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WO1999020797A1 - PRODUCTION DE POLYPEPTIDES HETEROLOGUES EN L'ABSENCE DE FONCTION DE DEGRADATION DE L'ARNm INDUITE PAR MUTATION NON-SENS - Google Patents

PRODUCTION DE POLYPEPTIDES HETEROLOGUES EN L'ABSENCE DE FONCTION DE DEGRADATION DE L'ARNm INDUITE PAR MUTATION NON-SENS Download PDF

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Publication number
WO1999020797A1
WO1999020797A1 PCT/US1998/022365 US9822365W WO9920797A1 WO 1999020797 A1 WO1999020797 A1 WO 1999020797A1 US 9822365 W US9822365 W US 9822365W WO 9920797 A1 WO9920797 A1 WO 9920797A1
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nonsense
nmd2
gene
compound
nmd2p
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PCT/US1998/022365
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English (en)
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Feng He
Allan S. Jacobson
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University Of Massachusetts
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Priority to AU11140/99A priority Critical patent/AU1114099A/en
Publication of WO1999020797A1 publication Critical patent/WO1999020797A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6897—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids involving reporter genes operably linked to promoters
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/37—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi
    • C07K14/39—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts
    • C07K14/395—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from fungi from yeasts from Saccharomyces
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer

Definitions

  • the invention relates to nonsense-mediated mRNA decay function.
  • mRNA transcripts vary in the length of time (transcript half- life) that they exist in a cell prior to being degraded by cellular proteins specific for that purpose. In some cases, degradation occurs rapidly such that very little protein is produced.
  • the yeast cell Sac char omyces cerevisiae, a commonly used cellular system for the production of recombinant proteins, has a biological pathway that specifically degrades mRNA transcripts containing a non-coding triplet sequence (nonsense or stop codons) in the transcript.
  • a non-coding triplet sequence nonsense or stop codons
  • the destabilizing nonsense codon occurs within the 5 '-proximal portion of the transcript (reviewed in Peltz et al . , Prog. Nucl . Acids Res. Mol. Biol. (1994) 4_7: 271-297) .
  • the translation process stops at the nonsense codons prior to reaching the end of the transcript's coding sequence resulting in the production of a truncated protein that may not possess normal biological activity.
  • the cell has developed a biochemical system to degrade transcripts containing mutations that create stop codons early in the coding sequence.
  • a nonsense codon can be a useful means of coding for an alternate amino acid when a nonsense codon is engineered into the coding sequence to produce an altered protein which is then screened for enhanced biological activity.
  • Suppressor strains e.g., SUFl -1
  • do not allow maximal expression of a nonsense codon-containing transcript (Leeds et al . , (1991) Genes & Dev. .5:2303-2314) .
  • Nonsense-mediated mRNA decay is a phenomenon in which nonsense mutations, e.g., point or frame shift mutations that create a stop codon in the reading frame, in a gene can enhance the decay rate of the mRNA transcribed from that gene.
  • nonsense mutations e.g., point or frame shift mutations that create a stop codon in the reading frame
  • Nonsense-mediated mRNA decay has been studied extensively in the yeast Saccharomyces cerevisiae where it has been shown that degradation of mRNA via this pathway is most likely to occur in the cytoplasm and is linked to translation.
  • Evidence in support of these conclusions includes the following: 1) unstable, nonsense-containing mRNAs are stabilized in a strain harboring an amber suppressor tRNA (Losson and Lacroute, (1979) Proc. Nat'l. Acad. Sci . USA 76.: 5134-5137 ; Gozalbo and Hohmann, (1990) Curr. Genet. 12:77-79); 2) nonsense- containing mRNAs are ribosome-associated (Leeds et al .
  • the UPF1 gene has been cloned and sequenced, (Leeds et al . , (1992) Mol. Cell Biol. 12:2165-2177) and shown to be: 1) non-essential for viability; 2) capable of encoding a 109 kD protein with a so-called zinc finger, nucleotide (GTP) binding site, and RNA helicase motifs (Leeds et al . , (1992) Mol. Cell. Biol. 12:2165- 2177; Altamura et al . , (1992) J. Mol. Biol. 224:575-587: Koonin, (1992) Trends Biochem. Sci.
  • Upflp Upflp protein
  • Suppression of nonsense-mediated mRNA decay in upfl deletion strains does not appear to result simply from enhanced read-through of the termination signal (Leeds et al . , (1991) Genes & Dev. 5.: 2303-2314) , nor does it appear to be specific for a single nonsense codon.
  • upfl mutants degrade nonsense-containing transcripts at a slower rate allowing synthesis of sufficient read-through protein to permit cells to grow under nutrient-deficient conditions that are nonpermissive for UPF1 + cells.
  • the invention relates to the discovery of a gene, NMD2, named after its role in the Nonsense-Mediated mRNA Decay pathway, and the protein, Nmd2p, encoded by the N D2 gene.
  • Nmd2p is shown herein to bind to Upflp.
  • a C- terminal fragment of the protein is also shown to bind to Upflp and, when overexpressed in the host cell, the fragment inhibits the function of Upflp, thereby inhibiting the nonsense-mediated mRNA decay pathway.
  • the components of the nonsense-mediated mRNA decay pathway monitor the fidelity of translation, terminating translation and accelerating decay when a premature nonsense codon-containing mRNA is detected. Interference with the components thus alters both the decay process and the fidelity process. Inhibition of the nonsense- mediated mRNA decay pathway is a useful means of treating disorders caused by the presence of nonsense mutations.
  • the invention further relates to the inhibition of the nonsense-mediated mRNA pathway to produce a heterologous recombinant protein or polypeptide in a host cell or to increase the production of an endogenous protein useful to a host cell or organism.
  • a codon of the gene encoding the recombinant protein is mutated to encode a nonsense codon. Expression of this recombinant protein is enhanced by stabilizing the nonsense codon- containing mRNA transcript in a host cell in which the nonsense-mediated mRNA decay pathway is inhibited.
  • Insertion of a nonsense codon into the gene of interest is useful to produce an altered heterologous protein by amino acid substitution at the nonsense codon in a suppressor host strain. Insertion of a nonsense codon further allows the controlled expression of a protein that may be toxic to the cell by controlling the timing of nonsense-mediated mRNA decay pathway inhibition. Insertion of a nonsense codon also allows the production of an N-terminal fragment of a heterologous protein in increased yield when the nonsense codon-containing transcript is expressed in a host strain that is not a suppressor of nonsense codons.
  • the invention further provides methods of increasing expression of nonsense codon-containing transcripts by inhibiting the nonsense-mediated mRNA decay pathway by overexpressing the C-terminal fragment of Nmd2p in the same cell that is also expressing the heterologous protein. Overexpression of the C-terminus of Nmd2p is not deleterious to the cell since its expression provides specific stabilization of transcripts having a stop codon early in the transcript and does not affect the stability of other transcripts.
  • the invention features a method of substantially inhibiting the nonsense-mediated mRNA decay pathway by providing a cell (such as a yeast cell) and mutating the N D2 gene such that essentially no functional ⁇ md2p is produced.
  • an insertional mutation which prevents synthesis of the Nmd2p results in an inhibited nonsense-mediated mRNA decay pathway without affecting the viability of the cell as described herein.
  • the invention also features a method of substantially inhibiting the nonsense-mediated mRNA decay pathway by providing a cell (such as a yeast cell) and mutating the UPF1 gene such that essentially no functional Upflp is produced.
  • a cell such as a yeast cell
  • mutating the UPF1 gene such that essentially no functional Upflp is produced.
  • an insertional mutation which prevents synthesis of the Upflp results in an inhibited nonsense-mediated mRNA decay pathway without affecting the viability of the cell as described herein.
  • the invention features a method of inhibiting the nonsense-mediated mRNA decay pathway by providing a cell and transforming the cell with a vector encoding NMD2 operably linked to regulatory sequences for constitutive or inducible expression of the antisense transcript.
  • Such an antisense transcript hybridizes to essentially all of the NMD2 sense transcript preventing translation and the production of functional Nmd2p, thereby inhibiting the nonsense-mediated mRNA decay pathway.
  • hybridizing to essentially all of the sense NMD2 transcript is meant that a sufficient amount of the sense transcript is bound by antisense transcript to inhibit translation such that substantially no functional Nmd2p protein is produced.
  • the invention features a method of inhibiting the nonsense-mediated mRNA decay pathway by providing a cell and transforming the cell with a vector encoding UPF1 operably linked to regulatory sequences for constitutive or inducible expression of the antisense transcript.
  • Such antisense transcript hybridizes to a sufficient portion of the UPF1 sense transcript to prevent translation production of functional Upflp, thereby inhibiting the nonsense mediated mRNA decay pathway.
  • the invention also features a substantially pure
  • the D ⁇ A of the invention is at least 90% identical to SEQ ID ⁇ O:l, and is preferably from the yeast Saccharomyces cerevisiae .
  • the DNA encodes an amino acid sequence of Nmd2p (SEQ ID NO: 2) .
  • the amino acid sequence of the invention is at least 90% identical to the amino acid sequence of SEQ ID NO : 2.
  • the invention also features the substantially pure DNA sequence of the 3' terminus (SEQ ID NO: 3) of NMD2.
  • the 3 ' terminus encodes the carboxy terminal fragment (SEQ ID NO: 4) of Nmd2p, which fragment, when overexpressed in a yeast cell, binds to Upflp and inhibits the nonsense-mediated mRNA decay pathway.
  • the invention features a vector containing a DNA sequence (SEQ ID NO:l) encoding a polypeptide (SEQ ID NO: 2) .
  • the coding sequence is under the transcriptional control of regulatory sequences that are activated and deactivated by an externally applied condition such as temperature, or an externally supplied chemical agent.
  • an externally applied condition such as temperature, or an externally supplied chemical agent.
  • the invention further features a vector containing a DNA sequence (SEQ ID NO: 3) encoding a polypeptide (SEQ ID NO: 4) which polypeptide, when overexpressed in a cell, inhibits the nonsense-mediated mRNA decay pathway.
  • a DNA sequence SEQ ID NO: 3
  • SEQ ID NO: 4 polypeptide which polypeptide, when overexpressed in a cell, inhibits the nonsense-mediated mRNA decay pathway.
  • the coding sequence is under the transcriptional control of regulatory sequences that are activated and deactivated by an externally applied condition such as temperature or an externally supplied chemical agent.
  • an externally applied condition such as temperature or an externally supplied chemical agent.
  • the invention also features a host cell containing the DNA of SEQ ID NO:l or SEQ ID NO: 3 or fragments thereof.
  • the invention also features cells harboring vectors containing the DNA of SEQ ID NO:l or SEQ ID NO: 3 or fragments thereof .
  • the invention features substantially pure nonsense-mediated mRNA decay pathway protein, Nmd2p (SEQ ID NO:2) , and fragments thereof from a yeast cell, preferably from the genus Saccharomyces .
  • the invention also features a substantially pure nonsense-mediated mRNA decay pathway protein Nmd2p C- terminal fragment (SEQ ID NO: 4) and fragments thereof which bind to the nonsense-mediated mRNA decay pathway protein, Upflp, and which when overexpressed in a cell, substantially inhibit the nonsense-mediated mRNA decay pathway in the cell.
  • the invention further features a cell containing a vector expressing a polypeptide containing the Nmd2p carboxy terminal fragment (SEQ ID NO:4) , which fragment binds to the nonsense-mediated mRNA decay pathway protein, Upflp, and, when overexpressed in the cell, substantially inhibits the nonsense-mediated mRNA decay pathway in the cell.
  • the invention features methods of producing a heterologous polypeptide from an mRNA transcript in which the transcript contains at least one nonsense codon within a transcript destabilizing 5' portion.
  • the method involves providing a cell in which the nonsense-mediated mRNA decay pathway is substantially inhibited by 1) overexpression of a polypeptide containing the Nmd2p carboxy terminal fragment (SEQ ID NO:4) ; or 2) mutation of NMD2 or UPFl (e.g., insertional mutagenesis) resulting in inhibition of the nonsense-mediated mR ⁇ A decay pathway of the cell; or 3) expression of NMD2 or UPFl antisense mR ⁇ A which hybridizes to the sense transcript of N D2 or UPFl , respectively, inhibiting translation and, thereby inhibiting the nonsense-mediated mRNA decay pathway.
  • Expression in this cell of a nonsense codon-containing gene encoding the heterologous polypeptide provides a transcript whose stability is enhanced at least two-fold compared to a wild-type
  • the invention features antibodies that are raised against and bind specifically to Nmd2p, a protein having the amino acid sequence of SEQ ID NO:2, or a polypeptide having the amino acid sequence of SEQ ID NO:4.
  • the antibodies can be polyclonal or monoclonal .
  • the invention further features a method of screening a candidate host cell for the presence or absence of 1) Nmd2p, 2) a C-terminal fragment of Nmd2p, 3) a polypeptide of SEQ ID N0:2, or 4) a polypeptide of SEQ ID N0:4, including fragments or analogs thereof.
  • the method also can be used to determine relative amounts of each of the proteins in a cell .
  • the screening method is useful for isolating a host strain in which heterologous protein production is to be optimized. The method first involves lysis of a clonal population of cells suspected of containing Nmd2p or Nmd2p fragment. Antibody to Nmd2p or Nmd2p fragment is contacted with proteins of the lysate.
  • Presence, relative abundance, or absence of Nmd2p or Nmd2p fragment in the lysate is determined by the binding of the antibody. Possible detection methods include affinity chromatography, Western blotting, or other techniques well known to those of ordinary skill in the art.
  • a heterologous polypeptide produced by the methods of the invention can be a particular fragment of a protein or polypeptide.
  • a nonsense codon is incorporated into the DNA sequence encoding the protein or polypeptide at a position within a transcript destabilizing 5' portion of the sequence at a desired transcriptional stop site.
  • Expression of the DNA in a cell having an inhibited nonsense-mediated mRNA decay pathway results in a substantially increased half-life for the nonsense codon-containing transcript.
  • An advantage of this method is the stabilization of the transcript allowing an increased amount of the protein fragment to be produced relative to the amount produced in a wild-type host strain.
