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PTRM Student Presentation Dan Wang 10-9-2008

PTRM Student Presentation Dan Wang 10-9-2008. Gentamicin treatment promotes dystrophin expression in mdx myotubes. Immunohistochemistry of myotubes from primary cell culture from muscles

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PTRM Student Presentation Dan Wang 10-9-2008

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  1. PTRM Student Presentation Dan Wang 10-9-2008

  2. Gentamicin treatment promotes dystrophin expression in mdx myotubes • Immunohistochemistry of myotubes from primary cell culture from muscles • Dystrophin was detected by mAb to the C-Ter of dystrophn, followed by a rhodamine-conjugated anti-mouse igG

  3. Dosage-dependent manner There is a window of gentamicin dosage in which misreading can lead to the restoration of full-length dystrophin synthesis.

  4. Gentamicin treatment can protect mdx muscle from contraction-induced injury Eccentric contraction protocol mdx EDL (extensor digitorum longus)muscles G: gentamicin Gi200: injection, 200% dose equivalent GP: infusion pumps D: DHB treatment F: females

  5. Reduction in the damaged sarcolemma Procion orange (fluorescent dye) can enter muscle cells only through membrane disruptions. Mice were subjected to eccentric contractions in the presence of Procion orange.

  6. Gentamicin treatment protected muscle from injury through the restoration of dystrophin dystrophin -sarcoglycan Tibialis anterior (TA) muscle cross-sections

  7. Gentamicin treatment increased both dystrophin and -sarcoglycan levels C57 controls mdx muscle (untreated) mdx muscle (Gi200) mdx muscle (Gi50F) mdx muscle (GP200) Immunoblot analysis of TA muscles for dystrophin and -sarcoglycan.

  8. Gentamicin treatment decreased the serum creatine kinase (CK) level Treatment: Gi200M

  9. PTC124 suppresses nonsense stop codons in vitro HEK293 cells stably transfected with LUC-190 gene HeLa cell-free extract containing synthetic LUC-190 mRNA Differences in transcript levels? PTC124

  10. Full-length dystrophin is produced in PTC124-treated cultured myotubes

  11. PTC124 treatment on the mdx mouse - the regimen

  12. PTC124 treatment rescued the dystrophic phenotype in muscles of the mdx mouse a. Force per cross-sectional area of treatment b. Eccentric contraction assay c. Serum creatine kinase changes d. Western blot of quadriceps and TA muscles

  13. Partial restoration of dystrophin to the membrane in skeletal muscles

  14. PTC124 demonstrates little off-target activity at the level of transcription or mRNA stability • Levels of 22 transcripts changed significantly • Not NMD substrates • Low expressing cellular mRNAs

  15. PTC124 does not promote readthrough of normal termination codons

  16. Conclusions • Gentamicin and PTC124 induced readthrough of premature stop codons both in vitro and in vivo • Full-length, functional proteins (dystrophin) were produced after treatment (10-20% of WT) • Dystrophic phenotype was partially rescued in the mdx mouse • Optimized dosage and regimen • PTC124 has no observed effect on normal stop codons

  17. Discussion • Efficiency of readthrough at premature stop codons • Stop codon context (other genetic diseases caused by nonsense mutations) • Dosage effect • Derivatives of compounds • Rescue of the disease phenotype • How much protein is needed? • Any problem with the inserted amino acid? • Combination with other approaches • Side effect • Mechanism of PTC124 activity • Understand how the cells can distinguish a premature stop codon from a normal one.

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