Development of Novel BIV-based Lentiviral Vectors

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，因为它们能够在体外和体内有效地转导各种未分裂的靶细胞。然而，大多数数据来自对人类免疫缺陷病毒(HIV)的研究，HIV是艾滋病的病原体。高级视觉疗法公司(AVT)开发了一种基于牛免疫缺陷病毒(BIV)的新型慢病毒载体系统，BIV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伪型BIV载体在体外有效地转导了多种未分裂细胞，在体内转导了视网膜和神经细胞，导致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有效地阻断了体内视网膜新生血管的形成，表明该载体适合临床应用。AVT目前正在将这种媒介系统应用于治疗威胁人类视力的眼病。慢病毒载体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，以便高效、可预测地生产、放大和制造载体。目前，以磷酸钙为载体的瞬时转染法主要用于慢病毒载体的生产，但临床应用并不理想。不能产生稳定的包装细胞系是因为细胞系发育所需的两种成分，Gag/Pol和VSV-G Env是细胞毒性的，不能在细胞系中结构性表达。这项第一阶段研究的目标是开发一种稳定的包装细胞系，该细胞系能够构成高滴度的基于BIV的慢病毒载体，并且易于放大和生产。其目的有三个：1.鉴定和消除载体中的核保留序列，以建立一个独立于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BIV载体在体内是否能有效地转导视网膜细胞；3.建立稳定的慢病毒载体包装细胞系。特定目标1的完成将通过从载体中消除病毒RRE序列来改善载体的安全性，并通过消除对Rev.的需要来促进包装细胞系的产生。特定目标2还旨在促进包装线的开发。AVT以前已经解决了GAG/POL的细胞毒性问题，并设计出了能够结构性表达这些蛋白质的细胞系。具体目标2的完成将通过使用GP64信封来解决信封的细胞毒性问题。最后，在特定目标3中创建包装细胞系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.