The Use of Recombinant Adeno-Associated ViralVectors in Gene Therapy   to Treat Epilepsy               Omega Cantrell
Outline   Introduction    ◦   What is normal?    ◦   What is epilepsy?    ◦   Current treatments    ◦   rAAV    ◦   Neuro...
Objectives Why is epilepsy a good target? How does gene therapy work? What is the best transgene for this?             ...
Outline   Introduction    ◦ What is normal?    ◦   What is epilepsy?    ◦   Current treatments    ◦   rAAV    ◦   Neurotr...
What is normal? Neurotransmissions EEG readings                       http:/www.epilepsy.org.au/images/ElectroEncephalog...
Outline   Introduction    ◦ What is normal?    ◦ What is epilepsy?    ◦ Current treatments    ◦ rAAV    ◦ Neurotransmitte...
Epilepsy   Definition   Affected species   Statistics   Types of epilepsy   Causes                        http://brai...
Normal                                            Epilepsyhttp:/www.epilepsy.org.au/images/ElectroEncephalogram.png   http...
Outline   Introduction    ◦ What is normal?    ◦ What is epilepsy?    ◦ Current treatments    ◦ rAAV    ◦ Neurotransmitte...
Current Treatments   Medications    ◦ Mode of action   Surgery    ◦ What kinds of epilepsy      does this treat?    ◦ Ho...
Outline   Introduction    ◦ What is normal?    ◦ What is epilepsy?    ◦ Current treatments    ◦ rAAV    ◦ Neurotransmitte...
How is a viral vector constructed? Life cycle manipulation Removal of rep and cap genes    ◦ Why?  Rep’s effect   Addi...
http://schoolnet.gov.mt/biology/virus%20life%20cycle.gif                                                      13
How is an rAAV vector constructed?  Life cycle manipulation  Removal of rep and cap genes  Addition of beneficial compo...
Molecular Neurology (2007)                         15
How is an rAAV vector constructed?  Life cycle manipulation  Removal of rep and cap genes  Addition of beneficial compo...
Recombinant Adeno-AssociatedVirus (rAAV) What is it? Characteristics Advantages What should be kept in mind when using...
Molecular Neurology (2007)                         18
Recombinant Adeno-AssociatedVirus (rAAV) What is it? Characteristics Advantages What should be kept in mind when using...
http://media.wiley.com/CurrentProtocols/HG/hg1209/hg1209-fig-0001-1-full.gif                                              ...
How is it delivered? Intraparenchymally Small volumes, at low flow rates Diffusion to targets Are there any limitation...
http://www.scielo.br/img/revistas/gmb/v31n1/01f3.gif                                                       22
Outline   Introduction    ◦   What is normal?    ◦   What is epilepsy?    ◦   Current treatments    ◦   rAAV    ◦ Neurotr...
Galanin (GAL) 29 amino acids In CNS Highly expressed in  hippocampus Inhibitors excitatory  neurotransmitters         ...
Neuropeptide Y (NPY) 36 amino acids In CNS, nervous  tissue Inhibits excitatory  neurotransmitters                     ...
Outline   Introduction    ◦   What is normal?    ◦   What is epilepsy?    ◦   Current treatments    ◦   rAAV    ◦   Neuro...
27www.chemicalbook.com/CASGIF142846-71-7.gif
http://upload.wikimedia.org/wikipedia/commons/2/2e/Gray739-emphasizing-hippocampus.png                                    ...
http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png                                                 ...
http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png                                                 ...
Lin et al. (2003) rAAV-NSE-GAL or rAAV-empty Decrease in seizure activity Decrease in number of seizures               ...
Lin et al. (2003)                    32
Lin et al. (2003) rAAV-NSE-GAL or rAAV-empty Decrease in seizure activity Decrease in number of seizures               ...
Lin et al. (2003)                    34
Mazarati et al. (1998) Perforant path stimulation 30 minutes before or after PPS Decreased time in seizure activity whe...
http://www.nature.com/neuro/journal/v10/n3/images/nn0307-271-F1.gif                                                       ...
Mazarati et al. (1998) Perforant path stimulation 30 minutes before or after PPS Decreased time in seizure activity whe...
Mazarati et al. (1998)                         38
Mazarati et al. (1998) Perforant path stimulation 30 minutes before or after PPS Decreased time in seizure activity whe...
Mazarati et al. (1998)                         40
Mazarati and Wasterlain (2002)   rAAV-GAL rats spent less time in seizures                                Mazarati and Wa...
Haberman et al. (2003) Seizure threshold reduced by rAAV-FIB-  GAL Given doxycycline, threshold returned to  baseline A...
43http://upload.wikimedia.org/wikipedia/commons/0/00/Gray685.png
Haberman et al. (2003) Seizure threshold reduced by rAAV-FIB-  GAL Given doxycycline, threshold returned to  baseline A...
Haberman et al. (2003)                         45
Outline   Introduction    ◦   What is normal?    ◦   What is epilepsy?    ◦   Current treatments    ◦   rAAV    ◦   Neuro...
www.chemicalbook.com/CASGIF113662-54-7/gif                                          47
Richichi et al. (2004) Used two serotypes of rAAV-NSE-NPY;  kainic acid to induce seizures Onset delayed almost twofold...
Richichi et al. (2004)                         49
Richichi et al. (2004) Used two serotypes of rAAV-NSE-NPY;  kainic acid to induce seizures Onset delayed almost twofold...
Richichi et al. (2004)                         51
Mazarati & Wasterlain (2002)   30 minutes PPS to induce seizure activity    ◦ Ten minutes after, injected with vector No...
http://www.nature.com/neuro/journal/v10/n3/images/nn0307-271-F1.gif                                                       ...
http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png                                                 ...
http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png                                                 ...
Mazarati & Wasterlain (2002)   30 minutes PPS to induce seizure activity    ◦ Ten minutes after, injected with vector No...
Mazarati and Wasterlain (2002)                                 57
Mazarati and Wasterlain (2002)                                 58
Mazarati and Wasterlain (2002)                                 59
Outline   Introduction    ◦   What is normal?    ◦   What is epilepsy?    ◦   Current treatments    ◦   rAAV         Wha...
Galanin transgene Strong neurotropism Prevents initiation of SE Drastic reduction in seizure activity and  number of se...
Neuropeptide Y transgene Reduced number of seizures Delay in seizure onset No significant decrease in time spent in  se...
CONCLUSIONS          63
Mazarati and Wasterlain (2002)                                 64
What’s next? Much more research needs to be done What needs to be determined? Injection should be minimally invasive O...
References   Haberman, R.P., R.J. Samulski, and T. J. McCown. 2003. Attenuation of    seizures and neuronal death by aden...
Questions?         67
Questions?         68
Upcoming SlideShare
Loading in...5
×