  • a heterologous protein that is normally toxic to a cell is produced by controllably inhibiting the nonsense- mediated mRNA decay pathway and thereby, controlling the stability of a nonsense codon-containing transcript for the toxic protein.
  • Inhibition of the nonsense-mediated mRNA decay pathway is accomplished, for example, by the inducible expression of the C-terminus of the Nmd2p only when protein production is desired (e.g., at optimal cell density of the culture) .
  • Inhibition of the nonsense- mediated mRNA decay pathway substantially increases the half-life of the transcript containing a nonsense codon in a transcript destabilizing 5' portion of the transcript thereby increasing translation and production of the protein when desired.
  • the cell expressing the heterologous protein can be a nonsense suppressor cell in which the suppressor mechanism is controllably expressed and substitutes the naturally occurring amino acid at the site of a nonsense codon.
  • An altered heterologous polypeptide is produced in a nonsense suppressor cell by substituting an amino acid at the position of a nonsense codon, which amino acid does not naturally occur at that position.
  • An amino acid is substituted which alters a target biological activity of the protein in the cell.
  • the nonsense-mediated mRNA pathway is inhibited to increase production of the altered heterologous polypeptide from a transcript containing a nonsense codon in a transcript destabilizing 5' portion of the transcript.
  • Alteration in biological activity includes increased binding affinity to a target molecule such as a receptor, antibody, or decreased toxicity of the protein to the host strain in which the protein is produced.
  • a target molecule such as a receptor, antibody, or decreased toxicity of the protein to the host strain in which the protein is produced.
  • substantially reduction in toxicity is meant that expression of the altered heterologous polypeptide allows the cell growth rate to be at least two-fold greater than the growth rate in the presence of the natural toxic heterologous polypeptide, or allows sufficient cell growth for production of the altered heterologous protein.
  • An advantage of the invention is the ability to increase heterologous protein production and direct amino acid substitution to a desired codon position using a nonsense codon and producing the protein in a suppressor mutant such that a known amino acid is substituted in each suppressor host.
  • Stabilization of the mRNA transcript by inhibiting the nonsense-mediated mRNA decay pathway increases the half-life of the transcript (decreases its decay rate) thereby allowing increased translation from the transcript.
  • the nonsense codon is present in a transcript destabilizing 5' portion of the transcript.
  • the transcript containing the nonsense codon decays rapidly in the presence of an unaltered wild-type nonsense-mediated mRNA decay pathway, and decays at least two-fold more slowly in the presence of a nonsense-mediated mRNA decay pathway inhibited by the method of the invention.
  • the invention also includes a substantially pure polypeptide that specifically binds to the Upflp protein, wherein the binding causes inhibition of the nonsense mediated mRNA decay pathway.
  • the invention features substantially pure nucleic acids (and vectors containing them) which hybridize under stringent conditions to the nucleotide sequence of SEQ ID N0:1 or SEQ ID NO: 3 or their complementary sequences, wherein the nucleic acid encode an Nmd2p polypeptide or a carboxy terminal fragment of an Nmd2p polypeptide that inhibits the nonsense-mediated mRNA decay pathway in a cell, respectively.
  • the invention features a method of determining whether a candidate compound, e.g., a small molecule or nucleic acid, modulates the nonsense- mediated mRNA decay pathway by a) obtaining a cell (e.g., from a mammal such as a human) containing a mutation in a specific nonsense mutation-containing gene; b) incubating the cell with the candidate compound under conditions and for a time sufficient for the cell to express nonsense- mediated mRNA decay pathway genes in the absence of the candidate compound; and c) measuring expression (e.g., RNA or protein) of the nonsense mutation-containing gene, or activity of the gene product in the presence and in the absence of the candidate compound, wherein a difference in expression or activity indicates that the compound modulates nonsense-mediated mRNA decay.
  • a cell e.g., from a mammal such as a human
  • expression e.g., RNA or protein
  • the cell can be, for example, a yeast cell containing a nonsense mutation in a gene such that the ability of the cell to grow in a selective medium depends on the functionality of the nonsense-mediated decay pathway.
  • the gene containing the nonsense mutation can be selected from the group consisting of tyr7, leu2, and CANl, and the nonsense-mediated decay pathway gene can be NMD2 , UPFl, UPF3 , RENT1, HUPF1 , or homologs thereof .
  • the invention features a method for treating a mammal, e.g., a human, having a disorder involving a nonsense mutation by administering to the mammal a therapeutically effective amount of a compound that inhibits the nonsense-mediated mRNA decay pathway.
  • a compound that inhibits the nonsense-mediated mRNA decay pathway can cause decreased expression of UPFl, UPF3, NMD2 , RENTl, HUPFl, or their homologs, or decreased activity of Upflp, Upf3p, or Nmd2p or their homologs.
  • the compound can be the C-terminal fragment of Nmd2p or an antisense oligonucleotide.
  • the disorder can be breast cancer, polycystic kidney disease I, polycystic kidney disease II, Niemann-Pick disease, adenomatous polyposis coli, cystic fibrosis, Fanconi's anemia, hemophilia, hypercholesterolemia, neurofibromatosis, ornithine transcarbamylase deficiency, retinoblastoma, glycogen storage disease, McArdle disease, cancer, Tay- Sachs disease, Cowden disease, Wilson disease, or ⁇ - thalassaemia.
  • the invention also features method for treating a patient with a disorder associated with excessive expression or activity of an NMD2 gene, the method involving administering to the patient a compound which inhibits expression of NMD2.
  • a "substantially pure DNA” is a DNA that is not immediately contiguous with (i.e., covalently linked to) both of the coding sequences with which it is immediately contiguous (i.e., one at the 5' end and one at the 3' end) in the naturally-occurring genome of the organism from which the DNA of the invention is derived.
  • the term therefore includes, for example, a recombinant DNA which is incorporated into a vector, into an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR (polymerase chain reaction) or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequences .
  • polypeptide is any chain of amino acids, regardless of length or post-translational modification (e.g., glycosylation) .
  • inhibitorted nonsense-mediated mRNA decay pathway decreased turnover of a nonsense codon- containing mRNA transcript in which the half-life of the nonsense codon-containing mRNA is at least two-fold greater in a nonsense-mediated mRNA decay pathway altered by the methods of the invention relative to its half-life in a wild type cell. Techniques for measuring mRNA half- life are described herein and in Parker et al . (1991) Meth. Enzymol . 194:415-423. The pathway can also be inhibited by increased read-through of nonsense codon- containing mRNAs.
  • a "transcript destabilizing 5' portion” is a 5' proximal region of an mRNA transcript in which region the presence of a nonsense codon results in an increased rate of transcript degradation by at least two-fold compared to the normal transcript in a wild-type organism. Determination of a transcript destabilizing 5' portion is readily performed by one of ordinary skill in the art. The half-life of the transcript from each altered DNA is compared to the wild-type transcript by standard techniques. An approximately two-fold or more decrease in half-life for the altered transcript in a cell expressing wild-type nonsense-mediated mRNA decay pathway activity indicates that the nonsense codon is in a transcript destabilizing region. The region 5' proximal of the most downstream destabilizing nonsense codon position is considered a transcript destabilizing 5' portion.
  • Nmd2p is the protein encoded by a gene, NMD2, which is involved in the nonsense-mediated mRNA decay pathway (e.g., SEQ ID NO:l depicts the NMD2 gene of Saccharomyces cerevisiae which encodes the Nmd2p depicted in SEQ ID NO: 2) .
  • Upflp is the protein encoded by a gene, UPFl, which is involved in the nonsense-mediated mRNA decay pathway (e.g., Figs. 5A and 5B (SEQ ID NO:7) depicts a UPFl nucleic acid sequence of Saccharomyces cerevisiae which encodes the Upflp depicted in Fig. 6 (SEQ ID NO:8); GenBank Accession No. M76659; Leeds et al . (1992), supra) .
  • Upf3p is the protein encoded by a gene, UPF3, which is involved in the nonsense-mediated mRNA decay pathway (e.g., Fig. 7 (SEQ ID NO:9) depicts a C7PF3 nucleic acid sequence of Saccharomyces cerevisiae which encodes the Upf3p depicted in Fig. 8 (SEQ ID NO:10);
  • a "substantially pure polypeptide” is a polypeptide, e.g., a nonsense-mediated mRNA decay pathway polypeptide or fragment thereof, that is at least 60%, by weight, free from the proteins and naturally-occurring organic molecules with which it is naturally associated.
  • the preparation is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight, nonsense-mediated mRNA decay pathway polypeptide or fragment.
  • a substantially pure nonsense- mediated mRNA decay pathway polypeptide or fragment thereof is obtained, for example, by extraction from a natural source; by expression of a recombinant nucleic acid encoding a nonsense-mediated mRNA decay pathway polypeptide or fragment thereof; or by chemically synthesizing the polypeptide or fragment. Purity can be measured by any appropriate method, e.g., column chromatography, polyacrylamide gel electrophoresis, or HPLC analysis.
  • a "carboxy terminal fragment of Nmd2p" is the sequence including amino acid 326 to amino acid 1089 (SEQ ID NO: 4) or a fragment thereof. The carboxyl terminus is any polypeptide including SEQ ID NO: 4 or a fragment thereof that substantially inhibits nonsense-mediated mRNA decay in a cell when the fragment is expressed above endogenous level, as described herein.
  • substantially inhibit nonsense-mediated mRNA decay is meant to cause an increase by at least two-fold in the half-life of an mRNA of interest in the presence of an inhibiting agent (e.g., a chemical agent, a polypeptide fragment, or like substance) that interferes with the functioning of the proteins of the nonsense- mediated mRNA pathway.
  • an inhibiting agent e.g., a chemical agent, a polypeptide fragment, or like substance
  • An “overexpressed polypeptide” is a polypeptide which, when produced by the in vivo expression of a DNA sequence to produce that polypeptide, is produced in a quantity at least two-fold greater than the quantity of the same polypeptide expressed from the endogenous transcription /translation regulatory elements of the DNA sequence of interest .
  • the endogenous regulatory elements are those of the native gene.
  • substantially increased transcript stability is meant an increase in the half-life of an mRNA transcript by at least two-fold in the presence of an inhibited nonsense-mediated mRNA decay pathway.
  • the half-life of an mRNA transcript can be measured by extracting at various time points total mRNA from a cell expressing the gene of interest. This is followed by determining the abundance of a transcript over time by Northern analysis using a labelled (e.g., radiolabelled probe) nucleic acid probe to visualize the transcript.
  • Increased transcript stability can also be inferred from increased expression of a polypeptide from the gene of interest in the presence of an inhibited nonsense- mediated mRNA pathway.
  • a particular protein e.g., Nmd2p or Upflp
  • a particular protein e.g., Nmd2p or Upflp
  • the nonsense- mediated mRNA decay pathway is inhibited, resulting in at least a two-fold increase in the stability of mRNA transcripts containing a nonsense codon in a transcript destabilizing 5' portion.
  • operably linked is meant that a gene and a regulatory sequence (s) are connected in such a way as to permit gene expression when the appropriate molecules (e.g., transcriptional activator proteins) are bound to the regulatory sequence (s).
  • “Inducible regulatory sequences” are regulatory sequences (e.g., transcriptional regulatory sequences) whose function is initiated by the introduction of one or more external agents to the cell culture medium and whose function is inhibited by the removal of the external agents.
  • binds a molecule that binds to a particular entity, e.g., an Nmd2p polypeptide, but which does not substantially recognize or bind to other molecules in a sample, e.g., a biological sample, which includes the particular entity, e.g., Nmd2p.
  • Figs. 1A to 1C are a representation of the DNA sequence (SEQ ID NO:l) and the deduced amino acid sequence (SEQ ID NO: 2) of NMD2. Cloning of the N D2 gene and determination of its D ⁇ A sequence are described herein. The predicted amino acid sequence is indicated in single-letter code and shown below each line of D ⁇ A sequence. Position number 1 corresponds to the A of the ATG initiation codon.
  • the N D2 open reading frame is interrupted by an intron of 113 nucleotides in which the conserved 5' splice site [GUAUGU] , branchpoint [UACUAAC] , and 3' splice site [AG] are underlined.
  • Transcription initiation sites at nucleotides -56, -60, - 64, and -67 were determined by primer extension analysis and are indicated by vertical arrows.
  • the putative TATA box and Abflp binding consensus sequence, located between positions - 219 to -213 and -198 to -186 in the NMD2 promoter region are respectively underlined by dashed lines. Double underlined residues fit the consensus for a bipartite nuclear localization signal (Dingwall and Laskey, (1991) Trends Biochem. Sci. 16 . :478-481) .
  • the positions where FLAG- or MYC-epitope tag sequences were inserted are indicated by lollipops and the position where the original GAL4 -NMD2 fusion begins is indicated by an arrow with a right angle stem.
  • the bent arrow also indicates the start of the D ⁇ A sequence from nucleotide 1089 to nucleotide 3383 (SEQ ID NO: 3) encoding the carboxyl terminal amino acid sequence from amino acid 326 to amino acid 1089 (SEQ ID NO: 4) of Nmd2p, a peptide fragment which, when overexpressed, binds to Upflp and inhibits the nonsense-mediated mRNA decay pathway.
  • Figs. 2A to 2C are diagrams illustrating insertion and deletion experiments performed to assess the active regions of NMD2 gene. DNA fragments associated with NMD2 function are indicated.
  • Fig. 2A is a restriction map of the nmd2 : :HIS3 allele.
  • Fig. 2B is a restriction map of the NMD2 gene.
  • Fig. 2C is a diagram of the results of a complementation analysis to determine functional portions of ⁇ md2p.
  • Figs. 3A to 3C are representations of autoradiograms .
  • Fig. 3A is reproduced from a Southern analysis of wild type and HIS3 -disrupted NMD2 associated with NMD2 gene disruption.
  • Fig. 3B is reproduced from a Northern analysis of the stability of different nonsense- containing PGK1 alleles in NMD2 and nmd2: : HIS3 haploid yeast strains.
  • Fig. 3C is reproduced from a Northern analysis of CYH2 pre-mRNA and mRNA transcript stability. Figs.
  • FIGS. 4A to 4B are representations of Northern analysis autoradiograms which record the CYH2 transcript stability phenotypes associated with disruption of both the NMD2 and UPFl genes or overexpression of ⁇ md2p fragments.
