Novel Strategy for BIV Vector Site-Specific Integration

描述（由申请人提供）：基于慢病毒的基因转移系统代表了一种有前途的基因递送技术，因为它们整合到靶细胞的基因组中并介导转移基因的持续表达。迄今为止，大多数研究使用了基于人类免疫缺陷病毒（HIV）的载体，HIV是一种人类病原体，也是艾滋病的病原体。Advanced Vision Therapies，Inc. (AVT)已经开发了一种基于牛免疫缺陷病毒（BIV）的专有慢病毒载体系统，BIV是一种与人类疾病无关的动物慢病毒。BIV载体联合收割机将基于HIV的载体的转导效率与基于动物的慢病毒载体系统的安全性优点相结合。BIV载体能够在体外转导多种非分裂细胞，在体内转导视网膜、神经元和脾细胞，导致持续的转基因表达。重要的是，BIV载体介导的抗血管生成转基因的递送在相关啮齿动物模型中有效地阻断视网膜新生血管形成，表明AVT载体适合于临床应用。使用整合载体的限制是与染色体插入位点相关的基因表达变异的可能性和整合位点选择导致的正常基因功能破坏的可能性。事实上，插入诱变最近已经成为现实，因为在接受致命疾病X连锁SCID治疗的儿童中发现了三种与基因治疗相关的白血病。使用基于HIV的载体进行的插入位点定位研究发现，虽然插入是一个非特异性事件，但它是非随机的。目前，不存在能够有效的、位点特异性整合的慢病毒载体。这样的系统将具有立即的临床实用性。在这里，我们建议开发和评估一种新的系统来介导定向BIV整合。为了实现位点特异性整合，我们建议将缺乏其天然细胞DNA结合活性的修饰的BIV整合酶融合到位点特异性DNA结合蛋白，目的是将整合引导到特定的靶位点。这个关键项目有三个具体目标。具体目标1。鉴定缺乏细胞DNA结合活性的BIV整合酶突变体。将生成一系列整合酶突变体，并在新型体外试验中评价其是否缺乏非特异性DNA结合活性。具体目标2。修饰的BIV整合酶-DNA结合融合蛋白的构建。来自特异性目的1的最有前途的整合酶突变体将与新型DNA结合蛋白融合，并在体外评估整合酶和特异性DNA结合功能。具体目标3。将整合酶融合蛋白掺入BIV载体中并评价位点特异性整合。将生成编码修饰的整合酶的载体，并将载体插入位点作图并与未修饰的载体的那些进行比较。II期研究将集中于在眼部新生血管的啮齿动物模型中评价位点特异性整合和载体功效。

DESCRIPTION (provided by applicant): Lentivirus-based gene transfer systems represent a promising gene delivery technology, as they integrate into the genome of the target cell and mediate sustained expression of the transferred gene. To date, the majority of studies have used vectors based on the Human Immunodeficiency Virus (HIV), a human pathogen and the causative agent of AIDS. Advanced Vision Therapies, Inc. (AVT) has developed a proprietary lentiviral vector system based on the bovine immunodeficiency virus (BIV), an animal lentivirus not associated with human disease. The BIV vectors combine the transduction efficiency of the HIV-based vectors with the safety advantages of animal-based lentiviral vector systems. The BIV vectors are capable of transducing a variety of non-dividing cells in vitro, and retinal, neuronal, and spleen cells in vivo, leading to sustained transgene expression. Importantly, BlV-vector mediated delivery of an anti-angiogenic transgene efficiently blocked retinal neovascularization in a relevant rodent model, suggesting that the AVT vector is suitable for clinical applications. A limitation to the use of integrating vectors is the potential for variations in gene expression associated with the chromosomal insertion site and the potential for disruption of normal gene function caused by integration site selection. Indeed, insertional mutagenesis has recently become a reality following the discovery of three gene therapy-related leukemias in children treated for the fatal disease, X-linked SCID. Insertion site mapping studies using HIV-based vectors have found that while insertion is a non-specific event, it is nonrandom. Currently, a lentiviral vector capable of efficient, site- specific integration does not exist. Such a system would have immediate clinical utility. Here, we propose to develop and evaluate a novel system to mediate directed BIV integration. To achieve site-specific integration, we propose to fuse a modified BIV integrase lacking its native cellular DNA binding activity to a site-specific DNA binding protein with the intent of directing integration to specific target site. There are three specific aims to this pivotal project. Specific Aim 1. Identification of BIV integrase mutants that lack celluar DNA binding activity. A series of integrase mutants will be generated and evaluated for lack of non-specific DNA binding activity in a novel in vitro assay. Specific Aim 2. Construction of modified BIV integrase-DNA binding fusion proteins. The most promising integrase mutants from Specific Aim 1 will be fused to a novel DNA binding protein and evaluated for integrase and specific DNA binding functions in vitro. Specific Aim 3. Incorporation of the integrase fusion protein into the BIV vectors and evaluation of site-specific integration. Vectors encoding the modified integrase will be generated and vector insertion sites mapped and compared to those of the unmodified vector. Phase II studies will focus on the evaluation of site-specific integration and vector efficacy in rodent models of ocular neovascularization.