US 20040023207A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2004/0023207 A1 Polansky (43) Pub. Date: Feb. 5, 2004

(54) ASSAYS FOR DRUG DISCOVERY BASED ON Publication Classification MICROCOMPETITION WITH A FOREIGN POLYNUCLEOTIDE (51) Int. Cl." ...... C12O 1/70; C12O 1/68; C12N 15/85; C12N 15/86 (52) U.S. Cl...... 435/5; 435/6; 435/455; 435/456 (76) Inventor: Hanan Polansky, Rochester, NY (US) (57) ABSTRACT Correspondence Address: A recent discovery showed that microcompetition between a Hanan Polansky foreign polynucleotide and a cellular polynucleotide is a risk 3159 S. Winton Rd. factor for Some of the major chronic diseases. The invention Rochester, NY 14623 (US) uses this novel discovery to present assays for Screening compounds based on their effectiveness in modulating Such microcompetition. The effective compounds can be used in (21) Appl. No.: 10/211,295 treatment of these chronic diseases. The invention also presents assays for Screening compounds that can be used in treatment of chronic diseaseS resulting from other foreign (22) Filed: Aug. 1, 2002 polynucleotide-type disruptions. Patent Application Publication Feb. 5, 2004 Sheet 1 of 6 US 2004/0023207 A1

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ASSAYS FOR DRUG DSCOVERY BASED ON the cellular Rb , a pharmaceutical composition can be MICROCOMPETITION WITH A FOREIGN administered to the Subject that reduces the copy number of POLYNUCLEOTDE the p300/cbp virus by, for instance, reducing viral replica tion, reduces binding of a p300/cbp transcription factor, Such BACKGROUND OF THE INVENTION as GABP, to the p300/cbp virus, increases expression of the p300/cbp transcription factor, increases binding of the p300/ 0001. The cause of many cases of the major chronic cbp transcription factor to the Rb by, for instance, diseases is unknown. Therefore, treatment is focused on Stimulating of the p300/cbp transcription clinical Symptoms associated with the disease rather than the factor, or increases expression of Rb, through, for instance, cause. As a result, in many cases, the treatment shows transfection of an exogenous Rb gene, reduced degradation limited efficacy and Serious negative side effects. of the Rb , or administration of exogenous Rb protein 0002 Recently, the National Cancer Institute (NIH Guide (see more examples below). In the case of another foreign 2000") announced a program aimed to "reorganize the polynucleotide-type disruption, for example, the composi "front-end,' or gateway, to drug discovery in cancer. The tion may reverse the effects of Such disruption. For instance, new approach promotes a three Stage discovery process, microcompetition with a p300/cbp virus reduces expression first, discovery of the molecular mechanisms underlying of Rb. A can also reduce the expression of Rb. neoplastic transformations, cancer growth and metastasis, Therefore, Such mutation is a foreign polynucleotide-type Second, Selection of a novel molecular target within the disruption. Microcompetition with a p300/cbp virus can discovered biochemical pathway associated with the disease result in cancer, and, therefore, a mutation in the Rb pro State; finally, design of a new drug that modifies the Selected moter that reduces Rb expression can also result in cancer. target. The program encourages moving away from Screen To ameliorate the Symptoms of cancer resulting from Such ing based on a clinical effects, Such as tumor cell shrinkage, mutation in the Rb gene, a pharmaceutical composition can either in Vivo or in Vitro, to Screening, or drug design, based be administered to the Subject that Stimulates complex on molecular effects. According to the NCI, Screening by a formation between a p300/cbp transcription factor and Rb. desired clinical effect identified drugs that traditionally 0006. In second aspect, the invention provides assays for demonstrated clear limitations in patients, while Screening Screening test compounds to find compounds which modu by a desired molecular effect should produce more effica late microcompetition between a foreign polynucleotide and cious and Specific drugs. a cellular polynucleotide, an effect of Such microcompeti 0003. The best drugs reverse the molecular events that tion, or an effect of another foreign polynucleotide-type cause a disease. Following the discovery of microcompeti disruption. tion between foreign polynucleolitides and cellular as 0007 A further aspect of the invention provides methods the cause of many chronic disease cases, the present inven for determining the risk of developing the molecular, cellu tion presents methods for treating chronic diseases, methods lar and clinical Symptoms associated with a chronic disease. for evaluating the effectiveness of a compound for use in The method may include detecting in a biological Sample modulating the progression of chronic diseases, and meth obtained from a Subject at least one of the following: (i) a ods for determining whether a Subject has a chronic disease, foreign polynucleotide, Specifically, a p300/cbp virus (ii) or has an increased risk of developing clinical Symptoms modified expression or bioactivity of a gene Suceptible to asSociated with Such disease. microcompetition with a foreign polynuleotide, Specifially, a p300/cbp regulated gene (iii) presence of a genetic lesion in BRIEF SUMMARY OF THE INVENTION a gene Suceptible to microcompetition with a foreign polyn 0004. In one aspect, the invention presents methods for culetide, Specifically, a gene encoding a p300/cbp factor, a treating chronic diseases. In a preferred embodiment, the p300/cbp regulated gene, p300/cbp factor kinase or p300/ methods feature administration to a Subject a therapeutically cbp phosphatase, or p300/cbp agent (iv) presence of a effective amount of a pharmaceutical or nutraceutical com genetic lesion in a DNA binding box of a p300/cbp tran position that attenuates microcompetition between a foreign Scription factor. polynucleotide and a cellular polynucleotide, attenuates an effect of Such microcompetition, or attenuates an effect of BRIEF DESCRIPTION OF THE FIGURES another foreign polynucleotide-type disruption. A pharma 0008 FIG. 1 shows the observed relative CAT activity as ceutical or nutraceutical composition may include, but not a function of the relative concentration of the competitor limited to, Small molecule (organic or inorganic), polynucle plasmid pX1.0 to the test plasmid pSV2CAT. otide, polypeptide or antibody. 0009 FIG.2 shows the observed relative CAT activity as 0005 For example, to ameliorate a disease symptom a function of the relative concentration of the competitor resulting from microcompetition between a foreign poly plasmid pSV2neo to the test plasmid pSV2CAT, and the nucleotide and a cellular polynucleotide, a pharmaceutical relative concentration of the competitor plasmid pA10neo to composition can be administered to the Subject that reduces the test plasmid pSV2CAT, in either Ltk- or ML fibroblast the cellular copy number of the foreign polynucleotide, cells. reduces complex formation between the foreign polynucle otide and a cellular transcription factor, increases complex 0010 FIG. 3 shows the observed relative CAT activity, formation between the microcompeted cellular transcription expressed as the ratio between CAT activity in the presence factor and the cellular polynucleotide, or reverses an effect of pSV2neo and CAT activity in the absence of pSV2neo, as of microcompetition on the expression or activity of a a function of the molar ratio of pSV2Neo to hMT-IIA-CAT. polypeptide with expression regulated by the cellular poly 0011 FIG. 4 shows accumulation of triglyceride assayed nucleotide. For example, in the case of a p300/cbp virus and by oil red staining in untreated F442A cells or in cells US 2004/0023207 A1 Feb. 5, 2004 transfected with either a vector expressing the SV40 large T 0023 a) Microcompetition Related Elements or the “empty vector p7.IPNeo. 0024 (1) Microcompetition 0012 FIG. 5 shows the observed relative CAT activity as a function of the molar ratio between the competitor plasmid 0.025 Definition CMV-Bgal and the test plasmid PDGF-B-CAT, or between 0026 Assume the DNA sequences DNA and DNA-bind the competitor plasmid SV40-fgal and the test plasmid the transcription complexes C and C, respectively. If C PDGF-B-CAT. and C include the same transcription factor, DNA and DNA, are called “microcompetitors." A special case of 0013 FIG. 6 shows the observed HSL mRNA in undif microcompetition is two DNA sequences which bind the ferentiated confluent controls and in differentiated 3T3-L1 Same transcription complex. cells transfected with the pZipNeo vector. 0027 Notes: 0014 FIG. 7 shows the observed number of viable cells following transfection with either the pBARB vector or the 0028 1. Transcription factors include transcription coac “empty vector” pSV-neo. tivators. 0015 FIG. 8 shows the observed luc activity following 0029 2. Sharing the same environment, such as cell, or transfection with 20 ng plRES-AR, pcDNA-AR or pSG5 chemical mix, is not required to be regarded microcompeti AR plasmids which express AR, 500 ng MMTV-luc which tors. For instance, two genes which were shown once to bind highly expresses luc following AR Stimulation of the the same transcription factor are regarded microcompetitors MMTV promoter, and increasing amounts of the empty independent of their actual physical environment. To empha expression vector, where luc activity in the presence of 650 Size Such independence, the terminology "Susceptible to ng pCEM-7Zf(+) was arbitrarily set to 1. microcompetition' may be used. 0016 FIG. 9 shows the observed luc activity following 0030 Exemplary Assays transfection with 20 ng pSG5-AR, 20 ng pS40-f-galactosi 0031) 1. If DNA and DNA are endogenous in the cell of dase (BGAL) and increasing amounts of the empty vector interest, assay the transcription factors bound to the DNA pSG5, where luc and BGAL activities in the presence of 650 Sequences (see in "Detailed description of Standard proto pGEM-7Zf(+) were arbitrarily set to 1. cols' below, the Section entitled “Identifying a polypeptide 0017 FIG. 10 shows the observed number of cells over bound to DNA or ”) and compare the two time following transfection with either the pcDNA3 vector Sets of polypeptides. If the two Sets include a common carrying an antisense to the macrophage inflammatory pro transcription factor, DNA and DNA are microcompetitors. tein 1-c. (MIP-1C) or with the “empty” pcDNA3 vector. 0032 2. In assay 1, if DNA and/or DNA are not 0018 FIG. 11 shows the observed volume of tumors endogenous, introduce DNA and/or DNA to the cellby, for injected with the vector encoding the icon, Volume of instance, transfecting the cell with plasmids carrying DNA uninjected tumors in the icon treated mice, Volume of tumors and/or DNA, infecting the cell with a virus that includes injected with the empty vector pcDNA3.1 (+), and volume of DNA, and/or DNA, and mutating endogenous DNA to uninjected tumors in the empty vector injected mice, over produce a sequence identical to DNA and/or DNA. time, following injection of SCID mice s.c. in both rear 0033) Notes: flanks with the human prostatic cancer line c4-2. 0034) 1. Introduction of exogenous DNA and/or DNA 0019 FIG. 12 shows the observed volume of tumors is a Special case of modifying the cellular copy number of a injected with the vector encoding the icon, Volume of DNA sequence. Such introduction increases the copy num uninjected tumors in the icon treated mice, Volume of tumors ber from Zero to a positive number. Generally, copy number injected with the empty vector pcDNA3.1 (+), and volume of may be modified by means Such as the ones mentioned uninjected tumors in the empty vector injected mice, over above, for instance, transfecting the cell with plasmids time, following injection of SCID mice s.c. in both rear carrying a DNA sequence of interest, infecting the cell with flanks with the human melanoma line TF2. a virus that includes the DNA sequence of interest, and mutating endogenous DNA to produce a sequence identical DETAILED DESCRIPTION OF THE to the DNA sequence of interest. INVENTION 0035 2. Assume DNA and DNA microcompete for the 0020 A. Introduction of Invention transcription factor F. ASSaying the copy number of at least one of the two Sequences, that is, DNA and/or DNA, is 0021 1. Detailed description of new elements regarded as assaying microcompetition for F, and observing 0022. The following sections present descriptions of ele a change in the copy number of at least one of the two ments used in the present invention. Following each defi Sequences is regarded as identification of modified micro nition, one or more exemplary assays are provided to competition for F. illustrate to one skilled in the art how to use the element. 0036 3. Assume the transcription factor F binds the DNA Each assay may include, as its own elements, Standard box DNA. Consider a specific DNA sequence, DNA methods in molecular biology, microbiology, cell biology, which includes a DNA box, then: cell culture, transgenic biology, recombinant DNA, immu nology, pharmacology, and toxicology, well known in the F-DNA=f(DNAF F-affinity, F-avidity) art. Details of the standard methods are available further 0037. The concentration of F bound to DNA is a func below. tion of the DNA copy number, the concentration of F in the US 2004/0023207 A1 Feb. 5, 2004 cell, F affinity and avidity to its box. Using f, a change in 0051 Examples microcompetition can be defined as a change in DNA, 0052 Antibodies specific for L. may be used in immu and a change in F-DNA as an effect of Such change. noprecipitation, Western blot or immunoaffinity to quantify 0038 4. Note that under certain conditions (fixed F), the levels of L before and after treatment. fixed F-affinity, fixed F-avidity, and limiting transcription factor (see below)), there is a “one to one” relation between 0053) See also examples below. F-DNA and DNA). Under such conditions, assaying 0054 (3) Limiting Transcription Factor FDNA is regarded assaying microcompetition. 0039 Examples 0055) Definition 0056 Assume the transcription factor F binds DNA. F is 0040 See studies in the section below entitled “Micro called “limiting in respect to DNA." if a decrease in competition with a limiting transcription complex.” microavailability of F to DNA decreases the concentration 0041) (2) Microavailable of F bound to DNA (“bound F"). 0042. Definition 0057. Notes: 0.058 1. The definition characterizes “limiting” by the 0.043 Let L and L be two molecules. Assume L can relationship between the concentration of microavailable F take S=(1 . . . n) shapes. Let L. denote L in shapes, and and the concentration of Factually bound to DNA. Accord let Li) denote concentration of Li. If L can bind L2, an ing to the definition, “limiting” means a direct relationship increase (or decrease) in L.) in the environment of L is between a decrease in microavailable F and a decrease in called “increase (or decrease) in microavailability of L to bound F, and “not limiting means no Such relationship L.” Microavailability of L is denoted L. A shape that between the two variables. For instance, according to this does not bind L is called “microunavailable to L.” definition, a decrease in microavailable F with no corre 0044) Let s=(1 . . . m) denote the set of all L. that can sponding change in bound F, means “not limiting.” bind L2. Any increase (or decrease) in the Sum of Lover 0059 2. Let G denote a DNA sequence of a certain gene. all S=(1 . . . m) is called "increase (or decrease) in Such DNA sequence may include coding and non coding microavailability of L to L.” Microavailability of L to La regions of a gene, Such as exons, introns, promoters, enhanc is denoted L. ers, or other segments positioned 5' or 3' to the coding region. ASSume the transcription factor F binds G. An assay 0045. Notes: can measure changes in G mRNA expression instead of changes in the concentration of bound F. ASSume F trans 0.046 1. A molecule in a complex is regarded in a activates G. Since F is necessary for transcription, a different shape relative to the same molecule uncomplexed, decrease in maf decreases FG, which, in turn, decreases or free. G, transcription. However, an increase in concentration of F 0047 2. Consider an example of an antibody against Lt. bound to G does not necessarily increase transcription if a specific shape of L. Assume the antibody binds Li in the binding of F is necessary but not Sufficient for transactiva region contacting L. ASSume the antibody binds a Single tion of G. region of Li, and that antibody binding prevents formation 0060 Exemplary Assays of the La L2 complex. By binding Li, the antibody changes the shape of L from L to Li (from exposed to hidden 0061 1. Identify a treatment that reduces F by trying different treatments, assaying F following each treatment, contact region). Since L does not bind L2, the decrease in and choosing a treatment which reduces F. ASSay the Li) decreases,L, or the microavailability of L to L2. If, concentration of F bound to DNA (see “Basic protocols”) on the other hand, the antibody converts Li to Li, a shape in a biological System (e.g. cell of interest). Use the iden which also forms the L-L complex with the same prob tified treatment to reduce F. Following treatment assay ability, Li is fixed. The decrease in Li) is equal to the again the concentration of bound F. A decrease in the increase in L.), resulting in a fixed sum of Ll computed concentration of F bound to DNA indicates that F is over all which bind L. limiting in respect to DNA. 0.048 Exemplary Assays 0062 2. Transfect a recombinant expression vector car 0049. The following assays identify a change in rying the gene expressing F. Expression of this exogenous F 3. will increase the intracellular concentration of F. Following following treatment. transfection: 0050) 1. Assay in a biological system (e.g., cell, cell lysate, chemical mixture) the concentrations of all L., 0063 (a) Assay the concentration of F bound to DNA. where S is a shape that can bind L. Apply a treatment to the An increase in concentration of bound F indicates that F is System which may change L. Following that treatment limiting in respect to DNA. assay again the concentrations of all L., where S is a shape 0064 (b) If DNA is the gene G, assay G transcription. that can bind L2. Calculate the Sum of Loveralls, before An increase in G transcription indicates that F is limiting in and after treatment. An increase (or decrease) in this Sum respect to G (Such an increase in transcription is expected indicates an increase (or decrease) in L. if binding of F to G is sufficient for transactivation). US 2004/0023207 A1 Feb. 5, 2004

0065 3. Contact a cell with antibodies which reduce F. or G, indicates that F is limiting in respect to G (see Following treatment: Hottiger 1998, ibid, FIG. 6D). 0.066 (a) Assay the concentration of F bound to DNA. A 0.077 6. Call the box which binds F the “F-box.” Trans decrease in concentration of bound F with any antibody fect a cell with DNA, another DNA sequence, or G, concentration indicates that F is limiting in respect to DNA. another gene, carrying a wild type F-box. Transfect another 0067 (b) If DNA is the gene G, assay G transcription. cell with with DNA or G, after mutating the F-box in the A decrease in G transcription with any antibody concen transfected DNA or G. tration indicates that F is limiting in respect to G. 0078 (a) Assay the concentration of F bound to DNA. 0068. See Kamei 1996 which used anti-CBP immuno Attenuated decrease in the concentration of F bound to glubulin G (IgG). (Instead of antibodies, Some Studies used DNA, with the wild type but not the mutated F-box indicates E1A, which, by binding to p300/cbp, also converts the shape that F is limiting in respect to DNA. from microavailable to microunavailable). 0079 (b) If DNA1 is the gene G, assay G transcription. Attenuated decrease in G transactivation with the wild type 0069. 4. Modify the copy number of DNA, another DNA but not the mutated F-box indicates that F is limiting in Sequence, or G, another gene, which also bind F (by, for respect to G. instance, transfecting the cell with DNA or G, See above). 0080) If DNA1 is the gene G, a mutation in the F-box 0070 (a) Assay the concentration of F bound to DNA. A results in diminished binding of F to DNA or G, and an decrease in concentration of F bound to DNA indicates that attenuated inhibitory effect on G transactivation. In Kamei F is limiting in respect to DNA. 1996 (ibid), in the RAR AF2 domain that inhibit 0071 (b) If DNA is the gene G, assay G transcription. binding of CBP, and other coactivator , abolished A decrease in G transcription indicates that F is limiting in AP-1 repression by nuclear receptors. respect to G. 0081 7. Let t and t be two transcription factors which 0072) If DNA is the gene G, competition with DNA or bind F. Let G and G be two genes transactivated by the t-F G, which also bind F, reduces the concentration of F bound and tR complexes, respectively. to G and, therefore, the resulting transactivation of G in 0082 (a) Transfect a cell of interest with t and assay G. any concentration of DNA or G. In respect to G, binding transcription. If the increase in It reduces transcription of of F to DNA or G reduces microavailability of F to G, G, F is limiting in respect to G. Call t F. the microavail since F bound to DNA or G is microunavailable for able shape of F in respect to G. The increase in It binding with G. increasest F), which, in turn, reducest F. The decrease 0073. This assay is exemplified in a study reported by in the shape of F microavailable to G reduces transactiva Kamei 1996 (ibid). The study used TPA to stimulate tran tion of G. In Hottiger 1998 (ibid), t is RelA, t is GAL4 Scription from a promoter containing an AP-1 Site. AP-1 Stat2(TA) and G is GAL4-CAT. See results of the increase interacts with CBP. CBP also interacts with a liganded in t on G transactivation shown in Hottiger (1998, ibid) retinoic acid receptor (RAR) and liganded glucocorticoid FIG. 6A. (b) Transfect F and assay the concatenation of F receptor (GR) (Kamei 1996, ibid, FIG. 1). Both RAR and bound to G, or transactivation of G. If the increase in F GR exhibited ligand-dependent repression of TPA stimu decreases the inhibitory effect of t, F is limiting in respect lated transcription. Induction by TPA was about 80% to G (see Hottiger 1998 (ibid), FIG. 6C showing the effect repressed by treatment with retinoic acid or dexamethasone. of p300/cbp transfection). (c) ASSay the concentration of t, In this study, G is the gene controlled by the AP1 promoter. t and F. If t and t have a high molar exceSS compared to In respect to this gene, the CBP-liganded-RAR complex is F, Fis limiting in respect to G (see Hottiger 1998, (ibid)). (4) the microunavailable form. An increase in CBP-liganded Microcompetition for a Limiting Factor RAR decreases the concentration of microavailable CBP. In 0083) Definition another exemplary study by Hottiger 1998, the two genes are HIV-CAT, which binds NF-kB, and GAL4-CAT, which 0084 Assume DNA and DNA microcompete for the binds the GAL4-Stat2(TA). NF-kB binds transcription factor F. If F is limiting in respect to DNA and p300/cbp. The GAL4-Stat2(TA) fusion protein includes the DNA, DNA and DNA are called “microcompetitiors for Stat2 transactivation domain which also binds p300/cbp. a limiting factor.” The Study showed a close dependent inhibition of gene 0085 Exemplary Assays activation by the transactivation domain of Stat2 following 0086 1. The assays 4-7 in the section entitled “Limiting transfection of a RelA expression vector (Hottiger 1998, transcription factor” above, can be used to identify micro ibid, FIG. 6A). competition for a limiting factor. 0074) 5. Transfect F and modify the copy number of 0087 2. Modify the copy number of DNA and DNA DNA, another DNA sequence, or G, a another gene, which (by, for instance, co-transfecting recombinant vector carry also bind F (by, for instance, transfecting the cell with DNA ing DNA and DNA, see also above). or G, See also above). Following transfection: 0088 (a) Assay DNA, protection against enzymatic 0075 (a) Assay concentration of F bound to DNA. digestion (“DNase footprint assay”). A change in protection Attenuated decrease in concentration of F bound to DNA indicates microcompetition for a limiting factor. indicates that F is limiting in respect to DNA. 0089 (b) Assay DNA electrophoretic gel mobility 0.076 (b) If DNA1 is the gene G, assay G transcription. (“electrophoretic mobility shift assay”). A change in mobil Attenuated decrease in G transactivation caused by DNA ity indicates microcompetition for a limiting factor. US 2004/0023207 A1 Feb. 5, 2004

0090 3. If DNA is a segment of a promoter or enhancer, 0106 Exemplary Assays or can function as a promoter or enhancer, independently, or in combination of other DNA sequences, fuse DNA to a 0107 1. Compare the sequence of Pn with the sequence, reporter gene such as CAT or LUC. Co-transfect the fused or Sequences of the published, or Self Sequenced Standard DNA, and DNA. ASSay for expression of the reporter gene. of R. If the Sequence is not a Segment of the Specifically, assay transactivation of reporter gene following Standard genome, Pn is foreign to R. an increase in DNA copy number. A change in transacti Vation of the reporter gene indicates microcompetition for a 0108) 2. Isolate DNA from O (for instance, from a limiting factor. specific cell, or a virus). Try to hybridize Pn to the isolated DNA. It Pn does not hybridize, it is foreign. 0.091 4. A special case is when DNA is the entire cellular genome responsible for normal cell morphology and 0109) Notes: function. Transfect DNA, and assay cell morphology and/or function (Such as, binding of extracellular protein, cell 0110) 1. Pn can still be foreign if it hybridizes with DNA replication, cellular oxidative stress, gene transcription, etc). from a specific O Specimen. Consider, for example, the case A change in cell morphology and/or function indicates of integrated viral . Viral Sequences integrated into microcompetition for a limiting factor. cellular genomes are foreign. To increase the probability of correct identification, repeat the assay with N>1 Specimens 0092) Notes: of O (for instance, by collecting N cells from different 0093 1. Preferably, following co-transfection of DNA representatives of R). Define the genome of R as all DNA and DNA, verify that the polynucleotides do not produce Sequences found in all O Specimens. Following this defini mRNA. If the sequences transcribe mRNA, block translation tion, integrated Sequences which are only Segments of of proteins with, for instance, an antisense oligonucleotide certain O Specimens are identified as foreign. Note that the specific for the exogenous mRNA. Alternatively, verify that test is dependent on the N population. For instance, a colony the proteins are not involved in binding of F to either which propagates from a Single cell might include a foreign Sequence. Also, Verify that co-transfection does not mutate polynucleotide in all daughter cells. Therefore, the NSpeci the F-boxes in DNA and DNA, and that the sequences do mens should include genomes (or cells) from different not change the methylation patterns of their F-boxes. lineages. Finally, check that DNA and DNA do not contact each other in the F-box region. 0.111) 2. A polynucleotide can also be identified as poten 0094) Examples tially foreign if it is found episomally in the nucleus. If the DNA is found in the cytoplasm, it is most likely foreign. 0.095 See studies in the section below entitled “Micro Also, a large enough polynucleotide can be identified as competition with a limiting transcription complex.” foreign if many copies of the polynucleotide can be 0096 (5) Foreign to observed in the nucleus. Finally, if Pn is identical to Sequences in genomes of other organisms, Such as viruses or 0097. Definition 1 bacteria, known to invade R cells, and Specifically nuclei of 0.098 Consider an organism R with standard genome O. R cells, Pn is likely foreign to R. Consider O. a segment of O. If a polynucleotide Pn is 0112 Definition 2 different from O for all O. in O, Pn is called “foreign to R.” 0113 Consider an organism R. If a polynucleotide Pn is 0099) Notes: immunologically foreign to R, Pn is called “foreign to R.” 0100) 1. As an example for different organisms consider the list of standard organisms in the PatentIn 3.1 software. 0114) Notes: The list includes organisms Such as, homo Sapiens (human), 0115 1. In Definition 1, the comparison between 0, the mus musculus (mouse), Ovis aries (sheep), and gallus gallus genome of the R organism, and Pn is performed logically by (chicken). the observer. In definition 2, the comparison is performed 0101) 2. Astandard genome is the genome shared by most biologically by the immune System of the organism R. representatives of the same organism. 0116 2. Definition 2 can be generalized to any compound 0102) 3. A polynucleotide and DNA sequence (see above) or Substance. A compound X is called foreign to organism R, are interchangeable concepts. if X is immunologically foreign to R. 0103 4. In multicellular organism, such as humans, the 0117 Exemplary Assays Standard genome of the organism is not necessarily found in 0118 1. If the test polynucleotide includes a coding every cell. The genomes found in Sampled cells can vary as region, incorporate the test polynucleotide in an expressing a result of Somatic mutations, viral integration, etc (See plasmid and transfer the plasmid into organism R, through, definition below of foreign polynucleotide in a specific cell). for instance, injection (see DNA-based immunization pro 0104 5. Assume Pn expresses the polypeptide Pp. If Pn tocols). An immune response against the expressed polypep is foreign to R, then Pp is foreign to R. tide indicates that the polynucleotide is foreign. 0105 6. When the reference organism is evident, instead 0119 2. Inject the test polynucleotide in R. An immune of the phrase “a polynucleotide foreign to organism R, the response against the injected polynucleotide indicates that “foreign polynucleotide’ phrase might be used. the test polynucleotide is foreign. US 2004/0023207 A1 Feb. 5, 2004

0120 Examples 0142. Notes: 0121 Many viruses, nuclear, such as Epstein-Barr, and 0.143 1. “Natural to” and “foreign to” are mutually cytoplasmic, Such as , express proteins which are exclusive. A polynucleotide cannot be both foreign and antigenic and immunogenic in their respective host cells. natural to R. If a polynucleotide is natural, it is not foreign 0122 Definition 3 to R, and if a polynucleotide is foreign, it is not natural to R. 0123 Consider an organism R with standard genome O. 014.4 2. If Pn is a gene natural to R, then, the its gene Consider Os, a segment of O. If a polynucleotide Pn is product is also natural to R. chemically or physically different than O for all O. in O, Pn 0145 3. The products of a reaction carried out in a cell is called “foreign to R.” between gene products natural to the cell, under normal conditions, are natural to the cell. For instance, cellular 0124 Notes: Splicing by factors natural to the cell produce Splice products 0.125 1. In Definition 3, the observer compares O, the natural to the cell. genome of the R organism, with Pn using the molecules chemical or physical characteristics. 0146 Exemplary Assays 0147 1. Compare the sequence of Pn with the sequence, 0.126 Exemplary assays or Sequences of the published, or Self Sequenced Standard 0127. In general, many assays in the “Detection of a genome of R. If the Sequence is a Segment of the Standard genetic lesion” Section below compare a test polynucleotide genome, Pn is natural to R. and a wild-type polynucleotide. In these assay, let Obe the 0148 2. Isolate DNA from O (for instance, from a wild-type polynucleotide and use the assays to identify a specific cell, or a virus). Try to hybridize Pn to the isolated foreign polynucleotide. Consider the following examples. DNA. If Pn hybridizes, it is natural. 0128 1. Compare the electrophoretic gel mobility of O. and the test polynucleotide. If mobility is different, the 0149) Notes: polynucleotides are different. 0150) 1. Hybridization with DNA from a specific O Specimen of R is not conclusive evidence that Pn is natural 0129 2. Compare the patterns of restriction enzyme to R. Consider, for example, the case of integrated viral cleavage of O and the test polynucleotide. If the patterns are genomes. Viral Sequences integrated into cellular genomes different, the polynucleotides are different. are foreign. To increase the probability of correct identifi 0.130 3. Compare the patterns of methylation of O. and cation, repeat the assay with N>1 specimens of O (for the test polynucleotide (by, for instance, electrophoretic gel instance, by collecting N cells from different representatives mobility). If the patterns are different, the polynucleotides of R). Define the genome of R as all DNA sequences found are different. in all O Specimens. Following this definition, integrated Sequences which are only Segments of certain O Specimens 0131 Definition 4 are identified as foreign. Note that the test is dependent on 0132) Consider an organism R with standard genome O. the N population. For instance, a colony which propagates Let Pn denote the copy number of Pnin O. Consider a cell from a single cell might include a foreign polynucleotide in Cell. Let Pn), denote the copy number of Pn in Celli. If all daughter cells. Therefore, the N specimens should Pn>Pn), Pn is called “foreign to Cell.” include genomes (or cells) from different lineages. 0133) Note 0151 (7) Empty Polynucleotide 0134) 1. Pn is the copy number of all Pnin Cell, from 0152 Definition all Sources. For instance, Pn includes all Pn Segments in 0, 0153 Consider the Pn polynucleotide. Consider an all Pn segments of viral DNA in the cell (if available), all Pn organism R with genome OR Let Pp(Pn), and Pp(O) denote segments of plasmid DNA in the cell (if available), etc. a gene product (polypeptide) of a Pn or O gene, respec 0135 1. If Pn=0, the definition is identical to definition tively. If Pp(Pn)-Pp(Os) for all Pp(Pn), Pn will be called an 1 of foreign polynucleotide. “empty polynucleotide' in respect to R. 0.136 Exemplary Assays 0154) Notes: 0137) 1. Sequence the genome of Cell. Count the number O155 1. A vector is a specific example of a polynucle of time Pn appears in the genome. Compare the result to the otide. number of times Pn appears in the published standard 0156 2. A vector that includes a non coding polynucle genome. If the number is greater, Pn is foreign to Cell. otide natural to R is considered empty in respect to the R. 0138 2. Sequence the genome of Cell and a group of (“natural to” is the opposite of “foreign to.” Note: A natural other cells Cell,..., Cell. If Pnli-Pnl= ... =Pn), polynucleotide means, a polynucleotide natural to at least Pn is foreign to Cell. one organism. An artificial polynucleotide means a poly nucleotide foreign to all known organisms. A viral enhancer 0139 (6) Natural to is a natural polynucleotide. A plasmid with a viral enhancer 0140. Definition fused to a human gene is artificial.) 0141 Consider an organism R with standard genome O. O157 3. A vector that includes a coding gene natural to Q, If a polynucleotide Pn is a fragment of O, Pn is called an organism different from R, can Still be considered empty “natural to R. in respect to R. For instance, a vector that includes the US 2004/0023207 A1 Feb. 5, 2004 bacterial chloramphenicol transacetylase (CAT), bacterial period of time. If the polynucleotide ShowS limited replica neomycin phosphotransferase (neo), or the firefly luciferase tion, no transcription, or a limited Set of transcripts, the (LUC) as reporter genes, but no human coding gene is polynucleotide is latent. considered empty in respect to the humans if it does not express a gene natural to humans. 0177 2. Introduce, or identify a foreign polynucleotide in a host cell. ASSay the cell over an extended period of time, 0158 Exemplary Assays if the cell shows no lytic Symptoms, the polynucleotide is 0159) 1. Identify all gene products encoded by Pn. Com latent. pare to the gene products of O. If all gene products are 0178 Examples different, Pn is considered empty in respect to the R. 0179. Using PCR, a study (Gonelli 2001') observed 0160 Examples persistent presence of viral human herpes virus 7 (HHV-7) 0.161 pSV2CAT, which expresses the chloramphenicol DNA in biopsies from 50 patients with chronic gastritis. The acethyltransterase (CAT) gene under the control of the SV40 study also observed no U14, U17/17, U31, U42 and U89/90, promoter/enhancer, pSV2neo, which expresses the neogene HHV-7 Specific transcripts highly expressed during replica under the control of the SV40 promoter/enhancer, HSV-neo, tion. Based on these observations, the Study concluded that which expresses the neomycin-resistance gene under control “gastric tissue represents a site of HHV-7 latent infection of the murine Harvey Sarcoma virus long terminal repeat and potential reservoir for viral reactivation.” To test the (LTR), pZIP-Neo, which expresses the neomycin-resistant effect of treatment on the establishment of latent herpes gene under control of the Moloney Simplex virus, type 1 (HSV-1) in Sensory neurons, another long terminal repeat (LTR), are considered empty polynucle study (Smith 2001) assays the expression of the latency otides, or empty vectors, in respect to humans and in respect associated transcript (LAT), the only region of the viral to the respective virus. See more examples below. genome transcribed at high levels during the period of viral latency. A recent review (Young 2000) discusses the limited 0162 Note: These vectors can be considered as “double” sets of Epstein-Barr viral (EBV) gene products expressed empty, empty in respect to humans, and empty in respect to during the period of Viral latency. the respective virus. 0163 (8) Latent Foreign Polynucleotide 0180 (9) Partial Description 0164. Definition 0181 Definition 0.165 Consider Pn, a polynucleotide foreign to organism 0182 Let C be a characteristic of a system. Let the set Ci, i=(1 ... m) be the set of characteristics providing a complete R. Pn will be called latent in a Cell of R if over an extended description of the System. Any Subset of Ci, i=(1 . . . m) is period of time, either: called a “partial description' of the System. 0166 1. Pn produces no Pn transcripts. 0183 Exemplary Assays 0167 2. Denote the set of gene products expressed by Pn in Cell, with Cell. Pp(Pn) and the set of all possible gene 0.184 1. Chose any set of characteristics describing the products of Pn with All Pp(Pn), then, Cell System and assay these characteristics. Pp(Pn)C All Pp(Pn), that is, the set of Pn gene products 0185. Examples expressed in Cell is a Subset of all possible Pn gene products. 0186 Assaying blood pressure, blood triglycerides, glu 0168 3. Pn shows limited or no replication. cose tolerance, body weight, etc. 0169. 4. Pn is undetected by the host immune system. 0187 (10) Equilibrium 0170 5. Cell shows no lytic symptoms. 0188 Definition 0171 6. R shows no macroscopic symptoms. 0189 If a system persists in a state Sto over time, Sto is 0172) Notes: called equilibrium. 0173 1. A virus in a host cell is a foreign polynucleotide. 0190. Note: According to the definition, a virus is considered latent if, 0191 The system related definitions can be modified to over an extended period of time, it either shows partial accommodate partial descriptions. For example, consider a expression of its gene products, no viral mRNA, limited or description of a System which includes only a proper Subset no replication, is undetected by the host immune System, of Ci, i=(1 ... m). If the values measured for the subset of causes no lytic Symptoms in the infected cell, or causes no characteristics in Sto persist over time, the probability that macroscopic Symptoms in the host. St, is an equilibrium is greater than Zero. However, since the 0.174 2. The above list of characterizations is not exhaus values are measured only on a Subset of Ci, i=(1... m), the tive. The medical literature includes more aspects of latency probability is less than 1. Overall, an increase in the size of that can be added to the definition. the Subset of characteristics increases the probability. 0175 Exemplary Assays 0.192 Exemplary Assays 0176 1. Introduce, or identify a foreign polynucleotide in 0193 1. Assay the values of the complete (sub) set of the a host cell. ASSay the polynucleotide replication, or tran System characteristics. Repeat the assays over time. If the Scription, or mRNA, or gene products over an extended values persist, the System is (probably) in equilibrium. US 2004/0023207 A1 Feb. 5, 2004

