviruses, have been used to on the stage of development of the embryonal target cell. The introduce a variety of genes into many different cell types, 50 specific line(s) of any animal used to practice this invention including neural cells, epithelial cells, retinal cells, endothe are selected for general good health, good embryo yields, lial cells, lymphocytes, myoblasts, hepatocytes, bone marrow good pronuclear visibility in the embryo, and good reproduc cells, in vitro and/or in vivo (see for example, review by tive fitness. In addition, the haplotype is a significant factor. Federico (1999) Curr. Opin. Biotechnol. 10:448; Eglitis et al., For example, when transgenic mice are to be produced, (1985) Science 230:1395-1398; Danos and Mulligan, (1988) 55 strains such as C57BL/6 or FVB lines are often used (Jackson PNAS USA 85:6460-6464; Wilson et al., (1988) PNAS USA Laboratory, Bar Harbor, ME). Preferred strains such as 85:3014-3018; Armentano et al., (1990) PNAS USA C57BL/6 or DBA/1 may be selected. The line(s) used to 87:6141-6145; Huber et al., (1991) PNAS USA 88:8039 practice this invention may themselves be transgenics, and/or 8043; Ferry et al., (1991) PNAS USA 88:8377-8381: may be knockouts (i.e., obtained from animals which have Chowdhury et al., (1991) Science 254:1802-1805; van 60 one or more genes partially or completely Suppressed). Beusechem et al., (1992) PNAS USA 89:7640-7644; Kay et In one embodiment, the construct comprising a nucleic al., (1992) Human Gene Therapy 3:641-647; Dai et al., acid encoding a contrast protein is introduced into a single (1992) PNAS USA 89:10892-10895; Hwu et al., (1993) J. stage embryo. The Zygote is the best target for microinjection. Immunol. 150:4104-4115; U.S. Pat. No. 4,868,116; U.S. Pat. In the mouse, the male pronucleus reaches the size of approxi No. 4,980,286; PCT Application WO 89/07136; PCT Appli 65 mately 20 micrometers in diameter which allows reproduc cation WO 89/02468; PCT Application WO 89/05345; and ible injection of 1-2 plof DNA solution. The use of Zygotes as PCT Application WO92/07573). a target for gene transfer has a major advantage in that in most US 8,084,017 B2 27 28 cases the injected DNA will be incorporated into the host genetic material inserted will not exceed about 10 picoliters. gene before the first cleavage (Brinster et al. (1985) PNAS The physical effects of addition must not be so great as to 82:4438-4442). As a consequence, all cells of the transgenic physically destroy the viability of the Zygote. The biological animal will carry the incorporated transgene. This will in limit of the number and variety of DNA sequences will vary general also be reflected in the efficient transmission of the 5 depending upon the particular Zygote and functions of the transgene to offspring of the founder since 50% of the germ exogenous genetic material and will be readily apparent to cells will harbor the transgene. one skilled in the art, because the genetic material, including Normally, fertilized embryos are incubated in suitable the exogenous genetic material, of the resulting Zygote must media until the pronuclei appear. At about this time, the be biologically capable of initiating and maintaining the dif nucleotide sequence comprising the transgene is introduced 10 ferentiation and development of the Zygote into a functional into the female or male pronucleus as described below. In organism. Some species such as mice, the male pronucleus is preferred. The number of copies of the transgene constructs which are It is most preferred that the exogenous genetic material be added to the Zygote is dependent upon the total amount of added to the male DNA complement of the Zygote prior to its exogenous genetic material added and will be the amount being processed by the ovum nucleus or the Zygote female 15 which enables the genetic transformation to occur. Theoreti pronucleus. It is thought that the ovum nucleus or female cally only one copy is required; however, generally, numer pronucleus release molecules which affect the male DNA ous copies are utilized, for example, 1,000-20,000 copies of complement, perhaps by replacing the protamines of the male the transgene construct, in order to insure that one copy is DNA with histones, thereby facilitating the combination of functional. As regards the present invention, there will often the female and male DNA complements to form the diploid be an advantage to having more than one functioning copy of Zygote. each of the inserted exogenous DNA sequences to enhance Thus, it is preferred that the exogenous genetic material be the phenotypic expression of the exogenous DNA sequences. added to the male complement of DNA or any other comple Any technique which allows for the addition of the exog ment of DNA prior to its being affected by the female pro enous genetic material into nucleic genetic material can be nucleus. For example, the exogenous genetic material is 25 utilized so long as it is not destructive to the cell, nuclear added to the early male pronucleus, as soon as possible after membrane or other existing cellular or genetic structures. The the formation of the male pronucleus, which is when the male exogenous genetic material is preferentially inserted into the and female pronuclei are well separated and both are located nucleic genetic material by microinjection. Microinjection of close to the cell membrane. Alternatively, the exogenous cells and cellular structures is known and is used in the art. genetic material could be added to the nucleus of the sperm 30 Reimplantation is accomplished using standard methods. after it has been induced to undergo decondensation. Sperm Usually, the Surrogate host is anesthetized, and the embryos containing the exogenous genetic material can then be added are inserted into the oviduct. The number of embryos to the ovum or the decondensed sperm could be added to the implanted into a particular host will vary by species, but will ovum with the transgene constructs being added as soon as usually be comparable to the number of offspring the species possible thereafter. 35 naturally produces. Introduction of the transgene nucleotide sequence into the Transgenic offspring of the Surrogate host may be screened embryo may be accomplished by any means known in the art for the presence and/or expression of the transgene by any Such as, for example, microinjection, electroporation, or lipo suitable method. Screening is often accomplished by South fection. Following introduction of the transgene nucleotide ern blot or Northern blot analysis, using a probe that is sequence into the embryo, the embryo may be incubated in 40 complementary to at least a portion of the transgene. Western vitro for varying amounts of time, or reimplanted into the blot analysis using an antibody against the protein encoded by Surrogate host, or both. In vitro incubation to maturity is the transgene may be employed as an alternative or additional within the scope of this invention. One common method in to method for screening for the presence of the transgene prod incubate the embryos in vitro for about 1-7 days, depending uct. Typically, DNA is prepared from tail tissue and analyzed on the species, and then reimplant them into the Surrogate 45 by Southern analysis or PCR for the transgene. Alternatively, host. the tissues or cells believed to express the transgene at the For the purposes of this invention a Zygote is essentially the highest levels are tested for the presence and expression of the formation of a diploid cell which is capable of developing into transgene using Southern analysis or PCR, although any tis a complete organism. Generally, the Zygote will be comprised Sues or cell types may be used for this analysis. of an egg containing a nucleus formed, either naturally or 50 Alternative or additional methods for evaluating the pres artificially, by the fusion of two haploid nuclei from a gamete ence of the transgene include, without limitation, Suitable or gametes. Thus, the gamete nuclei must be ones which are biochemical assays such as enzyme and/or immunological naturally compatible, i.e., ones which resultina viable Zygote assays, histological stains for particular marker or enzyme capable of undergoing differentiation and developing into a activities, flow cytometric analysis, and the like. Analysis of functioning organism. Generally, a euploid Zygote is pre 55 the blood may also be useful to detect the presence of the ferred. If an aneuploid Zygote is obtained, then the number of transgene product in the blood, as well as to evaluate the effect chromosomes should not vary by more than one with respect of the transgene on the levels of various types of blood cells to the euploid number of the organism from which either and other blood constituents. Alternatively, MRI can be used gamete originated. to visualize transgene expression. In addition to similar biological considerations, physical 60 An alternative method for generating transgenic animals ones also govern the amount (e.g., Volume) of exogenous involves the in vivo or ex vivo (in vitro) transfection of male genetic material which can be added to the nucleus of the animal germ cells with a desired nucleic acid (see e.g., U.S. Zygote or to the genetic material which forms a part of the Pat. No. 6,316,692). In one approach, the nucleic acid is Zygote nucleus. If no genetic material is removed, then the delivered in situ to the gonad of the animal (in vivo transfec amount of exogenous genetic material which can be added is 65 tion). The transfected germ cells are allowed to differentiate limited by the amount which will be absorbed without being in their own milieu, and then animals exhibiting integration of physically disruptive. Generally, the Volume of exogenous the nucleic acid into the germ cells are selected. The selected US 8,084,017 B2 29 30 animals may be mated, or their sperm utilized for insemina formed, the partner may or may not be transgenic and/or a tion or in vitro fertilization to produce transgenic progeny. knockout; where it is transgenic, it may contain the same or a The selection may take place after biopsy of one or both different transgene, or both. Alternatively, the partner may be gonads, or after examination of the animal’s ejaculate to a parental line. Where in vitro fertilization is used, the fertil confirm the incorporation of the desired nucleic acid 5 ized embryo may be implanted into a surrogate host or incu sequence. Alternatively, male germ cells may be isolated bated in vitro, or both. Using either method, the progeny may from a donor animal and transfected, or genetically altered in be evaluated for the presence of the transgene using methods vitro. Following this genetic manipulation, transfected germ described above, or other appropriate methods. cells are selected and transferred to the testis of a suitable The transgenic animals produced in accordance with the recipient animal. Before transfer of the germ cells, the recipi 10 present invention will include exogenous genetic material ent testis are generally treated in one, or a combination, of a encoding a contrast agent. Further, the sequence will prefer number of ways to inactivate or destroy endogenous germ ably be attached to a regulatory sequence that allows the cells, including by gamma irradiation, by chemical treatment, expression of the transgene. Contrast agent produced in situ by means of infectious agents such as viruses, or by autoim may be visualized by MRI. mune depletion or by combinations thereof. This treatment 15 8. MRI Methodologies facilitates the colonization of the recipient testis by the altered In general, contrast agents of the invention are designed for donor cells. Animals that carry suitably modified sperm cells use in MRI detection systems. In the most common imple may be allowed to mate naturally, or alternatively their sper mentation of MRI, one observes the hydrogen nucleus (pro matozoa are used for insemination or in vitro fertilization. ton) in molecules of mobile water contained in Subject mate In an exemplary embodiment, a transgenic animal may be rials. The Subject material is placed in a large static magnetic produced by in vitro infection of a single-cell embryo with a field. The field tends to align the magnetic moment associated lentiviral vector. See e.g., Lois et al., Science 295: 868-872 with the hydrogen nuclei in water along the field direction. (2002). The nuclei are perturbed from equilibrium by pulsed radio Retroviral infection can also be used to introduce the trans frequency (RF) radiation set at the Larmor frequency, which gene into a non-human animal. The developing non-human 25 is a characteristic frequency proportional to the magnetic embryo can be cultured in vitro to the blastocyst stage. During field strength where protons resonantly absorb energy. Upon this time, the blastomeres can be targets for retroviral infec removing the RF, the nuclei induce a transient Voltage in a tion (Jaenich, R. (1976) PNAS 73:1260-1264). Efficient receiver antenna; this transient Voltage constitutes the nuclear infection of the blastomeres is obtained by enzymatic treat magnetic resonance (NMR) signal. Spatial information is ment to remove the Zona pellucida (Manipulating the Mouse 30 encoded in both the frequency and/or phase of the NMR Embryo, Hogan eds. (Cold Spring Harbor Laboratory Press, signal by selective application of magnetic field gradients that Cold Spring Harbor, 1986). The viral vector system used to are Superimposed onto the large static field. The transient introduce the transgene is typically a replication-defective Voltages are generally digitized, and then these signals may retrovirus carrying the transgene (Jahner et al. (1985) PNAS be processed by, for example, using a computer to yield 82:6927-6931; Van der Putten et al. (1985) PNAS 82:6148 35 images. 