WO2002070744A2 - Test de criblage de banque adénovirale destiné à des procédés et à des gènes régulateurs e2f, et compositions destinées au criblage de composés - Google Patents

Test de criblage de banque adénovirale destiné à des procédés et à des gènes régulateurs e2f, et compositions destinées au criblage de composés Download PDF

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WO2002070744A2
WO2002070744A2 PCT/EP2002/002511 EP0202511W WO02070744A2 WO 2002070744 A2 WO2002070744 A2 WO 2002070744A2 EP 0202511 W EP0202511 W EP 0202511W WO 02070744 A2 WO02070744 A2 WO 02070744A2
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ceus
ceu
host
nucleic acid
adenovhal
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WO2002070744A3 (fr
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Helmuth Hendrikus Gerardus Van Es
Godefridus Augustinus Maria Michiels
Peter Herwig Maria Tomme
Reginald Christophe Xavier Brys
René Bernards
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Galapagos Genomics B.V.
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Publication of WO2002070744A3 publication Critical patent/WO2002070744A3/fr

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    • C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
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    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • G—PHYSICS
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    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
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    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
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    • C12N2710/00011—Details
    • C12N2710/10011—Adenoviridae
    • C12N2710/10311—Mastadenovirus, e.g. human or simian adenoviruses
    • C12N2710/10341—Use of virus, viral particle or viral elements as a vector
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    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • the invention relates to high throughput methods for identifying the function of sample nucleic acids and their products.
  • the ultimate goal of the Human Genome Project is to sequence the entire human genome.
  • the expected outcome of this effort is a precise map of the 70,000- 100,000 genes that are expressed in man.
  • ESTs Expressed Sequence Tags
  • cDNA complementary DNA
  • cDNAs are believed to represent approximately 50,000 different human genes expressed in a variety of tissues including liver, brain, spleen, B-cells, kidney, muscle, heart, alimentary tract, retina, and hypothalamus, and the number is growing daily.
  • Recent initiatives like that of the Cancer Genome Anatomy project support an effort to obtain full-length sequences of clones in the Unigene set (a set of cDNA clones that is publicly available).
  • commercial entities propose to validate 40,000 full-length cDNA clones. These individual clones will then be available to any interested party. The speed by which the coding sequences of novel genes are identified is in sharp contrast to the rate by which the function of these genes is elucidated. Assigning functions to the cDNAs in the databases, or functional genomics, is a major challenge in biotechnology today.
  • Gene chip technology has the potential to allow the monitoring of gene expression through the measurement of mRNA expression levels in cells of a large number of genes in only a few hours.
  • CeUs cultured under a variety of conditions can be analyzed for their mRNA expression patterns and compared to provide insight into their function and relationship to disease states.
  • pRb tumor-suppressor pathway One of the hallmarks of many disease states is the deregulation of the pRb tumor-suppressor pathway, either by mutation of pRb, its upstream regulator p 1 ⁇ 4 ", or by over expression of cyclin D, which associates with cyclin-dependent kinases (Cdks) that phosphorylate and thereby inactivate pRb (Weinberg, (1995) Cell 81:323- 30).
  • Cdks cyclin-dependent kinases
  • deregulation of the pRb pathway also underlies other human proliferative disorders such as the vascular disorders atherosclerosis and restenosis (Dzau, et al. (1996) Proc. Natl. Acad.
  • E2F transcription factors act as a potent oncogene in transformation assays (Johnson, et al. (1994) Proc. Natl. Acad. Sci.
  • E2F In addition to its effect on proliferation, E2F also plays a critical role in the regulation of apoptosis. E2F-1 deficient mice develop a broad spectrum of tumors, suggesting that E2F may act as either an oncogene or a tumor suppressor, depending on the context in which activity is analysed (Yamasaki, et al. (1996) Cell 85:537-48). Increase of E2F expression following DNA damage also provides evidence that E2F can induce growth arrest and apoptosis (Sears and Nevins, (2002) J. Biol. Chem. in press; Blattner, et al. (1999) Mol. Cell. Biol. 19:3704-13). As proliferation and apoptosis are antagonistic processes, both activation and inhibition of E2F can result in either tumorigenesis or apoptosis, depending on the cellular context.
  • E2F transcription factors are heterodimers containing a subunit encoded by the E2F gene family and a subunit encoded by the DP family of genes.
  • E2F-1 through 6 six E2F genes (E2F-1 through 6) and two DP genes (DP-1 and DP-2) have been found in mammahan cells.
  • E2F and DP proteins contain highly conserved DNA-binding and dimerization domains (Helin, (1998) Curr. Opin. Genet. Dev. 8:28-35).
  • the carboxy- terminal portion of E2F1-5 contains a potent transactivation domain, but no equivalent activity has been found in E2F-6 or in DP proteins.
  • E2F heterodimers are regulated by interactions with members of the retinoblastoma gene family (pRb, pl07 and pl30).
  • E2F1-3/DP complexes bind to pRb
  • E2F-4/DP heterodimers interact withpRb and pi 07
  • E2F-5 is preferentially bound by pi 30.
  • the association of E2Fs withpRb family members as well as their relative importance varies with specific stages of the cell cycle (Dyson, (1998) Genes Dev. 12:2245-62).
  • pl30/E2F complexes are primarily found in quiescent or differentiated cells and pl07/E2F complexes are most prevalent in S phase cells.
  • pRb/E2F complexes can be found in quiescent or differentiated cells, but are most evident as cells progress from Gl into S phase.
  • the progression through the mammalian cell cycle is cooperatively regulated by several classes of cyclin- dependent kinases (Cdks) and their regulatory subunits: the cyclins (reviewed in Sherr, (1994) Cell 79:551-5).
  • the cyclins display a sequential appearance as cells move from quiescence (GO) into the first gap phase (Gl), through initiation of DNA synthesis (S), and via the second gap phase (G2) to mitosis (M).
  • Cdk4 The activity of Cdk complexes depends on their expression levels, association with cyclins, phosphorylation status and the association with specific Cdk-inhibitors (CKIs).
  • the CKIs can be divided into two classes based on their structures and targets.
  • the first class involves the INK4a family including pl ⁇ 11 ⁇ 43 , plSTM** pi 8 ⁇ and pl Q TM ⁇ that act as inhibitors of D-type cyclins by inhibiting their catalytic partners: Cdk4 and
  • the second class consists of the Cip/Kip proteins p21 cipl , p27 Kipl and p57 ⁇ ip2 whose actions regulate cyclin D-, cyclin E- and A-dependent kinases, by binding to both the cyclin and Cdk subunits (Harper, et al. (1993) Cell 75:805-16; Polyak, et al. (1994) Genes Dev. 8:9-22).
  • cyclin D-dependent kinases When quiescent cells enter the ceh cycle, activated cyclin D-dependent kinases trigger the phosphorylation of the retinoblastoma tumor-suppressor protein Rb, and the related family members pi 07 and pl30 (Beijersbergen and Bernards, (1996) Biochim. Biophys. Ada 1287:103-20; Xiao, et al. (1996) Proc. Natl. Acad. Sci. USA 93:4633-7). Once pRb is primed with phosphates, Rb is further phosphorylated by cyclin E/Cdk2 complexes in late Gl phase (Lundberg and Weinberg, (1998) Mol. Cell. Biol. 18:753-61).
  • the phosphorylation of the Rb family members results in the release and activation of the E2F/DP transcription factors, which play a central role in the control of cell proliferation.
  • Inactivation of Rb, and subsequent activation of the E2F transcription factors at the Gl/S boundary irreversibly commits the cehs to complete the mitotic cycle (See FIG. 45 for schematic representation of Gl to S transition in the mammahan cell cycle).
  • E2F complexes bind to specific binding sites in the promoter regions of a number of cellular genes involved in D ⁇ A synthesis and regulation of the cell cycle, including D ⁇ A polymerase- , dhfr, thymidine kinase, MCM genes, orcl, cdk2, cdc2, cdc6, cyclin A, cyclin E, c-myc and b- yb (reviewed in Muller and Helin, (2000) Biochim. Biophys. Ada 1470:M1-12).
  • E2F complexes There appear to be three generic types of E2F complexes: activator E2F complexes, in which the E2F activation domain promotes transcription; inhibited E2F complexes, in which the activation domain is masked by pRb-family proteins to give a complex that is essentially inert; and repressor E2F complexes, in which Rb-family proteins that are recruited to the DNA by E2F, assemble a repressor activity.
  • the activation of E2F target genes may either result from transcriptional activation or loss of active repression on the promoter regions. As noted above, depending on the cellular context, this activation of E2F target genes can result in either proliferation or apoptosis.
  • E2F-mediated transcriptional activation remains unresolved. Possibly, E2F can regulate transcription via the recruitment of either TBP or CBP to E2F regulated promoters (Hagemeier, et al. (1993) Nucleic Acids Res. 21:4998-5004; Trouche, et al. (1996) Nucleic Acids Res. 24:4139-45). Also, although the Rb/E2F-mediated repression mechanism is unclear, a putative role for both HDACs and the SWI/SNF nucleosome-remodeling complexes in this mechanism has been suggested (Luo, et al. (1998) CeU 92:463-73; Trouche, et al. (1991) Proc. Natl. Acad. Sci. USA 94:11268-73). Thus, E2F binding sites serve to repress as well as to activate cellular promoters, depending on the nature of the E2F complexes found in the ceh.
  • adeno vhal mediated gene transfer of the retinoblastoma family proteins in a rat carotid artery model demonstrated that the inhibition of E2F activity resulted in reduced smooth muscle cell proliferation and prevented restenosis after angioplasty (Claudio, et al. (1999) Circ. Res. 85: 1032-9). Also, it was shown with in vivo adenoviral gene therapy that directed over expression of the pi 6 gene efficiently inhibited the pathology in an animal model of rheumatoid arthritis (Taniguchi, et al. (1999) Nat Med 5:760-7).
  • ex-vivo gene therapy of human bypass grafts with E2F decoy oligodeoxynucleotides demonstrated that inhibition of E2F-mediated cell proliferation in these vein grafts lowered the failure rates of human primary bypass vein grafting (Mann, et al. (1999) Lancet 354:1493-8).
  • therapeutic strategies may also take advantage of E2F-mediated cell death pathways. For instance, E2F can induce expression of pl9 AEP (DeGregori, et al. (1997) Proc. Natl. Acad. Sci.
  • E2F is also a substrate for the kinase ATM, which is activated by DNA damage (Lin, et al. (2001) Genes Dev. 15: 1833-45). Phosphorylation of E2F blocks proteosome-mediated degradation of E2F, thus increasing E2F levels in the ceh In addition, phosphorylation of E2F itself may disrupt pRb/E2F complexes (Fagan, et al.
  • Yeast expression systems have been developed which are used to screen for naturally secreted and membrane proteins of mammahan origin (Klein, et al. (1996) Proc. Natl. Acad. Sci. USA 93(14):7108-13). This system also allows for collapsing of large hbraries into hbraries with certain characteristics that aid in the identification of specific genes and gene products.
  • One disadvantage of this system is that genes encoding secreted proteins are primarily selected.
  • a second disadvantage is that the library may be biased because the technology is based on yeast as a heterologous expression system and there will be gene products that are not appropriately folded.
  • transgenic mice or knockout mice Other current strategies include the creation of transgenic mice or knockout mice.
  • a successful example of gene discovery by such an approach is the identification of the osteoprotegerin gene. DNA databases were screened to select ESTs with features suggesting that the cognate genes encoded secreted proteins. The biological functions of the genes were assessed by placing the corresponding full- length cDNAs under the control of a liver-specific promoter. Transgenic mice created with each of these constructs consequently have high plasma levels of the relevant protein. Subsequently, the transgenic animals were subjected to a battery of qualitative and quantitative phenotypic investigations.
  • the present invention relates to methods, and compositions for use therein, for identifying, in a high throughput setting, unique nucleic acids involved in apoptosis- associated processes in ceUs using hbraries of vectors comprising such nucleic acids.
  • the present invention relates to a method of identifying a unique nucleic acid capable of altering E2F activity in a ceU, wherein said unique nucleic acid is present in a library, said method comprising: (a) providing a library of a multitude of unique expressible nucleic acids, said library including a multiphcity of compartments, each of said compartments consisting essentiaUy of one or more adenoviral vector comprising at least one unique nucleic acid of said library in an aqueous medium, wherein said adenoviral vector is capable of introducing said nucleic acid into a host ceU, is capable of expressing the product of said nucleic acid in said host ceU, and is deleted in a portion of the adenoviral genome necessary for rephcation thereof in said host ceU; (b) transducing a multiphcity of host ceUs with at least one adenoviral vector comprising at least one unique nucleic acid from said library; (c) incubating said
  • the host ceU transduced with said recombinant adenoviral vector is observed for a change in E2F activity, and if such activity change is identified, a apoptosis-associated function is assigned to the product(s) encoded by the sample nucleic acids.
  • the present method also comprises: (a) growing a plurality of ceU cultures containing at least one ceU, said one ceU expressing adenoviral sequence consisting essentiaUy of El-region sequences and expressing one or more functional gene products encoded by at least one adenoviral region selected from an E2A region and an E4 region; (b) transfecting, under conditions whereby said recombinant adenovirus vector library is produced, said at least one ceU in each of said plurality of ceU cultures with i) an adapter plasmid comprising adenoviral sequence coding, in operable configuration, for a functional Inverted Terminal Repeat, a functional encapsidation signal, and sequences sufficient to aUow for homologous recombination with a first recombinant nucleic acid, and not coding for El region sequences which overlap with El region sequences in said at least one ceU, for El region sequences which overlap with El region sequences in a first recombin
  • a further aspect of the present assay methods is deterrnining whether the expression product of the nucleic acid capable of altering E2F activity is secreted by said ceU, comprising: (a) infecting producer ceUs in a medium with an adenoviral vector comprising a unique nucleic acid capable of altering E2F activity; (b) combining said medium with test ceUs that have not been infected with said vector; and (c) deteinhning if E2F activity is altered in said test ceUs.
  • Another aspect of the present invention relates to a method for identifying a drug candidate compound useful in the treatment of a disease state related to E2F- disregulation, said method comprising: (a) contacting a first subpopulation of host ceUs transfected with polynucleotide, identified in the above-described method of the invention, with one or more of said test compound, and (b) identifying, from said one or more test compounds, a candidate compound that alters E2F activity in said first subpopulation of transfected host ceUs relative to a second subpopulation of said transfected host ceUs that have not been contacted with said test compound.
  • Another means of detecting candidate compounds comprises selecting a compound that induces either an increase or decrease in the expression of mRNA encoded by a polynucleotide comprising a sequence of SEQ ID NO: 13 in said first subpopulation of transfected host ceUs relative to the expression of said mRNA in a second subpopulation of transfected host ceUs that has not been contacted with such compound.
  • a further aspect of the present method comprises first determining the binding affinity of said one or more test compound to (1) the polynucleotide identified in accordance with the present methods invention, or (2) the corresponding antisense sequences thereof, or (3) an expression product of said sequences, by contacting one or more test compound therewith.
  • the present method is useful for identifying compounds that are suitable as drug candidate compounds, the pharmaceutical apphcation of which is related to whether the aforesaid assay results in either an increase or a decrease in E2F activity, or the mRNA expression of the above-identified polynucleotides, in the host cells. If a test compound alters E2F activity, then the compound is useful for the treatment of apoptosis-associated disorders.
  • the present invention also relates to pharmaceutical compositions and methods of treatment comprising the polypeptides or polynucleotides described hereinbelow. Other aspects and more detaUed description of the present invention are provided in the foUowing sections.
  • FIG. 1 Construction of pBS.PGK.PCRI.
  • pBS.PGK.PCRI encodes the human phosphoglycerate kinase (PGK) promoter operatively linked to adenovirus 5 (Ad5) El nucleotides 459-916.
  • Ad5 nucleotides 459-916 are amplified by the polymerase chain reaction (PCR) with primers Ea-1 (SEQ ID NO: 1) and Ea-2 (SEQ ID NO:2), digested with Cla I, and cloned into the Clal-Eco Y sites of pBluescript (Stratagene), resulting in pBS.PCRI.
  • the PGK promoter is excised from pTN by complete digestion with Seal and partial digestion with EcoRI and cloned into the corresponding sites ofpBS.PCRI, resulting in pBS.PGK.PCRI.
  • FIG. 2 Construction of pIG.ElA.ElB.X. plG.ElAElB.X encodes Ad5 nucleotides 459-5788 (E1A and EIB regions) operatively linked to the human PGK promoter. plG.ElAElB.X also encodes Ad5 pIX protein. plG.ElAElB.X is constructed by replacing the Scal-BspEl fragment of pAT-X/S with the corresponding fragment of pBS.PGK.PCRI.
  • FIG. 3A Construction of pAT-PCR2-NEO.
  • EIB promoter and initiation codon (ATG) of the EIB 21 kDa protein are PCR amplified with primers Ea-3 (SEQ ID NO:3) and Ep-2 (SEQ ID NO:4), where Ep-2 introduces an Ncol site (5 -CCATGG) at the 21 kDa protein initiation codon.
  • the PCR product (PCR1I) is digested with Hpal and Ncol and hgated into the corresponding sites of pAT-X S, producing pAT-X/S-PCR2.
  • Ncol-Stu ⁇ fragment of pT ⁇ containing the ⁇ eo R and a portion of the HBV poly(A) site is hgated into the Nc ⁇ l-N ⁇ sites of pAT-X/S-PCR2, producing pAT-PCR2- ⁇ EO.
  • FIG. 3B Construction of pIG.ElA.NEO.
  • pIG.ElA.NEO encodes Ad5 nucleotides 459-1713 operatively hhked to the human PGK promoter. Also encoded is the EIB promoter functionaUy linked to the neomycin resistance gene (Neo R ) and the hepatitis B virus (HBV) poly(A) signal.
  • the AUG codon of the EIB 21 kDa protein functions as the initiation codon of Neo R .
  • the HBV poly(A) signal of ⁇ AT-PCR2-NEO see FIG.
  • FIG. 4 Construction of pIG.ElA.ElB.
  • pIG.ElA.ElB contains the Ad5 nucleotides 459-3510 (El A and EIB proteins) operatively linked to the PGK promoter and HBV poly(A) signal.
  • This plasmid is constructed by PCR amplification of the N-terminal amino acids of the EIB 55 kDa protein with primers Eb-1 (SEQ ID NO:5) and Eb-2 (SEQ ID NO:6), which introduces mXhol site, digested with Bgttl and cloned into the Bgl ⁇ -Nrul sites of pAT-X/S, producing pAT-PCR3.
  • the Xbal- Xhol fragment of pAT-PCR3 is replaced with fhe-Xb ⁇ l-S ⁇ H fragment (containing the HBNpolytA) site) ofpIG.ElA.NEO to produce pIG.ElA.ElB.
  • FIG.5 Construction of pIG EO.
  • pIG. ⁇ EO contains the ⁇ eo R operatively linked to the EIB promoter.
  • pIG.NEO was constructed by ligating the Hpal-Scal fragment of pAT.PCR2.NEO. ⁇ (A) or pIG.ElA.NEO, which contains the EIB promoter and Neo R into the EcoRV-Scal sites of pBS.
  • FIG. 6 Transformation of primary baby rat kidney (BRK) cehs by adenoviral packaging constructs.
  • Subconfluent dishes of BRK ceUs are transfected with 1 or 5 ⁇ g of either pIG.NEO, pIG.ElA.NEO, pIG.ElA.ElB, plG.ElAElB.X, pAd5XhoIC, or pIG.ElA.NEO plus pDC26, which expresses the Ad5 EIB gene under control of the SV40 early promoter.
  • foci are visible, cehs are fixed, Giemsa stained and the foci counted. The results shown are the average number of foci per 5 rephcate dishes.
  • FIG. 7 Western blot analysis of A549 clones transfected with pIG.ElA.NEO and human embryonic retinoblasts (HER) ceUs transfected with pIG.ElA.ElB (PER clones). Expression of Ad5 E1A and EIB 55 kDa and 21 kDa proteins in transfected A549 ceUs and PER ceUs is determined by Western blot with mouse monoclonal antibodies (Mab) M73, which recognizes E1A gene products, and Mabs AIC6 and C1G11, which recognize the EIB 55 kDa and 21 kDa proteins, respectively. Mab binding is visualized using horseradish peroxidase-labeUed goat anti-mouse antibody and enhanced chemUuminesence. 293 and 911 ceUs serve as controls.
  • Mob mouse monoclonal antibodies
  • FIG. 8 Southern blot analysis of 293, 911 and PER ceU lines. CeUular DNA is extracted, Hwdlll digested, electrophoresed, and transferred to ⁇ ybond N+ membranes (Amersham). Membranes are hybridized to radio labehed probes generated by random priming of the Sspl-Hin ⁇ T ⁇ fragment of pAd5. SalB (Ad5 nucleotides 342-2805).
  • FIG. 9 Transfection efficiency of PER.C3, PER.C5, PER.C6 and 911 ceUs.
  • CeUs are cultured in 6-weU plates and transfected in duphcate with 5 ⁇ g pRSV.lacZ by calcium-phosphate co-precipitation. Forty-eight hours post-transfection, ceUs are stained with X-Gal, and blue ceUs are counted. Results shown are the mean percentage of blue ceUs per weU. • » 1 / U
  • FIG. 10 Construction ofadenovhal vector, pMLPI.TK.
  • pMLPI.TK is designed to have no sequence overlap with the packaging construct pIG.El A.E1B.
  • pMLPI.TK is derived frompMLP.TK by deletion of the region of sequence overlap with plG.ElA.ElB and deletion of non-coding sequences derived from lacZ.
  • SV40 ⁇ oly(A) sequences of pMLP.TK are PCR amplified with primers SV40-1 (SEQ ID NO:7), which introduces a BamHI U ⁇ Q, and SV40-2 (SEQ ID NO:8), which introduces a Bgl ⁇ . site.
  • Ad5 sequences 2496 to 2779 are PCR amplified with primers Ad5-1 (SEQ ID NO:9), which introduces a Bg ⁇ Ti site, and Ad5-2 (SEQ ID NO: 10). Both PCR products are Bgl ⁇ l digested, hgated, and PCR amplified with primers SV40- 1 and Ad5-2. This third PCR product is BamHI and Aflill digested and hgated into the corresponding sites of pMLP.TK, producing pMLPI.TK.
  • FIG. 11A New adenoviral packaging construct, pIG.ElA.ElB, does not have sequence overlap with new adenoviral vector, pMLPI.TK. Regions of sequence overlap between the packaging construct pAd5XhoIC, expressed in 911 ceUs, and adenoviral vector pMLP.TK, that can result in homologous recombination and the formation of RCA are shown, In contrast, there are no regions of sequence overlap between the new packaging construct pIG.ElA.ElB, expressed in PER.C6 ceUs, and the new adenoviral vector pMLPI.TK.
