Gene engineering using CRISPR/Cas9 mutagenesis to eliminate latent HIV-1

描述（由申请人提供）：高效抗逆转录病毒疗法（HAART）大大降低了HIV-1感染的发病率和死亡率。然而，这些药物需要每天终身服用。HIV-1耐药菌株的发展和每日服药的坚持困难仍然是一个问题。即使药物有效地抑制了HIV-1，病毒仍然处于整合后潜伏状态，一旦药物治疗中断，这种潜伏状态就会迅速出现。因此，迫切需要针对新的治疗靶点和潜在宿主的其他方法。一种相对较新的方法是对细胞进行基因修饰，使其能够抵抗HIV-1感染。在临床前和临床研究中最先进的方法是敲低HIV-1共受体CCR5。基因修饰的其他目标，如HIV-1基因组本身，已经被考虑过，但有效的传递是限制因素。抗艾滋病毒的功效既需要有效地进入细胞，而且一旦进入细胞，就需要有效地进行基因修饰。锌指核酸酶（ZNFs）、转录激活物样效应核酸酶（TALENs）和归巢内切酶等系统已经实现了高效的基因修饰活性。这些强大的基因组编辑核酸酶已被用于在基因组DNA中产生各种突变，包括替换、缺失和插入，以及基因修饰的转基因动物。然而，设计针对特定基因组位点的核酸酶是相对困难和耗时的。最近，在细菌和古细菌中发现了另一种基因组编辑方法。与之前的基因组修饰活动相比，该系统的优点是简单-切割特定目标序列所需的全部是与目标同源的引导RNA （gRNA）和核酸酶Cas9。该CRISPR/Cas9系统已适用于哺乳动物细胞的基因组修饰，包括高效地生成携带多个基因突变的小鼠。最近，CRISPR/Cas系统被用于敲除潜伏的HIV-1原病毒。尽管在体外具有有效的活性，但上述所有方法都受到HIV-1储存库细胞递送效率低下的限制。在这个提议中要测试的总体假设是，将单个大分子封装在薄聚合物外壳中的纳米技术平台可以有效地将CRISPR/Cas9组件传递到靶细胞中，并使HIV-1前病毒突变，从而终止复制。

DESCRIPTION (provided by applicant): Highly active antiretroviral therapy (HAART) has substantially decreased the morbidity and mortality of HIV-1 infection. However, these drugs require daily, life-long administration. Development of resistant strains of HIV-1 and adherence difficulties with daily drug administration are still a problem. Even when the drugs effectively suppress HIV-1, the virus persists in a post-integration latent state which rapidly emerges once drug treatment is interrupted. Therefore, there is an urgent need for additional approaches directed to new therapeutic targets and targeting latent reservoirs. A relatively recent approach is to gene modify cells such that they are resistant to HIV-1 infection. The approach most advanced in preclinical and clinical studies is knockdown of the HIV-1 co-receptor, CCR5. Other targets for gene modification, such as the HIV-1 genome itself, have been considered, but efficient delivery is the limiting factor. Anti-HIV efficacy requires both efficient delivery into ells and, once delivered, efficient activity for gene-modification. Efficient gene-modification activity has been achieved by a number of systems including zinc-finger nucleases (ZNFs), transcription activator-like effector nucleases (TALENs) and homing endonucleases. These powerful genome editing nucleases have been used to create various mutations including substitutions, deletions and insertions in genomic DNA and for gene- modified transgenic animals. However, the design of nucleases directed to specific genomic sites is relatively difficult and time consuming. Recently, another genome editing method was identified in bacteria and archaea. The advantage of this system over previous genome modifying activities is simplicity- a guide RNA (gRNA) with homology to the target and a nuclease, Cas9, are all that is required to cleave specific target sequences. This CRISPR/Cas9 system has been adapted for genome-modification of mammalian cells including generation of mice bearing mutations in multiple genes with high efficiency. Recently the CRISPR/Cas system has been used to knockout latent HIV-1 provirus. Despite the efficient in vitro activity, all of the above methods ae limited for therapy by inefficient delivery to HIV-1 reservoir cells. The overall hypothesis to be tested in this proposal is that a nanotechnology platform whereby single macromolecules are encapsulated into a thin polymer shell can be used to effectively deliver CRISPR/Cas9 components into target cells and mutagenize the HIV-1 provirus such that replication is aborted.