6152). Transfection is easily and efficiently obtained by cul The invention now being generally described, it will be turing the blastomeres on a monolayer of virus-producing more readily understood by reference to the following cells (Van der Putten, supra; Stewart et al. (1987) EMBO J. examples, which are included merely for purposes of illus 6:383-388). Alternatively, infection can be performed at a tration of certain aspects and embodiments of the present later stage. Virus or virus-producing cells can be injected into 40 invention, and are not intended to limit the invention. the blastocoele (Jahner et al. (1982) Nature 298:623-628). Most of the founders will be mosaic for the transgene since EXAMPLES incorporation occurs only in a Subset of the cells which formed the transgenic non-human animal. Further, the Example 1 founder may contain various retroviral insertions of the trans 45 geneat different positions in the genome which generally will NMR of K562 Cells Over-Expressing Ferritin: segregate in the offspring. In addition, it is also possible to Simulated Tumor Studies introduce transgenes into the germ line by intrauterine retro viral infection of the midgestation embryo (Jahner et al. We describe data showing the feasibility of using of an (1982) supra). 50 over-expression of intra-cellular metal-binding polypeptides A fourth type of target cell for transgene introduction is the as a potent MRI contrast agent. These initial results focus on embryonal stem cell (ES). ES cells are obtained from pre ferritin in living human myeloid leukemia (K562) cells. implantation embryos cultured in vitro and fused with To investigate the sensitivity of ferritin in modulating the embryos (Evans et al. (1981)Nature 292:154-156: Bradley et NMR properties of K562 cells, we synthesized simulated al. (1984) Nature 309:255-258; Gossler et al. (1986) PNAS 55 “tumor samples. These consisted of K562 cells that were 83: 9065-9069; and Robertson et al. (1986) Nature 322:445 stimulated to produce varying amounts of excess intra-cellu 448). Transgenes can be efficiently introduced into the ES lar ferritin in vitro. Cells were then suspended in low-melting cells by DNA transfection or by retrovirus-mediated trans point agarose to form Small pellets. The spin-lattice relax duction. Such transformed ES cells can thereafter be com ation rate (1/T) and the spin-spin relaxation rate (1/T) were bined with blastocysts from a non-human animal. The ES 60 measured in the pellets to quantify the impact of ferritin. cells thereafter colonize the embryo and contribute to the (Modulation of these relaxation times give rise to image con germ line of the resulting chimeric animal. For review see trast in MRI.) In the same cells used for the samples, we Jaenisch, R. (1988) Science 240: 1468-1474. assayed the total ferritin content using ELISA (Enzyme In general, progeny of transgenic animals may be obtained LinkedImmuno-Sorbent Assay). by mating the transgenic animal with a suitable partner, or by 65 For the experiment, samples consisted of K562 cells that in vitro fertilization of eggs and/or sperm obtained from the were stimulated to over-express ferritin by a 16 hour incuba transgenic animal. Where mating with a partner is to be per tion with varying concentrations of ferric ammonium citrate US 8,084,017 B2 31 32 (FAC) in RPMI culture media supplemented with 2% fetal shown in FIGS. 4 and 5. Human light and heavy chain ferritin calf serum. After incubation, cells were washed. For each cDNA having defective iron regulatory elements were used. FAC concentration, 107 cells were counted for the NMR Using standard molecular biology techniques both trans sample and 10° cells we set aside for the ELISA assay (Alpha genes were placed under the control of the immediate early Diagnostics Int. Inc., San Antonio, Tex.)). Cells used for the promoter of the CMV. The integrity of the transgenes was NMR samples were re-suspended in 50 ul of low melting confirmed by electrophoresis of DNA fragments following point agarose in a small plastic tube. The 1/T and 1/T digestion with various restriction enzymes and by DNA measurements were performed at room temperature using a sequencing. Bruker Minispec relaxometer (Bruker Instruments, Billerica, Introduction of Ferritin via Transfection Mass.). Cells used for the ELISA were treated with lysis 10 9L cells (Fischer 344 rat gliosarcoma) were incubated in buffer and the consistency of the total amount of released DMEM supplemented with 10% fetal bovine serum (FBS), protein was confirmed using a bicinchoninic acid protein penicillin, Streptomycin, and glutamine. Cells were plated in quantitation assay (Pierce Inc., Rockford, Ill.). Ferritin con 24-well plates one day before transfection to achieve 60-80% centration was calculated as an average over the cell pellet 15 confluence. The cells were rinsed with serum-free DMEM Volume. and then covered with the same solution. A DNA mixture was The correlation between the NMR changes and ferritin content is shown in FIG. 1. The results show substantial prepared as follows. The reagent lipofectamineTM (Invitro changes in the relaxation times with modest increases in gen, Carlsbad, Calif.) was combined with equal amounts of ferritin expression over background; these changes are easily LF and HF DNA in serum-free DMEM. The reagent PlusTM observed using MRI (below). These simulated tumors have a (Invitrogen, Carlsbad, Calif.) was added to the DNA solution cell density of 200 cells/nl. to increase transfection efficiency. The DNA mixture was added to the cells, and then incubated for 3 hours at 37°C., Example 2 after which DMEM containing 10% FBS was added. Cells were collected 48 or 96 hours post-transfection and counted. Toxicity Studies 25 In addition, control samples were prepared by incubating 9L cells under identical conditions as above, except that no DNA The ferritin synthesis temporarily perturbs the cell's iron was added to the lipofectamineTM PlusTM DMEM mix metabolism. Although the adverse effects of this on the cells ture. Upon harvesting after 48 or 96 hours no significant long-term health have yet to be fully determined in vivo, differences in cell numbers were observed between samples indications from various invitro experiments have shown that 30 incubated with the DNA reporters and the control samples. ferritin overexpression is not harmful in a variety of cell lines, Thus, there was no apparent toxicity associated with the con especially for transient expression. This was confirmed in our trast proteins. experiments in K562 cells described in Example 1 above. For To assay the ferritin increase after transfection, 9L cells each FAC concentration (and control), cells before and after were prepared as described above. The intracellular proteins the incubation period were counted 3-times using a hemocy 35 were extracted using the M-PERTM extraction Reagent tometer and the results were averaged. FIG. 2 shows the (Pierce Biotechnology, Mountain View, Calif.) and the fer percent cells remaining after the 16 hour period of ferritin ritin content was assayed using an ELISA kit (Alpha diag loading. In the simulated tumors, ferritin increases of greater nostics, San Antonio, Tex.). The results typically showed a than 10-times over baseline levels only resulted in a cell loss ferritin concentration ~3 ng/ml in the transfected cells and a of order 20%. The ferritin increase required to provide 40 negligible (~0.0 ng/ml) amount of human ferritin in the non observable MRI contrast is only of order 2-4. transfected cells. (The 9L cell line is from rat, and the anti body used in the ELISA detects only human ferritin with no Example 3 cross-reactivity.) The intracellular iron content was measured in transfected MRI of Simulated Tumors 45 and control cells to confirm an increased iron-uptake with transgene expression. For these experiments 20x10 cells Ferritin over-expression in the simulated tumors is readily were plated and transfected using the methods described visualized using MRI. FIG. 3 shows a MRI image slice above. Control cells were also prepared as described above through three pellets used in the NMR experiments. In this with no DNA added to the incubation solution. Cells were image, contrast is predominately T-weighted. In FIG. 3, (a) 50 collected 96 hours post transfection and counted. Using stan is the control, and (b)-(c) are the samples containing a ferritin dard methods 2001 Blood 97 (9), 2863 cells were washed in increase of 2.7 and 4, respectively (see FIG. 1). Images were PBS, and pellets were dissolved in an acid solution and acquired simultaneously using a Bruker 7-Tesla MRI system treated with a batophenantroline sulconate solution. The light with TE/TR=45/2000 ms, 128x128 image points, and a 1 absorption of the Solution was read at 535 nm using a spec mm-thick slice. The pellet size was approximately 4 mm in 55 trophotometer and the iron concentration was calculated. The diameter. results indicate a factor of -1.5 increase in the net iron content of the transfected cells compared control. Example 4 Measurement of 1/T in pellets of transfected cells was performed. Cells (20x10) were transfected with the trans MRI Studies of Cells Comprising Recombinant 60 genes as described above. Cells were collected 96 hours post Ferritin transfection, washed twice with PBS, and transferred to a 0.2 ml micro-centrifuge tubes. Cells were again centrifuged and Both the light and heavy ferritin transgenes, denoted LF the supernatant discarded. NMR measurements were per and HF, respectively, were introduced into variety of cell lines formed on the pellets at 4° C. using a 20 MHz. Bruker (e.g. K562 and Rat 9L gliosarcoma) using lipid-based trans 65 Minispec NMR analyzer (Bruker Instruments, Billerica, fection methods and by using viruses. The results were ana Mass.). The results typically show a factor of ~15% increase lyzed using ELISA, NMR, and MRI. Typical results are in 17T in the transfected cells over control. US 8,084,017 B2 33 34 Using the same cell pellets that were prepared for the above rinsed, and the effects of the contrast genes were assayed. NMR experiments, we confirmed that the 1/T changes due to FIG. 5 shows typical MRI data of two pellets, infected and the expression of the contrast proteins provided satisfactory uninfected (control), 9L cells. These data were acquired using contrast in MR images. The micro-centrifuge tubes contain a T-weighted 2DFT spin-echo sequence in a similar manner ing the pellets were placed in an MRI apparatus and imaged as the transfection experiments above. The left pellet is the using a standard T-weighted two-dimensional Fourier trans control and the right pellet contains cells infected with LF and form (2DFT) spin-echo pulse sequence. FIG. 4 displays typi HF transgenes. Image contrastis clearly apparent between the cal data and shows a high-resolution MRI slice through two two samples. pellets acquired simultaneously; the left pellet is the control and the pellet on the right contains cells expressing the con- 10 Example 5 trast proteins. Image contrast is clearly apparent between the two samples. Introduction of a Nucleic Acid Encoding a Contrast Introduction of Ferritin via a Viral Vector Protein In Vivo Contrast proteins have also been introduced into cells via a viral vector. Infected cells were characterized using ELISA, 15 This experiment is designed to demonstrate the delivery of NMR, and MRI. The MRI data shows distinct contrast contrast agent of the invention in vivo. between cells infected with the contrast proteins and unin- In this example, two tumor samples are transplanted onto a fected (control) cells. For these experiments the LF and HF nude mouse. An HSV delivery is engineered to contain a transgenes were each incorporated into separate replication nucleic acid construct comprising the coding sequences for defective adenoviruses. These viruses were constructed using 20 the human ferritins represented in SEQID Nos: 2 and 4. One the commercially available Adeno-XTM expression system tumor sample is injected with the HSV--ferritin vector, while (Clontech, Palo Alto, Calif.) following the manufacture's the other tumor sample is injected with an “empty' HSV instructions. The transgene expression was controlled using vector. The mouse is subjected to MRI, and the contrast the CMV promoter. A HEK-293 cell line was used for pro- between the HSV--ferritin sample and the “empty” HSV duction of viral stocks. When the cytopathic effect was evi- 25 sample is compared. dent in the HEK-293 cells due to viral production, cells were Incorporation by Reference collected, lysed, and the Supernatants were collected. These All of the patents, publications and sequence database Supernatants are adenovirus-rich and were used to infect entries cited herein are hereby incorporated by reference. mammalian cells to demonstrate MRI contrasting effects. 9L Also incorporated by reference are the following: Trinder et cells were incubated in DMEM supplemented with 10% FBS, 30 al., Int. J. Biochem. & Cell Biol. 35:292-296 (2003); Flem penicillin, streptomycin, and glutamine. Cells (-20x10) ing et al., Proc. Natl. Acad. Sci. USA 99: 10653-10658 were plated in 24-well plates one day before infection to (2002); and Fleming et al., Proc. Natl. Acad. Sci. USA 97: achieve 60-80% confluence. The cells were then rinsed with 2214-2219 (2000). serum-free DMEM and then covered with the same solution. Equivalents Equal volumes of both the LF and HFadenovirus from each 35 Those skilled in the art will recognize, or be able to ascer of the respective supernatants were added to the 9L cells. The tain using no more than routine experimentation, many virus and cells were incubated in serum-free media for 0.5 equivalents to the specific embodiments of the invention hour, and then FBS was added to the DMEM to give 10% described herein. Such equivalents are intended to be encom FBS. After a 48 hours incubation the cells were harvested, passed by the following claims.

