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插入突变风险的方法，并使潜在危险基因产物的短期表达成为可能。在最近的研究中，nilv在体内被证明能有效地诱导抗原特异性免疫反应，目前正被用作递送锌指核酸酶介导位点特异性基因编辑的平台。然而，需要对NILV系统进行进一步的改进，以使这种有前途的基因传递系统适用于人类临床试验。这些改进包括：a)增加外源基因表达；b)减少非法载体整合；c)开发高效的非免疫原性基因调控系统；d)建立高效的载体生产系统。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计划。在目标1中，我们建议开发一种新的NILV，删除宿主LSF-YY1和AP-4转录抑制复合物识别的顺式抑制元件。这一提议的修饰将减轻nilv典型的转录沉默，并将提高它们的功效。目标2将侧重于以选择性剪接为前提的新型诱导型NILV系统的开发。与目前使用的诱导系统相比，新型剪接调节的nilv不包含合成的反激活因子，这可能会诱导细胞介导的免疫反应。在目标3中，我们将重点研究一种新型多嘌呤通道（PPT）删除NILV的表征和体内测试，它显示出减少非法整合。在最后一个目标中，我们将建立第一个缺乏整合酶的稳定包装细胞系，它将产生具有目标1-3中所述改进的高滴度nilv。所有包含不同修饰的新载体将携带改良的人因子IX (hFIX) cDNA。这些载体将通过瞬时转染或新型稳定包装细胞系产生，它们治疗FIX缺陷的能力将在血友病B小鼠模型中进行测试。

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. PUBLIC HEALTH RELEVANCE: Nonintegrating lentiviral vectors present a means of reducing the risk of insertional mutagenesis in nondividing cells and enabling short-term expression of potentially hazardous gene products. However, several limitations inherent to this promising system limit its utilization in human clinical trials. The goal of the proposed research is to advance the NILV system to the point at which it will be considered suitable for human clinical trials. To this end, we propose a four-aim research plan. In aim 1, we will improve the NILVs' gene expression. Aim 2 will focus on the development of a nonimunogenic inducible lentiviral vector system. In aim 3, we will test the ability of a novel vector with reduced illegitimate integration to support therapeutic levels of transgene expression in vivo. In the last aim, to facilitate large-scale NILV production, we will establish the first stable packaging cell line for NILV.