Compositions and methods related to protein displacement therapy for myotonic distrophy

Abstract

Disclosed are compositions and methods related to the interaction of polyCUG and polyCCUG repeat RNA and proteins that bind to these repetitive RNA sequences. Also disclosed are methods of treating DM1 or DM2 comprising inhibiting the interaction of poly(CUG) exp or poly(CCUG) exp RNA with muscleblind proteins, or by causing improvement of spliceopathy in myotonic dystrophy.

1 . A method of screening for an agent that inhibits the interaction of a protein and a ligand comprising the steps of a) capturing the ligand to a substrate; b) admixing a labeled protein with the ligand; c) contacting an agent with the mixture of step b; d) determining the level of the label; and e) comparing the amount of the label relative to a control; wherein a decrease in the level of the label indicates an agent that inhibits the interaction. 
     
     
         2 . The method of  claim 1 , wherein the protein is a polyCUG or polyCCUG repeat RNA interacting protein or a protein that is sequestered by polyCUG or polyCCUG repeats. 
     
     
         3 . The method of  claim 2 , wherein the polyCUG or polyCCUG repeat interacting protein or sequestered protein is MBNL1, MBNL2, or MBNL3. 
     
     
         4 . The method of  claim 1 , wherein the ligand is RNA comprising a polyCUG or polyCCUG repeat. 
     
     
         5 . The method of  claim 4 , wherein the polyCUG or polyCCUG repeat is flanked by RNA sequence from the DMPK gene or sequence that permits capture to a substrate. 
     
     
         6 . The method of  claim 4 , wherein the polyCUG or polyCCUG repeat ligand is bound to the substrate by binding to a complementary nucleic acid which is bound to the substrate. 
     
     
         7 . The method of  claim 6 , wherein the complementary nucleic acid is biotinylated. 
     
     
         8 . The method of  claim 7 , wherein the biotinylated nucleic acid is bound to the substrate through the binding of biotin with streptavidin bound to the substrate. 
     
     
         9 . The method of  claim 6 , wherein the complementary nucleic acid is an oligodeoxynucleotide (ODN). 
     
     
         10 . The method of  claim 1 , wherein the substrate is a polystyrene plate. 
     
     
         11 . The method of  claim 1 , wherein the label is a fluorescent label. 
     
     
         12 . The method of  claim 11 , wherein the level of fluorescence is determined by a fluorometer or fluorescence plate reader. 
     
     
         13 . A method of screening for an agent that inhibits the interaction of a protein and a ligand comprising the steps of a) mixing a protein bound to a substrate with a labeled ligand; b) contacting an agent with the mixture of step a; c) determining the level of the label; and d) comparing the amount of the label relative to a control; wherein a decrease in the level of the label indicates an agent that inhibits the interaction. 
     
     
         14 . The method of  claim 13 , wherein the ligand comprises polyCUG or polyCCUG repeat RNA. 
     
     
         15 . The method of  claim 13 , wherein the protein is a polyCUG or polyCCUG repeat RNA interacting protein or a protein that is sequestered by polyCUG or polyCCUG repeats. 
     
     
         16 . The method of  claim 15 , wherein the polyCUG or polyCCUG repeat interacting protein or sequestered protein is MBNL1, MBNL2, or MBNL3. 
     
     
         17 . The method of  claim 16 , wherein the substrate is a nitrocellulose filter. 
     
     
         18 . The method of  claim 13 , wherein the label is a fluorescent label. 
     
     
         19 . A method of screening for an agent that inhibits the interaction of a first protein and a first recognition element on a nucleic acid comprising the steps of a) administering an agent to a cell comprising the first protein, a second protein, and a nucleic acid comprising the first recognition element adjacent to a second recognition element, wherein the first protein binds the first recognition element and the second protein binds the second recognition element; and b) detecting co-localization of the first and second protein, wherein a decrease in co-localization of the first and second protein relative to a control indicates an agent that inhibits the interaction. 
     
     
         20 . The method of  claim 19 , wherein the first protein is MBNL1, MBNL2, or MBNL3 and the first recognition element is polyCUG repeat RNA. 
     
     
         21 . The method of  claim 19 , wherein the second protein is MS2 and the second recognition element is an MS2 coat protein RNA recognition element. 
     
     
         22 . The method of  claim 19 , wherein at least one of the first and second proteins comprises a donor fluorescent dye and at least one of the first and second proteins comprises an acceptor dye, wherein excitation of the donor fluorescent dye results in a fluorescent emission that excites the acceptor dye if the first and second proteins are co-localized. 
     
