Jon Moulton

Publications

• 4.66

  Impact points

  Morpholinos and their peptide conjugates: therapeutic promise and challenge for Duchenne muscular dystrophy.

  Hong M Moulton, Jon D Moulton

  Biochimica et biophysica acta. 02/2010; 1798(12):2296-303.

  Exon-skipping efficacies of phosphodiamidate morpholino oligomers (PMOs) or the conjugates of PMOs with cell-penetrating peptides (PPMOs) have been tested in various animal models of Duchenne muscular dystrophy (DMD), including mdx mice, utrophin-dystrophin double-knockout mice, and CXMD dogs, as we... [more] Exon-skipping efficacies of phosphodiamidate morpholino oligomers (PMOs) or the conjugates of PMOs with cell-penetrating peptides (PPMOs) have been tested in various animal models of Duchenne muscular dystrophy (DMD), including mdx mice, utrophin-dystrophin double-knockout mice, and CXMD dogs, as well as in DMD patients in clinical trials. The studies have shown that PMOs can diffuse into leaky muscle cells, modify splicing of DMD transcripts, induce expression of partially functional dystrophin, and improve function of some skeletal muscles. PMOs are non-toxic, with a report of mdx mice tolerating a 3g/kg dose, and no drug-related safety issue in human has been reported. However, because of their poor cell uptake and rapid renal clearance, large and frequently repeated doses of PMOs are likely required for functional benefit in some skeletal muscles of DMD patients. In addition, PMOs do not enter cardiomyocytes sufficiently to relieve heart pathology, the efficacy of delivery to various muscles varies greatly, and delivery across the tissue of each skeletal muscle tissue is patchy. PPMOs enter cells at far lower doses, enter cardiomyocytes in useful quantities, and deliver more evenly to myocytes both when different muscles are compared and when assessed at the level of single muscle tissue sections. Compared to PMOs, far lower doses of PPMOs can restore dystrophin sufficiently to reduce disease pathology, increase skeletal and cardiac muscle functions, and prolong survival of animals. The biggest challenge for PPMO is determining safe and effective doses. The toxicity of PPMOs will require caution when moving into the clinic. The first PPMO-based DMD drug is currently in preclinical development for DMD patients who can benefit from skipping exon 50.
• 1.74

  Impact points

  Gene knockdowns in adult animals: PPMOs and vivo-morpholinos.

  Jon D Moulton, Shan Jiang

  Molecules (Basel, Switzerland). 02/2009; 14(3):1304-23.

  Antisense molecules do not readily cross cell membranes. This has limited the use of antisense to systems where techniques have been worked out to introduce the molecules into cells, such as embryos and cell cultures. Uncharged antisense bearing a group of guanidinium moieties on either a linear pep... [more] Antisense molecules do not readily cross cell membranes. This has limited the use of antisense to systems where techniques have been worked out to introduce the molecules into cells, such as embryos and cell cultures. Uncharged antisense bearing a group of guanidinium moieties on either a linear peptide or dendrimer scaffold can enter cells by endocytosis and subsequently escape from endosomes into the cytosol/nuclear compartment of cells. These technologies allow systemic administration of antisense, making gene knockdowns and splice modification feasible in adult animals; this review presents examples of such animal studies. Techniques developed with PPMOs, which are an arginine-rich cell-penetrating peptide linked to a Morpholino oligo, can also be performed using commercially available Vivo-Morpholinos, which are eight guanidinium groups on a dendrimeric scaffold linked to a Morpholino oligo. Antisense-based techniques such as blocking translation, modifying pre-mRNA splicing, inhibiting miRNA maturation and inhibiting viral replication can be conveniently applied in adult animals by injecting PPMOs or Vivo-Morpholinos.

• Using Morpholinos to control gene expression.

  Jon D Moulton, Yi-Lin Yan

  Current protocols in molecular biology / edited by Frederick M. Ausubel ... [et al.]. 07/2008; Chapter 26:Unit 26.8.

  Morpholino oligonucleotides are stable, uncharged, water-soluble molecules that bind to complementary sequences of RNA, thereby inhibiting mRNA processing, read-through, and protein binding at those sites. Morpholinos are typically used to inhibit translation of mRNA, splicing of pre-mRNA, and matur... [more] Morpholino oligonucleotides are stable, uncharged, water-soluble molecules that bind to complementary sequences of RNA, thereby inhibiting mRNA processing, read-through, and protein binding at those sites. Morpholinos are typically used to inhibit translation of mRNA, splicing of pre-mRNA, and maturation of miRNA, although they can also inhibit other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA. However, unmodified Morpholinos do not pass well through plasma membranes and must therefore be delivered into the nuclear or cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by proteins and do not undergo protein-mediated catalysis; nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos.
• 12.92

  Impact points

  Targeted inhibition of miRNA maturation with morpholinos reveals a role for miR-375 in pancreatic islet development.

