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WO2006039399A2 - Decroissance de l'arnm mediee par staufen 1 (stau1) - Google Patents

Decroissance de l'arnm mediee par staufen 1 (stau1) Download PDF

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WO2006039399A2
WO2006039399A2 PCT/US2005/035011 US2005035011W WO2006039399A2 WO 2006039399 A2 WO2006039399 A2 WO 2006039399A2 US 2005035011 W US2005035011 W US 2005035011W WO 2006039399 A2 WO2006039399 A2 WO 2006039399A2
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staul
substance
mrna
smd
upfl
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WO2006039399A3 (fr
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Lynne E. Maquat
Yoon Ki Kim
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University Of Rochester
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4702—Regulators; Modulating activity
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    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering nucleic acids [NA]

Definitions

  • Mammalian Staufenl is an RNA binding protein that binds to extensive RNA secondary structures, primarily through one or more double-stranded RNA-binding domains (Marion et al., 1999; Wickham et al., 1999).
  • Staufen functions in the transport and localization of bicoid and oskar mRNAs to, respectively, the anterior and posterior poles of oocytes, and prospero mRNA during asymmetric divisions of embryonic neuroblasts (Broadus et al., 1998; Li et al., 1997; Matsuzaki et al., 1998; Schuldt et al., 1998; Shen et al., 1998; St Johnston, 1995).
  • Drosophila Staufen also functions in the translational derepression of oskar mRNA once the mRNA has been localized to the posterior pole of an oocyte (Ephrussi et al., 1991; Kim- Ha et al., 1995; Kim-Ha et al., 1991 ; Micklem et al., 2000).
  • the Staul gene is ubiquitously expressed and generates protein isoforms having apparent molecular weights of 55 and 63 kDa (Kiebler et al., 1999; Marion et al., 1999; Monshausen et al., 2001; Wickham et al., 1999).
  • the 55-kDa isoform associates with 4OS and 60S ribosomal subunits and co-localizes with the rough endoplasmic reticulum (Luo et al., 2002; Marion et al., 1999; Wickham et al., 1999).
  • Staul interacts with telomerase RNA, suggesting that it functions during DNA replication, cell division or both, possibly by influencing telomerase RNA processing or RNP assembly or localization (Bachand et al., 2001; Le et al., 2000).
  • this invention in one aspect, relates to methods of treating subjects with conditions that result from or modified by affecting Staul -mediated mRNA decay and to screening for and manufacturing those therapeutic agents that modulate Staul mediated mRNA decay.
  • Figure 1 shows that Human Upfl interacts with human Staufen (Stau)l in a yeast two-hybrid analyses, in vitro binding assays, and immunopurifications (IPs) of Staul -HA 3 from Cos cells.
  • Figure IA shows yeast two-hybrid screening. Human Upfl as bait interacts with human Staul from the pMyr-cDNA library (upper). Negative and positive controls were provided by Stratagene. The region of Staul that interacts with Upfl was mapped to reside within the double-stranded RNA binding domain (dsRBD)4 and tubulin binding domain (TBD) (lower).
  • Figure IB shows GST pull-down assays. E. coli lysates that expressed GST-Upfl (+) were mixed with E.
  • Cos cells were transiently transfected with a plasmid that expressed the 55-kDa isoform of Staul-HA 3 .
  • RNA and protein were purified from the lysate before and after IP using anti-HA antibody or, to control for the specificity of the IP, rat (r) IgG.
  • RNase A was added to half of each sample prior to IP.
  • SMG7 mRNA was analyzed using RT-PCR to demonstrate that the RNase A digestion was complete (lower). The four left-most lanes represent 2-fold serial dilutions of RNA and demonstrate that the RT-PCR is semi-quantitative. Western blotting was used to detect the specified proteins (upper).
  • Figure IE shows IP of cellular Upf3/3X. Lysate from untransfected Cos cells was immunopurified using anti-Upf3/3X antibody or, as a control for nonspecific IP, normal rabbit serum (NRS). Western blotting was used to analyze the specified proteins. Upf3 co-migrated with Ig heavy chains and, thus, could not be analyzed.
  • Figure IF shows IP of FLAG-Upfl .
  • Lysates of HeLa cells that did (+ pCI- neo-FLAG-UPFl) or did not (- pCI-neo-FLAG-UPFl) stably express FLAG-Upfl was analyzed either before or after IP using anti-Flag antibody by Western blotting for the specified proteins.
  • FIG. 2 shows that down-regulating cellular Staul has no detectable effect on the EJC-dependent NMD of Gl 39Ter or GPxI 46Ter mRNA.
  • HeLa cells were transiently transfected with Staul siRNA, Upf3X siRNA, or a nonspecific Control siRNA. Two days later, cells were re-transfected with pmCMV-Gl and pmCMV-GPxl test plasmids, either nonsense-free (Norm) or nonsense-containing (Ter), and the phCMV-MUP reference plasmid. After an additional day, protein and RNA were purified.
  • Figure 2A shows the Western blot analysis of the siRNA-mediated down-regulation of Staul or Upf3X, where the level of eIF3b served to control for variations in protein loading.
  • Figure 2B shows the RT-PCR analysis of the level of Gl mRNA (left) or GPxI mRNA (right), which was normalized to the level of MUP mRNA. Normalized levels of Norm mRNA in the presence of each siRNA were defined as 100%. Levels in three independently performed experiments did not vary by more than 7%.
  • Figure 3 shows that tethering Staul to the FLuc mRNA 3' UTR reduces FLuc mRNA abundance.
  • Figure 3 A shows the schematic representations of firefly (F) and renilla (R) luciferase (Luc) expression plasmids pcFLuc-MS2bs, pcFLuc, and pRLuc, where X8 specifies eight tandem repeats of the MS2 coat protein binding site.
  • HeLa cells were co- transfected with the specified pcFLuc-MS2bs reporter plasmid, the pRLuc reference plasmid, as well the specified effector plasmid. Two days after transfection, protein and RNA were purified. Western blotting using anti-HA antibody demonstrates effector expression (left).
  • RT-PCR demonstrates that tethered Staul reduces FLuc-MS2bs mRNA abundance (right). Numbers below the figure represent the levels of FLuc or FLuc-MS2bs mRNA, which were normalized to the level of RLuc mRNA. Each normalized level of FLuc or FLuc-MS2bs mRNA was then calculated as a percentage of the normalized level of FLuc or FLuc-MS2bs mRNA that was obtained in the presence of pMS2-HA or pcNMS2, which was defined as 100%. RT-PCR results in at least three independently performed experiments did not vary by more than 9%.
  • Figure 4 shows that the abundance of FLuc-MS2bs mRNA is reduced by only specific proteins, such as MS2-HA-Staul or MS2-Upfl , but not MS2-HA-eIF4AIII, myc-
  • FIG. 4 A shows the western blot analysis of MS2-HA, MS2-HA-Staul and MS2-HA- eIF4Ai ⁇ expression using anti-HA antibody (left), MS2-Upfl, myc-Upfl and endogenous Upfl expression using anti-Upfl antibody (upper right), and MS2-HA, MS2-HA-Staul and Stau-HA 3 expression using anti-Staul antibody (lower right). Endogenous Staul was detectable with enhanced chemiluminescence.
  • Figure 4B shows RT-PCR analysis of the levels of FLuc-MS2bs and RLuc mRNAs.
  • Figure 5 shows that siRNA-mediated down-regulation of cellular Upfl but not Upf2 or UpfiX inhibits the reduction in FLuc-MS2bs mRNA abundance that is mediated by tethered Staul .
  • Figure 5 A and 5B show that HeLa cells were transiently transfected with the specified siRNA. Two days later, cells were transfected with the pcFLuc-MS2bs reporter plasmid, the pRLuc reference plasmid, and the specified effector plasmid. Alternatively, cells were transfected with a pmCMV-Gl test plasmid (either Norm or Ter) and the phCMV-MUP reference plasmid. After an additional two days, protein and RNA were purified.
  • pmCMV-Gl test plasmid either Norm or Ter
  • Figures 5A-5C show that Western blotting was used to quantitate the extent of down-regulation.
  • Figures 5A-5B shows that RT-PCR was used to quantitate the effects of siRNA on Gl mRNA abundance, as in Figure 2B.
  • Figures 5A-5C (right) show that RT-PCR was used to quantitate the effects of siRNA on mRNA abundance that were mediated by tethering the specified protein as in Figure 3B.
  • mRNA levels in at least two independently performed experiments did not differ by more than 10 %.
  • Figure 6 shows evidence that Staul reduces mRNA abundance in a way that depends on an upstream termination codon that is located upstream of the Staul binding site.
  • HeLa cells were transfected as described in the legend to Figure 3 using the specified test, reference and effector plasmids.
  • Figure 6A shows schematic representations of the pcFLuc-MS2bs, pcFLuc (UAA->CAA)-MS2bs, pcGl-MS2bs, and pcGl(UAA ⁇ UAC)- MS2bs test plasmids (the latter two were called pc/3-6bs and pcj3UAC-6bs, respectively, in Lykke-Andersen et al., 2000).
  • Figure 6B shows the quantitation of MS2-HA and MS2-HA- Staul expression.
  • This figure is like Figure 3, except that pcFLuc(UAA-»CAA)-MS2bs was used as the reporter plasmid.
  • Figure 6C is like Figure 3, except that pcGl-MS2bs or pcGl(UAA-»UAC)-MS2bs was the reporter plasmid, and phCMV-MUP was the reference plasmid. Results represent two independently performed experiments and do not vary by more than 11%.
  • Figure 7 shows that Staul binds Arfl mRNA.
  • Figure 7 A shows the IP of Staul - HA 3 . 293 cells were transiently transfected with a plasmid that expressed Staul-HA 3 or, to control for nonspecific IP, Staul -6xHis. Two days later, cells were lysed, a fraction of cell lysates was immunopurified using anti-HA antibody, and Staul was identified before and after IP using Western blotting and anti-Staul antibody. Asterisks denote the 63-kDa and 55-kDa isoforms of endogenous Staul.
  • Figure 7B shows the identification of Arfl mRNA in Staul -containing RNP.
  • Biotin-labeled cRNA was synthesized from RNA that had been immunopurified using anti-HA antibody from Staul-HA 3 - or Staul -6xHis-expressing cells or poly(A) + RNA from untransfected cells.
  • the histogram represents the amount of hybridized Arfl mRNA that was either immunopurified using anti-HA antibody from
  • FIG. 7C shows RT-PCR of Arfl mRNA in Staul -containing RNPs. 293 cells were transiently transfected with a plasmid that expressed either Staul-HA 3 or, as a control, Staul-6xHis. As in 1OA, except that immunopurified RNA was purified, and Arfl mRNA was amplified using RT-PCR (two left-most lanes). Alternatively, lysates from untransfected 293 cells were immunopurified using with anti-Staul antibody (right) or an unrelated ascites fluid for the analysis of Arfl mRNA.
  • Figure 8 shows that Staul binds the 3' UTR of Arfl mRNA and reduces its abundance in a mechanism that involves Upfl .
  • Figure 8 A shows schematic representations of the Arfl gene and the pSport-Arfl and pSport-Arfl ⁇ (3'UTR) cDNA expression plasmids.
  • Figure 8B is a parallel study to Figure 4A, except that HeLa cells were transiently transfected with the specified siRNA. Two days later, cells were re-transfected with a pSport-Arfl test plasmid and the reference phCMV-MUP plasmid. After an additional day, protein and RNA were isolated for Western analysis and RT-PCR, respectively.
  • FIG. 8D shows IP of Staul -HA 3 .
  • Cos cells were transiently transfected with the Staul -HA 3 expression vector, pSport-Arfl or pSport- Arfl ⁇ (3 'UTR), and phCMV-MUP. Notably, cells were transfected with only half as much pSport-Arfl ⁇ (3'UTR) relative to pSport-Arfl in order to compensate for the difference in the level of product mRNA.
  • Figure 9 shows a comparable increase in the abundance of Arfl mRNA that derives from pSport-Arfl is obtained using two different Staul siRNAs and two different Upfl siRNAs. As in Figure 8B; however, Staul (A) and Upfl (A) siRNAs were analyzed in parallel to, respectively, the Staul and Upfl siRNAs that were analyzed in the Figure 8B.
  • Figure 9 A shows that the western blotting demonstrates down-regulation of Staul (left), and RT-PCR demonstrates that down-regulating Staul increases the level of Arfl mRNA abundance (right).
  • Figure 9B is like 9A, except that Up ⁇ was down-regulated. Results represent two independently performed experiments and do not vary by more than 12%.
  • FIG 10 shows that Staul binds within an ⁇ 230-nt region of the Arfl mRNA 3' UTR, and this region reduces the half- life of Arfl mRNA in a Staul -mediated mechanism.
  • Figure 1OA shows the schematic representations of the various Arfl mRNAs harboring deletions within the 3' UTR. Numbering is relative to the first nucleotide of endogenous Arfl mRNA, which is defined as 1. The upper-most construct represents full-length mRNA.
  • Figure 1OB shows that the 293 cells were transfected with the Staul -HA 3 expression vector and the specified pSport-Arfl test plasmid. One-tenth of each IP was used to determine IP efficiencies using Western blotting (upper).
  • Figure 1OC shows that the L cells were transfected with mouse (m)Staul, mUpfl, mUpf2, or Control siRNA. Two days later, cells were re- transfected with the pfos-Arfl-SBS test plasmid (upper left) and the phCMV-MUP reference plasmid in the absence of serum.
  • Serum was added to 15% after an additional 24 hr, and protein was purified from the cytoplasmic fraction for Western blotting (second- from-top, left and right).
  • RNA was purified from the nuclear fraction for RT-PCR analysis at the specified times (second-from-bottom, left and right).
  • the level of pfos-Arfl-SBS mRNA was normalized to the level of MUP mRNA. Normalized levels were calculated as a percentage of the normalized level of fos- Arfl -SBS mRNA at 30 min in the presence of each siRNA, which was defined as 100. Normalized levels represent the average of two independently performed experiments and are plotted as a function of time after serum addition (bottom, left and right).
  • Figure 11 shows that inserting the Staul binding site (SBS; nts 622-924) of the Arfl mRNA 3'UTR within FLuc mRNA results in a Staul -dependent reduction in FLuc mRNA halflife, whereas inserting a different region (No SBS; nts 899-1144) of the Arfl mRNA 3'UTR does not.
  • Figure 1 IA shows schematic representations of pcFLuc-SBS and pcFLuc-No SBS test plasmids.
  • the Arfl SBS maintains the distance and sequence of the minimized Staul binding site (nts 688-919) relative to the normal termination codon.
  • Figure 12 shows the 3' UTR of PAICS mRNA, which encodes phosphoribosylaminoimidazole carboxylase and phosphoribosylaminoimidazole succinocarboxamide synthetase activities, also binds Staul, and down-regulating Staul increases PAICS mRNA abundance.
  • Figure 12A is like Figure 1OB, except that 293 cells were transfected with test plasmid pSport-PAICS or, as a negative control, pCDNA3-RSV- CK2A2, which encodes casein kinase 2 alpha prime polypeptide.
  • Figure 12B is like Figure 8D, except that pSport-PAICS or pSport-PAICS ⁇ (3'UTR) was the test plasmid.
  • the small amount of MUP mRNA that was detected in the IP represents background since (i) MUP mRNA was never detected in other IPs and (ii) a comparison of the levels of PAICS and MUP mRNAs before and after IP shows a 110-fold enrichment of PAICS mRNA relative to MUP mRNA after IP.
  • Figure 12C is like Figure 8B, except that pSport-PAICS was the test plasmid. Results represent two independently performed experiments and do not vary by more than 2%.
  • Figure 13 shows that down-regulating Staul has no effect on the half-life of fos- Arfl ⁇ (3'UTR) mRNA.
  • Figure 13 was conduct in the same manner as Figure 1 IB, except that the test plasmid was pfos-Arfl ⁇ (3'UTR). Results represent two independently performed experiments, and RT-PCR quantitations did not vary by more than 19%.
  • Figure 14 shows the models for EJC-dependent NMD and SMD of Arfl mRNA in mammalian cells.
  • Figure 14A shows the recruitment of Upfl to one of the four EJCs of Arfl mRNA via Upf2 and Upf3/3X.
  • EJC exon junction complex
  • EJC consists of the NMD factor Upf3 or UpOX, each of which binds U ⁇ f2, and Upf2 is thought to subsequently bind Upfl .
  • EJC-dependent NMD occurs if translation terminates prematurely more than -50-55 nucleotides upstream of any exon-exon junction, which would be -20-25 nucleotide upstream of the corresponding EJC (only the 3'-most of which is shown).
  • Figure 14B shows the recruitment of Upfl to the 3' UTR of Arfl mRNA via Staul. Staul, which binds the 3' UTR of Arfl mRNA, recruits Upfl independently of an EJC. Data indicate that SMD occurs when translation terminates properly.
  • Staul binding site would reside more than ⁇ 20-25 nts downstream of the normal termination codon in order to elicit SMD. This is consistent with the finding that Staul binds at least 67-nt downstream of this codon.
  • FIG. 15 Demonstration that 11 of 12 transcripts that were upregulated in human cells depleted of Staul using three independently performed microarray analysis are upregulated using RT-PCR and transcript-specific primers.
  • RNAs analyzed were identical to the RNAs from Control and Staul siRNA-treated samples analyzed in Figure 17.
  • the level of each test transcript was normalized to the level of SMG7 mRNA, which is insensitive to Staul siRNA (data not shown) and served to control for variations in RNA recovery. Numbers below each lane specify the fold change in the level of each test transcript in cells treated with Stau siRNA relative to Control siRNA, the latter of which was defined as 1.
  • Figure 17 shows that c-JUN, SERPINEl and JX7R mRNAs are increased in abundance in human cells depleted of either Upfl or Staul but not Upf2.
  • HeLa cells were transiently transfected with Staul, Staul (A), Upfl, Upfl (A) or Upf2 siRNA or, to control for nonspecific depletion, Control siRNA.
  • Staul Staul
  • Upfl Upfl
  • Upfl Upfl
  • Upf2 siRNA Upf2 siRNA
  • Figure 17A shows Western blot analysis, where the level of Vimentin serves to control for variations in protein loading, and the normal level of Staul, Upfl or Upf2 is determined in the presence of Control siRNA.
  • Figure 17B shows RT-PCR analysis of the level of endogenous c-JUN mRNA (upper), SERPJJSfEl mRNA or JL7R mRNA (lower), each of which is normalized to the level of endogenous SMG7 mRNA.
  • the normalized level of each mRNA in the presence of Control siRNA is defined as 1.
  • RT-PCR results are representative of three independently performed experiments that did not differ by the amount specified.
  • FIG. 18 shows that Staul binds within the 3'UTR of c-JUN, SERPINEl and JX7R mRNAs.