  • Figs. 5A and 5B are a representation of the nucleic acid sequence of UPFl (SEQ ID NO: 7) .
  • Fig. 6 is a representation of the deduced amino acid sequence of Upflp (SEQ ID NO: 8) .
  • Fig. 7 is a representation of the nucleic acid sequence of UPF3 (SEQ ID NO: 9).
  • Fig. 8 is a representation of the deduced amino acid sequence of Upf3p (SEQ ID NO: 10) .
  • This invention relates to a DNA sequence, a protein, and methods useful in inhibiting the nonsense- mediated mRNA decay pathway in a cell, preferably in a yeast cell or a human cell, e.g., by stabilizing an mRNA transcript which contains a nonsense codon.
  • the nonsense codon is in a transcript destabilizing 5' portion of the transcript. Stabilization of the transcript allows increased translation and increased production of a heterologous protein of interest.
  • the protein of interest can be a full-length protein if the nonsense codon is suppressed.
  • the protein of interest can be a desired N-terminal fragment of a protein if the nonsense codon is not suppressed.
  • Inhibition of the decay of transcripts from the nonsense mutation-containing gene can ameliorate the effects of disorders caused by the presence of a nonsense codon. This can be accomplished by inhibiting a component of the nonsense-mediated decay pathway (e.g., Nmd2p, Upflp, or Upf3p) with, for example, compounds that bind to Nmd2p, compounds that interfere with the interaction between NMD2 and other molecules in the nonsense-mediated RNA decay pathway (e.g., Upflp or Upf3p) , or compounds that inhibit the expression of nonsense-mediated mRNA decay pathway genes.
  • Antisense therapy or ribozyme therapy are other methods of inhibiting the expression of components of the nonsense mediated decay pathway.
  • Antisense Constructs and Therapies Treatment regimes based on an "antisense” approach involve the design of oligonucleotides (either DNA or RNA) that are complementary to nonsense-mediated mRNA decay pathway mRNAs (e.g., transcripts from NMD2 or UPFl) . These oligonucleotides bind to the complementary mRNA transcripts and prevent translation. Absolute complementarity, although preferred, is not required.
  • a sequence "complementary" to a portion of an RNA is a sequence sufficiently complementary to be able to hybridize with the RNA, forming a stable duplex, within the environment of a cell; in the case of double-stranded antisense nucleic acids, a single strand of the duplex DNA may be tested, or triplex formation may be assayed.
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid. Generally, the longer the hybridizing nucleic acid, the more base mismatches with an RNA it may contain and still form a stable duplex (or triplex, as the case may be) .
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex.
  • the invention also encompasses nucleic acid molecules (DNA and RNA) that hybridize under stringent conditions to a nucleic acid molecule encoding a nonsense-mediated decay pathway polypeptide.
  • the cDNA sequences described herein can be used to identify these nucleic acids, which include, for example, nucleic acids that encode homologous polypeptides in other species, and splice variants of nonsense-mediated decay pathway genes (e.g., an NMD2) in humans or other mammals. Accordingly, the invention features methods of detecting and isolating these nucleic acid molecules.
  • a sample for example, a nucleic acid library, such as a cDNA or genomic library
  • an NMD2-specific probe for example, a fragment of SEQ ID NO:l that is at least 25 or 50 nucleotides long
  • the probe will selectively hybridize to nucleic acids encoding related polypeptides (or to complementary sequences thereof) .
  • the term "selectively hybridize” is used to refer to an event in which a probe binds to nucleic acids encoding a nonsense-mediated mRNA decay pathway gene such as NMD2 (or to complementary sequences thereof) to a detectably greater extent than to nucleic acids encoding other proteins (or to complementary sequences thereof) .
  • the probe which can contain at least 25 (for example, 25, 50, 100, or 200 nucleotides) can be produced using any of several standard methods (see, for example, Ausubel et al., "Current Protocols in Molecular Biology, Vol. I,” Green Publishing Associates, Inc., and John Wiley & Sons, Inc., NY, 1989).
  • the probe can be generated using PCR amplification methods in which oligonucleotide primers are used to amplify an NMD2-specific nucleic acid sequence (for example, a nucleic acid encoding the chemokine-like domain) that can be used as a probe to screen a nucleic acid library and thereby detect nucleic acid molecules (within the library) that hybridize to the probe .
  • an NMD2-specific nucleic acid sequence for example, a nucleic acid encoding the chemokine-like domain
  • One single-stranded nucleic acid is said to hybridize to another if a duplex forms between them. This occurs when one nucleic acid contains a sequence that is the reverse and complement of the other (this same arrangement gives rise to the natural interaction between the sense and antisense strands of DNA in the genome and underlies the configuration of the "double helix") . Complete complementarity between the hybridizing regions is not required in order for a duplex to form; it is only necessary that the number of paired bases is sufficient to maintain the duplex under the hybridization conditions used.
  • hybridization conditions are of low to moderate stringency. These conditions favor specific interactions between completely complementary sequences, but allow some non-specific interaction between less than perfectly matched sequences to occur as well.
  • the nucleic acids can be "washed” under moderate or high conditions of stringency to dissociate duplexes that are bound together by some non-specific interaction (the nucleic acids that form these duplexes are thus not completely complementary) .
  • the optimal conditions for washing are determined empirically, often by gradually increasing the stringency.
  • the parameters that can be changed to affect stringency include, primarily, temperature and salt concentration. In general, the lower the salt concentration and the higher the temperature, the higher the stringency. Washing can be initiated at a low temperature (for example, room temperature) using a solution containing a salt concentration that is equivalent to or lower than that of the hybridization solution. Subsequent washing can be carried out using progressively warmer solutions having the same salt concentration. As alternatives, the salt concentration can be lowered and the temperature maintained in the washing step, or the salt concentration can be lowered and the temperature increased. Additional parameters can also be altered. For example, use of a destabilizing agent, such as formamide, alters the stringency conditions.
  • nucleic acids In reactions where nucleic acids are hybridized, the conditions used to achieve a given level of stringency will vary. There is not one set of conditions, for example, that will allow duplexes to form between all nucleic acids that are 85% identical to one another; hybridization also depends on unique features of each nucleic acid.
  • the length of the sequence, the composition of the sequence (for example, the content of purine-like nucleotides versus the content of pyrimidine- like nucleotides) and the type of nucleic acid (for example, DNA or RNA) affect hybridization.
  • An additional consideration is whether one of the nucleic acids is immobilized (for example, on a filter) .
  • An example of a progression from lower to higher stringency conditions is the following, where the salt content is given as the relative abundance of SSC (a salt solution containing sodium chloride and sodium citrate; 2X SSC is 10-fold more concentrated than 0.2X SSC) .
  • Nucleic acids are hybridized at 42°C in 2X SSC/0.1% SDS (sodium dodecylsulfate; a detergent) and then washed in 0.2X SSC/0.1% SDS at room temperature (for conditions of low stringency); 0.2X SSC/0.1% SDS at 42°C (for conditions of moderate stringency); and 0. IX SSC at 68°C (for conditions of high stringency) .
  • Washing can be carried out using only one of the conditions given, or each of the conditions can be used (for example, washing for 10-15 minutes each in the order listed above) . Any or all of the washes can be repeated. As mentioned above, optimal conditions will vary and can be determined empirically.
  • a second set of conditions that are considered “stringent conditions” are those in which hybridization is carried out at 50°C in Church buffer (7% SDS, 0.5% NaHP0 4 , 1 M EDTA, 1% BSA) and washing is carried out at 50°C in 2X SSC.
  • a particular polypeptide or nucleic acid molecule is said to have a specific percent identity to a reference polypeptide or nucleic acid molecule of a defined length, the percent identity is relative to the reference polypeptide or nucleic acid molecule.
  • a peptide that is 50% identical to a reference polypeptide that is 100 amino acids long can be a 50 amino acid polypeptide that is completely identical to a 50 amino acid long portion of the reference polypeptide. It might also be a 100 amino acid long polypeptide which is 50% identical to the reference polypeptide over its entire length.
  • many other polypeptides will meet the same criteria. The same rule applies for nucleic acid molecules.
  • the length of the reference polypeptide sequence will generally be at least 16 amino acids, preferably at least 20 amino acids, more preferably at least 25 amino acids, and most preferably 35 amino acids, 50 amino acids, or 100 amino acids.
  • the length of the reference nucleic acid sequence will generally be at least 50 nucleotides, preferably at least 60 nucleotides, more preferably at least 75 nucleotides, and most preferably 100 nucleotides or 300 nucleotides.
  • non-identical positions are preferably, but not necessarily, conservative substitutions for the reference sequence.
  • Conservative substitutions typically include substitutions within the following groups: glycine and alanine; valine, isoleucine, and leucine; aspartic acid and glutamic acid; asparagine and glutamine; serine and threonine; lysine and arginine; and phenylalanine and tyrosine.
  • Sequence identity can be measured using sequence analysis software (for example, the Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, WI 53705) , with the default parameters as specified therein.
  • Oligonucleotides that are complementary to the 5' end of the message are generally most efficient for inhibiting translation.
  • sequences complementary to the 3' untranslated sequences of mRNAs have also been shown to be effective for inhibiting translation (Wagner, Nature, 372:333, 1984) .
  • oligonucleotides complementary to either the 5' or 3 ' non-translated, non-coding regions of a nonsense-mediated mR ⁇ A decay gene e.g., the human homolog of ⁇ MD2
  • Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
  • Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation, but could be used in accordance with the invention. Whether designed to hybridize to the
  • antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects, the oligonucleotide is at least 10 nucleotides, or at least 50 nucleotides in length.
  • vi tro studies are usually performed first to assess the ability of an antisense oligonucleotide to inhibit gene expression. In general, these studies utilize controls that distinguish between antisense gene inhibition and nonspecific biological effects of oligonucleotides. In these studies levels of the target RNA or protein are usually compared with that of an internal control RNA or protein. Additionally, it is envisioned that results obtained using the antisense oligonucleotide are compared with those obtained using a control oligonucleotide.
  • control oligonucleotide is of approximately the same length as the test oligonucleotide, and that the nucleotide sequence of the oligonucleotide differs from the antisense sequence no more than is necessary to prevent specific hybridization to the target sequence.
  • the oligonucleotides can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double-stranded.
  • the oligonucleotide can be modified at the base moiety, sugar moiety, or phosphate backbone, for example, to improve stability of the molecule or hybridization.
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo) , or agents facilitating transport across the cell membrane (as described, e.g., in Letsinger et al . , Proc . Nat ' l . Acad. Sci . USA 86:6553, 1989; Lemaitre et al . , Proc . Nat 'l . Acad . Sci . USA 84:648, 1987; PCT Publication No. WO 88/09810) or the blood-brain barrier (see, for example, PCT Publication No.
  • the oligonucleotide can be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent.
  • the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil , 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5- carboxymethylaminomethyl-2-thiouridine, 5-carboxymethyl- aminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2 , 2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3 -methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl- 2-thiouracil , beta-D-man
  • the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
  • the antisense oligonucleotide may also include at least one modified phosphate backbone selected from the group consisting of a phosphorothioate, a phosphorodithioate, a phosphoramidothioate , a phosphoramidate , a phosphordiamidate, a methylphosphonate , an alkyl phosphotriester, and a formacetal, or an analog of any of these backbones.
  • the antisense oligonucleotide can include an ⁇ -anomeric oligonucleotide.
  • oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al., Nucl . Acids . Res . 15:6625, 1987).
  • the oligonucleotide is a 2 ' -O-methylribonucleotide (Inoue et al . , Nucl . Acids Res . 15:6131, 1987), or a chimeric RNA-DNA analog (Inoue et al . , FEBS Lett . 215:327, 1987).
  • Antisense oligonucleotides of the invention can be synthesized by standard methods known in the art, e.g., by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
  • an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
  • phosphorothioate oligonucleotides can be synthesized by the method of Stein et al . (Nucl . Acids Res . 16:3209, 1988)
  • methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al . , Proc . Nat 'l . Acad. Sci . USA 85:7448, 1988).
  • antisense nucleotides complementary to the coding region of a nonsense-mediated mR ⁇ A decay gene could be used, those complementary to the transcribed untranslated region are most preferred. These include antisense oligonucleotides, 20-30 nucleotides in length, complementary to sequences downstream of the cap site or 5' to the initiator AUG of the respective mR ⁇ As . In yeast ⁇ MD2 mRNA, these regions include the mRNA sequences AAUGCUUAAAUAAUCUAAUAUUGUAUCUGC (SEQ ID NO: 11) and UCUGCAUUGAUAAUAUCAUUGGACAGAAAUU (SEQ ID NO: 12; He and Jacobson, Genes & Dev. 9: 437-454, 1995).
  • these regions include the sequences GGCGGCUCGGCACUGUUACCUCUCGGUCCG (SEQ ID NO: 13) and AACCGGCCCGAGGGCCCUACCCGGAGGCACC (SEQ ID NO: 14) ; Perlick et al . , (1996) Proc. Nat. Acad. Sci. USA 93:10928-10932, 1996; Applequist et al . , (1997) Nucleic Acids Res. 25:814-821).
  • the antisense molecules should be delivered to cells that express nonsense-mediated mRNA decay proteins in vivo.
  • antisense molecules can be injected directly into the tissue site, or modified antisense molecules, designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • modified antisense molecules designed to target the desired cells (e.g., antisense linked to peptides or antibodies that specifically bind receptors or antigens expressed on the target cell surface) can be administered systemically.
  • a recombinant DNA construct comprises an antisense oligonucleotide placed under the control of a strong pol III or pol II promoter.
  • RNAs that will form complementary base pairs with the endogenous nonsense-mediated mRNA decay pathway transcript and thereby prevent translation of that mRNA.
  • a vector can be introduced in vivo such that it is taken up by a cell and directs the transcription of an antisense RNA.
  • Such a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
  • Vectors can be constructed by recombinant DNA technology methods standard in the art .
  • Vectors can be plasmid, viral, or others known in the art, used for replication and expression in mammalian cells.
  • Expression of the sequence encoding the antisense RNA can be by any promoter known in the art to act in mammalian, preferably human cells.
