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

描述（由申请人提供）：美国约有250万人患有癫痫，使癫痫成为最普遍的神经系统疾病之一（Hauser and Hesdorffer, 1990）。虽然目前，抗癫痫药物有效地控制了大约70%的人口的癫痫发作，但其余30%的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，手术切除是最后的选择，对于那些符合手术条件的患者，高达66%的患者在术后2年内没有癫痫发作（Spencer et al, 2005）。然而，长期预后更不确定，如果癫痫发作部位影响到与关键功能相关的大脑部位，切除手术就不是可行的选择（McIntosh et al., 2004; Ojemann, 1997）。最近，一些研究已经建立了显著影响体内癫痫敏感性的基因治疗方法。利用腺相关病毒（AAV）载体，神经活性肽甘丙肽的表达和组成性分泌可以在体内预防癫痫发作（Haberman et al., 2003; McCown, 2006），而前体神经肽Y的表达也可以显著减弱体内癫痫发作活动（Richichi et al., 2004）。然而，目前，这些病毒载体基因疗法的临床应用将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规避神经外科干预的潜在手段。Maheshri等人（2006）表明，通过易出错PCR在AAV 2衣壳中引入随机变化，随后的定向进化导致AAV突变体缺乏肝素结合或逃避对AAV 2的体内免疫反应。在初步研究中，Samulski实验室已经证明，DNA洗牌技术与定向进化的结合产生了具有独特体内趋向性的新型突变AAV载体。由于癫痫发作损害了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重组来开发突变AAV文库。然后，在kainic酸诱导的癫痫发作后，突变文库将被静脉注射，并通过定向进化技术，将识别出新的突变AAV载体，这些载体将在癫痫发作后穿过受损的血脑屏障并转导神经元。这种突变AAV载体的成功创造可能会彻底改变顽固性癫痫的治疗。提出的研究重点是开发一种新的基因治疗方法来治疗神经系统疾病。使用定向进化技术，我们将选择特定的基因传递载体，可以通过外周静脉注射进入癫痫受损的大脑。如果成功，这种新方法将极大地推进基因疗法在神经疾病治疗中的应用。

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.