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

用于癫痫基因治疗的腺相关病毒载体的定向进化

基本信息

批准号：
7289549
负责人：
Thomas J. McCown
金额：
$ 15.74万
依托单位：
UNIV OF NORTH CAROLINA CHAPEL HILL
依托单位国家：
美国
项目类别：
财政年份：
2007
资助国家：
美国
起止时间：
2007-07-01 至 2009-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7289549
关键词：
Antiepileptic Agents Area Attenuated Blood - brain barrier anatomy Brain Capsid Capsid Proteins Cells Cellular Tropism Chickens Class Confocal Microscopy DNA Shuffling Deoxyribonuclease I Deoxyribonucleases Dependovirus Development Digestion Epilepsy Event Excision Exhibits Galanin Gene Delivery Generalized seizures Genes Green Fluorescent Proteins Heparin Binding Hippocampus (Brain)Hour Hybrids Immune response Immunohistochemistry Infusion procedures Injection of therapeutic agent Intervention Intractable Epilepsy Kainic Acid Laboratories Lead Libraries Light Methods Neurons Operative Surgical Procedures Outcome Patients Peptides Peripheral Personal Satisfaction Pharmaceutical Preparations Plasmids Polymerase Chain Reaction Population Process Protocols documentation Qualifying Rattus Risk Seizures Serotyping Site Slice Structure Techniques Testing Time Transfection Tropism Uncertainty Vertebral column Viral Vector Week adeno-associated viral vector attenuation base beta Actin clinical application directed evolution gene therapy in vivo intravenous administration mutant nervous system disorder novel novel strategies outcome forecast piriform cortex preproneuropeptide Y prevent promoter recombinant virus vector

项目摘要

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.

描述（由申请人提供）：在美国，癫痫症困扰着大约 250 万人，使癫痫症成为最流行的神经系统疾病之一（Hauser 和 Hesdorffer，1990）。尽管目前的抗癫痫药物有效控制了大约 70% 的该人群的癫痫发作，但其余 30% 的药物并不能充分控制癫痫发作（Kwan 和 Brodie，2000）。在这些棘手的病例中，手术切除提供了最后的选择，对于那些符合手术资格的患者，高达 66% 的患者在术后 2 年内保持无癫痫发作（Spencer 等，2005）。然而，长期预后更加不确定，如果癫痫发作部位影响与关键功能相关的大脑部位，则切除手术不是可行的选择（McIntosh 等，2004；Ojemann，1997）。最近，一些研究已经建立了显着影响体内癫痫敏感性的基因治疗方法。使用腺相关病毒（AAV）载体，神经活性肽甘丙肽的表达和组成型分泌可以预防体内癫痫发作（Haberman et al., 2003; McCown, 2006），同时前原神经肽 Y 的表达也可以预防癫痫发作。显着减弱体内癫痫发作活动（Richichi et al., 2004）。但目前，这些病毒载体基因疗法的临床应用需要通过神经外科手术直接注射到大脑中。 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。马赫什里等人。 (2006) 表明，通过易错 PCR 在 AAV 2 衣壳中引入随机变化，随后的定向进化产生了缺乏肝素结合或逃避对 AAV 2 体内免疫反应的 AAV 突变体。在初步研究中，Samulski 实验室表明DNA改组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。由于癫痫发作会损害癫痫活动区域的血脑屏障，因此我们建议通过 AAV 衣壳 1-9 序列的 DNA 改组来开发突变 AAV 文库。然后在红藻氨酸诱导癫痫发作后，将通过静脉注射施用突变体库。通过定向进化技术，将鉴定出新型突变 AAV 载体，在癫痫发作后，它们会穿过受损的 BBB 并转导神经元。这种突变 AAV 载体的成功创建可能会彻底改变难治性癫痫病的治疗。拟议的研究重点是开发一种治疗神经系统疾病的新型基因治疗方法。使用定向进化技术，我们将选择特定的基因传递载体，在外周静脉注射后可以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系统疾病治疗中的应用。