  • Such promoters can be inducible or constitutive. Suitable promoters may include, but are not limited to: the SV40 early promoter region (Bernoist et al . , Nature 290:304, 1981); the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al .
  • Ribozyme molecules designed to catalytically cleave nonsense-mediated mRNA decay pathway mRNAs can also be used to prevent translation of these mRNAs and expression of nonsense-mediated mRNA decay pathway mRNAs (see, e.g., PCT Publication
  • WO 90/11364 Saraver et al . , Science 247:1222, 1990. While various ribozymes that cleave mRNA at site-specific recognition sequences can be used to destroy specific mRNAs, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'. The construction and production of hammerhead ribozymes is well known in the art (Haseloff et al . , Nature 334:585, 1988).
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the nonsense-mediated mR ⁇ A decay mR ⁇ A, i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mR ⁇ A transcripts.
  • ribozyme sites in a nonsense-mediated mR ⁇ A decay pathway protein include 5'-UG-3' sites which correspond to the initiator methionine codon.
  • UG- containing sequences are located throughout the yeast ⁇ MD2 mRNA, including those surrounding codon 3 (AGGAUGGACG) (SEQ ID NO: 15), codons 17-18 (CUUGGAAUGGCGAAGAA) (SEQ ID NO: 16), codon 121 (CUUUUGAGAAC) (SEQ ID NO: 17), codon 203 (UAUUGCGA) , and codon 404 (AUAUUUGGACAA) (SEQ ID NO: 18), among others.
  • the ribozymes of the present invention also include RNA endoribonucleases (hereinafter "Cech-type ribozymes”), such as the one that occurs naturally in Tetrahymena Thermophila (known as the IVS or L-19 IVS RNA) , and which has been extensively described by Cech and his collaborators (Zaug et al . , Science 224:574, 1984; Zaug et al . , Science, 231:470, 1986; Switzerland et al . , Nature 324:429, 1986; PCT Application No. WO 88/04300; and Been et al . , Cell 47:207, 1986).
  • Cech-type ribozymes such as the one that occurs naturally in Tetrahymena Thermophila (known as the IVS or L-19 IVS RNA) , and which has been extensively described by Cech and his collaborators (Zaug et al . , Science 224:574, 1984; Zau
  • the Cech-type ribozymes have an eight base-pair sequence that hybridizes to a target RNA sequence, whereafter cleavage of the target RNA takes place.
  • the invention encompasses those Cech-type ribozymes that target eight base-pair active site sequences present in nonsense-mediated mRNA decay pathway proteins.
  • the ribozymes can be composed of modified oligonucleotides (e.g., for improved stability, targeting, etc.), and should be delivered to cells which express a nonsense-mediated mRNA decay pathway gene in vivo, e.g., heart, skeletal muscle, thymus, spleen, and small intestine.
  • a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous nonsense-mediated mRNA decay pathway messages and inhibit translation.
  • the therapeutic NMD2 antisense or ribozyme nucleic acid molecule construct is preferably applied to the site of the target area (for example, a hematopoetic stem cell in the case of j ⁇ -thalassemia, delivered by injection) , but can also be applied to tissue in the vicinity of the target area or even to a blood vessel supplying the target area.
  • the target area for example, a hematopoetic stem cell in the case of j ⁇ -thalassemia, delivered by injection
  • antisense or ribozyme NMD2 expression is directed from any suitable promoter (e.g., the human cytomegalovirus, simian virus 40, or metallothionein promoters) , and its production is regulated by any desired mammalian regulatory element.
  • promoter e.g., the human cytomegalovirus, simian virus 40, or metallothionein promoters
  • enhancers known to direct preferential gene expression in hematopoetic stem cells can be used to direct antisense NMD2 expression in a patient with j ⁇ -thalassemia.
  • NMD2 antisense or ribozyme therapy is also accomplished by direct administration of an antisense NMD2 or ribozyme RNA to a target area.
  • This mRNA can be produced and isolated by any standard technique, but is most readily produced by in vi tro transcription using an antisense NMD2 DNA under the control of a high efficiency promoter (e.g., the T7 promoter).
  • Administration of antisense NMD2 RNA to target cells is carried out by any of the methods for direct nucleic acid administration described above .
  • Nonsense-mediated mRNA Decay Pathway Expression Endogenous nonsense-mediated mRNA decay can also be reduced by inactivating or "knocking out" the nonsense-mediated mRNA decay pathway gene or its promoter using targeted homologous recombination (see, e.g., U.S. Patent No. 5,464,764).
  • a mutant, non- functional NMD2 nucleic acid sequence (or a completely unrelated DNA sequence) flanked by DNA homologous to the NMD2 gene can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express NMD2 in vivo.
  • Such approaches are particularly suited for use in the agricultural field where modifications to ES (embryonic stem) cells can be used to generate animal offspring with an inactive nonsense-mediated mRNA decay.
  • this approach can be adapted for use in humans.
  • the recombinant DNA constructs may be directly administered or targeted to the pertinant cells in vivo using appropriate viral vectors.
  • endogenous nonsense-mediated mRNA decay pathway gene expression can be reduced by targeting deoxyribonucleotide sequences complementary to the regulatory region of the nonsense-mediated mRNA decay pathway gene (i.e., an NMD2 promoter and/or enhancers) to form triple helical structures that prevent transcription of an NMD2 gene in target cells in the body (Helene, Anticancer Drug Res . 6:569, 1981; Helene et al . , Ann . N. Y. Acad. Sci . 660:27, 1992; and Maher, Bioassays 14:807, 1992) .
  • deoxyribonucleotide sequences complementary to the regulatory region of the nonsense-mediated mRNA decay pathway gene i.e., an NMD2 promoter and/or enhancers
  • Vectors to be used as described above include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, or other viral vectors with the appropriate tropism for Nmd2p-expressing cells (e.g., cells with activated nonsense-mediated mRNA decay pathways) can be used as a gene transfer delivery system for a therapeutic antisense nucleic acid construct or other nucleic acid construct that inhibits expression of a nonsense-mediated mRNA decay pathway gene (e.g., NMD2) expression.
  • a therapeutic antisense nucleic acid construct or other nucleic acid construct that inhibits expression of a nonsense-mediated mRNA decay pathway gene (e.g., NMD2) expression.
  • Retroviral vectors are particularly well developed and have been used in clinical settings [Rosenberg et al . , N. Engl . J. Med 323:370, (1990); Anderson et al . , U.S. Pat. No. 5,399,346] .
  • Non-viral approaches can also be employed for the introduction of therapeutic DNA into malignant cells.
  • an antisense NMD2 nucleic acid can be introduced into a carcinoma cell by the techniques of lipofection (Feigner et al . , Proc . Natl . Acad . Sci . USA 84:7413, (1987); Ono et al . , Neurosci . Lett . 117:259, (1990); Brigham et al . , Am. J. Med. Sci . 298:278, (1989); Staubinger and Papahadjopoulos, Meth . Enz . 101:512, 1983) ; polylysine conjugation methods (Wu and Wu, J. Biol .
  • the invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
  • the examples illustrate the invention by describing the NMD2 gene, the Nmd2 protein, and its C- terminal fragment .
  • Methods of substantially inhibiting the nonsense-mediated mRNA decay pathway in a cell, and methods of producing heterologous proteins and fragments of proteins are also described. These methods can inhibit the nonsense-mediated mRNA decay pathway to increase transcript stability.
  • the nonsense-mediated mRNA decay pathway can be affected, e.g., there can be increased read-through of nonsense codon-containing mRNAs. Inhibition of the nonsense- mediated mRNA decay pathway is useful for treating disorders involving a nonsense mutation.
  • GAL4p Upstream Activating Sequence
  • the first hybrid was cloned into a plasmid (such as pMA424; (Ma, J. and Ptashne, M. (1988) Cell 5J5:443-446) in which the entire UPFl coding region was fused in-frame to the Gal4p DNA binding domain (amino acids 1-147 of Gal4p) .
  • Construction of plasmid pMA424- UPF1 was performed by a three-fragment ligation.
  • Second hybrids were encoded by S. cerevisiae genomic DNA libraries in plasmids pGAD(l-3) (Chien et al . (1991) Proc. Nat'l. Acad. Sci USA 8 . : 9578-9582) fused, in the three reading frames, to sequences encoding the Gal4p transcriptional activation domain (amino acids 768-881) . Both were cotransformed into a Saccharomyces cerevisiae strain that contained an integrated GALl -LacZ reporter construct (such as the S.
  • the GGY1::171 yeast strain was cotransformed with both pMA424- UPFl and a library containing genomic DNA fragments fused to the GAL4 activation domain. After 3-4 days of growth on SD-His-Leu plates at 30°C, His + Leu + transformants were replica-plated to SSX plates and were incubated until blue colonies appeared as described in Rose et al . (1990) Methods in Yeast Genetics: A Laboratory Course Manual , Cold Spring Harbor Laboratory, Cold Spring Harbor, NY) .
  • the isolated library plasmids were retransformed into the original GGY1::171 strain with either: 1) pMA424- UPFl , a GAL4 DNA-binding domain- UPFl fusion plasmid; 2) pMA424, the GAL4 DNA binding domain vector only; 3) pMA424 - CEP1 , a GAL4 DNA-binding domain- CEP1 fusion plasmid; or 4) pMA424-LAM5, a GAL4 DNA-binding domain-LAM5 fusion plasmid, where CEP1 and LAM5 genes are negative control genes whose gene products are known not to bind to UPFl gene product .
  • Plasmids that yielded blue colonies only with the pMA424- UPFl fusion were characterized further by restriction mapping, Southern analysis, and sequence analysis (see e.g., Sambrook et al . , (1989), Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York) . DNA sequences were compared to existing sequence databases using the FASTA program (Devereux et al . , (1984) Nucleic Acids Res. 12.:387-395) . Colonies expressing detectable ⁇ -galactosidase activity were sought by screening approximately 400,000 transformants .
  • the library plasmids from the remaining colonies were rescued and tested for specificity by retransforming them into the original strain with either: 1) the GAL4-UPF1 fusion; 2) the GAL4 DNA binding domain vector only; 3) an unrelated fusion, GAL4 -CEP1 ; or 4) an unrelated fusion, GAL4-LAM5 (Bartel et al., (1993) Biotechniques 14.: 920-924) .
  • Forty-two plasmids that yielded blue colonies only with GAL4 -UPF1 fusion plasmid-containing strains were characterized further by restriction mapping, Southern analysis, and partial DNA sequence analysis using standard techniques (see e.g., Sambrook et al . , 1989, supra .
  • CEP1 was obtained from Richard Baker of the University of Massachusetts Medical Center, Worcester, MA; pMA424-LAW5 was obtained from Stanley Fields and Paul Bartel of State University of New York, Stony Brook, N.Y.) .
  • Individual Leu + His + transformants were selected and streaked on synthetic medium plates lacking histidine and leucine.
  • -galactosidase activity assays were performed by replica-plating the transformants onto SSX plates containing X-Gal. Cells were incubated at 30°C for 24-48 hours for development of blue color.
  • Southern blot analysis of the isolated plasmids was performed by first extracting total yeast genomic DNA according to the method of Holm et al . (1986) Gene 42.: 169-173. After restriction digestion, DNA was electrophoresed on 0.8% agarose gels, transferred and cross-linked to Zetaprobe membranes (BioRad, Richmond, CA) as described in Sambrook et al . (1989), supra . Filters were prehybridized for 2-3 hours at 42°C in 5X SSPE, 40% formamide, 5X Denhardt's solution, 0.1% SDS, and 4 mg/ml salmon sperm DNA.
  • a radiolabeled NMD2 probe (1.2 kb Clal-EcoRI fragment), generated by random priming, was added and filters were hybridized overnight at 42°C. Filters were washed twice in IX SSC, 0.1% SDS at room temperature and once in 0. IX SSC, 0.1% SDS at 58°C before analyzing on a Betagen Blot Analyzer
  • DNA sequences were determined by the method of Sanger et al . , (1978) Proc. Nat'l. Acad. Sci. USA 24:5463-5467. Overlapping fragments of the NMD2 gene were subcloned in Bluescript and sequenced by annealing oligonucleotide primers specific to the T3 or T7 promoter regions of the plasmid or by using oligonucleotide primers which annealed within the subcloned inserts.
  • Nine different genes were isolated by the following procedure. An S. cerevisiae genomic DNA library of Sau3A partial fragments constructed in YCp50 was used (Rose et al . (1987) Gene j50.:237-243) .
  • ⁇ md2p showed a specific dependency on Upflp in the two- hybrid system.
  • Cells expressing a GAL4 activation domain-N D2 fusion demonstrated strong -galactosidase activity when simultaneously expressing a GAL4 D ⁇ A- binding domain- UPFl fusion, but had no detectable ⁇ - galactosidase activity when co-transformed with plasmids encoding only the GAL4 D ⁇ A-binding domain-LAM5 fusion.
  • Further evidence for the specificity of the interaction (s) was obtained by analyzing the effects of specific deletions within the UPFl portion of the GAL4 D ⁇ A-binding domain- LTPFl fusion. Deletions in all but one segment of the UPFl coding region eliminated ⁇ md2p-Upflp interaction in the two-hybrid assay.
  • the GAL4 activation domain -NMD2 plasmid recovered in the two hybrid screen contained only a fragment of the NMD2 gene.
  • a 1.2 kb Clal- EcoRI fragment downstream of the GAL4 activation domain in the fusion plasmid was used to screen a yeast YCp50 genomic D ⁇ A library (Rose et al . , (1987) supra) . Two independent clones with identical restriction patterns were isolated.
  • restriction mapping, Southern analysis, and subsequent testing for complementation of an NMD2 chromosomal deletion the NMD2 gene was localized to a 5.2 kb Xbal -Sail D ⁇ A fragment as shown in Figs. 2A to 2C.
  • FIG. 2A A restriction map of the nmd2 : : HIS3 allele is shown in Fig. 2A.
  • the left arrow in Fig. 2A represents the HIS3 gene and indicates the direction of transcription.
  • the right arrow of Fig. 2A represents the NMD2 open reading frame.
  • a restriction map of the ⁇ MD2 gene is shown in Fig. 2B.