0194 Examples 0213) Notes: 0.195 Regular physicals include standard tests, such as 0214) 1. Using the above definitions it can be said that a blood count, cholesterol levels, HDL cholesterol, triglycer disruption is an exogenous event that produces a chronic ides, kidney function tests, thyroid function tests, liver disease. function tests, minerals, blood Sugar, uric acid, electrolytes, 0215 2. A disruption is a disturbance with a persisting resting electrocardiogram, an exercise treadmill test, vision effect. testing, and audiometry. When the values in these tests remain within a narrow range over time, the medical con 0216 Exemplary Assays dition of the subject can be labeled as a probable equilib 0217 1. Take a biological System (e.g., cell, whole organ rium. Other tests performed to identify deviations from ism, etc). ASSay a set of characteristics. Compare the results equilibrium are mammograms and prostate cancer Screen with the values of the same characteristics in healthy con IngS. trols. Verify that the system is in healthy equilibrium. Apply 0196) (11) Stable Equilibrium a chosen treatment to the System. Following treatment, assay the same characteristics again. If Some values deviate from 0197) Definition the values of healthy controls, continue to assay these characteristics over time. If the values continue to deviate 0198 Consider an equilibrium E. If, after small distur over time, the treatment produced a chronic disease, and, bances, the System always returns to Eo, the equilibrium is therefore, can be considered a disruption. called “stable.” If the system moves away from Eo after small disturbances, the equilibrium is called “unstable.” 0218. Examples 0219 Genetic knockout, carcinogens, infection with per 0199 Exemplary Assays sistent viruses (e.g., HIV, EBV), etc. 0200) 1. Take a biological system (e.g., cell, whole organ 0220 (14) Foreign Polynucleotide-Type Disruption ism, etc). ASSay a set of characteristics. Verify that the (Cause of Disruption) System is in equilibrium, that is, the values of these char acteristics persist over time. Apply treatment to the System 0221) Definition and assay the Set of characteristics again. Repeat assaying 0222 Let Pp be a polypeptide. Assume microcompetition over time. If the treatment changed the values of the with a foreign polynucleotide Pn directly, or indirectly characteristics, and within a reasonable time the values reduces (or increases) Pp bioactivity. A disruption that returned to the original levels, the equilibrium is stable. directly, or indirectly reduces (or increases) Pp bioactivity is called “foreign polynucleotide-type disruption.” 0201 (12) Chronic Disease 0223) Notes: 0202) Definition 0224) 1. The first “indirectly in the definition means that 0203 Let a healthy biological system be identified with a Pp can be downstream from the gene microcompeting with certain stable equilibrium. A stable equilibrium different Pn. The second “indirectly” means that Pp can be down from the healthy system equilibrium is called “chronic Stream from the gene, or polypeptide, directly affected by the disease.” exogenous event. According to the definition, if both micro competition with a foreign polynucleotide and an exogenous 0204) Notes: event increase, or both decrease bioactivity of Pp, the 0205 1. In chronic disease, in contrast to acute disease, exogenous event can be considered as a foreign polynucle the System does not return to the healthy equilibrium on its otide-type disruption. OW. 0225 2. Microcompetition with a foreign polynucleotide is a Special case of foreign polynucleotide-type disruption. 0206 Exemplary Assays 0226) 3. Treatment is a special case of an exogenous 0207 1. Take a biological system (e.g., cell, whole organ eVent. ism, etc). ASSay a set of characteristics. Compare the results 0227 4. A foreign polynucleotide-type disruption can with the values of the same characteristics in healthy con first affect a gene or a polypeptide. For instance, a mutation trols. If some values deviate from the values of healthy is an effect on a gene. Excessive protein phosphorylation is controls, and the values continue to deviate over time, the equilibrium of the System can be characterizes as chronic an effect on a polypeptide. disease. 0228 Exemplary Assays 0208 Examples 0229) 1. Take a biological System (e.g., cell, whole organ ism, etc). ASSay a set of characteristics. Compare the results 0209 High blood pressure, high body weight, hypergly with the values of the same characteristics in healthy con cemia, etc. trols to verify that the system is in a healthy equilibrium. Modify the copy number of Pn, a polynucleotide of interest 0210 (13) Disruption (by, for instance, transfection, infection, mutation, etc, See above). Identify a gene with modified expression. ASSume 0211 Definition the assays show decreased expression of G. Take another 0212 Let a healthy biological system be identified with a Specimen of the System in healthy equilibrium and apply a certain stable equilibrium. Any exogenous event which chosen treatment to the healthy Specimen. Following treat produces a new stable equilibrium is called “disruption.” ment, assay G expression. Continue to assay G expression US 2004/0023207 A1 Feb. 5, 2004 over time. If G expression is persistently decreased, the 0252) Notes: exogenous event can be considered a foreign polynucle 0253 Consider, as an example, microcompetition otide-type disruption. between a cell and a viral polynucleotide, including the 0230. Examples entire Viral genome. Pp. can be any viral or cellular protein 0231. A mutation in the leptin receptor, a mutation in the which increase or decreases viral replication. leptin gene, etc (see more examples below). 0254 Exemplary Assays 0232 (15) Disrupted (Gene, Polypeptide) (Result of Dis 0255 1. Take a biological System (e.g., cell, whole organ ruption) ism, etc). Apply a treatment to the System that modifies Pp. bioactivity, for instance, by increasing expression of a 0233. Definition foreign or cellular gene encoding Pp. ASSay Pn copy 0234) Let Pp be a polypeptide. If a foreign polynucle number. If the copy number changed, Pp. and the gene otide-type disruption modifies (reduces or increases) Pp encoding. Pp., are in a Pn disruptive pathway. bioactivity, Pp and the gene encoding Pp are called "dis 0256 Examples rupted.” 0257 Consider a GABP virus. The viral proteins which 0235. Notes: increase viral replication increase the copy number of viral 0236 1. Pp can be downstream from G, the microcom N-boxes in infected cells. According to the definition, these peted gene. proteins belong to a disruptive pathway. See Specific examples below. 0237 Exemplary Assays 0258 b) p300/cbp Related Elements 0238 1. Take a biological System (e.g., cell, whole organ ism, etc). Modify the copy number of Pn, a polynucleotide 0259 (1) p300/cbp of interest, (by, from instance, transfection, infection, muta 0260 Definition tion, etc, See above). ASSay bioactivity of genes and polypeptides in the treated System and controls to identify 0261) A member of the p300/cAMP response element genes and polypeptides with modified bioactivity relative to (CREB) binding protein (CBP) family of proteins is called controls. These genes and polypeptides are disrupted. p300/cbp. 0239 Examples 0262) Notes: 0240 See studies in the section below entitled “Micro 0263 1. For reviews on the p300/cbp family of proteins, competition with a limiting transcription complex. See also see, for instance, Vo 20017, Blobel 2000, Goodman 2000, all GABP regulated genes below. Hottiger 2000", Giordano 1999'', Eckner 1996. 0264. 2. CREB binding protein (CBP, or CREBBP) is 0241 (16) Disrupted Pathway also called RTS, Rubinstein-Taybi syndrome protein, and 0242 Definition RSTS. 0243 Let the polypeptide Pp be disrupted. A polypeptide 0265 3. See sequences of p300/cbp genes and p300/cbp Pp; which functions downstream or upstream of Pp, and the proteins in the List of Sequences below. gene encoding Pp, are considered a polypeptide and gene, respectively, in a Pp. “disrupted pathway.” 0266 Exemplary assays 0267 1. p300/cbp may be identified using antibodies in 0244 Exemplary Assays binding assays, oligonucleotide probes in hybridization 0245 1. Take a biological System (e.g., cell, whole organ assays, transcription factors such as GABP, NF-kB, E1A in ism, etc). Apply a treatment to the System that modifies Pp: binding assays, etc. (see protocols for binding and hybrid bioactivity. Assay Pp bioactivity. If the bioactivity of Pp. ization assays below). changed, Pp. is in a Pp disrupted pathway. 0268 Examples 0246 2. Take a biological System (e.g., cell, whole organ 0269. See examples of below. ism, etc). Apply a treatment to the System that modifies Pp. bioactivity. Assay Pp bioactivity. If the bioactivity of Pp. 0270 (2) p300/cbp Polynucleotide changed, Pp. is in a Pp disrupted pathway. 0271) Definition 0247 Examples 0272 Assume the polynucleotide Pn binds the transcrip tion complex C. If C contains p300/cbp, Pn is called 0248. See examples below. “p300/cbp polynucleotide.” 0249 (17) Disruptive Pathway 0273 Exemplary Assays 0250) Definition 0274) 1. Take a cell of interest. Modify the copy number 0251 Consider a polypeptide Pp and a foreign poly of Pn (by, for instance, transfection, infection, mutation, etc., nucleotide Pn. If a change in bioactivity of Pp increases or See also above). Use assays described in the Section entitled decreases Pn copy number, Pp. and the gene encoding. Pp. “Identifying a polypeptide bound to DNA or protein com are considered a polypeptide and a gene in a Pn “disruptive plexes,” or Similar assays, to test if the protein-Pin complexes pathway.” contain p300/cbp. US 2004/0023207 A1 Feb. 5, 2004 10

0275 2. See more assays below. 0281 Exemplary Assays 0276 Examples 0282) 1. Use assays describe in the section entitled “Iden 0277) See below in p300/cbp virus and p300/cbp regu tifying a polypeptide bound to DNA or protein complexes,” lated gene. or Similar assays, to test whether the complexes which 0278 (3) p300/cbp Factor contain F also contain p300/cbp. 0279 Definition 0283 Examples 0280 Assume the transcription factor F binds the com plex C. If C contains p300/cbp, F is called “p300/cbp 0284. The following table lists some cellular and viral factor.” p300/cbp factors.

p300/cbp Gene factor symbol Other names References Cellular

AML.1 RUNX1. acute myeloid leukemia 1 protein (AML1); Kitabayashi CBFA2 core-binding factor C.2 subunit (CBFC.2); 1998.13 AML.1 AML-1; Polyomavirus enhancer binding protein 2CB subunit (PEBP2CB); PEA2C.B.; SL3-3 enhancer factor 1, CB subunit; SL3/AKV core-binding factor CB subunit; SEF1; runt-related transcription factor 1; RUNX1; CBFA2 A-Myb MYBL1 Myb-related protein A; v-myb avian Facchinetti AMYB myeloblastosis viral oncogene homolog 199714 like 1 ATF1 ATF1 activating transcription factor 1 (ATF1): Goodman 2000 TREB36 TREB36 protein; cAMP-dependent Goodman 2000 transcription factor ATF-1 ATF2 ATF2 Activating transcription factor 2 (ATF2); Goodman 2000 CREB2 cAMP response element binding protein 1 Goodman 2000, CREBP1 (CRE-BP1); HB16; cAMP-dependent Duyndam 1999' transcription factor ATF-2; TREB7; CREB2 ATF4 ATF4 activating transcription factor 4 (ATF4); Goodman 2000 CREB2 DNA-binding protein TAXREB67; tax Goodman 2000, TAXREB67 responsive enhancer element B67 Yukawa 1999 (TAXREB67); TXREB; cAMP response element-binding protein 2 (CREB2); cAMP-dependent transcription factor ATF 4. CCAAT?enhancer binding protein related activating transcription factor (mouse); ApCREB2 (Aplysia) BRCA1 BRCA1 Breast cancer type 1 susceptibility protein Goodman 2000 PSCP (BRCA1) Goodman 2000 C/EBPB CEBPB CCAAT/enhancer binding protein B Goodman 2000 TCF5 (C/EBPB); nuclear factor NF-IL6 (NFIL6); Goodman 2000, transcription factor 5: CRP2; LAP: Mink 19977 IL6DBP; CEBPB, TCF5 c-Fos FOS proto-oncogene protein c-fos; cellular Goodman 2000 GOST oncogene fos; G0/G1 Switch regulatory Goodman 2000, protein 7: V-fos FBJ murine Osteosarcoma Sato 1997 viral oncogene homolog; FOS; GOST Sato 1997 MHC2TA MHC class II transactivator; MHC2TA; Goodman 2000 CITA CITA Goodman 2000, C2TA Sisk 2000 Sisk 2000 AP1 JUN transcription factor AP-1; proto-oncogene Goodman 2000 c-Jun (c-Jun); p39; V-jun avian sarcoma Goodman 2000, virus 17 oncogene homolog Hottiger 2000 Hottiger 2000 c-Myb MYB Myb proto-oncogene protein; MYB; v-myb Goodman 2000 avian myeloblastosis viral oncogene Goodman 2000, homolog Hottiger 2000 Hottiger 2000 CREB CREB1 cAMP-respone-element-binding protein Hottiger 2000 (CREB) Hottiger 2000 CRX CRX cone-rod homeobox (CRX); CRD; cone Yanagi 2000' CORD2 rod dystrophy 2 (CORD2) CRD US 2004/0023207 A1 Feb. 5, 2004 11

-continued p300/cbp Gene factor symbol Other names References CID CI-D cubitus interruptus dominant (CID) Goodman 2000 Goodman 2000 DBP DBP D-site binding protein (DBP); albumin D Lamprecht box-binding protein; D site of albumin 1999.19 promoter (albumin D-box) binding protein; TAXREB3O2 E2F1 E2F1 retinoblastoma binding protein 3 (RBBP Goodman 2000 RBBP3 3); PRB-binding protein E2F-1; PBR3; Goodman 2000, retinoblastoma-associated protein 1 Marzio 2000? (RBAP-1) E2F2 E2F2 transcription factor E2F2 Marzio 2000 Marzio 2000 E2F3 E2F3 transcription factor E2F3; KIAAO075 Marzio 2000 KIAAO075 Marzio 2000 Egr1 EGR1 early-growth response factor-1 (Egr1); Silverman ZNF225 Krox-24 protein: Z1F268; nerve growth 199821 factor-induced protein A; NGFI-A; transcription factor ETR103; zinc finger protein 225 (ZNF225); AT225; TIS8; GOS30; ZIF-268 ELK1 ELK1 ets-domain protein ELK-1 Hottiger 2000 Hottiger 2000 ERC ESR1 estrogen receptor C. (ERC); estrogen Kim 20012, NR3A1 receptor 1; estradiol receptor Wang 2001’, ESR Speir 2000’, Hottiger 2000 Hottiger 2000 ERB ESR2 estrogen receptor B; ESR2; NR3A2; Kobayashi NR3A2 ESTRB 2OOO25 ESTRB ER81 Ets translocation variant 1 (ETV1) Papoutsopoulou 2OOO26 Ets1 ETS1 C-ets-1 protein; v-ets avian erythroblastosis Goodman 2000 virus E2 oncogene homolog 1; p54 Goodman 2000, Jayaraman 199927 EtS2 ETS2 C-ets-2 protein; human erythroblastosis Jayaraman 1999 virus oncogene homolog 2; V-ets avian Jayaraman 1999 erythroblastosis virus E2 oncogene homolog 2 GABPC GABPA GA binding protein, C. subunit (GABPA); Bannert 1999' E4TF1A GABP-alpha subunit; transcription factor E4TF1-60; nuclear respiratory factor-2 subunit alpha (NRF-2A) GABPB1 GABPB1 GA binding protein beta-1 chain Bannert 1999 GABPB (GABPB1); GABP-beta-1 subunit: Bannert 1999 E4TF1B transcription factor E4TF1-53; nuclear respiratory factor-2 subunit beta 2 (NRF 2B) GABPB2 GABPB1 GA binding protein beta-2 chain Bannert 1999 GABPB (GABPB2); GABP-beta-2 subunit: Bannert 1999 E4TF1B transcription factor E4TF1-47 GATA1 GATA1 globin transcription factor 1; GATA Goodman 2000 GF1 binding protein 1 erythroid transcription Goodman 2000 ERYF1 factor; ERYF1; GF1; NF-E1 NFE1 G3 GLI3 zinc finger protein GLI3: PAP-A; GCPS; Goodman 2000 GLI-Kruppel family member GLI3 (Greig Goodman 2000 cephalopolysyndactyly syndrome); Pallister-Hall syndrome (PHS) GR NR3C1 glucocorticoid receptor (GR); nuclear Pfitzner 1998 GRL receptor subfamily 3, group C, member 1 Pfitzner 1998, GCR (NR3C1); GRL Hottiger 2000 Hottiger 2000 HIF1C. HIF1A hypoxia-inducible factor-1 C. (HIF1C); Goodman 2000 ARNT interacting protein; member of PAS Goodman 2000, protein 1; MOP1 Bhattacharya 1999', Kallio 19983, Ema 1999, Hottiger 2OOO Hottiger 2000 US 2004/0023207 A1 Feb. 5, 2004 12

-continued p300/cbp Gene factor symbol Other names References heaptocyte nulcear factor-1 C.; HNF-4- Goodman 2000 C.; transcription factor HNF-4; transcription Goodman 2000, factor 14; MODY; maturity onset diabetes Soutoglou of the young 1; MODY1; HNF4A: 2OOO32 NR2A1; TCF14; HNF IRF-3 IRF3 interferon regulatory factor-3 (IRF-3) Goodman 2000 Goodman 2000, Yoneyama 19983 JunB JUNEB transcription factor JunB; proto-oncogene Goodman 2000 JunB Goodman 2000 Mdm2 MDM2 mouse double minute 2; human homolog Goodman 2000 of -binding protein (Mdm2); ubiquitin Goodman 2000 protein ligase E3 Mdm2; EC 6.3.2.-: p53 binding protein Mdm2; oncoprotein Mdm2; double minute 2 protein; Ham2 MEF2C MEF2C myocyte enhancer factor 2C (MEF2C); Sartorelli 1997 myocyte-specific enhancer factor 2C; Sartorelli 1997 MADS box transcription enhancer factor 2 polypeptide C MITF microphthalmia-associated transcription Goodman 2000 factor Goodman 2000, Sato 1997 MyoD MYOD1M myoblast determination protein 1 (MyoD); Yuan 1996 Ref, YF3 myogenic factor MYF-3; myogenic factor Sartorelli 1997 3: PUM NFAT1 NFAT1 nuclear factor of activated T cells, Garcia NFATC2 cytoplasmic 2: T cell transcription factor Rodriguez NEATP NFAT1: NFAT pre-existing subunit; NF 1998, Sisk ATp 2OOO37 NFYB NF-Y protein chain B (NF-YB); nuclear Li 19983, HAP3 transcription factor Y subunit beta; C-CP1, Faniello 1999 CP1; CCAAT-binding transcription factor subunit A (CBF-A); CAAT-box DNA binding protein subunit B NFYA NF-Y protein chain A (NF-YA); CCAAT. Lil 1998 HAP2 binding transcription factor subunit B Lil 1998 (CBF-B); CAAT-box DNA binding protein subunit A; nuclear transcription factor Y C. RelA. RELA NF-kB RelA, transcription factor po5; Hottiger 1998', NFKB3 nuclear factor NF-kappa-B, p65 subunit; v Gerritsen 1997', rel avian reticuloendotheliosis viral Speir 2000', oncogene homolog A; nuclear factor of Hottiger 2000 kappa light polypeptide gene enhancer in Hottiger 2000 B-cells 3 (p65) P/CAF P/CAF p300/cbp-associated factor Goodman 2000 Goodman 2000 p/CIP TRAM-1 p300/cbp interacting protein (p/CIP); Goodman 2000 NCOA3 thyroid hormone receptor activator Goodman 2000 AB1 molecule; DJ1049g16.2; nuclear receptor coactivator 3 (thyroid hormone receptor activator molecule TRAM-1; receptor associated coactivator RAC3; amplified in breast cancer AIB1; ACTR PPARY PPARG peroxisome proliferator activated receptor Y Iannone 2001, NR1C3 (PPARG); PPAR-gamma; PPARG1; Kodera 2000' PPARG2 MRG1 CITED2 Cbp/p300-interacting transactivator 2; Bhattacharya MRG1 MSG-related protein 1; melanocyte 1999 specific gene 1; MRG1 protein Bhattacharya 1999, Han 2001 NFE2 nuclear factor, erythroid-derived 2 45 kDa Goodman 2000 subunit; NF-E2 45 kDa subunit (p45 NF Goodman 2000 E2); leucine zipper protein NF-E2 cellular tumor antigen p53; tumor Goodman 2000 suppressor p53:, phosphoprotein p53: Li Goodman 2000, Fraumeni syndrome Avantaggiati 1997 Van Order 19997, Hottiger 2000 Hottiger 2000 US 2004/0023207 A1 Feb. 5, 2004 13

-continued p300/cbp Gene factor symbol Other names References p73 TP73 tumor protein p73; p53-like transcription Goodman 2000 P73 factor; p53-related protein Goodman 2000 Pit-1 POU1F1 pituitary-specific positive transcription Goodman 2000 PIT1 factor 1: PIT-1; growth hormone factor 1, Goodman 2000 GHF1 GHF-1; POU domain, class 1, transcription factor 1 RSK1 RPS6KA1 90-kDA ribosomal S6 kinase, ribosomal Goodman 2000 RSK1 protein S6 kinase alpha 1: EC 2.7.1.-: S6K- Goodman 2000, alpha 1; 90 kDa ribosomal protein S6 Hottiger 2000 kinase 1: p.90-RSK1, ribosomal S6 kinase Hottiger 2000 1; RSK-1; pp.90RSK1; HU-1 RSK3 RPS6KA2 Ribosomal protein S6 kinase alpha 2: EC Hottiger 2000 RSK3 2.7.1.-: S6K-alpha 2: 90 kDa ribosomal Hottiger 2000 protein S6 kinase 2:... p90-RSK 2; ribosomal S6 kinase 3: RSK-3; pp.90RSK3; HU-2 RSK2 RPS6KA3 ribosomal protein S6 kinase alpha 3: EC Hottiger 2000 RSK2 2.7.1.-: S6K-alpha 3: 90 kDa ribosomal Hottiger 2000 ISPK1 protein S6 kinase 3; p90-RSK3; ribosomal S6 kinase 2: RSK-2: pp.90RSK2: Insulin stimulated protein kinase 1; ISPK-1; HU 2:, HU-3 RARY RARG retinoic acid receptor Y (RARY); retinoic Hottiger 2000 NR1B3 acid receptor gamma-1, RAR-gamma-1: Hottiger 2000, RARC; retinoic acid receptor gamma-2; Yang 2001 RAR-gamma-2 RNA DDX9 ATP-dependent RNA A; nuclear Goodman 2000 helicase NDH2 DNA helicase II (NDH II); DEAD-box Goodman 2000 A. protein 9; leukophysin (LKP) RXRC. RXRA retinoic acid receptor RXR-C. Goodman 2000 NR2B1 Goodman 2000, Yang 2001 Yang 2001 ELK4 ELK4 ETS-domain protein ELK-4; serum Goodman 2000 SAP1 response factor accessory protein 1 (SAP- Goodman 2000, 1); SRF accessory protein 1 Hottiger 2000 Hottiger 2000 SF-1 NRSA1 steroidogenic factor 1 (STF-1, SF-1); Goodman 2000 FTZF1 steroid hormone receptor AD4BP: Fushi Goodman 2000 AD4BP tarazu factor (Drosophila) homolog 1; SF1 FTZ1; ELP; NR5A1 (nuclear receptor subfamily 5, group A, member 1) Smad3 MADH3 mothers against decapentaplegic Goodman 2000 SMAD3 (Drosophila) homolog 3 (SMAD 3); Goodman 2000, MAD3 mothers against DPP homolog 3; Mad3; Janknecht 1998', hMAD-3; mMad3; JV15-2: hSMAD3 Feng 1998, Pouponnot 1998 Pouponnot 1998 Smada. MADH4 mothers against decapentaplegic de Caestecker', SMAD4 (Drosophila) homolog 4 (SMAD 4); Pouponnot 1998 DPC4 mothers against DPP homolog 4: deletion Pouponnot 1998 target in pancreatic carcinoma 4, hSMAD4 Smad1 MADH1 mothers against decapentaplegic Pearson 1999, SMAD1 (Drosophila) homolog 1 (SMAD 1); Pouponnot MADR1 mothers against DPP homolog 1; Mad- 1998.53 BSP1 related protein 1; transforming growth factor-beta signaling protein-1; BSP-1; hSMAD1; JV4-1 Smad3 MADH2 mothers against decapentaplegic Pouponnot 1998 SMAD2 (Drosophila) homolog 2 (SMAD 2); Pouponnot 1998 MADR2 mothers against DPP homolog 2; Mad related protein 2; hMAD-2: JV18-1; hSMAD2 SRC-1 SRC1 steroid receptor coactivtor - 1 (SRC-1); F- Goodman 2000 NCOA1 SRC-1; nuclear receptor coactivator 1 Goodman 2000, (NCOA-1); SRC1 Hottiger 2000 Hottiger 2000 SREBP1. SREBF1 sterol regulatory element binding protein-1 Goodman 2000 SREBP1 (SREBP-1); sterol regulatory element- Goodman 2000, binding transcription factor 1 Oliner 1996 US 2004/0023207 A1 Feb. 5, 2004 14

-continued p300/cbp Gene factor symbol Other names References SREBP2 SREBF2 sterol regulatory element binding protein-2 Goodman 2000 SREBP2 (SREBP-2); sterol regulatory element- Goodman 2000, binding transcription factor 2 Oliner 1996 Oliner 1996 Stat-1 STAT1 signal transducer and activator or Goodman 2000 transcription - 1C/B; transcription factor Goodman 2000, ISGF-3 components p91/p84; signal Paulson 1999, transducer and activator of transcription 1, Hottiger 1998 91kD (STAT91) Hottiger 1998, Gingras 1999 Gingras 1999, Zhang 1996 Stat-2 STAT2 signal transducer and activator or Goodman 2000 transcription - 2 (STAT2); ; signal Goodman 2000, transducer and activator of transcription 2, Paulson 1999 113kD (STAT113): p113 Paulson 1999, Hottiger 1998 Hottiger 1998, Gringras 1999 Gringras 1999, Bhattacharya 1996, Hottiger 2OOO Hottiger 2000 Stat-3 STAT3 signal transducer and activator or Paulson 1999 APRF transcription - 3; acute-phase response Paulson 1999, factor Hottiger 1998 Hottiger 1998 Stat-4 STAT4 signal transducer and activator or Paulson 1999 transcription - 4 Paulson 1999 Stat-5 STATS signal transducer and activator or Paulson 1999 STATSA transcription - 5A (STAT5A); MGF; signal Paulson 1999 STATSB transducer and activator or transcription - check, 5B (STATSB); STAT5 Gingras 1999 Gingras 1999, Pfitzner 1998 Stat-6 STAT6 signal transducer and activator or Paulson 1999 ranscription - 6 (STAT6); IL-4 Stat; Paulson 1999 D12S1644 check, Gingras 1999 TAL1 TAL1 T-cell acute lymphocytic leukemia-1 Goodman 2000 SCL protein; TAL-1 protein; STEM cell protein; Goodman 2000 TCL5 T-cell leukemia/lymphoma-5 protein TBP TBP TATA box binding protein (TBP); Goodman 2000 TFIID ranscription initiation factor TFIID; Goodman 2000 TF2D TATA-box factor; TATA sequence binding protein; SCA17: GTF2D1; HGNC:15735; GTF2D TFIIB TFIIB ranscription factor IIB (TFIIB, TF2B); Goodman 2000 TF2B ranscription initiation factor IIB; general Goodman 2000, GTF2B ranscription factor IIB (GTFIIB, GTF2B) Hottiger 2000 Hottiger 2000 THRA THRA hyroid hormone receptor C. (THRA); C- Hottiger 2000 NR1A1 erbA-alpha; c-erbA-1; EAR-7; EAR7; Hottiger 2000 THRA1 AR7; avian erythroblastic leukemia viral ERBA1 (v-erb-a) oncogene homolog; ERBA; THRA1:THRA2: THRA3; EAR-7.1/EAR 7.2 THRB THRB thyroid hormone receptor B1 (THRB); Hottiger 2000 NR1A2 thyroid hormone receptor, beta; avian Hottiger 2000 THR1 erythroblastic leukemia viral (v-erb-a) ERBA2 oncogene homolog 2: THRB1; THRB2; ERBA2; NR1A2; thyroid hormone receptor B2 (THRB) Twist TWIST Twist related protein; H-twist; Goodman 2000 acrocephalosyndactyly 3 (Saethre-Chotzen Goodman 2000, syndrome); twist (Drosophila) homolog; Hamamori acrocephalosyndactyly 3 (ACS3) 199960 YY1 YY1 Ying Yang 1 (YY1); transcriptional Goodman 2000 repressor protein YY1; delta transcription Goodman 2000 factor; NF-E1; UCRBP; CF1; Yin Yang 1; DELTA: YY1 transcription factor US 2004/0023207 A1 Feb. 5, 2004 15

-continued p300/cbp Gene factor symbol Other names References Viral

E1A Goodman 2000 Goodman 2000, Hottiger 2000 Hottiger 2000 EBNA2 EBV Goodman 2000 Goodman 2000 Py LT polyomavirus large T antigen Goodman 2000 Goodman 2000 SV40 LT simian virus 40 large T antigen, TAg Goodman 2000 Goodman 2000, Hottiger 2000 Hottiger 2000 HPV E2 human papillomavirus E2 Goodman 2000 Goodman 2000 HPV E6 human papillomavirus E6 Goodman 2000 Goodman 2000, Hottiger 2000 Hottiger 2000 Tat HIV-1 Goodman 2000 Goodman 2000, Hottiger 2000 Hottiger 2000 Tax Human T-cell leukemia virus type 1 Goodman 2000 Goodman 2000, Hottiger 2000 Hottiger 2000 Bacterial JMY H. pylori Goodman 2000 (cag) Goodman 2000

