USOO8569002B2

(12) United States Patent (10) Patent No.: US 8,569,002 B2 Lum et al. (45) Date of Patent: Oct. 29, 2013

(54) MULTIPLEXEDLUCIFERASE REPORTER Grin et al., “Investigations in the imidazole series: Reaction of ASSAY SYSTEMS 2-aminobenzothiazoles with C.—haloketones.” Chemistry of Hetero cyclic Compounds, 9:1149-1152, 1972. (75) Inventors: Lawrence Lum, Dallas, TX (US); Jones and Stanforth, "ChemInform Abstract: The Vilsmeier Reaction Ozlem Kulak, Dallas, TX (US) of Fully Conjugated Carbocycles and Heterocycles.” Cheminform, 28: No. doi:10.1002/chin. 1997 15268, 1997. (73) Assignee: The Board of Regents of the University Mase and Murase, "Nucleophilic substitution reactions on sulfur by of Texas System, Austin, TX (US) n-butyllithium 2.” Heterocycles, 26:3159-3164, 1987. Mase et al., “Imidazo[2.1-bbenzothiazoles. 2. New immunosup (*) Notice: Subject to any disclaimer, the term of this pressive agents.” Journal of Medicinal Chemistry, 29(3):386-94, patent is extended or adjusted under 35 1986. U.S.C. 154(b) by 320 days. Maseet al., “Nucleophilic substitution reactions on sulfur by n-butyl lithium. 2...” Heterocycles, 26(12):3159-64, 1987. (21) Appl. No.: 12/908,754 Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN 1188029-32-4 (accessed Aug. 13, 2010). (22) Filed: Oct. 20, 2010 Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN 1188215-75-9(accessed Aug. 13, 2010), calculated using ACD/Labs (65) Prior Publication Data Software version 11.02 1994-2010. Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN US 2012/O1 O2581 A1 Apr. 26, 2012 11882 15-85-1 (accessed Aug. 13, 2010), calculated using ACD/Labs Software version 11.02 1994-2010. (51) Int. Cl. Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN CI2O I/66 (2006.01) 1188029-25-5 (accessed Aug. 13, 2010). CI2O I/68 (2006.01) Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN GOIN33/567 (2006.01) 1188217-51-7 (accessed Aug. 13, 2010), calculated using ACD/Labs (52) U.S. Cl. Software version 11.02 1994-2010. USPC ...... 435/8; 435/6.1; 435/7.21 Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN 1188217-64-2 (accessed Aug. 13, 2010), calculated using ACD/Labs (58) Field of Classification Search Software version 11.02 1994-2010. USPC ... 435/8 Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN See application file for complete search history. 24247-14-1 (accessed Aug. 13, 2010). Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN (56) References Cited 352200-23-8 (accessed Aug. 13, 2010). U.S. PATENT DOCUMENTS Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN 38956-27-3 (accessed Aug. 13, 2010), calculated using ACD/Labs 5,814,471 A 9, 1998 Wood ...... 435.8 Software version 11.02 1994-2010. 7,601,846 B2 10/2009 Cottam et al...... 548.151 Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN 419557-50-9 (accessed Aug. 13, 2010), calculated using ACD/Labs FOREIGN PATENT DOCUMENTS Software version 11.02 1994-2010. Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN JP 2001.048786 2, 2001 7178-23-6 (accessed Aug. 13, 2010), calculated using ACD/Labs WO WO O2, 14321 2, 2002 Software version 11.02 1994-2010. OTHER PUBLICATIONS Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN 940394-25-2 (accessed Aug. 13, 2010), calculated using ACD/Labs “BioLuxTM Cyprodina Luciferase Assay Kit: Instruction Manual Software version 11.02 1994-2010. E3309” New England BioLabs, Ipswich, MA, Dec. 2009. Scifinder; Chemical Abstracts Service: Columbus, OH, 2010, RN “BioLuxTM Gaussia Luciferase Assay Kit: Instruction Manual 940438-95-9 (accessed Aug. 13, 2010), calculated using ACD/Labs E3300” New England BioLabs, Ipswich, MA, Nov. 2009. Software version 11.02 1994-2010. Angelopoulos et al., "Bayseian model averaging for ligand discov ery,” Journal of Chemical Information and Modeling, 49(6): 1547 Primary Examiner — Valarie Bertoglio 1557, 2009. (74) Attorney, Agent, or Firm — Parker Highlander PLLC Barchechath et al., “Inhibitors of apoptosis in lymphocytes: Synthe sis and biological evaluation of compounds related to pifithrin-O.” (57) ABSTRACT Journal of Medicinal Chemistry, 48(20):6409-6422, 2005. El-shorbagi et al., “Imidazo[2,1-bbenzothiazoles. I.” Chemical & Inhibitors of luciferase enzymes are disclosed and find use in Pharmaceutical Bulletin, 36(12):4760-8, 1988. multiplexed assays using multiple luciferases and multiple Ei-Shorbagiet al., “Imidazo[2.1-bbenzothiazoles. II. Synthesis and inhibitors, in both in vitro and in vivo embodiments. anti-inflammatory activity of some imidazol2.1-bbenzothiazoles.” Chemical & Pharmaceutical Bulletin, 37(11):2971-5, 1989. 18 Claims, 6 Drawing Sheets U.S. Patent Oct. 29, 2013 Sheet 1 of 6 US 8,569,002 B2

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Sample

lysate medium D-luciferin |

FL FL inhibitor coelenterazine R RLFL or RLls (to inhibit RFL or RLS | escaped RL) colenterazine GL-KDEL GL GL -signal rSequence allow insecaySidnal to deca | allow RSidnal to die oxyluciferin oxyluciferin CL-KDEL C CL-signalOr sequence

FL: luciferase (cytoplasmic) RL: Renilla luciferase (cytoplasmic GL: Gaussia luciferase (Secreted) GL-KDEL: Gaussia luciferase with ER retention signal Gl-signal sequence: Gaussia luciferase expressed in cytoplasm CL: Cypridina luciferase (secreted) CL-KDEL: Cypridina luciferase with ER retention signal CL-signal sequence: Cypridina luciferase expressed in cytoplasm coelenterazine: Substrate for RL and GL luciferin: substrate for FL oxyluciferin: substrate for CL

FIG 5 US 8,569,002 B2 1. 2 MULTIPLEXEDLUCFERASE REPORTER chemiluminescence from each of the plurality of distinct ASSAY SYSTEMIS luciferase molecules, wherein other of the plurality of the distinct luciferase molecules are inhibited or lack substrate. STATEMENT REGARDING FEDERALLY The plurality of distinct luciferase molecules may be pro SPONSORED RESEARCH ORDEVELOPMENT 5 vided sequentially or simultaneously. The plurality of distinct luciferase molecules may comprise 2, 3, 4, 5 or 6 distinct This invention was made in part with government Support luciferase molecules. The plurality of distinct luciferase under Grant No. 1 RO1GMO76398 from the National Insti inhibitors may comprise 2, 3, or 4 distinct luciferase inhibi tutes of Health. The United States Government has certain tOrS. rights in the invention. 10 The plurality of distinct luciferase molecules may com prise two or more or each of firefly luciferase, renilla TECHNICAL FIELD luciferase, gaussia luciferase, cypridina luciferase, gaussia luciferase lacking a secretion signal sequence or having an The present invention relates to the fields of molecular endoplasmic retention sequence, or cypridina luciferase lack biology, biochemistry, and drug screening. More particularly, 15 ing a secretion signal sequence or having an endoplasmic the present invention relates to the use of multiple luciferases retention sequence. The plurality of luciferases may be and multiple inhibitors thereof to create a multiplexed system expressed from cells in culture. The method may further permitting analysis of multiple targets at the same time and in comprise separating the cells from cell medium, Such as by Sequence. disrupting the cells to form a cell lysate. The substrates may comprise two or all three of D-lu BACKGROUND OF THE INVENTION ciferin, oxyluciferin, and coelenterazine. The inhibitor may be selective or specific for renilla luciferase, such as: Reporter genes have become an invaluable tool in studies of gene expression. They are widely used in biomedical and pharmaceutical research and also in molecular biology and 25 C biochemistry. A key feature in reporter assays is an expression cassette comprising a coding region for marker of interestand a transcriptional regulatory region, i.e., a promoter regulatory 1N region, that drives expression of the reporter. Any agent can then be tested for direct or indirect effects on the promoter, 30 O )- thereby identifying agents with potential value in directing 8. S-N control of products naturally driven by the promoter regula | tory region. Common reporters are B-galactosidase, B-glucu O ronidase and luciferase, which rely upon various readouts including luminescence, absorbance and fluorescence. 