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AVS 406 Presentation

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Presentation to accompany review paper written for AVS 406 - "The Use of Recombinant Adeno-Associated Viral Vectors in Gene Therapy to Treat Epilepsy".

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AVS 406 Presentation

  1. 1. The Use of Recombinant Adeno-Associated ViralVectors in Gene Therapy to Treat Epilepsy Omega Cantrell
  2. 2. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 2
  3. 3. Objectives Why is epilepsy a good target? How does gene therapy work? What is the best transgene for this? 3
  4. 4. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 4
  5. 5. What is normal? Neurotransmissions EEG readings http:/www.epilepsy.org.au/images/ElectroEncephalogram.png 5
  6. 6. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 6
  7. 7. Epilepsy Definition Affected species Statistics Types of epilepsy Causes http://brain.fuw.edu.pl/~suffa/SW/SW_patt.gif 7
  8. 8. Normal Epilepsyhttp:/www.epilepsy.org.au/images/ElectroEncephalogram.png http://brain.fuw.edu.pl/~suffa/SW/SW_patt.gif 8
  9. 9. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 9
  10. 10. Current Treatments Medications ◦ Mode of action Surgery ◦ What kinds of epilepsy does this treat? ◦ How is it done? http://static.guim.co.uk/sys- images/Guardian/Pix/pictures/2009/4/6/1239055717363/Cross- section-of-the-huma-001.jpg 10
  11. 11. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 11
  12. 12. How is a viral vector constructed? Life cycle manipulation Removal of rep and cap genes ◦ Why?  Rep’s effect Addition of beneficial components ◦ Triple plasmid transfection method; what is added?  Helper  Transgene  Promoter 12
  13. 13. http://schoolnet.gov.mt/biology/virus%20life%20cycle.gif 13
  14. 14. How is an rAAV vector constructed?  Life cycle manipulation  Removal of rep and cap genes  Addition of beneficial components ◦ Triple plasmid transfection method; what is added?  Helper  Transgene  Promoter 14
  15. 15. Molecular Neurology (2007) 15
  16. 16. How is an rAAV vector constructed?  Life cycle manipulation  Removal of rep and cap genes  Addition of beneficial components ◦ Triple plasmid transfection method; what is added?  Helper  Transgene  Promoter 16
  17. 17. Recombinant Adeno-AssociatedVirus (rAAV) What is it? Characteristics Advantages What should be kept in mind when using this vector? 17
  18. 18. Molecular Neurology (2007) 18
  19. 19. Recombinant Adeno-AssociatedVirus (rAAV) What is it? Characteristics Advantages What should be kept in mind when using this vector? 19
  20. 20. http://media.wiley.com/CurrentProtocols/HG/hg1209/hg1209-fig-0001-1-full.gif 20
  21. 21. How is it delivered? Intraparenchymally Small volumes, at low flow rates Diffusion to targets Are there any limitations? 21
  22. 22. http://www.scielo.br/img/revistas/gmb/v31n1/01f3.gif 22
  23. 23. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 23
  24. 24. Galanin (GAL) 29 amino acids In CNS Highly expressed in hippocampus Inhibitors excitatory neurotransmitters www.chemicalbook.com/CASGIF142846-71-7.gif 24
  25. 25. Neuropeptide Y (NPY) 36 amino acids In CNS, nervous tissue Inhibits excitatory neurotransmitters www.chemicalbook.com/CASGIF113662-54-7/gif 25
  26. 26. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 26
  27. 27. 27www.chemicalbook.com/CASGIF142846-71-7.gif
  28. 28. http://upload.wikimedia.org/wikipedia/commons/2/2e/Gray739-emphasizing-hippocampus.png 28
  29. 29. http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png 29
  30. 30. http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png 30
  31. 31. Lin et al. (2003) rAAV-NSE-GAL or rAAV-empty Decrease in seizure activity Decrease in number of seizures 31
  32. 32. Lin et al. (2003) 32
  33. 33. Lin et al. (2003) rAAV-NSE-GAL or rAAV-empty Decrease in seizure activity Decrease in number of seizures 33
  34. 34. Lin et al. (2003) 34
  35. 35. Mazarati et al. (1998) Perforant path stimulation 30 minutes before or after PPS Decreased time in seizure activity when given before PPS 35
  36. 36. http://www.nature.com/neuro/journal/v10/n3/images/nn0307-271-F1.gif 36
  37. 37. Mazarati et al. (1998) Perforant path stimulation 30 minutes before or after PPS Decreased time in seizure activity when given before PPS More GAL-positive neurons in treated rats 37
  38. 38. Mazarati et al. (1998) 38
