Gene Therapy for Neurological Disorders

University dissertation from Lund University, Faculty of Medicine

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. 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.

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