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Chemogenetics, Induced Neurons and Pluripotent Stem Cells: Towards Advanced Gene and Cell Therapies Targeting Epilepsy

Avaliani, Natalia LU (2015) In Lund University Faculty of Medicine Doctoral Dissertation Series 2015:118.
Abstract
The complexity of the central nervous system and existence of the blood-brain barrier often causes difficulties for traditional pharmacological treatments of neurological diseases. This thesis explores the feasibility and potential for novel gene and cell therapy approaches, which hold better promise for neurological disorders, while particularly targeting epilepsy.

Epilepsy is a multifactorial neurological disorder affecting 1% of the population. Available pharmacological therapies are merely symptomatic, and have severe side effects, while failing to adequately control seizures in one third of the patients, predominately in those with temporal lobe epilepsy (TLE). Targeted silencing of the pathological circuits by expressing... (More)
The complexity of the central nervous system and existence of the blood-brain barrier often causes difficulties for traditional pharmacological treatments of neurological diseases. This thesis explores the feasibility and potential for novel gene and cell therapy approaches, which hold better promise for neurological disorders, while particularly targeting epilepsy.

Epilepsy is a multifactorial neurological disorder affecting 1% of the population. Available pharmacological therapies are merely symptomatic, and have severe side effects, while failing to adequately control seizures in one third of the patients, predominately in those with temporal lobe epilepsy (TLE). Targeted silencing of the pathological circuits by expressing therapeutic genes, or increasing the inhibition by introducing new populations of GABA-releasing neurons, might prove therapeutic for epilepsy, by counteracting seizures and even modifying the disease.

Gene therapy offers localized, cell-type specific alteration of neuronal excitability, but on-demand seizure suppression can only be achieved by tools allowing external temporal control. One such recently developed chemogenetic technology is based on viral expression of modified muscarinic G-protein coupled receptors, specifically activated by otherwise inert clozapine-N-oxide (CNO). In paper I, we explored if such modified receptor, hM4Di, which selectively activates Gi pathway, thereby causing neuronal inhibition, could suppress epileptiform activity upon CNO application. This approach proved effective for localized suppression of neuronal excitability and seizure-like events in an in vitro model of TLE, organotypic hippocampal slice cultures (OHSC), without altering intrinsic properties of the hM4Di-expressing neurons, demonstrating the therapeutic potential of this technology.

In papers II and III we characterized two different cell sources with the prospect of cell replacement therapy: induced Pluripotent Stem cells (iPS) and induced Neurons (iN). These two patient specific alternative cell sources offer a solution to ethical and immunogenicity issues, related to embryonic stem cell use.

Already six weeks after grafting in OHSCs, iPS-derived neuroepitelial-like stem cells (lt-NES), predominately differentiating to GABA-ergic neurons, displayed functional neuronal properties and certain rate of synaptic integration. In vivo studies showed that longer differentiation time (up to 24 weeks) was needed for the grafts to fully mature and extensively integrate into the host synaptic network. The grafted cells still retained some of the distinct electrophysiological features, however, such as high input resistance.

Next, we studied long-term survival of human foetal fibroblast-derived induced neurons (iN) in rodent hippocampus. Human iNs survive and maintain neuronal profile up to six months post-grafting, developing more elaborate neuronal morphology and complex dendritic arborisation over time. Graft-derived neurons with mature neuronal properties could be observed at six months, although a portion of non-converted fibroblasts, as well as asynchronous neuronal conversion was apparent among the grafts. Improvements in conversion, survival and integration rate of iN cells are required before these cells can offer a better alternative to iPS or stem cells. While showing potential as candidates for cell replacement therapy, both characterised cell types have to be further tested in relevant epilepsy model systems to demonstrate their therapeutic effect.

