The main research challenge for our group is the development of gene transfer vectors for use in the CNS.
These vectors should give for long-term gene expression in the desired cell type. Preferably, the vectors allow for the level of transgene expression to be regulated. Moreover, the vectors should not be toxic to the host brain and be tolerated by the brain for extended periods of time.
Cell specific vectors may be used in gene therapy to limit transgene expression to a specific cell type. This is achieved by using the promoter of a gene that is exclusively expressed in a sought cell type. This will limit the transgene expression to that cell type even though the vector is transferred to several cell types. By using a promoter of a gene that is also up-regulated in a certain disease state, the vector could also be made disease specific and auto-regulated. This is possible since the expression of the transgene will mimic the expression of the endogenous gene as it changes with the state of the disease.
We use promoters of genes that have previously been described in literature as cell specific or an exclusive part of the direct or indirect pathway of the basal ganglia circuitry. We also use promoters of genes that have been shown to be highly expressed in patients with Parkinson’s disease (see e.g. Miller et al Neurobiol Dis. 2006 Feb;21(2):305-13).The developed vectors are validated using the 6-OHDA model of Parkinson’s disease.
Changing the gene expression in specific cell types of striatum could be a way to modulate the de-coordinated motor functions of PD patients. Now a technology is emerging, using so-called artificial transcription factors, which could enable us to much more readily modify or alter a cell’s existing genes.
Transcription factors are typically composed of a DNA binding domain, responsible for specific contacts with DNA, and an effector domain that mediates activation or repression of targeted genes. As for the DNA binding domain, zinc finger proteins are utilized as they bind into the DNA helix at a specific set of three bases. By making polydactyl zinc fingers, in this case six proteins ordered in a head to tail fashion, they will recognize an 18 base pairs sequence. This is thought to be long enough for specificity in the human genome. Linked to these zinc fingers are either the effector domains VP64 or KRAB.
However, a hurdle in the development of ATFs is that the polydactyl zinc fingers must be tailored for each target gene, which only a few labs can do at the moment. Through collaboration with senior researcher Torbjörn Gräslund at KTH we are combining the ATFs with cell specific vectors. Thus, we are now in a position where we can regulate a genes expression, up or down, simultaneously in a tissue. Our goal is to normalize the disregulated pathways of Parkinsonian striatum i.e to up-regulate the indirect pathway and down-regulate the direct pathway.
Development and screening of different siRNAs to target and normalize GAD67 expression in experimental Parkinson's disease
In Parkinson’s disease (PD) the projection neurons of the striatum become dysregulated due to the loss of dopamine as the dopamine neurons of the substantia nigra die. As a consequence, most of the enkephalinergic neurons become over-active in response to the dopamine receptor agonist-induced dysregulation and both GAD67 and GABA are up-regulated in Parkinson’s. The aim of the project is to develop cell-specific lentiviral vectors that express siRNAs (smiRNAs & shRNAs) directed against the GAD67 transcript specifically in the enkephalinergic striatal neurons of the indirect pathway to normalize GAD67 expression and to evaluate the functional outcome of the knock-down using molecular techniques (qPCR and WB) in a rat model of PD providing a new therapeutic approach for Parkinson’s.
Pro-inflammatory molecules and cell-specific lentiviral vectors in a rat model
Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, likely associated with dysregulation of oxidation of catechols, such as dopamine (DA) and 6-hydroxydopamine (6-OHDA), and resulting in oxidative stress. The involvement of proinflammatory molecules in pathogenesis of Parkinson's disease has been suggested. We will design different shRNA expressing lentiviral constructs directed against proinflammatory molecules and the shRNA containing cell-specific lentiviral vectors will be used for viral propagation and subsequent transfection of specific cell lines to validate their effectiveness in vitro. The most effective candidates will then be used for in vivo experiments to evaluate the possibility of using cell-specific lentiviral vectors expressing shRNAs for the development of a new therapeutic rat model of PD.
Developing new regulable lentiviral vectors for use in the brain.RNA interference, tetracycline-inducible and Cre/lox P systems have emerged as powerful technologies for downregulation of specific genes in cells and animals. However, these systems are not always tight but leaky.
A new method has been developed that conditionally controls the protein function (Banaszynski et al, Cell. 2006 Sep 8;126(5):995-1004). This method is potentially faster and more tunable than traditional methods and offers great potential for assessing the role of proteins in physiological environment.
This alternative strategy consists of using a small protein domain, termed a destabilizing domain (DD), which confers instability to fusion protein (DD-YFP) in cultured cells. Instability is counteracted when a ligand binds this domain.
The challenge in this project is to validate this novel approach in vivo, by using a lentiviral expression system in order to achieve an optimal expression of fusion protein (DD-YFP) in the striatum, efficiently stabilized by the ligand in a dose and time dependent manner.
The advantage of this method is: first, the possibility to fuse the destabilizing domain to a `neuroprotective´ protein such as GDNF, and second, the tight and reversible regulation of its expression and function, conferred by the ligand.
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Last modified: 2009-04-07