0285) The two major lists are from reviews by Goodman 0293) Note: 2000 and Hottiger 2000. 0294 Preferably, verify that co-transfection did not 0286 Mutations in some of these p300 factors are cur induce a change in cellular microcompetition, a mutation in rently associated with chronic diseases. HNF4A with the gene promoter, or a change in methylation of gene MODY, ESR1 with breast cancer and bronchial asthma, GR promoter. with cortisol resistance, etc. Consider the following defini 0295 3. Transfect a cell with the gene promoter fused to tion. a reporter gene, such as CAT or LUC. Contact the cell with an antibody against p300/cbp (or with a protein Such as 0287 (4) p300/cbp Regulated (Gene, Polypeptide) E1A). ASSay in the antibody treated cell and in the untreated controls. If reporter gene expression is 0288) Definition higher or lower in the antibody treated cell, the gene is 0289 Assume the gene G is transactivated, or suppressed p300/cbp regulated. by the transcription complex C. If C contains p300/cbp, the 0296 4. Select a cell which expresses a gene of interest. gene G, and the polypeptide encoded by G, are called Contact the cell with an antibody against p300/cbp (or with “p300/cbp regulated.” a protein Such as E1A). ASSay gene expression in both the 0290 Exemplary assays treated cell and in the untreated controls. If gene expression is higher or lower in the antibody treated cell, the gene is 0291 1. Co-transfect a cell with the gene promoter fused p300/cbp regulated. to a reporter gene, Such as CAT or LUC, and a vector expressing p300/cbp. ASSay reporter gene expression in the 0297 5. Perform chromatin assembly of the gene pro p300/cbp transfected cell and in control cells transfected moter, for instance, with chromatin assembly extract from with the fused gene promoter along with an “empty' plas Drosophila embryos. Add a transcription factor during the mid. If reporter gene expression is higher or lower in the chromatin assembly reactions. After the chromatin assembly p300/cbp transfected cell, the gene is p300/cbp regulated. reaction is complete add the p300/cbp proteins. Allow time for the interaction of the proteins with the chromatin tem 0292 2. Select a cell which expresses the gene of interest plate. Perform in vitro transcription reaction. Measure the and transfect it with a vector expressing p300/cbp. ASSay concentration of the RNA products, by for instance, primer endogenous gene expression in the p300/cbp transfected cell extension analysis. Compare to the RNA products before the and in control cells transfected with an “empty' plasmid. If addition of the p300/cbp proteins. If the addition of p300/ gene expression is higher or lower in the p300/cbp trans cbp increased the concentration of the RNA products, the fected cell, the gene is p300/cbp regulated. gene is p300/cbp regulated. US 2004/0023207 A1 Feb. 5, 2004

0298 6. See more assays below. phosphorylation,” or Similar assays, to uncover a change in 0299| Examples phosphorylation of the p300/cbp factor of interest. An increase in phosphorylation indicates that L is a p300/cbp 0300 Direct evidence shows transactivation of certain factor kinase, and a decrease indicates that L is a p300/cbp promoters by p300/cbp (Manning 2001, Kraus 1999', factor phosphatase. Kraus 1998). 0308 Example 0301 Indirect evidence is available in studies with p300/ cbp factors. Consider, for example, the p300/cbp factor 0309 Ras, Raf, MEK1, MEK2, MEK4, ERK., JNK, three GABP. GABP binds promoters and enhancers of many classes of ERK inactivators: type 1/2 Serine/threonine phos cellular genes including fleukocyte integrin (CD18) (ROS phatases, Such as PP2A, tyrosine-specific phosphatases (also marin 1998), interleukin 16 (IL-16) (Bannert 19997), interleukin 2 (IL-2) (Avots 1997), interleukin 2 receptor called protein-tyrosine phosphatase, denoted PTP), Such as P-chain (IL-2Rf8) (Lin 1993), IL-2 receptor Y-chain (IL-2 PTP1B, and dual specificity phosphatases, such as MKP-1 yc) (Markiewicz 1996'), human secretory interleukin-1 which affect phosphorylation of a number of transcription receptor antagonist (secretory IL-1 ra) (Smith 19987), ret factors, for instance, GABP, NF-kB. See also below. inoblastoma (Rb) (Sowa 1997'), human thrombopoietin 0310 (6) p300/cbp Agent (TPO) (Kamura 19977), aldose reductase (Wang 19937), neutrophil elastase (NE) (Nuchprayoon 19997, 0311 Definition Nuchprayoon 19977), folate binding protein (FBP) (Sada Sivan 199477), cytochrome c oxidase subunit Vb (COXVb) 0312 Assume the polynucleotide Pn binds the transcrip (Basu 19937, Sucharov 19957), cytochrome c oxidase tion complex C. Assume C contains p300/cbp. If a molecule subunit IV (Carter 1994, Carter 1992), mitochondrial L. Stimulates or Suppresses binding of C to Pn, L is called transcription factor A (mtTFA) (Virbasius 1994), B subunit “p300/cbp agent.” Specifically, Such an agent can Stimulate of the FoF1 ATP synthase (ATPsyn?) (Villena 1998), or suppress binding of p300/cbp to a p300/cbp factor, prolactin (prl) (Ouyang 199684) and the oxytocin receptor binding of p300/cbp to DNA, or binding of a p300/cbp (OTR) (Hoare 1999) among others. For some of these factor to DNA. genes, for instance, CD18, COXVb, COXIV, GABPbinds to the promoter while for others, for example IL-2 and 0313 Exemplary Assays ATPsyn?, GABP binds an enhancer. More examples see below. 0314) 1. Contact a System (for instance, whole organism, cell, cell lysate, chemical mixture) with a test molecule L. 0302) Another p300/cbp factor is NF-Y (see above). Use assays described in the Section entitled "ASSaying Mantovani 1998, provides a list of genes which include a binding to DNA, or similar assays, to uncover a change in NF-Y binding site (Mantovani 1998, ibid, Table 1). For the binding of the C to DNA. Specifically, assay for binding listed genes, the table indicates whether the referenced between p300/cbp and DNA, or p300/cbp and p300/cbp Studies report the presence of a proven binding site for a factor, or p300/cbp factor and DNA. transcription factor close to the NF-Y binding site, whether cross-competition data with bona fide NF-Y binding sites are 0315 Examples available, whether EMSA Supershift experiments with anti NF-Y antibodies were performed, and whether the studies 0316 Examples of p300/cbp agents include Sodium performed in vitro or in vivo transactivation studies with butyrate (SB), trichostatin A (TSA), trapoxin (for SB, TSA NF-Y. Some of the genes listed in the paper are MCH II, Ii, and trapoxin see in Espinos 1999), phorbol ester (phorbol Mig, GP91 Phox, CD10, RAG-1, IL4, Thy-1, globin C, C, y' 12 myristate 13-acetate, PMA, TPA), thapsigargin (for PMA Y, Coll C2 (I) O.1 (I), osteopontin, BSP, apoA-I, aldolase B, and thapsigargin see Shiraishi 2000, for PMA see Herrera TAT, Y-GT, SDH, fibronectin, arg lyase, factor VIII, factor X, 1998, Stadheim 1998'), retinoic acid (RA, vitamin A) MSP, ALDH, LPL, EXOKII, FAS, TSP-1, FGF-4, C1-chim, (Yen 1999''), interferon-y (IFNY) (Liu 1994', Nishiya Tr Hydr, NaKATPsea-3, PDFGB, FerH, MHC IA2 B8, 1997), heregulin (HRG, new differentiation factor, NDF, Cw2Ld and B7, MDR1, CYP1A1, c-JUN, Grp78, Hsp70, neuregulin, NRG) (Lessor 1998', Marte 1995, Sepp ADH2, GPAT, FPP, HMG, HSS, SREBP2, GHR, CP2, Lorenzino 1996, Fiddes 1998'), zinc (Zn) (Park 1999, B-actin, TK, Topolo., I, II, III, IV, cdc25, cdc2, cyclA, Kiss 1997), copper (Cu) (Wu 1999', Samet 1998', cyclB1, E2F1, PLK, RRR2, HisH2B, H is H3. both studies also show phosphorylation of ERK1/2 by Zn), estron, estradiol (Migliaccio 1996', Ruzycky 1996', 0303 (5) p300/cbp Factor Kinase (p300/cbp Factor Phos Nuedling 1999''), interleukin 1(3 (IL-1 B) (Laporte 1999', phatase) Larsen 1998'), interleukin 6 (IL-6) (Daeipoou 1993'7), 0304) Definition tumor necrosis factor a (TNFC) (Leonard 1999''), trans 0305 Assume F is a p300/cbp factor. If a molecule L forming growth factor B (TGFB) (Hartsough 1995'., Stimulates phosphorylation or dephosphorylation of F, L is Yonekura 1999'', oxytocin (OT) (Strakova 1998', Cop called “p300/cbp factor kinase” or “p300/cbp factor phos land 1999'', Hoare 1999''). All studies show phosphory phatase', respectively. lation of ERK1/2 by these agents. See more agents below. 0317. Other examples include agents which modify oxi 0306 Exemplary Assays dative stress, Such as, diethyl maleate (DEM), a glutathione 0307 1. Contact a system (for instance, organism, cell, (GSH)-depleting agent, and N-acetylcysteine (NAC), an cell lysate, chemical mixture) with a test molecule L. Use antioxidant and a precursor of GSH synthesis. See more assays described in the Section entitled "ASSaying protein agents below. US 2004/0023207 A1 Feb. 5, 2004

0318 (7) Foreign p300/cbp. Polynucleotide 0334 Ample evidence exists which supports the binding of GABP to the N-boxes in these viral enhancers. For 0319 Definition instance, Flory, et al., 1996' show binding of GABP to the 0320 Assume Pn is a polynucleotide foreign to organism HIV LTR, Douville, et al., 1995 show binding of GABP R. If Pn is a p300/cbp polynucleotide, Pn is called “p300/cbp to the promoter of ICP4 of HSV-1, Bruder, et al., 1991''' polynucleotide foreign to R.' and Bruder, et al., 1989 show binding of GABP to the adenovirus EIA enhancer element I, Ostapchuk, et al., 0321 Exemplary Assays 1986" show binding of GABP (called EF-1A in this paper) 0322 Combine assays in the p300/cbp polynucleotide to the polyomavirus enhancer and Gunther, et al., 1994'7 and foreign polynucleotide Sections above. show binding of GABP to Mo-MuLV. 0323 Examples 0335) Other studies demonstrate competition between these viral enhancers and enhancers of other viruses. Scholer 0324 See examples in “p300/cbp virus' below. and Gruss, 1984' show competition between the Moloney 0325 (8) p300/cbp Virus Sarcoma Virus (MSV) enhancer and SV40 enhancer and also competition between the RSV enhancer and the BK 0326 Definition Virus enhancer. 0327 Assume Pn is a p300/cbp polynucleotide. If Pn is 0336 Another p300/cbp factor is NF-Y (see above). a Segment of the genome of a virus V, V is called a Mantovani 1998 (ibid), provides a list of viruses which “p300/cbp virus.” include a NF-Y binding site (Table 1). The list includes HBV 0328 Exemplary Assays S, MSV LTR, RSV LTR, ad EIIL II, Ad MK, CMV gpUL4, HSV IE110k, VZV ORF62, MVM P4. 0329) 1. Verify that Pn is a p300/cbp polynucleotide (see assays above). Compare the sequence of Pn with the 0337 More Exemplary Assays for Identification of a Sequence of the published V genome. If the Sequence is a Polynucleotide Pn as a p300/cbp polynucleotide: segment of the V genome, Pn is a p300/cbp virus. If the V 0338 1. Take a cell of interest. Modify the copy number genome is not published, its Sequence can be determined of Pn in the cell (by, for instance, transfection, infection, empirically. mutation, etc, See also above). ASSay binding of all p300/cbp factors to Pn. If a p300/cbp factor binds Pn, Pn is a p300/cbp 0330 2. Verify that Pn is a p300/cbp polynucleotide (see polynucleotide. assays above) by hybridizing Pn to the V genome. If Pn hybridizes, Pn is a p300/cbp virus. 0339 2. Assay binding of a p300/cbp factor to endog enous DNA or to exogenous DNA following introduction to 0331 Examples the cell of interest. Modify the copy number of Pnin the cell. 0332 Direct evidence shows transactivation of certain Assay binding of the p300/cbp factor again. If binding viruses by p300/cbp. See, for instance, Subramanian 2002' changed, Pn is a p300/cbp polynucleotide. on Epstein-Barr virus, Banas 2001, Deng 2000' on HIV 0340. 3. Identify a binding site on Pn for p300/cbp or a 1''", Cho 2001''' on SV40 and polyomavirus, Wong p300/cbp factor by computerized Sequence analysis. 1994'', on adenovirus type 5. See also Hottiger 2000', a review on viral replication and p300/cbp. 0341. 4. Take a cell of interest. Co-transfect a vector expressing Pn (or change the copy number of Pn in the cell 0333 Indirect evidence is available in studies with p300/ through other means), and a promoter of a p300/cbp regu cbp factors. Consider, for instance, the p300/cbp factor lated gene fused to a reporter gene. ASSay reporter gene GABP. Since GABPbinds p300/cbp (see above), a complex expression and compare to cells co-transfected with an on DNA which includes GABP, also includes p300/cbp. The empty plasmid. If expression in the Pn transfected cell is DNA motif (A/C)GGA(A/T)(G/A), termed the N-box, is the different than controls, Pn is a p300/cbp polynucleotide. core binding sequence for GABP. The N-box is the core binding Sequence of many viral enhancers including the 0342 5. Take a cell of interest which express a p300/cbp polyomavirus enhancer area 3 (PEA3) (Asano 1990'), regulated gene. Modify the copy number of Pn in the cell adenovirus E1A enhancer (Higashino 1993'), Rous Sar (by, for instance, transfection, infection, mutation, etc, See coma Virus (RSV) enhancer (Laimins 1984''), Herpes also above). ASSay expression of the p300/cbp regulated Simplex Virus 1 (HSV-1) (in the promoter of the immediate gene and compare to cells with an unmodified copy number early gene ICP4) (LaMarco 1989, Douville 1995'), of Pn (for instance in cells transfected with an empty Cytomegalovirus (CMV) (IE-1 enhancer/promoter region) plasmid). If expression in the Pn transfected cell is different (Boshart 1985'), Moloney Murine Leukemia Virus (Mo than controls, Pn is a p300/cbp polynucleotide. MuLV) enhancer (Gunther 1994''), Human Immunodefi 0343 6. Take a cell of interest. Infect the cell with a ciency Virus (HIV) (the two NF-KB binding motifs in the p300/cbp virus. Modify the copy number of Pn in the cell HIV LTR) (Flory 19967), Epstein-Barr virus (EBV) (20 (by, for instance, transfection, infection, mutation, etc, See copies of the N-box in the +7421/+8042 or iP/enhancer) also above). ASSay viral replication and compare to cells (Rawlins 1985') and Human T-cell lymphotropic virus with unmodified copy number of Pn (for instance, in cells (HTLV) (8 N-boxes in the enhancer (Mauclere 1995 129) infected with a non p300/cbp virus). If viral replication is and one N-box in the LTR (Kornfeld 1987)). Moreover, different, Pn is a p300/cbp polynucleotide. Some Viral enhancers, for example SV40, lack a precise N-box, but still bind the GABP transcription factor (Bannert 0344 7. Compare the sequence of Pn to the genome of a 1999.13). p300/cbp virus using a Sequence alignment algorithm Such US 2004/0023207 A1 Feb. 5, 2004 as BLAST. If a segment of the Pn sequence is identical (or 0359 4. Assay the effect of an agent on binding of homologous) to a segment in viral genome, Pn is a p300/cbp p300/cbp to a p300/cbp factor. polynucleotide. A polynucleotide of at least 18 nucleotides should be Sufficient to ensure Specificity and validate align 0360 Specifically, take a biological system (e.g. cell, ment. whole organism, etc). Modify the copy number of Pn(by, for instance, transfection, infection, mutation, etc, See above). 0345) 8. Try to hybridize Pn to the genome of a p300/cbp Call this cell the Pn cell. Assay binding of p300/cbp to a virus. If Pn hybridizes to the viral genome, Pn is a p300/cbp p300/cbp factor (see above). Contact the biological system polynucleotide. Hybridization conditions should be suffi with an agent of interest. ASSay again the binding of p300/ ciently Stringent to permit Specific, but not promiscuous, cbp to a p300/cbp factor. If binding is higher or lower hybridization. Such conditions are well known in the art. compared to binding in Pn cells not contacted with the agent, the agent is a modulator. 0346) c) Agents Related Elements 0361 5. Assay the effect of an agent on expression of a 0347 (1) Modulator disrupted gene and/or polypeptide. 0348 Definition 0362 Specifically, take a biological System (e.g. cell, 0349 Consider a polynucleotide Pn. An agent, or treat whole organism, etc). Modify the copy number of Pn(by, for ment (called agent for short), is called “modulator” if the instance, transfection, infection, mutation, etc, See above). agent modifies microcompetition with Pn, modifies at least Call this cell the Pn cell. Identify a disrupted gene and/or one effect of microcompetition with Pn, or modifies at least polypeptide (See assays above). Contact the biological Sys one effect of another foreign polynucleotide-type disruption. tem with an agent of interest. ASSay the bioactivity of the disrupted gene and/or polypeptide. If the bioactivity of the 0350) Notes: disrupted gene and/or polypeptide is higher or lower com 0351 1. A treatment such as irradiation can also be a pared to the bioactivity in Pn cells not contacted with the modulator In principle, according to the definition, any agent, the agent is a modulator. foreign polynucleotide-type disruption is a modulator. 0363) EXAMPLES 0352 Exemplary Assays 0364 See below in constructive/disruptive. 0353 1. Assay the effect of an agent on Pn copy number. 0365 (2) Constructive/Disruptive 0354) Specifically, take a biological system (e.g. cell, whole organism, etc). Modify the copy number of Pn(by, for 0366) Definition instance, transfection, infection, mutation, etc, See above). 0367 A modulator, which attenuates or accentuates Call this cell the Pn cell. Assay the Pn copy number in the microcompetition with a foreign polynucleotide, attenuates Pn cell (see above). Contact the biological system with an or accentuates at least one effect of microcompetition with agent of interest. ASSay again the Pn copy number. If the Pn a foreign polynucleotide, or attenuates or accentuates at least copy number is higher or lower compared to the copy one effect of another foreign polynucleotide-type disruption, number in Pn cells not contacted with the agent, the agent is is called “constructive” or “disruptive,” respectively. a modulator. 0355 2. Assay the effect of an agent on binding of 0368. Notes: p300/cbp to Pn, directly or in a complex. 0369 1. A modulator can be both constructive and dis ruptive. 0356 Specifically, take a biological system (e.g. cell, whole organism, etc). Modify the copy number of Pn(by, for 0370 2. Consider a gene Suppressed by microcompeti instance, transfection, infection, mutation, etc, See above). tion with a foreign polynucleotide. Consider Such a gene in Call this cell the Pn cell. Assay binding of p300/cbp to Pn a cell without a foreign polynucleotide. Now consider a (See above). Contact the biological System with an agent of mutation which reduces the gene bioactivity. An agent which interest. Assay again the binding of p300/cbp to Pn. If the Stimulates expression of Such mutated gene will also be binding is higher or lower compared to binding in Pn cells called constructive. If, on the other hand, the mutation not contacted with the agent, the agent is a modulator. Stimulates the gene bioactivity, an agent which Suppresses its bioactivity will also be called constructive. 0357 3. Assay the effect of an agent on binding of a p300/cbp factor to Pn. 0371 3. A constructive agent can be an agonist, if it Stimulates expression of a gene Suppressed by microcom 0358 Specifically, take a biological system (e.g. cell, petition with a foreign polynucleotide, or if is stimulates whole organism, etc). Modify the copy number of Pn(by, for bioactivity of a polypeptide encoded by Such a gene. A instance, transfection, infection, mutation, etc, See above). constructive agent can also be an antagonist if it inhibits Call this cell the Pn cell. Assay binding of a p300/cbp factor expression of a gene Stimulated by microcompetition with a to Pn (see above). Contact the biological system with an foreign polynucleotide, or inhibits the bioactivity of a agent of interest. ASSay again the binding of the p300/cbp polypeptide encoded by Such a gene. factor to Pn. If binding is higher or lower compared to binding in Pn cells not contacted with the agent, the agent is 0372 4. A foreign polynucleotide-type disruption can be a modulator. constructive. US 2004/0023207 A1 Feb. 5, 2004

0373) Exemplary Assays Sassa, I Glenn Sipes. The following lists includes more 0374 1. See assays in Modulator section above. In these books with standard methods. Basic DNA and RNA Proto assay if either; cols (Methods in Molecular Biology, Vol 58), edited by Adrian J Harwood, Humana Press, 1994, DNA-Protein 0375 (a) Pn copy number in the Pn cell contacted with Interactions: Principles and Protocols (Methods in Molecu the agent is higher relative to Pn cells not contacted by lar Biology, Volume 148), edited by Tom Moss, Humana the agent; Press, 2001; Transcription Factor Protocols (Methods in 0376 (b) binding of p300/cbp to Pn in the Pn cell Molecular Biology), edited by Martin J Tymms, Humana contacted with the agent is higher compared to binding Press, 2000; Gene Transcription: A Practical Approach, in Pn cells not contacted with the agent; edited by BD Hames, and S J Higgins, IRL Press at Oxford 0377 (c) binding of p300/cbp factor to Pn in the Pn University Press, 1993; Gene Transcription, DNA Binding cell contacted with the agent is higher compared to Proteins: Essential Techniques, edited by Kevin Docherty, Jossey Bass, 1997; Gene Probes Principles and Protocols binding in Pn cells not contacted with the agent; (Methods in Molecular Biology, 179), edited by Marilena 0378) (d) binding of p300/cbp to a p300/cbp factor in Aquino de Muro and Ralph Rapley, Humana Press, 2001; the Pn cell contacted with the agent is higher or lower Gene Isolation and Mapping Protocols (Methods in Molecu compared to binding in Pn cells not contacted with the lar Biology Vol 68), edited by Jackie Boultwood and Jac agent, queline Boultwood, Humana Press, 1997; Gene Targeting Protocols (Methods in Molecular Biology, Vol 133), edited 0379 (e) bioactivity of the disrupted gene and/or by Eric B Kmiec and Dieter C Gruenert, Humana Press polypeptide in the Pn cell contacted with the agent is 2000; Epitope Mapping Protocols (Methods in Molecular higher (for genes and/or polypeptides with Suppressed Biology, Vol 66), edited by Glenn E Morris, Humana Press, bioactivity) compared to the bioactivity in Pn cells not 1996; Protein Targeting Protocols (Methods in Molecular contacted with the agent: Biology, Vol 88), edited by Roger A Clegg, Humana Press, 0380 the agent is constructive. 1998; Monoclonal Antibody Protocols (Methods in Molecu lar Biology, 45), edited by William C Davis, Humana Press, 0381) If the effect is in the opposite direction, the agent 1995; Immunochemical Protocols (Methods in Molecular is disruptive. Biology Vol 80), edited by John D. Pound, Humana Press, 0382) Examples 1998; Immunoassay Methods and Protocols (Methods in Molecular Biology), edited by Andrey L. Ghindilis, Andrey 0383 Antiviral drugs, sodium butyrate, garlic, etc. See RPavlov and Plamen B Atanassov, Humana Press, 2002; In more examples in Treatment Section below. situ Hybridization Protocols (Methods in Molecular Biol 0384 2. Detailed Description of Standard Elements ogy, 123), edited by Ian A Darby, Humana Presse, 2000; Bioluminescence Methods & Protocols, edited by Robert A 0385) a) General Comments Larossa, Humana Press, 1998; Affinity Chromatography: 0386 The elements of the present invention may include, Methods and Protocols (Methods in Molecular Biology), as their own elements, Standard methods in molecular biol etided by Pascal Bailon, George KEhrlich, Wen-Jian Fung, ogy, microbiology, cell biology, transgenic biology, recom wo Berthold and Wolfgang Berthold, Humana Press, 2000; binant DNA, immunology, cell culture, pharmacology, and Protocols for Oligonucleotide Conjugates: Synthesis and toxicology, well known in the art. The following Sections Analytical Techniques (Methods in Molecular Biology, Vol provide details for Some Standard methods. Complete 26), edited by Sudhir Agrawal, Humana Press, 1993; RNA descriptions are available in the literature. For instance, See Isolation and Characterization Protocols (Methods in the “Current Protocols' series published by John Wiley & Molecular Biology, No. 86). edited by Ralph Rapley and Sons. The following list provides a sample of books in the David L. Manning, Humana Press, 1998; Protocols for series: Current Protocols in Cell Biology, edited by: Juan S. Oligonucleotides and Analogs: Synthesis and Properties Bonifacino, Mary Dasso, Jennifer Lippincott-Schwartz, Joe (Methods in Molecular Biology, 20), edited by Sudhir B Harford, and Kenneth M Yamada; Current Protocols in Agrawal, Humana Press, 1993; Basic Cell Culture Protocols Human Genetics, edited by: Nicholas CDracopoli, Jonathan (Methods in Molecular Biology, 75), edited by Jeffrey W L Haines, Bruce R Korf, Cynthia C Morton, Christine E Pollard and John M Walker, Humana Press, 1997; Quanti Seidman, J G Seidman, Douglas R Smith; Current Protocols tative PCR Protocols (Methods in Molecular Medicine, 26), in Immunology, edited by: John E Coligan, Ada M Kruis edited by Bernd Kochanowski and Udo Reischl, Humana beek, David H Margulies, Ethan M Shevach, and Warren Press, 1999; In situ PCR Techniques, edited by Omar Strober; Current Protocols in Molecular Biology, edited by: Bagasra and John Hansen, John Wiley & Sons, 1997; PCR Frederick M Ausubel, Roger Brent, Robert E. Kingston, Cloning Protocols: From Molecular Cloning to Genetic David D Moore, J G Seidman, John A. Smith, and Kevin Engineering (Methods in Molecular Biology, No 67), edited Struhl, Current Protocols in Nucleic Acid Chemistry, edited by Bruce A White, Humana Press, 1996; PRINS and In situ by: Serge L. Beaucage, Donald E. Bergstrom, Gary D Glick, PCR Protocols (Methods in Molecular Biology, 71), edited Roger A Jones; Current Protocols in Pharmacology, edited by John R Gosden, Humana Press, 1996; PCR Protocols: by: S J Enna, Michael Williams, John W Ferkany, Terry Current Methods and Applications (Methods in Molecular Kenakin, Roger D Porsolt, James P Sullivan; Current Pro Biology, 15), edited by Bruce AWhite, Humana Press 1993; tocols in Protein Science, edited by: John E Coligan, Ben M Transmembrane Signaling Protocols (Methods in Molecular Dunn, Hidde LPloegh, David WSpeicher, Paul TWingfield: Biology, Vol 84), edited by Dafna Bar-Sagi, Humana Press, Current Protocols in Toxicology edited by: Mahin Maines 1998; Chemokine Protocols (Methods in Molecular Biology, (Editor-in-Chief), Lucio G Costa, Donald J Reed, Shigeru 138), edited by Amanda E I Proudfoot, Timothy N C Wells US 2004/0023207 A1 Feb. 5, 2004 20 and Chris Power, Humana Press, 2000; Baculovirus Expres 0388 More details regarding the presented exemplary sion Protocols (Methods in Molecular Biology, Vol 39), protocols, and details of other protocols that can be used edited by Christopher D Richardson, Humana Press, 1998; instead of the presented protocols, are available in the cited Recombinant Gene Expression Protocols (Methods in references, and in the books listed above. The contents of all Molecular Biology, 62), edited by Rocky S Tuan, Humana references cited in the application, including, but not limited Press, 1997: Recombinant Protein Protocols: Detection and to, abstracts, papers, books, published patent applications, Isolation (Methods in Molecular Biology, Vol 63), edited by issued patents, available in paper format or electronically, Rocky S Tuan, Humana Press, 1997; DNA Repair Protocols: are hereby expressly and entirely incorporated by reference. Eukaryotic Systems (Methods in Molecular Biology, Vol 113), edited by Daryl S Henderson, Humana Press, 1999; 0389. The following sections first present protocols for DNA Sequencing Protocols, editors Hugh G Griffin and formulation of a drug candidate, then protocols, that as Annette M Griffin, Humana Press, 1993; Protein Sequencing elements of above assays, can be used to test a drug Protocols (Methods in Molecular Biology, No. 64), edited by candidate for a desired biological activity during drug dis Bryan John Smith, Humana Press, 2001; Gene Transfer and covery, development and clinical trials. The assays can also Expression Protocols (Methods in Molecular Biology, Vol be used for diagnostic purposes. Finally, the following 7), edited by EJ Murray, Humana Press, 1991; Transgenesis Sections also present protocols for effective use of a drug as Techniques, Principles and Protocols (Methods in Molecular treatment. Biology, 180), edited by Alan R Clarke, Humana Press, 0390 b) Formulation Protocols 2002; Regulatory Protein Modification Techniques and Pro tocols (Neuromethods, 30), edited by Hugh C Hemmings, 0391) One aspect of the invention pertains to administra Humana Press, 1996; Downstream Processing of Proteins tion of a molecule of interest, equivalent molecules, or Methods and Protocols (Methods in Biotechnology, 9), homologous molecules, isolated from, or Substantially free edited by Mohamed A Desai, Humana Press. 2000: DNA of contaminating molecules, as treatment of a chronic dis Vaccines Methods and Protocols (Methods in Molecular CSC. Medicine, 29), edited by Douglas B Lowrie and Robert 0392 (1) Definitions Whalen, Humana Press, 1999; DNA Arrays Methods and Protocols (Methods in Molecular Biology, 170), edited by 0393 (a) Molecule of Interest Jang B Rampal, Humana Press, 2001; Drug-DNA Interac 0394. The terms “molecule of interest” or “agent,” is tion Protocols, editor Keith Fox, Humana Press, 1997. In understood to include Small molecules, polypeptides, poly vitro Mutagenesis Protocols, edited Michael K. Trower, nucleotides and antibodies, in a form of a pharmaceutical or Humana Press, 1996; In vitro Toxicity Testing Protocols nutraceutical. (Methods in Molecular Medicine, 43), edited by Sheila O'Hare and C K Atterwill, Humana Press, 1995; Mutation 0395 (b) Equivalent Molecules Detection: A Practical Approach (Practical Approach Series 0396 The term “equivalent molecules” is understood to (Paper), No 188), edited by Richard G H Cotton, E. Edkins include molecules having the same or Similar activity as the and S Forrect, Irl Press, 1998; Virus Pro molecule of interest, including, but not limited to, biological tocols (Methods in Molecular Medicine, 10), edited by S activity, chemical activity, pharmacological activity, and Moira Brown and Alasdair R MacLean, Humana Press, therapeutic activity, in vitro or in Vivo. 1997; HIV Protocols (Methods in Molecular Medicine, 17), edited by Nelson Michael and Jerome H Kim, Humana 0397 (c) Homologous Molecules Press, 1999; Cytomegalovirus Protocols (Methods in Molecular Medicine, 33), edited by John Sinclair, Humana 0398. The term “homologous molecules” is understood Press, 1999; Antiviral Methods and Protocols (Methods in to include molecules with the same or Similar chemical Molecular Medicine, 24), edited by Derek Kinchington and Structure as the molecule of interest. Raymond F Schinazi, Humana Press, 1999; Epstein-Barr 0399. In one exemplary embodiment, homologous mol Virus Protocols (Methods in Molecular Biology Vol 174), ecules may be Synthesized by chemical modification of a edited by Joanna B Wilson and Gerhard HW May, Humana molecule of interest, for instance, by adding any of a number Press, 2001; Adenovirus Methods and Protocols (Methods in of chemical groups, including but not limited to, Sugars (i.e. Molecular Medicine, Vol 21), edited by William S M Wold, glycosylation), phosphates, acetyls, methyls, and lipids. Humana Press, 1999; Molecular Methods for Virus Detec Such derivatives may be derived by the covalent linkage of tion, edited by Danny L Wiedbrauk and Daniel H Farkas, these or other groups to Sites within a molecule of interest, Academic Press, 1995; Diagnostic Virology Protocols or in the case of polypeptides, to the N-, or C-termini, or (Methods in Molecular Medicine, No 12), edited by John R polynucleotides, to the 5' or 3' ends. Stephenson and Alan Warnes, Humana Press, 1998. A more extensive list of books with detailed description of standard 0400. In one exemplary embodiment, homologous methods is available at the Promega web site: http://www polypeptides or homologous polynucleotides include promega.com/catalog/category.asp?catalog'765Fname= polypeptides or polynucleotides that differ by one or more Promega%5FProducts &category%5Fname= amino acid, or nucleotides, respectively, from the polypep Books&description%5Ftext=Books&Page=1. The Promega tide or polynucleotide of interest. The differences may arise from Substitutions, deletions or insertions into the initial list includes 260 books. Sequence, naturally occurring or artificially formulated, in 0387 For each element, one or more exemplary protocols vivo or in vitro. Techniques well known in the art may be are presented. All examples included in the application applied to introduce mutations, Such as point mutations, should be considered as illustrations, and, therefore, should insertions or deletion, or introduction of premature transla not be construed as limiting the invention in any way. tional Stops, leading to the Synthesis of truncated polypep US 2004/0023207 A1 Feb. 5, 2004 tides. In every case, homologs may show attenuated activi polynucleotide encoding another polypeptide which facili ties compared to the original molecules, exaggerated tates purification, for instance, a polypeptide with readily activities, or may express a Subset or SuperSet of the total available antibodies, Such as VP6 protein, a activities elicited by the original molecule. In these ways, vaccinia virus capsid protein, or the bacterial GST protein. homologs of constructive or disruptive polypeptides or When expressed, the facilitator polypeptide enables purifi polynucleotides have biological activities either diminished cation of the polypeptide of interest and immunological or expanded compared to the original molecule. In every identification of host cells which express it. In the case of case, a homolog may, or may not prove more effective in GST fusion proteins, purification may be achieved by use of achieving a desired therapeutic effect. Methods for identi glutathione-conjugated Sepharose beads in affinity chro fying homologous polypeptides or polynucleotides are well matographic techniques well known in the art (See, for known in the art, for instance, molecular hybridization instance, Ausubel 1998'). techniques, including, but not limited to, Northern and Southern blot analysis, performed under variable conditions 0407. In a related exemplary embodiment, the fusion of temperature and Salt, can formulate nucleic acid polypeptide includes a polyamino acid tract, Such as the Sequences with different levels of Stringency. Suitable pro polyhistidine/enterokinase cleavage Site, which confers tocols for identifying homologous polypeptides or poly physical properties that inherently enable purification. In nucleotides are well known in the art (see for instance. this example, purification may be achieved through nickel Sambrook 2001''' and above listed books of standard pro metal affinity chromatography. Once purified, the polyhis tocols). Homologous polypeptides or polynucleotides can tidine tract included to enable purification can be removed also be generated, for instance by a Suitable combinatorial by treatment with enterokinase in vitro to release the approach. polypeptide fragment of interest. 04.01. It is well known in the art that the ribonucleotide 0408 For molecules synthesized by an organism, for triplets, termed codons, encoding each amino acid, comprise instance, polypeptides or polynucleotides Synthesized by a set of Similar Sequences typically differing in their third human Subjects, in a preferred exemplary embodiment, a position. Variations, known as degeneracy, occur naturally, purified polynucleotide or polypeptide is free of other mol and in practice mean that any given amino acid may be ecules Synthesized by Same organism, accomplished, for encoded by more than one codon. For instance, the amino example, by expression of a human gene in a non-human acids arginine, Serine and leucine can be encoded by 6 host cell. codons. As a result, in one exemplary embodiment, homolo 04.09 The following sections present standard protocols gous DNA and RNA polynucleotides can be produced which for the formulation of certain types of agents. encode the same polypeptide of interest. 0402. In another exemplary embodiment, a set of 0410 (2) Small Molecules homologous polypeptides may be generated by incorporat 0411 One aspect of the invention pertains to administra ing a population of Synthetic oligodeoxyribonucleotides into tion of a Small molecule of interest, equivalent Small mol expression vectors already carrying additional portions of ecules, or homologous Small molecules, isolated from, or the polypeptide of interest. The Site into which the oligo Substantially free of contaminating molecules, as treatment nucleotide-gene fusion is incorporated must include appro of a chronic disease. priate transcriptional and translational regulatory Sequences flanking the inserted oligonucleotides to permit expression 0412. The following sections present standard protocols in host cells. Once introduced into an appropriate host cell, for formulation of Small molecules. the resulting collection of gene-oligonucleotide recombinant 0413 (a) Production vectors expresses polypeptide variants of the polypeptide of interest. The expressed polypeptide may be separately puri 0414. Small molecules, organic or inorganic, may be fied by cloning the vector bearing host cells, or by employ synthesized in vitro by any of a number of methods well ing appropriate bacteriophage vectors, Such as gt-11 or its known in the art. Those Small molecules, and otherS Syn derivatives, and Screening plaques with antibodies against thesized in Vivo, may by purified by, for instance, liquid or the polypeptide of interest, or against an immunological tag thin layer chromatography, high performance liquid chro matography (HPLC), electrophoresis, or Some other Suitable included in the recombinants. technique. 0403 (d) Isolated 04.04 The terms “isolated from, or substantially free of 0415 (3) Polypeptides contaminating molecules' is understood to include a mol 0416) Another aspect of the invention pertains to admin ecule containing less than about 20% contaminating mol istration of a polypeptide of interest, equivalent polypep ecules, based on dry weight calculations, preferably, leSS tides, or homologous polypeptides, isolated from, or Sub than about 5% contaminating molecules. Stantially free of contaminating molecules, as treatment of a 04.05) The terms “isolated” or “purified” do not refer to chronic disease. materials in a natural State, or materials separated into elements without further purification. For example, Separat 0417. The following sections present standard protocols ing a preparation of nucleic acids by gel electrophoresis, by for the formulation of polypeptides. itself, does not constitute purification unless the individual 0418 (a) Production molecular species are Subsequently isolated from the gel matrix. 0419 (i) In Vitro 0406. In one exemplary embodiment, a polynucleotide 0420. In one exemplary embodiment, a polypeptide of encoding a polypeptide of interest is ligated into a fusion interest is produced in Vitro by introducing into a host cell US 2004/0023207 A1 Feb. 5, 2004 22

