This grant was originally funded under a RFA that included a specific emphasis on the development of a "tool- kit" or technological advances in the area of gene transfer to the nervous system to facilitate both basic science research and successful translation to the clinic. The current application builds on our progress during the tenure of the award and now proposes some exciting developments with themes of astrocytic targeting and physiological autoregulatory strategies to increase the overall utility, effectiveness and safety of gene transfer in the CNS for both basic science and clinical applications. We propose here both basic vector characterization experiments as well as in vivo proof of concept experiments in well established models of neurological disease. In this grant application, we build on the promising finding that one AAV variant has strong tropism for astrocytes, and this vector thereby allows us to pursue a hypothesis that we have been wanting to test for many years. In brief, in 1992 we demonstrated using human microdialysis that adenosine was released in the conscious human brain, and that during an ictal event rose to concentrations that would likely arrest the seizure and cause post-ictal refractoriness. Moreover, we noted that in the interictal state, the basal concentration of adenosine was lower in the epileptogenic hippocampus, consistent with the astrogliosis which characterizes such hippocampi. At that time, we wished to genetically manipulate adenosine levels, specifically increase extracellular levels to increase local inhibition and test the hypothesis that such an intervention would have strong anti-epileptic effect. The key protein which regulates brain adenosine levels is adenosine kinase which is exclusively expressed in astrocytes, hence genetic manipulation required an astrocytic tropic vector. The astrogliosis which characterizes human temporal lobe epilepsy is associated with increased expression of adenosine kinase and has been proposed as a critical component of epileptogenesis. Here, we propose to knockdown adenosine kinase using our new isolate and test this in an epilepsy model. A second component of the grant is to build on another development made during the original award period. We have constructed a new regulatory system in which transgene expression is linked to the physiology of the cell. Specifically, we incorporate a second expression cassette in which a microRNA to the transgene is driven by a promoter which is suppressed in pathophysiological situations, but active in the normal physiological state. Hence, this approach couples the disease phenotype to gene expression and provides for the potential of tight physiological autoregulation. We propose to use this system to regulate expression of the adenosine A1 receptor in a spontaneous seizure model, an approach which directly complements our adenosine kinase knockout experiments.
Neurological diseases represent a significant challenge on the way to cure. To improve on neurological treatments, our laboratory has developed the tools and expertise to target a specific brain region, and within that brain region, we can now target specific cells. Here, we propose to use this new skill, to modify the function of so-called glial cells, often thought of the "glue of the brain", but now we know these cells have important functions in diseases like epilepsy. We will genetically modify these cells to see if we can reduce seizures in an animal model of epilepsy with a second part of the project developing a safer way to control our genetic intervention.
|Young, D; Fong, D M; Lawlor, P A et al. (2014) Adenosine kinase, glutamine synthetase and EAAT2 as gene therapy targets for temporal lobe epilepsy. Gene Ther 21:1029-40|
|Charbel Issa, Peter; De Silva, Samantha R; Lipinski, Daniel M et al. (2013) Assessment of tropism and effectiveness of new primate-derived hybrid recombinant AAV serotypes in the mouse and primate retina. PLoS One 8:e60361|
|Kalev-Zylinska, Maggie L; Symes, Wymond; Little, Kevin C E et al. (2013) Stroke patients develop antibodies that react with components of N-methyl-D-aspartate receptor subunit 1 in proportion to lesion size. Stroke 44:2212-9|
|Blouin, Ashley M; Stern, Anna L; Han, Sungho et al. (2013) Neuronal activity-regulated pentraxin expressed in medial prefrontal cortex neurons is not necessary for extinction of heroin self-administration. Behav Pharmacol 24:332-6|
|Carruthers, Katherine H; During, Matthew J; Muravlev, Alexander et al. (2013) Fat grafting as a vehicle for the delivery of recombinant adenoassociated viral vectors to achieve gene modification of muscle flaps. Ann Plast Surg 70:726-31|
|Lin, En-Ju D; Choi, Eugene; Liu, Xianglan et al. (2011) Environmental enrichment exerts sex-specific effects on emotionality in C57BL/6J mice. Behav Brain Res 216:349-57|
|Cao, Lei; Choi, Eugene Y; Liu, Xianglan et al. (2011) White to brown fat phenotypic switch induced by genetic and environmental activation of a hypothalamic-adipocyte axis. Cell Metab 14:324-38|
|Fitzsimons, Helen L; Riban, Veronique; Bland, Ross J et al. (2010) Biodistribution and safety assessment of AAV2-GAD following intrasubthalamic injection in the rat. J Gene Med 12:385-98|
|Kalev-Zylinska, Maggie L; Symes, Wymond; Young, Deborah et al. (2009) Knockdown and overexpression of NR1 modulates NMDA receptor function. Mol Cell Neurosci 41:383-96|
|Lawlor, Patricia A; Bland, Ross J; Mouravlev, Alexandre et al. (2009) Efficient gene delivery and selective transduction of glial cells in the mammalian brain by AAV serotypes isolated from nonhuman primates. Mol Ther 17:1692-702|
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