Lentiviral Transgenesis of the Aqueous Outflow Tract

描述(由申请人提供)：眼内高压是原发性开角型青光眼的主要危险因素，主要由小梁网房水流出障碍引起。表达谱分析和位置克隆正在为这种房水流出失调的遗传基础提供深入的见解。为了了解它们的致病相关性及其在青光眼基因治疗中的治疗价值，这类筛选中涉及的基因需要在小梁网络中以一种受调控的方式稳定表达。然而，在这种独特的、复杂的、有丝分裂静止的组织中，控制转基因有效传递、稳定性和扩展表达的因素得到的研究很少。以前解决这个问题的方法已经能够瞬时过度表达。由于青光眼是一种慢性病，这一限制是根本的。 这一建议的重点是通过在小梁网络中建立稳定和受调控的转基因表达来解决这一根本问题。我们已经使用归一化载体的严格比较来证明Lenti-逆转录病毒(慢病毒)载体介导β-半乳糖苷酶报告基因有效地转导到人的小梁网络。相反，我们证实了肿瘤逆转录病毒载体不能转导小梁网络。此外，基于猫免疫缺陷病毒(FIV)的慢病毒载体在对人体组织进行基因修饰方面与基于HIV-1的慢病毒载体一样有效。 我们假设，基于FIV的慢病毒载体可以在小梁网络中建立稳定的、受调控的转基因表达。绿色荧光蛋白编码的慢病毒载体将被用来在灌流培养和两个大型动物模型中测试和量化转基因在人眼网络中的表达。通过两种不同的策略来建立小梁网络中的调控表达。将确定编码标记基因的慢病毒载体对流出设施的影响，并将其与转导与青光眼发病机制有关的基因myoclin变体的载体的效果进行比较。

DESCRIPTION (provided by applicant): Intraocular hypertesion, the principal risk factor for primary open angle glaucoma, is caused primarily by impairment of aqueous humor outflow through the trabecular meshwork. Expression profiling and positional cloning are yielding insights into the genetic basis of this aqueous outflow dysregulation. In order to understand their pathogenic relevance or their therapeutic value for glaucoma gene therapy, genes implicated in such screens need to be stably expressed in a regulated manner in the trabecular meshwork. However, the factors that govern efficient delivery, stability and extended expression of transgenes in this unique, complex, mitotically-quiescent tissue have received very little study. Previous approaches to this problem have been capable of transient over-expression. Because glaucoma is a chronic disease, this limitation is fundamental. The focus of this proposal is to solve this fundamental problem by establishing stable and regulated transgene expression in the trabecular meshwork. We have used rigorous comparisons of normalized vectors to demonstrate that lenti-retroviral (lentiviral) vectors mediate efficient transduction of a beta-galactosidase reporter gene into human trabecular meshwork. In contrast, we established that onco-retroviral vectors do not transduce the trabecular meshwork. Moreover, feline immunodeficiency virus (FIV)-based lentiviral vectors were as effective as HIV-1 based lentiviral vectors in genetically modifying this human tissue. We hypothesize that FIV-based lentiviral vectors can establish stable, regulated transgene expression in the trabecular meshwork. eGFP-encoding lentiviral vectors will be used to test and quantify transgene expression in the meshwork in human eyes in perfusion culture and in two large animal models. Regulated expression in the trabecular meshwork will be established with two different strategies. Effects on outflow facility of lentiviral vectors encoding marker genes will be determined and compared to effects of vectors transducing variants of myocilin, a gene implicated in glaucoma pathogenesis.