Uploaded byOmega Cantrell
AVS 406 Presentation
This document discusses using recombinant adeno-associated viral (rAAV) vectors to deliver galanin and neuropeptide Y transgenes to the brain for the treatment of epilepsy via gene therapy. It first provides background on normal brain function, epilepsy, current treatments, and rAAV vectors. It then summarizes several studies that delivered rAAV vectors containing galanin or neuropeptide Y to rat models of epilepsy, finding reductions in seizure activity, onset, and duration. The document concludes that galanin and neuropeptide Y show promise as transgenes for epilepsy gene therapy but that further research is still needed.
AVS 406 Presentation
- 1. The Use ofRecombinant Adeno-Associated Viral Vectors in Gene Therapy to Treat Epilepsy Omega Cantrell
- 2. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 2
- 3. Objectives Why isepilepsy a good target? How does gene therapy work? What is the best transgene for this? 3
- 4. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 4
- 5. What is normal? Neurotransmissions EEG readings http:/www.epilepsy.org.au/images/ElectroEncephalogram.png 5
- 6. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 6
- 7. Epilepsy Definition Affected species Statistics Types of epilepsy Causes http://brain.fuw.edu.pl/~suffa/SW/SW_patt.gif 7
- 8. Normal Epilepsy http:/www.epilepsy.org.au/images/ElectroEncephalogram.png http://brain.fuw.edu.pl/~suffa/SW/SW_patt.gif 8
- 9. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 9
- 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. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 11
- 12. How is aviral 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
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- 14. How is anrAAV 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
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- 16. How is anrAAV 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. Recombinant Adeno-Associated Virus (rAAV) What is it? Characteristics Advantages What should be kept in mind when using this vector? 17
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- 19. Recombinant Adeno-Associated Virus (rAAV) What is it? Characteristics Advantages What should be kept in mind when using this vector? 19
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- 21. How is itdelivered? Intraparenchymally Small volumes, at low flow rates Diffusion to targets Are there any limitations? 21
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- 23. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 23
- 24. Galanin (GAL) 29amino acids In CNS Highly expressed in hippocampus Inhibitors excitatory neurotransmitters www.chemicalbook.com/CASGIF142846-71-7.gif 24
- 25. Neuropeptide Y (NPY) 36 amino acids In CNS, nervous tissue Inhibits excitatory neurotransmitters www.chemicalbook.com/CASGIF113662-54-7/gif 25
- 26. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 26
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- 31. Lin et al.(2003) rAAV-NSE-GAL or rAAV-empty Decrease in seizure activity Decrease in number of seizures 31
- 32. Lin et al.(2003) 32
- 33. Lin et al.(2003) rAAV-NSE-GAL or rAAV-empty Decrease in seizure activity Decrease in number of seizures 33
- 34. Lin et al.(2003) 34
- 35. Mazarati et al.(1998) Perforant path stimulation 30 minutes before or after PPS Decreased time in seizure activity when given before PPS 35
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- 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. Mazarati et al.(1998) 38
- 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. Mazarati et al.(1998) 40
- 41. Mazarati and Wasterlain(2002) rAAV-GAL rats spent less time in seizures Mazarati and Wasterlain (2002) 41
- 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
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- 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. Haberman et al.(2003) 45
- 46. Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 46
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- 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. Richichi et al.(2004) 49
- 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. Richichi et al.(2004) 51
- 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
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- 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. Mazarati and Wasterlain(2002) 57
- 58. Mazarati and Wasterlain(2002) 58
- 59. Mazarati and Wasterlain(2002) 59
- 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. Galanin transgene Strongneurotropism Prevents initiation of SE Drastic reduction in seizure activity and number of seizures observed 61
- 62. Neuropeptide Y transgene Reduced number of seizures Delay in seizure onset No significant decrease in time spent in seizure activity 62
- 63. CONCLUSIONS 63
- 64. Mazarati and Wasterlain(2002) 64
- 65. What’s next? Muchmore research needs to be done What needs to be determined? Injection should be minimally invasive Obtain FDA approval for use in humans 65
- 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. Questions? 67
- 68. Questions? 68
Editor's Notes
- #6 Negative feedback (Na/K); neurons fire 30 times/second brain waves are small
- #8 “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)
- #11 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)
- #19 Single strand DNA, capsid; very small, able to infect wide variety of cell types/host organisms
- #20 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)
- #22 Limited diffusion area
- #27 Done in hippocampus of brain, specifically, dorsal hippocampus
- #33 Average25 minutes in controls, 13.5 in treated ~55% decrease
- #35 Average 25 seizures observed in controls, 15 in treated (60% decrease)
- #36 Perforant path = input pathway to hippocampus
- #37 Input pathway, very neuron dense; stimulation = seizures (highlight hippocampus!)
- #38 Varying concentrations of galanin given
- #39 For same concentration (0.5 nmol), saw 95% decrease in seizure duration
- #40 In 2-mm slice of hippocampus, saw 16 positive neurons 24 h after PPS; none in control at any point
- #42 Average 590 minutes in seizure activity for control, less than 10 in gal-treated
- #43 FIB = fibronectinsecretory sequence (promoter), vector given in inferior collicular cortex of brainstem
- #45 Doxycycline = antibiotic; initial difference: 60% higher in treated than control, after removal of doxy: 30% higher in treated than controls, appeared to continue rising
- #46 In vitro, no detectable amount of GAL, but 32 ng/mL in treated cells
- #49 Serotypes = subtypes, based on glycoprotein markers; used 2 and 1/2
- #51 SE = status epilepticus (prolonged seizure activity – lasting more than a few minutes)
- #53 Injected with vector in dentate gyrus of hippocampus
- #57 15 minutes in control, 13 in treated (no significant difference)
- #62 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
- #63 Delay from 11.5 m (control) to ~6 m (treated); average length of one seizure 15 m in control, 13 m in treated
- #64 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);
- #65 590 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