SEQUENCE LISTING

<16 Os NUMBER OF SEO ID NOS: 22

<21 Os SEQ ID NO 1 &211s LENGTH: 955 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs SEQUENCE: 1 ggggagacgt tott.cgc.cga gagtc.gtcgg ggtttcctgc titcaiacagtg cttggacgga 60 acccgg.cgct cqttic cc cac cc.cggc.cggc cqcc catago cago'cct cog to acct ctitc 12O accgcaccct cqgactg.ccc caaggcc.ccc gcc.gc.cgct C cagcgc.cgcg cagccaccgc 18O

cgc.cgc.cgcc gcct CtcCtt agtcgc.cgcc atgacgaccg cgt.ccacct C go aggtgcgc 24 O

Cagaact acc accaggactic agaggcc.gcc at Calacc.gcc agat caacct ggagct ctac 3 OO

gcc to citacg tttacctgtc. catgtc.ttac tactittgacc gcigatgatgt ggctittgaag 360

aactittgcca aatactittct tcaccaatct catgaggaga gggalacatgc tigagaaactg 42O

atgaagctgc agaac Caacg aggtggc.cga atct tcc ttic aggatat cala gaalaccagac 48O

tgtgatgact gggagagcgg gctgaatgca atggagtgtg cattacattt ggaaaaaaat 54 O US 8,084,017 B2 35 36 - Continued gtgaat cagt cact actgga actgcacaaa Ctggccactg acaaaaatga ccc.ccatttg tgttgactt cattgaga caca ttacctgaat gag caggtga aagc.cat caa agaattgggit 660 gaccacgtga cca acttgcg Caagatggga gcgc.ccgaat Ctggcttggc ggaat at ct c 72 O tittgacaa.gc acaccctggg agacagtgat aatgaaagct aagcct cqgg ctaattitcc c

Catagc.cgtggggtgactitc cctggtcacc aaggcagtgc atgcatgttggggttt CCtt 84 O taccttitt ct ataagttgta CCaaaa.catc. cacttaagtt ctittgatttg taccatt cot 9 OO tcaaataaag aaatttggta CCCtcaaaaa. aaaaaaaaaa. aaaaaaaaaa aaaaa. 955

<210s, SEQ ID NO 2 &211s LENGTH: 183 212. TYPE: PRT &213s ORGANISM: Homo sapiens

<4 OOs, SEQUENCE: 2

Met Thir Thir Ala Ser Thir Ser Glin Val Arg Glin Asn His Glin Asp 1. 1O 15

Ser Glu Ala Ala Ile Asn Arg Glin Ile Asn Lieu. Glu Lell Tyr Ala Ser 25 3 O

Wall Tyr Lieu Ser Met Ser Tyr Tyr Phe Asp Arg Asp Asp Wall Ala 35 4 O 45

Lell Lys Asin Phe Ala Lys Tyr Phe Lieu. His Glin Ser His Glu Glu Arg SO 55 60

Glu His Ala Glu Lys Lieu Met Lys Lieu. Glin Asn Glin Arg Gly Gly Arg 65 70 7s

Ile Phe Lieu. Glin Asp Ile Llys Llys Asp Asp Trp. Glu Ser 85 90 95

Gly Luell Asn Ala Met Glu. Cys Ala Lieu. His Lieu. Glu Asn. Wall Asn 105 11 O

Glin Ser Lieu. Lieu. Glu Lieu. His Lys Lieu Ala Thr Asp Lys Asn Asp Pro 115 12 O 125

His Luell Cys Asp Phe Ile Glu Thr His Tyr Lieu Asn Glu Glin Val Lys 13 O 135 14 O

Ala Ile Lys Glu Lieu. Gly Asp His Wall. Thir Asn Lell Arg Lys Met Gly 145 150 155 160

Ala Pro Glu Ser Gly Lieu Ala Glu Tyr Lieu Phe Asp His Thir Lieu. 1.65 17O 17s Gly Asp Ser Asp Asn. Glu Ser 18O

SEQ ID NO 3 LENGTH: 860 TYPE: DNA ORGANISM: Homo sapiens

< 4 OOs SEQUENCE: 3 tgcatcaaaa agctittattt c catttggtc Caaggcttgt taggatagitt aagaaagctg 6 O

Cctattggct ggagggagag gct taggcag aagcc ctatt actittgcaag gggcc ctitca 12 O gaagtc.gctg ggct cagaag gct cittagt c gtgcttgaga gtgagcctitt Caagagata 18O citcgcc cago cca.gc.ctic cq ggccacccag CCtgttggagg ttggit Caggit ggt cacc cat 24 O cittcttgata agctt cactt cct catctag gaagtgagtic tccaggaagt cacagagatg 3OO gggg.tc.cgtg cgggcagaac cCagggcatg aagat coaaa agggcctggit t cagctttitt 360