  39. 39. Mazarati et al. (1998) Perforant path stimulation 30 minutes before or after PPS Decreased time in seizure activity when given before PPS More GAL-positive neurons in treated rats 39
  40. 40. Mazarati et al. (1998) 40
  41. 41. Mazarati and Wasterlain (2002) rAAV-GAL rats spent less time in seizures Mazarati and Wasterlain (2002) 41
  42. 42. Haberman et al. (2003) Seizure threshold reduced by rAAV-FIB- GAL Given doxycycline, threshold returned to baseline After removal, threshold increased again GAL in cells after seizures: higher GAL in vitro in rAAV-FIB-GAL cells 42
  43. 43. 43http://upload.wikimedia.org/wikipedia/commons/0/00/Gray685.png
  44. 44. Haberman et al. (2003) Seizure threshold reduced by rAAV-FIB- GAL Given doxycycline, threshold returned to baseline After removal, threshold increased again GAL in cells after seizures: higher GAL in vitro in rAAV-FIB-GAL cells 44
  45. 45. Haberman et al. (2003) 45
  46. 46. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 46
  47. 47. www.chemicalbook.com/CASGIF113662-54-7/gif 47
  48. 48. Richichi et al. (2004) Used two serotypes of rAAV-NSE-NPY; kainic acid to induce seizures Onset delayed almost twofold No SE in treated animals, at least 60 minute episodes in control group NPY found only in neurons 48
  49. 49. Richichi et al. (2004) 49
  50. 50. Richichi et al. (2004) Used two serotypes of rAAV-NSE-NPY; kainic acid to induce seizures Onset delayed almost twofold No SE in treated animals, average of 87 minute episodes in control group 50
  51. 51. Richichi et al. (2004) 51
  52. 52. Mazarati & Wasterlain (2002) 30 minutes PPS to induce seizure activity ◦ Ten minutes after, injected with vector No significant difference in time spent in total seizure activity Treated animals: ~4 hours in seizure activity SSSE decreased to <20 minutes total 52
  53. 53. http://www.nature.com/neuro/journal/v10/n3/images/nn0307-271-F1.gif 53
  54. 54. http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png 54
  55. 55. http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png 55
  56. 56. Mazarati & Wasterlain (2002) 30 minutes PPS to induce seizure activity ◦ Ten minutes after, injected with vector No significant difference in time spent in one seizure, but: 10 hours in seizure activity (controls), ~4 hours for NPY-treated animals SSSE decreased to <20 minutes total 56
  57. 57. Mazarati and Wasterlain (2002) 57
  58. 58. Mazarati and Wasterlain (2002) 58
  59. 59. Mazarati and Wasterlain (2002) 59
  60. 60. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV  What is it?  How is it contructed? ◦ Neurotransmitters StudiesImplications Conclusions 60
  61. 61. Galanin transgene Strong neurotropism Prevents initiation of SE Drastic reduction in seizure activity and number of seizures observed 61
  62. 62. Neuropeptide Y transgene Reduced number of seizures Delay in seizure onset No significant decrease in time spent in seizure activity 62
  63. 63. CONCLUSIONS 63
  64. 64. Mazarati and Wasterlain (2002) 64
  65. 65. What’s next? Much more research needs to be done What needs to be determined? Injection should be minimally invasive Obtain FDA approval for use in humans 65
  66. 66. References Haberman, R.P., R.J. Samulski, and T. J. McCown. 2003. Attenuation of seizures and neuronal death by adeno-associated virus vector galanin expression and secretion. Nature Medicine. 9(8): 1076-1080. Lin, E.D., C. Richichi, D. Young, K. Baer, A. Vezzani, and M.J. During. 2003. Recombinant AAV-mediated expression of galanin in rat hippocampus suppresses seizure development. European Journal of Neuroscience. 18: 2087-2092 Mazarati, A.M and C.G. Wasterlain. 2002. Anticonvulsant effects of four neuropeptides in the rat hippocampus during self-sustaining status epilepticus. Neuroscience Letters. 331: 123-127. Mazarati, A.M., H. Liu, U. Soomets, R. Sankar, D. Shin, H. Katsumori, Ü. Langel, and C.G. Wasterlain. 1998. Galanin modulation of seizures and seizure modulation of hippocampal galanin in animal models of status epilepticus. Journal of Neuroscience. 18(23): 10070-10077. Richichi, C, E.D. Lin, D. Stefanin, D. Colella, T. Ravizza, G. Grignaschi, P. Veglianese, G. Sperk, M.J. During, and A. Vezzani. 2004. Anticonvulsant and antiepileptogenic effects mediated by adeno-associated virus vector neuropeptide Y expression in the rat hippocampus. Journal of Neuroscience. 24(12): 3051-3059. 66
  67. 67. Questions? 67
  68. 68. Questions? 68

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