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Gene Therapy for Neurological Disorders

Tönnesen, Jan LU (2010) In Lund University, Faculty of Medicine, Doctoral dissertation series 2010:17.
Abstract (Swedish)
Popular Abstract in Danish

Genterapi er en attraktiv terapiform mod neurologiske lidelser som Parkinsons sygdom og epilepsi af flere grunde. Gener med en terapeutisk effekt kan introduceres lokalt ved præcise injektioner i det syge væv, hvor genproduktet kan frigives fra nerveceller i respons til aktivitet i det omgivende væv. Ved genterapi omgås den forhindring som blod-hjernebarrieren udgør, ligesom der ikke er tab af terapeutisk effekt på grund a metabolisering af genproduktet, som det kan være tilfældet med konventionelle systemisk og oralt administrerede medikamenter. To strategier kan anvendes til at levere et terapeutisk gen; direkte injektion af an genvektor i det syge væv, såkaldt in vivo genterapi, eller... (More)
Popular Abstract in Danish

Genterapi er en attraktiv terapiform mod neurologiske lidelser som Parkinsons sygdom og epilepsi af flere grunde. Gener med en terapeutisk effekt kan introduceres lokalt ved præcise injektioner i det syge væv, hvor genproduktet kan frigives fra nerveceller i respons til aktivitet i det omgivende væv. Ved genterapi omgås den forhindring som blod-hjernebarrieren udgør, ligesom der ikke er tab af terapeutisk effekt på grund a metabolisering af genproduktet, som det kan være tilfældet med konventionelle systemisk og oralt administrerede medikamenter. To strategier kan anvendes til at levere et terapeutisk gen; direkte injektion af an genvektor i det syge væv, såkaldt in vivo genterapi, eller alternativt kan genet overføres til celler i laboratoriet, inden disse transplanteres ind i det syge væv, såkaldt ex vivo genterapi. En forudsætning for ex vivo genterapi er tilgængelighed stamceller, som kan generere nerveceller til at integrere i det syge væv og udtrykke det terapeutiske gen.

I de første tre artikler er fokus på stamcelleterapi indenfor Parkinsons sygdom. Stadig mere avancerede genetiske metoder anvendes til at udvikle og undersøge stemcellefødte dopaminerge nerveceller i relation til behandling af Parkinsons sygdom. Artikel I anvender genetisk immortalisering af humane neurale stamceller til at muliggøre ubegrænset ekspansion af stamcellelinien. Under de givne forhold udvikles disse stamceller dog ikke til fuldt funktionelle nerveceller. I artikel II og III præsenteres en protokol til at udvikle fuldt funktionelle dopaminerge nerveceller fra neurale stamceller fra mus, via genetisk introduktion af den dopaminerge faktor Wnt5a i stamcellerne. De dopaminerge nerveceller beskrives ved brug af elektrofysiologiske teknikker, for at afdække deres funktionelle egenskaber. Genetisk introducerede, optisk kontrollerede membranproteiner anvendes til at belyse den funktionelle integration af stamceller efter transplantation ind i et værtsvæv. Sådanne optogenetiske proteiner kan adressere transplanterede celler og værtsceller uafhængigt af hinanden, og med millisekund præcision aktivere eller inhibere disse, i respons til optisk aktivering med definerede lyskilder. Gennem denne nylige optogenetiske teknik demonstreres det, at værtsnervecellerne og de transplanterede dopaminerge neuroner kan kommunikere og påvirke hinandens aktivitet.

Artikel IV benytter også optogenetisk cellekontrol, men som et in vivo genterapeutisk redskab, ikke som et undersøgelsesværktøj, som beskrevet ovenfor. En eksperimentel epilepsimodel etableres, baseret på elektrisk stimuleringer, som bevirker epileptiform aktivitet i et vævspræparat. Et optogenetisk protein introduceres genetisk i nervecellerne i præparatet, og gennem optisk aktivering af dette kan nervecellerne inhiberes øjeblikkeligt. Når nervecellerne inhiberes optisk ved opståen af epileptiform aktivitet i vævet, kan den epileptiforme aktivitet reduceres til 20-50% af kontrolværdier uden inhibering. Taget i betragtning at mere end 30% af epilepsipatienter stadig oplever anfald på trods af medicinsk behandling, og at antiepileptisk medicin ofte ledsages af kognitive bivirkninger, er disse data motiverende for fremtidig forskning i optogenetik som en terapiform indenfor epilepsi.

