Development of Novel BIV-based Lentiviral Vectors

描述（由申请人提供）：基于慢病毒的基因转移系统是一种很有前途的基因传递技术，因为它们能够在体外和体内有效地转导各种非分裂靶细胞。然而，大多数数据来自人类免疫缺陷病毒（HIV）的研究，HIV是艾滋病的病原体。先进视觉疗法公司（AVT）开发了一种基于牛免疫缺陷病毒（BIV）的新型慢病毒载体系统，这是一种与任何已知人类疾病无关的动物慢病毒。我们的初步数据表明，VSV-G假型BIV载体在体外有效地转导了多种非分裂细胞和体内视网膜和神经细胞，从而实现了持续的转基因表达。此外，BIV载体介导的抗血管生成转基因在体内有效地阻断了视网膜新生血管的形成，表明该载体适合临床应用。AVT目前正将这一载体系统应用于治疗威胁人类视力的眼病。慢病毒载体的临床应用和商业化的主要限制之一是缺乏稳定的载体包装细胞系，以实现高效、可预测的载体生产、规模化和制造。目前，磷酸钙介导的瞬时转染用于慢病毒载体的生产，不适合临床应用。不能产生稳定的包装细胞系是由于细胞系发育所需的两种成分，Gag/Pol和VSV-G Env具有细胞毒性，不能在细胞系中组成性表达。这项I期研究的目标是开发一种稳定的包装细胞系，该细胞系可以产生高滴度的基于hbv的慢病毒载体，并且可以扩大规模和生产。有三个具体目标：1。鉴定和消除载体中的核保留序列，生成独立于Rev/RRE的基因转移系统；2. 验证以杆状病毒GP64包膜蛋白为假型的基于biv的载体在体内有效地转导视网膜细胞；和3。目的：制备稳定的慢病毒载体包装细胞系。特异性目标1的完成将通过消除载体上的病毒RRE序列来提高载体的安全性，并通过消除对rev的需要来促进包装细胞系的生成。特异性目标2也旨在促进包装线的开发。AVT先前已经解决了Gag/Pol细胞毒性问题，并设计了组成性表达这些蛋白质的细胞系。特异性目标2的完成将通过启用GP64包膜来解决包膜细胞毒性问题。最后，在特定目标3中包装细胞系的创建将克服将慢病毒载体应用于人类疾病治疗中最关键的限速步骤。

DESCRIPTION (provided by applicant): Lentiviral based gene transfer systems represent a promising gene delivery technology due to their ability to efficiently transduce a variety of non-dividing target cells in vitro and in vivo. However, most data are from studies using Human Immunodeficiency Virus (HIV), the causative agent of AIDS. Advanced Vision Therapies, Inc. (AVT) has developed a novel lentiviral vector system based on bovine immunodeficiency virus (BIV), an animal lentivirus not associated with any known human disease. Our preliminary data showed that VSV-G pseudotyped BIV vector efficiently transduced a variety of non-dividing cells in vitro and retinal and neuronal cells in vivo, leading to sustained transgene expression. Furthermore, BIV vector mediated delivery of an anti-angiogenic transgene efficiently blocked retinal neovascularization in vivo, suggesting that the vector is suitable for clinical applications. AVT is currently applying this vector system to the treatment of human sight-threatening eye diseases. One of the major limitations to the clinical application and commercialization of lentiviral vectors is the lack of a stable vector packaging cell line for efficient, predictable vector production, scale-up and manufacturing. Currently, calcium phosphate mediated transient transfection is being used for lentiviral vector production, which is not ideal for clinical use. The inability to generate a stable packaging cell line is due to the fact that two components required for the cell line development, Gag/Pol and VSV-G Env, are cytotoxic and cannot be constitutively expressed in a cell line. The goal of this Phase I study is to develop a stable packaging cell line that constitutively produces high titer BIV-based lentiviral vectors and that is amenable to scale-up and manufacturing. There are three specific aims: 1. To identify and eliminate nuclear retention sequences in the vector to generate a gene transfer system that is independent of Rev/RRE; 2. To verify that BIV-based vectors pseudotyped with the Baculovirus GP64 envelope protein efficiently transduce retinal cells in vivo; and 3. To generate a stable lentiviral vector packaging cell line. Completion of specific aim 1 will improve the vector safety profile by eliminating the viral RRE sequence from the vector and will facilitate packaging cell line generation by eliminating the need for Rev. Specific aim 2 is also designed to facilitate packaging line development. AVT has previously solved the problem of Gag/Pol cytotoxicity and has engineered cell lines that constitutively express these proteins. Completion of specific aim 2 will solve the problem of envelope cytotoxicity by enabling the use of the GP64 envelope. Finally, the creation of a packaging cell line in specific aim 3 will overcome the most critical, rate-limiting step in applying lentiviral vectors to the treatment of human disease.