35 Luciferase is a generic term for the class of oxidative whereina is substituted or unsubstituted alkyl (C-Co), Sub enzymes used in and is distinct from a pho stituted or unsubstituted alkenyl (C-C), substituted or toprotein. The name is derived from Lucifer, the root of which unsubstituted alkynyl (C-C). Substituted or unsubstituted means “light-bearer. The advantages of a luciferase assay are alkyoxy (C-C), amido or halide, b is H. Substituted or the high sensitivity, the absence of luciferase activity inside 40 unsubstituted alkyl (C-C), substituted or unsubstituted most of the cell types, the wide dynamic range, rapidity and alkenyl (C-C), Substituted or unsubstituted alkynyl (C- low costs. One example is the firefly luciferase from the firefly Co), Substituted or unsubstituted alkyoxy (C-C), amido or pyralis, and this protein requires no post-transla halide, and c is H. Substituted or unsubstituted alkyl (C-C), tional modification for enzyme activity; it is not even toxic in substituted or unsubstituted alkenyl (C-C), substituted or high concentration (in vivo) and can be used in pro- and 45 unsubstituted alkynyl (C-C). Substituted or unsubstituted eukaryotic cells. Firefly luciferase catalyzes the biolumines alkyoxy (C-C), amido or halide. Alternatively, the inhibi cent oxidation of the luciferin in the presence of ATP mag tor may be selective or specific for firefly luciferase, such as: nesium and oxygen. In many assay formats, it is useful to examine multiple effects using distinct reporter cassettes. This is termed “mul 50 tiplexing, i.e., using multiple readouts to simultaneous assess various effects. By using multiplexed assays, one can acquire information much more quickly. While there exist differentluciferase enzymes, current assays only permit look ing at two different reporters within the confines of a single 55 assay. Thus, there remains a need to develop reagents that wherein a is H, substituted or unsubstituted alkyl (C-C), permit higher levels of multiplex luciferase assays. substituted or unsubstituted alkenyl (C-C), substituted or unsubstituted alkynyl (C-C), Substituted or unsubstituted SUMMARY OF THE INVENTION alkyoxy (C-C), amido or halide. The inhibitor may be 60 cross-reactive with renilla luciferase and firefly luciferase. Thus, in accordance with the present invention, there is In another embodiment, there is provided a biological sys provided a method of detecting a plurality of distinct tem comprising (a) a plurality of distinct luciferase molecules luciferase molecules comprising (a) providing a plurality of and substrates therefor; and (b) a plurality of distinct distinct luciferase molecules and substrates therefor; (b) pro luciferase inhibitors, wherein the plurality of inhibitors viding a plurality of distinct luciferase inhibitors, wherein the 65 inhibit at least two of the plurality of distinct luciferase mol plurality of inhibitors inhibit at least two of the plurality of ecules. The plurality of distinct luciferase molecules may distinct luciferase molecules; (c) sequentially measuring comprise 2, 3, 4, 5 or 6 distinct luciferase molecules. The US 8,569,002 B2 3 4 plurality of distinct luciferase inhibitors may comprise 2, 3, or inhibitors, wherein the plurality of inhibitors inhibit at least 4 distinct luciferase inhibitors. The plurality of distinct two of the plurality of distinct luciferase molecules; (c) luciferase molecules comprises two or more or each of firefly sequentially measuring chemiluminescence from each of the luciferase, renilla luciferase, gaussia luciferase, cypridina plurality of distinct luciferase molecules, wherein other of the luciferase, gaussia luciferase lacking a secretion signal plurality of the distinct luciferase molecules are inhibited or sequence or having an endoplasmic retention sequence, or lack substrate. The cell may be a cancer cell. The plurality of Cypridina luciferase lacking a secretion signal sequence or distinct luciferase molecules may be provided sequentially or having an endoplasmic retention sequence. simultaneously. The plurality of distinct luciferase molecules The system may further comprise cells that express the may comprise 2, 3, 4, 5 or 6 distinct luciferase molecules. The plurality of luciferase molecules. The Substrates may com 10 plurality of distinct luciferase inhibitors may comprise 2, 3, or prise two or all three of D-luciferin, oxyluciferin, and 4 distinct luciferase inhibitors. coelenterazine. The inhibitor may be selective or specific for The plurality of distinct luciferase molecules may com renilla luciferase, such as: prise two or more or each of firefly luciferase, renilla luciferase, gaussia luciferase, cypridina luciferase, gaussia 15 luciferase lacking a secretion signal sequence or having an C endoplasmic retention sequence, or cypridina luciferase lack ing a secretion signal sequence or having an endoplasmic retention sequence. The Substrates may comprise two or all 1N three of D-luciferin, oxyluciferin, and coelenterazine. The inhibitor may be selective or specific for renilla luciferase, 8. -N Such as:

25 whereina is substituted or unsubstituted alkyl (C-Co), Sub 1N stituted or unsubstituted alkenyl (C-C), substituted or unsubstituted alkynyl (C-C), Substituted or unsubstituted ( )- alkyoxy (C-C), amido or halide, b is H. Substituted or 30 unsubstituted alkyl (C-C), substituted or unsubstituted alkenyl (C-C), substituted or unsubstituted alkynyl (C- Co). Substituted or unsubstituted alkyoxy (C-C), amido or halide, and c is H. Substituted or unsubstituted alkyl (C-C), substituted or unsubstituted alkenyl (C-C), substituted or 35 whereina is substituted or unsubstituted alkyl (C-Co), Sub unsubstituted alkynyl (C-C), Substituted or unsubstituted stituted or unsubstituted alkenyl (C-C), substituted or alkyoxy (C-C), amido or halide. Alternatively, the inhibi unsubstituted alkynyl (C-C), Substituted or unsubstituted tor may be selective or specific for firefly luciferase, such as: alkyoxy (C-C), amido or halide, b is H. Substituted or unsubstituted alkyl (C-C), substituted or unsubstituted 40 alkenyl (C-C). Substituted or unsubstituted alkynyl (C- Co), Substituted or unsubstituted alkyoxy (C-C), amido or halide, and c is H. Substituted or unsubstituted alkyl (C-C), substituted or unsubstituted alkenyl (C-C), substituted or unsubstituted alkynyl (C-C), Substituted or unsubstituted 45 alkyoxy (C-C), amido or halide. Alternatively, the inhibi tor may be selective or specific for firefly luciferase, such as: wherein a is H, substituted or unsubstituted alkyl (C-C), substituted or unsubstituted alkenyl (C-C), substituted or unsubstituted alkynyl (C-C). Substituted or unsubstituted alkyoxy (C-C), amido or halide. The inhibitor may be 50 cross-reactive with renilla luciferase and firefly luciferase. In yet another embodiment, there is provided a kit com prising (a) a plurality of distinct luciferase Substrates; and (b) a plurality of distinct luciferase inhibitors. The plurality of distinct luciferase Substrates may comprise D-luciferin, oxy 55 wherein a is H, substituted or unsubstituted alkyl (C-C), luciferin, and coelenterazine. The plurality of distinct substituted or unsubstituted alkenyl (C-C), substituted or luciferase inhibitors may comprise inhibitors for firefly unsubstituted alkynyl (C-C), Substituted or unsubstituted luciferase and renilla luciferase. The kit may further comprise alkyoxy (C-C), amido or halide. The inhibitor may be cells for expression of luciferase molecules. The kit may cross-reactive with renilla luciferase and firefly luciferase. further comprise a plurality of expression constructs compris 60 Providing may comprise introducing into the non-human ing sequences for expressing a plurality of luciferase mol a cell previously transfected or transformed with one ecules. or more expression cassettes comprising one or more promot In a further embodiment, there is provided a method of ers driving the plurality of luciferase molecules, such as into imaging a cell in vivo comprising (a) providing a non-human a cancer cell. Providing may also comprise use of a transgenic animal comprising a cell expressing a plurality of distinct 65 non-human animal generated from stem cells transfected or luciferase molecules; (b) providing to the animal (i) transformed with one or more expression cassettes compris luciferase substrates; and (ii) a plurality of distinct luciferase ing one or more promoters driving the plurality of luciferase US 8,569,002 B2 5 6 molecules. Providing may alternatively comprise transfect of control and with no activity against FLuc, GLuc, or CLuc, ing or transforming a cell of the non-human animal in vivo are shown. A general scaffold that Supports specific RLuc with one or more expression cassettes comprising one or inhibitory activity emerges. (FIG.3B) A general scaffold that more promoters driving the plurality of luciferase molecules, Supports specific FLuc inhibitory activity is shown. Such as by transfecting or transforming with viral infection of 5 FIG. 4. A multiplexed screening platform premised upon the non-human animal. sequential analysis of multiple luciferase-based read-outs. The method may further comprise providing to the non HEK293 cells transfected with indicated FLuc, RLuc or human animal a candidate Substance, for example, wherein GLuc-KDEL reporters driven by a CMV promoter were lysed step (c) is performed after provision of the candidate sub and luciferase activities analyzed with the following stance, or wherein step (c) is performed before provision of 10 sequence of addition for substrate and inhibitors: 1) luciferin, the candidate Substance, and further comprising, after provi 2) FLI1, 3) coelenterazine (reveals both RLuc and GLuc sion of the candidate Substance, sequentially measuring activities), and 4) an RLI (to reveal GLuc signal). GL-KDEL chemiluminescence from each of the plurality of distinct protein is a secreted protein but harbors an endoplasmic luciferase molecules, wherein other of the plurality of the reticulum retention signal and is therefore found in the cell distinct luciferase molecules are inhibited or lack substrate. 