Denne afhandling præsenterer ny viden indenfor eksperimentel forskning og terapi, og vil bidrage til at udvikle fremtidige stamcelle- samt genterapier mod neurologiske sygdomme. (Less)
Abstract
Gene therapy is an attractive strategy for neurological disorders, such as Parkinson’s disease and epilepsy, for several reasons. Introduction of genes with a therapeutic potential can be achieved locally, by accurate injections in the compromised tissue, and release of a transgene therapeutic substance can be regulated by the surrounding host neural circuitry. The gene therapy approach circumvents any issues pertaining to drug transport across the blood-brain barrier, or biometabolism of drugs before reaching their target, as can happen with conventional chemical drugs when administered orally or systemically. Two strategies are available for providing gene therapy. One is direct injection of the gene vector into the targeted tissue,... (More)
Gene therapy is an attractive strategy for neurological disorders, such as Parkinson’s disease and epilepsy, for several reasons. Introduction of genes with a therapeutic potential can be achieved locally, by accurate injections in the compromised tissue, and release of a transgene therapeutic substance can be regulated by the surrounding host neural circuitry. The gene therapy approach circumvents any issues pertaining to drug transport across the blood-brain barrier, or biometabolism of drugs before reaching their target, as can happen with conventional chemical drugs when administered orally or systemically. Two strategies are available for providing gene therapy. One is direct injection of the gene vector into the targeted tissue, referred to as in vivo gene therapy; the other is introduction of cells that are previously transduced to express the gene of interest, referred to as ex vivo gene therapy. A prerequisite for successfully applying neural ex vivo gene therapy is stem cell sources of neurons, which can be transfected with the therapeutic gene, and upon injection, integrate functionally in a diseased tissue and express the gene. This thesis explores and develops experimental gene therapeutic approaches to enhance therapies in neurological disorders.

Through the first three papers the focus is on stem cell therapy for Parkinson’s disease. Progressively more advanced genetic tools are applied to develop and describe stem cell-derived dopaminergic neurons for use in experimental Parkinson’s disease. In Paper I, human neural stem cells are genetically immortalized, to enable unlimited expansion of the cells. However, these cells fail to reach maturity under the presented circumstances. Paper II and III introduces a protocol for successfully generating mature dopaminergic neurons from mouse neural stem cells, by genetically introducing the dopaminergic factor Wnt5a in the cells. The resulting dopaminergic neurons are characterized with electrophysiological tools, to gain information on their functional properties. Genetically introduced, optically controlled membrane proteins are used to investigate the integration of the dopaminergic neurons in a host tissue after transplantation. Such optogenetic probes can independently target host or transplanted cells, and with millisecond resolution activate or inhibit these, in response to optical activation with defined light-sources. Through this novel optogenetic approach, it is demonstrated that the host tissue neurons and the transplanted dopaminergic neurons can communicate, and influence the activity of each other.

Paper IV applies optogenetic cell control, but utilizes it as an in vivo gene therapeutic tool, instead of as an investigative tool as above. We establish an experimental epilepsy model based on electrical stimulations, which elicit excessive, synchronized neural activity in an experimental tissue preparation. An optogenetic probe is genetically introduced to the neurons of the tissue preparation, and through optical activation of the probe, the neurons can be instantly inhibited. When optically inhibiting neurons at the onset of epileptiform activity, the duration of this activity was reduced to 20-50 % of the duration in control neurons. Considering that more than 30 % of epilepsy patients do not respond well to available anti-epileptic drugs, and that these drugs commonly has adverse cognitive side effects, the suggested role of optogenetic cell control in epilepsy holds great promises, though this research is still at an early stage.