by any of a number of means well known in the art (see promote and regulate expression in mammalian cells, and protocols below) a recombinant expression vector carrying genes encoding Selectable markers. The list of appropriate a polynucleotide, preferably obtained from vertebrates, vectors includes, but is not limited to, pcDNA/neo, pcDNA/ especially mammals, encoding a polypeptide of interest, amp, pRSVneo, pZIPneo, and a host of others. Many viral equivalents of Such polypeptide, or homologous polypep derivatives are also available, for instance, pHEBo, derived tides. The recombinant polypeptide is engineered to include from the Epstein-Barr virus, BPV-a derived from the bovine a tag to facilitate purification. Such tags include fragments papillomavirus, and the plRCX system (BD Biosciences of the GST protein, or polyamino acid tracts either recog nized by Specific antibodies, or which convey physical Clonetech, Inc.). The use of mammalian expression vectors properties facilitating purification (See also below). Follow is well known in the art (see, for example, Sambrook 2001, ing culture under Suitable conditions, the cells are lysed and ibid, chapters 15 and 16). Similarly, many vectors are the expressed polypeptide purified. Typical culture condi available for expression of recombinant polypeptides in tions include appropriate host cells, growth medium, anti yeast, including, but not limited to, YEP24, YEP5, YEP51, biotics, nutrients, and other metabolic byproducts. The pYES2. The use of expression vectors in yeast is well known expressed polypeptide may be isolated from either a host in the art. cell lysate, culture medium, or both depending on the 0425. In addition to mammalian and yeast expression expressed polypeptide. Purification may involve any of Systems, a System of vectors is available which permits many techniques well known in the art, including but not expression in insect cells. The System, derived from bacu limited to, gel filtration, affinity chromatography, gel elec loviruses, includes pacUW-1 based vectors (for instance, trophoresis, ion-exchange chromatography, and others. pAcUW1), pVL-based vectors (for instance, pVL1292 and 0421) Polynucleotides, both mRNA and DNA, can be pVL1393), and pBlueBac-based vectors which carry the extracted from prokaryotic or eukaryotic cells, or whole gene encoding 3-galactosidase to facilitate Selection of host animals, at any developmental Stage, for instance, adults, cells harboring recombinant vectors. juveniles, or embryos. Polynucleotides may be isolated, or cloned from a gehomic library, cDNA library, or freshly 0426 (ii) In Situ isolated nucleic acids, using protocols well known in the art. For instance, total RNA is isolated from cells, and mRNA 0427. In another exemplary embodiment, a polypeptide converted to cDNA using oligo dT primers and viral reverse of interest is expressed in Situ by administering to an animal transcriptase. Alternatively, a polynucleotide of interest may or human Subject by any of a number of means well known be amplified using PCR. In any case, the initial nucleic acid in the art (see protocols below) a recombinant expression preparation may include either RNA or DNA and the pro vector carrying a polynucleotide encoding the polypeptide tocols chosen accordingly. The resulting DNA is inserted of interest, equivalent polypeptides, or homologous into an appropriate vector, for instance, bacterial plasmid, polypeptides. recombinant virus, cosmid, or bacteriophage, using proce 0428. In the present invention, such vectors may be used dures well known in the art. as therapeutic agents to introduce polynucleotides into cells 0422 Nucleotide sequences are considered functionally that express constructive or disruptive polypeptides (for linked if one Sequence regulates expression of the other. To exemplary applications See, for instance, Friedmann facilitate expression of a polypeptide of interest, the cloning 1999.14). vector Should include Suitable transcriptional regulatory 0429. It is critical that the potential effects of microcom Sequences well known in the art, for instance, promoter, petition between the enhancer, or other polynucleotide enhancer, polyadenylation Site, etc., functionally linked to Sequences carried in the delivery vector, and cellular genes the polynucleotide expressing the polypeptide of interest. In be considered and manipulated where needed. AS an one exemplary embodiment, an expression vector is con example consider a case where the polypeptide of interest Structed to carry a polynucleotide, a naturally occurring binds an enhancer carried by the vector, for instance, a Sequence, a gene, a fusion of two or more genes, or Some delivery vector that expresses GABP under control of a other Synthetic variant, under control of a regulatory promoter that includes an N-box. In one exemplary embodi Sequence, Such that when introduced into a cell expresses a ment, the vector expresses, in situ, a high enough concen polypeptide of interest. tration of the polypeptide of interest Such that any binding of 0423 Both viral and nonviral gene transfer methods may the polypeptide to the enhancer Sequences within the Vector be used to introduce desirable polynucleotides into cells. itself is negligible. In other words, the vector expresses Viral methods exploit natural mechanisms for viral attach enough free polypeptides to produce the desired biological ment and entry into target cells. Nonviral methods take activity in treated cells. In another example, the polypeptide advantage of normal mammalian transmembrane transport is not a transcription factor, but the delivery vector carries a mechanisms, for example, endocytosis. Exemplary proto polynucleotide that microcompetes with cellular genes for a cols employ packaging of deliverable polynucleotides in cellular transcription factor, for instance, a vector that liposomes, encasement in Synthetic viral envelopes or poly expresses Rb and microcompetes with cellular genes for GABP. In an exemplary embodiment, the delivery vector lysine, and precipitation with calcium phosphate (See also also includes a polynucleotide Sequence that expresses the below). microcompeted transcription factor, or is delivered in con 0424 The variety of Suitable expression vectors is vast junction with another vector that expresses the microcom and growing. For example, mammalian expression vectors peted transcription factor. In the example, the Rb vector typically include prokaryotic elements which facilitate includes a Sequence that expresses GABP, or is delivered in propagation in the laboratory, eukaryotic elements which conjunction with a vector that expresses GABP. US 2004/0023207 A1 Feb. 5, 2004

0430 (4) Polynucleotides profoundly detrimental effect than three bases of mismatch in a 100 base oligonucleotide. Inadequate complementarity 0431 Another aspect of the invention pertains to admin results in ineffective inhibition, or unwanted binding to istration of a polynucleotide as antisense/antigene, Sequences other than the polynucleotide of interest. In the ribozyme, triple helix, homologous nucleic acids, peptide latter case, inadvertent effects may include unwanted inhi nucleic acids, or microcompetitiors, equivalent polynucle bition of genes other than a gene of interest. Specificity and otides, or homologous polynucleotides, isolated from or binding avidity are easily determined empirically by meth Substantially free of contaminating molecules as treatment ods known in the art. for a chronic disease. 0432. The following sections present standard protocols 0438. Several methods are suitable for the delivery of for the formulation of Such polynucleotides. Since antisense/ antisense/antigene agents. In one exemplary embodiment, a antigene, ribozyme, triple helix, homologous nucleic acids, recombinant expression plasmid is engineered to express peptide nucleic acids, and microcompetition agents are antisense RNA following introduction into host cells. The nucleic acid based, they share protocols for their Synthesis, RNA is complementary to a unique portion of DNA or mechanisms of delivery and potential pitfalls in their use mRNA sequence of interest. In an alternative embodiment, including, but not limited to, Susceptibility to extracellular chemically derivatized Synthetic oligonucleotides are used and intracellular nucleases, instability and the potential for as antisense/antigene agents. nonspecific interactions. In consideration of these common 0439 Such oligonucleotides may contain modified nucle issues, the general methods for the formulation and delivery, otides to attain increased Stability once exposed to cellular as well as caveats regarding the use of nucleic agents, nucleases. Examples of modified nucleotides include, but described first, apply Similarly to each Subsequent agent. are not limited to, nucleotides carrying phosphoramidate, 0433 (a) Antisense/Antigene phosphorothioate and methylphosphonate groups. 0434. In the present invention, the terms “antisense” and 0440 Whichever sequence of the polynucleotide of inter “antigene' polynucleotides is understood to include natu est is targeted by antisense/antigene agents, in Vitro Studies rally or artificially generated polynucleotides capable of in should be undertaken first to determine the effectiveness and situ binding to RNA or DNA, respectively. Antisense bind Specificity of the agent. Control treatments should be ing to mRNA may modify translation of bound mRNA, included to differentiate between effects specifically elicited while antigene binding to DNA may modify transcription of by the agent and non-Specific biological effects of the bound DNA. Antisense/antigene binding may modify bind treatment. Control polynucleotides should have same length ing of a polypeptide of interest to RNA or DNA, for instance and nucleotide composition as the agent with the base binding of an antigene to a foreign N-box may reduce Sequence randomized. binding of cellular GABP to the foreign N-box resulting in 0441 Antisense/antigene agents can be oligonucleotides attenuated microcompetition between the foreign polynucle of RNA, DNA, mixtures of both, chemical derivatives of otide and a cellular gene for GABP. AntiSense/antigene either, and single or double stranded. Nucleotides within the binding may also modify, i.e., decrease or increase, expres oligonucleotide may carry modifications on the nucleotide Sion of a polypeptide of interest. base, the Sugar or the phosphate backbone. For example, 0435 Binding, or hybridization of the antisense/antigene modifications to the nucleotide base involves a number of agent, may be achieved by base complementarity, or by compounds including, but not limited to, hypoxanthine, interaction with the major groove of the cellular DNA Xanthine, 2-methyladenine, 2-methylguanine, 7-methylgua duplex. The techniques and conditions for achieving Such nine, 5-fluorouracil, 3-methylcytosine, 2-thiocytosine, interactions are well known in the art. 2-thiouracil, 5-methylcytosine, 5-methylaminomethyluracil, and a host of others well known in the art. Modifications are 0436 The target of antisense/antigene agents has been generally incorporated to increase Stability, e.g. infer resis thoroughly studied and is well known in the art. For tance to cellular nucleases, Stabilize hybridization, or instance, the antisense preferred target is the translational increase Solubility of the agent, increased cellular uptake, or initiation site of a gene of interest, from approximately 10 Some other appropriate action. nucleotides upstream to approximately 10 nucleotides downstream of the translational initiation site. Oligonucle 0442. In a related exemplary embodiment, adducts of otides targeting the 3' untranslated mRNA regions are also polypeptides, to target the agent to cellular receptors in Vivo, effective inhibitors of translation. Therefore, oligonucle or other compounds which facilitate transport into the target otides targeting the 5' or 3' UTRs of a polynucleotide of cell are included. Additional compounds may be adducted to interest may be used as antisense agents to inhibit transla the antisense/antigene agent to enable crossing of the blood tion. AntiSense agents targeting the coding region are leSS brain barrier, cleavage of the target Sequence upon binding, effective inhibitors of translation but may be used when or to intercalate in the duplex which results from hybrid appropriate. ization to Stabilize that complex. Any Such modification, intended to increase effectiveness of the antisense/antigene 0437 Effective synthetic agents are typically between 20 agent, is included in the present invention. and 30 nucleotides in length. However, to be effective, a complementary Sequence must be Sufficiently complemen 0443). Similarly, the antisense/antigene agent may include tary to bind tightly and uniquely to the polynucleotide of modifications to the phosphate backbone including, but not interest. The degree of complementarity is generally under limited to, phosphorothioates, phosphordamidate, meth stood by those skilled in the art to be measured relative to the ylphosphonate, and others. The agent may also contain length of the antisense/antigene agent. In other words, three modified Sugars including, but not limited variants of ara bases of mismatch in a 20 base oligonucleotide has a more binose, Xylulose and hexose. US 2004/0023207 A1 Feb. 5, 2004 24

0444. In another exemplary embodiment, the antisense/ sizer, or 5'-phosphate ON (cyanoethyl phosphoramidite) antigene agent is an alpha anomeric oligonucleotide capable chemistry developed by Clonotech Laboratories, Inc. In of forming parallel, rather than antiparallel, hybrids with a each of these procedures, oligonucleotides are Synthesized cellular mRNA of interest. from a Single nucleotide using a Series of deprotection and 0445. It is common for antisense agents to be targeted ligation Steps. The underlying chemistry of the reactions is against the coding regions of an RNA of interest to effect Standard practice and the availability and accessibility of translational inhibition. In a preferred embodiment, anti automated Synthesizers bring these Synthetic technologies Sense agents are targeted instead against the transcribed but within the grasp of anyone skilled in the art. untranslated region of an RNA transcript. In this case, rather 0450 Despite the ease of synthesis, the selection of than achieving translational inhibition, it is likely that oli effective antisense agents involves the identification of a gonucleotides hybridized to the target transcript will lead to Suitable target for the agent. This proceSS is simplified mRNA degradation through a pathway mediated by RNaseH Somewhat by the many Software programs available, Such or Similar cellular enzymes. as, for example, Premier Primer 5, available from Premier 0446 For optimal efficacy, the antisense/antigene agents Biosoft International or Primer 3, available online at http:// must be delivered to cells carrying the polynucleotide of www-5 enome.wi.mit.edu/cgi-bin/primer/primer3.cgi. interest in vivo. Several delivery methods are known in the Alternatively, antisense agents may be designed manually art, including but not limited to, targeting techniques by a Scientist skilled in the art. Relevant aspects of the design employing polypeptides linked to the antisense/antigene process which need attention include Selection of the target agent which bind to Specific cellular receptors. In this region to which the antisense agent will bind. Ideally it will instance the agents may be provided Systemically. Alterna be the gene promoter, if the target is DNA, or the translation tively the agents may be injected directly into the tissue of initiation site if the target is an mRNA. Attention also needs interest, or packaged in a virus, including , to be paid to the length of the agent, typically at least 20 chosen because its host range includes the target cell. In nucleotides are needed for Specificity. Shorter oligonucle every case, the agent must enter the target cell to be otides carry the risk of non-Specific binding and therefore may lead to undesired side effects. Also, the agents must be effective. composed of a Sequence that will not promote hybridization 0447 Antisense/antigene methodologies often face the between the oligonucleotides in the agent during applica problem of achieving Sufficient intracellular concentration tion. Taken together, these considerations are well known of the agent to effectively compete with cellular transcrip and are addressed by Standard procedures well known in the tion and/or translation factors. To overcome this challenge, art. those skilled in the art introduce recombinant expression vectors carrying the antisense/antigene agent. Once intro 04.51 Longer antisense agents may be produced within duced into the target cell, expression of the antisense/ the target cell from recombinant expression vectors. In one antigene agent from the incorporated RNA polymerase II or exemplary embodiment, the desired antisense-encoding III promoter results in Sufficient intracellular concentrations. Sequences can be incorporated into an appropriate expres Vectors can be chosen to integrate into the host cell chro Sion vector Selected because it contains the regulatory mosomes, thereby becoming Stable through multiple rounds Sequences necessary to ensure expression in the target cell of cell division, or vectors may be used which remain un type. Selection of the Sequence composition of the antisense integrated and therefore are lost when the target cell divides. agent must take into account the same considerations used to In either case, the primary goal is attaining levels of tran design shorter oligonucleotides as described in the previous Scription that produce Sufficient antisense/antigene agents to paragraph including, but not limited to, binding Specificity be effective. The choice of a Suitable vector and the devel for the target Sequence and minimizing interactions between opment of an effective antisense construct involves tech the expressed agents. Techniques for the design and con niques Standard in the art. Struction of appropriate recombinant expression vectors are well known to those skilled in the art. 0448 Antisense/antigene expression man be regulated by any promoter known to be active in mammalian, especially 0452 Control agents, whether synthetic oligonucleotides human, cells and may be either constitutively active or or longer antisense agents expressed in Vivo by expression inducible. Regardless of the promoter chosen, it is important vectors, are employed to validate the efficacy and Specificity to test for the effect of any enhancer regions intrinsic to those of the therapeutic agents. Each control agent should have the promoters as they may participate in microcompetition with Same nucleotide composition and length as the therapeutic cellular genes. In the case of inducible promoters, the agent but the Sequence should be random. Employment of biological effects of the expressed antisense can be dis this agent will permit the determination of whether any cerned from any effect the promoter has on microcompeti effects observed after treatment with the therapeutic agent tion by assaying any bioactivity with and without induced are indeed specific. Specificity will reduce the potential for gene expression. Suitable promoters, inducible or not, are binding to targets other than those desired, thereby reducing well known in the art (see, for example, Jones 1998'). asSociated unwanted Side effects. 0449 Antisense agents may be prepared using any of a 0453 Purification of Oligonucleotides: The efficacy of number of methods commonly known to those skilled in the Synthetic oligonucleotide agents is impacted by their purity. art. In on exemplary embodiment, oligonucleotides, up to Under typical conditions, approximately 75% of the synthe approximately 50 nucleotides in length, may be Synthesized sis products are full length while the remaining 25% of the using automated processes employing Solid phase, e.g. con oligonucleotides are shorter. This proportion of full length to trolled pore glass (CPG) technology, Such as that used on the Shorter products varies with the length of the desired prod Applied Biosystems model 394 medium throughput synthe uct. The Synthesis of longer oligonucleotides is less efficient, US 2004/0023207 A1 Feb. 5, 2004

and therefore the Synthesis products contain a Smaller pro effects that otherwise may arise from delivery of the thera portion of full length products, than that of shorter ones. peutic agent to the incorrect cell type. However, this advan Unwanted, shorter Synthesis products have reduced Speci tage may be negated if the multiplicity of infection is too ficity compared to the full length products and are therefore high and non-specific infection is thereby promoted. This undesirable in a therapeutic formulation due to their reduced potential problem may be avoided by thoroughly testing any Specificity which in turn leads to an increased risk of Side Viral deliver agent, using techniques well known in the art, effects. prior to its clinical administration. 0454. In one exemplary embodiment, full length oligo 0459 (b) Ribozymes nucleotides greater than 50 bp in length are purified by Virtue 0460. While antisense agents act by either inhibiting of their size. Gel permeation chromatography is used to transcription or translation of the target gene, or by inducing Separate full length products from the shorter Synthetic enzyme-mediated transcript degradation by RNase H or a byproducts. In a complementary exemplary embodiment, Similar enzyme, ribozymes offer an alternative approach. full length synthetic oligonucleotides shorter than 50 bp may Ribozymes are RNA molecules which natively bind to and be purified by liquid chromatography using charged resins cleave target transcripts. Typical ribozymes bind to and Such as hydroxyapatite or nucleic acid Specific resins Such as cleave RNA at specific sites, however hammerhead RPC-5 (which is composed of trioctylmethylamine adsorbed ribozymes cleave target transcripts at Sites directed by onto hydrophobic plastic particles). This latter technique flanking nucleotide Sequences which bind to the target Site. exploits both hydrophobic and ion eXchange methods to The use of hammerhead ribozymes is preferred because the achieve high reagent purity and is amenable to use in HPLC. only Sequence requirement for their activity is the UG 0455 Regardless of the method of purification used, the dinucleotide arranged in the 5'-3' orientation. Hammerhead desired oligonucleotides are concentrated by precipitation technologies are well known in the art (See, for example with ice cold ethanol followed by lyophilization and disso Doherty 2001'', or Goodchild2000'). In a preferred lution in an appropriate carrier for treatment. Carrier Selec embodiment, the Sequence targeted by the ribozyme lies tion is another important component of agent formulation. It near the 5' end of the transcript. That will result cleavage of is essential that the carrier used is first tested for biological the transcript near the translation initiation site thereby activity in the target cell type. This control measure, well blocking translation of a full-length protein. known to those skilled in the art, will ensure that any effects 0461) Ribozymes identified in Tetrahymena thermophila, observed upon administration of the nucleic acid agent are which employ an eight active Site which duplexes indeed due to the agent and not the carrier in which it is with the target RNA molecule, are included in this invention. administered (on purification of oligonucleotides see, for This invention includes those ribozymes, described and instance, Deshmukh (1999''). characterized by Cech and coworkers (i.e. IVS or L-191VS 0456 Delivery of Oligonucleotides: Methods for effec RNA), which target eight base-pair Sequences in a gene of tive administration of antisense agents vary with the agent interest and any others which may be effective in inhibiting used. In one exemplary embodiment, Synthetic oligenucle expression of a disrupted gene or a gene in a disrupting otides are delivered by Simple diffusion into the target cells. pathway. For the catalytic Sequence of these agents See, for Advantages of this delivery method include the ability to instance, U.S. Pat. No. 5,093,246, incorporated entirely administer the agent Systemically, for example by intrave herein by reference. Any ribozyme or hammerhead nous injection. This method, while effective carries Several ribozyme molecules that targets RNA sequences expressed risks, not the least of which is the potential to introduce by a foreign polynucleotide, disrupted gene or gene in a oligonucleotides into cells other than those of the desired disrupted pathway are included in this invention. target. Another disadvantage involves the risk of degrada tion by nucleases in blood and interstitial fluid. This second 0462 Ribozymes, being RNA molecules of specific disadvantage may be partially avoided by modification of Sequence, may be Synthesized with modified nucleotides the Synthetic oligonucleotide in Such a way, for example by which enable better targeting to the host cell of interest or incorporated modified nucleotides Such as those carrying which improve stability. As described above for conven phosphorothioate or methyl phosphonate moieties, which tional antisense agents, the preferred method of delivery involves introduction into the target cell, a recombinant renders them relatively resistant to exonuclease degradation. expression vector encoding the ribosome. Inclusion of an 0457. In a related embodiment those same agents may be appropriate transcriptional promoter will ensure Sufficient delivered by way of liposome mediated transfection as expression to cleave and disrupt transcripts of foreign DNA described by Daftary and Taylor (2001'). This method or disrupted genes or genes in a disrupting pathway. The enhances diffusion into the target cell by encasing the catalytic nature of ribozymes permits their effective use at antisense agent in a lipophilic liposome. However, this concentrations below those needed for traditional antisense method too has drawbacks. While cellular uptake is agents. enhanced, the ratio of liposome components to DNA must be 0463 Identification of ribozyme cleavage sites within a carefully controlled in order to maximize delivery efficiency. transcript of interest is accomplished with any of a number This technique is commonly employed and is well known to of computer algorithms which Scan linear oligonucleotide those skilled in the art. Sequences for alignments with a query Sequence. The iden 0458 In another exemplary embodiment, antisense tified Sequence, commonly containing the trinucleotide expressing viral vectors may be used to confer target cell sequences GUC, GUA or GUU, will serve as the nucleus of Specificity. In Some cases, Viral delivery agents may be a longer Sequence of approximately 20 nucleotides in length. Selected which include the target cell type in their respective That longer Sequence will be examined, again with appro host range. This delivery method minimizes unwanted Side priate computer algorithms well known in the art, for their US 2004/0023207 A1 Feb. 5, 2004 26 potential to form Secondary Structures which may interfere that Such modifications interfere with base pairing, addi with the action of targeted ribozyme agents. Alternatively, tional adducts, Such as derivatives of the base intercalating empirical assays employing ribonucleases may be used to agent acridine, may be incorporated into the therapeutic probe the accessibility of identified target Sequences. agent to restore desirable binding properties to the triplex 0464 Ribozymes comprise a unique class of oligonucle forming oligonucleotide. Alternatively, if the intracellular otides which bind to specific ribonucleic acid targets and target is an mRNA, C-5 propyne pyrimidines may be promote their hydrolysis. The design of ribozyme agents is included in the Synthetic oligophosphorothioate agent to well known to those skilled in the art. In order to prepare increase its binding affinity for mRNA and therefore effective ribozyme agents, initially a Suitable target Sequence decrease the concentration required for effectiveness. must be identified which conferS Specificity to the agent in 0471. The affinity of triplex agents for their respective order to minimize unwanted Side effects and maximize targets may be assessed by electrophoretic gel retardation efficacy. Once that target is identified the ribozyme agent is assays. The formation of triplex Structures will retard migra Synthesized using Standard oligonucleotide Synthesis proce tion through an electrophoretic gel. Similarly, the Stability of dures Such as those exemplified herein. Delivery to the target any tripleX agent binding to its target can be assessed by UV cell may be accomplished by direct transfection eX Vivo or melting experiments. In these assays tripleX agents are by liposome-mediated transfection. mixed with their intended target in Vitro and the resulting 0465 Ensuring the purity and efficacy of ribozyme agents triplexes are heated (with, for example, a Haake cryother may be more important than for other nucleic acid agents mostat) while monitoring their UV absorbance (with, for because their intended effects, namely the hydrolysis of example, a Kontron-Uvikon 940 spectrophotometer) (on target Sequences, are irreversible. In this light extensive design of triplex forming oligonucleotides See, for instance, preclinical testing is essential to minimize unwanted Side Francois (1999'')). effects. These risks are, however, outweighed by the poten 0472 Triplex forming agents are simply oligonucleotides tial effectiveness of ribozyme agents. designed to form triple helices with the target intracellular 0466 (c) Triple Helix nucleic acid. Accordingly, their Synthesis, purification and delivery parallels the procedures described herein for other 0467. In a related embodiment, synthetic single-stranded oligonucleotide agents. Each of these processes is com deoxyribonucleotides can be chosen which form triple heli monly known to those skilled in the art. ceS according to the Hoogsteen base pairing rules. The rules necessitate long Stretches of either purines or pyrimidines on 0473 (d) Homologous Recombination Agents one strand of the DNA duplex. In either case, triplexes are 0474 Binding of factors to foreign polynucleotides formed, with pyrimidines pairing with purines within the (either DNA or RNA), or polynucleotides of disrupted target Sequence and Vice versa, which inhibit transcription of genes, or polynucleotides of a gene in a disrupted or the target Sequence. The effectiveness of a targeted triplex disrupting pathway, or expression of a foreign gene, or a forming oligonucleotide may be enhanced by including a disrupted gene, or a gene in a disrupted or disrupting “switchback' motif composed of alternating 5'-3' and 3'-5' pathway can also be reduced by mutating the DNA, inacti regions of purines and pyrimidines. This “Switchback' Vating, or "knocking out the gene or its promoter using reduces the length of the required purine or pyrimidine tract in the target because the oligonucleotide can form duplexes targeted homologous recombination. alternatively with each Strand of the target Sequence. 0475. In one exemplary embodiment, a polynucleotide of interest flanked by DNA homologous to the polynucleotide 0468 Triple helix forming agents are oligonucleotides interest (encompassing either the coding or regulatory which have been designed to interact with cellular nucleic regions of the polynucleotide) can be introduced into cells acids and form triple helices. The resulting structure may be carrying the same Sequence. Homologous recombination targeted by intracellular degradation pathways or may pro mediated by the flanking Sequences disrupts expression of vide a steric block to nucleic acid replication, transcription the polynucleotide of interest and result in reduced expres or translation depending on the target. Sion. The technique is frequently used by those skilled in the 0469 Triplex agent formulation begins with selection of art to engineer transgenic animals that produce offspring an appropriate target Sequence within the cells to be treated. with Same disruption. However, the same approach may be That target may be within the cellular DNA or RNA or used in humans by administering the engineered construct within that of an exogenous Source Such as an infecting into target cells. Regardless of expression vector platform Virus. Suitable target Sequences should contain long chosen, it is important to recognize and control for any Stretches of homopyrimidines or homopurines and the most microcompetition effects that may be elicited by transcrip effective targets contain alternative Stretches of each. If the tional enhancers carried by the viral vectors (See also above). target is double stranded DNA, the most effective targets Control experiments must be carried out which study the Surround and include the transcriptional regulatory regions. biological activity of a non-recombinant viral vector to Formation of a triplex between the agent and the target will reveal any effects its intrinsic enhancers have on the target inhibit the binding of RNA polymerase or other requisite biological activities. transcriptional regulatory factors which otherwise bind the promoter and upstream regulatory regions. 0476 Nucleic acid agents for homologous recombination are designed to interact with Specific cellular DNA targets 0470 Triplex agents may be synthesized to be more and undergo recombination. The Specificity of the therapeu resistant to cellular and extracellular nucleases by the inclu tic agent is conferred by the nucleotide Sequences at its Sion of modified nucleotides Such as those containing phos termini, they must be complementary to adjacent cellular phorothioate or methyl phosphonate groups. In the event targets and bind them through Watson-Crick base pairing. US 2004/0023207 A1 Feb. 5, 2004 27

