Directed Evolution of Adeno-Associated Virus Vectors for Seizure Gene Therapy

描述（由申请人提供）：在美国，癫痫折磨着大约250万人，使得癫痫成为最普遍的神经系统疾病之一（豪瑟和Hesdorffer，1990）。尽管目前抗癫痫药物有效控制了约70%的该人群的癫痫发作，但对于其余30%的人群，药物不能充分控制癫痫发作（Kwan和Brodie，2000）。在这些难治性病例中，手术切除提供了最终选择，并且对于那些符合手术条件的患者，高达66%的患者在手术后2年内保持无癫痫发作（Spencer等人，2005年）。然而，长期预后更不确定，并且如果癫痫发作发生的部位撞击与关键功能相关的脑部位，则切除手术不是可行的选择（McIntosh等人，2004; Ojemann，1997）。最近，一些研究已经建立了基因治疗的方法，显着影响癫痫发作的敏感性在体内。使用腺相关病毒（AAV）载体，神经活性肽甘丙肽的表达和组成性分泌可以防止体内癫痫发作活性（Haberman et al.，2003; McCown，2006），而前原神经肽Y的表达也显著减弱体内癫痫发作活性（Richichi等人，2004年）。然而，目前，这些病毒载体基因疗法的临床应用需要直接神经外科注射到大脑中。AAV定向进化的最新进展提供了一种潜在的手段来规避神经外科干预。Maheshri等（2006）显示，通过易错PCR在AAV 2衣壳中引入随机变化，随后的定向进化导致缺乏肝素结合或逃避对AAV 2的体内免疫应答的AAV突变体。在初步研究中，Samulski实验室已经表明，DNA改组技术与定向进化的组合产生了具有独特体内向性的新型突变体AAV载体。由于癫痫发作损害癫痫发作活动区域的血脑屏障，我们建议通过AAV衣壳1-9序列的DNA改组来开发突变体AAV文库。然后在红藻氨酸诱导的癫痫发作后，将静脉内施用突变体文库，并通过定向进化技术，将鉴定出新的突变体AAV载体，其在癫痫发作活动后穿过受损的BBB和BBB神经元。这种突变体AAV载体的成功创建可能会彻底改变难治性癫痫发作疾病的治疗。拟议的研究集中在开发一种新的基因治疗方法用于治疗神经系统疾病。利用定向进化技术，我们将选择特定的基因递送载体，这些载体可以在外周静脉注射后进入癫痫发作受损的大脑。如果成功的话，这种新的方法可以大大推进基因治疗在神经系统疾病治疗中的应用。

DESCRIPTION (provided by applicant): Epilepsy afflicts approximately 2.5 million people in the U.S., making epilepsy one of the most prevalent neurological disorders (Hauser and Hesdorffer, 1990). Although current, anti-epileptic medication effectively controls the seizures in approximately 70% of this population, for the remaining 30% medications do not adequately control the seizures (Kwan and Brodie, 2000). In these intractable cases, surgical resection offers a final option, and for those patients that qualify for surgery, up to 66% remain seizure free for 2 years post- surgery (Spencer et al., 2005). However, longer term prognosis is more uncertain, and if the site of seizure genesis impinges upon brain sites associated with critical functions, resective surgery is not a viable option (McIntosh et al., 2004; Ojemann, 1997). Recently, several studies have established gene therapy approaches that significantly influence seizure sensitivity in vivo. Using adeno-associated virus (AAV) vectors, the expression and constitutive secretion of the neuroactive peptide, galanin, can prevent seizure activity in vivo (Haberman et al., 2003; McCown, 2006), while the expression of prepro-neuropeptide Y also significantly attenuates seizure activity in vivo (Richichi et al., 2004). At present, though, the clinical application of these viral vector gene therapies will require direct neurosurgical injection into the brain. Recent developments in AAV directed evolution provide a potential means to circumvent neurosurgical intervention. Maheshri et al. (2006) showed that by introducing random changes in the AAV 2 capsid by error prone PCR, subsequent directed evolution resulted in AAV mutants that lacked heparin binding or evaded the in vivo immune response to AAV 2. In preliminary studies, the Samulski laboratory has shown that the combination of DNA shuffling techniques with directed evolution produces novel mutant AAV vectors with unique in vivo tropisms. Because seizures compromise the blood-brain barrier in areas of seizure activity, we propose to develop a mutant AAV library through DNA shuffling of AAV capsid 1-9 sequences. Then after kainic acid-induced seizures, the mutant library will be administered i.v. and through directed evolution techniques, novel mutant AAV vectors will be identified that following seizure activity, cross the compromised BBB and transduce neurons. The successful creation of such mutant AAV vectors could revolutionize the treatment of intractable seizure disorders. The proposed studies focus upon developing a novel gene therapy method for the treatment of neurological disorders. Using directed evolution techniques, we will select specific gene delivery vehicles that can access a seizure-impaired brain after peripheral intravenous administration. If successful, this novel approach could greatly advance the application of gene therapy to the treatment of neurological disorders.