15 lysate as opposed to the culture medium. The non-human animal may be a mouse. FIG. 5. A general schematic for conducting multi-lu It is contemplated that any method or composition ciferase assays using FLIs, RLFL, and RLIS. described herein can be implemented with respect to any other method or composition described herein. DETAILED DESCRIPTION OF THE INVENTION The use of the word “a” or “an' when used in conjunction with the term “comprising in the claims and/or the specifi I. DEFINITIONS cation may mean "one.” but it is also consistent with the meaning of “one or more.” “at least one.” and “one or more Unless defined otherwise, technical and scientific terms than one.” used herein have the same meaning as commonly understood These, and other, embodiments of the invention will be 25 by one of ordinary skill in the art to which this invention better appreciated and understood when considered in con belongs. For purposes of the present invention, the following junction with the following description and the accompany terms are defined below. ing drawings. It should be understood, however, that the fol An inhibitor is defined as a molecule that is able to reduce lowing description, while indicating various embodiments of the activity of a luciferase molecule to no more than about the invention and numerous specific details thereof, is given 30 20% of the normal activity level, as measured in a similar by way of illustration and not of limitation. Many substitu assay without the inhibitor. tions, modifications, additions and/or rearrangements may be A selective inhibitor is one that inhibits a particular made within the scope of the invention without departing luciferase by at least X-fold over any other luciferase, or one from the spirit thereof, and the invention includes all such that has less than Y% inhibition of any other luciferase. Substitutions, modifications, additions and/or rearrange 35 A specific inhibitor is one that inhibits a particular mentS. luciferase by at least Z-fold over any other luciferase, or one that has less than Y% inhibition of any other luciferase. BRIEF DESCRIPTION OF THE DRAWINGS When used in the context of a chemical group, “hydrogen means —H; “hydroxy' means —OH: “oxo” means —O; The following drawings form part of the present specifica 40 “halo' means independently —F. —Cl, Bror —I: "amino” tion and are included to further demonstrate certain aspects of means —NH. (see below for definitions of groups containing the present invention. The invention may be better understood the term amino, e.g., alkylamino); "hydroxyamino” means by reference to one or more of these drawings in combination —NHOH: “nitro” means —NO; imino means —NH (see with the detailed description of specific embodiments pre below for definitions of groups containing the term imino, sented herein. 45 e.g., alkylimino); "cyano' means —CN; “azido” means FIGS. 1A-D. Identification of FL and RL inhibitors from —N; in a monovalent context “phosphate” means —OP(O) high-throughput chemical library screens. (FIG. 1A) Sche (OH), or a deprotonated form thereof; in a divalent context matic of two screens using the UTSW chemical library to “phosphate” means —OP(O)(OH)O— or a deprotonated identify firefly luciferase (FLuc) and Renilla luciferase form thereof; “mercapto' means —SH; “thio’ means —S: (RLuc) inhibitors. (FIG.1B) Cell lines used in various screen 50 “thioether” means —S ; "sulfonamido” means —NHS ing steps of the screens. (FIG. 1C) Structure and in vitro IC50 (O) - (see below for definitions of groups containing the of firefly luciferase inhibitors 1 and 2 (FLI1 and FLI2). (FIG. term sulfonamido, e.g., alkylsulfonamido); "Sulfonyl' means 1D) Structure and in vitro IC50 of Renilla luciferase inhibitor —S(O) - (see below for definitions of groups containing the 1 (RLI1). term Sulfonyl, e.g., alkylsulfonyl); 'Sulfinyl' means FIG. 2. Analysis of FLIs and RLI specificity and Identifi 55 —S(O)— (see below for definitions of groups containing the cation of second generation RL inhibitors with specificity for term sulfinyl, e.g., alkylsulfinyl); and “silyl means —SiH, RL activity. Chemical structures of FLI- and RLI-related (see below for definitions of group(s) containing the term compounds tested in vitro for Renilla, firefly, Guassia, and silyl, e.g., alkylsilyl). Cypridina luciferase activity (RLuc, FLuc, GLuc, and CLuc The term "amido” (acylamino), when used without the activity, respectively). Note that RLI has both RLuc and FLuc 60 “substituted modifier, refers to the group —NHR, in which inhibitory activity (henceforth called RLFLI) whereas other R is acyl, as that term is defined above. A non-limiting compounds tested have either FLuc or RLuc inhibitory activ example of an acylamino group is NHC(O)CH. When the ity alone. No inhibitors of GLuc or CLuc were identified from term amido is used with the “substituted” modifier, it refers to this approach. groups, defined as NHR, in which R is substituted acyl, as FIG. 3A-B. Assembling a chemical scaffold that supports 65 that term is defined above. The groups —NHC(O)OCH and specific RL and FL inhibitory activity. Compounds related to —NHC(O)NHCH are non-limiting examples of substituted RLFLI that inhibited RL (FIG.3A) activity greater than 20% amido groups. US 8,569,002 B2 7 8 The term “alkyl” when used without the “substituted” II. LUCIFERASES modifier refers to a non-aromatic monovalent group with a saturated carbon atom as the point of attachment, a linear or Luciferase is a generic term for the class of oxidative branched, cyclo, cyclic or acyclic structure, no carbon-carbon enzymes used in bioluminescence and is distinct from a pho double or triple bonds, and no atoms other than carbon and toprotein. One famous example is the firefly luciferase from hydrogen. The groups, —CH (Me), —CH2CH (Et), . “Firefly luciferase' as a laboratory reagent —CH2CHCH (n-Pr), —CH(CH) (iso-Pr), —CH(CH) usually refers to P. pyralis luciferase, although recombinant (cyclopropyl), —CHCHCHCH (n-Bu), —CH(CH) luciferases from several other species of are also CHCH (sec-butyl), —CH2CH(CH) (iso-butyl), commercially available. —C(CH) (tert-butyl), —CHC(CH) (neo-pentyl), 10 In luminescentreactions, light is produced by the oxidation cyclobutyl, cyclopentyl, cyclohexyl, and cyclohexylmethyl of a luciferin (a pigment): are non-limiting examples of alkyl groups. The term "substi luciferin--O-->oxyluciferin+light tuted alkyl refers to a non-aromatic monovalent group with The most common luminescent reactions release CO as a a Saturated carbon atom as the point of attachment, a linear or 15 product. The rates of this reaction between luciferin and oxy branched, cyclo, cyclic or acyclic structure, no carbon-carbon gen are extremely slow until they are catalyzed by luciferase, double or triple bonds, and at least one atom independently Sometimes mediated by the presence of cofactors such as selected from the group consisting of N, O, F, Cl, Br, I, Si, P. calcium ions or ATP. The reaction catalyzed by firefly and S. The following groups are non-limiting examples of luciferase takes place in two steps: substituted alkyl groups: —CH2OH. —CHCl, —CHBr, —CHSH, -CF, -CHCN, -CHC(O)H, -CHC(O) luciferin-i-ATP->luciferyl adenylate--PP, OH, -CHC(O)OCH, -CHC(O)NH, -CHC(O) NHCH, -CHC(O)CH, -CHOCH, CHOCHCF, luciferyl adenylate--O-->oxyluciferin-i-AMP+light —CHOC(O)CH, -CH-NH, CHNHCH, -CHN The reaction is very energetically efficient: nearly all of the (CH), —CHCHCl, —CH2CH2OH. —CHCF, 25 energy input into the reaction is transformed into light. As a —CHCHOC(O)CH, CHCH-NHCOC(CH), and comparison, the incandescent light bulb loses about 90% of —CHSiCCH). its energy to heat. Photon emission can be detected by light The term “alkenyl' when used without the “substituted sensitive apparatus Such as aluminometer or modified optical modifier refers to a monovalent group with a nonaromatic microscopes. This allows observation of biological pro carbon atom as the point of attachment, a linear or branched, 30 CCSSCS. cyclo, cyclic or acyclic structure, at least one nonaromatic A subtle structural difference in luciferase has been dis carbon-carbon double bond, no carbon-carbon triple bonds, covered to be the cause of the change in bioluminescence and no atoms other than carbon and hydrogen. Non-limiting emission color from a yellow-green to red. The structure of examples of alkenyl groups include: —CH=CH (vinyl), wild-type luciferase