  • FIG. 11B New adenoviral packaging construct pIG.ElA.NEO, does not have sequence overlap with new adenoviral vector pMLPI.TK. There are no regions of sequence overlap between the new packaging construct pIG.ElA.NEO and the new adenoviral vector pMLPI.TK that can result in homologous recombination and the formation of RCA.
  • FIG. 12 Generation of recombinant adenovirus, IG.Ad.MLPI.TK.
  • Recombinant adenovirus IG.Ad.MLPI.TK is generated by co-transfection of 293 ceUs with S ⁇ tl linearized pMLPI.TK and the right arm of CM digested, wdd-type Ad5 DNA. Homologous recombination between linearized pMLPI.TK and wdd-type Ad5 DNA produces IG.Ad.MLPI.TK DNA, which contains an El deletion of nucleotides 459-3510. 293 ceUs transcomplement the deleted Ad5 genome, thereby permitting rephcation of the IG.Ad.MLPI.TK DNA and its packaging into virus particles.
  • FIG. 13 Rationale for the design of adeno viral-derived recombinant DNA molecules that duplicate and replicate in ceUs expressing adenoviral rephcation proteins.
  • the terminal polypeptide (TP) attached to the 5'-terminus is indicated by closed circles.
  • the right arm of the adenoviral genome can be purified by removal of the left arm by restriction enzyme digestion. FoUowing transfection of the right arm into 293 or 911 ceUs, adenoviral DNA polymerase (white arrow) encoded on the right arm wiU produce only single-stranded forms.
  • FIG. 14 Adenoviral genome rephcation. The adenoviral genome is shown in the top left panel.
  • the origins or rephcation are located within the left and right ITRs at the genome ends. DNA rephcation occurs in two stages. Rephcation proceeds from one ITR, generating a daughter duplex and a displaced parental single-strand that is coated with adenoviral DNA binding protein (DBP, open circles) and can form a panhandle structure by annealing of the ITR sequences at both termini.
  • the panhandle is a substrate for DNA polymerase (Pol: white arrows) to produce double- stranded genomic DNA.
  • rephcation proceeds from both ITRs, generating two daughter molecules, thereby obviating the requirement for a panhandle structure.
  • FIG. 16 Diagram of pICLhac.
  • pICLhac contains aU the elements of pICL (FIG.19) but also contains the HP/asp sequence in site in an orientation that wih produce the hairpin structure shown in FIG. 15, foUowing linearization by Aspl 18 digestion and transfection into ceUs expressing adenoviral E2 proteins.
  • FIG. 17 Diagram ofpICLhaw. pICLhaw is identical to pICLhac (FIG. 16) except that the inserted HP/asp sequence is in the opposite orientation.
  • FIG. 18 Schematic representation of pICLI.
  • pICLI contains aU the elements of pICL (FIG. 19) but also contains an Ad5 ITR in the Aspl 1& site.
  • FIG. 19 Diagram of pICL.
  • pICL is derived from the foUowing: (i) nucleotides 1-457, Ad5 nucleotides 1-457 including the left ITR, (ii) nucleotides 458- 969, human Cytomegalovirus (CMV) enhancer and immediate early promoter, (hi) nucleotides 970-1204, SN40 19S exon and truncated 16/19S intron, (iv) nucleotides 1218-2987, firefly luciferase gene, (v) nucleotides 3018-3131, SN40 tandem polyadenylation signals from the late transcript, (vi) nucleotides 3132-5620, pUC12 sequences including an Aspl IS site, and (vii) ampiciUin resistance gene in reverse orientation.
  • CMV Cytomegalovirus
  • FIG. 20 Shows a schematic overview of the adenoviral fragments cloned in pBr322 (plasmid) or pWE15 (cosmid) derived vectors.
  • the top line depicts the complete adeno iral genome flanked by its ITRs (filled rectangles) and with some restriction sites indicated. Numbers foUowing restriction sites indicate approximate digestion sites (in kb) in the Ad5 genome.
  • FIG. 21 Drawing of adapter plasmid pAd L420-HSA
  • FIG. 22 Drawing of adapter plasmid pAd/Chp
  • FIG. 23 Schematic representation of the generation of recombinant adenoviruses using a plasmid-based system.
  • the genome organization of Ad5 is shown with filled boxes representing the different early and late transcription regions and flanking ITRs.
  • the middle of the figure represents the two DNAs used for a single homologous recombination whUe the bottom of the figure represents the recombinant virus after transfection into packaging ceUs
  • FIG. 24 Drawing of minimal adenoviral vector pMN/L420H
  • FIG. 25 Helper construct for rephcation and packaging of minimal adenoviral vectors. Schematic representation of the cloning steps for the generation of the helper construct pWE/AdD5'.
  • FIG. 26 Evidence for SN40-LargeT/ori mediated rephcation of large adenoviral constructs in COS-1 ceUs.
  • FIG. 26A shows a schematic representation of construct pWE/Ad.D5'. The location of the SN40 ori sequence and the fragments used to prepare probes are indicated.
  • FIG. 26B shows an autoradiogram of the Southern blot hybridized to the adenoviral probe.
  • FIG. 26C shows an autoradiogram of the Southern blot hybridized to the pWE probe. Lane 1 , marker lane: I D ⁇ A digested with EcoRI and HzndlH.
  • Lane 4 is empty. Lanes 2, 5, 7, 9, 11, 13, 15, and 17 contain undigested D ⁇ A and Lanes 3, 6, 8, 10, 12, 14, 16 and 18 contain Mbol digested D ⁇ A.
  • AU lanes contain D ⁇ A from COS-1 ceUs transfected with pWE.pac (lanes 2 and 3), pWE/Ad.D5' construct #1 (lanes 5 and 6), #5 (lanes 7 and 8) and #9 (lanes 9 and 10), pWE/Ad.Aflll-rlTR (lanes 11 and 12), pMV/CMV- LacZ (lanes 13 and 14), pWE.pac digested with P cl (lanes 15 and 16), or pWE/Ad.Aflll-rlTR digested with Pad (lanes 17 and 18) as described in the text.
  • FIG. 27 Production of E1/E2A deleted adenoviral vectors and its efficiency in miniaturized PER.C6/E2A based production system.
  • FIG. 28 Average titers produced in a 96-weU plate as measured using a PER.C6/E2A based plaque assay.
  • FIG. 30 Percentage of weUs showing CPE formation after transfection of PER.C6/E2A ceUs with pCLIP-LacZ, purified by 6 different protocols.
  • Qiagen standard alkaline lysis foUowed by Qiagen plasmid purification
  • AlkLys alkaline lysis foUowed by isopropanol precipitation, and solubihzation in TE buffer
  • AL + R ⁇ ase alkaline lysis foUowed by isopropanol precipitation, and solubihzation in TE buffer containing R ⁇ ase at 10 microgramper ml
  • AL+R+phenol alkaline lysis foUowed by isopropanol precipitation, and solubihzation in TE buffer containing RNase at 10 microgramper ml, foUowed by phenol/chloroform extraction and ethanol precipitation
  • cetyltrimethylammoniumbromide (CTAB) Standard CTAB plasmid isolation
  • FIG. 31 Effect of using digested plasmid for transfection without phenol- chloroform extraction. The results of aU experiments are depicted and expressed as percentage of weUs showing CPE formation.
  • FIG. 32 Stability of adenoviral vectors produced in miniaturized format and incubated for up to three weeks at three different temperatures and measured using a plaque forming assay for adenoviral vectors.
  • FIG. 33 Efficiencies of virus generation in percentages of CPE after virus generation of several adenoviruses (El and E2A deleted) containing cDNAs in antisense (AS) orientation.
  • FIG. 34A-M Plasmid maps of adenoviral adapter plasmids. These adenoviral adapter plasmids are particularly useful for the construction of expression hbraries in adenoviral vectors such as the subject of this apphcation. They have very rare restriction sites for the linearization of adapters and hbraries of adapters (with transgenes inserted) and wih not inactivate the adapter by digestion of the inserts.
  • the cosmid containing pIPspAdapt5- or pCLIP-fppoI-polynew-derived adenoviral DNA can be used for in vitro hgations.
  • Double stranded ohgonucleotides containing compatible overhangs are hgated between the l-Ceul and PI-Scel sites, between l-Ceul and 1-Ppo ⁇ , between I-Scel and PI-Scel, and between I-Scel and I- Ppol.
  • the Pad restriction endonuclease is subsequently used not only to linearize the construct after ligation and liberate the left- and right ITRs, but also to eliminate non-recombinants .
  • FIG. 34N Percentage of weUs showing CPE formation after transfection of PER.C6/E2A ceUs with pCLIP-LacZ and the adapter plasmid pIPspAdapt2.
  • FIG. 35 Percentage of virus producing weUs (CPE positive) in a 96-weU plate of PER.C6/E2A ceU after propagation of the freeze/thawed transfected ceU lysates.
  • Helper molecules used for cotransfection are (1) pWE/Ad.Aflll-rlTRsp, (2) pWE/Ad.AflII-rITRsp.dE2A, (3) pWE/Ad.Aflll-rlTRsp.dXba, and (4) pWE/Ad.AflII-rITR.
  • FIG. 36 (A and B): Schematic overview of constructing an arrayed adenovhal cDNA expression hbrary.
  • FIG. 37 Comparison of co transfections of different adapter plasmids and pWE/Ad.AfhT-rITRDE2A on 384-weU plates with co transfections on 96-weU plates.
  • the percentage of virus producing weUs (CPE positive weUs) scored at different time points as indicated after propagation of the freeze/thawed transfected ceUs to new PER.C6 E2A ceUs 5 days after transfection (upper panel) or 7 days after transfection (lower panel) is shown.
  • FIG. 38 The percentage of virus producing weUs (CPE positive wells) scored at different time points as indicated after changing the medium of the transfected ceUs 7 days after transfection (A); after no medium change (B); and after standard propagation of the freeze/thawed transfected ceUs to new PER.C6/E2A ceUs (C).
  • FIG. 39 (A, B, and C): The percentage of virus producing ceUs (CPE positive) scored after propagation of the freeze/thawed transfected ceUs to new PER.C6/E2A ceUs, in three different experiments using PER.C6/E2A ceUs for transfections with indicated confluency at time of transfection. CeU numbers from each flask in each experiment were counted. The ceUs from these flasks were used to seed 96-weU plates for transfection with adenoviral adapter and helper DNA molecules.
  • FIG. 40 The use of adenovhal expression vectors as a semi-stable expression system for assays with a delayed readout of phenotype after infection with an adenovhal expression hbrary.
  • Transgene used Green Fluorescent Protein (EGFP, Clontech).
  • a crude PER.C6/E2A production lysate is used at a multiplicity of infection (MOI) of about 500-1000.
  • FIG. 41 The use of polyethylenimine (PEI) for generating adenoviral vectors in miniaturized format. Transfection efficiency, virus formation (CPE), and proliferation (toxicity) are depicted,
  • FIG. 42 Effect of temperature PEI at time of transfections on CPE efficiency.
  • W Warm (room temperature) and C: Cold (4°C).
  • FIG. 43 Effect of PEI transfection volume on transfection efficiencies.
  • FIG. 44 Washing of PER.C6/E2A ceUs with serum free medium before applying hpofectamine-DNA complex can be omitted.
  • FIG. 45 Progression from Gl to S phase in the mammahan ceU cycle.
  • FIG. 46 Schematic representation of the construction of adenoviral Placenta hbrary.
  • FIG. 47 Schematic representation of pGL3-TATA-6xE2F-luc. The E2F binding sites are depicted as arrows over SEQ ID NO: 12.
  • FIG. 48 Schematic representation of pIPspAdapt8-L61Ras.
  • FIG. 49 Schematic representation of pIpSpAdApt3-E2F2.
  • FIG. 50 Schematic representation of pIpSpAdApt3-E2F3.
  • FIG. 51 Schematic representation of pIpSpAdApt ⁇ -pl ⁇ 31 ⁇ .
  • FIG. 52 Schematic representation of pIpSpAdApt6-p27 KIP .
  • FIG. 53 Schematic representation of pIpSpAdApt ⁇ -EGFP.
  • FIG. 54 Schematic representation of ⁇ CLIPPac-L61Ras.
  • FIG. 55 Schematic representation of pIpSpAdApt6-LacZ.
  • FIG. 56 Schematic representation of the various E2F reporter ceU lines tested + controls.
  • FIG. 57 Schematic representation of the optimalization infection conditions E2F-reporter ceU line IC5. Assay at different MOI.
  • FIG. 58 Schematic representation of the optimization of infection conditions E2F reporter ceU line 1C5. Assay at 48 or 72 hours after infection.
  • FIG. 59 Schematic representation of the optimization of infection conditions E2F reporter ceU line 1C5. High/Low serum conditions.
  • FIG. 60 Schematic representation of rescreen: reporter assay on ceU line IC5 with first hits from 1500 screen.
  • FIG. 61 Schematic representation validation (transient reporter) of hits from rescreen (1500).
  • FIG. 62 Schematic representation of reporter assay in 384-weUs format with control viruses from control virus plate.
  • FIG. 63 Schematic representation of the performance of control viruses that were implemented in the 11 ,000 hbrary virus reporter screen.
  • FIG. 64 Schematic representation of the results obtained for 51 hits in the first screen and rescreen at approximate MOIs of 600 and 2000.
  • FIG. 65 Comparison of the results of the hits obtained in first 11,000 screen and retested in rescreen.
  • FIG. 66 Schematic representation vahdation (transient reporter) of hits from rescreen (11,000).
  • FIG. 67 Nucleotide (FIG. 67A) and deduced amino acid sequences (FIG. 67B) of Hit Hl-9.
  • FIG. 68 Hl-9 induces E2F activity in transient reporter assay.
  • U2OS ceUs were transiently transfected with E2F-luciferase (marked as (E2F)6) or pGL3
  • FIG. 69 Optimization virus ratios for co-infections on U2OSwt ceUs.
  • FIG. 70 Optimization virus ratios for co-infections on U2OSwt ceUs.
  • FIG. 71 FiU up experiment on HUVEC ceUs by co-infections with increasing amounts of empty virus.
  • FIG. 72 Co-infection of HUVEC ceUs with viruses from the placenta hbrary.
  • FIG. 73 Co-infection of HUVEC ceUs with viruses from the placenta hbrary.
  • Abnormal ceU death means an apoptosis-associated disorder which disorder is characterized by increased ceU death due to malfunctioning of apoptotic ceU death mechanisms.
  • neuro -degenerative diseases such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, and cerebeUar degeneration
  • Apoptosis means ceU death by means of the ceU's regulatory mechanism, otherwise referred to as “programmed” ceU death.
  • Apoptosis-associated disorders means any human or animal disease or disorder, affecting any one or any combination of organs, cavities, or body parts, which disorder is characterized by prohferative disorders or abnormal ceU death related to the malfunctioning of apoptotic ceUular regulation.
  • Carrier means a non-toxic material used in the formulation of pharmaceutical compositions to provide a medium, bulk and/or useable form to a pharmaceutical composition.
  • a carrier may comprise one or more of such materials such as an excipient, stabilizer, or an aqueous pH buffered solution.
  • physiologicaUy acceptable carriers include aqueous or sohd buffer ingredients including phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptide; proteins, such as serum albumin, gelatin, or i muno globulins; hydrophilic polymers such as polyvinylpyrrohdone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and/or nonionic surfactants such as TWEENTM, polyethylene glycol (PEG), and PLURONICSTM.
  • aqueous or sohd buffer ingredients including phosphate, citrate, and other organic acids; antioxidants including ascorbic acid; low mole
  • Compound is used herein in the context of a “test compound” or a “drug candidate compound” described in connection with the screening assays of the present invention.
  • these compounds comprise organic or inorganic compounds, derived syntheticaUy or from natural sources.
  • the compounds include inorganic or organic compounds such as polynucleotides or hormone analogs that are characterized by relatively low molecular weights.
  • Other biopolymeric organic test compounds include ribozymes, peptides comprising from about 2 to about 40 amino acids and larger polypeptides comprising from about 40 to about 500 amino acids, such as antibodies or antibody conjugates.
  • Disease means the overt presentation of symptoms(t. e.
  • disease refers to a genetic or environmental risk of- or propensity for developing such symptoms or abnormal clinical indicators.
  • Diseases associated with defects in E2F activation include, but are not limited to apoptosis-associated disorders, which include proliferative disorders and abnormal ceU death diseases.
  • Expressible nucleic acid means a nucleic acid coding for a proteinaceous molecule, an RNA molecule, or a DNA molecule.
  • Hybridization refers to any process by which a strand of nucleic acid binds with a complementary strand through base pahing.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R ⁇ t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a sohd support (e.g., paper, membranes, filters, chips, pins or glass shdes, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • stringent conditions refers to conditions that permit hybridization between polynucleotides and the claimed polynucleotides.
  • Stringent conditions can be encloseded by salt concentration, the concentration of organic solvent, e.g., formamide, temperature, and other conditions weU known in the art.
  • stringency can be increased by reducing the concentration of salt, increasing the concentration of formamide, or raising the hybridization temperature.
  • “Mammal” means any animal classified as a rriammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, hamsters, rats, mice, cattle pigs, goats, sheep, etc,
  • Polynucleotide means a polynucleic acid, in single or double stranded form, and in the sense or antisense orientation, complementary polynucleic acids that hybridize to a particular polynucleic acid under stringent conditions, and polynucleotides that are homologous in at least about 60 percent of its base pahs, and more preferably 70 percent of its base pahs are in common.
  • the polynucleotides include polyribonucleic acids, polydeoxyribonucleic acids, and synthetic analogues thereof.
  • the polynucleotides are described by sequences that vary in length, that range from about 10 to about 5000 bases, preferably about 100 to about 4000 bases, more preferably about 250 to about 2500 bases.
  • a preferred polynucleotide embodiment comprises from about 10 to about 30 bases in length.
  • a special embodiment of polynucleotide is the polyribonucleotide of from about 10 to about 22 nucleotides, more commonly described as smaU interfering RNAs (siRNAs).
  • Proliferative disorders means an apoptosis-associated disorder which disorder is characterized by single or multiple local abnormal or uncontroUed proliferation of ceUs, groups of ceUs, or tissues, whether benign or malignant, and which ceUs may also be described as “neoplastic”.
  • proliferative disorders or diseases that can be treated, prevented, and/or diagnosed by nucleic acids, polypeptides or antibodies of the present invention include, but are not limited to, various types of sohd and hquid tumor growth, such as retinoblastoma, osteosarcoma, adeno carcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, and teratocarcinoma, tumors of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gaU bladder, gangha, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis, thymus, thyroid, and uterus, hyperplasias of the thyroid, endometrium, pituitary gland and adrenal gland, hpodystrophia, lymphoproliferative diseases, psori
  • Proliferative induction means the induction of proliferation in ceUs (not characterized as “neoplastic"), groups of ceUs, or tissues, whether or not it occurs in vivo or ex vivo.
  • diseases that can be treated, prevented or diagnosed by nucleic acids, polypeptides or antibodies of the present invention include, but are not limited to, anemia, lymphocytopenia, thrombopenia, and neutropenia.
  • transplantation e.g., of Langerhans ceUs
  • tissue repair repair and bone replacement tissue repair repair and bone replacement
  • corrective surgery might greatly benefit from an induction of proliferation in ceUs, groups of ceUs, or tissues.
  • induction of proliferation in cardiac myocytes can also be beneficial to prevent or treat hypertrophy.
  • Treatment means an intervention performed with the intention of preventing the development or altering the pathology of a disorder. Accordingly, “treatment” refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those aheady with the disorder as weU as those in which the disorder is to be prevented.
  • Administration "in combination with” or “admixture with” one or more further therapeutic agents includes simultaneous (concurrent) and consecutive administration in any order,
  • the present invention in one embodiment, provides methods that use a hbrary of expressible nucleic acids comprising a multiphcity of compartments.
  • Each compartment comprises at least one vehicle including at least one nucleic acid of the hbrary, whereby the vehicle is capable of introducing at least one nucleic acid into a ceU such that it can be expressed.
  • Another advantage of the hbrary is that it includes a multiphcity of compartments each including at least one nucleic acid. When a compartment includes only one nucleic acid, then it is known that the unique nucleic acid in the distinct compartment is responsible for whatever change in phenotype is observed.
  • At least one compartment includes at least two vehicles. EspeciaUy with, but not limited to, large hbraries, it becomes advantageous to reduce the number of compartments to reduce the number of screening assays that need to be performed. In such cases, hbraries are provided that include more than one vehicle. If after screening, a certain effect is correlated to a certain compartment, the vehicles in the compartment may be analysed separately in an additional screening assay to select the vehicle including the nucleic acid the expression of which exerts the effect. In addition, the presence of more than one vehicle in a compartment may be advantageous when a hbrary containing one vehicle per compartment is screened for a nucleic acid capable of exerting an effect in combination with one particular other nucleic acid.
  • the other nucleic acid may then be provided to the ceh by adding a vehicle including the particular other nucleic acid to aU compartments prior to performing the screening assay.
  • the vehicle may include at least two nucleic acids.
  • the hbrary used i the method may use any kind of ceU.
  • the ceU is a eukaryotic ceU, especiaUy a mammahan ceU.
  • ceUs examples include hepatomas: HepG2; keratinocytes: HCAT1; osteosarcomas: U2OS, SaOS; cervixcarcinoma: Hela; breast tumor: MCF7, T47D, MDA-MB-468; pancreatic tumor: BxPC3, HP AC; colon carcinoma: COLO205, HT29; melanomas: SK-MEL-2, M14; leukemia ceUs: K562, TF1, Daubi, Raji; central nervous system: SF-268; lung tumor: A549, SW1573; prostate: PC-3, DU-145; bladder: HT-1376; stomach: Hs740.T; and kidney: Caki-1,
  • the ceUs are divided over a number of compartments each including at least one vehicle including at least one nucleic acid from the hbrary.
  • the number of compartments preferably corresponds to the multiphcity of compartments in the hbrary.
  • a vhal element may include a vhal nucleic acid aUowing the packaging of at least one nucleic acid into a vehicle, where the vehicle is a virus particle.
  • the vhal element is derived from an adenovirus
  • the vehicle includes an adenoviral vector packaged into an adenovhal capsid, or a functional part, derivative, and/or analogue thereof.
  • Adenovirus biology is also comparatively weU known on the molecular level. Many tools for adenovhal vectors have been and continue to be developed, thus making an adenovhal capsid a preferred vehicle for incorporating in a hbrary of the invention.
  • the vehicle includes adenoviral fiber proteins from at least two adenoviruses.
  • the nucleic acid derived from an adenovirus includes the nucleic acid encoding an adenoviral late protein or a functional part, derivative, and/or analogue thereof.