CtcCagggcc atggcagctt t catggcgtc tggggttitta c cc cact cat citt cagotgg

US 8,084,017 B2 39 40 - Continued gcatggcaga at at cit ctitt gacaa.gcaca C cctgggaca C9gtgatgag agctaagctg 72 O actt CCCCaa agccacgtga Ctt tactggt cactgaggca gtgcatgcat gtcaggctgc citt cat cit tt tctataagtt gcaccaaaac atctgcttaa gttctittaat ttgtaccatt 84 O tott Caaata aagaattittg gtaccc 866

SEQ ID NO 6 LENGTH: 182 TYPE : PRT ORGANISM: Mus musculus

< 4 OOs SEQUENCE: 6

Met Thir Thir Ala Ser Pro Ser Glin Val Arg Glin Asn Tyr His Glin Asp 1. 1O 15

Ala Glu Ala Ala Ile Asn Arg Glin Ile Asn Lieu. Glu Lell Tyr Ala Ser 25 3 O

Wall Tyr Leu Ser Met Ser Cys Tyr Phe Asp Arg Asp Asp Wall Ala 35 4 O 45

Lell Lys Asn Phe Ala Lys Tyr Phe Lieu. His Glin Ser His Glu Glu Arg SO 55 60

Glu His Ala Glu Lys Lieu Met Lys Lieu. Glin Asn Glin Arg Gly Gly Arg 65 70 7s

Ile Phe Luell Glin Asp Ile Llys Llys Pro Asp Arg Asp Asp Trp Glu Ser 85 90 95

Gly Luell Asn Ala Met Glu. Cys Ala Lieu. His Lieu. Glu Ser Wall Asn 105 11 O

Glin Ser Luell Lieu. Glu Lieu. His Lys Lieu Ala Thr Asp Lys Asn Asp Pro 115 12 O 125

His Luell Asp Phe Ile Glu Thr Tyr Tyr Lieu. Ser Glu Glin Val Lys 13 O 135 14 O

Ser Ile Glu Lieu. Gly Asp His Wall. Thir Asn Lell Arg Met Gly 145 150 155 160

Ala Pro Glu Ala Gly Met Ala Glu Tyr Lieu Phe Asp His Thir Lieu. 1.65 17O 17s

Gly His Gly Asp Glu Ser 18O

<210s, SEQ ID NO 7 &211s LENGTH: 920 &212s. TYPE: DNA &213s ORGANISM: Mus musculus

<4 OO > SEQUENCE: 7

Cagcgc.cttg gaggtoccgt. ggatctgtgt acttgct tca acagtgtttg aacggaacag 6 O accc.ggggat tcc cactgta citcgct tcca gcc.gc.ctitta caagt ct ct c Cagtc.gcagc 12 O

Ctc.cgggacc atctocticgc tgc ctitcago tcc tagg acc agtctgcacc gtct citt cqc 18O ggittagct Co tact coggat Cagc.catgac citct cagatt cgt.ca.gaatt att coaccoga 24 O ggtggaagct gcc.gtgalacc gcctgtcaa cittgcacctg cgggcct cot acaccitacct 3OO

Ctct Ctgggc ttctttitttg atcgggatga cgtggct Ctg galagg.cgtag gcc actt citt 360 cc.gcga attg gcc.gaggaga agcgc.gaggg cgcggagcgt. ctic ct coagt ttcagaacga tCgcgggggc cgtgcact ct tccaggatgt gCagaa.gc.ca t ct caagatg aatggggtaa alacc Caggag gcc atggaag Ctgccttggc Catggagaag aacct gaatc aggcc ct citt 54 O ggatctgcat gcc ctgggitt ctg.ccc.gcac ggaccct cat citctgtgact t cctgaaag US 8,084,017 B2 41 42 - Continued c cactatotg gataaggagg taaact cat Caagaagatg ggcaac catc tgaccaacct 660

gcggggccac alaccagogca gactggcgc.g CCC caggggt Ctctgggcga 72 O gtat ct ctitt gag.cgc.ctica ct citcaagca cgact aggag gcct ctd tac Ctt coaaggg gctic cc cc ct ctgctctgca cca.gc.ccgcc Ctgggacctic cacctgaatg aac Ctect Caa 84 O gccact aggc agctttgtaa ccgt.cctic ca gcct ctdtca agt cttggac caagtaaaaa 9 OO taaagcttitt tgaga.ccc.cg 92 O

SEQ ID NO 8 LENGTH: 183 TYPE : PRT ORGANISM: Mus musculus

< 4 OOs SEQUENCE: 8

Met Thir Ser Glin Ile Arg Glin Asn Ser Thir Glu Wall Glu Ala Ala 1. 5 15

Wall Asn Arg Luell Wall Asn Lieu. His Luell Arg Ala Ser Thir Tyr Lieu. 2O 25 3 O

Ser Luell Gly Phe Phe Phe Asp Arg Asp Asp Wall Ala Lell Glu Gly Val 35 4 O 45

Gly His Phe Phe Arg Glu Lieu. Ala Glu Glu Arg Glu Gly Ala Glu SO 55 60

Arg Luell Luell Glu Phe Glin Asn Asp Arg Gly Gly Arg Ala Luell Phe Glin 65 70 8O

Asp Wall Glin Ser Glin Asp Glu Trp Gly Thir Glin Glu Ala 85 90 95

Met Glu Ala Ala Lel Ala Met Glu Lys Asn Luell Asn Glin Ala Lieu. Luell 105 11 O

Asp Luell His Ala Lel Gly Ser Ala Arg Thir Asp Pro His Luell 115 12 O 125

Phe Luell Glu Ser His Tyr Lieu. Asp Glu Wall Lys Lell Ile 13 O 135 14 O

Met Gly Asn His Lel Thir Asn Lieu. Arg Arg Wall Ala Gly Pro Gln Pro 145 150 155 160

Ala Glin Thir Gly Ala Pro Glin Gly Ser Luell Gly Glu Luell Phe Glu 1.65 17O 17s

Arg Luell Thir Luell Lys His Asp 18O

<210s, SEQ ID NO 9 &211s LENGTH: 869 &212s. TYPE: DNA &213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 9 ggaagactgt aaaagt cittg tcattttgtt Cagtgaagtic CCCtcattca cat caccaag 6 O gatgatgaca gtct ct coag ticgcc.gcagc Ctc.cgggacc atctoctitgc cgc.ct tccgg 12 O t cctaggacc agc.ca.gc.ccc gtctt.cgcgg ttagctic cat act coggat.c agc catgacc 18O t ct cagattic gtcagaatta titccaccgaa gtggaagctg cc.gtgaaccg cctggtcaac 24 O ttgcacctgc gggcct citta caccitacctic t ct ctgggct totttitttga tcgggatgac 3OO gtggctttgg aaggcgtagg C cactitct tc cgc gaattgg cc.gaggagaa gcgc.gagggc 360 gcggagcgtC t cct caagtt gcagaacgaa cgcgggggcc gtgcact citt cCaggatgtg

Cagaagcc at Ctcaagatga gtggggtaaa accctggagg c catccaagc tgccttgcgc 48O US 8,084,017 B2 43 44 - Continued

Ctggagaaga acctgaacca ggc cctottg gatctgcacg CCCtgggctic toccgcaca 54 O gaccct cacc tctgtgacitt Cttggaaagc cact tcc togg ataaggaggt gaagct catc aagaagatgg gcaaccacct gacca acctic cgtagggtgg Cagggccaca accagtgcag 660 actggcgtgg cc.caggcatc tctgggcgag tat ct ctittg agcgcct cac tctgaagicac 72 O gact aggc ct ctgtgc ctitc Caaggggctic cct cotctgc tctgcaccoga cc.gc.cticago acct CCaCCC gaatgaac ct ctaaag.ccac taggcagctt tgtaaccocc Ctggagcct c 84 O t cccaagt ct tggaccalagt aaaaataaa. 869

SEQ ID NO 10 LENGTH: 183 TYPE : PRT ORGANISM: Mus musculus

< 4 OOs SEQUENCE: 1.O

Met Thir Ser Glin Ile Arg Glin Asn Tyr Ser Thr Glu Wall Glu Ala Ala 1. 5 1O 15

Wall Asn Arg Lieu Val Asn Lieu. His Lieu. Arg Ala Ser Thir Tyr Lieu. 25 3 O

Ser Luell Gly Phe Phe Phe Asp Arg Asp Asp Val Ala Lell Glu Gly Val 35 4 O 45

Gly His Phe Phe Arg Glu Lieu Ala Glu Glu Lys Arg Glu Gly Ala Glu SO 55 60

Arg Luell Luell Llys Lieu. Glin Asn. Glu Arg Gly Gly Arg Ala Luell Phe Glin 65 70

Asp Wall Glin Llys Pro Ser Glin Asp Glu Trp Gly Thir Luell Glu Ala 85 90 95

Ile Glin Ala Ala Lieu. Arg Lieu. Glu Lys Asn Lieu. Asn Glin Ala Lieu. Luell 105 11 O

Asp Luell His Ala Lieu. Gly Ser Ala Arg Thr Asp Pro His Luell 115 12 O 125

Phe Luell Glu Ser His Phe Lieu. Asp Lys Glu Val Lys Lell Ile 13 O 135 14 O

Met Gly Asn His Lieu. Thir Asn Lieu. Arg Arg Val Ala Gly Pro Gln Pro 145 150 155 160

Wall Glin Thir Gly Val Ala Glin Ala Ser Lieu. Gly Glu Luell Phe Glu 1.65 17O 17s

Arg Luell Thir Lieu Lys His Asp 18O

<210s, SEQ ID NO 11 &211s LENGTH: 830 &212s. TYPE: DNA <213> ORGANISM: Rattus norvegicus <4 OOs, SEQUENCE: 11 cgacagtgct talacggaac acc cct c cqa CCC cc.gc.cgg cc.gctttgag 6 O cctgagcc ct ttgcaactitc cgctic cagcg tcqcctic cqc gcct cqc cca 12 O gcc.gc.cat ca tgaccaccgc Caagtgcgc.c agaactacca cCaggacticg 18O gaggctgc.ca t caaccocca gat caacctg gagttgt atg cct cotacgt. citatctgtc.c 24 O atgtc.ttgtt attittgaccg ggatgatgttg gcc ctgaaga actittgccaa a tact t t c to 3OO