  Wigard P Kloosterman, Anne K Lagendijk, René F Ketting, Jon D Moulton, Ronald H A Plasterk

  PLoS biology. 09/2007; 5(8):e203.

  Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function ph... [more] Several vertebrate microRNAs (miRNAs) have been implicated in cellular processes such as muscle differentiation, synapse function, and insulin secretion. In addition, analysis of Dicer null mutants has shown that miRNAs play a role in tissue morphogenesis. Nonetheless, only a few loss-of-function phenotypes for individual miRNAs have been described to date. Here, we introduce a quick and versatile method to interfere with miRNA function during zebrafish embryonic development. Morpholino oligonucleotides targeting the mature miRNA or the miRNA precursor specifically and temporally knock down miRNAs. Morpholinos can block processing of the primary miRNA (pri-miRNA) or the pre-miRNA, and they can inhibit the activity of the mature miRNA. We used this strategy to knock down 13 miRNAs conserved between zebrafish and mammals. For most miRNAs, this does not result in visible defects, but knockdown of miR-375 causes defects in the morphology of the pancreatic islet. Although the islet is still intact at 24 hours postfertilization, in later stages the islet cells become scattered. This phenotype can be recapitulated by independent control morpholinos targeting other sequences in the miR-375 precursor, excluding off-target effects as cause of the phenotype. The aberrant formation of the endocrine pancreas, caused by miR-375 knockdown, is one of the first loss-of-function phenotypes for an individual miRNA in vertebrate development. The miRNA knockdown strategy presented here will be widely used to unravel miRNA function in zebrafish.

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• Using morpholinos to control gene expression.

  Jon D Moulton

  Current protocols in nucleic acid chemistry / edited by Serge L. Beaucage ... [et al.]. 02/2007; Chapter 4:Unit 4.30.

  Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they ... [more] Morpholino oligonucleotides are stable, uncharged, water-soluble molecules used to block complementary sequences of RNA, preventing processing, read-through, or protein binding at those sites. Morpholinos are typically used to block translation of mRNA and to block splicing of pre-mRNA, though they can block other interactions between biological macromolecules and RNA. Morpholinos are effective, specific, and lack non-antisense effects. They work in any cell that transcribes and translates RNA, but must be delivered into the nuclear/cytosolic compartment to be effective. Morpholinos form stable base pairs with complementary nucleic acid sequences but apparently do not bind to proteins to a significant extent. They are not recognized by any proteins and do not undergo protein-mediated catalysis; nor do they mediate RNA cleavage by RNase H or the RISC complex. This work focuses on techniques and background for using Morpholinos.
• 6.63

  Impact points

  Arginine-rich cell-penetrating peptides with uncharged antisense oligomers.

  Hong M Moulton, Jon D Moulton

  Drug discovery today. 11/2004; 9(20):870.
• 3.45

  Impact points

  Peptide-assisted delivery of steric-blocking antisense oligomers.

  Hong M Moulton, Jon D Moulton

  Current opinion in molecular therapeutics. 05/2003; 5(2):123-32.

  Cationic transport peptides conjugated to steric blocking antisense oligomers (oligos) increase oligo uptake in eukaryotic cell lines, bacteria and mice. Recent reports of arginine-rich transport peptide conjugates strongly suggest that the mechanism of uptake is primarily endocytotic and that previ... [more] Cationic transport peptides conjugated to steric blocking antisense oligomers (oligos) increase oligo uptake in eukaryotic cell lines, bacteria and mice. Recent reports of arginine-rich transport peptide conjugates strongly suggest that the mechanism of uptake is primarily endocytotic and that previous assay techniques produced confounding artifacts that led to the old non-endocytotic, membrane-penetrating peptide model. The artifacts result from fixing cells for fluorescent microscopy and from using non-trypsinized cells for flow cytometry. Fixing cells redistributes the peptide or peptide-oligo conjugates associated with the outside of cell membranes and trapped in endosomes, giving apparent diffuse cytosolic and nuclear fluorescence. Cationic peptides bound to the outer surface of cells, if not removed, skew fluorescence data obtained by flow cytometry, leading to the earlier conclusions. Upregulation assays now provide a tool for comparing the efficacy of conjugates, measuring oligo uptake by quantitating antisense activity of conjugates. These assays, developed in cell culture and mouse models, are faster and have higher signal-to-noise ratios than downregulation assays. Thus, a convenient and effective method now exists to screen transport peptides.

• Photoadaptation rates of Synechococcus WH7803 cultures at two iron concentrations

  Jon D. Moulton

  Degree: Ph.D.

  Supervisor: Dr. John Rueter

• Antisense Morpholino Oligomers and Their Peptide Conjugates

  Moulton, Moulton

  pages 43-79;

  ISBN: 978-0-85404-116-9