and red mutant (S286N) luciferase from -CH=CHCH, -CH=CHCHCH, CH-CH=CH, 35 the Japanese Genji-Botaru (Luciola cruciata) in complex (allyl), —CH-CH=CHCH, and -CH=CH-CHs. The with an intermediate analogue 5'-O-N-(dehydroluciferyl)- term “substituted alkenyl refers to a monovalent group with Sulfamoyladenosine (DLSA) was examined and studies a nonaromatic carbonatom as the point of attachment, at least showed that the wild-type luciferase complexed with DLSA one nonaromatic carbon-carbon double bond, no carbon-car exhibited a closed form of the active site, where the side bontriple bonds, a linear or branched, cyclo, cyclic or acyclic 40 chain of amino acid isoleucine 288 moved towards the ben structure, and at least one atom independently selected from Zothiazole ring of DLSA, creating a rigid hydrophobic the group consisting of N, O, F, Cl, Br, I, Si, P. and S. The pocket. The closed form wild-type luciferase bound the groups, -CH=CHF, -CH=CHC1 and -CH=CHBr, are excited State of oxyluciferin in a highly rigid and nonpolar non-limiting examples of Substituted alkenyl groups. microenvironment, minimizing energy loss before emitting The term “alkynyl' when used without the “substituted 45 yellow-green light. The S286N luciferase complexed with modifier refers to a monovalent group with a nonaromatic DLSA exhibited an open form of the active site, where the carbon atom as the point of attachment, a linear or branched, amino acid side chain of isoleucine 288 did not move towards cyclo, cyclic or acyclic structure, at least one carbon-carbon the benzothiazole ring of DLSA, creating a less rigid and less triple bond, and no atoms other than carbon and hydrogen. hydrophobic microenvironment. The open form S286N The groups, —C=CH, —C=CCH. —C=CCHs and 50 luciferase had a less rigid microenvironment allowing some —CH2C=CCH are non-limiting examples of alkynyl energy loss from the excited state of oxyluciferin, which groups. The term “substituted alkynyl refers to a monovalent resulted in the emission of low-energy red light. group with a nonaromatic carbonatom as the point of attach A variety of organisms regulate their light production using ment and at least one carbon-carbon triple bond, a linear or different luciferases in a variety of light-emitting reactions. branched, cyclo, cyclic or acyclic structure, and at least one 55 The most famous are the fireflies, although the enzyme exists atom independently selected from the group consisting of N. in organisms as different as the Jack-O-Lantern mushroom O. F. Cl, Br, I, Si, P, and S. The group, —C=CSi (CH), is a (Omphalotus Olearius) and many marine creatures. In fire non-limiting example of a Substituted alkynyl group. flies, the oxygen required is Supplied through a tube in the The term “alkoxy' when used without the “substituted abdomen called the abdominal trachea. The luciferases of modifier refers to the group —OR, in which R is an alkyl, as 60 fireflies, of which there are over 2000 species, and of the that term is defined above. Non-limiting examples of alkoxy Elateroidea (fireflies, click and relatives) in general, groups include: —OCH —OCHCH —OCH2CHCH are diverse enough to be useful in molecular phylogeny. The —OCH(CH), —OCH(CH), —O— cyclopentyl, and most thoroughly studied luciferase is that of the Photinini —O— cyclohexyl. The term “substituted alkoxy' refers to firefly Photinus pyralis, which has an optimum pH of 7.8. the group —OR, in which R is a substituted alkyl, as that term 65 Also well studied is the luciferase from Renilla reniformis. is defined above. For example, —OCHCF is a substituted In this organism, the luciferase is closely associated with a alkoxy group. luciferin-binding protein as well as a green fluorescent pro US 8,569,002 B2 9 10 tein (GFP). Calcium triggers release of the luciferin (co of a particular enzyme can be used to detect enzyme activity elenterazine) from the luciferinbinding protein. The substrate in coupled or two-step luciferase assays. Such substrates have is then available for oxidation by the luciferase, where it is been used to detect caspase activity and cytochrome P450 degraded to coelenteramine with a resultant release of energy. activity, among others. In the absence of GFP, this energy would be released as a 5 photon of blue light (peak emission wavelength 482 nm). Whole animal imaging (referred to as in Vivo or, occasion However, due to the closely associated GFP, the energy ally, ex vivo imaging) is a powerful technique for studying released by the luciferase is instead coupled through reso cell populations in live , such as mice. Different types nance energy transfer to the fluorophore of the GFP, and is of cells (e.g., bone marrow stem cells, T-cells) can be engi Subsequently released as a photon of green light (peak emis- 10 neered to express a luciferase allowing their non-invasive sion wavelength 510 nm). The catalyzed reaction is: visualization inside a live animal using a sensitive charge coelenterazine+O->coelenteramine--CO+photon of couple device camera (CCD camera). This technique has light been used to follow tumorigenesis and response of tumors to Newer luciferases have recently been identified that, unlike 15 treatment in animal models. However, environmental factors Renilla or Firefly luciferase, are naturally secreted molecules. and therapeutic interferences may cause some discrepancies One such example is the Metridia luciferase (MetLuc) that is between tumor burden and bioluminescence intensity in rela derived from the marine copepod Metridia longa. The M. tion to changes in proliferative activity. The intensity of the longa secreted luciferase gene encodes a 24 kDa protein signal measured by in vivo imaging may depend on various containing an N-terminal Secretory signal peptide of 17 factors, such as D-luciferin absorption through the perito amino acid residues. The sensitivity and high signal intensity neum, blood flow, cell membrane permeability, availability of of this luciferase molecule proves advantageous in many co-factors, intracellular pH and transparency of overlying reporter studies. Some of the benefits of using a secreted tissue, in addition to the amount of luciferase. reporter molecule like MetLuc is its no-lysis protocol that 25 Luciferase can be used in blood banks to determine if red allows one to be able to conduct live cell assays and multiple blood cells are starting to break down. Forensic investigators assays on the same cell. can use a dilute solution containing the enzyme to uncover Gaussia luciferase (GLuc) is a 20 kDa luciferase from the traces of blood remaining on Surfaces at a crime scene. marine cocepod Gaussia princeps. This luciferase, which 30 Luciferase is a heat sensitive protein that is used in Studies on does not require ATP, catalyzes the oxidation of the substrate protein denaturation, testing the protective capacities of heat coelenterazine in a reaction that produces light (480 nm), and shock proteins. The opportunities for using luciferase con has considerable advantages over other luminescent reporter tinue to expand. genes. It is normally secreted from the cells and its secretion signal also functions very efficiently in mammalian cells. 35 III. LUCIFERASE INHIBITORS GLuc offers the advantage of a greatly increased biolumines cent signal relative to the commonly used firefly (Fluc) and In accordance with the present invention, a variety of Renilla luciferases (RLuc). GLuc was determined to emit luciferase inhibitors are now provided. As shown in FIG. 2, a light with a specific activity of 4.2x10° photons/s/mol, the number of inhibitors have been tested and can be used in highest reported activity for any characterized luciferase. 