GKA deel 1 college 6

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GKA deel 1 college 6

  1. 2. Genetische therapiën <ul><li>Anti-sense therapie </li></ul><ul><li>Triplex-strategie </li></ul><ul><li>Gentherapie </li></ul><ul><li>RNAi </li></ul><ul><li>Gen-repair </li></ul><ul><ul><li>Via oligonucleotide </li></ul></ul><ul><ul><li>Via zink-vinger geïnduceerde klieving </li></ul></ul>
  2. 3. Targeted gene correction Ye et al., Mol. Med. Today, 1998 <ul><li>Ideal gene therapy: </li></ul><ul><ul><li>Genetic defect is corrected in genome </li></ul></ul><ul><ul><li>No immune response from vector </li></ul></ul><ul><ul><li>Synthetic drug-like molecule </li></ul></ul><ul><ul><li>No cell-specificity </li></ul></ul><ul><ul><li>Only dysfunctional cells are targeted </li></ul></ul><ul><li>Homologous recombination? </li></ul><ul><li>Homologous recombination </li></ul><ul><li>In vitro very inefficient </li></ul>
  3. 4. Gene repair Ye et al., Mol. Med. Today, 1998 <ul><li>Single gene diseases </li></ul>
  4. 5. DNA repair pathways Ye et al., Mol. Med. Today, 1998 <ul><li>Every second, mutations are corrected </li></ul><ul><ul><li>Can repair machinery be targeted to gene defect? </li></ul></ul>
  5. 6. Structure of synthetic repair molecule Ye et al., Mol. Med. Today, 1998 <ul><li>Chimeraplast </li></ul><ul><li>a-e wrong in text </li></ul><ul><ul><li>a: mutator </li></ul></ul><ul><ul><li>b: RNA sequence </li></ul></ul><ul><ul><li>c: cap </li></ul></ul><ul><ul><ul><li>Concatemers </li></ul></ul></ul><ul><ul><ul><li>Stability </li></ul></ul></ul><ul><ul><ul><li>T-A, T-G basepairs </li></ul></ul></ul><ul><ul><li>d: nick </li></ul></ul><ul><ul><li>e: RNA-DNA hybrid </li></ul></ul><ul><li>Transfection with cationic liposomes </li></ul>
  6. 7. Structure of synthetic repair molecule Ye et al., Mol. Med. Today, 1998 <ul><li>Chimeraplast </li></ul><ul><li>a-e wrong in text </li></ul><ul><ul><li>a: mutator </li></ul></ul><ul><ul><li>b: RNA sequence </li></ul></ul><ul><ul><li>c: cap </li></ul></ul><ul><ul><ul><li>Concatemers </li></ul></ul></ul><ul><ul><ul><li>Stability </li></ul></ul></ul><ul><ul><ul><li>T-A, T-G basepairs </li></ul></ul></ul><ul><ul><li>d: nick </li></ul></ul><ul><ul><li>e: RNA-DNA hybrid </li></ul></ul><ul><li>Transfection with cationic liposomes </li></ul>
  7. 8. Relationship between gene repair and gene therapy <ul><li>Ye et al., Mol. Med. Today, 1998 </li></ul>
  8. 9. Biochemical approach Ye et al., Mol. Med. Today, 1998 <ul><li>Faster than cell culturing </li></ul><ul><li>Repair in mammalian cell-free extract </li></ul><ul><ul><li>Bacteria lack ability to repair </li></ul></ul>
  9. 10. Result of mutation-correction Ye et al., Mol. Med. Today, 1998 <ul><li>Left all cells, right GFP-expressing cells </li></ul>
  10. 11. Some examples of gene repair Ye et al., Mol. Med. Today, 1998
  11. 12. 2nd example of gene repair: Zinc-finger nuclease Urnov et al., Nature 435, 2005, 646-651 <ul><li>Combination of transcription factor and restriction enzyme </li></ul><ul><li>Correction in 15-20% of T-cells </li></ul>From: K.A. High Nature 435, 2005, 577-578
  12. 13. Gene repair by homologous recombination Nature 2005, Urnov et al.
  13. 14. Gene repair by homologous recombination Nature 2005, Urnov et al. Homology-directed repair X-ray induced DSB Sister chromatid Donor DNA (plasmid) ZFN-induced DSB ZFN-driven homology- directed repair:
  14. 15. Gene repair by homologous recombination Nature 2005, Urnov et al. <ul><li>ZFN: Zinc-finger nuclease </li></ul><ul><li>7% of cells corrected </li></ul>Homology-directed repair X-ray induced DSB Sister chromatid Donor DNA (plasmid) ZFN-induced DSB ZFN-driven homology- directed repair:
  15. 16. Genetic therapies for Duchenne patients? Tidball and Spencer, Nature Med 9, 2003, 997-998 <ul><li>Patients make no dystrophin, due to non-sense mutation </li></ul><ul><ul><li>Dystrophin is an essential muscle protein </li></ul></ul><ul><ul><li>‘ Dystrophine’ is functional without exon 23 </li></ul></ul>
  16. 17. Einde genetische therapieën ============
  17. 18. 2nd example of gene repair: Zinc-finger nuclease Urnov et al., Nature 435, 2005, 646-651 <ul><li>Combination of transcription factor and restriction enzyme </li></ul><ul><li>Correction in 15-20% of T-cells </li></ul>From: K.A. High Nature 435, 2005, 577-578
  18. 19. Gene repair by homologous recombination Nature 2005, Urnov et al. <ul><li>ZFN: Zinc-finger nuclease </li></ul><ul><li>7% of cells corrected </li></ul>Homology-directed repair X-ray induced DSB Sister chromatid Donor DNA (plasmid) ZFN-induced DSB ZFN-driven homology- directed repair:

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