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）已大幅降低HIV-1感染的发病率和死亡率。然而，这些药物需要每天、终身给药。HIV-1耐药株的发展和每日给药的依从性困难仍然是一个问题。即使药物有效地抑制HIV-1，病毒仍然处于整合后潜伏状态，一旦药物治疗中断，病毒就会迅速出现。因此，迫切需要针对新的治疗靶点和靶向潜在储库的其他方法。一种相对较新的方法是对细胞进行基因修饰，使其对HIV-1感染具有抵抗力。临床前和临床研究中最先进的方法是敲除HIV-1辅助受体CCR 5。基因修饰的其他目标，如HIV-1基因组本身，也曾被考虑过，但有效的递送是限制因素。抗HIV功效需要有效递送到细胞中，并且一旦递送，需要有效的基因修饰活性。有效的基因修饰活性已经通过许多系统实现，包括锌指核酸酶（ZNF）、转录激活因子样效应物核酸酶（TALEN）和归巢核酸内切酶。这些强大的基因组编辑核酸酶已用于在基因组DNA中产生各种突变，包括取代、缺失和插入，以及用于基因修饰的转基因动物。然而，针对特定基因组位点的核酸酶的设计相对困难且耗时。最近，在细菌和古细菌中发现了另一种基因组编辑方法。该系统相对于先前基因组修饰活性的优点是简单-与靶具有同源性的向导RNA（gRNA）和核酸酶Cas9是切割特定靶序列所需的全部。这种CRISPR/Cas9系统已被用于哺乳动物细胞的基因组修饰，包括高效地产生携带多个基因突变的小鼠。最近，CRISPR/Cas系统已被用于敲除潜伏的HIV-1前病毒。尽管有有效的体外活性，但所有上述方法由于向HIV-1储库细胞的低效递送而限制了治疗。该提案中要测试的总体假设是，将单个大分子封装到薄聚合物壳中的纳米技术平台可用于有效地将CRISPR/Cas9组分递送到靶细胞中并诱变HIV-1前病毒，从而使复制中止。

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.