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.

1. The Use of Recombinant
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 is epilepsy 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 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. http://schoolnet.gov.mt/biology/virus%20life%20cycle.gif
13

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. Molecular Neurology (2007)
15

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. Recombinant Adeno-Associated
Virus (rAAV)
 What is it?
 Characteristics
 Advantages
 What should be kept in mind when using
this vector?
17

18. Molecular Neurology (2007)
18

19. Recombinant Adeno-Associated
Virus (rAAV)
 What is it?
 Characteristics
 Advantages
 What should be kept in mind when using
this vector?
19

20. http://media.wiley.com/CurrentProtocols/HG/hg1209/hg1209-fig-0001-1-full.gif
20

21. How is it delivered?
 Intraparenchymally
 Small volumes, at low flow rates
 Diffusion to targets
 Are there any limitations?
21

22. http://www.scielo.br/img/revistas/gmb/v31n1/01f3.gif
22

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)
 29 amino 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

27. 27
www.chemicalbook.com/CASGIF142846-71-7.gif

28. http://upload.wikimedia.org/wikipedia/commons/2/2e/Gray739-emphasizing-hippocampus.png
28

29. http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png
29

30. http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png
30

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

36. http://www.nature.com/neuro/journal/v10/n3/images/nn0307-271-F1.gif
36

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

43. 43
http://upload.wikimedia.org/wikipedia/commons/0/00/Gray685.png

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

47. www.chemicalbook.com/CASGIF113662-54-7/gif
47

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

53. http://www.nature.com/neuro/journal/v10/n3/images/nn0307-271-F1.gif
53

54. http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png
54

55. http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png
55

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
 Strong neurotropism
 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?
 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. 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.
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67. Questions?
67

68. Questions?
68