Emerging Therapies For Dmd

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Emerging Therapies For Dmd

  1. 1. Sriharsha Gowtham
  2. 2. <ul><li>Connects actin to sacrolemmal plasma membrane </li></ul><ul><li>Likely a shock absorber as muscle contracts </li></ul><ul><ul><ul><ul><ul><li>(Finkel) </li></ul></ul></ul></ul></ul>
  3. 3. <ul><li>Mutations produce stop codons UAA, UAG or UGA in mRNA (Welch et al.) </li></ul><ul><li>Instability of transcript or truncation of protein can occur (Mendell and Dietz) </li></ul><ul><li>ptcbio.com </li></ul>
  4. 4. <ul><li>Can cause 5%-70% of cases of most inherited diseases (Welch et al.) </li></ul><ul><li>Cause 5-13% of DMD cases (Nelson et al.) </li></ul>
  5. 5. <ul><li>Degradation of truncated proteins </li></ul><ul><li>The nonsense-mediated mRNA decay pathway of the ribosome. This determines the decay rate of altered transcript during pre mRNA modification and extension of truncated protein </li></ul><ul><li>NMD pathway acts with the ribosome during “pioneering round of translation” as opposed to “productive translation” </li></ul><ul><li>Ribosome uses the exon-junction complex to determine the location of the nonsense mutation </li></ul><ul><ul><ul><ul><ul><li>(Schmitz and Famulok) </li></ul></ul></ul></ul></ul>
  6. 6. <ul><li>To suppress the ribosome’s ability to read nonsense codons (Welch et al.) </li></ul><ul><li>Results in only one change in amino acid </li></ul><ul><li>Should help for other diseases with premature stop codons such as cystic fibrosis (Finkel) </li></ul><ul><li>ptcbio.com </li></ul>
  7. 7. <ul><li>Two high through-put screens for a total of 800,000 low molecular weight compounds </li></ul><ul><li>1 st used human embryonic kidney cells(HEK293) transfected with defective luciferase reporter gene </li></ul><ul><li>2 nd used in vitro assay of luciferase mRNA and rabbit reticulocyte lysate </li></ul><ul><li>Non-toxic compounds that rescued the luciferase expression were isolated. </li></ul><ul><li> (Welch et. al Supp. info.) </li></ul>.
  8. 8. <ul><li>PTC124 (C15H9FN2O3) isolated. </li></ul><ul><li>no structural similarity to aminoglycosides(potentially toxic compounds shown to have worked before) </li></ul><ul><li>Active at low concentrations- non-toxic </li></ul><ul><ul><ul><ul><ul><li>(Welch et al) </li></ul></ul></ul></ul></ul>
  9. 9. <ul><ul><ul><ul><li>Welch et al. </li></ul></ul></ul></ul>
  10. 10. <ul><li>PTC124 treated in cells of human DMD patients and mdx mice (contained premature UAA codon in Exon 23 of dystrophin gene) . </li></ul><ul><li>Antibody for dystrophin protein used </li></ul><ul><li>Read through of nonsense codon successful </li></ul><ul><li>40-60% of normal dystrophin: myosin in humans, 35% in mdx samples </li></ul>
  11. 11. <ul><ul><ul><ul><ul><li>(Welch et al.) </li></ul></ul></ul></ul></ul>
  12. 12. <ul><li>PTC124 dosing in mdx mice: oral, intraperitoneal or both for 2-8 weeks </li></ul><ul><li>All three dosings improved strength </li></ul><ul><li>Protection from contraction-induced injury increased </li></ul><ul><li>Serum creatine kinase decreased </li></ul><ul><li>Full length dystrophin detected in western blots </li></ul><ul><li>Membrane localization determined with immunohistological assays. </li></ul>
  13. 13. <ul><ul><ul><ul><ul><li>(Welch et al) </li></ul></ul></ul></ul></ul>
  14. 14. <ul><li>Microarray analysis of HEK293 cells treated with PTC124 or gentamicin showed few transcripts changed in expression vs control </li></ul><ul><li>Do normal stop codons differ from nonsense mutations? </li></ul><ul><li>Luciferase transcript with nonsense mutation and treated with PTC124 showed full length protein and no read through. </li></ul>
  15. 15. <ul><ul><ul><ul><ul><li>(Welch et al) </li></ul></ul></ul></ul></ul>
  16. 16. <ul><li>Reads through the mutation but not correct stop codon. </li></ul><ul><li>Possibly due to the secondary structure of mRNA near triplet codon (Finkel, Nelson et al.) </li></ul><ul><li>Or it prevents nonsense-mediated decay. (Nelson et al.) </li></ul>
  17. 17. <ul><li>Welch et al. claims that PTC124 induces read through of nonsense codons. </li></ul><ul><li>Auld et al claims that PTC124 stabilizes the protein with nonsense codon. </li></ul><ul><li>Many aspects of the experiment differed including the concentrations of PTC124 used by Auld et al. </li></ul><ul><li>Since PTC124 shows differing efficacy with differing nonsense codons, it is likely inducing read through. </li></ul><ul><li>The molecular target of PTC124 is unknown </li></ul><ul><ul><ul><ul><ul><li>Nelson et al. </li></ul></ul></ul></ul></ul>
  18. 18. <ul><li>After it was clear that no normal stop codon read through occurred in humans (Phase 1), a study regarding cystic fibrosis was conducted (Phase 2). </li></ul><ul><li>Nonsense codon was successfully suppressed </li></ul><ul><li>A study on DMD patients was conducted (Phase 3) showing dose dependent improvement. </li></ul><ul><li>Drug is being considered for Hemophilia A and B </li></ul><ul><ul><ul><ul><ul><li>Finkel </li></ul></ul></ul></ul></ul>
  19. 19. <ul><li>Finkel RS. Read-Through Strategies for Suppression of Nonsense Mutations in Duchenne/ Becker Muscular Dystrophy: Aminoglycosides and Ataluren (PTC124). Journal of Child Neurology. 25(9) 1158-1164 </li></ul><ul><li>Mendell JT and Dietz HC . When the Message Goes Awry: Disease-Producing Mutations that Influence mRNA Content and Performance. Cell, Vol. 107, 411–414, November 16, 2001 </li></ul><ul><li>Nelson SF, Crosbie RH, et al. Emerging genetic therapies to treat Duchenne muscular dystrophy. Curr Opin Neurol. 2009 October ; 22(5): 532–538. </li></ul>
  20. 20. <ul><li>Schmitz A and Famulok M. Chemical Biology: Ignore the Nonsense. Nature  447, 42-43 (3 May 2007) </li></ul><ul><li>Welch EM, Barton ER, Zhuo J, et al. PTC124 targets genetic disorders caused by nonsense mutations. Nature 2007;447:87–91 </li></ul><ul><li>http://www.ptcbio.com/3.1.1_genetic_disorders.aspx </li></ul>

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