  • An adenovhal late protein for instance an adenovhal fiber protein, may be favorably used to target the vehicle to a certain ceU or to induce enhanced dehvery of the vehicle to the ceU.
  • the nucleic acid derived from an adenovirus encodes for essentiaUy aU adenovhal late proteins, enabling the formation of entire adenovhal capsids or functional parts, analogues, and/or derivatives thereof.
  • the nucleic acid derived from an adenovirus includes the nucleic acid encoding adenovirus E2A or a functional part, derivative, and/or analogue thereof.
  • the nucleic acid derived from an adenovirus includes the nucleic acid encoding at least one E4-region protein or a functional part, derivative, and/or analogue thereof, which facilitates, at least in part, rephcation of an adenovhal derived nucleic acid in a ceU.
  • the nucleic acid derived from an adenovirus includes the nucleic acid encoding at least one El -region protein or a functional part, derivative, and/or analogue thereof.
  • the presence of the adenovhal nucleic acid encoding an El- region protein facilitates, at least in part, rephcation of the nucleic acid in a ceU.
  • the rephcation capacity is favored in certain applications when screening is done for expressible nucleic acids capable of irradiating tumor ceUs. In such cases, rephcation of an adenovhal nucleic acid leading to the amplification of the vehicle in a mammal including tumor ceUs may lead to the irradiation of metastasized tumor ceUs.
  • the presence of an adenovhal nucleic acid encoding an El -region protein may facilitate, at least in part, amplification of the nucleic acid in a ceU for the amplification of vehicles including the adenovhal nucleic acid.
  • the vehicle further includes a nucleic acid including an adeno-associated virus terminal repeat or a functional part, derivative, and/or analogue thereof which aUows the integration of at least one nucleic acid in a ceU.
  • the present invention provides a method for identifying apoptosis-associated functions of the unique nucleic acids present in a hbrary, the functions of which are for the most part unknown, or at least not completely understood.
  • This method transduces multiple subpopulations of ceUs, each subpopulation present in a discrete compartment of the hbrary, with at least one vehicle including at least one nucleic acid from the hbrary, culturing the ceUs whUe aUowing for expression of the nucleic acid, and determining the expressed function.
  • the hbrary is screened for the presence of expressible nucleic acids capable of influencing, at least in part, the activity of E2F.
  • the present method preferably utilizes a set of adapter plasmids by inserting a set of cDNAs, DNAs, ESTs, genes, synthetic ohgonucleotides, or a hbrary of nucleic acids into El -deleted adapter plasmids; cotransfecting an El -complementing ceU line with the set or hbrary of adapter plasmids and at least one plasmid having sequences homologous to sequences in the set of adapter plasmids and which also includes aU adenovhal genes not provided by the complementing ceU line or adapter plasmids necessary for rephcation and packaging to produce a set or hbrary of recombinant adenovhal vectors preferably in a miniaturized, high throughput setting.
  • the plasmid-based system is used to rapidly produce adenovhal vector hbraries that are preferably rephcation competent adenovirus ("RCA")-free for high throughput screening.
  • adenovhal vector hbraries that are preferably rephcation competent adenovirus ("RCA")-free for high throughput screening.
  • RCA rephcation competent adenovirus
  • Each step of the method can be performed in a multiweU format and automated to further increase the capacity of the system.
  • This high throughput system facihtates expression analysis of a large number of sample nucleic acids from human and other organisms both in vitro and in vivo and is a significant improvement over other avaUable techniques in the field.
  • the method permits the amplification of the vehicles including the unique nucleic acids present in a hbrary, Such amplification maybe achieved culturing the ceU with the vehicle, aUowing the amplification of the vehicle, and harvesting vehicles amplified by the ceU.
  • the ceU is a primate ceU thereby enabling the amplification of vehicles including vhal elements that aUow rephcation of the vehicle nucleic acid.
  • the ceU includes a nucleic acid encoding an adenovhal El -region protein thereby aUowing, among other things, the amplification of vehicles including vhal elements derived from adenovirus including adenovhal nucleic acids including a functional deletion of at least part of the El -region.
  • the ceU is a PER.C6 ceU (ECACC deposit number 96022940) or a functional derivative and/or analogue thereof.
  • a PER.C6 ceU (or a functional derivative and/or analogue thereof) aUows the rephcation of adenovhal nucleic acid with a deletion of the El-coding region without concomitant production of RCA in instances wherein the adenovhal nucleic acid and chromosomal nucleic acid in the PER.C6 ceU or functional derivative and/or analogue thereof do not include sequence overlap that aUows for homologous recombination between the adenovhal and chromosomal nucleic acid leading to the formation of RCA.
  • the ceU further includes nucleic acid encoding adenovirus E2A and/or an adenovhal E4- region protein or a functional part, derivative, and/or analogue thereof.
  • This aU ows the rephcation of adenovhal nucleic acid with functional deletions of nucleic acid encoding adenovirus E2A and/or an adenovhal E4-region protein, thereby inhibiting rephcation of the adenovhal nucleic acid in a ceU not including nucleic acid encoding adenovirus E2A and/or an adenovhal E4-region protein or a functional part, derivative and/or analogue thereof, for instance a ceU capable of displaying a certain function.
  • the vehicle nucleic acid does not include sequence overlap with other nucleic acids present in the ceU, leading to the formation of vehicle nucleic acid capable of replicating in the absence of El-region encoded proteins.
  • the method is preferably implemented using a multiphcity of compartments in a multiweU format.
  • a multiweU format is very suited for automated execution of at least part of the methods of the invention.
  • the present invention uses high throughput generation of recombinant adenovhal vector hbraries containing one or more sample nucleic acids, foUowed by high throughput screening of the adenovhal vector hbraries in a host to alter the phenotype of the host as a means of assigning a function to expression product(s) of the sample nucleic acids.
  • Libraries of El -deleted adenoviruses are generated in a high throughput setting using nucleic acid constructs and transcomplementary packaging ceUs.
  • the sample nucleic acid hbraries can be a set of distinct defined or undefined sequences or can be a pool of undefined or defined sequences.
  • the first nucleic acid construct is a relatively smaU and easy to manipulate adapter plasmid containing, in an operable configuration, at least a left ITR, a packaging signal, and an expression cassette with the sample nucleic acids.
  • the second nucleic acid construct contains one or more nucleic acid molecules that partiaUy overlap with each other and/or with sequences in the first construct.
  • the second construct also contains at least aU adenovirus sequences necessary for rephcation and packaging of a recombinant adenovirus not provided by the adapter plasmid or packaging cells.
  • the second nucleic acid construct is deleted in El -region sequences and optionaUy E2B region sequences other than those required for virus generation and/or E2A, E3 and/or E4 region sequences.
  • Co transfection of the first and second nucleic acid constructs into the packaging ceUs leads to homologous recombination between overlapping sequences in the first and second nucleic acid constructs and among the second nucleic acid constructs when it is made up of more than one nucleic acid molecule.
  • GeneraUy the overlapping sequences are no more than 5000 bp and encompass E2B region sequences essential for virus production.
  • Recombinant vhal DNA is generated with an E 1 -deletion that is able to rephcate and propagate in the E 1 -complementing packaging ceUs to produce a recombinant adenovhal vector hbrary.
  • the adenovhal vector hbrary is introduced in a high throughput setting into a host which is grown to aUow sufficient expression of the product(s) encoded by the sample nucleic acids to permit detection and analysis of its biological activity.
  • the host can be cultured ceUs in vitro or an animal or plant model. Sufficient expression of the product(s) encoded by the sample nucleic acids alters the phenotype of the host.
  • the altered phenotype is analyzed and identified and a function is thereby assigned to the product(s) of the sample nucleic acids.
  • the plasmid-based adenovhal vector systems described here provide for the creation of large gene-transfer hbraries that can be used to screen for novel genes applicable to human diseases, such as those discussed in more detaU herein. Identification of a useful or beneficial biological effect of a particular adenovhal mediated transduction can result in a useful gene therapeutic product or a target for a smaU molecule drug for treatment of such human diseases.
  • the entire process lends itself to automation especiaUy when implemented in a 96-weU or other multi-weU format.
  • the high throughput screening using a number of different in vitro assays, provides a means of efficiently obtaining functional information in a relatively short period of time.
  • the member(s) of the recombinant adenovhal hbraries that exhibit or induce a desired phenotype in a host in vitro or in situ are identified to reduce the hbraries to a manageable number of recombinant adenovhal vectors or clones that can be tested in vitro in an animal model.
  • Another distinct advantage of the present hbrary is that the adenovhal hbraries produced are capable of being RCA-free. RCA contamination throughout the hbraries could become a major obstacle, especiaUy if hbraries are continuously amplified for use in multiple screening programs,
  • a further advantage of the subject invention is rnihimization of the number of steps involved in the process. The methods of the subject invention do not require cloning of each individual adenovirus before use in a high throughput-screening program.
  • Other systems include one or more steps in E. coli to achieve homologous recombination for the various adenovhal plasmids necessary for vector generation (Chartier, et al (1996) J. Virol.
  • a further advantage of the adenovhal hbrary is the abihty of recombinant adenoviruses to efficiently transfer and express recombinant genes in a variety of mammahan ceUs and tissues in vitro and in vivo, resulting in the high expression of the transferred sample nucleic acids.
  • the abihty to productively infect quiescent ceUs further expands the utility of the recombinant adenovhal hbraries.
  • high expression levels ensure that the prpduct(s) of the sample nucleic acids wiU be expressed to sufficient levels to induce a change that can be detected in the phenotype of a host and aUow the function of the product(s) encoded by the sample nucleic to be determined.
  • the sample nucleic acids can be genomic DNA, cDNA, previously cloned DNA, genes, ESTs, synthetic double stranded ohgonucleotides, or randomized sequences derived from one or multiple related or unrelated sequences.
  • the sample nucleic acids can also be directly expressed as polypeptides, antisense nucleic acids, or genetic suppressor elements (GSE).
  • GSE genetic suppressor elements
  • the sample nucleic acid sequences can be obtained from any organism including mammals (for example, man, monkey, mouse), fish (for example, zebrafish, pufferfish, salmon), nematodes (for example, C. elegans), insects (for example, Drosophila), yeasts, fungi, bacteria, and plants.
  • sample nucleic acids are prepared as synthetic ohgonucleotides using commerciaUy avaUable DNA synthesizers and kits.
  • the strand coding the open reading frame of the polypeptide or product of the sample nucleic acid and the complementary strand are prepared individuaUy and annealed to form double- stranded DNA. Special annealing conditions are not required.
  • the sequences of the sample nucleic acids can be randomized or not through mutagenizing or methodologies promoting recombination.
  • the sample nucleic acids code for a product(s) for which a biological activity is unknown.
  • the phrase biological activity is intended to mean an activity that is detectable or measurable either in situ, in vivo, or in vitro. Examples of a biological activity include but are not limited to altered viability, morphologic changes, apoptosis induction, DNA synthesis, tumorigenesis, disease or drug susceptibility, chemical responsiveness or secretion, and protein expression.
  • the sample nucleic acids preferably contain compatible ends to facilitate ligation to the vector in the correct orientation and to operatively link the sample nucleic acids to a promoter.
  • the ends compatible to the vector can be designed into the ohgonucleotides.
  • the sample nucleic acid is an EST, genomic DNA, cDNA, gene, or previously cloned DNA
  • the compatible ends can be formed by restriction enzyme digestion or the hgation of linkers to the DNA containing the appropriate restriction enzyme sites.
  • the vector can be modified by the use of linkers.
  • the restriction enzyme sites are chosen so that transcription of the sample nucleic acid from the vector produces the desired product, i.e., polypeptide, antisense nucleic acid, or GSE.
  • the El -deleted dehvery vector or adapter plasmid is digested with the appropriate restriction enzymes, either simultaneously or sequentiaUy, to produce the appropriate ends for directional cloning of the sample nucleic acid whether it be synthetic double-stranded ohgonucleotides, genomic DNA, cDNA, ESTs, or a previously-cloned DNA. Restriction enzyme digestion is routinely performed using commerciaUy avaUable reagents according to the manufacturer's recommendations and varies according to the restriction enzymes chosen.
  • a distinct set or pool of sample nucleic acids is inserted into El-deleted adapter plasmids to produce a corresponding set or hbrary of plasmids for the production of adenovhal vectors
  • An example of an adapter plasmid is pMLPI.TK, which is made up of adenovhal nucleotides 1-458 foUowed by the adenovhal major late promoter, functionaUy linked to the herpes simplex virus thymidine kinase gene, and foUowed by adenovhal nucleotides 3511-6095.
  • Other examples of adapter plasmids are pAd/L420-HSA (FIG. 21) andpAd/Chp (FIG.
  • pAd/L420-HSA contains adenovhal nucleotides 1-454, the L420 promoter linked to the murine HSA gene, a poly-A signal, and adenovhal nucleotides 3511-6095.
  • pA ⁇ VCLIP is made from pAd/L420-HSA by replacement of the expression cassette (L420-HSA) with the CMV promoter, a multiple cloning site, an intron, and a poly-A signal.
  • the vector and sample nucleic acids are purified by gel electrophoresis.
  • the nucleic acids can be extracted from various gel matrices by, for example, agarose digestion, electro elution, melting, or high salt extraction.
  • the digested nucleic acids can be purified by chromatography (e.g. , Qiagen or equivalent DNA binding resins) or phenol-chloroform extraction foUowed by ethanol precipitation,
  • the optimal purification method depends on the size and type of the vector and sample nucleic acids. Both can be used without purification.
  • the magnitude of expression can be modulated using promoters (CMV immediately early, promoter, SV40 promoter, or retrovirus LTRs) that differ in theh transcriptional activity.
  • CMV immediate early, promoter, SV40 promoter, or retrovirus LTRs a promoter that differ in theh transcriptional activity.
  • a strong promoter such as the CMV immediate early promoter and optionahy one or more enhancer element(s) results in higher expression aUowing the use of a lower multiphcity of infection to alter the phenotype of a host.
  • the option of using a lower multiphcity of infection increases the number of times a hbrary can be used in situ, in vitro, and in vivo.
  • a regulatable promoter particularly one which is repressed during virus production but can be turned on or is active during functional screenings with the adenovhal hbrary, to provide temporal and/or ceU type specific control throughout the screening assay.
  • sample nucleic acids that are members of the hbrary and are toxic, inhibitory, or in any other way interfere with adenovhal rephcation and production can still be produced as an adenovhal vector (see WO 97/20943).
  • this type of promoter are the API -dependent promoters which are repressed by adenovhal El gene products, resulting in shut off of sample nucleic acid expression during adenovhal hbrary production (see van Dam, et al. (1990) Mol. Cell. Biol. 10(11):5857-64).
  • Such a promoter can be made using combinatorial techniques or natural or adapted forms of promoters can be included.
  • tissue-regulated promoters include promoters with specific activity or enhanced activity in the hver, such as the albumin promoter (Tronche, et al. (1990) Mol. Biol. Med. 7(2):173-85).
  • Another approach to enhanced expression is to increase the half- life of the mRNA transcribed from the sample nucleic acids by including stabilizing sequences in the 3' untranslated region.
  • a second nucleic acid construct a helper plasmid having sequences homologous to sequences in the El -deleted adapter plasmids, which carries aU necessary adenovhal genes necessary for rephcation and packaging, also is prepared.
  • the helper plasmid has no complementing sequences that are expressed by the packaging ceUs that would lead to production of RCA.
  • the helper plasmids, adapter plasmid, and packaging ceU have no sequence overlap that would lead to homologous recombination and RCA formation.
  • GeneraUy the region of overlap encompasses E2B region sequences that are requhed for late gene expression.
  • the amount of overlap that provides for the best efficiency without appreciably decreasing the size of the hbrary insert can be determined emphicaUy.
  • the sequence overlap is generaUy 10 bp to 5000 bp, more preferably 2000 to 3000 bp.
  • the size of the sample nucleic acids or DNA inserts in a deshed adenovhal hbrary can vary from several hundred base pahs (e.g. , sequences encoding neuropeptides) to more than 30 kb (e.g, titin).
  • the cloning capacity of the adenovhal vectors produced using adapter plasmids can be increased by deletion of additional adenovhal gene(s) that are then easUy complemented by a derivative of an El- complementing ceU line.
  • candidate genes for deletion include E2, E3, and/or E4.
  • regions essential for adenovhal rephcation and packaging are deleted from the adapter and helper plasmids and expressed, for example, by the complementing ceU line.
  • genes in this region do not need to be complemented in the packaging ceU for in vitro models; in in vivo models, the impact upon immunogenicity of the recombinant virus can be assessed on an ad hoc basis.
  • the set or hbrary of specific adapter plasmids or pool(s) of adapter plasmids is converted to a set or hbrary of adenovhal vectors.
  • the adapter plasmids containing the sample nucleic acids are linearized and transfected into an El -complementing ceU line.
  • the adapter plasmids are preferably seeded in microtiter tissue culture plates with 96, 384, 1,536 or more weUs per plate, together with helper plasmids having sequences homologous to sequences in the adapter plasmid and containing aU adenovhal genes necessary for rephcation and packaging. Recombination occurs between the homologous sequences shared by adapter and helper plasmids to generate an El -deleted, rephcation-defective adenovhal genome that is rephcated and packaged, preferably, in an El -complementing ceU line.
  • helper plasmid If more than one helper plasmid is used, recombination between homologous regions shared among the helper plasmids and recombination between the helper plasmids and adapter plasmid results in the formation of a rephcation-defective recombinant adenovhal genome.
  • the regions of sequence overlap between the adapter and helper plasmids are at least about a few hundred nucleotides or greater. Recombination efficiency wiU increase by increasing the size of the overlap .
  • the El -functions provided by the trans complementing packaging ceh permit the rephcation and packaging of the El -deleted recombinant adenovhal genome.
  • the adapter and/or helper plasmids preferably have no sequence overlap amongst themselves or with the complementing sequences in the packaging ceUs that can lead to the formation of RCA.
  • at least one of the ITRs on the adapter and helper plasmids is flanked by a restriction enzyme recognition site not present in the adenovhal sequences or expression cassette so that the ITR is freed from vector sequences by digestion of the DNA with that restriction enzyme. In this way, initiation of rephcation occurs more efficiently.
  • the sample nucleic acids can be hgated to "mhhmar adenovhal vector plasmids.
  • the so-caUed "minimal" adenovhal vectors retain at least a portion of the vhal genome that is required for encapsidation of the genome into virus particles (the encapsidation signal).
  • the minimal vectors also retain at least one copy of at least a functional part or a derivative of the ITR, that is DNA sequences derived from the termini of the linear adenovhal genome that are requhed for rephcation.
  • the rninimal vectors preferably are used for the generation and production of helper- and RCA-free stocks of recombinant adenovhal vectors and can accommodate up to 38 kb of foreign DNA.
  • the helper functions of the rrhhimal adenovhal vectors are preferably provided in trans by encapsidation-defective, rephcation-competent DNA molecules that contain aU the vhal genes encoding the requhed gene products, with the exception of those genes that are present in the complementing ceU or genes that reside in the vector genome.
  • Packaging of the "minimal'' adenovhal vector is achieved by co transfection of an El -complementing ceU line with a helper virus or, alternatively, with a packaging deficient replicating helper system.
  • the packaging deficient replicating helper is amplified foUowing transfection and expresses the sequences requhed for rephcation and packaging of the minimal adenovhal vectors that are not expressed by the packaging ceU line.
  • the packaging deficient replicating helper is not packaged into adenovhal particles because its size exceeds the capacity of the adenovhal virion and/or because it lacks a functional encapsidation signal.
  • the packaging deficient replicating helper, the rrunimal adenovhal vector, and the complementing ceU line preferably, have no region of sequence overlap that permits RCA formation.
  • the replicating, packaging deficient helper molecule always contains aU adenovhal coding sequences that produce proteins necessary for rephcation and packaging, with or without the coding sequences provided by the packaging ceU line.
  • Rephcation of the helper molecule itself may or may not be mediated by adenovhal proteins and ITRs.
  • Helper molecules that rephcate through the activity of adenovhal proteins contain at least one ITR derived from adenovirus, The E2-gene products can be expressed by an El-dependent or an El -independent promoter. Where only one ITR is derived from an adenovirus, the helper molecule also preferably contains a sequence that anneals in an intramolecular fashion to form a hairpin-like structure.
  • the adenovhal DNA polymerase recognizes the ITR on the helper molecule and produces a single-stranded copy. Then, the 3'-terminus intramolecularly anneals, forming a hairpin-like structure that serves as a primer for reverse strand synthesis.
  • the product of reverse strand synthesis is a double-strand hairpin with an ITR at one end.
  • This ITR is recognized by adenovhal DNA polymerase that produces a double-stranded molecule with an ITR at both termini (see e.g., FIG. 13) and becomes twice as long as the transfected molecule (in our example it duphcates from 35 Kb to 70 Kb).
  • Duphcation of the helper DNA enhances the production of sufficient levels of adenovhal proteins.
  • the size of the duphcated molecule exceeds the packaging capacity of the adenovhal virion and is, therefore, not packaged into adenovhal particles.
  • the absence of a functional encapsidation signal in the helper molecule further ensures that the helper molecule is packaging deficient.
  • the produced adenovhal proteins mediate rephcation and packaging of the co transfected or co-infected rrrinimal vectors.
  • the helper construct When the rephcation of the helper molecule is independent of adenoviral E2- proteins, the helper construct preferably contains an origin of rephcation derived from SV40. Transfection of this DNA, together with the minimal adenovhal vector in an El -containing packaging ceU line that also inducibly expresses the SV40 Large T protein or as much S V40 derived proteins as necessary for efficient rephcation, leads to rephcation of the helper construct and expression of adenovhal proteins. The adenovhal proteins then initiate rephcation and packaging of the co -transfected or co- infected minimal adenovhal vectors. Preferably, there are no regions of sequence overlap shared by the rephcation-deficient packaging constructs, the minimal adenovhal vectors, and the complementing ceU lines that may lead to the generation ofRCA.
  • each amplification step on El -complementing ceUs is preceded by transfection of any of the described helper molecules since nhnimal vectors by themselves cannot rephcate on El -complementing cells.
  • a ceU line that contains aU the adenovhal genes necessary for rephcation and packaging, which are stably integrated in the genome and can be excised and rephcated conditionaUy can be used (Valerio and Einerhand, International patent Apphcation PCT/NL9800061).
  • Transfection of nucleic acid into ceUs is requhed for packaging of recombinant vectors into virus particles and can be mediated by a variety of chemicals including hposomes, DEAE-dextran, polybrene, and phosphazenes or phosphazene derivatives (WO 97/07226).
  • Liposomes are avaUable from a variety of commercial supphers and include DOTAP ⁇ (Boelninger-Mannheim), Ti ⁇ "-50, Transfectam*, ProFection* (Promega, Madison, WI), and LipofectAmine ⁇ , Lipofectin*, LipofectAce* (GibcoBRL, Gaithersburg, MD).