  • the NMD2 open reading frame and direction of transcription are indicated by an open arrow interrupted by a stippled box that indicates the position of the intron.
  • the box labeled probe indicates the DNA fragment used for screening the genomic DNA library.
  • the black box represents a segment from the cloning vector YCp50 and the restriction site abbreviations are: B, BamHI ; C, Clal; E, EcoRI; H, Hindlll; P, PstI; S, Sail; Xb, Xbal.
  • Fig. 2C lines represent DNA fragments which were subcloned into an appropriate vector (such as pRS315) . These constructs were transformed into the yeast strain HFY1300, or equivalent, which contains a partial chromosomal deletion of NMD2 and lacks nonsense- mediated mRNA decay activity (see also, Figs. 3A and 3B) . Total RNA was isolated from these transformants and Northern analysis was performed using a radiolabeled probe derived from the CYH2 gene (He et al . , (1993) Proc.
  • the 1.7 kb Xbal -Clal fragment was used to probe PrimeClone blots (American Type Culture Collection, Rockville, MD) containing characterized fragments of most of the S. cerevisiae genome (ATCC accession number 7155) known to lie on the right arm of chromosome VIII (Riles et al., (1993) Genetics 134 . : 81-150) .
  • This fragment is located between the put2 and CUP1 loci at a map position approximately 260 kb from the left telomere (Riles et al. , (1993) supra) .
  • the complete sequence of the NMD2 gene was determined (SEQ ID ⁇ O:l) .
  • This interpretation of the NMD2 sequence relies on the prediction of a 113 -nucleotide intervening sequence that commences at position +7 and divides the gene into two exons (Figs. 1A-1C) . Four observations support the existence of this intron.
  • the sequence contains all three of the standard consensus sequences expected of an intron (5' splice site [GUAUGU] , branchpoint [UACUAAC] , and 3' splice site [AG] ) (Figs. 1A-1C) .
  • this intron is located at the 5' end of the NMD2 gene (six nucleotides downstream from the predicted initiator ATG; Figs. 1A-1C) .
  • the expression of a 127 kD polypeptide was detected when the FLAG or c-MYC sequences were inserted adjacent to the putative initiator ATG (FLAG-2 -N D2 or C-MYC-NMD2 alleles) , but not when the FLAG sequence was inserted adjacent to the second ATG (FLAG-1-NMD2 allele) .
  • the second ATG is located within the putative intron, 37 nucleotides downstream of the predicted intron branchpoint, and is in frame with the major downstream open reading frame but not with the first ATG.
  • NMD2 transcript was consistent with the predicted open reading frame.
  • a putative TATA box required for most R ⁇ A polymerase II transcription (Struhl (1987) Cell.
  • Structural features of the NMD2 protein inferred from the sequence analysis include a highly acidic internal fragment (36.8% aspartic acid and 25.6% glutamic acid) from residues 843 to 975 near the C-terminus and a possible bipartite nuclear localization signal at the N-terminus of the protein (i.e., within residues 26 to 29 and 42 to 46) (Figs. 1A- 1C; Dingwall and Laskey, (1991) supra) .
  • Comparison of the Nmd2p sequence with those in the Swissprot and Pir protein sequence databases using the FASTA or TFASTA comparison programs (Devereux et al .
  • Nmd2p three domains of Nmd2p have substantial similarity to regions of other proteins.
  • the first domain spanning Nmd2p amino acids 1 to 390, has 17.7% sequence identity and 47% similarity with translational elongation factor 2 (Eftlp and Eft2p) from S. cerevisiae (Perentesis et al . , (1992) J. Biol. Chem. 267 :1190-1197) .
  • the second domain from amino acids 400 to 810 in Nmd2p, shares 19.5% sequence identity and 42.6% similarity with the S. cerevisiae mitochondrial RNase P protein Rpm2p (Dang and Martin (1993) J. Biol. Chem. 26£:19791-19796) .
  • the third domain encompassing the acidic stretch from amino acids 820 to 940, has 34% sequence identity and 63.2% similarity with human and mouse nucleoproteins (Lapeyre et al . , (1987) Proc. Natl . Acad. Sci. 84:1472- 1476; Bourbon et al . , (1988) J. Mol. Biol. 200:27-638) and 34% identity and 65% similarity to the mammalian polymerase I transcriptional factors hUBF and mUBF (Jantzen et al . , (1990) Nature 3_4_4: 830-836 ; Hisatake et al., (1991) Nucleic Acids Res. 19:4631-4637). In hUBF and mUBF this domain has been shown to be important for interaction with other proteins (Jantzen et al . , (1990) supra) and, as described below, is also true for Nmd2p.
  • Example 4 NMD2 Disruption Does Not Affect Cell Viability and Selectively Stabilizes Nonsense-containing mRNAs
  • NMD2 gene disruption experiment was performed to assess the cellular requirement for ⁇ md2p.
  • the nmd2 : : HIS3 disruption described in Fig. 2A was constructed.
  • Plasmid Bs-nmd2 : :HIS3 encodes the same NMD2 disruption and contains a 0.6 kb Clal-Xbal fragment in the 5' -end of NMD2 , a 1.7 kb Xbal-Clal fragment of HIS3 and a 1.2 kb Clal -EcoRI fragment in the NMD2 coding region in Bluescript .
  • a Sacl - Sall fragment carrying the nmd2 : :HIS3 allele was isolated from plasmid Bs-nmd2 : :HIS3 and used to transform the yeast diploid strain W303 for homologous recombination into one of the NMD2 alleles. His + transformants were sporulated and tetrads were individually dissected. Four viable spores were obtained from each tetrad analyzed. Genomic DNAs from parental diploid and progeny haploid strains were isolated, digested with EcoRI . Confirmation of integration is shown by the Southern analysis of Fig.
  • lane PI contains DNA isolated from the homozygous NMD2/NMD2 diploid strain W303; lane P2 contains DNA isolated from a diploid nmd2 : -. HIS3/NMD2 His + transformant of W303
  • lanes 1A to ID contain DNA isolated from the progeny of four viable spores dissected from the same tetrad represent the wild-type and disrupted alleles of NMD2, respectively. Other bands in the figure are not specific to NMD2.
  • Haploid strains containing the nmd2 : :HIS3 disruption were compared to isogenic NMD2 strains for their ability to grow on different carbon sources (glucose, galactose, and glycerol) at temperatures ranging from 18°C to 37°C and no differences in growth rates were detected between mutant and wild-type strains. These data indicate that NMD2 is non-essential for cell viability. Since disruption of the NMD2 gene was not lethal, the activities of the nonsense-mediated mR ⁇ A decay pathway in both NMD2 and nmd2 : :HIS3 strains were compared.
  • Yeast centromere plasmids carrying six different PGKl nonsense alleles were constructed previously (Peltz et al . , (1993) supra) . These plasmids were transformed into NMD2 and nmd2 : :HIS3 strains and the abundance of PGKl nonsense-containing mRNAs was assessed by Northern analysis as shown in Fig. 3B. Disruption of the NMD2 gene stabilizes PGKl mR ⁇ As containing early nonsense mutations.
  • Total R ⁇ A was isolated from these strains and analyzed by Northern blotting using a radiolabeled oligonucleotide probe complementary to the tag sequence located in the 3' untranslated region of PGKl nonsense- containing mRNAs (Peltz et al . , (1993) Genes & Dev. supra) .
  • the location of the nonsense mutation in each PGKl transcript is presented as a percentage of the PGKl protein-coding region that is translated before the mutation is encountered (Peltz et al . , (1993) supra) . Decay rates of mRNA were measured as previously described (Herrick et al . , (1990) supra ; Parker et al . ,
  • Nonsense mutations in the 5' two-thirds of the PGKl coding region reduced the abundance of the corresponding mRNAs 5- to 20-fold (Peltz et al . , (1993) supra) .
  • the abundance of PGKl mRNAs with nonsense mutations in the downstream third of the coding region is unaffected.
  • Disruption of the .NMD2 gene restored wild- type levels to all four of the PGKl transcripts normally subject to nonsense-mediated mRNA decay (Fig. 3B) .
  • Strain HFY2000 was transformed with pRS315 (or similar yeast shuttle plasmid; (Sikorski and Hieter, (1989) Genetics 122:19-27) or pRS315 -NMD2 (X-S) (containing a 5.2 kb Xbal-Sall fragment of NMD2 in pRS315) and a plasmid harboring a PGKl allele with a nonsense mutation at the Bgrlll site (Peltz et al . , (1993) supra) .
  • the region of Nmd2p that interacts with Upflp was determined by generating 5' and 3' deletions of the original NMD2 fragment, fusing them in-frame to the GAL4 activation domain, and assaying the resultant constructs for interaction with Upflp using the two-hybrid system. Fusions encoding either 237 or 477 amino acids from the amino-terminus of the original fragment demonstrated no detectable -galactosidase activity. However, fusions encoding either 526 or 286 amino acids from the carboxyl- terminus of the original fragment did demonstrate detectable -galactosidase activity. These results indicate that the acidic C-terminal domain of ⁇ md2p interacts with Upflp.
  • Nmd2p as a Upflp-interacting protein in a two-hybrid screen and the observation that disruption of the NMD2 gene yielded a nonsense-mediated mR ⁇ A decay phenotype equivalent to that obtained in strains harboring upfl mutations suggests that Upflp and ⁇ md2p interact with each other in vivo and that they perform different functions in the same decay pathway.
  • This conclusion is strengthened by the finding that double mutants in which both the UPFl and NMD2 gene products are functionally absent produce strains that have essentially identical phenotypes with regard to the half-lives of test mR ⁇ A transcripts such as CYH2 pre- mR ⁇ A.
  • Upflp and ⁇ md2p must function in closely related steps of the nonsense-mediated mRNA decay pathway.
  • Nmd2p A truncated form of Nmd2p was expressed in both the nucleus and cytoplasm and activity was functionally localized within the cell to the cytoplasm.
  • the original GAL4 activation domain-N D2 fusion plasmid encodes 764 amino acids of the C-terminal segment of ⁇ md2p (SEQ ID NO: 1
  • HFY1200 was derived from W303 by standard techniques (see, e.g., Rothstein, R. (1991) "Targeting, disruption, replacement, and allele rescue: integrative DNA transformation in yeast . " , in Methods in Enzvmology 194: Guide to Yeast Genetics and Molecular Biology, C. Guthrie and G. Fink, eds . , Academic Press, pp. 281-301).
  • RNAs were isolated from each of the following strains: HFY3002 ( UPF1/NMD2) ; HFY3005 ( upflA /NMD2) ; HFY3008 ( UPFl/nmd2A ) and HFY3001 (upflA/nmd ⁇ ) (see Table 2) .
  • R ⁇ As were analyzed by Northern blotting using a radiolabeled CYH2 fragment as probe.
  • HFY2206 Same as HFY2000 but containing [pRS315-NMD2 (X-S)] [pRIPPGKBgl UAG]
  • HFY3008 Same as HFY2000 but containing [pRS315] [pRS314- UPFl] The strains listed in Table 2 were prepared in this study. See Peltz et al . (1993), supra , for a description of the "pRIPPGK " plasmids listed above.
  • truncated Nmd2p in the cytoplasm results in a dominant-negative nonsense-mediated mRNA decay phenotype as shown in Fig. 4B.
  • the yeast strain HFY1200 which is wild-type for both UPFl and NMD2 was transformed with pGAD2F-NMD2-ADHt , pGAD2F-NMD2-ADHp , pGAD2F, pGAD2F-NMD2-ADHt- ⁇ LS , pGAD2F-.NMD2-ADHp- ⁇ NLS, respectively (see Table 3) .
  • Total RNA was isolated from these transformants and analyzed by Northern blotting using a CYH2 DNA fragment as probe.
  • Lane 1 contained RNA isolated from HFY1300 (control) ; RNA in other lanes was from transformants of HFY1200 harboring the following plasmids; lane 2, pGAD2F-N D2-ADHt ; lane 3, pGAD2F-NMD2- ADHp; lane 4, pGAD2F; lane 5, pGAD2F-. ⁇ MD2-ADHt- ⁇ LS; lane 6, pGAD2F-NMD2-ADHp- ⁇ NLS . Overexpression of truncated
  • NMD2 fusion protein localized to the nucleus had no effect on the accumulation of the CYH2 pre-mR ⁇ A (Fig. 4B, lanes 2 and 3) .
  • Expression of the cytoplasmically localized fusion protein caused an accumulation of CYH2 pre-mR ⁇ A in a dosage dependent manner, i.e., expression of the fusion protein from the stronger promoter led to a greater accumulation of the CYH2 pre-mR ⁇ A than expression from the weaker promoter (Fig. 4B, lanes 5 and 6) .
  • Nonsense-mediated mRNA decay pathway function cf a host cell i.e., a prokaryotic or eukaryotic cell such as a yeast cell
  • a host cell i.e., a prokaryotic or eukaryotic cell such as a yeast cell
  • the antisense oligonucleotide can be directly introduced into the cells in a form that is capable of binding to the NMD2 sense transcripts.
  • a vector containing se ⁇ uence which, once within the host cells, is transcribed into the appropriate antisense mRNA can be the species administered to the cells .
  • An antisense nucleic acid that hybridizes to the mRNA cf the target gene can decrease or inhibit production of the polypeptide product encoded by the gene by forming a double-stranded segment on the normally single-stranded mRNA transcript, thereby interfering with translation. It may be preferable to select sequences for antisense applications that do not contain nonsense codons as these may stimulate rather than inhibit the nonsense-mediated mRNA decay pathway.
  • the sequence can be operably linked to appropriate expression control sequences and introduced into host cells by standard techniques well known to those of ordinary skill in the art.
  • An effective amount of the expressed antisense transcript is produced such that translation of the NMD2 sense mRNA transcript is inhibited.
  • UPFl mRNA antisense transcript or a fragment thereof which binds to the UPFl sense transcript inhibiting translation and thereby, inhibiting the nonsense-mediated mRNA pathway.
  • Antisense transcript production can be constitutive or controlled, as desired, according to the transcription regulatory sequences operably linked to the .NMD2 or UPFl DNA sequences for the production of antisense transcript.