  • Cos cells were transiently transfected with three plasmids: (i) pcFLuc-c-JUN 3'UTR, pcFLuc-SERPINEl 3'UTR and pcFLuc-IL7R 3'UTR test plasmids, which encode FLuc mRNA in which all of the FLuc 3'UTR has been replaced by, respectively, part of the c- JUN 3'UTR or all of the SERPINEl or IL7R 3'UTR; (ii) the Staul-HA 3 expression vector; (iii) the pcFLuc-SBS test plasmid, which encodes FLuc mRNA in which the 3'UTR has been replaced by the 231 -nucleotide Staul binding site (SBS) of Arfl mRNA (Kim et al., 2005)
  • FIG. 18A shows schematic representations of pcFLuc-c-JUN, pcFLuc-SERPINE 1 , pcFLuc-IL7R and pcFLuc-SBS test plasmids.
  • Figure 18B shows Western blotting using anti( ⁇ )-HA or anti-Calnexin demonstrated that Staul -HA 3 but, as expected, not Calnexin was immunopurified.
  • Figure 18C shows RT-PCR analysis demonstrates that c-JUN, SERPINEl and IL7R 3'UTRs, like the Arfl SBS, bind Staul-HA 3 , whereas MUP mRNA does not. Results are representative of three independently performed experiments.
  • Figure 19 shows that depleting cells of Staul or Upfl increases the half-life of FLuc mRNA when the 3'UTR consists of the Staul binding site of the c-JUN, SERPME1 or IL7R 3'UTR.
  • L cells were transfected with mouse (m)Staul, mUpfl or Control siRNA.
  • Figure 19B shows Western blotting of mStaul or mUpfl, where the level of ⁇ -actin serves to control for variations in protein loading. mStaul was depleted to 35% its normal level, and mUpfl was depleted to 30% of its normal level.
  • Figure 19C shows RT-PCR analysis of the SMD candidate mRNAs. For each time point, the level of each mRNA transcribed from the fos promoter is normalized to the level of MUP mRNA. Normalized levels are calculated as a percentage of the normalized level of each mRNA transcribed at 30 minutes in the presence of each siRNA, which is defined as 100 (left). These levels are plotted as a function of time after serum addition (right).
  • FIG. 26 shows that GAP43 mRNA is an SMD target.
  • Figure 2OA shows RT- PCR demonstrating that depleting cells of Staul or Upfl increases the cellular abundance of GAP43 mRNA.
  • Figure 2OB shows IP revealing that Staul binds to the 3' UTR of GAP43 mRNA and, as a positive control Arfl SBS.
  • Cos cells were transfected with three plasmids: (i) pcFLuc-GAP43 3 ' UTR (left), (ii) the Staul - HA 3 expression vector; (iii) pcFLuc-SBS (left), and (iv) phCMV-MUP and analyzed as described in the legend to Figure 2 except that FLuc-GAP43 3' UTR and FLuc-Arfl SBS mRNAs were analyzed instead of the other FLuc mRNA derivatives.
  • Figure 2OC shows Western blot (upper right) and RT-PCR analysis (lower left) demonstrating that depleting cells of Staul or Upfl increases the half-life of FLuc mRNA when the 3'UTR consists of the Staul binding site of GAP43 mRNA.
  • L cells were transfected with mStaul, mUpfl or Control siRNA. Two days later, cells were re-transfected with the pfos-FLuc-GAP43 3' UTR test plasmid and the phCMV-MUP reference plasmid in the absence of serum. Subsequent treatment and analyses were as described in the legend to Figure 19 except that fos-FLuc- GAP43 3 ' UTR mRNA was analyzed instead of the other fos-FLuc mRNA derivatives. Results are representative of two independently performed experiments.
  • Ranges may be expressed herein as from “about” one particular value, and/or to "about” another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about,” it will be understood that the particular value forms another embodiment. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint.
  • Primers are a subset of probes which are capable of supporting some type of enzymatic manipulation and which can hybridize with a target nucleic acid such that the enzymatic manipulation can occur.
  • a primer can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art which do not interfere with the enzymatic manipulation.
  • Probes are molecules capable of interacting with a target nucleic acid, typically in a sequence specific manner, for example through hybridization. The hybridization of nucleic acids is well understood in the art and discussed herein. Typically a probe can be made from any combination of nucleotides or nucleotide derivatives or analogs available in the art.
  • chemical refers to any reagent that can bind, mutate, dissolve, cleave, initiate a reaction that results in a new substance, or alter the confirmation of a target substance.
  • An example of a chemical would be a small molecule.
  • pioneer round of translation or “pioneering round” refers to an initial round of mRNA translation prior to steady-state translation.
  • the round is characterized by the presence of CBP80 and CBP20 as the CAP binding proteins, PABP2 as a polyadenylation binding protein, and involves Upf2 and Upf3 or UpOX.
  • Staul is involved in mRNA decay. Also, Staul -mediated mRNA decay involves the nonsense-mediated mRNA decay (NMD) factor Upfl .
  • NMD nonsense-mediated mRNA decay
  • the expression of protein-encoding genes requires a series of steps in which pre-mRNA is processed to mRNA in the nucleus before mRNA is translated into protein in the cytoplasm. These steps are subject to quality control to ensure that only completely processed mRNA is exported to the cytoplasm (reviewed in Maquat and Carmichael, 2001).
  • One form of quality control called mRNA surveillance or NMD.
  • NMD in mammalian cells is generally a splicing-dependent mechanism that degrades newly synthesized mRNAs that prematurely terminate translation more than 50-55 nucleotides upstream of an exon-exon junction as a means to prevent the synthesis of potentially harmful truncated proteins (reviewed in Frischmeyer and Dietz, 1999; Hentze and Kulozik, 1999; Li and Wilkinson, 1998; Maquat, 2004a; Maquat, 2004b; Wilusz et al, 2001). By so doing, NMD precludes the synthesis of the encoded truncated proteins, which can function in deleterious ways (see, e.g., Inoue et al., 2004). NMD also targets naturally occurring mRNAs such as certain selenoprotein mRNAs and an estimated one-third of alternatively spliced mRNAs, some of which encode functional protein isoforms (Maquat, 2004b).
  • the dependence of NMD on splicing reflects the deposition of an exon junction complex (EJC) of proteins -20-24 nucleotides upstream of splicing-generated exon-exon junctions (Kataoka et al., 2000; Kim et al., 2001; Le Hir et al., 2000a; Le Hir et al., 2000b; Lykke-Andersen et al., 2001).
  • EJC exon junction complex
  • This EJC includes NMD factors Up ⁇ (also called UpOa) or Up ⁇ X (also called Upfib), Upf2 and, presumably, Upfl (Gehring et al., 2003; Kim et al., 2001; Lejeune et al., 2002; Lykke-Andersen et al., 2000; Lykke-Andersen et al., 2001).
  • Up ⁇ and Up ⁇ X appear to play a comparable role in NMD (Kim et al., 2001 ; Lykke-Andersen et al., 2001), although different isoforms of Up ⁇ can form distinct protein complexes (Ohashi et al., 2002).
  • EJC EJC
  • Y14 RNPSl, SRml ⁇ O, REF/Aly, UAP56, Mago, Pinin, and eIF4Am
  • Ferraiuolo et al., 2004 Kataoka et al., 2000; Kim et al., 2001 ; Le Hir et al., 2001 ; Le Hir et al., 2000b; Luo, et al., 2001 ; Palacios et al., 2004; Shibuya et al., 2004; Tarn et al., 2003).
  • EJCs are present on mRNA that is bound at the cap by the mostly nuclear cap binding protein (CBP)80-CBP20 heterodimer, which is consistent with data indicating that NMD targets CBP80-bound mRNA during a pioneer round of translation (Chiu et al., 2004; Ishigaki et al., 2001 ; Lejeune et al., 2002; Lejeune et al., 2004).
  • CBP nuclear cap binding protein
  • Staul binds the NMD factor Upfl and the 3' untranslated region (UTR) of mRNA that encodes ADP-ribosylation factor (Arf)l.
  • URR 3' untranslated region
  • Rhf ADP-ribosylation factor
  • Staul mediates Arfl mRNA decay in a mechanism that differs from NMD by occurring independently of splicing or Upf2 and Up ⁇ X.
  • SMD Staul -mediated mRNA decay
  • Staul plays no detectable role in the EJC-dependent NMD of either /3-globin or glutathione peroxidase 1 mRNA.
  • the invention described herein in one aspect relates to methods of treating a disorder in a subject comprising administering to the subject a substance, wherein the substance modulates Staul -mediated mRNA decay (SMD).
  • SMD Staul -mediated mRNA decay
  • treating can refer to any method that improves a disorder in a subject.
  • the improvement can include but is not limited to a decrease in one or more symptoms of the disorder such that the disorder is reduced.
  • Treating is understood to include small improvements in the disorder up to and including the complete ablation of the disorder.
  • the treatment can result in a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction in the disorder.
  • a disorder means any inherited or acquired disease or condition associated with Staul mediated mRNA decay that has a negative effect relative to the wild-type state.
  • the disorder can be inherited genetic disorder.
  • Inherited genetic disorders are disorders that result from the presence of an abherent gene or genes that alter the way an interaction, system or pathway works.
  • system refers to any cell, organism, or in vitro assay or culture. Such a system includes components necessary for SMD activity. Such components can include, for example, but are not limited to Staul and Upfl.
  • the disorder is a genetic disorder
  • the genetic disorder can be selected from the group consisting of cystic fibrosis, hemophilia, mucopolysaccharidoses, muscular dystrophy, anemia, glycolytic enzyme deficiency, connective tissue disorder, DNA repair disorder, dementia, Sandhoff disease, epidermolysis bullosa simplex, insulin resistance, maple syrup urine disease, hereditary fructose intolerance, inherited immunodeficiency, inherited cancer, carbohydrate metabolism disorder, amino acid metabolism disorder, lipoprotein metabolism disorder, lipid metabolism disorder, lysomal enzymes disorder, steroid metabolism disorder, purine metabolism disorder, pyrimidine metabolism disorder, metal metabolism disorder, porphyrin metabolism disorder, and heme metabolism disorder.
  • a disorder can be dementia.
  • Dementias can include but are not limited to Alzheimer's, Lewy Body dementia, Binswanger's dementia, or dementias associated with Parkinson's Disease, progressive supranuclear palsy, Huntingdon's disease, Pick's disease, Creutzfeldt- Jakob disease, Gerstmann-Straussler-Scheinker disease, AIDS, or trauma.
  • disorders can also include acquired disorders.
  • Acquired disorders are disorders that result from some external insult or injury, or from an unknown mechanism that is not derived from a genetic characteristic.
  • acquired disorder is meant any disorder that lacks a clear genetically inherited link.
  • acquired disorders can result from chemical exposure, radiation exposure, or random mutation in a gene that was not present in the subject earlier.
  • an acquired disorder can comprise a cancer. 44.
  • methods of the invention wherein the disorder is an acquired disorder.
  • the acquired disorder is, by way of example but not by way of a limitation, a cancer. Such a cancer may be related to mutations such as mutations in p53 or BRCA-I .
  • the disclosed compositions can be used to treat any disease where uncontrolled cellular proliferation occurs such as cancers.
  • a representative but non-limiting list of cancers that the disclosed compositions can be used to treat is the following: lymphoma, B cell lymphoma, T cell lymphoma, leukemia, carcinoma, sarcoma, glioma, blastoma, neuroblastoma, plasmacytoma, histiocytoma, melanoma, mycosis fungoide, hypoxic tumor, myeloma, metastatic cancer, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, liver cancer, colon cancer, cervical cancer, breast cancer, epithelial cancer, renal cancer, genitourinary cancer, pulmonary cancer, esophageal carcinoma, hematopoietic cancers, testicular cancer, and colorectal cancer.
  • SMD occurs in or as a result of the pioneering round of translation.
  • SMD can also occur during steady- state translation.
  • steady-state translation can target eIF4E-bound mRNA.
  • subject refers to any cell, tissue, system, or organism used to study or treat a disorder relating to SMD, including, for example, human patients with conditions that result from SMD or cell lines used to study aspects of SMD.
  • subject is a mammal. It is specifically contemplated that mammal can include but is not limited to human, monkey, mouse, dog, pig, rabbit, and cow.
  • the disclosed methods function by modulating SMD.
  • modulation or “modulating” is meant either an increase or a decrease in SMD activity.
  • Staufen 1 can modulate mRNA transcripts through the stabilization or destabilization of an mRNA.
  • SMD levels are increased or decreased in a subject with a condition resulting from a mutation that generates a nonsense codon (including but not limited to a nonsense mutatation) depends on the particular mRNA that is affected, the binding of Staufenl and, where binding of Staul occurs.
  • a decrease in SMD activity could increase the amount of mRNA that is available for translation.
  • modulation can be measured by, e.g., comparing the level of the mRNA harboring a premature or alternative termination codon relative to an unaffected cellular RNA to the level of that mRNA lacking a premature or alternative termination codon to the same unaffected cellular RNA. Subsequent nonsense suppression could be used to increase the amount of the encoded full-length protein. Conversely, an increase in SMD activity would decrease the amount of mRNA that is available for translation so as to reduce production of the encoded truncated protein.
  • the disclosed methods can be used to modulate the expression of genes by modulating SMD (e.g., to decrease mRNA from a truncated protein, one would increase SMD activity. A decrease in SMD activity would be used to increase the amount of mRNA of a truncated protein available).
  • SMD can also target mRNAs that do not have a premature or alternative termination codon, providing another means by which gene expression could be regulated.
  • a decrease in Staul -mediated mRNA decay can refer to any change that results in a smaller amount of Staul mRNA mediated decay.
  • a decrease can refer to a reduction in an activity.
  • a substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed.
  • a decrease can include, but is not limited to, a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction.
  • a decrease can but does not have to result in the complete ablation of a substance or activity. Therefore, for example, a decrease in SMD would result in the presence of more gene products with early or alternative termination sites (i.e., a decrease in SMD activity).
  • the term “inhibits” or “inhibition” refers to any degree of decrease as compared to a control. Thus, for example, “inhibition” can refer to a 10% " 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% reduction as compared to a control.
  • an increase in Staul - mediated mRNA decay can refer to any change that results in a larger amount of a Staul mediated mRNA decay activity.
  • an increase in the amount in SMD of a particular mRNA can include but is not limited to a 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 100% increase. It is understood and herein contemplated that an increase in SMD would result in a subsequent decrease in the amount of gene products with early or alternative termination sites. 51. It is understood and herein contemplated that Staul can bind mRNA at AU rich elements (ARE).
  • ARE AU rich elements
  • Staul binds mRNA at Type HI AREs.
  • the binding of Staul to AREs can affect the level of gene expression for the mRNA transcript to which it binds. It is also understood and herein contemplated that the binding of Staul to AREs can have a stabilizing effect. Therefore, herein disclosed is the ability of Staul to bind stabilizing or destabilizing elements.
  • methods wherein the modulation of SMD results in the stabilization of mRNA are also disclosed are methods wherein the modulation of SMD results in the destabilization of mRNA.
  • a disorder in a subject comprising administering to the subject a substance, wherein the substance modulates Staul -mediated mRNA decay.
  • Modulation of Staul -mediated mRNA decay can result in a decrease in SMD.
  • the decrease in SMD would increase the abundance of the nRNA containing a premature or alternative termination codon.
  • the disclosed methods can be used to modulate the level of mRNA or protein expression.
  • Staul can increase or decrease the abundance of an mRNA independently of SMD.
  • not all SMD activity occurs through the involvement of Staul and Upfl, but can be Upfl independent.
  • modulating the level of mRNA is meant that the abundance of a target transcript can be increased or decreased by the expression of Staul .
  • the ability to modulate the abundance of target genes can be achieved in conjunction with or independently of Upfl . It is understood that such effect can be either direct or indirect.
  • direct effect is meant that Staul acts on the targeted mRNA itself and by “indirect effect” is meant that Staul acts by an intermediary, including for example, a nucleic acid.
  • Staul down-regulation can result either directly or indirectly in the down reulation of target mRNA levels. It is understood that whether the effect is direct or indirect will depend on the target gene. One of skill in the art will be able to determine whether the effect is direct or indirect given the target gene.
  • identifying genes modulated by the down-regulation of Staul comprising a) incubating a substance that down- regulates SMD with a stably transfected cell comprising a reporter gene with a nonsense- mutation and Staul, and b) assaying the amount of mRNA present for a gene of a microarray, wherein a increase or decrease in the amount of mRNA relative to the amount of mRNA in the absence of the substance indicates a gene that is modulated by Staul activity. It is understood that these methods can be used to identify genes that are up- regulated or down regulated by the down-regulation of Staul as well as identify those genes whose abundance increases or decreases with the down-regulation of Staul .
  • RNA small interfering RNA
  • a componenet of SMD such as Staul or Upfl.
  • methods of identifying genes modulated by the down- regulation of Staul comprising a) transfecting a small interfering RNA (siRNA) that down- regulates SMD into a cell comprising Staul and one or more selected genes comprising one or more nonsense-mutations, and b) assaying the amount of protein expressed of the gene being screened or mRNA present for the gene being screened, wherein a increase or decrease in the amount of protein or mRNA relative to the amount of protein or mRNA in the absence of the siRNA indicates a gene that is modulated by Staul activity.
  • the siRNA can be for example, Staul siRNA.
  • the methods can also be used to identify genes that are stabilized or destabilized by the down-regulation of Staul.
  • Staul can modulate mRNA levels independently of Upfl and SMD, dependent modulation can also occur.
  • methods of identifying genes modulated by the down-regulation of Upfl comprising a) transfecting a small interfering RNA (siRNA) that down-regulates SMD into a cell comprising Upfl and one or more selected genes comprising one or more nonsense-mutations, and b) assaying the amount of protein expressed of the gene being screened or mRNA present for the gene being screened, wherein a increase or decrease in the amount of protein or mRNA relative to the amount of protein or mRNA in the absence of the siRNA indicates a gene that is modulated by Upfl activity.
  • siRNA small interfering RNA
  • Also disclosed are method of identifying genes modulated by the down- regulation of SMD comprising a) transfecting a small interfering RNA (siRNA) that down- regulates SMD into a cell comprising UPfI, Staul, and one or more selected genes comprising one or more nonsense-mutations, and b) assaying the amount of protein expressed of the gene being screened or mRNA present for the gene being screened, wherein a increase or decrease in the amount of protein or mRNA relative to the amount of protein or mRNA in the absence of the siRNA indicates a gene that is modulated by SMD activity.
  • the siRNA can be, for example, Upfl siRNA or Staul siRNA.
  • RNA levels can be measured by microarray or RT-PCR.
  • Protein expression can be measured by Western blot. It is understood and herein contemplated that the disclosed methods of identifying genes can be used with any method of measuring schoolein expression or mRNA levels known to those of skill in the art.
  • Also disclosed are methods of modulating the level of an mRNA comprising administering to a subject an effective amount of a substance that modulates Staul, wherein the modulation Staul directly or indirectly modulates the mRNA. Also disclosed are methods of treating a disorder in a subject comprising administering to the subject a substance, wherein the substance modulates Staul wherein the modulation of Staul modulates that level of mRNA abundance of another gene.