  • the hpids form vesicles that associate with the nucleic acid and facilitate its transfer into ceUs by fusion of the vesicles with ceU membranes or by endocytosis.
  • Other transfection methods include electroporation, calcium phosphate coprecipitation, and microinjection. If transfection conditions for a given ceU line have not been estabhshed or are unknown, they can be determined empirieatty (Maniatis, et al. Molecular Cloning, pages 16.30-16.55).
  • the yield of recombinant adenovhal virus vectors can be increased by denaturing the double stranded plasmid DNA before transfection into an El complementing ceU line. Denaturing can occur by heating double-stranded DNA at, for example, 95-100°C, foUowed by rapid cooling using various ratios of the adapter and helper plasmids that have overlapping sequences. Also, a PER.C6 derivative that stably or transiently expresses E2A (DNA binding protein) and or E2B gene (pTP- Pol) could be used to increase the adenovhal production per weU by increasing the rephcation rate per ceU.
  • E2A DNA binding protein
  • E2B gene pTP- Pol
  • recombinase protein(s), recombinase DNA expression construct(s), z.e.,recombinase from Kluyveromyces waltii (Ringrose, et ⁇ l. (1997) Eur. J. Biochem. 248(3):903-12), or any other gene or genes encoding factors that can increase homologous recombination efficiency can be used.
  • the inclusion of 0.1-100 mM sodium butyrate during transfection and/or rephcation of the packaging ceUs can increase vhal production.
  • These improvements wiU result in improved vhal yields per microtiter weU. Therefore, the number and type of assays that can be done with one hbrary wiU increase and may overcome variability between the various genes or sample nucleic acids in a hbrary.
  • the ceU lines used for the production of adenovhal vectors that express El region products includes, for example, 293 ceUs, PER.C6 (ECACC 96022940), or 911 ceUs. Each of these ceU lines has been transfected with nucleic acids that encode for the adenovhal El region. These ceUs stably express El region gene products and have been shown to package El-deleted recombinant adenovhal vectors. Yields of recombinant adenovirus obtained on PER.C6 ceUs are higher than obtained on 293 ceUs.
  • growth medium of the ceU culture contains sodium butyrate in an amount sufficient to enhance production of the recombinant adenovhal vector Hbrary.
  • the plurahty of ceUs further includes at least one of an adenovhal preterminal protein and a polymerase complementing sequence.
  • the plurahty of ceUs further includes an adenovhal E2 complementing sequence.
  • the E2 complementing sequence is an E2A complementing sequence or an E2B complementing sequence.
  • the plurahty of ceUs further includes a recombinase protein, whereby the homologous recombination leading to rephcation- defective, recombinant adenovirus is enhanced.
  • the recombinase protein is a Kluyveromyces waltii recombinase.
  • the plurahty of ceUs further includes a nucleotide sequence coding for a recombinase protein.
  • the recombinase protein is Kluyveromyces waltii recombinase.
  • in vitro assays can be complemented by using an electronic version of the sequence database on which the adenovhal hbrary is buUt.
  • This aUows for example, protein motif searching whereby new members of a fanhly can be linked to known members of the same family with known functions.
  • HMMs Hidden Markow Models
  • aUows the establishment of novel families by identifying essential features of a famUy and budding a model of what the members should look like.
  • the clones can be purified by, for example, using adenovirus tagged in the Hi loop of the knob domain of the fiber.
  • large scale HPLC analysis can be used in a semipreparative fashion to yield partiaUy purified adenovhal samples for in vivo or in vitro experiments when more purified adenovhal preparations are deshed. Therefore, the described method and reagents aUow rapid transfer of a coUection of genes in in vivo studies of a limited number of animals, which otherwise would be unfeasible.
  • the automation of the steps of the procedure using robotics wiU further enhance the number of genes and sample nucleic acids that can be functionated.
  • aspects of the present invention include methods of assay and compositions used therein for the identification of compounds useful for the treatment of disease states that involve apoptosis-associated processes.
  • the methods and compositions of the present invention are based on the identification of the polypeptides and polynucleotides discovered by the adenovhal hbrary screening methods described hereinabove.
  • Examples of polynucleotides and polypeptides identified by the methods of the present invention are the polynucleotide of SEQ ID NO: 13, polypeptides comprising an amino acid sequence encoded by the polynucleotide of SEQ ID NO: 13 and the polypeptide comprising the amino acid sequence of SEQ ID NO: 14.
  • the invention includes both naturaUy occurring and recmbinant forms of SEQ ID NO: 13 and SEQ ID NO: 14 as weU as methods of their production.
  • Methods of detecting the polynucleotide of SEQ ID NO: 13 include probing with polynucleotides complementary to SEQ ID NO: 13 (northern and southern hybridization) and amplifying using the polymerase chain reaction.
  • Methods of detection of the polypeptide of SEQ ID NO: 14 and polypeptides encoded by SEQ ID NO: 14 inlcude the use of epitope tags as weU as immunodetectioon.
  • smaU molecule compounds can be identified as drug candidates for pharmaceutical development.
  • the present invention also relates pharmaceutical compositions and methods of treatment comprising these polypeptides and polynucleotides.
  • Screening assays for drug candidates are designed to identify compounds that bind or complex with the polypeptides encoded by the genes identified herein, or otherwise interfere with the interaction of the encoded polypeptides with other ceUular proteins.
  • Such screening assays wiU include assays amenable to high- throughput screening of chemical hbraries, making them particularly suitable for identifying smaU molecule drug candidates.
  • SmaU molecules contemplated include synthetic organic or inorganic compounds, including peptides, preferably soluble peptides, (poly)peptide-i munoglobuhh fusions, antibodies including, without limitation, poly- and monoclonal antibodies and antibody fragments, single-chain antibodies, anti-idiotypic antibodies, and chimeric or humanized versions of such antibodies or fragments, as weU as human antibodies and antibody fragments.
  • the assays can be performed in a variety of formats, including protein-protein binding assays, biochemical screening assays, immunoassays and ceU based assays, which are weU characterized in the art.
  • Isolated antibodies that specificaUy bind to a polypeptide of SEQ ID NO: 14 or a polypeptide encoded by a polynucleotide of SEQ ID NO: 13 can be generated by methods known in the art and screened as drug candidates.
  • Types of antibodies include, but are not limited to polyclonal antibodies, monoclonal antibodies, chimeric antibodies, single chain antibodies, Fab fragments, F(ab) 2 fragments, and humanized antibodies.
  • Assays involve the contacting, under conditions and for a time sufficient to aUow interaction, of the drug candidate with a polypeptide or a polynucleotide that alters E2F activity.
  • binding assays the interaction is binding and the complex formed can be isolated or detected in the reaction mixture.
  • the polypeptide or polynucleotide that alters E2F activity or the drug candidate is immobilized on a sohd phase, e.g., on a microtiter plate, by covalent or non-covalent attachments.
  • Non-covalent attachment generaUy is accomplished by coating the sohd surface with a solution of the polypeptide or polynucleotide and drying.
  • an immobilized antibody e.g., a monoclonal antibody, specific for the polypeptide or polynucleotide to be immobilized can be used to anchor it to a sohd surface.
  • the assay is performed by adding the non-immobilized component, which may be labeUed by a detectable label, to the immobilized component, e.g., the coated surface containing the anchored component.
  • the non-reacted components are removed, e.g.,by washing, and complexes anchored on the sohd surface are detected.
  • the originaUy non-immobilized component carries a detectable label, the detection of label immobilized on the surface indicates that complexing occurred.
  • complexing can be detected, for example, by using a labeUed antibody specificaUy binding the imn obilized complex. If the candidate compound interacts with but does not bind to a polypeptide or polynucleotide that alters E2F activity, its interaction with that molecule can be assayed by methods weU known for detecting interactions. Such assays include traditional approaches, such as, cross-linking, co- immunoprecipitation, and co-purification through gradients or chromato graphic columns.
  • the assay mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the identified gene product alters E2F activity.
  • the mixture components can be added in any order that provides for the requisite activity. Incubation may be performed at any temperature that facihtates optimal binding, typicaUy between about 4°C and 40°C, more commonly between about 15 C C and 40°C. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening, and are typicaUy between about 0.1 and 10 hours, preferably less than 5 hours, more preferably less than 2 hours.
  • a separation step is often used to separate bound and unbound components. Separation may, for example, be effected by precipitation (e.g., TCA precipitation, irnmunoprecipitation, etc.), immobilization (e.g., on a sohd substrate), foUowed by washing.
  • the bound protein is conveniently detected by taking advantage of a detectable label attached to it, e.g.,by measuring radioactive emission, optical or electron density, or by indirect detection using, e.g., antibody conjugates.
  • Suitable compounds that bind to the polypeptide or polynucleotide include polypeptide or polynucleotide fragments or smaU molecules, e.g., peptidomimetics. Such compounds prevent interaction and proper complex formation.
  • SmaU molecule compounds which are usuaUy less than 10 kD molecular weight, are preferable as therapeutics since they are more likely to be permeable to ceUs, are less susceptible to degradation by various ceUular mechanisms, and are not as apt to ehcit an immune response as would proteins or polypeptides.
  • SmaU molecules include but are not limited to synthetic organic or inorganic compounds.
  • Non-limiting examples include proteins, peptides, glycoproteins, glycopeptides, glycohpids, polysaccharides, oligosacchardies, nucleic acids, bioorganic molecules, peptidomimetics, pharmacological agents and theh metabohtes, transcriptional and translation control sequences, and the like.
  • a preferred technique for identifying compounds that bind to the polypeptide or polynucleotide utilizes a chimeric substrate (e.g., epitope-tagged fused or fused immunoadhesin) attached to a sohd phase, such as the weU of an assay plate.
  • a chimeric substrate e.g., epitope-tagged fused or fused immunoadhesin
  • sohd phase such as the weU of an assay plate.
  • optionaUy labeUed e.g., radio labeUed
  • the invention further discloses methods for assessing toxicity of a test compound, said method comprising treating a biological sample containing nucleic acids with the test compound; hybridizing the nucleic acids of the treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide of SEQ ID NO: 13 under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide comprising a polynucleotide sequence of a polynucleotide of SEQ ID NO: 13 or fragment thereof; quantifying the amount of hybridization complex; and comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • the invention further discloses arrays, including microarrays, comprising different nucleotide molecules affixed in distinct physical locations on a sohd substrate, wherein at least one of said nucleotide molecules comprises a first ohgonucleotide or polynucleotide sequence specificaUy hybridizable with at least 30 contiguous nucleotides of a target polynucleotide, said target polynucleotide having a sequence of SEQ ID NO: 13.
  • the present method identifies compounds useful in abrogation of E2F activity by selecting test compounds that exhibit binding affinity to a polynucleotide comprising a sequence of SEQ ID NO: 13.
  • the determination of binding affinities of such test compounds for the present polynucleotides employs in vitro assay methods known in the art.
  • the most preferred test compound also selectively bind the polynucleotides of the present invention.
  • test compounds that exhibit binding affinity are contacted with a first subpopulation of host cells transfected with the polynucleotide for which the test compound has affinity.
  • the host ceUs are preferably primary ceUs, more preferably human primary ceUs, and most preferably HUVEC ceUs.
  • the host ceUs are transfected with the polynucleotide using methods known in the art, for example, as described above in connection with the adenovhal vectors transfection.
  • a second subpopulation of transfected host ceUs is not contacted with the test compound exhibiting binding affinity and is used as a control.
  • the first and second subpopulations of ceUs are then examined for E2F activity to determine if E2F activity has been altered in the first subpopulation relative to the second control subpopulation.
  • E2F activity may be detected by a variety of methods known in the art, including expression of a reporter gene operably linked to multiple E2F binding sites.
  • a reporter sequence is "operably hhked" to a transcription factor binding site (e.g., E2F binding site) when the transcription factor is capable of directing transcription of the reporter sequence upon binding of the transcription factor to the transcription factor binding site.
  • Reporter genes include, but are not limited to, genes encoding for luciferase, EGFP, Renilla luciferase, and alkaline phosphatase. Compounds that alter E2F activity are candidates for pharmaceutical development as anti-pro liferative or anti-apoptotic drugs.
  • a further method for identifying a compound useful in the treatment of apoptosis-associated disorders selects test compounds that exhibit binding affinity to a polypeptide comprising a sequence of SEQ ID NO: 14.
  • the assay methods are sirnUar to those described above, except that the target is the polypeptide in contrast to the polynucleotide.
  • the host ceUs are transfected with an expression vector encoding the polynucleotide that encodes the polypeptide using methods known in the art.
  • the expression vector maybe any suitable expression vector that can express the polypeptide in the host ceU.
  • Preferred expression vectors include adenovhal vectors described herein to transfect such ceUs.
  • a second subpopulation of transfected host ceUs are not contacted with the test compound exhibiting binding affinity, and is used as a control.
  • the fhst and second subpopulations of ceUs are then examined for E2F activity to determine if E2F activity has been altered in the first subpopulation relative to the second control subpopulation.
  • one or more test compounds are contacted with a corresponding number of one or more subpopulations of host ceUs transfected with an expression vector encoding a polynucleotide identified in the hbrary screening methods.
  • polynucleotides to be used in this assay include a polynucleotide comprising a sequence of SEQ ID NO: 13.
  • the host ceUs may be any of the host ceU types used in the methods described above.
  • the transfection may be performed using methods known in the art.
  • Compounds that alter E2F activity in the first subpopulation of ceUs that have been transfected (or transduced) with the expression vector relative to a second subpopulation of host ceUs that have not been contacted with a test compound are selected as drug candidates for pharmaceutical development for the treatment of apoptosis-associated disorders.
  • Another method for identifying drug candidate compounds is based on the measurement, in the ceUular mRNA population of the host ceUs, of mRNA encoded by the polynucleotide comprising a sequence of SEQ ID NO: 13.
  • the level of mRNA expression can be measured by a variety of methods known in the art.
  • a drug candidate compound may be selected by comparing the mRNA expression level in the first subpopulation of host ceUs relative to expression of the mRNA in a second subpopulation of host ceUs that have not been contacted with a test compound.
  • a decrease in the mRNA expression of the above-referenced polynucleotide would identify a compound candidate for pharmaceutical development for the treatment of apoptosis-associated disorders. Identification of Test Compounds that Bind to SEQ ID NOS: 13 or 14
  • the present method identifies compounds useful in the treatment of apoptosis-associated disorders by selecting test compounds that exhibit binding affinity to a polynucleotide comprising a sequence of SEQ ID NO: 13 or to a polypeptide comprising a sequence of SEQ ID NO: 14.
  • One such method is based on polypeptide binding and contacts a test compound with a polypeptide identified in the above-described adenovhal hbrary screening methods. Examples of such polypeptides include SEQ ID NO: 14.
  • the binding affinity of the test compound for the polypeptide is then determined using methods known in the art.
  • the binding affinity may be in a nanomolar to micromolar concentrations, with nanomolar concentration preferred.
  • a further aspect of this method contacts a test compound that exhibits binding affinity to the target polypeptide with a first subpopulation of host ceUs.
  • the host ceUs maybe any ceUs that aUow activation of E2F.
  • Preferred ceUs include immortal ceUs, such as neoplastic cehs.
  • Drug candidate compounds are selected from test compounds that bind to the aforesaid polypeptide and that induce an increase in expression of mRNA corresponding to a polynucleotide comprising a sequence of SEQ ID NO: 13 in the first subpopulation relative to expression of mRNA in a second subpopulation of host ceUs that has not been contacted with the test compound.
  • Another aspect of the present method comprises the contacting of a test compound that exhibits binding affinity for the polypeptide with a first subpopulation of host ceUs transfected with an expression vector encoding such polypeptide.
  • a test compound that exhibits binding affinity for the polypeptide
  • a first subpopulation of host ceUs transfected with an expression vector encoding such polypeptide.
  • Such fhst subpopulation of host ceUs is examined to determine if E2F activity is enhanced in the fhst subpopulation relative to a second subpopulation that is not contacted with such compound.
  • the first subpopulation of host ceUs ma be transfected with a lower MOI than used in the adenovhal Hbrary assay method described above, for example, using an MOI lower than that used in the hbrary screening method.
  • the method can be adapted using an MOI titration to determine the activity of the test compound.
  • Exemplary MOIs can range from 0-10%, 10-20%, 20-50% of the standard MOI.
  • the present method is capable of a more sensitive determination of compounds that induce E2F activity.
  • Compounds that exhibit binding affinity for the polypeptide and enhance E2F activity in the fhst subpopulation of host ceUs treated with said compound relative to a control untreated subpopulation of host ceUs are selected as drug candidate compounds.
  • the control subpopulation of host ceUs is preferably transfected using the same MOI as the first subpopulation of host ceUs.
  • one or more test compounds are contacted with a corresponding number of one or more first subpopulations of host ceUs transfected with an expression vector encoding a polynucleotide identified in the hbrary screening methods.
  • expression vectors to be used include expression vectors comprising a polynucleotide sequence of SEQ ID NO: 13.
  • the test compounds in accordance with this method may or may not have been previously identified as having any binding affinity to the aforesaid polypeptides or polynucleotides.
  • a drug candidate compound is selected from those compounds that enhance E2F activity in the first subpopulation of host ceUs relative to a second subpopulation of host ceUs that have not been contacted with such compound.
  • a drug candidate compound is selected from those compounds that induce an increase in expression of mRNA encoded by a polynucleotide identified using the above-described Hbrary screening method in a first subpopulation of ceUs relative to expression of said mRNA in a second subpopulation of host ceUs that has not been contacted with such test compound.
  • the preferred mRNA populations measured in this method are encoded by a polynucleotide comprising a sequence of SEQ ID NO: 13.
  • the level of expression of mRNA can be measured by a variety of methods known in the art.
  • a third population of ceUs comprising primary ceUs are contacted with test compounds that exhibit binding affinity to said target polypeptide or polynucleotide.
  • Test compounds that alter E2F activity in the neoplastic host ceUs and are not toxic to the primary ceUs are selected preferentiaUy.
  • Such compounds are candidates for drug development.
  • a particularly preferred drug candidate comprises compounds that induce apoptosis in the neoplastic host cells and that do no affect the primary ceU hosts.
  • the adenoviruses can be tested in appropriate animal models.
  • animal models that can be used include models for Alzheimer's disease, arteriosclerosis, cancer metastasis, and Parkinson's disease.
  • transgenic animals which have altered expression of endogenous or exogenous genes including mice with gene(s) that have been inactivated, animals with cancers implanted at specific sites, human bone marrow chimeric mice such as NOD-SCID mice, and the like can be used.
  • the stocks of candidate adenoviruses can be increased by passaging the adenoviruses under the appropriate transcomplementing conditions.
  • adenovhal vectors or mixtures of pre-selected pools of adenovhal vectors can be apphed or administered at appropriate sites such as lung in non-human primates (Sene, et al. (1995) Hum. Gene Ther. 6(12): 1587-93) and brain of normal and apoE deficient mice (Robertson, et al. (1998) Neuroscience 82(l):171-80.) for Alzheimer's disease (Walker, et al. (1997) Brain Res. Brain Res. Rev.
  • adenovhal vectors or mixtures of pre-selected pools of adenovhal vectors can also be injected in the blood stream for hver disease models including hver fahure and WUson disease (Cuthbert, (1995) J. Investig. Med. 43(4):323-36; Karrer, et al. (1984) Curr. Surg. 41(6):464-7) and tumor models including metastases models (Esandi, et al. (1991) Gene Ther.
  • selected adenovhal vectors can be injected directly into the bone marrow of human chimeric NOD-SCID mice (Dick, et al. (1997) Stem Cells 15 Suppl. 1:199-203; Mosier, et al. (1988) Nature 335(6187):256-9).
  • FinaUy selected adenovirus can be apphed locaUy, for example, in vascular tissue of restenosis animal models (Karas, et al. (1992) J. Am. Coll. Cardiol. 20(2):467-74).
  • animal models of apoptosis-associated disorders can be used to test the efficacy of the drug candidate compounds, including the polypeptides, nucleic acids, antibodies, and agonists and antagonists of the target molecules.
  • the in vivo nature of such models makes them particularly predictive of responses in human patients.
  • Animal models include both non-recombinant and recombinant (transgenic) animals.
  • Non-recombinant animal models include, for example, rodent, e.g., murine models. Examples of animal models that exhibit the apoptosis-associated condition and that are useful in testing the efficacy of candidate therapeutic agents are described hereafter.
  • transgenic animal models can be engineered by introducing the coding portion of the genes identified herein into the genome of animals of interest, using standard techniques for producing transgenic arhmals.
  • a transgenic animal is one containing a "transgene" or genetic material integrated into the genome introduced into the animal itself or an ancestor of the animal at a prenatal stage (e.g., embryonic stage).
  • Animals that can serve as a target for transgenic manipulation include, without limitation, mice, rats, rabbits, guinea pigs, sheep, goats, pigs, and non-human primates, e.g., baboons, chimpanzees and monkeys.
  • transgenic animals include those that carry the transgene only in part of theh ceUs ("mosaic animals").
  • the transgene can be integrated either as a single transgene, or in concatamers, e.g., head-to-head or head-to-taU tandems.
  • Selective introduction of a transgene into a particular ceU type is also possible by foUowing, for example, the technique of Lakso, et al. (1992) Proc. Natl. Acad. Sci. USA 89(14):6232-36.
  • the expression of the transgene in transgenic animals can be monitored by standard techniques. For example, Southern blot analysis or PCR amplification can be used to verify the integration of the transgene. The level of mRNA expression can then be analyzed using techniques such as in situ hybridization, Northern blot analysis, PCR, or immunocytochemistry, The animals are further examined for signs of tumor or cancer development.
  • "knock out" animals can be constructed which have a defective or altered gene encoding gene identified in the screen, as a result of homologous recombination between the endogenous gene encoding the gene and altered genomic DNA encoding the same polypeptide introduced into an embryonic ceU of the animal.
  • cDNA encoding an identified gene can be used to clone genomic DNA encoding that polypeptide in accordance with estabhshed techniques.
  • a portion of the genomic DNA encoding an identified gene can be deleted or replaced with another gene, such as a gene encoding a selectable marker that can be used to monitor integration.
  • the vector is introduced into an embryonic stem ceU line (e.g., by electroporation) and ceUs in which the introduced DNA has homologously recombined with the endogenous DNA are selected (see e.g. , Li, et al. (1992) Cell 69(6): 915-26).
  • ceUs are then injected into a blastocyst of an animal (e.g., a mouse or rat) to form aggregation chimeras (see e.g., Bradley, (1987) in Terato carcinomas and Embryonic Stem CeUs: A Practical Approach, E. J. Robertson, ed. IRL, Oxford, 113-1521).
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term to create a "knock out" animal.