Catcaatcto atgaagagag ggalacatgct gagaaactga tgaagctgca galaccagcga 360 ggtggacgaa tct tcc tigca ggatataaag aaacctgacc gtgatgactg ggaga.gcggg US 8,084,017 B2 45 46 - Continued

Ctgaatgcaa. tggagtgttgc actgcacttg gaaaagagtg tgaat cagtic act actggaa

Cttcacaaac tggctactga Caagaatgat CCC Cactitat gtgactt cat tgagacgcat 54 O tacctgaatg agcaggtgaa atcCattaala galactgggtg accacgtgac caacttacgc aagatgggag c cc ctdaatc tgg catggca gaatat citct ttgacaa.gca Caccctggga 660 cacggtgatg agagctaagc tgacgtc.ccc aaggc catgt gacitt tactg gtcactgagg 72 O

Cagtgcatgc atgtcaggct gcc tittat ct tittctataag ttgcaccaaa a catctgctt aaaagttctt taatttgtac catttgttca aataaagaat tittgg taccc 83 O

<210s, SEQ I D NO 12 &211s LENGT H: 182 212. TYPE : PRT <213> ORGANISM: Rattus norvegicus

<4 OOs, SEQUENCE: 12

Met Thir Thr Ala Ser Pro Ser Glin Val Arg Glin Asn His Glin Asp 1. 5 1O 15

Ser Glu Ala Ala Ile Asn Arg Glin Ile Asn Lieu. Glu Lell Tyr Ala Ser 25 3 O

Tyr Val Tyr Leu Ser Met Ser Cys Tyr Phe Asp Arg Asp Asp Wall Ala 35 4 O 45

Lieu Lys Asn Phe Ala Lys Tyr Phe Lieu. His Glin Ser His Glu Glu Arg SO 55 60

Glu. His Ala Glu Lys Lieu Met Lys Lieu. Glin Asn Glin Arg Gly Gly Arg 65 70 7s

Ile Phe Lieu. Glin Asp Ile Llys Llys Asp Asp Trp Glu Ser 85 90 95

Gly Lieu. Asn Ala Met Glu. Cys Ala Lieu. His Lieu. Glu Ser Wall Asn 105 11 O

Glin Ser Lieu. Lieu. Glu Lieu. His Lys Lieu Ala Thr Asp Lys Asn Asp Pro 115 12 O 125

His Lieu. Cys Asp Phe Ile Glu Thr His Tyr Lieu Asn Glu Glin Val Lys 13 O 135 14 O

Ser Ile Llys Glu Lieu. Gly Asp His Wall. Thir Asn Lell Arg Met Gly 145 150 155 160

Ala Pro Glu Ser Gly Met Ala Glu Tyr Lieu Phe Asp His Thir Lieu. 1.65 17O 17s Gly His Gly Asp Glu Ser 18O

<210s, SEQ I D NO 13 &211s LENGT H: 552 212. TYPE : DNA <213> ORGANISM: Rattus norvegicus <4 OOs, SEQUENCE: 13 atgacct citc agatt.cgt.ca gaattatt co accgaagtgg aagctg.ccgt. galacc.gc.ctg 6 O gtcaacttgc acctg.cgggc Ctect tacacc tacct ct citc. tgggctt citt ttittgat cqg 12 O gatgacgtgg Ctttggaagg cgtaggccac ttct tcc.gcg aattggc.cga ggagaag.cgc 18O gagggcgc.cg agcgtc. tcct Caagttgcag aacgaacgcg actict tccag 24 O gatgtgcaga agc catctica agatgagtgg ggtaaaaccc tggaggc cat ggaagctgcc 3OO ttggcc ctgg agaagaac ct galaccaggcc citc.ttggatc tgcacgc cct gggct Ctgcc 360 cgcacagacic citcaccitctg tgactitcttg gaaagcc act tcc tiggataa ggaggtgaag

US 8,084,017 B2 51 52 - Continued gatgggcttgttittcttgcc aatgaggit ct gaaatggagg to Cttctgct ggataaaatg 312 O aggttcaact gttgattgca ggaataaggc cittaatatgt taacct cagt gtcatttatg 318O aaaagagggg accagaagcc aaagacittag tatattittct titt cotctgt ccct tcc ccc 324 O ataagcct co atttagttct ttgttattitt tdtttct tcc aaag.cacatt gaaagagaac 33 OO cagttt cagg tdtttagttg cagacticagt ttgtcagact ttaaagaata atatgctgcc 3360 aaattittggc caaagtgtta at Cttagggg agagctttct gtc.cttittgg cactgagata 342O tittattgttt attitat cagt gacagagttc actataaatg gtgtttittitt aatagaatat 3480 aattatcgga agcagtgcct tccataatta tdacagttat actgtcggitt ttttittaaat 354 O aaaag.cagca totgctaata aaacccaa.ca gatactggaa gttittgcatt tatggtcaac 36OO acttaagggit tittagaaaac agc.cgt.ca.gc caaatgtaat taataaagt talagctaag 366 O atttagagat gaattaaatt taattagggg ttgctaagaa gcgag cactg accagataag 372 O aatgctggitt titcctaaatg cagtgaattg tdaccaagtt ataaatcaat gtcacttaaa 378 O ggctgtgg tag tactic ct gc aaaattitt at agcticagttt atccaaggtg taactictaat 384 O tcc catttgc aaaattitcca gtacctttgt cacaatccta acacattatic gggagcagtg 3900 tott coataa totataaaga acaagg tagt ttttacctac cacagtgtct gitat cqgaga 396 O cagtgat citc catatgttac actaagggitg taagtaatta t cqggaacag tdttt cocat 4 O2O aattitt ctitc atgcaatgac atc.ttcaaag cittgaagatc gttagtatict aacatgitatic 4 O8O c caact cota taattic ccta t ctitt tagtt ttagttgcag aaa cattttgtggtcattaa 414 O gcattgggtg gigtaaattica accactgtaa aatgaaatta ctacaaaatt togaaatttag 42OO cittgggttitt tdttacctitt atggtttct c caggit cotct acttaatgag atagoag cat 426 O acatttataa totttgct at togacaagt cattittaattta t cacattatt togcatgttac 432O citcc tataaa cittagtgcgg acaagttitta atccagaatt gaccttittga cittaaag cag 438 O agggactittg tatagalaggt ttgggggctg tdgggaagga gag to cc ctgaaggit ctgac 4 44 O acgtctgcct acc catt.cgt gigtgat caat taaatgtagg tatgaataag titcgaagctic 4500 cgtgagtgaa ccatcatata aacgtgtagt acagctgttt gtcat agggc agttggaaac 456 O ggcct cotag ggaaaagttc at agggit ct c titcaggttct tagtgtcact tacctagatt 462O tacago: ct ca cittgaatgtg to act actica cagt citctitt aatcttcagt tittat ctitta 468O atct cotctt ttatcttgga citgacattta gcgtagctaa gtgaaaaggit catagct gag 474. O attic ctggitt C9ggtgttac gcacacgtac ttaaatgaaa gcatgtggca tott catcgt. 48OO ataacacaat atgaatacag gigcatgcatt ttgcago agit gag to tcttic agaaaac cct 486 O tittctacagt tagggttgag titact tcc ta t caa.gc.cagt acgtgctaac aggct caata 492 O titcc togaatgaaatat caga citagtgacaa got cotgg to ttgagatgtc. ttct cqttaa 498O ggagtagggc Cttittggagg taalaggtata 5010

<210s, SEQ ID NO 16 &211s LENGTH: 760 212. TYPE : PRT <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 16 Met Met Asp Glin Ala Arg Ser Ala Phe Ser Asn Lieu. Phe Gly Gly Glu 1. 5 15

Pro Lieu. Ser Tyr Thr Arg Phe Ser Lieu Ala Arg Glin Val Asp Gly Asp 25 3 O US 8,084,017 B2 53 54 - Continued

Asn Ser His Wall Glu Met Luell Ala Wall Asp Glu Glu Glu Asn Ala 35 4 O 45

Asp Asn Asn Thir Lys Ala Asn Wall Thir Lys Pro Lys Arg Ser Gly SO 55 60

Ser Ile Gly Thir Ile Ala Wall Ile Wall Phe Phe Luell Ile Gly 65 70

Phe Met Ile Gly Tyr Lell Gly Lys Gly Wall Glu Pro Lys Thir 85 90 95

Glu Glu Arg Lell Ala Gly Thir Glu Ser Pro Wall Arg Glu Glu Pro 105 11 O

Gly Glu Asp Phe Pro Ala Ala Arg Arg Luell Trp Asp Asp Luell 115 12 O 125

Arg Lys Luell Ser Glu Lell Asp Ser Thir Asp Phe Thir Ser Thir Ile 13 O 135 14 O

Lys Luell Luell Asn Glu Asn Ser Wall Pro Arg Glu Ala Gly Ser Glin 145 150 155 160

Asp Glu Asn Lell Ala Lell Wall Glu ASn Glin Phe Arg Glu Phe 1.65 17O

Luell Ser Lys Wall Trp Arg Asp Glin His Phe Wall Ile Glin Wall 18O 185 19 O

Asp Ser Ala Glin Asn Ser Wall Ile Ile Wall Asp Lys Asn Gly Arg 195

Lell Wall Luell Wall Glu Asn Pro Gly Gly Wall Ala Ser 21 O 215 22O

Ala Ala Thir Wall Thir Gly Luell Wall His Ala Asn Phe Gly Thir Lys 225 23 O 235 24 O

