A Novel BIV vector pseudotyped with thogoto virus gp75

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。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。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。慢病毒通常用异源病毒包膜进行假型化，以拓宽载体嗜性。最广泛使用的包膜来源于水泡性口炎病毒糖蛋白（VSV-G）。然而，VSV-G有几个局限性，包括细胞毒性和人补体灭活。因此，已经探索了多种替代包膜用于假型化慢病毒载体，包括杆状病毒gp 64。AVT最近成功地产生了gp 64包膜蛋白假型BIV载体。获得了高载体滴度，载体稳定，并且重要的是，gp 64可以在细胞中组成型表达而没有毒性（在BIV生产细胞系的开发中的重要考虑）。然而，gp 64被人补体灭活。在第一阶段的申请中，AVT将探索使用一种来自Thogoto病毒的新型包膜。Thogoto病毒通过蜱虫转移到人类血液中，因此可能对人类补体具有抗性。有趣的是，Thogoto gp 75包膜糖蛋白显示出与杆状病毒gp 64包膜糖蛋白的显著同源性，这可能是由于Thogoto对节肢动物和人类宿主的向性。AVT已经成功地产生了高滴度的BIV载体假型与托戈托病毒gp 74包膜糖蛋白。该I期项目将重点关注对这种新型载体的进一步评价。第一阶段项目有三个具体目标。具体目的1：评价Thogoto病毒gp 75-假型BIV载体的细胞嗜性。将使用Thogoto gp 75-假型化GFP评估一组细胞系和原代细胞的转导效率。具体目标2：在啮齿动物中评价Thogoto假型BIV载体。VSV-G和杆状病毒gp 64包膜糖蛋白假型载体在啮齿动物视网膜下注射后特异性地抑制RPE细胞。将在啮齿动物中评价Thogoto假型化载体的嗜性。具体目的3：确定Thogoto假型BIV载体的人补体抗性。II期研究将进一步评估BIV载体的物理性质，包括稳定性、Thogoto gp 75细胞毒性和载体纯化策略。

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. 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. 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. Lentiviruses are routinely pseudotyped with heterologous viral envelopes to broaden vector tropism. The most widely used envelope is derived from the vesciular stomatitis virus glycoprotein (VSV-G). However, VSV-G has several limitations including cytotoxicity and inactivation by human complement. Therefore, a variety of alternative envelopes have been explored for use in pseudotyping lentiviral vectors including the baculovirus gp64. AVT was recently successful in the generation of a gp64 envelope protein-pseudotyped BIV vector. High vectors titers were obtained, the vectors were stable, and importantly, gp64 can be constitutively expressed in cells without toxicity (an important consideration in the development of a BIV producer cell line). However, gp64 is inactivated by human complement. In this Phase I application, AVT will explore the use of a novel envelope derived from the Thogoto virus. Thogoto virus is transferred to human blood through ticks, and therefore, may display resistance to human complement. Interestingly, the Thogoto gp75 envelope glycoprotein displays significant homology to the baculovirus gp64 envelope glycoprotein, potentially due to the tropism of Thogoto for arthropods and human hosts. AVT has successfully generated a high titer BIV vector pseudotyped with the Thogoto virus gp74 envelope glycoprotein. This Phase I project will focus on the further evaluation of this novel vector. There are 3 specific aims for this Phase I project. Specific Aim 1: Evaluation of cellular tropism of Thogoto virus gp75- pseudotyped BIV vectors. A panel of cell lines and primary cells will be evaluated for transduction efficiency using Thogoto gp75-pseudotyped GFP. Specific Aim 2: Evaluation of Thogoto-pseudotyped BIV vectors in rodents. Both VSV-G and baculovirus gp64 envelope glycoprotein- pseduotyped vectors specifically transduce RPE cells following a subretinal injection in rodents. The tropism of the Thogoto-pseudotyped vector will be evaluated in rodents. Specific Aim 3: Determination of the human complement resistance of the Thogoto-pseudotyped BIV vector. Phase II studies will further evaluate BIV vector physical properties, including stability, Thogoto gp75 cytotoxicity, and vector purification strategies.