  • Progeny harboring the homologously recombined DNA in theh germ ceUs can be identified by standard techniques and used to breed animals in which aU ceUs of the animal contain the homologously recombined DNA. Knockout animals can be characterized for instance, by theh abihty to defend against certain pathological conditions and by theh development of pathological conditions due to absence of the identified gene.
  • Codons maybe selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • Other reasons for substantiaUy altering a nucleotide sequence without altering the encoded arnino acid sequences include the production of RNA transcripts having more deshable properties, such as a greater half-life, than transcripts produced from the naturaUy occurring sequence.
  • the invention also encompasses production of DNA sequences that encode derivatives or fragments of the polypeptide encoded by the nucleic acid sequence identified using the methods of the present invention, enthely by synthetic chemistry.
  • the synthetic sequence maybe inserted into any of the many avaUable expression vectors and ceU systems using reagents weU known in the art.
  • synthetic chemistry maybe used to introduce any deshed mutations.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and, in particular, to those shown in SEQ ID NO: 13, and fragments thereof under various conditions of stringency (See, e.g., Wahl and Berger, (1987) Methods Enzymol. 152:399-407; Kirnmel, (1987) Methods Enzymol.
  • stringent salt concentration wiU ordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, and most preferably less than about 250 mM NaCl and 25 mM trisodium citrate.
  • Low stringency hybridization can be obtained in the absence of organic solvent, e.g., formamide, whUe high stringency hybridization can be obtained in the presence of at least about 35% formamide, and most preferably at least about 50% formamide.
  • Stringent temperature conditions wiU ordinarily include temperatures of at least about 30°C, more preferably of at least about 37°C, and most preferably of at least about 42°C. Varying additional parameters, such as hybridization time, the concentration of detergent, e.g., sodium dodecyl sulfate (SDS), and the inclusion or exclusion of carrier DNA, are weU known to those skiUed in the art. Various levels of stringency are accomphshed by combining these various conditions as needed,
  • wash stringency conditions can be defined by salt concentration and by temperature. As above, wash stringency can be increased by decreasing salt concentration or by increasing temperature.
  • stringent salt concentration for the wash steps wiU preferably be less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.
  • Stringent temperature conditions for the wash steps wiU ordinarUy include temperature of at least about 25°C, more preferably of at least about 42°C, and most preferably of at least about 68"C.
  • wash steps wiU occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, wash steps wiU occur at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a most preferred embodiment, wash steps wiU occur at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additional variations of these conditions are readUy apparent to those skiUed in the art. Polynucleic Acids Identified by the Present Invention
  • the present invention further relates to the polynucleotides identified in the practice of the method invention described hereinabove, more particularly, those isolated nucleic acids found capable of altering E2F activity.
  • the polynucleotides having the sequences of SEQ ID NO: 13 comprise polynucleotides of the present invention.
  • the present invention also utilizes antisense nucleic acids that can be used to down-regulate or block the expression of polypeptides capable of altering E2F activity in vitro, ex vivo, or in vivo.
  • the down regulation of gene expression using antisense nucleic acids can be achieved at the translational or transcriptional level.
  • Antisense nucleic acids of the invention are preferably nucleic acid fragments capable of specificaUy hybridizing with aU or part of a nucleic acid encoding a polypeptide capable of altering E2F activity or the corresponding messenger RNA.
  • antisense nucleic acids maybe designed or identified which decrease expression of the nucleic acid sequence capable of altering E2F activity by inhibiting splicing of its primary transcript. With knowledge of the structure and partial sequence of a nucleic acid capable of altering E2F activity, such antisense nucleic acids can be designed and tested for efficacy.
  • the antisense nucleic acids are preferably ohgonucleotides and may consist entirely of deoxyribo -nucleotides, modified deoxyribonucleotides, or some combination of both.
  • the antisense nucleic acids can be synthetic oHgonucleo tides.
  • the ohgonucleotides maybe chemicaUy modified, if deshed, to improve stability and/or selectivity. Since ohgonucleotides are susceptible to degradation by intraceUular nucleases, the modifications can include, for example, the use of a sulfur group to replace the free oxygen of the phosphodiester bond. This modification is caUed a phosphorothioate linkage.
  • Phosphorothioate antisense ohgonucleotides are water soluble, polyanionic, and resistant to endogenous nucleases.
  • the RNA- DNA duplex activates the endogenous enzyme ribonuclease (RNase) H, which cleaves the mRNA component of the hybrid molecule.
  • RNase ribonuclease
  • antisense ohgonucleotides with phosphoramidite and polyamide (peptide) linkages can be synthesized. These molecules should be very resistant to nuclease degradation.
  • chemical groups can be added to the 2' carbon of the sugar moiety and the 5 carbon (C-5) of pyrimidines to enhance stabihty and facilitate the binding of the antisense oHgonucleotide to its target site. Modifications may include 2' deoxy, O-pentoxy, O-propoxy, O-methoxy, fluoro, methoxyethoxy phosphoro-thioates, modified bases, as weU as other modifications known to those of skiU in the art.
  • Antisense nucleic acids can be prepared by expression of aU or part of a sequence selected from the group consisting of SEQ ID NO: 13, in the opposite orientation. Any length of antisense sequence is suitable for practice of the invention so long as it is capable of down-regulating or blocking expression of a nucleic acid capable of altering E2F activity. Preferably, the antisense sequence is at least about 20 nucleotides in length.
  • the preparation and use of antisense nucleic acids, DNA encoding antisense RNAs and the use of ohgo and genetic antisense is known in the art.
  • One approach to determining the optimum fragment of a nucleic acid sequence capable of altering E2F activity in an antisense nucleic acid treatment method involves preparing random cDNA fragments of a nucleic acid capable of altering E2F activity by mechanical shearing, enzymatic treatment, and cloning the fragment into any of the vector systems described herein. Individual clones or pools of clones are used to infect ceUs expressing the polypeptide and effective antisense cDNA fragments are identified by monitoring expression at the RNA or protein level.
  • a variety of viral-based systems including retroviral, adeno-associated vhal, and adenovhal vector systems may aU be used to introduce and express antisense nucleic acids in ceUs.
  • Antisense synthetic ohgonucleotides maybe introduced into the body of a patient in a variety of ways, as discussed below.
  • Ribozymes are catalytic RNA molecules (RNA enzymes) that have separate catalytic and substrate binding domains.
  • the substrate binding sequence combines by nucleotide complementarity and, possibly, nonhydrogen bond interactions with its target sequence.
  • the catalytic portion cleaves the target RNA at a specific site.
  • the substrate domain of a ribozyme can be engineered to dhect it to a specified mRNA sequence. The ribozyme recognizes and then binds a target mRNA through complementary base-pairing.
  • the ribozyme acts enzymaticaUy to cut the target mRNA. Cleavage of the mRNA by a ribozyme destroys its abihty to dhect synthesis of the corresponding polypeptide. Once the ribozyme has cleaved its target sequence, it is released and can repeatedly bind and cleave at other mRNAs.
  • Ribozyme forms include a hammerhead motif, a hairpin motif, a hepatitis delta virus, group I intron or RNaseP RNA (in association with an RNA guide sequence) motif or Neurospora VS RNA motif.
  • Ribozymes possessing a hammerhead or hairpin structure are readUy prepared since these catalytic RNA molecules can be expressed within ceUs from eukaryotic promoters (Chen, et al. ( 1992) Nucleic Acids Res. 20:4581 -9) .
  • a ribozyme of the present invention can be expressed in eukaryotic ceUs from the appropriate DNA vector. If deshed, the activity of the ribozyme may be augmented by its release from the primary transcript by a second ribozyme (Ventura, et al. (1993) Nucleic Acids Res. 21 :3249-55).
  • Ribozyme may be chemicaUy synthesized by combining an oHgodeoxyribonucleotide with a ribozyme catalytic domain (20 nucleotides) flanked by sequences that hybridize to the target mRNA after transcription.
  • the oHgodeoxyribonucleotide is ampHfied by using the substrate binding sequences as primers.
  • the amplification product is cloned into a eukaryotic expression vector.
  • Ribozymes are expressed from transcription units inserted into DNA, RNA, or vhal vectors. Transcription of the ribozyme sequences are driven from a promoter for eukaryotic RNA polymerase I (pol I, RNA polymerase II (pol II), or RNA polymerase III (pol III). Transcripts from pol II or pol III promoters wiU be expressed at high levels in aU ceUs; the levels of a given pol II promoter in a given ceU type wUl depend on nearby gene regulatory sequences.
  • Prokaryotic RNA polymerase promoters are also used, providing that the prokaryotic RNA polymerase enzyme is expressed in the appropriate ceUs (Gao and Huang, (1993) Nucleic Acids Res. 21 :2867-72). It has been demonstrated that ribozymes expressed from these promoters can function in mammahan ceUs (Kashani-Sabet, et al. (1992) Antisense Res. Dev. 2:3-15).
  • the ribozyme sequence of the present invention is inserted into a plasmid DNA vector, a retrovirus vector, an adenovirus DNA vhal vector or an adeno-associated virus vector.
  • DNA may be dehvered alone or complexed with various vehicles.
  • the DNA, DNA/vehicle complexes, or the recombinant virus particles are locaUy administered to the site of treatment, as discussed below.
  • recombinant vectors capable of expressing the ribozymes are locaUy dehvered as described below, and persist in target ceUs. Once expressed, the ribozymes cleave the target mRNA.
  • Ribozymes may be administered to a patient by a variety of methods. They may be added directly to target tissues, complexed with cationic hpids, packaged within hposomes, or dehvered to target ceUs by other methods known in the art. Localized administration to the deshed tissues ma be done by catheter, infusion pump or stent, with or without incorporation of the ribozyme in biopolymers. Alternative routes of dehvery include, but are not limited to, intravenous injection, intramuscular injection, subcutaneous injection, aerosol inhalation, oral (tablet or pUl form), topical, systemic, ocular, intraperitoneal and/or intrathecal dehvery. DetaUed descriptions of ribozyme dehvery and administration are provided in Sullivan et ⁇ l. WO 94/02595.
  • the present invention also relates to methods for expressing a polypeptide or polynucleotide identified as capable of altering E2F activity as a gene therapeutic.
  • the vhal vectors used in the gene therapy methods of the present invention are rephcation defective.
  • Such rephcation defective vectors wiU usuaUy lack at least one region that is necessary for the rephcation of the virus in the infected ceU. These regions can either be eliminated (in whole or in part), or be rendered non-functional by any technique known to a person skiUed in the art. These techniques include the total removal, substitution, partial deletion or addition of one or more bases to an essential (for rephcation) region. Such techniques maybe performed in vitro (on the isolated DNA) or in situ, using the techniques of genetic manipulation or by treatment with mutagenic agents.
  • the rephcation defective virus retains the sequences of its genome, which are necessary for encapsidating, the vhal particles.
  • Retroviruses are integrating viruses that infect dividing ceUs, and theh construction is known in the art.
  • Retroviral vectors can be constructed from different types of retrovirus, such as, MoMuLV ("murine Moloney leukemia virus” MSV ("murine Moloney sarcoma virus"), HaSV ("Harvey sarcoma virus”); SNV ("spleen necrosis virus”); RSV ("Rous sarcoma virus”) and Friend virus.
  • MoMuLV murine Moloney leukemia virus
  • MSV murine Moloney sarcoma virus
  • HaSV Harmonic sarcoma virus
  • SNV spleen necrosis virus
  • RSV Ra sarcoma virus
  • Friend virus Friend virus.
  • Lentivhus vector systems may also be used in the practice of the present invention.
  • adeno-associated viruses are utilized.
  • the AAN viruses are D ⁇ A viruses of relatively smaU size that integrate, in a stable and site-specific manner, into the genome of the infected ceUs. They are able to infect a wide spectrum of ceUs without inducing any effects on ceUular growth, morphology or differentiation, and they do not appear to be involved in human pathologies.
  • Promoters that may be used in the expression vectors of the present invention include both constitutive promoters and regulated (inducible) promoters.
  • the promoters may be prokaryotic or eukaryotic depending on the host.
  • prokaryotic (including bacteriophage) promoters useful for practice of this invention are lad, lacZ, T3, T7, lambda P r , P b and trp promoters.
  • eukaryotic (including vhal) promoters useful for practice of this invention are ubiquitous promoters (e.g.,HPRT, vimentin, actin, tubulin), intermediate filament promoters (e.g.,desmin, neurofilaments, keratin, GFAP), therapeutic gene promoters (e.g.,MDR type, CFTR, factor VIII), tissue-specific promoters (e.g., actin promoter in smooth muscle ceUs, or Fit and Flk promoters active in endothehal ceUs), including animal transcriptional control regions, which exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar ceUs (Swift, et al.
  • ubiquitous promoters e.g.,HPRT, vimentin, actin, tubulin
  • intermediate filament promoters e.g.,desmin, neurofilaments, keratin, GFAP
  • therapeutic gene promoters
  • mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast ceUs
  • albumin gene control region which is active in hver
  • alpha-fetoprotein gene control region which is active in hver
  • alpha 1-antitrypsin gene control region which is active in the hver (Kelsey, et al (1987) Genes and Devel, 1:161-71), beta-globin gene control region which is active in myeloid ceUs (Mogram, et al.
  • promoters which may be used in the practice of the invention include promoters which are preferentiaUy activated in dividing ceUs, promoters which respond to a stimulus (e.g., steroid hormone receptor, retinoic acid receptor), tetracycline-regulated transcriptional modulators, cytomegalovhus immediate-early, retro vhal LTR, metaUothionein, SV-40, Ela, and MLP promoters.
  • a stimulus e.g., steroid hormone receptor, retinoic acid receptor
  • tetracycline-regulated transcriptional modulators e.g., cytomegalovhus immediate-early, retro vhal LTR, metaUothionein, SV-40, Ela, and MLP promoters.
  • Additional vector systems include the non-viral systems that facilitate introduction of DNA encoding the polypeptides capable of altering E2F activity, the polynucleotides encoding these polypeptides, or antisense nucleic acids into a patient.
  • a DNA vector encoding a deshed sequence can be introduced in vivo by Hpofection.
  • Synthetic cationic hpids designed to limit the difficulties encountered with Hposome mediated transfection can be used to prepare hposomes for in vivo transfection of a gene encoding a marker (Feigner, et. al. (1987) Proc. Natl. Acad. Sci. USA 84:7413-7); see Mackey, et al. (1988) Proc.
  • cationic hpids may promote encapsulation of negatively charged nucleic acids, and also promote fusion with negatively charged ceU membranes (Feigner and Ringold, (1989) Nature 337:387-8).
  • Particularly useful Hpid compounds and compositions for transfer of nucleic acids are described in International Patent Pubhcations WO 95/18863 and WO 96/17823, and in U.S. Patent No. 5,459,127.
  • hpofection to introduce exogenous genes into the specific organs in vivo has certain practical advantages and directing transfection to particular ceU types would be particularly advantageous in a tissue with ceUular heterogeneity, for example, pancreas, hver, kidney, and the brain.
  • Lipids ma be chemicaUy coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins for example, antibodies, or non-peptide molecules could be coupled to Hposomes chemicaUy.
  • a cationic ohgopeptide e.g., International Patent Publication WO 95/21931
  • peptides derived from DNA binding proteins e.g., International Patent Pubhcation WO 96/25508
  • a cationic polymer e.g., International Patent Pubhcation WO 95/21931.
  • the present invention also relates to the polypeptides, or subfragments thereof, which have been identified by the practice of the present method invention as capable of altering E2F activity.
  • polypeptides include for example, the polypeptides that are encoded by nucleic acids, including, for example, SEQ ID NO: 14, or which comprise antibodies capable of binding to such polypeptides encoded by such nucleic acids.
  • the polypeptides of the present invention may be prepared by recombinant technology methods, isolated from natural sources, or prepared syntheticaUy, and maybe of human, or other animal origin,
  • the polypeptides of the present invention maybe unglycosylated or modified subsequent to translation. Such modifications include glycosylation, phosphorylation, acetylation, myristoylation , methylation, isoprenylation, and palmitoylation.
  • Preferred glycosylated polypeptides are produced in mammahan ceUs, and most preferably in human ceUs, a particular embodiment of which are the PER.C6 ceUs.
  • the nucleic acid encoding the polypeptide is inserted into a suitable vector, which is inserted into a suitable host ceU.
  • the polypeptide produced by the resulting host ceU is recovered and purified.
  • the polypeptides are characterized by amino acid composition and sequence, and biological activity. Other ways to characterize the polypeptides include reproducible single molecular weight and/or multiple set of molecular weights, chromatographic response and elution profiles,
  • the present invention also provides antibodies directed against polypeptides capable of altering E2F activity. These antibodies may be monoclonal antibodies or polyclonal antibodies.
  • the present invention includes chimeric, single chain, and humanized antibodies, as weU as FAb fragments and the products of an FAb expression hbrary, and Fv fragments and the products of an Fv expression Hbrary.
  • polyclonal antibodies may be used in the practice of the invention. Methods of preparing polyclonal antibodies are known to the skiUed artisan. Polyclonal antibodies can be raised in a mammal, for example, by one or more injections of an immunizing agent and, if deshed, an adjuvant. TypicaUy, the immunizing agent and/or adjuvant wiU be injected in the mammal by multiple subcutaneous or intraperitoneal injections. The immunizing agent may include the identified gene product or a fusion protein thereof. Antibodies may also be generated against the intact protein or polypeptide, or against a fragment, derivative, or epitope of the protein or polypeptide, by using for example a Hbrary of antibody variable regions, such as a phage display hbrary.
  • immunogenic proteins include but are not limited to keyhole limpet hemocyanin, serum albumin, bovine thyro globulin, and soybean trypsin inhibitor.
  • adjuvants that may be employed include Freund's complete adjuvant and MPL-TDM adjuvant
  • the irrmiunization protocol may be selected by one skiUed in the art without undue experimentation.
  • the antibodies maybe monoclonal antibodies.
  • Monoclonal antibodies may be prepared using methods known in the art.
  • the monoclonal antibodies of the present invention may be "humanized” to prevent the host from mounting an immune response to the antibodies,
  • a "humanized antibody” is one in which the complementarity deterrnining regions (CDRs) and/or other portions of the Hght and/or heavy variable domain framework are derived from a non- human immunoglobulin, but the rernaining portions of the molecule are derived from one or more human immunoglobulins.
  • CDRs complementarity deterrnining regions
  • Humanized antibodies also include antibodies characterized by a humanized heavy chain associated with a donor or acceptor unmodified hght chain or a chimeric hght chain, or vice versa.
  • the humanization of antibodies maybe accompHshed by methods known in the art (see, e.g.,Mark and Padlan, (1994) "Chapter 4. Humanization of Monoclonal Antibodies", The Handbook of Experimental Pharmacology Vol. 113, Springer- Verlag, New York).
  • Transgenic animals may be used to express humanized antibodies.
  • Human antibodies can also be produced using various techniques known in the art, including phage display hbraries (Hoogenboom and Winter, (1991) J. Mol Biol. 227:381-8; Marks, et al (1991). J.
  • the antibodies may be monovalent antibodies.
  • Methods for preparing monovalent antibodies are weU known in the art. For example, one method involves recombinant expression of immunoglobulin hght chain and modified heavy chain, The heavy chain is truncated generaUy at any point in the Fc region so as to prevent heavy chain crosslinking. Alternatively, the relevant cysteine residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking.
  • Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens.
  • a particularly preferred aspect of the present invention is an antibody that binds to a polypeptide capable of altering E2F activity and that is used to inhibit the activity of the polypeptide in a patient.
  • Antibodies as discussed above are also useful in assays for detecting or quantitating levels of a polypeptide capable of altering E2F activity. In one embodiment, these assays provide a clinical diagnosis and assessment of such polypeptides in various disease states and a method for monitoring treatment efficacy,
  • a polypeptide of the invention would have an activity that alters E2F activity; a nucleic acid would be able to rephcate, translate a message, or hybridize to a complementary nucleic acid; a vector would be able to transfect a target ceU; an antibody would bind a polypeptide identified by the present invention.
  • a preferred biologicaUy compatible composition is an aqueous solution that is buffered using, e.g., Tris, phosphate, or HEPES buffer, containing salt ions. UsuaUy the concentration of salt ions wiU be simUar to physiological levels.
  • BiologicaUy compatible solutions may include stabilizing agents and preservatives.
  • the biocompatible composition is a pharmaceuticaUy acceptable composition.
  • Such compositions can be formulated for administration by topical, oral, parenteral, intranasal, subcutaneous, and intraocular, routes. Parenteral administration is meant to include intravenous injection, intramuscular injection, intraarterial injection or infusion techniques.
  • the composition maybe administered parenteraUy in dosage unit formulations containing standard, weU known non-toxic physiologicaUy acceptable carriers, adjuvants and vehicles as deshed.
  • compositions for oral administration can be formulated using pharmaceuticaUy acceptable carriers weU known in the art in dosages suitable for oral administration. Such carriers enable the pharmaceutical compositions to be formulated as tablets, piUs, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • Pharmaceutical compositions for oral use can be prepared by combining active compounds with sohd excipient, optionaUy grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if deshed, to obtain tablets or dragee cores.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl-pyrrohdone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinyl-pyrrohdone, carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • compositions that can be used oraUy include push-fit capsules made of gelatin, as weU as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with a fiUer or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate, and, optionaUy, stabilizers.
  • the active compounds may be dissolved or suspended in suitable Hquids, such as fatty oUs, Hquid, or hquid polyethylene glycol with or without stabilizers.
  • composition of the present invention comprise a rephcation defective recombinant vhal vector and the polynucleotide identified by the present invention and a transfection enhancer, such as poloxamer.
  • a poloxamer is Poloxamer 407, which is commerciaUy available (BASF, Parsippany, NJ) and is a non-toxic, biocompatible polyol.
  • a poloxamer impregnated with recombinant viruses maybe deposited directly on the surface of the tissue to be treated, for example during a surgical intervention. Poloxamer possesses essentiaUy the same advantages as hydrogel while having a lower viscosity.
  • the formulation herein may also contain more than one active compound as necessary for the particular indication being treated, preferably those with complementary activities that do not adversely affect each other.
  • the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent.
  • cytotoxic agent cytokine or growth inhibitory agent.
  • Such molecules are suitably present in combination in amounts that are effective for the purpose intended.
  • the formulations to be used for in vivo administration must be sterUe. This is readUy accompHshed by filtration through sterUe filtration membranes.
  • the active ingredients may also be entrapped in microcapsules prepared, for example, by interfacial polymerization, for example, hydroxymethylceUulose or gelatin-microcapsules and ⁇ oly-(methylmethacylate) microcapsules, respectively, in cohoidal drug dehvery systems (for example, hposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules) or in macroemulsions.
