Semi-Synthetic, Site-Specifically Integrating Lentivirus

描述（由申请人提供）：本项目的总体目标是开发克服病毒载体局限性的新方法。 近年来，通过逆转录病毒转导X-SCID儿童的造血干细胞，首次治愈了遗传性疾病，但同一试验也导致了随机整合的逆转录病毒激活癌基因后的白血病综合征。 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，因为它们甚至可以在非分裂细胞中提供复杂遗传结构的长期基因表达。 然而，重要的安全性问题和生产障碍仍然存在：（i）被有复制能力的慢病毒（RCL）污染，（ii）随机插入诱变引起的肿瘤发生，以及（iii）GMP级、高滴度、稳定的慢病毒包装细胞系不可用。 根据广泛的初步结果，本建议将试图纠正这些重要问题。 在特定目标1中，我们将构建半合成慢病毒载体，其中将重组包膜蛋白或融合肽添加到在不存在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，并通过避免假型化包膜的细胞毒性，极大地促进了稳定的慢病毒包装细胞系的设计。 该策略通过超顺磁性纳米颗粒对病毒体的先前磁化而变得有效。 在具体目标2中，我们将测试这样的假设，即慢病毒载体可以通过用来自phiC 31噬菌体的位点特异性整合酶取代慢病毒整合酶而被工程化以在人类基因组的“非危险”染色体位点处位点特异性地整合，该噬菌体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”的工程原理以及慢病毒文库和筛选筛选的能力，以获得在人类细胞内具有更大内在活性和整合特异性的phiC 31整合酶突变体。 在Specific Aim 4中，我们将联合收割机结合之前的发现，在GMP条件下逐步构建一种包装细胞系，该细胞系可产生高滴度半合成慢病毒载体，能够在人体细胞中进行非危险的位点特异性整合。 这些方法将最终评估其将复杂的β-珠蛋白基因转移到鼠和人造血干细胞中的能力。

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.