  • Inhibition of the nonsense-mediated mRNA pathway using antisense transcripts to inhibit translation of a protein of the pathway is useful to enhance the stability of a nonsense codon-containing transcript which encodes a heterologous polypeptide to be produced in yeast cells or to enhance the production of a mutated endogenous polypeptides useful to the host cell or host organism.
  • Antisense transcripts are also useful for treating genetic disorders involving a nonsense mutation.
  • a vector able to express antisense transcripts for a gene in the nonsense-mediated mRNA decay pathway e.g., NMD2 or UPFl
  • NMD2 or UPFl a gene in the nonsense-mediated mRNA decay pathway
  • Example 7 Production of Heterologous Protein or Polypeptide in a Yeast Cell Inhibited in the Nonsense-Mediated mRNA Pathway
  • a protein or polypeptide of interest is produced by providing an expression vector encoding a gene for a heterologous protein.
  • the expressed transcript of the gene encodes a nonsense codon in a transcript destabilizing 5' portion of the transcript such that the transcript is at least 2 fold less stable in a wild-type strain than in a nonsense-mediated mRNA decay-inhibited host strain.
  • Nonsense-mediated mRNA decay is inhibited by 1) mutating the NMD2 gene such that no functional ⁇ md2p is produced; 2) overexpressing a C-terminal fragment of Nmd2p such that the fragment binds to UPFl inhibiting its function; or 3) expressing sufficient NMD2 or UPFl antisense transcript to hybridize to NMD2 or UPFl sense transcript preventing its translation into functional ⁇ md2p or Upflp, respectively. All of these methods can be carried out by standard procedures .
  • the host strain used is also an amino acid substitution suppressor strain.
  • the suppressor strain is chosen such that a specific amino acid (dictated by the type of suppressor mutation in the host strain) is substituted at the nonsense codon.
  • the substituted amino acid can be an amino acid encoded by the natural codon at that site.
  • the substituted amino acid can be different from the naturally encoded amino acid if it is desired to test the effect of that amino acid on the conformation or activity of the encoded protein.
  • heterologous protein to be expressed is toxic to the host cell
  • inhibition of the nonsense- mediated mRNA decay pathway can be controlled by the inducible expression of, for example, Nmd2p C-terminal fragment or NMD2 antisense transcript .
  • Controllable inhibition of the decay pathway allows transcript stabilization and translation a point in the host yeast cell culture growth such that maximum production of the toxic protein occurs prior to the death of the host cells.
  • the protein or fragment is isolated from the yeast host cells by standard protein purification methods.
  • Example 8 Production of Antibody to ⁇ md2p or a C-terminal Fragment of Nmd2p
  • Nmd2p or Nmd2p C-terminal fragment polypeptide of the invention can be produced by first transforming a suitable host cell with the entire NMD2 gene (for the production of Nmd2p) or with a partial NMD2 sequence (encoding the C-terminal part of Nmd2p) , respectively, cloned into a suitable expression vehicle followed by expression of the desired protein or polypeptide.
  • the precise host cell used is not critical to the invention.
  • the polypeptide can be produced in a prokaryotic host (e.g., E. coli) or in a eukaryotic host (e.g., Saccharomyces cerevisiae) .
  • the method of transformation of the cells and the choice of expression vehicle will depend on the host system selected. Methods described herein provide sufficient guidance to successfully carry out the production, purification and identification of Nmd2p or THE Nmd2p C-terminal fragment.
  • Nmd2p or Nmd2p C-terminal fragment (or fragment or analog thereof) is expressed, it is isolated, e.g., using immunoaffinity chromatography.
  • an anti-Nmd2p or anti- (Nmd2p C-terminal fragment) antibody can be attached to a column and used to isolate Nmd2p or Nmd2p C-terminal fragment, respectively. Lysis and fractionation of Nmd2p or Nmd2p C-terminal fragment-containing host cells prior to affinity chromatography can be performed by standard methods.
  • the recombinant protein can, if desired, be further purified, e.g, by high performance liquid chromatography (see e.g., Fisher, Laboratory Techniques In Biochemistry and Molecular Biology, eds., Work and Burdon, Elsevier, (1980) .
  • Nmd2p or fragments thereof, particularly short fragments which inhibit the nonsense-mediated mRNA decay pathway can also be produced by chemical synthesis, by standard solution or solid phase peptide synthesis techniques .
  • Substantially pure Nmd2p or Nmd2p C-terminal fragment can be used to raise antibodies.
  • the antibodies are useful for screening, by Western blot analysis, host stains overexpressing Nmd2p or Nmd2p C-terminal fragment, thereby identifying candidate strains which produce a desired amount of Nmd2p or Nmd2p C-terminal fragment.
  • Antibodies directed to the polypeptide or interest, Nmd2p or NMd2p C-terminal fragment, are produced as follows. Peptides corresponding to all or part of the polypeptide of interest are produced using a peptide synthesizer by standard techniques, or are isolated and purified as described above. The peptides are coupled to KLH with M-maleimide benzoic acid N- hydroxysuccinimide ester. The KLH-peptide is mixed with Freund's adjuvant and injected into animals, e.g., guinea pigs or goats, to produce polyclonal antibodies.
  • Monoclonal antibodies can be prepared using the polypeptide of interest described above and standard hybridoma technology (see, e.g., Kohler et al . , Nature (1975) 256: 495; Kohler et al . , Eur. J. Immunol. (1976) .6:292, Kohler et al . , Eur. J. Immunol. (1976) 6 . : 511; Hammerling et al . , in Monoclonal Antibodies and T Cell Hybridomas, Elsevier, NY, (1981) , which are incorporated herein by reference) . Antibodies are purified by peptide antigen affinity chromatography.
  • antibodies are tested for specific Nmd2p or Nmd2p C-terminal fragment binding by Western blot or immunoprecipitation analysis by standard techniques.
  • chimeric antibodies In addition, techniques developed for the production of "chimeric antibodies" (Morrison et al . , Proc . Natl . Acad. Sci . USA, 81:6851, 1984; Neuberger et al., Nature, 312-604, 1984; Takeda et al . , Nature, 314:452, 1984) can be used. These methods involve splicing the genes from a mouse antibody molecule of appropriate antigen specificity together with genes from a human antibody molecule of appropriate biological activity.
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine having a variable region derived from a murine mAb and a human immunoglobulin constant region.
  • comparative genomics are used to identify murine homologs of NMD2.
  • the sequence of the complete yeast NMD2 protein is compared to existing databases of random cDNA sequences (Bassett, D.J., et al., Trends Genet . 1:4372-373, 1995). Fragments with significant homology to NMD2 are used as nucleic acid probes in subsequent screens of murine genomic DNA and cDNA libraries as described herein. Full-length genes and cDNAs having substantial homology to NMD2 are then further characterized as described herein.
  • Two-hybrid screens can also be used to identify murine homologs of NMD2.
  • Mouse genes encloding proteins that interact with yeast UPFl or UPF3 proteins He et al., Mol . Cell . Biol . 17:1580-1594, 1997), or human homologs of the yeast UPFl protein (Perlick et al . , Proc. Natl. Acad. Sci. USA 93:10928-10932, 1996; Applequist et al., Nucleic Acids Res. 25:814-821, 1997), are identified using the two-hybrid method (Fields and Song, Nature 340:245-246, 1989; Chien et al . , Proc. Natl. Acad.
  • DNA encoding the UPF protein is cloned and expressed from plasmids harboring GAL4 or lexA DNA-binding domains and co-transformed into cells harboring lacZ and HIS3 reporter constructs along with libraries of cDNAs that have been cloned into plasmids harboring the GAL4 activation domain. Libraries used for such co-transformation include those made from B-cells and T-cells since such cells may have high activities of the nonsense-mediated mRNA decay pathway.
  • Another method for identifying murine homologs of NMD2 utilize complementation of yeast NMD2 mutants.
  • Yeast UPFl mutants incapable of nonsence-mediated mRNA decay suppress the growth defects of cells harboring nonsense mutations in the LEU2 or TYR7 genes (Leeds et al., Mol. Cell. Biol. 12:2165, 1992; Peltz et al . , Prog. Nucleic Acids Res. Molec. Biol. 47:271-298, 1994). Comparable effects are observed with NMD2 mutants, or in cells harboring the NMD2 dominant-negative fragment.
  • NMD2 mutants or cells harboring the NMD2 dominant-negative fragment, are transformed with mammalian cDNA libraries cloned into yeast plasmid vectors. The transformants are analyzed for restoration of the wild-type pattern, i.e., failure to grow in the absence of leucine or tyrosine.
  • the transformed cells are screened for their ability to grow in the presence of 100 ⁇ g/ml of the growth inhibitor canavanine.
  • This drug enters cells via arginine permease, a protein encoded by the CANl gene.
  • Cells harboring the canl-nonsense mutation are resistant to 100 ⁇ g/ml canavanine if they are wild-type for nonsense-mediated mRNA decay, but sensitive to it if they also harbor an NMD2 mutation. Hence, restoration of NMD2 function by exogenous DNA is assessed by plating cells on canavanine.
  • Plasmids are isolated from the cells surviving on canavanine, the plasmids are sequenced, and the sequences analyzed to confirm murine sequence that complements mutant nmd2 (e.g., the cell is not a revertant) .
  • PCR with degernate oligonucleotides is also a method of identifying murine NMD2 homologs.
  • Homologs of the NMD2 gene are identified in other, non-murine, species are compared to identify specific regions with a high degree of homology. These regions of high homology are selected for the design of PCR primers that maximize possible base-pairing with heterlogous genes. Construction of such primers involves the use of oligonucleotide mixtures that account for degeneracy in the genetic code, i.e., allow for the possible base changes in murine NMD2 genes that do not affect the amino acid sequence of the NMD2 protein. Such primers are used to amplify and clone possible NMD2 gene fragments from mouse DNA.
  • yeast NMD2 protein sequenced and those encoding protein fragments with high degrees of homology to fragments of yeast NMD2 protein are used as nucleic acid probes in subsequent screens of murine genomic DNA and cDNA libraries. Full-length genes and cDNAs having substantial homology to yeast NMD2 are identified in these screens.
  • Example 10 Identification of Human Homologs of Yeast NMD2
  • the human homolog of the yeast N D2 gene is useful for the elucidation of the biochemical pathways of nonsense-mediated mR ⁇ A decay in mammals as well as for the development of treatments for genetic disorder involving nonsense mutations.
  • Several approaches can be used to isolate human NMD2 genes including a two-hybrid screen, complementation of yeast ⁇ MD2 mutants by expression libraries of cloned human cDNAs, polymerase chain reactions (PCR) primed with degenerate oligonucleotides, low stringency hybridization screens of human libraries with the yeast NMD2 gene, and database screens for homologous sequences .
  • PCR polymerase chain reactions
  • the human NMD2 gene can also be identified by appropriate application of the above methods based on homology with the mouse NMD2 gene homolog. Methods of screening for and identifying human homologs of ⁇ MD2 are described above (e.g., Example 8).
  • the murine homolog of NMD2 can be used instead of the yeast Nmd2 sequence to probe a human cDNA or genomic DNA library for homologous sequences.
  • the human NMD2 gene product e.g., human Nmd2p
  • the human NMD2 gene is placed in an expression vector and the gene expressed in an appropriate cell type. Human Nmd2p is isolated from such cell lines using methods known to those in the art, and used in the assays described below.
  • Example 11 Methods of Screening for Molecules that Inhibit the Nonsense-mediated mRNA Decay Pathway
  • the following assays are designed to identify compounds that are effective inhibitors of the nonsense- mediated mRNA decay pathway.
  • Such inhibitors may act by, but are not limited to, binding to an Ndm2p (e.g., from yeast, mouse or human) , binding to intracellular proteins that bind to an Nmd2p, compounds that interfere with the interaction between Nmd2p and nonsense mutation- containing mRNA, compounds that modulate the activity of an NMD2 gene, or modulate the expression of an NMD2 gene or an Nmd2p.
  • Assays can also be used to identify molecules that bind to nonsense-mediated mRNA decay pathway gene regulatory sequences (e.g., promoter sequences), thus modulating gene expression. See e.g., Platt, 1994. J “ . Biol . Chem. 269:28558-28562, incorporated herein in its entirety.
  • the compounds which may be screened by the methods described herein include, but are not limited to, peptides and other organic compounds (e.g., peptidomimetics) that bind to a nonsense-mediated mRNA decay pathway protein (e.g., that bind ton an Nmd2p) , or inhibit its activity in any way.
  • Such compounds may include, but ar not limited to, peptides; for example, soluble peptides, including but not limited to members of random peptide libraries; (see, e.g., Lam et al . , 1991. Nature 354:82-94; Houghten et al . , 1991. Nature 354:84-86), and combinatorial chemistry-derived molecular libraries made of D-and/or L- amino acids, phosphopeptides (including, but not limited to, members of random or partially degenerate, directed phosphopeptide libraries; see e.g., Songyang et al . , 1993. Cell 72:767-778) , and small organic or inorganic molecules .
  • peptides for example, soluble peptides, including but not limited to members of random peptide libraries; (see, e.g., Lam et al . , 1991. Nature 354:82-94; Houghten et al . , 1991. Nature 3
  • Organic molecules are screened to identify candidate molecules that affect expression of a nonsense- mediated mR ⁇ A decay (e.g., ⁇ MD2) gene or some other gene involved in the nonsense-mediated mRNA decay pathway (e.g., by interacting with the regulatory region or transcription factors of a gene) .
  • Compounds are also screened to identify ones that affect the activity of such proteins, (e.g., by inhibiting Nmd2p activity) or the activity of a molecule involved in the regulation of, for example, Nmd2p.
  • Nmd2p compounds likely to interact with the active site of a protein
  • the active site of an Nmd2p molecule can be identified using methods known in the art including, for example, analysis of the amino acid sequence of a molecule, from a study of complexes of Nmd2p, with its native ligand (e.g., Upflp).
  • Chemical or X-ray crystallographic methods can be used to identify the active site of Nmdl2p by the location of a bound ligand such as Upflp.
  • the three-dimensional structure of the active site is determined. This can be done using known methods, including X-ray crystallography which may be used to determine a complete molecular structure. Solid or liquid phase NMR can be used to determine certain intra- molecular distances. Other methods of structural analysis can be used to determine partial or complete geometrical structures . Geometric structure can be determined with an Nmd2p bound to a natural (e.g., Upflp) or artificial ligand which may provide a more accurate active site structure determination.
