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AVS 406 Presentation
 

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".

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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  • 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

AVS 406 Presentation AVS 406 Presentation Presentation Transcript

  • 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 ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 2
  • Objectives Why is epilepsy a good target? How does gene therapy work? What is the best transgene for this? 3
  • Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 4
  • What is normal? Neurotransmissions EEG readings http:/www.epilepsy.org.au/images/ElectroEncephalogram.png 5
  • Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 6
  • Epilepsy Definition Affected species Statistics Types of epilepsy Causes http://brain.fuw.edu.pl/~suffa/SW/SW_patt.gif 7
  • Normal Epilepsyhttp:/www.epilepsy.org.au/images/ElectroEncephalogram.png http://brain.fuw.edu.pl/~suffa/SW/SW_patt.gif 8
  • Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 9
  • 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
  • Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 11
  • 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
  • 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 components ◦ Triple plasmid transfection method; what is added?  Helper  Transgene  Promoter 14
  • Molecular Neurology (2007) 15
  • 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
  • Recombinant Adeno-AssociatedVirus (rAAV) What is it? Characteristics Advantages What should be kept in mind when using this vector? 17
  • Molecular Neurology (2007) 18
  • Recombinant Adeno-AssociatedVirus (rAAV) What is it? Characteristics Advantages What should be kept in mind when using this vector? 19
  • http://media.wiley.com/CurrentProtocols/HG/hg1209/hg1209-fig-0001-1-full.gif 20
  • How is it delivered? Intraparenchymally Small volumes, at low flow rates Diffusion to targets Are there any limitations? 21
  • http://www.scielo.br/img/revistas/gmb/v31n1/01f3.gif 22
  • Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 23
  • Galanin (GAL) 29 amino acids In CNS Highly expressed in hippocampus Inhibitors excitatory neurotransmitters www.chemicalbook.com/CASGIF142846-71-7.gif 24
  • Neuropeptide Y (NPY) 36 amino acids In CNS, nervous tissue Inhibits excitatory neurotransmitters www.chemicalbook.com/CASGIF113662-54-7/gif 25
  • Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 26
  • 27www.chemicalbook.com/CASGIF142846-71-7.gif
  • http://upload.wikimedia.org/wikipedia/commons/2/2e/Gray739-emphasizing-hippocampus.png 28
  • http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png 29
  • http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png 30
  • Lin et al. (2003) rAAV-NSE-GAL or rAAV-empty Decrease in seizure activity Decrease in number of seizures 31
  • Lin et al. (2003) 32
  • Lin et al. (2003) rAAV-NSE-GAL or rAAV-empty Decrease in seizure activity Decrease in number of seizures 33
  • Lin et al. (2003) 34
  • Mazarati et al. (1998) Perforant path stimulation 30 minutes before or after PPS Decreased time in seizure activity when given before PPS 35
  • http://www.nature.com/neuro/journal/v10/n3/images/nn0307-271-F1.gif 36
  • 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
  • Mazarati et al. (1998) 38
  • 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
  • Mazarati et al. (1998) 40
  • Mazarati and Wasterlain (2002) rAAV-GAL rats spent less time in seizures Mazarati and Wasterlain (2002) 41
  • 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
  • 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 After removal, threshold increased again GAL in cells after seizures: higher GAL in vitro in rAAV-FIB-GAL cells 44
  • Haberman et al. (2003) 45
  • Outline Introduction ◦ What is normal? ◦ What is epilepsy? ◦ Current treatments ◦ rAAV ◦ Neurotransmitters Studies ◦ Galanin-focused studies ◦ NPY-focused studies Implications Conclusions 46
  • 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 No SE in treated animals, at least 60 minute episodes in control group NPY found only in neurons 48
  • 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 No SE in treated animals, average of 87 minute episodes in control group 50
  • Richichi et al. (2004) 51
  • 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
  • http://www.nature.com/neuro/journal/v10/n3/images/nn0307-271-F1.gif 53
  • http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png 54
  • http://www.brainybehavior.com/blog/wp-content/uploads/2008/11/gray747.png 55
  • 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
  • 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  What is it?  How is it contructed? ◦ Neurotransmitters StudiesImplications Conclusions 60
  • Galanin transgene Strong neurotropism Prevents initiation of SE Drastic reduction in seizure activity and number of seizures observed 61
  • Neuropeptide Y transgene Reduced number of seizures Delay in seizure onset No significant decrease in time spent in seizure activity 62
  • 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 Obtain FDA approval for use in humans 65
  • 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
  • Questions? 67
  • Questions? 68