In summary, this thesis adds new knowledge and experimental basis for development of gene- and cell-based therapies for neurological disorders. (Less)
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author
supervisor
opponent
  • Associate Professor, PhD Silberberg, Gilad, Department of Neuroscience, Karolinska Institute, Stockholm, Sweden
organization
publishing date
type
Thesis
publication status
published
subject
keywords
Chemogenetics, iPS, iN, electrophysiology, gene and cell therapy, Epilepsy
in
Lund University Faculty of Medicine Doctoral Dissertation Series
volume
2015:118
pages
75 pages
publisher
Department of Clinical Sciences, Lund University
defense location
A1005, Segerfalksalen, Wallenberg Neurocentrum, Sölvegatan 19, 223 62 Lund, Sweden
defense date
2015-11-06 13:15
ISSN
1652-8220
ISBN
978-91-7619-197-2
language
English
LU publication?
yes
id
d33cc485-af20-4281-a46f-9efaf828d81a (old id 8171362)
date added to LUP
2015-11-18 15:05:04
date last changed
2017-09-19 14:58:15
@phdthesis{d33cc485-af20-4281-a46f-9efaf828d81a,
  abstract     = {The complexity of the central nervous system and existence of the blood-brain barrier often causes difficulties for traditional pharmacological treatments of neurological diseases. This thesis explores the feasibility and potential for novel gene and cell therapy approaches, which hold better promise for neurological disorders, while particularly targeting epilepsy.<br/><br>
Epilepsy is a multifactorial neurological disorder affecting 1% of the population. Available pharmacological therapies are merely symptomatic, and have severe side effects, while failing to adequately control seizures in one third of the patients, predominately in those with temporal lobe epilepsy (TLE). Targeted silencing of the pathological circuits by expressing therapeutic genes, or increasing the inhibition by introducing new populations of GABA-releasing neurons, might prove therapeutic for epilepsy, by counteracting seizures and even modifying the disease.<br/><br>
Gene therapy offers localized, cell-type specific alteration of neuronal excitability, but on-demand seizure suppression can only be achieved by tools allowing external temporal control. One such recently developed chemogenetic technology is based on viral expression of modified muscarinic G-protein coupled receptors, specifically activated by otherwise inert clozapine-N-oxide (CNO). In paper I, we explored if such modified receptor, hM4Di, which selectively activates Gi pathway, thereby causing neuronal inhibition, could suppress epileptiform activity upon CNO application. This approach proved effective for localized suppression of neuronal excitability and seizure-like events in an in vitro model of TLE, organotypic hippocampal slice cultures (OHSC), without altering intrinsic properties of the hM4Di-expressing neurons, demonstrating the therapeutic potential of this technology.<br/><br>
In papers II and III we characterized two different cell sources with the prospect of cell replacement therapy: induced Pluripotent Stem cells (iPS) and induced Neurons (iN). These two patient specific alternative cell sources offer a solution to ethical and immunogenicity issues, related to embryonic stem cell use. <br/><br>
Already six weeks after grafting in OHSCs, iPS-derived neuroepitelial-like stem cells (lt-NES), predominately differentiating to GABA-ergic neurons, displayed functional neuronal properties and certain rate of synaptic integration. In vivo studies showed that longer differentiation time (up to 24 weeks) was needed for the grafts to fully mature and extensively integrate into the host synaptic network. The grafted cells still retained some of the distinct electrophysiological features, however, such as high input resistance. <br/><br>
Next, we studied long-term survival of human foetal fibroblast-derived induced neurons (iN) in rodent hippocampus. Human iNs survive and maintain neuronal profile up to six months post-grafting, developing more elaborate neuronal morphology and complex dendritic arborisation over time. Graft-derived neurons with mature neuronal properties could be observed at six months, although a portion of non-converted fibroblasts, as well as asynchronous neuronal conversion was apparent among the grafts. Improvements in conversion, survival and integration rate of iN cells are required before these cells can offer a better alternative to iPS or stem cells. While showing potential as candidates for cell replacement therapy, both characterised cell types have to be further tested in relevant epilepsy model systems to demonstrate their therapeutic effect. <br/><br>
In summary, this thesis adds new knowledge and experimental basis for development of gene- and cell-based therapies for neurological disorders.},
  author       = {Avaliani, Natalia},
  isbn         = {978-91-7619-197-2},
  issn         = {1652-8220},
  keyword      = {Chemogenetics,iPS,iN,electrophysiology,gene and cell therapy,Epilepsy},
  language     = {eng},
  pages        = {75},
  publisher    = {Department of Clinical Sciences, Lund University},
  school       = {Lund University},
  series       = {Lund University Faculty of Medicine Doctoral Dissertation Series},
  title        = {Chemogenetics, Induced Neurons and Pluripotent Stem Cells: Towards Advanced Gene and Cell Therapies Targeting Epilepsy},
  volume       = {2015:118},
  year         = {2015},
}