  • cohoidal drug dehvery systems for example, hposomes, albumin microspheres, microemulsions, nano-particles and nanocapsules
  • Sustained-release preparations may be prepared. Suitable examples of sustained-release preparations include semipermeable matrices of sohd hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Patent No.
  • copolymers of L- glutamic acid and ⁇ -ethyl-L-glutamate non-degradable ethylene-vinyl acetate
  • degradable lactic acid-glycoHc acid copolymers such as the LUPRON DEPOTTM (injectable microspheres composed of lactic acid-glycoHc acid copolymer and leuprohde acetate)
  • poly-D-(-)-3-hydroxybutyric acid WhUe polymers such as ethylene-vinyl acetate and lactic acid-glycoHc acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods.
  • encapsulated antibodies When encapsulated antibodies remain in the body for a long time, they may denature or aggregate as a result of exposure to moisture at 37°C, resulting in a loss of biological activity and possible changes in immunogenicity. Rational strategies can be devised for stabilization depending on the mechanism involved. For example, if the aggregation mechanism is discovered to be intermolecular S-S bond formation through thio-disulfide interchange, stabilization may be achieved by modifying sulfhydryl residues, lyophihzing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.
  • the present invention provides methods of treatment, which comprise the administration to a human or other animal of an effective amount of a composition of the invention.
  • a therapeuticaUy effective dose refers to that amount of protein, polynucleotide, peptide, or its antibodies, agonists or antagonists, which amehorate the symptoms or condition.
  • Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in ceU cultures or experimental animals, e.g., ED50 (the dose therapeuticaUy effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from ceU culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with Httle or no toxicity.
  • the dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the therapeuticaUy effective dose can be estimated initiaUy either in ceU culture assays or in animal models, usuaUy mice, rabbits, dogs, or pigs.
  • the animal model is also used to achieve a deshable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated.
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the deshed effect. Additional factors which may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, deshed duration of treatment, method of administration, time and frequency of adnhhistration, drug combinations), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions might be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Antibodies according to the invention may be dehvered as a bolus only, infused over time or both administered as a bolus and infused over time.
  • Those skiUed in the art may employ different formulations for polynucleotides than for proteins. SimUarly, dehvery of polynucleotides or polypeptides wiU be specific to particular ceUs, conditions, locations, etc.
  • recombinant viruses may be used to introduce both DNA encoding polypeptides capable of altering E2F activity as weU as antisense polynucleotides.
  • Recombinant viruses according to the invention are generaUy formulated and administered in the form of doses of between about 10 4 and about 10 14 pfu, In the case of AANs and adenoviruses, doses of from about 10 6 to about 10 11 pfu are preferably used.
  • the term pfu (“plaque-forming unit”) corresponds to the infective power of a suspension of virions and is determined by infecting an appropriate ceU culture and measuring the number of plaques formed.
  • Ribozymes according to the present invention ma be administered in a pharmaceuticaUy acceptable carrier. Dosage levels maybe adjusted based on the measured therapeutic efficacy.
  • the methods for decreasing the expression of a polypeptide capable of altering E2F activity and correct those conditions in which polypeptide activity contributes to a disease or disorder associated with an undesirable level of E2F activity include but are not limited to administration of a composition comprising an antisense nucleic acid, administration of a composition comprising an intraceUular binding protein such as an antibody, administration of a molecule that inhibits the activity of the polypeptide, for example, a smaU molecular weight molecule, including administration of a compound that down regulates expression at the level of transcription, translation or post-translation, and administration of a ribozyme which cleaves mRNA encoding the polypeptide.
  • the present invention in a particular embodiment, relates to a composition comprising an antisense polynucleotide that is used to down-regulate or block the expression of polypeptides capable of altering E2F activity.
  • the nucleic acid encodes antisense RNA molecules.
  • the nucleic acid is operably linked to signals enabling expression of the nucleic acid sequence and is introduced into a ceU utilizing, preferably, recombinant vector constructs, which wUl express the antisense nucleic acid once the vector is introduced into the ceU.
  • suitable vectors includes plasmids, adenoviruses, adeno-associated viruses, retroviruses, and herpes viruses.
  • the vector is an adenovirus.
  • the vector is a rephcation defective adenovirus comprising a deletion in the El and/or E3 regions of the virus.
  • the antisense sequence comprises aU or a portion of a polynucleotide complementary to SEQ ID NO: 13.
  • the antisense nucleic acid is synthesized and maybe chemicaUy modified to resist degradation by intraceUular nucleases, as discussed above.
  • Synthetic antisense ohgonucleotides can be introduced to a ceU using hposomes. CeUular uptake occurs when an antisense oHgonucleotide is encapsulated within a hposome. With an effective dehvery system, low, non-toxic concentrations of the antisense molecule can be used to inhibit translation of the target mRNA.
  • hposomes that are conjugated with ceU-specific binding sites dhect an antisense oHgonucleotide to a particular tissue.
  • Another aspect of the present invention relates to the down-regulation or blocking of the expression of a polypeptide capable of altering E2F activity by the induced expression of a polynucleotide encoding an intraceUular binding protein that is capable of selectively interacting with the polypeptide identified by the present method invention
  • An intraceUular binding protein includes any protein capable of selectively interacting, or binding, with the polypeptide in the ceU in which it is expressed and neutralizing the function of the polypeptide.
  • the intraceUular binding protein is a neutralizing antibody or a fragment of a neutralizing antibody. More preferably, the intraceUular binding protein is a single chain antibody.
  • WO 94/02610 discloses preparation of antibodies and identification of the nucleic acid encoding a particular antibody. Using a polypeptide capable of altering E2F activity or a fragment thereof, a specific monoclonal antibody is prepared by techniques known to those skiUed in the art. A vector comprising the nucleic acid encoding an intraceUular binding protein, or a portion thereof, and capable of expression in a host ceU is subsequently prepared for use in the method of this invention.
  • the activity of a polypeptide capable of altering E2F activity can be blocked by administration of a neutralizing antibody into the circulation.
  • a neutralizing antibody can be administered dhectly as a protein, or it can be expressed from a vector that also codes for a secretory signal.
  • smaU molecule compounds inhibit the activity of a polypeptide that alters E2F activity. These low molecular weight compounds interfere with the polypeptide's enzymatic properties or prevent its appropriate recognition by ceUular binding sites.
  • the present invention also involves the use of smaU molecule compounds to down regulate expression of a polypeptide that is capable of altering E2F activity at the level of transcription, translation or post-translation. Reporter gene systems may be used to identify such inhibitory compounds.
  • These inhibitory compounds may be combined with a pharmaceuticaUy acceptable carrier and administered using conventional methods known in the art.
  • the methods for increasing the expression or activity of a polypeptide capable of altering E2F activity polypeptide include, but are not limited to, administration of a composition comprising the polypeptide, administration of a composition comprising an expression vector that encodes the polypeptide, administration of a composition comprising a compound that enhances the enzymatic activity of the polypeptide and administration of a compound that increases expression of the gene encoding the polypeptide.
  • the level of activity is increased through the administration of a composition comprising the polypeptide.
  • This composition maybe administered in a convenient manner, such as by the oral, topical, intravenous, intraperitoneal, intramuscular, subcutaneous, intranasal, or intradermal routes.
  • the composition may be administered dhectly or it may be encapsulated (e.g., in a Hpid system, in amino acid microspheres, or in globular dendrimers).
  • the polypeptide may, in some cases, be attached to another polymer.
  • the intraceUular concentration of a polypeptide capable of altering E2F activity is increased through the use of gene therapy, which is through the administration of a composition comprising a nucleic acid that encodes and directs the expression of the polypeptide.
  • the polypeptide is cloned into an appropriate expression vector. Possible vector systems and promoters are discussed above.
  • the expression vector is transferred into the target tissue using one of the vector dehvery systems disclosed herein.
  • This transfer is carried out either ex vivo in a procedure in which the nucleic acid is transferred to ceUs in the laboratory and the modified ceUs are then administered to the human or other animal, or in vivo in a procedure in which the nucleic acid is transferred dhectly to ceUs within the human or other animal.
  • an adenovhal vector system is used to dehver the expression vector. If deshed, a tissue specific promoter is utilized in the expression vector as described above.
  • Non- vhal vectors ma be transferred into ceUs using any of the methods known in the art, including calcium phosphate co-precipitation, Hpofection (synthetic anionic and cationic hposomes), receptor-mediated gene dehvery, naked DNA injection, electroporation and bio-balHstic or particle acceleration.
  • the activity of the polypeptide is enhanced by agonist molecules that increase the enzymatic activity of the polypeptide or increase its appropriate recognition by ceUular binding sites.
  • enhancer molecules may be introduced by the same methods discussed above for the administration of polypeptides.
  • the level of a polypeptide capable of altering E2F activity is increased through the use of smaU molecular weight compounds, which upregulate expression at the level of transcription, translation, or post- translation. These compounds maybe administered by the same methods discussed above for the administration of polypeptides.
  • the activity of the polypeptide is inhibited by antagonist molecules that decrease the enzymatic activity of the polypeptide or decrease its appropriate recognition by ceUular binding sites.
  • antagonist molecules that decrease the enzymatic activity of the polypeptide or decrease its appropriate recognition by ceUular binding sites.
  • the level of a polypeptide capable of altering E2F activity is decreased through the use of smaU molecular weight compounds, which downregulate expression at the level of transcription, translation, or post-translation. These compounds maybe administered by the same methods discussed above for the administration of polypeptides.
  • the subject invention discloses methods and compositions for the high throughput dehvery and expression in a host of sample nucleic acid(s) encoding product(s) of unknown function. Methods are described for infecting a host with the adenoviral vectors that express the product(s) of the sample nucleic acid(s) in the host, identifying an altered phenotype relating to the modulation of E2F activity in the host by the product(s) of the sample nucleic acids, and thereby assigning a function to the product(s) encoded by the sample nucleic acids.
  • the sample nucleic acids can be, for example, synthetic ohgonucleotides, DNAs, or cDNAs and can encode, for example, polypeptides, antisense nucleic acids, or GSEs.
  • the methods can be fuUy automated and performed in a multiweU format to aUow for convenient high throughput analysis of sample nucleic acid Hbraries.
  • the foUowing examples describe the construction and screening, using a E2F transcriptional assay, of an arrayed adenovhal vector human placenta cDNA.
  • cDNA Hbrary Construction of the primary cDNA Hbrary is performed as foUows.
  • mRNA emanating from a 12 week old human placenta is used for the (ohgo dT- primed) generation of the first strand cDNA using the Superscript II method (Life Technologies).
  • cDNAs are dhectionaUy cloned (Sa - Notl) into the pIPspAdapt ⁇ vector (described in WO 99-64582).
  • the cD ⁇ A Hbrary is then transformed into Esche ⁇ chia coli (DH10B). 5' sequencing analysis on 167 clones revealed that 98.8% of the plasmids from the Hbrary contained inserts and that 24% of the inserts are fuU length cDNAs.
  • Parts of the bacteria transformed with the primary cDNA Hbrary are plated onto an LB agar growth medium (+ lOO ⁇ g/ml ampiciUin) contained in Bio-assay dishes (Life Technologies). These bio-assay dishes are then incubated at 37°C for 18 hrs, Bacteria are plated at a density of 1500 cfu/plate, thereby aUowing recognition and automatic picking of individual colonies by a QPix apparatus (Genetix), This device picked individual bacterial colonies and further inoculated 300 ⁇ l of hquid LB growth medium (+ 100 ⁇ g/ml ampiciUin) in 96-weU plates.
  • a second step in the construction of the adenovhal cDNA hbrary is the arrayed purification of DNA of individual plasmids from the primary cDNA hbrary in amounts sufficient for adenovirus generation.
  • a bacterial culture is prepared as foUows.
  • the glycerol stock plates are thawed and 3 ⁇ l of the bacterial culture is transferred to a 96-weU plate filled with 280 ⁇ l of hquid LB growth medium (+ 100 ⁇ g/ml ampiciUin) using a CybiWeU robot (CyBio).
  • the (3) buffers for performing alkaline lysis are purchased from Qiagen.
  • a fhst step the bacterial peUet is resuspended into 60 ⁇ l of buffer PI.
  • 60 ⁇ l of buffer P2 is added to the resuspended bacterial ceUs and a mixing step and 5 min incubation time are apphed to achieve complete ceU lysis.
  • FinaUy 60 ⁇ l of buffer P3 is added and a mixing step apphed for precipitation of proteins and genomic DNA.
  • the 96-weU plates are centrifuged (40 min, 3220 rcf).
  • the supernatant (100 ⁇ l) is coUected and transferred to new N-bottom 96-weU plates containing 80 ⁇ l of isopropanol (for precipitation of the plasmid D ⁇ A) using a CybiWeU robot (CyBio).
  • the plates containing the peUet are discarded.
  • the 96-weU plates are centrifuged (45 min, 2700 rcf) and the supernatant discarded by decanting in a waste container.
  • the peUet is washed with 100 ⁇ l of 70% ethanol and the 96-weU plates are centrifuged again (10 min, 2700 rcf).
  • the plasmid D ⁇ A preparations contained in 96-weU plates are quantified. For this purpose, 5 ⁇ l of plasmid D ⁇ A is pipetted from the D ⁇ A plates and transferred to a 96-weU plate containing 100 ⁇ l of TE buffer. Then 100 ⁇ l of 'quantification solution' is added.
  • This solution is prepared by dissolving 2 ⁇ l of SybrGreen (Molecular Probes) into 10 ml of TE Buffer. After a mixing step, measurement is performed in a Fluorimeter (Fluostar, BMG) with the foUowing settings: emission : 485 nm; excitation: 520 nm , gain: 35.
  • a standard curve is generated by performing a measurement using different dUutions (in TE buffer) of a standard D ⁇ A sample (lambda D ⁇ A). By fitting results for the individual D ⁇ A samples on this curve, D ⁇ A concentration per weU is calculated. The mean D ⁇ A concentration per weU for each 'D ⁇ A plate' is calculated. On average, a D ⁇ A concentration of 20 ng/ ⁇ l of D ⁇ A is obtained.
  • cD ⁇ As produced from the placenta tissue are cloned into the pIPspAdApt6 plasmid.
  • This adapter plasmid contains the 5' part (bp 1-454 and bp 3511-6093) of the adenovirus serotype 5 genome (in which the El A gene is deleted and a CMV promoter, multiple cloning site and SV40-derived poly adenylation signal have been inserted).
  • cosmid contains the main part of the adenovirus serotype 5 genome (bp 3534-35953) from which the E2A gene is deleted.
  • the Per.C6/E2A packaging ceU hne is derived from human embryonic retina ceUs (HER) transfected with plasmids mediating the expression of the El and E2A genes.
  • this adapter plasmid is cotransfected into a packaging ceU line Per.C6/E2A with the cosmid.
  • the complete Ad5 genome is reconstituted by homologous recombination.
  • the helper and adapter plasmids contain homologous sequences (bp 3535-6093), which are a substrate for this recombination event.
  • the El and E2A gene products, which are requhed for adenovhal repHcation, are provided by the Per.C6/E2A ceU line in trans.
  • the adenovhal genes integrated into the genome of the Per.C6/E2A ceU line and the reconstituted adenovhal genome share no homologous sequences, which renders the reversion to rephcation competent adenovhal particles vhtuaUy impossible.
  • the DNA plates that are prepared and quantified as described above, are used for transfection of the Per.C6/E2A ceU line. Prior to this transfection, the plasmids contained in these plates are linearized by digestion with the Yl-Pspl restriction enzyme (New England Biolabs).
  • the transfection of the Per.C6/E2A ceUs with the linearized adapter and helper plasmids is set up as foUows. 0.1867 ⁇ l of linearised helper plasmid (containing 93 ng of DNA) is mixed with 1.11 ⁇ l of serum free 2xDMEM (Life Technologies).
  • helper mix 0.597 ⁇ l of Lipofectamine (Life Technologies) is mixed to 1.11 ⁇ l of 2xDMEM to form a hpo mix.
  • 1.3 ⁇ l of helper mix and 1 ,7 ⁇ l Lipo mix are pipetted using a CyBi-WeU robot (CyBio, equipped with a dropper). The plates are then incubated for approximately 1 hour at room temperature before addition of 28.5 ⁇ l of serum-free DMEM. Mixing is performed by pipetting up and down the mix three times (CyBi WeU robot).
  • the final virus propagation step aims to obtain a higher percentage of weUs showing CPE and more homogenous virus titers.
  • Viruses are propagated according to foUowing procedure.
  • the transfection plates stored at -80°C are thawed at room temperature for about 1 hour.
  • 20 ⁇ l of the supernatant is transferred onto Per.C6/E2A ceUs seeded in 96-weU plates at a density of 2.25xl0 4 ceUs/weU in 180 ⁇ l of DMEM + 10%FBS.
  • needles of the dispenser are disinfected and sterihsed by pipetting up 60 ⁇ l of 5% bleach three times.
  • the traces of bleach present in the needles are removed by 3 successive washes with 70 ⁇ l of sterUe water.
  • CeUs are incubated at 34°C, 10% CO 2 during approximately 10 days and the number of weUs showing CPE is scored. On average, the number of weUs showing CPE increases by 10% as compared to the original scoring after transfection, which represents 65% to 75% of the total number of weUs processed.
  • the plates are stored at -80°C until ahquots are made.
  • FIG. 46 A schematic representation of the Hbrary construction is shown in FIG. 46.
  • CeUs are cultured in Dulbecco's modified eagle's medium containing 10% fetal calf serum (FBS) supplemented with peniciUin/streptomycin.
  • FBS fetal calf serum
  • CeUs are spHt 1:5, 1:50, 1:100, 1:200, 1:500.
  • Day 4 Medium is replaced with medium containing 1 ⁇ g/ml puromycin and is refreshed every third day.
  • Day 22 Medium is removed and the plates are incubated at 37°C for 4 minutes in PBS. Colonies are picked using a p200 pipette and transferred to a 24- weUs plate containing medium with puromycin. 50 colonies from #1 , 25 from #2 and 25 from #3 (transfection table) are isolated. Medium is refreshed every second day foUowing day 22.
  • Day 36 100 clones grown up from day 22 are spht 1:4 and reseeded in 24- weUs plates. Medium is changed every second day foUowing this.
  • Viruses used are ⁇ E1/ ⁇ E2A adenoviruses transducing E2F2; E2F3; pi 6 ⁇ p27 ]P l ⁇ cZ; EGFP (aU generated from pIPspAdApt plasmids); and empty virus (generated from pIPspAdApt 6).
  • Adenovhal constructs transducing E2F2 and E2F3 are created by digestion of the parental plasmids containing HA-E2F2 and HA-E2F3 cDNAs (Xu, et al. (1995) Proc. Natl. Acad. Sci. USA 92:1357-61) with BamHI and Hindl ⁇ i, isolation of the inserts over an agarose gel, and hgation of the insert fragments in BamHIIHindlll- digested pIPspAdApt 3 (see WO99/64582), to generate pIPs ⁇ AdApt3-E2F2 and pIPspAdApt3-E2F3, respectively (FIG. 49 and FIG. 50).
  • Adenovhal constructs transducing pl ⁇ 11 ⁇ 43 and p27 I > are created by Hindl ⁇ l- Xhol digestion of the parental plasmids containing pl ⁇ 11 ⁇ 43 and p27-HA cDNAs (Beijersbergen, et al. (1995) Genes Dev.
  • the adenovhal construct transducing L61Ras is created by digestion of the parental construct pMT2SM-L61Ras (Schaap, et al (1993) J. Biol. Chem. 268:20232- 6) with Sail, blunting of the overhang with Klenow polymerase and dNTP's, and digestion with EcoRI.
  • the isolated insert fragment is hgated in pAd5CLIPPac, which is digested with Hindl ⁇ l, blunted with Klenow polymerase and dNTP's, and redigested with EcoRI, resulting inpAd5ChpPac-L61Ras (FIG. 54).
  • the isolated insert fragment is also hgated in Hp ⁇ lvEcoRI-digested pIPspAdApt 8, leading to pIPspAdApt8-L61Ras (FIG. 48).
  • pIPspAdApt6-lacZ (FIG. 55), is constructed by digestion of pIPspAdApt ⁇ with Kpn and BamHI, foUowed by insertion of the correspondingly digested and purified nls-/ ⁇ cZ gene from pCLIP-lacZ (WO 00/52186).
  • pIPspAdApt6- ⁇ GFP is constructed by releasing the EGFP insert by HindTTl-
  • ⁇ E1/ ⁇ E2A adenoviruses are generated from these adapter plasmids by co- transfection of the helper plasmid pWEAd5AflII-rITR.dE2A in PER.C6/E2A packaging ceUs, as described (WO99/64582).
  • the 5 E2F-luciferase reporter ceU lines are seeded at 5x10 3 ceUs per weU in 96-weU plates and incubated overnight at 37°C in a humidified incubator at 10% CO 2 in 100 ⁇ l of DMEM supplemented with 10% heat inactivated FBS. The next day, ceUs are infected with control viruses, transducing pi ⁇ 1 ⁇ 4 , ⁇ 27 KIP , E2F2, E2F3, EGFP, and Empty, at a known MOI of 100 in dup ⁇ cate. 24 hours after infection, the medium of the 96-weU plates is replaced with 100 ⁇ l of fresh medium.
  • the medium is removed from the wells.
  • the ceUs are washed once with Phosphate Buffered Saline and frozen at -20°C in 100 ⁇ l of PBS. After thawing and resuspension of the ceU lysate, 100 ⁇ l of Steady-Glo
  • ceU line 1C5 gave the best activation of the luciferase reporter after infection with E2F2- or E2F3-transducing viruses, whUe repression by pl ⁇ 1 ⁇ 4 " or p27 ⁇ p could also be scored (see also FIG. 56). Further experiments to optimise the set up of the assay are therefore performed with ceU Hne lC5.
  • U2OS-1C5 ceUs are seeded per weU in a 96-weU plate, using DMEM with 10% heat inactivated FBS and 1 ⁇ g/ml puromycin (Clontech) (hereinafter referred to as U2OS medium), and incubated overnight at 37°C in a humidified incubator at 10% CO 2 . After 24 hours, ceUs are infected with adenoviruses transducing E2F2, E2F3, p l 6 iNK4a 5 p27 ⁇ i ⁇ LacZ5 EGFP and empty _ M0I used ⁇ 5 20, 100 and 500. AU experiments are done in triphcate.
  • Infections are aUowed for 24 hours after which the medium is replaced with fresh U2OS medium. After a further 24 hours, ceUs are washed with Phosphate Buffered Saline (PBS) and frozen at -20°C in 100 ⁇ l of PBS. After thawing and resuspension of the ceU lysate, 75 ⁇ l of each weU is transferred to a fresh plate, 75 ⁇ l of Steady-Glo (Promega) is added and incubated for 15 minutes at room temperature.