0477 Formulation of these agents involves careful selec Specific binding than that of DNA. These are among many tion of the desired cellular target. The nucleotide Sequence favorable properties of PNA and include, in addition, of that target must be available in public or private Sequence increased Stability and exhibit improved hybridization prop databases. The agent itself may be comprised of a Synthetic erties compared to their DNA analogs. While the mechanism oligonucleotide or a recombinant nucleic acid carried in a of PNA action is currently not fully understood, for example Suitable vector. PNA-RNA hybrids are not targets for RNase H degradation as are DNA-RNA hybrids, it is likely that they inhibit 0478. In one exemplary embodiment, a synthetic oligo translation by blocking the binding of RNA polymerase or nucleotide may be used for homologous recombination in order to interrupt the coding Sequence or regulatory other critical factors to the target mRNA. Sequences of the target gene. The oligonucleotide is 0484. In this light, it is important to select targets which designed to include nucleotides at its termini which are include the translation initiation codon. Other target Sites complementary to those of the target Sequence and the further downstream on the mRNA may be effective at central regions may contain any Sequence that is neither inhibiting translation by interfering with ribosome transit complementary to the target Sequence nor carry an in-frame although the role of this activity will need to be determined insertion into the target Sequence. empirically for each agent developed. In any case the actual mechanism of action, while interesting, is not necessary to 0479. In a related embodiment, a longer sequence of ascertain as long as the agent is effective and does not induce nucleic acid may be used. The Sequence of interest, which is undesired Side effects. intended to either interrupt a cellular gene or insert addi tional coding capacity into it, is flanked by Sequences 0485 Homopurines are best targeted by homopyrimidine homologous to the cellular target. That entire DNA fragment PNAS with stretches of greater than 8bp providing suitable is then inserted into an appropriate prokaryotic or viral targets within double stranded DNA. The synthesis of PNA vector for delivery to the target cells. Once inside the cell the agents is achieved using automated Solid-phase techniques agent will bind to and recombine with the target gene. employing Boc-, Fmoc- or Mmt-protected monomers. Alter natively, commercial Sources of custom Synthetic PNAS, 0480 (e) Peptide Nucleic Acids including Applied Biosystems (Foster City, Calif.) may be 0481. In various embodiments, hybridization of the exploited to minimize in-house expenses and expertise (on nucleic acid agents described herein may be enhanced by the design of PNA see, for instance, Nielsen 1999''). substitution of amino acids for the deoxyribose of the 0486 (5) Antibodies and nucleic acid backbone. This Substitution, thereby creating peptide nucleic acids (see, for example, Hyrup 1996'). 0487. Another aspect of the invention pertains to the This modification leads to a reduction of the Overall negative administration of an antibody of interest, equivalent of Such charge on the backbone and therefore reduces the need for antibody, homolog of Such antibody, as treatment of a counter ions to permit Sequence-specific hybridization of chronic disease. two Strands of negatively charged polynucleotides. Peptide 0488 For example, using standard protocols, one skilled nucleic acids can be Synthesized using techniques well in the art can use immunogens derived from a foreign known in the art Such as the Solid phase protocols described polynucleotide, foreign polypeptide, disrupted gene, dis by Hyrup and Nielsen (1996, ibid), and Perry-O'Keefe rupted polypeptide, gene or polypeptide in a disruptive or 1996', included herein in their entirety by reference. disrupted pathway, to produce anti-protein, anti-peptide 0482 Oligonucleotides so modified can be used in the antisera, or monoclonal antibodies (see, for example, Har Same therapeutic techniques as unmodified homologs. They low and Lane 1999'', Sambrook 1989''). can be used as antisense agents designed to interfere with the 0489 Animals which have been injected with an immu expression of a foreign polynucleotide, a disrupted gene, or nogenic agent can Serve as Sources of antisera containing a gene in a disrupted pathway. Similarly, by virtue of their polyclonal antibodies. Monoclonal antibodies, if desired, enhanced hybridization qualities, peptide nucleic acids can may be prepared by isolating lymphocytes from the immu be used, for example, as primers for the PCR, for S1 nized animals and fusing them, in vitro with immortal, nuclease mapping of Single Stranded regions and for other oncogenically transformed cells. Clonal lines from the enzyme-based techniques. Similarly, peptide nucleic acids resulting Somatic cell hybrids, or hybridomas, can be used as may be modified by the addition of lipophilic moieties to Sources of monoclonal antibodies Specific for the immuno enhance the cellular uptake of therapeutic oligonucleotide gen of interest. Techniques for developing hybridomas and agents. In related embodiments, peptide nucleotide agents for isolating and characterizing monoclonal antibodies are may be Synthesized as chimeras comprised of peptide well known in the art (see for instance, Kohler 1975 and nucleic acids and unmodified DNA. This configuration Zola 2000). exploits the advantages of a peptide nucleic acid while the 0490. In the context of this invention, “antibody” refers DNA portion of the molecule can serve as a substrate for to entire molecules or their fragments which react specifi cellular enzymes. cally with polypeptides or polynucleotides of interest, 0483) Peptide Nucleic Acid (PNA) is a DNA analog in whether they are monospecific, bispecific or chimeras which which the Sugar-phosphate backbone contains a pseudopep recognize more than two antigenic determinants. Those tide rather than the Sugars characteristic of DNA. Like DNA, skilled in the art employ well known methods for producing PNA agents bind complementary nucleic acid Strands Specific antibodies and for fragmenting Same. While Several thereby mimicking the behavior of DNA. This activity is methods are known to produce antibody fragments, pepsin, enhanced by the neutral, rather than negatively charged, for example, is used to treat whole antibody molecules to backbone of PNA which promotes more tenacious and more produce F(ab) fragments. These fragments can be further US 2004/0023207 A1 Feb. 5, 2004 28 dissociated with chemicals, Such as beta mercaptoethanol or column with 100 mM glycine (pH 3.0) and monitoring dithiothreotol, which reduce intra and intermolecular disul protein elution spectrophotometrically. fide bridges resulting in the release of Fab fragments. 0496. In an alternative embodiment, antibodies are puri 0491. Once produced, isolated and characterized, anti fied by binding to an affinity column comprised of antigen bodies, or fragments thereof, which bind to antigenic deter croSS-linked to an appropriate Solid Support. Bound antibod minants of interest may be used for diagnostic and analytical ies may be eluted by any of a number of methods and may purposes. For example, they may be used in immunohis include the use of an elution buffer containing glycine at low tochemical assays to assess expression levels of polynucle (e.g. 3.0) pH or 3M potassium thiocyanate and 0.5M otides or polypeptides of interest. They may also be NHOH. Due to the varied mechanisms involved with employed in other immunoassays, including but not limited antibody-antigen interactions, the actual optimal elution to, Western blots, immunoaffinity chromatography, and conditions must determined empirically. immunoprecipitation carried out to quantify protein levels in 0497. The therapeutic efficacy of polyclonal compared to cells or tissueS of interest. The assays, individually or monoclonal antibodies cannot be predicted. Each has together, may also be used by one skilled in the art to Strengths and weaknesses. For example, polyclonal antibod measure the concentration a protein of interest before and ies necessarily target multiple antigenic determinants on the after therapy to assess therapeutic efficacy. target antigen. This feature may increase reactivity but, at 0492 Similarly, it is common in the art to use specific the same time, may decrease specificity. On the other hand, antibodies to Screen libraries of recombinant expression monoclonal antibodies are exquisitely specific for a single vectors for those expressing a protein or polypeptide of antigenic determinant on the target antigen. This specificity interest. Suitable expression vectors are commonly derived greatly reduces the risk of unwanted reactivity with other from bacteriophage, including, for example, 2.gt11 and its antigens, and the associated Side effects, yet carries the risk derivatives. Identification of expression vectors, from that the target antigenic determinant may be inaccessible in among a library of Similar recombinants, can lead to the the cellular environment, either due to the natural folding of identification of vectors expressing a polypeptide of interest the protein or through interactions with other cellular mol which may then itself be used in diagnostic or therapeutic ecules. In every case, the efficacy of any antibody agent must assays. In a preferred embodiment, antibodies specific for a be determined empirically using a variety of techniques well particular polypeptide, protein or antigenic determinant car known to those skilled in the art. ried thereon, will croSS react with homologous counterparts 0498 Antibody production is necessarily preceded by the from different Species to facilitate antibody characterization isolation and purification of appropriate antigens. Cellular and assay development. proteins may be purified by any of a number of techniques 0493 Antibodies may serve as effective therapeutic well known to those skilled in the art. In one exemplary agents for the inactivation of Specific cellular proteins or for embodiment, cells expressing the desired antigen are lysed targeting other therapeutic agents to cells expressing par in the presence of non-ionic detergents and the resulting ticular Surface antigens to which an antibody may bind. lysate is Subjected to purification. That lysate is then frac Polyclonal antibodies are prepared in a Suitable host organ tionated by precipitation in the presence of ammonium ism, typically rabbit, goat or horse, by injecting the appro Sulfate. Sequentially higher concentrations of ammonium priate purified antigen into the host. Following a regimen of sulfate are used to derive protein mixtures which differ by repeated challenges by the desired antigen, using protocols their solubility in ammonium sulfate. Each fraction is then well known to those skilled in the art, serum is drawn from assessed for the presence of the desired antigen. the host and assayed for the presence of antibodies. Once a 0499. The fraction carrying the protein of interest is Suitable response is detected, additional Serum is removed, subjected to further purification by any of a number of well perhaps leading to eXSanguination of the producing organ known methods. For instance, if an antibody against the ism, and the desired antibodies are purified. protein is available, the protein may be purified by affinity 0494 Monoclonal antibodies may be prepared by any chromatography using a resin of Substrate, typically number of techniques well known to those skilled in the art. Sepharose, dextran or Some similar insoluble polymer, to In one exemplary embodiment, cells expressing the desired which the antibody is conjugated. The protein mixture target antigen are fused with immortalized cells in vitro. The containing the desired antigen is exposed to the resin under resulting hybridomas are cultured and clonal lines are conditions which promote antibody-antigen interactions. derived using Standard tissue culture techniques. Each Adventitiously bound proteins are washed from the resin resulting clone is assayed for expression of antibodies with an excess of binding buffer and the antigens are eluted against the desired antigen, typically but not necessarily by with buffer containing an ionic detergent Such as Sodium ELISA dodecylsulfate (SDS). 0500. In an alternative embodiment, crude fractions of 0495 Antibodies may be purified by a number of chro cellular proteins are further purified using methods well matographic techniques. In one exemplary embodiment, known in the art involving ion eXchange or molecular antibodies may be bound to S. aureuS protein AcroSS-linked exclusion chromatographic techniques. The purity of anti to a Suitable Support resin (e.g. Sepharose). The crude gens isolated by any technique may be assessed by electro antibody preparation is slowly applied to the chromato phoresis through denaturing polyacrylamide gels followed graphic column under conditions which permit antibody by Visualization by Staining. protein A interactions. The resin is then washed with Several column volumes of buffer to remove adventitiously bound 0501) c) Assay Protocols and trapped proteins, leaving only Specifically bound anti 0502. One aspect of the invention pertains to assaying the bodies on the column. Those are eluted by washing the effect of an agent on a molecule of interest, equivalent US 2004/0023207 A1 Feb. 5, 2004 29 molecules, or homologous molecules during drug discovery, 0519 Selectin E (NM 000450) development, use as treatment, or during diagnosis. 0520 Integrin alpha (NM 000885) 0503 (1) Definitions 0521) Hormone-sensitive lipase (NM 005357) 0504 (a) Molecule of Interest 0522 TGF-beta 1 (A18277) 0505) The term “molecule of interest” is understood to include, but not limited to, p300/cbp, p300/cbp polynucle 0523) ICAM-1 (X84737) otides, p300/cbp factors, p300/cbp regulated genes, p300/ 0524 GM-CSF (AJ224149) cbp regulated polypeptides, p300/cbp factor kinases, p300/ cbp factor phophatases, p300/cbp agents, foreign p300/cbp 0525 CD8 antigen (NM 004931) polynucleotides, p300/cbp viruses, disrupted genes, dis 0526 CD11A antigen integrin alpha L (XM 008099) rupted polypeptides, genes in disrupted pathways, polypep tides in disrupted pathways, genes in disruptive pathways, 0527 CD11b (NM 000632) polypeptides in disruptive pathways. 0528 CD11C (NM 000887) 0506 Every gene and protein mentioned in this invention 0529) CD28 (AHO02636) is uniquely defined by its Sequence as published in public databases. See, for instance, the Sequences in the nucleotide 0530 CD34 antigen (CD34) (NM 001773) and protein sequence databases at NCBI (also known as 0531 CD40 (XM 009624) , the name of the Search and retrieval System), Gen Bank, the NIH genetic sequence database, DDBJ, the DNA 0532 CD40 ligand (X67878 S50586) DataBank of Japan, EMBL, the European Molecular Biol 0533) CD44 (NT 024229) ogy Laboratory database (GenBank, DDBJ and EMBL comprise the International Nucleotide Sequence Database 0534) CD54 (NT 01.1130 NT 004939) Collaboration), SWISS-PROT, the protein knowledgebase, 0535 CD58 (XM 001325) and TrEMBL, the computer-annotated supplement to SWISS-PROT (see also the search and retrieval system 0536 CD62L (NT 004939) Expasy), PROSITE, the database of protein families and 0537) CD69 antigen (BC007037) domains, and TRANSFAC, the database of transcription factors. By a gene it is meant the coding and non coding 0538 CD80 antigen (CD28 antigen ligand 1 B7-1 regions, the promoters, enhancers, and the 5' and 3' UTRs. antigen) (XM 002948) Published Sequences are considered Standard information 0539 CD86 antigen (CD28 antigen ligand 2 B7-2 and are well known in the art. In one exemplary embodi antigen) (XM 002802) ment, Sequences for certain genes and proteins of interest in this invention are listed in the following section. For most 0540 Interleukin 1 beta (IL1B) (NM 000576) genes, the list includes the human Sequence. However, 0541 Interleukin 1 receptor antagonist (IL1-RA) homologous sequences (see definition below) are available (XM 010756 P18510 NM 000577 AJO05835 in the above databases for other organisms, Such as mouse, BCOO9745 M55646 M63099 X52O15X53296 X64532 rat, etc. The following listed Sequences should be regarded X84348 AF043143) as illustrations, and, therefore, should not be construed as limiting the invention in any way. 0542) Interleukin 2 (IL2) (AF359939) 0543) Interleukin 2 receptor beta (IL2R) 0507 List of Sequences (XM 009962) 0508) Metallothionein IIA (J00271 V00594x97260 S52379 P02795) 0544) Interleukin 4 (IL4) (AF3.95008) 0509 Interferon gamma (AF330164) 0545) Interleukin 5 (IL5) (AF353265) 0510 Platelet-derived growth factor B chain (PDGFB) 0546) Interleukin 6 (IL6) (AF048692) (Y14326 XM 009997) 0547 Interleukin 10 (IL10) (XM 001409) 0511 Platelet-derived growth factor alpha polypeptide 0548 Interleukin 12A (NM 000882) (PDGFA) (NM 002607) 0549 Interleukin 12B (NM 002187) 0512 Neuregulin 1 (NRG1) (NM 013964) 0550 Interleukin 13 (IL13) (AF377331) 0513) Heregulin-beta1 (M94166) 0551) Interleukin 16 (NM 004.513) 0514) TNF-alpha (AB048818) 0552 Aldose reductase (BCO10391) 0515 TNF-beta (Lymphotoxin) (D12614) 0553 Neutrophil elastase (AC004799) 0516. Oxytocin receptor (OXTR) (NM 000916x 0554) Folate binding protein (FBP) (X62753) 80282 M25650) 0555 Cytochrome c oxidase subunit Vb (Cox Vb) 0517 Kappa light chain nuclear factor NFKB (M19961) (L01459) 0556) Cytochrome c oxidise subunit IV (Cox IV) 0518) Selectin P (NM 003005) (BC008704) US 2004/0023207 A1 Feb. 5, 2004 30

0557. Transcription factor A mitochondrial (TFAM) 0589) Insulin (BC005255) (NM 012251) 0590 Leptin (Lep) (U65742) 0558 ATP synthase beta (NM 001686) 0591) Leptin receptor db form (OB-Rdb) (U58863) 0559) Prolactin (PRL) (XM 004269) 0592) Myelin basic protein (MBP) (XM 008797) 0560 Retinoic acid receptor beta (RARB) (XM 003071) 0593) RANTES (AF088219) 0561 Choline acetyltransferase (CHAT) 0594) MIP-1 alpha/RANTES receptor (E13385) (XM 011848) 0595 MIP-1 beta (NT 01.0795) 0562 Cholinergic receptor nicotinic beta polypeptide 0596) Chemokine (C-C motif) receptor 5 (CCR5) 4 (CHRNB4) (NM 000750) (NM 000579) 0563) RAF1 (NM 002880) 0597) Thioredoxin (TXN) (XM 015718) 0564) Nicotinic acetylcholine receptor (AChR) 0598. Thrombopoietin (XM 002815) (X17104) 0599 Polyomavirus (NC 001515 NC 001516) 0565 Acetylcholine receptor delta subunit (X55019 X53091 X53516) 0600 JC virus (J02226 J02227 NC 001699) 0566 Cholinergic receptor nicotinic epsilon polypep 0601) SV40 (J02400 J02402-3 J02406-10 J04139 tide (XM 008520) M24874 M24914 M28728 V01380 NC 001669) 0602 BK virus (NC001538 VO1108 J02038 strain 0567 PKC alpha (X52479) dunlop V01109 J02039 strain MM JO2038 KO0058 0568 v-Ha-ras (XM 006146) V01108 strain dunlop M23122 strain AS) 0569 v-fos FBJ murine osteosarcoma viral oncogene 0603 Lymphotropic polyomavirus (K02562) homolog (FOS) (NM 005252) 0604 Human adenovirus type 2 (NC 001405) 0570 Cvtochromey P450 monoxygenase CYP2J2 0605 Human adenovirus 5 (NC001406 M73260 (U37143) M29978) 0571 Fibronectin (EO1162) 0606 Human adenovirus type 5 E1A enhancer 0572 Vascular cell adhesion molecule 1 (VCAM-1) (M13156) (X53051) 0607 Human adenovirus 17 (NC 002067 AF108105) 0573 PECAM1 (NM 000442) 0608 Human adenovirus 40 (L19443) 0574) MCP-1 (Y18933) 0609 Human herpesvirus 1 (NC 001806x14112 0575 AP-2 (X77343) D00317 D00374S40593) 0576 Apob-100 (M14162) 0610 Human herpesvirus 2 (NC 001798) 0577 Actin beta (ACTB) (XM 004814) 0611 Human herpesvirus 3 (NC 001348) 0578) GAPDH (NTO09731) 0612 Human herpesvirus 4 (NC 001345) 0579 Cyclin-dependent 0613 Human herpesvirus 5 (NC 001347 X04650 kinase 4 (CDK4) D00328 D00327 X17403 (strain AD169) M17956 (NM 000075) M21295 U33331 D63854 KO1263 M60321 XO3922 0580 Cyclin-dependent kinase 2 (CDK2) M11298 M18921) (XM 006726) 0614 Human herpesvirus 6 (NC001664 X83413 0581 Human cyclin D1 (M64349) (U1102 variant A) AB021506 (variant B strain HST)) 0582 Human cyclin D2 (X68452) 0615 Human herpesvirus 6B (NC 000898 AF157706 0583) Human cyclin A1 (NM 003914) L13162 L14772 L16947 (strain Z29)) 0584) Skeletal muscle alpha-actin (ACTA1) 0616) Human herpesvirus 7 (NC 001716 U43400 (JI) (AF182035) AF037218 (strain RK)) 0617 Epstein-Barr virus (EBV) (V01555 J02070 0585) Retinoic acid receptor alpha (BCO08727) KO1729-30 VO554 X00498-99 X00784 (strain B95-8) 0586 Transforming growth factor-beta (TGF-beta) L07923 X58140 D10059) (X02812 J05114) 0618. (NC 001407) 0587 Beta-1-adrenergic receptor (ADRB 1) (AF 169007) 0619 Y73 sarcoma virus (NC 001404) 0588 Adrenergic beta-2-receptor Surface (ADRB2) 0620) Human coxsackievirus A (NC 001429) (NM 000024) 0621 Coxsackievirus B3 (NC 001473)

US 2004/0023207 A1 Feb. 5, 2004 33

0710) MyoD (P15172 AF027148 BC000353 X17650 0738) STAT4 (Q14765) X56677) 0739 STAT5A (P42229 L41142 U43185) 0711) RelA (Q04206 BC011603 BCO14095 L19067 M62399 Z22948 Z22951) 0740 STAT5B (P51692 U47686 U48730) 0712 NFAT1 (Q13469 AL035682 U43341 U43342) 0741 STAT6 (P42226 AF067572 AF067573 0713 NF-YB (P25208 BC005316 BC005317 AFO67574 AFO67575 BCOO4973 BCOO5823O U16031 BC007035 L06145 X59710) U66574) 0714 NF-YA (P23511 NM 021705 AKO25201 0742 TAL1 (P17542 AJ131016 AL135960 M29038 AL031778 M59079x59711) M61108 S53245 X51990) 0715) P/CAF (Q92831) 0743) TBP (P20226 AL031259 M34960 M55654 0716) p/CIP (Q9Y6Q9 AL0344180 Q9UPG4) X54993) 0717 MRG1 (Q99967 AF109161 AF129290 0744) TF2B (Q00403 AL445991 S44.184) BC004377 U65093) 0745) THRA (P10827 BC000261 BC002728 J03239 0718 NFE2 (Q16621 BC005044 L13974 L24122 M24748 M24899 X55005 X55074 Y00479) S77763) 0746 THRB (P10828 M26747 X04707 P37243) 0719 p53 (PO, AF052180 AF066082 AF135121 AF136271 AF307851 BCOO3596 KO3199 M13121 0747 TWIST (Q15672 U80998 X91662 X99268 M14694 M14695 M22881 M22898 U94788 XO1405 Y10871) XO2469 X541560 X60010 X600110 X6OO12 X60013 0748 IRF3 (Q14653 AF112181 AXO15330 X6OO14 X6OO15 X6OO16 X6OO17 X60018 X6OO19 AXO15339 BC009395 U86636Z56281) X60020) 0749) YY1 (P25490 AF047455 M76541 M77698 0720 p73 (015350 AF077628 AL136528 Y11416) Z14077) 0721 RSKI (NM 002953 AL109743 BC014966 0750 PPARG (P37231 NM 015869 BC006811 L07597 Q15418) D83233 L40904 U63415 U79012 X90563) 0722) RSK3 (AL022069 AXO19387 BC002363 0751) AR (P10275 AF162704 L294.96 M20132 L07598 X85106) M2O260 M21748 M23263 M27430 M34233 M35851 0723) RSK2 (P51812 L07599 U08316) M581.58 S79366 ST793.68 M27424 M27425 M27426 0724) PITI (P28069 D10216 D12892 L18781 X62429 M27427 M27428 M27429 M35845 M35846 M35847 X72215) M35848 M35849 M35850) 0725 RARG (P13631 AJ250835 L12060 M24857 0752 SRD5A1 (P18405 AF052126 AF113128 M38258 M57707 P22932) ALOO8713 BCOO6373 BCOOTO33 BCOO8673 M32313 M68886 M68882 M68883 M68884 M68885 0726) RXRA (AFO52092 BC007925 BC009882 AF073302 AF073304) U66306 X52773) 0753) (b) Equivalent Molecules 0727) DDX9 (Q08211 L13848 U03643 Y10658) 0754) The term “equivalent molecules” is understood to 0728 ELK4 (P28324 NM 001973 M85164 M85165) include molecules having the same or Similar activity as the 0729 SF-1 (Qi3285 D842060 D84207 D84208 molecule of interest, including, but not limited to, biological D842090 D84210 D88155 U76388) activity and chemical activity, in vitro or in Vivo. 0730 SMAD4 (Q13485 AF0454470 BC002379 0755) (c) Homologous Molecules U44378) 0756. The term “homologous molecules” is understood 0731) SMAD1 (Q15797 BC001878 U548260 U57456 to include molecules with the same or Similar chemical U59423 U59912) Structure as the molecule of interest (see exemplary embodi ments above). 0732) SMAD2 (Q15796 AF027964 BC014840 0757. The following section presents standard assays U59911 U65019 U68018 U78733) which can be used, in conjunction with the assays in the new 0733 SMAD3 (Q92940 U68019 U76622) elements Section, to test the effect of an agent on a molecule 0734) SRCI (AJO00882 NM 0037430 AJO00881 of interest. U19177 U19179 U40396 U59302 U90661) 0758) (d) During 0735 SREBP1 (P36956 U00968) 0759. The term “during drug discovery, development, use as treatment, or during diagnosis” is understood to include, 0736. SREBP2 (Q12772 U02031 Z99716) but not be limited to, drug Screening, rational design, opti 0737) STAT3 (P40763 AJO12463 BC000627 mization, in laboratory or clinical trials, in Vitro or in vivo BC014.482 L29277) (see exemplary embodiment below). US 2004/0023207 A1 Feb. 5, 2004 34

0760 (2) Assaying Protein Concentration extracted from the Solution by mixing with phenol/chloro form/isoamyl alcohol followed by extraction with chloro 0761 (a) UV Absorbance form/isoamyl alcohol. Nucleic acids in the resulting protein 0762. In one exemplary embodiment, cellular protein deficient Solution are precipitated by addition of Salt, typi concentration is measured by Virtue of its absorbance of cally Sodium acetate or ammonium acetate, and ethanol. ultraviolet light at the wavelength of 280 nm (Ausubel After a brief incubation of the mixture at -20° C., the 1999''). To calibrate the reagents used, and to validate the insoluble nucleic acids are removed by centrifugation, dried, Spectrophotometer, a Standard curve is established using and redissolved in a sterile, RNase free Solution of Tris and protein Solutions of known concentration. Typically Solu EDTA. Contaminating DNA is removed from the lysate by tions of bovine Serum albumin, a commonly available pro treatment with RNase-free DNase I. Degraded DNA is tein, are used to establish the Standard curve. Cells are lysed removed by precipitation of the intact RNA with salt and in a detergent-rich buffer to liberate membrane associated ethanol. The dried, purified RNA is dissolved in Tris-EDTA and intracellular proteins. Following lysis, insoluble mate and quantified by virtue of its absorbance of light at 260 nm. rials are removed by centrifugation. The absorbance of UV Since the molar extinction coefficient of RNA at 260 nm is light by the Supernatant, which contains Soluble proteins of well known, the concentration of RNA in the Solution can be unknown concentration, is then measured and compared to determined directly. the Standard curve. Comparison of the data obtained from the cellular extracts with those represented by the Standard 0770 (b) Northern Blot curve provides an indication of cellular protein concentra 0771. The concentration of a particular RNA species can tion. also be determined. In one exemplary embodiment, the amount of mRNA which encodes a protein of interest, for 0763) (b) Bradford Method instance, p300, GABP, CBP, within a population of cells is 0764. In another exemplary embodiment, protein concen measured by Northern blot analysis (Ausubel 1999, Ibid, tration is determined using the Bradford method (Sapan Gizard 2001). Total cellular RNA is isolated and sepa 1999, Ausubel 1999, Ibid). A standard curve is con rated by electrophoresis through agarose under denaturing Structed using Solutions of known protein concentration conditions, typically in a gel containing formaldehyde. The mixed with coomassie brilliant blue. Following a brief RNA is then transferred to, and immobilized upon a charged incubation at room temperature, the absorbance of light at nylon membrane. The membrane is incubated with a solu 595 nm is measured and a Standard curve is constructed. tion of detergent and excess of low molecular weight DNA, Cells are lysed as described above, the lysate is mixed with typically isolated from Salmon Sperm, to prevent adventi coomassie brilliant blue and the absorbance measured in a tious binding of the gene Specific, for instance, p300-, manner identical to that of the Standard curve. Comparison GABP-, CBP-specific, radiolabeled DNA probe to the mem of the values obtained from the cellular extract with those of brane. Radiolabeled cDNA probes representing the protein, the Solutions of known concentration reveals the concentra e.g., p300, GABP, CBP, transcript are then hybridized to the tion of cellular proteins. membranes and bound probe is visualized by autoradiogra 0765 (c) Immunoaffinity Chromatography phy. 0766 To measure concentration of a specific cellular 0772 (c) -Polymerase Chain protein, for instance, p300, GABP or CBP, additional steps Reaction (RT-PCR) are employed to purify the protein away from other cellular 0773) In another exemplary embodiment, the amount of proteins. One exemplary embodiment involves the use of mRNA encoding a protein of interest, for instance, p300, Specific antibodies targeted against the protein of interest to GABP, CBP, expressed by a population of cells is measured remove it from the cellular lysate. Specific antibodies, for by first isolating RNA from cells and preparing cDNA by instance, anti-p300, anti-GABP or anti-CBP, are chemically binding oligo deoxythymidine (dT) to the polyadenylated bound to a resin and contained within a vertical glass or mRNA within the prepared RNA. Reverse transcriptase is plastic column. Cell lysate is passed over that resin to permit then used to extend the bound oligo dT primers in the antibody-antigen interactions, thereby allowing the protein presence of all four deoxynucleotides to create DNA copies to bind to the immobilized antibodies. Efficient removal of of the mRNA. The cDNA population is then amplified by the the protein of interest from the cell lysate is accomplished by polymerase chain reaction in the presence of oligonucleotide using an excess of antibody. Protein bound to the column is primerS Specific for the Sequence of the gene or RNA of removed which releases the bound protein. The eluted interest and Taq DNA polymerase. The amplification prod protein is collected and its concentration determined by an ucts can be visualized by gel electrophoresis followed by assay for protein concentration Such as those exemplified Staining with ethidium bromide and exposure to ultraViolet above. light. Quantification can be achieved by adding a radiola 0767 (3) Assaying mRNA Concentration beled deoxynucleotide to the PCR reaction. Radiolabel incorporated into the amplification products is visualized by 0768 (a) UV Absorbance autoradiography and quantified by densitometric analysis of 0769. In certain embodiments, RNA concentration is the autoradiograph or by direct phosphorimager analysis of measured by absorption of ultraViolet light at a wavelength the electrophoretic gel. of 260 nm (Manchester 1995'7, Davis 1986, Ausubel 1999, Ibid). RNA is purified from cells by first lysing the 0774) (d) S1 Nuclease Protection cells in a detergent rich buffer. Proteins in the cellular lysate 0775. In a related exemplary embodiment, expression of are degraded by incubation overnight at 65 C. with pro RNA encoding a protein of interest, for instance, p300, teinase K. After enzymatic degradation, proteins are GABP, CBP, can be assessed by hybridizing isolated cellular US 2004/0023207 A1 Feb. 5, 2004

RNA with a radiolableled synthetic DNA sequence homolo lized polynucleotide or polypeptide of interest, or test com gous to the 5' terminus of the RNA of the protein of interest. pound were labeled before the reaction, the label may be The synthetic deoxyribonucleotide, less than 40 nucleotides used to detect the anchored complexes. If the components in length, is labeled at it 5' end with T4 polynucleotide were not prelabeled, a label may be added during or after kinase and Y- PATP. Once the oligonucleotide is bound to complex formation, for instance, an antibody directed the RNA, the mixture is incubated in the presence of the against the nonimmobilized polynucleotide or polypeptide Single Strand-Specific nuclease S1. Any unhybridized, and of interest, or test compound, can be added to the Surface therefore single stranded, molecules of RNA or DNA are coated wells. degraded, leaving the DNA-RNA hybrids of the protein of interest intact. The undegraded hybrids are removed from 0784. In a variation of this assay, the polynucleotide or the Solution by precipitation with ammonium acetate and polypeptide of interest is anchored to the a Solid Surface and ethanol and resolved by nondenaturing gel electrophoresis. the nonimmobilized test compound is added to the Surface Radiolabeled bands on the gel are then visualized by auto coated wells. radiography. The radiolabel can be quantified by densito 0785. In another variation of this assay, the reactions are metric analysis of the autoradiographs or by phosphorimager performed in a liquid phase, and the complexes are removed analysis of the electrophoretic gels themselves. from the reaction mixture by immunoaffinity chromatogra 0776 (4) Assaying Polynucleotide Copy Number phy, or immunoprecipitation, as described herein. 0777) (a) S1 Nuclease Protection 0786) (b) Detection of Binding to DNA 0778 This same technique can be used to quantify the 0787. In one exemplary embodiment, DNA fragments level of any nucleic acid, naturally expressed or exogenous, carrying a known, or Suspected binding domain for a within a population of cells. In every case the Sequence of polypeptide of interest, for instance, p300, GABP, etc., are the Single Stranded Synthetic oligonucleotide must be purified by gel electrophoresis and labeled with T4 poly designed So that it is complementary to the 5' terminal nucleotide kinase in the presence of YP-ATP (Bulman et Sequence of the Species to be measured. al. 2001). Labeled DNA is then added to a solution contain ing the polypeptide of interest under conditions, ionic and 0779) (b) Real Time PCR thermal, which permit formation of DNA-polypeptide com 0780. In another exemplary embodiment, DNA copy plexes. The Solution is then maintained for a period of time number can be measured using real time PCR (Heid Sufficient for the reaction to complete. Following comple 1996'). This technique employs oligonucleotides doubly tion, the mixture is separated by electrophoresis through labeled. At the 5' ends they carry a reporter dye that nondenaturing polyacrylamide in parallel to labeled, but fluoresces upon excitation by the appropriate wavelength of otherwise unreacted test DNA. Following electrophoresis, light. At the 3' end they carry a quencher dye that Suppresses the labeled DNA is detected by autoradiography or by the fluorescence of the first dye. These oligonucleotides are phosphorimager analysis. Formation of complexes is prepared So that their Sequence is complementary to the detected by the shift in electrophoretic mobility (see also region of interest which lies between the forward and below). reverse PCR primers. Once hybridized to the DNA sequence 0788. The assay detects polypeptide-DNA complexes of interest, the close proximity of the quencher dye and the formed by direct binding of the polypeptide of interest with fluorescent dye Suppresses the fluorescent emissions of the DNA, or by indirect binding through intermediary polypep reporter dye. However, during the process of PCR, Taq tides, as long as the intermediary polypeptides are present in polymerase cleaves the reporter dye from the oligonucle the reaction mixture. Further, the magnitude of the gel shift otide and releases it. Once removed from the nearby provides a Semi-quantitative measure of the relative con quencher dye, fluorescence is permitted. Free fluorescent centration of the polypeptide-DNA binding in the assay dye is quantified with a fluorimeter and is directly related to mixture. AS Such, changes in concentration can also be the number of molecules of interest present prior to PCR. detected. 0781 (5) Detection of Binding 0789 (i) Affinity Chromatography 0782) (a) General 0790. In one exemplary embodiment, binding of a 0783. In one exemplary embodiment, an assay to identify polypeptide of interest, that is, disrupted polypeptide, or compounds that bind to a polynucleotide or polypeptide of polypeptide in a disrupted or disruptive pathway, Such as interest involves binding of a test compound to Wells of a p300, GABP, CBP, to DNA is measured by first expressing microtiter plate by covalent or non-covalent binding. For fragments of the polypeptide of interest as GST (glutathione instance, the assay may anchor a specific test compound to sulfonyl transferase) fusion proteins in E. coli (Gizard 2001, a microtiter plate Substrate using a mono or polyclonal Ibid). The expressed polypeptides are then bound to glu immobilized antibody. A Solution of the test compound can tathione coupled Sepharose. Radiolabeled DNA fragments, also be used to coated the Solid Surface. Then, the nonim carrying P, representing the polypeptide binding site, are mobilized polynucleotide or polypeptide of interest may be incubated with protein-bead complexes and Subsequently added to the Surface coated wells. After Sufficient time is washed three times to remove adventitiously bound DNA. allowed for the reaction to complete, the residual compo Any DNA bound to the immobilized polypeptide of interest nents are removed by, for instance, Washing. Care should be are released by boiling in presence of the ionic detergent taken not to remove complexes anchored on the Solid SDS. Liberated radiolabeled DNA is quantified by liquid Surface. Anchored complexes may be detected by Several Scintillation counting, or by direct measurement of Ceren methods known in the art. For instance, if the nonimmobi kOV radiation. US 2004/0023207 A1 Feb. 5, 2004 36

