Novel Lentiviral Vector with Regulatable Gene Expression

描述（由申请人提供）Advanced Vision Therapies，Inc.（AV-T）专注于治疗导致失明的眼部疾病。主要疾病适应症是湿性年龄相关性黄斑变性（AMD），并立即衍生应用于糖尿病增殖性视网膜病变和糖尿病黄斑水肿。疾病病理生理学的特征在于脉络膜或视网膜的异常新生血管形成，导致血管渗漏和出血，并最终导致失明。专注于抑制这一过程的疗法在临床试验中显示出希望;然而，这些疗法必须反复直接注射到眼睛中。需要一个更好的交付系统。AVT开发了一种基于牛免疫缺陷病毒（BIV）的最先进的基因传递系统，BIV是一种非人类病原体的动物病毒。AVT正在将BIV载体系统与新型抗血管生成转基因相结合，以快速开发用于眼部应用的上级产品。这些转基因包括T2-TrpRS，其作用机制基于VEGF功能的抑制，和激肽抑制素，其作用机制导致内皮细胞功能的抑制。这两个过程对于血管生成过程都是至关重要的。 目前，AVI正在利用指导治疗性转基因的组成型表达的载体。转录调控系统的引入将大大提高许多基因治疗策略的实用性、安全性和有效性，包括AVT所追求的眼部治疗。AVT已经开发了一种新的嵌合配体诱导的转录系统，当在无肠腺病毒载体的背景下进行测试时，该系统已经显示出减少眼部新生血管形成的功效。然而，该系统尚未在BIV载体中进行测试。这个关键项目有三个具体目标。1)双BIV载体系统的产生和体外评价。基因调控系统有两个组成部分，一个新的他莫昔芬诱导的转录因子，和响应启动子驱动转基因表达。将这些组分掺入两个单独的载体中，并在体外评价标记基因诱导。2)包含两种系统组件的单个BIV载体的生成和体外评价。3)眼部递送至小鼠后可诱导载体功能的评估。将通过视网膜下注射将Aim 2的载体递送至正常小鼠，并且在他莫昔芬递送后在活动物中定性评价标记基因诱导，并在视网膜全载片中定量评价。

DESCRIPTION (provided by applicant) Advanced Vision Therapies, Inc. (AV-T) is focused on the treatment of ocular diseases that cause blindness. The primary disease indication is wet age-related macular degeneration (AMD), with immediate spin-off applications to diabetic proliferative retinopathy and diabetic macular edema. Disease pathophysiology is characterized by abnormal neovascularization of the choroid or retina resulting in vessel leakage and hemorrhage, and ultimately to blindness. Therapies focused on inhibiting this process are showing promise in clinical trials; however, these therapeutics must be repeatedly injected directly into the eyes. A better delivery system is needed. AVT developed a state-of-the-art gene delivery system based on the bovine immunodeficiency virus (BIV), an animal virus that is not a human pathogen. AVT is combining the BIV vector system with novel, anti-angiogenic transgenes to rapidly develop a superior product for ocular application. These transgenes include T2-TrpRS, whose mechanism of action is based on inhibition of VEGF-function, and kininostatin, whose mechanism of action results in inhibition of endothelial cell function. Both of these processes are critical for the angiogenic process. Currently, AVI is utilizing vectors that direct constitutive expression of the therapeutic transgene. The incorporation of a transcription regulation system would greatly enhance the utility, safety, and efficacy of many gene therapy strategies, including the ocular therapies pursued by AVT. AVT has developed a novel, chimeric ligand-inducible transcriptional system, which has shown efficacy in reducing ocular neovascularization when tested in the context of a gutless adenoviral vector. However, this system has not been tested in a BIV vector. There are three specific aims for this pivotal project. 1) Generation and in vitro evaluation of a two BIV vector system. The gene regulation system has two components, a novel tamoxifen-inducible transcription factor, and the responsive promoter driving transgene expression. These components will be incorporated into two separate vectors and evaluated in vitro for marker gene induction. 2) Generation and in vitro evaluation of a single BIV vector that contains both system components. 3) Evaluation of inducible vector function following ocular delivery to mice. The vector of Aim 2 will be delivered to normal mice via subretinal injection, and marker gene induction will be qualitatively evaluated in live animals following tamoxifen delivery, and quantitatively assessed in retinal whole mounts.