  • PBS Phosphate Buffered Saline
  • E2F3 gives the highest induction of the E2F-luciferase reporter. Repression by pi g iNK4a and p27 ⁇ > j s more difficult to monitor, but the highest repression is also seen with the highest MOI.
  • ceUs are infected with adenoviruses transducing E2F2, E2F3, pl6 iN ⁇ 4a? p27 ⁇ i ⁇ LacZ> EGFP and empty> M0I used aiQ 100 and 500 experiment are done in triphcate on the two plates. Infections are aUowed for 24 hours after which the medium is replaced with fresh U2OS medium. After a further 24 hours, one of the plates is washed with PBS and frozen at -20°C in 100 ⁇ l of PBS. The remaining plate is washed and frozen 24 hours later.
  • Results are summarized in FIG. 58.
  • activation of the E2F-reporter by E2F2 and E2F3 is comparable between 48 hours and 72 hours infection time.
  • repression by l ⁇ 11 ⁇ 4* and p27 ⁇ p is more pronounced after 48 hours compared to 72 hours. It therefore is concluded that the optimal length of infection is 48 hours.
  • U2OS-1C5 ceUs are seeded per weU in a 96-weU plate, using U2OS medium, and incubated overnight at 37°C in a humidified incubator at 10% CO 2 . A total of 3 plates are seeded.
  • plate 1 is infected with adenoviruses transducing E2F3, pl ⁇ 0 ⁇ 4 ", p27 KIP , LacZ, EGFP, empty, and pCHp-L61Ras.
  • MOI used are 100 and 500, each in triphcate, using half of the plate. Infections are duphcated on the second half of the plate. The same layout is used to infect plate two and three.
  • aU weUs from plate 2 are co-infected with MOI 20 of adenovirus transducing E2F2
  • whUe aU weUs of plate 3 are co-infected with MOI 100 of adenovirus transducing E2F2.
  • Infections are aUowed for 24 hours after the medium on the first half of the plates is replaced with fresh U2OS medium, whUe on the second half of the plates, it is replaced with U2OS-medium containing 2% FBS. After a further 24 hours, aU plates is washed with PBS and frozen at -20°C in 100 ⁇ l of PBS .
  • L61Ras seems to co-operate with E2F2 in that the fold induction over empty virus is dramaticaUy increases when co-infected with MOI 20 or 100 of E2F2.
  • the induction by L61 Ras, co-infected with MOI 20 or 100 of E2F2 is even 5 fold higher than the induction by E2F3 after co-infection with MOI 20 or 100 of E2F2, whUe induction of L61Ras in the absence of E2F2 is less than that of E2F3. This suggests some synergism between the Ras- and E2F-pathways.
  • U20S 1C5 reporter ceUs are seeded at a density of 5x10 3 ceUs per weU in a 96-weU plate and incubated overnight at 37°C in a humidified incubator at 10% C0 2 in 100 ⁇ l of DMEM supplemented with 10% heat inactivated FBS.
  • the medium of the 96-weU plates is replaced with 100 ⁇ l of fresh medium. 48 hours after infection, the medium is removed from the weUs and the ceUs are washed once with Phosphate Buffered Saline and frozen at -20°C in 100 ⁇ l of PBS.
  • E2F2 and E2F3 expression causes a 1.5 to 3.1 increase of the luciferase signal, compared to empty virus control.
  • E2F2 and E2F3 expression causes a 2.3 to 8.3 fold induction of the luciferase signal, compared to empty virus control.
  • induction by E2F2 and E2F3 is between 7.1 and 10.9 fold empty virus.
  • U2OS 1C5 reporter ceUs are seeded in 96-weU plates at a density of 5x10 3 ceUs per weU using 100 ⁇ l of DMEM supplemented with 10% heat inactivated FBS.
  • ceUs are infected in triphcate using an MOI of 100 and 500 and a total infection volume of 20 ⁇ l.
  • Infections are done with the potential hits as identified in the first round of screening (see example 4) and randomly picked viruses from the same plates as control.
  • a titer of 5x10 9 virus particles (vp) per ml for the propagated viruses from the Hbrary.
  • Known titers are used for the control viruses transducing E2F2, E2F3, pl ⁇ 1 ⁇ 42 , p27 ⁇ p , LacZ, EGFP and empty, Viruses transducing E2F2, p1 gr N ⁇ 4 an em t v> are included on aU 96-weU plates.
  • the medium of the 96-weU plates is replaced with 100 ⁇ l of fresh medium.
  • the medium is removed from the weUs and the ceUs are washed once with Phosphate Buffered Saline supplemented with 1 mM Ca 2+ and 1 mM Mg 2+ (PBS ++ ), and frozen away at -20°C in 100 ⁇ l of PBS ++ .
  • the E2F-luciferase reporter construct and a control reporter construct are transiently transfected in wUdtype U2OS ceUs. Particle titers of these viruses are determined by real-time PCR, as described (Ma, et al. (2001) J. Virol. Methods 93:181-8).
  • viruses encoding potential hits are used to infect U2OS ceUs transiently transfected with the E2F-luciferase reporter construct, or the pGL3- basic control reporter construct.
  • CeUs are infected with the viruses at MOI of 100 and 500. 6-wells of a 96-weU plate are used for each MOI for aU viruses. CeUs are incubated further for 48 hours at 37°C in a humidified incubator at 10% CO 2 . 48 hours after infection, the medium is removed from the weUs and the ceUs are washed once with PBS and frozen away at -20°C in 100 ⁇ l of PBS.
  • Results are presented relative to empty virus control in FIG. 61. Neither of the potential hits, nor the control viruses, modulated expression of the transfected pGL3-basic control reporter construct (data not shown).
  • Control plates are prepared that contain different control pIPspAdApt viruses transducing the foUowing transgenes: E2F2, E2F3, pl ⁇ TM 4 ", ⁇ 27 mp , GFP or the empty virus (defined as the virus with empty MCS) or no virus at aU. These viruses are propagated according to the protocol apphed for the Phenoselect hbrary.
  • Day 0 TC treated 96-weU plates are seeded with Per.C6/E2A ceUs at a density of 2.25x10 4 ceUs per weU in 200 ⁇ l medium.
  • Day 1, 48 weUs per plate are infected with 20 ⁇ l of one type of control virus emanating from a larger batch preparation. After 7 days, fuU CPE is obtained.
  • the aHquots are sealed with a seal (Nunc Cat No 236366) and stored at -80°C untU use.
  • the control plates are tested according to the screening protocol. 8 ⁇ l of virus crude lysate is pipetted from a control plate using a 96 channel Hydra dispenser (Robbins Scientific) and 1 ⁇ l is dispensed in positions Al, A2, Bl and B2 of a white 384-weU plate (Greiner) in which U2OS 1C5 reporter ceUs are seeded at a density of 1250 ceUs/weU (20 ⁇ l medium per weU).
  • U2OS reporter ceUs 1C5 are cultured in DMEM containing 10% of heath inactivated FBS and 1 ⁇ g/ml puromycin. Performing the assay, U2OS ceU cultures are strictly kept subconfluent.
  • T175 flasks seeded day -3 are treated with trypsin/EDTA (2ml of trypsin/EDTA mix/flask) to detach ceUs.
  • CeUs (resuspended in 10 ml culture medium/T175 culture flask) are counted. CeUs are then resuspended in culture medium at a density of 6.25x10 4 ceUs/ml for further seeding.
  • White tissue culture treated 384-weU plates are seeded at a density of 1.25x10 4 cells per weU, 20 ⁇ l per weU, using a multidrop (Labsystems).
  • the virus Hbrary aliquot plates (384-weU format) to be processed (10 plates per day) are put in a laminar ahflow cabinet for 1 hour for thawing. Plates are put at 4°C untU further processing.
  • syringes are cleaned by performing 3 washing steps with 20 ⁇ l of 5% bleach. The syringes are then rinsed 3 times with 25 ⁇ l of sterUe deionized water.
  • the control viruses are added to the plates as foUows: for every weU of the 96-weU control plate, one ⁇ l of virus crude lysate is transferred to 1 weU, B2 quadrant, on 8 to 10 384-weU plates containing the reporter ceUs infected with the hbrary viruses. (This position is left uninfected during infection of the reporter ceUs with the Hbrary viruses.) Addition of the control viruses is also performed using the Hydra dispenser,
  • luciferase substrate (Steady Glow, Promega) is freshly prepared according to the protocol of the manufacturer. 15 ⁇ l of luciferase substrate is added to the weUs using the Hydra dispenser. This operation is performed in a laminar ahflow cabinet and under subdued hght conditions. The dispenser is programmed to fill its syringes with 70 ⁇ l of substrate and to sequentiaUy dispense 15 ⁇ l to the Al, A2, Bl and B2 quadrants. The syringes are then refilled for processing the next plate without intermediate washing step.
  • the plates are sealed (Nunc cat N° 236366) and put on a rotary shaker for 30 min. Plates are then sequentiaUy inserted into a lu inometer (Lumicount, Packard) for readout.
  • the apparatus is used with the foUowing settings: Gain 150, PMT voltage 1100 V, 0.3 sec reading time. Time in between substrate addition and readout is not aUowed to exceed 1 hour. Data are stored in Excel sheet format (Microsoft).
  • the screening is performed in 4 series of 10x384-weU virus ahquot plates, which represents 15360 weUs. As the virus production efficiency for the Phenoselect hbrary reached on average 70% of the total amount of weUs, this represents approximately 10750 viruses.
  • control data inserted in 96 positions of the B2 quadrant in 8 to 10 assay plates per screen are extracted and compUed. Background signal levels associated with the Empty virus and the standard deviation on this measurement are determined.
  • the results obtained for the weUs infected with the various control viruses are analysed in order to evaluate the quahty of the screening.
  • a typical result for the weUs infected with the control viruses during one out of the 4 runs of the screening is shown in FIG. 63.
  • AU mean values are sorted. Viruses causing an increase of the signal are considered as hits provided these mediated a signal superior to the cut off value.
  • the cut off value for samples identified as E2F activators is defined as being the mean plus three times the standard deviation of the signal obtained for the weUs in which ceUs are infected with the empty virus. Library viruses that mediated a lower signal as the empty virus-infected weUs are considered as hits provided these mediated a signal of at least half of the signal of the 8 neighbor Hbrary viruses.
  • Example 8 Rescreen of hits from 11.000 screen
  • viruses scored as hit two ⁇ l of virus crude lysate is recovered from the weU of the original 384-weU ahquot plates that are used for performing the screening. These ahquot plates are stored at -80°C and thawed for a second time for removal of this 2 ⁇ l aUquot.
  • the viruses of the hits are propagated by using the 2 ⁇ l aHquots of crude virus to infect 2.25x10 4 Per.C6/E2A ceUs seeded in 96-weU plates (200 ⁇ l of DMEM + 10%> FBS). After appearance of complete CPE, these 96-weU plates undergo a single freeze-thaw cycle.
  • 1 ⁇ l of the undUuted virus crude lysate ahquots (emanating from the repropagation step) as weU as 1 ⁇ l from a 3-fold dUution of these aHquots are used to infect the 1C5 U2OS reporter ceU line seeded in 384-weU plates. (This corresponds to MOI of approximately 2000 and 600, respectively).
  • luciferase substrate is added and readout is performed. Results of the rescreen are compared to the results of the original screening (FIG.
  • the cut off value for samples identified as E2F activators is defined as being the mean plus three times the standard deviation of the signal obtained for the weUs in which cells are infected with the empty virus.
  • the viruses mediating a signal lower as the non-infected weUs are scored as repressors. Applying these cut off values, 27 of the hits are confirmed as activators and 21 hits are confirmed as repressors for the higher MOI.
  • the strongest hits are generaUy confirmed
  • the moderate activators between 4.5 and 6 fold empty virus
  • the weak activators are generaUy not confirmed.
  • Particle titers of these viruses are determined by real-time PCR, as described (Ma, et al. (2001) J. Virol. Methods 93:181-8).
  • Results are calculated as fold activation compared to empty virus.
  • E2F2 and E2F3 stimulate the E2F-luciferase reporter 4.5-9 times compared to empty virus wlhle pl ⁇ TM 1 ' 4 and p27 ⁇ p repressed luciferase activity 0.4-0.2 times compared to empty virus in a MOI-dependent manner.
  • Other control viruses like EGFP hardly influenced luciferase activity.
  • Two potential hits stimulate both the E2F reporter and the pGL3-basic control reporter (compare FIG. 66A and FIG. 66B), and are discarded.
  • Two potential hits (H89 and HI -92) stimulate both the E2F reporter and the pGL3- promoter control reporter to equal relative levels and are also discarded.
  • Two potential hits are retained that repressed E2F-reporter activity more than 0.6 times empty vector control (Hl-35 and Hl-96), while not influencing the pGL3- basic or pGL3-promoter control reporters (see FIG. 66A).
  • Several other potential repressors are discarded since they also seemed to influence the pGL3 -promoter control reporter (data not shown).
  • fragments of the cDNAs expressed by the hit adenoviruses are ampHfied by PCR using primers complementary to sequences flanking the MCS of the pAdapt plasmid.
  • the foUowing protocol is apphed to obtain these PCR fragments.
  • Day 0, Per.C6/E2A ceUs are seeded in 96-weU plates at a density of 2.25x10 4 ceUs per weU, in 200 ⁇ l of Per.C6/E2A medium. CeUs are incubated overnight at 39°C, 10% CO 2 .
  • Day 1 ceUs are infected with the hit viruses using 2 ⁇ l of crude ceU lysate material from the repropagation step.
  • CeUs are then incubated at 34°C, 10% CO 2 untU appearance of starting of CPE (as revealed by the swelling and rounding up of the ceUs, typicaUy 2 to 3 days post infection).
  • the supernatant is removed from the ceUs and 50 ⁇ l of lysis buffer (lx Expand High Fidehty buffer with MgCl 2 (Roche Molecular Biochemicals Cat No 1332465) supplemented with 1 mg/ml proteinase K (Roche Molecular Biochemicals Cat No 745 723) and 0.45% Tween-20 (Roche Molecular
  • Biochemicals, Cat No 1335465) is added to the ceUs. CeU lysates are then transferred to sterUe micro centrifuge tubes and incubated at 55°C for 2 hrs foUowed by a 15 min inactivation step at 95°C.
  • 5 ⁇ l of the ceU lysates is then added to a PCR master mix composed of 5 ⁇ l lOx Expand High Fidehty buffer +MgCl 2 , l ⁇ l of dNTP mix (lOmM for each dNTP), l ⁇ l of pChp-FOR primer (lO ⁇ M stock, sequence: 5' GGT GGG AGG TCT ATA TAA GC), l ⁇ l of pAdapt-REV primer (lO ⁇ M stock, sequence: 5' GGA CAA ACC ACA ACT AGA ATG C), 0.75 ⁇ l of Expand High Fidehty DNA polymerase (3.5 U/ ⁇ l, Roche Molecular Biochemicals) and 36,25 ⁇ l of H 2 O.
  • a PCR master mix composed of 5 ⁇ l lOx Expand High Fidehty buffer +MgCl 2 , l ⁇ l of dNTP mix (lOmM for each dNTP), l ⁇ l of pChp
  • PCR is performed using a PE Biosystems Gen Amp PCR system 9700 as foUows: the PCR mixture (50 ⁇ l in total) is incubated at 95°C for 5 min; at 95°C for 30 sec; 55°C for 30 sec; 68°C for 4 min, and this is repeated for 35 cycles. A final incubation at 68°C is apphed for 7 min.
  • the amplification products are resolved on a 0.8% agarose gel containing 0,5 ⁇ g/ml ethidium bromide and theh length estimated by comparison with the migration of a standard DNA ladder.
  • 15 ⁇ l of PCR mixture is mixed with lO ⁇ l of 6x gel loading Buffer.
  • the PCR products obtained are also used as template for sequencing using the aforementioned pChp-FOR primer.
  • Hit Hl-9 is detected as an activator in the E2F screen. According to a
  • the DNA sequence of hit Hl-9 (FIG. 67A; SEQ ID NO: 13) is identical (100% identity) with the pubHc cDNA sequence referenced by GenBank accession number NM_017710. However, as compared to this pubhc sequence, a 5'- terminal fragment of 571 bps is missing in Hl-9.
  • GenBank accession number XM_002079 gives a better match at the 5' end of Hl- 9, although there are sthl 86 bps less in the Hl-9 sequence.
  • ORF number 1 encoding a protein of 485 amino acids, most likely wiU be the coding sequence of this cDNA (FIG. 67B; SEQ ID NO: 14).
  • a search for homology in GenBank results in a perfect match with the amino acid sequence referenced by GenBank accession number NP_060180, which contains the translated product of the ORF in NM_017710.
  • NP_060180 describes a hypothetical protein, FLJ20203, of 697 amino acids.
  • the N- terminal 212 amino acids of NPJ360180 are not encoded by ORFl.
  • the C-terminal part gives a perfect match.
  • the match with the sequence in XP_002079 (containing the translated product of the ORF in XM_002079) is 100%, both in sequence as in length.
  • U2OS ceUs are grown in Dulbecco's modified eagle's medium containing 10% fetal calf serum (FBS) and supplemented with peiucUlin/streptomycin (100 units/ml; (Gibco-BRL) and glutamine (Gibco-BRL)(abbreviated U2OS medium) on T80 culture flasks until 50 % confluency is reached.
  • CeUs are washed in PBS and trypsinized in 1 ml of Trypsin/EDTA (Gibco-BRL) for 5 minutes at 37°C and cohected in 10 ml of culture medium. Subsequently ceUs are washed in 10 ml of PBS and resuspended in electroporation buffer (2mM HEPES (pH 7.2), 15mM
  • DNA mixes are prepared in a total volume of 10 ⁇ l containing reporter plasmid (5 ⁇ gram), effector plasmid (0 ⁇ gram, 0.5 ⁇ gram (only for Hl-9 in pIPsAdapt ⁇ , or 2.5 ⁇ gram; adjusted to 2.5 ⁇ gram with empty pIPspAdapt6), and 0.1 ⁇ gram of RenUla (pRL-CMV; Promega).
  • Reporter plasmids are either E2F-luciferase reporter construct, or the pGL3-promoter control reporter construct (Promega)(see Example 9).
  • Effector plasmids are pIPspAdApt3-E2F2 (see Example 3); pIPspAdApt6-EGFP (see Example 3); or Hl-9 in pIPspAdapt ⁇ (see above).
  • 9 DNA mixtures are prepared and added to 100 ⁇ l of the ceU suspension.
  • Electroporation is performed on a Gene Pulser II electroporator including RF module (BioRad) at 140 Volt, 40 Khertz, 1.5 msecond per pulse, 1.5 second delay, total 15 pulses.
  • Example 13 Analysis of hits for activity as secreted proteins
  • producer ceUs are infected by the viruses of the adenovhal hbrary prepared as described hereinabove.
  • the producer ceUs may be infected with viruses identified as "hits" in the E2F activity assay.
  • Another population of ceUs are infected with control viruses that induce or do not induce E2F activity.
  • the conditioned media from each infected producer ceU population are harvested 2 or 4 days post infection (dpi) and added to freshly seeded primary human ceUs. If the conditioned medium contains secreted proteins that induce E2F activity, this wih be identified after adding the conditioned medium to the ceUs and by analyzing E2F activity.
  • HeLa or U2OS producer ceUs are cultured in DMEM 10% FBS. 1000-5000 HeLa cells weU or 1000-5000 U2OS ceUs/weU (384-weU plate) are plated in 60 ⁇ l medium. Four hours later, the ceUs are infected with l ⁇ l of adenovhal stock solutions. Two or 3 days later, 384-weU plates containing 1000 primary ceUs/weU are seeded in 30 ⁇ l of medium. One day after seeding the primary ceUs, the primary ceUs are infected with adenovirus containing the E2F-reporter of Example 16, below, using the conditions described in Example 17, below.
  • Phage displaying human FAb fragments encompassing the hght and heavy variable and constant regions are employed to isolate antibodies that bind to the protein identified herein (characterized by SEQ ID NO: 14).
  • a human FAb phage display hbrary is constructed in a phage display vector such as pCESl a vector derived frompCANTAB6 (McCafferty, et al. (1994) Appl. Biochem. Biotech. 47:157- 73).
  • the hbrary is constructed in the filamentous E. coli phage ml 3 and the FAb sequences are displayed as N-terminal fusion proteins with the minor coat protein pill.
  • the Hbrary can have a complexity of more or less than 10 10 different sequences.
  • Three types of targets can be used to select for polypeptide-displaying phages that bind to the amino acid epitopes encoded by the sequences of SEQ ID NO: 13.
  • Fhst a predicted extraceUular or otherwise accessible domain encoded by sequences of SEQ ID NO: 13 is synthesized as a synthetic peptide.
  • the N-terminus of this peptide is biotinylated and foUowed by three amino acid linker residues KRR, foUowed by the predicted sequence of encoded by sequences of SEQ IDNO: 13, respectively.
  • a fusion protein is made of a portion of or the complete polypeptide encoded by sequences of SEQ ID NO: 13 in frame with the ORF of glutathione-S- transferase (GST) or maltose-binding protein or His6 or another tag and expressed in E. coli.
  • GST glutathione-S- transferase
  • His6 or another tag is fused in frame with the ORF of SEQ ID NO: 13 and expressed in a mammaUan expression system such as PER.C6/E2A. Fusion proteins are then purified using, for example, N ⁇ NTA columns for His6-tagged proteins (Qiagen) or glutathione resin (Pharmacia) for GST-tagged proteins.
  • a pool of FAb displaying phage is selected out of a complex mixture of a high number of different FAb displaying phages in four rounds by theh abihty to bind with significant affinity to a biotinylated peptide or to a purified fusion protein that has been expressed in E. coli or in a mammahan expression system such as PER.C6/E2A.
  • the coUection of selected FAb displaying phage is further decreases by the next selection procedure: the FAb displaying phage are further selected in three rounds for theh abihty to bind to polypeptides encoded by sequences of SEQ ID NO: 13 present in ceU lysates from ceUs overexpressing SEQ ID NO: 13.
  • biotinylated peptide 250 ⁇ l of FAb hbrary (or eluted phage from the previous round) is mixed with 250 ⁇ l 4% non fat dry milk in PBS and equilibrated while rotating at RT for 1 hour. Subsequently biotinylated peptide (20-500 nM in H 2 O) is added.
  • This mix is incubated on the rotator at RT for 1 hour before 250 ml equilibrated streptavidin-dynabeads in 2% non fat dry milk in PBS is added. After incubation on a rotator at RT for 15 min the beads with the bound phage are washed 5 times with PBS/2% non fat dry milkl/0.1% Tween, 5 times with PBS /0.1% Tween and 5 times with PBS. Then the phage are eluted by incubation with 0.1M Triethylamine on a rotator at RT for 10 min and neutrahsed in 1 M Tris-HCl (pH 7.4). The eluted phages are titered and amplified in E.coli bacteria, e.g. TGI , before the next selection.