0791) (ii) Electrophoretic Gel Mobility Shift Assay 0798. In another variation of the assay, peptide fragments 0792. In another exemplary embodiment, binding of a of the binding domains, instead of full length complex polypeptide of interest, or a group of polypeptides to DNA components are used. Several methods well known in the art is assessed by electrophoretic gel mobility shift assay can be used to identify and isolate binding domains. For (Gizard 2001, Ibid, Ausubel 1999, Ibid, Nuchprayoon instance, one method entails mutating a gene and Screening 1999''). Radiolabeled DNA carrying the polypeptide bind for a disruption in normal binding of the polypeptide ing Site, for instance, the p300 binding Site, or N-box, is encoded by the gene by co-immunoprecipitation or immu mixed with the recombinant polypeptide, for instance, p300, noaffinity. If the polypeptide ShowS disrupted binding, GABP, expressed as GST fusion protein. Subsequent reso analysis of the gene Sequence can reveal the binding domain, lution by electrophoresis through nondenaturing polyacry or the region of the polypeptide involved in binding. Another lamide gels in parallel with labeled DNA alone, reveals a approach partially proteolyzes a labeled polypeptide shift in electrophoretic mobility only if the polypeptide is anchored to a Solid Surface. Non bound fragments are bound to DNA in the DNA/polypeptide mixtures. If the removed by Washing leaving a labeled polypeptide compris DNA binding site is unknown, or one is Suspected to be ing the binding domain immobilized on the Solid Surface. carried in a collection of DNA fragments, this assay can be The polypeptide fragments bound to the immobilized pro performed to test for, and potentially affirm the presence of teins are than isolated and analyzed by amino acid Sequenc ing, using for instance the Edman degradation procedure Such a binding Site. (Creighton 1983'). Another approach expresses specific 0793 (6) Detection of Binding Interference fragments of a polynucleotide, or gene, and tests the frag 0794. A polynucleotide or polypeptide of interest may ments for binding activity. bind with one or many cellular or extracellular proteins in vivo. Compounds that interfere with, or disrupt the binding 0799. In another embodiment, an assay uses a complex may include, but are not limited to, antisense oligonucle with one component labeled. However, binding to the com otides, antibodies, peptides, and Similar molecules. plex quenches the Signal generated by the label (see, for 0795. In one exemplary embodiment, binding interfer instance, U.S. Pat. No. 4,109,496). A test compound which ence of a test compound is assessed by adding the compound disrupts the complex, for instance, by displacing a part of the to a mixture containing a polynucleotide or polypeptide of complex, restores the Signal. This assay can be used to interest and a binding partner. After enough time is allowed identify compounds which either interfere with complex for the reaction to be completed, the complex concentration formation, or disrupt an already formed complex. in the test reaction mixture is compared to a control mixture 0800 Specifically, a test compound can interfere with prepared without the test compound, or with a placebo. A binding between a disrupted gene or polypeptide, or a gene decreased concentration in the test reaction indicates inter or polypeptide in a disruptive or disrupted pathway, for ference. Reactants may be added at different orders regard instance, a microcompeted or mutated gene or polypeptide, less of the method used. For example, a test compound may and their binding partner. The assay may be especially useful be added to the reaction mixture before adding the poly in identifying compounds capable of interfering in binding nucleotide or polypeptide of interest and their binding reactions between foreign polynucleotides and cellular partners, or at the same time. A test compound that can polypeptides without interfering in binding between cellular disrupt an already formed complex, for instance, by displac polynucleotide and cellular polypeptides. The assay is also ing a complex component, can be added to the reaction especially useful in identifying compounds capable of inter mixture after complex formation. The interference assay can fering in binding between mutant cellular polynucleotide or be conducted in two ways, in liquid, or in Solid phases, as polypeptide and normal cellular polynucleotide or polypep described above. tide without interfering in binding between normal poly 0796. In another embodiment, a polynucleotide or nucleotide or polypeptides. polypeptide of interest is prepared for immobilization by fusion to glutathione-S-transferase (GST), while maintain 0801 (7) Identification of a Polypeptide Bound to DNA ing the binding capacity of the fusion protein. Another or Protein Complex complex component, a cellular polynucleotide or polypep 0802 (a) Immunoprecipitation tide, or extracellular protein, can be purified, and then utilized in developing a monoclonal antibody using methods 0803. In one exemplary embodiments, the identity of a well known in the art. The GST-polynucleotide fusion bound polypeptide, for instance, p300, GABP, CBP, is protein is coupled to glutathione-agarose beads and exposed confirmed by reacting antibodies Specific to the polypeptide to the other complex component in the presence or absence of interest with polypeptides bound to DNA. For example, of a test compound. After Sufficient time has been allowed p300-specific antibodies are mixed with the polypeptide for the reaction to complete, unbound components are DNA complexes and incubated overnight at 4 C. Immune removed, for instance, by Washing, and the labeled mono complexes are then precipitated by the addition of a Sec clonal antibody is added. Bound radiolabeled antibody is ondary antibody directed against the primary p300-specific then measured to quantify the extent of complex formation. antibody. Precipitated antibody-antigen complexes are Inhibition of complex formation by a test compound resolved by denaturing gel electrophoresis and the constitu decreases measured radioactivity. AS above, a test com ent proteins are visualized by Staining with coomassie pound capable of complex disruption can also be added after brilliant blue. complex formation. 0804. In a related exemplary embodiment, the interaction 0797. In one variation of the assay, the fusion protein is between a polypeptide of interest, for instance, p300, GABP, mixed with the other complex component in liquid, that is, CBP, and other cellular proteins, Such as transcription fac without Solid glutathione-agarose beads. tors, may be detected by co-immunoprecipitation of the US 2004/0023207 A1 Feb. 5, 2004 37 polypeptide of interest with antibodies specific to the 0816 5. Gross chromosomal rearrangement of a dis polypeptide, for instance, p300-specific antibodies. For rupted gene, or gene in a disrupted pathway, for instance, example, in the case of p300, cellular protein extracts are translocation. incubated with purified p300-GST fusion proteins to enable protein-protein interactions. p300-specific antibodies are 0817 6. Allelic loss of disrupted gene, or gene in a then added and the mixture is incubated overnight at 4 C. disrupted pathway. Immune complexes are precipitated by addition of a Sec 0818 7. Different than wild-type mRNA concentration of ondary antibody directed against the primary p300 antibod a disrupted gene, or gene in a disrupted pathway. ies and the precipitates are resolved by electrophoresis on denaturing polyacrylamide gels. Proteins are Subsequently 0819 8. Irregular splicing pattern of mRNA transcript of detected by Staining with coomassie brilliant blue. a disrupted gene, or gene in a disrupted pathway. 0805) (b) Antibody Supershift Assay 0820) 9. Irregular post-transcriptional modification of an mRNA transcript other than Splicing, for instance, editing, 0806. In a related exemplary embodiment, DNA-protein capping or polyadenylation, of a disrupted gene or gene in complexes are detected by electrophoretic gel mobility shift a disrupted pathway. assay (Gizard 2001, Ibid, Ausubel 1999, Ibid). Radiolabeled DNA carrying the polypeptide binding Site, for instance, 0821 10. Different than wild-type concentration of a p300 binding site, or N-box, is mixed with a recombinant disrupted polypeptide, or polypeptide in a disrupted path polypeptide, for instance, p300, or GABP, expressed as GST way. fusion protein. Subsequent resolution by electrophoresis 0822 11. Irregular post-translational modification of a through nondenaturing polyacrylamide gels in parallel with disrupted polypeptide, or a polypeptide in a disrupted path labeled DNA alone, reveals a shift in electrophoretic mobil way. ity only if the polypeptide is bound to DNA in the DNA/ polypeptide mixture. To identify the bound polypeptide, a 0823. Many assays are known in the art for detection of specific antibody is reacted to the DNA/polypeptide mixture the above, or other irregularities associated with a genetic prior to electrophoresis. Bound antibody molecules cause a lesion. Consider the following exemplary assays. Also con further change in gel mobility, namely a SuperShift, and sider the exemplary assays discussed in the following serve to identify the polypeptide bound to DNA. reviews on detection of genetic lesions, Kristensen 2001''", 0807 (8) Identification of a DNA Consensus Binding Tawata 2000, Pecheniuk 2000, Cotton 19937, Site Prosser 1993, Abrams 1990, Forrest 1990'7. 0808 (a) PCR and DNA Sequencing 0824 (a) Sequencing 0809. In one exemplary embodiment, DNA fragments are 0825. In one exemplary embodiment, a polynucleotide of prepared containing potential polypeptide binding sites, interest can be sequenced using any Sequencing techniques either wild-type or variants, flanked by DNA fragments of known in the art to reveal a lesion by comparing the test known nucleotide Sequence. The fragments are then reacted Sequence to wild-type control, known mutant Sequence, or with the polypeptide-GST fusion proteins immobilized on Sequences available in public databases. Sepharose beads. After washing to remove adventitiously 0826. An introduction to sequencing is available in Gra bound DNA, bound fragments are eluted by heating in ham 2001'''. Exemplary sequencing protocols are available presence of a detergent. The eluted fragments are amplified in Rapley 19967. Recent sequencing methods are available by the polymerase chain reaction (PCR) using primers in Marziali 20017, Dovichi 20017, Huang 19997, Specific for the flanking DNA sequences. The nucleotide Schmalzing 19997, Murray 1996'77, Cohen 19967; Grif Sequence of the amplification products is then determined by fin 1993''. Automated sequencing methods are available in any Sequencing method known in the art, for instance, the Watts 2001, MacBeath 2001, Smith 1996. For clas dideoxy chain termination Sequencing method of Sanger sical sequencing methods see Maxam 1977, Sanger 1977 (Sanger 1977'), using as sequencing primer one of the two (Ibid). PCR primers. Several sequence variants of the binding site are likely to be identified. Together they can be used to 0827) (b) Restriction Enzyme Cleavage Patterns establish a consensus DNA sequence for the polypeptide 0828. In another exemplary embodiment, patterns of binding Site. restriction enzyme cleavage are analyzed to reveal lesions in 0810 (9) Detection of a Genetic Lesion a polynucleotide of interest. For example, Sample and con trol DNA are isolated, amplified, if necessary, digested with 0811 Existence of a genetic lesion can be determined by one or Several restriction endonucleases, and the fragments observing one or more of the following irregularities. Separated by gel electrophoresis. Sequence Specific 08.12 1. Deletion of at least one nucleotide from a ribozymes are then used to detect Specific mutations by disrupted gene, or gene in a disrupted pathway. development or loSS of a ribozyme cleavage Site. 0813 2. Addition of at least one nucleotide to a disrupted 0829 (c) Protection from Cleavage Agents gene, or a gene in a disrupted pathway. 0830. In another exemplary embodiment, cleavage 0814) 3. Substitution of at least one nucleotide to a agents, Such as certain Single-Strand Specific nucleases, disrupted gene, or gene in a disrupted pathway. hydroxylamine, osmium tetroxide or piperidine, are used to 0815 4. Irregular modification of a disrupted gene, or detect mismatched base pairs in nucleic acid hybrids com gene in a disrupted pathway, Such as change in DNA prised of either RNA/RNA or RNA/DNA duplexes. Wild methylation patterns. type and test DNA or RNA, with one or the other molecule US 2004/0023207 A1 Feb. 5, 2004 38 labeled with radioactivity, are mixed under conditions per trophoresis,” (DGGE) (Myers 1985B, Ibid) is commonly mitting formation of heteroduplexes between the two spe used to detect differences between Similar oligonucleotides. cies. Following hybridization, the duplexes formed are Prior to analysis, test DNA is often modified by addition of treated with an agent capable of cleaving Single, but not up to 40 base pairs of GC rich DNA through PCR. The double Stranded nucleic acids. Examples include, but are not relatively stable region, termed “GC clamp, ensures only limited to S1 nuclease, piperidine, hydroxylamine and partial denaturation. A variation of the assay employs a RNase H, in the case of RNA/DNA heteroduplexes. Since temperature rather than chemical gradient of denaturant. mismatches between wild-type and mutant oligonucleotide result in Single Stranded regions, mismatch Sites are Suscep 0837 (f) Selective Oligonucleotide Hybridization tible to digestion. Once cleaved, the nucleic acid fragments are separated according to Size by native polyacrylamide gel 0838. In another embodiment, selective hybridization electrophoresis. Genetic lesion are detected by, for instance, involves the use of Synthetic oligonucleotide primers pre observing different fragment sizes in test relative to wild pared to carry a known mutation in a central position. type DNA or RNA. Primers are then mixed with test DNA under conditions permitting hybridization for perfectly matched molecules 0831 Examples of such assay in practice are available in (Lipshutz 1995', Guo 1994'', Saiki 1989''). The allele Saleeba 1992, Takahashi 1990, Cotton 1988, Myers specific oligonucleotide (ASO) hybridization method can be 1985A'7, Myers 1985B. used to test a Single mutation per reaction mixture, or many 0832 (d) Mismatched Base Pairs Recognition different mutations if the ASO is first immobilized on a suitable membrane. The technique, termed “dot blotting.” 0833. In another exemplary embodiment, mismatch permits rapid Screening of many mutations when nonimmo cleavage reactions are carried out using one or more proteins bilized DNA is first radiolabeled to permit visualization of capable of recognizing mismatched base pairs. The proteins the immobilized hybrids. are typically components of the naturally occurring DNA mismatch repair mechanism. In a preferred embodiment, the 0839 (g) Allele Specific Amplification mutY enzyme derived from E. coli cleaves the adenine at a G/A mismatch (Xu 1996189). The enzyme thymidine DNA 0840 Under certain conditions, polymerase extension glycosylase, isolated from the human cell line HeLa, cleaves occurs only if there is a perfect match between primer and the thymidine at G/T mismatches (Hsu 1994.190). In prac the 3' terminus of the 5", left-most or upstream region of a tice, a probe is used comprising the wild-type Sequence of Sequence of interest. Therefore, in another embodiment, interest. The probe is hybridized to DNA, or cDNA corre allele specific amplification, a Selective PCR amplification sponding to mRNA of interest. Once duplex formation has based assay, a Synthetic oligonucleotide primer is prepared reached completion, a DNA mismatch repair enzyme is carrying a mutation at the center, or extreme 3' end of the added to the reaction, and the products of the cleavage are primer, such that mismatch between primer and test DNA detected by, for instance, Separating reactants by denaturing prevents, or reduces efficiency of the polymerase extension polyacrylamide gel electrophoresis. during amplification (Efremov 1991'7, Gibbs 1989''). A mutation in the test DNA is detected by a change in 0834) (e) Alterations in Electrophoretic Mobility amplification product concentration relative to controls, or, 0835. In another exemplary embodiment, variations in in Special cases, by the presence or absence of amplification electrophoretic mobility are used to identify genetic lesions, products. by Standard techniques, Such as Single Strand conformation 0841) A variation of the assay introduces a novel restric polymorphism (SSCP) (Miterski 2000', Jaeckel 1998', tion endonuclease recognition site in the expected mutation Cotton 1993, Ibid, Hayashi 1992'). Dilute preparations of region to permit detection by restriction endonuclease cleav radiolabeled single-stranded DNA fragments of test and age of the amplification products (See also above). control nucleic acids, Separately, are denatured by heat and permitted to renature Slowly. Upon renaturation, Single 0842 (h) Protein Truncation Test Stranded nucleic acids in the dilute Solutions form Secondary Structures. Each molecule forms internal base paired regions 0843. Another embodiment uses the protein truncation depending on each molecule Sequence. Consequently, wild test (PTT). If a mutation introduces a premature translation type and mutant Sequences, otherwise identical except for stop site, PTT offers an effective detection assay Geisler regions of mutation, form different Secondary Structures. 2001199, Moore 2000', van der Luijt 1994'', Roest Each preparation is separated in adjacent lanes by electro 1993'). In this assay, RNA is isolated from sample cells or phoresis through native polyacrylamide gels while preserv tissue and converted to cDNA by reverse transcriptase. The ing the Secondary Structure formed during renaturation. sequence of interest is amplified by the PCR, and the Alterations in electrophoretic mobility reveal differences products are Subjected to another round of amplification between wild-type and mutant oligonucleotides as Small as with a primer carrying a promoter for RNA polymerase, a Single nucleotide differences. Following electrophoresis the Sequence for translation initiation. The products of the second round of PCR are subjected to transcription and radiolabeled nucleic acids are detected by autoradiography translation in Vitro. Electrophoresis of the expressed or by phosphorimager analysis. A variation of this assay polypeptides through sodium dodecyl sulfate (SDS) con employs RNA rather than DNA. taining polyacrylamide gels reveals the presence of trun 0836. In a related exemplary embodiment, wild-type and cated Species arising from the presence of premature trans mutant DNA molecules are separated by electrophoresis lation Stop Sites. In a variation of this assay, if the Sequence through polyacrylamide gels containing a gradient of dena of interest is contained within a single exon, DNA rather turant. The method, termed “denaturing gradient gel elec than cDNA can used as PCR amplification template. US 2004/0023207 A1 Feb. 5, 2004 39

0844 (i) General Comments troblotting (Burnett 1981'). Prior to incubation with anti body, the membrane is incubated with blocking buffer 0845 Any tissue or cell type expressing a sequence of containing the nonionic detergent Tween 20 and nonfat dry interest may be used in the described assayS. For instance, milk as a Source of protein to later block adventitious bodily fluids, such as blood obtained by Venipuncture or binding of specific antibodies to the nylon membrane. The Saliva, or non-fluid Samples, Such as hair, or skin, may be immobilized proteins are then reacted with anti-phosphoty used. Samples of fetal polynucleotides collected from mater rosine antibodies and Visualized after reaction with a Sec nal blood, amniocytes derived from amniocentesis, or chori ondary antibody conjugated to horse radish peroxidase. onic Villi obtained for prenatal testing, can also be used. Exposure to hydrogen peroxide in presence of the chro 0846 Pre-packaged diagnostic kits containing one or mogenic indicator diaminobenzidine produces visible bands more nucleic acid probes, primer Set, and antibody reagent where Secondary antibodies are bound, thereby enabling may be useful in performing the assayS. Such kits are their localization. designed to provide an easy to use instrument especially 0857. A variation of this assay can be performed with Suitable for use in the clinic. antibodies directed against phosphothreonine (for instance, 0847 The assays may also be applied in situ directly on those available from Santa Cruz Biotechnology, catalog the tissue to be tested, fixed or frozen. Typically, Such tissue number sc-5267) or a host of phosphorylated molecules. is obtained in biopsies, or Surgical procedures. In Situ Sources of available phosphoprotein specific antibodies analysis precludes the need for nucleic acid purification. include, but are not limited to, Santa Cruz, Biotechnology of 0848 While the exemplary assays described so far pri Santa Cruz, California, Calbiochem of San Diego, Calif. and marily permit the analysis of one nucleic acid Sequence of Chemicon International, Inc. of Temecula, California. interest, they may be also used to generate a profile of 0858. The protein phosphorylation detection assays may multiple Sequences of interest. The profile may be generated, be employed before and/or after treatment with an agent of for example, by employing Northern blot analysis, a differ interest to detect changes in phosphorylation Status of a ential display procedure, or reverse transcriptase-PCR (RT polypeptide, or group of polypeptides. Moreover, detection PCR). of changes in phosphorylation Status of polypeptides of interest may be used to monitor efficacy of a therapeutic 0849. In addition to nucleic acid assays, antibodies directed against a mutated polynucleotide, or polypeptide treatment or progression of a chronic disease. product of a mutated polynucleotide may be used in various 0859) (b) Immunoprecipitation assays (see below). 0860. In one complementary embodiment, the relative 0850 (10) Assaying Methylation Status of DNA levels of phosphorylated and nonphosphorylated forms of any particular protein may be measured. The levels of the 0851) (a) Sodium Bisulfite Method phosphorylated forms are measured as described above. 0852. In one exemplary embodiment, the methylation Nonphosphorylated proteins are measured by first immuno Status of DNA sequences can be determined by first isolating precipitating all forms of the protein of interest with a cellular DNA, and then converting unmethylated cytosines Specific antibody directed toward that protein. The immune into uracil by treatment with Sodium bisulfite, leaving complexes are then analyzed by Western blotting as methylated cytosines unchanged. Following treatment, the described. Comparison of the levels of total protein of bisulfite is removed, and the chemically treated DNA is used interest to those of the phosphorylated forms provide Some as a template for PCR. Two parallel PCR reactions are insight into the relative levels of each form of the polypep performed for each DNA sample, one using primerS Specific tide of interest. for the DNA prior to bisulfite treatment, and one using primers for the chemically modified DNA. The amplifica 0861 (12) Assaying Gene Activation and Suppression tion products are resolved on native polyacrylamide gels and 0862 (a) Co-Transfection with Report Gene to Identify visualized by staining with ethidium bromide followed by Transactivators UV illumination. Amplification products detected from the Sodium bisulfite treated Samples indicate methylation of the 0863. In one exemplary embodiment, interactions original Sample. between regulatory proteins and a DNA sequence of interest can be revealed through co-transfection of two recombinant 0853 Specifically, this assay can be used to asses the vectors. The first vector carries a full length cDNA for the methylation status of DNA binding sites of a polypeptide of regulatory factor driven by a promoter known to be active in interest, such as GABP, p300, CBP, etc. the transfected cells. The Second recombinant vector carries a reporter gene driven by the DNA sequence of interest. 0854 (11) Assaying Protein Phosphorylation Examples of Suitable reporter genes include chlorampheni 0855) (a) Western Blot with Antiphosphotyrosine col acetyltransferase (CAT), luciferase or f3-galactosidase 0856. In one exemplary embodiment, protein phospho (Virts 2001?'"). Detection of reporter gene expression by rylation is measured using antiphosphotyrosine antibodies methods known in the art (see examples below) indicates (for instance, antibodies available from Santa Cruz Biotech transactivation of the DNA sequence of interest by the nology, catalog numbers sc-508 or sc-7020). Cultured cells regulatory factor. are lysed by boiling in detergent-containing buffer. Proteins 0864 Transfection of appropriate recombinant vectors contained in the cell lysate are separated by electrophoresis can be mediated either with calcium phosphate (Chen through SDS polyacrylamide gels followed by transfer to a 1988') or DEAE-dextran (Lopata 19849). In one exem nylon membrane by electrophoresis, a process termed elec plary embodiment, exponentially growing cells are exposed US 2004/0023207 A1 Feb. 5, 2004 40 to precipitated DNA. A DNA solution, prepared in 0.25M 0874) (b) Co-Transfection with Reporter Gene to Identify CaCl is added to an equal volume of HEPES buffered saline Trans-Acting Repressors and incubated briefly at room temperature. The mixture is 0875. These assays can be applied to assess trans-acting then placed over cells and incubated overnight to permit factors which potentially repress rather than Stimulate DNA adsorption and absorption into the cells. The next day reporter gene expression. In this embodiment, putative the cells are washed and cultured in complete growth repression factors are expressed from a recombinant vector medium. in cells which carry a reporter gene driven by a constitu tively active promoter which may interact with the repres 0865. In a related exemplary embodiment, calcium chlo Sion factor. The assays described above are applied to ride precipitation is replaced with DEAE-dextran as a carrier determine whether expression of the repression factor for the DNA to be transfected. Growth medium is made reduces reporter gene activity. 2.5% with respect to fetal bovine serum (FBS) and 10 uM with respect to chloroquine. The medium is prewarmed, and 0876 (13) Assaying Gene Expression Levels DNA is added prior to addition of DEAE-dextran. The mixture is then added to exponentially growing cells, and 0877) (a) Northern Blot Analyses incubated for 4 hours to allow DNA adsorption. The trans 0878. In one exemplary embodiment, the relative expres fection medium is replaced by a 10% solution of DMSO Sion levels of a gene of interest are measured by Northern causing the DNA to enter the cells. The cells are incubated blot analysis (Ausubel 1999, Ibid). RNA is isolated from for 2-10 hours. The DMSO solution is then replaced by untreated cells and cells after treatment with an agent growth medium, and the cells are incubated until assayed for expected to modulate gene expression. The RNA is sepa exogenous gene expression. rated by electrophoresis through a denaturing agarose gel, typically incorporating the denaturant formaldehyde, and 0866 CAT transferred to a nylon membrane. Immobilized RNA is hybridized to a radiolabeled DNA probe representing the 0867 Detection of CAT gene expression is achieved by gene of interest. Bound radiolabel is visualized by autora mixing lysates of the cells in which the reporter gene has diography. Levels of bound radiolabel can be quantified by been co-transfected along with a recombinant vector carry Scanning the resulting autoradiograph with a densitometer ing the putative activating factor with "C-labeled chloram and integrating the area under the traces. Alternatively, phenicol (Gorman 1982-7). incorporated radiolabel can be quantified by phosphorim ager analysis of the blot itself. 0868 Acetylated and unacetylated forms of the com pound, the latter resulting from enzymatic degradation of the 0879 (b) RT-PCR Substrate by expressed CAT, are separated by thin layer 0880. In a related embodiment, RNA is isolated from chromatography and Visualized by autoradiography. Quan similarly treated cells. The RNA is then subjected to reverse titation of each radiolabeled Species is attained by densito transcription (RT) and amplification by the polymerase metric analysis of the autoradiograph, or by direct phospho chain reaction (PCR) in the presence of radiolabeled deoxy rimager analysis of the chromatograph. nucleotides. The amplification products are resolved by gel electrophoresis and Visualized by autoradiography. Levels of 0869 Luciferase incorporated radiolabel can be quantified by Scanning the 0870 Detection of expressed luciferase is achieved by resulting autoradiograph with a densitometer and integrating exposure of transfected cell lysates to the luciferase Sub the area under the traces. Alternatively, incorporated radio Strate luciferin in presence of ATP, magnesium and molecu label can be quantified by phosphorimager analysis of the lar oxygen (Luo 2001'). The presence of luciferase results electrophoretic gel. in transient release of light detected by luminometer. 0881 (14) Assaying viral replication 0871 B-Galactosidase 0882) (a) Viral Titer 0872 Detection of B-galactosidase gene expression is 0883. In one exemplary embodiment, viral replication is achieved by mixing cell lysates with a chromogenic Sub measured by titration of infectious particles on cultured host Strate for the enzyme, Such as o-nitrophenyl-B-D-galacto cells. Virus replication is permitted in host cells, with or pyranoside (ONPG), or a chemiluminescent substrate con without chemical treatment, or with or without co-expres taining 1.2 dioxetane. Products of the catalytic degradation Sion of a regulatory gene, for a measured period of time. The of the chromogenic Substrate are easily visualized, or alter cells are lysed by exposure to a hypotonic Solution, and the natively, quantified by Spectrophotometry, while the prod lysates are Subjected to a Series of dilutions in isotonic ucts of the chemiluminescent Substrate are detected by buffer. Several concentrations of cell lysate are Separately luminometer. The latter assay is especially Sensitive and can plated onto cultured host cells. The culture cells are incu detect minute levels, or minute changes in levels of B-ga bated until the cytopathic effects (CPE) are evident. The lactosidase reporter gene expression. cultured cells are then fixed and Stained with a contrast enhancing dye, Such as crystal violet, to facilitate identifi 0873. These assays were applied to demonstrate binding cation of Viral plaques. Several culture plates are counted, of GABP to the promoter regions of a number of genes and the number of plaques multiplied by the appropriate including the retinoblastoma gene (Sowa 1997'), CD 18 dilution factor, representing the dilution from the original (Rosmarin 1998'), cytochrome C oxidase Vb (Sucharov cell lysate. The result reveals the viral titer of the original 1995') and the prolactin gene (Ouyang 1996') cell lysate. US 2004/0023207 A1 Feb. 5, 2004

