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，主要由通过小梁网流出的房水受损引起。表达谱分析和定位克隆正在深入了解这种水流出失调的遗传基础。为了了解它们的致病相关性或它们对青光眼基因治疗的治疗价值，涉及这种筛选的基因需要在小梁网中以受调控的方式稳定表达。然而，在这个独特的，复杂的，有丝分裂静止组织中，控制转基因的有效递送，稳定性和延长表达的因素很少受到研究。以前解决这个问题的方法已经能够瞬时过表达。由于青光眼是一种慢性疾病，这种限制是根本性的。 该提案的重点是通过在小梁网中建立稳定且可调节的转基因表达来解决这一根本问题。我们已经使用了严格的标准化载体的比较，以证明慢病毒逆转录病毒（慢病毒）载体介导的β-半乳糖苷酶报告基因到人类小梁网的有效转导。相反，我们确定肿瘤逆转录病毒载体不包裹小梁网。此外，猫免疫缺陷病毒（FIV）为基础的慢病毒载体是有效的HIV-1为基础的慢病毒载体在遗传修饰这一人类组织。 我们假设，FIV为基础的慢病毒载体可以建立稳定的，调节的转基因表达的小梁网。编码eGFP的慢病毒载体将用于在灌注培养中和两个大型动物模型中测试和定量人眼网状结构中的转基因表达。将用两种不同的策略建立小梁网中的调节表达。将确定编码标记基因的慢病毒载体对流出设施的影响，并与转导肌球蛋白（一种与青光眼发病机制有关的基因）变体的载体的影响进行比较。

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.