VP22 AND TAT mediated gene therapy for the CNS

VP22 和 TAT 介导的中枢神经系统基因治疗

• 批准号: 6780658
• 负责人: RAJENDRA KUMAR-SINGH
• 金额: $ 29.9万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-04-01 至 2008-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6780658
• 关键词: Adenoviridae; Alphaherpesvirinae; biotechnology; cyclic GMP; electroretinography; flow cytometry; gene therapy; green fluorescent proteins; immunocytochemistry; laboratory mouse; northern blottings; phosphodiesterases; polymerase chain reaction; protein transport; retina degeneration; tissue /cell culture; transfection /expression vector; virus genetics; virus protein; visual photoreceptor; western blottings

DESCRIPTION (provided by applicant): Adenovirus (Ad) mediated gene transfer is a promising technology for the treatment of many genetic disorders of the Central Nervous System (CNS). Many of the drawbacks that exist with first-generation Ad vectors have been resolved through the use of 'gutted' or 'helper-dependent' vectors. The genetic basis of neurodegeneration is currently best understood in the retina. In addition, relative to the rest of the CNS, the physical accessibility of the eye makes the retina an excellent model system for studying gene therapy for the CNS. Upon ocular delivery, Ad vectors primarily infect the retinal pigment epithelium (RPE) and the Mueller cells of the retina. However, the diseases that most frequently cause blindness are associated with the expression of mutant proteins in the photoreceptor neurons. In our preliminary studies we have found that adenovirus-delivered green fluorescent protein fused to the full length Herpes Simplex Virus (HSV) tegument protein VP22 can translocate from infected cells to uninfected cells in culture or from the RPE to photoreceptors in vivo. This has led to the hypothesis we wish to test in this study: Can HSV VP22 be used to deliver therapeutic proteins to photoreceptor neurons via the RPE? This will be answered using a mouse model of inherited retinal degeneration (rd). Specifically, in this study we propose to 1) Construct an adenovirus vector expressing the protein transduction domains (PTD) of HSV VP22 fused to GFP and compare their ability to traffic GFP in cell culture and in murine retina. 2) Construct an adenovirus vector expressing a fusion between beta PDE and the PTD of HSV VP22. Assess the ability of this virus to express a functional PDE. 3) Administer a gutted adenovirus vector expressing either a VP22-beta PDE fusion to the retina of rd mice (which have a naturally occurring mutation in beta PDE) and assess the effects upon photoreceptor degeneration.

描述(申请人提供)：腺病毒(Ad)介导的基因转移是治疗许多中枢神经系统(CNS)遗传性疾病的一种很有前途的技术。第一代广告载体存在的许多缺点已经通过使用“空的”或“依赖于助手的”载体得到了解决。神经变性的遗传基础目前在视网膜中得到了最好的理解。此外，相对于中枢神经系统的其他部分，眼睛的物理可及性使视网膜成为研究中枢神经系统基因治疗的极佳模型系统。在眼部递送时，Ad载体主要感染视网膜色素上皮(RPE)和视网膜的Mueller细胞。然而，最常导致失明的疾病与光感受器神经元中突变蛋白的表达有关。在我们的初步研究中，我们发现腺病毒携带的绿色荧光蛋白与全长单纯疱疹病毒(HSV)被膜蛋白VP22融合后，在培养中可以从感染细胞转移到未感染细胞，或者在体内从RPE转移到光感受器。这导致了我们希望在这项研究中测试的假设：HSV VP22能否通过RPE将治疗性蛋白输送到光感受器神经元？这将使用遗传性视网膜变性(RD)的小鼠模型来回答。 具体地说，在本研究中，我们建议1)构建表达HSV VP22蛋白转导结构域(PTD)与GFP融合的腺病毒载体，并比较它们在细胞培养和小鼠视网膜中转运GFP的能力。2)构建表达β-PDE和HSV VP22 PTD融合蛋白的腺病毒载体。评估该病毒表达功能性PDE的能力。3)将表达VP22-βPDE融合的内切腺病毒载体注射到RD小鼠的视网膜(其βPDE具有自然发生的突变)，并评估其对光感受器退化的影响。