Asp Phe Glu Asp Lell Thir Pro Wall ASn Gly Ser Ile Wall Ile 245 250 255

Wall Arg Ala Gly Lys Ile Thir Phe Ala Glu Wall Ala Asn Ala Glu 26 O 265 27 O

Ser Luell Asn Ala Ile Gly Wall Luell Ile Met Asp Glin Thir Phe 27s 285

Pro Ile Wall Asn Ala Glu Lell Ser Phe Phe Gly His Ala His Luell Gly 29 O 295 3 OO

Thir Gly Asp Pro Tyr Thir Pro Gly Phe Pro Ser Phe Asn His Thir Glin 3. OS 310 315

Phe Pro Pro Ser Arg Ser Ser Gly Luell Pro ASn Ile Pro Wall Glin Thir 3.25 330 335

Ile Ser Arg Ala Ala Ala Glu Luell Phe Gly Asn Met Glu Gly Asp 34 O 345 35. O

Pro Ser Asp Trp Thir Asp Ser Thir Arg Met Wall Thir Ser 355 360 365

Glu Ser Asn Wall Lell Thir Wall Ser ASn Wall Lell Glu Ile 37 O 375

Lys Ile Luell Asn Ile Phe Gly Wall Ile Gly Phe Wall Glu Pro Asp 385 390 395 4 OO

His Wall Wall Wall Gly Ala Glin Arg Asp Ala Trp Gly Pro Gly Ala 4 OS 41O 415

Ala Ser Gly Wall Gly Thir Ala Luell Luell Luell Lell Ala Glin Met 425 43 O

Phe Ser Asp Met Wall Lell Asp Gly Phe Glin Pro Ser Arg Ser Ile 435 44 O 445

Ile Phe Ala Ser Trp Ser Ala Gly Asp Phe Gly Ser Wall Gly Ala Thir US 8,084,017 B2 55 56 - Continued

450 45.5 460

Glu Trp Luell Glu Gly Tyr Lieu Ser Ser Lieu. His Lell Ala Phe Thr 465 470 47s 48O

Ile Asn Lieu. Asp Lys Ala Val Leu Gly Thr Ser Asn Phe Llys Val 485 490 495

Ser Ala Ser Pro Leu Lleu Tyr Thr Lieu. Ile Glu Thir Met Glin Asn SOO 505

Wall His Pro Val Thr Gly Glin Phe Leu Tyr Glin Asp Ser Asn Trp 515 525

Ala Ser Val Glu Lys Lieu. Thr Lieu. Asp Asn Ala Ala Phe Pro Phe 53 O 535 54 O

Lell Ala Ser Gly Ile Pro Ala Wall Ser Phe Phe Glu Asp 5.45 550 555 560

Thir Asp Pro Tyr Lieu. Gly Thr Thr Met Asp Thir Glu Lieu. 565 st O sts

Ile Glu Arg Ile Pro Glu Lieu. Asn Llys Val Ala Arg Ala Ala Ala Glu 585 59 O

Wall Ala Gly Glin Phe Val Ile Llys Lieu. Thir His Asp Wall Glu Luell Asn 595 605

Lell Asp Glu Arg Tyr Asn. Ser Gln Lieu. Lieu. Ser Phe Wall Arg Asp 610 615

Lell Asn Glin Tyr Arg Ala Asp Ile Lys Glu Met Gly Lell Ser Lieu. Glin 625 630 635 64 O

Trp Luell Tyr Ser Ala Arg Gly Asp Phe Phe Arg Ala Thir Ser Arg Lieu. 645 650 655

Thir Thir Asp Phe Gly Asn Ala Glu Llys Thr Asp Arg Phe Wall Met Lys 660 665 67 O

Luell Asn Asp Arg Val Met Arg Val Glu Tyr His Phe Luell Ser Pro 675 685

Wall Ser Pro Lys Glu Ser Pro Phe Arg His Wall Phe Trp Gly Ser 69 O. 695 7 OO

Gly Ser His Thir Lieu. Pro Ala Lieu. Lieu. Glu Asn Lell Luell Arg Llys 7 Os 71O

Glin Asn Asn Gly Ala Phe Asn. Glu Thir Lieu. Phe Arg Asn Glin Lieu Ala 72 73 O 73

Lell Ala Thir Trp. Thir Ile Glin Gly Ala Ala Asn Ala Lell Ser Gly Asp 740 74. 7 O

Wall Trp Asp Ile Asp Asn. Glu Phe 7ss 760

<210s, SEQ ID NO 17 &211s LENGTH: 2513 &212s. TYPE: DNA <213> ORGANISM: Homo sapiens <4 OOs, SEQUENCE: 17

Ctgcaggctt Caggagggga Cacaa.gcatg gag.cggctitt ggggtctatt ccagaga.gc.g 6 O caacaactgt c cc caagatc citct cagacc gtc.taccago gtgttggalagg CCC ccggaaa 12 O gggCacct g aggaggalaga ggalagacggg gaggaggggg cggagacatt gg.cccacttic 18O tgc.cccatgg agctgagggg c cctdagc cc Ctgggcticta gacccaggca gccaaacctic 24 O attic cctggg cggcagcagg acggagggct gcc cc ct acc tggtcCtgac ggc cctgctg 3OO atct tcactg gggcct tcct actgggctac gagggtcCtg cCaggcgtgc 360 ggagacitctg tgttggtggit Cagtgaggat gtcaactato agcct gacct ggattitccac

US 8,084,017 B2 59 60 - Continued

1O 15

Arg Ser Ser Glin Thir Wall Glin Arg Wall Glu Gly Pro Arg Lys Gly 2O 25 3 O

His Luell Glu Glu Glu Glu Glu Asp Gly Glu Glu Gly Ala Glu Thir Luell 35 4 O 45

Ala His Phe Pro Met Glu Luell Arg Gly Pro Glu Pro Luell Gly Ser SO 55 60

Arg Pro Arg Glin Pro Asn Lell Ile Pro Trp Ala Ala Ala Gly Arg Arg 65 70

Ala Ala Pro Lell Wall Lell Thir Ala Luell Luell Ile Phe Thir Gly Ala 85 90 95

Phe Luell Luell Gly Tyr Wall Ala Phe Arg Gly Ser Glin Ala Gly 105 11 O

Asp Ser Wall Luell Wall Wall Ser Glu Asp Wall ASn Glu Pro Asp Luell 115 12 O 125

Asp Phe His Glin Gly Arg Lell Trp Ser Asp Lell Glin Ala Met Phe 13 O 135 14 O

Lell Glin Phe Luell Gly Glu Gly Arg Luell Glu Asp Thir Ile Arg Glin Thir 145 150 155 160

Ser Luell Arg Glu Arg Wall Ala Gly Ser Ala Gly Met Ala Ala Luell Thir 1.65 17s

Glin Asp Ile Arg Ala Ala Lell Ser Arg Glin Lell Asp His Wall Trp 18O 185 19 O

Thir Asp Thir His Tyr Wall Gly Luell Glin Phe Pro Asp Pro Ala His Pro 195

Asn Thir Luell His Trp Wall Asp Glu Ala Gly Wall Gly Glu Glin Luell 21 O 215 22O

Pro Luell Glu Asp Pro Asp Wall Pro Tyr Ser Ala Ile Gly Asn 225 23 O 235 24 O

Wall Thir Gly Glu Lell Wall Ala His Tyr Gly Arg Pro Glu Asp Luell 245 250 255

Glin Asp Luell Arg Ala Arg Gly Wall Asp Pro Wall Gly Arg Luell Luell Luell 26 O 265 27 O

Wall Arg Wall Gly Wall Ile Ser Phe Ala Glin Wall Thir Asn Ala Glin 28O 285

Asp Phe Gly Ala Glin Gly Wall Luell Ile Pro Glu Pro Ala Asp Phe 29 O 295 3 OO

Ser Glin Asp Pro Pro Lys Pro Ser Luell Ser Ser Glin Glin Ala Wall Tyr 3. OS 310 315

Gly His Wall His Lell Gly Thir Gly Asp Pro Thir Pro Gly Phe Pro 3.25 330 335

Ser Phe Asn Glin Thir Glin Luell Lys Gly Pro Wall Ala Pro Glin Glu 34 O 345 35. O

