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）：癫痫病在美国约250万人，使癫痫成为最普遍的神经系统疾病之一（Hauser and Hesdorffer，1990）。尽管目前的抗癫痫药有效地控制着大约70％的人群的癫痫发作，但其余30％的药物不能充分控制癫痫发作（Kwan and Brodie，2000）。在这些棘手的病例中，手术切除提供了最终选择，对于那些有资格有手术的患者，多达66％的人在手术后2年保持无癫痫发作（Spencer等，2005）。然而，长期预后更不确定，如果癫痫发作的部位影响与关键功能相关的大脑部位，则切除手术不是一个可行的选择（McIntosh等，2004； Ojemann，1997）。最近，一些研究建立了基因治疗方法，从而显着影响体内癫痫发作敏感性。使用与腺癌相关的病毒（AAV）载体，神经活性肽的表达和组成性分泌可以防止体内癫痫发作活性（Haberman等，2003; McCown，2006），而前尿素肽y的表达也显着降低了疗程的表达。但是，目前，这些病毒载体基因疗法的临床应用将需要直接在大脑中进行神经外科注射。 AAV定向进化的最新发展提供了一种潜在的手段来规避神经外科手术干预措施。 Maheshri等。（2006年）表明，通过在AAV 2上引入易误PCR的随机变化，随后的定向进化导致AAV突变体缺乏肝素结合或避免了对AAV 2的体内免疫反应。向上主义。由于癫痫发作会损害癫痫发作区域中的血脑屏障，因此我们建议通过对AAV CAPSID 1-9序列的DNA进行开发一个突变的AAV文库。然后，在天贺酸诱导的癫痫发作后，将向静脉内施用突变库。通过定向的进化技术，将确定新型的突变体AAV载体，癫痫发作活动后，越过受损的BBB并传播神经元。这种突变的AAV载体的成功创造可能会彻底改变治疗顽固性癫痫疾病的治疗。拟议的研究集中于开发一种用于治疗神经系统疾病的新型基因治疗方法。使用定向的进化技术，我们将选择特定的基因递送车辆，这些递送车在外周静脉内给药后可以进入癫痫发作的大脑。如果成功的话，这种新颖的方法可以大大提高基因治疗在神经系统疾病治疗中的应用。