Semi-Synthetic, Site-Specifically Integrating Lentivirus

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。虽然近年来看到了第一次通过逆转录病毒转导X-SCID儿童的造血干细胞来治愈遗传病，但同一试验也导致了由随机整合的逆转录病毒激活癌基因而导致的白血病综合征。防止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，因为即使在未分裂的细胞中，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)被复制能力强的慢病毒(RCL)污染，(Ii)通过随机插入突变致癌，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，本提案将试图纠正这些重要问题。在具体目标1中，我们将构建一个半合成的慢病毒载体，在没有任何病毒被膜基因的情况下，将重组包膜蛋白或融合多肽添加到包装的慢病毒颗粒中。这种方法几乎消除了RCL污染的可能性，并通过避免假分型包膜的细胞毒性，极大地促进了稳定的慢病毒包装细胞系的设计。这一策略通过超顺磁性纳米粒子对病毒粒子的先前磁化而变得有效。在具体目标2中，我们将测试这样一个假设，即慢病毒载体可以通过工程技术在人类基因组的“非危险”染色体位置上整合，方法是将慢病毒整合酶替换为来自phiC31噬菌体的位置特异性整合酶，该整合酶已被证明在人类细胞中起作用。在具体目标3中，我们将应用“体外进化”的工程学原理，结合慢病毒文库和选择筛选的能力，获得在人类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，我们将结合先前的发现，逐步建立GMP条件下的包装细胞系，该包装细胞系能够产生能够在人类细胞中进行非危险的部位特异性整合的高滴度半合成慢病毒载体。这些方法将最终评估它们将复杂的β-珠蛋白基因转移到小鼠和人类造血干细胞的能力。

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.