  • a natural e.g., Upflp
  • artificial ligand which may provide a more accurate active site structure determination.
  • Modeling methods that may be used are, for example, parameterized models specific to particular biopolymers such as proteins or nucleic acids, molecular dynamics models based on computing molecular motions, statistical mechanics models based on thermal ensembles, or combined models.
  • standard molecular force fields representing the forces between constituent atoms and groups are necessary, and can be selected from force field known in physical chemistry.
  • Information on incomplete or less accurate structures determined as above can be incorporated as constraints on the structures computed by these modeling methods.
  • candidate modulating compounds can be identified by searching databases containing compounds along with information on their molecular structure.
  • the compounds identified in such a search are those that have structures that match the active site structure, fit into the active site, or interact with groups defining the active site.
  • the compounds identified by the search are potential nonsense-mediated mRNA decay pathway modulating compounds .
  • These methods may also be used to identify improved modulating compounds from an already known modulating compound or ligands.
  • the structure of the known compound is modified and effects are determined using experimental and computer modelling methods as described above.
  • the altered structure may be compared to the active site structure of a nonsense-mediated mRNA decay protein (e.g., an Nmd2p) to determine or predict how a particular modification to the ligand or modulating compound will affect its interaction with that protein.
  • Systematic variations in composition such as by varying side groups, can be evaluated to obtain modified modulating compounds or ligands of preferred specificity or activity.
  • Examples of molecular modelling systems are the QUANTA programs, e.g., CHARMm, MCSS/HOOK, and X-LIGAND, (Molecular Simulations, Inc., San Diego, CA) .
  • QUANTA analyzes the construction, graphic modelling, and analysis of molecular structure.
  • CHARMm analyzes energy minimization and molecular dynamics functions.
  • MCSS/HOOK characterizes the ability of an active site to bind a ligand using energetics calculated via CHARMm.
  • X-LIGAND fits ligand molecules to electron density of protein- ligand complexes. It also allows interactive construction, modification, visualization, and analysis of the behavior of molecules with each other. Articles reviewing computer modelling of compounds interacting with specific protein can provide additional guidance.
  • libraries of known compounds including natural products, synthetic chemicals, and biologically active materials including peptides, can be screened for compounds that are inhibitors or activators of the nonsense-mediated mRNA decay pathway.
  • Example 12 In vi tro Screening Assays for Compounds that Bind to Nonsense-mediated Decay Proteins and Genes
  • vi tro systems can be used to identify compounds that interact with (e.g., bind to) nonsense-mediated decay pathway proteins or genes encoding those proteins (e.g., a UPFl, UPF3 , or NMD2 gene). Such compounds are useful, for example, for modulating the activity of these entities, elaborating their biochemistry, or treating disorders involving nonsense mutations. These compounds can be used in screens for compounds that disrupt normal function, or may themselves disrupt normal function.
  • Assays to identify compounds that bind nonsense- mediated decay pathway proteins involve preparation of a reaction mixture of the protein and the test compound under conditions sufficient to allow the two components to interact and bind, thus forming a complex which can be removed and/or detected.
  • Screening assays can be performed using a number of methods.
  • a nonsense-mediated RNA decay pathway protein from an organism e.g., UPFl , NMD2, UPF3 , NMD3 , or DBP2 protein
  • peptide, or fusion protein can be immobilized onto a solid phase, reacted with the test compound, and complexes detected by direct or indirect labeling of the test compound.
  • the test compound can be immobilized, reacted with the nonsense-mediated decay pathway molecule, and the complexes detected.
  • Microtiter plates may be used as the solid phase and the immobilized component anchored by covalent or noncovalent interactions.
  • Non-covalent attachment may be achieved by coating the solid phase with a solution containing the molecule and drying.
  • an antibody for example, one specific for NMD2 or UPFl , is used to anchor the molecule to the solid surface.
  • Such surfaces may be prepared in advance of use, and stored.
  • the non-immobilized component is added to the coated surface containing the immobilized component under conditions sufficient to permit interaction between the two components.
  • the unreacted components are then removed (e.g., by washing) under conditions such that any complexes formed will remain immobilized on the solid phase.
  • the detection of the complexes may be accomplished by a number of methods known to those in the art.
  • the nonimmobilized component of the assay may be prelabeled with a radioactive or enzymatic entity and detected using appropriate means. If the non-immobilized entity was not prelabeled, an indirect method is used. For example, if the non-immobilized entity is an Nmd2p, an antibody against the Nmd2p is used to detect the bound molecule, and a secondary, labeled antibody used to detect the entire complex.
  • a reaction can be conducted in a liquid phase, the reaction products separated from unreacted components, and complexes detected (e.g., using an immobilized antibody specific for a nonsense-mediated mRNA decay pathway protein) .
  • Cell-based assays can be used to identify compounds that interact with nonsense-mediated mRNA decay pathways proteins.
  • Cell lines that naturally express such proteins or have been genetically engineered to express such proteins e.g., by transfection or transduction of a UPFl or NMD2 DNA
  • test compounds can be administered to cell cultures and the amount of mRNA derived from a nonsense mutation-containing gene analyzed, e.g., by Northern analysis . An increase in the amount of RNA transcribed from such a gene compared to control cultures that did not contain the test compound indicates that the test compound is an inhibitor of the nonsense-mediated mRNA decay pathway.
  • the amount of a polypeptide encoded by a nonsense mutation-containing gene, or the activity of such a polypeptide can be analyzed in the presence and absence of a test compound. An increase in the amount or activity of the polypeptide indicates that the test compound is an inhibitor of the nonsense- mediated mRNA decay pathway.
  • An alternative method of identifying small molecules that inhibit nonsense-mediated mRNA decay involves evaluating the effect of test compounds on yeast cells that contain conditional mutations.
  • the conditional mutations permit growth in the presence (or absence) of a specific factor when the nonsense-mediated mRNA decay pathway is not functional. For example, in the absence of functional NMD2, cells harboring leu2 or tyr7 nonsense mutations gain the ability to grow in the absence of leucine or tyrosine, respectively.
  • a test compound that effectively inhibits expression of the wild-type NMD2 gene in a cell harboring one or more of these mutations permits the cell to grow under the restrictive condition (e.g., in the absence of leucine or tyrosine, or in the presence of canavanine) .
  • yeast cells that contain leu2-3 or tyr7-l nonsense mutations are grown in the presence and absence of test compounds .
  • Compounds that promote the growth of cells in the presence of leucine or tyrosine, respectively, are candidate compounds to be used as drugs that inhibit the nonsense-mediated mRNA decay pathway.
  • This type of test can also be performed in yeast cells that lack functional NMD2 and express human NMD2 cDNA, thus restoring the nonsense-mediated mRNA decay pathway in a yeast cell with a human gene product to provide an in vi tro model that can be used to identify candidate compounds that may be effective in humans.
  • Candidate compounds can also be screened using cells containing a nonsense mutation in the CANl gene.
  • Canavanine is a growth inhibitor that requires the presence of arginine permease, a protein encoded by the CANl gene, to enter cells.
  • Cells harboring the canl-100 nonsense mutation are resistant to 100 ⁇ g/ml canavanine if they are wild-type for the nonsense-mediated mRNA decay pathway, but sensitive if they harbor an NMD2 mutation.
  • yeast cells that have the canl-100 mutation and are wild-type for the nonsense-mediated mR ⁇ A decay pathway are incubated in the presence and absence of 100 ⁇ g/ml canavanine. If a candidate compound inhibits cell growth in canavanine, the compound is a candidate drug for inhibiting nonsense- mediated mR ⁇ A decay.
  • Candidate inhibitory compounds can be tested in tissue culture cells. For example, nonsense-containing /3-globin mR ⁇ As are rapidly degraded in culture cells
  • RNA 1:453-4605 Such rapid decay would be reversed by candidate drugs that are effective at inhibiting the mR ⁇ A decay pathway.
  • Culture cells expressing nonsense mutation-containing globin genes are incubated with a candidate compound. Lysates are prepared from treated and untreated cells and Western blotted according to known methods. The blots are probed with antibodies specific for the amino or carboxy terminus of 3-globin and the amount of each quantitated. An increase in the amount of carboxy-terminal ⁇ -globin in treated compared to untreated cells indicates that the candidate compound is inhibiting nonsense-mediated mR ⁇ A decay and is a candidate for a drug to treat disorders associated with nonsense mutations.
  • Example 13 Assays for Compounds that Interfere with Upflp/ ⁇ md2p or Upf3p/Nmd2p Interactions
  • Assays for compounds that interfere with the interactions of Nmd2p with its binding partners can be based on both biochemical and genetic approaches.
  • interaction of a Upflp and an Nmd2p or Upf3p is monitored using methods described above, or by more automated methods.
  • the latter include the use of devices such as the BIAcore ® (Pharmacia Biosensor, Uppsala, Sweden) , a surface plasmon resonance detector that measures the interactions of very small amounts of proteins (Szabo et al . , (1995) Curr. Opin. Struct. Biol. 5_:699-705).
  • the BIAcore provides rapid (e.g., within seconds) graphical output of data indicating whether two molecules have interacted, and the affinity and kinetics of that interaction.
  • it provides a suitable method of screening for compounds that interfere with the interaction between the molecules of interest.
  • the chip is coated with carboxymethylated dextran and the protein of interest (e.g., an Nmd2p) is linked to the coating via the protein's primary amine groups using carbodiimide coupling.
  • the chip After washing, the chip is inserted into the sensor, and a solution containing the partner protein (in this case, a Upflp) , is pumped over the surface of the chip. Interaction, as surface plasmon resonance, is detected optically in real time (readouts may be collected at 0.1 second intervals) . The kinetic rates of association and, after removing unbound free protein (in this case, Upflp) , dissociation are measured. Compounds that are candidates to interfere with the interaction between Nmd2p and Upflp are added either with Upflp (to test for interference with association) , or after washing out the Upflp (to test the ability of the candidate compound to enhance the rate of dissociation between Nmd2p and Upflp) .
  • a solution containing the partner protein in this case, a Upflp
  • a comparison between the association and dissociation rates of Nmd2p and Upflp in the presence and absence of the candidate compound indicates whether the compound affects either rate .
  • Candidate compounds that decrease the rate of association or increase the rate of dissociation are compounds that are to be tested further for their ability to interfere with nonsense-mediated RNA decay.
  • the protocol can also be performed by covalently binding Upflp to the chip and using Nmd2p as the free partner in the assay. Compounds that interfere with the interaction between Nmd2p and Upf3p can be tested as described for Nmd2p and Upflp.
  • a genetic approach using a two-hybrid assay can also be used to screen for compounds that interfere with the interaction between components of the nonsense- mediated mRNA decay pathway.
  • the two-hybrid system is a genetic assay in yeast cells that can detect protein :protein interaction (Fields and Song (1989) Nature 340:245-246; Chien et al . , (1991) Proc. Nat. Acad. Sci. USA 88.: 9578-9582; Fields and Sternglanz, (1994) Trends Genet. 10:286-292) .
  • the method is based on the observation that the DNA binding and transcriptional activation functions of the GAL4 protein (Gal4p) can reside on two distinct chimeric polyeptides and still activate transcription from a GAL UAS (upstream activation sequence) , provided that the two polypeptides can interact with each other.
  • GAL4p DNA binding and transcriptional activation functions of the GAL4 protein
  • two plasmids encoding chimeric sequences are constructed.
  • the nucleic acid sequence containing the entire UPFl (or UPF3) coding region, or fragments thereof is fused in-frame to the Gal4p DNA binding domain or the lexA binding domain.
  • the other plasmid contains the NMD2 gene, or fragments thereof, fused to sequences encoding the Gal4p transcriptional activation domain (amino acids 768-881) .
  • the nonsense-mediated mRNA decay pathway genes can be from a yeast or another organism.
  • Plasmids encoding both hybrid molecules are cotransformed into a Saccharomyces cerevisiae strain that contains an integrated GALl -lacZ reporter construct and an integrated GAL1 -HIS3 reporter construct.
  • the transformed yeast are plated and those colonies expressing detectable -galactosidase activity (blue colonies on X-gal plates) and HIS3 activity (detected by resistance to 3-aminotriazole [5-80 mM] ; 3-AT) are indicative of interaction.
  • the lacZ assay is a quantitative assay for enzymatic activity.
  • the HJS3 assay provides a colony growth assay, e.g., resistance to different concentrations of aminotriazole .
  • the effect of a compound on the interaction can also be detected via HIS3 activity, e.g., compounds that prevent the transformed yeast cells from growing in the presence of 3 -AT are candidate compounds for interfering with the interaction between the two proteins (the product of the HIS3 gene antagonizes the latter drug) .
  • Compounds identified as above, or other candidate compounds that inhibit mRNA-mediated decay pathway proteins in vi tro may be useful for treating disorders caused by nonsense mutations. These compounds can be tested in in vivo assays, for example, in animal models of genetic disorders involving nonsense mutations.
  • One such model uses mice transgenic for and that express human ⁇ -globin genes. These mice have been shown to be subject to nonsense-mediated mRNA decay (Lim et al . , Mol. Cell. Biol. 12:1149-1161, 1992).
  • Candidate compounds predicted to inhibit the nonsense-mediated mRNA decay pathway are administered to animals containing nonsense mutations and assayed for inhibition of the nonsense-mediated mRNA decay pathway.
  • Such assays may be indirect or inferential, for example, inhibition would be indicated by improved health or survival of the animal.
  • Assays may also be direct.
  • inhibition would be indicated by a change in the expression of a disease gene (e.g., nonsense codon- containing gene) as measured, e.g., by Northern analysis of tissue removed from an animal treated with a candidate compound.
  • An increase in the amount of disease gene mRNA present in the sample from treated animals compared to untreated control would indicate that the candidate compound is inhibiting the nonsense-mediated mRNA decay pathway.
  • the polypeptide encoded by the disease gene can be measured. For example, an increase in the amount of polypeptide indicates that the candidate compound is inhibiting the nonsense-mediated mRNA decay pathway.
