A Novel BIV vector pseudotyped with thogoto virus gp75

描述(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，因为它们整合到目标细胞的基因组中，并介导转移基因的持续表达。高级视觉疗法公司(AVT)开发了一种基于牛免疫缺陷病毒(BIV)的专利慢病毒载体系统，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优势。重要的是，BLV载体介导的抗血管生成转基因有效地阻断了相关啮齿动物模型中的视网膜新生血管，表明AVT载体适合于临床应用。慢病毒通常与异源病毒包膜一起假型，以扩大载体的趋向性。最广泛使用的包膜来自水泡性口炎病毒糖蛋白(VSV-G)。然而，VSV-G有几个局限性，包括细胞毒性和人类补体的灭活。因此，包括杆状病毒gp64在内的各种可供选择的包膜已被探索用于慢病毒载体的伪分型。AVT最近成功地构建了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可以在细胞中无毒性地组成性表达(这是发展BIV产生细胞系的一个重要考虑因素)。然而，gp64被人类补体灭活。在这一阶段的应用中，AVT将探索使用从托戈托病毒衍生的新型信封。托戈托病毒通过扁虱传播到人的血液中，因此可能对人类补体产生抵抗力。有趣的是，Thogoto gp75包膜糖蛋白与杆状病毒gp64包膜糖蛋白显示出显著的同源性，这可能是由于Thogoto对节肢动物和人类宿主的趋向性。AVT已经成功地获得了与托戈托病毒gp74包膜糖蛋白伪型的高滴度BIV载体。这个第一阶段的项目将集中于对这一新型载体的进一步评估。这一阶段的项目有三个具体目标。具体目的1：评价托果托病毒gp75伪型BIV载体的细胞亲和性。将使用Thogoto gp75伪型GFP评估一组细胞系和原代细胞的转导效率。具体目的2：评价Thogoto伪型BIV载体在啮齿动物中的应用。VSV-G和杆状病毒gp64包膜糖蛋白伪多型载体都能在啮齿动物视网膜下注射后特异性地转导RPE细胞。Thogoto伪型载体的趋向性将在啮齿类动物身上进行评估。特异性目标3：测定Thogoto假型BIV载体的人类补体抵抗力。第二阶段研究将进一步评估BIV载体的物理性质，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.