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很有前途的基因传递技术，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。先进视觉疗法公司（AVT）开发了一种专有的慢病毒载体系统，该系统基于牛免疫缺陷病毒（BIV），一种与人类疾病无关的动物慢病毒。BIV载体结合了基于hiv的载体的转导效率和基于动物的慢病毒载体系统的安全性优势。重要的是，在相关的啮齿动物模型中，blv载体介导的抗血管生成转基因有效地阻断了视网膜新生血管的形成，这表明AVT载体适合临床应用。慢病毒通常用异源病毒包膜进行假型，以扩大载体的向性。最广泛使用的包膜来源于水疱性口炎病毒糖蛋白（VSV-G）。然而，VSV-G有一些局限性，包括细胞毒性和人类补体的失活。因此，各种替代包膜已被探索用于假分型慢病毒载体，包括杆状病毒gp64。AVT最近成功地生成了gp64包膜蛋白假型BIV载体。获得了高滴度的载体，载体稳定，重要的是，gp64可以在细胞中组成性表达而没有毒性（这是开发BIV产生细胞系的重要考虑因素）。然而，gp64被人补体灭活。在这个I期申请中，AVT将探索使用从Thogoto病毒衍生的新型包膜。蜱虫病毒通过蜱虫传播到人的血液中，因此可能对人体补体产生抗药性。有趣的是，Thogoto gp75包膜糖蛋白与杆状病毒gp64包膜糖蛋白具有显著的同源性，这可能是由于Thogoto对节肢动物和人类宿主的趋向性。AVT已成功地生成了一种高滴度的以Thogoto病毒gp74包膜糖蛋白为假型的BIV载体。这个第一阶段的项目将集中于对这个新载体的进一步评估。第一期项目有三个具体目标。特异性目的1:Thogoto病毒gp75假型BIV载体的细胞趋向性评价。一组细胞系和原代细胞将使用Thogoto gp75伪型GFP评估转导效率。专项目标2：啮齿动物thogoto -pseudotype BIV载体的评价。VSV-G和杆状病毒gp64包膜糖蛋白伪多型载体在啮齿类动物视网膜下注射后特异性转导RPE细胞。将在啮齿动物中评估thogoto -伪型载体的向性。特异性目的3：测定thogoto伪型BIV载体的人类补体耐药性。II期研究将进一步评估BIV载体的物理特性，包括稳定性、Thogoto gp75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.