  • the nonsense-mediated mRNA decay pathway can be inhibited by overexpressing the C-terminal truncated form of an Nmd2p in a cell (such as a yeast cell) .
  • Other methods for inhibiting the nonsense-mediated mRNA decay pathway include disruption or mutation of an NMD2 gene or NMD2 antisense transcript expression.
  • a transcript for a heterologous protein which contains at least one stop codon within a transcript-destabilizing 5' portion will be specifically stabilized when expressed in a host cell inhibited in a nonsense-mediated mRNA decay pathway.
  • Such stabilization allows translation of the stabilized transcript in a yeast suppressor mutant to produce a full-length peptide with an amino acid inserted at the position of the nonsense codon.
  • the inserted amino acid is specific to the suppressor mutant host in which the heterologous gene and the Nmd2p C-terminus are expressed.
  • the relevant properties of each of the mutant heterologous proteins are compared to the properties of the wild-type protein, and altered heterologous proteins having desired properties are collected. Such properties may include, but are not limited to, protein receptor binding, antibody binding, enzymatic activity, three dimensional structure, and other biological and physical properties known to those of ordinary skill in the arts of biochemistry and protein chemistry.
  • the invention is also useful in the production of heterologous protein fragments by inserting into the DNA a stop codon within a transcript-destabilizing 5' portion of the coding sequence at a site at which translation is to stop thereby producing an N-terminal protein fragment. This can be done using site-directed mutagenesis. PCR or oligonucleotides containing the desired sequence are used to alter a specfic codon in a gene of interest cloned into an expression plasmid using methods known in the art. Fragments useful in pharmaceutical or other applications can be isolated in large quantities if so desired by techniques well known to those of ordinary skill in the art. Methods of Treating Disorders Involving Nonsense Mutations
  • the invention also encompasses the treatment of disorders, especially in mammals, caused by nonsense mutations.
  • a broad range of genetic disorders associated with a nonsense mutation can be treated by the methods described herein. Without limiting the invention by committing to any particular theory, a substantial number of genetic disorders are attributable to the presence of a premature translational termination codon (e.g, nonsense codon) within the coding region of specific genes (e.g., certain cases of -thalassaemia, breast cancer, polycystic kidney disease I, and Duchenne muscular dystrophy) .
  • a premature translational termination codon e.g, nonsense codon
  • specific genes e.g., certain cases of -thalassaemia, breast cancer, polycystic kidney disease I, and Duchenne muscular dystrophy
  • Table 4 gives examples of specific sites of nonsense mutations associated with cancers such as breast cancer (BRCA1 and BRCA2) , colorectal cancer (non-polyposis) , retinoblastoma, adrenocortical carcinoma, and Li-Fraumeni syndrome.
  • Table 4 also gives specific examples of nonsense mutations associated with other disorders: Duchenne muscular dystrophy, polycystic kidney disease I, polycystic kidney disease II, Fanconi anemia, haemophilia A, hypercholesterolemia, neurofibromatosis 1, Tay-Sachs disease, glycogen storage disease III, cystic fibrosis, adenomatous polyposis coli, and -thalassaemia.
  • Cowden disease (Liaw et al . , (1997) Nat. Genet. 16:64), Maple syrup urine disease (Fisher et al . , (1993) Am. J. Hum. Genet. 5_2.:414) , Wilson disease (Thomas et al . (1995) Nature Genet. 9_:210), Niemann-Pick disease (Schuchman et al . , (1995) Hum. Mut . 6 . :352), Turcot syndrome (Hamilton et al., (1995) N. Engl . J. Med. 332:839) , McArdle disease (Tsujino et al .
  • nonsense codons not only interrupt translation, but also promote enhanced decay of transcripts from genes containing nonsense mutations.
  • inhibitors of this pathway identified as described above are useful for treating disorders involving nonsense mutations.
  • Treatment is designed to reduce the level of endogenous nonsense-medidated mRNA decay pathway gene expression (e.g., expression of an NMD2 , UPFl, UPF3 , or homologs thereof) using, e.g., antisense or ribozyme approaches to inhibit or prevent translation of a nonsense-mediated mRNA decay pathway mRNA transcript; triple helix approaches to inhibit transcription of the gene; or targeted homologous recombination to inactivate or "knock out" a gene or its endogenous promoter.
  • endogenous nonsense-medidated mRNA decay pathway gene expression e.g., expression of an NMD2 , UPFl, UPF3 , or homologs thereof
  • antisense or ribozyme approaches to inhibit or prevent translation of a nonsense-mediated mRNA decay pathway mRNA transcript
  • triple helix approaches to inhibit transcription of the gene
  • targeted homologous recombination to inactivate or "knock out" a gene or its endogen
  • the antisense, ribozyme, or DNA constructs described herein can be administered directly to the site containing the target cells; e.g., heart, skeletal muscle, thymus, spleen, and small intestine.
  • Effective Dose Toxicity and therapeutic efficacy of the polypeptides of the invention and the compounds that modulate their expression or activity can be determined by standard pharmaceutical procedures, using either cells in culture or experimental animals to determine the LD 50 (the dose lethal to 50% of the population) and the ED 50 (the dose therapeutically effective in 50% of the population) .
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD 50 /ED 50 .
  • Polypeptides or other compounds that exhibit large therapeutic indices are preferred.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans .
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED 50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized.
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC 50 (that is, the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful doses in humans.
  • Levels in plasma can be measured, for example, by high performance liquid chromatography. Dosages are from about 0.1 to 500 mg per day.
  • compositions for use in accordance with the present invention can be formulated in a conventional manner using one or more physiologically acceptable carriers or excipients.
  • the compounds and their physiologically acceptable salts and solvates may be formulated for administration by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, parenteral or rectal administration.
  • the pharmaceutical compositions may take the form of, for example, tablets or capsules prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (for example, pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose) ; fillers (for example, lactose, microcrystallme cellulose or calcium hydrogen phosphate) ; lubricants (for example, magnesium stearate, talc or silica) ; disintegrants (for example, potato starch or sodium starch glycolate) ; or wetting agents (for example, sodium lauryl sulphate) .
  • binding agents for example, pregelatinised maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose
  • fillers for example, lactose, microcrystallme cellulose or calcium hydrogen phosphate
  • lubricants for example, magnesium stearate, talc or silica
  • disintegrants for example, potato starch or sodium starch glycolate
  • Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or they may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable additives such as suspending agents (for example, sorbitol syrup, cellulose derivatives or hydrogenated edible fats) ; emulsifying agents (for example, lecithin or acacia) ; non-aqueous vehicles (for example, almond oil, oily esters, ethyl alcohol or fractionated vegetable oils) ; and preservatives (for example, methyl or propyl- p-hydroxybenzoates or sorbic acid) .
  • the preparations may also contain buffer salts, flavoring, coloring and sweetening agents as appropriate.
  • Preparations for oral administration may be suitably formulated to give controlled release of the active compound.
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds for use according to the present invention are conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • a suitable propellant for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas.
  • the dosage unit may be determined
  • the compounds may be formulated for parenteral administration by injection, for example, by bolus injection or continuous infusion.
  • Formulations for injection may be presented in unit dosage form, for example, in ampoules or in multi-dose containers, with an added preservative.
  • the compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, for example, sterile pyrogen-free water, before use.
  • the compounds may also be formulated in rectal compositions such as suppositories or retention enemas, for example, containing conventional suppository bases such as cocoa butter or other glycerides .
  • the compounds may also be formulated as a depot preparation.
  • Such long acting formulations may be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection.
  • the compounds may be formulated with suitable polymeric or hydrophobic materials (for example as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives, for example, as a sparingly soluble salt .
  • compositions may, if desired, be presented in a pack or dispenser device which may contain one or more unit dosage forms containing the active ingredient.
  • the pack may for example comprise metal or plastic foil, such as a blister pack.
  • the pack or dispenser device may be accompanied by instructions for administration.
  • the therapeutic compositions of the invention can also contain a carrier or excipient, many of which are known to skilled artisans.
  • Excipients which can be used include buffers (for example, citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer) , amino acids, urea, alcohols, ascorbic acid, phospholipids, proteins (for example, serum albumin) , EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol .
  • the nucleic acids, polypeptides, antibodies, or modulatory compounds of the invention can be administered by any standard route of administration.
  • administration can be parenteral, intravenous, subcutaneous, intramuscular, intracranial, intraorbital, opthalmic, intraventricular, intracapsular, intraspinal, intracisternal, intraperitoneal , transmucosal , or oral.
  • the modulatory compound can be formulated in various ways, according to the corresponding route of administration.
  • liquid solutions can be made for ingestion or injection; gels or powders can be made for ingestion, inhalation, or topical application. Methods for making such formulations are well known and can be found in, for example, "Remington's Pharmaceutical Sciences.” It is expected that the preferred route of administration will be intravenous.

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Abstract

La présente invention concerne la découverte d'un gène, le NMD2, ainsi nommé pour son rôle dans la voie de dégradation de l'ARNm induite par mutation non-sens, et de la protéine, Nmd2p, codée par le gène NMD2. L'invention concerne également la séquence d'acides aminés de Nmd2p et la séquence nucléotidique du gène NMD2 codant pour cette dernière. La Nmd2p se lie à une autre protéine dans la voie de dégradation, l'Upf1p. Un fragment C-terminal de la protéine se lie également à l'Upf1p et, lorsqu'il est surexprimé dans la cellule hôte, ce fragment inhibe la fonction de l'Upf1p et, de ce fait, la voie de dégradation de l'ARNm induite par mutation non-sens. L'invention se rapporte aussi à des procédés permettant d'inhiber la voie de dégradation de l'ARNm induite par mutation non-sens afin de stabiliser les ARNm transcrits contenant un codon non-sens qui devrait normalement entraîner une augmentation de la vitesse de dégradation des produits de transcription. La stabilisation d'un produit de transcription est utilisée pour produire une protéine recombinée ou d'un fragment de cette dernière. L'invention concerne également des procédés permettant d'identifier des molécules inhibant la voie de dégradation de l'ARNm induite par mutation non-sens, et l'utilisation de ces molécules dans le traitement de troubles associés aux mutations non-sens.
PCT/US1998/022365 1997-10-21 1998-10-21 PRODUCTION DE POLYPEPTIDES HETEROLOGUES EN L'ABSENCE DE FONCTION DE DEGRADATION DE L'ARNm INDUITE PAR MUTATION NON-SENS WO1999020797A1 (fr)

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001044516A3 (fr) * 1999-12-14 2002-06-06 Tularik Inc Procedes permettant de tester des composes qui inhibent la fin de translation prematuree et la degradation de l"arn mediee par les non-sens
US7291461B2 (en) 2002-06-21 2007-11-06 Ptc Therapeutics, Inc. Methods for identifying small molecules that modulate premature translation termination and nonsense mRNA decay
EP2251437A1 (fr) * 2009-05-13 2010-11-17 Hannelore Breitenbach-Koller Procédé d'identification de composés qui contrôlent l'activité translationnelle des protéines ribosomales dans l'expression mRNA différentielle
US7927791B2 (en) 2002-07-24 2011-04-19 Ptc Therapeutics, Inc. Methods for identifying small molecules that modulate premature translation termination and nonsense mediated mRNA decay

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
DARLING T. N., ET AL.: "PREMATURE TERMINATION CODONS ARE PRESENT ON BOTH ALLELES OF THE BULLOUS PEMPHIGOID ANTIGEN 2/ TYPE XVII COLLAGEN GENE IN FIVE AUSTRIAN FAMILIES WITH GENERALIZED ATROPHIC BENIGN EPIDERMOLYSIS BULLOSA.", JOURNAL OF INVESTIGATIVE DERMATOLOGY, NATURE PUBLISHING GROUP, US, vol. 108., no. 04., 1 April 1997 (1997-04-01), US, pages 463 - 468., XP002915632, ISSN: 0022-202X, DOI: 10.1111/1523-1747.ep12289718 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2001044516A3 (fr) * 1999-12-14 2002-06-06 Tularik Inc Procedes permettant de tester des composes qui inhibent la fin de translation prematuree et la degradation de l"arn mediee par les non-sens
JP2003516767A (ja) * 1999-12-14 2003-05-20 テュラリク インコーポレイテッド 早期翻訳終結およびナンセンス媒介性ｒｎａ崩壊を阻害する化合物を検定する方法
US7026122B2 (en) 1999-12-14 2006-04-11 Ptc Therapeutics, Inc. Methods of assaying for compounds that inhibit premature translation termination and nonsense-mediated RNA decay
AU784947B2 (en) * 1999-12-14 2006-08-03 Ptc Therapeutics, Inc. Methods of assaying for compounds that inhibit premature translation termination and nonsense-mediated RNA decay
EP2036991A1 (fr) * 1999-12-14 2009-03-18 PTC Therapeutics, Inc. Procédé pour analyser des composés qui inhibent la fin précoce de la translation et la dégradation de l'ARN à médiation non sens
JP4683808B2 (ja) * 1999-12-14 2011-05-18 ピーティシー・セラピューティックス・インコーポレイテッド 早期翻訳終結およびナンセンス媒介性ｒｎａ崩壊を阻害する化合物を検定する方法
US7291461B2 (en) 2002-06-21 2007-11-06 Ptc Therapeutics, Inc. Methods for identifying small molecules that modulate premature translation termination and nonsense mRNA decay
US7927791B2 (en) 2002-07-24 2011-04-19 Ptc Therapeutics, Inc. Methods for identifying small molecules that modulate premature translation termination and nonsense mediated mRNA decay
EP2251437A1 (fr) * 2009-05-13 2010-11-17 Hannelore Breitenbach-Koller Procédé d'identification de composés qui contrôlent l'activité translationnelle des protéines ribosomales dans l'expression mRNA différentielle
WO2010130447A1 (fr) * 2009-05-13 2010-11-18 Hannelore Breitenbach-Koller Procédé d'identification d'interventions qui régulent l'activité de traduction de protéines ribosomales dans l'expression d'arnm différentielle
US11959145B2 (en) 2009-05-13 2024-04-16 Hannelore Breitenbach-Koller Method for identifying interventions that control the translational activity of ribosomal proteins in differential mRNA expression

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