Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
PLoS One. 2011 Feb 9;6(2):e16825. doi: 10.1371/journal.pone.0016825.
Current antiviral therapy does not cure HIV-infected individuals because the virus establishes lifelong latent infection within long-lived memory T cells as integrated HIV proviral DNA. Here, we report a new therapeutic approach that aims to cure cells of latent HIV infection by rendering latent virus incapable of replication and pathogenesis via targeted cellular mutagenesis of essential viral genes. This is achieved by using a homing endonuclease to introduce DNA double-stranded breaks (dsb) within the integrated proviral DNA, which is followed by triggering of the cellular DNA damage response and error-prone repair. To evaluate this concept, we developed an in vitro culture model of viral latency, consisting of an integrated lentiviral vector with an easily evaluated reporter system to detect targeted mutagenesis events. Using this system, we demonstrate that homing endonucleases can efficiently and selectively target an integrated reporter lentivirus within the cellular genome, leading to mutation in the proviral DNA and loss of reporter gene expression. This new technology offers the possibility of selectively disabling integrated HIV provirus within latently infected cells.
目前的抗病毒疗法无法治愈 HIV 感染者，因为病毒会将其整合的 HIV 前病毒 DNA 作为长寿命记忆 T 细胞中的终身潜伏感染而建立。在这里，我们报告了一种新的治疗方法，旨在通过靶向病毒基因的细胞诱变使潜伏病毒无法复制和致病，从而治愈潜伏感染的细胞。这是通过使用归巢内切酶在整合的前病毒 DNA 内引入 DNA 双链断裂（dsb）来实现的，随后触发细胞 DNA 损伤反应和易错修复。为了评估这一概念，我们开发了一种病毒潜伏的体外培养模型，其中包含一个整合的慢病毒载体，具有易于评估的报告系统来检测靶向诱变事件。使用该系统，我们证明归巢内切酶可以有效地、选择性地靶向细胞基因组中的整合报告慢病毒，导致前病毒 DNA 突变和报告基因表达丧失。这项新技术为选择性地使潜伏感染细胞内的整合 HIV 前病毒失活提供了可能。