O884) (b) In Situ PCR mixed with the Substrate 1-napthyldiphenylphosphine 0885. In a related exemplary embodiment, a latent, low (NDPP). Any resulting oxidized form of the substrate, copy number virus can be detected with the polymerase ONDPP, can be quantified by high performance liquid chain reaction in situ (Staskus 1994''). Cells grown either chromatography (HPLC). ONDPP concentration provides in Suspension culture or on a Solid Substrate are fixed and an indirect measure of the oxidation capacity of the cell permeabilized. PCR reaction components, including Syn lysate. thetic primers complementary to the gene of interest, Taq 0894 (b) H2DCFDA as Indicator polymerase, deoxyribonucleotides, are then added to the cells and subjected to thermal cycling typical of PCR. The 0895. In another exemplary embodiment, the production amplification products, retained in each cell, are detected by of cellular reactive oxygen Species can be detected by in situ hybridization with appropriately labeled DNA probes. mixing cell lysates with 2,7'-dichlorodihydrofleuoescein An exemplary detection method involves hybridization with diacetate (H2DCFDA) (Brubacher 2001'). In the presence radiolabeled probes followed by autoradiography. Similarly, of cellular esterases, H2DCFDA is deacetlyated to produce hybridization probes may be nonradioactively labeled by 2,7-dichlorodihydrofleuoescein (H2DCF), an oxidant-sen including digoxygenin-11-dUTP into the PCR reaction. Sitive indicator. Increased cellular oxidation excites the Incorporated label is detected either enzymatically or chemi fluorogenic indicator. Increased Sensitivity can be attained cally. by using H2DCF directly, but caution must be exercised by one skilled in the art to ensure that none of the experimental 0886 (15) Assaying Cell Morphology and Function buffers contain contaminants, Such as metals, which may 0887) (a) Light Microscopy lead to Spontaneous fluorescence. 0888. In one exemplary embodiment, the morphology of 0896) d) Optimization Protocols cells is ascertained by microScopic examination. Living and 0897. Once a single constructive or disruptive agent dead cells are distinguished by treating cells with the Stain (polynucleotide, polypeptide, Small molecule, etc.) is iden trypan blue (Schuurhuis 2001?'"). Living cells, with intact tified in the manner described above, variant agents can be cellular membranes, exclude trypan blue while dead cells, formulated that improve upon the original agent. with leaky, or perforated outer membranes, permit trypan blue to enter the cytoplasm. Following treatment, examina 0898 The expression “variant agents . . . that improve tion by phase contrast microScopy reveals the proportion of upon the original agent' is understood to include, but not be dead VS. living cells. Similarly, cellular morphology can be limited to, agents that increase therapeutic efficacy, increase ascertained by examination with phase contrast microscopy, prophylactic potential, increase, or decrease Stability in Vivo with or without prior Staining, with, for example, crystal or in Storage, or increase the number, or variety of post Violet, to enhance contrast. Such examination reveals mor translational modifications in Vivo, including, but not lim phologies common to known cell types, and concomitantly ited to, phosphorylation, acetylation and glycosylation, rela reveals irregularities present in the cell population under tive to the original agent. examination. 0899 Variant agents are not limited to those produced in 0889 (b) Functional Assessment by Immunocytochem the laboratory. They may include naturally occurring Vari istry ants. For example, variants with increased Stability, due to alterations in ubiquitination or modifications of other target 0890. In a related exemplary embodiment, the functional Sites conferring resistance to proteolytic degradation. Status of a given cell population may be determined by treatment with Specific antibodies. Cells are dehydrated and 0900 e) Treatment Protocols fixed with a Series of methanol washes using increasing concentrations of methanol. Once fixed, the cells are 0901) (1) Introduction exposed to cell-type specific antibodies. Examples of Suit 0902. According to the present invention, a polypeptide able antibodies include, but are not limited to, anti-filaggrin has a constructive effect if it attenuates microcompetition for epidermal cells, anti-CD4 for T cells, thymocytes and with a foreign polynucleotide or attenuates at least one effect monocytes, and anti-macrosialin for macrophages. After of microcompetition with a foreign polynucleotide, or one incubation with differentiation-specific marker antibodies, effect of another foreign polynucleotide-type disruption. For fluorescently labeled Secondary antibodies Specific for the example, a constructive polypeptide can reduce copy num first antibody are added. Bound Secondary antibodies are ber of the foreign polynucleotide, Stimulate expression of a Visualized by illumination with light of appropriate wave GABP regulated gene, increase bioactivity of a GABP length to excite the bound fluorochrome followed by micro regulated protein, through, for instance, GABP phosphory Scopic examination. The use of different antibodies, each lation and/or increase bioavailability of a GABP regulated conjugated to a different fluorochrome, permits the identi protein, through, for instance, a reduction in copy number of fication of multiple differentiation-Specific antigens Simul microcompeting foreign polynucleotides which bind GABP. taneously in the same population of cells. A constructive polypeptide can also, for example, inhibit expression of a microcompetition Suppressed gene, Such as, 0891 (16) Assaying Cellular Oxidation Stress tissue factor, androgen receptor, and/or inhibit replication of 0892) (a) Cellular Indicators a p300/cbp virus (see more examples below). 0893. In one exemplary embodiment, oxidation stress 0903 Agents of the present invention are designed to within a population of cells can be measured by assaying the address and ameliorate Symptoms of chronic diseases, Spe activity levels of certain indicatorS Such as lipid hydroper cifically, diseaseS resulting from microcompetition between oxides (Weyers 2001). Cell lysates are prepared and a foreign polynucleotide and cellular genes. For instance, US 2004/0023207 A1 Feb. 5, 2004 42 introduction of an oligonucleotide agent into a cell may unavoidable, they may be minimized by delivery of the disrupt this microcompetition and restore normal regulation therapeutic agent directly to target cells or tissues, thereby and expression of a microcompeted gene. Agents directed avoiding Systemic exposure. against a foreign polynucleotide may reduce binding or 0908. Those skilled in the art recognize that animal or cellular transcription factors to the foreign polynucleotide cell culture models are imperfect predictors of the efficacy of by, for instance, reducing the copy number of the foreign any treatment in humans. Factors affecting efficacy include polynucleotide, or its affinity to the transcription factor, route of administration, achievable Serum concentration and resulting in increased microavailability of the factors formulation of the therapeutic agent (i.e. in pill or injectable towards normal levels. Alternatively, binding of the tran forms, administered orally or intramuscularly, with accom Scription factors to cellular genes can be stimulated. In yet panying carrier, formulation of an agent adducted with a another exemplary embodiment, insufficient, or excessive Specific antibody and injected directly into the target tissue, expression of a cellular gene in a Subject can be modified by etc.). Regardless of the method of delivery or formulation of administration of nucleic acids or polypeptides to the Subject the therapeutic agent, it is important to monitor plasma that return the concentration of a cellular polypeptide of levels using a Suitable technique, Such as atomic absorption interest towards normal levels. Spectroscopy, enzyme linked immunosorbant assay (ELISA), or high performance liquid chromatography 0904. The following section describes standard protocols (HPLC) among others. for determining effective dose, and for agent formulation for use. Additional Standard protocols and background informa 0909 (3) Formulation for Use tion are available in books, Such as In vitro Toxicity Testing 0910 Those skilled in the art recognize a host of standard Protocols (Methods in Molecular Medicine, 43), edited by formulations for the agents described in this invention. Any Sheila O'Hare and C K Atterwill, Humana Press, 1995; suitable formulation may be prepared for delivery of the Current Protocols in Pharmacology, edited by: S J Enna, agent by injection, inhalation, transdermal diffusion or insuf Michael Williams, John W Ferkany, Terry Kenakin, Roger D flation. In every case, the formulation must be appropriate Porsolt, James P Sullivan; Current Protocols in Toxicology, for the means and route of administration. edited by: Mahin Maines (Editor-in-Chief), Lucio G Costa, Donald J Reed, Shigeru Sassa, I Glenn Sipes; Remington: 0911 Oligonucleotide agents, e.g. antisense oligonucle The Science and Practice of Pharmacy, edited by Alfonso R otides or recombinant expression vectors, may be formu Gennaro, 20" edition, Lippincott, Williams & Wilkins Pub lated for localized or Systemic administration. Systemic lishers, 2000; Pharmaceutical Dosage Forms and Drug administration may be achieved by injection in a physiologi Delivery Systems, by Howard C Ansel, Loyd V Allen, cally isotonic buffer including Ringer's or Hank's Solution, Nicholas G Popovich, 7" edition, Lippincott Williams & among others. Alternatively, the agent may be given orally Wilkins Publishers, 1999; Pharmaceutical Calculations, by by delivery in a tablet, capsule or liquid Syrup. Those skilled Mitchell J Stoklosa, Howard C Ansel, 10" edition, Lippin in the art recognize pharmaceutical binding agents and cott, Williams & Wilkins Publishers, 1996; Applied Biop carriers which protect the agent from degradation in the harmaceutics and Pharmacokinetics, by Leon Shargel, digestive System and facilitate uptake. Similarly, coatings Andrew B C Yu, 4" edition, McGraw-Hill Professional for the tablet or capsule may be used to ease ingestion Publishing, 1999; Oral Drug Absorption: Prediction and thereby encouraging patient compliance. If delivered in ASSessment (Drugs and the Pharmaceutical Sciences, Vol liquid Suspension, additives may be included which keep the 106), edited by Jennifer B Dressman, Hans Lennernas, agent Suspended, Such as Sorbitol Syrup and the emulsifying Marcel Dekker, 2000, Goodman & Gilman's The Pharma agent lecithin, among others, lipophilic additives may be cological Basis of Therapeutics, edited by Joel G Hardman, included, Such as oily esters, or preservatives may be used Lee E Limbird, 10" edition, McGraw-Hill Professional to increase shelf life of the agent. Patient compliance may be Publishing, 2001. See also above referenced. further enhanced by the addition of flavors, coloring agents or Sweeteners. In a related embodiment the agent may be 0905) (2) Effective Dose provided in lyophilized form for reconstitution by the patient 0906 Compounds can be administered to a subject, at a or his or her caregiver. therapeutically effective dose, to treat, ameliorate, or prevent 0912. The agents described herein may also be delivered a chronic disease. Carefull monitoring of patient Status, via buccal absorption in lozenge form or by inhalation via using either Systemic means, Standard clinical laboratory nasal aerosol. In the latter mode of administration any of assays or assayS Specifically designed to monitor the bioac Several propellants, including, but not limited to, trichlorof tivity of a foreign polynucleotide, is necessary to establish luoromethane and carbon dioxide, or delivery methods, the therapeutic dose and monitor its effectiveness. including but not limited to a nebulser, can be employed. Similarly, compounds may be included in the formulation 0907 Prior to patient administration, techniques standard which facilitate transepithelial uptake of the agent. These in the art are used with any agent described herein to include, among others, bile Salts and detergents. Alterna determine the LDso and EDso (lethal dose which kills one tively, the agents of this invention may be formulated for half the treated population, and effective dose in one half the delivery by rectal Suppository or retention enema. Those population, respectively) either in cultured cells or labora skilled in the art recognize suitable methods for delivery of tory animals. The ratio LDs/EDso represents the therapeutic controlled doses. indeX which indicates the ratio between toxic and therapeu tic effects. Compounds with a relatively large indeX are 0913) In related embodiments, the agents may be formu preferred. These values are also used to determine the initial lated for depot administration, Such as by implantation, Via therapeutic dose. While unwanted Side effects are Sometimes regulated pumps, either implanted or worn extracorporally US 2004/0023207 A1 Feb. 5, 2004 or by intramuscular injection. In these instances the agent with appropriately labeled probes may be performed in Situ may be formulated with hydrophobic materials, Such as an on isolated cells or tissues removed from the patient. Suit emulsification in a pharmaceutically permissible oil, bound able techniques are described, for example, Sambrook 2001 to ion eXchange resins or as a sparingly Soluble Salt. (ibid), incorporated herein in its entirety by reference. Con trol cells and tissues are compared in parallel to validate any 0914. In every case, therapeutic agents destined for positive findings in clinical Samples. administration outside of a clinical Setting may be packaged in any Suitable way that assures patient compliance with 0922) If the nucleic acid molecules specific to foreign regard to dose and frequency of admimistration. polynucleotides or disrupted genes, or genes in disrupted or disruptive pathways are in low concentration, preferred 0915 Administration of the agents included in this inven diagnostic methods employ Some means of amplification. tion in a clinical situation may be achieved by a number of Examples of suitable procedures include the PCR, ligase means including injection. This method of Systemic admin chain reaction, or any of a number of other Suitable methods istration may achieve cell-type specific targeting by using a well known in the art. nucleic acid agent, described herein, modified by addition of a polypeptide which binds to receptors on the target cell. 0923. In one exemplary embodiment of a diagnostic Additional specificity may be derived from the use of technique employing nucleic acid hybridization, RNA from recombinant expression vectors which carry cell- or tissue the cell of interest is isolated and converted to cDNA (using type specific promoters or other regulatory elements. In the enzyme reverse transcriptase of avian or murine origin). contrast to Systemic injection more Specific delivery may be Once cDNA is prepared, it is amplified by the PCR, or a achieved by means of a catheter, by Stereotactic injection, by Similar method, using a Sequence Specific oligonucleotide electorporation or by transdermal electrophoresis. Many primer of 20-30 nucleotides in length. Incorporation of Suitable delivery techniques are well known in the art. radiolabeled nucleotides during amplification facilitates detection following electrophoresis through native polyacry 0916. In an alternative embodiment the therapeutic agent lamide gels by autoradiography or phosphorimager analysis. may be administered by infection with a recombinant virus If Sufficient amplification products are attained, they may be carrying the agent. Similarly cells may be engineered eX Visualized by Staining of the electrophoretic gel by ethidium Vivo which express the agent. Those cells may themselves become the pharmaceutical agent for implantation into the bromide or a Similar compound well known in the art. Site of interest in the patient. 0924 (2) Detection and Quantification of Polypeptides 0917 f) Diagnosis Protocols 0925) Antibodies directed against foreign polypeptides, disrupted polypeptides, or polypeptides in disrupted or dis 0918 Diagnosis may be achieved by a number of meth ruptive pathways, may also be used for the diagnosis of ods, well known in the art, using as reagents Sequences of a chronic disease. Diagnostic protocols may be employed to foreign polynucleotide, disrupted gene or polypeptide, or a detect variations in the expression levels of polypeptides or gene or polypeptide in a disruptive or disrupted pathway, or RNA transcripts. Similarly, they may be used to detect antibodies directed against Such polynucleotides or polypep Structural variation including nucleic acid mutations and tides. Those reagents may be used to detect and quantify the changes in the Sequence of encoded polypeptides. The latter copy number, level of expression or persistence of expres may be detected by changes in electrophoretic mobility, Sion products of a foreign polynucleotide, disrupted gene or indicative of altered charge, or by changes in immunoreac gene Susceptible to microcompetition with a foreign poly tivity, indicative of alterations in antigenic determinants. nucleotide. 0926 For diagnositic purposes, protein may be isolated 0919 Diagnostic methods may employ any suitable tech from the cells or tissueS of interest using any of many nique well known in the art. These include, but are not techniques well known in the art. Exemplary protocols are limited to, commercially available diagnostic kits which are described in Molecular Cloning: A Laboratory Manual, 3rd Specific for one or more foreign polynucleotides, a specific Ed (Third Edition) By Joe Sambrook, Peter MacCallum and disrupted gene, a disrupted polypeptide, a gene or polypep David Russell (Cold Spring Harbor Laboratory Press 2001), tide in a disruptive or disrupted pathway, or an antibody incorporated herein by reference in its entirety. against Such polynucleotides or polypeptides. Well known advantages of commercial kits include convenience and 0927. In a preferred embodiment, detection of a foreign reproducibility due to manufacturing Standardization, qual polypeptide molecule, or a cellular disrupted polypeptide ity control and validation procedures. molecule, or a polypeptide in a disruptive or disrupted pathway is achieved with immunological methods, includ 0920 (1) Detection and Quantification of Polynucle ing immunoaffinity chromatography, radial immunoassays, otides radioimmunoassay, enzyme linked immunSorbant assay, etc. 0921. In one exemplary embodiment, nucleic acids, DNA These techniques, quantitative and qualitative, all well or RNA, are isolated from a cell or tissue of interest using known in the art, exploit the interaction between Specific procedures well known in the art. Once isolated, the pres antibodies and antigenic determinants on the target mol ence of a foreign polynucleotide may be ascertained by any ecule. In each assay, polyclonal or monoclonal antibodies, or of a number of procedures including, but not limited to, fragments thereof, may be used as appropriate. Southern blot hybridization, dot blotting and the PCR, 0928 Immunological assays may be employed to analyze among others. Mutations in those polynucleotides may be histological preparations. In a preferred embodiment, tissue detected by Single Strand conformation analysis, allele Spe or cells of interest are treated with a fluorescently labeled cific oligonucleotide hybridization and related and comple specific antibody or an unlabeled antibody followed by mentary techniques. Alternatively nucleic acid hybridization reaction with a Secondary fluorescently labeled antibody. US 2004/0023207 A1 Feb. 5, 2004 44

Following incubation for Sufficient time and under appro used to both diagnose disease and to guide and gauge priate conditions for antibody-antigen interaction, the label therapy. For example, in atherOSclerosis, Such disrupted may be visualized microscopically, in the case of either polypeptides may include, but are not limited to CD18 or tissueS or cells, or by flow cytometry, in the case of indi tissue factor (see more details in examples below). vidual cells. These techniques are particularly Suitable for antigens expressed on the cell Surface. If they are not on the 0935 Under these circumstances, antibodies, mono cell Surface, the cells or tissue to be analyzed must be treated clonal or polyclonal, which Specifically interact with pro to become permeable to the diagnostic antibodies. In addi teins expressed on the cell Surface may be used for the tion to the detection of antigens on the material Studied, the diagnosis of chronic disease and for monitoring treatment distribution of that antibody will become evident upon efficacy. In this embodiment, an appropriate antibody or microscopic examination. All immunological assays involve antibody fragment is labeled with a radioactive, fluorescent, the incubation of a biological Sample, cells or tissue, with an or other Suitable tag prior to reaction with the biomaterial to appropriately specific antibody or antibodies. These and be assayed. Conditions for reaction and Visualization are well known in the art and permit analyses to be carried out other Suitable diagnostic methods are familiar to those in vitro as well as in situ. In a preferred embodiment, skilled in the art. antibody fragments are used for in Situ or in vitro assays 0929. In an alternative embodiment, immunological tech because their Smaller Size leads to more rapid accumulation niques may be employed which involve either immobilized in the tissue of interest and more rapid clearing from that antibodies or immobilizing the cells to be analyzed on, for tissue following the assay. A number of Suitable and appro example, Synthetic beads or the Surface of a plastic dish, priate labels may be used for the assays in this invention that typically a microtiter plate (See above). are well known in the art. 0930 Immobilization of antibodies or cells to be ana 0936 g) Clinical Trials lyzed is achieved through the use of any of Several Substrates well known in the art including, but not limited to, glass, 0937. Another aspect of current invention involves moni dextran, nylon, cellulose, and polypropylene, among others. toring the effect of a compound on a treated Subject in a The actual shape or configuration of the Substrate may vary clinical trial. In Such a trial, the copy number of a foreign to Suite the desired assay. For example, polystyrene may be polynucleotide, its affinity to cellular transcription factors, formed into tissue culture or microtitre plates, dextran may the expression or bioactivity of a disrupted gene or polypep be formed into beads Suitable for column chromatography, tide, or expression or bioactivity of a gene or polypeptide in or polyacrylamide may be coated onto the inner Surface of a disrupted or disruptive pathway, may be used as an a glass test tube or bottle. These and related carriers and indicator of the compound effect on a disease state. configurations are well known and can be tested for utility 0938 For example, to study the effect of a test compound by those skilled in the art. in a clinical trial, blood may be collected from a Subject 0931) Detection of bound antibodies is achieved by label before, and at different times following treatment with such ing, either directly or indirectly, through the use of a a compound. The copy number of a foreign polynucleotide secondary antibody specific for the first. The label may be may be assayed in monocytes as described above, or the either a chromophore which responds to excitation by a levels of expression of a disrupted gene, Such as tissue Specific wavelength of light, thereby producing fluorescence factor, may be assayed by, for instance, Northern blot or it may be an enzyme which reacts with a chromogenic analysis, or RT-PCR, as described in this application, or by Substrate to produce detectable reaction products. Common measuring the concentration of the protein by one of the florescent labels include fluorescineisothiocyanate (FITC), methods described above. In this way, the copy number, or rhodamine and trans-1-bromo-2,5-bis-(3-hydroxycarbonyl expression profile of a gene of interest or its mRNA, may Serve a Surrogate or direct biomarker of treatment efficacy. 4-hydroxy)styrylbenzene (BSB), among others. Enzymes Accordingly, the response may be determined prior to, and commonly conjugated with antibodies include, but are not at various times following compound administration. The limited to, alkaline phosphatase, horse radish peroxidase and effects of any therapeutic agent of this invention may be B-galactosidase. Other alternatives are available and well Similarly Studied if, prior to the Study, a Suitable Surrogate or known in the art. direct biomarker of efficacy, which is readily assayable, was 0932. In a related embodiment, the antibody is labeled identified. with a fluorescent metal, for example Eu, which can be attached directly to the primary or Secondary antibody in an 0939 B. Examples immunoassay. Alternatively, the antibody may be labeled 0940. The current view holds that, in vivo, viral proteins with a chemiluminescent compound, Such as luminol, iso are the Sole mediators of viral effects on the host cell. Such luminol or imidazole or a bioluminiscent compount, Such as proteins include, for example, the papillomavirus type 16 E6 luciferin or aequorin. Subsequent reaction with the appro and E7 oncoproteins, SV40 large T antigen, Epstein-Barr priate Substrate for the labeling compound produces light virus BRLF1 protein, and adenovirus E1A. The possibility which is detectable visually or by fluorimetry. that presence of viral DNA in the host cell can directly impact cell function, independent of , is typi 0933 (3) Imaging of Diseased Tissues cally ignored. The viral “protein-dependent' view is so 0934 Under suitable circumstances, foreign polypep ingrained in current research that in many cases, when a tides, polypeptides expressed from disrupted genes, or from “protein-independent' effect on cellular gene expression, or genes in a disruptive or disrupted pathway, may be detected other cell functions, presents itself in the laboratory, the on the Surface of affected cells or tissues. In these instances effect is ignored. As a result, the significance of Such effect, the level and pattern of expression may be visualized and and Specifically, its relation to disease is overlooked. Note US 2004/0023207 A1 Feb. 5, 2004 that the effect of viral DNA on the cellular genome in cases 0947) (2) Mercola 1985 of viral DNA integration which may result in mutations, 0948. The plasmid pSV2CAT expresses the chloram deletions or methylation of host cell DNA, cannot be con phenicol acethyltransferase (CAT) gene under the control of sidered “protein-independent” since it is mediated by viral the SV40 promoter/enhancer. The pX1.0 plasmid contains proteins, Such as, HIV-1 IN protein, or . the murine immunoglobulin heavy-chain (Ig H) enhancer. The following examples illustrate the invention. More The pSV2neo expresses the neogene under the control of examples can be found in patent application PCT/US01/ the SV40 promoter/enhancer. The pa10neo and pSV2neo 05314, incorporated herein in its entirety by reference. are identical except that pA10neo lacks most of the SV40 enhancer. 0941 1. Foreign Polynucleotides and Aberrant Transcrip tion 0949. A study (Mercola 1985°) cotransfected a constant amount of pSV2CAT into murine plasmacytoma P3x63-Ag8 0942) a) Introduction cells, as test plasmid, with increasing amounts of pX1.0 as competitor plasmid. A plasmid lacking both reporter gene 0943 Microcompetition between a foreign polynucle and enhancer Sequences was added to produce equimolar otide and a cellular gene for a limiting cellular transcription amounts of plasmid DNA in the transfected cells. FIG. 1 complex result in aberrant transcription of the cellular gene. illustrates the observed relative CAT activity as a function of If the limiting complex Stimulates the gene transcription, microcompetition with the foreign polynucleotide reduces the relative concentration of the competitor plasmid (Mer transcription. If the limiting complex Suppresses the gene cola 1985, ibid, FIG. 4A). transcription, microcompetition with the the foreign poly 0950 An increase in concentration of the contransfected nucleotide increases transcription. Aberrant transcription murine immunoglobulin heavy-chain (H) enhancer can result in abberant gene expression, abnormal gene decreased expression from the plasmid carrying the SV40 product activity, and irregular cell function. Consider the viral enhancer. Microcompetition between viral and cellular following observations. heavy-chain enhancers decreased expression of the gene under control of the viral enhancer. Based on these obser 0944 b) Examples vations, Mercola, et al., (1985, ibid) concluded that in the plasmacytoma cells the heavy chain enhancer competes for 0945) (1) Scholer 1984 a trans-acting factor required for the SV40 enhancer func 0946 The plasmid pSV2CAT expresses the chloram tion. phenicol acethyltransferase (CAT) gene under the control of 0951. In another experiment, the Study cotransfected a the SV40 promoter/enhancer. A study (Scholer 1984'7) first constant amount of pSV2CAT, as test plasmid, with increas transfected an increasing amount of pSV2CAT in CV-1 ing amount of pSV2neo, as competitior plasmid, in either cells. CAT activity reached a plateau at 0.3 pmol pSV2CAT Ltk- or ML fibroblast cells. To isolate the effect of the viral DNA per dish. Based on this observation, the study con enhancer, the Study also cotransfected a constant amount of cluded that CV-1 cell contain a limited concentration of the test plasmid pSV2CAT with increasing amount of the cellular factor needed for pSV2CAT transcription. Next, the enhancerless pa10neo plasmid. FIG. 2 illustrates the study cotransfected a constant concentration of pSV2CAT observed relative CAT activity as a function of the relative with increasing concentrations of pSV2neo, a plasmid iden concentration of the competitor plasmid (Mercola 1985, tical to pSV2CAT, except the reporter gene is neomycin ibid, FIG. 4B). phosphotransferase (neo). The addition of pSV2neo resulted 0952) An increase in concentration of the contransfected in a linear decrease of the CAT signal (Scholer 1984, ibid, SV40 viral enhancer decreased expression from the plasmid FIG. 2B). Next, the study cotransfected pSV2CAT with a also carrying the SV40 enhancer. An increase in concentra plasmid that included all SV40 early control elements except tion of a plasmid lacking the enhancer did not affect the test for the 72-bp enhancer. No competition was observed. A plasmid reporter gene activity. point mutation in the 72-bp enhancer, which abolished the enhancer functional activity, also eliminated competition. 0953. Overall, the study concluded that “in vivo compe Based on these observations, Scholer, et al., (1984, ibid) tition experiments revealed the presence of a limited con concluded that “taken together, our data indicate that a centration of molecules that bind to the heavy-chain limited amount of the cellular factors required for the enhancer and are required for its activity. In the plasmacy function of the SV40 72-bp repeats is present in CV-1 cells. toma cell, transcription dependent on the SV40 enhancer Increasing the number of functional SV40 enhancer ele was also prevented with the heavy-chain enhancer as com ments Successfully competes for these factors, whereas other petitor, indicating that at least one common factor is utilized elements necessary for Stable transcription did not show by the heavy-chain and SV40 enhancers.” (3) Scholer 1986 such an effect.” The study also observed competition 0954) Another study (Scholer 1986°) cotransfected between pSV2CAT and pSrM2A, a plasmid which harbors CV-1 monkey kidney cells with a constant amount of a the Moloney murine sarcoma virus (MSV) enhancer (Scho plasmid containing the hMT-IIA promoter (-286 nt relative ler 1984, ibid, FIG. 5A and B). Note, that except the to the start of transcription to +75 nt) fused to the bacterial enhancers, the transcriptional control elements in pSV2CAT gene encoding chloramphenicol acetyltransferase (hMT and pSrM2A are same. Based on these observations, Scho IIA-CAT) along with increasing concentrations of a plasmid ler, et al., (1984, ibid) concluded that “one class of (a containing the viral SV40 early promoter and enhancer limiting) cellular factor(s) is required for the activity of fused to the bacterial gene conferring neomycin resistance different enhancers. Furthermore, BK (BK virus) and RSV (pSV2neo). FIG. 3 presents the observed relative CAT (Rous sarcoma virus) enhancers also interact with the same activity (expressed as the ratio between CAT activity in the class of molecule(s).” presence of pSV2neo and CAT activity in the absence of US 2004/0023207 A1 Feb. 5, 2004 46 pSV2neo) as a function of the molar ratio of pSV2Neo to early region of the SV40 genome expressing the SV40 large hMT-IIA-CAT. The figure illustrates the effect of competi tumor antigen and the SV40 Small tumor antigen. The cells tion between the two plasmids on the relative CAT activity. were also transfected with HSV-neo and pZIP-neo as A 2.4-fold molar excess of the plasmid containing the viral “empty” controls. Following transfection, the study cultured enhancer reduced CAT activity by 90%. No competition was the cells under differentiation inducing conditions, and mea observed with the viral plasmid after deletion of the SV40 Sured glycerophosphate dehydrogenase (GPD) activity as enhancer Suggesting that elements in the viral enhancer are marker of differentiation. The results are presented in the responsible for the observed reduction in reporter gene following table (Higgins 1996°, Table 1, first four lines). expression. 0955) (4) Cherington 1988 0956 pZIP-neo expresses the neomycin-resistant gene GPD activity under control of the Moloney murine leukemia virus long Vector Cell line (U/mg of protein) terminal repeat (LTR) (Cepko 1984). None L1 2,063 1599 HSV-neo L1-HNeo 1,519 1,133 0957 Another study inserted the wild-type early region ZIP-neo L1-ZNeo 1,155 1,123 of SV40 into the “empty” pZIP-Neo plasmid and labled the PVUO L1-PVUO 47, 25 new plasmid, which expressed the SV40 large T antigen, “wild-type” (WT). The study transfected 3T3-F442Apreadi pocytes with either WT or pZIPneo. Accumulation of trig 0965 Transfection of PVU0 and expression of the large lyceride, assayed by oil red Staining, was used as marker of and Small T antigens resulted in a Statistically significant differentiation. Seven days postconfluence, the number of decrease in GPD activity. Transfection of the “empty” Staining of cells was recorded. Consider the following figure vectors, HSV-neo and ZIP-neo, although less effective than (Cherington 1988', FIG. 4A, B and C). Darker staining PVU0, also reduced GPD activity. In a t-test, assuming indicates increased differentiation. (A) marks untreated unequal variances, the p-value for the difference between the F442A cells, (B) marks cells transfected with pZIP-neo, (C) HSV-neo vector and no vector is 0.118, and the p-value for marks cells transfected with WT. Consider FIG. 4. the difference between ZIP-neo and no vector is 0.103. Given that the sample includes only two observations, a 0958 Transfection with WT, the vector expressing the p-value around 10% for vectors carrying two different LTRs SV40 large T antigen, reduced differentiation. Compare indicates a trend. The observations demonstrate the effect of triglyceride Staining in (C) and (A). Transfection with the microcompetition with HSV-neo and Zip-Neo, the “empty “empty vector, although leSS So than transfection with the vectors,” on cell differentiation. WT vector, also reduced differentiation. Compare triglycer ide staining in (B) relative to (A) and (C). The results 0966 (7) Gordeladze 1997 demotstrate the effect of microcomopetition on cell differ 0967. The effect of microcompetition on hormone sen entiation. stivie lipase (HSL) transcription can be demonstrated by 0959 (5) Adam 1996 combining observations from two studies. Swiss mouse embryo 3T3-L1 fibroblasts can be induced to differentiate 0960 Another study (Adam 1996’) cotransfected into adipocyte-like cells. Undifferentiated cells contain very JEG-3 human choriocarcinoma cells with a constant con low level of HSL activity, while differentiated adipocyte-like centration of a plasmid carrying CAT reporter gene under the cells show much higher activity (a 19-fold increase relative control of the platelet derived growth factor-B (PDGF-B) to undifferentiated cells) (Kawamura 1981). 3T3-L1 promoter/enhancer (PDGF-B-CAT), and increasing concen preadipocytes were induced to differentiate by incubation trations of a Second plasmid containing either the human with insulin (10 ug/ml), dexamethasone (10 nM), or cytomegalovirus promoter/enhancer fused to B-galactosi iBuMexan (0.5 mM) for 8 consecutive days following cell dase (CMV-fgal), or the SV40 early promoter and enhancer confluency. HSL mRNA was measured in undifferentiated elements fused to Bgal (SV40-fgal). FIG. 5 present the confluent controls and differentiated 3T3-L1 cells trans observed relative CAT activity as a function of the molar fected with the pzipNeo vector. Although differentiated ratio between the plasmids carrying fgal and CAT (based on 3T3-L1 cells usually show significant HSL activity, the Adam 1996, ibid, FIG. 1). 3T3-L1 differentiated cells transfected with pZipNeo 0961. The results demonstrate the negative, concentra showed decreased HSL mRNA (Gordeladze 19977, FIG. tion-dependent effect of microcompetition between the 11. Compare pZipNeo and Wtype columns in FIG. 6). CMV promoter/enhancer, or SV40 promoter/enhancer, and pZipNeo carries the Moloney murine leukemia virus LTR the PDGF-B promoter. which microcompeted with HSL promoter. The results demostrate the effect of microcompetition with the viral LTR 0962 (6) Higgins 1996 on HSL transcription. 0963 HSV-neo is a plasmid that expresses the neomycin 0968 (8) Awazu 1998 resistance gene under control of the murine Harvey Sarcoma 0969 A study (Awazu 1998?) transfected HuH-7 virus long terminal repeat (LTR) (Armelin 1984°). pZIP human hepatoma cells with pBARB, a plasmid in which the neo expresses the neomycin-resistant gene under control of B-actin promoter regulates the expression of the Rb gene, the Moloney murine leukemia virus long terminal repeat and the Simian virus (SV40) promoter regulates the expres (LTR) (Cepko 1984, ibid). Sion of the neomycin-resistance (neo) gene. The study also 0964) A study (Higgins 1996’) transfected murine 3T3 transfected the cells with the pSV40-neo plasmid, which L1 preadipocytes with PVU0, a vector carrying an intact only includes the SV40 promoter and neo gene. Since US 2004/0023207 A1 Feb. 5, 2004 47 pSV40-neo does not include the O-actin promoter and Rb virus that infects bacteria). Second note that a decrease in the gene, the Study considered the pSV40-neo plasmid as Size of the insert, increases the copy number of the insert “empty” and used it as control. The cell were incubated in plasmid resulting in accentuated microcompetition with the IS-RPMI, with or without 5% FBS, and the number of viable test plasmid. cells were counted at the indicated times. FIG. 7 Summa 0975. The study also measured the effect of cotransfec rizes the results (Awazu 1998, ibid, FIG. 2A). The SD is tion on the activity of the androgen receptor (AR). The study about the size of the triangular Symbols. transfected COS-7 cells with 20 ng plRES-AR, pcDNA-AR 0970) The result demonstrate the effect of microcompe or pSG5-AR plasmids which express AR, 500 ng MMTV tition with pSV40-neo, the “empty vector” on cell prolif luc which highly expresses luc following AR Stimulation of eration. the MMTV promoter, and increasing amounts of the empty expression vector. The pGEM-7Zf(+) plasmid was used 0971 (9) Hofman 2000 instead of the expression plasmid to maintain a 650 ng final 0972. The pSG5 plasmid includes the early SV40 pro concentration of cotransfected DNA. Transfected cells were moter to facilitate in vivo expression, and the T7 bacterioph treated with 10 nM R1881, an AR ligand, and luciferase age promoter to facilitate in vitro transcription of cloned activity was measured. The luc activity in the presence of inserts. Both the pcDNA1.1 and pRESneo plasmids 650 ng pGEM-7Zf(+) was arbitrarily set to 1, and the includes the human cytomegalovirus (CMV) immediate relative luc activity was calculated. FIG. 8 presents the early (IE) promoter and enhancer. results (Hofman 2000, ibid, FIG. 5a). 0973) Another study (Hofman 2000’) constructed a 0976. According to Hofman, et al., (2000, ibid) “The Series of pSG5 based vectors by cloning certain Sequences MMTV-luciferase response was strongly reduced in the into the EcoRI restriction site (“insert plasmid,” see list in presence of increasing concentrations of the empty expres table below). The inserts varied in length measured in base Sion vector and the reduced receptor activities were propor pair (bp). The study cotransfected each insert plasmid (650 tional to AR expression levels.” The decrease in MMTV-luc ng) with pSG5-luc (20 ng), as test plasmid, in COS-7 cells. transcription resulted from decreased transcription of the AR The test plasmid pSG5-luc was also cotransfected with the gene expressed by the plRES-AR, pcDNA-AR, and pSG5 pGEM-7Zf(+) plasmid, or with herring sperm DNA. AR plasmids (see also Hofman 2000, ibid, FIG. 5b). Trans Luciferase (luc) activities were measured. Luc activity in fection with the calcium phosphate precipitation method, presence of the empty pSG5 vector was arbitrarily set to 1. TM instead of FuGENE-6TM, produced similar results. The following table presents the observed relative luc activ 0977 Finally, the study transiently cotransfected COS-7 ity in every experiment (Hofman 2000, ibid, FIG. 3a). cells with 20 ng pSG5-AR, 20 ng pS40-f-galactosidase (BGAL) and increasing amounts of the empty pSG5 vector. pGEM-7Zf(+) was used to maintain the DNA concentration Luc activity at a constant level. Luc and GAL activities in the presence Size of insert from pSG5-luc of 650 pGEM-7Zf(+) were arbitrarily set to 1, and the Plasmid (bp) (fold increase) relative luc activity was calculated. FIG. 9 present the pGEM7zf+ 72 results (Hofman 2000, ibid, FIG. 7a). herring 71 pSG5-NuRIP183 4,776 47 0978 Based on these observations, Hofman, et al., (2000, pSG5-TIF2 4,395 40 ibid) concluded that “The most likely explanation is that the pSG5-NuRIP183D1 4,326 36 total amount of transfected expression vectors largely pSG5-NuRIP183D2 3,723 33 exceeds the capacity of the transcriptional machinery of the pSG5-NuRIP183D3 3,219 3O pSG5-NuRIP183D4 2,684 28 cell. For that reason, competition occurs between the recep pSG5-NuRIP183D5 2,400 25 tor construct and the cotransfected construct.” pSG5-NuRIP183D6 1889 22 pSG5-ARA70 1,800 2O 0979 (10) Choi 2001 pSG5-TIF2.5 738 7 pSG5-DBI 259 3 0980) Another study (Choi 2001230) stably transfected pSG5 O 1. the human MM-derived cell line ARH with the pcDNA3 vector carrying an antisense to the macrophage inflamma tory protein 1C.. (MIP-1C) (AS-ARH). As control, the study 0974) Based on these observations Hofman, et al., (2000, transfected other ARH cells with the “empty' pcDNA3 ibid) concluded that “Remarkably, the measured luciferase vector (EV-ARH). To measure the effect of the antisense on activity tended to be inversely related to the length of the cell growth, the study cultured 105 non-transfected (wild insert in the cotransfected pSG5-constructs.” Moreover, type), empty vector, and MIP-1a antisenese (antisense) “We can conclude from these data that the SV40 promoter transfected ARH cells in six-well plates with RPMI-1640 driven expression of nuclear receptor or of luciferase in media containing 10% FBS. At days 3 and 5, the cells were COS-7 cells is inhibited to various degrees by cotransfec sampled, stained and counted. FIG. 10 present the results tion, with maximal inhibition in the presence of the empty (Choi 2001, ibid, FIG. 2a). After 5 days in culture, the expression vector and minimal inhibition in the presence of number of cell transfected with the empty vector was larger pSG5 constructs containing large inserts.' First note that the than the non-transfected cells. The Study also measured pGEM-7Zf(+) plasmid and the herring sperm DNA do not MIP-1C. expression in vivo. Wild type, empty vector trans include a human Viral promoter or enhancer. The promoters fected, and antisense transfected ARH cell were infused in pGEM-7Zf(+) is the bacteriophage SP6 and bacterioph intravenously into SCID mice (n=10 per group). The mice age T7 RNA polymerase promoters (a bacteriophage is a were Sacrificed when they became paraplegic. Femurs and US 2004/0023207 A1 Feb. 5, 2004 48 vertebrae were removed, and bone marrow plasma was posed of a mutated mouse factor VII (mfVII) targeting obtained. Expression of hMIP-1C. was measured with domain, and the Fc effector domain of an IgG1 Ig(mfVII/Fc ELISA kits. The following table summarizes the results icon), with the severe combined immunodeficient (SCID) according to data points in Choi 2001 (ibid) FIG. 3a. mouse model of human prostatic cancer. First, the Study injected the SCID mice s.c. in both rear flanks with the Wild type Empty vector P value human prostatic cancer line c4-2. Femur 193.33 591.2O O.042 0984. The injection resulted in skin tumors. Then, on Vertebrae 389.44 1031.25 O.O59 days 0,3,6,9,12,15,33,36.39, and 42, the study injected into Combined 291.39 786.78 O.O12 the skin tumor on one flank, either the pcDNA3.1(+) vector carrying the icon (four mice), or the empty vector (four 0981 Expression of hMIP-1C. in mice femur was signifi mice). The tumor on the other flank was left uninjected. The cantly higher after infusion with cells transfected with the Study measured tumor Volume in the injected and non empty vector relative mice infused with non-transfected injected flanks. FIG. 11 presents the results (Hu 2001, ibid, cells. In mice vertebrae, the expression of hMIP-1C. was FIG.3). O denotes tumors injected with the vector encoding borderline higher in mice infused with the cells transfected the icon, O-uninjected tumors in the icon treated mice, with the empty vector relative to mice infused with non A-tumors injected with the empty vector, A-uninjected transfected cells. The combined data from the femur and the tumors in the empty vector injected mice. vertebrae shows a Statistically significant effect of transfec 0985 The experiment was repeated with the human tion with the empty vector on MIP-1C. expression. melanoma line TF2 instead of the human prostatic cancer 0982) The pcDNA3 vector carries the cytomegalovirus line C4-2. The results are presented in FIG. 12 (Hu 2001, (CMV) promoter. The observations demonstrate the effect of ibid, FIG. 5) microcompetition with pcDNA3, the “empty” vector, on cell 0986. In both experiments, injection of the “empty vec proliferation and on MIP-1C. expression in vivo. Note that tor' Stimulated tumor growth. Compare tumors injected with the pcDNA3 vector carries the cytomegalovirus (CMV) empty vector (A) and uninjected tumors in the empty vector promoter. injected mice (A). (11) Hu 2001 0987) c) Summary 0983. Another study (Hu 2001") measured the efficacy 0988. The following table summarizes the studies above. and safety of an immunoconjugate (icon) molecule, com Promoter means promoter/enhancer.

