Development of Intravenous AAV Vectors for Intractable Epilepsy

描述(申请人提供)：在美国大约有300万人患有癫痫，虽然目前的抗癫痫药物有效地控制了大约70%的人的癫痫发作，但药物不能充分控制其余30%的人的癫痫发作(Kwan和Brodie，2000)。由于只有不到10%的药物难治性癫痫患者考虑进行手术切除，相当数量的癫痫患者基本上没有有效的治疗选择(Engel等人；1992，Siegel，2004)。即使推出了许多新药，耐药癫痫的数量并没有减少，这促使Loscher和Schmidt(2011)指出，“现有证据表明，癫痫的药物治疗的有效性和耐受性并没有实质性的改善”。Gray等人(2010)最近的研究为这个问题提供了一个潜在的解决方案。使用急性边缘癫痫模型，Gray等人。(2010)表明衣壳DNA改组和定向进化可以识别一种新的嵌合腺相关病毒(AAV)克隆(#83)，该克隆在静脉注射时选择性地穿过急性癫痫发作，损害血脑屏障并转导中枢神经系统中的细胞。为了实现这一方法的全部潜力，我们假设在慢性边缘癫痫模型中额外的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，一个基于容易出错的克隆83的聚合酶链式反应，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。这些文库将被静脉注射到有记录的慢性自发癫痫发作活动的大鼠体内。随后，神经元将从海马区和梨状皮质分离，突变克隆将被拯救。在体内验证转导效果和外周生物分布后，最有效的克隆将与经过验证的治疗性神经肽盒一起包装，并将产生重组病毒。这些新型载体将被静脉注射到有自发性癫痫发作活动的大鼠身上，以评估其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的能力。如果成功，这一发现将极大地改变目前的癫痫治疗模式，并对治疗难治性癫痫人群产生重大影响。

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.