  • the disclosed methods make use of substances administered to a subject to achieve a desired effect.
  • “substance” can refer to any agent, compound, functional nucleic acid, siRNA, peptide, protein, antibody, or small molecule.
  • one embodiment of the disclosed methods is a method of treating a subject with a substance, wherein the substance is Staul or a complex comprising Staul and Upfl.
  • Staul can be administered directly or by transferr with a subject with a Staul encoding nucleic acid or by administering to the subject a compound that modulates SMD.
  • a substance that modulates SMD wherein the substance is an antibody or fragment thereof that modulates SMD.
  • the antibody can be an antibody that binds Upfl or Staul and affects SMD activity.
  • specifically disclosed is antibody, wherein the antibody binds Staul.
  • an antibody, wherein the antibody binds Upfl .
  • a substance that modulates SMD can also be a nucleic acid. Therefore specifically disclosed and herein contemplated is a substance, wherein the substance is a vector comprising a nucleic acid that encodes an SMD modulator. Also disclosed is a vector comprising a nucleic acid that encodes an SMD modulator. Also disclosed is a cell comprising the disclosed vectors.
  • siRNA can bind any factor that modulates SMD.
  • an siRNA specifically disclosed is an siRNA, wherein the siRNA binds Upfl .
  • a substance comprising siRNA, wherein the siRNA binds Staul is also disclosed.
  • binds or “interacts” means to affect a substance either directly or indirectly through cooperative function, competitive inhibition, non ⁇ competitive inhibition, binding, or contacting the substance, a target molecule, an accessory molecule, or alternative portion of a system so as to effect at least one function.
  • the interaction can be stimulatory or cooperative in nature having an additive or synergistic effect.
  • the interaction can also result in the inhibition of a process or target molecule.
  • a system comprising the components for SMD with Staul.
  • methods of facilitating Staul -mediated mRNA decay comprising contacting with Upfl a system, wherein the system comprises the components for SMD.
  • methods of facilitating Staul -mediated mRNA decay comprising contacting with Staul and Upfl, a system comprising the components for SMD.
  • compositions can also be administered in vivo in a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vector, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like, although topical intranasal administration or administration by inhalant is typically preferred.
  • topical intranasal administration means delivery of the compositions into the nose and nasal passages through one or both of the nares and can comprise delivery by a spraying mechanism or droplet mechanism, or through aerosolization of the nucleic acid or vector. The latter may be effective when a large number of animals is to be treated simultaneously.
  • Administration of the compositions by inhalant can be through the nose or mouth via delivery by a spraying or droplet mechanism.
  • compositions can also be directly to any area of the respiratory system (e.g., lungs) via intubation.
  • the exact amount of the compositions required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disorder being treated, the particular nucleic acid or vector used, its mode of administration and the like. Thus, it is not possible to specify an exact amount for every composition. However, an appropriate amount can be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.
  • Parenteral administration of the composition is generally characterized by injection.
  • I ⁇ jectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced.
  • receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, type of ligand, ligand valency, and ligand concentration. Molecular and cellular mechanisms of receptor-mediated endocytosis has been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).
  • compositions can be used therapeutically in combination with a pharmaceutically acceptable carrier.
  • Pharmaceutical carriers are known to those skilled in the art. These most typically would be standard carriers for administration of drugs to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The compositions can be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.
  • Pharmaceutical compositions may include carriers, thickeners, diluents, buffers, preservatives, surface active agents and the like in addition to the molecule of choice. Pharmaceutical compositions may also include one or more active ingredients such as antimicrobial agents, antiinflammatory agents, anesthetics, and the like.
  • the pharmaceutical composition may be administered in a number of ways depending on whether local or systemic treatment is desired, and on the area to be treated. Administration may be topically (including ophthalmically, vaginally, rectally, intranasally), orally, by inhalation, or parenterally, for example by intravenous drip, subcutaneous, intraperitoneal or intramuscular injection.
  • the disclosed antibodies or other agents can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, or transdermally.
  • Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like.
  • Preservatives and other additives may also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders.
  • Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.
  • compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavorings, diluents, emulsifiers, dispersing aids or binders may be desirable.
  • compositions may potentially be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid, glyco
  • the dosage ranges for the administration of the compositions are those large enough to produce the desired effect in which the symptoms disorder are effected.
  • the dosage should not be so large as to cause adverse side effects, such as unwanted cross- reactions, anaphylactic reactions, and the like.
  • the dosage will vary with the age, condition, sex and extent of the disease in the patient and can be determined by one of skill in the art.
  • the dosage can be adjusted by the individual physician in the event of any counterindications. Dosage can vary, and can be administered in one or more dose administrations daily, for one or several days.
  • the disclosed methods and compositions can also be used for example as tools to isolate and test new drug candidates for a variety of diseases. They can also be used for the continued isolation and study, for example, the cell cycle. There use as exogenous DNA delivery devices can be expanded for nearly any reason desired by those of skill in the art.
  • compositions and methods can be used to evaluate the expression of genes involved in SMD and in particular in or as a result of the pioneer round of translation. Specifically contemplated are methods wherein mRNA from a system comprising a nonsense-mutation is assayed using a micro array. Genes identified as having significantly (as determined by the manufacturers specifications of the array) increased or decreased expression are comodulators of SMD.
  • chips where at least one address is the sequences or part of the sequences set forth in any of the nucleic acid sequences disclosed herein. Also disclosed are chips where at least one address is the sequences or portion of sequences set forth in any of the peptide sequences disclosed herein.
  • chips where at least one address is a variant of the sequences or part of the sequences set forth in any of the nucleic acid sequences disclosed herein. Also disclosed are chips where at least one address is a variant of the sequences or portion of sequences set forth in any of the peptide sequences disclosed herein.
  • a substance that is able to modulate SMD is useful for the treatment and study of SMD related disorders.
  • methods of screening for a substance that modulates Staul -mediated mRNA decay (SMD) comprising incubating the substance to be screened with a Staul mRNA decay complex and assaying for a change in SMD.
  • An increase or decrease in SMD activity indicates a modulating substance.
  • SMD complex is any combination of one or more of the essential components of SMD.
  • screening methods wherein the complex comprises Upfl and Staul.
  • screening methods wherein the complex comprises one of Upfl and Staul. 81.
  • a method of screening for a substance that modulates Staul - mediated mRNA decay comprising incubating the substance to be screened with a stably transfected cell comprising a reporter gene with a nonsense-mutation and Staul, and assaying the amount of SMD in the cell, wherein a increase or decrease in the amount of mRNA relative to the amount of mRNA in the absence of the substance indicates a substance that modulates SMD activity.
  • SMD Staul - mediated mRNA decay
  • a substance that inhibits Staul -mediated mRNA decay comprising incubating the substance with Upfl and Staul forming a substance-Upfl -Staul mixture, and assaying the amount of Upfl -Staul complex present in the mixture, a decrease in the amount of Upfl-Staulcomplex relative to the amount of Upfl -Staul complex in the absence of the substance indicating the substance inhibits SMD.
  • SMD Staul -mediated mRNA decay
  • Also disclosed are method of screening for a substance that promotes Staul -mediated mRNA decay (SMD) comprising incubating the substance with Upfl and Staul forming a substance- Upfl -Staul mixture, and assaying the amount of Upfl-Staul complex present in the mixture, wherein a increase in the amount of Upfl-Staul complex relative to the amount of Upfl-Staul complex in the absence of the substance indicates that the substance promotes SMD.
  • SMD Staul -mediated mRNA decay
  • Staul has at least 80%, 85%, 90%, or 95% identity to the sequence set forth in SEQ ID NO: 2, or an SMD- active fragment thereof.
  • Upfl has at least 80%, 85%, 90%, or 95% identity to the sequence set forth in SEQ ID NO: 4, or an SMD- active fragment thereof.
  • SMD Staul- mediated mRNA decay
  • methods of screening for a substance that modulates Staul- mediated mRNA decay comprising, administering a substance to a system, wherein the system comprises the components for SMD activity, and assaying the effect of the substance on the amount of SMD activity in the system, a change in the amount of SMD activity present in the system compared to the amount of SMD activity in the system in the absence of the substance indicates the substance is a modulator.
  • SMD Staul-mediated mRNA decay
  • Also disclosed are methods of making a substance that modulates Staul -mediated mRNA decay (SMD) activity comprising admixing a substance identified by the disclosed screening methods with a pharmaceutically acceptable carrier.
  • the agents that can be used to modulate SMD can function by inhibiting the binding of Staul to its binding site.
  • a substance that competitively binds a Staul binding site can inhibit SMD. Therefore, specifically disclosed are methods of identifying an agent that binds a Staul binding site comprising contacting the agent to be screened with the Staul binding site. Also disclosed are methods of identifying an agent that binds a Staul binding site wherein the Staul binding site comprises SEQ ID NO: 56.
  • compositions can be used as targets for any combinatorial technique to identify molecules or macromolecular molecules that interact with the disclosed compositions in a desired way.
  • the nucleic acids, peptides, and related molecules disclosed herein can be used as targets for the combinatorial approaches.
  • the molecules identified and isolated when using the disclosed compositions, such as, Staul are also disclosed.
  • the products produced using the combinatorial or screening approaches that involve the disclosed compositions, such as, Staul are also considered herein disclosed.
  • Combinatorial chemistry includes but is not limited to all methods for isolating small molecules or macromolecules that are capable of binding either a small molecule or another macromolecule, typically in an iterative process.
  • Proteins, oligonucleotides, and sugars are examples of macromolecules.
  • oligonucleotide molecules with a given function, catalytic or ligand-binding can be isolated from a complex mixture of random oligonucleotides in what has been referred to as "in vitro genetics" (Szostak, TIBS 19:89, 1992).
  • Combinatorial techniques are particularly suited for defining binding interactions between molecules and for isolating molecules that have a specific binding activity, often called ap tamers when the macromolecules are nucleic acids.
  • RNA molecule is generated in which a puromycin molecule is covalently attached to the 3 '-end of the RNA molecule.
  • An in vitro translation of this modified RNA molecule causes the correct protein, encoded by the RNA to be translated.
  • the puromycin a peptidyl acceptor which cannot be extended, the growing peptide chain is attached to the puromycin which is attached to the RNA.
  • the protein molecule is attached to the genetic material that encodes it. Normal in vitro selection procedures can now be done to isolate functional peptides.
  • nucleic acid manipulation procedures are performed to amplify the nucleic acid that codes for the selected functional peptides.
  • new RNA is transcribed with puromycin at the 3 '-end, new peptide is translated and another functional round of selection is performed.
  • protein selection can be performed in an iterative manner just like nucleic acid selection techniques.
  • the peptide which is translated is controlled by the sequence of the RNA attached to the puromycin. This sequence can be anything from a random sequence engineered for optimum translation (i.e. no stop codons etc.) or it can be a degenerate sequence of a known RNA molecule to look for improved or altered function of a known peptide.
  • the two-hybrid system is a powerful molecular genetic technique for identifying new regulatory molecules, specific to the protein of interest (Fields and Song, Nature 340:245-6 (1989)). Cohen et al. modified this technology so that novel interactions between synthetic or engineered peptide sequences could be identified which bind a molecule of choice.
  • the benefit of this type of technology is that the selection is done in an intracellular environment.
  • the method utilizes a library of peptide molecules that attached to an acidic activation domain.
  • Combinatorial libraries can be made from a wide array of molecules using a number of different synthetic techniques. For example, libraries containing fused 2,4- pyrimidinediones (United States patent 6,025,371) dihydrobenzopyrans (United States Patent 6,017,768and 5,821,130), amide alcohols (United States Patent 5,976,894), hydroxy- amino acid amides (United States Patent 5,972,719) carbohydrates (United States patent 5,965,719), l,4-benzodiazepin-2,5-diones (United States patent 5,962,337), cyclics (United States patent 5,958,792), biaryl amino acid amides (United States patent 5,948,696), thiophenes (United States patent 5,942,387), tricyclic Tetrahydroquinolines (United States patent 5,925,527), benzofurans (United States patent 5,919,955), isoquinolines (Uni
  • combinatorial methods and libraries included traditional screening methods and libraries as well as methods and libraries used in interative processes.
  • the disclosed compositions can be used as targets for any molecular modeling technique to identify either the structure of the disclosed compositions or to identify potential or actual molecules, such as small molecules, which interact in a desired way with the disclosed compositions.
  • the nucleic acids, peptides, and related molecules disclosed herein can be used as targets in any molecular modeling program or approach.
  • Computer modeling technology allows visualization of the three-dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule.
  • the three-dimensional construct typically depends on data from x-ray crystallographic analyses or NMR imaging of the selected molecule.
  • the molecular dynamics require force field data.
  • the computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule-compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
  • antibodies is used herein in a broad sense and includes both polyclonal and monoclonal antibodies. In addition to intact immunoglobulin molecules, also included in the term “antibodies” are fragments or polymers of those immunoglobulin molecules, and human or humanized versions of immunoglobulin molecules or fragments thereof, as described herein. The antibodies are tested for their desired activity using the in vitro assays described herein, or by analogous methods, after which their in vivo therapeutic and/or prophylactic activities are tested according to known clinical testing methods.
  • the term "monoclonal antibody” as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., the individual antibodies within the population are identical except for possible naturally occurring mutations that may be present in a small subset of the antibody molecules.
  • the monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, as long as they exhibit the desired antagonistic activity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl. Acad. ScL USA, 81:6851-6855 (1984)).
  • Monoclonal antibodies of the invention can be prepared using hybridoma methods, such as those described by Kohler and Milstein, Nature, 256:495 (1975).
  • a hybridoma method a mouse or other appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent.
  • the lymphocytes may be immunized in vitro, e.g., using the Staul or Upfl described herein.
  • the monoclonal antibodies may also be made by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.).
  • DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of murine antibodies).
  • Libraries of antibodies or active antibody fragments can also be generated and screened using phage display techniques, e.g., as described in U.S. Patent No. 5,804,440 and U.S. Patent No. 6,096,441.
  • In vitro methods are also suitable for preparing monovalent antibodies.
  • Digestion of antibodies to produce fragments thereof, particularly, Fab fragments can be Ii ⁇ Il ..; Ui S ⁇ :.:::» ,/iller; ⁇ admir& !';,:* H, J ...Ii IL.
  • digestion can be performed using papain. Examples of papain digestion are described in WO 94/29348 and U.S. Pat. No. 4,342,566.
  • Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment. Pepsin treatment yields a fragment that has two antigen combining sites and is still capable of cross-linking antigen.
  • the fragments can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the antibody or antibody fragment is not significantly altered or impaired compared to the non-modified antibody or antibody fragment. These modifications can provide for some additional property, such as to remove/add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.
  • the antibody or antibody fragment must possess a bioactive property, such as specific binding to its cognate antigen.
  • Functional or active regions of the antibody or antibody fragment may be identified by mutagenesis of a specific region of the protein, followed by expression and testing of the expressed polypeptide.
  • antibody or “antibodies” can also refer to a human antibody and/or a humanized antibody.
  • Many non-human antibodies e.g., those derived from mice, rats, or rabbits
  • are naturally antigenic in humans and thus can give rise to undesirable immune responses when administered to humans. Therefore, the use of human or humanized antibodies in the methods of the invention serves to lessen the chance that an antibody administered to a human will evoke an undesirable immune response.
  • the human antibodies of the invention can be prepared using any technique. Examples of techniques for human monoclonal antibody production include those described by Cole et al. ⁇ Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77, 1985) and by Boemer et al. (J. Immunol., 147(l):86-95, 1991). Human antibodies of the invention (and fragments thereof) can also be produced using phage display libraries (Hoogenboom et al., J. MoI. Biol, 227:381, 1991; Marks et al., J. MoI. Biol, 222:581, 1991).
  • the human antibodies of the invention can also be obtained from transgenic animals.
  • transgenic, mutant mice that are capable of producing a full j
  • a humanized form of a non-human antibody is a chimeric antibody or antibody chain (or a fragment thereof, such as an Fv, Fab, Fab', or other antigen-binding portion of an antibody) which contains a portion of an antigen binding site from a non-human (donor) antibody integrated into the framework of a human (recipient) antibody.
  • a humanized antibody residues from one or more complementarity determining regions (CDRs) of a recipient (human) antibody molecule are replaced by residues from one or more CDRs of a donor (non-human) antibody molecule that is known to have desired antigen binding characteristics (e.g., a certain level of specificity and affinity for the target antigen).
  • CDRs complementarity determining regions
  • donor non-human antibody molecule that is known to have desired antigen binding characteristics
  • Fv framework (FR) residues of the human antibody are replaced by corresponding non-human residues.
  • Humanized antibodies may also contain residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences.
  • a humanized antibody has one or more amino acid residues introduced into it from a source which is non-human.
  • humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.
  • Humanized antibodies generally contain at least a portion of an antibody constant region (Fc), typically that of a human antibody (Jones et al., Nature, 321 :522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), and Presta, Curr. Opin. Struct. Biol, 2:593-596 (1992)).
  • Fc antibody constant region
  • humanized antibodies can be generated according to the methods of Winter and co-workers (Jones et al., Nature, 321:522-525 (1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al., Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody.
  • Methods that can be used to produce humanized antibodies are also described in U.S. Patent No. 4,816,567, U.S. Patent No. 5,565,332, U.S. Patent No. 5,721,367, U.S. Patent No. 5,837,243, U.S. Patent No. 5, 939,598, U.S. Patent No. 6,130,364, and U.S. Patent No. 6,180,377.
  • Antibodies of the invention are preferably administered to a subject in a pharmaceutically acceptable carrier as described above. Effective dosages and schedules for administering the antibodies may be determined empirically, and making such determinations is within the skill in the art. Those skilled in the art will understand that the dosage of antibodies that must be administered will vary depending on, for example, the subject that will receive the antibody, the route of administration, the particular type of antibody used and other drugs being administered. Guidance in selecting appropriate doses for antibodies is found in the literature on therapeutic uses of antibodies, e.g., Handbook of Monoclonal Antibodies, Ferrone et al., eds., Noges Publications, Park Ridge, NJ., (1985) ch. 22 and pp.
  • a typical daily dosage of the antibody used alone might range from about 1 ⁇ g/kg to up to 100 mg/kg of body weight or more per day, depending on the factors mentioned above.
  • the efficacy of the therapeutic antibody can be assessed in various ways well known to the skilled practitioner. Specifically, SMD can be assessed directly or indirectly as taught herein.
  • the nucleic acids of the present invention can be in the form of naked DNA or RNA, or the nucleic acids can be in a vector for delivering the nucleic acids to the cells, whereby the antibody-encoding DNA fragment is under the transcriptional regulation of a promoter, as would be well understood by one of ordinary skill in the art.
  • the vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms. As one example, delivery can be via a liposome, using commercially available liposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art.