40 accordance with the present invention to inhibit firefly Cypridina luciferase (CLuc) is isolated from the marine luciferase, renilla luciferase or both. FIG. 3 shows one of the ostracod Cypridina noctiluca and is efficiently secreted from inhibitors from FIG. 2, which is a cross-inhibitor, and a num mammalian cells. CLuc differs from Fluc in the form of ber of additional inhibitors that are specific or selective for luciferin it uses as a substrate (Cypridina luciferin), it its 45 renilla luciferase, as well as a generic structure indicating the independence from ATP to achieve light emission. CLuc does core for such inhibitors. not react with coelenterazine, a common Substrate of marine A. Firefly Luciferase luciferases. This allows the simultaneous detection of both of Firefly luciferase selective inhibitors are identified as com FLuc, Rluc, or GLuc with CLuc expressed from the same pounds 32, FLI1 (also named compound 5), and FLI2. A cells provided the cell-derived samples can be divided and 50 scaffold for such compounds is shown below and in FIG. 3: independently analyzed for each enzymatic activity, or if a method for sequentially measuring individual enzymatic activities from the same cell-derived sample is in place. Luciferase can be produced in the lab through genetic 55 8. a NN engineering for a number of purposes. Luciferase genes can be synthesized and inserted into organisms or transfected into S cells. Mice, silkworms, and potatoes are just a few organisms that have already been engineered to produce the protein. In wherein a is H, alkyl substituted or unsubstituted (C-C), biological research, luciferase commonly is used as a reporter 60 substituted or unsubstituted alkenyl (C-C), substituted or to assess the transcriptional activity in cells that are trans unsubstituted alkynyl (C-C), Substituted or unsubstituted fected with a genetic construct containing the luciferase gene alkyoxy (C-C), amido or halide. under the control of a promoter of interest. Luciferase can B. Renilla Luciferase also be used to detect the level of cellular ATP in cell viability as Renilla luciferase selective inhibitors are identified as com assays or for kinase activity assays. Additionally, pro-lumi pounds 13, 23, 11, 12, 25, 35, 19, 42, 34, 16, 37 and 20. A nescent molecules that are converted to luciferin upon activity scaffold for such compounds is shown below and in FIG. 3: US 8,569,002 B2 11 12 sequences necessary for the transcription and possibly trans C lation of an operably linked coding sequence in a particular host cell. In certain embodiments, the nucleic acid encoding a gene 1N product is under transcriptional control of a promoter. A O )- “promoter” refers to a DNA sequence recognized by the 8. -N synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. The phrase “under transcriptional control’ means that 10 the promoter is in the correct location and orientation in relation to the nucleic acid to control RNA polymerase ini wherein a is H, substituted or unsubstituted alkyl (C-C), tiation and expression of the gene. substituted or unsubstituted alkenyl (C-C), substituted or The term promoter will be used here to refer to a group of unsubstituted alkynyl (C-C), Substituted or unsubstituted 15 transcriptional control modules that are clustered around the alkyoxy (C-C), amido or halide, wherein b is H. Substi initiation site for RNA polymerase. Much of the thinking tuted or unsubstituted alkyl (C-C). Substituted or unsub about how promoters are organized derives from analyses of stituted alkenyl (C-Co), Substituted or unsubstituted alkynyl several viral promoters, including those for the HSV thymi (C-Co), Substituted or unsubstituted alkyoxy (C-Co.), dine kinase (tk) and SV40 early transcription units, or from amido or halide, and c is H, substituted or unsubstituted alkyl the cytomegalovirus (CMV). These studies, augmented by (C-Co), Substituted or unsubstituted alkenyl (C-C), Sub more recent work, have shown that promoters are composed stituted or unsubstituted alkynyl (C-Co), Substituted or of discrete functional modules, each consisting of approxi unsubstituted alkyoxy (C-C), amido or halide. mately 7-20 bp of DNA, and containing one or more recog C. Cross-Inhibitors nition sites for transcriptional activator or repressor proteins. 25 Other promoters that can be used, include IPTG-inducible An inhibitor effective on both renilla and firefly luciferase promoter. are identified as compound RFLI/6. At least one module in each promoter functions to position the start site for RNA synthesis. The best known example of IV. ASSAY COMPONENTS this is the TATA box, but in some promoters lacking a TATA 30 box. A. Expression Constructs In the bacterial genome, there are several conserved fea The present invention may involve using expression con tures in a bacterial promoter: the start site or point, the 10-35 structs to luciferase molecules in the context of Screening bp sequence upstream of the start site, and the distance assays. In certain embodiments, it is contemplated that the between the 10-35 bp sequences upstream of the start site. expression construct comprises nucleic acid sequences 35 The start point is usually (90% of the time) a purine. Upstream encoding luciferase polypeptides, as discussed above. Gen of the start site is a 6 bp region that is recognizable in most erally, such methods involve the generation of expression promoters. The distance varies from 9-18bp upstream of the constructs containing, for example, a heterologous nucleic start site, however, the consensus sequence is TATAAT. acid sequence encoding the luciferase and a means for its Another conserved hexamer is centered at 35 bp upstream of expression, replicating the vector in an appropriate cell. 40 the start site. This consensus sequence is TTGACA. Addi Luciferase enzymes may be fused to proteins of interest in tional promoter elements regulate the frequency of transcrip order to interrogate changes in the proteins’ half-lives in tional initiation. The spacing between promoter elements fre response to various cellular perturbations including addition quently is flexible, so that promoter function is preserved of signaling proteins (such as Wnt, Hh, FGF proteins), chemi when elements are inverted or moved relative to one another. cals, or siRNAS. Luciferase-fusion proteins co-expressed in 45 By employing a promoter with well-known properties, the cells by introduction of appropriate expression constructs level and pattern of expression of the protein of interest fol could also be used to examine protein-protein interactions, lowing transfection or transformation can be optimized or when a specific protein of interest is immunoprecipitated or assessed. Further, selection of a promoter that is regulated in otherwise isolated from cells expressing multiple luciferase response to specific physiologic signals can permit inducible fusion proteins and co-purified luciferase activities measured 50 expression of the gene product. In certain embodiments, the as a determinant of interaction between luciferase-fusion pro promoter will be selected based on an interest in the regula teins and the target protein. tion of a coding sequence naturally associated with the pro Yet further, in certain embodiments, it is contemplated that moter, and the ability of candidate substance to alter the nucleic acid or proteinaceous sequences may be co-expressed activity of that promoter, once linked to a luciferase coding with other selected nucleic acid or proteinaceous sequences 55 region, will be assessed. in the same host cell. Co-expression may be achieved by In certain embodiments of the invention, the cells contain co-transfecting the host cell with two or more distinct recom expression cassettes of the present invention, a cell may be binant vectors. Alternatively, a single recombinant vector selected using a selectable marker in the expression construct. may be constructed to include fused coding regions for Usually the inclusion of a drug selection marker aids in clon luciferase and other products. 60 ing and in the selection of transformants, for example, genes 1. Regulatory Sequences that confer resistance to neomycin, puromycin, hygromycin, As used in the present invention, the term “expression DHFR, GPT. Zeocin and histidinol are useful selectable vector” refers to any type of genetic construct comprising a markers. Alternatively, enzymes such as herpes simplex virus nucleic acid sequence coding for luciferase polypeptides. In thymidine kinase (tk) or chloramphenicol acetyltransferase Some cases, DNA molecules are then translated into a protein, 65 (CAT) may be employed. Immunologic markers also can be polypeptide, or peptide. Expression vectors can contain a employed. The selectable marker employed is not believed to variety of “control sequences.” which refer to nucleic acid be important, so long as it is capable of being expressed US 8,569,002 B2 13 14 simultaneously with the nucleic acid encoding a gene prod mins or other growth promoting Substances. The addition of uct. Further examples of selectable markers are well known to peptone provides a readily available source of nitrogen and one of skill in the art. carbon. Furthermore, different media results in different In addition to control sequences that govern transcription growth rates and different stationary phase densities. A rich and translation, vectors and expression vectors may contain media results in a short doubling time and higher cell density nucleic acid sequences that serve other functions such as at a stationary phase. Minimal media results in slow growth replications of origin, transcription termination signals, mul and low final cell densities. Efficient agitation and aeration tipurpose cloning sites, internal ribosome entry site, etc. It is increases final cell densities. A skilled artisan will be able to contemplated in the present invention, that virtually any type determine which type of media is best suited to culture a of vector may be employed in any known or later discovered 10 specific type of microorganism. For example, since 1927, the method to deliver nucleic