     
         23 . The method of  claim 19 , wherein at least one of the first and second proteins comprises a first half of a split beta galactosidase protein and at least one of the first and second proteins comprises a second half of the split beta galactosidase protein, wherein hydrolysis of a beta galactosidase substrate results in fluorescence of luminescence if the first and second proteins are co-localized. 
     
     
         24 . The method of  claim 19 , wherein at least one of the first and second proteins comprises a first half of a split fluorescent protein and at least one of the first and second proteins comprises a second half of the split fluorescent protein, wherein excitation of the split fluorescent protein results in a fluorescent emission if the first and second proteins are co-localized. 
     
     
         25 . The method of  claim 24 , wherein the split fluorescent protein is Venus fluorescent protein (VFP). 
     
     
         26 . A method of screening for an agent that improves spliceopathy comprising the steps of a) introducing an agent into a cell comprising of a splicing regulator, overexpressed polyCUG or polyCCUG repeat RNA, and spliceopathy reporter construct, wherein the reporter construct comprises a gene susceptible to polyCUG or polyCCUG repeat induced spliceopathy flanked by one or more genes encoding a labeled protein; and b) measuring the level of the labeled protein; and c) comparing the ratio of labeled protein, wherein an increase of labeled protein indicates an agent that improves spliceopathy. 
     
     
         27 . The method of  claim 26 , wherein the gene susceptible to polyCUG or polyCCUG is flanked by genes encoding first and second labeled protein, wherein the first and second proteins are differentially labeled; and wherein the method further comprises d) comparing the ratio of the first labeled protein to the second labeled protein, wherein a high ratio indicates an agent that improves spliceopathy. 
     
     
         28 . The method of  claim 27 , wherein the first and second labeled proteins are, YFP and Y•CFP, respectively. 
     
     
         29 . The method of  claim 26 , wherein the gene susceptible to polyCUG or polyCCUG is flanked by a gene encoding a single labeled protein, and wherein an increase of the labeled protein indicates an agent that improves spliceopathy. 
     
     
         30 . The method of  claim 29 , wherein the labeled protein is GFP 
     
     
         31 . The method of  claim 26 , wherein the splicing regulator is MBLN1, MBNL2, MBNL3, CUG-BP1, or ETR-3. 
     
     
         32 . The method of  claim 26 , wherein the spliceopathy susceptible gene is SERCA1 exon 22 or the fetal exon of TNNT3. 
     
     
         33 . The method of  claims 26 , wherein the level of fluorescence is determined by fluorometry or Fluorescence Resonance Energy Transfer (FRET). 
     
     
         34 . A method of screening for an agent that improves spliceopathy comprising the steps of a) introducing an agent into a cell comprising of a splicing regulator, overexpressed polyCUG or polyCCUG repeat RNA, and spliceopathy reporter construct, wherein the reporter construct comprises a gene susceptible to polyCUG or polyCCUG induced spliceopathy, flanked by the luciferase gene; and b) measuring the luferase activity, wherein an increase of luciferase activity indicates an agent that improves spliceopathy. 
     
     
         35 . A method treating myotonic dystrophy (DM) in a subject in need thereof comprising administering to the subject an agent that inhibits the interaction of MBNL1 with polyCUG exp  mRNA. 
     
     
         36 . The method of  claim 35 , wherein the DM is myotonic dystrophy type 1. 
     
     
         37 . The method of  claim 35 , wherein the agent is a morpholino. 
     
     
         38 . The method of  claim 37 , wherein the morpholino is CAG25. 
     
     
         39 . The method of  claim 38 , wherein the morpholino is set forth in SEQ ID NO: 3. 
     
     
         40 . The method of  claim 37 , wherein the morpholino is an antisense oligonucleotide (AON). 
     
     
         41 . The method of  claim 37 , wherein the morpholino is set forth in SEQ ID NO: 4 or SEQ ID NO: 6. 
     
     
         42 . The method of  claim 35 , wherein the agent is a PNA-CAG. 
     
     
         43 . The method of  claim 42 , wherein the PNA-CAG consists of 2, 3, 4, or 5 CAG repeats. 
     
     
         44 . The method of  claim 35 , wherein the agent is an aminoglycosidic antibiotic compound. 
     
     
         45 . A method treating myotonic dystrophy in a subject in need thereof comprising administering to the subject an agent that corrects spliceopathy. 
     
     
         46 . The method of  claim 45 , wherein the sliceopathy results in channelopathy. 
     
     
         47 . The method of  claim 45 , wherein the agent is a morpholino. 
     
     
         48 . The method of  claim 47 , wherein the morpholino is CAG25. 
     