  • LIPOFECTIN LIPOFECTAMINE
  • SUPERFECT Qiagen, Inc. Hilden, Germany
  • TRANSFECTAM Promega Biotec, Inc., Madison, WI
  • nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Arlington, AZ).
  • vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome (see e.g., Pastan et al., Proc. Natl. Acad. ScL U.S.A. 85:4486, 1988; Miller et al., MoI. Cell. Biol.
  • a viral system such as a retroviral vector system which can package a recombinant retroviral genome
  • the recombinant retrovirus can then be used to infect and thereby deliver to the infected cells nucleic acid encoding a broadly neutralizing antibody (or active fragment thereof) of the invention.
  • the exact method of introducing the altered nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors. Other techniques are widely available for this procedure including the use of adenoviral vectors (Mitani et al., Hum. Gene Ther.
  • AAV adeno-associated viral
  • lentiviral vectors Non-deficiency virus vectors
  • pseudotyped retroviral vectors Agrawal et al., Exper. Hematol. 2A:12>Z-1A1, 1996.
  • Physical transduction techniques can also be used, such as liposome delivery and receptor- mediated and other endocytosis mechanisms (see, for example, Schwartzenberger et al., Blood 87:472-478, 1996). This invention can be used in conjunction with any of these or other commonly used gene transfer methods.
  • the dosage for administration of adenovirus to humans can range from about 10 7 to 10 9 plaque forming units (pfu) per injection but can be as high as 10 12 pfu per injection (Crystal, Hum. Gene Ther. 8:985-1001, 1997; Alvarez and Curiel, Hum. Gene Ther. 8:597-613, 1997).
  • a subject can receive a single injection, or, if additional injections are necessary, they can be repeated at six month intervals (or other appropriate time intervals, as determined by the skilled practitioner) for an indefinite period and/or until the efficacy of the treatment has been established.
  • Parenteral administration of the nucleic acid or vector of the present invention is generally characterized by injection.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions, solid forms suitable for P Il I! / U :!3 U :::::» ⁇ • ⁇ êt:::!; :::::» Ul .1. ,1, solution of suspension in liquid prior to injection, or as emulsions.
  • a more recently revised approach for parenteral administration involves use of a slow release or sustained release system such that a constant dosage is maintained. See, e.g., U.S. Patent No. 3,610,795, which is incorporated by reference herein.
  • suitable formulations and various routes of administration of therapeutic compounds see, e.g., Remington: The Science and Practice of Pharmacy (19th ed.) ed. A.R. Gennaro, Mack Publishing Company, Easton, PA 1995.
  • Staul can include proteins or nucleic acid sequences having at least 80%, 85%, 90%, or 95% identity to the sequence set forth in SEQ ED NO: 1 or 2, or fragment thereof. Also disclosed are methods of the invention, wherein the Upfl or a nucleic acid encoding Upfl has at least 80%, 85%, 90%, or 95% identity to the sequence set forth in SEQ ID NO: 3 or 4, or fragment thereof.
  • SEQ ID NO: 1 sets forth a particular sequence of a Staul encoding nucleic acid
  • SEQ ID NO: 2 sets forth a particular sequence of the protein encoded by SEQ ID NO: 1, an Staul protein.
  • variants of these and other genes and proteins herein disclosed which have at least, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 percent homology to the stated sequence.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • nucleic acid based there are a variety of molecules disclosed herein that are nucleic acid based, including for example the nucleic acids that encode, for example Staul and Upfl, as well as various functional nucleic acids.
  • the disclosed nucleic acids are made up of, for example, nucleotides, nucleotide analogs, or nucleotide substitutes. Non-limiting examples of these and other molecules are discussed herein. It is understood that for example, when a vector is expressed in a cell, that the expressed mRNA will typically be made up of A, C, G, and U.
  • an antisense molecule is introduced into a cell or cell environment through for example exogenous delivery, it is advantagous that the antisense molecule be made up of nucleotide analogs that reduce the degradation of the antisense molecule in the cellular environment.
  • a nucleotide is a molecule that contains a base moiety, a sugar moiety and a phosphate moiety. Nucleotides can be linked together through their phosphate moieties and sugar moieties creating an internucleoside linkage.
  • the base moiety of a nucleotide can be adenin-9-yl (A), cytosin-1-yl (C), guanin-9-yl (G), uracil- 1-yl (U), and thymin-1-yl (T).
  • the sugar moiety of a nucleotide is a ribose or a deoxyribose.
  • the phosphate moiety of a nucleotide is pentavalent phosphate.
  • An non-limiting example of a nucleotide would be 3'- AMP (3'-adenosine monophosphate) or 5'-GMP (5'-guanosine monophosphate).
  • a nucleotide analog is a nucleotide which contains some type of modification to either the base, sugar, or phosphate moieties. Modifications to the base moiety would include natural and synthetic modifications of A, C, G, and T/U as well as different purine or pyrimidine bases, such as uracil-5-yl (.psi.), hypoxanthin-9-yl (T), and 2-aminoadenin-9-yl.
  • a modified base includes but is not limited to 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5-halo particularly 5-bromo, 5-trifluoromethyl and other 5-substituted uracils and cytosines
  • Nucleotide analogs can also include modifications of the sugar moiety. Modifications to the sugar moiety would include natural modifications of the ribose and deoxy ribose as well as synthetic modifications. Sugar modifications include but are not limited to the following modifications at the 2' position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-, S- or N-alkynyl; or O-alkyl-0-alkyl, wherein the alkyl, alkenyl and alkynyl may be substituted or unsubstituted Ci to Qo, alkyl or C 2 to Ci 0 alkenyl and alkynyl.
  • 2' sugar modiifcations also include but are not limited to -O[(CH 2 ) n O] m CH 3 , -O(CH 2 ) n OCH 3 , -O(CH 2 )n NH 2 , -O(CH 2 ) n CH 3 , -O(CH 2 ) n -ONH 2 , and -O(CH 2 ) n ON[(CH 2 ) n CH 3 )J 2 , where n and m are from 1 to about 10.
  • sugars Similar modifications may also be made at other positions on the sugar, particularly the 3' position of the sugar on the 3' terminal nucleotide or in 2'-5' linked oligonucleotides and the 5' position of 5' terminal nucleotide. Modified sugars would also include those that contain modifications at the bridging ring oxygen, such as CH 2 and S. Nucleotide sugar analogs may also have sugar mimetics such as cyclobutyl moieties in place of the pentofuranosyl sugar.
  • Modified phosphate moieties include but are not limited to those that can be modified so that the linkage between two nucleotides contains a phosphorothioate, chiral phosphorothioate, phosphorodithioate, phosphotriester, aminoalkylphosphotriester, methyl and other alkyl phosphonates including 3'-alkylene phosphonate and chiral phosphonates, phosphinates, phosphoramidates including 3'-amino phosphoramidate and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates.
  • these phosphate or modified phosphate linkage between two nucleotides can be through a 3'-5' linkage or a 2'-5' linkage, and the linkage can contain inverted polarity such as 3'-5' to 5'-3' or 2'-5' to 5'-2'.
  • Various salts, mixed salts and free acid forms are also included.
  • nucleotides containing modified phosphates include but are not limited to, 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111; 5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is herein incorporated by reference.
  • nucleotide analogs need only contain a single modification, but may also contain multiple modifications within one of the moieties or between different moieties.
  • Nucleotide substitutes are molecules having similar functional properties to nucleotides, but which do not contain a phosphate moiety, such as peptide nucleic acid (PNA). Nucleotide substitutes are molecules that will recognize nucleic acids in a Watson- Crick or Hoogsteen manner, but which are linked together through a moiety other than a phosphate moiety. Nucleotide substitutes are able to conform to a double helix type structure when interacting with the appropriate target nucleic acid.
  • PNA peptide nucleic acid
  • Nucleotide substitutes are nucleotides or nucleotide analogs that have had the phosphate moiety and/or sugar moieties replaced. Nucleotide substitutes do not contain a standard phosphorus atom. Substitutes for the phosphate can be for example, short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages.
  • morpholino linkages formed in part from the sugar portion of a nucleoside
  • siloxane backbones sulfide, sulfoxide and sulfone backbones
  • formacetyl and thioformacetyl backbones methylene formacetyl and thioformacetyl backbones
  • alkene containing backbones sulfamate backbones
  • sulfonate and sulfonamide backbones amide backbones; and others having mixed N, O, S and CH 2 component parts.
  • conjugates can be chemically linked to the nucleotide or nucleotide analogs.
  • conjugates include but are not limited to lipid moieties such as a cholesterol moiety (Letsinger et al., Proc. Natl. Acad. Sci. USA, 1989, 86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem.
  • a thioether e.g., hexyl-S-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al., Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol (Oberhauser et al., Nucl.
  • Acids Res., 1990, 18, 3777-3783 a polyamine or a polyethylene glycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), or adamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36, 3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta, 1995, 1264, 229-237), or an octadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol. Exp.
  • Functional nucleic acids are nucleic acid molecules that have a specific function, such as binding a target molecule or catalyzing a specific reaction.
  • Functional nucleic acid molecules can be divided into the following categories, which are not meant to be limiting.
  • functional nucleic acids include antisense molecules, aptamers, ribozymes, triplex forming molecules, and external guide sequences.
  • the functional nucleic acid molecules can act as affectors, inhibitors, modulators, and stimulators of a specific activity possessed by a target molecule, or the functional nucleic acid molecules can possess a de novo activity independent of any other molecules.
  • Antisense molecules are designed to interact with a target nucleic acid molecule through either canonical or non-canonical base pairing. The interaction of the antisense molecule and the target molecule is designed to promote the destruction of the target molecule through, for example, RNAseH mediated RNA-DNA hybrid degradation. Alternatively the antisense molecule is designed to interrupt a processing function that normally would take place on the target molecule, such as transcription or replication. Antisense molecules can be designed based on the sequence of the target molecule. Numerous methods for optimization of antisense efficiency by finding the most accessible regions of the target molecule exist. Exemplary methods would be in vitro selection experiments and DNA modification studies using DMS and DEPC.
  • antisense molecules bind the target molecule with a dissociation constant (k d ) less than 10 "6 . It is more preferred that antisense molecules bind with a k d less than 10 "8 . It is also more preferred that the antisense molecules bind the target moelcule with a k d less than 10 "10 . It is S-" i,;; Ii / 11...11 :;» u a../ ,3 !b. u ,;. ». ,:; «.. also preferred that the antisense molecules bind the target molecule with a kd less than 10 " .
  • Aptamers are molecules that interact with a target molecule, preferably in a specific way.
  • aptamers are small nucleic acids ranging from 15-50 bases in length that fold into defined secondary and tertiary structures, such as stem-loops or G-quartets.
  • Aptamers can bind small molecules, such as ATP (United States patent 5,631,146) and theophiline (United States patent 5,580,737), as well as large molecules, such as reverse transcriptase (United States patent 5,786,462) and thrombin (United States patent 5,543,293).
  • Aptamers can bind very tightly with k d S from the target molecule of less than 10 "12 M.
  • the aptamers bind the target molecule with a k d less than 10 "6 . It is more preferred that the aptamers bind the target molecule with a k d less than 10 " . It is also more preferred that the aptamers bind the target molecule with a k ⁇ j less than 10 "10 . It is also preferred that the aptamers bind the target molecule with a k d less than 10 '12 . Aptamers can bind the target molecule with a very high degree of specificity.
  • aptamers have been isolated that have greater than a 10000 fold difference in binding affinities between the target molecule and another molecule that differ at only a single position on the molecule (United States patent 5,543,293). It is preferred that the aptamer have a k d with the target molecule at least 10 fold lower than the k d with a background binding molecule. It is more preferred that the aptamer have a k d with the target molecule at least 100 fold lower than the k d with a background binding molecule. It is more preferred that the aptamer have a k d with the target molecule at least 1000 fold lower than the k d with a background binding molecule.
  • the aptamer have a k d with the target molecule at least 10000 fold lower than the k d with a background binding molecule. It is preferred when doing the comparison for a polypeptide for example, that the background molecule be a different polypeptide.
  • Representative examples of how to make and use aptamers to bind a variety of different target molecules can be found in the following non-limiting list of United States patents: 5,476,766, 5,503,978, 5,631,146, 5,731,424 , 5,780,228, 5,792,613, F Il Il / IUi :'3 IUi :::::n ,, ⁇ ' ,.::;i; 1 Ia U ...Il L
  • siRNA referes to double-stranded RNAs that can induce sequence-specific post-transcriptional gene silencing, thereby decreasing or even inhibiting gene expression.
  • an siRNA triggers the specific degradation of homologous RNA molecules, such as mRNAs, within the region of sequence identity between both the siRNA and the target RNA.
  • mRNAs homologous RNA molecules
  • WO 02/44321 discloses siRNAs capable of sequence-specific degradation of target mRNAs when base-paired with 3' overhanging ends. The direction of dsRNA processing determines whether a sense or an antisense target RNA can be cleaved by the produced siRNA endonuclease complex.
  • siRNAs can be used to modulate transcription, for example, by silencing genes such as Staul and Upfl .
  • the effects of siRNAs have been demonstrated in cells from a variety of organisms, including Drosophila, C. elegans, insects, frogs, plants, fungi, mice and humans (for example, WO 02/44321; Gitlin et al., Nature 418:430-4, 2002; Caplen et al., Proc. Natl. Acad. Sci. 98:9742-9747, 2001 ; and Elbashir et al., Nature 411 :494-8, 2001).
  • siRNAs are directed against certain target genes to down regulate gene expression.
  • Staul or Upfl expression can be down regulated by specifically targeting the siRNA to Staul or Upfl.
  • Staul protein and Upfl protein As discussed herein there are numerous variants of the Staul protein and Upfl protein that are known and herein contemplated. In addition, to the known functional Staul and Upfl strain variants, there are derivatives of the Staul and Upfl proteins which also function in the disclosed methods and compositions. Protein variants and derivatives are well understood to those of skill in the art and in can involve amino acid sequence modifications. For example, amino acid sequence modifications typically fall into one or more of three classes: substitutional, insertional or deletional variants. Insertions include amino and/or carboxyl terminal fusions as well as intrasequence insertions of single or multiple amino acid residues.
  • Insertions ordinarily will be smaller insertions than those of amino or carboxyl terminal fusions, for example, on the order of one to four residues.
  • Immunogenic fusion protein derivatives such as those described in the examples, are made by fusing a polypeptide sufficiently large to confer immunogenicity to the target sequence by cross-linking in vitro or by recombinant cell culture transformed with DNA encoding the fusion. Deletions are characterized by the removal of one or more amino acid residues from the protein sequence. Typically, no more than about from 2 to 6 residues are deleted at any p il Il ,• ⁇ II...I' ::::i ⁇ i ⁇ ,.. ⁇ :::::n / constructive..:!.
  • Deletions or insertions preferably are made in adjacent pairs, i.e. a deletion of 2 residues or insertion of 2 residues. Substitutions, deletions, insertions or any combination thereof may be combined to arrive at a final construct. The mutations must not place the sequence out of reading frame and preferably will not create complementary regions that could produce secondary mRNA structure. Substitutional variants are those in which at least one residue has been removed and a different residue inserted in its place. Such substitutions generally are made in accordance with the following Tables 1 and 2 and are referred to as conservative substitutions.
  • Substantial changes in function or immunological identity are made by selecting substitutions that are less conservative than those in Table 2, i.e., selecting residues that differ more significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the area of the substitution, for example as a sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site or (c) the bulk of the side chain.
  • substitutions which in general are expected to produce the greatest changes in the protein properties will be those in which (a) a hydrophilic residue, e.g. seryl or threonyl, is substituted for (or by) a hydrophobic residue, e.g.
  • an electropositive side chain e.g., lysyl, arginyl, or histidyl
  • an electronegative residue e.g., glutamyl or aspartyl
  • the replacement of one amino acid residue with another that is biologically and/or chemically similar is known to those skilled in the art as a conservative H-" Ii Ii ,.• ⁇ 'i..,!' .::;:n u,..n :::;» . ⁇ ⁇ • .3 ,;::: ⁇ > u ,,, ⁇ ii,, substitution.
  • a conservative substitution would be replacing one hydrophobic residue for another, or one polar residue for another.
  • the substitutions include combinations such as, for example, GIy, Ala; VaI, He, Leu; Asp, GIu; Asn, GIn; Ser, Thr; Lys, Arg; and Phe, Tyr.
  • Such conservatively substituted variations of each explicitly disclosed sequence are included within the mosaic polypeptides provided herein.
  • Substitutional or deletional mutagenesis can be employed to insert sites for N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).
  • Deletions of cysteine or other labile residues also may be desirable.
  • Deletions or substitutions of potential proteolysis sites, e.g. Arg is accomplished for example by deleting one of the basic residues or substituting one by glutaminyl or histidyl residues.
  • Certain post-translational derivatizations are the result of the action of recombinant host cells on the expressed polypeptide. Glutaminyl and asparaginyl residues are frequently post-translationally deamidated to the corresponding glutamyl and asparyl residues. Alternatively, these residues are deamidated under mildly acidic conditions. Other post-translational modifications include hydroxylation of proline and lysine, phosphorylation of hydroxyl groups of seryl or threonyl residues, methylation of the o- amino groups of lysine, arginine, and histidine side chains (T.E. Creighton, Proteins: Structure and Molecular Properties, W. H. Freeman & Co., San Francisco pp 79-86 [1983]), acetylation of the N-terminal amine and, in some instances, amidation of the C-terminal carboxyl.
  • variants and derivatives of the disclosed proteins herein are through defining the variants and derivatives in terms of homology/identity to specific known sequences.
  • SEQ ID NO: 2 sets forth a particular sequence of Staul
  • SEQ ID NO: 4 sets forth a particular sequence of a Upfl protein.
  • variants of these and other proteins herein disclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95% homology to the stated sequence.
  • the homology can be calculated after aligning the two sequences so that the homology is at its highest level.
  • Another way of calculating homology can be performed by published algorithms.
  • Optimal alignment of sequences for comparison may be conducted by the local homology algorithm of Smith and Waterman Adv. Appl. Math. 2: 482 (1981), by the homology alignment algorithm of Needleman and Wunsch, J. MoL Biol. 48: 443 (1970), by the search for similarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A. 85: 2444 (1988), by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WT), or by inspection. 147.
  • the same types of homology can be obtained for nucleic acids by for example the algorithms disclosed in Zuker, M.
  • nucleic acids that can encode those protein sequences are also disclosed. This would include all degenerate sequences related to a specific protein sequence, i.e. all nucleic acids having a sequence that encodes one particular protein sequence as well as all nucleic acids, including degenerate nucleic acids, encoding the disclosed variants and derivatives of the protein sequences.
  • each particular nucleic acid sequence may not be written out herein, it is understood that each and every sequence is in fact disclosed and described herein through the disclosed protein sequence. For example, one of the many nucleic acid sequences that can encode the protein sequence set forth in SEQ ID NO:2 is set forth in SEQ ED NO:1.