Trp Glin Gly Ser Lell Lell Gly Ser Pro His Lell Gly Pro Gly Pro 355 360 365

Arg Luell Arg Luell Wall Wall Asn Asn His Arg Thir Ser Thir Pro Ile Asn 37 O 375

Asn Ile Phe Gly Cys Ile Glu Gly Arg Ser Glu Pro Asp His Wall 385 390 395 4 OO

Wall Ile Gly Ala Glin Arg Asp Ala Trp Gly Pro Gly Ala Ala Lys Ser 4 OS 41O 415

Ala Wall Gly Thir Ala Ile Lell Luell Glu Luell Wall Arg Thir Phe Ser Ser 42O 425 43 O US 8,084,017 B2 61 - Continued Met Val Ser Asn Gly Phe Arg Pro Arg Arg Ser Lieu Lleu Phe Ile Ser 435 44 O 445 Trp Asp Gly Gly Asp Phe Gly Ser Val Gly Ser Thr Glu Trp Lieu. Glu 450 45.5 460 Gly Tyr Lieu. Ser Val Lieu. His Lieu Lys Ala Val Val Tyr Val Ser Lieu. 465 470 47s 48O Asp Asn Ala Val Lieu. Gly Asp Asp Llys Phe His Ala Lys Thir Ser Pro 485 490 495 Lieu. Lieu. Thir Ser Lieu. Ile Glu Ser Val Lieu Lys Glin Val Asp Ser Pro SOO 505 51O Asn His Ser Gly Glin Thr Lieu. Tyr Glu Glin Val Val Phe Thr Asn Pro 515 52O 525 Ser Trp Asp Ala Glu Val Ile Arg Pro Lieu Pro Met Asp Ser Ser Ala 53 O 535 54 O Tyr Ser Phe Thr Ala Phe Val Gly Val Pro Ala Val Glu Phe Ser Phe 5.45 550 555 560 Met Glu Asp Asp Glin Ala Tyr Pro Phe Lieu. His Thr Lys Glu Asp Thr 565 st O sts Tyr Glu Asn Lieu. His Llys Val Lieu. Glin Gly Arg Lieu Pro Ala Val Ala 58O 585 59 O Glin Ala Val Ala Glin Lieu Ala Gly Glin Lieu. Lieu. Ile Arg Lieu. Ser His 595 6OO 605 Asp Arg Lieu. Lieu Pro Lieu. Asp Phe Gly Arg Tyr Gly Asp Val Val Lieu 610 615 62O Arg His Ile Gly ASn Lieu. ASn Glu Phe Ser Gly Asp Lieu Lys Ala Arg 625 630 635 64 O Gly Lieu. Thir Lieu. Glin Trp Val Tyr Ser Ala Arg Gly Asp Tyr Ile Arg 645 650 655 Ala Ala Glu Lys Lieu. Arg Glin Glu Ile Tyr Ser Ser Glu Glu Arg Asp 660 665 67 O Glu Arg Lieu. Thir Arg Met Tyr Asn Val Arg Ile Met Arg Ile Pro Lieu. 675 68O 685 Ser Ala Glin Val Glu Phe Tyr Phe Leu Ser Glin Tyr Val Ser Pro Ala 69 O. 695 7 OO Asp Ser Pro Phe Arg His Ile Phe Met Gly Arg Gly Asp His Thr Lieu. 7 Os 71O 71s 72O Gly Ala Lieu. Lieu. Asp His Lieu. Arg Lieu. Lieu. Arg Ser Asn. Ser Ser Gly 72 73 O 73 Thr Pro Gly Ala Thr Ser Ser Thr Gly Phe Glin Glu Ser Arg Phe Arg 740 74. 7 O Arg Glin Lieu Ala Lieu. Lieu. Thir Trp Thir Lieu. Glin Gly Ala Ala Asn Ala 7ss 760 765 Lieu. Ser Gly Asp Val Trp Asn. Ile Asp Asn. Asn. Phe 770 775 78O

<210s, SEQ ID NO 19 &211s LENGTH: 2292 &212s. TYPE: DNA <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 19 atgatggat.c aagc.ca.gatc agcattct ct a acttgtttg gtggggalacc attgt catac 6 O accc.ggttta gccttgct cq gcaagtagat ggagatalaca gtcatgtgga gatgaaactg 12 O gctgcagatg aagaagaaaa to cacaat alacatgaagg Ctagtgtcag aaaac ccaag 18O

US 8,084,017 B2 65 66 - Continued

Met Met Asp Glin Ala Arg Ser Ala Phe Ser ASn Lell Phe Gly Gly Glu 1O 15

Pro Luell Ser Tyr Thir Arg Phe Ser Luell Ala Arg Glin Wall Asp Gly Asp 25 3 O

Asn Ser His Wall Glu Met Luell Ala Ala Asp Glu Glu Glu Asn Ala 35 4 O 45

Asp Asn Asn Met Lys Ala Ser Wall Arg Pro Lys Arg Phe Asn Gly SO 55 60

Arg Luell Phe Ala Ala Ile Ala Luell Wall Ile Phe Phe Luell Ile Gly 65 70

Phe Met Ser Gly Tyr Lell Gly Tyr Lys Arg Wall Glu Glin Lys Glu 85 90 95

Glu Wall Lys Lell Ala Glu Thir Glu Glu Thir Asp Ser Glu Thir 105 11 O

Met Glu Thir Glu Asp Wall Pro Thir Ser Ser Arg Lell Tyr Trp Ala Asp 115 12 O 125

Lell Lys Thir Luell Lell Ser Glu Luell Asn Ser Ile Glu Phe Ala Asp 13 O 135 14 O

Thir Ile Glin Lell Ser Glin Asn Thir Thir Pro Arg Glu Ala Gly 145 150 155 160

Ser Glin Asp Glu Ser Lell Ala Tyr Ile Glu Asn Glin Phe His 1.65 17O 17s

Glu Phe Phe Ser Wall Trp Arg Asp Glu His Wall Ile 18O 185 19 O

Glin Wall Lys Ser Ser Ile Gly Glin Asn Met Wall Thir Ile Wall Glin Ser 195

Asn Gly Asn Luell Asp Pro Wall Glu Ser Pro Glu Gly Wall Ala Phe 21 O 215 22O

Ser Pro Thir Glu Wall Ser Gly Luell Wall His Ala Asn Phe Gly 225 23 O 235 24 O

Thir Asp Phe Glu Glu Luell Ser Tyr Ser Wall Asn Gly Ser Luell 245 250 255

Wall Ile Wall Arg Ala Gly Glu Ile Thir Phe Ala Glu Wall Ala Asn 26 O 265 27 O

Ala Glin Ser Phe Asn Ala Ile Gly Wall Luell Ile Met Asp Asn 27s 285

Phe Pro Wall Wall Glu Ala Asp Luell Ala Luell Phe Gly His Ala His 29 O 295 3 OO

Lell Gly Thir Gly Asp Pro Thir Pro Gly Phe Pro Ser Phe Asn His 3. OS 310 315

Thir Glin Phe Pro Pro Ser Glin Ser Ser Gly Luell Pro Asn Ile Pro Wall 3.25 330 335

Glin Thir Ile Ser Arg Ala Ala Ala Glu Luell Phe Gly Lys Met Glu 34 O 345 35. O

Gly Ser Cys Pro Ala Arg Trp Asn Ile Asp Ser Ser Cys Luell Glu 355 360 365

Lell Ser Glin Asn Glin Asn Wall Luell Ile Wall Lys Asn Wall Luell 37 O 375

Glu Arg Arg Ile Lell Asn Ile Phe Gly Wall Ile Gly Glu Glu 385 390 395 4 OO

Pro Asp Arg Wall Wall Wall Gly Ala Glin Arg Asp Ala Luell Gly Ala 4 OS 415

Gly Wall Ala Ala Lys Ser Ser Wall Gly Thir Gly Lell Lell Luell Luell 42O 425 43 O US 8,084,017 B2 67 68 - Continued

Ala Glin Val Phe Ser Asp Met Ile Ser Lys Asp Gly Phe Arg Pro Ser 435 44 O 445

Arg Ser Ile Ile Phe Ala Ser Trp Thr Ala Gly Asp Phe Gly Ala Wall 450 45.5 460

Gly Ala Thr Glu Trp Lieu. Glu Gly Tyr Lieu Ser Ser Lell His Lieu Lys 465 470 47s 48O

Ala Phe Thir Tyr Ile Asn Lieu. Asp Llys Val Val Lell Gly Thir Ser Asn 485 490 495

Phe Llys Val Ser Ala Ser Pro Leu Leu Tyr Thr Lell Met Gly Lys Ile SOO 505

Met Glin Asp Val Lys His Pro Val Asp Gly Lys Ser Lell Arg Asp 515 525

Ser Asn Trp Ile Ser Llys Val Glu Llys Lieu. Ser Phe Asp Asn Ala Ala 53 O 535 54 O

Tyr Pro Phe Leu Ala Tyr Ser Gly Ile Pro Ala Wall Ser Phe Cys Phe 5.45 550 555 560

Cys Glu Asp Ala Asp Tyr Pro Tyr Leu Gly Thr Arg Lell Asp Thr Tyr 565 st O sts

Glu Ala Lieu. Thr Glin Llys Val Pro Gln Lieu. Asn Glin Met Wall Arg Thr 585 59 O

Ala Ala Glu Val Ala Gly Glin Lieu. Ile Ile Llys Lell Thir His Asp Wall 595 605

Glu Lieu. Asn Lieu. Asp Tyr Glu Met Tyr Asn Ser Lys Lell Luell Ser Phe 610 615

Met Lys Asp Lieu. Asn Glin Phe Lys Thr Asp Ile Arg Asp Met Gly Lieu. 625 630 635 64 O

Ser Lieu Gln Trp Lieu. Tyr Ser Ala Arg Gly Asp Phe Arg Ala Thr 645 650 655

Ser Arg Lieu. Thir Thr Asp Phe His Asn Ala Glu Thir Asn Arg Phe 660 665 67 O

Val Met Arg Glu Ile Asn Asp Arg Ile Met Lys Wall Glu His Phe 675 685

Lieu. Ser Pro Tyr Val Ser Pro Arg Glu Ser Pro Phe Arg His Ile Phe 69 O. 695 7 OO

Trp Gly Ser Gly Ser His Thr Lieu. Ser Ala Lieu Wall Glu Asn Lieu Lys 7 Os 71O 71s 72O

Lieu. Arg Gln Lys Asn. Ile Thr Ala Phe Asn. Glu Thir Lell Phe Arg Asn 72 73 O 73

Glin Lieu Ala Lieu Ala Thir Trp Thr Ile Glin Gly Wall Ala Asn Ala Lieu 740 74. 7 O

