Lentiviral Transgenesis of the Aqueous Outflow Tract

描述（由申请人提供）：眼内高血压是原发性开角型青光眼的主要危险因素，主要由房水通过小梁网流出受损引起。表达谱和位置克隆对这种水流出失调的遗传基础产生了深刻的见解。为了了解它们在青光眼基因治疗中的致病相关性或治疗价值，这些筛选涉及的基因需要在小梁网络中以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，控制转基因的有效传递、稳定性和扩展表达的因素却很少得到研究。以前解决这一问题的方法都有过表达的能力。因为青光眼是一种慢性疾病，这种限制是基本的。

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.