  • nucleic acid sequences that encode this particular derivative of the Staul or Upfl are also disclosed including for example SEQ ID NO:1 and SEQ ID NO:3 which set forth two of the degenerate nucleic acid sequences that encode the particular polypeptide set forth in SEQ ID NO:2 and 4, respectively. It is also understood that while no amino acid sequence indicates what particular DNA sequence encodes that protein within an organism, where particular variants of a disclosed protein are disclosed herein, the known nucleic acid sequence that encodes that protein in the particular Staul from which that protein arises is also known and herein disclosed and described. 150. There are a number of compositions and methods which can be used to deliver nucleic acids to cells, either in vitro or in vivo.
  • nucleic acids can be delivered through a number II' 11 ' II,,,-,. Sf ' 1 I,..! 1 Il «...!' I 1 • ⁇ ' l> 1' "Mi ..11 II,. of direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.
  • direct delivery systems such as, electroporation, lipofection, calcium phosphate precipitation, plasmids, viral vectors, viral nucleic acids, phage nucleic acids, phages, cosmids, or via transfer of genetic material in cells or carriers such as cationic liposomes.
  • Transfer vectors can be any nucleotide construction used to deliver genes into cells (e.g., a plasmid), or as part of a general strategy to deliver genes, e.g., as part of recombinant retrovirus or adenovirus (Ram et al. Cancer Res. 53:83-88, (1993)).
  • plasmid or viral vectors are agents that transport the disclosed nucleic acids, such as Staul and Upfl into the cell without degradation and include a promoter yielding expression of the gene in the cells into which it is delivered.
  • Viral vectors are, for example, Adenovirus, Adeno-associated virus, Herpes virus, Vaccinia virus, Polio virus, ADDS virus, neuronal trophic virus, Sindbis and other RNA viruses, including these viruses with the HIV backbone. Also preferred are any viral families which share the properties of these viruses which make them suitable for use as vectors. Retroviruses include Murine Maloney Leukemia virus, MMLV, and retroviruses that express the desirable properties of MMLV as a vector. Retroviral vectors are able to carry a larger genetic payload, i.e., a transgene or marker gene, than other viral vectors, and for this reason are a commonly used vector. However, they are not as useful in non-proliferating cells.
  • Adenovirus vectors are relatively stable and easy to work with, have high titers, and can be delivered in aerosol formulation, and can transfect non-dividing cells.
  • Pox viral vectors are large and have several sites for inserting genes, they are thermostable and can be stored at room temperature.
  • a preferred embodiment is a viral vector which has been engineered so as to suppress the immune response of the host organism, elicited by the viral antigens.
  • Preferred vectors of this type will carry coding regions for Interleukin 8 or 10.
  • Viral vectors can have higher transaction (ability to introduce genes) abilities than chemical or physical methods to introduce genes into cells.
  • viral vectors II TM!!:,,,, i
  • ,.' %,. ⁇ > .,,..Il 1...I' mull ,.' I' Ii II...I1 mil,, . together11, contain, nonstructural early genes, structural late genes, an RNA polymerase ED transcript, inverted terminal repeats necessary for replication and encapsidation, and promoters to control the transcription and replication of the viral genome.
  • viruses typically have one or more of the early genes removed and a gene or gene/promotor cassette is inserted into the viral genome in place of the removed viral DNA. Constructs of this type can carry up to about 8 kb of foreign genetic material.
  • the necessary functions of the removed early genes are typically supplied by cell lines which have been engineered to express the gene products of the early genes in trans.
  • a retrovirus is an animal virus belonging to the virus family of Retro viridae, including any types, subfamilies, genus, or tropisms.
  • Retroviral vectors in general, are described by Verma, LM. , Retroviral vectors for gene transfer. In Microbiology-1985, American Society for Microbiology, pp. 229-232, Washington, (1985), which is incorporated by reference herein. Examples of methods for using retroviral vectors for gene therapy are described in U.S. Patent Nos. 4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136; and Mulligan, (Science 260:926-932 (1993)); the teachings of which are incorporated herein by reference.
  • a retrovirus is essentially a package which has packed into it nucleic acid cargo.
  • the nucleic acid cargo carries with it a packaging signal, which ensures that the replicated daughter molecules will be efficiently packaged within the package coat, hi addition to the package signal, there are a number of molecules which are needed in cis, for the replication, and packaging of the replicated virus.
  • a retroviral genome contains the gag, pol, and env genes which are involved in the making of the protein coat. It is the gag, pol, and env genes which are typically replaced by the foreign DNA that it is to be transferred to the target cell.
  • Retrovirus vectors typically contain a packaging signal for incorporation into the package coat, a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the LTRs that enable the insertion of the DNA state of the retrovirus to insert into the host genome.
  • a packaging signal for incorporation into the package coat a sequence which signals the start of the gag transcription unit, elements necessary for reverse transcription, including a primer binding site to bind the tRNA primer of reverse transcription, terminal repeat sequences that guide the switch of RNA strands during DNA synthesis, a purine rich sequence 5' to the 3' LTR that serve as the priming site for the synthesis of the second strand of DNA synthesis, and specific sequences near the ends of the
  • gag, pol, and env genes allow for about 8 kb of foreign sequence to be inserted into the viral genome, become reverse transcribed, and upon replication be packaged into a new retroviral particle.
  • II,, arri ⁇ iint of nucleic acid is sufficient for the delivery of a one to many genes depending on the size of each transcript. It is preferable to include either positive or negative selectable markers along with other genes in the insert.
  • a packaging cell line is a cell line which has been transfected or transformed with a retrovirus that contains the replication and packaging machinery, but lacks any packaging signal.
  • the vector carrying the DNA of choice is transfected into these cell lines, the vector containing the gene of interest is replicated and packaged into new retroviral particles, by the machinery provided in cis by the helper cell. The genomes for the machinery are not packaged because they lack the necessary signals.
  • viruses have been shown to achieve high efficiency gene transfer after direct, in vivo delivery to airway epithelium, hepatocytes, vascular endothelium, CNS parenchyma and a number of other tissue sites (Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin. Invest. 92:381-387 (1993); Roessler, J. Clin. Invest.
  • Recombinant adenoviruses achieve gene transduction by binding to specific cell surface receptors, after which the virus is internalized by receptor-mediated endocytosis, in the same manner as wild type or replication-defective adenovirus (Chardonnet and Dales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology 12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985); Seth, et al., J. Virol. 51 :650-655 (1984); Seth, et Et Ci- It ••' 1 U I 1 «...1 1' .' ....I' I' ! 1..J i ...Il II,, al., Mol. Cell. Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991); Wickham et al., Cell 73:309-319 (1993)).
  • a viral vector can be one based on an adenovirus which has had the El gene removed and these virons are generated in a cell line such as the human 293 cell line. In another preferred embodiment both the El and E3 genes are removed from the adenovirus genome.
  • AAV adeno-associated virus
  • This defective parvovirus is a preferred vector because it can infect many cell types and is nonpathogenic to humans.
  • AAV type vectors can transport about 4 to 5 kb and wild type AAV is known to stably insert into chromosome 19. Vectors which contain this site specific integration property are preferred.
  • An especially preferred embodiment of this type of vector is the P4.1 C vector produced by Avigen, San Francisco, CA, which can contain the herpes simplex virus thymidine kinase gene, HSV-tk, and/or a marker gene, such as the gene encoding the green fluorescent protein, GFP. 160.
  • the AAV contains a pair of inverted terminal repeats (ITRs) which flank at least one cassette containing a promoter which directs cell- specific expression operably linked to a heterologous gene.
  • ITRs inverted terminal repeats
  • Heterologous in this context refers to any nucleotide sequence or gene which is not native to the AAV or Bl 9 parvovirus.
  • the vectors of the present invention thus provide DNA molecules which are capable of integration into a mammalian chromosome without substantial toxicity.
  • the inserted genes in viral and retroviral usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
  • a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements.
  • HSV herpes simplex virus
  • EBV Epstein-Barr virus
  • Other useful systems include, for example, replicating and host-restricted non-replicating vaccinia virus vectors.
  • compositions can be delivered to the target cells in a variety of ways.
  • the compositions can be delivered through electroporation, or through lipofection, or through calcium phosphate precipitation.
  • the delivery mechanism chosen will depend in part on the type of cell targeted and whether the delivery is occurring for example in vivo or in vitro.
  • compositions can comprise, lipids such as liposomes, such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes.
  • liposomes such as cationic liposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes.
  • Liposomes can further comprise proteins to facilitate targeting a particular cell, if desired.
  • Administration of a composition comprising a compound and a cationic liposome can be administered to the blood afferent to a target organ or inhaled into the respiratory tract to target cells of the respiratory tract.
  • liposomes see, e.g., Brigham et al. Am. J. Resp. Cell. MoI. Biol. 1 :95-100 (1989); Feigner et al. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.4,897,355.
  • the compound can be administered as a component of a microcapsule that can be targeted to specific cell types, such as macrophages, or where the diffusion of the compound or delivery of the compound from the microcapsule is designed for a specific rate or dosage. 168.
  • delivery of the compositions to cells can be via a variety of mechanisms.
  • delivery can be via a liposome, using commercially available liposome preparations such as UPOFECTTN " , LIPOFEcf AMINE (GIBCO-BRL, Inc., Gaithersburg, MD), SUPERFECT (Qiagen, Inc.
  • nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a SONOPORATION machine (ImaRx Pharmaceutical Corp., Arlington, AZ).
  • the materials may be in solution, suspension (for example, incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type via antibodies, receptors, or receptor ligands.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Senter, et al., Bioconjugate Chem., 2:447-451, (1991); Bagshawe, K.D., Br. J. Cancer, 60:275-281, (1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, et al., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., Cancer Immunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem. Pharmacol,
  • Vehicles such as "stealth” and other antibody conjugated liposomes (including lipid mediated drug targeting to colonic carcinoma), receptor mediated targeting of DNA through cell specific ligands, lymphocyte directed tumor targeting, and highly specific therapeutic retroviral targeting of murine glioma cells in vivo.
  • the following references are examples of the use of this technology to target specific proteins to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187, (1992)).
  • receptors are involved in pathways of endocytosis, either constitutive or ligand induced. These receptors cluster in clathrin-coated pits, enter the cell via clathrin-coated vesicles, pass through an acidified endosome in which the receptors are sorted, and then either recycle to the cell surface, become stored intracellularly, or are degraded in lysosomes.
  • the internalization pathways serve a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligand, and receptor-level regulation.
  • Nucleic acids that are delivered to cells which are to be integrated into the host cell genome typically contain integration sequences. These sequences are often viral related sequences, particularly when viral based systems are used. These viral intergration systems can also be incorporated into nucleic acids which are to be delivered using a non- nucleic acid based system of deliver, such as a liposome, so that the nucleic acid contained in the delivery system can be come integrated into the host genome.
  • Other general techniques for integration into the host genome include, for example, systems designed to promote homologous recombination with the host genome. These systems typically rely on sequence flanking the nucleic acid to be expressed that has enough homology with a target sequence within the host cell genome that recombination between the vector nucleic acid and the target nucleic acid takes place, causing the delivered nucleic acid to be integrated into the host genome. These systems and the methods necessary to promote homologous recombination are known to those of skill in the art.
  • compositions can be administered in a pharmaceutically acceptable carrier and can be delivered to the subject's cells in vivo and/or ex vivo by a variety of mechanisms well known in the art (e.g., uptake of naked DNA, liposome fusion, intramuscular injection of DNA via a gene gun, endocytosis and the like).
  • cells or tissues can be removed and maintained outside the body according to standard protocols well known in the art.
  • the compositions can be introduced into the cells via any gene transfer mechanism, such as, for example, calcium phosphate mediated gene delivery, electroporation, microinjection or proteoliposomes.
  • the transduced cells can then be infused (e.g., in a pharmaceutically acceptable carrier) or homo topically transplanted back into the subject per standard methods for the cell or tissue type. Standard methods are known for transplantation or infusion of various cells into a subject.
  • the nucleic acids that are delivered to cells typically contain expression controlling systems.
  • the inserted genes in viral and retroviral systems usually contain promoters, and/or enhancers to help control the expression of the desired gene product.
  • a promoter is generally a sequence or sequences of DNA that function when in a relatively fixed location in regard to the transcription start site.
  • a promoter contains core elements required for basic interaction of RNA polymerase and transcription factors, and may contain upstream elements and response elements. « ⁇ •' 11 Ii ./ u ..a ⁇ :,;» ,••' ,:J 3 U ,JI ii,
  • Preferred promoters controlling transcription from vectors in mammalian host cells may be obtained from various sources, for example, the genomes of viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
  • viruses such as polyoma, Simian Virus 40 (SV40), adenovirus, retroviruses, hepatitis-B virus and most preferably cytomegalovirus, or from heterologous mammalian promoters, e.g. beta actin promoter.
  • the early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment which also contains the SV40 viral origin of replication (Fiers et al., Nature, 273: 113 (1978)).
  • the immediate early promoter of the human cytomegalovirus is conveniently obtained as a HindUl E restriction fragment (Greenway, PJ. et al., Gene 18: 355-360 (1982)).
  • promoters from the host cell or related species also are useful herein.
  • Enhancer generally refers to a sequence of DNA that functions at no fixed distance from the transcription start site and can be either 5' (Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3' (Lusky, M.L., et al., MoI. Cell Bio, 3: 1108 (1983)) to the transcription unit. Furthermore, enhancers can be within an intron (Banerji, J.L. et al., Cell 33: 729 (1983)) as well as within the coding sequence itself (Osborne, T.F., et al., MoI. Cell Bio. 4: 1293 (1984)).
  • Enhancers function to increase transcription from nearby promoters. Enhancers also often contain response elements that mediate the regulation of transcription. Promoters can also contain response elements that mediate the regulation of transcription. Enhancers often determine the regulation of expression of a gene. While many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, ⁇ -fetoprotein and insulin), typically one will use an enhancer from a eukaryotic cell virus for general expression.
  • Preferred examples are the SV40 enhancer on the late side of the replication origin (bp 100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers.
  • the promotor and/or enhancer may be specifically activated either by light or specific chemical events which trigger their function.
  • Systems can be regulated by reagents such as tetracycline and dexamethasone.
  • reagents such as tetracycline and dexamethasone.
  • irradiation such as gamma irradiation, or alkylating chemotherapy drugs.
  • the promoter and/or enhancer region can act as a constitutive promoter and/or enhancer to maximize expression of the region of the transcription unit to be transcribed, hi certain constructs the promoter and/or enhancer region be active in all eukaryotic cell types, even if it is only expressed in a particular type of cell at a particular time.
  • a preferred promoter of this type is the CMV promoter (650 bases).
  • Other preferred promoters are SV40 promoters, cytomegalovirus (full length promoter), and retroviral vector LTF. 179. It has been shown that all specific regulatory elements can be cloned and used to construct expression vectors that are selectively expressed in specific cell types such as melanoma cells.
  • the glial fibrillary acetic protein (GFAP) promoter has been used to selectively express genes in cells of glial origin.
  • Expression vectors used in eukaryotic host cells may also contain sequences necessary for the termination of transcription that may affect mRNA expression. These regions are transcribed as polyadenylated segments in the untranslated portion of the mRNA encoding tissue factor protein. The 3' untranslated regions also include transcription termination sites. It is preferred that the transcription unit also contain a polyadenylation region. One benefit of this region is that it increases the likelihood that the transcribed unit will be processed and transported like mRNA.
  • the identification and use of polyadenylation signals in expression constructs is well established. It is preferred that homologous polyadenylation signals be used in the transgene constructs.
  • the polyadenylation region is derived from the SV40 early polyadenylation signal and consists of about 400 bases. It is also preferred that the transcribed units contain other standard sequences alone or in combination with the above sequences improve expression from, or stability of, the construct.
  • the viral vectors can include a nucleic acid sequence encoding a marker product. This marker product is used to determine if the gene has been delivered to the cell and once delivered is being expressed.
  • Preferred marker genes are the E. CoIi lacZ gene, which encodes ⁇ -galactosidase, and green fluorescent protein.
  • the marker may be a selectable marker.
  • suitable selectable markers for mammalian cells are dihydrofolate reductase (DHFR), thymidine kinase, neomycin, neomycin analog G418, hydromycin, and puromycin.
  • DHFR dihydrofolate reductase
  • thymidine kinase thymidine kinase
  • neomycin neomycin analog G418, hydromycin
  • puromycin puromycin.
  • selectable markers When such selectable markers are successfully transferred into a mammalian host cell, the transformed mammalian host cell can survive if placed under selective pressure.
  • These cells lack the ability to grow without the addition of such nutrients as thymidine or hypoxanthine. Because these cells lack certain genes necessary for a complete nucleotide synthesis pathway, they cannot survive unless the missing nucleotides are provided in a supplemented media.
  • An alternative to supplementing the media is to introduce an intact DHFR or TK gene into cells lacking the respective genes, thus altering their growth requirements. Individual cells that were not transformed with the DHFR or TK gene will not be capable of survival in non-supplemented media.
  • the second category is dominant selection which refers to a selection scheme used in any cell type and does not require the use of a mutant cell line. These schemes typically use a drug to arrest growth of a host cell. Those cells that have a novel gene would express a protein conveying drug resistance and would survive the selection. Examples of such dominant selection use the drugs neomycin, (Southern P. and Berg, P.,_J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan, R.C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B. et al., MoI. Cell. Biol. 5: 410-413 (1985)).
  • the three examples employ bacterial genes under eukaryotic control to convey resistance to the appropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid) or hygromycin, respectively.
  • Others include the neomycin analog G418 and puramycin.
  • kits that are drawn to reagents that can be used in practicing the methods disclosed herein.
  • the kits can include any reagent or combination of reagents discussed herein or that would be understood to be required or beneficial in the practice of the disclosed methods.
  • the kits could include primers to perform the amplification reactions discussed in certain embodiments of the methods, as well as the buffers and enzymes required to use the primers as intended.
  • the kits could include systems comprising the essential elements of SMD activity.
  • compositions disclosed herein and the compositions necessary to perform the disclosed methods can be made using any method known to those of skill in the art for that particular reagent or compound unless otherwise specifically noted.
  • the invention also provides substances made by methods of the invention. 187.
  • a modulator of Staul -mediated mRNA decay comprising a) administering a substance to a system, wherein the system comprises SMD activity, b) assaying the effect of the substance on the amount of SMD activity in the system, c) selecting a substance which causes a change in the amount of SMD activity present in the system compared to the amount of SMD activity in the system in the absence of substance, and d) synthesizing the substance.
  • SMD Staul -mediated mRNA decay
  • Staul-HA 3 also co-immunopurifled with Barentsz and CBP80, although in an RNase A-sensitive manner, but did not co-immunopurify with eIF4E or a non ⁇ specific control, Vimentin, which is a component of intermediate filaments ( Figure ID, upper).
  • the co-IP of Staul with Barentsz had previously been shown to be RNase A-sensitive (Macchi et al., 2003).