  • E.coli bacteria e.g. TGI
  • the FAb phages or antibodies derived thereof are used as diagnostic tools, for example in immunohistochemistry, as research tools, for example in affinity chromatography, as therapeutic antibodies dhectly, or for the generation of therapeutic antibodies by generating anti-idiotypic antibodies.
  • Example 15 Screeriing for compounds that alter E2F activity
  • Polynucleotides of SEQ ID NO: 13 or polypeptides of SEQ ID NO: 14 are attached to the bottom of the weUs of a 96-weU plate by incubating the polypeptide or polynucleotide in the weUs overnight at 4°C.
  • the weUs are first coated with composition ofpolylysine that facihtates binding of the polypeptide or polynucleotides.
  • FoUowing attachment of the biopolymer samples from a hbrary of test compounds are added to the weUs and incubated for a sufficient time and temperature to facilitate binding using an appropriate binding buffer known in the art. FoUowing this incubation, the weUs are washed with an appropriate washing solution at 4°C.
  • the stringency of the washing steps is varied by increasing or decreasing salt and/or detergent concentrations in the wash. Detection of binding is accompHshed by using antibodies (RIA, ELISA), biotintylation, bio tin-strep tavidin binding, and radio isotopes.
  • the concentration of the sample Hbrary compounds is also varied to calculate a binding affinity by Scatchard analysis.
  • Binding to the polypeptide or polynucleotides identifies a "lead compound”. Once a lead compound is identified the screening process is repeated using compounds chemicaUy related to the lead compound to identify compounds with the tightest binding affinities. Selected compounds having binding affinity are further tested in one of the two foUowing assays.
  • E2F Transcriptional Assay Compounds that bind to the polynucleotide or polypeptide are tested for theh effects on E2F activity, In general, a ceU that expresses a polynucleotide of SEQ ID NO: 13 is treated with a binding compound. The treatment with the compound can occur pre-transfection with the polynucleotide sequence (see day 0 and 1 below), post-transfection (see days 1 to 4 below), or concurrently with transfection (see day 1 below). After transfection and incubation with the compound, E2F activity is assessed.
  • control viruses or viruses comprising SEQ ID NO: 13 are added to the hCAR transfected weUs according to the foUowing procedure: Plates harbouring control viruses or SEQ ID NO: 13 are aUowed to thaw at room temperature. Two ⁇ l of control virus or SEQ ID NO: 13 virus are transferred to the 384-weU plate containing the U20S ceUs using a HydralOO 96 channel dispenser.
  • the viruses from the control plates are screened in dupHcate, whUe the viruses from the PhenoSelect Hbrary are screened in singular fashion.
  • the plates containing the freshly infected ceUs are then incubated at 37°C. Three days after infecting the ceUs, plates are analyzed for E2F activity.
  • the binding compounds identified in the previous step can be added on Day 0, Day 1, or on any of the days after transfection with the virus containing SEQ ID NO: 13.
  • mRNA Expression Assay On day 0, 1000 U2OS ceUs are seeded in 60 ⁇ l medium in the weUs of a black 384-weU plate with clear bottom (Costar or Nunc).
  • the ceUs are plated in dupHcate so that RNA is isolated from a first set of plates whUe E2F activity is assessed in the second set of plates.
  • the binding compound is added to the medium of both sets of plates at a concentration ranging from 1 nM to ImM.
  • the second set of plates is analyzed for E2F activity.
  • the ceUs of the first set are lysed and the RNA from the ceUs is extracted. Extraction is performed as described in Maniatis, et al.
  • RNA isolated from the ceUs is used as template for PCR using primers specific to SEQ ID NO: 13 to determine if the compound induces mRNA expression.
  • the above experiment can be done at a larger scale in 96- or
  • CeU lines almost always have mutations in ceU cycle regulatory pathways, which prevent the isolation of novel regulators that function upstream of the mutated proteins. Therefore, the use of an E2F-reporter in primary ceUs leads to the isolation of more hits from these screens, as primary ceUs in general do not have mutations in ceU cycle regulatory pathways.
  • an adenovhal E2F-reporter construct is generated to use primary ceUs for the isolation novel modulators of E2F activity.
  • the adenovhal E2F-reporter is co-infected into the targets ceUs together with the individual Hbrary viruses.
  • An adenovhal E2F-reporter also aUows using multiple primary ceUs during screening, which enhances the isolation of ceU-specific modulators.
  • the pGL3-TATA-E2F-luc construct is digested with Sail, which cuts 5' to the E2F-dependent promoter, and Notl, which cuts downstream of the luciferase/poly(A) signal.
  • Sail which cuts 5' to the E2F-dependent promoter
  • Notl which cuts downstream of the luciferase/poly(A) signal.
  • the 'overhangs are blunted by filling in with Klenow polymerase enzyme in the presence of d ⁇ TPs.
  • a pGL3- TATA-luc construct, without E2F-binding sites, is treated in a sirnUar manner.
  • the vector fragment is further digested with Xmnl.
  • the insert fragments are isolated on a 0.8% agarose gel, and purified using a QIAquick gel extraction kit (Qiagen).
  • the adapter plasmid pIPspAdapt 6 is digested with Bgl ⁇ l, blunted by filling in with Klenow polymerase enzyme in the presence of d ⁇ TPs, and redigested with SnaBl. FoUowing phosphatase treatment to prevent reHgation of the vector, the vector fragment is isolated on a 0.8% agarose gel, and purified using a QIAquick gel extraction kit (Qiagen).
  • Both insert fragments frompGL3-TATA-E2F-luc and frompGL3-TATA-luc, are hgated to the adapter fragment using lx Hgation buffer and T4 DNA hgase (New England Biolabs). FoUowing transformation into E. coli and selection on ampicUHn- agar plates, single colonies are inoculated to prepare miniprep DNA. Correct clones are obtained that contained the reporter fragments in both orientations in pIPspAdapt, as determined by restriction enzyme analysis and sequence analysis.
  • ⁇ E 1/ ⁇ E2A adenoviruses are generated from these adapter plasmids by co- transfection of the helper plasmid pWEAd5AflII-rITR.dE2A in PER.C6/E2A packaging ceUs, as described (WO99/64582),
  • a transient co-infection assay is developed for isolation of novel modulators of E2F activity in primary ceUs. Optimization of this new assay is done on U2OS wUd type ceUs. These ceUs are co-infected with an adenovhal E2F-reporter (referred to as pGL3E2F reporter) and the ⁇ E1/ ⁇ E2A control adenoviruses, as mentioned in example 3, transducing E2F3, pl ⁇ 1 ⁇ 43 and EGFP. To study the specificity towards an E2F-dependent promoter, ceUs are also co-infected with thepGL3-TATA-luc reporter, which does not contain E2F binding sites (referred to as pGL3basic reporter). The ceUs are co-infected with reporter and control virus in different ratios to study optimal co-infection conditions.
  • pGL3E2F reporter adenovhal E2F-reporter
  • ⁇ E1/ ⁇ E2A control adenoviruses
  • U2OS wUd type ceUs are seeded at 5x10 3 ceUs per weU in 96-weU plates and incubated overnight at 37°C in a humidified incubator at 10% CO 2 in 100 ⁇ l of DMEM supplemented with 10% heat inactivated FBS.
  • ceUs are co-infected with pGL3E2F reporter virus at MOIs of 250, 500 and 750 and control viruses at MOIs of 0, 250, 500 and 750.
  • Each MOI of reporter virus is combined with each the four different MOIs of the control viruses.
  • Empty virus is added to aU samples in order to obtain a final total MOI of 1500.
  • the final volume is set to 20 ⁇ l with culture medium. AU experiments are performed in tripHcate.
  • a BSA standard curve is prepared as foUows (differs from protocol):
  • screens are normaUy performed using Hbrary viruses with unknown titers (using an assumed titer of 2xl0 9 vp/ml, there can be some variation between the weUs in the total amount of virus that is added to the ceUs), to study the influence of different virus concentrations on the assay performance, a fill up experiment is performed.
  • This assay is done on primary Human UumbUical Vein Endothehal ceUs (HUVEC) using the conditions as described for U2OS wt ceUs.
  • HUVEC ceUs are seeded at 5x10 3 ceUs per weU in 96-weU plates and incubated overnight at 37°C in a humidified incubator at 10% CO 2 in 100 ⁇ l EBM-2 supplemented medium (Clonetics CC-4176).
  • ceUs are co-infected with pGL3E2F reporter virus at a known MOI of 250 and empty virus at a known MOI of 0, 500, 750, 1250, 2250, 4750 and 9750, ah in a total volume of 20 ⁇ l.
  • HUVEC ceUs are also co-infected withpGL3E2F reporter virus at a known MOI of 250 and control viruses at a known MOI of 750 for comparison.
  • AU conditions are performed in triplicate.
  • AU components of the CBQCA protein kit (Molecular Probes, C-6667) are prepared as described in the protocol.
  • a BSA standard curve is prepared as described before.
  • Results are expressed as average luminescence values normalized for protein concentrations (see FIG. 71).
  • a MOI dependent repression of the luciferase signal is observed when empty virus is added to the pGL3E2F reporter. Co-infection of empty virus with a known MOI of 4750 or higher leads to repression of the signal. Therefore Hbrary viruses with a real titer that is much higher than the assumed titer of 2xl0 9 vp/ml can be identified as false positive repressors. However, these false positive repressors are excluded after the rescreen that is done with real titers.
  • Hbrary viruses are picked and used to co-infect the HUVEC primary ceUs. These Hbrary viruses are previously used on the stable U2OS 1-5 E2F-reporter ceU Hne and do not yield any positive or negative modulators.
  • HUVEC ceUs are seeded at 5x10 3 ceUs per weU in 96- weU plates and incubated overnight at 37°C in a humidified incubator at 10% CO 2 in 100 ⁇ l EBM-2 supplemented medium (Clonetics CC-4176).
  • AU components of the CBQCA protein kit (Molecular Probes, C-6667) are prepared as described in the protocol.
  • a BSA standard curve is prepared as described before.
  • Ten ⁇ l of each BSA dUution and 10 ⁇ l of the ceU lysates are transferred to a fresh 96-weU plate.
  • 5 ⁇ l 20 mM KCN and 10 ⁇ l 2mM ATTO-TAG is added.
  • the reactions are protected from the Hght by covering with aluminum foU.
  • the plates are incubated for at least 1 hour (max. 5 hours) at RT with shaking. Fluorescence is measured on the FLUOstar Galaxy of BMG with excitation at 485/12 nm and emission at 525/20 nm.
  • the optimal gain is set using the plate that contained the standard curve. AU other plates are measured using the same gain.
  • Results are expressed as average luminescence values normalized for protein concentrations (see FIG 72 and FIG. 73). Empty vhus gave an average luciferase reading of 48.9 relative Hght units (luminescence per microgram of protein). E2F3 expression causes a 12.8 times increase of the relative luciferase signal, compared to empty virus control, pl ⁇ 1 ⁇ 4 " expression causes a signal 2.3 times decreased as compared to empty virus control. CeUs that are only infected with pGL3E2F reporter show to have a 2.9 times increase of the relative luciferase signal compared to empty virus control.
  • weUs that are co-infected with crude lysates from the placenta hbrary from weUs that do not show CPE, These weUs are excluded from aU calculations.
  • the average signal of the Hbrary is 60.3, with a standard deviation of 12.1.
  • one of the Hbrary viruses induce readings higher than the average of the hbrary plus 4 times the standard deviation, 108.9.
  • the lowest value measured for the hbrary viruses is 32.8, which is stiU 1.5 times higher than the signal of p 1 ⁇ 11 ⁇ 43 .
  • the hCAR cDNA is isolated using a PCR methodology.
  • the foUowing hCAR-specific primers are used:
  • HuCARfor 5'-GCGAAGCTTCCATGGCGCTCCTGCTGTGCTTCG-3' (SEQ ID NO:15)
  • HuCARrev 5'-GCGGGATCCATCTATACTATAGACCCATCCTTGCTC-3' The 5' primer contains a Hind ⁇ U. site, and the 3' primer a BamHI site.
  • the hCAR cDNA is PCR amplified from a HeLa ceU cDNA Hbrary (Quick clone, Clontech). A single fragment of 111 bp is obtained and digested with the Hindlll and BamHI restriction enzymes.
  • pIPspAdapt ⁇ vector (described in U.S. Patent No. 6,340,595) is digested with the same enzymes, gel-purified and used to Ugate to the digested PCR hCAR fragment.
  • SEQ ID NO: 13 Down regulation and over expression of SEQ ID NO: 13 are tested in transgenic animal models.
  • knockout animals preferably mice, are generated according to estabhshed procedures.
  • One or more exons of the genes encoding SEQ ID NO: 13 are deleted by homologous recombination in mouse ES ceUs.
  • ES ceUs have been isolated from a limited number of homozygous strains of inbred lab mice weU-suited to derive knock-out mice and are weU known for those skiUed in the art, Removal ofone or more exons is checked by techniques such as southern blotting and the diploid state of ES ceUs is checked by cytogenetic techniques.
  • the foUowing procedure is foUowed: subclone SEQ ID NO: 13 into a eukaryotic expression plasmid, downstream of a ubiquitously expressed promoter or, preferably, downstream of a promoter aUowing for expression only in a specific compartment of the body.
  • the plasmid containing the above-mentioned promoter and SEQ ID NO: 13 is then used to derive transgenic mice according to estabhshed procedures. Homozygous mouse strains, weU suited to derive transgenic mice, such as the FVB strain are used.
  • Exogenous expression of SEQ ID NO: 13 is analysed using southern blot, aUowing an estimation of the copy number of the expression cassette, integrated in the mouse genome and also by northern or western blotting, e.g., with antibodies produced in example 14.
  • the effect of the exogenous expression of SEQ ID NO: 13 on proliferation and apoptosis and on cancer is analyzed as described above for knockout animals.
  • Example 19 Transfection of hematopoietic stem ceUs with activators of E2F activity
  • Hits that activate E2F e.g., Hl-9 and therefore stimulate proliferation of ceUs, are used to stimulate the proliferation of, for example, hematopoietic stem ceUs for gene therapy, e.g., for treating sickle ceU anemia, thalassemia, or severely combined immuno-difficiencies (SCID)
  • Hematopoietic stem ceUs represent attractive targets for genetic modification since theh progeny make up the entire spectrum of the hematopoietic system.
  • stem ceUs due to the inherent quiescent nature of stem ceUs, gene transfer is limited since stable integration of retroviruses, the most currently used expression and transfer system in gene therapy, requhes ceU division. Moreover, there is a need for increasing the proportion of geneticaUy modified stem cells through ex vivo expansion before transplanting them back into the bone marrow. Furthermore, methodology for enriching pluripotent stem ceUs in culture could also have a major impact on treatment of blood and immune system disorders. For example, bone marrow transplantation is often the only option for persons having hematopoietic and immune system dysfunctions causes by chemotherapy or radiation therapy. Therefore, expansion of primitive stem ceUs in culture is a major advance for aU aspects of bone marrow transplantation as weU as gene therapy applications.
  • CD34+ positive ceUs are infected with adenoviruses (International AppHcation No. PCT/EP01/11086) transducing Hl-9 sequence, which activates E2F, CD34 does not appear on normal, mature human lymphoid or myeloid ceUs and is used for the identification of early progenitor and stem ceUs of the human hematopoietic system.
  • Hl-9 sequence induces proliferation of the CD34+ ceUs.
  • the thus expanded CD34+ population are subsequently used to reconstitute the bone marrow.
  • One major advantage is that the proliferation of the CD34+ ceUs in vitro guarantees a more efficient transfer and integration of retroviruses for gene therapy purposes.
  • retro vhal infection An alternative to adenovhal infection is retro vhal infection.
  • retro vhal particles obtained after transfection in a retroviral packaging ceU line, are used to infect the CD34+ ceUs.
  • retrovirus integrates into the genome of the target ceUs, this leads to the stable expression of the transgene, which is not shut down.
  • the retrovirus is equipped with an inducible promoter such that expression is shut down after transplantation into the bone marrow.
  • Hl-9 sequence it is advantageous for the Hl-9 sequence to function only in some ceUs types and not in other ceUs types. This aUows the in vivo use of viruses transducing this sequence, for example in tissue repah (e.g.,bone repah and bone replacement) and corrective surgery, without the need to purify the target ceUs away from ceUs that are not aUowed to proliferate.
  • tissue repah e.g.,bone repah and bone replacement
  • gccactgtag cagcaaggtc tgtgacagca aatcctacaa gagcaaggag ccccatgagt 480
  • agggcaagga tatgatggaa gaggaagccc cagaggagcg ggagagcact gaggccaccc 600 agagcaggac tgtcaggacc accagaaagg gagagatgcc tgtttcagga ttggcagtgg 660
  • aggatgagga tgatgcaacc agtaccagca atgcagacca gctgtctgac catggggacc 1500 ttctgtctga agaggagctg gatgaatgag actctgggaa tcatctacac aggaccaaac 1560

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Abstract

L'invention concerne les domaines de la génétique moléculaire et de la médecine. Cette invention concerne notamment le domaine de la génomique fonctionnelle. Elle concerne aussi des procédés et des moyens d'identification d'acides nucléiques et des polypeptides codés par ces acides nucléiques présentant une fonction liée à la voie, lesquels ont été isolés lors d'un test de criblage à haut débit au moyen de l'activité du facteur de transcription E2F en tant qu'afficheur. Les composés identifiés sont des cibles de médicament convenant au traitement de maladies humaines.
PCT/EP2002/002511 2001-03-07 2002-03-06 Test de criblage de banque adénovirale destiné à des procédés et à des gènes régulateurs e2f, et compositions destinées au criblage de composés WO2002070744A2 (fr)

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EP01870038A EP1239038A1 (fr) 2001-03-07 2001-03-07 Essais à haut rendement pour l'identification de modulateurs de l'activité de E2F
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US28259001P 2001-04-09 2001-04-09
US60/282,590 2001-04-09
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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2256197A1 (fr) 2004-06-14 2010-12-01 Galapagos N.V. Procédés d'identification, et composés utiles pour le traitement de maladies dégéneratives et inflammatoires
EP2360474A2 (fr) 2004-06-21 2011-08-24 Galapagos N.V. Méthodes et moyens pour le traitement de l'ostéoarthrite
EP2360478A1 (fr) 2004-10-15 2011-08-24 Galapagos N.V. Cibles et composés moléculaires et procédés d'identification afferents utilisés dans le traitement de maladies de dégénérescence des articulations et de maladies inflammatoires
US8637257B2 (en) 2007-06-20 2014-01-28 Galapagos Nv Molecular targets and compounds, and methods to identify the same, useful in the treatment of bone and joint degenerative diseases

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WO1999050405A1 (fr) * 1998-03-31 1999-10-07 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
WO1999054461A2 (fr) * 1998-04-17 1999-10-28 Metagen Gesellschaft Für Genomforschung Mbh Sequences d'acide nucleique humaines provenant d'une tumeur de l'endometre
WO1999055886A1 (fr) * 1998-04-24 1999-11-04 Genova Pharmaceuticals Corporation Decouverte de genes fonctionnels
WO1999060407A1 (fr) * 1998-05-15 1999-11-25 Cancer Research Campaign Technogoly Limited Analyses, techniques et dispositifs permettant de moduler l'activite de e2f
WO1999064582A2 (fr) * 1998-06-12 1999-12-16 Introgene B.V. Criblage de fonction genique a haut rendement a l'aide de banques d'applications genomiques fonctionnelles
US6048693A (en) * 1996-10-16 2000-04-11 Bittech, Inc. Phenotypic assays of cyclin/cyclin-dependent kinase function
WO2000056881A1 (fr) * 1999-03-23 2000-09-28 Human Genome Sciences, Inc. 48 proteines humaines secretees
WO2001004296A1 (fr) * 1999-07-12 2001-01-18 Mcgill University Polypeptides rbp1 et leurs utilisations

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Publication number Priority date Publication date Assignee Title
US6048693A (en) * 1996-10-16 2000-04-11 Bittech, Inc. Phenotypic assays of cyclin/cyclin-dependent kinase function
WO1998033819A1 (fr) * 1997-01-30 1998-08-06 New York University Recepteurs cellulaires des adenovirus du sous-groupe c et des virus coxsackie du groupe b
WO1999050405A1 (fr) * 1998-03-31 1999-10-07 Genetics Institute, Inc. Proteines secretees et polynucleotides les codant
WO1999054461A2 (fr) * 1998-04-17 1999-10-28 Metagen Gesellschaft Für Genomforschung Mbh Sequences d'acide nucleique humaines provenant d'une tumeur de l'endometre
WO1999055886A1 (fr) * 1998-04-24 1999-11-04 Genova Pharmaceuticals Corporation Decouverte de genes fonctionnels
WO1999060407A1 (fr) * 1998-05-15 1999-11-25 Cancer Research Campaign Technogoly Limited Analyses, techniques et dispositifs permettant de moduler l'activite de e2f
WO1999064582A2 (fr) * 1998-06-12 1999-12-16 Introgene B.V. Criblage de fonction genique a haut rendement a l'aide de banques d'applications genomiques fonctionnelles
WO2000056881A1 (fr) * 1999-03-23 2000-09-28 Human Genome Sciences, Inc. 48 proteines humaines secretees
WO2001004296A1 (fr) * 1999-07-12 2001-01-18 Mcgill University Polypeptides rbp1 et leurs utilisations

Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2256197A1 (fr) 2004-06-14 2010-12-01 Galapagos N.V. Procédés d'identification, et composés utiles pour le traitement de maladies dégéneratives et inflammatoires
EP2256198A1 (fr) 2004-06-14 2010-12-01 Galapagos N.V. Procédés d'identification, et composés utiles pour le traitement de maladies dégénératives et inflammatoires
EP2270160A1 (fr) 2004-06-14 2011-01-05 Galapagos N.V. Procédés d'identification, et composés utiles pour le traitement de maladies dégéneratives et inflammatoires
EP2360474A2 (fr) 2004-06-21 2011-08-24 Galapagos N.V. Méthodes et moyens pour le traitement de l'ostéoarthrite
EP2386856A2 (fr) 2004-06-21 2011-11-16 Galapagos N.V. Procédés et moyens pour le traitement de l'arthrose
EP2360478A1 (fr) 2004-10-15 2011-08-24 Galapagos N.V. Cibles et composés moléculaires et procédés d'identification afferents utilisés dans le traitement de maladies de dégénérescence des articulations et de maladies inflammatoires
US8637257B2 (en) 2007-06-20 2014-01-28 Galapagos Nv Molecular targets and compounds, and methods to identify the same, useful in the treatment of bone and joint degenerative diseases

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