Scholer Mercola Scholer Cherington Adam Higgins 1984 1985 1986 1988 1996 1996 Viral promoter effect Viral SV40 SV40 SV40 CMV on cotransfected promoter MSV SV40 (exogenous) gene BK expression/activity RSV Plasmid pSV2CAT pSVCAT pSV2neo pCMV pSV2neo fgal fgal Gene SV40 murine hMT-IIA PDGF-B MSV IgH

RSV Viral promoter effect Viral on cellular promoter (endogenous) gene expression/activity Plasmid Gene Viral promoter effect Viral MMTV HSV on cellular function promoter MMTV Plasmid pZIP-neo HSV-neo pZIP-neo Function Cell Cell Differen- Differen tiation tiation Study includes Yes Yes Yes No Yes No reference to effect of (1) (2) (3) (4) viral promoter (empty vector) Study includes No No No No No No reference to disease US 2004/0023207 A1 Feb. 5, 2004 49

-continued Gordeladze Awazu Hofman Choi Hu 1997 1998 2OOO 2OO1 2001

Viral promoter effect Viral MMTV SV40 on contransfected promoter CMV (exogenous) gene expression/activity Plasmid pZip-Neo pSG5 PRES pcDNA psV40 Gene HSL SV40 CMV Viral promoter effect Viral CMV on cellular promoter (endogenous) gene expression/activity Plasmid pcDNA3 Gene hMIP-1C. Viral promoter effect Viral SV40 CMV CMV on cellular function promoter Plasmid psV40-neo pcDNA3 pcDNA3 Function Cell Cell Tumor growth growth growth Study includes No No Yes No No reference to effect on (5) (6) (8) (9) viral promoter (empty vector) Study includes No No No No reference to disease

0989 References in the study to the effect of the viral the control of Viral enhancers: Initial calibration experiments promoter (empty vector): to determine the appropriate reference plasmid and the optimal relative molar concentration may be worthwhile in 0990 (1) Scholer 1984: The study measured and dis order to avoid erroneous interoperations of Such transfection cussed competition between different Viral enhancers in data.” The authors interpret the data in the narrow context of contransfected Studies. For instance, the Study reports com laboratory techniques, specifically, reference plasmids. No petition between the SV40 and MSV enhancers. The com relation is Suggested between microcompetition, endog petition between viral enhancers was also observed in Hof man 2000 (see above). The study includes no discussion enous gene expression, cellular function, or disease. relating the effect of Such competition with endogenous gene 0994) (5) Gordeladze 1997 (ibid): The only reference to expression, cellular function, and disease. the difference between pre-differentiated and post-differen tiated 3T3-L1 cells transfected with the pZipNeo “empty 0991) (2) Mercola 1985: The study measured and dis vector” is the following sentence: “However, post-differen cussed competition between SV40 enhancer and the cotrans tiated vector transfected cells exhibited a non-significant fected Ig H enhancer. The Study includes no discussion alteration compared to corresponding pre-differentiated relating the effect of Such competition with endogenous gene cells.” Note that, although the authors used the term “non expression, cellular function, and disease. Significant alteration, the paper reports no quantitative 0992 (3) Scholer 1986 (ibid): The study measured and analysis of the blot in FIG. 11, and specifically, no statistical discussed competition between SV40 enhancer and the analysis that can justify the use of the term “significant.” cotransfected hMT-IIA promoter. The study includes no Contrary to the authors conclusion, a Visual inspection of the discussion relating the effect of Such competition with blot in FIG. 11 shows a decline of HSL mRNA in the endogenous gene expression, cellular function, and disease. post-differentiated compared to the pre-differentiated cells transfected with the empty vector. 0993 (4) Adam 1996 (ibid): The study measured and discussed microcompetition between the CMV and SV40 0995 (6) Awazu 1998: The study does not compare promoter/enhancer and the cotransfected PDGF-B pro between nontransfected (HuH-7 wild) and empty vector moter/enhancer. Based on the observations, the Study con transfected (HuH-7 neo) cells. It is interesting that the study cluded that "Of more general interest, these results indicate called both the “Hu-7 wild and “Hu-7 neo’ the non that care should be exercised when using commonly avail transfected cells. In particular, the Study does not mention a able reporter gene constructs to Standardize transfection relation between microcompetition, endogenous gene efficiencies. It is possible that the importance of Some expression, cellular function, or disease. potential gene regulatory Sequences could be under esti 0996 (7) Hofman 2000 (ibid): Measured competition mated, or overlooked entirely, given certain combinations of between different viral enhancers in cotransfected experi reference plasmid co-transfection conditions and cell-types. ments. In the discussion the authors remark that “Whether Moreover, "The results we present here indicate a warning this competition occurs at the level of transcription initiation note for the use of co-transfected reference plasmids under or at a later Step is not clear.” Moreover, based on the US 2004/0023207 A1 Feb. 5, 2004 50 observations, the study concluded that “Moreover, it is treated MT-null mice showed dose-related nephromegaly, recommended to limit the amount of (co)transfected expres and following exposure, reduced renal function compared to Sion plasmid and to avoid the use of empty expression wild type (Qu 2002233). MT-I+II knock out (MTKO) mice plasmid as a control. Finally, one should be aware of Similar showed higher Susceptibility to autoimmune encephalomy misleading results in other experimental Set-ups base on elitis (EAE) compared to wild type (Penkowa 2001234), and cotransfection.” Similar to Adam 1996 (see above), the increased Susceptibility to the immunosuppresseive effects authors interpret the data in the narrow context of laboratory of ultraviolet B radiation and cis-urocanic acid (Reeve Set-ups, Specifically, the use of empty vectors as controls in 2000’). MT-I/II null mice also showed increased liver and cotransfection Studies. No relation is Suggested between the kidney damage following chronic exposure to inorganic observed microcompetition, endogenous gene expression, arsenicals (Liu 2000). cellular function, or disease. 1006 (2) PDGF-B Deficiency and Disease 0997 (8) Choi 2001: The study includes comparisons between antisense transfected cells and either empty vector 1007. In mice, a PDGF-B deficiency is embryonic lethal transfected cells or wild type cells as controls. The Study and is associated with cardiovascular, renal, placental and does not include a comparison between the empty vector hematological disorders. Specifically, mice show formation transfected and the wild type cells, that is, between the two of hemorrhage, microaneurysm, and microVessel leakage. “controls.” The mice also show lack of kidney glomerular mesangial cells and microvascular pericytes, and reduced or complete 0998 (9) Hu 2001: The study does not compare between loss of vascular smooth muscle cells (SMC) around small the tumors injected with the control (empty) vector and the and medium sized arteries. uninjected tumors in control mice. The only reference to the effect of the empty vector as reported in FIGS. 3 and 5 is 1008 The mice also show dilated heart and aorta, anemia the following sentence: “In mice injected with the control and thrombocytopenia (Kaminski 2001’, Lindahl vector, the tumors on both flanks grew continuously, and the 1997238). mice died or had to be euthanized by day 57.” 1009 (3) HSL Deficiency and Disease (Adipocyte 0999 Conclusion: these studies demonstrate the commit Hypertrophy) ment of the research community to the “protein-dependent' 1010 HSL knockout mice were generated by homolo paradigm. Each Study used two types of plasmids, one with gous recombination in embryonic Stem cells. Cholesterol a gene of interest, for instance, cellular Rb or viral Tantigen, ester hydrolase (NCEH) activities were completely absent an another with a reporter gene under control of a viral from both brown adipose tissue (BAT) and white adipose promoter/enhancer. The Second plasmid was considered tissue (WAT) in mice homozygous for the mutant HSL allele “empty,” and was, therefore, used as control. All Studies (HSL-/-). The cytoplasmic area of BAT adipocytes was above report observations which clearly show a significant increased 5-fold in HSL-/- mice (Osuga 2000239, FIG.3a) effect of the “empty' plasmid on gene expression, and the median cytoplasmic areas in WAT was enlarged progression, cell proliferation or cell differentiation. How 2-fold (Ibid, FIG. 3b). The HSL knockout mice showed ever, Some of these Studies include no reference to these adipocyte hypertrophy. HSL-deficient mice are normogly observation, the observations are completely ignored. More cemic and normoinsulinemic under basal conditions. How over, even the studies which discuss the effect of the empty ever, after overnight fast, the mice showed reduce concen vector, miss the relation between microcompetition and tration of circulating free fatty acids (FFAS) relative to disease. control and heterozygous mice. Moreover, an intraperitoneal 1000 2. Aberrant Transcription and Disease glucose tolerance test of the HSL-null mice revealed insulin resistance (Roduit 200120). HSL-deficient male mice are 1001) a) Introduction infertile (Chung 2001241). HSL-deficient mice also showed 1002. It is a well known fact that aberrant transcription, other defects associated with mobilization of triglycerides resulting from, for instance, a mutation or hypermethylation, (TG), diglycerides (DG) and cholesteryl esters (Haernmerle may result in disease. Consider, for instance, the Online 2002A, Haemmerle 2002B'). Mendelian Inheritance in Man (OMIMTM) database which 1011 c) Summary catalogs specific mutations and their association with genetic disorders. The following examples demonstrate the 1012 Microcompetition between a foreign polynucle effect of controlled mutation in three specific genes, MT, otide and a cellular gene for a limiting transcription complex PDGF-B, and HSL on the subject health. result in aberrant transcription of the cellular gene. Aberrant transcription results in disease. Therefore, microcompetition 1003 b) Examples between a foreign polynucleotide and a cellular gene for a 1004 (1) MT-I or MT-II Deficiency and Disease (Weight limiting transcription complex results in disease. When the Gain) foreign polynucleotide persists in the host cell for an extended period of time, microcompetition between the 1005 Mice with mutated MT-I and MT-II genes are foreign polynucleotide and the cellular gene results in a apparently phenotypically normal, despite reduced expres chronic disease. Sion of the metallothionein genes. The disruption shows no adverse effect on their ability to reproduce and rear off 1013 3. Limiting Transcription Factors Spring. However, after weaning, MT-null mice consume a) Examples more food and gain weight at a higher rate compared to 1014) controls. The majority of adult male mice in the MT-null 1015 The coactivator p300 is a 2,414-amino acid protein colony showed moderate obesity (Beattie 1998.232). Lead initially identified as a binding target of the E1A oncopro US 2004/0023207 A1 Feb. 5, 2004 tein. cbp is a 2,441-amino acid protein initially identified as Epstein-Barr virus (EBV) (20 copies of the N-box in the a transcriptional activator bound to phosphorylated cAMP +7421/+8042 oriP/enhancer) (Rawlins 19857) and Human response element (CREB) binding protein (hence, cbp). T-cell lymphotropic virus (HTLV) (8 N-boxes in the p300 and cbp share 91% sequence identity and are func enhancer (Mauclere 1995'7") and one N-box in the LTR tionally equivalent. Both p300 and cbp are members of a (Kornfeld 19877)). Note that some viral enhancers, for family of proteins collectively referred to as p300/cbp. example SV40, lack a precise N-box, but still bind the 1016 Although p300/cbp are widely expressed, their GABP transcription factor (Bannert 1999''). cellular availability is limited. Several studies demonstrated inhibited activation of certain transcription factors resulting 1021 Ample evidence exists Supporting binding of from competitive binding of p300/cbp to other cellular or GABP to the N-boxes in these viral enhancers. For instance, viral proteins. For example, competitive binding of p300 or Flory, et al., (1996'7") showed binding of GABP to the HIV CBP to the glucocorticoid receptor (GR), or retinoic acid LTR, Douville, et al., (19957) showed binding of GABP to receptor (RAR), inhibited activation of a promoter depen the promoter of ICP4 of HSV-1, Bruder, et al., (1991°7') and dent on the AP-1 transcription factor (Kamei 1996244). Bruder, et al., (198977) showed binding of GABP to the Competitive binding of cbp to STAT1C. inhibited activation adenovirus E1A enhancer element I, Ostapehuk, et al., of a promoter dependent on both the AP-1 and etS transcrip (1986278) showed binding of GABP (called EF-1A in their tion factors (Horvai 1997"). Competitive binding of p300 paper) to the polyomavirus enhancer and Gunther, et al., to STAT2 inhibited activation of a promoter dependent on (1994?7) showed binding of GABP to Mo-Mul V. Other the NF-kB RelA transcription factor (Hottiger 1998'). Studies demonstrate competition between the above viral Other studies also demonstrated limited availability of p300/ enhancers and enhancers of other viruses. Scholer and cbp, see, for instance, Pise-Masison 2001''", Banas 2001''", Gruss, (1984) showed competition between the Moloney Wang 2001?', Ernst 2001, Yuan 2001, Ghosh Sarcoma Virus (MSV) enhancer and SV40 enhancer and 2001’’, Li 2000253, Nagarajan 2000’, Speir 2000’, competition between the RSV enhancer and the BK virus Chen 2000’, and Werner 2000°7. enhancer. 1017 4. Transcription Factors Microcompeted by For eign Polynucleotides 1022 Other cellular transcription factors also form com plexes on viral DNA, and transactivate or Suppress viral 1018) a) Examples transcription. For instance, AML1 binds the polyomavirus 1019. One example of a foreign polynucleotide typically (Chen 1998?), Mo-MLV (Lewis 1999, Sun 1995), found in host cells is viral DNA. Several cellular transcrip and SL3 retrovirus (Martiney 1999A", Martiney tion factors form complexes on Viral DNA, and transactivate 1999B°), NF-AT binds HIV-1 (NFAT1 binds the NF-kB or Suppress viral transcription. Consider GAbinding protein site in the viral LTR) (Cron 2000), HNF4C. binds the (GABP), also called Nuclear Respiratory Factor 2 (NRF (Wang 1998.7), the Smad3/Smad4 com 2)’, E4 Transcription factor 1 (E4TF1), and Enhancer plex binds the Epstein-Barr virus (Liang 2000), ets1 Factor 1A(EF-1A)', as an example. The literature lists five binds the human cytomegalovirus (Chen 2000), NF-YB subunits of GABP: GABPC, GABPB1, GABPB2 (together binds the human cytomegalovirus (Huang 1994''), hepa called GABPB), GABPy1 and GABPy2 (together called titis B virus (Lu 1996', Bock 1999''), minute virus (Gu GABPy). GABPC. is an ets-related DNA-binding protein 1995), adenovirus (Song 1998'), and varicella-zoster which binds the DNA motif (A/C)GGA(A/T)(G/A), termed virus (Moriuchi 1995'), ATF-2 binds the human T-cell the N-box. GABPC forms a heterocomplex with GABPB leukemia type 1 (HTLV-1) (Xu 1996', Xu 1994?7), and which stimulates transcription efficiently both in vitro and in hepatitis B virus (Choi 1997'), p53 binds the polyomavi vivo. GABPC also forms a heterocomplex with GABPY, but rus (Py) (Kanda 1994''), human CMV (Allamane 20013, the heterodimer does not stimulate transcription. The degree Deb 2001'), human immunodeficiency virus type 1 (HIV of transactivation by GABP appears to correlate with the 1) (Deb 2001, ibid), and the Hepatitis B virus (Lee 1998', relative intracellular concentrations of GABPB and GABPy. Ori 1998), YY-1 binds the human papillomavirus type 18 An increase in GABPB relative to GABPY increases tran (HPV-18) (Jundt 1995'), NF-kB binds HIV (Hottiger scription, while an increase of GABPY relative to GABPB 1998, ibid), Stat2 binds HIV (Hottiger 1998, ibid), and decreases transcription. The degree of transactivation by C/EBPB binds the Hepatitis B virus (Lai 1999, Gilbert GABP is, therefore, a function of the ratio between GABPB 2000), and HIV-1 (LTR) (Honda 1998.7), and the glu and GABPY. Control of this ratio within the cell regulates cocorticoid receptor (GR) binds the mouse mammary tumor transcription of genes with binding sites for GABP (Suzuki virus LTR (Pfitzner 1998, ibid). 199826). 1023 Note that all the above mentioned transcription 1020. The N-box is the core binding sequence of many factors bind the limiting coactivator p300/cbp (Bannert Viral enhancers including the polyomavirus enhancer area 3 1999, Kitabayashi 1998, Garcia-Rodriguez 1998, (PEA3) (Asano 1990), adenovirus E1A enhancer Sisk 2000', Soutoglou 2000, Janknecht 1998, Feng (Higashino 1993), Rous Sarcoma Virus (RSV) enhancer 1998:14, Pouponnot 1998:15, Jayaraman 1999, Li (Laimins 1984), Herpes Simplex Virus 1 (HSV-1) (in the 19987, Duyndam 1999, Avantaggiati 1997' Van promoter of the immediate early gene ICP4) (LaMarco Order 1999, Hottiger 1998, Gerritsen 1997', Hotti 1989, Douville 1995°), Cytomegalovirus (CMV) (IE-1 ger 1998, ibid, Paulson 1999, ibid, Gringras 1999, ibid, enhancer/promoter region) (Boshart 1985), Moloney Bhattacharya 1996, Mink 1997, Pfitzner 1998, ibid). Murine Leukemia Virus (Mo-MuLV) enhancer (Gunther Since p300/cbp is limiting, a transcription complex that 1994), Human Immunodeficiency Virus (HIV) (the two includes p300/cbp is also limiting. For instance, since p300/ NF-KB binding motifs in the HIV LTR) (Flory 1996’), cbp is limiting, GABPp300/cbp is also limiting. US 2004/0023207 A1 Feb. 5, 2004 52

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protein transcription factor or by ectopically expressed responsive transcription from the Xenopus hsp70 pro steroidogenic factor 1.J. Virol. 2000 June;74(11):5032 moter in vivo. EMBO J. Nov. 2, 1998;17(21):6300-15. 9. 1337 Duyndam M. C. van Dam H. Smits P H, 1326 7 Honda Y. Rogers L, Nakata K, Zhao BY, Verlaan M. van der Eb AJ, Zantema A. The N-terminal Pine R, Nakai Y, Kurosu K, Rom WN, Weiden M. Type transactivation domain of ATF2 is a target for the 1 interferon induces inhibitory 16-kD CCAAT/en co-operative activation of the c-jun promoter by p300 hancer binding protein (C/EBP)beta, repressing the and 12S E1A. Oncogene. Apr. 8, 1999;18(14):2311-21. HIV-1 long terminal repeat in macrophages: pulmonary 1338 ' Avantaggiati M L, Ogryzko V, Gardner K, tuberculosis alters C/EBP expression, enhancing HIV-1 Giordano A, Levine A S, Kelly K. Recruitment of replication. J Exp Med. Oct. 5, 1998;188(7): 1255-65. p300/CBP in p53-dependent signal pathways. 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Jun. 1, 1998;17(11):3124 of activated T cells (NFAT)-dependent transactivation 34. regulated by the coactivators p300/CREB-binding pro 1341 ° Gerritsen M E, Williams A J, Neish A S, tein (CBP). J Exp Med. Jun. 15, 1998;187(12):2031-6. Moore S, Shi Y, Collins T. CREB-binding protein/p300 1330) '' Sisk TJ, Gourley T. Roys S, Chang C H. are transcriptional coactivators of p65. Proc Natl Acad MHC class II transactivator inhibits IL-4 gene tran Sci USA. Apr. 1, 1997:94(7):2927-32. scription by competing with NF-AT to bind the coac 1342) 'Bhattacharya S, Eckner R, Grossman S, Old tivator CREB binding protein (CBP)/p300. J. Immunol. read E, Arany Z, D'Andrea A, Livingston D M. Coop Sep. 1, 2000; 165(5):25.11-7. eration of Stat2 and p300/CBP in signalling induced by 1331 ° Soutoglou E, Papafotiou G, Katrakili N, interferon-alpha. Nature. Sep. 26, Talianidis I. Transcriptional activation by hepatocyte 1996:383(6598):344-7. nuclear factor-1 requires Synergism between multiple 1343 '' Mink S, Haenig B, Klempnauer K H. Inter coactivator proteins. J Biol. Chem. Apr. 28, action and functional collaboration of p300 and C/EB 2000:275(17): 12515-20. Pbeta. Mol Cell Biol. 1997 November; 17(11):6609-17. 1332 Janknecht R, Wells NJ, Hunter T. TGF-beta We claim: Stimulated cooperation of Smad proteins with the coac 1. A method for evaluating the effectiveness of a com tivators CBP/p300. Genes Dev. Jul. 15, pound for use in modulating the progression of a chronic 1998; 12(14):2114-9. disease, the method comprising the Steps of 1333 '' Feng XH, ZhangY, Wu RY, Derynck R. The (a) Selecting a cell; tumor suppressor Smad4/DPC4 and transcriptional (b) introducing a polynucleotide foreign to said cell into adaptor CBP/p300 are coactivators for Smad3 in TGF Said cell; beta-induced transcriptional activation. Genes Dev. Jul. (c) incubating said cell in the presence and absence of said 15, 1998; 12(14):2153-63. compound; 1334) Pouponnot C, Jayaraman L, Massague J. Physical and functional interaction of SMADs and (d) assaying microcompetition between said foreign poly p300/CBP. J. Biol. Chem. Sep. 4, 1998:273(36):22865 nucleotide and a polynucleotide natural to Said cell; 8. (e) Selecting a compound that can modify microcompe 1335) Jayaraman G, Srinivas R, Duggan C, Ferreira tition between Said foreign polynucleotide and Said E, Swaminathan S, Somasundaram K, Williams J, polynucleotide natural to Said cell. Hauser C, Kurkinen M, Dhar R, Weitzman S, Buttice 2. The method of claim 1, wherein Said compound is G, Thimmapaya B. p300/cAMP-responsive element foreign to Said both organisms. 3. The method of claim 1, wherein said compound is a binding protein interactions with etS-1 and etS-2 in the Synthetic compound. transcriptional activation of the human Stromelysin 4. The method of claim 1, wherein Said introducing a promoter. J Biol. Chem. Jun. 11, 1999;274(24):17342 foreign polynucleotide into a cell is by changing the copy 52. number of a polynucleotide in a cell. 1336 7 Li Q, Herrler M, Landsberger N, Kaludov N., 5. The method of claim 1, wherein said introducing a Ogryzko V V, Nakatani Y, Wolffe AP. Xenopus NF-Y foreign polynucleotide into a cell is by transfecting a cell pre-Sets chromatin to potentiate p300 and acetylation with a foreign polynucleotide. US 2004/0023207 A1 Feb. 5, 2004

6. The method of claim 1, wherein Said introducing a (b) introducing said polynucleotide into a cell of Said foreign polynucleotide into a cell is by infecting a cell with Second organism: a virus, active or deactivated. 7. The method of claim 1, wherein said introducing a (c) incubating said cell in the presence and absence of said foreign polynucleotide into a cell is by chemically modify compound; ing a polynucleotide in the cell. (c) assaying microcompetition between said foreign poly 8. The method of claim 1, wherein said introducing a nucleotide and a polynucleotide natural to Said cell; foreign polynucleotide into a cell is by mutating a poly (d) Selecting a compound that can modify microcompe nucleotide in the cell. tition between Said foreign polynucleotide and Said 9. The method of claim 1, wherein said introducing a polynucleotide natural to Said cell. foreign polynucleotide into a cell is by modifying the 24. The method of claim 23, wherein said compound is binding affinity of avidity of a polynucleotide in the cell. foreign to Said both organisms. 10. The method of claim 1, wherein said cell is an animal 25. The method of claim 23, wherein said compound is a or human cell. Synthetic compound. 11. The method of claim 1, wherein said foreign poly 26. The method of claim 23, wherein said introducing said nucleotide is a viral polynucleotide. polynucleotide into a cell is by changing the copy number of 12. The method of claim 1, wherein said foreign poly a polynucleotide in a cell. nucleotide is a viral promoter. 27. The method of claim 23, wherein said introducing said 13. The method of claim 1, wherein said foreign poly polynucleotide into a cell is by transfecting a cell with a nucleotide is a viral enhancer. foreign polynucleotide. 14. The method of claim 1, wherein Said assaying micro 28. The method of claim 23, wherein said introducing said competition is assaying formation of a complex between a polynucleotide into a cell is by infecting a cell with a virus, transcription factor natural to Said cell and Said foreign active or deactivated. polynucleotide. 29. The method of claim 23, wherein said introducing said 15. The method of claim 1, wherein Said assaying micro polynucleotide into a cell is by chemically modifying a competition is assaying expression of a gene, or gene polynucleotide in the cell. fragment, where Said gene is under the control of Said 30. The method of claim 23, wherein said introducing said foreign polynucleotide. polynucleotide into a cell is by mutating a polynucleotide in 16. The method of claim 1, wherein Said assaying micro the cell. competition is assaying activity of a gene product of a gene, 31. The method of claim 23, wherein said introducing said or gene fragment, where said gene is under the control of polynucleotide into a cell is by modifying the binding Said foreign polynucleotide. affinity of avidity of a polynucleotide in the cell. 17. The method of claim 1, wherein Said assaying micro 32. The method of claim 23, wherein said cell is an animal competition is assaying formation of a complex between a or human cell. transcription factor natural to Said cell and Said polynucle 33. The method of claim 23, wherein said foreign poly otide natural to Said cell. nucleotide is a viral polynucleotide. 18. The method of claim 1, wherein Said assaying micro 34. The method of claim 23, wherein said foreign poly competition is assaying expression of a gene, or gene nucleotide is a viral promoter. fragment, where Said gene is under the control of Said 35. The method of claim 23, wherein said foreign poly natural polynucleotide. nucleotide is a viral enhancer. 19. The method of claim 1, wherein said assaying micro 36. The method of claim 23, wherein said assaying competition is assaying activity of a gene product of a gene, microcompetition is assaying formation of a complex or gene fragment, where said gene is under the control of between a transcription factor natural to Said cell and Said Said polynucleotide natural to Said cell. foreign polynucleotide. 20. The method of claim 1, wherein Said assaying micro 37. The method of claim 23, wherein said assaying competition is assaying copy number of Said foreign poly microcompetition is assaying expression of a gene, or gene nucleotide. fragment, where Said gene is under the control of Said 21. The method of claim 1 wherein said evaluating the foreign polynucleotide. effectiveness of a compound for use in modulating the 38. The method of claim 23, wherein said assaying progression of a chronic disease is evaluating the effective microcompetition is assaying activity of a gene product of a neSS of a compound for use in Stimulating or inhibiting the gene, or gene fragment, where said gene is under the control progression of a chronic disease. of Said foreign polynucleotide. 22. The method of claim 1, wherein Said Selecting a 39. The method of claim 23, wherein said assaying compound that can modify microcompetition is Selecting a microcompetition is assaying formation of a complex between a transcription factor natural to Said cell and Said compound that can decrease or increase microcompetition. polynucleotide natural to Said cell. 23. A method for evaluating the effectiveness of a com 40. The method of claim 23, wherein said assaying pound for use in modulating the progression of a chronic microcompetition is assaying expression of a gene, or gene disease, the method comprising the Steps of fragment, where Said gene is under the control of Said (a) Selecting a polynucleotide where said polynucleotide polynucleotide natural to Said cell. is natural to a certain first organism; and where Said 41. The method of claim 23, wherein said assaying polynucleotide is empty in respect to Said first organ microcompetition is assaying activity of a gene product of a ism; and where Said polynucleotide is foreign to a gene, or gene fragment, where said gene is under the control Second organism; of Said polynucleotide natural to Said cell. US 2004/0023207 A1 Feb. 5, 2004

42. The method of claim 23, wherein said assaying 45. A method for monitoring the efficacy of a compound microcompetition is assaying copy number of Said foreign (or other treatments Such as irradiation) in clinical trials for polynucleotide. the treatment of a chronic disease in an animal or human Subject, the method comprising assaying the effect of Said 43. The method of claim 23, wherein said evaluating the compound on microcompetition between a polynucleotide effectiveness of a compound for use in modulating the natural to Said Subject and a polynucleotide foreign to Said progression of a chronic disease is evaluating the effective Subject. neSS of a compound for use in Stimulating or inhibiting the 46. The method of claim 45, wherein said assay is carried progression of a chronic disease. out in a chemical mix. 44. The method of claim 23, wherein said selecting a 47. The method of claim 45, wherein said assay is carried compound that can modify microcompetition is Selecting a out in a cell. compound that can decrease or increase microcompetition.