Semi-Synthetic, Site-Specifically Integrating Lentivirus

半合成、位点特异性整合慢病毒

• 批准号: 6735800
• 负责人: Philippe Leboulch
• 金额: $ 25.95万
• 依托单位: BRIGHAM AND WOMEN'S HOSPITAL
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-09-19 至 2005-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6735800
• 关键词: Lentivirus; bacterial virus; bioengineering /biomedical engineering; biotechnology; bone marrow transplantation; directed evolution; gene delivery system; gene mutation; gene therapy; genetic library; genetic transduction; immunomagnetic separation; integrase; juvenile animal; laboratory mouse; nonhuman therapy evaluation; oncogenic virus; recombinant virus; synthetic nucleic acid; technology /technique development; thalassemia; transfection /expression vector; virus envelope; virus replication

DESCRIPTION (provided by applicant): The overall goal of this project is the development of novel approaches that overcome the limitations of viral vectors. While recent years have seen the first cure of a genetic disease by retroviral transduction of hematepoietic stem cells in X-SCID children, the same trial has also resulted in leukemic syndromes upon activation of an oncogene by the randomly integrated Retrovirus. Preventing similar adverse events has now become a main goal of the field of gene therapy. Among Retroviruses, Lentiviruses offer unique advantages because they can provide long-term gene expression of complex genetic structures even in non-dividing cells. However, important safety concerns and manufacturing hurdles remain: (i) contamination with a replication-competent Lentivirus (RCL), (ii) oncogenesis by random insertional mutagenesis, and (iii) the unavailability of GMP-grade, high-titer, stable lentiviral packaging cell-lines. On the basis of extensive preliminary results, this proposal will attempt to remedy these important issues. In Specific Aim 1, we will build a semi-synthetic lentiviral vector in which recombinant envelope proteins or fusiogenic peptides are added to lentiviral particles packaged in the absence of any viral envelope gene. This approach virtually eliminates the possibility of RCL contamination and greatly facilitates the design of stable lentiviral packaging cell-lines by avoiding the cytotoxicity of pseudotyping envelopes. This strategy was rendered efficient by the prior magnetization of virions by super-paramagnetic nanoparticles. In Specific Aim 2, we will test the hypothesis that lentiviral vectors can be engineered to integrate site-specifically at "non-dangerous" chromosomal sites of the human genome by substituting the lentiviral integrase with a site-specific integrase from the phiC31 bacteriophage, which has been shown to operate in human cells. In Specific Aim 3, we will apply the engineering principles of "in-vitro evolution" together with the power of lentiviral libraries and selection screens to derive mutants of the phiC31 integrase with greater intrinsic activity and integration specificity within human cells. In Specific Aim 4, we will combine the previous findings to build step-by-step under GMP conditions, a packaging cell-line that produces high-titer semi-synthetic lentiviral vectors capable of non-dangerous site-specific integration in human cells. These approaches will be ultimately evaluated for their capacity to transfer a complex beta-globin gene into murine and human hematopoietic stem cells.

描述（由申请人提供）：该项目的总体目标是开发克服病毒载体局限性的新方法。 尽管近年来首次通过逆转录病毒转导造血干细胞治疗 X-SCID 儿童的遗传性疾病，但同一试验也因随机整合的逆转录病毒激活癌基因而导致白血病综合征。 预防类似的不良事件现已成为基因治疗领域的主要目标。 在逆转录病毒中，慢病毒具有独特的优势，因为即使在非分裂细胞中，它们也可以提供复杂遗传结构的长期基因表达。 然而，重要的安全问题和制造障碍仍然存在：(i) 具有复制能力的慢病毒 (RCL) 污染，(ii) 随机插入突变导致肿瘤发生，以及 (iii) 无法获得 GMP 级、高滴度、稳定的慢病毒包装细胞系。 在广泛的初步结果的基础上，该提案将尝试解决这些重要问题。 在具体目标1中，我们将构建一种半合成慢病毒载体，其中将重组包膜蛋白或融合肽添加到在没有任何病毒包膜基因的情况下包装的慢病毒颗粒中。 这种方法实际上消除了 RCL 污染的可能性，并通过避免假型包膜的细胞毒性，极大地促进了稳定慢病毒包装细胞系的设计。 通过超顺磁性纳米粒子对病毒粒子的预先磁化，使该策略变得有效。 在具体目标 2 中，我们将测试以下假设：通过用来自 phiC31 噬菌体的位点特异性整合酶取代慢病毒整合酶，可以将慢病毒载体设计为在人类基因组的“非危险”染色体位点进行位点特异性整合。已被证明可以在人体细胞中发挥作用。 在具体目标 3 中，我们将应用“体外进化”的工程原理，结合慢病毒文库和选择筛选的力量，衍生出在人体细胞内具有更高内在活性和整合特异性的 phiC31 整合酶突变体。 在具体目标4中，我们将结合之前的发现，在GMP条件下逐步构建一种包装细胞系，产生高滴度的半合成慢病毒载体，能够无危险地定点整合到人体细胞中。 这些方法最终将评估其将复杂的β珠蛋白基因转移到小鼠和人类造血干细胞中的能力。