acids encoding a luciferase polypeptide. Where incorporation into an expression vectoris DIFCO manual has been used in the art as a guide for culture desired, the nucleic acid luciferase polypeptides may also media and nutritive agents for microbiology. comprise a natural intron or an intron derived from another Non-viral Methods. Suitable methods for nucleic acid gene. Such vectors may be viral or non-viral vectors as 15 delivery for transformation of an organelle, a cell, a tissue or described herein, and as known to those skilled in the art. an organism for use with the current invention are believed to 2. Vectors include virtually any method by which a nucleic acid (e.g., In particular embodiments of the invention, a plasmid vec DNA) can be introduced into an organelle, a cell, a tissue oran tor is contemplated for use to transform a host cell. In general, organism, as described herein or as would be known to one of plasmid vectors containing replicon and control sequences ordinary skill in the art. Such methods include, but are not that are derived from species compatible with the host cell are limited to, direct delivery of DNA such as by injection (U.S. used in connection with these hosts. The vector ordinarily Pat. Nos. 5,994,624, 5,981,274, 5,945,100, 5,780,448, 5,736, carries a replication site, as well as marking sequences that 524, 5,702,932, 5,656,610, 5,589,466 and 5,580,859, each are capable of providing phenotypic selection in transformed incorporated herein by reference), including microinjection cells. Plasmid vectors are well known and are commercially 25 (Harland and Weintraub, 1985; U.S. Pat. No. 5,789,215, available. Such vectors include, but are not limited to, pWL incorporated herein by reference); by electroporation (U.S. NEO, pSV2CAT, pCG44, PXT1, pSG (Stratagene) pSVK3, Pat. No. 5,384.253, incorporated herein by reference); by pBSK, pBR322, pcDNA3 and puC vectors. calcium phosphate precipitation (Graham and Van Der Eb, Yet further, prokaryote-based systems can be employed for 1973; Chen and Okayama, 1987: Rippe et al., 1990); by using use with the present invention to produce nucleic acid 30 DEAE-dextran followed by polyethylene glycol (Gopal, sequences, or their cognate polypeptides, proteins and pep 1985); by direct sonic loading (Fechheimer et al., 1987); by tides. Many such systems are commercially and widely avail liposome mediated transfection (Nicolau and Sene, 1982; able. One skilled in the art is aware of the various prokaryote Fraley et al., 1979; Nicolau et al., 1987; Wong et al., 1980; based expression systems. Exemplary systems from Kaneda et al., 1989: Kato et al., 1991); by microprojectile PROMEGA include, but are not limited to, pGEMEX(R)-1 35 bombardment (PCT Application Nos. WO 94/09699 and vector, pGEMX(R)-2 Vector, and Pinpoint control Vectors. 95/06128: U.S. Pat. Nos. 5,610,042; 5,322,783, 5,563,055, Examples from STRATAGENER) include, but are not limited 5,550,318, 5.538,877 and 5,538,880, and each incorporated to, pBK Phagemid Vector, which is inducible by IPTG, herein by reference); by agitation with silicon carbide fibers pSPUTK In vitro Translation Vector, pFT Expression sys (Kaeppler et al., 1990; U.S. Pat. Nos. 5,302,523 and 5,464, tems, Epicurian Coli(R) BL21 Competent Cells and plDualTM 40 765, each incorporated herein by reference); or by PEG Expression System. mediated transformation of protoplasts (Omirulleh et al., 3. Transfection/Transformation 1993; U.S. Pat. Nos. 4,684,611 and 4,952,500, each incorpo In order to effect expression of constructs, the expression rated herein by reference); by desiccation/inhibition-medi construct must be delivered into a cell. This delivery may be ated DNA uptake (Potrykus et al., 1985). Through the appli accomplished in vitro, as in laboratory procedures for trans 45 cation of techniques such as these, organelle(s), cell(s), forming cells lines using well developed procedures. Trans tissue(s) or organism(s) may be stably or transiently trans formation of cell lines can be achieved using a variety of formed. techniques, although the techniques generally fall into either In certain embodiments, a nucleic acid may be delivered to viral or non-viral methods. These techniques and modifica an organelle, a cell, a tissue or an organism via one or more tions are well known in the art. Thus, it is well within the 50 injections (i.e., a needle injection). Such as, for example, Scope of the present invention that a cell line may be trans either Subcutaneously, intradermally, intramuscularly, inter formed by any available transformation procedure or modi venously or intraperitoneally. Methods of injection of vac fication thereof. cines are well known to those of ordinary skill in the art (e.g., Once the cell is transformed with the vector, the cells are injection of a composition comprising a saline solution). Fur cultured such that the cells multiply resulting in production of 55 ther embodiments of the present invention include the intro the desired protein. In certain embodiments, the cells that are duction of a nucleic acid by direct microinjection. Direct transformed can be bacterial cells. Thus, a skilled artisan is microinjection has been used to introduce nucleic acid con cognizant that the development of microorganisms in culture structs into Xenopus oocytes (Harland and Weintraub, 1985). media is dependent upon a number of very important factors, In certain embodiments of the present invention, a nucleic e.g., the proper nutrients must be available; oxygen or other 60 acid is introduced into an organelle, a cell, a tissue or an gases must be available as required; a certain degree of mois organism via electroporation. Electroporation involves the ture is necessary; the media must be of the proper reaction; exposure of a suspension of cells and DNA to a high-voltage proper temperature relations must prevail; the media must be electric discharge. In some variants of this method, certain sterile; and contamination must be prevented. cell wall-degrading enzymes, such as pectin-degrading A satisfactory microbiological culture contains available 65 enzymes, are employed to render the target recipient cells Sources of hydrogen donors and acceptors, carbon, nitrogen, more Susceptible to transformation by electroporation than Sulfur, phosphorus, inorganic salts, and, in certain cases, Vita untreated cells (U.S. Pat. No. 5,384.253, incorporated herein US 8,569,002 B2 15 16 by reference). Alternatively, recipient cells can be made more In certain embodiments of the invention, a liposome may Susceptible to transformation by mechanical wounding. be complexed with a hemagglutinating virus (HVJ). This has Transfection of eukaryotic cells using electroporation has been shown to facilitate fusion with the cell membrane and been quite successful. Mouse pre-B lymphocytes have been promote cell entry of liposome-encapsulated DNA (Kaneda transfected with human kappa-immunoglobulin genes (Potter et al., 1989). In other embodiments, a liposome may be com et al., 1984), and rathepatocytes have been transfected with plexed or employed in conjunction with nuclear non-histone the chloramphenicolacetyltransferase gene (Tur-Kaspa et al., chromosomal proteins (HMG-1) (Kato et al., 1991). In yet 1986) in this manner. further embodiments, a liposome may be complexed or To effect transformation by electroporation in cells such as, employed in conjunction with both HVJ and HMG-1. In other for example, plant cells, one may employ either friable tis 10 embodiments, a delivery vehicle may comprise a ligand and a Sues, such as a Suspension culture of cells or embryogenic liposome. callus or alternatively one may transform immature embryos Still further, a nucleic acid may be delivered to a target cell or other organized tissue directly. In this technique, one via receptor-mediated delivery vehicles. These take advan would partially degrade the cell walls of the chosen cells by tage of the selective uptake of macromolecules by receptor exposing them to pectin-degrading enzymes (pectolyases) or 15 mediated endocytosis that will be occurring in a target cell. In mechanically wounding in a controlled manner. Examples of view of the cell type-specific distribution of various receptors, Some species which have been transformed by electropora this delivery method adds another degree of specificity to the tion of intact cells include maize (U.S. Pat. No. 5,384.253; present invention. Rhodes et al., 1995; DHalluin et al., 1992), wheat (Zhou et Certain receptor-mediated gene targeting vehicles com al., 1993), tomato (Hou and Lin, 1996), soybean (Christou et prise a cell receptor-specific ligandanda nucleic acid-binding al., 1987) and tobacco (Lee et al., 1989). agent. Others comprise a cell receptor-specific ligand to One also may employ protoplasts for electroporation trans which the nucleic acid to be delivered has been operatively formation of plant