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: 6877021
• 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 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6877021
• 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（由申请人提供）：腺病毒（Ad）介导的基因转移是一种治疗中枢神经系统（CNS）许多遗传性疾病的有前途的技术。第一代Ad载体存在的许多缺点已经通过使用“掏空”或“辅助依赖”载体得到解决。神经变性的遗传基础目前在视网膜中最好理解。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好模型系统。眼部递送后，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感光神经元中突变蛋白的表达有关。在我们的初步研究中，我们已经发现腺病毒递送的与全长单纯疱疹病毒（HSV）被膜蛋白VP 22融合的绿色荧光蛋白可以在培养物中从感染的细胞易位到未感染的细胞或在体内从RPE易位到光感受器。这导致了我们希望在这项研究中测试的假设：HSV VP 22可以用于通过RPE将治疗性蛋白质递送到感光神经元吗？这将通过遗传性视网膜变性（rd）的小鼠模型来回答。 具体而言，在本研究中，我们提出1）构建表达与GFP融合的HSV VP 22的蛋白转导结构域（PTD）的腺病毒载体，并比较它们在细胞培养物和小鼠视网膜中运输GFP的能力。2)构建表达β PDE和HSV VP 22的PTD之间的融合的腺病毒载体。评估该病毒表达功能性PDE的能力。3)将表达VP 22-β PDE融合体的去内脏腺病毒载体给予rd小鼠（其在β PDE中具有天然存在的突变）的视网膜，并评估对感光细胞变性的影响。