     
         49 . The method of  claim 48 , wherein the morpholino is set forth in SEQ ID NO: 3. 
     
     
         50 . The method of  claim 47 , wherein the morpholino targets the chloride ion channel ClC-1. 
     
     
         51 . The method of  claim 50 , wherein the morpholino is an antisense oligonucleotide (AON). 
     
     
         52 . The method of  claim 50 , wherein the morpholino is set forth in SEQ ID NO: 4 or SEQ ID NO: 5. 
     
     
         53 . The method of  claim 45 , wherein the agent is a peptide nucleic acid (PNA)-CAG. 
     
     
         54 . The method of  claim 53 , wherein the PNA-CAG consists of 2, 3, 4, or 5 CAG repeats. 
     
     
         55 . The method of  claim 45 , wherein the agent is an aminoglycosidic antibiotic compound. 
     
     
         56 . The method of  claim 55 , wherein the antibiotic compound is neomycin or gentamicin. 
     
     
         57 . A cell comprising the first protein, a second protein, and a nucleic acid comprising the first recognition element adjacent to a second recognition element, wherein the first protein binds the first recognition element and the second protein binds the second recognition element, wherein at least one of the first and second proteins comprises a first half of a split fluorescent protein and at least one of the first and second proteins comprises a second half of the split fluorescent protein, wherein excitation of the split fluorescent protein results in a fluorescent emission if the first and second proteins are co-localized. 
     
     
         58 . A kit comprising a polystyrene plate, polyCUG exp  mRNA, a capture oligodeoxynucleotide (ODN), and a muscleblind protein, wherein the muscleblind protein is labeled. 
     
     
         59 . The kit of  claim 58 , wherein the muscleblind protein is MBNL1, MBNL2, or MBNL3. 
     
     
         60 . The kit of  claim 58 , wherein the muscleblind protein is fluorescently labeled. 
     
     
         61 . A kit comprising a nitrocellulose filter plate, labeled polyCUG exp  mRNA, and a muscleblind protein. 
     
     
         62 . The kit of  claim 61 , wherein the muscleblind protein is MBNL1, MBNL2, or MBNL3. 
     
     
         63 . The kit of  claim 61 , wherein the polyCUG exp  mRNA is fluorescently labeled. 
     
     
         64 . An antisense oligonucleotide as set forth in SEQ ID NO: 3. 
     
     
         65 . An antisense oligonucleotide as set forth in SEQ ID NO: 4. 
     
     
         66 . An antisense oligonucleotide as set forth in SEQ ID NO: 5. 
     
     
         67 . The antisense oligonucleotide of  claim 64 , wherein the antisense oligonucleotide has a peptide nucleic acid backbone. 
     
     
         68 . A method of treating myotonic dystrophy in a subject in need thereof comprising administering to the subject the peptide nucleic acid of  claims 67 . 
     
     
         69 . The antisense oligonucleotide of claims  claim 64 , wherein the antisense oligonucleotide is a morpholino. 
     
     
         70 . A method of treating myotonic dystrophy in a subject in need thereof comprising administering to the subject the morpholino of  claims 69 . 
     
     
         71 . A method of screening for an agent that inhibits the interaction of a protein and a ligand comprising the steps of a) contacting an agent with the protein or labeled ligand; b) admixing the protein with labeled ligand; c) determining the level of the label bound to protein; and d) comparing the amount of the label relative to a control; wherein a decrease in the level of the label bound to protein relative to a control indicates an agent that inhibits the interaction. 
     
     
         72 . A method of screening for an agent that inhibits the interaction of a protein and a ligand comprising the steps of a) contacting an agent with the labeled protein or ligand; b) admixing the protein with ligand; c) determining the level of the label; and d) comparing the amount of the label relative to a control; wherein a decrease in the level of the label bound to protein indicates an agent that inhibits the interaction. 
     
     
         73 . A method of screening for an agent that inhibits the interaction of a protein and ligand comprising the steps of a) admixing the protein and labeled ligand; b) contacting an agent with the protein and labeled ligand; and c) comparing the amount of ligand bound to protein relative to a control; wherein a decrease in the level of the label bound to protein indicates an agent that inhibits the interaction. 
     
     
         74 . A method of screening for an agent that inhibits the interaction of a protein and a ligand comprising the steps of a) contacting the protein or labeled ligand with an agent; b) admixing the protein and labeled ligand in the presence of agent; and c) comparing the amount of ligand bound to protein relative to a control; wherein a decrease in the level of the label bound to protein indicates an agent that inhibits the interaction.