cells (Bates, 1994: Lazzeri, 1995). For attached. Several ligands have been used for receptor-medi example, the generation of transgenic Soybean plants by elec ated gene transfer (Wu and Wu, 1987; Wagner et al., 1990; troporation of cotyledon-derived protoplasts is described by 25 Perales et al., 1994; Myers, EPO 0273085), which establishes Dhir and Widholm in International Patent Application No. the operability of the technique. Specific delivery in the con WO 9217598, incorporated herein by reference. Other text of another mammalian cell type has been described (Wu examples of species for which protoplast transformation has and Wu, 1993; incorporated herein by reference). In certain been described include barley (Lazerri, 1995), sorghum (Bat aspects of the present invention, a ligand will be chosen to traw and Hall, 1991), maize (Bhattacharjee et al., 1997), 30 correspond to a receptor specifically expressed on the target wheat (He et al., 1994) and tomato (Tsukada, 1989). cell population. In other embodiments of the present invention, a nucleic In other embodiments, a nucleic acid delivery vehicle com acid is introduced to the cells using calcium phosphate pre ponent of a cell-specific nucleic acid targeting vehicle may cipitation. Human KB cells have been transfected with aden comprise a specific binding ligand in combination with a ovirus 5 DNA (Graham and Van Der Eb. 1973) using this 35 liposome. The nucleic acid(s) to be delivered are housed technique. Also in this manner, mouse L(A9), mouse C 127. within the liposome and the specific binding ligand is func CHO, CV-1, BHK, NIH3T3 and HeLa cells were transfected tionally incorporated into the liposome membrane. The lipo with a neomycin marker gene (Chen and Okayama, 1987), some will thus specifically bind to the receptor(s) of a target and rathepatocytes were transfected with a variety of marker cell and deliver the contents to a cell. Such systems have been genes (Rippe et al., 1990). 40 shown to be functional using systems in which, for example, In another embodiment, a nucleic acid is delivered into a epidermal growth factor (EGF) is used in the receptor-medi cell using DEAE-dextran followed by polyethylene glycol. In ated delivery of a nucleic acid to cells that exhibit upregula this manner, reporter plasmids were introduced into mouse tion of the EGF receptor. myeloma and erythroleukemia cells (Gopal, 1985). In still further embodiments, the nucleic acid delivery Additional embodiments of the present invention include 45 vehicle component of a targeted delivery vehicle may be a the introduction of a nucleic acid by direct Sonic loading. liposome itself, which will preferably comprise one or more LTKT fibroblasts have been transfected with the thymidine lipids or glycoproteins that direct cell-specific binding. For kinase gene by sonication loading (Fechheimer et al., 1987). example, lactosyl-ceramido, a galactose-terminal asialgan In a further embodiment of the invention, a nucleic acid glioside, have been incorporated into liposomes and observed may be entrapped in a lipid complex Such as, for example, a 50 an increase in the uptake of the insulin gene by hepatocytes liposome. Liposomes are vesicular structures characterized (Nicolau et al., 1987). It is contemplated that the tissue by a phospholipid bilayer membrane and an inner aqueous specific transforming constructs of the present invention can medium. Multilamellar liposomes have multiple lipid layers be specifically delivered into a target cell in a similar manner. separated by aqueous medium. They form spontaneously Viral delivery. The capacity of certain viral vectors to effi when phospholipids are Suspended in an excess of aqueous 55 ciently infect or enter cells, to integrate into a host cell Solution. The lipid components undergo self-rearrangement genome and stably express viral genes, have led to the devel before the formation of closed structures and entrap water and opment and application of a number of different viral vector dissolved solutes between the lipid bilayers (Ghosh and systems (Robbins et al., 1998). Viral systems are currently Bachhawat, 1991). Also contemplated is an nucleic acid com being developed for use as vectors for ex vivo and in Vivo gene plexed with Lipofectamine (Gibco BRL) or Superfect 60 transfer. For example, adenovirus, herpes-simplex virus, ret (Qiagen). rovirus and adeno-associated virus vectors are being evalu Liposome-mediated nucleic acid delivery and expression ated currently for treatment of diseases Such as cancer, cystic of foreign DNA in vitro has been very successful (Nicolau fibrosis, Gaucher disease, renal disease and arthritis (Robbins and Sene, 1982: Fraley et al., 1979; Nicolau et al., 1987). The and Ghivizzani, 1998: Imai et al., 1998: U.S. Pat. No. 5,670, feasibility of liposome-mediated delivery and expression of 65 488). The various viral vectors described below, present spe foreign DNA in cultured chick embryo, HeLa and hepatoma cific advantages and disadvantages, depending on the particu cells has also been demonstrated (Wong et al., 1980). lar gene-therapeutic application. US 8,569,002 B2 17 18 In particular embodiments, an adenoviral expression vec transcribing their RNA genomes, and integrating the reverse tor is contemplated for the delivery of expression constructs. transcribed DNA into the target cell genome, but are inca Adenoviruses comprise linear, double-stranded DNA, with a pable of replicating within the target cell to produce infec genome ranging from 30 to 35 kb in size (Reddy et al., 1998: tious retroviral particles (e.g., the retroviral genome trans Morrison et al., 1997: Chillon et al., 1999). An adenovirus ferred into the target cell is defective in gag, the gene expression vector according to the present invention com encoding virion structural proteins, and/or in pol, the gene prises a genetically engineered form of the adenovirus. encoding reverse transcriptase). Thus, transcription of the Advantages of adenoviral gene transfer include the ability to provirus and assembly into infectious virus occurs in the infect a wide variety of cell types, including non-dividing presence of an appropriate helper virus or in a cell line con cells, a mid-sized genome, ease of manipulation, high infec 10 taining appropriate sequences enabling encapsidation with tivity and the ability to be grown to high titers (Wilson, 1996). out coincident production of a contaminating helper virus. Further, adenoviral infection of host cells does not result in Herpes simplex virus (HSV) type I and type II contain a chromosomal integration because adenoviral DNA can rep double-stranded, linear DNA genome of approximately 150 licate in an episomal manner, without potential genotoxicity kb, encoding 70-80 genes. Wild type HSV are able to infect associated with other viral vectors. Adenoviruses also are 15 cells lytically and to establish latency in certain cell types structurally stable (Marienfeld et al., 1999) and no genome (e.g., neurons). Similar to adenovirus, HSV also can infect a rearrangement has been detected after extensive amplifica variety of cell types including muscle (Yeung et al., 1999), ear tion (Parks et al., 1997: Bettet al., 1993). (Derby et al., 1999), eye (Kaufman et al., 1999), tumors A common approach for generating an adenoviruses for (Yoon et al., 1999; Howard et al., 1999), lung (Kohut et al., use as a gene transfer vector is the deletion of the E1 gene 1998), neuronal (Gamido et al., 1999: Lachmann and (Er), which is involved in the induction of the E2, E3 and E4 Efstathiou, 1999), liver (Miyatake et al., 1999: Kooby et al., promoters (Graham and Prevec, 1995). Subsequently, athera 1999) and pancreatic islets (Rabinovitch et al., 1999). peutic gene or genes can be inserted recombinantly in place of For use in therapeutic gene delivery, HSV must be rendered the E1 gene, wherein expression of the therapeutic gene(s) is replication-defective. Protocols for generating replication driven by the E1 promoter or a heterologous promoter. The 25 defective HSV helper virus-free cell lines have been E1, replication-deficient virus is then proliferated in a described (U.S. Pat. Nos. 5,879,934; 5,851,826, each specifi “helper cell line that provides the E1 polypeptides in trans cally incorporated herein by reference in its entirety). One IE (e.g., the human embryonic kidney cell line 293). Thus, in the protein, Infected Cell Polypeptide 4 (ICP4), also known as present invention it may be convenient to introduce the trans alpha 4 or Vmw 175, is absolutely required for both virus forming construct at the position from which the E1-coding 30 infectivity and the transition from IE to later transcription. sequences have been removed. However, the position of Thus, due to its complex, multifunctional nature and central insertion of the construct within the adenovirus sequences is role in the regulation of HSV gene expression, ICP4 