  • Example 2 Down-Regulating Cellular Staul Has No Detectable Consequence to the FJC -Dependent NMD of Gl or GPxI mRNA 194.
  • Staul functions in EJC-dependent NMD
  • the effect of down-regulating the level of cellular Staul on the NMD of mRNAs for /3-globin (Gl) and glutathione peroxidase (GPx)I was examined using small interfering (si)RNAs.
  • si small interfering
  • HeLa cells were transfected with Staul siRNA, UpDX siRNA or a non-specific "Control" siRNA and, two days later, with three plasmids: (i) the pmCMV-Gl test plasmid that was either nonsense-free (Norm) or nonsense- containing (39Ter), (ii) the pmCMV-GPxl test plasmid, either Norm or 46Ter, and (iii) the phCMV-MUP reference plasmid. 195.
  • the level of each Staul isoform was down-regulated to 28% of normal, where normal is defined as the level in the presence of the non-specific Control siRNA, and the level of UpOX was down-regulated to 24% of normal ( Figure 2A).
  • Example 3 Tethered Staul Reduces mRNA Abundance in a Mechanism that Involves Upfl and an Upstream Nonsense Codon but Not Upf2 or UpOX 196.
  • Staul could elicit a type of EJC-independent mRNA decay based on the finding that it binds to Upfl .
  • fusions of each Upf protein and the bacteriophage MS2 coat protein were shown to reduce mRNA abundance when tethered to a series of MS2 coat protein binding sites that were located more than 50 nts downstream of the normal termination codon (Lykke- Andersen et al., 2000). Since the reduced mRNA abundance was dependent on the normal termination codon, it was attributed to NMD.
  • MS2-Upfl and MS2-Upf2 were also of no consequence to the level of FLuc mRNA that lacked the MS2bs but elicited a 2-to-5-fold reduction in the level of FLuc mRNA that harbored the MS2bs ( Figure 3B, right).
  • tethering an unrelated protein, HA-eIF4Ai ⁇ , or expressing Stau-HA 3 or myc-Upfl that could not be tethered failed to affect the abundance of FLuc-MS2bs mRNA ( Figure 8). 199.
  • the other combination consisted of the pcFLuc-MS2bs test plasmid, the pRLuc reference plasmid, and an effector plasmid that produces MS2-HA, MS2-HA-Staul, MS2, MS2-Upfl, MS2-Upf2 or MS2-Upf3.
  • ADP-ribosylation factor (Arf)l mRNA which encodes a Ras-related G protein that regulates membrane traffic and organelle structure (Donaldson and Jackson, 2000), was identified as a natural ligand of Staul using two methods.
  • Staul -containing RNPs were immunopurified from human 293 cells that transiently expressed either Staul -HA 3 or, as a control for IP specificity, Staul -6xHis using anti-HA antibody ( Figure 7A).
  • PSMD 12 NM_002816 proteasome (prosome macropain) 26S subunit non-ATPase 12
  • PRKAR2A BF246917 protein kinase cAMP-dependent regulatory type II alpha
  • PAICS AA902652 phosphoribosylaminoimidazole carboxylase phosphoribosylaminoimidazole succinocarboxamide synthetase
  • 293 cells were transiently transfected with a plasmid that expressed Staul -HA3 or, to control for nonspecific IP, Staul -6xHis.
  • Biotin-labeled cRNA was synthesized from RNA that had been immunopurif ⁇ ed using anti-HA antibody and hybridized to Affymetrix U133A microarrays. Changes of at least 2.5-fold were scored as Staul -interacting transcripts.
  • plasmids consisted of: (i) a pSport-Arfl or pSport-Arfl ⁇ (3'UTR) test plasmid, the latter of which lacks all nucleotides that reside downstream of the normal termination codon ( Figure 8A), and (ii) the phCMV-MUP reference plasmid.
  • the HeLa-cell Arfl gene contains four introns (Lee et al., 1992; Figure 8A) so that the resulting newly synthesized mRNA would harbor four EJCs.
  • Arfl cDNA within either pSport plasmid lacks all introns, the resulting newly synthesized mRNA would lack EJCs.
  • Cos cells were transiently transfected with the Staul -HA 3 expression vector, the pSport-Arfl or pSport- Arfl ⁇ (3 'UTR) test plasmid, and the phCMV-MUP reference plasmid. Notably, cells were transfected with only half as much pSport-Arfl ⁇ (3'UTR) as pSport-Arfl in order to compensate for the difference in the level of product mRNA. Cell extract was prepared two days later, and a fraction was immunopurified using anti-HA antibody or, as a control for nonspecific IP, rat (r) IgG.
  • the transcripts that we reported in Table 3 can be weak targets for decay.
  • the transcripts can be up-regulated by Staul binding.
  • microarray analysis was performed (Tables 4 and 5).
  • G protein 205184_at guanine nucleotide binding protein (G protein) gamma 4
  • MDR/TAP 203192_at ATP-binding cassette sub-family B
  • SMD can be utilized by mammalian cells to regulate the abundance of hundreds of cellular transcripts and, hence, expression of the encoded proteins.
  • Transcripts identified to be regulated by SMD have a broad range of cellular functions that include signal transduction, cell proliferation, cell metabolism, immune response, DNA repair, and transcriptional regulation. Therefore, SMD can play a key role in establishing and maintaining cellular homeostasis.
  • SMD naturally targets transcripts encoding the IL- 7 receptor (IL- 7R), c-JUN, and SERPINEl (also called PAIl). Down regulating cellular Staufenl or Upfl but not Upf2 increased the abundance of each cellular mRNA. Notably, the abundance of many transcripts was down-regulated in the cells depleted of Staul.
  • Example 5 A Minimized Staul Binding Site Resides > 67 Nucleotides Downstream of the Normal Termination Codon of Arfl mRNA and Mediates mRNA Decay 210.
  • a series of deletions was generated from the distal-most nucleotide of the Arfl 3' UTR within pSport- Arfl ( Figure 10A).
  • Each of the resulting test plasmids was transiently introduced into 293 cells together with the Stau-HA 3 expression vector. Cell extract was prepared two days later, and a fraction was immunopurified using anti-HA antibody. Western blotting of immunopurified protein revealed relative IP efficiencies ( Figure 1OB, upper).
  • Staul also binds within the 3' UTR downstream of nucleotide 919 or upstream of nucleotide 688
  • the finding that inserting the Staul binding site (SBS, nucleotides 622-924) within the 3' UTR of the heterologous FLuc mRNA reduced FLuc mRNA abundance ( Figure 11) corroborates that Staul indeed binds between nucleotides 689 and 919. It was concluded that Staul binds to the 3' UTR of Arfl mRNA at a position that is predicted to reduce mRNA abundance.
  • Down-regulating mUpfl relative to normal also increased the half-life of nucleus- associated fos-Arfl-SBS mRNA (ie., abrogated the nucleus associated NMD), whereas down-regulating mUpf2 or mUpOX relative to normal did not.
  • down- regulating mStaul and mUpfl also increased the half-life of a heterologous mRNA (fos- FLuc-Arfl) harboring the SBS, but had no effect on fos-FLuc-Arfl without the SBS.
  • Staul binds to an ⁇ 230-nt region of the 3' UTR of Arfl mRNA so as to recruit Upfl independently of other Upf proteins and, as a consequence, reduce Arfl mRNA half-life ( Figures 7, 8, 9, 10, 11). 213. It was found that Staul plays no detectable role in the EJC-dependent NMD of
  • Hrb27C inhibits the removal by nuclear splicing of intron 3 from P-element encoded transposase pre-mRNA in Drosophila (Hammond et al., 1997; Siebel et al., 1994) and also binds directly to Drosophila gurken mRNA so as to regulate gurken mRNA cytoplasmic localization and translation (Goodrich et al., 2004).
  • Barentsz and Y14 are essential for EJC-dependent NMD in mammalian cells (Ferraiuolo et al., 2004; Gehring et al., 2003; Palacios et al., 2004; Shibuya et al., 2004) and also function in localizing Drosophila oskar mRNA to the posterior pole of the oocyte (Hachet and Ephrussi, 2001 ; Hachet and Ephrussi, 2004; Mohr et al., 2001 ; van Eeden et al., 2001).
  • mammalian Staul which is a component of RNPs that are transported and localized within dendrites of mature hippocampal neurons (Kiebler et al., 1999; Kohrmann et al., 1999), is involved in a new type of EJC-independent NMD.
  • Staul In mammals, Staul is ubiquitously expressed, but its level of expression varies among tissues (Wickham et al., 1999; Marion et al., 1999; Monshausen et al., 2001). For example, it is highly expressed in brain, heart, liver, testis, pancreas and placenta, whereas it is generally expressed to lesser but varying degrees in other tissues. Additionally, differential splicing generates several isoforms that are not uniformly expressed among tissues. Staul also contains several putative phosphorylation sites that could regulate function, and Staul has been shown to interact with protein phosphate 1 (Monshausen et al., 2002).
  • Upfl phosphorylation influences Upfl function in NMD (Pal et al., 2001 ; Ohnishi et al., 2003, it is possible that Upfl phosphorylation also regulates its function or the function of Staul in SMD. Therefore, Staul functions can be modulated in different tissues according to the level of expression and the nature of each expressed isoform and, possibly, the degree of Upfl or Staul phosphorylation.
  • Staul is linked to mRNA transport in neurons (Kohrmann et al., 1999) and to HIV- 1 replication (Mouland et al., 2000). Its association with polysomes (Marion et al., 1999; Luo et al., 2002) suggests a role in the translational regulation Staul-bound mRNAs.
  • Staul is a component of large RNA granules (Krishevsky and Kosik, 2001; Kanai et al., 2004; Mallardo et al., 2003; Villace et al., 2004, Brendel et al., 2004; Ohashi et al., 2002) that contain ribosomes, mRNA and several other proteins. It is shown herein that several Staul-bound mRNAs encode key regulatory enzymes that control cell metabolism, proteins involved in organelle trafficking, cell division or the cell cycle, or both. Arfl is involved in protein trafficking, and it may modulate vesicle budding and uncoating within the Golgi apparatus.
  • PAICS controls steps 6 and 7 of the purine nucleotide biosynthetic pathway.
  • the level of PAICS mRNA varies with the cell cycle in synchronized rat 3Yl fibroblasts (Iwahana et al., 1995). This variation can be controlled by SMD.
  • GNE is a regulatory enzyme that regulates the first step in the biosynthesis of N-acetylneuraminic acid (NeuAc), which is a precursor to sialic acids (Hinderlich et al., 1997). Modification of cell surface molecules with sialic acid is essential for many biological processes, such as cell adhesion and signal transduction. Modulation of sialylation is involved in the tumorigenicity and metastatic behavior of malignant cells.
  • pGEX-UPFl was constructed by ligating the Notl/Klenow-filled EcoRI fragment from pCMV-Myc-UPFl that contains UPFl cDNA to the Notl/Klenow-filled BamHI fragment from pGEX-6p- 1 +NdeI (a gift from J. Wedekind).
  • pGEX-Staul was constructed by ligating the BamHI/NotI fragment from pGEX-6p-l+NdeI to a PCR-amplified fragment that had been digested with BgIH and Notl.
  • the PCR fragment was amplified using the human Staul cDNA expression vector phStaul 55 - HA 3 (Luo et al., 2002) and two primers: 5 '-
  • pRSET B-Staul was constructed by ligating the Klenow-filled Ndel/NotI fragment from pGEX-Staul to the Klenow-filled BgIH fragment from pRSET B (Invitrogen).
  • MS2 coat protein-encoding fragment was amplified using pET-MS2 (Coller et al., 1998) and two primers: 5'- CGCTACTAGCTAGCCGCCATGGCTTCTAACTTTACTCAGTTCGTTC-S ' (SEQ E) NO. 7) (sense) and 5'-
  • ATAAGAATGCGGCCGCCTAGGATCCAGCGTAGTCTGGAACGTCGTATGGGTAGA TGCCGGAGTTTGCTGCGATTG-3' (SEQ ID NO. 8) (antisense), where underlined and bold nucleotides specify the Nhel or Notl site and HA tag sequence, respectively.
  • the Staul cDNA-containing sequence was amplified using phStaul 55 -HA 3 and two primers: 5'- GAAGATCTAGAATGAAACTTGGAAAAAAACCAATGTATAAG-S' (SEQ ED NO. 5) (sense) and 5'- ATAAGAATGCGGCCGCTCAGCACCTCCCACACACAGACATTGGTCC-S' (SEQ ID NO. 6) (antisense), where underlined nucleotides specify the Bgi ⁇ or Notl site, respectively.
  • pMS2-HA that harbors an N-terminal MS2 coat protein followed by an HA tag
  • pCI-neo that had been digested with Nhel and Notl was ligated to the same PCR- amplified fragment that contains the MS2 coat protein-encoding sequence and was similarly digested with Nhel and Notl (see above).
  • pcFLuc-MS2bs which contains eight tandem repeats of the MS2 coat protein- binding sites within the 3'UTR of firefly luciferase (FLuc) cDNA, was generated from pq8- 8bs (Lykke- Andersen et al., 2000) by replacing /3-globin cDNA with FLuc cDNA.
  • pcFLuc which lacks the MS2 coat protein-binding sites, was generated from pcFLuc-8bs by cleaving with PspOMI and Notl followed by self-ligation after generating blunt ends using Klenow (New England Biolabs).
  • pRLuc which encodes renilla luciferase (RLuc) was generated from p21uc (Grentzmann et al., 1998) by precisely deleting FLuc cDNA.
  • AGGTCTAGATCGGAGCAGCAGCCTCTGAGGTGT-3' (SEQ ID NO. 9) (sense) and 5'- GGCTCGAGCTAATAGCTATAATTACAGTGCTTGTTTGTCGAAATG-S' (SEQ ID NO. 10) (antisense), where underlined nucleotides specify the Xbal and Xhol site, respectively.
  • the PCR product was digested with Xbal and Xhol and ligated to pSport ⁇ -gal (Invitrogen) that had been digested with Xbal and Xhol.
  • the resulting plasmid, pSport ⁇ -gal- Arfl was digested with Notl, purified, and self-ligated to generate pSport- Arfl .
  • pSport-Arfl ⁇ (3'UTR) pSport- Arfl was digested with Kpnl and Xhol. The resulting vector-containing fragment was ligated to a PCR-amplified fragment that contains the Arfl open translational reading frame and had been digested with Kpnl and Xhol. PCR was carried out using pSport- Arfl and two primers: 5 '-
  • GCTATTTAGGTGACACTATAGAAGGTAC-S' (SEQ ID NO. 11) (sense) and 5'- CCGCTCGAGTTCACTTCTGGTTCCGGAGCTGATTG-3' (SEQ ID NO. 12) (antisense), where underlined nucleotides specify the Hindi ⁇ site.
  • Deletions within the 3' UTR of Arfl cDNA were generated using pSport-Arfl, 5'-CATTTCGACAAACAAGCACTGTAATTATAGCTATTAG-S' (SEQ ED NO. 13) (sense) and one of the following antisense primers: 5'-CCAAGGACAAGCGAGTTGCG- 3'( ⁇ 1229-1794) (SEQ ID NO.
  • pfos- Arfl -SBS was digested with Ncol and EcoRI and ligated to a PCR-amplified fragment that contains Arfl -SBS cDNA.
  • PCR was carried out using pSport-Arfl and two primers : 5'- ACAACCATGGGGAACATCTTCGCCAACCTCTTC-3' (sense) (SEQ E) NO. 19) and 5'- CCGGAATTCTGGGCCTACATCCCCTCTCAGCACTGAAC-S ' (SEQ E) NO. 20) (antisense), where underlined nucleotides specify the Ncol or EcoRI site.
  • pMS2-HA-eE T 4AE[ which encodes N-terminal oligomerization- defective MS2 coat protein followed successively by an HA tag and full-length human eE ⁇ AEI cDNA
  • pCI-neo that had been digested with Nhel and Notl was ligated to two fragments: the Nhel/BamHI fragment from pMS2-HA-Staul that contains the MS2 coat protein-encoding sequence, and a PCR-amplified fragment that contains human eIF4A ⁇ i cDNA and had been digested with BamHI and Notl.
  • eD?4AIE was amplified using pcDNA3- HA-eIF4AE[ (Chiu et al., 2004) and two primers: 5'- CGCGGATCCATGGCGACCACGGCCACGATGGCGACC-3' (SEQ E) NO. 21) (sense) and 5 '- AT AAGAATGCGGCCGCTC AGAT AAGATC AGC AACGTTC ATCGG-3 ' (SEQ E) NO. 22) (antisense), where underlined nucleotides specify the BamHI or Notl site, respectively.
  • pcFLuc(UAA-»CAA)-MS2bs which lacks a termination codon upstream of the MS2 binding sites
  • pcFLuc-8bs that had been digested with Notl and EcoRV was ligated to a PCR-amplified fragment that contains C-terminus of FLuc in which the UAA codon was converted to a CAA codon and had been digested with Notl and EcoRV.
  • the PCR reactions were performed using pR/HCV/F (Kim et al., 2003) and two primers: 5'- TTGACCGCTTGAAGTCTTTAATTAAATAC-3' (SEQ E) NO.
  • pcFLuc-8bs were digested with Xbal and ligated to a Xbal-digested PCR-amplified fragment that had been generated using pSport-Arfl and two primers: 5'-
  • GCTCTAGAGGGCCCGAGGGGAACAGCTGGGCTGGCGACTGG-S' (antisense) for pcFLuc-Arfl No SBS.
  • Underlined nucleotides specify Xbal sites.
  • pfos-Arfl-SBS or pfos-Arfl-No SBS was digested with Ncol and EcoRI and ligated to a PCR-amplified fragment that contains Arfl-SBS cDNA or Arfl-No SBS cDNA.
  • PCR was carried out using pSport-Arfl and two primers : 5 '-AC AACCATGGGGAACATCTTCGCCAACCTCTTC-3 ' (sense) (SEQ ID NO: 57) and 5'-CCGGAATTCTGGGCCTACATCCCCTCTCAGCACTGAAC- 3' (antisense) (SEQ ID NO: 58) for Arfl-SBS cDNA or 5'- CCGGAATTCTC ACTTCTGGTTCCGGAGCTGATTGGAC-3' (SEQ ID NO: 59) (antisense) for Arfl-No SBS cDNA, where underlined nucleotides specify the Ncol or EcoRI site.
  • pSport-PAICS was purchased from ATCC (catalog # MGC-5024, NCBI Accession # BCOl 0273).
  • pSport-PAICS ⁇ (3'UTR) was generated using pSpoit-PAICS and two primers: 5'-CCAAGCTTACGCGTACCCAGCTTTC-S' (SEQ ID NO. 29) (sense) and 5'- CCCCTAAAAAATTCAATGGCATTCTTTC-S' (SEQ ID NO. 30) (antisense).
  • PCR amplification was carried out using PfU Ultra (Stratagene). The PCR product was incubated with Dpnl to digest the methylated template DNA, phosphorylated at the 5' ends using T4 polynucleotide kinase (Fermentas), and circularized by ligation.