Ser Gly Asp Ile Trp Asn. Ile Asp Asn. Glu Phe 760

<210s, SEQ ID NO 21 &211s LENGTH: 2859 &212s. TYPE: DNA <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 21 aaaaaaaaaa attgattgtt ttgcagtctg CCC.gcaa.cag tggggtttgt ggaaagattg 6 O agttcaggag giggcacaag Catggagcaa. cgttggggtc tactt.cggag agtgcaa.ca.g 12 O tggit coccaa gaccct ct ca gac catctac agacgc.gtgg aaggc cct ca gctggagcac 18O

Ctggaggagg alagacaggga ggaagggg.cg gagct tcctg cc.cagttctg CCC catggaa 24 O

US 8,084,017 B2 71 - Continued cagaaag.cgg ttittct tccc at cacaggcc cittctgtc.tt caggagcaaa gttc.cccata 27 OO tctagagact atctagatgc tigggatctga t cagct Ctct tagagagtga gatggacagc 276 O gtcatt attt tatgacacat gagctacggit atgtgagcag cccaagggga ttagatgtca 282O ataaac caat tdtaac ccca aaaaaaaaaa aaaaaaaaa 2859

<210s, SEQ ID NO 22 &211s LENGTH: 798 212. TYPE: PRT <213s ORGANISM: Mus musculus

<4 OOs, SEQUENCE: 22 Met Glu Glin Arg Trp Gly Lieu. Lieu. Arg Arg Val Glin Gln Trp Ser Pro 1. 5 1O 15 Arg Pro Ser Glin Thr Ile Tyr Arg Arg Val Glu Gly Pro Gln Leu Glu 2O 25 3 O His Lieu. Glu Glu Glu Asp Arg Glu Glu Gly Ala Glu Lieu Pro Ala Glin 35 4 O 45 Phe Cys Pro Met Glu Lieu Lys Gly Pro Glu. His Leu Gly Ser Cys Pro SO 55 60 Gly Arg Ser Ile Pro Ile Pro Trp Ala Ala Ala Gly Arg Lys Ala Ala 65 70 7s 8O Pro Tyr Lieu Val Lieu. Ile Thr Lieu. Lieu. Ile Phe Thr Gly Ala Phe Leu 85 90 95 Lieu. Gly Tyr Val Ala Phe Arg Gly Ser Cys Glin Ala Cys Gly Asp Ser 1OO 105 11 O Val Lieu Val Val Asp Glu Asp Val Asn Pro Glu Asp Ser Gly Arg Thr 115 12 O 125 Thir Lieu. Tyr Trp Ser Asp Lieu. Glin Ala Met Phe Lieu. Arg Phe Lieu. Gly 13 O 135 14 O Glu Gly Arg Met Glu Asp Thir Ile Arg Lieu. Thir Ser Lieu. Arg Glu Arg 145 150 155 160 Val Ala Gly Ser Ala Arg Met Ala Thir Lieu Val Glin Asp Ile Lieu. Asp 1.65 17O 17s Llys Lieu. Ser Arg Glin Llys Lieu. Asp His Val Trp Thr Asp Thir His Tyr 18O 185 19 O Val Gly Lieu. Glin Phe Pro Asp Pro Ala His Ala Asn Thr Lieu. His Trp 195 2OO 2O5 Val Asp Ala Asp Gly Ser Val Glin Glu Glin Lieu Pro Lieu. Glu Asp Pro 21 O 215 22O Glu Val Tyr Cys Pro Tyr Ser Ala Thr Gly Asn Ala Thr Gly Lys Lieu. 225 23 O 235 24 O Val Tyr Ala His Tyr Gly Arg Ser Glu Asp Lieu. Glin Asp Lieu Lys Ala 245 250 255 Lys Gly Val Glu Lieu Ala Gly Ser Lieu. Lieu. Lieu Val Arg Val Gly Ile 26 O 265 27 O Thir Ser Phe Ala Glin Llys Val Ala Val Ala Glin Asp Phe Gly Ala Glin 27s 28O 285 Gly Val Lieu. Ile Tyr Pro Asp Pro Ser Asp Phe Ser Glin Asp Pro His 29 O 295 3 OO Llys Pro Gly Lieu Ser Ser His Glin Ala Val Tyr Gly His Val His Leu 3. OS 310 315 32O Gly Thr Gly Asp Pro Tyr Thr Pro Gly Phe Pro Ser Phe Asin Glin Thr 3.25 330 335

Glin Phe Pro Pro Val Glu Ser Ser Gly Lieu Pro Ser Ile Pro Ala Glin US 8,084,017 B2 73 74 - Continued

34 O 345 35. O

Pro Ile Ser Ala Asp Ile Ala Asp Glin Luell Luell Arg Lys Luell Thir Gly 355 360 365

Pro Wall Ala Pro Glin Glu Trp Gly His Luell Ser Gly Ser Pro Tyr 37 O 375

Arg Luell Gly Pro Gly Pro Asp Luell Arg Luell Wall Wall Asn Asn His Arg 385 390 395 4 OO

Wall Ser Thir Pro Ile Ser Asn Ile Phe Ala Cys Ile Glu Gly Phe Ala 4 OS 415

Glu Pro Asp His Tyr Wall Wall Ile Gly Ala Glin Arg Asp Ala Trp Gly 425 43 O

Pro Gly Ala Ala Ser Ala Wall Gly Thir Ala Ile Lell Luell Glu Luell 435 44 O 445

Wall Arg Thir Phe Ser Ser Met Wall Ser Asn Gly Phe Arg Pro Arg Arg 450 45.5 460

Ser Luell Luell Phe Ile Ser Trp Asp Gly Gly Asp Phe Gly Ser Wall Gly 465 470

Ala Thir Glu Trp Lell Glu Gly Tyr Luell Ser Wall Lell His Luell Lys Ala 485 490 495

Wall Wall Wall Ser Lell Asp Asn Ser Wall Luell Gly Asp Gly Phe SOO 505

His Ala Lys Thir Ser Pro Lell Luell Wall Ser Luell Ile Glu Asn Ile Luell 515 525

Glin Wall Asp Ser Pro Asn His Ser Gly Glin Thir Lell Tyr Glu Glin 53 O 535 54 O

Wall Ala Luell Thir His Pro Ser Trp Asp Ala Glu Wall Ile Glin Pro Luell 5.45 550 555 560

Pro Met Asp Ser Ser Ala Ser Phe Thir Ala Phe Ala Gly Wall Pro 565 st O sts

Ala Wall Glu Phe Ser Phe Met Glu Asp Asp Arg Wall Pro Phe Luell 585 59 O

His Thir Glu Glu Asp Thir Glu Asn Luell His Met Luell Arg Gly 595 605

Arg Luell Pro Ala Wall Wall Glin Ala Wall Ala Glin Lell Ala Gly Glin Luell 610 615

Lell Ile Arg Luell Ser His Asp His Luell Luell Pro Lell Asp Phe Gly Arg 625 630 635 64 O

Gly Asp Wall Wall Lell Arg His Ile Gly ASn Lell Asn Glu Phe Ser 645 650 655

Gly Asp Luell Lys Glu Arg Gly Luell Thir Luell Glin Trp Wall Tyr Ser Ala 660 665 67 O

Arg Gly Asp Ile Arg Ala Ala Glu Luell Arg Lys Glu Ile Tyr 675 685

Ser Ser Glu Arg Asn Asp Glu Arg Luell Met Arg Met Asn Wall Arg 69 O. 695 7 OO

Ile Met Arg Wall Glu Phe Phe Luell Ser Glin Wall Ser Pro Ala 7 Os

Asp Ser Pro Phe Arg His Ile Phe Luell Gly Glin Gly Asp His Thir Luell 72 73 O 73

Gly Ala Luell Wall Asp His Lell Arg Met Luell Arg Ala Asp Gly Ser Gly 740 74. 7 O

Ala Ala Ser Ser Arg Lell Thir Ala Gly Luell Gly Phe Glin Glu Ser Arg 7ss 760 765 US 8,084,017 B2 75 76 - Continued Phe Arg Arg Glin Lieu Ala Lieu. Lieu. Thir Trp Thir Lieu. Glin Gly Ala Ala 770 775 78O Asn Ala Lieu. Ser Gly Asp Val Trp Asn. Ile Asp Asn. Asn. Phe 78s 79 O 79.

What is claimed: 4. The method of claim 1 or 3, wherein the ferritin is 1. A method of generating an image of a subject material 10 selected from a group consisting of a protein comprising SEQ comprising ID NO: 2 and a protein comprising SEQID NO: 4. providing a Subject material comprising a plurality of cells 5. The method of claim 3, wherein the cell is part of a cell wherein a subset of the cells express a recombinant culture. nucleic acid encoding an MRI-detectable amount offer 6. The method of claim 3, wherein the cell is part of an in ritin; and 15 vitro tissue. detecting the cells by magnetic resonance imaging caused 7. The method of claim 3, wherein the cell is part of a by the expressed ferritin. multicellular organism. 2. The method of claim 1, wherein the cells comprising the 8. The method of claim 3, wherein the cell is part of a measurable amount of ferritin are distinguishable from cells mammal. or other components of the material that do not comprise the 9. The method of claim3, wherein the cell is part of a plant. measurable amount of ferritin. 10. The method of claim 1 or 3, wherein the recombinant 3. A method of detecting the expression of a recombinant nucleic acid is operably linked to a regulatory sequence. nucleic acid encoding ferritin comprising 11. The method 10, wherein the regulatory sequence is providing a cell expressing a recombinant nucleic acid active in situ. encoding ferritin; and 25 12. The method of claim 10, wherein the regulatory detecting the cell by magnetic resonance imaging caused sequence is a constitutive regulatory sequence. by the expressed ferritin: 13. The method of claim 10, wherein the regulatory wherein the detection of ferritin by magnetic resonance sequence is exogenously regulated. imaging indicates that the nucleic acid encoding the ferritin is and/or has been expressed. k k k k k