has not critical to the invention. Alternatively, the E3 region, typically been the target of HSV genetic studies. portions of the E4 region or both may be deleted, wherein a Phenotypic studies of HSV viruses deleted of ICP4 indi heterologous nucleic acid sequence under the control of a 35 cate that such viruses will be potentially useful for gene promoter operable in eukaryotic cells is inserted into the transfer purposes (Krisky et al., 1998a). One property of adenovirus genome for use in gene transfer (U.S. Pat. Nos. viruses deleted for ICP4 that makes them desirable for gene 5,670,488; 5.932.210, each specifically incorporated herein transfer is that they only express the five other IE genes: ICP0, by reference). ICP6, ICP27, ICP22 and ICP47 (DeLuca et al., 1985), with In certain embodiments of the invention, the use of retro 40 out the expression of viral genes encoding proteins that direct viruses for gene delivery are contemplated. Retroviruses are viral DNA synthesis, as well as the structural proteins of the RNA viruses comprising an RNA genome. When a host cell virus. This property is desirable for minimizing possible del is infected by a retrovirus, the genomic RNA is reverse tran eterious effects on host cell metabolism or an immune scribed into a DNA intermediate which is integrated into the response following genetransfer. Further deletion of IE genes chromosomal DNA of infected cells. This integrated DNA 45 ICP22 and ICP27, in addition to ICP4, substantially improve intermediate is referred to as a provirus. A particular advan reduction of HSV cytotoxicity and prevented early and late tage of retroviruses is that they can stably infect dividing cells viral gene expression (Krisky et al., 1998b). with a gene of interest (e.g., a therapeutic gene) by integrating Adeno-associated virus (AAV), a member of the parvovi into the host DNA, without expressing immunogenic viral rus family, is a human virus that is increasingly being used for proteins. Theoretically, the integrated retroviral vector will be 50 gene delivery therapeutics. AAV has several advantageous maintained for the life of the infected host cell, expressing the features not found in other viral systems. First, AAV can gene of interest. infect a wide range of host cells, including non-dividing cells. A recombinant retrovirus of the present invention may be Second, AAV can infect cells from different species. Third, genetically modified in Such away that some of the structural, AAV has not been associated with any human or animal infectious genes of the native virus have been removed and 55 disease and does not appear to alter the biological properties replaced instead with a nucleic acid sequence to be delivered of the host cell upon integration. For example, it is estimated to a target cell (U.S. Pat. Nos. 5,858,744; 5,739,018, each that 80-85% of the human population has been exposed to incorporated herein by reference). After infection of a cell by AAV. Finally, AAV is stable at a wide range of physical and the virus, the virus injects its nucleic acid into the cell and the chemical conditions which lends itself to production, Storage retrovirus genetic material can integrate into the host cell 60 and transportation requirements. genome. The transferred retrovirus genetic material is then AAV has been engineered to deliver genes of interest by transcribed and translated into proteins within the host cell. deleting the internal non-repeating portion of the AAV As with other viral vector systems, the generation of a repli genome and inserting a heterologous gene between the ITRS. cation-competent retrovirus during vector production or dur The heterologous gene may be functionally linked to a het ing therapy is a major concern. Retroviral vectors suitable for 65 erologous promoter (constitutive, cell-specific, or inducible) use in the present invention are generally defective retroviral capable of driving gene expression in target cells. To produce vectors that are capable of infecting the target cell, reverse infectious recombinant AAV (rAAV) containing a heterolo US 8,569,002 B2 19 20 gous gene, a suitable producer cell line is transfected with a includes the primary Subject cell and its progeny. As used rAAV vector containing a heterologous gene. The producer herein, the terms “engineered and “recombinant cells or cell is concurrently transfected with a second plasmid harbor host cells are intended to refer to a cell into which an exog ing the AAV rep and cap genes under the control of their enous nucleic acid sequence, such as, for example, luciferase respective endogenous promoters or heterologous promoters. molecules or a construct thereof. Therefore, recombinant Finally, the producer cell is infected with a helper virus. cells are distinguishable from naturally occurring cells that do Once these factors come together, the heterologous gene is not contain a recombinantly introduced nucleic acid. In par replicated and packaged as though it were a wild-type AAV ticular embodiments of the present invention, the host cell is genome. When target cells are infected with the resulting a eukaryotic cell that has been transformed with one or more rAAV virions, the heterologous gene enters and is expressed 10 luciferase encoding constructs. in the target cells. Because the target cells lack the rep and cap C. Purification of Proteins genes and the adenovirus helper genes, the rAAV cannot In certain embodiments, luciferase proteins may be are further replicate, package or form wild-type AAV. expressed using expression systems and may further be puri Lentiviruses are complex retroviruses, which, in addition fied using standard techniques. Protein purification tech to the common retroviral genes gag, pol, and env, contain 15 niques are well known to those of skill in the art. These other genes with regulatory or structural function. The higher techniques involve, at one level, the crude fractionation of the complexity enables the virus to modulate its life cycle, as in cellular milieu to polypeptide and non-polypeptide fractions. the course of latent infection. Some examples of lentivirus Having separated the polypeptide from other proteins, the include the Human Immunodeficiency Viruses: HIV-1, HIV-2 polypeptide of interest may be further purified using chro and the Simian Immunodeficiency Virus: SIV. Lentiviral vec matographic and electrophoretic techniques to achieve partial tors have been generated by multiply attenuating the HIV or complete purification (or purification to homogeneity). virulence genes, for example, the genes env, vif, vpr, vpu and Analytical methods particularly Suited to the preparation of a nefare deleted making the vector biologically safe. pure peptide are ion-exchange chromatography, exclusion Other viral vectors such as poxvirus; e.g., vaccinia virus chromatography; polyacrylamido gel electrophoresis; iso (Gnant et al., 1999; Gnant et al., 1999), alpha virus; e.g., 25 electric focusing. A particularly efficient method of purifying sindbis virus, Semliki forest virus (Lundstrom, 1999), reovi peptides is fast protein liquid chromatography or even HPLC. rus (Coffey et al., 1998) and influenza A virus (Neumann et Certain aspects of the present invention concern the puri al., 1999) are contemplated for use in the present invention fication, and in particular embodiments, the Substantial puri and may be selected according to the requisite properties of fication, of an encoded protein or peptide. The term “purified the target system. 30 protein or peptide' as used herein, is intended to refer to a In certain embodiments, vaccinia viral vectors are contem composition, isolatable from other components, wherein the plated for use in the present invention. Vaccinia virus is a protein or peptide is purified to any degree relative to its particularly useful eukaryotic viral vector System for express naturally-obtainable state. A purified protein or peptide there ing heterologous genes. For example, when recombinant vac fore also refers to a protein or peptide, free from the environ cinia virus is properly engineered, the proteins are synthe 35 ment in which it may naturally occur. sized, processed and transported to the plasma membrane. Generally, “purified’ will refer to a protein or peptide com Vaccinia viruses as gene delivery vectors have recently been position that has been Subjected to fractionation to remove demonstrated to transfer genes to human tumor cells, e.g., various other components, and which composition Substan EMAP-II (Gnant et al., 1999), inner ear (Derby et al., 1999), tially retains its expressed biological activity. Where the term glioma cells, e.g., p53 (Timiryasova et al., 1999) and various 40 “substantially purified' is used, this designation will refer to mammalian cells, e.g., P-450 (U.S. Pat. No. 5,506,138). The a composition in which the protein or peptide forms the major preparation, growth a