Abstracts

Rationale: Given the high incidence of refractory epilepsy, novel therapeutic approaches and concepts are urgently needed. To date, viral mediated delivery and endogenous expression of antisense sequences as a strategy to prevent seizures has received little attention in epilepsy therapy development efforts. Here we validate adenosine kinase (ADK), the astrocyte-based key negative regulator of the brain s endogenous anticonvulsant adenosine, as therapeutic target for antisense-mediated seizure suppression. Methods: We developed adeno-associated virus 8 (AAV8)-based gene therapy vectors to selectively modulate ADK expression in astrocytes. Cell type selectivity was achieved by expressing an Adk-cDNA in sense (to overexpress ADK) or antisense (to knockdown ADK) orientation under the control of an astrocyte-specific gfaABC1D promoter. Viral vectors (10^12 genomic particles per ml) where injected into the CA3 hippocampal subfiled of wild-type mice (n=4) or spontaneously epileptic Adk-tg transgenic mice (n=4) that show a global, brain-wide overexpression of ADK (141% of normal). An AAV8-Gfa-null virus was used as control (n=4). After virus injection ADK expression was assessed histologically and biochemically. In addition, animals were subjected to 12-hour sessions of continuous intracranial EEG-monitoring using intrahippocampal bipolar electrodes placed into the CA3 region. Results: We demonstrate in wild-type mice that viral overexpression of ADK (145% of normal) within astrocytes of the CA3 region is sufficient to trigger spontaneous recurrent seizures in the absence of any other epileptogenic event. Seizures in the Adk-sense injected mice were frequent in terms of the average number of seizures per hour (6.6 0.21, ***p = 0.0001) and the long seizure duration (81.2 0.6 sec, ***p = 0.0002) compared to related events recorded in null virus injected control animals (1.38 0.38 seizures per hour, duration = 45.3 7.8 sec). These results were confirmed by western blot analysis of ipsi- and contralateral hippocampal protein extracts from Adk-SS injected mice (n = 2). Additionally, AAV8-mediated RNA interference almost completely abolished spontaneous recurrent seizures in Adk-tg mice (**p=0.006) compared to null virus injected Adk-tg mice. Conclusions: Our data demonstrate that over- or underexpression of astrocytic ADK in the absence any epileptogenic factor is sufficient to trigger or prevent seizures, respectively. This is the first study to use an antisense approach to validate ADK as a rational therapeutic target for the treatment of epilepsy and suggests that gene therapies based on the knock down of ADK might be a feasible approach to control seizures in refractory epilepsy.