  • pCDNA3-RSV-CK2A2 was constructed by inserting a BamHI-XhoI fragment from pOTB7-CK2A2 (ATCC catalog # MGC-10397, NCBI Accession # BC008812 ) into pCDNA3-RSV that had been digested using BamHI and Xhol.
  • Example 7 Yeast Two-Hybrid Analysis 238. Proteins that interact with human Upfl were identified using the CytoTrap Two- Hybrid System (Stratagene). The yeast strain cdc25H was transformed with pSos-Upfl and the pMyr library of HeLa-cell cDNAs (Stratagene). Transformants were processed following manufacturer instructions.
  • Example 8 siRNA-Mediated Down-Regulation of Human Upfl, Upf2, Upf3X or Staul
  • HeLa cells (2 x 10 6 ) were grown in DMEM medium (Gibco-BRL) containing 10% fetal bovine serum (Gibco-BRL) in 60-mm dishes and transiently transfected with 100 nM of in vitro-synthesized small interfering (si)RNA (Xeragon or Dharmacon) using Oligofectamine (Invitrogen).
  • Upfl, Upf2, UpBX or Staul were down-regulated using, respectively, 5'-r(GAUGCAGUUCCGCUCCAUU)d(TT)-3' (SEQ ID NO. 31), 5'- r(GGCUUUUGUCCCAGCCAUC)d(TT)-3' (SEQ ID NO.
  • Staul (A) siRNA consisted of 5 1 -r(GUUUGAGAUUGCACUUAAA)d(TT)-3 I (SEQ ID NO. 35), and Upfl (A) siRNA consisted of 5'- r(AACGUUUGCCGUGGAUGAG)d(TT)-3' (SEQ ID NO. 36).
  • HeLa cells (2 x 10 6 ) were transiently transfected using Lipofectamine Plus (Invitrogen) with 0.3 ⁇ g of the reporter plasmid pcFLuc or pcFLuc-MS2bs, 0.02 ⁇ g of the reference plasmid pRLuc, and 5 ⁇ g of one of the following effector plasmids: pMS2-HA, pMS2-HA-Staul, pcNMS2, pcNMS2-U ⁇ fl, pcNMS2-Upf2 or pcNMS2-Upf3. Cells were harvested two days later. Protein was purified from half of the cells using passive lysis buffer (Promega), and total RNA was purified from the other half using TRIzol Reagent (Invitrogen).
  • Ltk " cells (4 X lO 6 ) were transiently transfected with 200 nM of Control siRNA or 100 nM each of two different mouse Staul siRNAs [5'- r(CAACUGUACUACCUUUCCA)d(TT)-3' (SEQ ID NO. 37) or 5' r(AACGGUAACUGCCAUGAUA)d(TT)-3' (SEQ ID NO. 38)].
  • Cos-7 or HeLa cells were cultured as described above but in 150-mm dishes. Transfections and immunopurifications (IPs) were performed as described (Chiu et al., 2004;
  • Protein was electrophoresed in SDS-polyacrylamide, transferred to Hybond ECL nitrocellulose (Amersham), and probed with antibodies that recognize FLAG (Sigma), GST (Qiagen), HA (Roche), Upfl (Lykke-Andersen et al., 2000), Upf2 (Serin et al., 2001),
  • RNAs were quantitated using RT-PCR as described (Sun et al., 1998).
  • FLuc, ⁇ -G ⁇ and MUP mRNAs were amplified as described previously (Chiu et al., 2004; Lejeune et al., 2003), and RLuc mRNA was amplified using the primers 5 - ATGACTTCGAAAGTTTATG-S ' (SEQ ID NO. 39) (sense) and 5'- TTCAGATTTGATC AACGC A-3' (SEQ ID NO. 40) (antisense).
  • Cellular Arfl mRNA or Arfl mRNA that derived from a pSport vector was amplified using the primers 5 - AACCAACGCCTGGCTCGG-3' (SEQ ID NO. 41) (sense) and 5'-
  • RT-PCR products were electrophoresed in 5% polyacrylamide and quantitated by Phosphorlmaging (Molecular Dynamics).
  • RT-PCR analysis of Arfl mRNA was performed using One Step RT-PCR (Qiagen), the primer pair 5'-GCTATTTAGGTGACACTATAGAAGGTAC-S' (SEQ ID NO. 45) (sense) and 5'- CTCTGTC ATTGCTGTCC ACC ACG-3' (SEQ ID NO. 46) (antisense), and ethidium bromide staining.
  • FLuc-MS2bs mRNA or FLuc(UAA ⁇ CAA)-MS2bs mRNA was amplified using the primers 5'-CAACACCCCAACATCTTCG-S' (SEQ ID NO. 47) (sense) and 5'- CTTTCCGCCCTTCTTGGCC-3' (SEQ ID NO. 48) (antisense).
  • Gl(U AA- »U AC)-MS2bs was amplified using the primers 5 - AATACGACTCACTATAGGGA-3 ' (SEQ ID NO. 49) (sense), which anneals to the T7 promoter, and 5 '-GATACTTGTGGGCCAGGGCA-3 ' (SEQ ID NO. 50) (antisense).
  • FLuc- Arfl mRNA was amplified using the same T7 promoter primer (sense) and 5 - TCTAGAGGATAGAATGGCG-3 ' (SEQ ID NO. 51) (antisense).
  • PAICS mRNA was amplified using the primers 5'-AGCAGGCTGGTACCGGTCCG-S' (SEQ ID NO. 52) (sense) and 5 '-ACC AATGTTCAGTACCTCAG-3 ' (SEQ ID NO. 53) (antisense).
  • Example 14 Far-Western Blotting
  • FLAG-Upfl was purified from HeLa cells that had been stably transfected with pCI-neo-FLAG-UPFl as previously described (Pal et al., 2001).
  • GST-Upfl was purified from E. coli using Bulk GST Purification Modules (Amersham). 252. Lysates of E. coli that were or were not induced to express ⁇ xHis-Staul or GST-
  • HeLa-cell RNA from three independently performed transfections in which the level of cellular Staul was depleted to as little as 4% of normal (where normal is defined as the level in the presence of Control siRNA), was separately hybridized to microarrays. Sequences from 18,279 HeLa-cell transcripts were analyzed, representing 34% of the array probe sets, in all three hybridization experiments. It was observed that 124 transcripts, or 1.1 % of the HeLa-cell transcriptome that was analyzed, were upregulated at least 2-fold in all three transfections (Table 6).
  • Table 6 Transcripts upregulated in human cells depleted of Staul in three independently performed microarray analyses
  • I factor (complement) 8.78 IF 1555564_a_at fibronectin 1 7.98 FN1 216442_x_at interferon-induced protein with tetratricopeptide repeats 2 7.51 IFIT2 226757_at
  • FLJ20378 [Homo sapiens] 4.46 235629_at insulin-like growth factor binding protein 5 4.30 IGFBP5 211959_at integrin beta 3 (platelet glycoprotein MIa antigen CD61) 4.18 ITGB3 204627_s_at interferon-induced protein with tetratricopeptide repeats 4 4.13 IFIT4 229450_at alpha-actinin-2-associated LIM protein 3.97 ALP 210170_at transgelin 3.94 TAGLN 205547_s_at
  • Homo sapiens cDNA FLJ20914 fis clone ADSE00646 3.76 234597_at thrombospondin 1 3.73 THBS1 201108_s_at protein tyrosine phosphatase receptor type O 3.62 PTPRO 1554199_at cyclin-E binding protein 1 3.60 CEB1 219863_at
  • Homo sapiens transcribed sequences 2.61 229242_at v-jun sarcoma virus 17 oncogene homolog (avian) 2.58 JUN 201466_s_at integrin alpha 4 (antigen CD49D alpha 4 subunit of VLA-4 receptor) 2.56 ITGA4 205885_s_at
  • Homo sapiens cDNA FLJ46457 fis clone THYMU3020856 2.44 225007_at thymosin beta identified in neuroblastoma cells 2.44 TMSNB 205347_s_at prostaglandin E receptor 4 (subtype EP4) 2.42 PTGER4 204897_at chromosome 14 open reading frame 128 2.41 C14orf128 228889_at ganglioside-induced differentiation-associated protein 1 2.41 GDAP1 226269_at zinc finger protein 36 C3H type-like 1 2.38 ZFP36L1 211965_at uracil-DNA glycosylase 2.37 UNG 202330_s_at hypothetical protein MGC27277 2.37 MGC27277 242283_at
  • CDK5 regulatory subunit associated protein 1-like 1 2.34 CDKAL1 214877_at
  • cytokine receptor-like factor 2 2.32 227547_at aldehyde dehydrogenase 1 family member A3 2.32 ALDH 1 A3 203180_at proteasome (prosome macropain) subunit beta type 9 (large multifunctional protease 2) 2.29 PSMB9 204279_at
  • KIAA0143 protein 2.26 KIAA0143 212150_at guanylate binding protein 1 interferon-inducible 67kDa 2.26 GBP1 202270_at epiplakin 1 2.25 EPPK1 232164_s_at actinin alpha 1 2.24 ACTN 1 211160_x_at ring finger protein 20 2.22 RNF20 222683_at leucine rich repeat (in FLII) interacting protein 1 2.22 LRRFIP1 223492_s_at
  • 2.21 1556111_s_at hypothetical protein LOC150759 2.21 LOC150759 213703_at dihydropyrimidinase-like 3
  • 2.20 DPYSL3 201431_s_at type I transmembrane receptor (seizure-related protein) 2.20 PSK-1 233337_s_at zinc finger protein 90 homolog (mouse)
  • ZFP90 235698_at tumor protein p53 (Li-Fraumeni syndrome) 2.20 TP53 211300_s_at
  • LOC284120 Homo sapiens hypothetical LOC284120 (LOC284120) mRNA 2.13 241394_at suppressor of cytokine signaling 2 2.13 SOCS2 203373_at cyclin-dependent kinase 5 regulatory subunit 1 (p35) 2.11 CDK5R1 204995_at likely ortholog of mouse Sds3 2.11 SDS3 233841_s_at
  • TIA1 cytotoxic granule-associated RNA binding protein 2.00 TIA1 1554890 a at
  • MAX dimerization protein 1 0.50 MAD 228846_at ecotropic viral integration site 5 0.50 EVI5 209717_at
  • T-cell leukemia translocation altered gene 0.49 TCTA 203054_s_at ubiquitin-conjugating enzyme E2R 2 0.49 UBE2R2 226954_at hypothetical protein FLJ30794 0.49 FLJ30794 238029_s_at kelch-like 8 ⁇ Drosophila) 0.49 KLHL8 242648_at junction-mediating and regulatory protein 0.49 JMY 241985_at tripartite motif-containing 37 0.49 TRIM37 213009_s_at tubulin-tyrosine ligase 0.48 TTL 224896_s_at cystathionase (cystathionine gamma-lyase) 0.48 CTH 217127_at
  • SARIa gene homolog 2 (S. cerevisiae) 0.47 SARA2 1554482_a_at putative nucleic acid binding protein RY-1 0.47 RY1 212440_at TATA element modulatory factor 1 0.47 TMF1 227685_at hydroxysteroid (17-beta) dehydrogenase 12 0.46 HSD17B12 217869_at disabled homolog 2 mitogen-responsive phosphoprotein (Drosophila) 0.46 DAB2 201280_s_at
  • GABA(A) receptors associated protein like 3 0.45 GABARAPL3 211458_s_at nuclear receptor subfamily 4 group A member 2 0.45 NR4A2 204622_x_at
  • GABA(A) receptor-associated protein like 1 0.43
  • GABARAPL1 208868_s_at solute carrier family 11 proton-coupled divalent metal ion transporters
  • member 2 0.43
  • SMPDL3A 213624_at neuronal pentraxin I 0.43
  • NPTX1 204684_at fucosyltransferase 11 alpha (13) fucosyltransferase
  • DKFZp762C111 hypothetical protein DKFZp762C1112 0.43 2 225974_at sorting nexin 13 0.43 SNX13 227031_at fibronectin leucine rich transmembrane protein 2 0.42 FLRT2 204359_at
  • BRAMY2013590 0.42 225728_at protein tyrosine phosphatase type IVA member 1 0.42 PTP4A1 200732_s_at glutamate-cysteine ligase modifier subunit 0.42 GCLM 234986_at guanine nucleotide binding protein (G protein) alpha activating activity polypeptide olfactory type 0.42 GNAL 218178_s_at chromosome 13 open reading frame 1 0.42 C13orf1 230151_at heat shock 7OkDa protein 9B (mortalin-2) 0.42 HSPA9B 200690_at
  • Transcripts that were upregulated upon Staul depletion and, thus, represent possible SMD targets encode proteins that are involved in signal transduction, cell proliferation or both (Table 9).
  • Table 9 Selected examples of transcripts upregulated in human cells depleted of Staul in three independently performed microarray analyses
  • Rho GDP dissociation inhibitor (GDI) beta thymosin beta identified in neuroblastoma cells tissue factor pathway inhibitor 2
  • cell cycle chemokine (C-X-C motif) ligand 1 (melanoma growth stimulating activity alpha) chromosome 14 open reading frame 141 cyclin-dependent kinase 5 regulatory subunit 1 (p35) cyclin-E binding protein 1 dual specificity phosphatase 6 ets variant gene 1 growth associated protein 43 histone deacetylase 8 signaling lymphocytic activation molecule family member 1 spinal cord-derived growth factor-B suppressor of cytokine signaling 2 rumor protein p53 (Li-Fraumeni syndrome) v-jun sarcoma virus 17 oncogene homolog (avian) VIII. Apoptosis catenin (cadherin-associated protein) alpha-like 1 TIAl cytotoxic granule-associated RNA binding protein tumor protein p53 (Li-Fraumeni syndrome)
  • chemokine (C-X-C motif) ligand 1 melanoma growth stimulating activity alpha
  • fibronectin 1 growth associated protein 43
  • integrin beta 3 platelet glycoprotein Ilia antigen CD61
  • interferon-induced protein 44
  • PHD finger protein 11 transducin-like enhancer of split 4 E(spl) homolog Drosophila) transforming growth factor beta 1 induced transcript 1 tumor protein p53 (Li-Fraumeni syndrome) v-jun sarcoma virus 17 oncogene homolog (avian) zinc finger protein 90 homolog (mouse)
  • Rho GDP dissociation inhibitor beta signaling lymphocytic activation molecule family member 1 spinal cord-derived growth factor-B stathmin-like 3 suppressor of cytokine signaling 2 tenascin C (hexabrachion) thrombospondin 1 transducin-like enhancer of split 4 (E(spl) homolog Drosophil ⁇ ) transforming growth factor beta 1 induced transcript 1 type I transmembrane receptor (seizure-related protein)
  • Rho GDP dissociation inhibitor beta stathmin-like 3 suppressor of cytokine signaling 2 thrombospondin 1 transducin-like enhancer of split 4 (E(spl) homolog Drosophild) transgelin tumor protein p53 (Li-Fraumeni syndrome)
  • transcripts that were upregulated encode proteins that function in the immune response. Still others produce proteins that participate in cell adhesion, motility, the extracellular matrix, or other aspects of cell structure. A number encode factors that regulate transcription. Others encode proteins involved in RNA metabolism, including the TIAl cytotoxic granule- associated RNA binding protein, which regulates the alternative splicing of pre-mRNA that encodes the human apoptotic factor Fas(Forch et al., 2002) and translationally silences mRNAs that encode inhibitors of apotosis such as tumor necrosis factor a. (TNF-a)(Piecyk et al., 2000; Li et al., 2004).
  • RNA metabolic protein that was upregulated upon Staul depletion is Dcp2, which mediates transcript decapping(Wang et al., 2002). It is worth noting, however, that the abundance of an mRNA could be upregulated upon Staul depletion via a mechanism that involves an alteration in its half-life, such as SMD, or via the product of another mRNA that itself is directly regulated by Staul .
  • Staul or Upfl depletion increases the abundance of c-JUN, SERPINE and IL7R 3' mRNAs
  • Serine (or cysteine) proteinase inhibitor clade E Bait for tissue plasminogen activator, 3.2 3.8 (nexin plasminogen activator inhibitor type 1) urokinase, and protein C member 1 (SERPINEl) Interleukin 7 receptor (IL7R) Receptor for interleukin 7 3.0 2.1 v-jun sarcoma virus 17 oncogene homolog (avian) Proto oncogene 2.6 5.3 (c-JUN)
  • the three transcripts encode serine (or cysteine) proteinase inhibitor clade E (nexin plasminogen activator inhibitor type 1) member 1 (Serpinel), interleukin 7 receptor (IL7R), and v-jun sarcoma virus 17 oncogene homolog (avian) (c-jun).
  • serpinel serine proteinase inhibitor clade E
  • IL7R interleukin 7 receptor
  • v-jun sarcoma virus 17 oncogene homolog avian
  • RNAs small interfering (si) RNAs
  • Staul or Staul (A) siRNA which targeted a different Staul mRNA sequence
  • Upfl or Upfl(A) siRNA which targeted a different UPFl mRNA sequence
  • Upf2 siRNA which has no effect on SMD
  • protein and RNA were isolated for analysis using Western blotting and RT-PCR, respectively.
  • nucleotides 481-671 of the c-JUN 3' UTR which include the 151-nucleotide class IJJ (i.e., II-" Il Il .. • ' 'U i ::::ii !.J !:,;:i ⁇ ,,' ,,;:;
  • nucleotide 1 is defined as the nucleotide immediately 3' to the normal termination codon.
  • Cos cells were transfected with four test plasmids : pcFLuc-c- JUN 3 'UTR, pcFLuc-SERPHNEl 3'UTR, pcFLuc-IL7R 3'UTR and pcFLuc-Arfl SBS ( Figure 18A), the latter of which serves as a positive control for Staul-HA 3 binding since it contains the minimized Staul binding site (SBS) from the Arfl 3'UTR (Kim et al., 2005).
  • SBS Staul binding site
  • L cells were transfected with mouse (m)Staul siRNA, mUpfl siRNA or a nonspecific Control siRNA (Kim et al., 2005) and, two days later, with the four pfos-FLuc test plasmids and the phCMV-MUP reference plasmid.
  • the reference plasmid controls for variations in transfection Il « Il ..-' ⁇ O ::::::n i
  • serum was added, and protein and RNA were purified from, respectively, cytoplasmic and nuclear fractions after 0, 30, 45 and 60 minutes.
  • Staufen binding site exists within Drosophila bicoid mRNA (Ferrandon et al., 1994). Linker scanning mutations that disrupted the interaction of Staufen with this mRNA mapped to three noncontiguous regions: 148 nucleotides of stem IJJ, 89 nucleotides of the distal region of stem IV, and 88 nucleotides of the distal region of stem V (Ferrandon et al., 1994). Given the structural complexity of human Staufen binding site(s), it may be difficult to identify human Staul binding sites exclusively from our existing mRNA immunopurification and half-life data.
