Nonintegrating Lentiviral Vectors Towards Clinical Trials

描述（由申请人提供）：非整合慢病毒载体（NILV）提供了一种降低非分裂细胞中插入诱变风险并使潜在危险基因产物短期表达的方法。在最近的研究中，NILV被证明在体内诱导抗原特异性免疫应答方面是有效的，并且目前被用作递送锌指核酸酶以介导位点特异性基因编辑的平台。然而，需要在NILV系统中进行额外的改进，以使这种有前途的基因递送系统适合于人类临床试验。这些改进包括：a）增加附加型基因表达，B）减少非法载体整合，c）开发有效和非免疫原性的基因调控系统，和d）建立有效的载体生产系统。为了实现这些改进，我们在这里概述了一个由四个目标组成的研究建议。在目的1中，我们提出了开发一种新的NILV删除的顺式抑制元件识别的主机LSF-YY 1和AP-4转录抑制复合物。这种提出的修饰将减轻NILV典型的转录沉默，并将提高其功效。第二个目标是开发一种新的基于选择性剪接的可诱导NILV系统。与目前使用的诱导系统相反，新的剪接调节NILV不包含合成的反式激活因子，其可能诱导细胞介导的免疫应答。在目标3中，我们将专注于表征和体内测试的一种新的聚嘌呤道（PPT）删除NILV，表现出减少非法整合。在最后一个目标中，我们将建立第一个整合酶缺陷的稳定包装细胞系，其将产生具有目标1-3中所述的改进的高滴度NILV。所有含有不同修饰的新载体将携带改进的人因子IX（hFIX）cDNA。将通过瞬时转染或通过新型稳定包装细胞系生产载体，并将在血友病B小鼠模型中检测其治愈FIX缺陷的能力。

DESCRIPTION (provided by applicant): Nonintegrating lentiviral viral vectors (NILVs) present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling the short-term expression of potentially hazardous gene products. In recent studies, NILVs proved efficient at inducing an antigen specific immune response in vivo and are currently being used as a platform to deliver zinc finger nucleases to mediate site-specific gene editing. However, additional improvements in the NILV system are required to render this promising gene delivery system suitable for human clinical trials. These improvements include: a) increasing episomal gene expression, b) reducing illegitimate vector integration, c) developing an efficient and nonimmunogenic gene regulation system, and d) establishing an efficient vector production system. To achieve these improvements, we outline here a research proposal consisting of four aims. In aim 1 we propose to develop a novel NILV deleted of the cis inhibitory elements recognized by the host LSF-YY1 and AP-4 transcriptional repressor complexes. This proposed modification will alleviate the transcriptional silencing typical of NILVs and will improve their efficacy. Aim 2 will focus on the development of a novel inducible NILV system premised on alternative splicing. In contrast to currently used inducible systems, the novel splicing-regulated NILVs do not contain a synthetic transactivator, which could potentially induce a cell-mediated immune response. In aim 3, we will focus on characterization and in vivo testing of a novel polypurine tract (PPT) deleted NILV, which exhibits reduced illegitimate integration. In the last aim, we will establish the first integrase-deficient stable packaging cell line, which will produce high-titer NILVs bearing the improvements described in aims 1-3. All new vectors containing the different modifications will carry an improved human factor IX (hFIX) cDNA. The vectors will be produced either by transient transfection or by the novel stable packaging cell line, and their ability to cure FIX deficiency will be tested in a hemophilia B mouse model.