This thesis provides new knowledge on experimental research and therapy approaches, which will contribute to understand and develop stem cell and gene therapies for neurological disorders. (Less)
Please use this url to cite or link to this publication:
author
supervisor
opponent
  • professor El Manira, Abdel, Karolinska Institue, Department of Neuroscience
organization
publishing date
type
Thesis
publication status
published
subject
keywords
epileptiform activity, striatum, organotypic culture, electrophysiology, Wnt5a, synaptic connectivity., cell transplantation, hippocampus, neural stem cells, epilepsy, functional integration, Parkinson’s disease, optogenetics, Gene therapy
in
Lund University, Faculty of Medicine, Doctoral dissertation series
volume
2010:17
pages
168 pages
publisher
Lund University, Faculty of Medicine
defense location
Segerfalksalen
defense date
2010-03-13 09:15
ISSN
1652-8220
ISBN
978-91-86443-31-3
language
English
LU publication?
yes
id
8b888435-f7e4-4450-86cf-570560799567 (old id 1578793)
date added to LUP
2010-03-19 11:34:21
date last changed
2018-05-29 11:49:36
@phdthesis{8b888435-f7e4-4450-86cf-570560799567,
  abstract     = {Gene therapy is an attractive strategy for neurological disorders, such as Parkinson’s disease and epilepsy, for several reasons. Introduction of genes with a therapeutic potential can be achieved locally, by accurate injections in the compromised tissue, and release of a transgene therapeutic substance can be regulated by the surrounding host neural circuitry. The gene therapy approach circumvents any issues pertaining to drug transport across the blood-brain barrier, or biometabolism of drugs before reaching their target, as can happen with conventional chemical drugs when administered orally or systemically. Two strategies are available for providing gene therapy. One is direct injection of the gene vector into the targeted tissue, referred to as in vivo gene therapy; the other is introduction of cells that are previously transduced to express the gene of interest, referred to as ex vivo gene therapy. A prerequisite for successfully applying neural ex vivo gene therapy is stem cell sources of neurons, which can be transfected with the therapeutic gene, and upon injection, integrate functionally in a diseased tissue and express the gene. This thesis explores and develops experimental gene therapeutic approaches to enhance therapies in neurological disorders. <br/><br>
Through the first three papers the focus is on stem cell therapy for Parkinson’s disease. Progressively more advanced genetic tools are applied to develop and describe stem cell-derived dopaminergic neurons for use in experimental Parkinson’s disease. In Paper I, human neural stem cells are genetically immortalized, to enable unlimited expansion of the cells. However, these cells fail to reach maturity under the presented circumstances. Paper II and III introduces a protocol for successfully generating mature dopaminergic neurons from mouse neural stem cells, by genetically introducing the dopaminergic factor Wnt5a in the cells. The resulting dopaminergic neurons are characterized with electrophysiological tools, to gain information on their functional properties. Genetically introduced, optically controlled membrane proteins are used to investigate the integration of the dopaminergic neurons in a host tissue after transplantation. Such optogenetic probes can independently target host or transplanted cells, and with millisecond resolution activate or inhibit these, in response to optical activation with defined light-sources. Through this novel optogenetic approach, it is demonstrated that the host tissue neurons and the transplanted dopaminergic neurons can communicate, and influence the activity of each other.<br/><br>
Paper IV applies optogenetic cell control, but utilizes it as an in vivo gene therapeutic tool, instead of as an investigative tool as above. We establish an experimental epilepsy model based on electrical stimulations, which elicit excessive, synchronized neural activity in an experimental tissue preparation. An optogenetic probe is genetically introduced to the neurons of the tissue preparation, and through optical activation of the probe, the neurons can be instantly inhibited. When optically inhibiting neurons at the onset of epileptiform activity, the duration of this activity was reduced to 20-50 % of the duration in control neurons. Considering that more than 30 % of epilepsy patients do not respond well to available anti-epileptic drugs, and that these drugs commonly has adverse cognitive side effects, the suggested role of optogenetic cell control in epilepsy holds great promises, though this research is still at an early stage.<br/><br>
This thesis provides new knowledge on experimental research and therapy approaches, which will contribute to understand and develop stem cell and gene therapies for neurological disorders.},
  author       = {Tönnesen, Jan},
  isbn         = {978-91-86443-31-3},
  issn         = {1652-8220},
  keyword      = {epileptiform activity,striatum,organotypic culture,electrophysiology,Wnt5a,synaptic connectivity.,cell transplantation,hippocampus,neural stem cells,epilepsy,functional integration,Parkinson’s disease,optogenetics,Gene therapy},
  language     = {eng},
  pages        = {168},
  publisher    = {Lund University, Faculty of Medicine},
  school       = {Lund University},
  series       = {Lund University, Faculty of Medicine, Doctoral dissertation series},
  title        = {Gene Therapy for Neurological Disorders},
  volume       = {2010:17},
  year         = {2010},
}