  • Arfl , c-JUN, SERPINEl , IL7R and GAP43 mRNAs will not be the only transcripts among those upregulated upon Staul depletion that are targeted for SMD.
  • CYR61 mRNA which encodes the cysteine-rich angiogenic inducer 61, was upregulated upon Staul depletion in only two of our three microarray analyses and, thus, fell below the stringent criteria as a candidate target for SMD.
  • this mRNA may very well be targeted for SMD since it was also among the transcripts upregulated upon Upfl depletion (Forch et al., 2002).
  • Interferon-induced protein with tetrat ⁇ copeptide repeats 4 (IFIT4) 4 1 7 7 22 AI075407
  • KIAA0143 protein (KIAA0143) 2 0 2 2 44 AW470003
  • Rho GTPase activating protein 19 (ARHGAP 19) 2 1 2 2 00 U79256
  • pcFLuc-8bs (Kim et al., 2005) was digested with Xbal and ligated to one of four PCR-amplified fragments that contained the (i) c-JUN ARE HI plus 45 nucleotides that had been digested with Xbal, (ii) SERPINEl 3'UTR without the normal termination codon that had been digested with Xbal, (iii) EL7R without the normal termination codon that had been digested with Nhel or (iv) GAP43 3'UTR without the normal termination codon that had been digested with Xbal .
  • the c-JUN ARE fragment was amplified using the Human HeLa BDTM Marathon-Ready cDNA (BD Biosciences) and two primers: 5'- CGCTCTAGAGTGAGAACTCTTTCTGGCCTGCCTTCGTTAAC-S ' (SEQ ID NO: 63) (sense) and 5 ' -CGCTCT AGATTAC AAATGGTAAACTCAGAGTGCTCC-3 ' (SEQ ID NO: 64) (antisense), where underlined nucleotides specify the Xbal site.
  • the SERPINEl 3'UTR fragment was amplified using pCMV6-XL5-SERPINEl (Origene Technologies) and two primers: 5 '-CGCTCTAGAGTGACCCTGGGGAAAGACGCCTTC ATCTG-3 ' (SEQ ID NO: 65) (sense) and 5'-CGCTCTAGAGCTTCTATTAGATTACATTCATTTCAC-S ' (SEQ ID NO: 66) (antisense), where underlined nucleotides specify the Xbal site.
  • the IL7R 3'UTR fragment was amplified using Human HeLa BDTM Marathon-Ready cDNA and two primers: 5'-CGCGCTAGCGTGAAGTGTAAGAAACCCAGACTGAAC-S' (SEQ ID NO: 67) (sense) and 5'-CGCGCTAGCTTTTTTTCCTCTCATGCTCTCTTCCTGC-S' (SEQ ID NO: 68) (antisense), where underlined nucleotides specify the Nhel site.
  • the GAP43 fragment was amplified using HeLa-cell total cDNA prepared from Staul siRNA-treated cells and two primers: 5'-CGCTCTAGAGTGAACTCTAAGAAATGGCTTTCCACATC- 3' (sense) (SEQ ID NO: 141) and 5'- CGCTCTAGAGTGAGAATTCACTCGATATTTTGGACTCCTCAG-S' (antisense) (SEQ ID NO: 142), where underlined nucleotides specify the Xbal site.
  • pfos-Gl (Kim et al., 2005) was digested with EcoRI and Ncol and ligated to one of three PCR-amplified H I! H .. ⁇ i
  • Each PCR fragment was amplified using the pcFLuc-c-JUN 3'UTR, pcFLuc-SERPINEl 3'UTR, or pcFLuc-IL7R 3'UTR and two primers: 5'-CATGCCATGGAAGACGCCAAAAACATAAAGAAAGGC-S' (SEQ ID NO: 69) (sense) and 5 ' -CGGAATTC AGGCTGATC AGCGAGCTCTAGC ATTT AGG-3 ' (SEQ E) NO: 70) (antisense), where underlined nucleotides specify the Ncol and EcoRI site, respectively.
  • pSport-Arfl-SBS derivatives harboring ⁇ (250-300), ⁇ (200- 300), ⁇ (l 50-300), ⁇ (100-300) or ⁇ (50-300).
  • pSport was digested with Xbal and Hpal and ligated to one of five PCR-amplified fragments that had been digested with Xbal.
  • Each PCR fragment was amplified using pSport-Arfl-SBS, the common sense primer 5'- GCTATTT AGGTGACACTATAGAAGGTAC-S' (SEQ ID NO: 143) and the specific antisense primer: 5'-CTTTTTACAATAAAAAAAGCTGAGTAATAT-S' (SEQ ID NO: 144), 5'-TGCCTCATTGGAAACAAAAACTATTTACAT-S' (SEQ DD NO: 145), 5'- AGGCTGCGTCTGCC ACATTTAC-3' (SEQ ID NO: 146), 5'- ACCACGGAGGCAGCTTCTGG-3' (SEQ ID NO: 147), or 5'- ATGAGAGTAAAGC AGAGGGC AAG-3' (SEQ ID NO: 148).
  • PCR amplifications were carried out using Pfu Ultra (Stratagene). PCR products were incubated with Dpnl to digest the methylated template DNA, phosphorylated at the 5' ends using T4 polynucleotide kinase (Fermentas), and circularized by ligation.
  • CATAGGAGTACCACTTCCTGCCTCATTGG-3' antisense, Single and Double, where underlined nucleotides specify mutated nucleotides
  • SEQ ID NO: 156 or 5'- CCACGGAGGCAGAAAGTGGCACTCACACC-3' (antisense, Double)
  • CCAATGAGGCAGGAAGTGGTACTCCTATG-3' (sense, Single and Double, where underlined nucleotides specify mutated nucleotides) (SEQ ID NO: 158) or 5'- GGTGTGAGTGCCACTTTCTGCCTCCGTGG-3' (sense, Double) (SEQ ID NO: 159) and the common antisense primer 5'-GTGCCC ATGGGCCTAC ATCC-3 1 (SEQ ID NO: 160).
  • the resulting fragments were mixed with the same sense and antisense primers that amplified, respectively, the 5' and 3' fragments to generate a joined product.
  • PCR products were digested with Xbal, and inserted into the Xbal and Hpal sites of pSport. To construct ⁇ (Loop), a 5'fragment was amplified using the primer pair 5'- GCTATTTAGGTGACACTATAGAAGGTAC-3' (sense) (SEQ ID NO: 161) and 5'-
  • AATTCTCGAGAGGCAGCTTCTGGCACTC-3' antisense, where underlined nucleotides specify a Xhol site
  • SEQ ID NO: 162 AATTCTCGAGAGGCAGCTTCTGGCACTC-3' (antisense, where underlined nucleotides specify a Xhol site)
  • SEQ ID NO: 163 AATTCTCGAGAGGCAGCTTCTGGCACTC-3' (antisense, where underlined nucleotides specify a Xhol site)
  • SEQ ID NO: 163 antisense, where underlined nucleotides specify a Xhol site
  • pcFLuc-8bs was digested with Xbal and ligated to one of three Xbal-digested PCR-amplified fragments that had been generated using pcFLuc-c-JUN 3 'UTR and two primers: 5 '-
  • CGCTCTAGAGTGATTCGTTAACTGTGTATGTAC-S' (SEQ ID NO: 71) (sense) and 5'- CGCTCTAGAAAATTAAAAAATATATATATG-S ' (SEQ ID NO: 72) (antisense) for pcFLuc-c-JUN 3'UTR (500-539), 5'- CGCTCTAGAGTGAGATGAAAGCTGATTACTGTC-3' (SEQ ID NO: 73) (sense) and 5'- CGCTCTAGAAACTTACAAAGGCATGAAGC-3 ' (SEQ ID NO: 74) (antisense) for pcFLuc-c-JUN 3'UTR (540-587), or 5'-
  • pcFLuc-8bs was digested with Xbal and ligated to one of three Xbal-digested PCR-amplified fragments that had been generated using pcFLuc-c-JUN 3'UTR and two primers: 5'- CGCTCTAGAGTGATTCGTTAACTGTGTATGTAC-3' (sense) (SEQ ID NO: 165) and ii «,..,. Ii .. ⁇ ' 1 I.,,!' .,,;:i ⁇ n..,n :;;;;n ,.• • constructive;;:!> ::;::n i
  • pcFLuc-8bs was digested with Xbal and ligated to one of four Xbal-digested PCR-amplified fragments that had been generated using pcFLuc-SERPINEl 3'UTR and two primers: 5'-
  • pcFLuc-8bs was digested with Xbal and ligated to one of four Xbal-digested PCR-amplified fragments that had been generated using pcFLuc-SERPESfEl 3'UTR and two primers: 5'-
  • pcFLuc-8bs was digested with Xbal and ligated to one of three Xbal-digested PCR-amplifled fragments that had been generated using pcFLuc-GAP43 3'UTR and two primers: 5'- CGCTCTAGAGTGAACTCTAAGAAATGGCTTTCCA-3' (SEQ ID NO: 85) (sense) and 5'-CGCTCTAGATGTTTGACTTGGGATCTTTCCTGC-S' (SEQ ID NO: 86) (antisense) for pcFLuc-GAP43 3'UTR (1-206), or 5'-
  • pcFLuc-8bs was digested with Xbal and ligated to one of two Nhel-digested PCR-amplified fragments that had been generated using pcFLuc-IL7R 3'UTR and two primers: 5'-
  • Human HeLa cells (2 x 10 6 ) were propagated in DMEM medium (GIBCO- BRL) containing 10% fetal bovine serum (GIBCO-BRL) in 60-mm dishes and, where specified, transiently transfected with plasmid DNA, in vitro-synthesized siRNA, or both as described (Kim et al., 2005).
  • Monkey Cos cells which were used in experiment involving immunopurification, were similarly treated as were mouse L cells, which effectively support fos promoter induction upon the addition of serum after serum deprivation. All human and mouse siRNAs have been previously reported as has been the serum-induced transcription of fos-FLuc and the isolation of total-cell or nuclear protein and RNA (Kim et al., 2005).
  • Example 24 Western blotting and RT-PCR
  • HeLa-cell RNA was purified using TriZol reagent (Invitrogen) and deemed to be intact using an RNA 6000 Nano LabChip® (Agilent) together with a Bio analysesr 2100 and Biosizing software (Agilent). Biotin-labeled cRNAs were generated and hybridized to Ul 33 Plus 2.0 Array human gene chips (comprising more than 47,000 transcripts and variants).
  • Hybridized chips were scanned using an Affymetrix GeneChip® 3000 Scanner. Results were recorded using the GeneChip® Operating Software (GCOS) platform, which included the GeneChip® Scanner 3000 high-resolution scanning patch that enables feature extraction (Affymetrix). Notably, the Affymetrix Gene Expression Assay identifies changes that are greater than 2-fold with 98% accuracy (Wodicka et al., 1997). Arrays were undertaken using three independently generated RNA samples. Transcripts that showed at least a 2-fold increase in abundance with a p value of less than 0.05 in each of the three analyses were scored as potential SMD targets. 285. Throughout this application, various publications are referenced.
  • Nonsense but not missense mutations can decrease the abundance of nuclear mRNA for the mouse major urinary protein, while both types of mutations can facilitate exon skipping. MoI. Cell. Biol. 14, 6326-6336. Bono, F., Ebert, J., Unterholzner, L., Guttler, T., Izaurralde, E., and Conti, E. (2004). Molecular insights into the interaction of PYM with the Mago-Y14 core of the exon junction complex. EMBO Rep. 5, 304-310.
  • eIF4A3 is a novel component of the exon junction complex. RNA 10, 200-209.
  • mRNA stabilization by poly(A) binding protein is independent of poly(A) and requires translation. Genes Dev. 12, 3226- 3235. Curatola, A.M., Nadal, M.S. & Schneider, RJ. Rapid degradation of AU-rich element (ARE) mRNAs is activated by ribosome transit and blocked by secondary structure at any position 5 1 to the ARE. MoI. Cell. Biol. 15, 6331-6340 (1995).
  • Ferrandon, D., Elphick, L., Nusslein-Volhard, C. & St Johnston, D. Staufen protein associates with the 3'UTR of bicoid mRNA to form particles that move in a microtubule- dependent manner. Cell 79, 1221-1232 (1994).
  • RNA 4, 479-486 A dual-luciferase reporter system for studying recoding signals.
  • Drosophila Y14 shuttles to the posterior of the oocyte and is required for oskar mRNA transport. Curr. Biol. 77, 1666-1674.
  • Pre-mRNA splicing imprints mRNA in the nucleus with a novel RNA-binding protein that persists in the cytoplasm. MoI. Cell 6, 673-682.
  • RNA granules a link between RNA localization and stimulation-dependent translation. Neuron 32, 683-696.
  • exon-exon junction complex provides a binding platform for factors involved in mRNA export and nonsense- mediated mRNA decay. EMBO J. 20, 4987-4997.
  • Nonsense-mediated mRNA decay in mammalian cells involves decapping, deadenylating, and exonucleolytic activities. MoI. Cell 12, 675-687.
  • Nonsense-mediated mRNA decay A comparative analysis of different species. Curr. Genomics 5, 175-190. Maquat, L. E. (2004b). Nonsense-mediated mRNA decay: splicing, translation and mRNP dynamics. Nat. Rev. MoI. Cell. Biol. 5, 89-99.
  • a human sequence homologue of Staufen is an RNA-binding protein that is associated with polysomes and localizes to the rough endoplasmic reticulum. MoI. Cell. Biol. 19, 2212-2219. Matsuzaki, F., Ohshiro, T., JJceshima-Kataoka, H., and Izumi, H. (1998). miranda localizes staufen and prospero asymmetrically in mitotic neuroblasts and epithelial cells in early Drosophila embryogenesis. Development 125, 4089-4098.
  • RNA-binding protein Tsunagi interacts with Mago Nashi to establish polarity and localize oskar mRNA during Drosophila oogenesis. Genes Dev. 15, 2886-2899.
  • Piecyk, M. et al. TIA-I is a translational silencer that selectively regulates the expression of TNF-alpha. EMBOJ. 19, 4154-4163 (2000).
  • eIF4Am binds spliced mRNA in the exon junction complex and is essential for nonsense-mediated decay. Nat. Struct. MoI. Biol. 11, 346-351.
  • Mammalian staufen is a double-stranded-RNA- and tubulin-binding protein which localizes to the rough endoplasmic reticulum. MoI. Cell. Biol. 19, 2220-2230. Wilusz, C. J., Wang, W., and Peltz, S. W. (2001). Curbing the nonsense: the activation and regulation of mRNA surveillance. Genes Dev. 15, 2781-2785.

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Abstract

L'invention concerne des compositions et des méthodes de traitement de patients présentant des états résultant d'une décroissance de l'ARNm médiée par Stau1, de criblage et de production de ces agents thérapeutiques. L'invention concerne de nouveaux mécanismes de décroissance de l'ARNm faisant intervenir Staufen mammifère (Stau)1, le facteur Upf1 de décroissance d'ARNm à médiation nonsens (NMD), ainsi qu'un codon de terminaison. Il est montré que Stau1 se fixe directement au Upf1 et peut mettre en lumière une décroissance de l'ARNm lorsqu'il est attaché en aval d'un codon de terminaison. L'invention concerne également la nouvelle voie constituant un moyen permettant aux cellules de réguler négativement l'expression des ARNm liant Stau1.
PCT/US2005/035011 2004-09-29 2005-09-29 Decroissance de l'arnm mediee par staufen 1 (stau1) WO2006039399A2 (fr)

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EP2294216A4 (fr) * 2008-05-14 2011-11-23 Dermtech Int Diagnostic de mélanome et de lentigo solaire par analyse d'acides nucléiques
WO2012109476A3 (fr) * 2011-02-09 2012-10-04 The University Of Rochester Procédés et compositions associés à des sites de liaison à staufen 1 formés par des éléments alu en duplex
WO2018107096A1 (fr) * 2016-12-08 2018-06-14 University Of Utah Research Foundation Agents de la staufen1 et procédés associés
US11578373B2 (en) 2019-03-26 2023-02-14 Dermtech, Inc. Gene classifiers and uses thereof in skin cancers
US11946046B2 (en) 2018-06-14 2024-04-02 University Of Utah Research Foundation Staufen1 regulating agents and associated methods
US11976332B2 (en) 2018-02-14 2024-05-07 Dermtech, Inc. Gene classifiers and uses thereof in non-melanoma skin cancers
USD1097166S1 (en) 2015-05-01 2025-10-07 Dermtech, Llc Dermatology adhesive patch

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US6506559B1 (en) * 1997-12-23 2003-01-14 Carnegie Institute Of Washington Genetic inhibition by double-stranded RNA
US20040241845A1 (en) * 1998-05-22 2004-12-02 Luc Desgroseillers Mammalian staufen and use thereof

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US11753687B2 (en) 2008-05-14 2023-09-12 Dermtech, Inc. Diagnosis of melanoma and solar lentigo by nucleic acid analysis
US9057109B2 (en) 2008-05-14 2015-06-16 Dermtech International Diagnosis of melanoma and solar lentigo by nucleic acid analysis
US10407729B2 (en) 2008-05-14 2019-09-10 Dermtech, Inc. Diagnosis of melanoma by nucleic acid analysis
US11332795B2 (en) 2008-05-14 2022-05-17 Dermtech, Inc. Diagnosis of melanoma and solar lentigo by nucleic acid analysis
EP2294216A4 (fr) * 2008-05-14 2011-11-23 Dermtech Int Diagnostic de mélanome et de lentigo solaire par analyse d'acides nucléiques
US9206479B2 (en) 2011-02-09 2015-12-08 University Of Rochester Methods and compositions related to Staufen 1 binding sites formed by duplexing Alu elements
WO2012109476A3 (fr) * 2011-02-09 2012-10-04 The University Of Rochester Procédés et compositions associés à des sites de liaison à staufen 1 formés par des éléments alu en duplex
USD1097166S1 (en) 2015-05-01 2025-10-07 Dermtech, Llc Dermatology adhesive patch
US12409189B2 (en) * 2016-12-08 2025-09-09 University Of Utah Research Foundation STAUFEN1 agents and associated methods
WO2018107096A1 (fr) * 2016-12-08 2018-06-14 University Of Utah Research Foundation Agents de la staufen1 et procédés associés
US11723912B2 (en) * 2016-12-08 2023-08-15 University Of Utah Research Foundation Staufen1 agents and associated methods
US20240180953A1 (en) * 2016-12-08 2024-06-06 University Of Utah Research Foundation Staufen1 agents and associated methods
US11976332B2 (en) 2018-02-14 2024-05-07 Dermtech, Inc. Gene classifiers and uses thereof in non-melanoma skin cancers
US11946046B2 (en) 2018-06-14 2024-04-02 University Of Utah Research Foundation Staufen1 regulating agents and associated methods
US11578373B2 (en) 2019-03-26 2023-02-14 Dermtech, Inc. Gene classifiers and uses thereof in skin cancers

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