AVS 406 Presentation

133

Published on

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

0 Comments
0 Likes
Statistics
Notes
  • Be the first to comment

  • Be the first to like this

No Downloads
Views
Total Views
133
On Slideshare
0
From Embeds
0
Number of Embeds
0
Actions
Shares
0
Downloads
0
Comments
0
Likes
0
Embeds 0
No embeds

No notes for slide
  • Negative feedback (Na/K); neurons fire 30 times/second brain waves are small
  • “recurrent, unprovoked seizures”; affects dogs, cats, rats; most common neurological disorder in humans; affects 0.5-1% (higher in undeveloped parts of the world); neurons fire up to 500 times/sec (30 in normal)  hyperactivity results in seizures; types: focal (one area), includes temporal lobe epilepsy (most common for in adults, often resistant to treatment with medication), generalized (spreads across brain); can be caused by genetics, metabolic disorders, etc; brain waves are high, rapid spikes (more = more intense)
  • Block sodium channels (prevent hyperactivity); 1/3 resistant to medication, may be eligible for surgery: done after multiple failed attempts with Rxs; focal and generalized, but only certain subtypes; focal area removed (TLE), corpus callosum cut (generalized)
  • Single strand DNA, capsid; very small, able to infect wide variety of cell types/host organisms
  • Long latency period, little or no response from host’s immune system, can stably transduce (intro foreign genetic material into host cell), especially neurons; needs a helper virus to replicate! (sheds once in host cell)
  • Limited diffusion area
  • Done in hippocampus of brain, specifically, dorsal hippocampus
  • Average25 minutes in controls, 13.5 in treated  ~55% decrease
  • Average 25 seizures observed in controls, 15 in treated (60% decrease)
  • Perforant path = input pathway to hippocampus
  • Input pathway, very neuron dense; stimulation = seizures (highlight hippocampus!)
  • Varying concentrations of galanin given
  • For same concentration (0.5 nmol), saw 95% decrease in seizure duration
  • In 2-mm slice of hippocampus, saw 16 positive neurons 24 h after PPS; none in control at any point
  • Average 590 minutes in seizure activity for control, less than 10 in gal-treated
  • FIB = fibronectinsecretory sequence (promoter), vector given in inferior collicular cortex of brainstem
  • Doxycycline = antibiotic; initial difference: 60% higher in treated than control, after removal of doxy: 30% higher in treated than controls, appeared to continue rising
  • In vitro, no detectable amount of GAL, but 32 ng/mL in treated cells
  • Serotypes = subtypes, based on glycoprotein markers; used 2 and 1/2
  • SE = status epilepticus (prolonged seizure activity – lasting more than a few minutes)
  • Injected with vector in dentate gyrus of hippocampus
  • 15 minutes in control, 13 in treated (no significant difference)
  • Expressed only in neurons (also due to neuronal promoter); can both prevent SE from starting, and stop it if it’s already going (drugs can’t even do this); 77% decrease in seizure activity, 40% decrease in number observed
  • Delay from 11.5 m (control) to ~6 m (treated); average length of one seizure 15 m in control, 13 m in treated
  • Both are strong anticonvulsants, but GAL appears to be the best choice: 40% decrease in number of seizures observed, compared to 13% in NPY (based on studies reviewed); 77% decrease in time in activity (no data for NPY);
  • 590 in control, ~180 in NPY, <10 in GAL; hard to be able to dispute that GAL has a strong effect on seizure activity than NPY
  • Transcript of "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

    ×