Development of Intravenous AAV Vectors for Intractable Epilepsy

描述（由申请人提供）：癫痫困扰着美国约300万人，虽然目前的抗癫痫药物有效地控制了约70%的癫痫患者的发作，但药物不能充分控制其余30%的癫痫发作（Kwan和Brodie，2000）。因为少于10%的药物难治性癫痫患者被考虑进行手术切除，所以大量癫痫患者基本上没有有效的治疗选择（Engel等人; 1992，Siegel，2004）。即使引入了许多新药，耐药性癫痫的数量也没有减少，促使Loscher和施密特（2011）指出“现有证据表明癫痫药物治疗的疗效和耐受性没有实质性改善”。Gray等人（2010年）的最新研究为这一问题提供了一个潜在的解决方案。使用急性边缘癫痫发作模型，Gray等人（2010）显示衣壳DNA改组和定向进化可以鉴定新的嵌合腺相关病毒（AAV）克隆（#83），其在静脉内施用后选择性地穿过急性癫痫发作损害的血脑屏障和CNS中的转导细胞。为了实现这种方法的全部潜力，我们假设在慢性边缘癫痫发作模型中的额外DNA改组和定向进化将产生安全的、治疗有效的嵌合载体。将构建两个新的脑特异性AAV衣壳文库，一个基于克隆83的易错PCR，另一个由穿过癫痫损害的血脑屏障的独特的多个克隆组成。将文库静脉注射到记录有慢性自发性癫痫发作活动的大鼠中。随后，神经元将从海马和梨状皮质中分离出来，突变克隆将被拯救。在转导功效和外周生物分布的体内验证之后，将最有效的克隆与经证实的治疗性神经肽盒包装，并将产生重组病毒。这些新型载体将静脉内给予具有记录的自发性癫痫发作活动的大鼠，以评估减弱自发性边缘癫痫发作活动的能力，以及在急性癫痫发作后测试抗癫痫作用。如果成功，这些发现将极大地改变目前的癫痫治疗模式，并显着影响治疗难治性癫痫人群。

DESCRIPTION (provided by applicant): Epilepsy afflicts approximately 3 million people in the U.S., and although current, anti-epileptic medication effectively controls the seizures in approximately 70% of this population, medications do not adequately control seizures for the remaining 30% (Kwan and Brodie, 2000). Because fewer than 10% of patients with drug refractory epilepsy are considered for surgical resection, a substantial number of epilepsy patients essentially have no effective therapeutic options (Engel et al.; 1992, Siegel, 2004). Even with the introduction of many new drugs, the number of drug resistant epilepsies has not decreased, prompting Loscher and Schmidt (2011) to state that "the available evidence indicates that the efficacy and tolerability of drug treatment of epilepsy has not substantially improved". Recent studies by Gray et al (2010) have provided a potential solution to this problem. Using an acute limbic seizure model Gray et al. (2010) showed that capsid DNA shuffling and directed evolution could identify a novel, chimeric adeno-associated virus (AAV) clone (#83) which upon intravenous administration selectively crossed the acute seizure compromised blood-brain barrier and transduced cells in the CNS. In order to realize the full potential of this approach we hypothesize that additional DNA shuffling and directed evolution in a chronic limbic seizure model will produce safe, therapeutically effective, chimeric vectors. Two new brain specific AAV capsid libraries will be constructed, one based upon error prone PCR of clone 83 and the other composed of unique, multiple clones that cross the seizure compromised blood-brain barrier. The libraries will be injected intravenously into rats with documented chronic, spontaneous seizure activity. Subsequently, neurons will be dissociated from the hippocampus and the piriform cortex, and the mutant clones will be rescued. After in vivo validation of both transduction efficacy and peripheral biodistribution, the most effective clones will be packaged with proven therapeutic neuropeptide cassettes and recombinant virus will be produced. These novel vectors will be administered intravenously to rats with documented spontaneous seizure activity in order to assess the ability to attenuate spontaneous limbic seizure activity, as well as after acute seizures to test for anti-epileptogenic actions. If successful the findings would dramatically shift current epilepsy treatment paradigms and significantly impact the treatment refractory epileptic population.