VP22 AND TAT mediated gene therapy for the CNS

描述（由申请人提供）：腺病毒（Ad）介导的基因转移是治疗许多中枢神经系统（CNS）遗传疾病的一种很有前途的技术。第一代广告媒介存在的许多弊端已经通过使用“掏空”或“依赖帮手”的媒介得以解决。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绝佳模型系统。在眼部递送后，Ad载体主要感染视网膜色素上皮（RPE）和视网膜的穆勒细胞。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，我们发现腺病毒传递的绿色荧光蛋白与全长单纯疱疹病毒（HSV）被膜蛋白VP22融合，可以在培养中从感染细胞转移到未感染细胞，或在体内从RPE转移到光感受器。这导致了我们希望在本研究中验证的假设：HSV VP22可以通过RPE将治疗性蛋白传递给感光神经元吗？这个问题将在小鼠遗传性视网膜变性（rd）模型中得到解答。

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.